Sie sind auf Seite 1von 89

Self introduction

ZAHID MEHMOOD
Supervised by
Dr SAQIB ALI
Session 2015-2017
Biological activities of
roots, leaves, and flowers
of Taraxacum
Officinale
GENERAL INTRODUCTION

Plants used for food


as well as
medicine.
Plant used for
medicine are called
as medicinal plant.
Medicinal plants

Medicinal plants played a vital role in


the life of humans
for their betterment.
 to secure and save life.
Natural products isolated from plants are
used for treating number of diseases.
 Almost eighty thousands (80,000)
medicinal plants
 2,50,000 higher plants.
Herbs
Herbalism is a useful medicinal practice
supported by the use of plant or plant
extracts. Plants used for the flavoring of
food and medicinal values are called as
herbs.
There are four types of herbs.
Wild
 ornamental
Vegetable
 spice
Uses of Herbs
as a medicine in daily lives
developed countries depend on herbs for their health
 in rural areas herbs (medicinal plants) are the source of primary health
care.
for therapeutic purposes.
In rural areas due to deficient health care centers, lack of hospitals,
poverty, unemployment and lack of health care organizations, different
parts of plants such as roots, stem, leaves and flowers are used for
medicinal purposes.
PHYTOCHEMISTRY

Plant produce chemical compound due to their


normal metabolic activity and are called as
phytochemicals
Phytochemicals are of two types.

primary metabolites.
secondary metabolites.
Primary and Secondary
Metabolites

Primary metabolites
are present in all plants and contain
• protein
•carbohydrate
Secondary Metabolites
•Alkaloids
• Flavonoids
•steroids
•By refining many secondary metabolites various drugs
can be obtained such as insulin.
Alkaloids

Alkaloids are secondary metabolites .


produced by bacteria, fungi, animals
and plants.
It has nitrogen ring in its structure.
It has pharmacological activity
therefore can be used as medication,
recreational drugs and in the
entheogenic habit
Polyphenol

Polyphenol
Polyphenol are also called as phenolics having
a phenol ring in their structure e.g.
Anthocyanins.
Phytoestrogens.
Isoflavones
 tannins.
Terpenes

Terpenes
it is a class of organic compound.
 It is chief constituent of resins
By the chemical modification it can be transformed
into terpenoids.
IMPORTANCE OF MEDICINAL PLANTS IN PAKISTAN

.
Almost 6000 plant species are found in Pakistan so it
can be concluded that it has a diverse flora.
Almost 80% people of the rural areas use the
traditional method of herbal treatment.
IMPORTANCE OF MEDICINAL PLANTS IN PAKISTAN

In Azad Kashmir large numbers of medicinal plants are present, in the coniferous
forest of Neelum valley.
Lachrat and Kutla forest of Neelum valley .(Qamar et al., 2006). 345 species of
medicinal plant are present in Swat.
IMPORTANCE OF MEDICINAL PLANTS IN PAKISTAN
continue

Treatment of diseases
gastro intestinal
skin diseases
Throat
 fever
Aching etc (Sher et al., 2010).
THE THREATS TO THE MEDICINAL PLANTS

Medicinal plant bears intense biotic pressure imposed by


the activities of humans such as
Overgrazing
 deforestation
 overexploitation
 collection of plants in an unscientific way
Increasing population
 shrinkage of forest cover
 high rate of extraction of the medicinal plant
Overharvesting
 loss at the time of collection
Storage
unmonitored trade
 Loss Due To the Lack of
Awareness
Loss Due To the Weak Law
Enforcement
 Loss Due to the Dependence on
Medicinal Plants
Taraxacum Officinale


Biological
Activities

Antidiabetic
Antioxidant
SecondaryActivities

• By High
level
Damaging effect
to cell
membrane &
OXIDATIVE
STRESS
product of Chronic
metabolic ROS macromolecules
diseases

pathways
ischemia
cancer

suppress
Low level
Physiologic
al diabete
Pathologica s Parkinson's
l process

Oxidation in
body Antioxidants

synthetic Alzheimer's

Natural hypertension

BHT

Secondary metabolites (carotinoids,


BHA flavonoids, cinnamic acid, benzoic acid,
ascorbic acid, tocopherols, tocotrienols )
TBH
Q
Antimicrobial Activities

