Upload
doannhi
View
221
Download
0
Embed Size (px)
Citation preview
Chapter-3 General phytochemical analysis
25
3.1 OBJECTIVE
To perform proximate and phytochemical analysis for the collected plants Scoparia
dulcis Linn. and Achyranthes aspera Linn.
3.2 INTRODUCTION
Phytochemicals can be defined as the chemicals that are produced by plants.
Currently the term is being used only for those plant chemicals that may have health-
related effects but are not considered essential nutrients (proteins, carbohydrates, fats,
minerals, and vitamins). Plants have valuable source of natural products for
maintaining human health, especially in the past, with more intensive studies for
natural therapies. The use of plant compounds for pharmaceutical purposes has
gradually increased in Brazil and other countries. According to World Health
Organization (WHO) medicinal plants would be the best source to obtain a variety of
drugs (Ellof, 1998).
At the same time the usage of herbal drugs must and should be supported by
analytical methods and techniques relevant to the extraction, separation, purification,
identification, qualification and quantification of substances in the medicinal plants,
which would give more justification for its intended usage.
Medicinal plants contain components of therapeutic values, hence they are
used as remedies for human diseases. Some of them are also used for prophylactic
purposes. An increasing interest in herbal remedies has been observed in several parts
of Nigeria and many of the herbal remedies have been incorporated into orthodox
medicinal plant practice (Nostro et al, 2000). Many disease including malaria,
epilepsy, infantile convulsion, diarrhea, dysentery, fungal and bacterial infections are
being managed traditionally using medicinal plants for many years and now these
systems require screening for their defined properties (Sofowora, 1996).
3.3 LITERATURE REVIEW
Achyranthes aspera Linn. :
Achyranthes aspera Linn. seeds contain the compounds saponins A and B. They are
glycosides of oleanolic acid. The carbohydrate components are the sugars D-glucose,
L-rhamnose, D-glucuronic acid ( Saponin A). Saponin B is the ß-D-galactopyranosyl
ester of Saponin A (Hariharan and Rangaswami, 1970).
Chapter-3 General phytochemical analysis
26
Figure-3.1: Photograph of plant Scoparia dulcis Linn.
Figure-3.2: Photograph of plant Achyranthes aspera Linn.
Chapter-3 General phytochemical analysis
27
The chemical constituents of Achyranthes aspera Linn. are triterpenoid saponins
possessing oleanolic acid as aglycone, viz. A, B, C and D as major chemical
constituents. Other constituents of the plant are ecdysterone, long chain alcohol, viz.
17-penta triacontanol, 27-cyclohexyl heptaeosan-7-ol, 16-hydroxyl 26-methyl
heptacosan-2one and 36, 47-dihydroxy hen-pentacontan-4one. It also contains a water
soluble base, betaine. Different chemical constituents reported by different scientist
are Saponins from alcoholic extract of defatted seeds (Gopalanchari and Dhar, 1985),
Oleanic acid from seeds (Khastgir et al, 1958), Saponins A and B (Hariharan and
Rangaswami, 1970), Saponins C and D from unripe fruits (Sheshadri et al, 1981),
protein, Fe, Ca, phosphorous (Satyanaryana et. al. 1964), Achyranthine, N-methyl
pyrrolidine –3 carboxylic acid (Basu, 1957), Water soluble base, betaine (Kapoor and
Singh, 1967), Vitamin C (Hasan, 1962), Ecdysterone (Banerjee and Chandha, 1970),
Inokosterone ecdysterone in callus and tissue culture (Hiroshi et al, 1971), Enzyme
level (Purohit et al, 1980).
Achyranthes aspera Linn. plant roots reported to contain oleanolic acid,
saponins, amino acids and hentriacontane and a long chained carbohydrate is also
found. In the shoots an aliphatic dihydroxyketone 36, 37-dihydroxyhenpentacontan-4-
on and triacontanol were found (Batta & Rangaswami, 1973). Two long chain
compounds isolated from the shoots of Achyranthes aspera have been characterized
as 27-cyclohexylheptacosan-7-ol and 16-hydroxy.26-methylheptacosan-2- on by
chemical and spectral investigations (Misra et al, 1993).
Simple color reaction was performed for Achyranthes aspera Linn. stems as
an investigation for alkaloids and reported to contain alkaloids. The shoots extracted
with petrol forms yellow semi-solid mass. From this a pink colored essential oil 17-
pentatriacontanol was separated (Gariballa et al, 1983). The whole plant was extracted
with methanol, after the removal of the solvent the residue was extracted successively
with different solvents and isolated in butanol through column chromatography.
Ecdysterone, a phytoecdysone was isolated and characterized by its colour and special
chemical reactions. Contents (g/kg) reported as 0.25 (seeds), 0.09 (roots), 0.04 (stem,
leaves) (Banerji et al, 1971).
Anti viral activity and anticancer activity was reported for methanolic extract
of leaves (Chakraborty et al, 2002). Leaves extract reported to contain antimicrobial
Chapter-3 General phytochemical analysis
28
activity against Staphylococcus aureus, Bacillus subtilis, E. coli (Bashir et al, 1992).
Cardiac toxicity was also reported for Achyranthes aspera Linn. (Han & Un, 2003).
Scoparia dulcis Linn.:
Scoparia dulcis Linn. was qualitatively reported to contain alkaloids, tannins,
saponins, terpinoids, flavonoids, phenols and cardiac glycosides. Some of them are
reported as alkaloids 0.81%, phenols 0.04%, tannins 6.23%, flavonoids 0.88% and
saponins 0.00% approximately (Edeoga, Okwu and Mbaebie, 2005). Scoparia dulcis
found to posses antiviral, inhibitory and antitumor activity (Hayashi et al, 1993).
Scoparia dulcis Linn. was reported to contain antidiabetic activity, the plant identified
to contain some chemical compounds like Scopadulcic Acid A, Scopadulcic Acid B,
Scopadulciol, Scopadiol, Scopadulin, Scoparic Acid A, Scoparic Acid B, Scoparic
Acid C, 4-epi-scopadulcic Acid B, scoparic acid D, Dulcidiol, Acacetin, Apigenin,
Betulinic acid, cirsitakaoside, diacetyl scopadol, iso-dulcinol, Sitosterol, vitexin,
Scutellarein, hispidulin, aphidicolin, eugenyl beta-D-glucopyranoside, Daucosterol
(Rupjyoti Saikia et al, 2011).
