1. 1
Document heading
Pharmacognostic standardization and antioxidant activity of Vitis vinifera
L. seeds
Ghulam Mustafa Khan1
, Feroz Ahmad2*
1
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard University, New Delhi, India
2
Department of Pharmaceutical Sciences, Faculty of Applied Sciences and Technology, University of Kashmir, Hazratbal, Srinagar, J&K, India
Asian Pacific Journal of Tropical Biomedicine (2012)1-6
Asian Pacific Journal of Tropical Biomedicine
journal homepage:www.elsevier.com/locate/apjtb
*Corresponding author: Feroz Ahmad, Department of Pharmaceutical Sciences,
Faculty of Applied Sciences and Technology, University of Kashmir, Hazratbal, Srinagar,
J&K, India.
Tel: +91-9697359523
E-mail: ferozahmad85@gmail.com
Fundation Project: Supported by Faculty of Pharmacy, Jamia Hamdard University (
Grant No. mpharm/jh/08).
1. Introduction
Vitis vinifera L. (grapevine) is a large deciduous climber
belonging to family Vitaceae. It has long bifid tendrils,
leaves 7.5-15 cm long, more or less deeply 3-5 lobed,
margin irregularly and coarsely toothed. The seeds are pear
shaped, with a discoidal tubercle on the back from which a
low ridge runs over the top and down the ventral face. Vitis
vinifera L. is a native of west Asia and cultivated in many
parts of India especially in north-west India[1].
Grapes (Vitis vinifera) have been heralded for their
medicinal and nutritional value for thousands of years.
Egyptians ate grapes at least 6 000 years ago, and several
ancient Greek philosophers praised the healing power of
grapes usually in the form of wine. European folk healers
made an ointment from the sap of grapevines to treat skin
and eye diseases. Grape leaves were used to stop bleeding,
inflammation, and pain, such as the kind brought on by
hemorrhoids. Unripe grapes were used to treat sore throats,
and dried grapes (raisins) were used for constipation and
thirst. Antioxidants of natural origin have attracted special
interest because they can protect human body from free
radicals without producing toxic effects[2]. Grape seeds are
a particularly rich source of complex polymers of flavonoids
such as gallic acid, the monomeric flavan-3-ols catechin,
epicatechin, gallocatechin, epigallocatechin, epicatechin-
3-ogallate, dimeric, trimeric and even more polymeric
proanthocyanidins[3]. Furthermore, grape skins and seeds
contain flavonoids (catechin, epicatechin, procyanidins
and anthocyanins), phenolic acids (gallic and ellagic
acids) and stilbenes. Earlier studies have demonstrated
the potent free radical scavenger ability of grape seed
procyanidins. Grape seed extract of Vitis vinifera L. has
in vivo antioxidant property and could be as important as
vitamin E in preventing oxidative damage in tissues by
ARTICLE INFO ABSTRACT
Article history:
Received 15 April 2011
Received in revised form27 April 2011
Accepted 28 June 2011
Available online 28 June 2011
Keywords:
Grape seed
Pharmacognosy
Antioxidant
Phytochemical analysis
Vitaceae
Objective: To establish the standardization parameters and antioxidant activity of the seeds
of Vitis vinifera L., a herbal drug. Methods: Morphological, microscopical, physico-chemical
evulations, florescence analysis, TLC, HPLTC fingerprinting, preliminary phytochemical analysis,
antioxidant activity of the seeds of Vitis vinifera L. Results: Chemo-microscopy showed the
presence of testa, tegmen, endospermic cells, embryonal axis and endospermic cells. Physico-
chemical evaluation showed results with total ash (4.15 暲 1.01)% w/w, acid insoluble ash (0.39
暲 0.14)% w/w, water soluble ash (2.01 暲 0.21)% w/w, moisture content (15.76)% w/w, resin content
(0.84%), fat content (14.09%), bitterness value (3.066), swelling index (0), and no foaming index.
Behavior characteristics of the seed powder showed presence of carbohydrates, phenolics,
proteins, resins, lipids and flavonoids. The seeds have a significant antioxidant activity, total
phenolic content was found to be 780毺g/mg and the Inhibitory concentration (IC50) value of Vitis
vinifera seeds was found to be 5.73毺g/ml. Conclusions: Pharmacognostic standardization,
preliminary phytochemical and antioxidant evaluation of seeds of Vitis vinifera L. provides
useful information, which will help in authenticating the genuine plant along with the nature of
phytoconstituents present in it.
