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International Journal of Research in Pharmaceutical and Biomedical Sciences

ISSN: 2229-3701

_________________________________________Research Article

Comparative Standardization Study of Two Marketed


Ashwagandha Churna Formulation
Pallab Dasgupta and Amartya De*
BCDA College of Pharmacy and Technology, 78 Jessore Road (S), Hridaypur, Barasat,
Kolkata, West Bengal, India.
__________________________________________________________________________________
ABSTRACT
In the few decades, there has been exponentionally growth in the field of herbal medicines. Most of the
traditional system of medicine are effective but they lack standardization. So there is a need to develop a
standardization technique. Standardization of herbal formulation is essential in order to assess the quality,
purity, safety and efficacy of the drug. ashwagandha is a reputed drug mentioned in the scientific books of
Ayurveda for the treatment of stress, hypertension, sleeping disorders and also as rejuvenative. The present
research studt deals with the comparagtive standardization of two marketed ashwagandha churna formulation
from Dabur and Dhaka Oushodhalay. The standardization of this formulation, the organoleptic characters,
physical properties, the various physic-chemical properties such as moisture content, ash values, extractive
values were carried out. Thin layer chromatography and heavy metal content study were also carried out to
ascertain the quality, purity and safety of this herbal formulation.
Key Words: Standardization, Ashwagandha churna, Physico-chemical parameters, Thin layer chromatography.
and major medical illnesses. One example is
INTRODUCTION
foxglove, which contains cardiac glycosides, and
Nature always stands as a golden mark to
serves as a classic treatment for congestive heart
exemplify the outstanding phenomena of
failure. Even now, physicians still use many drugs
symbiosis. Today about 80% of people in
that possess botanical origins. Huxtable notes that
developing countries still relay on traditional
one-quarter of the prescriptions currently written in
medicine based largely on the different species of
the United States are for plant products, while one
plants for their primary health care. About 500% of
quarter are for agents based on botanical
plants with medicinal uses are mentioned in ancient
compounds. The therapeutic potential of herbal
literature and 800 plants have been used in
medicines cannot be ignored and is highlighted in
indigenous system of medicine. The various
the three examples provided next.7
indigenous system such as ayurveda, siddha, unani
use several plant species to treat different
Advantages of Herbal Medicine
ailments.1,2,3
They have large amount of use.
Herbal medicines make up an important
They have better patient tolerance as well as
component of the trend toward alternative
acceptance.
medicine. A Harvard study recently found that one
The medicinal plants have renewable source of
in three respondents acknowledged use of at least
cheaper medicines.
one alternative therapy within the past year.4
Improvements in the quality, efficacy and
Extrapolated, these findings suggest that up to
safety of herbal medicines with the
$13.7 billion were spent in 1990 alone for these
development of science and technology.
treatments.4 Tyler defines herbal medicines as
Prolong and apparently uneventful use of
"crude drugs of vegetable origin utilized for the
herbal medicines may offer testimony of their
treatment of disease states, often of a chronic
safety and efficacy.
nature, or to attain or maintain a condition of
they are cheap in cost.
improved health."5 Current demands for herbal
they are not harmful.
medicines have resulted in an annual market of
they are more effective than any synthetic
$1.5 billion and increasingly widespread
drug.
availability.6
Throughout the world herbal medicines have
provided many of the most potent medicines to
Potential Benefits of Herbal Drugs
the vast arsenal of drugs available to modern
Historically, herbal medicines have played a
medical science, both in crude form as well as
significant role in the management of both minor

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International Journal of Research in Pharmaceutical and Biomedical Sciences

ISSN: 2229-3701

a pure chemical upon which modern medicines


are constructed.[8,9]

Need of Standardization
The quality control of herbal crude drug &
formulation is important in justifying their
acceptability in modern system of medicines.
Standardization of synthetic drugs offers no
problem with very well defined parameters of
analysis. It is not uncommon to have as many as
five or more different herbal ingredients in one
single formulation. The batch to batch variation
starts from the collection of the raw materials itself
in absence of any reference standard for
identification.
WHO has emphasized the need to ensure quality
control of medicinal plants products by using
modern techniques and by applying suitable
standards and parameters.10-13
Standardized products and services are valuable
User 'confidence builders' being perceived as

safe

healthy

secure

high quality

flexible
Standardization brings important benefits to
business including a solid foundation upon which
to develop new technologies and an opportunity to
share and enhance existing practices.
Standardization also plays a pivotal role in assisting
Governments, Administrations, Regulators and the
legal profession as legislation, regulation and
policy initiatives are all supported by
standardization.

