Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

Estimates of sensitivity, specificity, false rates and expected proportion of

population testing positive in screening tests
Authors: ABSTRACT:
Journal of Research in Biology

Oyeka Ikewelugo Cyprian

Anaene 1, Okeh Uchechukwu
Marius 2, Igwebuike Victor
Onyiaorah3, Adaora Amaoge
Onyiaorah4 and Chilota
Chibuife Efobi 5 This paper proposes and presents indices used as measures to evaluate or
Institution: assess results obtained from diagnostic screening tests. These indices include
1. Department of Applied sensitivity, specificity, prevalence rates and false rates. We here present statistical
Statistics, Nnamdi Azikiwe methods for estimating these rates and for testing hypotheses concerning them. An
University, Awka Nigeria. estimate of the proportion of a population expected to test positive in a diagnostic
2. Department of Industrial screening test is also provided. Further interest is also to estimate the sensitivity and
Mathematics and Applied specificity of the test and then the false rates as functions of sensitivity and specificity
Statistics, Ebonyi State given knowledge or availability of an estimate of the prevalence rate of a condition in
University Abakaliki, Nigeria.
a population. The indices proposed ranges from -1 to 1 inclusively and therefore
3. Department of enables the researcher to determine if an association exists and if it exists between
Histopathology, Nnamdi test results and condition as well as whether it is positive and direct or negative and
Azikiwe University Teaching
indirect which will serve as an advantage over the traditional methods. The proposed
Hospital Nnewi Anambra State,
Nigeria. indices provide estimates of the test statistic. When the proposed measures are
applied, results indicate that it is easier to interpret and understand more than those
4. Department of obtained using the traditional approaches. In addition, the proposed measure is
Opthalmology, Enugu State
University Teaching Hospital shown to be at least as efficient and hence as powerful as the traditional methods
Park lane Enugu State, Nigeria. when applied to sample data.
5. Department of Haematology,
University of Port Harcourt
Teaching Hospital, Port
Harcourt, Rivers State Nigeria.

Corresponding author: Keywords:

Okeh Uchechukwu Marius Traditional odds ratio, prevalence, sensitivity, specificity, false rates.

Article Citation:
Email Id:
Oyeka Ikewelugo Cyprian Anaene, Okeh Uchechukwu Marius, Igwebuike Victor
Onyiaorah, Adaora Amaoge Onyiaorah and Chilota Chibuife Efobi
Estimates of Sensitivity, Specificity, False Rates and Expected Proportion of Population
Testing Positive in Screening Tests
Web Address: Journal of Research in Biology (2014) 4(8): 1498-1504
http://jresearchbiology.com/
documents/RA0391.pdf
Date:
Received: 06 Nov 2013 Accepted: 15 Jan 2014 Published: 15 Nov 2014

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1498-1504 | JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
In diagnostic screening tests indices used as
measures to evaluate or assess results obtained include,
sensitivity and specificity of the test and if the prevalence
rate of a condition of interest in a population is known or
can be estimated from a previous study, also the false
positive and false negative rates of the test as well as the
proportion of the population expected to test positive to
the condition (Fleiss, 1973; Pepe, 2003). Hence research
interest is often in statistical methods for estimating
sensitivity, specificity, false rates and the proportion of a
population expected to test positive to a condition in
these screening tests. The sensitivity of a test is the
proportion of subjects testing positive among the subjects
known or believed to actually have a condition in nature,
while the specificity of a test is the proportion of subjects
who actually test negative to a condition among the
subjects known or believed not to actually have the
condition in nature. False positive rate of a test is the
proportion of subjects who are known or believed not to
actually have a condition in nature among the subjects
testing positive, while false negative rate is the
proportion of subjects who are known or believed to
actually have a condition in nature among the subjects
who never-the-less test negative (Fleiss,1973;Greenberg
et al., 2001;Linn, 2004).Sensitivity and Specificity of a
test are independent of the population being studied and
hence independent of the prevalence rate of a condition
in the population. False rates of a test on the other hand
are functions of the prevalence rate of a condition in a
population and hence are dependent on the population of
interest (Fleiss, 1973;Linn, 2004).
We here present statistical methods for
estimating these rates and for testing hypotheses
concerning them. An estimate of the proportion of a
population expected to test positive in a diagnostic
screening test is also provided.
Given that a researcher collects a random sample
of n.1 subjects known or believed, perhaps on the basis of
1499
Anaene et al., 2014
previous results from a gold standard test to actually
have a certain condition in nature from a population and
also takes a second random sample of n.2 subjects from
the same population Keeping in mind that known or
believed not to actually have the same condition in
nature, thus giving a total random sample of size
n=n..=n.1+n.2 subjects to be studied. It is always treated
to confirm through a diagnostic screening test for
whether or not each sampled subjects have or does not
have the condition of interest. Further interest is also to
estimate the sensitivity and specificity of the test and
then the false rates as functions of sensitivity and
specificity given knowledge or availability of an estimate
of the prevalence rate of a condition in a population.
Now suppose B and B are respectively the
events that a randomly selected subject from a
population has and does not have a condition in nature.
Also let A and A be respectively the events that the
randomly selected subject tests positive, and negative to
the condition in the test. We here assume that the
prevalence rate P(B) of the condition in the population is
either known or can be reliably estimated from previous
studies. The results of such a screening test may be
presented in the form of a four fold Table (Table 1).
In Table 1 above, of the n=n.. sample subjects
studied, n.1 subjects are known or believed to have the
condition in nature, that is in B and n.2 are known or
B and
believed not to have the condition in nature, that is B .
Also n1. subjects respond positive that is in A and n2.
subjects respond negative, thatAisand
in A . Of the n.1
subjects in B, n11 subjects actually have the condition
and test positive that is in AB and n21 subjects actually
have the condition but test negative, that is in AB .
Of the n.2 subjects who are known or believed not to
have the condition in nature,n12 subjects who do not have
the condition test positive, that is in A B w h i l e
n22 subjects who do not have the condition in nature also
test negative that is in A B. In an actual screening
test usually only the total sample size n=n..,n.1 subjects
Journal of Research in Biology (2014) 4(8): 1498-1504
Anaene et al., 2014

Table 1.Format for Presentation of Results of a Diagnostic Screening Test

Screening Test Results Condition Present Condition Absent Total
(B) B (ni.)
Positive (A) n11 n12 n1.
Negative () n21 n22 n2.
Total (n.j) n.1 n.2 n..(=n)

in B, n11 subjects in AB,n.2 subjects in B

a n d n 2 2 known or believed not to have the condition in nature.
subjects in A B are observed and actually known. Notationally, we have that
The values n12 in AB and n21 in AB are not P(A) = P (AB) + P (AB ) 3
known and hence also are n1. and n2., the overall number Now to develop sample estimates of these indices,
of subjects who would test positive and negative sensitivity for instance, we may let,
respectively in the screening test. Hence only the known 1, if the ith randomly selected and screened
values namely total sample size n, the number of subject known or believed to actually
ui1
have a condition in nature tests positive
subjects, n.1 known to have the condition in nature, n11,
0, otherwise 4
the number of subjects who test positive among these for i 1, 2,...n.1 subjects
known to have the condition in nature, the number of Let
Let
subjects n.2 known not to have the condition in nature 1=1 P (ui1 =P1) ui1 1 5
and n22 subjects who test negative among the subjects and
and
known not to have the condition in nature are used here n1.
Wi =
W ui1
to estimate the required indices and test statistics. Now 1
i 1 6
the sensitivity (Se) and specificity (Sp) of a screening
test expressed in terms of conditional probabilities or Now the expected value and variances of ui1 are
specific rates of events A and B are respectively E ui1 : Var ui1 1
1 1 1 7
Se P ( A / B ); Sp P( A / B ) 1 1
The higher Se and Sp are more sensitive and Similarly the expected value and variance of are
specific is the screening test, the lower these rates, the respectively
weaker are the sensitivity and specificity of the test. The E W1
n1.
E (ui1 ) n.1 ;Var W1
n1.
Var (ui1 ) n.1 1 8
1 1 1
i 1 i 1 8
false positive rate and the false negative rate of a
screening test also expressed in terms of conditional Now 1 is the probability that a randomly
probabilities or specific rates of events A and B are selected and screened subject known or believed to have
respectively a condition in nature in a population tests positive; that is
1 P( A / B ) P( B ) 1 P( A / B ) P ( B ) the proportion of subjects testing positive among the
F ve P( B / A) ; F ve P( B / A) 2 2
P( A) P( A) subjects in the population known or believed to actually
Where P(A) consists of the probability of have a condition in nature. This is in fact a measure of
composition of the events AB and which is the the sensitivity Se of the screening test. The sample
probability of the union of events that a randomly estimate of 1 is
selected subject tests positive and is known or believed W1 f
1 Se 9
n.1 n.1
to have a condition in nature or tests positive and is

Journal of Research in Biology (2014) 4(8): 1498-1504 1500

 Anaene et al., 2014

Where f+ is the number of subjects who test condition given that the subject is known or believed not
positive among subjects in the population known or to actually have the condition in nature. In other words,
believed to have the condition of interest in nature. In 2 is the proportion of subjects testing negative among
+
other words, f is the number of 1s in the frequency the population of subject known or believed not to have a
distribution of the n.1values of 1s and 0s in ui1,for i=1,2, condition in nature. Thus 2 is actually a measure of the
+
,n.1.Hence f =n11 of Table 1. specificity Sp of the screening test. Its sample estimate is
The corresponding variance of is from equation (8) from equation (18)

Var W1 1 (1 1 ) (1 Se
Se ) W2 f
Var 1
Var Se 2 10 Se 19
n.11 n.1 n.1 10 n.2 n.2
-
A researcher may sometimes wish to test a null Where f is the number of subjects whose test
hypothesis that sensitivity of a screening test is at most negative among the n.2 subjects in the sampled
some value 10 = Se0 . That is the null hypothesis, population known or believed not to have a condition in
H 0 : Se Seo versus H1 : Se Seo(0 Seo 1) 11other words f - is the total number of 1s in the
nature. In
10 11
This null hypothesis may be tested using the test statistic frequency distribution of the n.2 values of 0s and 1s in
ui2,for i=1,2,n.2.Thus f - = n22 in Table 1. The variance
2
2
n.1 Se Seo 1 10
2
2
W1 n.1 Seo n.1
12
Var W1 (1
Se )
Se 1 (1 1 ) 12
of equation 2 Sp (18)
Which under Ho has approximately the chi-square Var (W2 ) 2 (1 2 ) (1 Sp
Sp )

Var 2 Var Sp
n.22 n.2 n.2 20 20
distribution with one (1) degree of freedom for
sufficiently large n.1.the null hypothesis Ho is rejected at A researcher may also wish to test a null hypothesis that
the level of significance if specificity Sp of a diagnostic screening test is at least
2 2 some value That is the null hypothesis
1 ;1 13 13
Ho : Sp Spo versus H1 : Sp Spo, (0 Sp 1)
21
Similarly to develop a sample estimate of the specificity This null hypothesis is tested using test statistic
Sp of a screening test, we may let W2 n.2 Spo
2
n.2 Sp Spo
2
n.2 2 20
2
2
22 22
1, if the ith randomly selected and screened Var W2 (1
Sp )
Sp 2 (1 2 )
subject in the population is known or believed not to actually
ui 2
have a condition in nature tests negative
Which under Ho has approximately the chi-
0, otherwise 14 square distribution with one (1) degree of freedom for
for i 1, 2,...n.2 subjects
sufficiently large n.2. The null hypothesis Ho is rejected
Define at the level of significance if equation (13) is satisfied,
15
2 P ui 2 1 otherwise Ho is accepted.15
And To develop sample estimate of the proportion of
n.2
W2 ui 2 16 a population expected to
16test positive to a condition in a
i 1
diagnostic screening test, we note that when expressed in
Now
terms of conditional probability using Bayes rule
E ui 2 2 ;Var ui 2 2 1 2 17 17
equation (3) becomes
And
P( A) P( A / B).P( B) P( A / B ).P( B ) P( A / B).P( B) 1 P( A / B ) 1 P( B) 23
E W2 n.2 2 ;Var W2 n.2 2 (1 2 ) 18 18
Or when expressed in terms of sensitivity Se and
Note that 2 is the probability that a randomly specificity Sp of the screening test and prevalence rate
selected and screened subject tests negative to the P(B) of a condition in a population becomes
1501 Journal of Research in Biology (2014) 4(8): 1498-1504
Anaene et al., 2014
P( A) SeP( B) (1 Sp).P( B) 1 P( A / B) 1 P( A / B) P( A / B) .P( B) 1 Sp 1 Se Sp .P( B) 24 The null hypothesis Ho is expected at the level of
The sampled estimate of P(A) is using equation ( 9) and significance if equation (13) is satisfied; otherwise Ho is
equation (19) in equation (24) accepted.
P ( A) P( A) ( B)
SeP ).P( B )
(1 Sp
1 Sp
1 Se .P( B)
Sp 25 The researcher may also wish to obtain sample estimates
The corresponding sample variance is of false rates in a diagnostic screening test if the
)( P( B))
Var ( P( A)) Var (Se 2 ) P(( B))
Var (Sp 2 )
2P( B).P( B).Cov SeSp 26 prevalence rate P(B) of a condition in a population is
It is easily shown that known or can be determined.
W1 W2 Now from equations (2) and (25), the sample estimate of
; Sp
Cov ( Se ) Cov ; 0
n.1 n.2 false positive rate in terms of sample estimates of
To prove this it is sufficient to show that sensitivity and specificity and the known or estimated
Cov ui1 ; ui 2 0 prevalence rate is
Now ).P( B ) 1
Sp 1 P( B)
(1 Sp
F ve 31
Cov ui1 ; ui 2 E ui1 .ui 2 E (ui1 ).E (ui 2 ) E ui1 .ui1 .P( B) 31
1. 2 .
P( A) 1 Sp 1 Se Sp

Now ui1 .ui1can assume only the values 1 and 0 . Similarly the sample estimate of false negative rate is
It assumes the value 1 if ui1 and ui2both assume the value from equations (2) and (25)
1 with probability it assumes the value 0 if assumes (1 ).P( B)
Se 1 P( B)
Se

F
the values 1 and ui2 assumes the value 0 or ui1 assumes
ve
P ( A)
Sp 1
Se .P( B)
Sp 32
the value 0 and ui2 assumes the value 1 with probability Where e and p are given in equation (30).
1(1-2) - 2(1-1) Hence Finally with further interest the researcher may use some
Cov ui1 ; ui 2 1 2 1 2 0 so that elementary calculus or apply Fiellers convenience
(1 Se
Se ) Sp
(1 Sp ) 1 (1 1 ) 2 (1 2 ) Theory to obtain approximate estimates of the variances
) Var ( Sp
Var ( P( A)) Var ( Se ) 27
n.1 n.2 n.1 n.2
of F ve and F ve and also test any desired hypotheses.
The researcher may also wish to test the null
hypothesis that the proportion P(A) of subjects in a ILLUSTRATIVE EXAMPLE
population expected to test positive to a condition in a It a clinician is collecting a random sample of 98
diagnostic screening test is at most some value Po(A). subjects from a certain population; twelve of whom are
That is the null hypothesis doubted for having prostrate cancer and 86 of whom are
Ho : P( A) Po( A) versus H1 : P( A) Po( A), (0 Po( A) 1) 28 assumed not to28have the disease. The clinicians interest
This null hypothesis is tested using the test statistic is to confirm through a diagnosis screening test whether
2 or not each of the sampled subjects are actually prostrate
2
P ( A) Po( A)
29
Var P ( A) 29 cancer positive or negative. The results of the screening
test are presented in Table 2.
Which under Ho has approximately the chi-square Now from Table 2 we have that the sample
distribution with one (1) degree of freedom where P(A) estimate of the sensitivity and specificity of the test are
and Var (P(A)) are given by equations (25) and (26) respectively
respectively and from Table 1 f
n f
n 98 4
98
Se 8.167 0.041 0.335
n.1 n.1 12
f1 n11 f2 n22 and
Se 1 ; Sp 2 30 30
n.1 n.1 n.2 n.2 n f
98 84
f n 98

Sp 1.140 0.857 0.977.
n.2 n.2 86

Journal of Research in Biology (2014) 4(8): 1498-1504 1502

 Anaene et al., 2014

These results show that the screening test is low Table 2: Result of Prostrate Cancer Screening Test
in sensitivity but has high specificity. Clinical diagnosis Present (B) Absent B
Now from equations (13) and (14) the sample estimates Prostrate Cancer n11=f + +=4 n12=f+ - =4 n1.= 6
of are respectively Positive (A) n21=f - +=4 n22=f - - =4 n2.=92
12 0.335 86 0.977 Negative () n.1=12 n.2=86 n..=n=98
0.898
98
and
12 1 0.335 86 1 0.977 expected to be correctly informed. This probably makes
0.102
98 more difficulty in understanding than the simple
Hence from equations (15) and (17) we have that information conveyed by the simple difference in rates,
0.898 0.102 0.796 0.796, namely, the proportion of subjects
With estimated variance obtained from equations (16) testing positive among subjects who have prostrate
and (18) as cancer or testing negative among subjects who do not
1 (0.796) 2 have prostrate cancer is 79.6 percent higher than the
Var ( ) Var ( ) 0.004
98 proportion of subjects testing positive among subjects
Hence the test statistic of no association between who do not have prostrate cancer or testing negative
screening test results and state of nature or condition among subjects who have the disease.
(Prostrate Cancer) of equation (19) are obtained from 1 1 1 1 1 1 1 1
Se(O) O 21.00
equation (20) as n11 n12 n21 n22 4 2 8 84
2 (21.00)(0.942) 19.782
2
0.796
0.634
158.500( P value 0.0000)
0.004 0.004 This measure of the error of O namely 19.782
Which with one (1) degree of freedom is highly is clearly much larger than the error of only
statistically significant indicating a strong degree of Se 0.004 0.063 of the estimated value of for our
association between screening test results and state of sample data. The chi-square test statistic for the
nature or condition (presence of Prostrate cancer in significance of O is
the population). Also since 0.796 n(n11n22n12 n21)2 98(94)(84)-(2)(8)2
X2= = =17.616 (p-value=0.0000)
is positive, the association is positive and direct. n1.n2.n1.n2 (6)(92)(12)(86)
It is commendable to compare the present results with Which is also statistically significant again leading to a
what would have been obtained if we have used the rejection of the null hypothesis of no association.
traditional odds ratio to analyze the data of Table 2. In However, the proposed method and the traditional odds
spite of odds ratios short comings as already pointed out ratio approach explained here (both) lead to a rejection of
above, when used in the analysis of screening test results. the null hypothesis, the relative sizes of the calculated
The sample estimate of the traditional odds ratio for the chi-square values suggest that the traditional odds ratio
data of Table 2 is method is less efficient and likely to lead to an
n11 n22 (4)(84) acceptance of a false null hypothesis (Type II Error)
O 21.00
n12 n21 (2)(8) more frequently and hence is likely to be less powerful
This means, for every subject who has prostrate cancer than the proposed method.
among tested subjects and erroneously informed that
they are free of the disease (21 subjects) among those
tested and found to have prostrate cancer would be
1503 Journal of Research in Biology (2014) 4(8): 1498-1504
Anaene et al., 2014

CONCLUSION
In this paper, we proposed, developed and
presented a statistical method for measuring the strength
of association between test results and state of nature or
condition in a population expressed to a diagnostic
screening test. The proposed measure is based on only
the sensitivity and specificity of the screening test which
are independent of the population of interest and
estimated using only observed sample values.
The proposed measure which ranges from -1 to 1
can be used to establish whether an association is strong
and direct, strong and indirect or zero estimates of the
standard error. Test statistics for the significance of the
proposed measure are provided. The proposed measure
of association is shown to be easier to interpret and
explain than the traditional odds ratio, and the sample
data used suggest that the measure is at least as efficient
and powerful as the traditional odds ratio.

REFERENCE
Baron JA and Sorensen HT. 2010. Clinical
epidemiology, in Teaching Epidemiology: A guide for
teachers in epidemiology, public health and clinical
medicine. eds. Olsen J; Saracci R and Trichopoulos D;
Oxford: Oxford University Press, 237-249.

Fleiss JL. 1973. Statistical Method for Rates and

Proportions. John Wiley, New York.

Greenberg RS, Daniels SR, Flanders WD, Eley JW

and Boring JR. 2001. Medical Epidemiology, London:
Submit your articles online at www.jresearchbiology.com
Lange-McGraw- Hill.
Advantages
Pepe MS. 2003. The Statistical Evaluation of Medical Easy online submission
Complete Peer review
Tests for Classification and Prediction. Oxford statistical Affordable Charges
series 28, Oxford: University Press, U.K. Quick processing
Extensive indexing
Shai Linn. 2004. A new Conceptual Approach to You retain your copyright
teaching the interpretation of clinical tests. Journal of submit@jresearchbiology.com
www.jresearchbiology.com/Submit.php
statistics education 12(3). www.amstat.org/publications/
jse/v12n3/linn.html.

Journal of Research in Biology (2014) 4(8): 1498-1504 1504

 Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

Ferulic acid modulates ultraviolet-B radiation mediated inflammatory

signaling in human dermal fibroblasts
Journal of Research in Biology

Authors: ABSTRACT:
Kanagalakshmi A1, Ultraviolet B (UVB 290-320 nm) participate in the development of the
Agilan B1, Mohana S1, cutaneous inflammatory response which includes a cascade of events that involves
Ananthakrishnan D2, increased expression of cyclooxygenase-2 (COX-2), release of tumor necrosis factor-
Velmurugan D2, alpha (TNF-) and other inflammatory cytokines. Peroxisome proliferator-activated
Karthikeyan R1, receptors (PPAR/) are considered to be potential targets for photo protection
Ganesan M1, Srithar G1 because they inhibit UVB mediated inflammatory responses. In this study, we
and Rajendra Prasad N1* investigated the effect of ferulic acid on UVB-radiation induced expression of TNF-
and COX-2 in human dermal fibroblasts (HDFa). Further, the action of ferulic acid on
Institution:
PPAR/ activation and its binding interaction with these proteins were analyzed by
1. Department of
Biochemistry and induced fit docking. We found that onetime UVB exposure (19.8 mJ/cm 2) showed
Biotechnology, Annamalai significantly increased the expressions of COX-2 and TNF- in HDFa after 4 h post-
University, Annamalainagar irradiation when compared to the control cells. Ferulic acid pretreatment for 30 min
608 002, India. before UVB exposure prevented UVB-induced overexpression of these inflammatory
markers. It has also been found that ferulic acid activates PPAR/ expressions in
2. Bioinformatics HDFa. Further, induced fit docking analysis showed that there was a greater binding
Infrastructure Facility interaction of ferulic acid with PPAR than PPAR. Thus, ferulic acid exhibits
(BIF),University of Madras, beneficial effects against UVB-induced inflammatory responses probably through
Chennai-25 down-regulating COX-2 and TNF- expressions and activating PPAR/ agonists.

Corresponding author: Keywords:

Rajendra Prasad N Ultraviolet B radiation, Ferulic acid, Human dermal fibroblasts, Inflammatory
markers, Photoprotection

Article Citation:
Email Id:
Kanagalakshmi A, Agilan B, Mohana S, Ananthakrishnan D, Velmurugan D,
Karthikeyan R, Ganesan M, Srithar G and Rajendra Prasad N.
Ferulic acid modulates ultraviolet-B radiation mediated inflammatory signaling in
human dermal fibroblasts
Journal of Research in Biology (2014) 4(8):1505-1515
Web Address:
http://jresearchbiology.com/ Dates:
documents/RA0488.pdf Received: 06 Oct 2014 Accepted: 25 Oct 2014 Published: 17 Nov 2014

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

Journal of Research in Biology 1505-1515 | JRB | 2014 | Vol 4 | No 8

An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
Epidemiological studies have showed that
ultraviolet (UV) radiation exposure mediates several
damaging effects that include melanoma and non-
melanoma skin cancers (Afaq and Santosh, 2012).
Although UVB radiation (280320 nm) be a small
portion of sun light that reaches the earth, it is considered
to be a most deleterious agent because it can penetrate
the skin to a depth of 160180 m and alters the skin
architecture (Chilampalli et al., 2011; Gregoris et al.,
2011). UVB is a strong pro-inflammatory agent with
profound effects on skin in part through its ability to
stimulate cytokine production. UVB exposure leads to
activation of many cytokines such as cyclooxygenase-2
(COX-2), tumor necrosis factor-a (TNF-) and
interleukin-6 (IL-6), (Alexia et al., 2003; Kondo et al.,
1993). These cytokines support the development of the
UVB-induced cutaneous inflammatory responses that is
observed in the skin as sunburn and erythema (Kondo
1999).
Peroxisome proliferator-activated receptor
(PPARa/) regulates inflammatory signaling and
cytokine activation in different experimental systems
(Zhang et al., 2004). PPARs belong to the nuclear
receptor super family, a family of ligand activated
transcriptional factors and it consists of three isotypes
(PPAR, and ). PPARs function as ligand dependent
transcription factors and can heterodimerize with retinoid
X receptors and then bind to PPAR-responsive elements
(PPRE) in target gene promoters, which usually leads to
transcriptional activation. Moreover, PPARs inhibits
inflammatory gene expression in experimental models
(Ricote et al., 1998). Previous studies provide strong
evidence for the role of PPAR in controlling
inflammation and suggest their potential as therapeutic
targets for inflammatory diseases (Kim et al., 2012).
Dietary phytochemicals offer exciting platforms
for the management of UV related disorders. Dietary
phytochemicals modulate UVB radiation-mediated
1506
Kanagalakshmi et al., 2014
damages by their antioxidant, anti-inflammatory and
immunomodulatory actions (Ramachandran and Prasad
2008). Ferulic acid (3-methoxy-4-hydroxycinnamic acid)
is a naturally occuring phenolic compound derived from
the phenylpropanoid pathway. It is commonly abundant
in fruits, vegetables and Cereals. (Prasad et al., 2011).
Ferulic acid recovers the antioxidant cell defense
system and stimulates cytoprotective enzymes due to its
resonance-stabilized phenoxy radical structure such as
phenolic nucleus and unsaturated side chain (Picone
et al., 2009). Ferulic acid can block the penetration of
UV radiation into the epidermis. This sunscreen ability
of ferulic acid can reduce UV-induced erythema (Saija
et al., 2000; Oresajo et al., 2008). Moreover, ferulic acid
could exert beneficial therapeutic effects of free radical-
related syndromes such as neurodegenerative disorders,
cancer, cardiovascular diseases and diabetes (Barone
et al., 2009). Recently, we found that ferulic acid inhibits
UVB mediated ROS generation, TBARS levels and
apoptosis in human dermal fibroblasts (Kanagalakshmi
and Prasad, 2014). In the present study, we examined the
beneficial effect of ferulic acid on the UVB mediated
inflammatory responses by targeting PPAR/ agonists
in human dermal fibroblasts.
MATERIALS AND METHDOS
Chemicals
HDFa cells were procured from Invitrogen
Bioservices, India. Low Serum Growth Supplement, fetal
bovine serum (FBS), human epidermal growth factor,
fibroblast growth factor, heparin, trypsin-EDTA and were
obtained from Invitrogen Bioservices, India. Ferulic acid,
monoclonal antibodies anti-TNF, anti-COX-2, -actin
anti-mouse and goat anti-mouse IgG-HRP polyclonal
antibody were purchased from Sigma chemical Co., St.
Louis, MO, USA. Bovine serum albumins (BSA), radio
immune precipitation assay (RIPA) buffer were
purchased from Himedia, Mumbai. All other analytical
Journal of Research in Biology (2014) 4(8): 1505-1515
Kanagalakshmi et al., 2014
grade chemicals, solvents and reagents were purchased
from SD Fine Chemical, Mumbai.
Culturing human skin fibroblasts
HDFa cells were maintained at 37C under 5%
CO2 condition in medium-106 supplemented with
2% v/v fetal bovine serum, 1 g/ml hydrocortisone,
10 ng/ml human epidermal growth factor, 3 ng/ml basic
fibroblast growth factor, 10 g/ml heparin and
antibiotics. The HDFa cells were cultured to grow for
7 days to obtain the maximum confluence for
experiments. Then, HDFa cells were harvested using
trypsin-EDTA, subcultured and the remaining cells were
used for photo protection experiments (Ramachandran
et al., 2010).
Study design
Cultured fibroblasts were divided into four
groups as follows:
Group 1: Normal fibroblasts without any treatment;
Group 2: Normal fibroblasts with 40 g/mL of FA;
Group 3: UVB-irradiated fibroblasts;
Group 4: UVB-irradiated fibroblasts pretreated with 40
g/mL of FA.
Treatment of the HDFa cells
Thirty minutes before UVB exposure, 40 g/mL
of ferulic acid was added to the HDFa cells. Trypan blue
dye exclusion test was carried out to find out the toxicity
and suitability of 40 g/mL of ferulic acid for
photoprotection studies. Before UVB exposure, the
HDFa cells were washed once with PBS solution. Mock-
irradiated HDFa showed no viability changes over the
30 min period of incubation (HDFa cells were
maintained at 37C under 5% CO2 condition in medium-
106 supplemented with 2% v/v fetal bovine serum, 1 g/
ml hydrocortisone, 10 ng/ml human epidermal growth
factor, 3 ng/ml basic fibroblast growth factor, 10 g/ml
heparin and antibiotics. The HDFa cells were cultured to
grow for 7 days to obtain the maximum confluence for
experiments. Then, HDFa cells were harvested using
trypsin-EDTA, subcultured and the remaining cells were
Journal of Research in Biology (2014) 4(8): 1505-1515
used for photoprotection experiments (Ramachandran
et al., 2010).
Irradiation procedure
HDFa cells were washed twice with PBS and
UVB irradiated in a thin layer of medium without FBS. A
battery of TL 20 W/20 fluorescent tubes (Heber
Scientific, Chennai, India) was used as UVB source,
which possess a wavelength range of 290320 nm,
peaked at 312 nm, and with an intensity of 2.2 mW/ cm2
for 9 min. The total UVB radiation exposure was
19.8 mJ/ cm2, with an average value of 1.52 103 mJ/
cell. Immediately after UVB exposure, the HDFa cells
were kept at 37C for 4 h at in 5% CO2 environment.
Irradiated HDFa cells were then washed with PBS, and
transferred to sterile centrifuge tubes for biochemical
analysis (Kanagalakshmi and Prasad 2014).
Western blot analysis for pro-inflammatory markers
expression
Western blot analysis was carried out for TNF
and COX-2 expressions in ferulic acid plus UVB-
irradiated HDFa. The results were normalized to -actin
gene expression. Treated HDFa cells were washed with
PBS and detached using 0.25% trypsin/EDTA solution.
Cell suspensions were centrifuged and the pellets were
lysed with an ice-cold lysis RIPA buffer containing a
protease inhibitor cocktail (SigmaAldrich, St. Louis,
MO, USA) for 30 min. The lysate was centrifuged at
4C at 13,000 rpm for 10 min and the supernatant was
used to determine protein concentration using Nanodrop
2000 (Thermo Scientific, USA). Cell extracts containing
50 g of proteins were subjected to electrophoresis on
12% SDS-PAGE gel and transferred to a PVDF
membrane using transblot semi-dry apparatus (Biorad,
USA). PVDF membranes were blocked with non-fat
milk (5% (w/v) for 6 h and then incubated overnight with
TNF and COX-2 antibodies (Sigma-Aldrich, USA), in
blocking solution at 37C. Then the membranes were
washed with TBST thrice with 10 min interval and
incubated with secondary antibody (diluted 1:2000) in
1507

blocking solution for 2 h at 37C. Then, the PVDF
membranes were washed with TBST thrice with 10 min
interval and the developed bands were detected using a
DAB solution. The images were acquired by Image
Studio software (LI-COR, USA) (Ramachandran et al.,
2012).
RNA isolation and real-time quantitative PCR.
The total RNA was extracted from the HDFa
cells using RNeasy Mini kit (Qiagen, USA) as per the
protocol recommended by the manufacturer. The mRNA
expression of PPAR/ in HDFa cells was determined
using real-time PCR, as described previously (Sharma
and Katiyar, 2010). RNA purification and quantity was
analyzed by nanodrop 2000 (Thermo Scientific, USA).
Experiments were run in triplicate to confirm
amplification integrity. Manufacturer-synthesized primer
pairs were used to measure the mRNA expression level
of PPARs. PCR cyclic condition 25C for 10 min; 42C
for 50 min; 75C for 15 min were used for cDNA
synthesis. The cyclic condition used for amplification
was 95C for 2 sec; 55C for 15 sec; and 68C for 20 sec
as prescribed by the primers manufacturer. The
expression levels of genes were normalized
18S mRNA expression level. The cyclic threshold (Ct)
for positivity of real-time PCR was determined based on
negative controls.
Molecular docking
Molecular docking was performed on Red Hat
Enterprise Linux EL5 workstation using Maestro
(Schrodinger LLC 2009, USA). GLIDE5.5 searches
were performed for understanding docking interactions
between ferulic acid and PPAR/. All molecular
modeling was carried out using OPLSAA (Optimized
Potential Liquid Simulation for All Atom) force field
(Glide, 2009). PyMOL (DeLano WL, 2002) software
employed for the analysis of hydrogen bond interactions.
Hydrophobic interactions were analyzed between protein
and ligand using Ligplot software (Wallace AC, 1995).
1508
Kanagalakshmi et al., 2014
Ligprep 2.3 module (Schrdinger, USA) was
employed for ferulic acid preparation. The
three dimensional crystal structure of PPAR / (PDB Id:
1K7L/ PDB Id: 3DZY) and Cox-2 (PDB Id: 6COX) were
downloaded from the Protein Data Bank (PDB)
(http://www.rcsb.org). Protein preparation wizard of
Schrodingers was used for PPAR/ and COX-2
preparation. Non-hydrogen atoms were minimized until
the average root mean square deviation reached default
value of 0.3. Sitemap 2.3 was used to understand
binding site in the ligand binding domain (LBD) of the
PPAR/ and COX-2 (Schrodinger Suite 2009).
Induced fit docking (IFD) was performed to
predict ferulic acid binding modes and structural
movements in the LBD region of PPAR/ and COX-2
using Glide and Prime modules. The prepared proteins
were loaded in the workstation and the Grid values were
calculated about 20 in order to cover all the active site
amino acids. The Vander Waals radii of nonpolar amino
acids and ligand atoms were scaled by a default value of
0.50. About 20 conformational images were created and
analyzed for the best conformational pose based on the
to docking score and glide energy.
RESULTS
Ferulic acid inhibits UVB-induced TNF- and COX-2
expressions in HDFa
Western blot analysis shows that there was an
overexpression of TNF- and COX-2 in the UVB
exposed HDFa (Figure 1). It indicates inflammatory
responses in HDFa cells as compared with control HDFa
cells. TNF- and COX-2 expression levels were
significantly down-regulated in ferulic acid pretreated
plus UVB irradiated HDFa (Figure 1).
Ferulic acid activates PPAR/ mRNA expression in
HDFa
Quantitative Real Time-PCR analyses were
adopted to analyze the activation of PPAR/ mRNA
expression in ferulic acid and/or UVB-irradiated HDFa
Journal of Research in Biology (2014) 4(8): 1505-1515
Kanagalakshmi et al., 2014

cells (Figure 2A,B). In this study mRNA levels of
PPAR/ were down-regulated in UVB-exposed HDFa.
Whereas, FA treatment prevented the UVB induced loss
of PPAR/ and up-regulated these mRNA expression in
HDFa cells.
Molecular docking with PPAR/
Ferulic acid was docked against PPAR (PDB
code: 1K7L). The glide energy score, docking score and
hydrogen bond interactions of PPAR with
cocrystallized ligand 2-(1-methyl-3-oxo-3-phenyl-
propylamino)-3-{4-[2-(5-methyl-
2-phenyl-oxazol -4-yl)-ethoxy]- phenyl}-propionic acid
was shown in the table. 1. The energy score of PPAR
with cocrystallized ligand was -86.02 (kcal/mol) and
with ferulic acid was -39.31 (kcal/mol). PPAR has a
common hydrogen bond interaction (Tyr 464 and
Ser 280) with co-crystallized ligand and ferulic acid.
The amino acid residues such as Phe 273, Cys 276 and
Ile 354 showed hydrophobic bond interactions with
ferulic acid and the co-crystallized ligand (Figure 3).
The energy score of PPAR with ligand retinoic
acid was -49.18 (kcal/mol) and with the ferulic acid it
was -40.44 kcal/mol. PPAR possess a common
hydrogen bond interaction (Arg 316) with retinoic acid
and ferulic acid. The amino acid residues such as Ala
its 272, Ile 268, Leu 326, Leu 309, Phe 313 and Ile 310
showed hydrophobic interactions with ferulic acid and
retinoic acid. The aminoacid Ala 271 was interacting
with ferulic acid through hydrogen bonding and
interacting with retinoic acid through hydrophobic
interaction (Figure 4).
Molecular docking with COX-2
The energy score of COX-2 with the
co-crystallized ligand 1-Phenylsulfonamide-3-

Table. 1. Induced fit docking results of PPAR, PPAR and Cox-2 with their cocrystallized ligands and ferulic acid.
Induced-fit docking was carried out using Schrodinger software. Ferulic acid interacts with PPAR, PPAR and COX-2
through hydrogen bonding and hydrophobic interactions. Ferulic acid possess greater binding interaction with PPAR
(glide energy -40.44) than PPAR (glide energy 39.31) when compared with their corresponding cocrystallized ligands.

Distance
Docking Score Glide Energy Hydrophobic Bond Hydrogen-Bond
Protein Compound/Ligand between donor
(Kcal/mol) (Kcal/mol) Interactions Interactions
and acceptor (A)

Gln 277, Ile 272,

2-(1-methyl-3-oxo-3- Ile 339, Phe 273,
phenyl-propylamino)-3 Leu 254, Cys 275, Leu Tyr 464 (O-H-O) 3.02
- {4-[2-(5-methyl-2- 347, Met 355, Leu 347,
-16.22 -86.02 His 440 (N-H-O) 2.98
phenyl-oxazol-4-yl)- Met 330, Val 332, Phe Ser 280 (O-H-O) 2.72
PPAR ethoxy]- phenyl}- 351, Leu 321, Ile 354,
propionic acid Cys 276
Phe 273, Cys 276, Leu Tyr 464 (O-H-O) 2.88
Ferulic acid -8.15 -39.31 460 and Ile 354 Ser 280 (O-H-O) 2.74
Tyr 314 (O-H-O) 2.79
Phe 346, Cys 432,
Ile 324, Ile 310, Ala 327 (N-H-O) 3.15
Phe 313, Leu 326, Arg 316 (N-H-O) 2.88
Retinoic Acid -13.93 -49.18
leu 309, ile 268, Arg 316 (N-H-O) 2.86
PPAR ala 272, Ala 271. Gln 275 (N-H-O) 2.62
Ala 272, Ile 310, Phe Asn 306 (O-H-O) 2.74
Ferulic acid -7.75 -40.44 313, Leu 309, Cys 269, Ala 271 (O-H-O) 2.69
Ile 268, Leu 326 Arg 316 (N-H-O) 3.04
Val 349, Ser 530, Ala
1-Phenylsulfonamide- Tyr 355 (O-H-N) 3.08
527, Gly 526, Val 523,
3-Trifluoromethyl-5- His 90 (N-H-O) 3.31
-11.68 -62.09 Ala 516, Leu 352, Ser
Parabromophenylpyra- Gln 192 (O-H-O) 3.25
353
Cox-2 zole Phe 518 (N-H-O) 3.32
Trp 387, Phe 518,Leu Tyr 385 (O-H-O) 3.22
Ferulic acid -7.95 -37.82 384, Ser353 and Val His 90 (N-H-O) 2.70
523 Leu 352 (O-H-O) 2.86

Journal of Research in Biology (2014) 4(8): 1505-1515 1509

 Kanagalakshmi et al., 2014

Figure 1. Effect of ferulic acid on UVB-induced activation of TNF and COX-2 in HDFa
cells. HDFa cells were exposed to UVB (19.8 mJ/cm2) with or without ferulic acid for 30
min. Cells were harvested at 4 h after UVB exposure, and the expression of TNF and
COX-2 were analyzed by Western blot. ferulic acid treatment down-regulated these
protein expressions when compared with UVB control group. The graph represents the
quantification results normalized to -actin levels.

