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Phytochemical Screening and Chromatographic Fingerprint Analysis of Vitex

altissima Linn. Leaf Extract by TLC and HPTLC Techniques

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International Journal of Scientific Research and Review ISSN NO: 2279-543X

Phytochemical Screening and Chromatographic Fingerprint Analysis of

Vitex altissima Linn. Leaf Extract by TLC and HPTLC Techniques.

E. Rajesh1, M. Gopalakrishnan2, G. Priya3 & T. Sekar4*.

1,2,3& 4 Post Graduate and Research Department of Botany, Pachaiyappa’s College, Chennai,
Tamil Nadu,India.

*Corresponding authors email: tsekar_bot@yahoo.com

ABSTRACT

Most of the traditional medicinal plants in India are not scientifically validated. Scientific
evaluation along with traditional knowledge is essential to obtain effective drugs for commercial
purpose. The present study was aimed to phytochemical screening, Thin Layer Chromatography
(TLC) and High performance Thin Layer Chromatography (HPTLC) analysis of hexane,
chloroform, ethyl acetate, ethanol and aqueous leaf extracts of Vitex altissima. Leaf extracts were
prepared according to the polarity of the solvents. i.e. hexane, chloroform, ethyl acetate, ethanol
and aqueous. Preliminary phytochemical screening involved the qualitative methods to detect the
presence of carbohydrate, flavonoids, saponins, alkaloids, phenols, etc. The present study is to
establish the chemical fingerprint through TLC & HPTLC studies were carried out as per the
standard method. The TLC shows ethanolic extracts of Vitex altissima at UV short and long
wavelength 254 nm and 366 nm using Toluene: Ethyl Acetate: Formic acid (5:1.5:0.5) ratio and
it gives the Rf values of the same. The HPTLC finger print profile was performed at 254nm and
13 peaks were observed. The results of qualitative phytochemical screening confirm the presence
of carbohydrate, phenols, flavonoids, etc. the HPTLC & TLC analysis developed to help in
proper identification and quantification of marker compounds.

KEY WORDS: Vitex altissima, HPTLC, TLC, phytochemical screening, extraction

I. INTRODUCTION

Medicinal Plants have long provided humanity with medicine with traditional products
once serving as the source of all drugs1.The rural population of the country is more disposed to
conventional ways of treatment, because of its easy accessible and cheaper value2. According to
world health Organization (WHO) more than 80% of the World’s population mostly in

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developing countries depend on traditional plant based medicines for their primary healthcare
needs3. India has about 45,000 plant species and among them many have been claimed to possess
medicinal properties. The need for scientific validation of these useful medicinal plants are very
essential 4, and standardization of the plant materials is need of the hour. Several pharmacopoeia
containing monographs of the plant materials describe only the physicochemical parameters.
Hence, the modern methods describing the identification and quantification of active constituents
in the plant material may be useful for proper standardization of herbals and its formulations.
Also the WHO has emphasized the need to ensure the quality of medicinal plant products using
modern technique and applying suitable standards5. HPTLC offers better resolution and
estimation of active constituents can be done with responsible accuracy in a shorter duration 6.

Vitex altissima Linn is large deciduous trees grow up to 35m tall, widely distributed
Indomalaysia, Indochina and Sri Lanka; in the Western Ghats; throughout South India, in
evergreen and deciduous forest. The bark is grayish in colour lenticellate, scaly blaze yellowish.
Branch lets young branchlets quadrangular, pubescent, lenticellate and the leaves compound,
usually trifoliate or rarely 5-foliate with 2 small leaflets, opposite, leaflets sessile, narrow elliptic,
apex acuminate, base cuneate, margin serrate of entire, chartaceous, gloucus and minute
pubescent or plabrous beneath, midrib canaliculated above, secondary nerves 10-20 pairs,
tertiary nerves reticulo-percurrent flower are inflorescence terminal panicles, minutely
pubescent, flowers zygomorphic, blue white tinged. The fruits and seeds are drupe, smooth,
globose 0.9 cm across purplish black, containing hard seeds7,8. Flavonoids, triterpenoids, lignans,
iredoids 9,10, n-Hexadecanoic acid, 9, 12-octadecadienoic acid, Squalence 11, Phytoconstituents
were present in this medicinal plant.

