Inter. J. of Phytotherapy / Vol4 / Issue 1 / 2014 / 1-6.

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Print ISSN - 2249-7730

International Journal of Phytotherapy

www.phytotherapyjournal.com

PHYSICO-CHEMICAL STANDARDIZATION AND

PHYTOCHEMICAL SCREENING OF POTENTIAL MEDICINAL
HERB: VETIVERIA ZIZANIOIDES (ROOTS)
Snigdha Mishra*1, Satish Kumar Sharma1, Aleza Rizvi2, Abhineet Chowdhary3
1

Department of Pharmacology, 3Department of Pharmacology, Sunder Deep Pharmacy College, Ghaziabad, India.
2
Department of Pharmacognosy, Integral University, Lucknow, India.

ABSTRACT
Vetiveria zizanioides L. Nash is commonly known as the Khas-Khas, Khasor Khusgrass in India, is a
perennial grass with thick fibrous adventitious roots which are aromatic. The roots of Vetiveria zizanioides, used in
curing different types of diseases like bronchitis, foul ulcers, sting bites, burn, inflammation, fungal infections and
rheumatism. Herbal drug standardization is an essential step in order to assess the quality of drugs, based on the
concentration of their active principle, physical and chemical standards. The present work attempts to evaluate the
physicochemical and preliminary phytochemical studies on the roots of Vetiveria zizanioides(Linn.) familyPoaceae. The herbal standardization was carried out on the basis of organoleptic properties, physical characteristics,
and physico-chemical properties. Physicochemical parameters including ash values, extractive values, moisture
content were evaluated. Preliminary phytochemical studies of the powdered fruit drug revealed the presences of
alkaloids, flavanoides, phytosterols, tannins, saponin, essential oils, proteins and carbohydrates. Thin layer
chromatographic studies also had been done on different extracts. The total ash, acid insoluble ash and water soluble
ash were found to be 6.33, 4.83 and 0.66% w/w respectively. The alcohol and water soluble extractive values (cold)
are 15.46, 14.20%w/w respectively. The pH of 1 and 10% aqueous solution was found to be 4 and 6.2 respectively.
Various morphological characteristics were also reported.
Key words: Vetiveria zizanioides, Khus, Standardization, Phytochemical, Physico-chemical, TLC.

INTRODUCTION
Vetiver grass is also cultivated for the production
of a commercially important essential oil used in
perfumery and aromatherapy [1]. This tufted grass grows
throughout the plains of India ascending up to an
elevation of 1200 m. Having wide ecological amplitude,
this grass grows in a wide variety of ecological habitats
covering all bio-geographic provinces of India. No
wonder that this is one grass which has been extensively
used by almost all the tribes [2-4]. Vetiver is the most
versatile, multifarious grass with immense potential. It is

a plant known for its ability to produce essential oil from

the roots which is especially used in the perfume industry.
Various tribes use the different parts of this grass for
several complications like mouth ulcer, boil, epilepsy,
burn, snakebite, fever, rheumatism, headache, etc.
Because of these activities khas has found vast
applications making it a green treasure [5]. The roots of
vetiver grow downwards upto 2 meters 4 meters in
depth. Presently countries like India, Haiti, and Reunion
are the largest producer of vetiver worldwide [6].

Corresponding Author:-Snigdha Mishra Email: snigdha2112@gmail.com

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MATERIAL AND METHODS

Collection and authentication of plant material
Roots of Vetiveria zizanioides L. Nash (5kg) was
purchased from whole sale supplier of plants opposite to
SampoornaNand Sanskrit VishwaVidyalay, Sigra
Varanasi, U.P. during the month of October 2012. Sample
of plant material was given to Department of Botany
Banaras Hindu University, Varanasi -221005, India for
identification and Taxonomic authentication. The text
report from BHU confirmed the authenticity of plant
sample. Specification no.: 2012/Poa/25.
Morphological
characteristics
General
appearance of the dried roots is given in fig.1. Detailed
study of the morphological characters can be helpful in
differentiating them. The macroscopy of a drug includes
its visual appearance to the naked eye. It depends to a
large extent on the part of the plant from which the drug is
obtained. For each particular morphological group a
particular systemic examination can be carried out. Some
organoleptic characteristics of dried roots of Vetiveria
zizanioides are described in Table 1.

