MATERIALS AND METHODS

3. MATERIALS AND METHODS 3.1. PHARMACOGNOSTICAL STUDIES 3.1.1. Collection of plant materials: Plant parts of Pedalium murex and Martynia annua for the proposed study were collected from Karaikal district, U.T of Puducherry and care was taken to select healthy plants and for normal organs. The identity of the plant specimens was confirmed by the use of local Floras (Hooker, 1885; Gamble, 1957; Nair and Henry, 1983; Matthew, 1983; Henry et al., 1987) and medicinal plant Treatises (Anonymous, 1992; Chatterjee and Pakrashi, 1994; Kirtikar and Basu, 1965). The botanical identify was authenticated by Dr. M. Jegadeesan, Professor, Department of Environmental and Herbal Science, Tamil University, Thanjavur. Herbarium specimens of these two taxa (Plate 1 4) were deposited at Tamil University Herbarium (TUH ).

Medicinally useful parts of the plants were studied in both fresh and dried conditions (Trease, 1961; Trease and Evans, 1983; Wallis, 1985; Kokate, 1994; Kokate et al., 1995). The required samples of different organs were cut and removed from the plant and fixed in FAA (Formalin 5 ml + Acetic acid 5 ml + 70 % Ethyl Alcohol 90 ml) as per the schedule given by Sass (1940). Infiltration

of the specimens was carried by gradual addition of paraffin wax (melting point 58-60C) Tertiary Butyl Alcohol (TBA) until TBA solution attained super saturation. The specimens were cast into paraffin blocks. 3.1.2. Sectioning The paraffin embedded specimens were sectioned with the help of Rotary Microtome. The thickness of the sections was 10-12 m. De-waxing of the sections was done by customary procedure (Johansen, 1940). The sections were stained with Toluidine blue as per method published by OBrian et al (1964). Since Toluidine blue is a polychromatic stain, the staining results were remarkably good and some cytochemical reactions were also obtained. The dye rendered pink colour to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the mucilage, blue to the protein bodies etc. Wherever, necessary sections were also stained with safranin and Fast-green and IKI (for starch). For studying stomatal morphology, venation pattern and trichome distribution, paradermal sections (sections taken parallel to the surface of leaf) as well as clearing of the leaf with 5 % sodium hydroxide or epidermal peeling by partial maceration employing Jeffreys maceration fluid (Sass, 1940) were employed. Glycerine mounted temporary preparations were

made for macerated materials. Powdered materials of different parts were cleared with NaoH and mounted in glycerine medium after staining. Different cell component were studied and measured. 3.1.3. Photomicrographs Microscopic descriptions of tissues are supplemented with

micrographs wherever necessary. Photographs of different magnifications were taken with Nikon Labphot 2 microscopic unit. For normal observations bright field was used. For the study of crystals, starch grains and lignified cells, polarized light was used as they appear bright against dark background. Magnifications of the figures are indicated by the scalebars. Descriptive terms of the anatomical features are as given in the standard anatomy books (Esau, 1960, 1964). 3.2. GLASSWARE AND CHEMICALS Good quality glassware and chemicals were used for all tests. They were washed with good detergent, rinsed in tap water and soaked in chromic acid clearing solution. Cleaning solution (Mahadevan and Sridhar, 1996) Potassium dichromate 60 g

Conc. H2So4 Distilled water

60 ml 1L

3.2.1. QUANTITATIVE MICROSCOPY (Kokate et al., 1995) A piece of leaf was cut in the middle portion and boiled in chloral hydrate solution or treated with chlorinated soda. Upper and lower epidermis were peeled out and mounted on glycerine on a glass slide. The slide was observed under microscope. The following measurements or determination were calculated according to the procedures laid by Salisbury (1927) and Kokate et al., (1995) using stage micrometer and camera lucida. i) ii) iii) iv) v) vi) Number of stomata present in l mm2 Total number of epidermal cells in l mm2 Total number of vein islet present in l mm2 Total number of vein termination in l mm2 Palisade ratio Number of trichomes in leaf, petiole and stem / mm2

