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International Journal
of Recent Scientific
International Journal of Recent Scientific Research Research
Vol. 5, Issue, 1, pp.142-147, January, 2014
ISSN: 0976-3031
RESEARCH ARTICLE
VOLATILE OIL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF ESSENTIAL OIL
OF TWO VARIETIES OF OCIMUM SANCTUM (L.) FROM DHAMETA (KANGRA)-A
NORTH INDIAN REGION
1Vivek Sharma*, 2Raghbir Chand Gupta, 3Bikram Singh, 1Harcharan Singh Dhaliwal
and 4Devendra Kumar Srivastava
1AkalSchool of Biotechnology, Eternal University Baru Sahib-173101 (H.P.) India
2Department of Botany, Punjabi University Patiala-147002 (Punjab) India
3N.P.P. Division, I.H.B.T. (CSIR) Palampur-176061 (H.P.) India
4Department of Biology, D.A.V. College Hoshiarpur-146001 (Punjab) India

ARTICLE INFO ABSTRACT

Article History:
Received 16th, December, 2013
Received in revised form 26th, December, 2013
Accepted 15th, January, 2014
Published online 28th, January, 2014
Key words:
Ocimum sanctum (L.); Essential oil; GC-MS;
Anti-microbial activity; Dhameta (Kangra
District); Himachal Pradesh (North India);
Hydroponic cultivation.
The GC-MS analysis of two varieties of Ocimum sactum (L.) has been done for the first
time from Dhameta (440m) of Kangra District of Himachal Pradesh from Northern India.
The essential oil analysis led to the identification of 26 constituents, representing 97.75% of
the compositions of oil in (OSW) and 19 constituents, representing 96.17% in (OSB).
Eugenol is one of the major constituent with very high percentage reported first time from
North Indian varieties of O. sanctum (L.). Furthermore, anti-microbial activities of oils were
also evaluated against seven bacterial strains. Both varieties showed maximum zone of
inhibition and MIC against Bacillus subtilis (MTCC-2451) and Pseudomonas fluorescence
(MTCC-664). Further our plan is to develop the hydroponic cultivation technology and
technique for different Ocimum species and varieties to increase the essential oil percentage
of this most astonishing herb.
© Copy Right, IJRSR, 2014, Academic Journals. All rights reserved.

INTRODUCTION contain ascorbic acid and carotene as well (Anonymous, 1991).

Ocimum sanctum (L.) belonging to family Lamiaceae is one of
the most astonishing herbs for having enormous medicinal
potentialities. It is widely used in Indian system of medicine and is
commonly known as Tulsi or Holy Basil and is known as "the
queen of herbs". The genus Ocimum comprises between 60 to 150
species of herbs and shrubs which are naturally found wild in the
tropical and subtropical regions of the world (Grayer et al., 2002).
Genus is native to India, Iran and now cultivated in Egypt, France,
Hungary, Italy, Morocco and U.S.A. In India, the plant is grown
throughout the country from Andaman and Nicobar islands to the
Himalayas up to 1800 meters above the sea level (Anonymous,
1991). Ocimum is an erect sweet-scented pubescent herb. Its
leaves are nearly round and margin being entire or toothed. These
are aromatic because of the presence of a kind of scented oil in
them. Its leaves, seeds and whole plant is useful. The plant shows
variation in the color of leaves. The color of leaves vary from
bright green to dark green and some time almost black. Though
the colors in the plants vary, but the reason behind it, especially in
basils are not being studied yet. Two main varieties of Ocimum
sanctum that are commonly known are-“Safed Tulsi” or “Sri
Tulsi” or “Rama Tulsi” bearing green leaves and “Kali Tulsi” or
“Krishna Tulsi” with deep magenta colored leaves. Black variety
has a stronger smell as compared to “Safed Tulsi”. Safed Tulsi is
actually very green while Krishna Tulsi is deep magenta, often
bordering on purple (Maimes, 2004). This aromatic volatile oil
mainly contains phenols, terpenes and aldehydes. Besides oil the
plant also contains alkaloids, saponines and tannins. The leaves
Ayurvedic practice recommends Tulsi in several formulations to
enhance immunity and metabolic functions as well as in the
management of respiratory problems (Gupta et al., 2002).