Ashwagandha churna of dabur

INTRODUCTION OF THE SAMPLE

Ashwagandha churna of sadhana ausadhalayadhaka

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735

International Journal of Research in Pharmaceutical and Biomedical Sciences

Sample name
Ashwagandha churna
Biological name
Withania somnifera
Family
Solanaceae
Vernacular name
In English
In Hindi
In Bengali
In Malayalam

2
Determination of physico-chemical
parameters
Moister content
Total ash
Acid insoluble ash
Water soluble ash
Water soluble extractive
Alcohol soluble extractive
Crude fiber contents
3
Quantitative estimation of selected
phyto- constituents
Total alkaloids
4
Evaluation of Churna
Powder fineness
Bulk density
Tap density
Angle of repose
Compressibility
Hausner ratio
5
Determination of Ph
6
Establishing the safety pertaining to
Heavy metals & Microbial load

Indian ginseng, Winter cherry


Ajagandha, Kanaje
Ashwagandha
Amukkuram

The main constituents of Ashwagandha1


a>
Alkaloids.
b>
Steroidal lactones.
Among the various alkaloids

Withanine is the main alkaloid.

The other alkaloids are:


1>
somniferine
2>
somnine
3>
somniferinine
4>
withananine
5>
pseudo-withanine
6>
tropine
7>
pseudotropine
8>
cuscohygrine
9>
anferine .

MATERIALS & METHODS


Developments of standardization parameters
for ASHWAGANDHA CHURNA

15,16

Use : Ashwagandha have


1>
Anti-carcinogenic effects in animal and
cell cultures
2>
It makes the anus tingle by decreasing the
expression of nuclear
factor-kappaB, suppressing intercellular tumor
necrosis factor, and potentiating apoptotic signaling
in cancerous cell lines.
Scientific
work
done
Standardization parameters
Churna-Ayurvedic drug.17

Evaluation
of
of Ashwagandha

PLAN OF WORK
Comparative standardisation of Ashwagandha
churna formulated by Dabour pharma. & Dhaka
ausadhalaya was planned to carry out development
of quality standards for the finished marketed
formulation.The method used for the comparative
standardization was planned to be carried out as
follows:
DEVELOPMENT OF STANDARDIZATION
PARAMETERS
FOR
ASHWAGANDHA
CHURNA
1 Study of organoleptic characters
i.
Colour
ii.
Odour
iii.
Taste

Vol. 3 (2) Apr Jun2012

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1 Study of Organoleptic Characters


The polyherbal formulation is studied for
organoleptic characters like color,odour and taste
using the sensory organs of our body.
2 PHYSICO-CHEMICAL ANALYSIS18
Determination of loss and drying
10 g of the sample(without preliminary drying) was
weighed and placed in a tarred evaporating dish. It
was dried at 105 C for 5 hours, and at 1 hour
interval until difference two successive weighings
corresponded to not more than 0.25%.
Determination of Total ash
About 2 to 3 g of sample was accurately weighed
in a tarred silica dish at a temperature not
exceeding 450 C until it was free from carbon.
Then it was cooled and weighed. The percentage of
total ash was calculated with reference to the airdried drug.
Determination of Acid insoluble ash
The total ash obtained was boiled for 5 minutes
with 25 ml of dilute hydrochloric acid; the
insoluble matter obtained was collected on an ash
less filter paper, washed with hot water and ignited
to constant weight. The percentage of acidinsoluble ash was calculated with reference to the
air dried drug.
Water-soluble Ash
The ash obtained in the determination of total ash
was boiled for 5 minutes with 25 ml of water. The
insoluble matter was collected on an ash less filter