Weight of total ash

% Total ash value =

Wt of acid insoluble ash

% acid insoluble =
ash value

X 100
Wt of crude drug taken
Wt of
total ash

% water soluble =
ash value

Wt of water
insoluble ash
X 100

Weight of crude rug taken

Phytochemical screening
The Phytochemical examination of both extract
(ethanolic and aqueous) was performed by the standard
methods and shown the presence of various
phytochemical
constituents
[9-13].
Preliminary
phytochemical screening which was performed to
establish a chemical profile of a crude drug.

Preparation of plant extract

The roots of the plant were dried under shade,
separated and made to dry powder. It was then passed
through the 40 mesh sieve. Then 150 g of weighed
powered roots of Vetiveria zizanioides was packed in
Soxhlet apparatus (Fig.2) and extracted with ethanol and
distilled water for 36 hrs and completion of extraction
was confirmed by pouring a drop of extract from the
thimble on a filter paper, which does not shown the
presence of any oil spot on that. Beside the hot extraction
process the plant roots were also kept for maceration
process (Fig. 3). After complete extraction the solvent
was evaporated and concentrated to dry residue. The
various colour& consistency of extracts of Vetiveria
zizanioides roots were observed. Determination of pH
was done by using Systronics Digital pH Meter, MKVI
with the preparation of 1% &10% extract solution.

Powder drug observation with different chemical

reagents
The powder drug with different chemical
reagents show different colour when seen on naked eye.
Result obtained is tabulated in table 5.
Thin Layer Chromatography
Slurry of silica gel G was prepared in distilled
water and poured over a glass plates to form a thin layer.
The prepared plates were air dried for setting and then
kept in an oven at 100-120C (30min) for activation [20].
The extracts were dissolved in respective solvents and
spotted over an activated plate (1cm above from the
bottom). The spotted plates were kept in a previously
saturated developing chamber containing mobile phase,
and allowed to run 3/4th of the height of the prepared
plate [21]. A number of developing solvent system was
tried, but the good result and the maximum number of
spots were obtained in the solvent system summarized in
the Table mentioned below. The plates were air dried and
number of spots were noted and Rf value were calculated.
Spots were visualized by respective spraying agents.
(1) For determination of Alkaloides (Table 6.1)
Spraying agent- Dragendroffs Reagent, Orange brown
color is produced
Solvent system- Methanol: Ammonium hydroxide
(2) For determination of Saponin (Table 6.2)
Spraying agent- Conc. HCl , Dark brown is produced
Solvent system- Butanol: Water (1: 1)
(3) For determination of Flavanoides (Table 6.3)
Spraying agent- Phenol Sulphuric Acid (H2SO4), Greenish
black colour is produced

Physicochemical evaluation
The crude dried plant material was subjected to
the physico-chemical evaluation. The various parameters
such as extractive value, moisture content, ash value
including acid insoluble and water soluble ash etc [7,8]
were evaluated by using standard procedures. The Loss
on Drying (L.O.D.), Ash Values likes (Total Ash, Acid
insoluble ash, Water soluble ash) Water soluble
extractive, alcohol soluble extractive of dried root powder
are given in table 3.Values were evaluated by following
formulas.
Loss in weight of the sample
% moisture content =

X 100
Weight of crude drug taken

X 100
Weight of sample

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Solvent system- Butanol: Acetic acid: Water: Ether

(9:6:1:3)
(4) For determination of Glycosides (Table 6.4)
Spraying agent- Chloroform, Orange color is produced
Solvent system- Ethyl acetate: Pyridine: Water (5:1:4).

Distance travelled by the solute front

Rf= ----------------------------------------------------Distance travelled by the solvent front
The result of Thin Layer chromatography was
reported in respective tables as mentioned below.

