3. MATERIALS AND METHODS

3.1. PHARMACOGNOSY

3.1.1. Collection of plant materials

Roots of Tephrosia purpurea Pers. and Tephrosia villosa Pers. were

collected in and around Thanjavur.

3.1.2. Plant Identifications

Collected specimen were carefully examined and identified with the help

of regional Floras (Gamble, 1967; Kirthikar and Basu, 1980; Mathew,

1983; Nair and Hendry, 1983 and Henry et al., 1987). The botanical

identity was authenticated by Dr. M. Jegadeesan, Professor and Head,

Dept. of Environmental and Herbal Sciences, Tamil University,

Thanjavur. Specimens were further confirmed with reference to

Herbarium sheet available in the Botanical Survey of India, Southern

Circle, and Coimbatore. A voucher specimen has been deposited at Tamil

University Herbarium for future reference (TUH 277-278).

3.1.3. Taxonomy

Systematic position of the plant specimen has been assigned as per the

angiosperm taxonomic classification of Bentham and Hooker (1862-

1883).

63

3.1.4. Anatomical and histochemical studies (Krishnamoorthy, 1988)

Free hand section of roots of T. purpurea and T. villosa were taken. Then

these sections were stained with different chemical reagents for the

localization of alkaloids, phytosterol, lipids, tannins and carbohydrates.

Destaining was made by washing with respective solvents of the stains.

Sections were mounted on slides using glycerin and covered with cover

glass and margins were sealed with nail polish and observed under

microscope.

3.1.5. Preparation of powder (Harborne, 1973)

The root was separated from the selected species of Tephrosia and dried

under shade. These dried materials were mechanically powdered, sheaved

using 80 meshes and stored in an airtight container. These powdered

materials were used for further physicochemical, phytochemical and

fluorescent analysis.

3.1.6. Analytical studies

The procedures recommended in Indian Pharmacopoeia (Anonymous,

1966; 1985; 1996) were followed for the determination total ash, water-

soluble ash, water-insoluble ash, acid- insoluble ash, sulphated ash and

loss on drying at 110oC.

64

a) Total ash value

5 g of plant powder was ignited in an electric furnace at 600oC in silica

crucible until the sample reaches a constant weight. Percentage of total

ash value was calculated.

b) Water – soluble ash value

Total ash obtained was heated upto 600oC with addition of 25 ml of water

for 10 minutes. It was filtered in an ashless filter paper (Whatman No. 41)

and the residue was ignited in the furnace to get a constant weight.

c) Acid - insoluble ash value

Total ash obtained was heated with addition of 25 ml of dil. HCl for 10

minutes. It was filtered in an ash less filter paper (Whatman No.41) and

the residue was ignited in the furnace to get a constant weight.

d) Sulphated ash value

1g of plant powder was ignited in an electric furnace until the drug gets

charred. The crucible was cooled and the residue was moistened with 1ml

of H2SO4, heated gently until the white fumes were no longer evolved and

ignited at 800oC ± 25

oC until all black particles disappear. The crucible

was allowed to cool; few drop of H2SO4 was added and again heated. The

ignition was carried as before, allowed to cool and then weighed. This

was repeated until the sample reaches a constant weight.

3.1.7. Solubility percentage (Kokate, 1994)

65

a) Alcohol

5 g of powdered material along with 100 ml of alcohol was shaken well

occasionally for the first 6 hours and kept undisturbed for 18 hours. The

liquefied extract thus obtained was concentrated in a vacuum pump and

the percentage was calculated with the weight of the drug powder taken.

b) Water

The procedure adopted for the solubility percentage of the plant powder in

alcohol is used with chloroform water instead of alcohol to get the water

solubility percentage.

3.1.8. Powder analysis

Fluorescent analysis was carried out by using the method of Chase and

Pratt, 1949. Behavior of different chemical reagents was carried out as

mentioned by Kokoshi et al. (1958)

3.1.9. Qualitative phytochemical analysis

Qualitative phytochemical analyses were done using the procedures of

Kokate (1994). Alkaloids, carbohydrates, tannins and phenols,

flavonoides, gums and mucilage, fixed oils and fats and saponins were

qualitatively analyzed.

66

a) Alkaloids

The extracts were dissolved in dil. H2SO4 and filtered. The filtrate was

treated with Mayer’s, Dragendroff’s, Hager’s and Wagner’s reagents

separately. Appearance of cream, orange brown, yellow and reddish

brown precipitates in response to the above reagents respectively indicate

the presence of alkaloids.

b) Carbohydrates

300 mg of 50 per cent alcoholic extracts were dissolved in water

and filtered. The filtrate was treated with con H2SO4 and then with

Molisch’s reagent. Appearance of pink or violet color indicates the

presence of carbohydrates. The filtrate was boiled with Fehling’s and

with Benedict solution. Formation of brick red precipitate in Fehling’s

and Benedict’s solution is the positive result for reducing sugars and non-

reducing sugars respectively.

c) Tannins and phenols

Small quantity of 50 per cent alcoholic extract was dissolved in water and

5 per cent ferric chloride solution or 10 per cent lead acetate solution was

added. Appearance of blue color with ferric chloride or precipitation with

other reagent indicates the presence of tannins and phenols.