Trifluoromethyl-5-Parabromophenylpyrazole was pro-inflammatory mediators and infiltration of

-62.09 kcal/mol and with ferulic acid the energy score
was found to be -37.82 kcal/mol. COX-2 posses a
common hydrogen bond interaction (His 90) with
co-crystallized ligand and Ferulic acid. The amino acid
Leu 352 was interacting with ferulic acid through
hydrogen bonding and interacting with co-crystallized
ligand through hydrophobic interaction. The amino acid
such as Ser 353 and Val 523 shows hydrophobic bond
interactions with ferulic acid and the co-crystallized
ligand (Figure 5).
DISCUSSION
UVB (280320 nm) causes acute inflammatory
skin damages including erythema, production of
1510
inflammatory cells (Lee et al., 2013). Previous studies
have demonstrated that UVB-induced oxidative stress
plays a critical role in the induction of proinflammatroy
cytokines including TNF- expression (Ramachandran
et al., 2012). TNF- can promote both DNA damage and
activation of nuclear factor kB (NF- kB) inducing the
formation of sunburned cells and leading to
photodamage in the skin (Muthusamy and Piva, 2010).
In the present study, TNF- was over expresed in the
UVB irradiated HDFa cells when compared to control
cells. The pre-administration of anti-inflammatory agents
was found to be an effective strategy for preventing
UVB-irradiation induced skin inflammation. In this
Journal of Research in Biology (2014) 4(8): 1505-1515
Kanagalakshmi et al., 2014

Figure 2. Ferulic acid activated UVB-induced PPAR/ mRNA expression in cultured human dermal
fibroblasts. Total RNAs were prepared after UV irradiation and subjected to real-time PCR. A), mRNA
expression of PPAR. B), mRNA expression of PPAR.

study, ferulic acid treatment diminished the UVB- acid can be converted to eicosonoids, forming multiple
induced overexpression of TNF- in HDFa. prostaglandins through the COX-2 pathway and
Previous studies have indicated that UVB ultimately leading to skin cell death. The expression of
mediated ROS over production and TNF activation COX-2 has been used as inflammatory marker for
resulted in cPLA2 synthesis and increasing the formation evaluating UVB irradiation induced skin inflammation.
of arachidonic acid. This overproduction of arachidonic In this report, we observed that UVB exposure increased

Figure 3. Binding interaction of ferulic acid and cocrystallized ligand with

PPAR. Ligplot image showing hydrogen bonding and hydrophobic
interactions of PPAR with its cocrystallized ligand (A) and ferulic acid (C).
Pymol view of LBD region of PPAR with cocrystallized ligand (B) and
ferulic acid (D). Ferulic acid has similar binding sites as compared with the
cocrystallized ligand on the LBD region of PPAR.

Journal of Research in Biology (2014) 4(8): 1505-1515 1511

 Kanagalakshmi et al., 2014

Figure 4. Binding interaction of ferulic acid and retinoic acid with PPAR.
Ligplot image showing hydrogen bonding and hydrophobic interactions of
PPAR with its cocrystallized ligand retinoic acid (A) and ferulic acid (C). Pymol
view of LBD region of PPAR with cocrystallized ligand (B) and ferulic acid (D
Ferulic acid has similar binding sites as compared with the cocrystallized ligand
on the LBD region of PPAR.

COX-2 protein expression in HDFa and ferulic acid
pretreatment restored the UVB induced expression of
COX-2 in HDFa. The inhibition of COX-2 expression by
ferulic acid may be caused by its phenol function, which
is associated with decreased anti-radical activity (Lee
et al., 2013). The hydrogen-donating ability of ferulic
acid was previously reported to correlate with COX-2
inhibitory activity (Hirata et al., 2005). Previously, we
showed ferulic acid scavenges free radicals and restored
UVB induced oxidative events (Prasad et al., 2007).
Molecular docking results show that ferulic acid directly
interacts with COX-2 protein.
It was reported that expression and activation of
PPAR blocks inflammation were induced by cytokine
production (Hirsch 2003, Blanquart 2003, Grimble
2002). PPARs regulate important cellular functions,
including cell differentiation, proliferation, and
inflammation. In this study, ferulic acid was found to be
an activator of PPAR and subsequently alleviates UVB
induced inflammatory markers expression. PPAR
possess a small polar and a hydrophobic residue in the
LBD that form hydrogen bonds and hydrophobic binding
interactions with ferulic acid and the cocrystallized
ligand. Arg 316 present in the LBD region of PPAR
was a common amino acid that interacts with both ferulic
acid and the cocrystallized ligand. Ferulic acid also
activates PPAR and the amino acid Tyr 464 and
Ser 280 were the common amino acids that are interact
with ferulic acid and the cocrystallized ligand. Further,
induced fit docking analysis showed that there was a
greater binding interaction of ferulic acid with PPAR
than PPAR. The binding interaction of these proteins
are due to increase in polarity, number of OH groups
present in the LBD, position of the OH group in the

1512 Journal of Research in Biology (2014) 4(8): 1505-1515

Kanagalakshmi et al., 2014

Figure 5. Binding interactions of ferulic acid and the cocrystallized ligand

(1-Phenylsulfonamide-3-Trifluoromethyl-5-Parabromophenylpyrazole) with COX-2. Ligplot
image showing hydrogen bonding and hydrophobic interactions of COX-2 with its
cocrystallized ligand retinoic acid (A) and ferulic acid (C). Pymol view of interaction of LBD
region of COX-2 with cocrystallized ligand (B) and ferulic acid (D). Ferulic acid has similar
binding sites as compared with the cocrystallized ligand on the LBD region of COX-2.

ferulic acid, based on the steric effect/inductive effect of
the ferulic acid. Ligand exposure is also considered to be
one of the reasons for strong binding interaction. A
recent study by Waku et al. (2009) shows that ligand
interact with LBD region and thereby activating PPAR
conformations. Covalent interactions stimulate alteration
of the side-chain network nearby created covalent bond
to create diverse transcriptional strengths. Thus, ferulic
acid exhibits beneficial effects against UVB-induced
inflammatory responses probably through down-
regulating COX-2 and TNF- expressions and activating
PPAR / agonists.
ACKNOWLEDGEMENT
The work was partly supported by University
Grants Commission, India (File No. 42-641/2013)
skin cancer. In: Sarkar, F.H. (Ed.) Nutraceuticals and
Cancer. Springer Publishers . chapter 14 295321.
Alexia GL, Richard LN, Sophie Gangloff C, Moncef
G. 2003. Differential regulation of TNF- alpha, IL-6 and
IL-10 in UVB-irradiated human keratinocytes via cyclic
AMP/protein kinase A pathway. Cytokine, 23 (4-5) : 138
149.
Barone E, Calabrese V, Mancuso C. 2009. Ferulic acid
and its therapeutic potential as a hormetin for age-related
diseases. Biogerontology 10 (2): 97108
Blanquart C, Barbier O, Fruchart JC, Staels B,
Glineur C. 2003. Peroxisome proliferator activated
receptors: regulation of transcriptional activities and
rules in inflammation. The journal of steroid
biochemistry and molecular biology. 85 (2-5): 267273.

REFERENCES Chilampalli C, Guillermo R, Zhang X, Kaushik RS,

Afaq F, Santosh KK. 2012. Dietary phytochemicals and Young A, Zeman D, Hildreth MB, Fahmy H,
chemoprevention of solar ultraviolet radiation-induced Dwivedi C. 2011. Effects of magnonol on UVB-induced

Journal of Research in Biology (2014) 4(8): 1505-1515 1513

 Kanagalakshmi et al., 2014

skin cancer development in mice and its possible Ko A, Schwarz T, Kirnbauer R, Urbanski A, Ansel JC
mechanism of action. BMC Cancer 11, 456. patricia p, john c. 1990. Human keratinocytes are a
source for tumor necrosis factor alpha: evidence for
DeLano WL. 2002. PYMOL a molecular graphics
synthesis and release upon stimulation with endotoxin or
system. DeLano Scientific: San Carlos, CA, USA. Web
ultraviolet light. J .Exp. Med.,172(6) : 16091614.
link: http://www.pymol.org
Kondo S, Kono T, Sauder DN, McKenzie R. 1993.
Glide. 2009. version 5.5, Schrodinger, LLC, New York,
IL-8 gene expression and production in human
NY.
keratinocytes and their modulation by UVB. J. Invest.
Gregoris E, Fabris S, Bertelle M, Grassato L, Dermatol., 101(5): 690694.
Stevanato R. 2011. Propolis as potential cosmeceutical
Kondo S. 1999. The roles of keratinocyte-derived
sunscreen agent for its combined photoprotective and
cytokines in the epidermis and their possible responses to
antioxidant properties. Int. J. Pharm., 405 (1-2):97-101.
UVA-irradiation. J .Invest. Dermatol., 4 (2) :177 183.
Grimble Robert F. 2002. Inflammatory status and
Lee C, Park GH, Ahn EM, Kim BA, Park C Jang JH.
insulin resistance. Current Opinion in Clinical Nutrition
2013. Protective effect of Codium fragile against UVB-
& Metabolic Care 5 (5): 551559.
induced pro-inflammatory and oxidative damages in
Hirata A, Murakami Y, Atsumi T, Shoji M, Ogiwara HaCaT cells and BALB/c mice. Fitoterapia., 86: 5463.
T, Shibuya K, Ito S, Yokoe I, Fujisawa S. 2005.
Lee CW, Ko HH, Lin CC, Chai CY, Chen WT, Yen
Ferulic acid dimer inhibits lipopolysaccharide-stimulated
F. 2013. Artocarpin attenuates ultraviolet B-induced skin
cyclooxygenase-2 expression in macrophages. InVivo.,
damage in hairless mice by antioxidant and anti-
19 (5): 849-53.
inflammatory effect. Food. Chem. Toxicol., 60: 123
Hirsch EC, Breidert T, Rousselet E, Hunot S, 129.
Hartmann A, Michel PP.2003. The Role of Glial
Muthusamy V and Piva TJ. 2010. The UV response of
Reaction and Inflammation in Parkinson's Disease Ann.
the skin: a review of the MAPK, NF kappa B and TNF
N. Y. Acad. Sci., 991: 214228.
alpha signal transduction pathways. Arch Dermatol Res.,
Kanagalakshmi A and Rajendra Prasad N. 2014. 302: 5-17.
Ferulic acid prevents ultraviolet-B radiation induced
Oresajo C, Stephens T, Hino PD, Law RM, Yatskayer
oxidative DNA damage in human dermal fibroblasts.
M, Foltis P, Pillai S, Pinnell SR. 2008. Protective
International Journal of Nutr. Pharmaco. Neurol. Dise., 4
effects of a topical antioxidant mixture containing
(4): 203-213.
vitamin C, ferulic acid, and phloretin against ultraviolet-
Kim JK, Sukyeong M, Myung Suk K, Mi-Bo Kim, Bo induced photodamage in human skin. J. Cosmet.
-Kyung Sa and Jae-Kwan Hwang. 2012. 5,7- Dermatol., 7(4): 290297.
Dimethoxyflavone, an activator of PPARac,
Picone P, Bondi ML, Montana G, Bruno A, Pitarresi
inhibitsUVB-induced MMP expression in human skin
G, Giammona G, Di Carlo M. 2009. Ferulic acid
fibroblast cells. Exp. Dermatol., 21 (3): 211-216.
inhibits oxidative stress and cell death induced by Ab

1514 Journal of Research in Biology (2014) 4(8): 1505-1515

Kanagalakshmi et al., 2014

oligomers: improved delivery by solid lipid Sharma SD and Katiyar SK. 2010. Dietary grape seed
nanoparticles. Free Radic. Res,. 43(11): 11331145. proanthocyanidins inhibit UVB-induced cyclooxygenase
-2 expression and other inflammatory mediators in UVB-
Prasad NR, Karthikeyan S, Kanimozhi G,
exposed skin and skin tumors of SKH-1 hairless mice.
Mahalakshmi R. 2011. Radiosensitizing effect of ferulic
Pharm. Res., 27(6): 1092-1102.
acid on human cervical carcinoma cells in vitro
Toxicol.in Vitro., 25(7): 13661375 Waku TT, Shiraki T, Oyama, Yoshito Fujimoto,
Prasad NR, Ramachandran S, Pugalendi KV, Menon Kanako Maebara, Narutoshi Kamiya. 2009.
VP. 2007. Ferulic acid inhibits UV-Binduced oxidative Structural insight into PPAR activation through
stress in human lymphocytes Nutr. Res., 27 (7): 559 covalent modification with endogenous fatty acids,
564. J. Mol. Biol., 385(1):188199.

Ramachandran S and Rajendra Prasad N. 2008.
Effect of ursolic acid, a triterpenoid antioxidant, on
ultraviolet-B radiation-induced cytotoxicity,
peroxidation and DNA damage in human lymphocytes.
Chem. Biolo. Inter., 176(2-3): 99107.
Ramachandran S and Rajendra Prasad N. 2012.
Sesamol modulates ultraviolet-B-induced apoptotic and
inflammatory signaling in human skin dermal fibroblasts.
Nutr. Pharmaco. Neurol. Dise., 2(1): 31-39.
Wallace AC, Laskowski RA, Thornton JM. 1995.
LIGPLOT: A program to generate schematic diagrams of
lipid proteinligand interactions. Prot. Eng,. 8(2):12734.
Zhang Q, Southall MD, Mezsick SM, Johnson C,
Murphy RC, Konger RL Travers JB. 2004. Travers JB
Epidermal peroxisome proliferator-activated receptor
gamma as a target for ultraviolet B radiation. J .Biol.
Chem., 280: 73-9.

Ramachandran S, Rajendra Prasad N, Karthikeyan

S. 2010. Sesamol inhibits UVB-induced ROS generation
and subsequent oxidative damage in cultured human skin
dermal fibroblasts. Arch Dermatol Res. 302: 733-44.

Ricote M, Li CA, Willson TM, Kelly CJ, Glass CK.

1998. The peroxisome proliferator-activated receptor-
gamma is a negative regulator of macrophage activation.
Nature. 391(6662): 79-82.
Submit your articles online at www.jresearchbiology.com
Saija A, Tomaino A, Trombetta D, Pasquale D,
Advantages
Uccella A, Barbuzzi T, Paolino D, Bonina F. 2000. In Easy online submission
vitro and in vivo evaluation of caffeic and ferulic acids as Complete Peer review
Affordable Charges
topical photoprotective agents. Int. J. Pharm., 199(1): Quick processing
3947. Extensive indexing
You retain your copyright
Schrodinger Suite. 2009. The induced Fit Docking submit@jresearchbiology.com
protocol Schrodinger, LLC, New York, NY. www.jresearchbiology.com/Submit.php

Journal of Research in Biology (2014) 4(8): 1505-1515 1515

 Journal of Research in Biology
An International Scientific Research Journal
Determining an effective way to control weeds in the Olam Palm, Kango
(Gabon) using herbicide treatment
Authors:
Journal of Research in Biology
Nguema Ndoutoumou P*,
Mbeang Beyeme AM,
Mouele Balimbi J and
Leboussy Ayenengoye SU
Institution:
University of Science and
Technology of Masuku,
National Higher Institute of
Agronomy and
Biotechnology (INSAB).
Poto-Poto BP 99 Franceville,
Gabon.
Corresponding author:
Nguema Ndoutoumou P
Email Id:
Web Address:
http://jresearchbiology.com/
documents/RA0487.pdf
Journal of Research in Biology
An International
Scientific Research Journal
ISSN No: Print: 2231 6280; Online: 2231- 6299
Original Research
ABSTRACT:
The fight against weeds in forest areas is a major constraint to
agricultural production; indeed, the loss of production there are around 25%. The
use of herbicides for control remains common. This study aims to propose an
appropriate herbicide against weeds in the young palms of central Gabon region.
The protocol is based on the observation and analysis of the effects of
three herbicide formulations (T1 = 30 ml Finish + 2g Ally; T2 = 50 ml Roundup +
2g Ally and T3 =100 ml of "mixing 150 ml of Finish + 150 ml water 2g + Ally") on
the dominant weeds and their impact on the growth of palm trees. The data are
collected on the time taken for drying of weeds, time of recurrence of weeds, the
growth of leaves and changing the size of the arrows.
The treatment T2 is the most effective way to induce speed drying of
weeds, their period of resurgence and growth of oil palm seedling. However, the
species Marantochloa purpurea Ridl., perennial broadleaf weed is the most common
resistant. This characteristic is related to the length of the leaves protecting the base
of the screw-to-weed herbicide and screw to advanced growth stage. In addition, this
species has a higher resistance compared to grasses (Centotheca lappacea L. and
Paspalum conjugatum Berg).
In conclusion, treatment with the molecules of glyphosate and
metsulfuron methyl are most suitable for weed control in the palm of OLAM Palm,
Kango.
Keywords:
Palm oil, efficiency, weeds, herbicides, growth, dryness, growth
Article Citation:
Nguema Ndoutoumou P, Mbeang Beyeme AM, Mouele Balimbi J and Leboussy
Ayenengoye SU
Determining an effective way to control weeds in the palm of Olam Palm Kango
(Gabon) herbicide treatment
Journal of Research in Biology (2014) 4(8): 1516-1525
Dates:
Received: 21 Oct 2014 Accepted: 04 Nov 2014 Published: 29 Nov 2014
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
1516-1525| JRB | 2014 | Vol 4 | No 8
www.jresearchbiology.com

INTRODUCTION
The oil palm (Elaeis guineensis Jacq.) is a
perennial plant belonging to the monocot family-
Arecaceae from Africa (Coulibaly, 1999).
According to Hekimian et al. (2002), it is
mainly grown for its fruit which has fats that are
extracted for multipurpose with a higher yield compared
to peanut and soybean. Fruit pulp provides edible oil,
fats and soaps. Almond oil palm gives exploitable
amount of yield in nature. The cake obtained after the
extraction of these oils is used for animal feed. Excess
sap of palm oil are collected in jars, ferment quickly
and give the palm wine. Its leaves are used for many
purposes: roofs, fences, straw etc., (Pantzaris, 1988;
Jacquemard, 1995; Ofosu-Budu and Sarpong, 2013 ).
This tropical plant with continued growth
requires a constant annual climate condition; any
deviation from the optimum leads to a decrease in the
production (Hekimian et al., 2002).
According to Adje and Affoyon (1994) and
Traore et al. (2010), in tropics, weeds are a major brake
on development in crop productivity through the
phenomena of direct competition for the various inputs.
The man had to fight as soon as he began to cultivate
plants for their needs. For the effectiveness of this
struggle, it is important to have a good knowledge of
weeds and herbicides (Montegut and Merlier, 1982;
Hornus et al. 1990; Dore, 2008). According to these
previously tested authors, weeds dissociate into three
groups, including grasses, sedges and broadleaf
weeds. This classification joins those by Barnes (1990),
Johnson (1997) and Traore et al. (2005 and 2010).
Weeding in a palm grove is basically to
eliminate weeds around the palm to reduce or limit
weed competition on the one hand, and to prevent the
cover crop cluttering the palm on the other hand. The
weeding may be chemical or manual. In general,
chemical weed control is achieved with Roundup or
Finish, where the active ingredient is glyphosate base. In
1517
Ndoutoumou et al., 2014
these products, the glyphosate concentration is between
120 g/l and 480 g/l, but the most common is 360 g/l
(Lemercier, 2009). These herbicides are foliar,
systemic, non- selective and effective in controlling
grasses.
Finally, herbicides having metsulfuron methyl
allow the inhibition of cell division in the shoots and
roots. They disrupt the physiology of the plant. The
ensuing block, prevents the growth of the plant
resulting in early senescence. It is rapidly absorbed by
plants at the roots and foliage level (Tissut et al., 2006;
Pipon, 2013).
In Gabon, the company Olam plans for the
production of 25,000 tons of oil for the country to
become the leading palm oil producer in Africa by 2020.
The plantations are still young. Several parameters,
including weed competition can undermine these
forecasts.
Indeed, production losses due to weeds affect
global agricultural production; the developing countries
are the most affected targets as they cant invest in high
volumes. Loss rates are higher (25% of production) when
compared to developed countries (5%), (Adou and Ipou
Ipou, 2007).
The application of herbicides has become a
common way to fight against weeds in the modern
agricultural practice (Mischler et al., 2007; Bertonnier
et al., 2012). The significance of this study lies precisely
based on the fact that the competition weeds with the
crop may limit its growth. It is the question of
determining impact of three formulations of herbicides
on weeds in the dominant Olam Palm Kango and then to
measure their impact on the growth of young oil palms.
It is therefore proposed to check the Olam Palm Kango
effective herbicide treatment for getting best suited to
combat the expectations for the conduct of the palm.
Journal of Research in Biology (2014) 4(8): 1516-1525
Ndoutoumou et al., 2014
MATERIALS AND METHODS
Site Characterization
The site Olam Palm Kango is located on the
002' latitude and 1012' longitude. The experimental
plot is located at 003' South latitude and 011' east
longitude.
It is an area of rainforest where a succession of
hills and plateaus are seen. The climate is equatorial
marked by two dry seasons and two rainy seasons in the
year. The months of March, April and December are
endowed with heavy rainfall above 300 mm average.
These are also the months which record the highest
temperatures (average above 27C). The driest months
are June, July and August.
Samples analyzed using a 52 cm soil pit shows
that the soil is acidic and ferralitic kind. The texture of
the soil is sandy loam.
Plant and technical equipment
Five weeds were identified in the experimental
plot. For the purposes of collecting and analyzing data,
they are coded as MH (weed) and assigned a number
from one to five. The following species are the dominant
weed in the palm of Olam Palm Kango:
Centotheca lappacea L. (Grass) = MH1
Marantochloa purpurea (Ridl.) Milne-Redh.
(Marantaceae) = MH2
Paspalum conjugatum Berg (Grass) = MH3
Dissotis rotundifolia (Sm.) Triana (Mlastomatace) = MH4
Mikania micrantha Kunth (Asteraceae) = MH5
The study covers a total of 36 oil palm
(Elaeis guineensis Jacq.), aged two years. These palms
are derived from crosses of improved varieties.
Preliminary work on the plantation took place a year in
advance.
Herbicides used in this study are: Roundup
(glyphosate 360 g/l), the Finish (glyphosate 360 g/l) and
Ally (metsulfuronmthyle). Ally is usually used in
combination with other herbicides. In the experiment, it
is sometimes associated with Roundup but now with
Journal of Research in Biology (2014) 4(8): 1516-1525
Finish. It is a selective herbicide for pre and post-
emergence that control broadleaf weeds, annual weeds
and some grasses.
100 ml beaker was used to contain the amounts
of herbicides removed from their original packaging,
using a 20 ml syringe. A backpack sprayer with a
capacity of 15 liters was used for processing around the
palm while a 200 liters drum was used as a reserve for
the preparation of boiled water.
METHODS
Selection and identification of weeds
Site selection was taken into account for
determining the diversity of weed flora in the oil palm
plantation region. Weeds were selected following the
enumeration of different species in the plot. The
following species have been identified with their
attendance: Centotheca lappacea (MH1, 72.2%),
Marantochloa purpurea (MH2, 55.5%), Paspalum
conjugatum (MH3, 83.3%), Dissotis rotundifolia (MH4,
75%) and Mikania micrantha (MH5, 25%). These rates
correspond to the number of times, the weeds were found
around 36 palms of the experimental site.
Species identification was done using a
magnifying glass and utilized the work of Johnson
(1997) for characters comparison. A final notice was
given by the National Herbarium of Gabon.
Treatments and experimental design
The test was conducted according to an
experimental device, completely randomized in blocks at
once. It consists of three blocks (B1, B2 and B3) and
three experimental units per block. Each unit has four
palm trees. Treatments are randomly assigned within the
blocks.
Weeds identified were subjected to the following
experimental treatments:
T1 : 30 ml Finish + 2g Ally
T2 : 50 ml Roundup + 2g Ally
1518
 Ndoutoumou et al., 2014

T3 : 100ml mixture "150ml Finish + 150ml
water + 2g Ally."
The mixtures were made in a water container
of 15 liter. These treatments are common in the Olam
Palm Kango.
Observed parameters and analysis
Drying weed
Drying weed corresponds to the period of first
yellowing weed in the perimeter of the spray.
Observations are made to the total drying of various
weeds. Five stages of dryness were selected:
The Resistance (R): No treatment effect on weeds;
Yellowing (Y): The beginning of desiccation;
The Significant Yellowing (YS): Transition between
yellowing and drying of weeds;
The Desiccation (D): Onset of senescence of weeds.
The Significant Dryness (DS): Total desiccation of weeds.
Recurrence weed
Recurrence weed is the observation period for
recurrence of any weed on the treated plot. Shades are
made according to the identified flora.
The growth parameters of the palm on one hand
are related to the number of sheets issued by the palm
plant, and on the other hand, the size of the arrow until
maturity. Finally, the time required for the arrows to
becoming true leaves was also determined.
Data collection is weekly for the different
parameters studied. Analyses of the size of the boom, the
number of leaves and respawning of weeds were made
using the XLSTAT software. Duncan test was used for
making comparison between means.
For the number of leaves and the size of the
arrow, an analysis of variance on repeated measurements
was made, thereby enabling monitoring of the evolution
of these parameters until the end of the experiment. For
the respawn of weeds, analysis of variance (ANOVA)
was performed.
For the statistical analysis of time taken for
drying weed, the five levels of dryness were scored from
0 to 4, respectively for levels R, J, JS, DS and D; then the
observations were transformed into log values (X + 1)
and compared using analysis of variance (ANOVA) and
on repeated measurements separated by the LSD at 5%
when the difference was significant. This analysis was
performed using SAS software.
RESULTS AND DISCUSSION
Drying time of weed
Figures 1 to 4 present the effects of treatments
T1, T2 and T3 on weeds MH1, MH2, MH4 and MH5, over
time.

1.8 1.4
1.6 1.2
Level of desiccation

1.4
Level of desiccation

1.2
1

1 0.8
MH2 T1 MH2 T1
0.8 0.6
0.6 MH2 T2 MH2 T2
0.4
0.4 MH2 T3 MH2 T3
0.2
0.2

0 0

Time (weeks) Time (weeks)

Figure 1 Evolution of drying MH1 Figure 2 Evolution of drying MH2

(Centotheca lappacea ) among treatments. (Marantochloa purpurea ) among treatments.

1519 Journal of Research in Biology (2014) 4(8): 1516-1525

Ndoutoumou et al., 2014
The effect of the three treatments (T 1, T2, T3) are
identical in MH1 (Centotheca lappacea), where the
superposition of the curves representing that effect over
time (Figure 1).
The effect of treatments on Centotheca lappacea
(MH1), Paspalum conjugatum Berg (MH3) and Mikania
micrantha (MH5) was achieved after three weeks to the
last level of dryness (DS). As these are short-cycle
species mainly following vegetative propagation, they
undergo rapid drying out three weeks after the different
treatments.
Dissotis rotundifolia (MH4) reacts differently to
other weeds (MH1, MH3 and MH5) with respect to the
three treatments. Indeed, in the sixth and seventh weeks,
all the three treatments have the same behavior. These
results are explained by the fact that Dissotis rotundifolia
is a perennial broadleaf and therefore more resistant to
herbicides compared to grasses.
Formulations T1 and T3 in the same way act on
Marantochloa purpurea (MH2). Their effectiveness on
this weed is less compared to that of T 2 with which they
will reach a higher level of drying (JS) after three weeks.
In general, Marantochloa purpurea is resistant to these
three treatments. It is a plant with large leaves and
rhizomes, where foliage limit absorption of the herbicide
and rhizomes promote regeneration.
1.8
1.6
1.4
Level desiccation
Level desiccation
1.2
1
0.8
MH2 T1
0.6
0.4 MH2 T2
0.2 MH2 T3
0
Time (weeks)
Figure 3 Evolution of drying MH4
(Dissotis rotundifolia) among treatments.
Journal of Research in Biology (2014) 4(8): 1516-1525
The overall results showed that the most resistant
weed is MH2 (Marantochloa purpurea). In addition, the
treatment T2 seems to be more effective in the fight
against these weeds.
From a statistical point of view, if we take into
account the probability effect of different sources of
variation, it appears at first as a highly significant
difference in response between the three treatments given
to five weed species. This result seems to be that the life
cycle of annual grasses are thus relatively short. More
recent to this, they reproduce only sexually, unlike
broadleaf weeds (Marantochloa purpurea) and have
longer life cycles.
In the second step, there is a significant
difference between three formulations applied to the
herbicides. The treatments do not work with the same
effectiveness on weeds. This difference is explained by
the fact that Roundup does not contain metsulfuron
methyl while the Finish contains enough active form of
this adjuvant.
Finally, the probability of interaction (treatment
x species) confirms the effect of differences between the
data for three treatments or behavior differences in weed
species which meet these three species treatments.
The speed of drying out is higher in grasses than
broadleaf weeds. Similarly, in general, the time and
1.8
1.6
1.4
1.2
1 MH2 T1
0.8
MH2 T2
0.6
MH2 T3
0.4
0.2
0
Time (weeks)
Figure 4 Evolution of drying MH5
(Mikania micrantha) among treatments.
1520
 Ndoutoumou et al., 2014

Size (m)
T1
T2
T3

Time (enjours)

Figure 5 Evolution of the size of the arrow.

dryness is greater when the treatment is accelerated with Number of sheets

T2. Analysis of the differences between treatments Growth in the number of leaves is not significant
revealed that for the "drying time of weed" parameter, during the period of observation. Indeed, this parameter
treatment T2 is much better than T3. does not vary between treatments received by palm
Treatment T1 occupies an intermediate position plants. However, experimental units having received the
between T2 and T3 in the analysis of interactions (species treatment T2 exhibit better growth compared with the
x treatment). Its effectiveness on Marantochloa purpurea other units which received other two treatments. Indeed,
remains lower compared to the effect of treatment T 2 of the growth of the arrows cause an increase in the number
the same species. The treatment T2 has a better efficacy of leaves; ultimately, the application of different
on weed control treatments than T 1 and T3. herbicides on weed formulations allow the increase of
Size of the arrow palm leaves.
The curves of the three treatments have a Respawn weed
constant gradual pace. However, regarding the Treatments followed by T1, T2 and T3 are
processing curve of T2 (Roundup + Ally) it is above the significantly different at the 5% level.
curves representing experimental units having received There are significant differences between
treatment with T1and T3. treatments T1 and T2, on one hand, and between
Indeed, the reduction of competition enable treatments T1 and T3, on the other hand, while the effects
palms to increase their export of mineral, water and soil of treatments on the respawn of weeds are similar to
as well as their ability to capture light for photosynthesis.
Table 1 Analysis of the differences between
This promotes the growth and development of the palm treatments for the respawn of weeds.
of the arrows. All treatments allow a better development
Treatments Means and standard
of the size of the arrows. In addition, weeds can be a deviations
habitat for some palm pests. Removing them allows T3 94.08a 23.12
expelling the pests of cultivated areas. Although various
T2 90.16a 10.69
treatments have a positive impact on the growth of the
size of the arrows of palms; treatment T 2 proves to be the T1 73.16b 09.99
best.

1521 Journal of Research in Biology (2014) 4(8): 1516-1525

Ndoutoumou et al., 2014
treatments T3 and T2.
The mean time for recurrence of palms receiving
treatment T2 and T3 treatments are very close. The effect
of treatment T2 and T3 on weeds can greatly delay the
time to recurrence weeds. Treatments T 2 and T3 are
effective in delaying the onset of weeds and promote the
harmonious growth of the oil palm. Nevertheless, one
can notice that the standard deviation of the respawn
units receiving treatment T 3 is very high (23.12).
Application of the treatment T 3 is obtained promptly to
recur weeds. The treatment T 3 does not allow time to
obtain a balanced and stable recurrence. Against by
standard deviation units, receiving treatment T 2 is low
and therefore stable (10,69), this treatment allows for
respawn close to the average. Indeed, the Finish contains
not only glyphosate as the active ingredient but also as
metsulfuron methyl builder. It is therefore possible that
these two substances in certain concentrations reduce the
effectiveness of the fight against weeds. While in the
treatment T2, the only source of the product is
Metsulfuron methyl Ally as Roundup has only
glyphosate as active ingredient. The treatment T 2 seems
more stable and allows for respawn which is almost
uniform.
Respawned weed and palms which received the
treatment T1 is 73 days on average. This duration is less
than three months. It is significantly lower than the
respawn of weeds in units that have received treatments
T2 and T3. The respawn in units that received the
herbicide formulations T2 and T3 are 90 days and
94 days respectively and correspond to three months on
average.
All treatments can slow the recurrence of weeds,
although the treatment T3 and T2 are used to obtain
respawn of longer weeds. Indeed, the stage of weed
growth is a key factor for weed control in an effective
way.
Journal of Research in Biology (2014) 4(8): 1516-1525
DISCUSSION
Resistance to treatment
In terms of drug resistance, it would be wise to
treat weeds in stage two or three leaves, so the treatment
frequency is quarterly. The different treatments were
applied while weeds had overtaken the juvenile stage
development. In fact, the observed resistance of Dissotis
rotundifolia could be explained. Indeed, early weed
control is important for effective control and yields. This
is consistent with the observations of Johnson (1997).
The conduct of palm monoculture promotes
weed resistance vis--vis herbicides (Mangara et al.,
2010). Marantochloa purpurea is the only weed that
truly has broad leaves and rhizomes. Indeed, perennials
are much more resistant to herbicides than grasses
(Johnson, 1997).
Finish, Round up and Ally are the main herbicide
used, they contain glyphosate (active ingredient) at a
concentration of 360 g/l. Some weeds such
Marantochloa purpurea may acquire resistance to
glyphosate due to the repeated use of the same herbicide.
Indeed, the work of Baylis (2000) showed that this fact
promotes weed resistance.
Determining the type of weeds is essential to
effectively fight against their impact on culture.
Perennial weeds have a high resistance compared to the
other. Resistance of Marantochloa purpurea is also
explained by the fact that it has a long life cycle.
Marantochloa purpurea can be propagated vegetatively,
unlike species Paspalum conjugatum, Centotheca
lappacea and Mikania micrantha which can reproduce
that sexually. This faculty of reproduction gives
Marantochloa purpurea a better resilience compared to
other weeds, such as stated by Aubert and Glachant
(2009).
Effectiveness of treatment T2 compared to T1 and T3
The treatment T2 (Roundup + Ally) is better
compared to the other two treatments. Indeed, it only
contains glyphosate Roundup outside its adjuvants. In
1522

Finish, it contains not only glyphosate but also
metsulfuron-methyl which is a sulfonylurea substance.
The combination of the Ally and Finish increased
the content of metsulfuron-methyl mixture. It thus seems
clear that the presence of metsulfuron-methyl in the
solution is a factor limiting the effectiveness of the
herbicide treatment. These results confirm the resistance
shown by weeds that deal with sulfonylurea herbicides
substances, up to 60-100%, according to Rodriguez
(2005) and Chauvel and Guillemain (2013).
Foliar and root herbicides should be applied
earlier. Indeed, three weeks after herbicide treatment,
different weeds have very different spectra of activities
in accordance with the work of Tissut et al. (2006).
The Roundup association and Ally (T 2) is a
combination of herbicides that can fight against a wide
range of weeds (perennial, annual or biennial). Due to its
composition, it is recommendable to young weeds.
Indeed, metsulfuron methyl by its mode of action or
essentially leaf and root penetration destroys stolons,
rhizomes and tubers. However, Roundup association and
Ally (T2) may be ineffective on some weeds; this was
evidentially proved by Valantin-Morison et al. (2008)
and Bertonnier et al. (2012).
Effects of herbicides on weed growth and oil palm
As in this study, the work of Echezona et al.
(2011) on plantain showed that the combination of
glyphosate (the active ingredient in Roundup and Finish)
and sawdust for weed control improves and facilitates
the growth of the crop. It is important to better
understand, weeds and herbicides for effective control,
according to Schaub (2010).
CONCLUSION
The three treatments that have proven effects on
weed control are encountered on this site. However,
broadleaf species such as Marantochloa purpurea are
the most resistant, while grasses Centotheca lappacea
and Paspalum conjugatum Berg are very sensitive to
1523
Ndoutoumou et al., 2014
three treatments. The treatment T 2 is significantly
different from the other two treatments. It has a long
respawn time of weeds, a different curve drying weeds
over other treatments and a greater influence on the
growth parameters of oil palm. This treatment combines
the molecules of glyphosate (Roundup) and metsulfuron
methyl (Ally) which is most suitable for weed control in
the palm of Olam Palm Kango.
These results would be improved by taking into
account the identification of other weeds present in the
field, the combination of herbicide treatment in round
weeding around the palm trees, the quarterly frequency
of herbicide treatment and the use of cover crop
(Pueraria javanica) with its various beneficial effects on
the culture.
REFERENCES
Adje AI and Af fo y o n R. 1994.
Chromolaena odorata (L.) dans les palmeraies du
Benin : incidence et mthodes de lutte. Bulletin de la
Recherche agronomique, 10.
Adou LMD and Ipou Ipou J. 2007.
Pteridium aquilinum, une ptridophyte envahissante des
cultures prennes du sud de la Cte dIvoire: quelques
notes ethnobotaniques. Tropicultura, 25 (4) : 232-234.
Aubert C and Glachant C. 2009. Caractristiques
biologiques des adventices. Disponible sur : http://
www.itab.asso.fr/downloads/programmes/centre -
rapport2009ann3_1.pdf (Consult le 21/12/2013).
Barnes DE. 1990. Common weeds of Malaysia and
their control. Edition Ancon. Singapour. p 17-230.
Baylis AD. 2000. Why glyphosate is a global herbicide:
strengths, weakness and prospect. Pest Management
sciences. 56(4) : 299-308.
Bertonnier L, Bonansea V, Bonnet F and Duran R
Camilo. 2012. Etude du glyphosate (Roundup).
Journal of Research in Biology (2014) 4(8): 1516-1525
Ndoutoumou et al., 2014
Janvier. p 3-30.
Chauvel B and Guillemain J-P. 2013. Histoire des
herbicides. Disponible sur :www.histoire-des-
herbicides.info/home (Consult le 29/11/2013).
Coulibaly H. 1999. Analyse technique de la ppinire
directe de palmier huile d'Ehania. Mmoire de fin
d'tude ITA. ESA/INPHB, Yamoussoukro, 45 p.
Dore T. 2008. Dynamique d'implantation et conduite
technique des Plantations villageoises de palmier huile
au Cameroun : Facteurs limitants et raisons des
pratiques. Thse de doctorat. Institut des Sciences et
Industries du Vivant et de lEnvironnement (Agro
Paris Tech). 148p.
Echezona B, Baiyeri K and Aindigh F. 2011.
Rendement et conomie de la production de la banane
plantain avec six techniques de gestion des mauvaises
herbes dans un agro- systme de la savane drive.
Tropicultura 29 (1) : 14-19.
Hekimian LC, Rouzire A, Schilling R and Taillez B.
2002. Les Plantes olagineuses.In Mmento de
lAgronome. Edition Quae. Paris. pp 877-927.
Hornus P, Nguimjeu E, Kouotou M and Kamga E.
1990. Entretien chimique des ronds de palmier huile.
Essais herbicides : glyphosate/ glufosinate; 1- Rsultats.
Olag. 45(2):57-68.
Jacquemard JC. 1995. Le palmier huile. (Collection
Le technicien dagriculture tropicale n 33).
Maisonneuve et Larose Ed., Paris. 206 p.
Johnson DE. 1997. Les adventices en riziculture en
Afrique de lOuest. Edit par lAssociation pour le
Dveloppement de la riziculture en Afrique de lOuest.
Bouak. 312 p.
Lemercier G. 2009. Glyphosate : mieux lutiliser pour
assurer sa prennit. FICHE. AGRI-MIEUX. pp 1-4.
Journal of Research in Biology (2014) 4(8): 1516-1525
Mangara A, Nda AAA, Traore K, Kehe M, Soro K
and Tour M. 2010. Etude phytocologique des
adventices en cultures dananas (Ananas comosus (L.)
Merr.) dans les localits de Bonoua et Ndouci en Basse
Cte dIvoire. Journal of Applied Biosciences 36: 2367-
2382.
Mischler P, Lheureux S, Sen O, Dumoulin F and
Menu P. 2007. Des parcelles plus propres avec moins
dherbicides grce des systmes de culture intgrs
fonds sur l'agronomie. Guide Pratique. Disponible sur :
http://www.agro -transfert- rt.org/index.php/fr/
component/docman/doc_download/52-des- parcelles-
plus-propres- avec-moins-dherbicides-guide-12p
Montegut J and Merlier H. 1982. Adventices
tropicales : flore aux stades plantule et adulte de 123
espces africaines ou pantropicales. Paris : Ministre
des relations extrieures. Coopration et dveloppement
p 490.
Ofosu-Budu K and Sarpong D. 2013. Croissance de
la filire de lhuile de palme au Ghana : implications
pour les petits agriculteurs et viabilit long terme.
In :Reconstruire le potentiel alimentaire de lAfrique de
lOuest, A. Elbehri (ed.),FAO/FIDA. Chapitre 11. 375-
419.
Pantzaris TP. 1988. Le livret des usages de lhuile de
palme. Porim Ed., Kuala Lumpur,Malaisie.
Pipon E. 2013. Identification et caractrisation
dinnovations agrocologiques en matire de gestion des
adventices mises en place par des agriculteurs. Mmoire
de fin dtudes. Agrocampus Ouest. Rennes France. 97 p.
Rodriguez A. 2005. Matriser les adventices en grandes
cultures. Alter-Agri., 68 : 4-7.
Schaub C. 2010. Mieux connatre les mauvaises
herbes pour mieux matriser le dsherbage. Disponible
sur: www. b as - rh i n. c h am b ag r i. f r/f i l ead mi n /
1524
 Ndoutoumou et al., 2014

documents. P 1-42.

