4. MATERIALS AND METHODS Plant Materials of TC AND FW: Fresh leaves of Talinum Cuneifolium Linn and Flemingia Wightiana

Transcription

1 4. MATERIALS AND METHODS 4.1. Plant Materials of TC AND FW: Fresh leaves of Talinum Cuneifolium Linn and Flemingia Wightiana Graham ex Wt. & Arn were collected from S.V.U campus, Tirumala gardens of Chittor district, Andhra Pradesh, India, in the month of October and authentified by Asst. Prof. Dr. K. Madhava Chetty, Dept. of Botany S.V.University, Tirupathi. A.P. Specimen vouchers of Talinum Cuneifolium and Flemingia wightiana were deposited at Department of Pharmacognosy (P.R.R.M.C.P- Kadapa) for further reference Ash values: The plant leaves were washed, cleaned and dried under shade. These dried materials were mechanically powdered in a grinder mixer to obtain a coarse powder and then passed through 40 mesh sieve. These powdered materials were used for physiochemical and phytochemical analysis. The procedure recommended in Indian Pharmacopoeia was followed for the determination of total ash, acid insoluble ash, water soluble ash and sulphated ash. All chemicals and solvents used for different studies were of analytical reagent. Total ash value: About 3 gm of dried powdered material was accurately weighed and taken into crucible. The powder was evenly spread as a fine layer and ignited gradually increasing the temperature to C until colorless, indicating

2 the absence of carbon. The crucible was cooled in a dessicator and weighed. The procedure was repeated to get a constant weight. The percentage of total ash was calculated with reference to air-dried material. Acid insoluble ash value: The ash obtained as described in the above method was boiled gently with 2 M hydrochloric acid for 5 minutes. The insoluble ash was collected on ash less filter paper and washed with hot water until the filtrate was neutral. The filter paper containing the insoluble was transferred into silica crucible and was ignited to a constant weight. The percent of acid insoluble ash was calculated with respect to air-dried drug. Water soluble ash value: 3 gms of powdered drug was accurately weighed and taken in a silica crucible, ignited until the substance was thoroughly charred, cooled and the residue was moistened with 1 ml of conc sulphuric acid, heated gently until white fumes are no longer evolved and ignited at C until all carbon particles have been disappeared. Cooled the crucible and added few drops of conc sulphuric acid. Then ignited as before and further allowed to cool and weighed. The procedure was repeated until constant weight was obtained. The percent of sulphated ash value was calculated with respect to air-dried drug. Determination of Sulphated ash Three grams of powdered drug was accurately weighed and taken in a silica crucible, ignited until the substance was thoroughly charred, cooled and

3 moistened the residue with 1ml of conc sulphuric acid, heated gently until white fumes are no longer evolved and ignited at 80±25 0 C until all carbon particles have been disappeared. Cooled the crucible and added few drops of conc sulphuric acid. Then ignited as before and further allowed to cool and weighed. The procedure was repeated until constant weight was obtained. The percentage of sulphated ash value was calculated with respect to air dried drug Elemental Analysis 105, 106 : The elements like sodium, potassium, magnesium, calcium, arsenic, lead, palladium and mercury were analyzed by Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES). Digestion and preparation of sample: 3 gm of dried powdered material was accurately weighed and taken into crucible and ignited gradually increasing the temperature to C until colorless ash was obtained. 1 gm of above ash was treated with 10 ml HNO3 and ignited at C until the volume of nitric acid was reduced to half. To the above solution 10 ml of nitric acid was added again and heated at C till the volume of nitric acid was reduced to 5 ml. The solution was cooled to room temperature, then treated with 3 ml perchloric acid and heated to convert to dense fume, which was passed through Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES).