Infectious diseases caused by microorganisms such as bacteria


and fungi and are treated by antibiotics. Use of antibiotics in
large amount led to the development of resistant by
microorganisms. As a result it became very important to
screen more medicinal plants for their effective biological
activities to overcome the resistance developed by microbes
against existing drugs (Verma et al., 2011). About 80% people
of South Africa use traditional medicinal plants for health care
purpose and only few of them were commercialized. It
becomes important to commercialize drugs isolated from
medicinal plant on large scale due to their therapeutic
purposes (Street et al., 2013).
Replacement of The Synthetic Antimicrobials

Plants extract showing


antimicrobial property becomes a
great interest. Medicinal plants
play a vital role for the formation
of new drugs as these have

less side effects


 low cost,
 more effectiveness compared to
synthetic drugs (Neethu et al.,
2016).
MATERIAL AND
METHODS
COLLECTION OF
PLANT MATERIAL

Taraxacum officinale locally


known as "Hund" were
collected from village Pirkot
Tehsil Hajira District Poonch, in
April 2017. The whole plant
was collected and antioxidant,
antimicrobial and antidiabetic
activity was performed.
PROCESSING OF PLANT SAMPLE

• Washing of the plant.


• Drying of the sample
• Converting sample into fine powder,
EXTRACTION OF PLANT
MATERIAL
Plant samples were collected and shade dried and ground to powder
with the help of grinder.

 Extraction and concentration of plant sample with


 n-Hexane.
 Chloroform
 Ethanol
 Water

Fractions were evaporated in rotary evaporator and these extracts


were used to study biological activities.
T. Officinale fractions

n-hexane

EtOH
CHCl3

H2O
ANTIDIABETIC ACTIVITY

Antidiabetic activity was carry out with albino mice


Balb-C weighing about 30-40g. These mice were kept in
the National Veterinary laboratory under the rules of
Ethics Committees in Islamabad during the entire study.
Adaptation period was 7 days prior to starting of the
experiment. Mice were kept in 10 separate cages, each
cage contain 5 mice. All mice have free admittance to
tap water and were fed with poultry no 09 feed. This
assay was performed according to protocol of Ahmed
et al.(2015).
Induction of Diabetes in Animal Models
All mice were divided into 10 groups (G).
 G-I was serving as negative control and was injected
intraperitoneally with citrate buffer (pH=4.5) per kg body weight of
mice.
 G-II to G-X was given 0.4ml of newly prepared Streptozotocin 7
mg/ml of citrate buffer. G-II serve as diabetic control for diabetes.
 G-III serves as positive control for antidiabetic drug Glucophage
1mg/ml citrate buffer pH 4.5.
 G-IV to G-X was experimental groups and was injected with fractions
of roots, leaves, and flowers at a dose of 1mg/kg body weight.
 Diabetes was confirmed by determining the concentration of glucose
in the blood (mg/dl) after 2 hours of feed ingestion.
 For the authentication of diabetes blood was tested for 3 successive
days after giving Streptozotocin.
Evaluation of Antidiabetic Activity

Fractions of root, leave and flower were


administrated intraperitoneally. For
determination of blood glucose concentration
blood was collected from the tail of the mice.
Following parameters were used for the
assessment of antidiabetic activity.
Body weight measurement and
Blood glucose level test

Body weight of the mice was measured in


normal condition and after the induction of
diabetes at the start and end of the experiment.

Blood glucose was measured by pre-calibrated


Glucometer
ANTIMICROBIAL ACTIVITY
 Antimicrobial activity was performed against different pathogenic strain of
bacteria and fungi.
 Bacterial strain like Acinetobacter sp, Staphylococcus aureus, Klebsiella
pneumonia, Pseudomonas aeruginosa was obtained from Applied Microbiology
and Biotechnology Laboratory, International Islamic University Islamabad.
 Antimicrobial activity was performed by agar well diffusion method. Inoculums of
all microbes were prepared in LB broth media gL˗1 (10 g tryptophan, 10 g NaCl,
and distilled water). LB agar media was prepared and autoclaved at 121˚C for 20
minute which was then cooled and poured in Petri plates under sterilized
conditions of laminar flow hood. Plates were then seeded with 20μl of microbial
strain and using a sterile glass rod. Plates were then allowed to dry. The wells of
6mm were bored and 60 μl of each sample was pipettedin each well and plates
were then incubated at 37˚C for 24 hours. After 24 hours zone of inhibition were
measured and expressed in millimeter.
 Clarithyromycin was used as positive control.