67

d) Gum and mucilage

About 10 ml of the extract was slowly added to 25 ml of absolute alcohol

under constant stirring. Precipitation indicates the presence of gum and

mucilage.

e) Fixed oils and fats

A drop of concentrated extract was pressed in between two filter papers

and kept undisturbed. Oil stain on the paper indicates the presence of oils

and fats.

f) Saponins

About 1ml of the extract was dissolved in 20 ml of water and shake in a

graduated cylinder for 15 minutes. Formations of one cm layer of foam

indicate the presence of saponins.

g) Phytosterol

The extract was treated with Lieberman Burchard under suitable

conditions. Appearance of blue-emerald green indicates the presence of

phytosterol and terpenes.

3.1.10. Preparation and concentration of Extracts (Successive)

(Anonymous, 1966).

The root powders of T. purpurea and T. villosa were extracted

successively using Soxhlet apparatus with petroleum ether 60-80°C,

benzene 60°C, chloroform 60°C, alcohol 78°C. Each time before

68

extracting next solvent powdered materials was dried in hot air oven

below 50°C. The extracts dried over anhydrous sodium sulphate, stored in

sealed vials in a refrigerator (5-8°C). Finally mark was macerated with

chloroform water for 24 hrs to obtain the aqueous extract. The extract was

concentrated by distilling of the solvent and then evaporation to dryness

on a water bath.

3.1.11. Cold Extraction Technique

The air-dried material was coarsely powdered to aid the extraction. They

were soaked in alcohol and kept for 48 hours. The extract thus obtained

was decanted and filtered. The clear extract was subsequently

concentrated using rotary vacuum evaporator. This method was done to

save the active principles if any that would have been otherwise

inactivated by a heating process that usually involved in any concentration

process. Pilot biological studies conducted using this extract and when

compared its effects with the heated extracts, the active principles

withstood the heating up to 65oC for least 24 hours. So subsequently the

extracts were concentrated over a boiling water bath on glass Petri-dishes

by free evaporation.

3.1.12. Preparation of Extract

50 per cent alcohol extract was prepared according to the methodology of

Indian Pharmacopoeia (Anonymous, 1996). The 50 per cent alc. extract

was subjected to pharmacological studies and GC/MS.

69

3.2. PHYTOCHEMISTRY

3.2.1. Quantitative phytochemical studies

a) Estimation of total terpenoid

100 g of plant powder were taken separately and soaked in alcohol for 24

hours. Then filtered, the filtrate was extracted with petroleum ether; the

ether extract was treated as total terpenoid (Ferguson, 1956)

b) Estimation of total alkaloid

This alcoholic extract of plant sample was treated with 0.1N HCl and

aqueous acidified layer thus obtained was partitioned with chloroform in a

separating funnel. The chloroform layer is rejected. The aqueous layer

was basified with ammonium hydroxide and then partitioned with

chloroform. The chloroform layer was concentrated and tested for

alkaloids with alkaloid testing reagents (Ferguson, 1956)

c) Estimation of Tannin-free total glycoside

100 g of air-dried powder were extracted with ethanol; water (2:1). The

aqueous ethanol extract thus obtained contains tannins which usually

interfere with the biological activities. Hence, this should be removed by

treating with 5 per cent neutral lead acetate reagent which precipitates the

tannins as lead tannate. The aqueous ethanolic solution was treated with

5per cent neutral lead acetate solution and the precipitated lead tannate

was filtered off. This process is repeated with until no more precipitate

was obtained. The clear filtrate now contained the excess un-precipitated

lead ions in solutions which were removed by passing H2S gas into the

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solution. This removed the lead ions as insoluble complex black lead

sulphide. The black precipitate was filtered and this process was usually

repeated until no more black precipitate was formed and the solution

strongly smelled of H2S. The solution, usually of syrupy consistency, was

concentrated over water-bath maintained at 55oC. This procedure also

removed the excess of H2S (Ferguson, 1956)

d) Estimation of total flavonoid

Isolation of flavonoid from the ethanol extract of root was carried out on

the basis of solubility. For isolation, distilled water (100 ml) was added to

the concentrated ethanol extract (50 ml). After about 1 hour precipitation

was observed. This precipitate was recovered by filtration. Further, the

precipitate was dissolved in chloroform (100 ml) by shaking for 15 min

and heated gently for 5 min and filtered in hot state. The chloroform

soluble fraction was discarded and insoluble fraction, left on filter paper

was dissolved in ethyl acetate soluble fraction was discarded and insoluble

fraction, left on filter paper was crystallized with methanol, thereafter the

residue obtained. For characterization positive result for Shinoda test

which is characteristic of flavonoids. (Jain et al., 2006)

e) Estimation of ascorbic acid (vitamin – C) (Ganguly, 1948)

Titration with Iodine (or) Chloramines T 2

Principle

For determination of ascorbic acid, 0.1N iodine solution (or)

chloramines-T solution have been recommended by Leonhardt and

71

Moeser Both these volumetric solution oxidize ascorbic acid to dehydro

ascorbic acid quantitatively. However Chloramine-T is prepared because

of it is better ability to retain it is titrate with iodine, the end point is not

well defined and starch solution used as indicator retards the reaction.

Titration with iodine is the U.S.P procedure.