3.3. ANALYTICAL METHODS 3.3.1. PREPARATION OF POWDER AND REAGENTS The whole plant parts of P. murex and M. annua were collected and dried under shade. These dried materials were mechanically powdered after keeping them in an oven at 350C for 24 hrs. These powdered materials were used for further physico-chemical, fluorescent and phyto-chemical analyses. The procedure recommended in Indian Pharmacopoeia (Anonymous, 1966; 1985; 1996) were followed for the determination of total ash, acid insoluble ash, water soluble ash, sulphated ash, loss on drying at 110C, Na+ and K+ content. Separately the plant materials were ground to course powder and passed through 80 m mesh sieve. These powdered materials were used for pharmacological evaluation. All chemicals and solvents used for different studies were of analytical reagent / spectroscopy grade. 3.3.2. STUDY ON ORGANOLEPTIC CHARACTERISTICS The organoleptic characteristics of powered samples namely their appearance and colour in day light, smell and their taste were also studied.

A. Loss on drying: The powdered samples were dried in an electrical oven at 1050C until reaches a constant weight. B. Determination of pH of Aqueous Solution: The powdered materials (80 m mesh) were suspended in glass distilled water. After 2hrs, filtered and the clear solution was measured for pH. C. Total Ash Value 5 gm of plant powder was ignited in an electric furnace at 500 5500C in silica crucible until the sample reaches a constant weight. D. Sulphated Value: The ash powder was moistened with 1 ml of H2So4 and ignited to 800 + 25C until reaches a constant weight. E. Water Soluble Ash Value The water insoluble matter was collected in an ashless filter paper and ignited in an electric furnace at 4500C in silica crucible until reaches a

constant value. The weight of insoluble matter was subtracted from the weight of the total ash to indicate the weight of water soluble ash. F. Acid Insoluble Ash Value Total ash obtained was heated with addition of 25 ml of dilute HCl for 10 min. It was filtered in an ashless filter paper (Whatman No. 41) and the residue was ignited in the furnace to get a constant weight. G. Determination of K+ and Na+ Content 5 g of the powder was ashed in a furnace at 500 550C. The ash was further heated with 25 ml of dilute HCl for 5 min. The solution was filtered through ashless filter paper, the container and filter paper were washed several times with distilled water. The acid filtrate and washings were made up to 100 ml in volumetric flask. The percentage of Na+ and K+ were calculated with reference to air dried powder. Na+ and K+ content were quantified by Flame Photometer based on Watad et al., (1986). H. Preparation of Extracts The percentage of extractive values in different solvents namely petroleum ether (60 80C), chloroform, acetone, methanol and water

calculated by successive extraction of the sample in a soxhlet extractor for 6 hrs each. Water Soluble Extractive Five gram of powdered plant materials (stem bark, root bark and leaves) was added to 50 ml of water at 80C in a stoppered flask. It was shaken well and allowed to stand for 10 min. It was cooled to 15C and 2 g of Kiesdghr was added and filtered. 5 ml of the filtrate was transferred to a tarred evaporating basin. The solvent was evaporated on a water bath for 30 minutes and then dried in a steam over water soluble extractive was calculated with reference to the air-dried powdered plant material. Alcohol Soluble Extractive Five gram of the air - dried powdered plant material (Stem bark, root bark and leaves), coarsely powdered was macerated with 100 ml of alcohol of the specified strength in a closed flask for 24 hrs, shaken frequently for 6h and allowed to stand for 18 hrs. It was filtered rapidly taking precautions against loss of alcohol and 25 ml of the filtrate was evaporated to dryness in a tarred flat bottomed shallow dish and dried at 105C and weighed. The percentage of alcohol soluble extractive was calculated with reference to the air-dried powered plant material.