Total Phenolic Contents

Minor modifications were made in Deo et al. (1957) for the


determination of total phenolic contents. Stock solutions of
plant extracts were made in DMSO (1 mg/1 mL). The
reaction mixture was made by mixing 50 μL of plant extracts
and 50 μl of 10 % Folin-Ciocalteu reagent and incubated for
5 minutes at room temperature. After incubation period, 50
μL of 10 % Na2CO3 was added and again incubated at room
temperature for 60 minutes in darkness. The absorbance
value was recorded at 760 nm. Phenolic contents were
determined against Gallic acid standard curve.
Total Flavonoid Contents

Minor modifications were made in Ghous et al. (1957) for the


determination of total flavonoid contents. The reaction mixture
comprising of 0.5 ml of extracts, 2mL distilled water, and 100 μL of
NaNO2(5%) was incubated for 10 minutes. 100 μL Solution of AlCl3 (10
%) was added to the reaction mixture and placed for further
incubation for 10 minutes. 4 % NaOH (2 mL) solution was mixed in
the above reaction
mixture and left for incubation for 20 minutes. The absorbance value
was recorded at 510 nm. Flavonoid contents were determined against
rutin standard curve.
EVALUATION OF ANTIOXIDANT
POTENTIAL

DPPH Assay

Ghous et al. (1957) with slight modifications was adopted For DPPH
scavenging activity. Plant extracts were dissolved in DMSO (5 mg/5 mL) to
prepare stock solution. DPPH solution was prepared in Methanol (0.004
%). 0.5 mL and 1 mL of plant extracts from the stock solution were added
to 3 mL and 2.5 mL of reaction mixture of DMSO and DPPH solution
respectively. The absorbance was measured at 517 nm after incubation
time (0, 15, and 30 minutes in darkness at the room temperature).
Following formula was used for the calculation of percent DPPH
scavenging activity.
Percent Scavenging activity = [(Ac- Ai)/ Ac] x 100
Here Ac = Absorbance value of control Ai= Absorbance value of extract.
ABTS Radical Cation Decolorizing Assay
Ghous et al. (1957) with slight modifications was adopted for the
experimental purposes. 3 mM solution of ABTS in water was
oxidized using 2.5 mM solution of potassium persulfate leaving in
darkness for 16 hours at the room temperature. 3 ml of DMSO was
used as blank and 2 mL of DMSO and 1 mL of ABTS+ were used as a
standard. Stock solution of plant extracts was prepared in DMSO (1
mg/5 ml). For the measurement of percent scavenging activity of
plant extracts, 20, 100, and 200 μL extracts were mixed with 1 ml of
ABTS+.. The samples were incubated for 20
minutes at room temperature. The absorbance of the mixtures was
measured at 734 nm. Following formula was used for the calculation
of percent scavenging activity. Percent scavenging activity = [(Ac-
Ai)/Ac] x 100
Where Ac = Absorbance value of control. Ai= Absorbance value of
extract.
RESULTS AND DISCUSSIONS
ANTIDIABETIC ACTIVITY

Antidiabetic activity of T. officinale root, leaves, and flower


fractions (n-hexane, ethanol, and water) was evaluated in
diabetic mice. Fifty mice were distributed in 10 separate cages
each having 5 mice and designated according to following
arrangement. G-I considered as negative control, G-II
considered as positive control or diabetic control. G-III
considered as antidiabetic control and G –IV to G-X were
considered as treatment groups.
Induction of Diabetes

All the groups were administrated with


Streptozotocin (STZ) except G-I was a
negative control and was given only
with citrate buffer, all other groups were
made diabetic.
Body Weight Measurement

