5 A] MATERIALS AND EQUIPMENTS:

Transcription

1 5 A] MATERIALS AND EQUIPMENTS: Materials used for the present study are listed in the following table. Table no. 19: List of chemicals /drugs used in study: NAME OF MATERIAL Petroleum ether Ethanol Acetone Methanol Chloroform Benzene Silica gel for TLC Silica for column Sodium dithionite Lowenstein Jensen Medium base W/O Starch Rifampicin Isoniazide Sodium hydroxide Hexane Diethyl ether Sodium chloride Glycerol Oleic acid Potassium nitrate Barium chloride Conc. Sulphuric acid Acetonitrile HPLC Glacial acetic acid Methylene blue Potassium dihydrogen phosphate Sulphanilamide N-1 napthyl ethylenediamine dihydrochloride MANUFACTURER / SUPPLIER Lupin Ltd, Mumbai. Lupin Ltd, Mumbai. Loba chemie, Mumbai. CDH Analytical Reagents, Mumbai. 99

2 Hydrochloric acid Nitric acid Disodium hydrogen phosphate Phenol Saffranin Maconky agar Malachite green Yoghel johnson agar Cetrimide agar Sabouraud dextrose agar Silymarin Paracetamol Anaesthetic ether SGOT and SGPT test kits Acid Phosphatase test kit Alkaline Phosphatase test kit MB/BACT Reconstitution fluid BacT/ALERT MP bottles B-sitosterol Linoleic acid Phloroglucinol Iodine Xylene Micro master Lab Pvt. Ltd. Micro lab Ltd. Span Diagnostics Pvt. Ltd. Surat, India Span Diagnostics Pvt. Ltd. Surat, India Span Diagnostics Pvt. Ltd. Surat, India Biomeriux India Pvt. Ltd Biomeriux India Pvt. Ltd Sigma-aldrich, Mumbai Sigma-aldrich, Mumbai. Ozone International, Mumbai. 100

3 5 B] EQUIPMENT / INSTRUMENTS Equipments and instruments used in the study are listed in table no. 20 Table no. 20: List of equipments used in study: EQUIPMENT/INSTRUMENTS MAKE AND MODEL BacT/ALERT 3D 60 System Biomeriux, India Pvt. Ltd FTIR Jasco FTIR-410 UV - Visible Spectrophotometer Jasco V550 Analytical Balance Schimadzu AUX 220 ph meter Equiptronics India EI-101 Laboratory centrifuge Remi Instruments KBr press Technosearch Instruments INC Incubator Remi Instruments Digital water bath Aditi associates, Mumbai Eddy s hot plate INCO, Ambala Colorimeter Remi Instruments Laminar Air Flow Klenzaids Bioclean Devices (P) Ltd Hot Air Oven Kumar,Dolphin,biotechniques, India Photo-micrographic equipment Kyowa-Getner,Videoplan-11 UP with Bio-plus- 55 software Vortex Mixer Remi Equipments Autoclave Lab Hospital Rotary Microtome Sipcon Optical Industries, Ambala NMR Bruker model AV 300MHZ HPTLC system Camag with Wincat GC-MS Hewlett Packard, GCD:1800 A. 101

4 5.1 PRELIMINARY PHYTOCHEMICAL INVESTIGATION COLLECTION AND PREPARATION OF EXTRACTS:- (1.2) Fresh leaves of Ricinus communis Linn. and Vitex negundo Linn. were collected from Sangli, and Miraj areas. The both plants were authenticated by Smt. U. S. Shinde, Botanist from Botany department of Willingdon College, Sangli. The leaves were washed and were shade dried to obtain coarse powder. This powder of each plant was subjected to different extraction procedures. The aqueous extract: The aqueous extract was prepared by maceration process. Dry powder was taken in stoppard container in which the powdered leaves were macerated with 1000 ml of 10% chloroform water for seven days with occasional shaking. The mixture of crude drug containing solvent is filtered until most of the liquid drains off. The filtrate and washings were combined to produce 1000 ml of the solution. After maceration the marc was pressed and filtrate was concentrated to get the residue on water bath. The aqueous extract obtained was weighed (1). Successive solvent extract: Sequential extraction was carried out with the same air dried powdered plant material in soxhlet assembly by using solvents of increasing polarity like petroleum ether, benzene, chloroform, acetone, ethanol and methanol at a temperature not exceeding the boiling point of the solvent. Finally, the powder was macerated with chloroform water. Each time before extracting with the next solvent, the powdered material was dried in oven below 50 0 C. Each extract was concentrated by distilling off the solvent and evaporated to dryness on water bath. Ethanolic extract: The air dried powdered plant material was extracted in soxhlet extractor with ethanol (1,2). All the extracts of both plants Ricinus communis Linn. and Vitex negundo Linn. were collected individually, dried well and observed for colour and consistency of each extract. The weights of all the extracts were taken individually and the percentage yield value of each extract was calculated. 102