Recent pharmacological studies have established the anti-diabetic
(Reddy et al., 2008), hepatoprotective (Ubaid et al., 2003), cardio-
protective (Sood et al., 2005), anti-fertility (Kantak and Gogate,
1992), adaptogenic (Ravindran et al., 2005), anti-carcinogenic
(Banerjee et al., 1996), immuno-modulatory (Mukherjee et al.,
2005), anti-inflammatory (Singh et al., 2008), radio-protective
(Subramanian et al., 2005), anti-microbial (anti-bacterial, anti-
fungal and anti-viral) (Prasad et al., 1986; Sinha and Gulati, 1990;
Suppakul et al., 2003b; Bozin et al., 2006; Mondal et al., 2007),
mosquito repellent/larvicidal and insecticidal properties of this
plant (Aslan et al., 2004; Anees, 2008; Trevisan, 2006). The
major chemical constituent Eugenol is known to possess potent
anti-cancer (Yoo et al., 2005) and anti-inflammatory (Sharma et
al., 1994) activity and induces dose-dependent hypotension and
bradycardia (Lahlou et al., 2004). O. sanctum L. is considered as
the most promising essential oil crop and used as a major aromatic
agent with applications in various industries such as the food,
pharmaceutical, cosmetic and aromatherapy industries (Charles
and Simon, 1990). Previous publication suggests that essential oil
may be isolated from O. sanctum but with absolute seasonal
variation (Laskar and Majumdar, 1988). The present day
information about the chemical composition of the plant is based
on the various studies that have been done in different parts of the
world such as: from South India (Kothari et al., 2004; Jirovetz et

* Corresponding author: Vivek Sharma

Akal School of Biotechnology, Eternal University Baru Sahib-173101 (H.P.) India
 International Journal of Recent Scientific Research, Vol. 5, Issue, 1, pp.142-147, January, 2014
al., 2003), Thailand (Lawrence, 1972), Northern Australia
(Brophy et al., 1993), Northeastern Brazil (Machado et al., 1999),
Poland (Kicel et al., 2005), Colombia (Vina and Murillo, 2003),
Mississippi (Zheljazkov et al., 2008), France (Anwar et al., 2005),
Canada (Bowes and Zheljazkov, 2004), Bangladesh (Mondello et
al., 2002), Cuba (Jorge et al., 1998). Beside this, some other
workers have also studied the composition of essential oil of
Ocimum spp. from different geographical regions of the world
(Skaltsa et al., 1987; Norr and Wanger, 1992; Ravid et al., 1997;
Zheljazkov et al., 2008; Vani et al., 2009; Nakatsu et al., 2000;
Laakso et al., 1990; Maheshwari et al., 1987; Lal et al., 2003;
Gangrade et al., 2000). To the best of our knowledge, no
comparative GC-MS analyses based on varieties of Ocimum
sanctum (L.) along with antimicrobial activity from this study area
of Northern India are reported till now.
EXPERIMENTAL
Plant Material
Fresh leaves of Ocimum sanctum (L.) were collected from
Dhameta (440m) of Kangra District of Himachal Pradesh from
Northern India, during the month of August, 2008 (Table 1).
Specimens were authenticated by the Botanical Survey of India
(BSI, Northern Circle), Dehradoon, Department of Biodiversity,
I.H.B.T. (CSIR) Palampur and Department of Botany, Punjabi
University, Patiala (Punjab) India.
Oil Distillation
Two hundred grams fresh sample of leaves from study area were
separated and ground, then immersed in water in a round bottom
flask and hydrodistilled for 4h in a full glass Clevenger-type
apparatus as recommended by British Pharmacopoeia giving
yellowish oils. The essential oil was dried over anhydrous sodium
sulphate (Merck) until the last traces of water were removed and
then stored in a dark glass bottle at 4 ºC prior to GC-MS analysis
(Adams, 1991).