The body weight of each mice before induction of STZ


was ranging from 30-33g. After the induction of STZ
body weight of the mice were began to increase
gradually and after the 15 days of the administration
of STZ about 10g body weights of each mice
increased (Figure4.1). The values are given in Table
4.1. Weight of the mice in each group was ranging
from 38-42g.
Table 4.1Weight of normal and diabetic mice of various groups

GROUPS Normal mice Diabetic mice


G-I 31.8±2.16 32.6±1.51
G-II 32±2.34 39.4±1.51
G-III 31.4±1.14 41.0±01
G-IV 31.4±1.94 40.6±2.30
G-V 31.6±1.14 42±1.87
G-VI 30.8±1.64 41.4±1.67
G-VII 30.6±0.89 40.8±1.30
G-VIII 31.2±1.30 42.4±0.89
G-IX 30.2±1.48 41.2±1.48
G-X 30.4±1.81 41±1.22
Body weights of the mice before and after
diabetes

180

160

140

120

100
NORMAL
80 DAY :05

60

40

20

0
G-I G-II G-III G-IV G-V G-VI G-VII G-VIII G-IX G-X
Before the induction of the STZ blood glucose of each mice was ranging from 106-112 mg/dL which is a normal concentration of glucose in

Glucose Measurement
• Before the induction of the STZ blood glucose of
each mice was ranging from 106-112 mg/dL
which is a normal concentration of glucose in
blood.
• Glucose level was measured with Glucometer on
every 5th day after the administration of STZ
intraperitoneally and it was observed that
glucose level increases.
• After 15th day blood glucose level increased up
to 250 mg/dL.
Concentration of glucose (mg/dL) in various groups of
mice after induction of STZ.
300

250

200

NORMAL

150 DAY :05


DAY:10
DAY:15
100

50

0
G-I G-II G-III G-IV G-V G-VI G-VII G-VIII G-IX G-X
Treatment f The Diabetic Mice

When mice become diabetic after induction of STZ,


 G-I and G-II was not treated because these were considered as negative control
and diabetic control respectively.
 G-III was treated with standard antidiabetic drug i.e. glimbenclamide.
 G-IV was administrated with n-hexane fraction of flower,
 G-V was treated with ethanolic fraction of flower
 G-VI with water fraction of flower.
 G-VII was treated with n-hexane fraction of leaves.
 G-VIII with ethanolic fraction of leave.
 GIX with water fraction of leaves .
 G-X was treated with water fraction of roots.
• It was observed that all parts of the
Taraxacum officinale showed activity against
diabetes. These fractions decreased blood
glucose level after administration of the
extracts (n-hexane, ethanol, and water) of
root, leave and flower. But n-hexane fraction
of flowers, water fraction of leave and water
fraction of root showed remarkable
antidiabetic activity almost equal to
antidiabetic drug
Concentration of glucose (mg/dL) in various groups of mice

Groups Day :01 Day :02 Day:03

G-I 113±3.56 117.2±3.56 113.2±3.18


G-II 113.2±4.94 256±4.49 256±4.42
G-III 256±5.09 154.8±8.43 126.2±3.86
G-IV 204±8.29 157.6±5.94 121.2±4.91
G-V 196.4±6.04 152.2±7.19 141.8±4.62
G-VI 209±8.31 153.2±8.52 137±3.94
G-VII 195.8±11.01 157.8±6.30 134.4±4.63
G-VIII 192.4±6.06 164.4±4.87 126.4±3.13
G-IX 195.6±3.56 152.6±3.13 126.6±4.84
G-X 200.8±5.29 153.4±8.08 120.8±2.92
Comparison of glucose level of extracts of root, leaves and
flowers of T. officinale on diabetic mice.

300

250

200

150

DAY :01
100
DAY :02
50 DAY:03

0
ANTIBACTERIAL ACTIVITY

To know antibacterial activity four


fractions of T. officinale fractions of n-
hexane, chloroform, ethanol and water
ware carried out with concentrations of
10mg/ml solvent each.
Comparison of The Results of All The
Fractions of T. Officinale With Standard.