4 Elemental analysis using Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES- BERTY Series II VARIAN): The elemental analysis of digested samples has been determined by ICP- OES. The elements like sodium, potassium, magnesium calcium, arsenic, lead, palladium and mercury have been analyzed. In this method, the sample in the form of a homogeneous liquid was introduced into plasma where the free atoms are capable of emitting characteristic wave length. The free atoms are excited to first excited state. Intensity of this spontaneous emission is measured. The intensity of the radiated emission can be measured by using- Icm = Aji x h x ji x N Where Aji = Transion probability for spontaneous emission h = Planks Constant ji = Frequency of Radiation N = Number of atoms in the exited state Preparation of Extracts: About 430 gm of powdered drug was extracted successively with hexane, chloroform, ethanol and water using soxhlet apparatus. The extraction was

5 carried out until the extractive becomes colorless. The solvent was completely removed from the marc in each case before the next extraction was carried out. The solvents were recovered from their extract by distillation under reduced pressure. The dried extract thus obtained was kept in a dessicator and was used for further experiments Preliminary Phytochemical Studies: Preliminary phytochemical analysis was done using the procedures of Khandelwal 107 and Kokate 108. Alkaloids, carbohydrates, fixed oils, flavonoids, glycosides, phytosterol/terpenes, proteins, saponins and tannins/phenols were qualitatively analysed. a) Detection of alkaloids: About 50 mg of solvent-free extract was stirred with little quantity of dilute hydrochloric acid and filtered. The filtrate was tested carefully with various alkaloid reagents as follows. i) Mayer s test To a few ml of filtrate, two drops of Mayer s reagent was added along the sides of the test tube. Formation of white or creamy precipitate confirms the test as positive. ii) Wagner s test

6 To a few ml of filtrate, few drops of Wagner s reagent were added along the sides of the test tube. Formation of reddish brown precipitate confirms the test as positive. iii) Hager s test To a few ml of filtrate, 1 or 2 ml of Hager s reagent was added. A prominent yellow precipitate indicates positive test. iv) Dragendroff s test To a few ml of filtrate, 1 or 2 ml of Dragendroff s reagent was added. A prominent reddish brown precipitate indicates positive test. b) Detection of carbohydrates: About 100 mg of the extract was dissolved in 5 ml of distilled water and filtered. The filtrate was subjected to the following tests. i) Molisch s test To 2 ml of filtrate, two drops of alcoholic solution of naphthol was added. The mixture was shaken well and 1 ml of concentrated sulphuric acid was added slowly along the sides of the test tube. The test tube was cooled in ice water and allowed to stand for few minutes. A violet ring at the junction indicates the presence of reducing sugars. ii) Fehling s test

7 1 ml of filtrate was boiled on a water bath with 1 ml each of Fehling s solution A and B. Formation of red precipitate indicates the presence of sugars. iii) Barfoed s test To 1 ml of the filtrate, 1 ml of Barfoed s reagent was added and boiled on a boiling water bath for 2 minutes. Red precipitate indicates the presence of sugars. iv) Benedict s test To 0.5 ml of filtrate, 0.5 ml of Benedict s reagent was added. The mixture was heated on a water bath for 2 minutes. Red precipitate indicates the presence of sugars. c) Detection of glycosides: For detection of glycosides, about 50 mg of extract was hydrolyzed with concentrated hydrochloric acid for 2 hrs on a water bath, filtered and the hydrolysate was subjected to the following tests. i) Borntrager s test To 2 ml of filtrate hydrolysate, 3 ml of chloroform was added and shaken, chloroform layer was separated and 10% ammonia solution was added to it. Formation of pink color indicates the presence of glycosides. ii) Legal s test

8 About 50 mg of the extract was dissolved in pyridine. Sodium nitroprusside solution was added and made alkaline using 10% sodium hydroxide solution. Presence of glycoside is indicated by a characteristic pink color. d) Detection of saponins: i) Foam or froth test A small quantity of extract was diluted with distilled water to 20 ml. The suspension was shaken in a graduated cylinder for 15 minutes. A two centimeter layer of foam or froth which is stable for 10 minutes indicates the presence of saponins. e) Detection of proteins and amino acids: About 100 mg of extract was dissolved in 10 ml of distilled water and filtered through Whatmann s No.1 filter paper and the filtrate was subjected to tests for proteins and amino acids. i) Millon s test To 2 ml of filtrate, few drops of Millon s reagent were added. A white precipitate indicates the presence of proteins. ii) Biuret s test 2 ml of filterate was treated with one drop of 2% copper sulphate solution, 1 ml of 95% of ethanol, followed by excess of potassium hydroxide pellets. Pink color in the ethanolic layer indicates the presence of proteins.