Gas Chromatography-Mass-Spectrometry
GC-MS (70ev) data were measured on GC-MS (QP 2010 series
Shimadzu, Tokyo, Japan) equipped with AOC 20i autosampler
and BP-20 capillary column (SGC International Ringwood,
Australia) of 30m length, 0.25mm i.d. and 0.25μm film thickness.
Temperature was programmed from 70-220ºC at a rate of
4ºC/min, held isothermally at 70ºC and 220ºC for 4 and 5min,
respectively. Mass spectrometer source temperature, 200ºC;
interface temperature, 220ºC; injector temperature, 220ºC. Sample
injection volume 2μL (diluted 5μL oil in 2mL dichloromethane,
HPLC grade); split ratio, 1:50 and mass scan, 50-600 amu.
Helium was used as a carrier gas with 1.1mL/min flow rate.
Identification of Components
The retention index was calculated for all volatile constituents
using a homologous series of n-alkanes. The components of oil
were identified by matching their mass-spectra with those stored
in the computer library such as Wiley (Mc Lafferty, 1989), New
York mass spectral (MS) library (Jennings and Shibamoto, 1980;
Adams, 1989), National Institute of Standards and Technology
(NIST) (Stein, 1990) and their retention indices (RI) either with
authentic compounds or with published data in the literature based
on retention indices of components on same phases of polar
columns such as: BP-20, CW-20M and DB Wax.
143
Microbial Strains for Antimicrobial Activity
The microorganism strains used in the agar disc diffusion method
were supplied by the Institute of Microbial Technology,
Chandigarh, India. Gram-positive bacteria: Bacillus subtilis
(MTCC-2451), Staphylococcus aureus (MTCC-740),
Staphylococcus epidermis (MTCC-435), Gram-negative bacteria:
Escherichia coli (MTCC-443), Salmonella typhimurium (MTCC-
1251), Pseudomonas fluorescence (MTCC-664) and
Acenetobactor calcoaceticus (MTCC-127).
Antimicrobial Screening
In vitro antibacterial activity of the O. sanctum essential oil was
studied against seven bacterial strains by the agar well diffusion
method as described by Perez and coworkers (Perez et al., 1990)
with certain modifications. Nutrient agar (Hi Media, India) was
used as the bacteriological medium. The antibacterial activity of
essential oils was taken at different concentrations (10, 20, 40 and
60μL/well). The nutrient agar was melted and cooled to 48-50oC
and a standardized inoculum of 1 × 106 CFU/mL, (0.5
McFarland) was then added aseptically to the molten agar and
poured into sterile petri dishes to give a solid plate. Wells were
prepared in the seeded agar plates. The test compound was
introduced in the well (8.5 mm). The plates were incubated
overnight at 37oC. The antimicrobial spectrum of the oils was
determined for the bacterial species in terms of zone sizes around
each well. The diameters of zone of inhibition produced by the
agent were compared with those produced by the commercial
control antibiotics, 20μL each of amoxicillin and ciprofloxacin
(5mg/mL of autoclaved distilled water). These are commonly
used antibiotics to treat infections caused by several Gram+ve and
Gram-ve bacteria. For each bacterial strain positive controls were
maintained. The experiment was performed three times to
minimize the error and the mean values were presented.
Minimal Inhibition Concentration
The essential oil that exhibited considerable activity was diluted
with nutrient broth (1:1) in a series of seven sets of three test tubes
for different microorganisms (Aboaba, 2006). An aliquot of 1mL
of the bacterial suspension (1x106) was inoculated into each tube.
The control tubes were inoculated with same quantity of sterile
distilled water and 75% ethanol. All tubes were incubated at 37oC
for 24hrs. The lowest concentration that did not permit any visible
growth when compared with the control was considered as the
minimum inhibitory concentration. The contents of all tubes that
showed no visible growth were cultured on nutrient agar,
incubated at 37oC for 24hrs. The minimum bactericidal
concentration was considered as the lowest concentration that
could not produce a single bacterial colony.