Contents lists available at ScienceDirect

2. Ghulam Mustafa Khan, Feroz Ahmad./Asian Pacific Journal of Tropical Biomedicine (2012)1-62
reducing the lipid oxidation and/or inhibiting the production
of free radicals[4]. The seeds and the leaves of the grapevine
are used in herbal medicine and its fruits are utilized as a
dietary supplement. Vitis vinifera L. seeds are reported to
posses antioxidant[4], cardioprotective[5], hepatoprotective[6],
anticarcinogenic[7], antimicrobial[8], antidiabetic[9], and CNS
effects[10].
Since grapevine (Vitis vinifera) is a dietary supplement, the
government bodies cannot compel the manufacturer to test
the safety of these materials. Given their potential benefits,
an independent demonstration of their safety appears
warranted. So, the present study was conducted to determine
the quality standards and antioxidant activity of the seeds of
Vitis vinifera L.
2. Materials and methods
2.1. Plant material
Grape seeds were bought from a local supermarket (Khari
bavli) in Delhi. They were dried for 1hour at 600C in an oven.
Seeds were identified as Vitis vinifera L. by M.P. Sharma,
Prof. Department of Botany Jamia Hamdard University, New
Delhi.
2.2. Pharmacognostic standardization
2.2.1. Macroscopic and microscopic analysis
Proper examination of the untreated sample of seeds of the
chosen plant was carried out under diffused sunlight and
artificial source similar to day light. The colour, size, odour,
taste, shape of the seeds were determined. Powder (60 mesh)
of the dried seeds was used for the observation of powder
microscopical characters. The powdered drug was separately
treated with phloroglucinol - hydrochloric acid (1:1) solution,
acetic acid, and iodine solutions to determine the presence
of lignified fibers, calcium oxalate crystals and starch grains
respectively[11].
2.2.2. Physico-chemical analysis
Physico-chemical parameters of the powdered material
such as total ash, water-soluble ash, acid-insoluble ash
were determined. The powered material was subjected to
cold extraction, hot extraction and successive extraction
using petroleum ether, chloroform, acetone, ethanol, water
and ethanol-water as solvents and respective extractive
values were determined. The moisture content was detected
by loss on drying method[12]. Bitterness value, swelling
index, foaming index were also determined. The microbial
load was determined as per WHO guidelines using nutrient
agar medium. 1% and 10% solutions of the grape seed powder
in distilled water were prepared and the pH of their filtrate
was measured with a standardized glass electrode[13]. Heavy
metal analysis for grape (Vitis vinifera) seeds was performed
at ITL Labs Pvt. Ltd, New Delhi, India, under the analysis
No. ITLR093-080307[14].
2.2.3. Fluorescence analysis
The fluorescence character of the plant powders (40 mesh)
was studied both in daylight and UV light (255 and 366 nm)
and after treatment with different reagents like sodium
hydroxide, picric acid, acetic acid, hydrochloric acid, nitric
acid, iodine and ferric chloride[15].
2.2.4. Behavior of seed powder with different reagents
The powdered drug was treated separately with different
reagents and acids like sodium hydroxide, picric acid,
hydrochloric acid, nitric acid, iodine, ferric chloride, and
the colour shown was noted by that treatment as such and
under the microscope[16].
2.2.5. Determination of resin content
The accurately weighed drug sample (5 g) was rapidly
refluxed with acetone (3 x 200 mL) for 6 hours to exhaust the
drug for the resin content. The excess solvent was removed
by distillation on a water bath. The residue so obtained
was suspended in water and transferred to a separating
funnel, repeatedly extracted the suspension with solvent
ether (2 x 200 mL) to extract all the resin contents. The ether
extracts were cooled and then dried over anhydrous sodium
sulphate and excess ether removed over a water bath. It was
transferred to a weighed beaker and the final weight was
noted[15].