Reagents

Iodine solution, 0.1N

14.085 g of reagent is dissolved in water to make 100 ml

Petroleum ether boiling ranges 40C Potassium Iodide. Reagent grade

Hydrochloric acid (5 g.1.122 - 1.124). Starch solution. A 1 per cent

aqueous solution.

Procedure

A measured volume of assay solution containing 75-250 mg of ascorbic

acid is transferred to a conical flask and diluted to 75 ml with water 1ml

of hydrochloric acid, 1 g of Potassium iodide and a few drops of starch

solution as indicator are added and the solution is titrated with 0.1 N

chloramines-T until a blue color appear in the titrating flask.

The solution can also be titrated using 0.1 N iodine solutions. Ascorbic

acid in Pharmaceutical preparations containing alcohol, glycerol and syrup

is determined by titration with iodine 12 vitamin A&B interfere with the

determination.

72

Calculation

Each milliliter of 0.1 N Chloramine -t T or 0.1N iodine solution

corresponds 8.806

3.2.2. GC-MS Studies

The 50 per cent alc. extract was examined in GC-MS for its chemical

composition by GC-MS engine model, GC–Clarus 500; Perkin Elmer and

Computer Mass Library (Wiley 138L) of 80,000 compounds with a GC

column Elite – 1 (100per cent Methyl Poly Siloxane). The other

conditions were as follows.

Injector: GC-Clarus – 500; Perkin Elmer; Carrier gas flow Helium 1

ml/min; Split ratio – 1:25; Sample injected 1µl; Oven temperature –

110deg – 2 min hold; Up to 270deg at the ratio of 5 deg/min – 4 min hold;

Injector temperature 250oC; Total GC- time 38 min; MS inlet line

temperature 200oC; Source temperature 200

oC; Electron energy 70eV;

Mass Scan 25-400; MS time 39 min.

3.2.3. TLC studies

TLC studies on 50 per cent alcoholic extracts of the powdered

drugs of T. purpurea and T. villosa were carried out.

TLC plates were prepared by using Silica Gel-G as adsorbent. 100 g

silica gel-G was mixed with sufficient quantity of distilled water to make

slurry. The slurry was immediately poured into a spreading the slurry on

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glass plates of required size. The thickness of the layer was fixed 1.5 mm

plates were allowed to air dry for one hour and layer was fixed by drying

at 110oC for two hours.

Using micropipette, about 10 ml of 1 per cent w/v solution of extraction

were loaded gradually over the plate. The loaded plate was eluted by

suitable mobile phase like TBA (t-BuOH – AcOH – H2O - 3:1:1 ratio)

BAW (n-BuOH- AcOH-H2O – 4:1:5 ratio – Upper phase), Ferostal

(AcOH – Con.HCl – H2O – 30:3:10 ratio) and MWF (Methanol-Water-

Formic acid- 40:57:3, v/v/v) reported by Jain et al., 2009. Before the

elution, the tank and extract were allowed 30 minutes for saturation with

mobile phase. The extracts showed separation into bands. The

chromatograms were observed under visible light and were photographed.

The Rf value of the band can be obtained by using the following formula.

Rf =

Distance traveled by the substance (cm)

Distance traveled by the mobile phase (cm)

3.3. PHARMACOLOGY

Pharmacological experiments involving animals described in the present

work were carried out and get approved by Local Animal Ethical

Committee of Dept. of Pharmacology, Periyar College of Pharmacy for

Women, Trichy.

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3.3.1. Toxicological Studies

Gross behavioural and acute oral toxicity studies

Gross behavioural and acute toxicity studies of the extracts were

determined as suggested by Turner (1965). The mice were divided into 6

groups and 6 mice in each, 50per cent ethanolic extract was administered

orally at different dose levels of 1, 2 and 3 g/Kg B.W. to the overnight

fasted animals. The group receiving Tween 80 (1 ml/Kg B.W.) was kept

as control. The animals were subjected to primary screening studies at ½,

1, 2 and 4h respectively was recorded. Behaviour of the animals and any

other toxic symptoms were also observed for 24, 48 and 72 h and the

animals were kept under observation upto 14 days after drug

administration to find our delayed mortality if any (Miller and Trainter,

1944; Gosh, 1991).

3.3.2. Anti-ulcer study (Ethanol induced model)

The gastric ulcer was induced in rats of either sex weighing between 130-

140 g by administrating absolute ethanol (8 ml/Kg B.W.). They were kept

in specially constructed cages to prevent coprophagia during and after

the experiment. The rats were divided in to nine groups each containing

six animals and fasted for 24 hrs and allowed free access to water only

and second group received ethanol orally. The third group received

ethanol and standard antiulcer drugs Ranitidine (150 mg/Kg B.W.). The

fourth, fifth, sixth, seventh, eighth and ninth groups were given absolute

alcohol and 50 per cent alc. extracts of T. purpurea and

T. villosa at a dose of 5, 10, 20 mg/Kg B.W. respectively. The drugs were

75

administered orally 30 min prior to the oral administration of absolute

ethanol. The animals were anesthetized 6 h later with ether and stomachs

were incised along the greater curvature, collected the gastric juice and

ulceration was recorded.

