3.1 objective - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/28821/10/10_chapter 3.pdf ·...

Chapter-3 General phytochemical analysis

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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).


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Figure-3.1: Photograph of plant Scoparia dulcis Linn.

Figure-3.2: Photograph of plant Achyranthes aspera Linn.


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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


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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


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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


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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


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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.


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.


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Water-soluble ash content:


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)


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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


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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).


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�� 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


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


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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


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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.


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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


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 (%) %



% RSD 2.9 2.6 2.5 5.0


Table 3.4. Percentage of ethanol extractable matter in Achyranthes aspera Linn.




% RSD 2.6 2.0 2.1 2.6


Table 3.5. Percentage of water extractable matter in Scoparia dulcis Linn.



Scoparia dulcis Linn 27.66 29.13 28.93 28.57

% RSD 2.7 3.7 4.6 2.8



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Table 3.6. Percentage of water extractable matter in Achyranthes aspera Linn.




% RSD 2.1 3.7 2.2 1.3


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


% RSD 3.4 2.4 6.5 1.1


Table 3.8. Total ash content of Achyranthes aspera Linn.

Sample % Total ash content



% RSD 3.7 1.5 5.1 2.3


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.


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Table 3.9 Acid insoluble ash content of Scoparia dulcis Linn.

Sample % Acid insoluble ash %



% RSD 2.4 5.7 3.2 5.6


Table 3.10. Acid insoluble ash content of Achyranthes aspera Linn.

Sample % Acid insoluble ash %



% RSD 7.5 8.1 7.3 1.9


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 %



% RSD 4.3 5.8 2.5 1.3


Table 3.12. Water soluble ash content of Achyranthes aspera Linn.

Sample % Water soluble ash %



% RSD 2.1 0.6 1.4 0.4



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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



% RSD 1.8 3.0 1. 4.6


Table 3.14. Percentage loss on drying of Achyranthes aspera Linn.

Sample % Loss on drying %



% 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


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


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


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


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.


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.


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.


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


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.


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.


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.


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


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 = = = = √√√√


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.


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 = = √√√√ = =


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.


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.

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