Traor K, Pene CB, Aman Kadio J and Ake S.

2005. Phyto-sociologie et diversit floristique du
primtre laeicole de la Me en basse Cte divoire
forestire. Agronomie Africaine, 17 (3) : 163-178.

Traor K, Soro D, Pene CB and Ake S. 2010. Flore

adventice sous palmeraie, dans la zone de savane
incluse a Dabou, basse cte divoire. Agronomie
Africaine, 22 (1) : 21 32.

Tissut M, Delval P, Mamarot J and Ravanel P. 2006.

Plantes, herbicides et dsherbage, ACTA, Paris, 600 p.

Valantin-Morison M, Guichard L and

Jeuffroy M-H. 2008. Comment matriser la flore
adventice des grandes cultures travers les lments de
l'itinraire technique? Innovations Agronomiques,
3 : 27-41.

Submit your articles online at www.jresearchbiology.com

Advantages
Easy online submission
Complete Peer review
Affordable Charges
Quick processing
Extensive indexing
You retain your copyright
submit@jresearchbiology.com
www.jresearchbiology.com/Submit.php

1525 Journal of Research in Biology (2014) 4(8): 1516-1525

 Journal of Research in Biology
An International Scientific Research Journal
Application of multivariate principal component analysis on dimensional
reduction of milk composition variables
Authors:
Journal of Research in Biology
Alphonsus C1, Akpa GN1,
Nwagu BI2, Abdullahi I2,
Zanna M3, Ayigun AE3,
Opoola E3, Anos KU3,
Olaiya O3 and Olayinka-
Babawale OI3
Institution:
1. Animal Science
Department, Ahmadu Bello
University, Zaria, Nigeria.
2. National Animal
Production Research
Institute, Shika-Zaria
3. Kabba College of
Agriculture, Ahmadu Bello
University, Kabba, Nigeria
Corresponding author:
Alphonsus C
Email Id:
Web Address:
http://jresearchbiology.com/
documents/RA0489.pdf
Journal of Research in Biology
An International
Scientific Research Journal
ISSN No: Print: 2231 6280; Online: 2231- 6299
Original Research
ABSTRACT:
Variable selection and dimension reduction are major prerequisites for
reliable multivariate regression analysis. Most a times, many variables used as
independent variables in a multiple regression display high degree of correlations. This
problem is known as multicollinearity. Absence of multicollinearity is essential for
multiple regression models, because parameters estimated using multi-collinear data
are unstable and can change with slight change in data, hence are unreliable for
predicting the future. This paper presents the application of Principal Component
Analysis (PCA) on the dimension reduction of milk composition variables. The
application of PCA successfully reduced the dimension of the milk composition
variables, by grouping the 17 milk composition variables into five principal
components (PCs) that were uncorrelated and independent of each other, and
explained about 92.38% of the total variation in the milk composition variables.
Keywords:
Principal component analysis, eigenvalues, communality
Article Citation:
Alphonsus C, Akpa GN, Nwagu BI, Abdullahi I, Zanna M, Ayigun AE, Opoola E,
Anos KU, Olaiya O and Olayinka-Babawale OI
Application of multivariate principal component analysis on dimensional reduction of
milk composition variables
Journal of Research in Biology (2014) 4(8): 1526-1533
Dates:
Received: 27 Oct 2014 Accepted: 15 Nov 2014 Published: 03 Dec 2014
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
1526-1533| JRB | 2014 | Vol 4 | No 8
www.jresearchbiology.com

INTRODUCTION
In recent times, many scientist, especially in the
field of dairy science have postulated the use of milk
composition variables as a tool for monitoring and
evaluation of energy balance (Friggens et al., 2007;
Lovendahl et al., 2010; Alphonsus, 2014), health
(Hansen et al., 2000; Pryce et al., 2001; Invartsen et al.,
2003; Cejna and Chiladek, 2005), fertility (Harris and
Pryce, 2004; Fahey, 2008) and nutritional status
(Kuterovac et al., 2005; Alphonsus et al., 2013) of dairy
cows. One way of validating this hypothesis is to assess
the relationship between the milk composition variables
and the parameters in question through multiple
regression analysis. However, the drawback in applying
multiple regression analysis to the milk composition
variables is that most of the milk composition variables
are highly correlated (Lovendahl et al., 2010; Alphonsus
and Essien, 2012).
A high degree of correlation among the
predictive variables increases the variance in estimates of
the regression parameters (Yu, 2010). This problem is
known as multicollinearity (Kleinbaum et al., 1998;
Fekedulegn et al., 2002; Leahy, 2001; Yu, 2008).
The problem with multicollinearity is that it
compromises the basic assumption of multiple regression
that state that the predictive variables are uncorrelated
and independent of each other and parameters estimated
using multi-collinear data are unstable and can change
with slight change in data, hence are unreliable for
predicting the future. When predictors suffer from
multicollinearity, using multiple regressions may lead to
inflation of regression coefficients. These coefficients
could fluctuate in signs and magnitude as a result of a
slight change in the dependent variables (Fekedulegn
et al., 2002).
Therefore, the first step to counteract this
problem of multicollinearity is the use of Principal
Component Analysis (PCA). Principal component
analysis is a multivariate statistical tool that is commonly
1527
Alphonsus et al., 2014
used to reduce the number of predictive variables as well
as solving the problem of multicollinearity (Bair et al.,
2006). It transforms the original independent variables
into newly uncorrelated variables called Principal
Components (PCs) (Lafi and Kaneene, 1992), so that
each PC is a linear combination of all the original
independent variables. It looks for a few linear
combinations of variables that can best be used to
summarize the data without loosing information of the
original variables (Lafi and Kaneen, 1992; Bair et al.,
2006)
This study therefore attempted to apply the
principle of Principal Component Analysis (PCA) on
variable selection and dimension reduction of milk
composition variables
MATERIALS AND METHODS
Experimental site
Data for this study were collected from 13
primiparous and 47 multiparous Friesian x Bunaji dairy
cows, at the dairy herd of National Animal Production
Research Institute (NAPRI) Shika-Zaria, located
between latitude 11 and 12N at an altitude of 640m
above sea level, and lies within the Northern Guinea
Savannah Zone (Oni et al., 2001). The cows were
managed during the rainy season on both natural and
paddocksown pasture, while during the dry season they
were fed hay and /or silage supplemented with
concentrate mixture of undelinted cotton seed cake and
grinded maize. They had access to water and salt lick ad-
libitum. Unrestricted grazing was allowed under the
supervision of herdsmen for 7 9 hours per day
(Alphonsus et al., 2013)
Milk composition measures
Cows were milked twice daily (morning and
evening) and milk yield was recorded on daily basis. The
milk sampled for the determination of fat, protein and
lactose percentages were taken once per week starting
from 4 days postpartum to the end of each lactation.
Journal of Research in Biology (2014) 4(8):1526-1533
Alphonsus et al., 2014
The milk samples were frozen immediately after
o
collection and stored at -20 C until analysed (Alphonsus
et al., 2013). The milk composition analysis was carried
out at the Food Science and Technology Laboratory of
Institute of Agricultural Research (IAR) in Ahmadu
Bello University, Zaria-Nigeria. The yield values and the
ratios were derived from the percentage values of fat,
protein and lactose (Friggens et al., 2007 Lvendahl et
al., 2010). The following milk composition measures
were calculated: Milk Fat Content (MFC), Milk Protein
Content (MPC), Milk Lactose Content (MLC), Milk Fat
Yield (MFY), Milk Protein Yield (MPY), Milk Lactose
Yield (MLY), Fat-Protein Ratio (FPR), Fat-Lactose
Ratio (FLR), Protein - Lactose Ratio (PLR), change in
Milk Yield (dMY), change in Milk Protein Content
(dMPC), change in Milk Fat Content (dMFC), change in
Milk Lactose Content (dMLC), change in Fat Protein
Ratio (dFPR), change in Fat Lactose Ratio (dFLR) and
change in Protein-Lactose Ratio (dPLR).
Statistical Analysis
The correlation matrix of all the
composition variables was first run using PROC CORR
procedure of SAS (2000) to determine the level of the
collinearity among milk composition variables.
Principal component analysis
Principal component analysis is a method for
transforming the variables in a multivariate data set
X2, X2,.Xn, into new variables, Y1, Y2,..Yn,
which are uncorrelated with each other and account for
decreasing proportions of the total variance of the
original variables, defined as
Y1 = P11X1 + P12X2 +. +P1nXn
Y2 = P21 X1 + P22X2 + + P2nXn
Y3 = Pn1X1 + Pn2X2 + . + PnnXn
With the coefficient being chosen so
Y1, Y2, .. Yn account for decreasing proportion of
the total variance of the original variables X1, X2 ..Xn
(Lafi and Kaneene, 1992).
Journal of Research in Biology (2014) 4(8): 1526-1533
The principal component analysis was run using
PROC Factor SAS software (SAS, 2002).
RESULTS AND DISCUSSION
Correlation matrix of the milk composition variables
The correlation matrix shows high degree of
correlation among the milk composition variables (Table
1). This strong correlation among the measured variables
is called multicollinearity (Kleinbaum et al., 1998;
Vaughan and Berry, 2005). Multicollinearity is a serious
problem in multiple regression analysis because it
violates the basic assumption of regression that requires
the predictors to be independent and uncorrelated with
each others. It also compromise the integrity and
reliability of the regression models (Kleinbaum et al.,
1998; Maitra and Yan, 2008).
The problem of multicollinearity is as a result of
redundancy of some variables. Redundancy in this case
means that some of the variables are strongly correlated
with one another, possibly because they are measuring
the same characteristic (http://support.sas.com/
milk publishing/publicat/chaps/55). For example, the
correlations between the milk composition yield
variables (MFY, MPY, MLY) were very strong (r =
0.943 to 0.989). Likewise, the correlations between the
rate of change d in milk composition variables (dMY,
dMFC, dMPC, dMLC) were very strong ranging from
0.980 to 0.992, and a lot of others. Therefore, given this
apparent redundancy, it is likely that these correlated
variables are measuring the same construct or have the
same characteristics. Therefore, it could be possible to
reduce these collinear variables into smaller number of
composite variable (artificial variables) called Principal
Components (PCs) that are independent and account for
most of the variation in the milk composition variables.
that The PCs can then be used for subsequent multiple
regression analysis. One way of achieving this is the use
of Principal Component Analysis (PCA).
Principal Component Analysis
The measured milk composition variables were
1528
1529
Table 1: Correlation co-efficients among milk yield and milk composition variables used for prediction of Energy Balance (EB)

*Milk AD- MFC MPC MLC MFY MPY MLY FPR FLR PLR DMY dMFC dMPC dMLC dFPR dFLR
variables MY
ADMY -
MFC -0.264 -
MPC -0.195 0.352 -
MLC -0.321 0.853 0.305 -
MFY 0.956 0.025 -0.054 -0.078 -
MPY 0.986 -0.189 -0.029 -0.275 0.966 -
MLY 0.939 0.019 -0.089 0.014 0.988 0.943 -
FPR -0.177 0.853 -0.079 0.773 0.063 0.191 0.078 -
FLR 0.183 0.044 0.169 -0.484 0.203 0.218 0.016 -0.048 -
PLR 0.240 -0.669 0.162 -0.889 0.056 0.272 -0.057 -0.841 0.579 -
dMY -0.669 0.037 0.232 -0.058 -0.681 -0.645 -0.728 -0.095 0.161 0.171 -
dMFC -0.674 -0.056 0.120 -0.085 -0.714 -0.668 -0.742 -0.133 0.056 0.144 0.980 -
dMPC -0.671 0.000 0.117 -0.068 -0.695 -0.666 -0.734 -0.118 0.118 0.123 0.985 0.989 -
dMLC -0.653 0.021 0.187 -0.084 -0.671 -0.634 -0.723 0.183 0.183 0.176 0.992 0.983 0.985 -
dFPR -0.182 -0.388 -0.002 -0.154 -0.279 -0.183 -0.220 -0.352 -0.352 -0.152 0.240 0.345 0.212 0.246 -
dFLR -0.129 -0.433 -0.336 0.017 -0.254 -0.196 -0.126 -0.284 -0.691 0.171 -0.021 0.142 0.063 -0.036 0.605 -
dPLR 0.070 -0.061 -0.391 0.147 0.038 -0.005 0.115 0.165 -0.385 -0.363 -0.297 -0.23 0.459 -0.321 -0.427 0.459
*
milk composition variables indicated by the following: Average Daily Milk Yield (ADMY), Milk Fat Content (MFC), Milk Protein Content (MPC), Milk
Lactose Content (MLC), Milk Fat Yield (MFY), Milk Protein Yield (MPY), Milk Lactose Yield (MLY), Fat Protein Ratio (FPR), Fat Lactose Ratio (FLR),
Protein Lactose Ratio (PLR). Variable abbreviations starting with d are the current minus the previous values of milk measures in question. Yield values are
in kilogram per day (kg/day), content values are in percentages (%) and ratios are unitless. The measures used were group mean averages.
2
cummulative percentages of variation explained with increasing number of PC indicated

Journal of Research in Biology (2014) 4(8): 1526-1533

Alphonsus et al., 2014
Alphonsus et al., 2014
Table 2: Relationships among milk composition measures1 expressed as loadings in a
principal component analysis.
Items a Principal components (PCs) h
PC1 PC2 PC3 PC4 PC5
Variable explained2 38.88 60.01 75.00 85.30 92.38 -
Average Daily Milk Yield -0.34 0.93 -0.02 0.00 -0.00 99.81
(ADMY)
Milk Fat Content (MFC) 0.02 0.13 0.85 -0.23 0.42 99.96
Milk Protein Content (MPC) -0.04 0.15 0.05 0.07 0.98 99.96
Milk Lactose Content (MLC) 0.05 0.13 0.82 0.08 0.37 99.89
Milk Fat Yield (MFY) -0.33 0.92 0.17 -0.05 0.08 99.88
Milk Protein Yield (MPY) -0.34 0.93 -0.01 -0.01 0.14 99.90
Milk Lactose Yield (MLY) -0.34 0.91 0.19 0.01 0.07 99.83
Fat-Protein Ratio (FPR) 0.05 0.04 0.90 -0.27 -0.25 99.97
Fat-Lactose Ratio (FLR) 0.10 -0.04 -0.15 -0.48 -0.05 99.97
Protein-Lactose Ratio (PLR) 0.03 -0.06 -0.86 -0.09 0.17 99.86
dMY 0.94 -0.33 0.01 -0.06 0.01 99.40
dMFC 0.94 -0.32 -0.02 0.06 -0.03 99.77
dMPC 0.94 -0.32 0.02 -0.06 -0.04 88.81
dMLC 0.95 -0.31 -0.01 -0.07 -0.02 99.77
dFPR -0.04 0.03 -0.25 0.81 0.04 99.94
dFLR -0.04 -0.05 -0.08 0.92 -0.03 99.95
dPLR -0.01 -0.09 0.22 0.02 -0.09 99.96
3
% variance 38.88 21.20 14.92 10.30 07.08 -
Eigen values 6.610 3.604 2.536 1.751 1.204 -
a
Variable abbreviations starting with d are the change variables signifying current minus
the previous values of milk measures in question. Yield values are in kilogram per day (kg/
day), content values are in percentages (%) and ratios are unitless.
2
cummulative percentages of variation explained with increasing number of PC indicated
3
percentage variance explained by each principal components
h= communality estimates is a variance in observed variables acounted for by a common
factor

subjected to Principal Component Analysis (PCA) using
one as a prior communality estimate. The principal axis
method was used to extract the components, and this was
followed by varimax (orthogonal) rotation. Only the first
five components accounted for a meaningful amount of
the total variance (92.38%) in the milk composition
variables. Also using eigenvalue criteria of one,
it was obvious that the first five components displayed
eigenvalues equal to or greater than one. Therefore, the
first five principal components were retained and used
for rotation and interpretation. The milk composition
variables and the corresponding factor loadings are
presented in Table 2. In interpreting the rotated factor
pattern, an item was said to load heavily on a given
component if the factor loading was 0.50 or greater.
Using these criteria, it was obvious that the change d
in milk composition variables (dMY, dMFC, dMPC,
dMLC) loaded heavily on the first Principal Component
(PC) which were subsequently labeled change
component. Also, the four milk composition yield
variables (ADMY, MFY, MPY, MLY) loaded heavily on
the second PC and were labeled yield component.
Other variables like MFC, MLC, FPR and FLR loaded
heavily on the third PC and were labeled mixed
component. Change in Fat-Protein Ratio (dFPR) and
Fat-Lactose Ratio (dFLR) loaded heavily on the fourth
PC and were labeled change in fat ratio component.
The last PC had only one variable (MPC) heavily loaded

Journal of Research in Biology (2014) 4(8): 1526-1533 1530

 Alphonsus et al., 2014

Table 3: Pearson correlation between the Principal components and milk composition variables

Variables i Principal Components (PCs)

PC1 PC2 PC3 PC PC5
Average daily milk yield -0.340 0.938** -0.016 0.000 0.004
(ADMY)
Milk fat content (mFc) 0.018 0.135 0.853** -0.233 0.317
Milk protein content (mPc) -0.043 0.146 0.059 0.015 0.981**

Milk lactose content(mLc) -0.052 0.128 0.825 0.085 0.373

Milk fat yield (mFy) -0.327 0.923** 0.168 -0.053 0.079

Milk protein yield (mPy) -0.341 0.928** 0.012 -0.005 0.137

Milk lactose yield (mLy) 10.342 0.909** 0.193 0.014 0.078

Fat-protein ratio (FPR) 0.046 0.039 0.900** -0.267 -0.246

Fat-lactose ratio (FLR) 0.098 -0.036 -0.153 -0.476 -0.046
Protein-lactose ratio (PLR) 0.029 -0.061 -0.859** -0.085 0.172
dmy 0.939** -0.328 0.008 -0.057 0.009
dmFc 0.942** -0.322 -0.021 0.069 -0.030
dmPc 0.941** -0.316 0.021 -0.062 -0.039
dmLc 0.944** -0.307 -0.008 -0.069 -0.022
dFPR -0.041 0.027 -0.254 0.811** 0.043
dFLR -0.044 -0.055 -0.082 0.921** -0.028
dPLR -0.005 -0.091 0.221 0.049 -0.087
PC1 1.000 0.000 0.000 0.000 0.000
PC2 0.000 1.000 0.000 0.000 0.000
PC3 0.000 0.000 1.000 0.000 0.000
PC4 0.000 0.000 0.000 1.000 0.000
PC5 0.000 0.000 0.000 0.000 1.000
I
Variable abbreviations starting with d are the current minus the previous values of milk measures in
question. Yield values are in kilogram per day (kg/day), content values are in percentages (%) and rati-
os are unitless. The measures used were group mean averages. ** = P < 0.001

on it, suggesting that MPC is not strongly correlated with
any of the measured milk composition variables (as can
be verified in Table 1) and could therefore be treated as
independent variable in subsequent multivariate analysis.
Since PCs are labeled according to the size of
their variances, the first Principal Component (PC)
explained larger amount of variation (38.88%) among
the variables, while the last PC explained the least
(07.08%). Also, the eigenvalues followed the same trend
as the percentage variance explained by each of the PCs.
The communality estimates, which tells us how much of
the variance in each of the original variables is explained
by the extracted PC was very high ranging from 83.30 to
99.71%. There was a clear grouping of the measured
variables evident by the loading pattern of the variables
on the PCs (the best loading of each variable is indicated
by the bolded values). Each variable loaded only on one
component. No variable loaded heavily on more than one
PC. This suggested that the milk composition variables
can be reduced into smaller composite variable without
losing much of the information.
The PCs displayed varying degrees of
correlations with the milk composition variables
(Table 3) and the correlation structure was similar to the
loading pattern of the milk composition variables on the
PCs. Thus, confirming the loading pattern of the
principal component analysis (Table 2). However, the
correlation among the PCs was zero. This shows that the
Principal component analysis resulted in orthogonal
solution whereby the PCs extracted were completely

1531 Journal of Research in Biology (2014) 4(8): 1526-1533

Alphonsus et al., 2014

Table 4: Descriptive statistics of the principal components

Principal components N Means S.D Min Max
(PCs)
PC1 60 0.00 1.000 -2.544 3.102
PC2 60 0.00 1.000 -3.001 2.573
PC3 60 0.00 1.000 -2.626 2.036
PC4 60 0.00 1.000 -5.045 2.563
PC5 60 0.00 1.000 -4.104 2.085
N= animals, S.D = standard deviation, Min =minimum, Max = maximum

uncorrelated and independent of each other. Also, the
PCs were standardized to have a mean of zero and
standard deviation of one (Table 4)
CONCLUSION
The Principal Component Analysis (PCA)
successfully reduced the dimensionality of the milk
composition variables, by grouping the 17 milk
composition variables into five Principal Components
(PCs) that were uncorrelated and independent of each
other, and explained about 92.38% of the total variation
in the milk composition variables. Therefore, PCA can
be used to solve the problem of multicollinearity and
variable reduction in multiple regression analysis
REFERENCES
Alphonsus C. 2014. Prediction of energy balance and
post-partum reproductive function using
composition measures in dairy cows. PhD Dissertation
Submitted to Department of Animal Science, Ahmadu
Bello University, Zaria-Nigeria.
Alphonsus C and Essien IC. 2012. The relationship
estimates amongst milk yield and milk composition
characteristics of Bunaji and Friesian Bunaji cows.
African Journal of Biotechnology. 11(36): 8790-8793.
Alphonsus C, Akpa GN, Nwagu BI, Barje PP,
Orunmuyi M, Yashim SM, Zana M, Ayigun AE and
Opoola E. 2013. Evaluation of Nutritional Status of
Friesian x Bunaji Dairy Herd Based on Milk
Composition Analysis .Journal of Animal Science
Advances. 3(5): 219-225.
Bair Eric, Trevor Hastie, Paul Debashis and Robert
Tibshirani. 2006. Prediction by supervised Principal
Components. Journal of the American Statistical
Association. 473 (19): 119-137.
ejna V and Chldek G. 2005. The importance of
monitoring changes in milk fat to protein ratio in
Holstein cows during lactation. Journal of Central
European Agriculture. 6: 539-545.
Fahey J. 2008. Milk protein percentage and dairy cow
fertility. University of Melbourne, Department of
Veterinary Science, VIAS, Sneydes Road 600, Werribee,
Victoria, Australia.P.12. Web link: http://
w w w . n h i a . o r g . a u / h t m l /
body_milk_protein__fertility.html 30/10/2008.
milk
Fekedulegn DB, Colbert JJ, Hicks Jr RR and
Schuckers ME. 2002. Coping with multicollinearity: An
example on application of Principal Components
Regression in Dendroecology. Research Paper NE-721,
Newton Square PA: United States Department of
Agriculture. Forest service. 1-48p Web link:
www.fs.fed.us/ne/morgantown/4557/dendrochron/
rpne721.pdf.
Friggens NC, Ridder C and Lvendahl P. 2007. On
the use of milk composition measures to predict the
energy balance of dairy cows. Journal of Dairy Science.
90(12): 5453-5467

Journal of Research in Biology (2014) 4(8): 1526-1533 1532


Hansen LB. 2000. Consequences of selection for milk
yield from a geneticist's viewpoint. Journal of Dairy
Science. 83(5): 1145-1450.
Harris BL and Pryce JE. 2004. Genetic and Phenotypic
relationships between milk protein percentage,
reproductive performance and body condition score in
New Zealand dairy cattle. Proceeding of the New
Zealand Society of Animal Production. 64: 127-131.
Ingvartsen KL, Dewhurst RJ and Friggens NC. 2003.
On the relationship between lactational performance and
health: Is it yield or metabolic imbalance that cause
production diseases in dairy cattle? A position paper.
Livestock Production Science. 83: 277308.
Klainbaum DG, Kupper LL and Muller KE. 1998.
Applied Regression Analysis and Multivariable
Methods. 3rd Edition (Colle Pacific Grove, CA).
Kuterovac K, Balas S, Gantner V, Jovanovac S,
Dakic A. 2005. Evaluation of nutritional status of dairy
cows based on milk analysis results. Italian Journal of
Animal Science. 4(3): 33-35.
Lafi SQ and Kaneene JB. 1992. An explanation of the
use of principal component analysis to detect and correct
for multicollinearity. Preventive Veterinary Medicine. 13
(4): 261-275.
Leahy K. 2001. Multicollinearity: When the solution is
the problem. In Olivia Parr Rud (Ed.) Data Mining
Cookbook (pp. 106 - 108). New York: John Wiley &
Sons, Inc.
Lvendahl P, Ridder C and Friggens NC. 2010.
Limits to prediction of energy balance from milk
composition measures at individual cow level. Journal of
Dairy Science. 93(5): 19982006.
Maitra S and Yan J. 2008. Principal Component
Analysis and Partial Least Squares: two dimension
1533
Alphonsus et al., 2014
reduction techniques for regression. Casualty Actuarial
Society, Discussion paper program. pp.79-90
Oni OO, Adeyinka IA, Afolayan RA, Nwagu BI
Malau-Aduli AEO, Alawa CBI and Lamidi OS. 2001.
Relationships between milk yield, post partum body
weight and reproductive performance in Friesian x
Bunaji Cattle. AsianAustralian Journal of Animal
Science. 14(11): 1505 1654.
Principal Component Analysis http://support.sas.com/
publishing/publicat/chaps/55 )
Pryce JE, Royal PC, Garnsworthy and Mao IL. 2001.
Fertility in the high-producing dairy cow. Livestock
Production Science. 86(1-3): 125-135.
SAS. 2000. SAS Users Guide Version 8.1. Statistical
Analysis system institute Inc, Cary, Nc, USA.
Vaughan TS and Berry KE. 2005. Using Monte Carlo
techniques to demonstrate the meaning and implications
of multicollinearity. Journal of Statistics Education, 13
(1): 1-9. Web link: www.amstat.org/publications/jse/
v13n1/vaughan.html
Yu CH. 2008. Multi-collinearity, variance inflation, and
orthogonalization in regression. Web link: http://
www.creative-wisdom.com/computer/sas/collinear.html
Yu CH. 2010. Checking assumptions in regression. Web
link: http://www.creativewisdom.com/computer/sas/
regression_assumption.html
Submit your articles online at www.jresearchbiology.com
Advantages
Easy online submission
Complete Peer review
Affordable Charges
Quick processing
Extensive indexing
You retain your copyright
submit@jresearchbiology.com
www.jresearchbiology.com/Submit.php
Journal of Research in Biology (2014) 4(8): 1526-1533
 Journal of Research in Biology
An International Scientific Research Journal
Diversity and conservation status of water birds at Upper lake,
Bhopal A Ramsar site in central India
Authors:
Journal of Research in Biology
Hina Veerwal,
Satish Balapure and
Vipin Vyas
Institution:
Department of
Environmental Sciences
and Limnology, Barkatullah
University, Bhopal.
Corresponding author:
Vipin Vyas
Email Id:
vyasvipin992@gmail.com
Web Address:
http://jresearchbiology.com/
documents/RA0485.pdf
Journal of Research in Biology
An International
Scientific Research Journal
ISSN No: Print: 2231 6280; Online: 2231- 6299
Original Research
ABSTRACT:
Wetlands support considerable biodiversity including water birds. In central
India, the Upper lake of Bhopal is designated as a Ramsar site and IBA status
concerning to its diverse avifauna. At present, a detailed study regarding avian
diversity of entire Upper lake is lacking. Considering the increasing population
pressure leading to land use changes in immediate catchment of Upper lake and
affecting the lake ecology, such monitoring is essential. The present work has been
carried out from June 2010 to June 2012. Monthly observations of birds were made
with the aim to identify and enlist various species of water birds of Upper lake. Total
68 species of water birds belonging to 14 families are reported with Anatidae as the
most dominating family with 16 species. Of these, 43 species were migratory and 25
species were residents. Also, 11 important species in terms of their conservation
status are reported. Of these, eight species of Near Threatened status, two species of
Vulnerable status and one species of Endangered status are reported. The presence of
internationally important birds, migratory, local migratory and resident species of
birds in this area indicates the importance of Upper Lake as a year round habitat for
water birds. The increasing human population in the area is placing strain on this
valuable inland freshwater resource and the related avifauna and thus more
conservation efforts are desperately needed.
Keywords:
Upper Lake, wetland, water birds, diversity, conservation status
Article Citation:
Hina Veerwal, Satish Balapure and Vipin Vyas
Diversity and conservation status of water birds at Upper lake, Bhopal A Ramsar site
in central India
Journal of Research in Biology (2014) 4(8): 1534-1542
Dates:
Received: 15 Oct 2014 Accepted:04 Nov 2014 Published: 03 Dec 2014
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
1534-1542| JRB | 2014 | Vol 4 | No 8
www.jresearchbiology.com

INTRODUCTION
Wetlands are shallow water areas which act as
transitional zones between terrestrial and aquatic systems
(Cowardin et al., 1979; Mitsch and Gosselink, 1986) and
support considerable biodiversity of organisms (Dudgeon
et al., 2006). Wetlands hold immense ecological
significance by providing suitable habitat to a vast
variety of faunal diversity. Water birds generally occupy
the position of top level consumers in aquatic food chain
and thus any changes in the habitat and food availability
directly affects them. Water birds are thus considered as
indicators of wetland habitat conditions (Kushlan, 1978).
Among the several kinds of wetlands such as marshes,
lagoons, bogs, fens, mangroves and other open water
bodies etc., urban lakes are at a large risk of habitat
degradation due to the prevailing anthropogenic pressure
in the surroundings which in turn affects the avian
diversity supported by them.
India possesses wide-ranging wetland habitats
that support numerous water birds, many of which are
migratory visiting the subcontinent from their breeding
Map Showing Sampling Stations of Upper Lake
S1- Prempura Ghat S2- VanVihar
S3- Takia Island S4- Kamla Park
S5- Inlet A S6- Inlet B
S7- KhanuGaon S8- BehtaGaon
S9- Deep Zone Urban S10-Deep Zone Rural
S11- BishenKhedi S12- GohraGaon
Figure 1 Map of the study area showing all the sampling stations
1535
Veerwal et al., 2014
grounds in the northern regions. In central India, the
Upper lake of Bhopal is an important water bird abode
for resident species as well as an equally prominent
staging and wintering site for numerous migratory
species. It is designated as a Ramsar site and IBA status
in concern to the fact that it supports a distinct and ample
population of water birds around the year. A variety of
water birds like cormorants, egrets and herons, storks
and ibises, crane, ducks, jacanas, lapwings, stilts,
sandpipers, gulls and terns, kingfishers etc. find refuge in
this lake (Vyas, 1992) emphasizing the overall
importance of this water body. The avian species
richness supported by Upper lake is largely due to the
presence of high food availability which attracts avifauna
to settle here (Vyas et al., 2010). At present, a detailed
study regarding avian diversity of entire area of Upper
lake is lacking which is essential considering the
increasing population pressure leading to changes in the
landuse of immediate catchment of Upper lake and
affecting the lake ecology. Thus, the present work has
been carried out with the aim to identify and enlist
S8 S6 S5
S7
S10
S9
S3
S4
S11
S2
S12
S1
Upper Lake
Polygon
Journal of Research in Biology (2014) 4(8): 1534-1542
Veerwal et al., 2014
Figure 2 Upper Lake of Bhopal
various species of water birds visiting and residing in the
Upper lake which may provide a baseline for the future
management of avian fauna in the area.
MATERIALS AND METHODS
The Upper lake is an east-westerly elongated
urban lake which receives water from the river Kolans
and from the direct rains, both during rainfall months
which is the main source of drinking water for the
residents of Bhopal. This lake was formed by
constructing an earthen dam across the Kolans river in
the 11th century by Raja Bhoj of Dhar. The catchment
area of the lake is 361 sq. km and the water spread area
is 30.72 km at FTL, its mean depth is 3.16 m while
maximum depth is 11.64 m. The excess water from
Upper lake flows into Kaliasot River which further meets
Betwa River and finally gets drained into the Yamuna
river. The altitude of Upper lake is about 503 m above
o
mean sea level and it is situated at 23 16 N latitude and
o
77 25 E longitude. It is an east westerly elongated
shallow lake bordered by human settlements on the
eastern and northern boundaries while its western
margins are intensively used for agriculture, on the
southern margin lies Van Vihar National Park. It has
irregular margins that support dense growth of
macrophytes and sustains well diversified aquatic flora
and fauna.
The study was conducted from June 2010 to June
2012 at the Upper Lake of Bhopal (Figure 1 and 2). The
Journal of Research in Biology (2014) 4(8): 1534-1542
sampling points, total 12 in number, were identified
carefully keeping in mind the subject of habitat features
and avifaunal occurrence and also such limitations as
approachability (Figure 1). Study was conducted with
special reference to diversity of birds. Monthly
observations of birds were made during the study where
birds were observed within 300 m transect using Nikon
Binoculars of 1050 magnification. Identification of the
water birds was done using standard taxonomic keys (Ali
and Ripley, 1988; Ali, 2002) and the checklist was
prepared as per Manakadan and Pittie (2001). On foot
surveys were done by walking across selected tracks
along the lake margins and the lake area was covered
using boats. Information from the local community and
on site observations were recorded on every visit and
accumulated at the end of the study. The status of the
birds is categorized as Resident (R), Migratory (M) and
Resident Migratory (RM) after Ali (2002) and the
threatened status is taken according to the Bird Life
International, (2014).
RESULTS AND DISCUSSION
The lake ecology of Upper Lake is under
constant threat due to various on going developmental
activities in the immediate catchment. In the urban
surroundings, a vast increase in developmental activities
and related waste dumping is observed whereas in the
rural areas, major changes in cropping patterns, including
extensive use of chemical fertilizers and pesticides has
1536
 Veerwal et al., 2014

Table 1 The residence and conservation status of bird diversity of Upper lake

S.No Family Scientific Name Common Name Status

Residence Conservation
Podicipitidae
1 Tachybaptus ruficollis Little Grebe R LC
Phalacrocoracidae
2 Phalacrocorax carbo Great Cormorant RM LC
3 Phalacrocorax fuscicollis Indian Shag RM LC
4 Phalacrocorax niger Little Cormorant RM LC
5 Anhinga melanogaster Darter RM NT
Ardeidae
6 Casmerodius albus Large Egret RM LC
7 Egretta garzetta Little Egret R LC
8 Mesophoyx intermedia Median Egret RM LC
9 Bubulcus ibis Cattle Egret RM LC
10 Ardea cinerea Grey Heron RM LC
11 Ardea purpurea Purple Heron RM LC
12 Butorides striatus Little Green Heron R LC
13 Nycticorax nycticorax Black-crowned Night-Heron R LC
14 Ardeola grayii Indian Pond-Heron R LC
15 Ixobrychus cinnamomeus Chestnut Bittern RM LC
Ciconiidae
16 Mycteria leucocephala Painted Stork RM NT
17 Anastomus oscitans Asian Openbill-Stork R LC
18 Ciconia episcopus White-Necked Stork R V
19 Ciconia ciconia European White Stork M LC
20 Ciconia nigra Black Stork M LC
21 Ephippiorhynchus asiaticus Black-Necked Stork R NT
Threskiornithidae
22 Threskiornis melanocephalus Oriental White Ibis R NT
23 Pseudibis papillosa Black Ibis R LC
24 Plegadis falcinellus Glossy Ibis RM LC
25 Platalea leucorodia Eurasian Spoonbill RM LC
Anatidae
26 Anser indicus Bar-headed Goose RM LC
27 Tadorna ferruginea Brahminy Shelduck RM LC

28 Sarkidiornis melanotos Comb Duck R LC

29 Dendrocygna javanica Lesser Whistling-Duck R LC

30 Anas acuta Northern Pintail M LC
31 Anas crecca Common Teal M LC
32 Anas poecilorhyncha Spot-billed Duck RM LC
33 Anas platyrhynchos Mallard RM LC
34 Anas strepera Gadwall M LC

become a common practice. The entry of pesticides from the rural margins and untreated sewage from both urban

1537 Journal of Research in Biology (2014) 4(8): 1534-1542

Veerwal et al., 2014

36 Anas clypeata Northern Shoveller M LC

37 Anas querquedula Garganey M LC
38 Rhodonessa rufina Red-crested Pochard M LC
39 Aythya ferina Common Pochard M LC
40 Aythya nyroca Ferruginous Pochard RM NT
41 Nettapus coromandelianus Cotton Teal R LC
Gruidae
42 Grus antigone Sarus Crane R V
Rallidae
43 Amaurornis phoenicurus White-breasted Waterhen R LC
44 Gallinula chloropus Common Moorhen RM LC
45 Porphyrio porphyrio Purple Moorhen R LC
46 Fulica atra Common Coot RM LC
Jacanidae
47 Metopidius indicus Bronze-winged Jacana R LC
48 Hydrophasianus chirurgus Pheasant-tailed Jacana R LC
Charadriidae
49 Vanellus indicus Red-wattled Lapwing R LC
50 Charadrius dubius Little Ringed Plover RM LC
51 Charadrius alexandrinus Kentish Plover RM LC
52 Tringa totanus Common Redshank RM LC
53 Tringa nebularia Common Greenshank M LC
54 Actitis hypoleucos Common Sandpiper RM LC
55 Tringa stagnatilis Marsh Sandpiper M LC
56 Tringa glareola Wood Sandpiper M LC
57 Calidris temminckii Temmincks Stint M LC
58 Numenius arquata Eurasian Curlew M NT
59 Limosa limosa Black-tailed Godwit M NT
Rostratulidae
60 Rostratula benghalensis Greater Painted-Snipe R LC
61 Gallinago gallinago Common Snipe RM LC
Recurvirostridae
62 Himantopus himantopus Black-winged Stilt R LC
Laridae
63 Sterna aurantia River Tern R NT
64 Sterna acuticauda Black-bellied Tern R E
65 Larus brunnicephalus Brown-headed Gull RM LC
Alcedinidae
66 Ceryle rudis Lesser Pied Kingfisher R LC
67 Alcedo atthis Small Blue Kingfisher RM LC
68 Halcyon smyrnensis White breasted Kingfisher R LC
Residenoncern; NT Near Threatened; V Vulnerable; E Endangeredce Status :R Resident; RM Resident
Migratory; M MigratoryConservation Status : LC Least

and rural surroundings is severely affecting water quality
of the lake (Nandi, 2003). As a result, this water bird
habitat is under immense pressure, which if not managed
and conserved properly, may deteriorate to a level where
its suitability to water birds may decline irreversibly. The
Upper lake supports many ecosystem services on which
many taxa depend. Thus changes in its wetland structure
will undoubtedly also affect the related biodiversity
especially the associated avifauna. The major threats to
avifauna in the area are (i) intense agriculture in the
surrounding villages which is seriously degrading the
natural water quality and thus habitat of water birds (ii)
uninterrupted effluent discharge leading to high nutrient
levels which may lead to increased eutrophication (iii)
sedimentation leading to reduced water storage that may
influence the open water area required by waterfowl (iv)
livestock grazing that disturbs the water birds (v)
unmanaged tourism and religious activities also
adversely affect bird population.
A list of birds recorded from Upper lake along