The collected gastric juice were analyzed for gastric volume, pH, free

acidity (Jeffery et al., 1992). Biochemical estimation like total protein,

hexose, hexosamine (Dische and Schettles, 1948; Dische and

Borentreund, 1950; Lowery et al., 1951; Winzler, 1958; Warren, 1959;

Debnath et al., 1974 and Goel et al., 1985) were also done. The mucosa

was fleshed with saline and stomach pinned on a frog board and scored.

The number of ulcer and length of each ulcer were measured.

3.3.2.1. Biochemical estimation

Estimation of free and total acidity, mucosal glycoproteins and pepsin

in gastric juice (Hawk, 1947 and Szabo et al., 1985)

a) Collection of gastric juice

Gastric juice was collected from the pylorus-ligated rats. The gastric juice

thus collected was centrifuged and the volume of gastric juice as well pH

of gastric juice was measured. The sodium (Na+) and potassium (K

+) ion

concentration of gastric juice was carried out in flame photometer (Jeffery

et al., 1991). Then the gastric juice was subjected to bio-chemical

estimation as follows.

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b) Determination of free and total acidity in gastric juice (Hawk,

1947)

1 ml of gastric juice was pipette into a 100ml conical flask; add 10ml of

distilled water. Note the pH of this solution using with the help of pH -

Meter, then added 2 to 3 drops of Topfer’s reagent and triturated with

0.01N NaOH ( which was previously standardized with 0.01N of oxalic

acid ) until all traces of the red color disappears and the color of solution

was yellowish orange. The volume of alkali added was noted. The

volume corresponds to free acidity. Then 2 to 3 drops of phenolphthalein

solution was added and titration was continued until a definite red tinge

reappears. Again the total volume of added was noted. The volume

corresponds to total acidity.

Acidity was calculated by using the formula:-

Volume of NaOH x Normality of NaOH x 100

Acidity = meq/l/100g

0.1

c) Estimation of Sodium (Na+) and Potassium (K

+) ion concentration

in gastric juice (Jeffery et al., 1991)

The estimation for sodium and potassium ions was carried out using

Systronics mediflame 127 – flame photometer.

Preparation of stock solution

1. Sodium stock solution was prepared by dissolving 2.542 g NaCl

in 1 liter of distilled water. It contains 1mg Na per ml (i.e. 1000

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ppm). Stock solution was diluted to give four solutions

containing 10, 5, 2.5 and 1 ppm of sodium ions.

2. Potassium stock solution was prepared by dissolving 1.909 g KCl

in 1 liter of distilled water. It contains 1mg potassium per ml

(i.e. 1000 ppm). Stock solution was diluted to give four solutions

containing 20, 10, 5 and 2 ppm of potassium ions.

Procedure

For sodium and potassium, the flame intensity corresponding to the

concentration of stock solution was noted using appropriate filters. The

results were plotted in a graph. The flame intensity of the gastric juice was

noted. The concentration of sodium and potassium ions was calculated

from the graph. The results are expressed in terms of mg / l.

d) Estimation of pepsin (Debnath et al., 1974)

For each determination four tubes (1) and (2) containing 5ml of substrate,

(3) and (4) containing 10ml TCA was placed in the water bath at 37oC.

The gastric juice was mixed with an equal volume of HCl at pH 2.1,

warmed to 37oC and added 1ml of mixture to each tube (1) and (4),

incubated for 15 minutes and at the end mixed the contents of tube (1)

with tube (3) and allowed to stand in the bath for about 4 minutes.

Contents of tube (1) and tube (3) give test and contents of tube (2) and

tube (4) gives blank. Both the contents were filtered after 25-30 minutes,

2ml of filtrate was pippeted into 10 ml of NaOH, mixed by gentle

rotation, then 1ml of phenol was added and again mixed by gentle

78

rotation. After 30 min, intensity of color was measure at 680 nm in

Systronics UV-VIS spectrophotometer- 180.

The difference between test and blank gives a measure of peptic activity.

As standard, mixed 2 ml of freshly prepared phenol solution containing 50

µg/ml with 10 ml of NaOH and 1 ml of phenol reagent was added. After

5-10 minutes, the color intensity was measured at 680 nm.

e) Estimation of total proteins (Lowry et al., 1951)

The dissolved protein in gastric juice was estimated in the alcoholic

precipitate obtained by adding 90 per cent alcohol with gastric juice in 9:1

ratio. Then 0.1 ml of alcoholic precipitate of gastric juice was dissolved

in 1 ml of 0.1N NaOH and from this 0.05 ml was taken in another test

tube, to this 4 ml of alkaline mixture was added and kept for 10 min.

Then 0.4 ml of phenol reagent was added and again 10 min was allowed

for color development. Reading was taken against blank prepared with

distilled water at 610 nm in Systronics UV-VIS spectrophotometer-180.

The protein content was calculated from standard curve prepared with

bovine albumin and was expressed in terms of µg/ ml of gastric juice.

f) Estimation of total carbohydrates (Goel et al., 1985)

The dissolved mucosubstance in gastric juice was estimated in the

alcoholic precipitate obtained by adding 90 per cent alcohol with gastric

juice in 9:1 ratio. Briefly the method consists of taking two aliquots of

gastric juice and treated as follows:

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A) To 1 ml of gastric juice, 9 ml of 90 per cent alcohol was added.

The mixture was kept for 10 minutes before it was centrifuged.