Preliminary phytochemical screening concluded on various successive extracts of
Scoparia dulcis Linn. powder indicated the presence of carbohydrates, steroids,
Triterpenoid glycosides, flavonoid glycoside, phytosterols, steroids, mucilage and
saponins (Vaishali Parekh et al, 2011)
Alpha amyrin is also quantified in Scoparia dulcis Linn. whole plant powder
extract by high performance liquid chromatography (Lakshmu naidu P.V et al, 2012)
3.4 MATERIALS AND METHODS
Scoparia dulcis Linn. was collected during the flowering seasons i.e., monsoon,
winter and summer from different geographical regions namely Visakhapatnam,
Srikakulam and Mumbai (India). Sample collection dates were mentioned as
described below for Scoparia dulcis Linn. Visakhaptanam on 17January2006,
Srikakulam on 15January2006 and Mumbai on 22January2006, Visakhaptanam on
11April2006, Srikakulam on 10April2006 and Mumbai on 7April2006,
Visakhaptanam on 5June2006, Srikakulam on 4June2006 and Mumbai on
10June2006. Sample collection dates for Achyranthes aspera Linn. Visakhaptanam on
Chapter-3 General phytochemical analysis
29
17January2006, Srikakulam on 15January2006 and Mumbai on 22January2006 and in
the other season it was dried and not obtainable.
The plant material was thoroughly washed to remove soil particles, dust, etc. and
properly drained to remove excess of water by spreading over newspaper for 6 hours
in shade, away from sunlight. The plant material was then placed in a preset oven at
45 ± 50C. The plant material was allowed to dry for 4 days, after drying, it was
powdered using an electric mixer-grinder and sieved through a BSS mesh No.85
sieve. The sieved powder was stored in commercially available airtight plastic
(Amber PET) containers and this powdered plant material was used for research work
in the present thesis. The plant material collected during monsoon, from above
different regions was used for the study.
Proximate analysis: Crude drugs before use in formulation was subjected to
proximate analysis, after which stored in quarantine store and they remained there for
long time. During storage proper ventilation, humidity controls, suitable temperature
and light conditions should be ensured to maintain their original pharmacological
action; however, it is observed that crude plant materials before processing, if are not
analyzed can lead to changes in their original characteristics. To avoid this crude
drugs should be tested for the following tests as per the USP and Indian Herbal
Pharmacopoeia (IHP).
1. Foreign organic matter
2. Ethanol soluble extractives
3. Water soluble extractives
4. Total ash content
5. Acid insoluble ash
6. Water soluble ash
7. Loss on drying
8. Percentage moisture content
Chapter-3 General phytochemical analysis
30
Foreign organic matter: Medicinal plant materials should be entirely free from
visible signs of contamination i.e. moulds, insects and other animal contamination,
including animal excreta. Any soil, stones, sand, dust and other foreign organic matter
must be removed before medicinal plant materials were cut or ground for testing.
Macroscopic examination can conveniently be employed for the determination of
foreign matter in whole or cut plant materials.
Procedure: The whole plant material was washed thoroughly with water to remove
the dust particles on the surface of the plant and the soil particles adhering to the
roots. Excess water was allowed to drain off by spreading the plant material on filter
paper. Then 500g of the washed and drained whole plant material was taken and
spread as a thin layer on a white, clean muslin cloth. Foreign matter was sorted by
visual inspection and by using magnifying lens (6x). The portions of the sorted
foreign matter were weighed and the contents of foreign matter in grams per 100g of
the sample were calculated. The procedure was carried out for a total of three sets.
Extractable matter: This method determines the amount of phytoconstituents
extracted with solvents from a given amount of medicinal plant material. Here
according to Indian Herbal Pharmacopoeia, ethanol and water were used as solvents
to determine the extractable matter.
Procedure: Accurately weighed 4g each whole plant material of Scoparia dulcis
Linn. and Achyranthes aspera Linn. were placed in glass-stoppered conical flasks
separately. To this 100 cm3 of water was added. The flask was shaken frequently for
six hours, and then allowed to stand for eighteen hours. The contents were filtered.
The filtrates were transferred to previously weighed clean beakers and evaporated to
dryness on a water-bath. After evaporation the extracts were dried at 105º C for six
hours and kept in desiccators for cooling. The beakers were weighed and percent
extractable matter in water was calculated. The above procedure was repeated twice.
Ethanol soluble extractable matter was determined by following the above procedure
except that ethanol was used instead of water, as extracting solvent. The experiment
was repeated three times.
Ash content: The ash remaining after the ignition of medicinal plant materials is
determined by three different methods, which measure total ash, Acid-insoluble ash
and Water-soluble ash. The total ash method is designed to measure the total amount
of material remaining after ignition. This includes both ‘physiological ash’ which is
Chapter-3 General phytochemical analysis
31
derived from the plant tissue itself, and ‘non-physiological ash’ which is the residue
of the extraneous matter (e. g. sand and soil) adhering to the plant surface. Acid-
insoluble ash is the residue obtained after boiling the total ash with dilute
hydrochloric acid and igniting the remaining insoluble matter. This measures the
amount of silica present as sand and siliceous earth. Water-soluble ash is the
difference in weight between the total ash and the residue after treatment of the total
ash with water.
Total ash content:
Apparatus: Silica dish, dessicator, air oven and muffle furnace.
Procedure: Accurately weighed 2g of the dried material was taken in a tarred silica
dish and was ignited with a flame of Bunsen burner for about 1 hr. The ignition was
completed by keeping it in a muffle furnace at 5500C ± 20
0C till grey ash was formed.
It was then cooled in desiccators and weighed. The process was repeated (ignition,
cooling and weighing) till the difference in the weight between two successive
weighing was less than 1 mg.
Acid insoluble ash content:
Chemicals: Dilute HCl , 5 N HCl, and AgNO3 solution.
Apparatus: Silica dish, dessicator, air oven and muffle furnace.
Procedure: Accurately weighed 2g of the dried material was taken in a
porcelain/silica dish and was ignited with a bunsen burner for about 1 hr. The
porcelain dish was kept in a muffle furnace at 5500C ± 20
0C till grey ash was
obtained. The ash was moistened with concentrated HCl and evaporated to dryness
after which it was kept in an electric air oven maintained at 1350C ± 2
0C for 3 hr.
After cooling, 25 cm3 of dilute HCl was added, and was kept covered with watch
glass and heated on a water bath for 10 minutes. It was then allowed to cool, filtered
through Whatman filter paper no. 41. The residue was then washed with hot water till
washings were free from chloride (as tested with AgNO3 solution). The filter paper
and the residue were put in a dish and ignited in a muffle furnace at 5500C ± 20
0C for
1 hr. The dish was removed and cooled in desiccators and weighed. The process was
repeated till the difference between two successive weights was found to be less than
1 mg.