Tested strain Standard n-Hexane Chloroform Ethanol Water

Zone of inhibition in mm
S. aureousR 30` 10 15 0 0

S. Aureous L 32 13 19 0

S. aureousF 31 13 19 0 0

E. coliR 30 26 25 15

E. coliL 40 35 35 0 0

E. coli F 38 17 26 0 30

S typhea. R 30 25 24 0 0

S typhea. L 30 15 26 10 20

S typhea. F 31 25 30 0 20

P aeruginosa.R 25 10 20 0 6

Paeruginosa. L 35 15 15 0 10

P aeruginosa. F 28 20 20 5 0
Comparison of the results of antibacterial activity of all the fractions of T.
officinale with standards

45
40
35
30
25
Standard
20
n-Hexane
15
Chloroform
10
Ethanol
5
Water
0

Psodomonas…

Psodomonas…

Psodomonas…
Salmonella…

Salmonella…

Salmonella…
R

L
F
E coli.

E coli.

E coli.
F

F
Staphylococcus

Staphylococcus

Staphylococcus
1

Staphylococcus Aureus

Zone of inhibition of chloroform fraction


of root, leaves, and flower (15mm, 19mm and
19mm) was more pronounced as compared to
rest of the fractions but less than standard
(30mm, 32mm and 31mm) as shown in Table
4.4 and Figure 4.5 fraction of ethanol and
water has no zone of inhibition against
staphylococcus aureus.
Measurement of zone of inhibition of
fractions against Staphylococcus aureus.

35

30

25

20 Standard

15 n-Hexane
Chloroform
10
Ethanol
5
Water
0
E. Coli

In this study it was observed that chloroform extract of T.


officinale parts i.e. root, leaves and flowers showed remarkable
activity (25mm, 35mm and 26mm) but less than standard
(30mm, 40mm, 38mm). The n-hexane (26mm, 35mm and
17mm) was less active as compared to chloroform, on the
other hand ethanolic fractions do not showed any activity but
water fraction of root and flowers showed zone of inhibition of
15mm and 30mm but water extract of leaf have no activity.
Measurement of zone of inhibition of various
extracts against E.coli

45

40

35

30

Standard
25
n-Hexane
Chloroform
20
Ethanol
Water
15

10

0
E coli. R E coli. L E coli. F
Salmonella Typhea

Chloroform extracts of all parts root, leave and flower (24mm, 26mm and 30mm) of
the T. officinale has pronounced activity as compared to rest of the extracts i.e. n-
hexane (25mm, 15mm and 25mm), ethanol (0mm, 10mm and 0mm), water (0mm,
20mm and 0mm). Ethanolic fractions of root and flower and water fractions of root
do not showed any activity. Chloroform fractions of flower have highest activity as
compared to root and leaf. All these have less activity as compared to standard
(30mm, 30mm and 31mm).
Measurement of zone of inhibition of various extracts against Salmonella
typhea.

35

30

25

20
S typhea R
S typhea L
15 S typhea F

10

0
Standard n-Hexane Chloroform Ethanol Water
Pseudomonas Aeruginosa

It is observed that chloroform fraction of root (20mm) and


flower (20mm) has more significant activity and n-hexane
fraction of root (10mm) and water fractions of leaves (10mm)
has same zone of inhibition, while n-hexane (20mm) and
chloroform fraction (20mm) of flower and chloroform of root
(20mm) has same activity. Ethanol (0 mm, 0 mm, and 5mm)
and water (6mm, 10mm,0mm) have very low zone of inhibition
but the activity of all the fractions were less than standard
Measurement of zone of inhibition of various extracts against
Pseudomonas aeruginosa.

40

35

30

25
pseudomonas aeruginosa R

20 pseudomonas aeruginosa
L
pseudomonas aeruginosa F
15

10

0
Standard n-Hexane Chloroform Ethanol Water
ANTIFUNGAL ACTIVITY

To determine antifungal activity of four


fractions of T. officinale i.e. n-hexane,
chloroform ,ethanol and water was carried out
with concentration of 10mg/mL solvent each.
.