2.2.6. Determination of fat content
A weighed quantity of sample (3 g) was extracted with
anhydrous ether in a continuous extraction apparatus for
six hours. The extract was filtered into a clean dry weighed
flask. The extraction flask was rinsed with small quantity of
ether; filtered and added to the weighed flask. The solvent
was evaporated and dried to constant weight at 105 毺g[17].
2.2.7. Preparation of extracts and preliminary phytochemical
analysis
After collection and authentication the plant materials
were shade dried and powdered separately. All plant
materials were passed through sieve no. 40 and used for
extraction. Petroleum ether, acetone, chloroform, alcoholic,
hydro alcoholic and aqueous extracts of the powdered plant
material were prepared[18]. Preliminary phytochemical
analysis of these different extracts was done using standard
methods[19].
2.2.8. TLC and HPTLC profile
Petroleum ether, chloroform and methanolic extracts
of Vitis vinifera were subjected to TLC with the aim of
identifying the individual substances in a mixture and also
for testing the purity. Solvent systems used for petroleum
ether extract was hexane: ethyl acetate: toluene as 8:2:2, for

3. Ghulam Mustafa Khan1, Feroz Ahmad./Asian Pacific Journal of Tropical Biomedicine (2012)1-6
3
chloroform extract it was hexane: ethyl acetate in the ratio
of 9:1 and for methanolic extract the solvent system used
was ethyl acetate: methanol: water as 77:8:8. Anisaldehyde
sulphuric acid was used as visualising agent[20]. The
methanolic extract were then taken and HPTLC was done
with toluene: ethyl acetate: formic acid (14:4:1) as solvent
system using CAMAG Linomat 5[21].
2.3. Antioxidant activity
2.3.1. Quantitative estimation of total phenolic contents
The total phenolic content of Vitis vinifera seeds was
determined with Folin- Ciocalteu method reagent in alkanlie
medium using gallic acid as standard[22]. The total phenolic
content was expressed in 毺g of gallic acid equivalents/mg of
extract.
2.3.2. Free radical scavenging activity
Free radical scavenging activity was measured by 1,
1-diphenyl-2-picryl-hydrazyl (DPPH). The antioxidant
activities of the ethanolic extract of Vitis vinifera seeds
were compared with standard vitamin C, butylated hydroxy
anisole (BHA) and butylated hydroxy toluene (BHT).
The activities were assessed on the basis of the radical
scavenging effect of the stable DPPH free radical[23].
3. Results
3.1. Pharmacognostic characters
3.1.1 Macroscopic characteristics
The seeds of Vitis vinifera are dark brown in colour and are
4-8 mm long. They have discoid apex, no odour, bitter taste
and smooth surface with a ridge on back (Figure 1).
Figure 1. Morphological characters of Vitis vinifera L. seeds
3.1.2. Microscopic characteristics
Seed coat which is the hardest structure present in this
seed was cut with great difficulty by keeping the seeds in
formalin solution for two months and the following structures
were seen: testa, tegmen, endospermic cells, embryonal axis
and endospermic cells as shown in Figure 2.
3.1.3. Physico-chemical results
The total, acid-insoluble and water soluble ash values
were found to be 4.15暲1.01, 0.39暲0.14 and 2.01 % w/w
respectively. The results of extractives values after cold
extraction, hot extraction and successive extraction are
given in Table 1. Bitterness value was found to be 3.066;
foreign matter content was found to be 0.40 %w/w; swelling
index was found to be 0.00; moisture content was found to be
15.76%; no foaming index was found of the drug with respect
to Glycyrrhiza glabra (foaming index 100). The resin and fat
contents of the seeds were found to be 0.84% (w/w) and 14.09%
(w/w) respectively.
Table 1.
Extractive values of Vitis vinifera seeds.
Extract
Individual extractive value
(% w/w)
Successive
extraction (% w/w)
Hot extraction Cold extraction
Petroleum ether 2.74 1.89 2.74
Chloroform 15.60 2.67 3.36
Acetone 18.43 3.77 4.12
Alcoholic 25.99 4.31 6.78
Hydro alcoholic 11.32 4.53 2.79
Aqueous 11.87 1.32 0.66
EA
EC
SC
TS
TG
EC
A
Figure 2. Microscopy of Vitis vinifera L. Seeds.