Chapter-3 General phytochemical analysis
32
Water-soluble ash content:
Apparatus: Silica dish, dessicator, air oven and muffle furnace.
Procedure: 25 cm3 of distilled water was added in a silica dish containing the 0.5g of
ash and boiled for ten minutes. The insoluble matter was collected on an ashless filter
paper. The residue was washed with hot water and ignited in a crucible for fifteen
minutes at a temperature not exceeding 450ºC. The water-soluble ash was calculated;
by subtracting the weight of this residue from the weight of the total ash.
Loss on drying:
Apparatus: Wide mouthed Stoppard weighing bottle, dessicator and air oven.
Procedure: 5g each of Scoparia dulcis Linn and Achyranthes aspera Linn. whole
plant powdered samples were weighed in wide mouthed stopper weighing bottle. The
bottle was then placed with lid open in an air oven maintained at 1050
C ± 20 C. The
sample was kept in an oven for 2 hr. The bottle was then removed, covered and placed
in a desiccators. The bottle was weighed after cooling to room temperature.
The bottle was again kept in the oven for 2 hrs and the above procedure was repeated
(heating, cooling and weighing) till the difference in the weight between two
successive weighing was less than 5 mg. Three readings for each sample were
recorded.
Moisture content (Karl-Fischer titrimetric method):
Instrument: Digital automatic karl fischer titrator.
Model : Veego / Matic – MD.
Reagents: Karl Fischer (K/F) reagent, methanol K/F grade, commercial grade
methanol (only for cleaning the dispensing system) and distilled water.
Procedure: The instrument was turned on and the speed of magnetic stirrer was
adjusted. Methanol was neutralized and the titre factor was determined by calibrating
the K/F reagent. This was done by adding 10 µl of distilled water with the help of a
microlitre syringe in the reaction vessel and completing the titration.
The calibration of the reagent was done in triplicate. The readings were noted and the
titre factor was calculated. The data for determination of titre factor reported in Table
and it was calculated using the following formula
Titer factor = mg of water added (wt.) / Reading in cm3 (vol)
Chapter-3 General phytochemical analysis
33
Sample analysis:
Exactly 100 mg of the plant powder was weighed and added to the titration vessel and
the titration was allowed to complete. The results obtained are given in result tables.
Percentage of moisture = Titre factor x reading x 100 / Weight of sample (in mg)
Selection of solvent for extraction and optimization studies:
Extraction the term used pharmaceutically, involves the separation of medicinally
active constituents from complex matrix of plant or animal tissues by the use of
selective solvents in standard extraction procedures. The products so obtained from
plants are relatively impure liquids, semisolids or powders intended only for oral or
external use. These include classes of preparations such as decoctions, infusions, fluid
extracts, tinctures, extracts (semisolid) and powdered extracts. Such preparations are
popularly known as galenicals.
The present research work concentrates primarily on basic extraction procedures for
crude drugs to obtain the therapeutically desirable portion and eliminate the unwanted
material by treatment with a selective solvent, known as the menstrum. The principal
methods of extraction are maceration, percolation, digestion, infusion and decoction.
The quality of the finished product can be enhanced by optimization of primary
extracts. The United State Pharmacopoeia (USP) provides general guidelines for
maceration and percolation under the heading of tinctures.
Choice of extracting solvent:
To obtain complete extraction of a given active principle from a solid material, the
ideal solvent is obviously one that has maximum selectivity, best capacity for
extraction in terms of coefficient of saturation of the product in the medium and its
compatibility with the properties of the material to be extracted.
In the present work different solvents, from non-polar to polar, were used to optimize
the extractive values of Scoparia dulcis Linn and Achyranthes aspera Linn.
Procedure:
Optimization of amount of solvent:
In this experiment, the amount of Scoparia dulcis Linn. and Achyranthes aspera Linn.
whole plant powder taken was kept constant throughout the experiment. In different
sets of volumetric flasks, accurately weighed Scoparia dulcis Linn. and Achyranthes
aspera Linn. whole plant powder was taken. In these different sets of volumetric
Chapter-3 General phytochemical analysis
34
flasks, different amounts of various solvents were added and kept for 1.0 hr. Then
these solvents were filtered through Whatman filter paper no. 41 in pre-weighed dry
beakers separately and solvents were evaporated on a water-bath to dryness. The dried
residue was then weighed and the percentage extractions were calculated. From the
percentage extraction values, the amount of solvent was optimized.
From the graph of volume of solvent (in cm3) versus percentage extraction as shown
in figure optimization of time of extraction, it was observed that the percentage
extraction levels remain constant after certain volume of a solvent used for extraction.
More or less Methanol and Ethanol had same % extraction. Hence methanol is
selected as solvent for extractions. The solvent volume and time of extraction were
optimized using the same.
Optimization of time:
For optimization of time of contact between powder and solvent, the optimized
volume of solvent was added to the sample and the contents in the flasks were filtered
after different time intervals. The above procedure was repeated and the percentage
extractive values were calculated. From the percentage extractive values, the
optimization time was determined. For optimization of the time, methanol was added
to the samples kept in different flasks. The contents in the flasks were filtered after
different time intervals and the percentage extractive values were calculated.
Optimization of number of extractions:
For optimization of the number of extractions, the optimized amount of selected
solvent was added to the sample in different sets of flasks and these flasks were kept
aside for the optimized time. Then the contents of the flasks were filtered separately
through Whatman filter paper no. 41 in pre-weighed dry beakers. The residues were
again taken in a flask and extracted repeatedly using optimized solvent and time. The
above procedure was repeated and the percentage extraction values were calculated.
From these values number of extractions was optimized.
Phytochemical analysis:
Plant constituents of medicinal importance form an extensively diverse group of
chemical compounds showing greater variation in solubility and stability. They can be
broadly classified as follows: (Camille et al, 1996).
Chapter-3 General phytochemical analysis
35
���� Fixed oils, fats and waxes (lipids)
���� Phenols
���� Tannins
���� Proteins
���� Alkaloids
���� Carbohydrates
���� Glycosides
���� Volatile oils
���� Resin and resin combinations
Extraction of phytochemicals:
The precise mode of extraction depends on the texture and type of the substances to
be isolated. The classical chemical procedure for obtaining constituents from dried
plant tissues is to continuously extract powered material in a ‘Soxhlet apparatus’ with
a range of solvents.
