You are on page 1of 34

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 (18621883).

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, watersoluble 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 25oC 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 Mayers, Dragendroffs, Hagers and Wagners 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 Molischs reagent. Appearance of pink or violet color indicates the presence of carbohydrates. The filtrate was boiled with Fehlings and with Benedict solution. Formation of brick red precipitate in Fehlings and Benedicts solution is the positive result for reducing sugars and nonreducing 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 60-80C, benzene 60C, chloroform 60C, alcohol 78C. Each time before

68

extracting next solvent powdered materials was dried in hot air oven below 50C. The extracts dried over anhydrous sodium sulphate, stored in sealed vials in a refrigerator (5-8C). 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

70

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, GCClarus 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 1l; 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 200oC; 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

73

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-WaterFormic 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. Distance traveled by the substance (cm) Rf = 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.

74

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 , 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 130140 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.

76

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 Topfers 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

77

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 g/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 g/ 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:

79

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 glassstoppered 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 (g/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.

80

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 (+) galactosemannose and has been expressed in g/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 acetylacetone 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 g/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

81

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 g/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

82

content of the sample was determined from the standard curve of sialic acid and has been expressed in terms of g/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, and 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: CottonPellet 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.

86

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. Tuberculins 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.

87

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 5g/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 animals 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 300 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

88

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

89

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 37C. 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 (1g)--Histamine (2 g) --Washing-Histamine (4g) 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.

90

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

91

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 g/ml was added when pouring

92

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 g) and Chloramphenicol (30 g) 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.

93

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 1 were tested against

94

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 g/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 1 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:

95

Students t test are computed for all the biochemical estimations, to find out statistical significance at 1 per cent and 5 per cent probability levels.

You might also like