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International Journal of Research in Pharmaceutical and Biomedical Sciences

paper and washed with hot water. The insoluble ash


was transferred into a tarred silica crucible and
ignited for 15 minutes at a temperature not
exceeding 450 C. The weight of the insoluble
matter was subtracted from the weight of the total
ash. The difference in weight was considered as the
water- soluble ash was calculated with reference to
the air dried drug.
Determination of Water-soluble extractive
5 g of test sample was weighed and macerated
with 100 ml of chloroform water in a closed flask
for twenty-four hours, shaking frequently during
six hours and allowing standing for eighteen hours.
it was filtered rapidly, taking precautions against
the loss of solvent.25 ml of the filtrate was taken
and evaporated to dryness in a tarred flat bottomed
shallow dish at 1050 C, to constant weight and
weighed the percentage of water soluble extractive
was calculated with reference to the air dried
sample.
Determination of Alcohol-soluble extractive
Procedure for water soluble extractive was
followed for the determination of alcohol soluble
extractive but 90% ethanol was used instead of
chloroform water.
3 Qulitative Phytochemical Screening19,20,21
Detection of alkaloids
50 mg of solvent free extract was hydrolysed with
dil.HCL and filtered. The filtrates were tested
carefully with various alkaloid test reagents as
follows
1. Dragendroffs test
To few ml of filtrates, 1 to 2 ml of dragendroffs
reagent was added. A prominent yellow precipitate
indicates the test is positive.
2.Wagners test
To few ml of filtrates, few drops of wagners
reagent were added by the side of the test tube. A
reddish-brown precipitate confirms the test as
positive.
3. Mayers test
To few ml of filtrates, few drops of mayers
reagent were added by the side of the test tube.
White or creamy precipitates if obtained indicates
the presence of alkaloids.
4.Hagers test
To few ml of filtrates, few drops of hagers reagent
were added. A prominent yellow precipitate
indicates the test is positive.
Quantitative
estimation
of
selected
phytoconstituents
Estimation of total
alkaloids22
3 gm of the extract was weighed accurately and
transferred into a 150 ml conical flask. 100 ml of a
mixture of 4 volumes of solvent ether and one
volume of alcohol was added. 5 ml of dilute

Vol. 3 (2) Apr Jun2012

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ammonia solution was added and shaken frequently


during 1 hour. The clear solution was decanted and
filtered through cotton into a separator. The residue
was washed further with 100 ml of ether alcohol in
5 lots of 20 ml of each. 30 ml of 1N sulfuric acid
was added to the total ether alcohol solution to
make it acidic to litmus. It was shaken well and
allow to separate. The lower acid layer was run off
into another separator. The extraction was
continued first with 25 ml then with successive
quantities of each of 20 ml of a mixture of 3
volumes of 0.5N H2SO4 and 1 volume of alcohol
until complete extraction of alkaloid was effected.
The combined acid extracts were washed first with
10 ml then with 2 successive quantities each of
chloroform. Each chloroform extract was washed
20 ml of same acid alcohol mixture contained in
the other separator.
The chloroform layer was rejected and the acid
layer was transferred from the 2nd separator to the
1st separator. The acid was make alkaline with
dilute ammonia solution and 5 ml was added in
excess. It was shaken first with 25 ml then with
successive quantities of each of 20 ml of
chloroform until complete extraction of the
alkaloid was affected. Each chloroform extract was
washed with 10 ml of water contained in the 2nd
separator and filtered through cotton in a 150 ml
conical flask. The
chloroform layer was
evaporated on a water bath and evaporation was
continued after adding 2 ml of alcohol. The process
was repeated using further 2 ml of alcohol and
residue was dried on a water bath for 5 min at 1050
C.
4 Determination of physical characteristics23
Bulk density
It is the ratio of given mass of powder and its bulk
volume. It is determined by transferring an
accurately weighed amount of powder sample to
the graduated cylinder with the aid of a funnel. The
initial volume was noted. The ratio of weight of the
volume it occupied was calculated.
Bulk density=w/v0 g/ml
Where,
W = mass of the powder
V0 = untapped volume
Tapped density
it is measured by transferring a know quantity
(25g) of powder into a graduated cylinder and
tapping it for a specific number of times. The initial
volume was noted. The graduated cylinder was
tapped continuously for a period of 10-15 min. The
density can be determined as the ratio of mass of
the powder to the tapped volume.
Tapped volume= w/vf g/ml
Where,
W = mass of the powder
Vf = tapped volume.