5 The solubility of thick paste extracts was checked in different solvents and solubilised according to their solubility in water, dimethyl formamide (DMF) and dimethyl sulphoxide (DMSO). The solubility of each extracts of Ricinus communis Linn. and Vitex negundo Linn. is reported in result. DMF and DMSO did not show any antimicrobial as well as antimycobacterial activity against the test microorganism during this work TRANSVERSE SECTION OF RICINUS COMMUNIS LINN. LEAF: Selection of drug sample for section cutting: Young leaves of Ricinus communis Linn. were taken, as it does not have a thick midrib and lamina, hence it was more convenient to obtain fine sections. Preparation of sample for section cutting: 1. Leaf sample was taken in a beaker and sufficient water was added so that the sample remains submerged. 2. Cut a part of leaf passing through midrib. 3. This cut off portion was not boiled, since the lamina of a leaf is very thin, section cutting is difficult. The surface area of the surface to be cut was increased by embedding the sample in a block of pith. This pith was obtained from potato. A cubical portion of pith was cut off and used. 4. The sections were transferred to a watch glass containing water with the help of brush rejected thick and opaque one. Staining and mounting of sections: Staining process: Cleaned watch glass was taken and staining solution i.e. Phloroglucinol and HCL was added in it. With the help of a brush, sections from water were transferred to staining solution and kept for 2-3 minutes. Picked up the sections after 2-3 minutes and transferred to watch glass containing plain water, so that excess stain was washed away. Then section was made ready for mounting on a slide. Mounting process: Cleaned glass slide was taken and section was transferred for the mounting, with the help of brush. 1-2 drops of water were added on the section with the help of dropper as the section is submerged in water. Cleaned cover slip was placed on section gently. When air bubbles were seen slightly lifts the cover slip and added drop of water and coverslip was replaced till air bubble was removed. With the help of blotting paper, excess water was wiped off outside the cover slip. Then slide was ready for 103

6 observation (3). The slide was observed under photomicrograhic equipment and photo was taken. The diagram of T. S. of Ricinus communis Linn. leaf along with description was reported in results PRELMINARY PHYTOCHEMICAL INVESTIGATION OF EXTRACTS OF RICINUS COMMUNIS LINN: All the extracts were subjected to preliminary organic qualitative analysis to identify nature of compounds present by chemical tests (3) ACUTE ORAL TOXICITY TESTING: In order to decide the dose of plant extract it was essential to go through the toxicity study of the extracts according to OECD guidelines. Hence for acute oral toxicity and LD 50 determination OECD guidelines 423 (Organization for economic co-operation and development) were followed. Limit test: The Limit test is primarily used in situations where available information indicating that the test material is likely to be nontoxic, i, e., having toxicity only above regulatory limit doses. Information about the toxicity of the test material can be gained from knowledge about similar tested mixtures or products, taking into consideration the identity and percentage of components known to be of toxicological significance. In those situations where there is little or no information about its toxicity, or in which the test material is expected to be toxic, the main test should be performed. A limit test at one dose level of 2000 mg/kg body weight may be carried out with five animals (three animals per step). Exceptionally a limit test at one dose level of 5000 mg/kg may be carried out with three animals. If test substance-related mortality is produced, further testing at the next lower level may need to be carried out. Preparation of dose: 200 mg/ml of each test extract was prepared and 1ml /100gm of body weight was administered to each animal. Procedure: Healthy young adult female Wistar albino rats / Swiss albino mice (females are generally slightly more sensitive) were randomly selected and marked to permit individual identification. The temperature in the experimental animal room was maintained at 22 0 C (± 3 0 C). Although the relative humidity was maintained from 50-60%. 104

7 Lighting was artificially maintained for 12 hrs dark and 12 hrs light. For feeding, conventional rodent laboratory diets was used with an unlimited supply of drinking water. Animals were kept in the cages for at least 5 days prior to dosing for acclimatization to the laboratory conditions (4). Animals were fasted 24 hrs prior to dosing. Test substances (acetone, ethanol, successive ethanol extracts of Ricinus communis Linn. and ethanolic extract of Vitex negundo Linn.) were administered in a single dose by gavage using orogastric tube. The substance is administered to the set of five female rats/five female mice at predetermined doses (2000 mg/kg body weight for limit test). Animals were observed individually after dosing at least once during the first 30 minutes, periodically during the first 4 hrs. (OECD/OCDE guidelines, 2001) 105