The supernatant was discarded. The precipitate was dissolved in

0.5 ml of 0.1N NaOH. To this 1.8 ml of 6N HCl was added. The

mixture was hydrolyzed in water bath at 100oC for 2 hours. The

hydrolysate was neutralized by 5N NaOH using phenolphthalein

as indicator and the volume was made up to 4.5 ml with distilled

water and using for the estimation of total hexoses, hexosamine

and fucose as described below.

B) To the other aliquot of 0.5 ml gastric juice, 4.5 ml of alcohol was

added. The mixture was shaken for 10 minutes and centrifuged

to obtain precipitate. The precipitate was dissolved in 0.5 ml of

0.1N H2SO4. This reconstituted solution was transferred to glass-

stoppered tubes and then hydrolyzed in a water bath at 100oC for

1 hour. After hydrolysis, the volume restored to 0.5 ml; 0.2 ml of

this hydrolyzes was used for the estimation of sialic acid.

After obtaining the concentration (µg/ml) of individual carbohydrates

namely hexose, hexosamine, fucose and sialic acid, the total carbohydrate

content was calculated by adding the concentration of individual

carbohydrates. Mucosubstances activity has been expressed as ratio of

total carbohydrates to total proteins.

g) Estimation of hexoses (Winzler, 1958)

To 0.4 ml of hydrolysate, 3.4 ml of Orcinol reagent was added. The

mixture was then heated in the boiling water bath 60oC for 15 minutes.

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This was then cooled under running tap water land intensity of the color

was read in Systronics UV-VIS spectrophotometer- 180 at 540 nm against

the blank by using distilled water instead of hydrolysate. Total hexoses

content was determined from the standard curve of D (+) – galactose-

mannose and has been expressed in µg/ml of gastric juice.

h) Estimation of hexosamine (Disch and Borentreund, 1983)

0.5 ml of the hydrolysate fraction was taken. To this 0.5 ml of acetyl-

acetone reagent was added. The mixture was heated in boiling water bath

at 60oC for 20 minutes, and then cooled under running tap water. 1.5 ml

of 90per cent alcohol was added and allowed for 30 minutes. The color

intensity was measured in Systronics UV-VIS spectrophotometer- 180 at

540 nm against blank prepared by using distilled water instead of

hydrolysate. Hexosamine content was determined from the standard

curve prepared by using D (+) – glucosamine hydrochloride and

concentration has been expressed in µg/ml of gastric juice.

i) Estimation of fucose (Dische and Schettles, 1948)

In this method, three test tubes were taken. In one tube 0.4 ml of distilled

water was taken to serve as control and in each of the other two 0.4 ml of

hydrolysate were taken. To all three tubes 1.8 ml of H2SO4: water (6:1)

added by keeping the test tubes in ice-cold water bath to prevent breakage

due to strong exothermic reaction. The mixture was then heated in boiling

water bath for exactly 3 min. The tubes were taken out and cooled. To the

blank and to one of the hydrolysate containing tube (unknown), 0.1 ml of

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cysteine reagent was added while cysteine regent was not added to the last

test tube containing the hydrolysate (unknown blank). It is then allowed

for 90 minutes to complete the reaction. The reading was taken in

Systronics UV-VIS spectrophotometer- 180 at 396 and 430 nm setting

zero with the distilled water. The optical density for the fucose in the

hydrolysate was calculated from the differences in the reading obtained at

396 and 430 nm and subtracting the values without cysteine. This was

read against standard curve prepared with D (+) – fucose content and was

expressed in terms of µg/ml of gastric juice.

(OD396 – OD430) unknown

– (OD396 – OD430) unknown blank

True optical Density =

(OD396 – OD430) water blank

j) Estimation of sialic acid (Warren, 1959)

To 0.5 ml of the hydrolysate in 0.1N H2SO4, 0.2 ml of sodium periodate

was added and mixed thoroughly by shaking. A time of 20 min was

allowed to elapse before addition of 1 ml of sodium arsenite solution to

this mixture. The brown color produced disappeared after shaking. Then 3

ml of thibarbituric acid was added and the mixture was heated in boiling

water bath for 15 minutes. After cooling the tubes, 4.5 ml of

cyclohexanone was added and through shaking was done for 15 seconds

till all the color was taken up by the cyclohexanone supernatant. The

mixture was centrifuged to get a clear pink layer of cyclohexanone. This

supernatant was pippeted out and intensity of color was measured in

Systronics UV-VIS spectrophotometer- 180 at 550 nm. The sialic acid

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content of the sample was determined from the standard curve of sialic

acid and has been expressed in terms of µg/ml of gastric juice.

3.3.3. Anti-inflammatory studies

The anti-inflammatory effects exerted by different doses of 50per cent alc.

extract of T. purpurea and T. villosa were evaluated by conducting the

following methods:

a) Carrageenan induced rat-paw oedema method.

b) Cotton-Pellet granuloma method.

a) Acute Anti-inflammatory Studies: Carrageenan Induced Rat-Paw

Oedema Method (Winter et al., 1962)

Carrageenan induced rat paw oedema method (Winter et. al., 1962) was

followed for acute anti-inflammatory study. The carrageenan, which was

used to provoke rat-paw oedema, was of Sigma grade with the following

particulars: carrageenan (gelatin, vegetable, Irish moss No. 1013; type-I)

believed to be a natural mixture of approximately 80per cent and 20 per

cent, κ and λ carrageenan respectively.