I. EXTRACTIVE VALUES The material, 5 g was weighed in a thimble and it was placed in a Soxhlet apparatus. Hexane was taken in the round bottom flask and hot extraction was carried out for 24 hrs. The extract in the round bottom flask was concentrated by distillation and the dry extract was weighed to get the hexane soluble fraction. The mare was used for successive extraction with chloroform and methanol. The percentage solubility in each case was calculated with reference to the powdered plant material (stem bark, root bark and leaves) taken initially. 80 m mesh powders of plants were extracted sequentially (Soxlet) with alcohol. The water extracts were purified separately. The extracts were evaporated under vaccum. Extractive value (%W/W) of plant materials in water and alcohol were calculated separately. J. FLUORESCENCE ANALYSIS Fluorescent characteristics of the plant powders as such and after treating them with chemical reagents were observed in day light as well as under UV radiation. Fluorescent analyses of all the plant powders were carried out according to the methods of Chase and Pratt (1949) and Kokoshi et al (1958). Behaviour of powdered plant materials with different chemical reagents was carried out as described by Kay (1938) and Johansen (1940).

3.4. QUALITATIVE PHYTOCHEMICAL SCREENING The different qualitative chemical tests were performed for establishing profile of given extract for its chemical composition. Qualitative phytochemical analyses were done using the procedures of Kokate (1994) and Kokate et al (1995). The following tests were performed on extracts to detect various phyto constituents present in them. 3.4.1. Detection of Alkaloids Solvent free extract, 50 mg was stirred with few ml of dilute hydrochloric acid and filtered. The filtrate was tested carefully with various alkaloidal regents as follows. A. Mayers Test To a few ml of filtrate, a drop or two of Mayers reagent were added by the side of the test tube. A white or creamy precipitate indicated the test as positive. Mayers Reagent Mercuric chloride (1 35 g) was dissolved in 60 ml of water and potassium iodide (5.0 g) was dissolved in 10 ml of water. The two solutions were mixed and made up to 100 ml with water.

B. Wagners test To a few ml of filtrate, few drops of Wagners reagent were added by the side of the test tube. A reddish brown precipitate confirmed the test as positive. Wagners reagent Iodine (1.27 g) and potassium iodide (2 g) was dissolved in 5 ml of water and made up to 100 mL with distilled water. C. Hagers test To a few ml of filtrate 1 or 2 ml of Hagers reagent (saturated aqueous solution of picric acid) were added. A prominent yellow precipitate indicated the test as positive. D. Dragendorffs test To a few ml of filtrate, 1 2 ml of Dragendorffs reagent were added. A prominent yellow precipitate indicated the test as positive.

Dragendorffs reagent Stock solution Bismuth carbonate (5.2 g) and sodium iodide (4 g) were boiled for a few min with 50 ml glacial acetic acid. After 12hrs, the precipitated sodium acetate crystals were filtered off using a sintered glass funnel. Clear, reddish brown filtrate, 40 ml was mixed with 160 ml ethyl acetate and 1 ml water and stored in amber coloured bottle. Working Solution Stock solution 910 ml) was mixed with 20 ml of acetic acid and made up to 100 ml with water. 3.4.2. Detection of Carbohydrates The extract (100 mg) was dissolved in 5 ml of water and filtered. The filtrate was subjected to the following tests. A. Molishs test To 2 ml of filtrate, two drops of alcoholic solution of -naphthol were added, the mixture was shaken well and 1 ml of concentrated sulphuric acid was added slowly along the sides of the test tube and allowed to stand. A violet ring indicated the presence of carbohydrates.

B. Fehlings test One ml of filtrate was boiled on water bath with 1 ml each of Fehling solutions A and B. A red precipitate indicated the presence of sugar. Fehlings Solution Fehlings solution A: Copper sulphate (34.66 g) was dissolved in distilled water and made up to 500 ml distilled water. Fehlings solution B: potassium sodium tartarate (173 g) and sodium hydroxide (50 g) was dissolved in water and made up to 500 ml. C. Barfoeds test To 1 ml of filtrate, 1 ml of Barfoeds reagent was added and heated on a boiling water bath for 2 min. Red precipitate indicated presence of sugar. Barfoeds reagent Copper acetate, 30 5 g was dissolved in 1-8 ml of glacial acetic acid. D. Benedicts test To 0.5 ml of filtrate, 0.5 ml of Benedicts reagent was added. The mixture was heated on a boiling water bath for 2 min. A characteristic colored precipitate indicated the presence so sugar.