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International Journal of Research in Pharmaceutical and Biomedical Sciences

a)
Compressibility index
it is the propensity of the powder to be
compressed.
Based on the apparent bulk density and tapped
density the percentage compressibility of the
powder can be determined using the following
formula.
Compressibility index=[(v0-vf)/v0] x 100,
Or
% compressibility=[(tapped density bulk
density)]/ tapped density] x 100
b)
Hausner ratio
it indicates the flow properties of the powder. The
ratio of tapped density to the bulk density of the
powder is called Hausner ratio.

ISSN: 2229-3701

Hausner ratio= Tapped density/bulk density


c)
Angle of repose
The internal angle between the surface of the pile
of powder and the horizontal surface is known as
the angle of repose.
The powder is passed through funnel fixed to a
burette at s height of 4 cm. A graph paper is placed
below the funnel on the table. The height and the
radius of the pile were measured. Angle of repose
of the powder was calculated using the formula
angle of repose= tan-1(h/r)
Where,
H=height of the pile
r = radius of the pile

SCALE OF FLOW ABILITY


S.NO

FLOW PROPERTIES

1
2
3
4
5
6
7

Excellent
Good
Fair
Possible
Poor
Very Poor
Very very
poor

ANGLE OF
REPOSE
25-30
31-35
36-40
41-45
45-46
55-56
>66

5 Determination of PH range
The powder sample of ashwagandha churna was
weighed to about 5g and immersed in 100 ml of
water in a beaker. The beaker was closed with
aluminum foil and left behind for 24 hour s in room
temperature. Later the supernatant solution was
decanted into another beaker and the pH of the
formulation was determined using a calibrated pH
meter.
6 TLC17
1.4 gm of sample was taken in 40 ml rectified spirit
(90%)
2.Shaking it for 18 hrs & boiled for 10 mins &
filtered.

COMPRESSIBILITY
INDEX (%)
<10
11-15
16-20
21-25
26-31
32-37
>38

HAUSNER
RATIO
1.00-1.11
1.12-1.18
1.19-1.25
1.26-1.34
1.35-1.45
1.46-1.59
>1.6

3.The filtrate was then evaporated &extracted


chloroform.
4.The soluble portion was filtered, concentrated &
made upto 10 ml in flask.
5.The solution was applied on aluminium plate
precoated with silica gel 60F254 of 0.2 mm
thickness using linomat IV applicator. The plate
was developed in toluene: Ethyl acetate: acetic acid
(6:4:0.5). After air drying the plate was visualized
in 0V-254 & 366nm.The plate was then dipped in
vanillin sulphuric acid & heated in hot air oven at
105o c till the spots appeared.

7 Heavy metals test24


For Cadmium
Experiment
NH4 OH added in the sample solution.
Potassium ferrocyanide added.

Observation
White ppt of cadmium hydroxide soluble in excess NH4 OH
White ppt of cadmium ferrocyanide.

Result
P/0 cadmium
P/0 cadmium

For Bismuth
Experiment
H2 S gass added in the sample solution
NH4 OH

Observation
Dark brown ppt soluble in hot dil. HNO3 but insoluble in NH4 S
white ppt insoluble in excess NH4 OH dissolved in dil. Hcl.