According to ayurvedic system of medicine, the plant is believed to pacify vitiated kapha,
Vata, inflammation, wounds, ulcers, allergy, eczema, prutitus, worm infestation, urinary system
diseases, stomatitis, emaciation and used to treat Cardiac disease, anorexia, blindness, leprosy
and worm infestation, digestive, wound healing 12, rheumatic swelling and chest pain and it has
also anti-inflammatory, antibacterial and antioxidant activities 13.

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2. MATERIALS AND METHODS

2.1 Collection and Authentication of the selected plant material

The medicinal properties of plants have been investigated in recent scientific

developments throughout the World, due to their potent pharmacological activities, the plant
Vitex altissima Linn. Leaf extract was selected for preliminary phytochemical screening and
TLC, HPTLC finger printing of various profiles like flavonoids, Phenols, carbohydrate, saponins
and alkaloids.

The leaves of selected plant were collected from Kolli hills, Namakkal District, Tamil
Nadu, and one of the co-author were collected the plant Dr. A. Selvaraju, Department of Botany,
SVM College, Uthangarai, Tamil Nadu. Subsequently identified by Dr. T. Sekar, Associate
Professor, Department of Botany, Pachaiyappa’s College, Chennai, Tamil Nadu.

2.2 Extraction of plant material:

The collected fresh leaves were dried under shade and then pulverized with an electric
blender .The pulverized powder was passed through sieve to clear the leaf veins and stored in air
tight container for further use. Two fifty grams of powdered plant material were soaked for the
sequential extraction in hexane, chloroform, ethyl acetate, ethanol, aqueous and incubated for 48
hrs with 750ml each. The extracts were filtered using Whatmann No.1 filter paper and filtrate
was concentrated using a vaccum rotary evaporator (Super fit- ROTAVAP. India) The sticky
extract was obtained and dried in an oven at 32ºC and stored in vials at 4ºC for further analysis
and yield percentage was calculated as 14,
WE-WC
Percentage yield = __________ X 100
WE
Where, WE - Weight of the Extract
WC - Weight of the tube without extract

2.3 Phytochemical analysis of leaf extracts

The extracts of leaves of Vitex altissima Linn was subjected to various tests to determine
the presence of different phytoconstituents as per the experimental protocols stated by Sofowora
(1993) 15, Trease (1989) 16, Evans (1997) 17, Mace (1963)18 and Kokate (1999) 19.
.

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2.4 Chromatographic Fingerprints Techniques

Thin Layer Chromatographic Method

The TLC profiling was performed as per described by Biradar et al., 201320. The purity
of each eluted sample was tested by using TLC method. It is a technique used to separate wide
range of compounds of biochemical interest. The ethanolic extract was subjected to thin layer
chromatography. Four grams of Vitex altissima powder were soaked in 40ml of 95% ethyl
alcohol and kept it overnight. The extracts are boiled on water bath for 10 minutes. Then filtered
and concentrated and made up to 10 ml standard volumetric flask 2µl, 4 µl, and 6 µl of this
solution was applied on (Merck) aluminium plate 60F254 precoated with silica gel of 0.2cm,
thickness and plate was developed with a solvent system of Toluene: Ethyl acetate: Formic acid
(5:1.5:0.5). The TLC plate was visualized under 254nm and UV 366nm and photos were taken.
Then the plate was scanned at 254nm before the plate was dipped in vanillin sulphuric acid and
kept in oven at 105°C till the color of the spots appeared.

2.5 Development of the chromatogram

After the application of the sample on the plate, the plates were kept in TLC glass
chamber (solvent saturated) than mobile phase was allowed to move through adsorbent phase up
to 3/4th of the plate. TLC was performed for alkaloids, flavonoids, tannins and phenols, solvent
system and confirmatory tests

2.6 High Performance Thin Layer Chromatography (HPTLC)

High Performance Thin Layer Chromatography (HPTLC) was carried out to verify the
presence of constituents in plant extract under investigation. HPTLC studies were carried out
following the method of Wagner et al 21.

2.7 Test solution preparation:

The given plant samples of 2 mg each weighed accurately in an electronic balance

dissolved is 30 µl of the respective solvent and centrifuged. This solution was used as test
solution for HPTLC analysis. The sample applications were prepared. The spot developments
were developed up to 90nm.