Benedicts reagent Sodium citrate (173 g) and sodium carbonate (100 g) were dissolved in 800 ml distilled water and boiled to make it clear. Capper sulphate (17.3 g) dissolved in 100 ml distilled water was added to it. 3.4.3. Detection of Glycosides For detection of glycosides, 50 mg of extract was hydrolysed with concentrated hydrochloric acid for 2 hrs on water both, filtered and the hydrolysate was subjected to the following tests. Borntragers test To 2 ml of filtered hydrolysate, 3 ml of chloroform was added and shaken, chloroform layer was separated and 10% ammonia solution was added to it pink colour indicated the presence of glycosides. Legals test Fifty mg of the extract was dissolved in pyridine, sodium nitroprusside solution was added and made alkaline using 10 % sodium hydroxide. Presence of glycoside was indicated by pink colour.

3.4.4. Detection of Saponins by Foam Test (Kokate, 1999) The extract (50 mg) was diluted with distilled water and made up to 20 ml. The suspension was shaken in a graduated cylinder for 15 min. A two cm layer of foam indicated the presence of saponins. 3.4.5. Detection of Proteins and Amino Acids The extract (100 mg) was dissolved in 10 ml of distilled water and filtered through whatman No.1 filter paper and the filtrate was subjected to tests for proteins and amino acids. Millons test To 2 ml of filtrate, few drops of Millons reagent were added. A white precipitate indicated the presence of proteins. Millons Reagent Mercury (1g) was dissolved in 9 ml of fuming nitric acid. When the reaction was completed, equal volume of distilled water was added. Biuret test An aliquot of 2 ml of filtrate was treated with one drop of 2 % copper sulphate solution. To this, 1 ml of ethanol (95%) was added, followed by

excess of potassium hydroxide pellets, pink colour in the ethanolic layer indicated the presence of proteins. Ninhydrin test Two drops of ninhudrin solution (10 mg of ninhydrin in 200 ml of actone) were added to two ml of aqueous filtrate. A characteristic purple colour indicated the presence of amino acid. Hopkins Cole test 2 Drops of Hopins Cole reagent was added to 2 ml of aqueous filtrate. A characteristic violet colour indicates the presence of protein. 3.4.6. Detection of Phytosterols A. Libermann Burchards test The extract (50 mg) was dissolved in 2 ml acetic anhydride. To this, one or two drops of concentrated sulphuric acid were added slowly along the side of the test tube. An array of colur changes showed the presence of phytosterols. B. The extract was treated with Salkowskis reagent. The yellowish colour with green fluorescence appearance indicate the presence of phytosterol in it.

3.4.7. Detection of Fixed Oils and Fats Spot test A small quanlity of extract was pressed between two filter papers. Oil stain on the paper indicated the presence of fixed oil. Saponification test A few drop of 0.5 N alcoholic potassium hydroxide solution were added to a small quantity of extract along with a drop of phenolphthalein. The mixture was heated on water bath for 2hrs. Formation of soap or partial neutralization of alkali indicated the presence of fixed oils and fats. 3.4.8. Detection of phenolic compounds and Tannins Ferric chloride test The extract (50 mg) was dissolved in 5 ml of distilled water. To this, few drops of neutral 5% ferric chloride solution were added. A dark green colour indicated the presence of phenolic compounds.

B. Gelatin test The extract (50 mg) was dissolved in 5 ml of distilled water and 2 ml of % solution of gelatin containing 10 % sodium chloride was added to it. White precipitate indicated the presence of phenolic compounds. C. Lead acetates test The extract (50 mg) was dissolved in distilled water and 3 ml of 10% lead acetate solution was added. A bulky white precipitate indicated the presence of phenol compounds. D. Alkaline reagent test An aqueous solution of the extract was treated with 10% ammonium hydroxide solution. Yellow fluorescence indicated the presence of flavonoids. E. Magnesium and hydrochloric acid reduction The extract (50 mg) was dissolved in 5 ml of alcohol and few fragments of magnesium ribbon and concentrated hydrochloric acid (Drop wise) were added. Presence of flavanol glycosides was inferred by the development of pink to crimson colour.