The rat paw oedema was provoked by sub-plantar injection of 0.1 ml 1 per

cent aqueous suspension of carrageenan in 0.9 per cent NaCl in the left

hind-paw. The hind-paw volume was measured by dipping the foot in the

mercury bath of the Plethysmograph – apparatus up to the anatomical

hairline on lateral malleolus (Goldenberg and Ilse, 1977). The

displacement of mercury made a rise in the colored fluid in the thin limb

83

of the Plethysmograph. Before commencing the experiment, the initial

level of the colored fluid in the thin limb was adjusted to zero. So the

increase in reading directly gave the volume of the foot.

Wistar albino rats weighing about 125-150 g were separated into 8 groups

of 6 in each. The animals were allowed with food and water ad libitum

(Chaudari et al., 1977). Each animal in a group was recognised by a mark

of dye on the fur. The initial paw volume was measured and recorded. 1st

group of animals were served as negative control and given 0.75per cent

CMC (5 ml/Kg B.W.). 2nd

group of animals were served as positive

control, which were given Dichlofenac sodium (5 mg/Kg B.W.).The

extracts were administered orally one hour before commencing the

experiments at a dose of 5, 10, 20 mg/Kg body weight. 1per cent

carrageenan suspension in normal saline was prepared 1 hour before use.

0.1 ml was injected under the plantar aponeurosis. 3 hours after the

carrageenan injection the hind-paw volumes were recorded. Thus these

studies were rather indicative of acute anti-inflammatory effect.

The differences between the initial and final paw volume indicated the

oedema volume due to inflammation. The percentage inhibitions produced

by the drugs or principles were calculated which are directly indicative of

the anti-inflammatory activity exerted. The paw volume was measured at

0, 1, 3, 5 hours after injection of carrageenan. Drug pre-treatment was

given one hour before the injection of carrageenan. Mean increase in paw

was measured and per cent inhibition was calculated.

84

100 – Odema Volume in the treated

% of Inhibition = X 100

Odema Volume in the Control

(Chu and Kovacs, 1977)

b) Sub Acute Anti-inflammatory studies: Cotton–Pellet Granuloma

Model

Sub-acute inflammation was produced by the method described by Winter

and Portar (1957) and Turner (1965). This method or technique rather

reveals the chronic (sub-acute) anti-inflammatory effects of a test

substance when it was administered for 7 days in seven divided doses

(Meir et al., 1950 and Winter and Porter, 1957). This method used here

was adopted from Sheth et al. (1972) with a slight modification of using

only male rats (Lassman et al., 1977)

Wistar albino rats weighing about 125 -150 g were divided into 6 groups

of 6 animals in each and were kept in separate cages one or two days

before commencing the experiments.

Using sensitive monoplane balance, which reads the weights to an

accuracy of 0.1 mg, Cotton pellets weighing exactly 10 mg each were

made. Each cotton pellet was rolled to an identical size. The pellets were

sterilized in an autoclave for 45 minutes under 15lbs/ inch 2 of steam

pressure.

The ventral portions of animals were shaved with scissors and swabbed

with alcohol. The animals were anaesthetized with anesthetic ether. 2 or

85

1cm skin incisions were made one on the mid thorax and the other on the

mid abdominal region. Using a pair of blunt artery forceps, a small

channel was made bilaterally and one cotton pellet was placed in each

channel, 1 in the axilla, one in each and 2 in the groin, one in each

subcutaneously. All the air in the channels was removed by gentle

pressing the incisions were closed with the stitches.

Treatment to the control, positive control and the test groups were

initiated a soon as the animals were recovered from anesthesia and

continued daily for 7 days. The dosage, treatment and grouping were done

as described in acute anti-inflammatory studies.

On the 8th

day the animals were sacrificed and the cotton pellets were

removed. They were weighed immediately after separating the adjoining

and masking subcutaneous fascia. Then they were dried in a hot air oven

maintained at 60oC for at least 24 hours and weighed. By subtracting dry

weight of granuloma cotton with initial by weight, the percentage

inhibition of granuloma was calculated.

The weight of the cotton pellet before implantation was subtracted from

the weight of dried granuloma pellets. The values were expressed as mean

± SEM.

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3.3.4. Anti-histamine activity

3.3.4.1. Instruments

1. Kymograph and smoked drum

2. Frontal lever

3. L. stand

4. T-rod

5. X-blocks

6. Screw clip

7. Marriotte bottle

8. Rubber tubes

9. Tuberculin’s syringe 26 no needle

10. Droppers

11. Thermometer

12. Thread and needle (non-stretch nylon)

13. Surgical gloves

14. Acrylic board

15. Dissection Kit: Scalpel, Forceps, Scissors, Dissection Pins, Tape

16. Microscope

17. Petridish

3.3.4.2. Chemical used

Histamine was purchased from Sigma-Aldrich, St. Louis, USA. Other

chemicals used include Sodium chloride (NaCl), Potassium Chloride

(KCl) Calcium Chloride (CaCl2), Magnesium Chloride (MgCl2) Sodium

bicarbonate (NaHCO3), Sodium dihydrogen phosphate (NaH2PO4) and

Glucose from Hi-Media laboratory Pvt Ltd, Mumbai, India.

