INTRODUCTION:
Plants
are rich resources of medicines and most of the modern medicines own their
origin from plant. Ethnobotanically, all over the world, plants are the chief
sources of medicines even today, which are mostly used by folklore,
traditional, complementary and alternative forms of medicines. Although the
history of use of plants as medicine is as old as mankind itself, their
scientific role and mechanism of action are being probed only recently.
This
knowledge helps one to understand the clinical aspects of such medicines such
as the molecules present, the half-life of such molecules, their bio-availability,
short and long term side effects if any, target cells or organs, their overall
role on the homeostasis of the body etc. The reports on these aspects on plants,
their phytochemicals and their roles are slowly increasing, which is a welcome
sign.1-18
The
present study is one step in this endeavour, wherein one medicinal plant of
importance, Arkaraga (Nirgundi) (Vitex negundo) is being reported on the
phytochemicals and antioxidant activities of its various extracts.
Vitex
genus has a number of species and all of them have medicinal roles and used
ethnobotanically all over the world. In Ayurveda Vitex negundo is known
as Nirgundi (meaning “protects the body from all diseases”) and as
“Sarvaroganivarini” (meaning “cures all diseases”). There are number of reports
and reviews on the ethnobotanical and medicinal aspects of this plants which
mention its wide range of applications. (Nautiyal et al, 2018; Ladda and
Magdum, 2012).19,20 The medicinal properties are well documented
which include anti-oxidant, anti-asthmetic, analgesic, antipyretic,
antibacterial, larvicidal, nephroprotective, hepatoprotective, anti- HIV
activity, anti-eosinophilic, anti-cancer and anti-snake venom activity etc.
(Rani and Sharma, 2013; Tandon and Gupta, 2005; Jignesh et al, 2009).21-23
The water extract of Vitex negundo leaves are given in combination along
with some other plants leaf extracts at the beginning of fifth month of
pregnancy. It is believed that this treatment is given for normal development
of foetus. The present study is to evaluate the antioxidant activities of Vitex
negundo leaves which were collected from Karaikudi, Tamil Nadu, India.
MATERIALS AND METHODS:
100 grams of dried powder of Vitex negundo leaves
was packed in separate round bottom flasks for sample extraction using ethanol,
methanol, hexane and distilled water as solvents. The extraction was conducted
with 300 ml of the solvent for a period of 72 hours. The extracts were decanted,
filtered, concentrated under reduced pressure to get semi solid residues which
were stored at 4 degree Celsius till further use.
Phyto-chemical Analysis:
The extracts prepared were analysed for the presence
of alkaloids, saponins, tannins, steroids, flavonoids, anthraquinones, triple
sugars, amino acids, proteins, glycosides and reducing sugars as per standard
protocols.24-25
ANTIOXIDANT STUDIES:
THE
RADICAL SCAVENGING EFFECTS:
A. DOT-PLOT
RAPID ASSAY:
The rapid screening assay was performed by the method
proposed by Soler-Rivas et al. (2000).27
PROCEDURE:
Aliquots of plant extracts were spotted carefully on
TLC plates and dried. The sheets bearing the dry spots were placed upside down
for 30-60 seconds in DPPH solution and the layer was dried. The stained silica
layer revealed a purple background with yellow spots, which showed radical
scavenging capacity.
TLC procedure was followed for a total of five
samples. The first one had 10% dilution whereas the subsequent four were having
serial dilutions by 10% each. To visualize the spots the amount of the above
five samples were charged with decreasing order of quantity, i.e. 100, 80, 60,
40 and 20
l respectively. Since the spots were visible normally,
we have deviated from the procedure by not keeping the TLC plates in Iodine
chamber or treating with Sulphuric acid.
B. DPPH SPECTROPHOTOMETRIC ASSAY:
The antioxidant activity of the plant extracts was
examined on the basis of the scavenging effect on the stable DPPH free radical
activity as per the method of Braca, 2002.28
PRINCIPLE:
DPPH radical reacts with an antioxidant compound that
can donate hydrogen and gets reduced. DPPH, when acted upon by an antioxidant,
is converted into diphenylpicryl hydrazine. This can be identified by the
conversion of purple to light yellow colour.
Chemicals and Reagents:
1,1-diphenyl-2-picrylhydrazyl (DPPH), Methanol,
Ascorbic acid and sample
Antioxidant activity (DPPH free radical scavenging
activity) determination:
DPPH solution (DPPH–1mg/ml in methanol) was freshly
prepared and kept in the dark at 4°C. 3.7ml of absolute methanol was added all
test tubes including blank. 100, 200, 300, 400, 500mg of ascorbic acid was
dissolved in 1ml of distilled water and 100μl of respective ascorbic acid
sample was added to the tubes marked as blank. The same concentration was
prepared for Vitex negundo water extract and 100μl of respective
samples were added to all tubes marked as tests. 100μl of distilled water was
added to the blank test tube. 200 μl of DPPH reagent was added to all the
test tubes including blank. All the test tubes were incubated at room
temperature and in dark for a period of 30 minutes. The mixture was left to
stand for 5 min and absorbance was measured spectrophotometrically at 517nm.