Antifungal activity of various crude


extracts of Taraxicum officinale

Zone of inhibition in mm
Tested strain Standard n-Hexane Chloroform Ethanol Water
MAB 4 (R)| 10 25 25 - 20
MAB 4 (L) 30 34 28 30 -
MAB 4 (F) 20 24 26 - 30
MAB 18 (R) 30 32 31 5 27
MAB 18 (L) 30 30 32 10 30
MAB 18 (F) 31 34 30 20 12
MAB 3 (R) 32 30 31 5 30
MAB 3 (L) 35 30 31 32 25
MAB 3 (F) 31 30 20 15 15
AB 4 (R) 34 36 35 5 26
AB 4 (L) 33 30 27 20 18
AB 4 (F) 10 25 28 25 26
AB 17 (R) 10 25 20 - -
AB 17 (L) 25 27 24 5 20
AB 17 (F) 25 26 25 20 25
Comparison of antifungal activity of various extract of T. officinale

40

35

30

25 Standard
n-Hexane
20
Chloroform
15
Ethanol
10 Water

0
MAB MAB MAB MAB MAB MAB MAB MAB MAB AB 4 AB 4 AB 4 AB 17 AB 17 AB 17
4 (R)| 4 (L) 4 (F) 18 (R) 18 (L) 18 (F) 3 (R) 3 (L) 3 (F) (R) (L) (F) (R) (L) (F)
MAB4
• n-hexane (25mm, 34mm, 26mm ), chloroform
(25mm, 28mm, 26mm) and ethanol (30mm)
extracts of leave has very pronounced activity,
but n-hexane (34mm) extract of leaves has
highest then other extracts of leaf. Water and
ethanol extract of root, flower and water extract
of leaf has no zone of inhibition. n-hexane extract
of leaf has high activity as compared to standard
but chloroform extracts of leaf is equal to that of
standard
Zone of inhibition of fractions of root, leaf and flower against
MAB4
40

35

30

25

MAB 4 (R)|
20
MAB 4 (L)
MAB 4 (F)
15

10

0
Standard n-Hexane Chloroform Ethanol Water
MAB18
• n-hexane extract of root (32mm), leaf(30mm),
and flower(34mm) showed good activity as
compared to chloroform (31mm, 32mm,
30mm). But chloroform has good activity as
compared to ethanol (0mm, 30mm, 0mm) and
water (27mm, 30mm, 12mm). Ethanol and
water showed less activity leaves showed
good activity in all fractions except ethanol as
compared to standard
Zone of inhibition of fractions of root,
leaves and flowers against MAB18.

40

35

30

25

MAB 18 (R)
20
MAB 18 (L)
MAB 18 (F)
15

10

0
Standard n-Hexane Chloroform Ethanol Water
MAB3
• n-hexane extract of root (30mm) leave (30mm) and
flower (30mm) have same activity and nearly equal to
chloroform extract (31mm, 31mm, 20mm), but the
activity of ethanol (0mm, 30mm, 0mm) and water
(30mm, 25mm, 15mm) was less then n-hexane and
chloroform but water has more pronounced activity.
Ethanol extract showed equal activity to n-hexane and
chloroform, ethanolic extracts of root showed very less
activity. n-hexane extract of leaf and flower showed
equal activity to that of standard and in case of
ethanolic leaf showed very high activity as compared
to standard and flower extracts
Zone of inhibition of fractions of root,
leaf and flower against MAB3

40

35

30

25

MAB 3 (R)
20
MAB 3 (L)
MAB 3 (F)
15

10

0
Standard n-Hexane Chloroform Ethanol Water
AB4
• n-hexane extract (36mm, 30mm, and 25mm) has more pronounced
activity than chloroform (35mm, 27mm, 28mm). Water extract (26mm,
18mm, and 26mm) showed relatively less activity as compared to n-
hexane and chloroform extract. Ethanol (5mm, 20mm, and 25mm) has
very little activity in case of root. But in case of leaf n-hexane showed very
good activity and chloroform has relatively less but more the ethanol and
water. Ethanol and water showed nearly equal but less than n-hexane and
chloroform. In case of flower all the extract showed nearly equal activity. It
is observed that amongst all the extracts n-hexane has remarkable activity
as compared to the roots extract and ethanol extract has less activity. It is
oblivious from the Table 4.5 and Figure 4.12 that root showed high activity
then flower and in case of chloroform flower showed high activity then
leaf in ethanol fraction flower showed high activity than leaf and leaves
showed high then root, in water root and flower showed equal activity
and more than leaf but less than standard
Zone of inhibition of fractions of root, leaf
and flower against AB4