Result
p/o bismuth
p/o bismuth

For Lead
Experiment
Dil Hcl added in sample solution.
KI is added in sample solution.

Vol. 3 (2) Apr Jun2012

Observation
of CaCl2 soluble in boiled
water & conc. Hcl.
Yellow ppt soluble in boiling water.

White ppt

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Result
p/o lead
p/o lead

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International Journal of Research in Pharmaceutical and Biomedical Sciences

RESULTS AND DISCUSSIONS


1)
DETERMINATION OF
ORGANOLEPTIC CHARACTERS
Sample
Ashwagandha churna of dabour
Color
Odor
Test

Yellowish white
Characteristics
Slightly bitter

SAMPLE
Ashwagandha Churna of Dhaka Ausadhalaya
Color
Odor
Test

Pale brown
Characteristics
Slightly bitter

2 Physico Chemical Standards


Ash Values Sample
Ashwagandha Churna of Dabour
Serial no

% of ash values

Type of ash

(S.E.M)

1.

Total ash

4.7 % 0.115

2.

Acid insoluble ash

1.4 % 0.17

SAMPLE
Ashwagandha Churna of Dhaka Ausadhalaya
Serial no

Type of

% of ash values

ash

(S.E.M)

1.

Total ash

3.5% 0.115

2.

Acid insoluble ash

1.5 % 0.12

DISCUSSIONS
The total ash value is an indicative of total amount
of inorganic material after complete incineration
and the acid insoluble ash value is an indicative of
silicate impurities, which might have arisen due to
improper washing of drug. The loss on drying
value obtained is an indicative of amount of
moisture content present in the drug. The extractive
values names water soluble and alcohol soluble
indicates the amount of active constituent in given
amount of plant material when extracted with
respective solvent, values obtained supports the
fact that drug is unexhausted which is contrary to
lower extractive value.
3 Qualitative Analysis
S.No.
1
2
3
4
5

% Loss on drying

7.20% 0.115

% Loss on drying

13.6% 0.115

Extractive Values
Sample
Ashwagandha Churna of Dabour
S.No.
1
2

NAME OF THE
SOLVENT
WATER
ALCOHOL

EXTRACTIVE
VALUE(% w/w)
1.6% 0.115
0.6% 0.12

S.No.
1

S.No.
1

EXTRACTIVE
VALUE(% w/w)
2.4% 0.115
1.4% 0.12

Vol. 3 (2) Apr Jun2012

TOTAL ALKALOID CONTENT


% 0.5

Determination of Physical
Characteristics of Powder
Bulk Density & Tap Density
Sample
Ashwagandha Churna of Dabour
S.No.

BULLK DENSITY
(S.E.M)
0.45 0.011

TAP DDENSITY
(S.E.M)
0.52 0.0115

Sample
Ashwagandha Churna of Dhaka
AUSADHALAYA

SAMPLE
Ashwagandha Churna of Dhaka Ausadhalaya
NAME OF THE
SOLVENT
WATER
ALCOHOL

TOTAL ALKALOID CONTENT


28.3% 0.5

Sample
Ashwagandha Churna of Dhaka Ausadhalaya

S.No.
1
2

Alcohol Extract
++

4 Quantitative Estimation

Estimation of Alkaloid Sample


Ashwagandha Churna of Dabour

Sample
Ashwagandha OF Dhaka Ausadhalaya
S.No.

Chemical Constituents
Alkaloids
Glycoside
Tannin
Saponin
Phyto sterol

DISCUSSIONS
The results of phytochemical tests were given in
the above table.
++ this indicates the presence of more amounts
of compounds.

Moisture Content/ Loss On Drying


Sample
Ashwagandha Churna of Dabour
S.No.

ISSN: 2229-3701

S.No.
1

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BULLK
DENSITY
(S.E.M)
0.35 0.01

TAP DDENSITY
(S.E.M)
0.44 0.01

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International Journal of Research in Pharmaceutical and Biomedical Sciences

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Carrs Index & Hausner Ratio


Sample
Ashwagandha Churna of Dabour
S.No.