RESULTS AND DISCUSSION
The extraction yield for the essential oil of two varieties of O.
sanctum L. from Dhameta, (440m) of Kangra District of
Himachal Pradesh was 0.8% and 1.3% for white variety (OSW)
and black variety (OSB) respectively. The essential oil analysis
led to the identification of 26 constituents, representing 97.75% of
the composition of oil in (OSW) and 19 constituents, representing
96.17% in (OSB). In the essential oil sample (OSW), some major
constituents reported were as follows: Eugenol (49.95%); β-
Elemene (39.27%); Germacrene-D (2.31%), etc. Whereas, in the
essential oil sample (OSB), the major constituents were: Eugenol
(47.33%); β-Elemene (23.14%); Caryophyllene oxide (20.30%),
etc. (Table 2). Few previous investigations on O. sanctum oil
 International Journal of Recent Scientific Research, Vol. 5, Issue, 1, pp.142-147, January, 2014
composition are not consistent with our results in which methyl Therefore such a high percentage of eugenol in both varieties of
eugenol was found to be the major compound from India (Kothari Ocimum from Indian origin has been first time detected and hence
et al., 2004; Vani et al., 2009). it can be called as new chemotype. Antimicrobial activity showed
that, the inhibition zones were found increased considerably when
Table 1 Collection details and essential oil yield of O.
the concentration rate increased. Therefore it can be said that
sanctum L. varieties from study area of North India
Collection Altitude Month &
quantity of the oil was important for inhibition effect. Among all
Species Place of Oil gram+ve bacterial growths, oil sample of white variety of O.
number of study year
name collection yield
of two area (m) of
(%)
sanctum (OSW) showed the maximum zone of inhibition against
varieties collection Bacillus subtilis (MTCC-2451) i.e. 25.7mm. On the other hand
Dhameta,
(Kangra OSW-
four different gram-ve bacterial strains were tested and among
August, these microorganisms, Pseudomonas fluorescence (MTCC-664)
Ocimum District) 0033 440 0.8
2008
sanctum Himachal showed maximum zone of inhibition i.e. 23.6mm, followed by
L. Pradesh, Acenetobactor calcoaceticus (MTCC-127) i.e. 16.7mm. The
North OSB-0066 August,
India
440
2008
1.3 minimum zone of inhibition was recorded against the Escherichia
coli (MTCC-443) strain i.e. 11.2mm at 60μL/well (Table 3). The
But it is reported that eugenol is the main component of O. minimal inhibition concentration (MIC) of oil sample of white
sanctum grown in Northeastern Brazil (Machado et al., 1999), variety of O. sanctum (OSW) was 2.2μL recorded in gram+ve
Bangladesh (Mondello et al., 2002), Cuba (Jorge et al., 1998), strains Bacillus subtilis (MTCC-2451) followed by a 2.5μL in
Germany (Laakso et al., 1990). In the present study the gram-ve strain Pseudomonas fluorescence (MTCC-664) (Table
percentage of methyl eugenol is found to be very less in (OSW) 4). Whereas, oil sample of black variety of O. sanctum (OSB)
sample i.e. 0.11% and in sample (OSB) it is not at all detected. showed the maximum zone of inhibition against Bacillus subtilis
Whereas, eugenol is the major constituent in both varieties of the (MTCC-2451) i.e. 29.4mm. On the other hand four other gram-ve
Ocimum presently measured from India, which is in corroboration bacterial strains were tested and among these microorganisms,
with the previous results from outside India as mentioned above. Pseudomonas fluorescence (MTCC-664) showed maximum zone
of inhibition i.e. 27.4mm. The minimum zone of inhibition was
Table 2 Comparison of percentage composition of the
recorded against the Escherichia coli (MTCC-443) strain i.e.