The above prepared sample solution were

applied on coated TLC plate using capillary tubes and
developed in a TLC chamber using suitable mobile phase.
The developed TLC plates were air dried. They were later
sprayed with different spraying reagent and were placed
in hot air oven for 1 min for the development of color in
separated bands. The R f values were calculated for
different samples.

RESULT AND DISCUSSION

Dried roots of Vetiveria zizanioides were
subjected to various analytical techniques. Botanical
parameters revealed that, the fresh roots were yellowish in
colour and dried roots were yellowish brown in colour.
Outer surface shows presence of markings & wrinkles
with yellow colored patches and characteristic in taste
(Table 1). Characteristics of the different extracts
(ethanolic and aqueous) were described in Table 2.

Table1. Organoleptic evaluation of roots of Vetiveria zizanioides

S.No
Character
When fresh
1
Colour
Pale yellow
2
Odour
Pleasant, aromatic
3
Taste
Characteristic
4
Shape
Twisted 2- 4m
5
Texture
Rough, non fragile

After shade drying

Yellowish brown
Pleasant, aromatic
Characteristic
twisted
Rough, non fragile(due to presence of essential oils)

Table 2. Color and consistency of ethanolic and aqueous extract of Vetiveria zizanioides
S.No
Extract
Color
Consistency
Ethanolic extract of
1
Yellow- brown
Semi solid (sticky)
Vetiveria zizanioides
Aqueous extract of Vetiveria
2
Dark brown
Semi solid (non sticky)
zizanioides

Solubility
Water insoluble
Water soluble

Table 3.Physico chemical parameters of dried roots of Vetiveria zizanioides

S.No
Particulars
Values
Total ash value
6.33% w/w
1
Ash Value
Water soluble ash
4.83% w/w
Acid insoluble ash
0.66% w/w
Hot extractive value (soxhlet)EEVZ
4.18% w/w
Hot extractive value of AEVZ
2.72% w/w
2
Extractive value
Cold extractive value(maceration) EEVZ
15.46% w/w
Cold extractive value(maceration) AEVZ
14.20% w/w
pH of Aqueous solution (1%)
4.0
3
Determination of pH
pH of Aqueous solution (10%)
6.2
4
Loss on drying (%w/w)
11.123% w/w
Note: AEVZ (Aqueous extract of Vetiveria zizanioides), EEVZ (Ethanolic extract of Vetiveria zizanioides)
Table 4. Preliminary Phytochemical Screening of ethanolic as well as aqueous extract of Vetiveria zizanioides
Sl.No
Test
Aqueous Extract
Ethanolic Extract
Test of sterols
1
Salkowaski Test
-ve
-ve
Libermann-Burchard Test
-ve
-ve
Tannins Test
2
Gelatin Solution
+ve
+ve
Catechin
+ve
+ve
Flavanoides Test
3
Shinoda Test
+ve
+ve

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Test of Proteins and amino acids

Millons Test
4
Biurate Test
Ninhydrine Test
Glycosides Test
Keller Killiani Test
5
Legal Test
Borntragger test
Balget Test
Phenolic Test
Ferric chloride Test
6
Lead Acetate Test
Gelatin test
2ml extract+ Dil HNO3
2ml extract + K2CrO4
Test for acidic compounds
7
Sodium Bi carbonate Test
Litmus test
Carbohydrates Test
Fehling Test
Molisch Test
8
Bendict test
Reducing sugar Test
Barford Test
Saponin Test
9
Saponification
Foam Test
Alkaloides Test
Dragandrof Test
10
Haggers Test
Wagnars Test
Mayers Test
Test for Volatile Oils
Characteristic Odour
11
Filter paper
Solubility test
(+ve) signifies present, (-ve) signifies absent.