9 iii) Ninhydrin test About 2 drops of ninhydrin solution was added to 2 ml of aqueous filtrate. A characteristic purple colour indicates the presence of amino acids. f) Detection of phytosterols and triterpenoids: i) Libermann Burchard s test The extract was dissolved in acetic anhydride, heated to boiling, cooled and then 1 ml of concentrated sulphuric acid was added along the side of test tube. Red, pink or Violet color at the junction of the liquids indicates the presence of steroids/triterpenoids and their glycosides. ii) Salkwoski test Few drops of concentrated sulphuric acid was added to the chloroform extract and shaken. On standing, red colour to the lower layer indicates the presence of steroids and golden yellow color indicates the presence of triterpenoids. g) Detection of phenolic compounds and tannins: i) Ferric chloride test About 50 mg of extract was dissolved in distilled water and to this few drops of neutral 5% ferric chloride solution was added. Formation of blue/green/violet color indicates the presence of phenolic compounds. ii) Gelatin test

10 A little quantity of extract was dissolved in distilled water and 2 ml of 1% solution of gelatin containing 10% sodium chloride was added to it. Development of white precipitate indicates the presence of phenolic compounds. iii) Lead acetate test A small quantity of extract was dissolved in distilled water and to this 3 ml of 10% lead acetate solution was added. A bulky white precipitate indicates the presence of phenolic compounds. h) Detection of flavonoids: i) Alkaline test: An aqueous solution of extract was treated with 10% ammonium hydroxide solution. Formation of yellow fluorescence indicates the presence of flavonoids. ii) Shinoda test (magnesium hydrochloric acid): To the test extract add magnesium turnings followed by concentrated hydrochloric acid. Flavonoids produce magenta, crimson red colour Antimicrobial Activity of EETC and EEFW (Disc Diffusion Method) Preparation of discs:

11 Whatmann s filter paper (size no.1) was pieced into small discs of onequarter inch diameter and was autoclaved for 1hr. Preparation of sample of different concentrations: 10 mg of the EETC, EEFW were weighed accurately and dissolved in 5 ml of DMSO and volume was made upto10 ml with DMSO (Stock A, 1000 µg/ml). From Stock A, a Sub Stock of Concentration 500 μg/ml was prepared. Sub stock solution was diluted and different conc. of 20, 40, 60, 80 and 100 μg/ml was prepared. All the dilutions were applied to autoclaved filter paper disc using micropipette. Discs were dried by evaporating the solvent and kept in sterilized bottle in refrigerator. Media preparation: Muller Hinton agar media was prepared as per procedure. All the media were cooled up to C and were poured into different size of sterilized Petri plate and kept in refrigerator. Before using the plate they were totally dried. Microorganisms used: The identified organism was collected from Rajeev Gandhi Institute of Medical Sciences, Kadapa and applied to Muller Hinton medium, for sensitivity test, by streaking on the medium with the help of swab.