essential oils of Ocimum sanctum L.varieties i.e. White
13.2mm at 60μL/well (Table 3). The minimal inhibition
(OSW) and Black (OSB)
S. RI Composition (%)
concentration (MIC) of oil sample of black variety of O. sanctum
Compound (OSB) was 1.5μL in gram-ve strain Pseudomonas fluorescence
No. (BP-20) OSW OSB
1. dl-Limonene 1199 0.07 0.03 (MTCC-664) followed by 2.1μL recorded in gram+ve strains
2. Octanal 1285 0.03 0.04 Bacillus subtilis (MTCC-2451) (Table 4).
3. 3-Hexen-1-ol 1382 0.14 0.05
4. 3-Octanol 1387 0.06 0.03 From these, it is concluded that the essential oil of both varieties
5. α-Cubebene 1445* 0.49 ND showed maximum zone of inhibition and minimal inhibition
6. α-Longipinene 1477 0.05 ND
7. α-Campholenal 1486 0.14 ND concentration against Bacillus subtilis (MTCC-2451) and
8. α-Funebrene 1501 0.06 ND Pseudomonas fluorescence (MTCC-664) bacterial strains, which
9. β-Bourbonene 1516 0.08 ND indicate that essential oil of both these varieties has capacity to
10. Linalool 1538 0.09 0.03 inhibit the growth of both gram+ve and gram-ve bacterial strains
11. trans-Caryophyllene 1587 0.06 ND
12. β-Elemene 1592 39.27 23.14 when used in a higher amount. According to few previous reports,
13. Methyl Eugenol 1639 0.11 ND the oil has been shown to have inhibitory effects on growth of
14. Aromadendrenepoxide-(I) 2221* 0.04 0.18 Mycobacterium tuberculosis and Micrococcus pyogenes var.
15. α-Humulene 1658 1.41 0.11 aureus (Anonymous, 1991), Anthobacter globiformis, Bacillus
16. Germacrene-D 1705 2.31 ND
17. β-Selinene 1714 0.60 0.99 megaterium, Eschrichia coli, Pseudomonas spp., Staphylococus
18. α-Selinene 1719 0.66 1.10 aureus, Staphylococcus albus and Vibrio cholerae (Rao and
19. Carveol 1825 0.21 ND Nigam, 1970; Dey and Choudhury, 1984). The essential oils of O.
20. Cubebol 1951 0.03 ND sanctum have been effective against both gram+ve and gram-ve
21. Caryophyllene oxide 1981 0.63 20.30
22. Elemol 2080 0.18 ND bacteria and properties were comparable with the effectiveness of
23. Veridiflorol 2086* ND 0.15 clove oil (Prasad et al., 1986; Phadke and Kulkurni, 1989). It was
24. β-Oplopenone 2091* ND 0.07 also observed that antimicrobial activity of O. sanctum was found
25. Spathulenol 2124 0.15 0.51 to be higher as compared to commonly available other species of
26. Eugenol 2177 49.95 47.33
27. Epi-globulol 2024 ND 0.06 Ocimum (Sinha and Gulati, 1990). More so, aqueous extract,
28. Globulol 2076 ND 0.03 alcoholic extract and seed oil of O. sanctum shown antimicrobial
29. Selin-11-en-4 alpha-ol 2253 0.89 1.70 properties against entire pathogens (Geeta et al., 2001; Singh et
30. Khusinol 2309 ND 0.32 al., 2005). Essential oil of O. sanctum reported to have shown
31. Isoeugenol 2770 0.04 ND
97.75 96.17 antimicrobial activity against Propionibacterium acues in in-vitro
OSW: Ocimum sanctum white study and minimum inhibitory concentration (MIC) value found
OSB: Ocimum sanctum black to be 3.0% v/v (Viyoch et al., 2006).