When investigating the complete phytochemical profile of a given plant species,
fractionation of crude extract is desirable, in order to separate the main classes of
constituents from each other prior to chromatographic analysis.
Procedure:
Following is the procedure based on varying polarity of solvents, which was
employed for determination of phytochemical profile of Scoparia dulcis Linn. and
Achyranthes aspera Linn. (Harbone, 1998).
a) 2g of the dried whole plant powder of Scoparia dulcis Linn and Achyranthes
aspera Linn. was separately soxhlet extracted with a mixture of methanol and
distilled water (50 cm3) in the volume ratio 4:1. The extract was cooled and
filtered through Whatman filter paper No. 41 into a dry and pre-weighed beaker.
b) The residue was extracted with 125 cm3 of (5 x 25 cm
3) of ethyl acetate and
filtered into a dry, pre-weighed beaker. The residue obtained after filtration
comprised plant fibers. Weight of the extract was noted down and the plant fibers
were dried in an oven at 60 ±50C and percent crude fiber was calculated.
c) The filtrate obtained from step (b) was evaporated to dryness on a water bath
maintained at 45±50C. After evaporation of ethyl acetate, the beaker was allowed
to cool to room temperature in a desiccator. After cooling, the weight of beaker
Chapter-3 General phytochemical analysis
36
containing the residue was noted down. The residue obtained was the neutral
extract and consisting of fats and waxes.
d) The filtrate obtained from step (a) was evaporated to approximately 1/10th
of its
volume by heating in a water bath maintained at a temperature less than 700C. It
was acidified with 2M H2SO4. The acidified filtrate was extracted using 75 cm3
(3x25 cm3) chloroform in a separating funnel. It was then transferred to a dry pre-
weighed beaker. Chloroform layer was evaporated to dryness on a water bath
maintained at 45±50C.
e) After evaporation of chloroform the beaker was allowed to cool to room
temperature in a dessicator. After cooling the weight of this beaker containing the
residue was noted down, the residue obtained was moderately polar extract and
consisting of terpenoids and phenolics.
f) The aqueous acid layer obtained from step (d) was basified (pH was adjusted to10
with 2M NaOH). It was then extracted with 60 cm3 (2x30 cm
3) of mixture of
chloroform and methanol in the volume ratio 3:1, followed by extraction with
40 cm3
(2x20 cm3) chloroform in a separating funnel. The aqueous basic layer was
transferred to a dry pre-weighed beaker. The aqueous basic layer was evaporated
to dryness on a water bath maintained at 700C. After evaporation of the solvent,
the beaker was allowed to cool to room temperature in a dessicator.
g) The weight of the beaker containing the residue was noted down. The residue
obtained was polar extract consisting of quaternary alkaloids and N-oxides.
h) The organic layer (chloroform and methanol) was transferred to a dry, pre-
weighed beaker, placed on a water bath maintained at 45±50C. After evaporation
of the solvent, the beaker was allowed to cool to room temperature in a desecrator.
After cooling, the weight of this beaker containing the residue was noted down.
The residue obtained was the basic extract consisting of alkaloids.
i) 2g of the dried whole plant powder of Scoparia dulcis Linn. and Achyranthes
aspera Linn. was extracted with 100 cm3 methanol (4x25 cm
3) in a dry, stoppered
conical flask. The extract was filtered into a dry, pre-weighed beaker. The filtrate
obtained was evaporated to dryness on a water bath maintained at 700C. After
evaporation, the beaker was allowed to cool to room temperature in a dessicator.
After cooling, the weight of beaker containing the residue was noted down. The
residue obtained was reconstituted in methanol to obtain a final concentration of
Chapter-3 General phytochemical analysis
37
100 mg/cm3. This extract was filtered through Whatman filter paper No. 41. The
filtrate obtained was the plant extract.
Each of the extracts obtained was spotted on HPTLC plate. The plate was
developed in previously optimized mobile phase and the chromatograms of
different extracts were compared with plant extract under same experimental
conditions.
Flow chart for extraction of phytochemicals:
HPTLC Analysis of phytochemical extracts:
Sample application:
The HPTLC separation of plant extract and extracts of separated phytochemicals was
performed on pre-coated silica gel 60 F254 HPTLC plates. Ten micro liters of the
extracts comprising alkaloids, fats and waxes, phenolics and terpenoids, basic extracts
and polar extract and plant extract were applied at a distance of 10 mm/ from the base
of chromatographic plate and 7mm width on HPTLC plate using Camag Linomat-IV
sample applicator.
Methodology:
The methodology followed for High Performance Thin Layered Chromatography was
mentioned in the Chapter-8.
The filtrate evaporated below 700C, acidified H2SO4.
extracted 75 cm3 (3x25 cm3) chloroform evaporated at
45±50C to get terpenoids and phenols
Residue 5 x25ml ethyl acetate extraction
Plant powder
Methanol + Water (4:1) (50 cm3)
Soxhlet extraction
Ethyl acetate evaporate to get
neutral extract and consisting of
fats and waxes
Plant fibers dried in an
oven at 60 ±50C.
The aqueous acid layer pH 10 with NaOH extracted
60 cm3 (2x30 cm3) chloroform and methanol
3:1,extraction 40 cm3 (2x20 cm3) chloroform
The aqueous basic layer evaporated at 700C
quaternary alkaloids and N-oxides Chloroform and methanol layer evaporated at 45±50C
basic extract consisting of alkaloids
Chapter-3 General phytochemical analysis
38
The individual chromatograms of all separated phytochemicals i. e. alkaloids, fats and
waxes, phenolics and terpenoids, basic extracts and polar extract are compared with
plant extract by overlay, for Scoparia dulcis Linn. and Achyranthes aspera Linn. were
shown in figure 3.9 and 3.10 respectively. Thus each peak in the chromatogram of
plant extract decides which phytochemicals they belong to.
3.5 RESULTS OBTAINED
Foreign organic matter observations and results:
It is observed that the percentage foreign organic matter in Scoparia dulcis Linn. and
Achyranthes aspera Linn. whole plant is between 0.032 % to 0.040%. The results are
given in Table 3.1. and Table 3.2.
Calculations:
% Foreign organic matter = (M1 - M) × 100
M2
Where
M = Weight of empty dish in g
M1 = Weight of dish with foreign matter in g
M2 = Weight of sample (whole plant material) in g
For Scoparia dulcis Linn.
(29.8148 - 29.6822) x 100 = 0.026 %
500
For Achyranthes aspera Linn.
(29.9342 - 29.7921) x 100 = 0.028 %
500
Table 3.1. Percentage foreign organic matter in Scoparia dulcis Linn.