Journal of Research in Biology (2014) 4(8): 1534-1542 1538


with their common names, residence and conservation
status is reported in Table 1. In the present study, 68
species of water birds belonging to 7 orders and 14
families are reported at Upper lake. Among these, the
most dominating family with 16 species is Anatidae as
also observed by Vyas (1992; Vyas et al., 2010) in
Upper lake and Balapure et al. (2012) in Barna reservoir
of M.P. The second dominant family in the present study
was Charadriidae with eleven species, Ardeidae with ten
species, Ciconiidae with six species, Phalacrocoracidae,
Threskiornithidae and Rallidae each with four species,
Laridae and Alcedinidae each with three species,
Jacanidae and Rostratulidae each with two species while
Podicipitidae, Gruidae and Recurvirostridae were all
represented by a single species each. Kumar and Gupta
(2009; 2013) have also noted that family Anatidae
dominated the wetland bird community at Kurukshetra
and Chhilchhila Wildlife Sanctuary, Haryana
respectively. Verma (2009) reported 68 waterbird species
Bharatpur, Bundh Baretha Reservoir where Anatidae,
Scolopacidae, and Ardeidae were recorded as the
dominant families. Total 39 water bird species belonging
to 16 families were reported by Das and Saikia (2011)
from Deeporbeel of Assam. Twenty eight species of
water birds were reported in three lakes viz. Rajura,
Godada and Dhanora lakes of Buldhana district in
Maharashtra, India by Joshi (2012).
The waterbird diversity was observed to be
Figure 3 SARUS CRANE at Upper lake of Bhopal
1539
Veerwal et al., 2014
higher during post monsoon and winter months. Deshkar
et al. (2010) have reported similar observations on
seasonal variations in species richness of birds. The high
diversity during winter is due to arrival of migratory
birds during this season and minimum during summer as
during this season all the migratory birds leave the area
and return to their home ground. Least number of avian
species was recorded in summer and monsoon which
may be due to departure of winter visitors, local
migration of resident birds, drying of sites making
habitats unsuitable for birds in summer season and the
commencement of heavy rains in monsoon season. Such
behaviour is also reported by Surana et al. (2007) in
Chimdilake of Nepal. Out of the total 68 species of
waterbirds, 43 species (63.24 %) were migratory birds
while 25 species (36.76 %) were resident birds. Of all the
43 migratory birds, 27 species (62.79 %) were local
migrants and 16 species (37.21 %) were long distance
migrants. Chinchkhede and Kedar (2012) reported 54
residents, 9 local migrants and 13 winter migrants among
total observed 76 water bird species in and around the
Koradi lake of Nagpur. Among all the 68 water bird
species using Upper Lake, 11 important species in terms
of their conservation status are reported (IUCN, 2014).
Of these 11 species, 8 species of Near Threatened status,
2 species of Vulnerable status and 1 species of
Endangered status are reported in this study. These
Near-threatened species are: Black-Necked Stork
(Ephippiorhynchus asiaticus), Black-headed Ibis
(Threskiornis melanocephalus) and River Tern (Sterna
aurantia) which are all resident water birds; Darter
(Anhinga melanogaster) (Figure 4), Painted Stork
(Mycteria leucocephala) and Ferruginous Pochard
(Aythya nyroca) which are resident migratory water
birds; Black tailed Godwit (Limosa limosa) and Eurasian
Curlew (Numenius arquata) which are migrant species of
water birds. Among the vulnerable species were 2 water
birds - White necked Stork (Ciconia episcopus) and
Sarus Crane (Grus antigone) (Figure 3) which are both
Journal of Research in Biology (2014) 4(8): 1534-1542
Veerwal et al., 2014
resident birds. One water bird species reported in the
endangered category is the Black-bellied Tern (Sterna
acuticauda) which is a resident bird. Asian Openbill
Stork (Anastomus oscitans) (Figure 5) is a resident water
bird of least concern.
Presence of such important birds as regular
visitors as well as some residents in this lake highlights
the importance of this area as a bird haven. Water birds
like Black-necked Stork and White necked Stork were
found along the shoreline areas of the lake with moderate
tree canopy which allowed these birds proper roosting
sites as well as protection from direct human disturbance.
Maheswaran and Rahmani (2001) have reported that
higher water level (> 60 cm) is not suitable for wading
birds, including the Black-necked Stork even though the
patch has more prey species. Sarus Crane and Black-
headed Ibis preferred marshy borders of the wetland with
more affinity towards surrounding agricultural fields.
Thus, these species demonstrated a tendency to endure
human presence up to some level. Darter and Painted
storks were found preferring areas with dense
surrounding vegetation but with least disturbance and
moderate water depth. The conversion of wetlands into
agricultural fields is altering the preferred habitats of
birds and thus negatively impacting their distribution
(Del Hoyo et al., 1996; Bird Life International, 2014).
The terns were mostly observed at deep water zones of
Figure 4 DARTER at Upper lake of Bhopal
Journal of Research in Biology (2014) 4(8): 1534-1542
lake with proper perching sites where the birds could
assemble to consume their prey which were for the most
part small fishes.
CONCLUSION
Upper lake is an ideal residence for innumerable
water birds with different needs. The presence of
migratory birds, local migratory and resident species of
birds equally utilizing this area as their abode for
continuing various lifecycle activities indicates the
importance of Upper Lake as a year round habitat for
water birds.
However, identification of this lake as a Ramsar
site and IBA is alone not sufficient to conserve the entire
biodiversity. In the present state of ecologically
unmanaged development, full protection to all the
existing habitats should be given with special attention
during the migratory period. Since the lake is a shared
resource, a common property to all the occupants
including animals and human being alike, it is imperative
to protect or conserve the entire ecosystem. For this, the
most notorious component of this arrangement and the
principal stakeholder involved in the deterioration
process i.e. the human community must be educated
about the importance of conserving the lake. With an
increasing human population placing strain on the
valuable inland freshwater resources and the related
Figure 5 OPENBILL-STORK at Upper lake, Bhopal
1540

avifauna due to rapid conversion of rural areas to urban
lands, by creating awareness in local people through
dispersion of fortifying environmental education
concerning disturbance effects on ecosystem especially
wildlife could aid in further conservation efforts.
ACKNOWLEDGEMENTS
We are thankful to the University Grants
Commission (UGC), Government of India, New Delhi,
for providing financial assistance in the form of award of
Junior/ Senior Research Fellowship to Hina Veerwal
which made completion of this work possible.
REFERENCES
Ali S. 2002. The Book of Indian Birds. Bombay Natural
History Society. Oxford University Press, 326.
Ali S and Ripley SD. 1988. Compact handbook of the
birds of India and Pakistan together with those of
Bangladesh, Nepal, Bhutan and Sri Lanka. Oxford
University Press 2nd Edition, Delhi, 890.
Balapure S, Dutta S and Vyas V. 2012. Avian
diversity in Barna \wetland of Narmada basin in central
India. Journal of Research in Biology. 2(5): 460-468.
Bird Life International 2014. IUCN Red List for birds.
Downloaded from http://www.birdife.org on 20/08/2014.
Chinchkhede KH and Kedar GT. 2012. Avifaunal
diversity of Koradi lake in Nagpur district of central
India. Journal of Research in Biology. 2(2): 070-076.
Cowardin LM, Carter V, Golet FC and La Roe ET.
1979. Classification of wetlands and deep water habitats
of the United States. U. S. Department of the Interior,
Fish and Wildlife Service, Washington, D. C. 131.
Das J and Saikia PK. 2011.Conservation threats to the
water birds in Deep or Beel. Assam Journal of Research
in Biology. 1(6): 435-439.
1541
Veerwal et al., 2014
Del Hoyo J, Elliott A and Sargatal J. 1996. Handbook
of the birds of the world, vol. 3: Hoatzin to Auks. Lynx
Edicions, Barcelona, Spain. p 821.
Deshkar S, Rathod J and Padate G. 2010. Avifaunal
diversity and water quality analysis of an inland wetland.
Journal of Wetlands Ecology. 4: 132.
Dudgeon D, Arthington AH, Gessner MO, Kawabata
Z, Knowler DJ, Leveque C, Naiman RJ, Prieur-
Richard AH, Soto D, Stiassny ML and Sullivan CA.
2006. Freshwater biodiversity: importance, threats, status
and conservation challenges. Biological Reviews. 81(2):
163-182.
Joshi PS. 2012. An annotated checklist of aquatic
avifauna of Rajura, Godada and Dhanora lakes of
Buldhana district of (M. S.) India. Science Research
Reporter. 2(1): 3033.
Kumar P and Gupta SK. 2009. Diversity and
abundance of wetland birds around Kurukshetra, India.
Our Nature. 7(1): 212217.
Kumar P and Gupta SK. 2013. Status of wetland birds
of Chhilchhila Wildlife Sanctuary, Haryana, India.
Journal of Threatened Taxa. 5(5): 39693976.
Kushlan JA. 1978. Feeding ecology of wading birds. In:
Wading Birds. Sprunt, A. IV, Ogden, J. C. and Winckler,
S. (Eds.), National Audubon Society. Research Report
No. 7. New York, 249-297.
Maheswaran G and Rahmani AR. 2001. Effects of
water level changes and wading bird abundance on the
foraging behaviour of black necked storks
Ephippiorhynchus asiaticus in Dudwa National Park,
India. Journal of Bioscience. 26(3): 373-382.
Manakadan R and Pittie A. 2001.Standardised
common and scientific names of the birds of the Indian
subcontinent. Buceros. 6(1): 1-37.
Journal of Research in Biology (2014) 4(8): 1534-1542
 Veerwal et al., 2014
Mitsch WI and Gosselink IG. 1986. Wetlands. Van
Nostrand Reinhold, New York. 539.

Nandi PK. 2003. Management of Upper Lake

Watershed. First Interagency Conference on Research in
the Watersheds, October 27 -30, Benson,
Arizona.www.tucson.ars.ag.gov/icrw/Proceedings/
Nandi.pdf

Surana R, Subba BR and Limbu KP. 2007. Avian

diversity during rehabilitation stage of Chimdi Lake,
Sunsari , Nepal. Our Nature. 5(1): 7580.

The IUCN Red List of Threatened Species. Version

2014.2.www.iucnredlist.org.

Verma A. 2009. Occurrence and abundance of water

birds at Bundh Baretha reservoir, Bharatpur, north-
western India. Indian Birds. 4(5): 150153.

Vyas V. 1992. Waterfowl community of Bhoj of Bhopal

with reference to its management and conservation.
Environment and Biodegradation. 155-162.

Vyas V, Vishwakarma M and Dhar N. 2010. Avian

Diversity of Bhoj Wetland: A Ramsar Site of Central
India. Our Nature. 8(1): 3439.

Submit your articles online at www.jresearchbiology.com

Advantages
Easy online submission
Complete Peer review
Affordable Charges
Quick processing
Extensive indexing
You retain your copyright
submit@jresearchbiology.com
www.jresearchbiology.com/Submit.php

Journal of Research in Biology (2014) 4(8): 1534-1542 1542

 Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

A review on ethnomedicinal, phytochemical and pharmacological studies

of Geodorum densiflorum (Lam.) Schltr. - an endangered orchid
Journal of Research in Biology

Authors: ABSTRACT:
Keerthiga M* and
Anand SP.

From the primitive period, medicinal plants have been used in treating
Institution: human diseases and the traditional herbal medicines have owned its distinct place in
1. Department of
medication because of their pharmacological importance. Geodorum densiflorum is a
Biotechnology, National
medicinally important endangered orchid which is widely used in the orient.
College (Autonomous),
Tiruchirappalli 01, Flavonoids, alkaloids and terpenoids, the primary constituents of G. densiflorum are
Tamil Nadu. mainly believed to be responsible for its wide therapeutic actions. The orchid was
traditionally recommended for various ailments such as wound healing, skin diseases,
2. PG and Research dysentery, diabetes, improving fertility in men, to cure carbuncles and to regularize
Department of Botany, menstrual cycle in women. The present review provides comprehensive information
National College on plant, ethnomedicinal, phytochemical and pharmacology profile and current
(Autonomous), research prospects of the orchid.
Tiruchirappalli 01, Tamil
Nadu

Corresponding author: Keywords:

Keerthiga M. Orchidaceae, Ethnomedicinal uses, Geodorum densiflorum

Article Citation:
Email Id:
keerthigamanohar@gmail.com
Keerthiga M and Anand SP.
A review on ethnomedicinal, phytochemical and pharmacological studies of
Geodorum densiflorum (Lam.) Schltr. - an endangered orchid
Journal of Research in Biology (2014) 4(8):1543-1548
Dates:
Web Address:
http://jresearchbiology.com/ Received:22 Aug 2014 Accepted: 12 Sep 2014 Published: 03 Dec 2014
documents/RA0474.pdf
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1543-1548| JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
Orchids belonging to the family Orchidaceae
display diversified in their floral morphology compared
to other angiospermic plants. Eventhough the family is
characterised as highly advanced group, they are at risk
for slight changes in environmental conditions will cause
the orchids as endangered one. Approximately 25,000 to
35,000 species within 600 800 genera of Orchidaceae
were distributed throughout the world (Chugh et al.,
2009). In India, Orchidaceae form 9% of our flora and
about 1,300 species with 140 genera was found in
Himalayas as their native (Yonzone et al., 2008).
Orchidaceae plants will grow in variety of habitats and
are mostly annual and perennial herbs and live either as
epiphytes, lithophytes and terrestrial. The people are
attracted by orchids because of their high ornamental
value but nowadays they are known for its medicinal
importance. In Chinese Materia Medica, a total of 365
plants including several orchids are grown primarily as
ornamentals, many of which are now used as herbal
medicines, food and other cultural components by tribes
in different parts of the world.
Geodorum densiflorum (Lam.) Schltr. belongs to
the family Orchidaceae, an endangered terrestrial orchid
(Datta et al., 1999) commonly known as Nodding
Swamp orchid. It is a glabrous and deciduous plant
which grows during spring and summer, becoming
deciduous in winter (Ramirez, 2007) and is widely
distributed in the tropical forests of south India
(Sudhakor et al., 2005) and in Bangladesh.
G.densiflorum is a medicinal orchid plant that has been
traditionally used for the treatment of various diseases.
The root paste is used as an insecticide and wound
healing medicine. The pseudobulb is used to regularize
menstrual cycle (Dash et al., 2008), for the treatment of
diabetes (Patil et al., 2005, Roy et al., 2002), and it is
applied externally to cure carbuncles (Nath et al., 2011),
etc. It is also possessing antimicrobial and antidiabetic
property (Saleha et al., 2010).
1544
Keerthiga and Anand, 2014
Intensive phytochemical investigations of the
leaves and pseudobulb of orchid have resulted in the
presence of flavonoids, terpenoids, alkaloids and
steroids. The isolated compounds display some of the
interesting pharmacological activities, such as
antibacterial, cytotoxicity, antioxidant, thrombolytic,
analgesic, sedative and anxiolytic properties. Despite
number of studies were reported on the evaluation of
biologically active components and their
pharmacological action, yet these studies were still
inadequate. Although there have been several claims
regarding the bioactive compounds which involved in the
biological actions of the plant, more scientific research
are needed to justify the therapeutic value of this orchid.
The present review gives a detailed account of the plant,
ethnomedicinal uses, phytochemical and pharmacology
profile along with the current research potential of the
orchid.
Systematic Description
Kingdom - Plantae
Division - Tracheophyta
Class - Magnoliopsida
Order - Asparagales
Family - Orchidaceae
Genus - Geodorum
Species -densiflorum (Lam.) Schltr. (ITIS Report,
www.itis.gov)
Plant Description
Terrestrial herb forming small clumps, 33-50 cm
tall. Pseudobulbs subglobose, crowded, subterranean to
partially emergent, erect, ovoid, 2-4 x 3-3.6 cm, fleshy,
often yellowish, enclosed by scarious sheaths. Stem
erect, 8 cm long, slender. Leaves
3-5, erect to prostrate, apical; enclosed in sheathing
bracts forming a pseudostem; 10-32 x 4-9 cm, suberect,
lanceolate, lanceolate-elliptic, or oblong-elliptic, acute to
acuminate, petiolate, blade; petiole 5-22 cm long, dark
green to yellowish, pleated, margins entire to undulate
Journal of Research in Biology (2014) 4(8): 1543-1548
Keerthiga and Anand, 2014

margins entire to undulate, apex acute to acuminate. are made into powder and 5 gm of the powder mixed
Inflorescence: shorter than the leaves, compact, with 200 ml cow milk is given orally twice a day for 15
subglobose axillary raceme, 200-300 mm tall, nodding in days or till it is cured (Tiwari et al., 2012).
flower, erect in bud and at seed dispersal; pendent, 8-10 Tubers are cleaned, powdered and taken with honey to
flowered; pedicels 5-10mm long. Flowers: 0.9-1.2 cm increase sperm density (Rahmatullah et al., 2010).
long, white to pale green with yellow and purple Rhizomes are consumed along with honey for two to
markings. Sepals and petals similar, gaping, linear- three months as a treatment for impotency (Patil et al.,
oblong, ovate-elliptic, acute, 5 to 7 veined. Dorsal sepal 2004).
lanceolate, lateral sepals free, lanceolate. Petals similar Phytochemical profile
to lateral sepals. Lip 1-1.3 x 0.8-1.1 cm, pink with dark G. densiflorum was investigated for the
red veins boat shaped; apex obscurely 2 rounded lobes, phytochemical chemical constituents of methanol, ethyl
margins weakly undulate; Column 3-4 mm long, weakly acetate and petroleum ether extracts of the roots which
curved, with a foot. Capsules dehiscent, obovoid, revealed the presence of carbohydrate, alkaloid,
pendant. Fruit elongated. (Figure 1, 2 and 3). glycoside and steroid (Habib et al., 2011).
Ethnobotanical Uses Phytochemical screening of G. densiflorum shows
To regularize menstrual cycle in women, the root paste presence of alkaloids, steroids, carbohydrates,
of G. densiflorum was taken orally by mixing along with flavonoids, tannin and saponins in different extract such
two drops of ghee and 5 ml of honey (Dash et al., as water, ethanol and chloroform and were reported
2008). having various pharmacological activities (Keerthiga et
Australians use the gum obtained from root stock for al., 2014).
joining parts of musical instruments. Fresh root stocks Various major, minor and trace elements
were crushed and rubbed on cattle to kill flies (Yonzone concentration of G. densiflorum were studied for
et al., 2011). determining pharmacological action against various
Tuber powder mixed with cow ghee is employed to diseases. Among the twenty six vital elements, the plant
treat dysentery (Mohammad, 2011). had maximum concentration of phosphorus. As stated by
Tuber is used to treat impotency in men. Dried tubers Hossen et al. (2014), Phosphorus participates in DNA

Figure 1. Habit of Geodorum densiflorum Figure 2. Vegetative stage of Geodorum densiflorum

Journal of Research in Biology (2014) 4(8): 1543-1548 1545


Figure 3. Flowers of Geodorum densiflorum (Lam.)
synthesis, energy metabolism and calcium absorption
and utilization.
Pharmacological profile
Antibacterial activity
In vitro antibacterial study of three different
extracts using water, ethanol and chloroform solvents
with various concentrations was tested for their
bioactivity. Maximum antibacterial activities were
recorded for ethanolic extract against Staphylococcus
aureus followed by Klebsiella pneumoniae but water
extracts didnt show any significant antibacterial activity
(Keerthiga et al., 2014)
The antibacterial screening of extracts revealed
that methanolic extract showed significant activity
against both Gram positive and Gram negative bacterium
than other extracts (Akter et al., 2010). Previous
investigation on the antibacterial activity of G.
densiflorum against thirteen pathogenic bacteria was
reported that ethyl acetate extract displayed the highest
inhibitory actions (Habib et al., 2011).
Antioxidant activity
G. densiflorum (Lam) Schltr. was investigated
for the antioxidant property of methanol, ethyl acetate
and petroleum ether extracts of the roots. In vitro
antioxidant activity of the extracts was performed using
various methods. DPPH radical scavenging and Nitric
1546
Keerthiga and Anand, 2014
Oxide (NO) scavenging activity showed that the
petroleum ether extract had high antioxidant capacity
than other extract. Reducing power and CUPRAC assay
revealed that ethyl acetate extract was having high
reducing activity (Habib et al., 2011).
Cytotoxic Activity
In vivo cytotoxicity studies were subjected and a
concentration dependent increase in percent mortality
was produced by the extracts. The study revealed the
presence of cytotoxic principles in these extractives
(Habib et al., 2011).
The cytotoxic activities of crude extract were
determined using brine shrimp lethality bioassay and on
comparison to vincristine sulfate as standard, ethyl
acetate extract showed a significant cytotoxic activity
(Hossain et al., 2012).
Thrombolytic activity
Thrombolytic activity of five traditional
medicinal plants was evaluated from which
G. densiflorum whole plant with different solvents viz:
ethanol, petroleum ether and ethyl acetate shows
moderate to negligible amount of clot lysis, i.e. 25.31%,
29.82%, and 19.06% (Hossen et al., 2014).
Analgesic activity
Khatun et al. (2013) evaluated peripheral
analgesic activity by conducting acetic acid induced
writhing test. The n-hexane and dichloromethane soluble
extract of the root of G. densiflorum was used for
analgesic activity in mice. At higher dose (400mg/
kg, p.o.), both extracts exhibited significantly (p<0.001)
moderate analgesic property compared to that of
diclofenac sodium which might be due to interaction
with the central and/or peripheral opiate system.
Sedative activity
Extracts from G. densiflorum using n-hexane and
dichloromethane was assessed by hole cross and open
field tests. Both extracts at doses of 200mg/kg and
400mg/kg, p.o. showed statistically significant (p<0.001
Journal of Research in Biology (2014) 4(8): 1543-1548
Keerthiga and Anand, 2014
and p<0.01) suppression of locomotor and exploratory
behaviours of mice compared to that of diazepam in the
hole cross test. In the open field test, all test samples
except 400mg/kg dose of dichloromethane soluble root
extract showed same results that were observed in the
hole cross test. The study done by Khatun et al. (2013)
revealed the sedative activity of the medicinal orchid.
Anxiolytic property
The root of G. densiflorum was reported to
possess anxiolytic activity in both n-hexane and
dichloromethane extracts as confirmed by the hole board
test (Khatun et al., 2013).
CONCLUSION
It is concluded that the plant extract possesses
antibacterial and antioxidant property and it can be used
effectively as a natural tool for treating different
infectious diseases. The orchid is also reported to possess
cytotoxic, thrombolytic, sedative, analgesic and
anxiolytic activities. The present review provided
combined information about ethnomedicinal,
phytochemical and pharmacological studies on G.
densiflorum. It is necessary to carry out more clinical and
pharmacological studies by the researchers to exploit the
potential of this plant. Further investigations to find out
novel drug lead compounds of industrial importance
could be a new platform for future researchers.
REFERENCES
Arjun Prasad Tiwari, Bhavana Joshi and Ansari AA.
2012. Less known ethno medicinal uses of some orchids
by the Tribal inhabitants of Amarkantak Plateau,
Madhya Pradesh, India. Nature and Science. 10(12): 33-
37.
Chugh S, Guha S and Rao IU. 2009. Micropropagation
of orchids: A review on the potential of different
explants. Scientia Horticulturae. 122(4): 507-520.
Dash PK, Sahoo S and Bal S. 2008. Ethno botanical
studies on orchids of Niyamgiri hill ranges Orissa, India.
Ethnobotanical leaflets.12: 70-78.
Journal of Research in Biology (2014) 4(8): 1543-1548
Datta KB, Kanjilal B and Desarker D. 1999. Artificial
seed technology: Development of a protocol
in Geodorum densiflorum (Lam.) Schltr. An endangered
orchid. Current Science. 76: 1142-1145.
Farjana Khatun, Nishat Nasrin, Shammee Monira,
Muhammad Asaduzzaman and Apurba Sarker Apu.
2013. Assessment of neuropharmacological and
analgesic potentials of Geodorum densiflorum (Lam.)
Schltr. root extracts in experimental animals.
Pharmacologyonline. 3: 16-22
Habib M R, Rana M S, Hasan M R, Imam M Z,
Hasan S M R and Saha A. 2011. Antioxidant,
cytotoxicity and antibacterial potential of different
extract of Geodorum densiflorum (Lam.) Schltr. root.
Journal of Herbal Medicine and Toxicology. 5 (1): 63-
70 .
http://www.itis.gov/servlet/SingleRpt/
SingleRptsearch_topic=all&search_value=Geodorum+
densiflorum&search_kingdom=every&search_span=exac
tly_for&categories=All&source=html&search_credRatin
g=All (Accessed on July 10, 2012).
Keerthiga M and Anand SP. 2014. Physicochemical,
preliminary phytochemical analysis and antibacterial
activity against clinical pathogens of medicinally
important orchid Geodorum densiflorum (Lam) Schltr.
International Journal of Pharmacy and Pharmaceutical
Sciences. 6(8): 558-561
Lokman Hossen Md, Azharul Islam SM, Md. Joynal
Abedin, Shirin Akter, Rasel OF, M. Monjur Ahasan,
Rajada Khatun and Ashrafun Nahar Monika 2014.
Elemental profile analysis of some traditional medicinal
plants of Bangladesh using PIXE Technique. Journal of
Nuclear and Particle Physics. 4(5): 137-141.
Moazzem Hossen SM, Irfan Newaz khan, Md
Mominul Islam Sarkar and Md Anwar Jahid. 2014.
Assessment of thrombolytic activity of five Bangladeshi
medicinal plants: potential source for thrombolytic
compounds. International Blood Research and Reviews
2(6): 262-269.
Mohammad HM. 2011. Therapeutic orchids: traditional
uses and recent advances An overview. Fitoterapia. 82
(2): 102-140.
1547
 Keerthiga and Anand, 2014

hills of West Bengal, India, Golden Research Thought.1

Mohammad Shahadat Hossain, Mohammed Abu
Sayeed, Mohammed Aktar Sayeed and Mohammad
Ehsanul Hoque Chowdhury. 2012. Investigation on in-
Vitro cytotoxic, antibacterial and phytochemical
screening of ethyl acetate extract of Geodorum
densiflorum (Lam.) Schltr. The Pharma Innovation. 1(8):
108-113.
(6): 121-22.
Yonzone, Rajendra and Kamran A. 2008.
Ethnobotanical uses of Orchids. Abstract in an
International Seminar Multidisciplinary Approaches in
Angiosperm Systematics Kalyani University, West
Bengal, India.

Mohammed Rahmatullah, Md Nur Kabidul Azam,

Md Ariful Haque Mollik, Md Mahamudul Hasan,
Azmal Ibna Hassan, Rownak Jahan, Farhana Jamal,
Dilruba Nasrin, Rasheda Ahmed, Md Mahbubur
Rahman and Mst Afsana Khatun. 2010. Medicinal
plants used by the Kavirajes of Daulatdia Ghat, Kushtia
district, Bangladesh. American-Eurasian Journal of
Sustainable Agriculture. 4(2): 219-229.

Nath M, Dutta BK and Hajra PK. 2011. Medicinal

plants in major diseases by Dimasa tribes of Barak
valley, Assam University. Journal of Science and
Technology: Biological and Environmental Sciences. 7
(1): 1 8-26.

Patil MV and Patil DA. 2004. Plants used in

reproductive ailments by tribals of Nasik district
(Maharashtra). Ancient science of life. XXIII (3): 1-4.

Patil MV and Patil DA. 2005. Ethnomedicinal practices

of Nasik district, Maharashtra. Indian Journal of
Traditional Knowledge. 4(3): 287-290.

Roy J and Banerjee N. 2002. Rhizome and shoot

development during in vitro propagation of Geodorum
densiflorum (Lam.) Schtr. Scientia Horticulturae. 94(1-
2): 181-192.

Salcha Akter, Mohammad Zafar Iman and Tahira

Akter. 2010. Antimicrobial activity of different extracts
Submit your articles online at www.jresearchbiology.com
of Geodorum densiflorum (Lam.) Schltr. pseudobulb.
Stamford Journal of Pharmaceutical Sciences. 3(2): 47 - Advantages
48. Easy online submission
Complete Peer review
Sudhakor Reddy. CH, Pattanaik C, Murthy MSR, Affordable Charges
and Reddy KN. 2005. EPTRI-ENVIS Newsletter; Quick processing
Extensive indexing
Hyderabad, India; 11(4).
You retain your copyright
Yonzone R., Lama D, Bhujel RB and Rai S. 2011. submit@jresearchbiology.com
Intraspecific colour variation in population of www.jresearchbiology.com/Submit.php
Dendrobium anceps Sw. (Orchidaceae) in Darjeeling

1548 Journal of Research in Biology (2014) 4(8): 1543-1548

 Journal of Research in Biology
An International Scientific Research Journal
Phytochemical and antibacterial studies on the aqueous extract
of Eucalyptus gomphocephala DC
Authors:
Journal of Research in Biology
Hayate Bouharb1 ,
Khalid El Badaoui1,
Ali Amechrouq2 and
Jalila El Amri1.
Institution:
1. Laboratory
of Environment and Health,
Faculty of Science,
University Moulay Ismail,
BP 11201 Zitoune Meknes,
Morocco.
2. Laboratory of
Chemistry and Molecular
Substances , Faculty of
Science, University Moulay
Ismail, BP 11201 Zitoune,
Meknes, Morocco
Corresponding author:
Hayate Bouharb
Email Id:
Web Address:
http://jresearchbiology.com/
documents/RA0494.pdf
Journal of Research in Biology
An International
Scientific Research Journal
ISSN No: Print: 2231 6280; Online: 2231- 6299
Original Research
ABSTRACT:
This work aims to find out the molecule responsible for the good activity
against the strains of Pseudomonas aeruginosa reflected in a pre work done on the
phytochemical study of aqueous extract of Eucalyptus gomphocephala. We have
initially processed the quantitative colorimetric determination by a UV-Vis
spectrophotometer for total polyphenols and flavonoids and a qualitative analysis by
high performance liquid chromatography (HPLC) coupled with mass spectroscopy.
Quantitative determinations of total polyphenols by the Folin-Ciocalteu reagent and
flavonoids by AlCl3 method revealed the richness of this extract in total polyphenols.
Qualitative analysis by HPLC / ESI-MS revealed the presence of gallic acid. This
molecule was tested by the agar diffusion method and the macrodilution method in
liquid medium, which showed greater activity than the aqueous extract. The results
obtained in this study suggest that the gallic acid may be used in the treatment of
infections caused by Pseudomonas aeruginosa .
.
Keywords:
Eucalyptus gomphocephala, quantitative analysis, qualitative analysis,
aqueous extract, gallic acid, Pseudomonas aeruginosa
Article Citation:
Hayate Bouharb, Khalid El Badaoui, Ali Amechrouq and Jalila El Amri.
Phytochemical and antibacterial studies on the aqueous extract of Eucalyptus
gomphocephala DC
Journal of Research in Biology (2014) 4(8): 1549-1556
Dates:
Received: 22 Nov 2014 Accepted: 29 Nov 2014 Published: 20 Dec 2014
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
1549-1556| JRB | 2014 | Vol 4 | No 8
www.jresearchbiology.com

INTRODUCTION
The genus Eucalyptus is native to Australia
where there are over 750 species (Elliot and Jones,
1983; Singh et al., 1999). These kinds of species
have the particularity to grow rapidly and are used to
drain the swampy soil. They are also used for timber
and firewood. Their essential oils are used in
pharmaceutical and cosmetic industries for the
manufacture of various products (Hyodo et al., 1992)
The genus Eucalyptus is known to be a rich
source of bioactive natural products, including terpenes,
tannins, flavonoids and derivatives of phloroglucinol
(Sing et al., 1999).The eucalyptol (1.8 cineole) is the
principle active essential oil of the genus Eucalyptus that
posses different pharmacological actions (IPD, 1996).
The introduction of Eucalyptus in Morocco
th
dates back to the early 19 century. They are spread
over more than 200,000 ha, or 41% of the total area of
artificial plantations, most of which is occupied by the
E. gomphocephala and E. camaldulensis (80% of the
planted area) (Marien, 1993).
The Eucalyptus gomphocephala is a plant very
interesting because it grows well on limestone, upto
the level of 45 meters (Boudy, 1952). The plant division
has many beneficial qualities and economic uses. It is
used in the national industrial wood production division
and also as a honey plant. It was widely used in
traditional medicine for its healing properties. In
Morocco, this plant is used in the treatment of cold,
bronchitis and as a fumigant against microbes.
The leaves of E. gomphocephala were reported
to have biological activities including antioxidant and
cytotoxic activities (Alsayed et al., 2010), antitumor
activity (Alsayed et al, 2012), insecticidal activity
(Barbouche et al., 2007; Guendouz et al., 2006),
antibacterial (Bouharb et al., 2014) and so on.
The study of the chemistry of plants is still a
burning issue despite its age. This is mainly due to the
fact that the plant kingdom is a major source of immense
1550
Bouharb et al., 2014
variety of bioactive molecules (Ferrari, 2002).
The purpose of this work is the phytochemical
study of aqueous extract of Eucalyptus gomphocephala
which showed good activity against strains of
Pseudomonas aeruginosa (Bouharb et al., 2014) and
to identify some compounds for highlighting the
molecule responsible for the antibacterial activity.
MATERIALS AND METHODS
Plant material
The plant was harvested in summer during the
month of July. The harvest was carried out at mid-day,
the collection was casually done by selecting adult tree
leaves on a same tree. These are dried in shade and
ground finely to make the plant material suitable for
elucidating the bioactive compound. The identification
of the plant has been made to the National Forestry
School of Engineering (Sale).
Preparation of the aqueous extract
100g of powder was extracted by heat reflux for
two hours in water and then filtration and evaporation
was carried out in a rotary evaporator at 60C; the
residue obtained is kept still until its use.
Phytochemical study
Quantitative analysis
Content of total polyphenols
Polyphenols are estimated by the Folin-Ciocalteu
(Wong et al., 2006). 1 ml aqueous extract dissolved in
distilled water was added to 1 ml of Folin-Ciocalteu
reagent and diluted 10 times. After 4 min, 800 l of
sodium carbonate solution (75g / l) was added in a
volumetric flask of 25ml. The absorbance was measured
at 765 nm after 2h of incubation. The concentration of
polyphenols were detected from the calibration ranges
established with gallic acid (0-200g / ml) and are
expressed in milligrams of gallic acid equivalent per
gram of dry matter MS (EAG mg / g) .
Determination of Flavonoids
The method of aluminum trichloride (Bahorun
Journal of Research in Biology (2014) 4(8): 1549-1556
Bouharb et al., 2014
et al., 1996) was used to quantify the flavonoids of
various extracts. Quercetin was used as a standard (0.1
g / l prepared in methanol). 1 ml of extract was added to
0.1 ml of the solution of AlCl3 (10% in methanol). 20 ml
distilled water was added to it and mixed with methanol
in a volumetric flask of 50ml. After 10 min, the
absorbance was read at 430 nm. The concentration of
flavonoids were detected from the calibration range
established with quercetin (0-35 g / ml) and are Antibacterial tests
expressed in milligrams of quercetin equivalents per
gram of dry matter (EQ mg / g).
Qualitative Analysis
Work on this section were performed on a
HPLC chromatographic system coupled to mass
spectrum in the laboratory of UATRS (CNRST, Rabat).
The HPLC used was LC Surveyor thermo-electron
brand, equipped with low-pressure quaternary pump with
integrated degasser in isocratic mode. The separated
molecules were then detected by two systems: a Photo
Diode Array spectrophotometer (PDA) and Surveyor
(spectral range from 190 to 800 nm) followed by a mass
spectrometer (LCQ Advantage MAX) comprising an ion
trap with ESI ionization. The ionization of samples was
made using negative polarity. The mass range was set
at: 50-2000. To analyze the aqueous extract of
Eucalyptus, HPLC-RP-C18 was used. The stationary
phase was a column (125 x 4.6mm) whereas, the mobile
phase was methanol / water (60/40), and the mobile
Figure 1 : Calibration curve of gallic acid for the determination of total polyphenols
Journal of Research in Biology (2014) 4(8): 1549-1556
Table 1: Identification of chemical profile of
the aqueous extract
N Temps de rtention Aire (%)
1 2.44 18.79
2 3.36 81.21
% Total 100
phase flow rate was 0.5 ml / min. The temperature was
adjusted to 40 C (Kuntie et al., 2007).
In vitro inhibition of bacterial growth of
Pseudomonas aeruginosa by the aqueous extract of E.
gomphocephala was investigated by solid medium
diffusion method and macrodilution method was done in
liquid medium (Bouharb et al., 2014). In this study, we
have tested the activity of pure molecule of gallic acid
(10mg / ml) against the bacterial strain.
RESULTS AND DISCUSSION
Quantitative study
For there are many important biological
activities in polyphenols and flavonoids, we have chosen
them among different phytochemicals for quantification.
Two straight calibration (Fig. 1 and 2) were
plotted for this purpose which are made with the
standard solutions at different concentrations. The
amounts of polyphenols and related flavonoids have
been reported in milligrams equivalent of the standard
used per gram of dry matter (ms) (mg EE / ms g) and
1551
 Bouharb et al., 2014

Figure 2: Calibration curve of quercetin for determination of flavonoids

determined by the type of equation: y = ax + b. The coefficient of determination (R2 = 0.995) (Figure 2).
assay of total polyphenols was made according to the The contents of total polyphenols and flavonoids in
method of Folin-Ciocalteu reagent. Gallic acid was used the aqueous extract of leaves of Eucalyptus
as a standard. The flavonoids assay was performed gomphocephala (mg / g of DM) are:
according to the method of aluminum trichloride and Polyphenol content: 10.64 1.94 mg / g DM
quercetin was used as a standard. Flavonoid content: 0.49 0.004 mg / g DM
The only work done on the levels of phenolic
Calibration curve for the determination of total
compounds in the species studied is that of Alsayed et al.
polyphenols
(2012). A comparison between the two studies is
This curve is established using gallic acid as
difficult because it is important to emphasize that the
reference. The formula for the linear regression of the
use of original plant is distinctly different from
curve y = 0.0048 x + 0.0198 is used with a correlation
geographical and climate zone as well as different
coefficient R2 = 0.991 (Figure 1).
extraction and assay methods which reduce the
Calibration curve for the determination of flavonoids
reliability of a comparison between the two studies.
The reference compound used in the preparation
Recent studies have shown that the level of phenolic
of this curve is quercetin. The formula for the linear
compounds change in a considerable way from one
regression of the curve is y = 0.0732*-0.0811 with a

Figure 3: Chromatogram of aqueous extract of Eucalyptus gomphocephala

1552 Journal of Research in Biology (2014) 4(8): 1549-1556

Bouharb et al., 2014

Figure 4: Mass spectrum of gallic acid in negative mode

species to another and within the same species (Ksouri et
al., 2012), because of external factors (temperature,
climate, etc.,) (Ksouri et al., 2008), genetic (variety and
the origin of species) (Ebrahimzadeh et al., 2008),
physiological (the degree of maturation of the plant,
nature of organs) (Maisuthisakul et al., 2007) and the
period of storage (Aganga and mosase, 2001).
Qualitative Study
The aqueous extract of E. gomphocephala was
analyzed by HPLC / ESI-MS to obtain information