A: Ordinary microscopic slide of the seed at 40X resolution; B:
Microtome slide. EA: Embryonal axis; EC: Endopermic cell; SC: Seed
coat; TS: Testa; TG: Tegmen.

4. Ghulam Mustafa Khan, Feroz Ahmad./Asian Pacific Journal of Tropical Biomedicine (2012)1-64
3.1.4. Microbial load, pH and heavy metal analysis
The pH of 1% and 10% solutions of Vitis vinifera seeds were
found to be 6.02 and 5.94 (at 22.5 曟) respectively. The
results of heavy metal analysis showed presence of lead (5.06
ppm) and arsenic (2.14 mg/kg), however, the levels were less
than the minimum approved levels. The seeds showed no
presence of cadmium and mercury. The results of microbial
load determination are given in Table 2.
Table 2.
Colony forming units on nutrient agar medium
Dilution of
stock
No. of colonies Colony characteristics
Drug Control Shape Colour Observation aid
1:1 23 Nil Round Off white Naked eye
1:10 10 Nil Round Off white Naked eye
1:100 Not visible Nil Nil Nil Naked eye
3.1.5. Fluorescence analysis
The powder of the seeds of Vitis vinifera (mesh size 40) was
examined under daylight and UV light. The observations are
recorded in Table 3.
Table 3.
Fluorescence analysis of powered seeds of Vitis vinifera
Reagents
Colour observed
in ordinary light
Colour observed under
ultraviolet light
254 nm 365 nm
Powder as such Dark brown Off white Black
Distilled water Dark brown Off white Black
5% NaOH Brown Black Black
Ferric chloride Green Light green Dark brown
Conc. H2SO4 Dark brown Off white Black
Conc. HCl No change Off white Black
Acetone No change Off white Black
Chloroform No change Off white Black
3.1.6. Preliminary phytochemical analysis
Preliminary phytochemical analysis mainly revealed the
presence of carbohydrates, phenolics, proteins, resins,
lipids, phenolics and flavonoids (Table 4).
3.1.7. TLC and HPTLC profile
In the petroleum ether extract three spots were observed
at Rf 0.3, 0.45 and0.67. Chloroform extract showed four spots
in the TLC profile at Rf 0.31, 0.36, 0.45 and 0.7. While the
methanolic extract showed five spots at Rf 0.11, 0.44, 0.55,
0.66 and 0.73 (Figure 3).
In HPTLC profile of the methanolic extract of the Vitis
vinifera seeds, it was observed the constituents of methanolic
extract appear at the Rf 0.06, 0.55 and 0.92.
3.2. Antioxidant activity of Vitis vinifera seeds
3.2.1. Total phenolic content
The amount of phenolic component was calculated as
gallic acid equivalent and was found to be 780.00 毺g/mg,
indicating considerable free radical scavenging activity.
3.2.2. Free radical scavenging activity
DPPH assay is widely used as a free radical to evaluate
the antioxidant activity of natural compounds. Table 5
demonstrates the antioxidant activity of Vitis vinifera seeds
using DPPH dye. Vitis vinifera at different doses, i.e. 1.25-10
毺g/mL showed free radical scavenging activity in dose
dependent manner. Maximum percentage inhibition of
DPPH radicals was about 80% at 10毺g/mL concentration.
The Inhibitory concentration (IC50) value of Vitis vinifera was
found to be 5.73毺g/mL and that of Vitamin C was 8.46毺g/mL,
BHA was 2.82毺g/mL and BHT 7.47毺g/mL (Figure 4).
4. Discussion
Plant materials are used throughout developed and
Table 4.
Preliminary phytochemical analysis of various extracts.
Chemical
Constituents
Petroleum ether extract Acetone extract Chlorofrorm extract Alcoholic extract Hydro-alcoholic extract Aqueous extract
Alkaloids - - - - - -
Carbohydrates - - - + + +
Glycosides - - - - - -
Phenolics - - - + + +
Flavonoids - + + + + +
Proteins - - + + + +
Saponins - - - - - -
Mucilage - - - - - -
Resins + + + + - -
Lipids + + + + - -
+: present; -: absent.