40

35

30

25

AB 4 (R)
20
AB 4 (L)
AB 4 (F)
15

10

0
Standard n-Hexane Chloroform Ethanol Water
AB17
• n-hexane extract of root (25mm), leaves (27mm) and flowers (26mm) has
more pronounced activity as compared to chloroform (20mm, 24mm,
25mm). AB17 strain is resistant to ethanolic (0mm, 5mm, 20mm) and
water 0mm, 20mm, 25mm) extract. Ethanol extract of leaves has little
activity. Ethanolic extract of flower and water extract of leaves and
chloroform extract has equal activity. Similarly n-hexane extract of roots,
chloroform extract of flower and water extract of flower has same activity.
Leaves extracts of ethanol has very little activity. According to the
Table(4.5) and Figure(4.13) n-hexane fraction of leaf showed high activity
than flower and root showed less than leaf and flower and all these
fraction showed good activity than standard, and in case of chloroform
fractions flower showed highest activity than leaf and root and also higher
than standard and leaf showed higher then root but less than standard
except flower. There is remarkable activity of flower in water extracts but
less than standard except flower which is equal to standard. Root do not
showed any activity in ethanol and water
ANTIOXIDANT ACTIVITIES

• To find antioxidant activities different procedures were used. Plant


extracts are a mixture of different compounds and these compounds have
diverse functional groups, chemical behavior and polarity, so when
different test were applied we get interesting and different results.
• DPPH radical scavenging activity
• ABTS cation radical
• All the procedures were important and commonly used to know the
extract antioxidant potential. DPPH assay, ABTS assay, total phenolic
compounds and total flavonoid content were used to test four extract of T.
officinale plant (n-hexane, chloroform, ethanol and water)at
concentrations of 1 mg/mL.The results of DPPH radical scavenging activity
and ABTS cation radical activity were compared with ascorbic acid.
Similarly the total phenolic compounds and total flavonoid contents were
compared with Gallic acid and rutin respectively.
ABTS Cation Radical Activity
• ABTS cation radical activity for four fractions of extracts of n-
hexane, chloroform, ethanol and water was carried out with
concentration of 1mg/ml. The results of all these fractions are
presented in Table 3.1.By comparing the results of these fractions
with standards it was confirmed that all fractions of T. officinale
extract were good for ABTS cation radical activity. Percentage
inhibition was calculated from y equation obtained from ascorbic
acid calibration curve as shown in the Figure 4.15 Maximum
activity of these fractions was observed in water leaf extracts with
ABTS values 0.052 and percentage inhibition of 96 %, root and
flower extracts showed 95 % each. Ethanolic fraction of root, leaf
and flower showed %age inhibition of 90 %, 95 % and 89 %
respectively. n-hexane fraction showed percentage inhibition of (39
%, 47 %, 38 %) while chloroform showed 62 %, 59 % and 52 %
Antioxidant activity of various extracts of T. officinale by the ABTS

Extract Absorbance %age inhibition Total ABTS activity


n-hexane
R 1.202 39 4.03
L 1.042 47 3.46
F 1.225 38 4.09
CHCl3
R 0.757 62 2.4
L 0.815 59 2.6
F 0.954 52 3.15
Ethanol
R 0.188 90 0.45
L 0.096 95 0.129
F 0.207 89 0.521
Water
R 0.083 95 0.08
L 0.074 96 0.052
ascorbic acid calibration curve

ascorbic acid calibration curve


0.4

0.35
y = 0.2835x + 0.0592
0.3 R² = 0.9129

0.25
absorbance

0.2

0.15

0.1

0.05

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
concentration mg/ml
The ABTS cation antioxidant activity of T. officinale extract.