3.4.10. Detection of Gum and Mucilage The extract (100mg) was dissolved in 10 ml of distilled water and to this, 25 ml of absolute alcohol was added with constant stirring. White or cloudy precipitate indicated the presence of gums and mucilages. 3.5. THIN LAYER CHROMATOGRAPHY (TLC) Thin layer chromatographic studies of ethanolic and chloroform extracts of the powdered drugs of Pedalium murex and Martynia annua were carried out. The extracts were prepared separately by allowing a small quantity each of the drugs to remain in alcohol or chloroform overnight. TLC plates were prepared by using silica GelG as adsorbent. 100 mg silica GelG was mixed with sufficient quantity of distilled water to make slurry. The slurry was immediately poured into a spreader and plates were prepared by spreading the slurry on the glass plates of the required size. The thickness of the layer was fixed as 1.5 mm. Plates were allowed to air dry for 1 hour and layer was fixed by drying at 105C for 1 hour. Using a syringe and needle, about 10 l of 1 % W/V solution of extracts were loaded gradually over the plate. The loaded plates were eluted by suitable elutent mixture containing 30 % of ethyl acetate in hexane. Before the elution, the tank was allowed 30 minutes for saturation with elutent mixture. The extracts showed

separation into bands. In alcoholic extract chromatogram, the bands were made visible by dipping the profile in pancal-D solution. The chromatograms were observed under both visible light and UV radiation (at 254 nm and 346 nm) and were photographed. 3.6. GC-MS SPECTRUM The plant samples of Pedalium murex and Martynia annua were made to work in Soxhlet apparatus for conquering its Alcoholic and Aqueous extracts. They were subjected to GC-MS examination under column Elite 1 (100% methyl polysiloxane). Gas chromatography combined with mass spectrum (GC-MS) of the purified isolated compounds was recorded by direct inlet method. Equipment Carrier gas Detector : : : GC Clarus 500 Perkin Elmer Helium 1 ml/min Mass Indicator Turbo mass gold Perkin Elmer Sample Injects Split Inlet line temperature Source temperature : : : : 1 Ml Ratio mode 10:1 200C 200C

Electron energy Mass scan

: :

70 V 25 400

at an oven temperature programme of 110 deg 9 min hold up to 280C at the rate of 5C/ min p min hold with the injection temperature at 250C (Total GC time 45 min) under MS programme With the time of 46 minutes was analysed for different compounds present in both the plant samples P. murex and M. annua. Extracts of hexane (10 g), chloroform (15 g) and methonal (25 g) and CD-1, CD-2, CD-3 and CD-4 of isolated compounds of P. murex and M. annua were dissolved in methanol (1 g/ml). These were applied on the precoated TLC plate with the help of sample applicator Linomat 2002) and developed in the suitable solvent system. The plates are viewed under short and long ultraviolet light (254 nm and 366 nm) respectively. The plates were scanned at 254 nm and 346 nm, spectrum and chromatogram were taken. The spectrum of isolated compounds was found to be super imposed with spectrum of the extracts. It was documented photographically.

3.7. PHARMACOGLOGICAL STUDIES Adult Wistar albino male rats (Body weight 170 200 g) were used for pharmacological evaluations for anti inflammatory and diuretic activities. The animals were supplied by the King Institute of Preventive Medicine, Guindy, Chennai. The animals were housed in polypropylene cages at 25+20C with relative humidify of 45 55 % under 12 h light; 12-h dark cycles. Rats were fed with standard laboratory animal feed (Amrut Laboratory animal feed, Sangli 416436) and water ad libitum. 3.7.1. Carrageenan-induced paw edema The rats were divided into six groups (n=8) and the first group served as negative control (received 0.75% CMC; 5ml/kg). Second group was administered diclofenac sodium (5 mg/kg) as a standard drug. Group 3 to 6 were fed alcoholic extracts. Paw edema was induced (Winter et al., 1962) by injecting 0.1 ml of 1 % carrageenan in physiological saline into the subplantar tissues of the left hind paw of each rat. The extract of the plants (250 and 500 mg/kg) were administered orally 30 min prior to carrageenan administration. The paw volume was measured at 60, 120, 180 and 240 min by the mercury displacement method using a plethysmograph. The