Sample % Foreign organic matter %
Mean Mumbai Visakhapatnam Srikakulam
Scoparia dulcis Linn. 0.031 0.026 0.028 0.028
% RSD 5.6 3.8 10.7 8.9
Note: Each observation is mean of three readings.
Chapter-3 General phytochemical analysis
39
Table 3.2. Percentage Foreign organic matter in Achyranthes aspera Linn.
Sample % Foreign organic matter
% Mean Mumbai Srikakulam Visakhapatnam
Achyranthes aspera Linn. 0.03 0.022 0.028 0.027
% RSD 3.8 7.1 5.4 15.6
Note: Each observation is mean of three readings.
Extractable matter observations and results:
It is observed that the percentage ethanol extractable matter in Scoparia dulcis Linn.
and Achyranthes aspera Linn. whole plant powder is between 21.00 % to 25.00 %
and percentage water extractable matter is between 28.00 % to 30.00 %. The results
are given in Table 3.3, 3.4, 3.5 and 3.6.
Table 3.3. Percentage of ethanol extractable matter in Scoparia dulcis linn.
Sample Extractive value (%) %
Mean Mumbai Visakhapatnam Srikakulam
Scoparia dulcis Linn. 21.29 23.42 22.95 22.55
% RSD 2.9 2.6 2.5 5.0
Note: Each observation is mean of three readings.
Table 3.4. Percentage of ethanol extractable matter in Achyranthes aspera Linn.
Sample Extractive value (%) %
Mean Mumbai Visakhapatnam Srikakulam
Achyranthes aspera Linn. 23.2 23.95 24.42 23.86
% RSD 2.6 2.0 2.1 2.6
Note: Each observation is mean of three readings.
Table 3.5. Percentage of water extractable matter in Scoparia dulcis Linn.
Sample Extractive value (%) %
Mean Mumbai Visakhapatnam Srikakulam
Scoparia dulcis Linn 27.66 29.13 28.93 28.57
% RSD 2.7 3.7 4.6 2.8
Note: Each observation is mean of three readings.
Chapter-3 General phytochemical analysis
40
Table 3.6. Percentage of water extractable matter in Achyranthes aspera Linn.
Sample Extractive value (%) %
Mean Mumbai Visakhapatnam Srikakulam
Achyranthes aspera Linn. 29.33 29.93 29.24 29.50
% RSD 2.1 3.7 2.2 1.3
Note: Each observation is mean of three readings.
Ash content observations and results:
It is observed that the percentage total ash content of Scoparia dulcis Linn and
Achyranthes aspera Linn whole plant powder is between 3.17% to 3.23% and 4.76%
to 4.98% respectively.
Table 3.7. Total ash content of Scoparia dulcis Linn.
Sample % Total ash content
% Mean Mumbai Visakhapatnam Srikakulam
Scoparia dulcis Linn. 3.23 3.17 3.17 3.19
% RSD 3.4 2.4 6.5 1.1
Note: Each observation is mean of three readings.
Table 3.8. Total ash content of Achyranthes aspera Linn.
Sample % Total ash content
% Mean Mumbai Visakhapatnam Srikakulam
Achyranthes aspera Linn. 4.76 4.98 4.89 4.88
% RSD 3.7 1.5 5.1 2.3
Note: Each observation is mean of three readings.
Acid insoluble ash content observations and results:
It is observed that the percentage acid insoluble ash content of Scoparia dulcis Linn.
and Achyranthes aspera Linn. whole plant powder is between 0.85 % to 0.95 %.and
0.255% to 0.265% respectively.
Chapter-3 General phytochemical analysis
41
Table 3.9 Acid insoluble ash content of Scoparia dulcis Linn.
Sample % Acid insoluble ash %
Mean Mumbai Visakhapatnam Srikakulam
Scoparia dulcis Linn. 0.85 0.91 0.95 0.90
% RSD 2.4 5.7 3.2 5.6
Note: Each observation is mean of three readings.
Table 3.10. Acid insoluble ash content of Achyranthes aspera Linn.
Sample % Acid insoluble ash %
Mean Mumbai Visakhapatnam Srikakulam
Achyranthes aspera Linn. 0.265 0.255 0.2599 0.2599
% RSD 7.5 8.1 7.3 1.9
Note: Each observation is mean of three readings.
Water-soluble ash content observations and results:
It is observed that the percentage water-soluble ash content of Scoparia dulcis Linn.
and Achyranthes aspera Linn. whole plant powder is found to be between 2.28 % to
2.34 %. and 4.14% to 4.17 % respectively.
Table 3.11. Water soluble ash content of Scoparia dulcis Linn.
Sample % Water soluble ash %
Mean Mumbai Visakhapatnam Srikakulam
Scoparia dulcis Linn. 2.34 2.30 2.28 2.31
% RSD 4.3 5.8 2.5 1.3
Note: Each observation is mean of three readings.
Table 3.12. Water soluble ash content of Achyranthes aspera Linn.
Sample % Water soluble ash %
Mean Mumbai Visakhapatnam Srikakulam
Achyranthes aspera Linn. 4.14 4.14 4.17 4.15
% RSD 2.1 0.6 1.4 0.4
Note: Each observation is mean of three readings.
Chapter-3 General phytochemical analysis
42
Loss on drying observations and results:
It is observed that the percentage of loss on drying of Scoparia dulcis Linn. and
Achyranthes aspera Linn. whole plant powder is between 8.00% to 9.00%
Table 3.13. Percentage loss on drying of Scoparia dulcis Linn.
Sample % Loss on drying
% Mean Mumbai Visakhapatnam Srikakulam
Scoparia dulcis Linn. 8.24 8.86 8.16 8.42
% RSD 1.8 3.0 1. 4.6
Note: Each observation is mean of three readings.
Table 3.14. Percentage loss on drying of Achyranthes aspera Linn.
Sample % Loss on drying %
Mean Mumbai Visakhapatnam Srikakulam
Achyranthes aspera Linn. 8.58 8.59 8.59 8.59
% RSD 1.2 3.9 3.5 0.1
Moisture content observations and results:
Table 3.15. Determination of moisture content (Titre Factor) of Scoparia dulcis Linn.
Sr. No. Weight of water
added in mg
Volume of reagent
added in cm3
Titer
factor
1. 10 1.85 5.41
2. 10 1.83 5.46
3. 10 1.86 5.38
Mean 5.42
Table 3.16. Determination of moisture content (Titre Factor) of Achyranthes aspera
Linn.