Figure 5: Mass Spectrum of gallic acid in the aqueous extract in negative mode

Journal of Research in Biology (2014) 4(8): 1549-1556 1553

 Bouharb et al., 2014

on the chemical nature of its constituents. Both standards seeds. Animal Feed Science and Technology. 91(1-2):
were used: gallic acid and quercetin. The results of this 107-113
study showed the absence of quercetin and the presence
Al-Sayed E, Martiskainen O, Bobrowska-
of large quantities of gallic acid (81.21%) (Table 1).
Hgerstrand M, Sinkkonen J, Trnquist K, Pihlaja
Antibacterial tests
K, Ayoub N and Singab AN. 2010. Phenolic
The results of antibacterial tests (Bouharb et
compounds from Eucalyptus gomphocephala with
al., 2014), from the aqueous extract of E.
potential cytotoxic and antioxidant activities. Natural
gomphocephala showed good activity against all
Product Communications. 5(10): 1639-1642
strains of P. aeruginosa with inhibition zones of 12 to 18
mm and the minimum inhibitory concentration (MIC) Al-Sayed E, Singab A, Ayoub N, Martiskainen O,
between 6.25 and 12.5 mg / ml. Sinkkonen J and Pihlaja K. 2012. HPLCPDAESI
The remarkable presence of gallic acid in the MS/MS profiling and chemopreventive potential of
aqueous extract prompted us to test this molecule Eucalyptus gomphocephala DC. Food Chemistry. 133
against P. aeruginosa. The results showed higher (3): 10171024
activity than the aqueous extract with MICs of 2.5 mg /
Bahorun T, Gressier B, Trotin F, Brunet C, Dine T,
ml. Gallic acid was demonstrated effectively by several
Vasseur J, Cazin JC, Pinkas, M, Luyckx M and
studies as antibacterial and especially against P.
Cazin M. 1996. Oxygen species scavenging activity of
aeruginosa. Ikuro et al. (2000) showed that the gallic
phenolic extracts from hawthron fresh plant organs and
acid and its esters were evaluated as inhibitors of the
pharmaceutical preparations. Arzneimittel-Forschung. 46
enzyme p-Hydroxybenzoate recombinant Hydroxylase
(11): 1086-1089.
(PHBH) a flavin-dependent monooxygenase
Nicotinamide Adenine Dinucloeotide Phosphate Barbouche N, Jeridi S and Ben Dhaou S. 2007.
(NADPH) from P. aeruginosa . Premkumar et al. (2010, Alternative de lutte biologique contre Sitophilus
2011) also has demonstrated antibacterial effect of the oryzae (Coleoptera : curculionidae) par lutilisation des
combination of polyphenolic compounds and gallic acid huiles essentielles et des poudres de plantes. Rev. INAT.
with antibiotics. Revue de l'Institut National Agronomique de Tunisie
(TN) 22(1): 85-96.
CONCLUSION
Boudy P. 1952. Guide du forestier en Afrique du nord.
In this study, gallic acid was shown to be a
Ed. La Maison Rustique. Paris. 505
promising molecule against P. aeruginosa. It is
recommended to do further research with different Bouharb H, K EL Badaoui, T Zair, H Shisseh, S
solvents and extraction methods on the Eucalyptus Chakir and Alaoui T. 2014. Antibacterial Evaluation
gomphocephala for identifying more active molecules. and Phytochemical Screening of Eucalyptus
gomphocephala DC against Pseudomonas aeruginosa.
REFERENCES Asian Journal of Pharmaceutical and Clinical Research.
Aganga AA and Mosase KW. 2001. Tannin content, 7(4): 264-267
nutritive value and dry matter digestibility of
Ebrahimzadeh MA, Pourmorad F and
Lonchocarpus capassa, Zizyphus mucronata,
Bekhradnia AR. 2008. Iron chelating activity, phenol
Sclerocarya birrea, Kirkia acuminata and Rhus lancea
1554 Journal of Research in Biology (2014) 4(8): 1549-1556
Bouharb et al., 2014

and flavonoid content of some medicinal plants from Ksouri R, Ksouri MW, Jallali I, Debez A, Magn
Iran. African Journal of Biotechnology. 7(18): 3188- C, Hiroko I and Chedly A. 2012. Medicinal halophytes:
3192. potent source of health Rfrences bibliographiques112
promoting biomolecules with medical, nutraceutical and
Elliot WR and Jones DL. 1983. Encyclopedia of
food applications. Critical Reviews in Biotechnology. 32
Australian Plants suitable for Cultivation. 4:528.
(4): 289-326
Ferrari J. 2002. Contribution la connaissance du
Kuntie V, Pejie N, Ivkovie B, Vugie Z, Ilie K,
mtabolisme secondaire des Thymelaeaceae et
Mieie S and Vukojevie V. 2007. Isocratic R-PHPLC
investigation phytochimique de lune delles: Gnidia
method for rutin determination in solid oral dosage
involucrata Steud. ex A. Rich. Thse de doctorat.
forms. Journal of pharmaceutical and biomedical
Lausanne, Facult des sciences 228
analysis. 43(2): 718-721.
Guendouz BA and Doumandji-Mitiche B. 2006.
Maisuthisakul P, Pongsawatmanit R and Maitree
Effect of plant extract of Eucalyptus gomphocephala,
Suttajit. 2007. Assessment of phenolic content and free-
Shinus molle and Melia azedarach on Schistocerca
radical scavenging capacity of some Thai indigenous
gregaria (FORSKL 1775) (Insectaorthoptera) Under
plants. Food Chemistry. 100(4): 1409-1418.
Laboratory conditions. Ninth Arab Congress of Plant
Protection, 19-23 November, Damascus, Syria Marien JN. 1993. Projet damlioration des Eucalyptus
au Maroc. Afocel ed. 114
Hyodo S, Etoh H, Yamashita N, Sakata K and Ina
K. 1992. Structure of resinosides from Eucalyptus Premkumar J, Sakharkar MK, Chu Sing L, Thean
resinifera as repellents against the blue mussel, Mytilus Hock T and Kishore RS. 2010. Activity and
edulis. Bioscience Biotechnology and Biochemistry. 56 interactions of antibiotic and phytochemical
(1): 138. combinations against Pseudomonas aeruginosa in vitro.
International Journal of Biological Sciences. 6(6): 556
Ikuro A, Kenji K and Hiroshi N. 2000.
568.
Antioxidative galloyl esters as enzyme inhibitors of p-
hydroxybenzoate hydroxylase. FEBS Letters. 483: 131- Premkumar J, Kishore RS, Chu Sing L, Vincent
134. TKC and Sakharkar MK. 2011. Insights into
antifolate activity of phytochemicals against
IPD. 1996. Indian Pharmacopoeia. Delhi: Government of
Pseudomonas aeruginosa. Journal of Drug Targeting. 19
India, Ministry of Health and Family Welfare-Controller
(3): 179-188
of Publications. 11: 310.
Singh AK, Monika K and Sushil K. 1999. Non-volatile
Ksouri R, Megdiche W, Falleh H, Trabelsi N,
constituents of Eucalyptus: A review on chemistry and
Boulaaba M, Smaoui A and Abdelly C. 2008.
biological activities. Journal of Medicinal Aromatic Plant
Influence of biological, environmental and technical
Sciences. 21(2): 375 407.
factors on phenolic content and antioxidant activities of
Tunisian halophytes. Comptes Rendus biologies. 331 Singh IP, Umehara K and ETOH H. 1999.
(11): 865-873 Macrocarpals in Eucalyptus sp. As attachment- inhibitors

Journal of Research in Biology (2014) 4(8):1549-1556 1555

 Bouharb et al., 2014

against the blue mussel. Natural Product Letters. 14(1):

11-15.

Wong SP, Leong LP and William Koh JH. 2006.

Antioxidant activities of extracts of selected plants. Food
chemistry. 99(4): 775-783

Submit your articles online at www.jresearchbiology.com

Advantages
Easy online submission
Complete Peer review
Affordable Charges
Quick processing
Extensive indexing
You retain your copyright
submit@jresearchbiology.com
www.jresearchbiology.com/Submit.php

1556 Journal of Research in Biology (2014) 4(8): 1549-1556

 Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

A comparative hydrobiological study on Authoorangal channel and

Srivaikuntam channel and their role as sustainable water resources
ABSTRACT:
Journal of Research in Biology

A hydrobiological study conducted in two different river channels of two

Authors: different taluks of south Tamil Nadu showed that the concentrations of Physico-
Esther Isabella Eucharista F
chemical parameters like turbidity, temperature, pH, dissolved oxygen, electrical
and Mohanraj Ebenezer
conductivity, total dissolved solids, total hardness, total alkalinity, calcium,
magnesium, sodium, potassium, chloride, sulphate, carbonate, bicarbonate, total
coliform and faecal coliform were deteriorating water quality rapidly due to intense
and excessive amounts of fertilizers, manures, pesticides and insecticides. In the
present investigation among the two channels, maximum values of the physical
parameters with a mean of turbidity 6.50.070 NTU, temperature 33.90.707 0C, pH
Institution: 8.70.707, dissolved oxygen 7.90.707 mg/l, electrical conductivity 3.080.632 ds/m,
PG and Research
total dissolved solids 15.7 3.162mg/l, total hardness 25.66.324 mg/l, and total
Department of Zoology,
alkalinity 50.11.140 mg/l was recorded. The maximum values of the chemical
St. Johns College,
Palayamkottai - 627002, parameters with a mean of calcium 10.01.702 mg/l, magnesium 11.60.378 mg/l,
Tamil Nadu. sodium 8.482.828 mg/l, potassium 0.230.012 mg/l, chloride 16.12.607 mg/l,
sulphate 11.93.224 mg/l, carbonate 0.30.070 mg/l and bicarbonate 2.80.050 mg/l
was noticed. The maximum values of the coliform parameters with a mean of total
coliform bacteria 3900.707 (MPN/100 ml) and faecal coliform 370.707 (MPN/100
ml) was also recorded. The results of this study point out the fact that the water
quality of both the channel waters are slowly deteriorating.

Keywords:
Corresponding author: Physico-chemical and coliform parameters, Authoorangal and Srivaikuntam
Esther Isabella Eucharista F.
river channels

Email Id:
Article Citation:
Esther Isabella Eucharista F. and Mohanraj Ebenezer
A comparative hydrobiological study on Authoorangal channel and Srivaikuntam
channel and their role as sustainable water resources
Journal of Research in Biology (2014) 4(8): 1557-1567

Dates:
Web Address:
http://jresearchbiology.com/ Received: 09 Aug 2014 Accepted: 4 Dec 2014 Published: 26 Dec 2014
documents/RA0473pdf
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1557-1567 | JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
Fresh water is an important natural resource
necessary for the survival of all ecosystems. In India,
ponds, lakes, rivers and ground water are used for
domestic and agricultural purposes (Hemant Pathak et
al., 2011). Agriculture is the chief source of livelihood
for the use of agriculture a great effect on the flow of
fresh water is necessary. The potable water should be
free from infectious agents and chemical constituents,
pleasant to taste, odour and usable for domestic purposes
(Saini et al., 2010). Physico-chemical parameters play a
significant role in determining the distributional pattern
and quantitative abundance of aquatic ecosystem
(Sayeswara et al., 2011). The physical and chemical
properties of fresh water bodies are based upon the
geomorphological and weathering processes (Sahni and
Yadav, 2012).
The presence of safe and reliable drinking water
is a significant prerequisite for a stable community (Sen
et al., 2011). Due to the growth of population and man-
made activities, the quality of water is deteriorating
everywhere. For this purpose, it is important to know
about different physico-chemical characteristics of water.
There has been no report on physico-chemical
parameters assessment in the Authoorangal channel and
Srivaikuntam channel. Hence, a periodical assessment is
Figure 1: Sampling Site Authoorangal
1558
Eucharista and Ebenezer, 2014
essential in monitoring the physico-chemical parameters
of Authoorangal channel and Srivaikuntam channel for
the welfare of surrounding human settlements. The
present study is an attempt to assess the water quality
with the following objectives:
Collection of baseline data on the structure and
function of the Authoorangal channel and Srivaikuntam
channel.
Assessment of seasonal changes in physico-chemical
parameters.
Analysis of seasonal fluctuations in the microbes.
MATERIALS AND METHODS
Study area of Authoorangal Channel
The Authoorangal channel has thirteen channel
inlets. It has the width of about 6m broad and 1.5m depth
(Figure 1). The channel is getting water from
Thamirabarani river. Further, it is passing through
Authoor and Sethukavaithan villages. In about 460
acres, the water is utilized for agricultural purposes. It
belongs to Tiruchendur taluk.
Study area of North main channel (Srivaikuntam)
The North main channel (Srivaikuntam) is spread
over a distance of 87 sq.km and 82 acres (Figure 2). The
channel has the width of 30 feet and 6 feet depth. It
consists of two channel inlets namely Mukoot and
Figure 2: Sampling Site Srivaikuntam
Journal of Research in Biology (2014) 4(8): 1557-1567
Eucharista and Ebenezer, 2014
Table 1: Monthly variations of physical parameters of Authoorangal channel in the year of 2011
YEAR-
PHYSICAL PARAMETERS
2011
Turbidity Tempera-
Month pH DO (mg/l) EC (ds/m) TDS (mg/l) TH (mg/l) TA (mg/l)
(NTU) ture 0C
January 4.00.252 21.10.935 7.30.196 7.90. 326 1.20. 078 11.00. 465 10.10.345 22.20. 677
February 4.40. 307 22.40. 196 7.90. 183 7.10. 111 3.00.058 11.10. 160 15.50.316 25.70. 080
March 4.10.040 23.50.223 7.70.066 7.30.094 0.490.029 11.50.353 4.30.050 28.00.948
April 4.00.707 23.70.241 7.70.230 6.60.215 0.490.010 11.30.330 3.30.134 26.20.322
May 3.90.037 25.90.583 8.50.612 5.60.168 0.310.005 12.00.400 6.60.276 33.30.583
June 4.00.316 25.70.241 7.80.193 5.50.067 1.270.013 12.30.083 4.10.031 29.80.083
July 4.30.221 24.50.223 7.40.126 5.80.126 0.270.023 13.10.333 3.00.707 30.30.377
August 3.40.141 24.81.414 7.40.126 6.30.134 0.300.044 13.23.049 16.23.391 33.21.140
September 3.40.178 24.90.707 7.32.302 5.72.024 0.210.032 12.31.898 13.01.140 40.26.324
October 4.51.414 23.76.324 7.90.707 7.60.707 3.080.632 14.90.707 25.66.324 45.512.747
November 5.91.303 22.23.224 7.71.140 7.91.140 2.020.707 15.73.162 23.93.178 50.11.140
December 6.20.317 21.11.000 8.00.707 7.20.707 0.210.010 14.31.303 3.11.000 30.40.100

Thamiraparani river. Further, the water is passing cleaned polythene two liter bottles without any air bubbles
through Varadharajapuram, Appankoil and Kaspa and after rinsing it with the sample waer in morning hours
villages. It belongs to Srivaikuntam taluk. between 8 am to 10 am. The temperature was recorded on
Water analysis the sot by mercury thermometer. The turbidity was
The two channels have been selected for
measured on spot by Nephelometer. The other parameters
investigation on the basis of geographical locations.
such as pH, Dissolved Oxygen (DO), Electrical
Samplings were made at monthly intervals from January
Conductivity (EC), Total Dissolved Solids (TDS), Total
2011 to December 2011. Samples were collected in
Hardness (TH), Total Alkalinity (TA), calcium,

Table 2: Seasonal variations of chemical and coliform parameters of Authoorangal channel in the year
YEAR- COLIFORM
CHEMICAL PARAMETERS
2011 PARAMETERS
Total Faecal
Ca Mg Na K Cl SO4 (mg/ CO3 HCO3 Col Col
Month
(mg/l) (mg/l) (mg/l) (mg/l) (mg/l) l) (mg/l) (mg/l) (MPN/ (MPN/
100ml) 100ml)
January 8.20.31 5.50.37 6.45000.14 0.2100.01 13.50.50 4.20.09 0.000.00 2.50.29 2408.94 210.58

February 9.90.36 11.60.18 8.10000.20 0.2300.01 16.00.15 0.00.00 0.240.05 2.70.13 2203.53 200.67

March 2.90.04 1.30.08 0.69560.00 0.0250.00 2.30.13 0.00.00 0.000.00 2.60.16 2302.02 221.14

April 2.60.15 1.20.18 0.59520.00 0.2200.03 2.20.32 0.00.00 0.000.00 2.50.15 2406.51 250.53

May 1.80.37 0.60.07 0.65210.13 0.0510.00 1.40.18 0.00.00 0.200.03 1.50.20 2507.07 230.66

June 2.90.07 451.58 0.66230.00 0.0520.00 2.30.03 3.00.31 0.000.00 2.80.05 2707.07 252.23

July 0.90.08 1.30.09 0.52170.00 0.0250.00 1.00.07 0.00.00 0.000.00 1.70.17 2643.40 242.28

August 2.80.70 1.50.35 0.68560.00 0.0270.00 2.20.31 2.50.70 0.200.07 2.60.32 2771.14 293.11

Septem-
2.80.70 1.30.32 0.56210.03 0.2300.03 2.30.32 2.60.33 0.000.00 2.60.33 27631.63 263.30
ber

October 10.01.7 11.63.16 8.48002.82 0.2100.03 16.12.60 11.93.22 0.200.07 2.70.70 27031.62 273.22

Novem-
9.80. 1.0 10.20.7 7.31000.70 0.2000.07 14.20.70 5.50.63 0.000.00 2.60.33 2610.70 213.17
ber
Decem-
2.00.70 3.00.70 0.56500.00 0.0280.00 2.50.07 0.00.00 0.200.07 2.80.31 2560.70 2601.00
ber

1559 Journal of Research in Biology (2014) 4(8): 1557-1567


magnesium, sodium, potassium and chloride, were
analyzed in the laboratory as per the standard methods
described by APHA (2005). Microbial studies were
analyzed by MPN method described by Papen and Van
Berg (1998).
Statistical analysis
Results obtained were subjected to statistical
analysis using SPSS (11.5).
RESULTS AND DISCUSSION
Turbidity
The present study indicated that the highest value
of turbidity was 6.2 0.317 NTU in the month of
December 2011 in the Authoorangal channel; Similarly
the lowest value of turbidity was reported as 3.4 0.141
NTU in the months of both August and September 2011.
In the Srivaikuntam channel, the highest value of
turbidity was reported as 6.5 0.070 NTU in the month
of August 2011. On the other hand, the lowest value of
turbidity was showed as 2.9 0.316 NTU in the month of
September 2011 (Table 3). The present investigations
reported the highest concentration of turbidity was due to
the interaction of agricultural fertilizers, manures,
insecticides and pesticides from the nearby banana field.
Water Temperature
The sun is the source of heat that warms most
water and its effect depends on the angle at which it
strikes the surface. There may also be a direct exchange
of heat between air and water and between substratum
and water (Macan, 1974). In the present investigation,
the temperature values in the station Authoorangal
channel from January to December 2011 were recorded.
The maximum value of water temperature was recorded
as 25.9 0.583oC in the month of May 2011. The
o
minimum value was recorded as 21.11.00 C in the
months of December and January (Table 1). In the
station Srivaikuntam channel, the maximum value was
o
recorded as 33.90.707 C in the month of May. The
o
minimum value was recorded as 10.10.707 C in the
1560
Eucharista and Ebenezer, 2014
month of December (Table 3). In this investigation, the
temperature values were maximum during summer and
minimum during winter. Sonawane (2011) reported that
the maximum value was recorded as 21.07 1.49 oC
during summer; the minimum value was recorded as
12.152.95oC during winter. In the present study, the
maximum value recorded during summer may be due to
low water level, more solar radiation and more
evaporation. The minimum value recorded during winter
may be due to high water level, less solar radiation and
less evaporation.
Hydrogen Ion Concentration (pH)
The acidity of water is gauged by its pH, which
is a measure of the concentration of the hydrogen ion
(H+). During the study period January December 2011,
the maximum value of pH was recorded as 8.50.612
during summer in the month of May 2011 in the
Authoorangal channel whereas the minimum value was
recorded as 7.30.196 in the months of January and
September 2011 (Table 1). The maximum pH value of
Srivaikuntam channel was recorded as 8.7 0.707 in the
month of February 2011. The minimum value was
recorded as 7.30.707 in the months of January and
September 2011 (Table 3). The present study revealed
that the pH was alkaline during summer season in
Authoorangal channel, on the other hand pH was alkaline
during winter season in Srivaikuntam channel. In the
present investigation, higher values may be due to
accumulation of ions owing to evaporation and
pesticides, insecticides from the nearby banana field.
Pathak et al. (2012) reported that the higher values may
be due to accumulation of ions owing to evaporation,
biological turnover and interaction with sediments.
Dissolved Oxygen (DO)
Dissolved oxygen is essential to the respiratory
metabolism of most aquatic organisms. Natural sources
of dissolved oxygen are derived from the atmosphere or
through photosynthetic production by aquatic plants. In
the analysis of dissolved oxygen during the study period,
Journal of Research in Biology (2014) 4(8): 1557-1567
Eucharista and Ebenezer, 2014
the maximum value was recorded as 7.90.326 mg/l in
the Authoorangal channel during the months of January
and November (Table 1). The minimum value was
recorded as 5.50.067 mg/l in the month of June. In the
Srivaikuntam channel, the maximum value was recorded
as 7.90.707 mg/l during the months of August and
November. The minimum value was recorded as
5.90.707 mg/l in the month of May (Table 3). The
present investigation indicated the dissolved oxygen
level concentration was high during rainy season. High
concentration of DO was due to the photosynthetic
activity of algal flora. Bhuiyan and Gupta (2007)
reported that the dissolved oxygen is mainly regulated by
photosynthetic activity of algal flora. The minimum
concentration level of DO was due to the depletion of
water and abundance of nutrients in the Authoorangal
channel and Srivaikuntam channel. Sonawane (2011)
reported that minimum concentration is probably due to
the decomposition of organic matter.
Electrical Conductivity (EC)
In the analysis of electrical conductivity during
the period of January to December 2011, the maximum
value was 3.080.632 ds/m in the Authoorangal channel
during the month of October. The minimum value was
recorded as 0.20.032 ds/m in the months of September
and December. In the Srivaikuntam channel, the
maximum value was recorded as 0.410.007 ds/m in the
months of February and May. The minimum value was
recorded as 0.20.031 ds/m in the month of November
(Table 3). This study reported that the electrical
conductivity was high due to the surface run off during
the rainy season in the Authoorangal channel. The study
indicated that the EC was high during the months of May
and February in the Srivaikuntam channel which may be
due to the greater content of ions in the water and
dissolved solids. Dutta and Chowhan (2009) reported
that water of higher conductivity may be used with
suitable amendments and precautions, but under normal
conditions, they are harmful to the soil structure and their
1561
continuous use will result in salinity hazard, with
ultimate effect on plant growth.
Total Dissolved Solids (TDS)
In this investigation, the maximum value of TDS
was noticed as 15.73.162 mg/l in the Authoorangal
channel in the month of November, whereas the
minimum value was noticed as 11.00.465 mg/l in the
month of January. The analysis of Sirvaikuntam channel
revealed the maximum value of TDS as 14.01.000 mg/l
in the month of May. The minimum value of TDS was
9.1 0.707mg/l in the month of February (Table 3). The
present study revealed the highest concentration of total
dissolved solids which may be due to the surface run off
and agricultural run off from the nearby banana field.
Sonawane (2011) reported that the higher concentration
of total dissolved solids ranged between 502 mg/l and
8626.25 mg/l. The ground water chemistry changes when
the water flows through the subsurface geological
environment having overall change in the major ions and
dissolved solids.
Total Hardness (TH)
The hardness of water is generally due to the
presence of calcium and Magnesium in the water. The
study recorded the maximum value of total hardness in
the Authoorangal channel as 25.66.324 mg/l in the
month of October 2011. Similarly, the minimum value of
hardness was 3.00.707 mg/l in the month of July 2011
(Table 1). But in the Srivaikuntam channel, the
maximum value of hardness was revealed as 13.90.707
mg/l in the month of November 2011. The minimum
value of total hardness was noticed as 3.00.707 mg/l in
the month of July 2011 (Table 3). The present
investigation noticed the highest concentration of total
hardness reflected by the nature of the agricultural
fertilizers, manures, insecticides and pesticides. Saini et
al. (2010) reported that the hardness of water reflects the
nature of the geological formation with which it has been
contacted.
Journal of Research in Biology (2014) 4(8): 1557-1567
 Eucharista and Ebenezer, 2014

Total Alkalinity (TA)
The present study indicated the
concentration of TA in the Authoorangal channel as
50.11.140 mg/l in the month of November 2011,
whereas the lowest concentration of TA was 22.20.677
mg/l in the month of January 2011. The analysis of
Srivaikuntam channel showed the highest concentration
of TA as 45.10.317 mg/l in the month of November
2011 and the lowest concentration of TA as 20.2
0.707mg/l in the month of January (Table 3). The present
study showed the presence of the highest concentration
of total alkalinity which was due to the surface run off
from banana field which contains the
concentration of calcium, carbonates and bicarbonates
ions during rainy season. Sahni and Silotia (2011)
reported that the alkalinity values correlate positively
with the pattern of rainfall and this implies surface run
off from the Mansagar lake.
Calcium
Waters that emerge from the earth are highly
charged with calcium bicarbonate and flow away as
streams which may deposit calcium carbonate (Macan,
1974). The values of calcium ions were given in Table 2
highest for Authoorangal channel which represented the highest
value of 10.01.702 mg/l in the month of October 2011.
Similarly, the lowest value was 0.90.086 mg/l in the
month of July 2011. Considering the Srivaikuntam
channel, the analysis of calcium ions was recorded as
3.60.633 mg/l in the month of March 2011 as the
highest value. The lowest value was recorded as
0.80.031mg/l in the month of May 2011. The
precipitation of agricultural pesticides, fertilizers and
manures from the banana field represented the highest
calcium concentration. Samson and Elangovan (2011)
highest reported that water more or less in equilibrium with the
materials in the drainage basin is characterized by higher
concentration of calcium.
Magnesium
The magnesium ion in the Authoorangal channel
was recorded as 11.60.378mg/l in the months of
February and October 2011 is the highest value.
Similarly, the lowest value was 0.60.070mg/l in the
month of May 2011 (Table 2). In the Srivaikuntam
channel, the highest value of magnesium ion was

Table 3: Monthly variations of physical parameters of Srivaikuntam channel in the year of 2011

YEAR-
PHYSICAL PARAMETERS
2011
Turbidity Tempera- DO TDS
Month pH EC (ds/m) TH (mg/l) TA (mg/l)
(NTU) ture 0C (mg/l) (mg/l)
January 3.00.707 13.11.000 7.30.707 7.10.707 0.260.007 9.20.707 6.10.317 20.20.707

February 3.00.707 12.40.707 8.70.707 7.30.633 0.410.007 9.10.707 5.51.000 22.70.707

March 4.10.707 15.01.000 7.80.707 7.60.707 0.350.007 10.50.707 4.30.707 23.00.707

April 4.00.707 23.70.070 7.70.070 6.60.317 0.310.007 10.30.635 3.30.707 21.20.707

May 3.60.317 33.90.707 8.40.707 5.90.707 0.410.007 14.01.000 6.60.317 23.30.317

June 3.10.707 33.70.070 7.70.633 6.50.707 0.270.007 12.30.322 4.10.707 22.80.317

July 3.50.707 29.50.633 7.60.707 6.80.707 0.270.007 13.10.317 3.00.707 31.30.317

August 6.50.070 27.80.633 7.40.707 7.90.707 0.390.007 13.20.317 4.20.707 30.20.707

September 2.90.316 26.80.707 7.30.707 7.70.317 0.210.007 12.70.317 6.00.707 39.20.317

October 3.30.317 17.00.707 7.90.707 7.60.317 0.280.010 13.90.707 8.60.317 37.50.317

November 3.20.317 15.20.317 7.70.317 7.90.707 0.20.031 10.70.633 13.90.707 45.10.317

December 3.60.317 10.10.707 7.60.317 7.20.317 0.290.007 12.30.317 13.10.707 23.40.317

1562 Journal of Research in Biology (2014) 4(8): 1557-1567

Eucharista and Ebenezer, 2014
1.60.317 mg/l in the month of October 2011. Similarly,
the lowest value of magnesium ion was 0.050.007 mg/l
in the month of December 2011. The present study
revealed the highest magnesium concentration due to
leachates from the banana field during rainy season.
Sonawane (2011) reported that the main source of
magnesium concentration is obtained from the leachates
of rocks in the catchments.
Sodium
The present analysis of sodium ion revealed the
highest value as 8.482.828mg/l in the month of October
2011. The lowest value of sodium ion 0.520.007mg/l in
the month of July 2011 was recorded in the
Authoorangal channel (Table 2). Similar results were
reported in the Srivaikutam channel (Table 4). The
highest concentration of sodium ion in both channels
represented the agricultural fertilizers, manures and the
detergents used by the humans for bathing and washing
clothes. Mary Kensa (2011) reported that the main
source of sodium is obtained from soaps and detergents,
used by the local residents for bathing and washing
purposes.
Potassium
The present study recorded the highest value of
potassium ion in the Authoorangal channel as
(0.230.012 mg/l) in the months of February and
September 2011 (Table 2). Likewise, the lowest value of
potassium ion was (0.0250.0009 mg/l) in the months of
March and July 2011 (Table 1). In proportion to
Srivaikuntam channel, the highest value of potassium ion
(0.23 0.007 mg/l) was recorded in the month of
September 2011. The lowest value of potassium ion was
recorded as 0.022 0.007 mg/l in the month of April
2011. The present investigations reported that the highest
concentration of potassium may be due to wash out of
slurry from the agriculture fields and sewage run off.
Gopalkrushna (2011) reported that the major source of
potassium in natural fresh water is due to weathering of
rocks but the quantities increase in the polluted water due
1563
to the disposal of waste water.
Chloride
Chloride is one of the most widely used
disinfectants. The highest concentration of chloride in
the Authoorangal channel was 16.12.607 mg/l during
October 2011. Likewise, the lowest value was recorded
as 100.070 mg/l in the month of July 2011 (Table 2).
With reference to the Srivaikuntam channel, the highest
concentration of chloride analyzed was 1.90.318 mg/l
in the month of March 2011. Similarly, the lowest
concentration of chloride was analyzed as 1.00.00 mg/l
in the months of July and December 2011. The present
study depicted that the highest concentration of chloride
(16.12.607 mg/l) may be due to the water sources that
come from organic wastes and refuse of leachates.
Prabakaran et al. (2013) reported that the high
concentration of chloride may be due to anthropogenic
activities, sewage contamination and decomposition of
organic wastes.
Sulphate
Sulphate occurs in certain igneous rock minerals
of the field spathoid group, but the most extensive and
important occurrences are in the evaporate sediments.
The highest concentration of sulphate in the
Authoorangal channel was found as 11.93.224 mg/l in
the month of October 2011 (Table 2). While comparing
with the Srivaikuntam channel, the highest concentration
of sulphate was reported as 11.90.707 mg/l during
October 2011 which is similar to the Authoorangal
channel. The highest concentration of sulphate in the
present investigation revealed the deposition of
agricultural fertilizers which are drained into the water
body from the catchment area. Suresh et al. (2013)
reported that sulphate enter into the water body from the
catchment area through surface run off. Since the study
area is bordered by agricultural lands where sulphate
fertilizers are used in plenty, relatively higher
concentrations of sulphate observed could be attributed
to the run off from these agricultural lands.
Journal of Research in Biology (2014) 4(8): 1557-1567
 Eucharista and Ebenezer, 2014

Carbonate Authoorangal channel was found as 2.80.050 mg/l in

The present study investigated the highest
concentration of carbonate in the Authoorangal channel
as 0.20.050 mg/l (Table 2) and was uniformly
distributed over the month of February, May, August,
October and December 2011. The Srivaikuntam channel
revealed the highest concentration of carbonate as
0.30.070 mg/l in the months of February and April
2011. The present investigation manifested the highest
concentration of carbonate which was due to the surface
run off from the agricultural fertilizers, manures and
pesticides as precipitation to where it exits the watershed.
Vasanthy and Velmurugan (2009) reported the presence
of temporary (carbonates and bicarbonates of Ca and
Mg) and also permanent hardness (sulphates, phosphates,
nitrates, chlorides of Ca and Mg). This may be attributed
to the geological reasons and surface run-off.
Bicarbonate
The highest concentration of bicarbonate in the
the months of June and December 2011(Table 2) but the
lowest concentration was 1.50.203 mg/l in the month of
May 2011. With reference to the Srivaikuntam channel,
the highest concentration of bicarbonate was recorded as
2.20.633 mg/l in the months of January, April, August,
September, November and December 2011 whereas the
lowest concentration of bicarbonate recorded was
1.50.317 mg/l in the month of May 2011. The present
findings revealed the highest concentration of
bicarbonate which may be due to the leachates from the
agricultural run off and human activities. Sonawane
(2011) reported that the highest concentration of
bicarbonates may be attributed to the rate of organic
decomposition during which Co2 is liberated, which
reacts with water to form HCo 3.
Total Coliform Bacteria
The analysis of total coliform bacteria revealed
the highest count as 2771.140 (MPN / 100ml) in the

Table 4: Seasonal variations of chemical and coliform parameters of Srivaikuntam channel in the year of
YEAR- COLIFORM
CHEMICAL PARAMETERS
2011 PARAMETERS
Total Faecal
Ca Mg Na Cl SO4 Co3 Hco3 Col Col
Month K (mg/l)
(mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (MPN (MPN/
/100ml) 100ml)
January 2.20.31 1.50.31 6.450.31 0.10.00 1.50.31 4.20.63 0.0000.00 2.20.63 2590.70 210.70

February 2.90.70 0.700.07 8.100.70 0.100.00 1.60.31 0.0000.00 0.30.37 2.10.70 2551.00 201.00

March 3.60.63 0.000.00 0.690.00 0.020.00 1.90.31 0.0000.00 0.0000.00 2.10.70 2570.70 270.70

April 2.60.31 1.20.31 0.590.00 0.020.00 1.20.31 0.0000.00 0.30.07 2.20.07 2570.70 250.70

May 0.80.03 0.60.07 0.650.00 0.050.00 1.70.32 0.0000.00 0.0000.00 1.50.31 3000.70 230.70

June 2.90.70 1.50.31 0.660.00 0.050.00 1.30.31 3.00.70 0.0000.00 2.10.70 2591.14 291.14

July 1.30.31 1.30.07 0.520.00 0.070.00 1.00.00 0.0000.00 0.0000.00 1.70.07 2550.70 230.70

August 2.80.31 1.50.31 0.680.00 0.020.00 1.20.31 2.50.31 0.20.03 2.20.31 2530.70 270.70

September 2.80.31 1.30.31 0.560.00 0.230.00 1.30.31 2.60.31 0.0000.00 2.20.31 2551.00 251.00

October 3.00.70 1.60.31 8.480.31 0.210.00 1.10.31 11.90.70 0.20.03 2.10.31 3500.70 300.70

November 2.80.31 1.20.31 7.310.31 0.200.03 1.20.31 5.50.31 0.0000.00 2.20.31 3900.70 370.70

December 2.00.70 0.050.00 0.560.00 0.050.00 1.00.00 0.0000.00 0.0000.00 2.20.31 3000.70 291.14

1564 Journal of Research in Biology (2014) 4(8): 1557-1567

Eucharista and Ebenezer, 2014
month of August 2011 in the Authoorangal channel. The
lowest counts of total coliform bacteria was recorded as
2203.535 (MPN/ 100ml) in the month of February 2011
(Table 2). In the Srivaikuntam channel, the highest
counts of total coliform bacteria was recorded as
3900.707 (MPN/ 100ml) in the month of November
2011 (Table 4). Similarly, the lowest counts revealed as
2530.707 (MPN/100ml) in the month of August 2011.
The present study revealed the highest counts of total
coliform which may be due to physico-chemical
parameters along with the agricultural decomposition of
organic matter from the catchment area. Sharma et al.
(2013) reported that all physico-chemical parameters of
sediments showed significant relationship with bacterial
count. Dark brown color of sediments, organic carbon
(1.62%) and high organic matter (2.79%) showed high
content of organic waste in the bottom sediments. pH of
the sediment sample showed alkaline nature (8.1) and
most of the isolated bacteria grow at slight pollution and
helped in the growth of pollution indicator bacteria such
as Escherichia coli, Pseudomonas sp., Enterobacter sp.
etc.
Faecal Coliform Bacteria
During the study, the highest counts of fecal
coliform bacteria was shown to be 293.114 (MPN/
100ml) in the month of August 2011, in the station
namely Authoorangal channel (Table 2). The lowest
count of faecal coliform bacteria was found to be
200.678 (MPN/ 100ml) in the month of February 2011.
The Table 4 represented the highest counts of fecal
coliform bacteria as 370.707 (MPN/ 100ml) in the
srivaikuntam channel in the month of November 2011.
The lowest count of faecal coliform bacteria was found
to be 201.0 (MPN / 100ml) in the month of February
2011 (Table 4). The present study indicated the highest
counts of fecal coliform bacteria owing to contamination
of humic and animal matter from where the bacteria can
be transported by surface run off. Adriana et al. (2012)
reported that in Serra do Cipo, the sites of PEI1and PEI2
1565
showed the highest counts (500 to 700 MPN 100 mL-1)
of fecal coliforms during the rainy season in 2000. These
sites receive large amounts of organic matter from non-
treated effluents from the farms and small towns
surrounding the region.
CONCLUSION
From the above investigations, it may be
concluded that most of the physico-chemical parameters
viz. turbidity, temperature, pH, dissolved oxygen,
electrical conductivity, total dissolved solids, total
hardness, total alkalinity, calcium, magnesium, sodium,
potassium, chloride, sulphate, carbonate, bicarbonate,
total coliform and faecal coliform were found within the
World Health Organization limits in the Authoorangal
channel and Srivaikuntam channel. The findings clearly
indicated that both the channel water bodies are slowly
deteriorating due to discharge of agricultural fertilizers,
manures, pesticides and insecticides from the nearby
banana field. The manifested results calls for the need to
conserve manage and restore the water bodies. Intensive
efforts such as regular monitoring, systematic assessment
can save the human health and provide sustainable
environment. There is a need for awareness among the
people to maintain the significance of highest quality and
purity levels of both Authoorangal and Srivaikuntam
channel water bodies.
REFERENCES
APHA. 2005. Standard Methods for examination of
st
water and waste water.21 ed. American Public Health
Association, Washington, DC.
Adriana Medeiros O, Beatriz Missagia S, Luciana
Brandao R, Marcos Callisto, Francisco Barbosa AR,
Carlos Rosa A. 2012. Water quality and diversity of
yeasts from tropical lakes and rivers from the Rio Doce
basin in Southeastern Brazil. Brazilian Journal of
Microbiology. 43(4):15821594 http://
Journal of Research in Biology (2014) 4(8): 1557-1567

dx.dol.org/10.1590/S1517-83822012000400043.
Bhuiyan JR and Gupta S. 2007. A comparative
hydrobiological study of a few ponds of Barak valley,
Assam and their role as sustainable water resources.
Journal of Environmental Biology. 28(4):799-802.
Hemant Pathak, Deepak Pathak and Sudhir Nagesh
Limaye SN. 2011. Seasonal study with interpretation of
the chemical characteristics of water pond in reference to
quality assessment: A case study. Analele Universitatii
din Oradea- seria geografie. 21(2):233-238. http://
istgeorelint.uoradae.ro/Reviste/Anale/anale.htm
Hemant Pathak, Deepak Pathak and Limaye SN.
2012. Studies on the physic-chemical status of two water
bodies at Sagar city under anthropogenic influences.
Advances In Applied Science Research. 3(1):31-44.
Kavita Sahni and Pooja Silotia. 2011. Physico-
chemical parameters of Mansagar lake. Jaipur. Journal of
Ecotoxicology and Environmental Monitoring. 21(4):
321-324.
Kavita Sahni and Sheela Yadav. 2012. Seasonal
variations in physic-chemical parameters of Bharawas
pond, Rewari, Haryana. Asian Journal of Experimental
Sciences. 26(1): 61-64.
Macan TT. 1974. Fresh water ecology-second edition,
Longman, London, p 343.
Mary Kensa V. 2011. Inter-relationship between physic-
chemical parameters and phytoplankton diversity of two
perennial ponds of Kulasekharam area, Kanyakumari
district, Tamil Nadu. Plant Sciences Feed. 1(8): 147-154.
Murhekar Gopalkrushna H. 2011. Assessment of
physic-chemical status of ground water samples in Akot
city. Research Journal of Chemical Sciences. 1(4):117-
124.
Papen and Von Berg R. 1998. A most probable number
1566
Eucharista and Ebenezer, 2014
method (MPN) for the estimation of cell numbers of
heterotrophic nitrifying bacteria in soil. Plant soil.
199:123-130.
Prabakaran.K, Rupa Pal and Chitra J. 2013.
Hydrobiological characteristics of freshwater habitats of
South Dum Dum municipality, North 24 Parganas
district, West Bengal, India. Journal of Academia and
Industrial Research volume 2,issue 3 August 2013.
Samson S and Elangovan K. 2011. Hydrobiological
analysis and estimation of groundwater quality in
Namakkal district Tamil Nadu. Journal of Ecotoxicology
and Environmental Monitoring .21(5): 415-430.
Sayeswara HA, Mahesh Anand Gowdar and
Manujunatha R. 2011. A preliminary study on
ecological characteristics of Sominkoppa pond,
Shivamogga, Karnataka, India. The ecoscan. 5(1&2):11-
14.
Sharma KK, Neha Antal, Sanjana Kaul, Aarti Devi,
Sarbject Kour and Arti Sharma. 2013. Bacteriological
quality assessment of a subtrophical temple pond of
Birpur, Jammu (J&K, India). Journal Of Environmental
Science, Toxicology and Food Technology. 3(4): 19-25.
Sonawane L. 2011. Physico-chemical profile of
Sukhana river, in Aurangabad (M.S.) India. The Ecoscan
5(3&4):121-126.
Subroto Dutta and Pinkey Chowhan. 2009.
Assessment of groundwater quality of Masuda Tehsil,
Ajmer district, Rajasthan. The Ecoscan. 3(3&4):281-
284.
Sujata Sen, Mrinal Kanti Paul and Madhab Borah.
2011. Study of some physic-chemical parameters of
pond and river water with reference to correlation study.
International Journal of ChemTech Research. 3(4):1802-
1807.
Suresh B, Manjappa S and Puttaiah ET. 2013.
Journal of Research in Biology (2014) 4(8): 1557-1567
Eucharista and Ebenezer, 2014

Dynamics of phytoplankton succession in Tungabhadra

river near Harihar, Karnataka (India). Journal of
Microbiology and Antimicrobials. 5(7):65-71.