5. Ghulam Mustafa Khan1, Feroz Ahmad./Asian Pacific Journal of Tropical Biomedicine (2012)1-6
5
developing countries as home remedies, over-the-counter
Methanolic Chloroform Pet. Ether Methanolic Chloroform Pet. Ether
Figure 3. HPTLC profile of different extracts of Vitis vinifera L. seeds
A: Chromatogram at daylight sprayed by anisalydehyde sulphuric
acid; B: Chromatogram scanned at 366 nm.
3A 3B
drug products and raw materials for the pharmaceutical
industry, and represent a substantial proportion of the
global drug market. It is therefore essential to establish
internationally recognized guidelines for assessing
their quality[24]. The quantitative determination of
some pharmacognostic parameters is useful for setting
standards for crude drugs[25]. Macroscopic or morphological
investigation gives an idea about specific characteristic
of crude drug and which will be considered as first step
for establishment of identity of crude drug. Microscopic
investigation provides detailed information about crude drug
by analyzing organized structure[26]. Microscopic study used
to identify closely similar varieties of crude drug which is
difficult to identify by their morphological characters and
also useful for identification of adulteration[27].
Pharmacognostic standardization including physico-
chemical evaluation is meant for identification,
authentication, and detection of adulteration and also
compilation of quality control of crude drugs[28]. Hot
alcoholic extract was found to be having highest extractive
value of 25.99%. The analysis of ash values suggested the
presence of inorganic substances in considerably normal
amounts. Heavy metal analysis and microbial load for the
drug was in normal values as set down by WHO[29]. The
moisture content of the seeds is high, thus the growth of
bacteria, fungi or yeast can occur easily, as the general
requirement for moisture content in crude drugs is not more
than 14% w/w[30]. When physical and chemical methods are
inadequate, the plant material may be identified from their
adulterants on the basis of fluorescence characteristics.
Behaviors of the powdered drug with different chemical
reagents and preliminary phytochemical analysis are
helpful for detection of various phytoconstituents[31]. The
height of the foam in every test tube was less than 1 cm,
so the foaming index is less than 100 (Glycyrrhiza glabra).
Preliminary phytochemical analysis mainly revealed the
presence of carbohydrates, phenolics, proteins, resins,
lipids and flavonoids. In the last two decades HPTLC method
has emerged as an important tool for the qualitative and
quantitative phytochemical analysis of herbal drugs and
formulations[32]. Vitis vinifera at different doses, i.e. 1.25-10
µg/mL showed free radical scavenging activity in dose
dependent manner. Maximum percentage inhibition of
DPPH radicals was about 80% at 10 µg/mL concentration.
The amount of phenolic components was calculated as gallic
acid equivalents and was found to be 780 µg/mg; indicating
considerable free radical scavenging activity. The IC50 values
show that the seeds of Vitis vinifera L. are better antioxidant
than butylated hydroxyl anisole but less effective as
compared to vitamin C and butylated hydroxyl toluene.
In conclusion, the present study was undertaken with
an aim of pharmacognostic standardization, preliminary
phytochemical and antioxidant evaluation of seeds of Vitis
vinifera L. providing useful information, which may help in
authenticating the genuine plant along with the nature of
phytoconstituents present in it. This work could be useful for
the adulterant resolution of doubtful specimen, compilation
of a suitable monograph for its proper identification.
Conflict of interest statement
Table 5.
DPPH scavenging activity at various concentrations of Vitis vinifera
seeds.
Concentration (µg/mL) % scavenging of DPPH*
1.25 17.43
2.5 25.68
5 43.11
6 49.54
7 58.71
8 71.55
9 76.14
10 79.81
*1, 1-diphenyl-2-picryl-hydrazyl.
9
8
7
6
5
4
3
2
1
0
IC50 values
2.82
7.47
8.46
5.74
IC50
BHA BHT vit.c V.vinifera
ug/mL
Figure 4. Histogram comparing Inhibitory concentration (IC 50) values
of Vitis vinifera L. seeds with Butylated Hydroxy Anisole (BHA),
Butylated Hydroxy Toluene (BHT) and Vitamin C as standards.

6. Ghulam Mustafa Khan, Feroz Ahmad./Asian Pacific Journal of Tropical Biomedicine (2012)1-66
We declare that we have no conflict of interest.
Acknowledgements
The research was funded by Faculty of Pharmacy, Jamia
Hamdard University (mpharm/jh/08).
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