Sr. No. Weight of water
added in mg
Volume of reagent
added in cm3
Titer
factor
1. 10 1.73 5.78
2. 10 1.78 5.61
3. 10 1.80 5.55
Mean 5.55
Chapter-3 General phytochemical analysis
43
Table 3.17. Moisture content of Scoparia dulcis Linn.
Sr. No. Weight of
powder in mg
Volume of reagent
added in cm3
%
Moisture
1. 100 1.23 6.66
2. 100 1.18 6.39
3. 100 1.15 6.23
Mean 6.43
Table 3.18. Moisture content of Achyranthes aspera Linn.
Sr. No. Weight of
powder in mg
Volume of reagent
added in cm3
%
Moisture
1. 100 1.08 6.24
2. 100 1.08 6.85
3. 100 1.07 5.93
Mean 6.34
Following are the parameters (Table 3.19) required for checking the quality of raw
material of whole plant powder of Scoparia dulcis Linn. and Achyranthes aspera
Linn. before going for processing. These quality control parameters depend on
cultivation, handling of the raw material and storage condition.
Table 3.19. Proximate analysis of Scoparia dulcis Linn. and Achyranthes aspera
Linn.
Sr.
No.
Parameter % Content
Scoparia dulcis
Linn.
Achyranthes aspera
Linn.
1 Foreign organic matter 0.028 0.027
2 Ethanol soluble
extractive 22.55 23.86
3 Water soluble extractive 28.57 29.50
4 Total ash 3.19 4.88
5 Acid-insoluble ash 0.90 0.26
6 Water soluble ash 2.31 4.15
7 Loss on drying 8.42 8.59
8 Moisture content 6.43 6.34
Chapter-3 General phytochemical analysis
44
Solvent optimization for extraction and acceptability:
Table 3.20. Optimization of extraction conditions for selecting suitable solvent
Scoparia dulcis Linn.
S. No. Solvent Volume
(cm3)
Time
(Min)
No. of
Extractions
(n)
Extractive
value (%) SD % RSD
1. Water 100 60 3 27.96 1.47 5.3
2. Methanol 100 60 3 11.91 1.4 11.8
3. Ethanol 100 60 3 12.51 0.99 7.9
4. Acetonitrile 100 60 3 8.75 0.67 7.7
5 Acetone 100 60 3 7.35 0.83 11.3
6 Chloroform 100 60 3 6.85 0.33 4.8
Note: All values are mean of three extractions.
Table 3.21. Optimization of extraction conditions for selecting suitable solvent
Achyranthes aspera Linn.
S. No. Solvent Volume
(cm3)
Time
(min.)
No. of
Extractions
(n)
Extractive
value (%) SD
%
RSD
1. Water 100 30 3 29.0 1.32 4.6
2. Methanol 100 60 3 14.40 0.9 6.3
3. Ethanol 100 60 3 15.13 1.18 7.8
4. Acetonitrile 100 60 2 10.24 0.91 8.9
5 Acetone 100 60 3 9.88 0.83 8.4
6 Chloroform 100 60 3 12.12 0.93 7.6
Chapter-3 General phytochemical analysis
45
Table 3.22. Optimization of amount of solvent for extraction Scoparia dulcis Linn.
0.0
5.0
10.0
15.0
0 100 200 300
Amt. in ml
% Extraction
Amt of solvent
Figure 3.3. Optimization of amount of solvent for extraction of Scoparia dulcis Linn.
Set No.
Weight of
powder
Amount of
solvent in
cm3
Extractive
value (%) SD % RSD
1 0.5 25 7.50 0.91 12.2
2 0.5 50 8.03 0.95 11.8
3 0.5 75 10.32 0.99 9.6
4 0.5 100 11.91 1.1 9.2
5 0.5 150 12.02 1.48 12.3
6 0.5 200 12.12 0.53 4.4
Chapter-3 General phytochemical analysis
46
Table 3.23. Optimization of amount of solvent for extraction Achyranthes aspera
Linn
Set
No.
Weight of
powder
in g
Amount of
solvent in cm3
Extractive
value (%)
SD % RSD
1 0.25 25 9.01 0.49 5.4
2 0.25 50 10.42 0.88 8.4
3 0.25 75 12.00 1.5 12.5
4 0.25 100 13.00 0.92 7.1
5 0.25 125 13.04 0.56 4.3
6 0.25 150 13.00 1.44 11.1
Figure 3.4. Optimization of amount of solvent for extraction of Achyranthes aspera
Linn.
Chapter-3 General phytochemical analysis
47
Optimization of time:
Table 3.24. Optimization of time of extraction for Scoparia dulcis Linn.
Set No.
Weight of
powder
in g
Time of
extraction
in min
Extractive
value (%) SD
%
RSD
1 0.5 30 7.74 0.66 8.5
2 0.5 60 11.77 1.63 13.8
3 0.5 90 12.42 1.04 8.4
4 0.5 120 13.11 0.85 6.4
5 0.5 180 13.71 0.75 5.5
6 0.5 240 13.76 0.46 3.4
0
5
10
15
0 100 200 300
Time in min
% Extraction
Time of Extraction
Figure 3.5. Optimization of time of extraction for Scoparia dulcis Linn.
Chapter-3 General phytochemical analysis
48
Table 3.25. Optimization of time of extraction for Achyranthes aspera Linn.
Set No.
Weight of
powder in
g.
Time in
minutes
Extractive
value (%)
SD % RSD
1 0.25 30 7.44 0.64 8.6
2 0.25 60 12.16 0.81 6.7
3 0.25 90 12.44 1.08 8.7
4 0.25 120 12.36 1.23 9.9
5 0.25 150 12.48 1.03 8.2
0
5
10
15
0 50 100 150 200
Time in min
% Extraction
Time of Extraction
Figure 3.6. Optimisation of time of extraction for Achyranthes aspera Linn.
Chapter-3 General phytochemical analysis
49
Optimization of number of extractions:
Table 3.26. Optimization of number of extractions for Scoparia dulcis Linn.
Set No.
Weight of
powder
in g
No. of
Extraction
Extractive value
(%) SD % RSD
1 0.5 1 13.74 0.67 4.9
2 0.5 2 15.08 1.35 9.0
3 0.5 3 16.85 0.57 3.4
4 0.5 4 17.51 0.31 1.7
5 0.5 5 17.62 0.15 0.9
6 0.5 6 17.68 0.19 1.1
0
5
10
15
20
0 2 4 6 8
nos of extraction
% extraction
nos of
extraction
Figure 3.7. Optimization of number of extraction for Scoparia dulcis Linn
Chapter-3 General phytochemical analysis
50
Table 3.27. Optimization of number of extractions for Achyranthes aspera Linn.