percentage inhibition of paw volume in drug treated groups were compared with the carrageenan control group. 3.7.2. Cotton Pellet-induced Granuloma The cotton pellet-induced granuloma in rats (DArcy et al., 1960) was studied. The animals were divided into ten groups of six animals in each group. Cotton pellets weighing 20 + 1 mg were autoclaved and implanted subcutaneously into both sides of the groin region of each rat. Group I served as control and received the vehicle. The extracts at concentration of 250 and 500 mg/kg were administered orally for groups II to IX animals for 7 days. Group X animals received diclofenac sodium at a dose of 5 mg/kg p.o. for the same period. On the 8th day, the animals were sacrified and the pellets together with the granuloma tissues were carefully removed, dried in an ovan at 60C, weighed and compared with control. 3.7.3. Statistical Analysis The results were expressed as mean + SEM. The differences between the means of the test and control were compared using one-way ANOVA followed by DMRT/ student t-test. The P values < 0.05 were considered statistically significant.

3.7.4. Test for Diuretic activity Group I served as plain control and administered with vehicle while groups II to IX were given dose (250, 500 mg/kg) aqueous and ethanol extract respectively, while the animals in group X were treated with furosemide in the dose of 4 mg/kg. dissolved in normal saline and administered intragastrically by gastric canula. Food and water were withdrawn 8 hrs before the administration of the drug (Taylor and Topliss, 1962; Amin et al., 1994). Immediately after dosing, all the animals were placed individually in metabolic cages and urine passed by the animals over a period of 6 hrs was collected in a jar. Total urine output was measured and concentration of sodium and potassium was determined by flame photometer. 3.8. ANTIMICROBIAL STUDIES The concentrate of all the extracts and isolated compounds were tested for antimicrobial activities against human pathogens.

3.8.1. Media preparation Bacterial Media (Muller Hinton Media) 36g of Muller Hinton Media (HiMedia) was mixed with distilled water and than sterilized in autoclave at 15 lb pressure for 15 minutes. The sterilized media were poured in to petridishes. The solidified plates were pored with 5 mm dia cork pore. The plates with wells were used for antibacterial studies. Fungal Media (PDA) 200g of potato slices were boiled with distilled water. The potato infusion was used as water solute of media preparation. 29g of dextrose was mixed with potato infusion. 20g of agar was added as a solidifying agent. This constituent ware mixed and autoclaved. The solidified plates were pored with 6mm diam. cork borer. Bacterial Strains The bacterial and fungal pathogenic strains were obtained from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India. Bacterial strains used were Escherichia coli,

Staphylococcus epidermidis, Klebsiela pneumoniae, Citrobactor diversus for in vitro studies. Fungal Strains Fungal strains were Aspergillus niger (MTCC 1344) Aspergillus flavus (MTCC 1973) and Candida albicans. Anti bacterial activity of the plant extract The aqueous extract of whole plants of Pedalium murex and Martynia were used throughout the study. The aqueous extract 10% 20% and 40% were tested against different bacterial pathogens such as Escherichia coli, Staphylococcus epidermis, Klebsielia pneumonia, Citrobactor diversus and Enterococcus feacalis for their antimicrobial activity. It was demonstrated by well diffusion assay. Antifungal activity of the plant extract The aqueous extract of 10 % 20 % and 40% were tested against different fungal pathogens such as Aspergillus flavus, Aspergillus niger and Candida albicans for their antifungal activity. It was demonstrated by well diffusion assay.

Well Diffusion Method: Antibacterial and antifungal activity of plant extract was tested using well diffusion method (Baneletal, 1996). The prepared culture plates were inoculated with different strains of bacteria and fungi using streak plate method. Wells were made on the agar surface with 5mm cork borer. The extracts were pound into the well using sterile syringe. The plates were incubated at 37+2C for 48 hrs for fungal activity and for 24 hrs for bacterial activity. The plates were observed for zone formation around the wells. 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 three replicates and the average values were tabulated.