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2.8 Photo Documentation:

The developed plate was dried by hot air oven to evaporate solvents from the plate. The
plate was kept in photo-documentation chamber using visible light, UV-254nm and UV-366 nm.

2.7 Derivatization

The developed plate was sprayed with respective spray reagent and dried at 100ºC in Hot
air oven. The plate was photo-documented in Visible light and UV 366nm mode using Photo-
documentation (CAMAG REPROSTAR 3) chamber.

2.8 Scanning

After derivatization, the plate was fixed in scanner stage (CAMAG TLC SCANNER 3)
and scanning was done at UV 366nm. The peak table, peak display and peak densitogram were
noted. The software 1.3.4 was used.

3. Results & Discussion:

3.1 Yield Percentage

Leaves of Vitex altissime were collected and shade dried. Dried leaves were coarsely
powdered and soaked in hexane, chloroform, ethyl acetate, ethanol and aqueous for 48hrs. Then
they were filtered and the filtrate was processed in vacuum evaporator to recollect the solvents
and extracts. The extracts were prepared by complete drying of solvent and the percentage of
yield (w/w) of the dried extracts were analyzed, whereas the highest yield was found to be in the
aqueous extract (36.24%) followed ethyl acetate (34%), chloroform (33.8%), ethanol (19.12%),
and lowest yield found in hexane (12%) (Table -1).

Table 1. Percentage of leaf part extracts values of Vitex altissima

Total weight of Percentage of total plant
Sl. No Solvents
plant extract (g) extract (%)
1 Hexane 3 12
2 Chloroform 8.45 33.8
3 Ethyl acetate 8.50 34
4 Ethanol 4.78 19.12
5 Aqueous 9.06 36.24

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3.2 Phytochemical screening:

The extract of leaf part of Vitex altissima was subjected to preliminary phytochemical
analysis viz., primary metabolities like, carbohydrates and secondary metabolite analysis like;
alkaloids, flavonoids, saponins, phenols, tannins, glycosides, terpenoids, coumarin,
phytosteroids, phytobatanins, anthraquinones were tested. Preliminary phytochemical analyses of
the leaf extract of Vitex altissima were tabulated in Table- 2.

In the extract of leaf of Vitex altissima, the carbohydrates were observed in hexane,
chloroform, ethyl acetate, ethanol and aqueous. Tannins were not observed in any solvent.
Saponins were observed in hexane, chloroform, ethanol and aqueous (more intense). Flavonoids
were observed in hexane and chloroform. Alkaloids were observed in hexane, chloroform, ethyl
acetate, ethanol and aqueous. Quinones were observed in ethanol (more intense) and aqueous.
Glycosides were not observed in any solvents. Cardiac glycosides were observed in ethyl acetate,
ethanol and aqueous. Terpenoids were observed in ethanol. Phenols were observed in ethyl
acetate and aqueous. Coumarins were observed in ethanol. Phytosteriods were observed in
hexane and chloroform. Phylobatannins and anthraquinones were found to be absent all solvent.

Table 2. Preliminary Phytochemical analysis on different solvent extracts of

Vitex altissima - Leaf part.
Sl.No Test Name Observation for different solvent extract
Hexane Chloroform Ethyl Ethanol Aqueous
acetate
1 Carbohydrates +++ +++ +++ +++ +
2 Tannins - - - - -
3 Saponins ++ + - ++ +++
4 Flavonoids ++ + - - -
5 Alkaloids ++ + ++ + +
6 Quinones - - - +++ +
7 Glycosides - - - - -
8 Cardiac glycosides - - + ++ +
9 Terpenoids - - - ++ -
10 Phenols - - ++ - +
11 Coumarin - - - ++ -
12 Phytosteroids + + ++ - - -
13 Phylobatannins - - - - -
14 Anthraquinones - - - - -
-absent, + present, ++ moderate, +++ strongly present

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According to Mathai, 200022, phytochemicals are accumulated in different parts of the

plants, such as in the roots, stems, leaves, flowers, fruits or seeds in different compositions and
are found to protect plants from disease and damage and also plays an important role in plant’s
colour, odour and flavor. Generally, the chemicals from plants can protect plant cells from
environmental hazards such as pollution, stress, drought, UV exposure and pathogenic attack
which are popularly known as phytochemicals.