Set No. Weight of
powder in g
Number of
extractions
Extractive
value (%)
SD % RSD
1 0.2500 1 12.16 0.35 2.9
2 0.2500 2 13.72 0.42 3.0
3 0.2500 3 14.40 0.69 4.8
4 0.2500 4 14.60 0.56 3.8
5 0.2500 5 14.66 0.58 3.9
0
5
10
15
20
0 2 4 6
nos of Extraction
% of Extraction
nos of extraction
Figure 3.8. Optimisation of number of extraction for Achyranthes aspera Linn.
Chapter-3 General phytochemical analysis
51
Phytochemical analysis:
Table 3.28. Percent phytochemicals from Scoparia dulcis Linn.
Sr.
No. Phytochemical extract
Extractive
value (%)*
SD % RSD
1 Neutral extract
(Fats and waxes) 1.7 0.1 5.9
2 Moderately polar extract
(Terpenoids and phenolics) 1.8 0.06 3.1
3 Basic extract
(Most alkaloids) 0.96 0.02 2.2
4 Polar extract
(Quaternary alkaloids and
N-oxides)
19.34 0.67 3.4
5 Fibers 76 1.32 1.7
Total 99.8
Table 3.29. Percent phytochemicals from Achyranthes aspera Linn.
Sr.
No. Phytochemical extract
Extractive
value (%)*
SD % RSD
1 Neutral extract
(Fats and waxes) 1.3 0.1 7.7
2 Moderately polar extract
(Terpenoids and phenolics) 1.13 0.12 10.2
3 Basic extract
(Most alkaloids) 0.70 0.1 14.3
4 Polar extract
(Quaternary alkaloids and
N-oxides)
21.24 0.9 4.2
5 Fibers 75.5 1.0 1.3
Total 99.87
* Each observation is mean of three readings.
Chapter-3 General phytochemical analysis
52
Results:
Separate extracts of the isolated phytochemical constituents and the plant extract in
methanol were prepared as discussed earlier. The comparison of phytochemical
constituents in isolated extracts and in plants extract was required to study their
equivalence. The data obtained after chromatographic scan under the same
chromatographic conditions was studied carefully. The Rf values were compared for
the data treatment.
The plant extract of Scoparia dulcis Linn. in methanol showed nine peaks at Rf values
0.22, 0.28, 0.33, 0.41, 0.45, 0.56, 0.65, 0.71 and 0.77 which are representative of
individual phytochemical type.
The alkaloid extract showed three peaks at Rf-values 0.34, 0.56 and 0.71 representing
the presence of different alkaloids.
The chromatogram of the basic extract showed eight peaks with Rf values 0.23, 0.28,
0.34, 0.46, 0.57, 0.66, 0.72 and 0.78.
The fats and waxes showed two peaks with Rf values 0.64 and 0.71.
Mid polar extract showed six peaks with Rf values 0.27, 0.33, 0.40, 0.51, 0.56 and
0.71 representing the presence of terpenoids.
Chapter-3 General phytochemical analysis
53
Figure 3.9: The overlay of densitometric chromatogram of Scoparia dulcis Linn.
Figure 3.10: The overlay of densitometric chromatogram of Achyranthes aspera
Linn.
Fats and waxes
Terpenoids
Plant extract
Alkaloids
Basic extracts
Fats and waxes
Terpenoids
Plant extract
Alkaloids
Basic extracts
Chapter-3 General phytochemical analysis
54
Table 3.30. Phytochemical analysis comparative results of characteristic spots
Scoparia dulcis Linn.
S No. Rf value Whole plant Alkaloid Fats &
Waxes
Phenolics &
Terpenoids
Basic
extract
1 0.22 √√√√ ✔✔✔✔ ✔✔✔✔ ✔✔✔✔ ✔✔✔✔
2 0.23 ✔✔✔✔ ✔✔✔✔ ✔✔✔✔ ✔✔✔✔ √√√√
3 0.27 ✔✔✔✔ ✔✔✔✔ ✔✔✔✔ √√√√ ✔✔✔✔
4 0.28 √√√√ ✔✔✔✔ ✔✔✔✔ ✔✔✔✔ √√√√
5 0.33 √√√√ ✔✔✔✔ ✔✔✔✔ √√√√ ✔✔✔✔
6 0.34 ✔✔✔✔ √√√√ ✔✔✔✔ ✔✔✔✔ √√√√
7 0.40 ✔✔✔✔ ✔✔✔✔ ✔✔✔✔ √√√√ ✔✔✔✔
8 0.41 √√√√ ✔✔✔✔ = = =
9 0.45 √√√√ = = = =
10 0.46 = = = = √√√√
11 0.51 = = = √√√√ =
12 0.56 √√√√ √√√√ = √√√√ =
13 0.57 = = = = √√√√
14 0.64 = = √√√√ = =
15 0.65 √√√√ = = = =
16 0.66 = = = = √√√√
17 0.71 √√√√ √√√√ √√√√ √√√√ =
18 0.72 = = = = √√√√
19 0.77 √√√√ = = = =
20 0.78 = = = = √√√√
Chapter-3 General phytochemical analysis
55
The plant extract of Achyranthes aspera Linn. in methanol showed seven peaks at Rf
values 0.23, 0.33, 0.45, 0.49, 0.60, 0.66 and 0.73 which are representative of
individual phytochemical type.
The alkaloid extract showed four peaks at Rf values 0.20, 0.30, 0.33 and 0.43
representing the presence of different alkaloids.
The chromatogram of the basic extract showed seven peaks with Rf values 0.25, 0.29,
0.35, 0.46, 0.61, 0.68 and 0.73.
The fats and waxes showed five peaks with Rf values 0.33, 0.59, 0.66, 0.71 and 0.78.
Mid polar extract showed ten peaks with Rf values 0.21, 0.24, 0.29, 0.35, 0.41, 0.49,
0.59, 0.61, 0.73 and 0.80 representing the presence of terpenoids.
Chapter-3 General phytochemical analysis
56
Table 3.31. Phytochemical analysis comparative results of characteristic spots in
Achyranthes aspera Linn.
SNo.