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3.3.4.3. Extract preparation

60 mg portion of both 50per cent alc. extracts were scraped off

from the bottom of the container and placed in a mortar and pestle. To this

added 2ml of distilled water and triturated well. This mixture was then

made up with 6ml of distilled water. This process gave a stock solution of

10 mg / ml. This solution was tested against the guinea pig ileum

preparation.

3.3.4.4. Histamine

A stock solution of 5µg/ml was made with tyrode solution. This

concentration was added to the bath and used as a standard drug.

3.3.4.5. Selection of animal’s species

The Healthy Adult male guinea pigs (460 g; Hartley strain) were

kept separately in individual polypropylene cage with stainless steel

hopper. The females were nulliparous and non-pregnant.

3.3.4.6. Housing and feeding

The temperature in the experimental animal room was 220 C ± 30

0 C.

Although the relative humidity was 30 per cent and preferably not

exceeding 70 per cent other than during room cleaning and the aim was

50-60 per cent. Lighting used artificially, the sequence being 12 hours

light and 12 hours dark. The animal was housed individually. For feeding,

conventional laboratory diets was used with an unlimited supply of

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drinking water. The study was performed under CPCSEA guidelines and

IAEC.

3.3.4.7. Preparation of animals

The animals were uniquely identified and kept in their cages for five days

prior to dosing for acclimatized to the laboratory conditions. During

acclimatization the animals were observed for ill health.

3.3.4.8. Perfusion Apparatus (Morgan et al., 1961)

In this system the tissue was suspended in a 2 X 20 cm (internal

dimensions) water-jacketed chamber with a coarse sintered glass filter

disk sealed into the lower portion. A mixture of moistened O2:CO2 (95:5)

was delivered by small diameter tubing to the lower portion of the

chamber by the aerator.

a) Methods

For the preparation of tissues, adult male guinea pigs (460 g; Hartley

strain) were killed by a blow to the head and exanguinated. The abdomen

region was opened and identified ileo-cecal junction. The lumen of ileum

were removed, the intact tissues and rubbed preparations in which the

blood had been removed by vigorously rubbing the luminal surface with

filter paper. A piece of ileum was excised (approximately 3-4 cm) by

using surgical suturing needle tied a thread at each end. One end of the

thread was tied to the hook of the aeration tube and the other to frontal

writing lever. The ileum was mounted in 30 ml organ bath under a load of

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500mg. The tissues were allowed to equilibrate for 90min in Tyrode

solution (composition in mM): NaCl 139.2, KCl 2.7, CaCl2 1.8, and

MgCl2 0.49, NaHCO3 11.9, NaH2PO4 0.4, glucose 5.5, pH 7.4 and gassed

with 5per cent CO2 and 95per cent O2 at 37°C. During the equilibration

period the bath fluid was exchanged every 10 min with fresh Tyrode

solution. All protocols were applied to both intact and rubbed

preparations. (Braunstein et al., 1988)

b) Drug injected order

Stabilization--Histamine (500ng) --Washing--Histamine (1µg)--Histamine

(2 µg) --Washing-Histamine (4µg) –Washing-- Sample (2mg) +Histamine

(500ng) --Washing--Sample (4mg) +Histamine (500ng)--Washing--

Sample (8mg) + Histamine (500ng) --Washing—

3.3.5. Analgesic activities

a) Acetic acid induced model (Chemical stimulus)

Mice were divided in to 8 groups (6). Group wise animal received T.

purpurea and T. villosa of 50per cent alc. extracts at a dose level of 5, 10,

20 control groups received normal saline and the reference group received

400 mg/Kg B.W. aspirin. Drug pretreatment was given one hr before i.p

injection of 0.6per centv/v acetic acid (10 ml/Kg B.W.). The severity of

pain response (writhing) was assessed by counting the number of writhes

(construction of abdomen) in mice (Koster et al., 1959). Number of

writhes per animal was counted during 15 minutes series beginning 5

minutes after injection of acetic acid.

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Analgesic activity was calculated as maximum possible effect (per cent

MPE) using the following relation.

100 x (Mean of writhes in control group-Mean of writhes

in treated group)

% MPE =

Mean of writhes in control group

b) Tail immersion method (Thermal stimulus)

All the mice were screened by expose to thermal stimulation. Those

showing positive response were divided into groups of six animals each.

Normal saline (control) 5, 10, 20 mg/Kg B.W. of the selected extracts

respectively and 1mg/Kg B.W. Fortral (Pentazocine) were administered

i.p. The time taken to withdraw the tail clearly out of the water was

considered as the reaction time with cut off time being 60 seconds. The

reading was immediately after administration of the drugs, and 60 min

later (Awe et al., 1998)

3.3.6. Anti-diarrhoeal studies (Castor oil induced)

The method described by Awounters et al. (1978) was followed.

Healthy albino rats of the either sex (160-190g) were divided into 8

groups of 6 animals each. They were fasted for 18hours prior to the test,

with free access to water. Group-1 received the vehicle (0.2 ml of 5per

cent Tween 80) and served as the control group. Groups 2 received

standard drug (Loperamide 3 mg/Kg B.W.), the third, fourth, fifth, sixth,

seventh and eight groups were given 50per cent alc. extract of the

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T. purpurea and T. villosa root at a dose of 5, 10 and 20 mg/Kg B.W.

respectively.