CARRS INDEX

HAUSNER RATIO

(S.E.M)

(S.E.M)

13.6 0.11

1.15 0.01

SAMPLE
Ashwagandha Churna of Both Dabour & Dhaka
Ausadhalaya.
For cadmium

Sample
Ashwagandha Churna of Dhaka
Ausadhalaya
CARRS INDEX

S.No.
1

HAUSNER RATIO

(S.E.M)

(S.E.M)

19.5 0.11

1.24 0.01

Angle of repose
Sample
Ashwagandha churna of dabour
S.No.
1

ANGLE OF REPOSE
34 1.15

SAMPLE
Ashwagandha Churna of Dhaka Ausadhalaya
S.No.

ANGLE OF REPOSE

38 1.15

DISCUSSIONS
From the all above values, it can be concluded that
the quality of ashwagandha churna of dabour is
GOOD & the quality of ashwagandha churna of
Dhaka Ausadhalaya is FAIR.
5

Determination of P H Sample
Ashwagandha Churna of Dabour
H

S.No
1

P (IN 1%)
8.4

P (IN 10%)
5.76

Sample
Ashwagandha Churna of Dhaka
Ausadhalaya
PH (IN 1%)
7.36

S.No.
1

PH (IN 10%)
6.40

Estimation of Crude Fiber


Sample
Ashwagandha Churna of Dabour
S.No
1

CRUDE FIBER
(S.E.M)
3.8 0.115

SAMPLE
Ashwagandha Churna of Dhaka
Ausadhalaya
S.No.
1

CRUDE FIBER (S.E.M)


4.5 0.115

Vol. 3 (2) Apr Jun2012

5 Heavy metal test

Experiment
NH4 OH added in the
sample solution.
Potassium ferrocyanide
added.

Observation
White ppt is
absent.
White ppt is
absent.

Result
Absence
cadmium
Absence
cadmium

of
of

For bismuth
Experiment
H2S gas added in the
sample solution
NH4 OH

Observation
Dark brown ppt is
absent.
White
ppt
is
absent.

Result
Absence
bismuth
Absence
bismuth

of
of

For lead
Experiment

Observation

Dil Hcl added in sample


solution.
KI is added in sample
solution.

Result

White ppt of CaCl2 is


absent.
Yellow ppt is absent.

Absence of
lead
Absence of
lead

DISCUSSION
From the heavy metal test it is concluded that both
the ashwagandha churna of dabour & dhaka
Ausadhalaya are free from heavy metals.
TLC VALUES
SAMPLE
Ashwagandha Churna of Dabour
Ashwagandha Churna of Dhaka
Ausadhalaya

R.F VALUES AT 366


nm
0.78
0.89

DISCUSSION
The TLC profile of both the formulation showed a
bright fluorescence at 366 nm and their Rf values
were found to be 0.78 & 0.89 respectively .
CONCLUSION
From
the
present
investigation
various
standardization
parameters
such
as
physicochemical standards like total ash, acid
insoluble ash, water & alcohol soluble extractive
values, loss on drying, phyto-chemical analysis,
flow properties, TLC profile and safety evaluation
were carried out, it can be concluded that the
formulation of Ashwagandha churna contains all
good characters of an ideal churna and it was found
to be harmless, more effective, and economic. The
comparison between the two marketed samples
have been done on the basis of the above
mentioned parameters which shows satisfactory
results, but the efficacy of the products can only be
judged by doing the pharmacology of which is
suggested as future scope of R & D. The study
shows that the contents of formulation presents

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International Journal of Research in Pharmaceutical and Biomedical Sciences

within the permissible limits as per WHO, all these


investigations are not specified in the standard
literature such as in pharmacopoeia, which could
helpful in authentication of Ashwagandha churna.
The result of present study will also serve as
reference monograph in the preparation of drug
formulation.

11

12

ACKNOWLEDGEMENT
Authors are very much grateful to BCDA College
of Pharmacy & Technology for providing
necessary facilities for completion of the research
work.

13

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