%age inhibition ABTS activity of plant extracts


120

100

80

60

40

20

0
n-hex CHCl3 EtOH water

y roots y leaves y flowers


DPPH Radical Scavenging Activity

• To check DPPH radical activity of T. officinale root, leaf,


and flower extract i.e. n-hexane, chloroform, ethanol,
and water were used in 1mg/ml concentration.
Percentage inhibition was calculated from y equation
obtained from ascorbic acid calibration curve as
showed in the Figure 4.17 .The results of all these
fractions obtained for DPPH radical activity are showed
in the Table 4.7. .The chloroform extracts of root and
ethanolic extracts of leaves showed maximum activity
then all other fractions. The overall results showed that
the chloroform root fraction (93%) and ethanolic
fraction of leaf has high antioxidant potential.
DPPH activity by using ascorbic acid as
a standard at 517nm
Extract Absorbance %age inhibition Total Dpph activity
n-hexane
R 0.208 91 0.52
L 0.274 89 0.75
F 0.422 83 1.279
CHCl3
R 0.158 93 0.34
L 0.263 89 0.71
F 0.277 88 0.76
Ethanol
R 0.223 91 0.53
L 0.163 93 0.64
F 0.247 91 0.51
Water
R 0.405 83 1.20
L 0.209 91 0.52
F 0.258 89 o.70
Ascorbic acid calibration curve.

ascorbic acid calibration curve


0.4

0.35
y = 0.2835x + 0.0592
0.3 R² = 0.9129

0.25
absorbance

0.2

0.15

0.1

0.05

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
concentration mg/ml
DPPH radical scavenging activity of
T. officinaleextracts.

%age inhibition DPPH activity of plant extracts


94

92

90

88

86

84

82

80

78
n-hex CHCl3 EtOH water

y roots y leaves y flowers


Total Flavonoids Content

Values of the flavonoid contents of various extracts (n-hexane,


chloroform, ethanol and water) are showed in Table 4.8. It is clear from
the Figure 4.20 and Table 4.8 that n-hexane root extract and water
extracts of root have high flavonoid contents in 100µL extracts of plant T.
officinale.
Total Flavonoids content by using
lutin as a standard at 510nm

Extract Absorbance Total Flavonoids content

n-hexane
R 0.833 0.11
L 0.196 0.00
F 0.402 0.05
CHCl3
R 0.200 0.02
L 0.576 0.07
F 0.633 0.08
Ethanol
R 0.321 0.04
L 0.576 0.07
F 0.633 0.05
Water
R 0.736 0.10
L 1.031 0.14
Rutin calibration curve

rutin calibration curve

1.2

1
y = 0.988x - 0.0074
R² = 0.9426
0.8
absorbance

0.6

0.4

0.2

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
concentration mg/ml
.

Flavonoids contents of root, leaf and flower of Taraxacum officinale

1.2

0.8

0.6
Total Flavonoids content
Absorbance

0.4

0.2

0
Total Phenolic Content

• Values of the phenolic contents of various


extracts (n-hexane, chloroform, ethanol and
water) are shown in Table 4.9.It is clear from
the Figure 4.21and Table 4.9 that n-hexane
root extract and water extracts of leaf have
high phenolic contents in 100µL extracts of T.
officinale.
Total phenolic content by using Gallic acid as a standard at
765nm.

Extract Absorbance Total phenolic content


n-hexane
R 0.833 0.21
L 0.196 0.13
F 0.402 0.09
CHCl3
R 0.200 0.17
L 0.576 0.14
F 0.633 0.08
Ethanol
R 0.321 0.06
L 0.576 0.07
F 0.633 0.05
Water
R 0.736 0.04
L 1.031 0.16
F 0.381 0.11
Gallic Acid Calibration Curve

gallic acid calibration curve


0.7

0.6
y = 0.545x + 0.0649
R² = 0.9557
0.5
absorbance

0.4

0.3

0.2

0.1

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
concentration mg/ml
.

Comparison of the phenolic contents in root, leaf


and flower of Taraxacum officinale.

1.2

0.8

0.6
Total phenolic content
Absorbance

0.4

0.2

0
•Thank you