Vasanthy M and Velmurugan R. 2009. Groundwater

quality assessment in and around port Blair Andaman
and Nicobar Islands. The Ecoscan. 3(3&4):247-250.

Yashoda Saini, Nagendra Bhardwaj and Radha

Gautam. 2010. Physico-chemical analysis of
groundwater of Jhotwara panchayat Samiti, Jaipur
(Rajasthan). The Ecoscan. 4(1):137-139.

Submit your articles online at www.jresearchbiology.com

Advantages
Easy online submission
Complete Peer review
Affordable Charges
Quick processing
Extensive indexing
You retain your copyright
submit@jresearchbiology.com
www.jresearchbiology.com/Submit.php.

1567 Journal of Research in Biology (2014) 4(8): 1557-1567

 Journal of Research in Biology
An International Scientific Research Journal
Ethnobotanical study of medicinal plants in the region El Hajeb
(central Morocco)
Authors:
Journal of Research in Biology
Jalila El Amri1, Khalid El
Badaoui1, Touria Zair2,
Hayate Bouharb1, Said
Chakir1and Taj El Molk
Alaoui1
Institution:
1. Laboratory of
Environment and Health,
Faculty of Sciences,
University Moulay Ismail ,
BP 11201 Zitoune, Meknes,
Morocco.
2. Laboratory of Chemistry
of Bioactive Molecules and
Environment , Faculty of
Sciences , University
Moulay Ismail, Zitoune BP
11201, Meknes, Morocco.
Corresponding author:
Jalila El Amri
Email Id:
Web Address:
http://jresearchbiology.com/
documents/RA0492.pdf
Journal of Research in Biology
An International
Scientific Research Journal
ISSN No: Print: 2231 6280; Online: 2231- 6299
Original research
ABSTRACT:
As a part of enhancement of the natural heritage of the region Meknes-
Tafilalet, an ethnobotanical study was conducted in the El Hajeb region. This study
describes the different uses of medicinal plants by the local population and their
impact on the degradation of plant biodiversity. And it also helped to create a
catalogue of different species in the region. On the basis of questionnaire prepared, a
survey was conducted among the local population and herbalists operating in the field
of medicinal and aromatic plant intermediaries. The analysis of results from 220
question cards, using data processing technique has enabled us:-
To identify 80 species distributed in 40 families with a significant representation
of the Lamiaceae family
To represent the frequency of use of cash by the local population surveyed at the
study area
To represent the frequency of use by age, gender, and according to the school.
Despite the richness of the studied medicinal and aromatic plants in this area, the
local population does not benefit from its natural resources.
Keywords:
Morocco, El Hajeb, medicinal plants, ethnobotany, Lamiaceae
Abbreviations:
AC: Circulatory apparatus; AD: Digestive System; AR : Respiratory; AU: Urinary; AG: Genital Apparatus;
AA: Hearing Aids; AV: Device Image; SN: Nervous System; SQ: Skeleton; P: Skin
Part used
PS: Underground Party; T: stem; F: Leaf; TF: Stem + Leaf; FL: Flower; FR: Fruit; PE: Whole plant
Method of Preparation
Inf: Infusion; D: Decoction; C: poultice; M: maceration; Inh: Inhalation; F: Friction; Inj: Injection; P: Powder;
N: Nature; D: Miscellaneous
Abundance:
(***): Abundant; (**): Means; (*): Rare ; (0): none
Article Citation:
Jalila El Amri, Khalid El Badaoui, Touria Zair, Hayate Bouharb, Said Chakir and Taj El
Molk Alaoui
Ethnobotanical study of medicinal plants in the region El Hajeb (central Morocco)
Journal of Research in Biology (2014) 4(8): 1568 1580
Dates:
Received: 20 Nov 2014 Accepted: 05 Dec 2014 Published: 27 Dec 2014
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
1568-1580 | JRB | 2014 | Vol 4 | No 8
www.jresearchbiology.com

INTRODUCTION
The ethnobotanical and ethnopharmacological
study of Moroccan medicinal plants show the relative
importance of such plants in the health system in
Morocco. The first serious study of the Moroccan
medicinal plants dated back in 1978 when book was
published entitled Traditional Medicine and Toxicology
west-Saharan Africa by Jamal Bellakhdar. This book
was an undeniable contribution of Moroccan
pharmacopoeia. Other studies have focused on regional
medicinal flora, the most important of which were
conducted by Bellakhdar et al. (1987) in the region of
Tata (southern Morocco), Benabid and Bellakhdar
(1987) in the region of Rif and Kahouadji (1995) in
Morocco oriental and Hmamouchi and Agoumi
(1993) have reported studies from
Morocco (Central plateau). Other studies have focused
on the study of biological activity of some Moroccan
medicinal plants (Abdelrhafour et al., 1993 a and b;
Bammi et al., 2000; Remmal, 1994).
In 1997, Bellakhdar published a document of
crucial importance entitled, Traditional Moroccan
Pharmacopoeia, a bibliographic research on ancient
arabic texts of medical material, developed by North
African. Andalusian authors have demonstrated that the
Moroccan pharmacopoeia showed a remarkable
continuity with the knowledge of the ancients, at least as
regards the nature of the remedies, since 77.7% of what
it employs are already mentioned in the texts taken as a
reference (Bammi et al., 2000) .
Our laboratory has been trying for
years conducting floristic and ethnobotanical research of
medicinal plants in the region of Mekns-Tafilalet, to
enhance these natural resources. In this sense, an
ethnobotanical study was conducted at the area of El
Hajeb, a province of the Meknes-Tafilalet region.
This study is to invent medicinal plants, for
identifying the different ways of use and to understand
the close relationship between plant species described in
1569
El Amri et al., 2014
the said region and the types of diseases that affect
human beings.
MATERIALS AND METHODS
Description of the study area
The province of El Hajeb, relatively new, spreads
over an area of 2,22,000 ha. It is limited:
To the north is the province of Meknes
To the south is the province of Ifrane
To the east is the province of Fez and Sefrou province
To the west is the province of Khmisset
The administrative division consists of three
circles, five cadats, twelve rural communes and four
urban municipalities.
The total population was estimated in 2004 as
2,40,436 with 64% rural and 36% urban inhabitants. The
number of households was estimated as 30,326 and the
number of farmers has been estimated as 18,800.
The area is characterized by semi-arid to sub-
humid climate with a average annual rainfall of 520 mm.
The minimum temperature reached was 2.8 C, while the
maximum temperature reached was 38.2 C.
Farming is the main source of population in as given
below
Total area: 2,22,000 ha
SAU: 1,46,000 ha
Bour: 1,25,500 ha
Irrigated: 44,700 ha
Forests: 31,300 ha
UAA consists mainly of cereal crops (78,000 ha),
market gardening and arboriculture 44.00 ha (22,000 ha)
primarily.
The choice of the region
The choice of the study has been focused on the
region El Hajeb for the following reasons:
The geographical position of the mass if that forms
the transition between the Rif Mountains and the
Atlas chain means.
Journal of Research in Biology (2014) 4(8): 1568 1580
El Amri et al., 2014
The abundance and diversity of plants in the
spontaneous state in this region.
The existence of traditional know-how on aromatic and
medicinal plants.
Knowledge of the area as we live in El Hajeb since
childhood.
Collection of plants
Collection of medicinal species was made on
land by a random sampling method. Taxonomic
identification was performed on comparison with the
herbarium specimens of the vegetation in the area and
verified by Mohamed, El Hajeb scientific institute in
Rabat Agdal.
The choice of investigators
For the choice of the survey population, we
considered the following criteria:
Age: Age between 19 and 70 years
Gender: Heterogeneity of respondents (women / men)
Level of activity performed: Local population, farmers,
herbalists, intermediate and healers.
Ethnobotanical surveys
To meet the goals of the ethnobotanical study,
surveys were conducted and exploratory outings were
done to familiarize themselves with the terrain, the local
vocabulary and recognition of species of WFP in the
region, their spatial distribution, and abundance. This
work lasted eight months.
On the basis of a questionnaire, a series of
surveys was carried out with the local population,
herbalists and intermediaries; 220 records
questionnaires were collected, of which 110 listings were
from the local population, 80 from the souks: City El
Hajeb, Ait Boubidmane (14 km), Agourai (30km), Ain
Lhnach (20 km) and 30 from intermediaries, herbalists,
farmers and traders.
Statistical analysis
Data collected on raw data sheets were processed
by MS excel; this helped to establish a herbal monograph
Journal of Research in Biology (2014) 4(8): 1568 1580
of El Hajeb and describe their methods of valuation by
the local population.
RESULTS AND DISCUSSION
The ethnobotanical information was included on
raw data sheets and transferred to a database after
collection, processed and analysed to obtain standardized
data for the following areas:
Frequency of use of medicinal and aromatic plants by
the local population surveyed at the study area: El
Hajeb
a) Frequency of use of PAM by gender:
The WFP operation in this region affects both
men and women, although women have more knowledge
about the species in relation to men (56% against 44%).
The data processing and the graph were shown on Figure
1. The results presented in the graph confirms the results
of other ethnobotanical work at the national level, which
says that more women are holders of traditional herbal
knowledge (Bellakhdar, 1997).
on
b) Frequency of use by age:
The ethnobotanical study revealed differences in
the age of the study population regarding the use of PAM
as shown in the graph in Figure 2.
A wide range of El Hajeb people over 50 years
of age, have a greater frequency of use of medicinal
plants (50%) compared to other age groups: [40-50], [ 30
-40], [20-30], [<20], with frequencies of use 20%, 16%,
1570

10%, 4%, respectively; This shows that knowledge of
users of WFP is usually acquired following a long
experience and passed from one generation to another,
but now the transmission is in danger, because it is not
always assured.
There is also some loss of information on the
WFP because of the reduction of plant resources of the
region and the distrust of some people, particularly
young people who tend to know little about the virtues of
plants and be reluctant to their use in traditional
medicine.
2. Frequency of use of PAM by level of schooling
El Hajeb is a region which has a lower level of
schooling. Illiteracy reached a high level, especially in
women with a higher percentage compared to men (HCP,
2014).
It should be noted that at this region most users
of PAM are illiterate (68% of the population). However,
1571
El Amri et al., 2014
people with primary level schooling - a percentage of use
of significant WFP is 20%, while those with the level of
secondary and university studies are found to be very
little users of PAM (Figure 3).
3. Frequency of medicinal plants according to their
origin
Most species of WFP in El Hajeb are
characteristics of the region and are spontaneous from
the forests and Douars with a frequency of 83.75%. Also
crops such as caraway, anise, spearmint, fenugreek,
lemongrass and garlic, do not have a low frequency of
availability - 16.25% (Figure 4).
4. Distribution of different plant parts used
From the survey, the local population uses
different parts of the plant in the treatment of diseases; it
can be the leaves, stems, flowers, fruit, bark of roots,
tubers and rhizomes.
The graph given in Figure 5 shows the most
commonly used plant parts in the area of El Hajeb. Stems
Journal of Research in Biology (2014) 4(8): 1568 1580
El Amri et al., 2014

were used by 33% of people followed by the stems with mint (Mentha suaveolens), oregano (Origanum
leaves (22%), fruit (20%) and underground parts (15%). compactum), pennyroyal (Mentha pulegium L), white
5. WFP and their methods of preparation horehound (Marrubium vulgare L.) with a frequency of
The method of preparation of the crude drug operation between 70% and 90%. Other species such
from a designated body of the plant is very diverse as German chamomile (Camomilla recutita L.),
(infusion, maceration, decoction, bouillon powder etc). Paronychia argenta, oleander (Nerium oleander),
The results shown in graph in Figure 6 are as follows: Corrigiola telephifolia, Euphorbia helioscopia,
Decoction is the most common method of Mercurialis annua, Malva sylvestris and Calendula
preparation with a percentage of 45%, followed by the officinalis represent frequencies between 40% and 60%

infusion preparation (19%), and other modes are less (Figure 7).
than 9%. 7. Botanical families represented in the study area
6. PAM very common in the region According to the survey and graph in Figure
Computer analysis revealed the most common 8, we find that families Lamiaceae and Asteraceae are
PAM in the study area tel que thyme the most represented with 11 and 10 species,
(Thymus ciliatus), round-leaved respectively, followed by the Apiaceae family (7),

Journal of Research in Biology (2014) 4(8): 1568 1580 1572


liliaceae (6), Fabaceae, and Solanaceae grasses
respectively (3), other families are less than two. The
lamiaceae are flavouring plants. WFP belonging to this
family are most commonly used for self-medication in
the region. These results confirm those of other
ethnobotanical studies throughout the Moroccan territory
(Bellakhdar , 1997).
8. The frequency of medicinal plants mostly used by
the local population
From the ethnobotanical study conducted it is
possible to highlight 13 most used medicinal plants by
the local population (Figure 9).
Plantago psyllium plant is most commonly used;
when it is not among the most common plants in the
region, it is due to its effectiveness in treating wounds,
followed by plants that are common as Thymus ciliatus,
Origanum compactum, Mentha pulegium, Paronychia
1573
El Amri et al., 2014
argenta, Camomilla recutilla L., Marrubium vulgare.L
and Mentha suaveolens.
9. Distribution of WFP as pathologies
According to the statistical analysis, we are
successful in revealing various pathologies most
frequently encountered in the El Hajeb region (Figure
10) .
Dermatoses occur by a percentage of 27%
followed by the digestive tract (22%), nervous system
(14%), urogenital tract (14%), hormonal and circulatory
system (9%), and other disorders less than 5%.
10. The number of species listed according to the
most common diseases
The number of species found in the study area
that treats a given disease were given in Figure 11. The
most common diseases in the study area are skin
diseases. 18 species treat these diseases, most of them are
Journal of Research in Biology (2014) 4(8): 1568 1580
El Amri et al., 2014
healing, the most represented are Plantago psyllium with
a percentage of 25.17%, followed by Corrigiola
telephifolia (14.42%), Euphorbia helioscopia (13.13%) ,
Rosmarinus officinalis (13.10%), Nerium oleander
(12.15%), Solanum sodomaeum L. (8%), Teucrium
capitatum .L (7.33%) and Silene vulgaris (6.7% ).
For diseases of the digestive system, 14 species
are used, followed by 15 species for the nervous system,
5 species for respiratory and 4 species for the urogenital
tract problem (Figure 12).
It is found that 15 species treat diseases of the
gastrointestinal tract, the most represented are
primarily Thymus ciliatus, Euphorbia helioscopia,
Origanum compactum, Solanum sodomeum L., Silefia
vulgaris, Teucrium capitalum. L, Ceratonia siliqua,
Arbutus unedo, Ajuga iva, Helosciadium nodiflorum ,
Carum carvi L and Pimpinella anisum L not developed neither technically nor economically.
and last Ziziphus lotus.
According to Figures 12 and 13, it was found
that two plants were used for curing both dermatoses and
digestive tract diseases. Both plants were chosen to study
their antimicrobial effect.
CONCLUSION
This study identified the aromatic and medicinal
plants that exist in the region of El Hajeb and their
various uses by the local population.
The results of this study showed that the area
holds significant potential in spontaneous WFP crop and
toxic. El Hajeb region has a low level of schooling. It
should be noted that at this region most of the users of
PAM are illiterate.
The information acquired from questionnaires
sheets and floristic surveys conducted in the field, helped
us to catalog 80 plant species belonging to 40 families.
The most common medicinal and aromatic plants
widely used in the study area are thyme, mint round
leaves, oregano and white horehound. These species
contain essential oils used mainly as a carminative,
Journal of Research in Biology (2014) 4(8): 1568-1580
antiseptic, stomachic and antitussive. Over-harvesting
led to their extinction which leads us to adopt a
management approach to the protection and preservation
of natural resources.
The ethnobotanical study revealed that the most
common disease prevalent in this area is dermatitis.
Foliage is the most used plant organ and decoction is the
most dominant mode. Also skin diseases are treated
locally which allowed us to describe the different uses of
medicinal plants by the local population. This study
helped us to understand the close relationship between
plant species and described the different types of
pathologies affecting population. Thus two plants used
both for skin diseases and diseases of the intestine were
chosen for future studies.
It is noted, that the exploitation of wild plants is
Therefore, local people do not benefit from the
development of natural resources and are not said to be
as top managers.
For sustainable management of aromatic and
medicinal resources and better utilization of medicinal
and aromatic plants at the local level, Provincial
Agriculture Officer (PAO) with others proposed the
following steps:
An organization of local people together
The integration of rural women in the exploitation of
medicinal and aromatic plants
Improved incomes of men and women
Creating self-employment
Improved quality of products of medicinal and
aromatic plants
Promotion markets for WFP nationally and
internationally
An organization from the sale of products derived
from PAM (dried plants, floral waters and essential
oils).
1574
 El Amri et al., 2014

Appendix 1: Photo of some of the most used plants in the region El Hajeb

Thymus ciliatus (Le thym) Nerium oleander (laurier rose) Mentha pulegium (La menthe pouliot)

Inula viscosa (Linule visqueuse) Marrubium vulgare L. (Le marrube Laurus nobilis L. (Le laurier noble)

Origanum compactum (Lorigan) Papaver rhoeas (Le coquelicot) Salvia officinalis L. (La sauge)

Calendula officinalis (Le souci offici- Camomilla recutita ( L.) (Camomille Lippia Triphylla (La verveine)

1575 Journal of Research in Biology (2014) 4(8): 1568 1580

El Amri et al., 2014

Appendix 1: Photo of some of the most used plants in the region El Hajeb (Contd)

Mentha longifolia L. (Menthe Rumex acetosa L. (Loseille)

Urtica dioica L. (Lortie)

Lavandula dentata (Lavande sauvage) Carum carvi L. (Le carvi)

Appendix 2 : Lot picture ( investigators and users )

Journal of Research in Biology (2014) 4(8): 1568-1580 1576

 El Amri et al., 2014

Appendix 3: Landscape picture of the region of El Hajeb

Appendix 4 : Field pictures (users, herbalists and healers )

User 1 User 2 Herbalist 1

Herbalist 2 Herbalist 3 Healer

1577 Journal of Research in Biology (2014) 4(8): 1568-1580
El Amri et al., 2014

Table I : Catalog of aromatic and medicinal plants of the region of El Hajeb

Family Botanical Name Use No species Toxic Plants Abundant Spontaneous

Name vernacular cited by Yes / no or
Bellakh- cultivated
dar plant
- To treat
Thymus zitra abdominal Yes Spontaneous
ciliatus pain plant
- As a flavor in
bread
-To treat fever,
Mentha mersita headache, Yes Spontaneous
lamiaces suaveolens typhoid plant
- As a flavor in
bread
Flavor milk,
tea and
Mentha manta coffee Enough Spontaneous
longifolia - Treating plant
colds
zaatar - Gastralgia
Origanum - Flavor bread Enough Spontaneous
compactum plant
Influenza,
Mentha L. Ber Fliyou cough,
pulegium headache and Yes Spontaneous
chills plant
- Flavor meals,
lamiaces burns
Salvia salmia - Flavor tea Spontaneous
officinalis L. - Antidiabetic Not plant
Mentha viridis Nanaa - Flavor tea
L. -Headache. + Yes Cultivated
Teucrium L. Lkhiyata Furuncle,
capitatium healing, + Yes Spontaneous
gastroenteritis plant
-gastralgie,
Rosma- Azir dysmenorrhea,
rinusofficinali skin care, coo- not Spontaneous
s L. ling, headache plant
Calendula jemmra - Freezing
officinalis L. them, acne Yes Spontaneous
plant
Anthemis babnouj Belly aches,
Aste- nobilis hair care and Enough Spontaneous
raceae facial plant
Inula mgrmn Tuberculosis, Yes Spontaneous
viscosa pneumonia, plant
healing
Atractylis Ddd Fumigation Deadly com- not Spontaneous
gummifera L. against mon in Mo- plant
microbes and rocco: Root
insects, (Bellakhdar,
abscess 1997)
Scolymus l-gernina -Consumption Yes Spontaneous
hispanicus l. plant
Echinops Taskra or -Consumption Yes Spontaneous
Aste- Chawkate plant
spinosus L.
raceae elhmar

Journal of Research in Biology (2014) 4(8): 1568-1580 1578

 El Amri et al., 2014
Paronychia Harras el Kidney - Yes Spontaneous
Argenta Hjar Lithiases plant
Anacyclus Krae Djaja -gastralgie not Spontaneous
clavatus plant

Rutaceae Ruta FIJL Fever, Yes Spontaneous

angustifo- typhoid, plant
lia headaches
-digestions
Apiaceae Carum karwiy difficiles- Yes Cultivated
carvi L. antispasmodic
gas
-gonflements
Apiaceae Eryngium
ilicifolium boumghiz Gastroenteri- not Spontaneous
al tis plant
cooling,
Apiaceae Daucus bouzfour headache not Spontaneous
crinitus plant

- Digestive
Lily Pimpinella Habat problems + Yes Cultivated
anisum L. Hlawa - Flavor bread

Corrigiola Serghina -Eczma, + not Spontaneous

telephiifo- Hemorrhoids plant
lia
Abscess,
Apocyna- Nerium Dafla cooling, Very toxic enough Spontaneous
ces oleander headache ac- (Kingsbury, plant
ne, burns 1964)
Laurus
Lauraceae nobilis L. Asa Musa - Flavor Not Spontaneous
sauces plant
-Cooling Toxic to
Papave- Papaver bel- -Toux + animals Yes Spontaneous
raceae rhoeas naaman -laryngite (Cooper plant
and
Johnson,
1984)
Lippia Lwiza - Flavor tea,
Ver- triphylla fever, + Not Cultivated
benaceae headache

REFERENCES Journal of Essential Oil Research. 5(5): 535-545.

Abdelrhafour Tantaoui-Elaraki, Nezha Lattaoui, ziza
Errifi and Bachir Benjilali. 1993a. Composition and
Bammi J, Gmira N, Douria A, Remmal A, Khelifa et R
antimicrobial activity of the essential oils of Thymus
and Manai M. 2000. A study of antibacterial and antiviral
broussonettii, T. zygis and T. satureioides. Journal of
activities of essential oils of oregano and clove. The first
Essential Oil Research. 5(1): 45-53.
African Congress on Biology and Haelth. Ferhat Abbas
University. Setif, Algeria.
Abdelrhafour Tantaoui-Elarakia, Hicham Ferhouta and
Aziza Errifia. 1993 b. Inhibition of the fungal asexual
Settaf A, EL Kabbaj S, Labhal A, Cherrah Y, Slaoui A
reproduction stages by three Moroccan essential oils.
and Hassar M. 2000. Herniaria hirsuta reduces biliary

1579 Journal of Research in Biology (2014) 4(8): 1568-1580

El Amri et al., 2014

cholesterol in dogs. Induced changes in bile composition, Palermo. 8: 1- 243.

Biologic et Sant. V.1 nI. p44-49.
Lahsissene H, Kahouadji A, Tijane M and Hseini S.
Remmal A. 1994. Activits antibactrienne et antivirale des 2009. Catalogue des plantes mdicinales utilises dans la
huiles essetztielles d'origan, de girofle et de thym. Thse de rgion de Zaer (Maroc occidental). Lejeunia, N 186.
Doctorat d'tat essciences naturelles. Unv. Sidi Mohammed
Ben Abdallh. Fs. Mehdioui R and Kahouadji A. 2007. Etude
ethnobotanique auprs de la population riveraine de la fort
Bellakhdar J. 1997. La pharmacope marocaine dAmsittne : cas de la Commune dImi nTlit (Province
traditionnelle. Mdecine arabe ancienne et savoirs dEssaouira). Bulletin de lInstitut Scientifique, Rabat,
populaires - Saint Etienne, Edit. Ibis Press. 764 p. section Sciences de la Vie, n29, 11-20.

Haut Commissariat au Plan (HCP). 2004. Recensement OMS and UICN et WWF 1993. Principes directeurs pour
gnral de la population et de lhabitat. Centre de lecture la conservation des plantes mdicinales, Gland, Suisse, 35
automatique des documents. p.

Kahouadji MS. 1995. Contribution une tude

ethnobotanique des plantes mdicinales dons le Maroc
oriental. Thse de troisime cycle. Universit Mohammed I.
facult des sciences Oujda. 206 p.

Mehdioui R and Kahouadji A. 2007. Etude

ethnobotanique auprs de la population riveraine de la fort
dAmsittne : cas de la Commune dImi nTlit (Province
dEssaouira). Bulletin de lInstitut Scientifique, Rabat,
section Sciences de la Vie. n29, 11-20.

Hmamouchi M and Et Agoumi A. 1993. Place des plantes

mdicinales dons' le systme de sant au Maroc. Premier
congres international des plantes mdicinales et
phytothrapie. Tunis. P17.
Submit your articles online at www.jresearchbiology.com

Benjilali B and ZRIRA S. 2005. Plantes aromatiques et Advantages

mdicinales : Atouts du secteur et exigences pour une Easy online submission
Complete Peer review
valorisation durable. Actes Editions. Institut Agronomique Affordable Charges
et Vtrinaire Hassan II 2005.environmental crisis and Quick processing
indigenous medical practices. Social Science and Medicine. Extensive indexing
You retain your copyright
4: 321-329.
submit@jresearchbiology.com
www.jresearchbiology.com/Submit.php
Fennane M and Ibn Tattou M. 1998. Catalogue des
plants rares, menaces ou endmiques du Maroc. Bocconea,

Journal of Research in Biology (2014) 4(8): 1568-1580 1580

 Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

ORIGINAL RESEARCH
urn:lsid:zoobank.org:pub:463695EC-9269-4EA7-8534-E8E8975A205D
urn:lsid:zoobank.org:act:238CD6D0-1548-4F46-99DE-802627090BD1

Puntius nigronotus, a new fish species (cypriniformes; cyprinidae) from

Kerala, India
Journal of Research in Biology

Author: ABSTRACT:
Mathews Plamoottil
Puntius nigronotus, a new fish species of the family Cyprinidae, is described
from Mananthavady River of Kerala, India. It is distinguished from its congeners by the
following combination of characters: body deeper, maxillary barbels shorter and never
Institution: reach nostrils, dorsal fin shorter, 9 branched rays in dorsal fin and 6 branched rays in
Assistant Professor in anal fin, last unbranched dorsal fin ray feebly ossified and flexible, 27 lateral line
Zoology, Govt. College, scales, pre pelvic distance greater, caudal peduncle wide and lateral line straight.
Chavara, Kollam, Kerala,
India

Keywords:
Western Ghats, Mananthavady River, New description, Puntius viridis

Corresponding author:
Mathews Plamoottil

Email Id:

Article Citation:
Mathews Plamoottil
Puntius nigronotus, a new fish species (cypriniformes; cyprinidae) from Kerala, India
Journal of Research in Biology (2014) 4(8): 1581-1588

Dates:
Web Address:
http://jresearchbiology.com/
Received: 06 July 2014 Accepted: 29 Nov 2014 Published: 31 Dec 2014
documents/RA0484.pdf
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1581-1588| JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
Species of genus Puntius are the most common
cyprinid fishes of India. Jayaram (1991) revised the
Puntius species of India and classified the genus into ten
groups and fourteen complexes. Pethiyagoda et al.
(2012) created five genera namely Puntius, Dawkinsia,
Systomus, Haludaria and Pethia based on taxonomic
analysis of 30 putative species of Indian and Sri Lankan
Puntius species.
Puntius species are characterized by the absence
of rostral barbels, smooth last unbranched dorsal fin ray,
presence of free uroneural, simple and accumulate gill
Figure 1. A fresh specimen of Puntius nigronotus, Holotype, ZSI FF 5285, 82.3 mm SL
Figure 2. Head region of Puntius nigronotus
1582
Plamoottil, 2014
rakers and presence of a post epiphyseal fontanelle. The
present fish described from the Mananthavady River of
Wayanad, Kerala, India, carries characters of the genus
Puntius but bears many features to separate it from its
relative species. So it is described here as a new species
Puntius nigronotus.
MATERIALS AND METHODS
Methods used are those of Jayaram (2002);
subunits of the head are presented as percentage of Head
Length (HL); head length and measurements of body
parts are given as percentage of Standard Length (SL).
Figure 3. Dorsal fin of P. nigronotus
Journal of Research in Biology (2014) 4(8): 1581-1588
Plamoottil, 2014

Table 1. Meristic Counts of Puntius nigronotus sp. nov. and its relative species
SL. Counts P. nigronotus P. viridis P. parrah P. madhusoodani P. chola P. dorsalis P. sophore
No. ZSI FF 4934 CRG/SAC 456- ZSI/ ZSI/F2730, ZSI FF
ZSI/ WGRC/ (n=4) 459 F2203, ZSI/ 4938
Holotype, IR/2382, 2383, (n=4) 4009 SRC4954 (n= 2)
ZSI FF 5285 ZSI FF 4932 (n=2) (n=3)
(n= 8)
Scale Counts
1 LLS 27 25- 26 25 25- 26 26 25-26 25

2 PDS 11 9 8 9 9 9 9

3 PRPLS 10 5 6 6 5-6 5-6 5

4 PRAS 18 10- 12 14 14 12-13 11-13 13

5 CPS 10 9-10 10 10 11-12 9-10 10

6 LL/D 5 5- 5 5 4 4-5 4-5 5

7 LL/V 3 3 3 3 3-3 2 3

8 LL/A 4 3 3 3 3 3 4

9 L/TR 5/3 5-5/3 5/4 5/3 5/4 5/2 5/4

Fin Ray Counts

10 D ii, 9 iii, 8 iii, 8 iii,7 iii,8 iii, 8 iii, 8

11 P i, 15 i, 14 i, 14 i, 14 i, 13-16 i, 14-15 i, 13- 14

12 V i, 8 i, 8 i, 8 ii, 8 i, 8 i, 7 i, 8

13 A ii, 6 iii, 5 ii, 5 ii, 6 iii, 5 iii, 5 iii, 5

14 C ii, 17 18- 19 19 19 19 17 18

Abbreviations used: Caudal Peduncle; LL/D- scales between lateral

line and dorsal fin; LL/V- scales between lateral
BDD Body depth at dorsal fin origin; IOW
line and ventral fin; LL/A- scales between
Inter Orbital Width; HD Head Depth; LLS
lateral line and anal fin; L/TR Lateral
Lateral Line Scales; PDS Pre Dorsal Scales;
Transverse Scales; D Dorsal fin rays; P
PRPLS Pre Pelvic Scales; PRAS Pre Anal
Pectoral fin rays; V Ventral fin rays; A Anal
Scales; CPS Circum Peduncular Scales; LCP
fin rays; C Caudal fin rays. ZSI/ WGRC-
Length of Caudal Peduncle; DCP Depth of
Zoological Survey of India, Western Ghats
Regional Centre, Kozhikode; ZSI- Zoological
Survey of India, Kolkata; CRG/SAC-
Conservation Research Group, St. Alberts
college, Cochin; ZSI/SRC- Zoological Survey
of India, Southern Regional Centre, Chennai;
UOK/AQB- department of aquatic biology and
fisheries, University of Kerala.
Figure 4. Puntius viridis, Paratype, ZSI FF 4932

Journal of Research in Biology (2014) 4(8): 1581-1588 1583

 Plamoottil, 2014

Table 2. Comparison of morphometric characters of P. nigronotus and its congeners

SL. P. viridis ZSI/ P. parrah P. madhusoodani
N0. Characters P. nigronotus WGRC/IR/2382, ZSI FF CRG/SAC 456- 459,
2383, ZSI FF 4934 (n= 4)
ZSI FF 5285 4932 (n= 8) (n=4)
1 Total length (mm) 105.1 91.2-103 86.5- 102.0 90.5- 118.3
2 Standard Length (mm) 82.3 72-81 65.5- 78.0 67.6- -91.4
% SL
3 Head length 29.5 26.4- 31.1 25.6-26.0 27.5-29.5
4 Head depth 22.1 19.7- 22.9 21.6-24.0 20.7-23.1
5 Head width 17.0 15.8- 17.8 15.4-17.6 15.0-16.7
6 Body depth at dorsal fin 35.8 31.5- 33.8 32.1- 33.1 34.5-36.2
7 Body depth at anal fin 23.7 21.1- 23.9 23.7-24.4 22.1-23.7
8 Body width at dorsal fin 20.0 16.2- 19.1 17.3-19.7 17.6-19.1
9 Body width at anal fin 12.2 10.8- 13.2 13.4- 15.2 11.7-14.5
10 Pre occipital distance 18.5 18.9- 23.0 20.5- 24.3 18.9-22.9
11 Distance from occiput to dorsal front 33.8 30.4- 31.7 24.3- 29.8 29.0-32.9
12 Pre-dorsal length 54.7 48.2- 54.8 50.0-52.1 49.3-50.6
13 Post-dorsal length 51.0 48.2- 54.8 48.7- 53.5 50.2-58.6
14 Pre-pectoral length 27.0 25.8- 29.7 27.0-28.2 26.2- 28.9
15 Pre-pelvic length 52.6 47.9- 50.0 47.2- 51.3 46.5-50.3
16 Pre-anal length 75.1 72.2- 76.6 70.3- 74.4 67.6-74.3
17 Length of dorsal fin 21.1 22.4- 26.5 22.1-24.4 25.2-28.7
18 Length of pectoral fin 19.4 16.7- 19.7 17.6-19.8 17.7- 19.1
19 Length of pelvic fin 17.9 17.3-20.3 20.3-21.4 20.7- 21.1
20 Length of anal fin 16.2 14.8- 18.9 13.3-16.8 19.2-21.5
21 Length of caudal fin 27.7 29.3- 30.0 28.4- 32.1 24.8- 27.0
22 Length of base of dorsal fin 18.2 17.6- 19.2 18.0-21.0 19.0-20.0
23 Length of base of anal fin 8.7 9.8- 11.1 12.0-15.4 9.0-12.0
24 Length of base of pectoral fin 5.1 4.1- 5.3 3.3-4.2 3.7- 4.1
25 Length of base of pelvic fin 4.9 5.0- 6.9 4.2-5.4 6.0-7.1
26 Length of base of caudal 14.6 13.5- 14.2 12.2-14.1 12.4- 13.8
27 Length of caudal peduncle 17.6 16.3- 17.8 19.1-21.2 12.6-17.5
28 Depth of caudal peduncle 14.0 13.5-14.5 12.9-13.5 12.8-14.6
29 LCP/DCP 79.3 77.0- 88.0 63.6-74.3 73.1- 84.6
30 Width of caudal peduncle 9.1 5.5- 7.4 4.1- 5.4 6.2-.6.6
31 Distance from pectoral to pelvic fin 25.7 21.0-21.6 20.4-20.9 22.8-25.0
32 Distance from pelvic to anal fin 24.3 23.8- 25.0 24.3- 26.8 25.0-28.9
33 Distance from anal to caudal fin 26.4 25.9- 27.5 27.7-29.6 25.5-27.0
34 Distance from anal to vent 0 2.6- 4.1 0 4.8-6.6
35 Distance from ventral to vent 22.5 19.1- 22.8 23.0-25.6 22.4-23.4
% HL
36 Head depth 74.9 68.2- 80.0 84.2-89.5 95.0-100.0
37 Head width 57.6 56.5- 63.2 60.0- 68.4 55.0-61.9
38 Eye diameter 32.1 26.1- 31.6 32.5-36.8 27.5-33.3
39 Inter orbital width 32.1 31.6- 40.9 42.1- 42.5 37.5-41.9
40 Inter narial width 23.0 23.9- 28.9 23.5-30.0 25.0-28.6
41 Snout length 24.7 22.7- 31.8 26.3-30.0 28.6-30.0
42 Width of gape of mouth 22.6 23.0- 27.3 28.9-30.0 25.0-27.6
43 Length of maxillary barbels 12.3 13.0- 21.1 15.0-17.6 14.3-15.0

RESULTS AND DISCUSSION Plamoottil, 1 January 2012.