Rf value Whole plant Alkaloid Fats & Waxes
Phenolics & Terpenoids
Basic extract
1 0.20 = √√√√ = = =
2 0.21 = = = √√√√ =
3 0.23 √√√√ = = = =
4 0.24 = = = √√√√ =
5 0.25 = = = = √√√√
6 0.29 = = = √√√√ √√√√
7 0.30 = √√√√ = = =
8 0.33 √√√√ √√√√ √√√√ = =
9 0.35 = = = √√√√ √√√√
10 0.41 = = = √√√√ =
11 0.43 = √√√√ = = =
12 0.45 √√√√ = = = =
13 0.46 = = = = √√√√
14 0.49 √√√√ = = √√√√ =
15 0.59 = = √√√√ √√√√ =
16 0.60 √√√√ = = = =
17 0.61 = = = √√√√ √√√√
18 0.66 √√√√ = √√√√ = =
19 0.68 = = = = √√√√
20 0.71 = = √√√√ = =
21 0.73 √√√√ = = √√√√ √√√√
22 0.78 = = √√√√ = =
Chapter-3 General phytochemical analysis
57
3.6 DISCUSSION OF RESULTS IN LIGHT OF OTHERS WORK
No literature reported for the content of foreign organic matter, ethanol soluble
extractives, water soluble extractives, total ash content, acid insoluble ash, water
soluble ash for the plants Scoparia dulcis and Achyranthes aspera.
No HPTLC method reported to identify the chemical constituents present in the whole
plant. Several methods reported that methanolic, ethanolic extracts were having
pharmacologically active. In accordance with the present data, the ethanol extracts
have more percentage of extraction.
The above data shows that the plants have more mid polar components that are
extractable in ethanol.
3.7 CONCLUSION
Type of chemical constituents present in the above two plants are identified.
Qualitative HPTLC method developed to identify the type of chemical constituents
present in the whole plant. The solvent required for extraction selected.
The maximum extraction of the whole plant powder of Scoparia dulcis Linn. and
Achyranthes aspera Linn. found in water are 27.96 and 29.0 respectively. The
optimum extraction conditions of different solvents are given in Table 3.20 and 3.21.
The % extraction for the solvent methanol for Scoparia dulcis and Achyranthes
aspera are 11.91 and 14.40 respectively.
The % extraction for the solvent ethanol for Scoparia dulcis and Achyranthes aspera
are 12.51 and 15.13 respectively.
Comparatively Ethanol is having more % extraction capacity rather than methanol.
Hence ethanol used for extraction as ethanol extracts can be used directly for oral
administration.
Even though water posses more extraction capacity it is not used because of its
susceptibility for microbial contamination.
These optimized parameters can be used for bulk production of whole plant powder
extracts of Scoparia dulcis Linn. and Achyranthes aspera Linn.
3.8 FUTURE PROSPECTS
The ethanol and mid polar extracts should be further separated using different
techniques such as flash chromatography followed by preparative column
chromatography.
Chapter-3 General phytochemical analysis
58
The separated components have to be purified, characterized and identified by
different techniques such as Mass, NMR and IR.
The purified components are to be screened for the anti diabetic and anti HIV activity.
3.9 REFERENCES
Banerjee A and Chadha MS. Phytochem., 1970; 9(7): 1671.
Banerji A, Chintalwar GJ, Joshi NK et al. Phytochemistry., 1971; 10: 2225-6.
Bashir A, El Sayed H, Amiri MH. Fitoterapia., 1992; 4: 371-5.
Basu NK, Singh K, Agrwal OP, J. Proc. Inst. Chemist., 1957; 29(1): 55.
Batta AK, Rangaswami S. Phytochemistry., 1973; 12: 214-6.
Camille Georges Wermuth. The Practice of Medicinal Chemistry. Academic press
Ltd. London, 1996; 100 -113.
Chakraborty A, Brantner A, Mukainaka T et al. Canc Lett., 2002; 177(1):
PubMed.11809524.
Edeoga H.O, Okwu D. E and Mbaebie B.O. African Journal of Biotechnology., 2005;
4 (7): 685-688. Available online at http://www.academicjournals.org/AJB ISSN 1684-
5315
Ellof J.N. J. Ethnopharmacol., 1998; 60: 1-6.
Gariballa Y, Iskander GM, Daw El Beit A. Fitoterapia., 1983; 54: 269-72.
Gopalanchari R, Dhar ML, J.Sci Indust. Res., 1985; 17(B):276.
Han ST, Un CC. Vet Hum Toxicol., 2003; 45 (4): 212-3.
Harbone J.B, Phytochemical Methods. A Guide to Modern Techniques of Plant
Analysis, Champan & Hall London UK, 3rd
edition, 1998;1-7.
Hariharan V, Rangaswami S. Phytochemistry., 1970; 9:409-414.
Hasan F, Pakistan J Sci Res., 1962:14(1): 4.
Chapter-3 General phytochemical analysis
59
Hayashi T, Okamuka K, Kawasaki M, Morita N. Phytochemistry., 1993; 35 (2): 353-
356.
Hiroshi H, Hisanon J, Takemota T. Chem. Pharm. Bull., 1971; 19(12): 438.
Indian Herbal Pharmacopoeia, A joint publication of Regional Research
Laboratory(CSIR) Jammu tawi and Indian Drugs Manufacturer’s Association., 1998;
2.
Kapoor VK, Singh HK. Ind. J. Pharm., 1967; 29(10): 281.
Khastgir H, Sengupta SK, Sengupta P. J Ind. Che. Soc., 1958; 35: 693.
Lakshmu Naidu PV, Kishore Kumar K, Sujatha S, Narasimha Raoa M., Journal of
Pharmacy Research., 2012; 5 (4) : 1970.
Misra TG, Singh RS, Pandey HS. Phytochemistry., 1993; 33(1): 221-3.
Nostro A, Germano MP, D'Angelo V, Marino A, Cannatelli MA. Lett. Appl.
Microbiol., 2000; 30(5): 379.
Purohit S, Bhattacharya I C. J. Sci. Ind. Res., 1980; 20C (8): 246.
Rupjyoti Saikia, M. Dutta Choudhury, A. Das Talukdar and Pankaj Chetia. Assam
University Jour. of Science & Technol., 2011; 7 (I): 173-180.
Satyanarayana MS, Sushila BA, Rao, AN, Vijaraghwan, PK. J. Food sci. tech., 1964;
1(12): 26.
Sheshadri V, Batt AK, Rangaswami S. Ind. J. Chem., 1981; 20B (9) : 773.
Sofowora A. J. Altern. Complement. Med., 1996; 2 (3): 365-372.
United States Pharmacopoeia, 24 NF (19). The United States Pharmacopoeia
Convention Inc. USA, 2000.
Vaishali Parekh, Saurabh Parmar, Urvashi Shah., International Journal of Universal
Pharmacy and Life Sciences., 2011: 1(2).