Methanol was used to set the absorbance zero.
The radical scavenging activities of the tested
samples, expressed as percentage of inhibition were calculated according to the
following equation (Yen and Duh, 1994).29 Per cent inhibition versus
concentration curve was plotted and the concentration of sample required for
50% inhibition was determined and represented as IC50 value for each of the
test solutions.
CALCULATION
FOR PERCENTAGE SCAVENGING ACTIVITY:
Absorbance at blank – Absorbance at test
% Scavenging
= ----------------------------------------------- × 100
Antioxidant
Absorbance at blank – absorbance at test
Determination of Reducing Property (Reducing power
assay):
The reducing power of the herbal medicine extract was
determined by a slightly modified method (Oyaizu, 1986).30 The
reducing ability of the drug extract was measured by the transformation of Fe3+
to Fe2+ in the presence of the extract at 400 to 680nm. Increased absorbance of
the reaction mixture indicates increased reducing power. 100mg of Vitex
negundo water extract was dissolved in 1ml of distilled water, from this
concentration 0.10, 0.25, 0.50 and 1ml was taken in tubes. It was mixed with
phosphate buffer (2.5ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe (CN6)]
(2.5 ml, 1 %). The mixtures were then incubated at 50 °C for 20 min. Aliquots
(2.5ml) of trichloroacetic acid (10 %) were added to each mixture, which were
then centrifuged for 10 min at 1000 rpm. The upper layer of the solutions (2.5ml)
were mixed separately with distilled water (2.5ml) and FeCl3 (0.5ml, 0.1 %),
and the absorbance levels were measured at 400 to 680 nm using a colorimeter.
RESULTS AND DISCUSSION:
The
phytochemical analysis results are shown in Table 1. Figures 1 to 4 indicate
the Dot-Plot Assay results for all the extracts of Vitex negundo leaves.
Table 2 indicates the DPPH scavenging activity of Vitex negundo water extracts
as compared with that of standard, Ascorbic acid. Figure 5 indicates the DPPH
scavenging activity of Vitex negundo water extracts. Figure 6 indicates
the DPPH scavenging activity of standard, ascorbic acid. Table 3 indicates the
FRAP assay results for all the extracts of Vitex negundo leaves. Table 4
indicates the FRAP assay results for Standards, Ascorbic acid and Quercetin.
Figure 7 indicates the FRAP assay results for all the extracts of Vitex negundo
leaves and for Standards, Ascorbic acid and Quercetin. Figure 8 indicates
the comparative IC50 values for FRAP assay for all the extracts of Vitex
negundo leaves and for Standards, Ascorbic acid and Quercetin.
DISCUSSION:
The
leaf extracts:
The
phytochemical analysis results in Table 1, indicated that flavonoids and
cardiac glycosides were present in all the extracts whereas amino acids and
proteins were absent in all of them. Alkaloids and triple sugars were present
in methanol and hexane fractions whereas triterpenoids were present in all but
water extract. Saponins and Tanins were observed in ethanol, methanol and water
extracts and Anthraquinones were observed in ethanol and water extracts. Steroids
were present in methanol and hexane fractions and absent in ethanol and water
fractions From the above-mentioned results, it is clear that Vitex negundo
leaf extracts show promising antioxidant potentials. The Dot Plot experiments
results indicated that for all the four extracts of Vitex negundo, i.e.
water, hexane, ethanol and methanol, there was a gradual increase in the
reactions with increase in concentration, as visualized by the colour changes.
i.e. purple to light yellow (Figure 1- 4). These results indicated that Vitex
shows antioxidant activities in all the concentrations observed.
The
IC50 value of DPPH scavenging activity was 111.16 as compared to that of the standard
(Ascorbic acid), which was 348.275 (Table 2 and Figures 5 and 6)
In
another antioxidant study (FRAP), it was observed that the antioxidant
potential of water extract was better than those of Ascorbic acid and Quercetin
(70.563, 85.162, 79.647, respectively). The ethanol and methanol extract also
showed promising antioxidant activities which were nearer to the values of the standards
(80.67 and 84.57). The hexane extract, however did not show good antioxidant
potential (214.175). It is interesting to find that all the extracts which
indicated good activity were polar or semi polar solvents. Hexane, which is a
nonpolar solvent, perhaps could not extract the biomolecules from the plant
source, which are responsible for the antioxidant activity. Incidentally, it is
a fact that usually the water extracts of Vitex negundo leaves are
given as medicine, thus indicating the genius of the complementary and
alternative medicine practitioners who had enough knowledge of the adjuvants
which could make the medicine molecules bioavailable to the body.
CONCLUSION:
From
the above results and discussion, we could surmise that the presence of flavonoids
in all the four extracts and the presence of alkaloids in hexane and ethanol fraction
give us a clue that the antioxidant potential of Vitex negundo could
be due to the presence of these two compounds. Further work is in progress in
this direction.
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