Puntius nigronotus sp. nov. Diagnosis
(Figures 1- 3; Table 1 and 2) Puntius nigronotus can be differentiated from all
Holotype: ZSI FF 5285, 82.3 mm SL, India: Kerala, its congeners in having blackish dorsal side, shorter
Mananthavady River, Wayanad, coll. Mathews maxillary barbels (12.3 % HL), shorter width of mouth

1584 Journal of Research in Biology (2014) 4(8): 1581-1588

Plamoottil, 2014
Figure 5. Puntius parrah, ZSI FF 4934, coll. Plamoottil
gape (22.6 5 % HL), a higher body (BDD 35.8 % SL), a
row of elongated tiny black dots present on dorsal fin,
the latter is shorter (21.1 % SL) and with 9 branched
rays, last unbranched dorsal fin ray feebly ossified and
flexible, 6 branched rays in anal fin, 27 lateral line
scales, greater pre pelvic distance (52.6 % SL), wide
caudal peduncle (9.1 % SL) and straight lateral line.
Description
General body shape and appearance is shown in
Figure 1- 3. Meristic counts are shown in Table 1 and
morphometric data in Table 2. Body laterally
compressed; dorsal profile convex; region from dorsal
front to occiput a little bent, after sinking down very
slightly goes straight to snout tip; post dorsal region
slightly concave. Ventral profile from the base of
pectoral fin to tip of pelvic fin straight, then abruptly
concave and then goes straight to caudal base. Eyes
situated considerably behind and above the angle of
jaws, protruding above the surface of head and distinctly
visible from below ventral side; inter orbital region
slightly convex; nostrils, in short tube, situated nearer to
Figure 7. Puntius dorsalis, ZSI FF 2730, coll. Day
Journal of Research in Biology (2014) 4(8):1581-1588
Figure 6. Puntius chola, ZSI FF 2203, coll. Hora
eyes than to snout tip; jaws equal; barbels one pair
maxillaries only, shorter than orbit, feeble and roughly
reach the lower margin of orbit but never reach nostrils;
mouth terminal, straight and protruding; width of gape of
mouth equal to inter narial distance; operculum soft and
flexible.
Dorsal fin originates considerably behind the
pectoral tip, a little in front of ventral origin and nearer to
caudal fin base than snout tip; its upper margin fairly
concave; dorsal fin with two undivided and nine
branched rays; first ray short and hard; second ray feebly
ossified and flexible, tip a little filamentous; its inner
margin slightly roughened but not serrated; last branched
ray divided to root; pectoral tip never reach ventral
origin; ventral fin originates just behind dorsal fin origin
and considerably behind pectoral tip; its tip never reach
anal fin origin and vent; two axillary scales present on
either side of base of ventral, one above the other, of this
the upper one soft and delicate and form 2 in length of
ventral fin; lower one short but more fleshy; anal fin
roughly rectangular, outer margin fairly concave, its tip
Figure 8. Puntius madhusoodani, Paratype, CRG- SAC 459
1585

Figure 9. Puntius sophore, ZSI FF 4938, coll. Plamoottil
never reach caudal base; no distance between anal fin
origin and vent; no prominent ridge on the base of anal
fin and dorsal fin; unbranched and branched rays of anal
fin soft; caudal lobes equal. Scales thin and soft; lateral
line straight and passes through the middle of body.
Lateral line scales 27.
Colouration
In live condition, back side deep black; upper
lateral side blackish brown; lower lateral side silvery
white; dorsal fin pale orange red; pectoral, pelvic and
anal fins whitish yellow; caudal fin dirty yellow; minute
bluish black dots present on operculum; a few tiny
elongated black spots present on dorsal fin rays; caudal
blotch small, diffuse and concentrated on one scale as an
aggregation of bluish black dots. After preservation in
formalin back and upper lateral side turned to reddish
brown and lower lateral side yellowish white and fins
became hyaline.
Etymology: The specific name nigronotus denotes the
color of the new fish. In Greek nigra means black and
notus means back; refers to the blackish dorsal side
of the fish.
Distribution:
Currently known only from the type locality in Kerala.
Comparisons
Puntius nigronotus shows similarity to Puntius
viridis, P. parrah and P. madhusoodani of Kerala and
Puntius dorsalis of Chennai and Puntius chola and
P. sophore of River Ganges (Figure 4- 9). The new fish
differs from Puntius viridis Plamoottil and Abraham
1586
Plamoottil, 2014
(2013) in having 27 lateral line scales (vs. 25/26 in P.
viridis), 11 pre dorsal scales (vs. 9), 9 branched rays in
dorsal fin (vs. 8), deeper body (35.8 % SL vs. 31.5-
33.8), greater distance (25.7 % SL vs. 21.0- 21.6) from
pectoral fin to pelvic fin, no distance from anal fin origin
to vent (vs. 2.6- 4.1 % SL) and wider caudal peduncle
(9.1 % SL vs. 5.5- 7.4). P. nigronotus differs from P.
parrah Day (1865, 1878, 1889) in having closely located
orbits (IOW 32.1 vs. 42.1- 42.5 % HL in P. parrah),
shorter maxillary barbels (12.3 % HL vs. 15.0- 17.6),
lower (HD 74.9 % HL vs.84.2- 89.5) and longer head
(29.5 % SL vs. 25.6- 26.0) and greater number of
branched dorsal fin rays (9 vs. 8) and lateral line scales
(28 vs. 25).
The new fish differs from Puntius chola
(Hamilton, 1822) in having 11 pre dorsal scales (vs. 9 in
P. chola), 10 pre pelvic scales (vs. 5/6), 27 lateral line
scales (vs. 26), 9 branched rays in dorsal fin (vs. 8), 6
branched rays in anal fin (vs. 5) and 4 scales between
lateral line and anal fin (vs. 3). Puntius nigronotus
differs from P. dorsalis Jerdon (1849) in many meristic
and morphometric characters. In P. dorsalis, a black
spot present at the posterior portion of the base of dorsal
fin (vs. absent in new species), 2 scales present
between lateral line and ventral fin (vs. 3), snout longer
(31.8-37.1 % HL vs. 24.7), dorsal fin with 8 branched
rays (vs. 9) and lateral line with 25/26 scales (vs. 27).
The new species can also be easily distinguished
from Puntius madhusoodani Kumar et al. (2011) in
many taxonomic characters. In P. madhusoodani 4
scales present between lateral line and dorsal fin (vs.
5), pre dorsal scales 9 (vs.11), dorsal fin longer (25.2-
28.7 % SL vs. 21.1), dorsal fin with 7 branched rays (vs.
9), lateral line scales 27 (vs. 25/26), snout longer (28.6-
30 % HL vs. 24.7), head deeper (head depth 95.0- 100.0
% HL vs. 74.9) and anal fin longer (19.2- 21.5 % SL vs.
16.2). The new species can be distinguished from
Puntius sophore (Hamilton, 1822) in having one pair of
maxillary barbels (vs. absent), smaller eyes (32.1 % HL
Journal of Research in Biology (2014) 4(8): 1581-1588
Plamoottil, 2014
vs. 34.7- 36.0), lower head (head depth at occiput 74.9 %
HL vs. 80.3- 86.7), lateral line scales 27 (vs. 25), 11 pre
dorsal scales (vs. 9) and 9 branched rays in dorsal fin (vs.
8).
CONCLUSION
Puntius nigronotus is a unique fish having
greater number of branched dorsal fin rays than all other
relative species; its lateral line scales and pre dorsal
scales are also greater in number than its congeners; their
last unbranched dorsal fin ray feebly ossified and
flexible; it was believed that branched rays in dorsal fin
of Puntius species are only eight (Pethiyagoda et al.,
2012); the present fish is an exception to this; Puntius
nigronotus have nine branched rays in dorsal fin and
additionally its last branched ray is divided to root; it is
expected that more aspects of its biology will be revealed
in near future based on studies on more number of
specimens.
Comparative material examined
Puntius viridis: Holotype, 21.08.2011, 81 mm
SL, Kallumkal, Manimala River, Kerala, India,
9200N, 76300E, coll. Mathews Plamoottil, ZSI/ Sukla Talai, Jhalwar, Rajasthan, ZSI/F 4009/2, coll. N.
WGRC/IR/2382. Paratypes, 21.08.2011, 5 examples, 72-
76 mm SL, Kallumkal, Manimala River, Kerala, India,
9200N, 76300E, coll. Mathews Plamoottil, ZSI/ Keval Singh; 3 examples, 50- 62 mm SL, Rajastan,
WGRC/ IR/2383; 10. 10. 2012, 2 examples, 63- 74 mm
SL, Manimala River at Kallumkal, Kerala, coll. Mathews
Plamoottil, ZSI FF 4932.
Puntius dorsalis: 27.10.95, 1 example, 62 mm
SL, Thunakadavu dam, Parambikulam wild life
sanctuary, Kerala, ZSI/WGRC/IR 8466, coll. P.M.
Sureshan, identified by K.C. Gopi; 23.2.2000, 2
examples, 56- 63 mm SL, Pampa River at Parumala,
Kerala, ZSI/WGRC/IR/10379, coll. K. C. Gopi; 11.02.
58; 1 example, 53 mm SL, Isteri tank, 7 miles north west
of Pondicherry, ZSI/F 2801, coll. A.G.K. Menon; 16.02.
1996, 2 examples, 52- 53 mm SL, Sethumadai canal,
Indira Gandhi Wild Life sanctuary, Tamil nadu, ZSI/
Journal of Research in Biology (2014) 4(8): 1581-1588
SRC/F 4954, coll. M.B. Reghunathan; undated, 1
example, Madras, ZSI/F 2730, coll. Francis Day;
undated, 1 example, 53 mm SL, Tunga River at
Shimoga, ZSI/F 12320/1, coll. H.S. Rao; undated, 5
examples, 55- 62 mm SL, Cauvery River, Coorg,
Karnataka, ZSI/F 12319/1, coll. C.R. Narayan Rao;
Puntius parrah: 10.01. 2012, 4 examples, 65.5-
78.0 mm SL, Arattupuzha, Karuvannoor River,
Irinjalakuda, Kerala, ZSI FF 4934, coll. Mathews
Plamoottil; 15.12.1994; 1 example, 60 mm SL, Kuruva
Island, Wayanad, ZSI/WGRC/IR/742, coll. C.
Radhakrishnan; 24.03.1997, 1 example, 44 mm SL,
Parambikulam WLS, ZSI/WGRC/IR/10696, coll. K. C.
Gopi; 10.8.2001, 2 examples, 100.0- 103.0 mm SL,
Achankoil River, UOK/AQB/F/ 102, coll. Bijukumar;
undated, 1 example, Karuvannoor River, Kerala, ZSI/F
2718 Syntype, coll. Francis Day; 08.05. 1977, 6
examples, 71 mm- 94 mm SL, Cauvery River at
Chunchanakatte, ZSI/SRC Uncat, coll. K. C. Jayaram.
Puntius chola: 08.11.1939, 1 example, 41.5 mm
SL, Soni Gaon Bheel, Lokpa, Batipara, Assam, ZSI/F
2203, coll. S.L. Hora; 1963, 1 example, 54 mm SL,
Majumdar & R.N. Bhargava; 18.03.1958, 2 examples,
32.5- 55 mm SL, Raxanal, Bihar, ZSI/F/2804/2, coll.
ZSI/F/4379/2, coll. Birla college, Pilani; 1 example, 71
mm SL, Mahanadi Irrigation Canal, Rudri, Orissa, ZSI/F
13082/1, coll. H.S. Rao.
Puntius madhusoodani: 17.11.2010, Holotype,
91.43mm SL, Manimala River, near Thirumoolapuram,
Thiruvalla, Kerala, CRG-SAC 456, coll. K.
Krishnakumar; 17. 11. 2010, 3 examples, 67.6 -
80.91mm SL, Manimala River, near
Thirumoolapuram,Thiruvalla, Pathanamthitta District,
CRG-SAC 457 459 paratypes, coll. K. Krishnakumar
and Benno Pereira.
Puntius sophore: 10.05.2012, 2 examples, 58-
59 mm SL, Serrampore, River Ganges, Kolkata, ZSI FF
1587
 Plamoottil, 2014

4938, Coll. Mathews Plamoottil; 20.06. 1963, 4

examples, 62.5- 70.0 mm SL, Sukla Talai, Jhalawar,
Rajasthan, ZSI/F 4008/2, coll. N. Majumdar & R. N.
Bhargava; 24.10.1939, 1 example, 40 mm SL,
Suwannee River, east of Hazaribagh Barhi Road, ZSI/F
13827, H.S. Rao; 22.06.1963, 4 examples, 66- 102 mm
SL, Gadhuli Talai, Shergarh, Rajasthan, ZSI/F 4023, SE
Rajastan Survey of ZSI; 30.06.1983, 4, examples, 58.0-
67.5 mm SL, Talbi, N. of Bimmal Railway station, ZSI/F
4029/2, S. E. Rajasthan Survey of ZSI.
Jayaram KC. 2002. Fundamentals of Fish Taxonomy.
Narendra Publishing House, Delhi. 174 p.
Jerdon TC. 1849. On the freshwater fishes of southern
India, Madras Journal of Literature and Science.15 (2):
302- 346.
Kumar KK, Pereira FGB and Radhakrishnan KV.
2011. Puntius madhusoodani (Teleostei: Cyprinidae), a
new species of barb from Manimala River, Kerala, South
India, Biosystematica. 5 (2): 31- 37.

ACKNOWLEDGMENTS Pethiyagoda R, Meegaskumbura M and Maduwage

The author acknowledges University Grants K. 2012. A synopsis of the South Asian fishes referred
Commission of India for sanctioning Faculty to Puntius (Pisces: Cyprinidae). Ichthyological
Development Programme to undergo this research. I am Exploration of Freshwaters. 23 (1): 69-95.
grateful to anonymous reviewers for their comments that
Plamoottil M and Abraham NP. 2013. Puntius viridis
helped to improve the manuscript.
(Cypriniformes, Cyprinidae), a new fish species from
Kerala, India, Journal of Research in Biology. 3 (7):
REFERENCES
1093-1104.
Day F. 1865. The Fishes of Malabar. Bernard Quaritch,
London. 293.

Day F. 1878. The fishes of India: being a natural history

of the fishes known to inhabit the seas and fresh waters
of India, Burma, and Ceylon. William and Norgate,
London. (suppl.) 556-574.

Day F. 1889. Fauna of British India including Ceylon

and Burma. Edited by W. T. Blanford. Fishes, by Francis
Day. 2, 8: London: Taylor and Francis, 1889. Fishes. I,
Taylor and Francis, London. 209-334.
Submit your articles online at www.jresearchbiology.com
Hamilton F. 1822. An Account of Fishes found in the Advantages
River Ganges and its branches. Edinburgh: Printed for Easy online submission
Complete Peer review
Archibald Constable and Company and Hurst, Robinson,
Affordable Charges
and Co. 90, Cheapside, London. 312-389. Quick processing
Extensive indexing
Jayaram KC. 1991. Revision of the genus Puntius You retain your copyright
Hamilton (Pisces: Cypriniformes, Cyprinidae, submit@jresearchbiology.com
Cyprininae). from Indian region. Records of Zoological www.jresearchbiology.com/Submit.php

Survey of India, Occasional Paper No. 135: 178.

1588 Journal of Research in Biology (2014) 4(8): 1581-1588

 Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

ORIGINAL RESEARCH

Ethnobotanical plants used as curatives for skin diseases in a Cauvery river

stretch, Namakkal district, Tamil Nadu, India
Authors: ABSTRACT:
Journal of Research in Biology

Thirumaran G1,
Ganesan CM2,
Nandakumar K1
and Paulsamy S2 The study area covers about 15 km stretch in the perennial river bed, Cauvery
which supports semi evergreen to dry deciduous type of forests. Ethnobotanical
information on 24 plant species was recorded during the extensive field survey carried
Institution:
1. PG and Research out in this stretch during 2012-2013. The information covers botanical names,
Department of Botany, vernacular names, family, plant parts used and the mode of usage.
Kandaswami Kandars
College, Velur, Namakkal,
Tamil Nadu, India.

2. Department of Botany,
Kongunadu Arts and Science
College, Coimbatore, Tamil
Nadu, India Keywords:
Cauvery river basin, Medicinal plants, Traditional uses

Corresponding author:
Thirumaran. G

Email Id:

Web Address:
http://jresearchbiology.com/
documents/RA0493.pdf
Article Citation:
Thirumaran G, Ganesan CM, Nandakumar K and Paulsamy S.
Ethnobotanical plants used as curatives for skin diseases in a Cauvery river stretch,
Namakkal district, Tamil Nadu, India
Journal of Research in Biology (2014) 4(8): 1589-1594
Dates:
Received: 21 Nov 2014 Accepted: 29 Nov 2014 Published: 31 Dec 2014

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1589-1594| JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
Plants are proved to be more reliable for
therapeutic uses. Still 70-80% of the people all over the
world rely on plants for their health care (Shanley and
Luz, 2003). India, the mega diversity nation, harbours
about 3000-3500 (15%) medicinal plants, out of 20000
available in the world. At global level, 90% of these
species are growing in various climatic areas (Farombi,
2003). Studies on medicinal plants availability,
biochemical compounds, mechanism of their action etc.,
are being made in India since few decades intensively. In
Tamil Nadu, many vegetation are being attempted for
this study by many workers (Banu et al., 2007;
Murugesan et al., 2007; Ignacimuthu et al., 2008;
Arunachalam et al., 2009; Balakrishnan et al., 2009;
Ayyanar et al., 2010; Maruthapandian and Mohan, 2010;
Shanmugam et al., 2011 and 2012).
The riparian ecosystem at Cauvery stretch in
Tamil Nadu is known to harbor many medicinal plants of
local uses. However, no documentation has been made in
this line. Therefore, the present study was aimed to
collect information on traditional uses of medicinal
plants used in preparation of herbal drugs for the
treatment of skin diseases by local people living in the
nearby villages of Cauvery river, Namakkal District,
Tamil Nadu.
STUDY AREA
The Cauvery river stretching between Nanjai
Edayar and Mohanur area of Namakkal district Tamil
Nadu, is situated at 1104 L and 7803 E. The altitude
is 130.45 above MSL. The soil is mostly sandy with
slightly acidic pH. The local people of nearby villages, in
addition to western medicine also use herbal plants for
their day to day life, as per the prescription made by
healers. The present study was undertaken to document
the traditional uses of medicinal plants available in this
stretch which comprises grassy patches, man-made
plantations and semi evergreen and dry deciduous
forests.
1590
Thirumaran et al., 2014
MATERIALS AND METHODS
Survey and collection of ethnomedicinal plants
used by the local people of Namakkal district
(Pandamangalam, Nanjai Edayar and Mohanur) were
made over a period of 12 months (2012-2013). Frequent
field trips were undertaken to the study areas for
collecting information regarding the medicinal plants
used by them from the elderly people. Experienced
healers are interrogated intensively to bring out the
information about the medicinal plants in various aspects
viz., plant parts used, medicinal uses, mode of
administration and the doses to be prescribed. The plant
specimens were collected for taxonomic identification
from different parts of the study area. Identification was
made with the help of The Flora of Presidency of
Madras (Gamble, 1915- 1935) and The Flora of Tamil
Nadu Carnatic (Mathew, 1983) and finally confirmed by
comparing with the authenticated specimens in the
Herbarium of Botanical Survey of India (Southern
Circle) Coimbatore district, Tamil Nadu. Voucher
specimens were deposited in the Herbarium of the
Research Department of Botany, Kandaswami Kandars
College, Velur, Namakkal, Tamil Nadu. Indian
Medicinal Plants (Kirtikar and Basu, 2012) and The
Wealth of India, (2006) were referred for further
information on medicinal uses.
Enumeration
In the following enumeration, the plants are
arranged alphabetically as per botanical name, local
name in parenthesis family name and a brief note on
plant parts, mode of utilization, dosage etc.
Aegle marmelos (L.) Corr. Serr. (Vilvam) Family:
Rutaceae.
One fresh fruit is crushed with seeds of
Strychnos nux vomica and Pongamia pinnata and boiled
with coconut oil for fifteen minutes. This extract is
applied on the affected parts twice a day for three days to
treat scabies.
Journal of Research in Biology (2014) 4(8): 1589-1594
Thirumaran et al., 2014

Calotropis gigantea (L.) R.Br. (Erukku) Family: wounds.

Asclepiadaceae. Cynodon dactylon L. Pers. (Arugam pull) Family:
Paste prepared from leaf powder of this species Poaceae.
is applied externally once a day for three days to cure the Five grams of fresh leaves is made into paste
lesions of eczema. with little amount of turmeric (Curcuma longa) and few
Cassia alata L. (Seemai agathi) Family: drops of water. This paste is applied externally twice a
Caesalpiniaceae. day for ten days to treat dermatophytosis.
Ten grams of fresh leaf is applied over infected Datura innoxia Mill. (Vellai umathai) Family:
skin twice a day for fifteen days to treat ringworm Solanaceae.
infection. Paste prepared from dried leaf powder with
Cassia fistula L. (Sarakonrai) Fa mily: coconut oil is applied externally once a day, continuously
Caesalpiniaceae. for eight to ten days to get relief from wounds.
About five grams of the powder of flower is Euphorbia tirucalli L. (Elaikalli) Family:
made into paste with lemon juice and applied externally Euphorbiaceae.
on the skin to treat eczema and freckles. Five milliliters of milky latex is externally
Celosia argentea L. (Kolikondai) Family: applied once a day for a period of two days to treat
Amaranthaceae. arthritis and cracked foot.
A paste is prepared from five grams root with Ficus religiosa L. (Arasu) Family: Moraceae.
water. This paste is applied externally once a day for ten About three grams of shade dried powder of bark
days to treat wounds. is mixed with water and taken orally once a day for ten
Clerodendron inerme (L.) Gaertn. Fruct. days to treat psoriasis.
(Naaraseengi) Family: Verbenaceae. Ficus racemosa L. (Athi) Family: Moraceae.
About two grams of fresh leaves is made into A decoction is prepared from ten grams of bark
paste with water and applied externally on the skin once powder mixed with water. This decoction is applied
a day, for one week to treat psoriasis. externally once a day for six days on the wounds.
Cocos nucifera L. (Thennai maram) Family: Lawsonia inermis L. (Maruthaani) Family:
Areaceae. Lythraceae.
A thick paste prepared from five milliliters of oil About fifty grams of bark powder is made into a
and two grams of turmeric (Curcuma longa) powder is paste with coconut oil. This paste is applied externally
applied externally twice a day for a period of three days once a day for seven days to treat skin diseases.
to treat cuts and wounds. Madhuca longifolia (Koen) Macbr. (Illuppai) Family:
Commelina benghalensis L. (Neerchedi) Family: Sapotaceae.
Commelinaceae. A decoction is prepared by boiling a hundred
Leaf paste prepared is applied once a day on the grams of stem bark and five hundred milliliters of water.
wounds for healing and to remove the poisonous spines This decoction is taken orally once a day, for one week
that had struck accidently on the body parts. to get relief from wounds and skin diseases.
Curcuma longa L. (Manjal) Family: Zingiberaceae. Melia dubia Cav. (Malai vembu) Family: Meliaceae.
A sufficient amount of dried powder of rhizome The fruits are made into paste and applied
is applied externally twice a day for three days to treat externally once a day, for a period of one week as

Journal of Research in Biology (2014) 4(8):1589-1594 1591

 Thirumaran et al., 2014

Senna alata Melia dubia Calotropis gigantea

Euphorbia tirucalli Pedalium murex Ficus religiosa

Curcuma longa Datura inoxia Mirabilis jalapa

Figure 1. Some common medicinal plant species of Cauvery riparian ecosystem

ointment to get cure from skin diseases. Pedalium murex L. (Aanai nerunjil) Family:
Mirabilis jalapa L. (Anthimalli) Family: Pedaliaceae.
Nyctaginaceae. Two milliliters of juice is prepared and mixed
About six grams of fresh leaves are made into with five milliliters of water. This juice is taken orally
paste with water. This paste is applied externally twice a once a day for fifteen days to treat psoriasis.
day, for three days to treat swellings in the skin. Pongamia pinnata (L.) Pierre. (Punga maram)
Euphorbia hirta L. (Arisi poondu) Family: Family: Fabaceae.
Euphorbiaceae. Ten grams of seed powder are boiled with
The latex obtained from the leaves is applied coconut oil and the extract is applied over the skin to
externally once a day for three days to treat pimples in treat skin diseases.
the face (Acne vulgaris). Hundred grams of bark powder are boiled with

1592 Journal of Research in Biology (2014) 4(8): 1589-1594

Thirumaran et al., 2014
Figure 2. Pie chart describing the percent plant species
used for various ailments
two hundred milliliters of coconut oil and the oil extract
is applied externally once a day to treat ringworm
infection, rashes, scabies, eczema and psoriasis.
Solanum torvum Sw. (Sundai) Family: Solanaceae.
The paste prepared from five grams of fresh root
with water is applied externally to get cure from chronic
wounds.
Thespesia populnea (L.) Sol. Ex Correa (Poovarasu)
Family: Malvaceae.
Two grams of senescent leaves are made into
paste and applied externally twice a day for one week to
get cure from chronic wounds.
Tridax procumbens L. (Thaneerpundu) Family:
Asteraceae.
Ten grams of whole plant are made into paste.
This paste is applied externally twice a day for one week
to treat cuts and wounds.
RESULTS AND DISCUSSION
The present study revealed that local people of
Namakkal district in the nearby villages of Cauvery
stretch were using 24 species of medicinally important
plants belonging to 20 families. The most commonly
used families were Solanaceae (2 species),
Euphorbiaceae (2 species), Moraceae (2 species) and
Journal of Research in Biology (2014) 4(8): 1589-1594
Caesalpiniaceae (2 species). They were using these
plants to treat various types of skin diseases, scabies,
eczema, wounds, acne vulgaris, ringworm infection,
freckles, psoriasis, cracked foot, swellings, rashes, etc.
Among different plant parts used, the leaves are
most commonly prescribed. They use the plant parts in
various forms, either raw or paste, powder, decoction and
juice for curing various skin disorders. In the above
mentioned data, 14 plants are used in the form of paste,
8 plants are used in the form of powder, decoction and
juice. The latex is used as such in 2 plants. External
application is also prescribed for the treatment of certain
skin diseases. The investigated 24 plant species can treat/
cure as many as 7 different skin diseases. About 9 plants
are used for wounds healing followed by 4 plants for
psoriasis, 3 plants for eczema, 2 plants for scabies, 2
plants for acne vulgaris, ringworm and healing the
cracked foot.
The healers have preferred to prepare
the formulations by combining several plants for their
synergetic effect to heal the ailments. The
medicinal plants prescribed by the healers need to be
systematically screened for their phytochemicals and
thus the scientific validation may help in the discovery of
new drugs from these medicinal plants.
CONCLUSION
From the study it is suggested that Cauvery river
stretch in Namakkal district, Tamil Nadu is a potential
ecosystem of medicinal plants as it is endowed with
many medicinal plants which are prescribed most
commonly by the local healers for various ailments. In-
depth studies are suggested all over the Cauvery river
banks in Tamil Nadu to explore the medicinal plants.
Phytochemical and pharmacological investigation should
also be made to have the scientific validation of such
valuable bioresources and confirm our traditional
knowledge on medicinal plants.
1593

REFERENCES
Arunachalam G, Karunanithi M, Subramanian N,
Ravichandran V and Selvamuthukumar S. 2009.
Ethnomedicines of Kolli hills at Namakkal district in
Tamilnadu and its significance in Indian systems of
medicine. International Journal of Pharmaceutical
Sciences Review and Research. 1(1): 1 15.
Ayyanar M, Sankarasivaraman K, Ignacimuthu S
and Sekar T. 2010. Plant species with ethnobotanical
importance other than medicinal in Theni district of
Tamilnadu, Southern India. Asian Journal
Experimental Biological Sciences. 1(4): 765 771.
Balakrishnan V, Prema P, Ravindran KC and Philip
Robinson J. 2009. Ethnobotanical studies among
villagers from Dharapuram taluk, Tamilnadu, India.
Global Journal of Pharmacology. 3(1): 8 14.
Farombi EO. 2003. African indigenous plants with
chemotherapeutic potentials and biotechnological
approach to the production of bioactive prophylactic
agents. African Journal of Biotechnology. 2(12):
662 671.
Ignacimuthu S, Ayyanar M and Sankarasivaraman
K. 2008. Ethnobotanical study of medicinal plants used
by Paliyar tribals in Theni district of Tamilnadu, India.
Fitoterapia. 79(7-8): 562 568.
Kirtikar KR and Basu BD. 2012. Indian Medicinal
plants. Second Edition in Eleven Volumes. Oriental
Enterprises, Dehradun, Uttaranchal, India. 3846p.
Laila banu NR, Sreeja S, Pinky VR, Prakash JW and
Jeenath Jasmine A. 2007. Medicinal plants used by the
rural people of Kattathurai, Kanyakumari District,
Tamilnadu. Journal of Basic and Applied Biology.
1: 18 22.
Matthew KM. The Flora of Tamilnadu carnatic. Vols. 1-
3. The Rapinat Herbarium, St. Josephs College,
1594
Thirumaran et al., 2014
Tiruchirapalli, India.
Maruthapandian A and Mohan VR. 2010.
Observation of ethnomedicinal plants from Sirumalai
hills in Western Ghats of Tamilnadu, India. Journal of
Herbal Medicine and Toxicology. 4: 89 92.
Murugesan M and Balasubramanian V. 2007. Ethno-
medico-botanical diversity of Irulas in Velliangiri Hills,
Coimbatore district, Tamilnadu, India. Journal of
Non-Timber Forest Products. 14: 105 110.
of Shanley P and Luz P. 2003. The impacts of forest
degradation on medicinal plant use and implication for
health care in Eastern Amazonia. Bio Science. 53:
573 584.
Rai LK, Pankaj Prasad and Sharma E. 2000.
Conservation threat to some important medicinal plants
of the Sikkim Himalaya. Biological Conservation. 93:
27-33.
Shanmugam S, Kalaiselvan M, Selvalumar P, Kuru
Suresh and Rajendran K. 2011. Ethnomedicinal plants
used to cure diarrhea and dysentery in Sivagangai district
of Tamil Nadu, India. International Journal of Research
in Ayurveda and Pharmacy. 2: 991 994.
Pullaiah T. 2002. Medicinal plants in India. Regency
Publications, New Delhi. Vol. 1-2
The Wealth of India 2006. Council for Scientific and
industrial Research, New Delhi. Vols.1-5
Submit your articles online at www.jresearchbiology.com
Advantages
Easy online submission
Complete Peer review
Affordable Charges
Quick processing
Extensive indexing
You retain your copyright
submit@jresearchbiology.com
www.jresearchbiology.com/Submit.php
Journal of Research in Biology (2014) 4(8): 1589-1594
 Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

ORIGINAL RESEARCH

Anesthetic efficacy of clove oil and its impact on hematological and

biochemical changes in Channa striatus (Bloch, 1793)
Authors: ABSTRACT:
Journal of Research in Biology

Jeyasheela P, Haniffa MA Channa striatus is one among the dominant group of air breathing
and Kavitha K freshwater fishes in Southeast Asian countries. In the present study, fish anesthetic
clove oil was used to study the hematological and biochemical changes at different
concentration (400 ppm, 450 ppm and 500 ppm) and time interval (0 h, 1h and 24 h)
in C. striatus. The induction and recovery time was noted for each treatment groups.
Institution: Erythrocyte count (T/L), Hemoglobin and Hematocrit values showed elevated levels
Centre for Aquaculture when compared to control. RBC, Hb and Ht values significantly increased 1 h after
Research and Extension anesthesia and returned to normal after 24 h. Anesthetic treated fishes exhibited
(CARE), St.Xaviers College marked decrease in WBCs when compared to control group. The rest of the indices
(Autonomous), (MCV, MCH, MCHC, Lymphocytes, Monocytes, Neutrophils) were at comparable levels
Palayamkottai, in all groups. The anesthetic treated fishes were found to show a significant increase
Tamil Nadu-627005, India in the concentration of glucose. The rest of the indices (TP, ALB, GLOB, ALT, AST) were
at comparable levels in all groups. Results of the study suggested that the use of clove
oil at the concentrations of 400, 450 and 500 ppm does not cause irreversible damage
on the blood parameters as well as biochemical profile in C. striatus.
Corresponding author:
Haniffa MA
Keywords:
C. striatus, anesthesia, induction and recovery time

Email Id:
Article Citation:
Jeyasheela P, Haniffa MA and Kavitha K
Anesthetic efficacy of clove oil and its impact on hematological and biochemical
changes in Channa striatus (Bloch, 1793)
Web Address: Journal of Research in Biology (2014) 4(8): 1595-1603
http://jresearchbiology.com/
documents/RA0497.pdf Dates:
Received: 09 Oct 2014 Accepted: 11 Nov 2014 Published: 31 Dec 2014

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1595-1603 | JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION:
Murrels, commonly called snakeheads are
important air breathing freshwater fishes and are highly
regarded as food fish in the South and Southeast Asian
countries. They belong to the family Channidae
(Ophiocephalidae) (Wee et al., 1982). Murrels can be
kept alive for several hours outside water under slight
moist condition which facilitates transportation to distant
markets in good condition. Even then often fishermen
meet heavy loss due to death of murrels during transport.
Hence, sedation or use of anesthetics can be beneficial
to calm excitable fish during bulk transportation of fish
stocks, especially over long distances and high density.
In aquaculture practices and research activities,
fish handling are a common source of stress. Hence, a
variety of anesthetics are used mainly in order to reduce
stress level and to prevent fish injury/ death during their
handling. The most commonly used fish anesthetics are
tricaine methanesulfonate (MS-222), benzocaine (ethyl
paraaminobenzoate) (Kiessling et al., 2009),
2-phenoxyethanol, metomidate (Weber, 2009), and
carbon dioxide (Pirhonen and Schreck, 2003).
Clove oil is a dark - brown liquid, a distillate of
flowers, stalks and leaves of the clove tree Eugenia
aromatica (Soto and Burhanuddin, 1995) having a
mild anesthetic effect on human (Nagababu and
Lakshmaiah, 1992; Taylor and Roberts, 1999) and
fish (Ross and Ross, 2008). Keene et al. (1998)
showed that the clove oil i s much less e x p e n s i v e
than other chemicals including MS222 and
recommended the same for fish transport.
Anesthesia may affect blood parameters and
hemolyse tissues (McKnight, 1966). Since,
hemodynamic is closely related to response of animal to
external environment (Fernandes and Mason, 2003). The
goal of this study was to assess efficacy of clove oil as an
anesthetic through the measurement of multiple blood
parameters of striped murrel Channa striatus. The
effect of different concentrations of clove oil on
1596
Jeyasheela, 2014
induction and recovery periods and mortality are also
studied.
MATERIALS AND METHODS:
The striped murrel, C. striatus acclimatized in
cement tanks (15mX3mX2m) for a period of two weeks
at CARE Aquafarm were used for this study. During this
period, the fishes were fed twice a day with chicken
intestine. Forty fishes (29.7 1.69 cm and 232 14 g)
were selected and were divided into four groups each
with 10 fishes based on the concentration of clove oil
(group I: control [0ppm], group II: 400ppm, group III:
450 ppm and group IV: 500 ppm). Test fishes were
starved for 24 hours prior to the experiment and
mortality rate (if any) was recorded regularly throughout
the course of the study. Stock solution of clove oil was
prepared by dissolving 1 ml of clove oil in 9 ml of tap
water. The test fishes were exposed to the respective
concentrations and they were anesthetized.
The induction and recovery period of fishes as a
function of concentration were recorded. Blood was
sampled from caudal vein of the anesthetized C. striatus
with heparin coated syringe. To stabilize the blood
samples, aqueous solution of heparin sodium salt (5000
U/ml) was added to blood (Svobodova et al., 1991). The
blood sample was divided into two aliquots; one part was
transferred to a 2 ml heparinized tube and stored in
refrigerator prior to hematological analyses. The other
part of aliquots was transferred to 1.5 ml microcentrifuge
tubes and centrifuged for 15 min. at 4C. The plasma
was removed and transferred to another 1.5 ml microtube
and stored frozen at 70C for biochemical analyses
(Fast et al., 2008).
Hemogram was established by estimation of total
erythrocyte count (RBC), total white blood cell count
(WBC), hematocrit (Ht), hemoglobin concentration (Hb),
erythrocyte indices (MCV, MCH, MCHC) and white
blood cell differential count (Campbell, 2004).
Biochemical indices of blood plasma included glucose
Journal of Research in Biology (2014) 4(8): 1595-1603
Jeyasheela, 2014
Time (min)
Concentration (ppm)
Figure 1: Effect of Clove oil on Induction and Recovery time of C. striatus
(GLU), Total Protein (TP), Albumin (ALB), Globulin
(GLOB), Aspartate amino Transferase (AST), and
Alanine amino Transferase (ALT). Statistical differences
between groups if any at each time point (0 h, 1 h
and 24 h) were tested using SPSS software and data
were presented a s m e a n SD.
RESULT AND DISCUSSION:
Water quality parameters were measured and
recorded as pH 7.0, chloride 200 ppm, total hardness
525 ppm, fluoride 0.5 mg/l, iron 0.5 mg/l, residual
chlorine 0 mg/l and nitrate 0.45 mg/l.
All the fish exposed to different concentrations
of clove oil recovered well and returned to normal
behaviour with respect to feeding, surfacing activity,
swimming and respond to external stimuli after the
anesthetic treatment. Furthermore, no mortality was
noticed within 48 h following recovery from anesthesia.
The technique anesthesia by immersion wa s
ap p li ed to provide the active ingredient into the fish
gills through water flow to travel through bloodstream
to the central nervous system. Thus, the fish goes
through several stages of anesthesia viz ; light sedation,
deep sedation, partial loss of equilibrium, total loss of
equilibrium, loss of reflex reactivity and ultimately
medullary collapse as described by Bowser (2001).
Induction of anesthesia was assumed to be
Journal of Research in Biology (2014) 4(8): 1595-1603
complete when the fish lost its response to external
stimuli. As per the results obtained, the optimum
concentration to anesthetize C. striatus was found to be
450 ppm. The fishes underwent anesthesia through six
stages as described by Bowser (2001). The first stage of
anesthesia was light sedation which involved: Slight loss
of reactivity to external stimuli; opercular rate slightly
decreased with normal equilibrium. This was followed
by the next stage i.e., deep sedation involving total loss
of reactivity to all but strong external stimuli; slight
decrease in opercular rate with normal equilibrium. The
signs of next stage included: Partial loss of muscle tone
and swimming was erratic; increased opercular rate;
reactivity only to strong tactile and vibration stimuli.
Total loss of muscle tone and equilibrium; slow but
regular opercular rate; loss of spinal reflexes especially
loss of equilibrium observed in the fourth stage. The
changes that were noticed in the fifth stage were total
loss of reactivity; opercular movements were slow and
irregular, heart rate was very slow and loss of all reflexes
was noticed. The final stage during anesthesia was
medullary collapse (stage of asphyxia) and the
anesthetized fishes showed opercular movements to
cease; cardiac arrest followed quickly. The time duration
for each stage was shown in the figure 1.
Increase in clove oil concentration resulted in
decrease in induction time whereas the recovery time
1597

increased significantly. However, the recovery and
induction time was also concentration dependent. The
mean induction time for C. striatus 400, 450 and 500
ppm clove oil was found to be 7.10 1.10 min., 2.36
0.42 min. and 2.020.52 min. respectively. The mean
recovery time for each concentration was 5.211.1 min.,
3.200.40 min. and 6.381.15 min. respectively. The
induction time was longer at the lower dose whereas it is
quick and short at higher dose. In the same manner, the
recovery time was quick in lower dose whereas it elapsed
for a longer duration at higher dose. For morphological
evaluations, biopsy and stripping, long handling periods
are required and hence, anesthetizing with clove oil
would be an added advantage which produces longer
recovery time (Seol et al., 2007; Park et al., 2009).
It was observed that if the exposure was
prolonged until the fish become anesthetized; the
recovery was concentration-independent and lasted for
about four minutes. In the present study, the tested
concentrations met the efficacy criteria specified by
Marking and Meyer (1985) and hence, dose of 450 ppm
of clove oil can be suggested for transport or grading of
C. striatus. This finding is also in accordance with the
results reported by Woody et al. (2002) where higher
dose produced a rapid and uniform response in all size
classes, suggesting that a dose of 400 ppm might be well
over the effective concentration for sockeye salmon
(Oncorhynchus nerka).
Similar observations were made by Inoue et al.
(2003) on juveniles of matrinxa, Brycon cephalus,
where, the recovery in the experiment with a prolonged
exposure was longer at all concentrations, but with 30
and 40 mg /L, it was still below 10 min. Our results
showed that increasing the anesthetic dose significantly
decreased induction time but prolonged recovery time.
This was in agreement with reports of Salmo salar smolts
(Iversen et al., 2003), Cyprinus carpio (Velisek et al.,
2005) and Silurus glanis (Velisek et al., 2006).
As reported by Matin et al. (2009), when the fish
1598
Jeyasheela, 2014
were put into induction tray containing clove oil, they
became excited and hypermotile followed by bubbling;
the gill and fin movements progressively decreased, the
fish lost equilibrium and started swimming laterally.
Finally, the fish became immobile with full loss of
equilibrium and consciousness. After transfer to recovery
tray, reappearance of gill movement was noticed first.
This was followed by fin and then tail movement. The
fish started moving laterally. Gradually full equilibrium
was regained and normal behaviour was restored at
5.211.1 min., 3.200.40 min. and 6.381.15 min. as a
function of 400 ppm, 450 ppm and 500 ppm clove oil
respectively. Similar, behavioural changes during
induction and recovery as a function of anesthesia have
been reported by McFarland (1960).
Hematological and biochemical profiles of blood
are necessary to provide vital information about internal
environment of organism. Erythrocyte count (T/L)
showed increased values in anesthetic treated groups
(1.18-1.21) when compared to control (1.14) (Table 1).
Similarly, Hemoglobin and Hematocrit also showed
elevated levels in anesthetic induced fishes (Hb: 59.93
64.826 g/dl and Ht: 0.179 0.194 l/l) as compared to
control (Hb: 59.58 63.24 g/dl and Ht: 0.178 0.189 l/l
respectively). RBC, Hb and Ht values significantly
increased 1 h after anesthesia and returned to normal 24
h post anesthesia.
Anesthetic treated fishes exhibited marked
decrease in WBCs (0.876 1.109 g/L) when compared
to control group (0.981 1.138 g/L) (Table 1). The rest
of the indices (MCV, MCH, MCHC, Lymphocytes,
Monocytes, Neutrophils) were at comparable levels in all
groups. Results of the study suggested that the use of
clove oil at the concentrations of 400, 450 and 500 ppm
does not cause irreversible damage on the blood
parameters in C. striatus.
Tort et al. (2002) reported clove oil altering
hematocrit concentrations in rainbow trout. Velisek et al.
(2005) observed the same in rainbow trout and carp, both
Journal of Research in Biology (2014) 4(8): 1595-1603
 Table 1: Effect of Clove Oil on Hematological Indices of C. striatus
S. Hematological Control 400 ppm 450 ppm 500 ppm
No. Indices
0h 1h 24 h 0h 1h 24 h 0h 1h 24 h 0h 1h 24 h
1 Erythrocyte 1.180.07 1.140.08 1.140.05 1.180.08 1.200.09 1.180.06 1.190.09 1.200.12 1.180.05 1.190.06 1.210.07 1.180.08
Jeyasheela, 2014