RI: Actual retention indices of components on same phases of columns (BP-20 and
*CW-20M)
RA: Percentage of components
ND: Not Detected

144
 International Journal of Recent Scientific Research, Vol. 5, Issue, 1, pp.142-147, January, 2014

Table 3 Antimicrobial activity of essential oil of two varieties of O. sanctum L. against gram +ve and gram -ve bacterial strains
Nature Diameter of inhibition zone (mm) of essential oil Control +ve
S. Microorganisms
of concentration used for antimicrobial analysis (μL/well) (n=3) (n=3)
No. (Bacterial source
bacterial OSW OSB Amoxicillin Ciprofloxacin
number)
strains 10μL 20μL 40μL 60μL 10μL 20μL 40μL 60μL 20μL 20μL
Bacillus
1. subtilis 5.3 9.3 16.4 25.7 8.2 10.3 18.8 29.4 44.2 55.3
(MTCC-2451)
Staphylococcus
2. aureus 3.5 4.8 9.5 14.4 6.2 7.0 12.1 17.3 38.0 46.5
Gram
(MTCC-740)
+ve
Staphylococcus
3. epidermis 5.9 7.2 8.9 15.2 6.6 10.1 11.7 19.0 35.4 56.3
(MTCC-435)
Escherichia
4. coli 1.8 3.5 5.3 11.2 3.1 5.3 7.2 13.2 49.0 52.5
(MTCC-443)
Salmonella
5. typhimurium 1.2 2.4 5.1 12.4 2.7 3.6 8.3 17.6 29.3 36.6
(MTCC-1251)
Pseudomonas
6. Gram fluorescence 4.9 8.1 12.6 23.6 10.4 7.8 14.5 27.4 45.8 58.5
-ve (MTCC-664)
Acenetobactor
7. calcoaceticus 2.1 5.3 9.3 16.7 7.5 4.3 11.6 18.8 38.6 48.0
(MTCC-127)
All values are mean of triplicates (n=3); Gram +ve: gram positive; Gram -ve: Gram negative; N. A: No Activity; OSW: Ocimum sanctum white; OSB: Ocimum sanctum black.

Table 4 Minimal inhibition concentration (μL) of essential oil of two varieties of O.sanctum L.
S. Nature of bacterial Microorganisms (Bacterial source MIC (μL)
No. strains number) OSW OSB
1. Bacillus subtilis (MTCC-2451) 2.2 2.1
2. Staphylococcus aureus (MTCC-740) 3.5 2.4
Gram +ve
3. Staphylococcus epidermis (MTCC-435) 3.0 4.2
4. Escherichia coli (MTCC-443) 5.2 6.1
5. Salmonella typhimurium (MTCC-1251) 6.1 5.2
6. Gram-ve Pseudomonas fluorescence (MTCC-664) 2.5 1.5
7. Acenetobactor calcoaceticus (MTCC-127) 7.3 2.5
MIC: Minimal Inhibition Concentration; Gram +ve: Gram positive; Gram-ve: Gram negative; OSW: Ocimum sanctum white; OSB:
Ocimum sanctum black.

CONCLUSION
Therefore, finally, it can be concluded that such a high percentage
of eugenol in the oil samples from Northern India has been first
time detected and variation in percentage of the active chemical
compounds present in O. sanctum L. varieties should find place in
treatment of various bacterial infections. The results from the
present investigation are very encouraging and indicate that herb
should be studied more extensively to explore its potential in the
treatment of infectious diseases as well. Further, our aim is to
develop hydroponic agriculture practices for different Ocimum
species and varieties by using organic products especially made
for hydroponic cultivation practices to increase the percentage of
some most important constituents in their essential oil. In future
we will work towards the hydroponic agricultural practices for
aromatic, endangered, essential oil bearing medicinal plants from
India, so as to maintain the quality and quantity of their essential
oil and also to protect threatened aromatic medicinal plant’s
germplasm as a whole.
Acknowledgments
The authors are grateful to the Director, IHBT (CSIR), Palampur
(Himachal Pradesh) India; Head, Department of Botany-SAP-II
DRS (UGC) Punjabi University, Patiala (Punjab) India and Vice
Chancellor, Akal School of Biotechnology, Eternal University,
Baru Sahib (Himachal Pradesh) India for providing necessary
facilities and support.
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