12 Application of disc on the Muller Hinton agar media: All the disc samples and standard samples were applied on microbes inoculated plate (Muller-Hinton) with the help of flame sterilized forceps. After application the petri dishes were inverted and incubated in incubator (37 0 C) for 24 hrs. Measurement of Zone of Inhibition: Petri dish was taken from the incubator (37 0 C) after 24 hrs and zone of inhibition was recorded on scale by measuring diameter. The reading was corrected by the formula. Zone of Inhibition = Diameter of - Diameter of Disc Sample/Standard/Control As diameter of disc =5 mm 4.7. Acute Toxicity Studies: An acute toxicity study was performed for Talinum Cuneifolium and Flemingia wightiana to determine LD50 using different doses of the extract according to the method described by Goush et al Anti-Inflammatory activity of EETC and EEFW 116,117,118,119 : The present anti-inflammatory activity was determined in albino rats of either sex according to the method of Winter et al 120. One hour after oral administration of both the extracts, edema was induced to all the groups by

13 injecting 0.1 ml of 1% carrageenan in 0.9% w/v in saline 121, 122 in the subplantar region of left hind paw of rats. Group-1: Normal rats treated with 2 ml/kg of 1% NaCMC. Group-2: Standard group received Ibuprofen 10 mg/kg i.p. Group-3: Received EETC 200 mg/kg in 1% NaCMC per orally. Group-4: Received EETC 400 mg/kg in 1% NaCMC per orally. Group-5: Received EEFW 200 mg/kg in 1% NaCMC per orally. Group-6: Received EEFW 400 mg/kg in 1% NaCMC per orally All the doses were administered per orally according to the body weight of the animals. Paw edema volume was measured with plethysmometer just before 0 (before drug challenge) 3 rd, 6 th and 24 th hour after administration of drug. The percent inhibition of inflammation were calculated by using the formula Percentage of inhibition inflammation= (A-B/A) X100 Where, A and B denote mean increase in paw volume of control and drug treated animals respectively Analgesic activity of EETC and EEFW: Test animal:

14 Male albino mice (20-30 gm) were used throughout the experiment. They were housed in standard cages by maintaining a temperature of 22±2 0 C and 12:12 hrs light dark cycle. The animals were provided with pellet diet and water ad libitum. The animals were fasted for 16 hrs before experimentation but allowed free access to water. Acetic acid induced abdominal writhing test: The acetic acid induced abdominal writhing test was performed as described by Koester et al 123, Fasted normal mice divided into 6 groups of 6 mice in each group. Group-1: control received 2 ml/kg of 1% NaCMC. Group-2: Standard group animals received Aspirin at an oral dose of 100 mg/kg. Group-3: Received EETC 200 mg/kg in 1% NaCMC per orally. Group-4: Received EETC 400 mg/kg in 1% NaCMC per orally. Group-5: Received EEFW 200 mg/kg in 1% NaCMC per orally. Group-6: Received EEFW 400 mg/kg in 1% NaCMC per orally. Animals were treated with scheduled doses as described above 60 min before acetic acid administration. The total number of writhings after intra peritoneal administration of 0.6% w/v acetic acid (0.1 ml for 10 gm) was recorded for 30 min, starting 5 min after the injection 124. Writhing response

15 means contraction of the abdominal muscle together with a stretching the hind limbs. The results of analgesic activity were expressed as the percentage reduction or inhibition of the abdominal writhings. Percentage inhibition = (control average test average) Control average X Isolation of Chemical Constitutents from EETC and EEFW : To the solution of ethanolic extract of Talinum Cuneifolium and Flemingia wightiana, 300 gm of silica gel of mesh was added and mixed by stirring with the help of a glass rod. The mixture was dried by the help of Buchi R-114 rotavapor. The dried silica gel extract was carefully layered on the column gel bed. The column was first eluted with n-hexane and then n-hexane with 5% increments of ethyl acetate. For each eluent mixture 1.0 litre volume was used and 300 ml fractions were collected in glass beakers. Collected fractions were concentrated using Buchi R-114 rotavapor. Chemical tests and TLC were used to analyze the fractions and those with similar properties were combined. These fractions were dried in pre-weighed beakers and their masses were determined. The results were shown in Table: 4.1& 4.2. Table: 4.1. Isolation of chemical constituents from Ethanolic Extract of Talinum Cuneifolium Fraction no. Eluent composition Weight of residue (g) Compound isolated 1-10 Hexane waxy residue % ethyl acetate in hexane waxy residue