Thirty minutes after the drug treatment, each rat was administered

1ml of castor oil orally and housed separately in metabolic cages, with

special provision to separate urine and faeces. The diarrheal episodes were

observed for 4 hours. Percent inhibition of diarrhoea was calculated using

the mean stool weight. Anti-diarrheal activity was determined in terms of

percentage protection.

3.3.7. Antimicrobial studies

3.3.7.1. Media preparation

a) Bacterial medium (Muller Hinton Agar)

36 g of Muller Hinton Media (Hi-Media) was mixed with distilled water

and then sterilized in autoclave at 15 lb pressure for 15 minutes. The

sterilized media were poured into petri dishes. The solidified plates were

pored with 6 mm dia. cork borer. The plates with wells were used for the

antibacterial studies.

b) Fungal medium (Potato Dextrose Agar)

200 gm of potato slices were boiled with distilled water. The potato

infusion was used as water source of media preparation. 20 g of dextrose

was mixed with potato infusion and the pH was adjusted to 5.6. 20 g of

agar was added as a solidifying agent. These constituents were mixed and

autoclaved. Streptomycin sulphate of 50 µg/ml was added when pouring

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into petriplates at molten condition. The solidified plates were pored with

6mm dia. cork borer.

3.3.7.2. Test against standard controls

Commercially available antibiotic disc Oflaxacin (20 µg) and

Chloramphenicol (30 µg) were used as standard control for the entire test

microorganism. The sensitivity patterns were recorded and the readings

were interpreted according to the critical diameter given by National

Committee for Clinical Laboratory Standards (NCCLS, 1997).

3.3.7.3. Bacterial inoculums

Bacterial inoculums were prepared by inoculating a loopful of test

organisms in 5 ml nutrient broth and incubated at 370C for 5 to 8hrs till a

moderate turbidity was developed. The turbidity was matched with 0.5

McFarland standard (WHO drug information, 1993) and the culture was

diluted with sterile distilled water if necessary which corresponds to the

cell density of 1.5 x 108 (cfu/ml).

3.3.7.4. Fungal inoculums

The in vitro method proposed by National Committee for Clinical

Laboratory Standard for testing Molds (NCCLS, 1997) was followed for

the present study. The fungal stock inoculum suspensions were prepared

from two day old culture grown on PDA medium. The fungal colonies

were covered with 10 ml of sterile distilled water and suspensions were

made by gently probing the surface with the tip of the Pasteur pipette.

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The resulting measure of conidia and hyphal fragments were withdrawn

and transferred to a sterile tube. Heavy particles were allowed to settle for

5-20 minutes, and the homogenous suspension were collected and mixed

with a vortex mixture. The density of these suspensions was adjusted with

a spectrophotometer at a wave length of 530 nm for 80-85per cent to

obtain the standard inoculums.

3.3.7.5. Antimicrobial assay (Bauer et al., 1996)

a) Antibacterial activity of the plant extract

Antibacterial and antifungal activity of the plant extract was tested using

well diffusion method (Bauer et al., 1996). The prepared culture plates

were inoculated with different selected strains of bacteria and fungi using

streak plate method. Wells were made on the agar surface with 6mm cork

borer. The extracts were loaded into the well using sterile syringe. The

plates were then kept in refrigerator for 15min. to allow diffusion of the

extracts into the media. The plates were incubated 24 hours at 37 ± 2oC

and 48 hours at 29 ± 1oC bacterial and fungal plates respectively. The

plates were observed for inhibition zone formation around the well. The

zone of inhibition was calculated by measuring the diameter of the

inhibition zone around the well (in mm) including the well diameter. The

readings were taken in three different fixed directions in all 3 replicates

and the average values were tabulated.

The 50 per cent alc. extract of T. purpurea and T. villosa was used

throughout the study. The extract of 10, 25 & 50 µ1 were tested against

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different bacterial pathogens such as Bacillus subtilis, Citrobacter

divergens, Klebsiella pneumonia, Staphylococcus aureus,

Staphylococcus epidermis for their antimicrobial activity. It was

demonstrated by well diffusion assay.

b) Antifungal activity of the plant extract

One ml volume of the inoculum was uniformly seeded on PDA plates

consist of 50 µg/ml of streptomycin sulphate. The in vitro antifungal

activity of the plant extracts were assessed by well diffusion method.

After 10 minutes, three wells of 6 mm size were made with the help of

cork borer and each concentration of the test drugs was aseptically loaded.

The plates were incubated at 29 ± 10C and were examined for the

appearance of fungal colony with inhibition zone.

The 50 per cent extract of 10, 25 & 50 µ1 were tested against different

fungal pathogens such as Aspergillus flavus, Aspergilus niger, Candida

albicans, Ganoderma lucidum, Mucor indicus for their antifungal

activity. It was demonstrated by well diffusion assay.

3.3.8. Statistical Analysis (Ghosh, 1984)

The raw data of the present study were subjected to simple statistical

analysis to draw meaningful interpretation and conclusion.

1. The standardization values of study drugs were expressed in

percentage (w/w).

2. For pharmacological studies:

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• Student’s ‘t’ test are computed for all the biochemical

estimations, to find out statistical significance at 1 per cent and

5 per cent probability levels.