(RBC) (T/L)
2 Hb (g/dl) 59.331.2 59.351.72 59.501.28 59.601.32 63.240.84 60.230.30 59.930.89 64.280.81 58.391.21 60.220.53 64.830.60 60.370.42

3 Ht (l/l) 0.170.003 0.140.005 0.160.004 0.180.004 0.190.003 0.180.30 0.180.003 0.190.002 0.180.004 0.180.002 0.190.002 0.180.001

4 MCV (fl) 151.918.98 156.2012.36 156.538.90 150.5910.23 157.7621.01 152.676.98 150.8017.26 160.4321.57 148.316.37 151.858.68 159.9337.22 153.2612.58

5 MCH (pg) 50.582.98 52.084.09 52.212.99 50.423.47 52.797.08 51.092.34 50.495.72 53.437.20 49.482.10 50.522.92 53.4412.36 51.124.12

6 MCHC (g/L) 332.960.53 333.440.52 333.550.69 334.820.71 334.600.63 334.630.52 334.800.34 333.050.44 333.630.3 332.270.45 334.150.57 333.550.53

7 Leukocytes 1.070.01 1.140.02 0.980.01 0.990.02 0.920.02 0.930.02 0.960.01 0.900.02 1.110.02 0.910.17 0.880.26 0.980.02
(WBC) (g/L)
8 Lymphocytes 83.43.05 83.62.41 84.12.71 81.61.14 81.21.14 83.11.58 821.58 80.41.14 81.80.84 811.58 80.61.14 82.41.14
%
9 Neutrophils 14.41.58 14.02.08 13.51.14 16.60.71 16.01.48 14.91.52 13.41.14 15.60.89 131.58 13.81.30 14.80.84 11.81.10

Journal of Research in Biology (2014) 4(8): 1595-1603

%
10 Monocytes % 2.20.84 2.41.14 2.41.52 1.80.84 2.80.84 2.01.0 1.60.89 3.01.22 2.20.84 1.20.45 2.60.89 2.00.71

Table 2: Effect of Clove Oil on Blood Plasma Indices of C. striatus

S.No Biochemical Control 400 ppm 450 ppm 500 ppm
Indices

0h 1h 24 h 0h 1h 24 h 0h 1h 24 h 0h 1h 24 h

1 Glucose 6.130.22 6.490.18 6.410.14 7.280.10 8.420.21 6.410.07 7.200.11 7.510.25 6.250.18 7.320.10 7.620.07 6.310.21
(mmol/L)

2 Total Protein 69.080.18 68.290.42 69.310.49 70.380.71 71.300.42 69.00.54 70.250.51 71.220.31 69.150.58 69.40.53 70.220.57 69.800.99
(g/L)
3 Albumin 4.090.19 4.510.28 4.240.40 6.260.32 5.320.50 5.110.25 7.960.30 7.080.42 5.380.23 13.110.61 15.370.24 11.330.37
(g/L)

4 Globulin 64.990.27 63.780.49 65.070.86 64.120.72 65.850.36 63.890.35 62.290.36 64.540.93 63.770.67 56.291.09 54.840.72 58.460.87
(g/L)
5 AST (IU/L) 8.330.28 6.60.45 7.340.44 7.600.35 5.040.20 8.310.33 6.270.27 8.180.30 14.140.32 12.330.33 16.260.45 18.180.26

6 ALP (IU/L) 12.440.38 14.520.36 13.420.39 8.590.36 12.630.29 19.850.34 13.790.33 10.490.30 13.390.31 16.520.33 21.540.15 23.070.26

1599

of them also exhibited significantly increased plasma
glucose concentrations after longer exposure periods (10
min.).
The increase in RBC level after anesthetic
treatment showed that fish's body is kept in touch with
insufficient gas when anesthetized. When oxygen
becomes a limiting factor, RBCs increased for carrying
more oxygen to the cells. Hemoglobin (Hb) is an
effective index of taking more oxygen in the blood and
hence Hb increased for obtaining more oxygen; the
average volume value (MCV) with a red blood cell
increases after anesthetization and showed that dissolved
oxygen amount decrease in fish's blood. So MCV is
strengthened by holding more oxygen, and keeping the
basic physiological function to fish's body; the average
concentration of hemoglobin of red blood cells (MCHC)
increase after anesthetization and show that dissolved
oxygen amount is decreasing in fish's blood, so MCHC
increase for combining more oxygen (Wu et al., 2000).
The respiratory actions lower resulting in
reduced O2 for circulation for breathing and survival
creating a hypoxic environment which
physiological changes altering the blood factors like
glucose and Hematocrit (Ht). Hyperglycemia (increased
blood glucose), elevated HB and Ht were similar to other
fishes anesthetized by Pirhonen and Schreck (2003);
Park (2009); Gomes et al. (2001). Reports by Velisek et
al. (2005) on Cyprinus carpio and Oncorhynchus mykiss
suggests that clove oil anesthesia at 30
concentration and 10 min exposure did not produce any
marked changes in the blood parameters after 24 h.
However Sudagar et al. (2009), reported that a 7-min
exposure to clove powder resulted in significant
reversible increase in Ht, Hb and RBC immediately after
anesthesia in Roach Rutilus rutilus.
Increase in the numbers of leukocyte (WBC)
increase after anesthetization showed the unexpected
changes of the living water environments or invasion of
outside material (Wu et al., 2000). The fish immune
1600
Jeyasheela, 2014
system was evaluated by the changes in White Blood
Cell (WBC). It showed a decline trend associated with
arresting in anesthetic. Increase in plasma cortisol
concentration which is a glucocorticoid hormone, is also
acting as an immunosuppressive (Fast et al., 2008), so it
could suppress humoral factors and lead in declining
circulating WBC along with elevating cortisol.
The anesthetic treated fishes were found to show
a significant increase in the concentration of glucose i.e.,
7.196 7.318 mmol/l at 0 h and 7.514 8.422 mmol/l at
1 h following anesthetic treatment. The glucose values of
anesthetic treated fishes returned back to control values
after 24 h post treatment (6.134 6.486 mmol/l). The
rest of the indices (TP, ALB, GLOB, ALT, AST) were at
comparable levels in all groups (Table 2).
These results revealed the significance of
exposure time and dosage on some physiological
indicators of anaesthetized fish. Post-exposure mortality
and lack of biochemical alteration in the present study
could be due to the short induction time after which
blood was collected. These results indicated the need to
results study possible physiological changes occurring in
different fish species exposed to different doses of clove
essence.
Plasma cortisol as well as glucose is a
physiological indicator of stress in fishes and their
interactive effects on metabolism during recovery from
stress have recently become a subject of more intense
mg/l study (Tytler and Hawkins, 1981; Woody, 2002; Weber
et al., 2009). Clove oil was found to block the activity of
cortisol, although not completely, in Brycon cephalus
(Inoue et al., 2005). Although the mechanism is not well
known, Iverson et al. (2003) suggested that it blocks
transmission of impulses to the Hypothalamus-Pituitary
Interregnal axis (HPI).
In our experiments with murrel, an increase in
blood plasma glucose immediately after clove oil
anesthesia was observed. Increased glucose level
returned to normal 24 h after anesthesia. Increased blood
Journal of Research in Biology (2014) 4(8): 1595-1603
Jeyasheela, 2014

plasma glucose level after anesthesia indicates that the Advances in Veterinary Anesthesia and Analgesia:
procedure caused some stress in the experimental fish. Companion Animals, International Veterinary
These findings are in accordance with results of Information Service, Ithaca, NY.
Holloway et al. (2004) and Velisek et al. (2005) who
Campbell TW. 2004. Hematology of lower vertebrates
also detected increase of glucose concentration in
In: Proceedings of the 55th Annual meeting of the
rainbow trout (Oncorhynchus mykiss) following clove oil
American College of Veterinary Pathologists (ACUPC).
anesthesia. On the other hand, Iverzen et al. (2003)
www.ivis.org/proceedings/AVCP/2004/Campbell/
found no change in the concentration of glucose in
ivis.pdf
Atlantic salmon (Salmo salar) following clove oil
anesthesia.
Fast MD, Hosoya S, Johnson SC and Afonso LOB.
2008. Cortisol response and immune- related effects of
CONCLUSION
Atlantic salmon (Salmo salar Linnaeus) subjected to
According to these results, clove oil at a
short- and long-term stress. Fish and Shellfish
concentration of 450 ppm could be an efficient and
Immunology 24(2):194-204.
relatively safe anesthetic agent, but further studies are
d o i : 10.1016/j.fsi.2007.10.009.
required to detect any possible toxicity effect on fish. In
conclusion, clove essence was found to be safe and can Fernandes MN and Mason AF. 2003. Environmental
be effectively and easily applied in used dosages to pollution and fish gill morphology. In: VAL, AL. and
anaesthetize various size groups of murrels with minimal KAPOOR, BG., Ed. Fish adaptations. Enfield: Science
disruption in the physiological indicators studied and Publishers. 203-231.
with zero mortality. Hence, considerations should be
Gomes LC, Chippari-Gomes AR, Lopes NP, Roubach
given to the use of clove essence as a replacement for
R and Araujo-Lima CARM. 2001. Efficacy of
synthetic forms of anesthetics.
Benzocaine as an Anesthetic in Juvenile Tambaqui
Colossoma macropomum. Journal of World Aquaculture
ACKNOWLEDGEMENT
Society 32(4):426-431.
We acknowledge the financial assistance
doi: 10.1111/j.1749-7345.2001.tb00470.x
received from ICAR-NAIP (F.No. 1(5)/2007-NAIP dt.
22 August 2008) to carry out this study. We are grateful Holloway AC, Keene JL, Noakes DG and Moccia
to Dr.T.J.Pandian, Visiting Professor, CAS Marine RD. 2004. Effects of clove oil and MS-222 on blood hor-
Biology, Annamalai University. mone profiles in rainbow trout Oncorhynchus mykiss,
Walbaum. Aquaculture Research 35(11):10251030.
REFERENCES doi: 10.1111/j.1365-2109.2004.01108.x
Anderson WG, McKinley RS and Colavecchia M.
Iversen M, Finstad B, MacKinley RS and Eliassen
1997. The use of clove oil as an anesthetic for rainbow
RA. 2003. The efficacy of metomidate, clove oil, Aqui-
trout and its effects on swimming performance. North
S and Benzoak as anaesthetics in Atlantic salmon
American Journal of Fisheries Management 17(2): 301-
(Salmo salar L.) smolts, and their potential stress-
307.
reducing capacity. Aquaculture 221: 549566.
Bowser PR. 2001. Anesthetic options for fish. In Recent
Inoue LAKA, Santos-Neto NC and Moraes G. 2003.

Journal of Research in Biology (2014) 4(8): 1595-1603 1601


Clove oil as anaesthetic for juveniles of matrinxa Brycon
cephalus (Gnther, 1869). Ciencia Rural. 33(5):943-947.
Inoue L, Afonso L, Iwama GK and Moares G. 2005.
Effects of clove oil on the stress response of matrinxa
(Brycon cephalus) subjected to transport. Acta
Amazonica 35(2):289-295. doi: http://
dx.doi.org/10.1590/S0044-59672005000200018.
Keene JL, Noakes DLG, Moccia RD and Soto GG.
1998. The efficacy of clove oil as an anesthetic for
rainbow trout, Oncorhynchus mykiss (Walbaum),
Aquaculture Research 29(2): 89-101.
doi: 10.1046/j.1365-2109.1998.00927.x
Kiessling A, Johansson D, Zahl IH and Samuelsen
OB. 2009. Pharmacokinetics, plasma cortisol and
effectiveness of benzocaine, MS-222 and isoeugenol
measured in individual dorsal aorta-cannulated Atlantic
salmon (Salmo salar) following bath administration
Aquaculture 286(3-4): 301308.
doi: 10.1016/j.aquaculture.2008.09.037
Matin SMA, Hossain MA and Hashim MA. 2009.
Clove oil anaesthesia in singhi (Heteropneustes fossilis)
and lata (Channa punctatus) fish. The Bangladesh
Veterinarian 26(2): 68-73
McFarland WN. 1960. The use of anaesthetics for the
handling and the transport of fishes. California Fish and
Game 46:407431.
Mcknight IMA. 1966. Hematological study on the
mountain white fish. Journal of the Fisheries Research
Board of Canada 23(1):45-64.
Munday PL and Wilson SK. 1997. Comparative
efficacy of clove oil and other chemicals
anaesthetization of Pomacentrus amboinensis, a coral
reef fish. Journal of Fish Biology 51(5): 931-938.
doi: 10.1111/j.1095-8649.1997.tb01532.x
Nagababu E and Lakshmaiah N. 1992. Inhibitory
1602
Jeyasheela, 2014
effects of eugenol on non-enzymatic lipid peroxidation in
rat liver mitochondria. Journal of Biochemistry
Pharmacology 43(11):23932400.
doi: 10.1016/0006-2952(92)90318-D
Park MO, Im SY, Seol DW and Park IS. 2009.
Efficacy and physiological responses of rock bream,
(Oplegnathus fasciatus) to anesthetization with clove oil.
Aquaculture 287(3-4): 427-430.
doi: 10.1016/j.aquaculture.2008.10.049
Pirhonen J and Schreck CB. 2003. Effects of
anesthesia with MS-222, clove oil and CO2 on feed
intake and plasma cortisol in steelhead trout
(Oncorhynchus mykiss). Aquaculture 220(1-4):507514.
doi: http://dx.doi.org/10.1016/S0044-8486(02)00624-5
Ross LG and Ross B. 2008. Anaesthetic and Sedative
Techniques for Aquatic Animals. 3rd Edn., Blackwell,
Oxford. ISBN:978-1-4051-4938-9
Seol DW, Lee J, Im SY and Park IS. 2007. Clove oil as
an anaesthetic for common octopus (Octopus minor,
Sasaki). Aquaculture Research 38(1):45 -49.
doi: 10.1111/j.1365-2109.2006.01622.x
Soto CG and Burhanuddin S. 1995. Clove oil as a fish
anaesthetic for measuring length and weight of rabbit
fish (Siganus lineatus). Aquaculture 136(1-2): 149152.
Sudagara M, Mohammadizarejabad A, Mazandarani
R and Pooralimotlagha S. 2009. The efficacy of clove
powder as an anesthetic and its effects on hematological
parameters on Roach (Rutilus rutilus). Journal of
Aquaculture feed science and nutrition 1(1): 1-5.
Svobodova Z and Vykusova B. 1991. Diagnostics,
in prevention and therapy of fish diseases and intoxications.
Manual for International Training Course on Freshwater
Fish Diseases and Intoxications, Research Institute of
Fish Culture and Hydrobiology. 270.
Taylor PW and Roberts S. D. 1999. Clove oil: An
Journal of Research in Biology (2014) 4(8): 1595-1603
Jeyasheela, 2014

alternative anaesthetic for aquaculture. North American

Journal of Aquaculture 61(2):150155.

Tort L, Puigcerver M, Crespo S and Padros F. 2002.

Cortisol and haematological response in sea bream and
trout subjected to the anaesthetics clove oil and 2-
phenoxyethanol. Aquaculture Research 33(11):907-910.

Tytler R and Hawkins AD. 1981. Vivisection,

anaesthetics and minor surgery. In Aquarium Systems
(A.D. Hawkins, eds.), Academic Press London, 247-278.

Veliesk J, Svobodova Z and Piackova V. 2005a.

Effects of clove oil anaesthesia on Rainbow Trout
(Oncorhynchus mykiss). Acta Veterinaria Brno 74:139-
146.

Velisek J, Svobodova Z, Piackova V, Groch L and

Nepejchalova L. 2005b. Effects of clove oil anaesthesia
on common carp (Cyprinus carpio L.). Veterinary
Medicine Czech 50(6):269275.

Velisek J, Wlasow T, Gomulka P, Svobodova Z and

Novotny L. 2006. Effects of Clove Oil anaesthesia on
european catfish (Silurus glanis L.). Acta Veterinaria
Brno 75(1): 99106.

Weber RA, Peleteiro JB, Martn LOG and Aldegunde

M. 2009. The efficacy of 2 phenoxyethanol, metomidate,
clove oil and MS-222 as anaesthetic agents in the
Senegalese sole (Solea senegalensis Kaup 1858).
Aquaculture 288(1-2):147-150.

Wee KL and Tacon AGJ. 1982. A preliminary study Submit your articles online at www.jresearchbiology.com
on the dietary protein requirement of juvenile Advantages
snakehead. Bulletin of the Japanese Society of Scientific Easy online submission
Complete Peer review
Fisheries 48(10):1463-1468. doi: 10.2331/suisan.48.1463
Affordable Charges
Quick processing
Woody CA, Nelson J and Ramstad K. 2002. Clove oil Extensive indexing
as an anaesthetic for adult sockeye salmon: field trials. You retain your copyright
Journal of Fish Biology 60(2):340-347. doi: 10.1111/ submit@jresearchbiology.com
j.1095-8649.2002.tb00284.x www.jresearchbiology.com/Submit.php

Journal of Research in Biology (2014) 4(8): 1595-1603 1603

 Journal of Research in Biology
An International Scientific Research Journal
Genetic variations of Adiantum incisum Forssk. revealed by ISSR markers
in the Western Ghats of Tamil Nadu, India
Authors:
Journal of Research in Biology
Abiya Chelliah D1,
John De Britto A2 and
Selvin Samuel A1
Institution:
1. Department of Botany,
St. Johns College,
Palayamkottai - 627 002,
Tamil Nadu.
2. Department of Botany,
St. Xaviers College,
Palayamkottai - 627 002,
Tamil Nadu.
Corresponding author:
Abiya Chelliah D
Email Id:
abiyachelliah@gmail.com
Web Address:
http://jresearchbiology.com/
documents/RA500.pdf
Journal of Research in Biology
An International
Scientific Research Journal
ISSN No: Print: 2231 6280; Online: 2231- 6299
Original Research
ABSTRACT:
Inter Simple Sequence Repeats (ISSR) markers were used to measure the
levels of genetic variation and patterns of the population structure within and among
the five populations of Adiantum incisum, a terrestrial fern in India. For this purpose, a
detailed study was conducted in three replicates at 2011-14 season in the collection
points of Western Ghats, South India. Five wild A. incisum accessions (maiden hair)
were evaluated for genotyping experiment. Results showed a significant variation
among genotypes and were classified based on this variation in four groups by genetic
cluster analysis. In the experiment, five ISSR primers amplified 63 polymorphic bands.
The genetic identity data among genotypes were calculated and varied from 0.4603 to
0.7460. The percentage of polymorphism showed superior genotype that could be
used for the conservation of species. ISSR proved to be a helpful marker for genotype
identification prediction within a closed group of inter specific population in the study
area.
Keywords:
ISSR analysis, Adiantum incisum Forssk, genetic variation, southern Western Ghats
Article Citation:
Abiya Chelliah D, John De Britto A and Selvin Samuel A.
Genetic variations of Adiantum incisum Forssk. revealed by ISSR markers in the Western
Ghats of Tamil Nadu, India
Journal of Research in Biology (2014) 4(8): 1604 1610
Dates:
Received: 10 Oct 2014 Accepted: 15 Nov 2014 Published: 31 Dec 2014
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
1604 1610 | JRB | 2014 | Vol 4 | No 8
www.jresearchbiology.com
 Abiya et al., 2014

INTRODUCTION possessing these capabilities will have a selective

The Indian subcontinent is endowed with an
advantage over other genotypes when colonizing new
amazing array of herbal plants which constitute the main
and distant habitats. This advantage is becoming more
resource base of the health care system in the country.
important in a world increasingly under the pressure of
Its rich vegetation wealth and diversity is undoubtedly
climate change and fragmentation of natural habitats.
due to the immense variety of the climate and altitudinal
Various studies on plants and animals have
variations coupled with the various ecological habitats.
shown that individuals with higher dispersal capacities
The Western Ghats of peninsular India is of great
tend to be found with greater frequency towards species
phyto-geographical importance which constitutes one of
range limits and that these enhanced capacities tend to
the 34 global biodiversity hotspots along with Sri Lanka,
have a genetic basis. Likewise, inbreeding rates often
on account of exceptional levels of plant endemism and
increase towards range margins.
higher levels of habitat loss.
Genetic diversity measurements are important for
The flora of Western Ghats comprises about
considering conservation of particular species. A decline
12,000 species ranging from unicellular cyanobacteria to
in genetic variation can undermine the ability of an
angiosperms. In this spectrum, the flowering plants
organism to respond to natural selection and
constitute about 27% of Indian flora with 4000 species of
consequently limits its evolutionary potential. Small
which, about 1500 species are endemic. Apart from
populations are often subjected to the loss of alleles
harbouring a rich diversity of the angiosperm flora, the
through genetic drift, or random fluctuations in allele
Western Ghats are also a rich repository of cryptogams
frequency. Thus, any study in genetic diversity of
such as pteridophytes, bryophytes, lichens, fungi and
Adiantum incisum has to address the above issues.
algae. The Western Ghats along with the Himalayas,
DNA markers have proved valuable in crop
Eastern Ghats and parts of Central India forms a major
breeding, especially in studies on genetic diversity and
centre for the distribution of the ferns and fern-allies
gene mapping. The commonly used Polymerase Chain
(MoEF, 2014).
Reaction (PCR) based DNA marker system are Random
Ferns in Western Ghats of South India, south of
Amplified Polymorphic DNA (RAPD), Amplified
Palghat gap constitute about one third of the fern flora of
Fragment Length Polymorphic (AFLP) and more
India. Most of them occur on streams and stream banks
recently Simple Sequences Repeats (SSRs) or
in evergreen forests and shoals above 800m while some
microsatellite (Gupta and Varshney, 2000). The major
occur on exposed roadsides and clearings. (Manickam,
limitations of these methods are low reproducibility of
1995).
RAPD, high cost of AFLP and the need to know the
In spite of its relatively infrequent occurrence,
flanking sequences to develop species specific primers
long distance colonization is of disproportionate
for SSR polymorphism.
importance to species range expansions. Long-distance
ISSR is such a DNA based marker system which
colonization requires plant species to possess a distinct
could be used for screening genetic variability. Changes
set of capabilities, not only related to the dispersal of
in DNA sequences and single base substitutions
propagules, but also to plant and population
including DNA conformation changes can be detected as
establishment upon arrival. This involves di-spore
shifts in electrophoretic mobility using these techniques.
characteristics, plant ontogenetic and morphological
In this present study, A. incisum Forssk, were
traits, as well as reproductive strategies. Genotypes
collected from Western Ghats of Tamil Nadu. A. incisum
1605 Journal of Research in Biology (2014) 4(8): 1604 1610
Abiya et al., 2014

Forssk, is found in the plains and lower slopes of the Table 1. Place of collection of the plants and their accession ID
hills of Punjab, Rajasthan, West Bengal, Tamil Nadu and S.No Species Accession ID Location

Maharashtra. It is used to cure hemicrania, cough and 1. POP 1 Kothayar

fever; it is applied externally for skin diseases and used 2. POP 2 Gundar
Adiantum inci-
as a substitute for A. capillus-veneris. This fern yields 3. sum POP 3 Thirugurankudi
4. POP 4 Kodaikanal
adiantone, isoadiantone, fernene, hentriacontane,
5. POP 5 Kadana dam
hentriacontanone-16 and beta-sitosterol on extraction
using different solvents. The plant extract of Adiantum ISSR amplification
incisum Forssk. (Adiantaceae) is also used in the ISSR amplification reactions were carried out in
treatment of cough, diabetes, and skin diseases 25-l volume containing 50 ng template DNA, 0.5 U Taq
(Manickam, 1995). DNA polymerase, 10 mM dNTP, 10 M primer in 1
The confusion prevailing at its species level is a reaction buffer that contained 10 mM Tris-HCl (pH 8.3),
great menace to the Pteridologists who try to identify it 50 mM KCl, 2.5 mM MgCl2, and 0.01% gelatine
for its valuable usages. The morphology remains similar
Table 2. ISSR primers for study species and their sequences
with that of the closely related species except minute
S. No Name of the Primer Sequences
characteristics with its chromosomal nature and primer
chemistry stands unique. Thus, designing a specific 1 B07 (CG)5AG
strategy for its species identification in spite its variation 2 B09 (ATG)3CA
3 G06 (GC)5CA
stands as a credential task irrefutably. Also, the superior
4 G04 (AT)6GC
genotype of the species was identified so that the 5 L03 (GC)4AT
conservation of the species made easy with special
initiative. (Williams et al., 1990). Amplifications were performed
in an Eppendorf Master Cycler gradient. Amplification
MATERIALS AND METHODS
conditions were one cycle at 94C for 4 min, and 94C
Study area
for 30 s, 55C for 45 s, followed by stepwise reduction of
Five Western Ghats regions viz., Kothayar,
1C for the first five cycles, and 72C for 2 min. In
Gundar, Thirugarankudi, Kodaikanal and Kadana Dam
subsequent 35 cycles, annealing temperature was
are selected for the study due to the availability of these
maintained at 50C, followed by one cycle of 7 min at
three species uniformly with no particular order.
72C. Amplified products were loaded on 2% agarose
DNA isolation
Table 3. Overall genetic variation statistics for all loci in
A. incisum samples were collected from the
Adiantum incisum
Western Ghats of Tamil Nadu, India. DNA was extracted
S. No. Parameters Values
from young leaves using the method described by
Dellaporta et al. (1983). The DNA isolated was purified 1. Observed numbers of 1.7778
alleles
using Phenol- Chloroform method and the concentration
2. Effective numbers of alleles 1.5887
of the DNA samples were determined using UV-
3. Neis (1973) gene diversity 0.3276
Spectrophotometer at the optical density of 260 nm and
4. Shannons Information Index 0.4741
280 nm; the DNA samples were diluted to 25 ng l1 for (Lewontin, 1972)
PCR amplification. 5. Overall percentage of polymorphism 77.78

Journal of Research in Biology (2014) 4(8): 1604 1610 1606

 Abiya et al,, 2014

gel and separated in 1 TBE buffer at 75 V. The gels reproducible, informative and easily scorable ISSR
were visualized under UV after staining with ethidium profiles. A total of 315 bands were scored, out of which
bromide and documented using a gel documentation and 180 were polymorphic bands and the number of bands
Table 4. ISSR Profile of A. incisum using selected primers ranged from 09 to 20 per primer (Table 4). The genetic
distance between the population ranged from 0.2930 to
S. ISSR Total Total Percentage
No. Primers Number Number of of 0.7758 and the genetic identity ranged from 0.4603 to
of Bands Polymorphic Polymor-
Bands phism (%) Table 6. Nei's original measures of genetic identity and
1 B07 63 20 31.74 genetic distance in Adiantum incisum
2 B09 63 09 14.28 POP 1 2 3 4 5
3 G04 63 12 19.04 ID
4 G06 63 10 15.87 1 ***** 0.5873 0.6190 0.4921 0.6190
5 L03 63 12 19.04
2 0.5322 ***** 0.4603 0.6190 0.4921
Total 99.97
3 0.4796 0.7758 ***** 0.6190 0.7460
4 0.7091 0.4796 0.4796 ***** 0.6508
image analysis system. The primers used for the ISSR
5 0.4796 0.7091 0.2930 0.4296 *****
analysis are listed out in Table 2.
Data Analysis 0.7460 (Table 06). The overall observed and effective
The gels from ISSR analysis were visualized at
numbers of alleles were 1.7778 and 1.5887 respectively
Table 5. Distance between and population length in and overall genetic diversity was 0.3276 (Table 3; Fig 2).
Adiantum incisum Forssk. The Shannans information index was found to be
Between And Length 0.4741. The overall percentage of polymorphism was
4 3 3.66294 77.78 (Table 3; Table 5).
3 pop1 26.61084 The number of polymorphic loci and percentage
3 pop2 26.61084 of polymorphism was calculated by using the software
4 2 7.54538 POPGENE package version 1.3.2. Among these five

2 1 8.07904 populations, populations 1, 4, and 5 (Kothayar,

Kodaikanal and Kadana Dam) showed high
1 pop3 14.64936
polymorphism. Considering these three populations,
1 pop5 14.64936
population 1 (Kothayar) showed highest polymorphism
2 pop4 22.72839
(Fig 1).
Hence, among the five accessions of A. incisum
gel documentation system (Alpha Imager 1200). Based
in the Kothayar accession (Pop 1) is considered as
on the primary data (presence or absence of bands) and
superior genotype, due to the high percentage of
pair wise genetic distance between samples was
polymorphism in ISSR analysis.
calculated using NTSYS and POPGENE packages Figure 1. UPGMA dendrogram of A. incisum based on Neis
Genetic distance derived using ISSR markers

RESULTS AND DISCUSSION

Analysis of five accessions of A. incisum Forssk.
revealed 63 polymorphic loci. Ten primers were
analyzed of which five primers (Table 2). generated

1607 Journal of Research in Biology (2014) 4(8): 1604 1610

Abiya et al., 2014

Dendrogram was drawn based on Nei's (1972) the predominant source of genetic variation than asexual
with the consideration of genetic distance. The reproduction (Mes, 1998; Van Der Hulst, 2000; Kjolner
et al., 2006). This finding is in good agreement with
Fig 2. Summary of genetic variation statistics for all loci in
Adiantum incisum other previous studies, which have nearly all used
Summary of genic variation statistics for all loci allozyme markers to infer the mating systems operating
B07-1
L03-12
L03-11 B07-2
B07-3
in natural populations of ferns.
L03-10 B07-4
L03-9 B07-5
L03-8 B07-6
L03-7 B07-7
L03-6 B07-8

Allozyme studies showed that populations of

L03-5 B07-9
L03-4 B07-10
L03-3 B07-11
L03-2 B07-12
L03-1
G06-10
B07-13
B07-14
diploid homosporous ferns are usually dominated by
G06-9 B07-15
G06-8 B07-16 Gene Diversity sexual random mating or outcrossing (Soltis and Soltis,
G06-7 B07-17
Mean Natural Selection Coefficient
G06-6
G06-5
B07-18
B07-19
1987; Ranker and Geiger, 2008). Secondly, gene flow
G06-4 B07-20
G06-3
G06-2
B09-1
B09-2
from the neighboring populations would slow the
G06-1 B09-3
G04-12
G04-11
B09-4
B09-5 diversification but only via sexual reproduction. Similar
G04-10 B09-6
G04-9 B09-7

patterns have been identified in a number of other fern

G04-8 B09-8
G04-7 B09-9
G04-6
G04-5 G04-1
G04-2
G04-4 G04-3

methodology followed is UPGMA based on neighbour
joining method of PHYLIP Version 3.5.
The dendrogram of A. incisum Forssk. (Fig 1)
produced 2 clusters. Cluster 2 was larger, containing
population 3, 5 and 4. Here, population 3 and 5 were
closely related together than population 4. In the first
cluster, population 1 and 2 formed a separate clade. It
was understood that there was considerable amount of
genetic variability between the populations 1, 2 and 3, 4,
5 of A. incisum Forssk.
The morphological variations were counter
confirmed by the genetic variations present in the plants
through ISSR markers. Specific primers that mediate
differentiation between the species were identified
successfully. The genetic relationship exemplified by the
molecular markers via DNA fingerprinting shows their
closeness and relativity. Out of ten primers, five revealed
consistent banding pattern and thus revealed variability
within the species.
The identification
relationship thus paves a way for better understanding of
the species in their position.
ISSR markers were used to compare genetic
differentiation within and between the selected species.
Analysis of genetic variability indicated all the five
populations examined, sexual recombination had been
species (Ranker, 1992; Ranker and Geiger, 2008)
Rumsey, 1999; Chen et al., 2010).
The reason for this genetic variation is most
likely arisen from differences in the DNA contents of the
progenitor species. This suggestion is consistent with the
occurrence of diploid apomicts in other ferns, including
taxa of the Adiantum incisum (Pravin, 2005). The clades
are constructed through Popgene 2.1 and related lineages
show strong patterns of reticulate evolution and higher
the genetic diversity of these species, greater is their
viability in the environment.
However, when evolutionary relationships were
considered using Phylogenetically Independent Contrasts
(PICs), no significant correlation was found. The
discrepancy between analyses is interesting, and
although the significance of the raw data should not be
discounted, it does highlight the importance of using
PICs to determine the evolutionary association of
statistically non-independent traits (Garland, 1992).
of a clear systematic Previous authors have pointed out that this
inference should be restricted to very close relatives, and
the distinction between diploids and their autoploid
offspring (Moran, 1982; Barrington, 1986).
Our findings are consistent with suggestions that
the high chromosome numbers and conserved
chromosome sizes reported for many homosporous ferns

Journal of Research in Biology (2014) 4(8): 1604 1610 1608


has contributed to the hypothesis that the evolution of
fern genomes is less dynamic than the evolution of
angiosperm genomes. This has been suggested to be due
to a higher retention rate of chromosomes and the
possible suppression of Transposable Elements (TEs) in
homosporous ferns (Barker, 2011; Bainard et al., 2011).
The inferred constancy of chromosome size is based on
physical measurements (Wagner and Wagner, 1980) and
the reported correlation between chromosome number
and genome size (Bainard et al., 2011).
It also confirms with the hypothesis that genome
size variation in homosporous ferns are driven by poly-
ploidisation. Our study provides evidence that the
genome evolution is occurring in these study plants.
Indeed, given the extent of hybridization and reticulate
evolution reported in homosporous ferns in general, it
seems likely that changes in genome size are probably
more widespread across ferns but may have been largely
overlooked due to the low level of sampling.
CONCLUSION
Genetic diversity between Adiantum incisum
species found on the Western Ghats region is identified.
ISSR markers proved amplification in the selected
species thus validates its genetic variation strategy
REFERENCES
Bainard JD, Henry TA, Bainard LD and Newmaster
SG. 2011. DNA content variation in monilophytes and
lycophytes: large genomes that are not endopolyploidy.
Chromosom Res. 19:763-775.
Barker MS. 2013: Karyotype and genome evolution
in pteridophytes. In Plant genome Divers Vol 2, Phys
Struct Behav Evol plant genomes. Edited by Leitch IJ,
Greilhuber J, Dolezel J, Wendel JF. Wien: Springer: 245-
253.
Barrington DS, Paris CA and Ranker TA. 1986.
Systematic inferences from spore and stomate size in the
ferns. Am Fern J. 76:149-159.
1609
Abiya et al., 2014
Chen YY, Han QX, Cheng Y, Li ZZ and Li W. 2010.
Genetic variation and clonal diversity of the endangered
aquatic fern Ceratopteris pteridoides as revealed by
AFLP analysis. Biochem Syst Ecol. 38:1129-1136.
Dellaporta SL, Wood J and Hicks JB. 1983. A plant
DNA minipreparation version II. Plant Molecular
Biology Reporter. 1: 19-21.
Garland TJ, Harvey PH and Ives AR.
1992. Procedures for the analysis of comparative data
using phylogenetically independent contrasts. Syst
Biol. 41:18-32.
Gupta PK and Varshney RK. 2000. The development
and use of microsatellite markers for genetics and plant
breeding with emphasis on bread wheat. Euphytica.
113:163185.
Kjolner S, Sstad SM and Brochmann C. 2006.
Clonality and recombination in the arctic plant Saxifraga
cernua. Bot. J. Linn. Soc. 152: 209217.
Manickam VS. 1995. Rare and endangered ferns of
Western Ghats of South India. FernGaz. 15(1): 1-10.
Mes THM. 1998 Character compatibility of molecular
markers to distinguish asexual and sexual reproduction.
Mol. Ecol. 7: 17191727.
MoEF. 2014. Indias Fifth National Report to the
Convention on Biological Diversity, Ministry Of
Environment and Forests, Government of India.
Moran RC. 1982. The Asplenium trichomanes complex
in the United States and adjacent Canada. Am. Fern
J. 72:5-11.
Ranker TA and Geiger JMO. 2008. Population
genetics. In: Ranker TA, Haufler CH, editors. pp. 107
133. Cambridge: Cambridge University Press.
Ranker TA. 1992 Genetic diversity, mating systems,
and interpopulation gene flow in neotropical Hemionitis
Journal of Research in Biology (2014) 4(8): 1604 1610
 Abiya et al., 2014

palmata L. (Adiantaceae). Heredity. 69: 175183.

Rumsey FJ, Vogel JC, Russell SJ, Barrett JA and

Gibby M. 1999. Population structure and conservation
biology of the endangered fern Trichomanes speciosum
Willd. (Hymenophyllaceae) at its northern distributional
limit. Biol. J. Linn. Soc. 66: 333344.

Sahaya Pravin, A. 2005. Studies on genetic variability

in some polymorphic species of ferns of Western Ghats,
India through isozymic analysis. Ph.D thesis,
Manonmaniam Sundaranar University, Tirunelveli,
Tamil Nadu, India.

Soltis DE and Soltis PS. 1987. Polyploidy and breeding

systems in homosporous Pteridophyta: a reevaluation.
Amer. Natur. 130: 219232.

Van der Hulst RGM, Mes THM, Den Nijs JCM and
Bachmann K. 2000. Amplified fragment length
polymorphism (AFLP) markers reveal that population
structure of triploid dandelions (Taraxacum officinale)
exhibits both clonality and recombination. Mol. Ecol. 9:
18.

Wagner W and Wagner F. 1980. Polyploidy in

pteridophytes. In Polyploidy, Biol Relev. Volume 13
edition. Edited by Lewis WH. New York: Plenum Press;
199-214.

Williams GK, Anne R Kubelik, Kenneth J Livak,

Antoni Rafaiski J and Scott V Tingey. 1990. DNA
polymorphism amplified by arbitrary primers are useful
genetic markers. Nucleic Acid Res.18: 6531-6535 Submit your articles online at www.jresearchbiology.com

Advantages
Easy online submission
Complete Peer review
Affordable Charges
Quick processing
Extensive indexing
You retain your copyright
submit@jresearchbiology.com
www.jresearchbiology.com/Submit.php.

1610 Journal of Research in Biology (2014) 4(8): 1604 1610