3.3 TLC of ethanolic extract of Vitex altissima Linn.

The TLC photo documentation shows the ethanolic extract of Vitex altissima at UV short
wavelength 254nm and wavelength 366nm using Toluene: Ethyl acetate: Formic acid (5:1.5:0.5) ratio and
its gives the Rf values. For 2µl concentration, 2 spots were observed at 254nm, 4 spots were observed at
366nm and 5 spots were observed derivatization with vanillin- sulphuric acid. Finally, for 6µl of
concentration, 4 spots were on served at 254nm and 5 spots were seen at 366nm and 6 spots were
observed derivatization with vanillin-sulphuric acid which was explained in Table 3 and Fig 1.

Table.3 Rf values and colour of spots of the investigated plant extracts.

S. No UV UV With Vanillin Sulphuric

254 nm 366 nm Acid spray reagent
Colour Rf Colour Rf Colour Rf
1 Green 0.14 Red 0.14 Violet 0.14
2 Green 0.17 Blue 0.17 Violet 0.21
3 Green 0.28 Red 0.24 Violet 0.24
4 Green 0.31 Red 0.26 Violet 0.29
5 Green 0.67 Fluorescent red 0.31 Violet 0.35
6 Green 0.71 Blue 0.34 Violet 0.44
Red 0.40 Violet 0.57
Red 0.42 Violet 0.62
Red 0.52 Green 0.67
Red 0.54
Fluorescent red 0.70
Red 0.74
Red 0.87
Fluorescent red 0.91

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Fig. 1 Chromatogram of TLC photo documentation of alcoholic extract of Vitex altissima

Normally, Rf values of the compounds are responsible for their polarity which are also
helps in selecting a suitable solvent system for further isolation of any compound from the plant
extracts using chromatographic and spectroscopic techniques20. Compound having high Rf value
in less polar solvent system shows low polarity while those with a low Rf value shows high
polarity (Talukdar et al 2010)23.

3.4 HPTLC of ethanolic extract of Vitex altissima:

The results from HPTLC finger print scanned at wavelength 254nm. Out of there
different concentration, 6µl was taken for further HPTLC study. The HPTLC finger print profile
was observed at 254nm and 13 peaks. Their peaks showed maximum peak area i. e 4th peak with

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Rf 0.26 value of 28.99% peak area and 12th peak with Rf 0.89 value with 27. 78 % of peak area
which was shown in Fig. 2.

Fig.3 HPTLC densitometric scan of alcoholic extract of Vitex altissima of UV254nm.

HPTLC fingerprint analysis plays an important role in determining quality of medicinal

plants and their related products because of its simplicity and reliability and also its outcome can
be directly evaluated by visual inspection (Reich et al 2006)24. The main use of HPTLC
fingerprint analysis is for identification and authentication of medicinal plants (Gunalan et al
2012)25.

The medicinal plants are rich in secondary metabolites and among the vast array of
bioactive compounds carbohydrate, alkaloids, flavonoids, saponins, phenols are in high interest.
Several methods are available for separating plant constituents; the chromatographic procedure is
the most commonly used techniques for general application. The present TLC and HPTLC

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studies confirmed the presence of active metabolites in the solvent extract of the study species
Vitex altissima 26.

The developed TLC and HPTLC methods will help the manufacture for quality control
and standardization of herbal formulations, such finger printing is useful in differentiating the
species from the adulterant and act as biochemical markers for this medicinally important plant
species in the Pharmaceutical industries and plant systematic studies 27, 28.

4. CONCLUSION:

In this present study of preliminary phytochemical screening showed the presence of

carbohydrate, alkaloid, saponin, flavonoid and phenols compounds. The preliminary TLC and
HPTLC analysis of ethanolic extracts of Vitex altissima confirmed the presence of various
phytochemicals. HPTLC finger printing profile is very important parameter of herbal drug
standardization for the proper identification of medicinal plants. These methods were also
employed to analysis commercial samples to illustrate their application in qualitative and
quantitative determination, demonstration their feasibility in the quality control of
phytoconstituents from mentioned herbal drugs and formulations. For further study, with the help
of developing analytical method pure active chemical compound should be isolated and
identified on the basis of standard protocol.

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