-ve
-ve
-ve

-ve
-ve
-ve

+ve
-ve
-ve
+ve

+ve
-ve
+ve
+ve

+ve
+ve
-ve
+ve
+ve

+ve
+ve
-ve
+ve
+ve

-ve
-ve

-ve
-ve

+ve
-ve
+ve
+ve
+ve

+ve
-ve
+ve
+ve
+ve

+ve
+ve

+ve
+ve

-ve
+ve
+ve
+ve

+ve
+ve
+ve
+ve

+ve
+ve
+ve

+ve
+ve
-ve

Table 5. Powdered drug analysis with different chemical reagents

S.No
Treatment
1
Powder as such
2
Powder+ Conc. HCl
3
Powder + Conc.HNO3
4
Powder + Conc.H2SO4
5
Powder + Glacial acetic acid
6
Powder + Iodine solution
7
Powder + 5%KOH
8
Powder + Picric acid
9
Powder + Ammonia
10
Powder + 5%ferric chloride solution
11
Powder + 5%NaOH
Table 6.1 TLC for evaluation of Alkaloides
Extract
Solvent system
Acetone: Water (5:5)
Aqueous
Acetone: Water (5:5)
Ethanolic

Detecting agent
Dragandrof s reagent
Dragandrof s reagent

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Observation
Yellowish brown
Reddish brown
Light Orange
Black
Light brown
Yellowish brown
Yellow
Dark Yellow
Yellowish Brown
Blackish Brown
Yellowish brown
No. of spots
2
2

Rf values
0.17, 0.61
0.13, 0.93

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Table 6.2 TLC for evaluation of Saponins

Extract
Solvent system
Butanol : Water (1:1)
Aqueous
Butanol : Water (1:1)
Ethanolic

Detecting agent
Conc. HCl
ConcHCl

Table 6.3 TLC for evaluation of Glycosides

Extract
Solvent system
Ethyl acetate: Pyridine : Water(5:1:4)
Aqueous
Ethyl acetate: Pyridine : Water
Ethanolic
Table 6.4 TLC for evaluation of Flavanoids
Extracts
Solvent system
Butanol: Acetic acid: Water:
Aqueous
Ether (9:6:1:3)
Butanol: Acetic acid: Water:
Ethanolic
Ether (9:6:1:3)

No. of spots
2
2

Detecting agent
Chloroform
Chloroform

Rf values
0.3, 0.72
0.3, 1.01

No. of spots
2
2

Rf values
0.60, 0.22
0.282, 1.25

Detecting agent

No. of spots

Rf values

Phenolic Sulphuric Acid

Phenol Sulphuric Acid

1.49

Fig. 2. Powder form of Vetiveria zizanioides

Fig. 1. Dried roots of Vetiveria zizanioides

Fig. 3. Assembly of soxhlet extraction

Physicochemical evaluation
Loss on drying, Ash Values likes (Total Ash,
Acid insoluble ash, Water soluble ash) Water soluble
extractive, alcohol soluble extractive values of powder of
dried roots are tabulated in Table 3.

Fig. 4. Maceration process

carbohydrates, alkaloids, tannins, essential oils etc. in
different extract obtained by using different solvents [19]
Results tabulated in table 4.

Thin Layer Chromatography

The different solvents of different polarities were
prepared as a mobile phase for developing a TLC system
for identification of constituents in aqueous and ethanolic
extracts. The solvent system, number of spots and Rf
values with detecting agents are shown in table 6.1 to 6.4.

Preliminary phytochemical screening

Preliminary phytochemical screening which was
performed to establish a chemical profile of a crude drug
was a part of chemical evaluation [14-18]. These
phytochemical tests shown the presence of saponins,

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CONCLUSION
Preliminary phytochemical as well as various
aspects of the roots sample were studied and described
along with physicochemical parameters and TLC profile
studies in authentification for quality control. The dried
roots of Vetiveria zizanioides exhibit a set of diagnostic
characters which will help to identify the drug and its
medicinal importance in dried condition.

ACKNOWLEDGEMENT
The authors express their sincere thanks to the
Mahamaya Technical University, Noida, Uttar Pradesh
and also grateful to the Chairman and Director of Sunder
Deep Pharmacy College, Ghaziabad for giving all
encouragement and valuable support to carry out the
research work.

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