16 % ethyl acetate in hexane White powder TC-1 (passed the test for steroids) % ethyl acetate in hexane waxy residue % ethyl acetate in hexane waxy residue % ethyl acetate in hexane waxy residue % ethyl acetate in hexane % ethyl acetate in hexane % ethyl acetate in hexane Yellow powder TC-2 (passed the test for flavonoids) % ethyl acetate in hexane Intangible mass % ethyl acetate in hexane Intangible mass % ethyl acetate in hexane Intangible mass % ethyl acetate in hexane % ethyl acetate in hexane % ethyl acetate in hexane % ethyl acetate in hexane Ethyl acetate Table: 4.2 Isolation of Chemical constitutents from Ethanolic Extract of Flemingia Wightiana

17 Fraction no. Eluent composition Weight of residue (g) Compound isolated 1-10 Hexane waxy residue % ethyl acetate in hexane waxy residue % ethyl acetate in hexane White powder FW-1 (passed the test for alkaloides) % ethyl acetate in hexane waxy residue % ethyl acetate in hexane waxy residue % ethyl acetate in hexane waxy residue % ethyl acetate in hexane % ethyl acetate in hexane % ethyl acetate in hexane % ethyl acetate in hexane Intangible mass % ethyl acetate in hexane Intangible mass % ethyl acetate in hexane Intangible mass % ethyl acetate in hexane Intangible mass % ethyl acetate in hexane % ethyl acetate in hexane % ethyl acetate in hexane Ethyl acetate Spectroscopic analysis

18 a) Infrared Spectroscopy The infrared region of the spectrum encompasses radiation with wave numbers ranging from about cm -1 or wave length from μ. Application and instrumentation point of view, the infra red region has been subdivided into near, middle and far infra red regions. Infra red radiation refers broadly to that part of electromagnetic spectrum which lies between the visible and microwave region. The large majority of analytical applications of infra red region are confined to the limited portion between 400cm -1 and 666cm -1 ( μm, the middle infrared region).near infrared region cm -1 ( μm), far infrared region cm Infra red spectrum is an important record which gives sufficient information about the structure 130. Infra red spectra are usually plotted as percentage transmittance rather than as absorbance as the ordinate. This makes absorption bands appear as dips in the curve rather than as maxima as in the case of UV and visible spectrum 129. Each dip in the spectra is called a band or peak and represents absorption of infra red radiation at that frequency by the sample. The transmittance is 0% if all the radiation is absorbed and the transmittance is 100% for no absorption 129. Infra red absorption occurs when the frequency of the alternating electric field that is associated with the incident radiation matches a possible change in vibrational or rotational frequency of absorbing molecule. When a match

19 occurs, EMR can be absorbed by the molecule causing a change in amplitude of vibration or a change in the rotation. In order for electromagnetic radiation be absorbed by a molecule, it is necessary the molecule to undergo a change of dipole moment during the absorption. If no change in the distribution of charge in the molecule occurs, the varying charge in the electric component of the radiation has nothing with which interact and cannot transfer energy to the molecule. Molecules that have a completely symmetrical charge distribution and in which no change in dipole moment occurs when the molecule vibrates with different amplitude or rotates at a different rate do not absorb infra red radiation. Substances that are transparent to infrared radiation primarily monoatomic and homonuclear diatomic gases such as He, Ne, Cl2, N2 and O2. Nearly all other substances absorb radiation in the infrared region 131. b) 1 H Nuclear Magnetic Resonance Spectroscopy A radially different type of interaction between and electromagnetic forces can be observed by subjecting a substance simultaneously to two magnetic fields, one stationary and the other varying at some radio frequency. At a particular combination of field, energy is observed by a change in the signal developed by a radiofrequency detector and amplifier. This energy absorption can be related to the magnetic dipolar nature of spinning nuclei. This technique known as nuclear magnetic resonance ( 1 H NMR); thus involves the magnetic energy of nuclei when they are placed in a

20 magnetic field and the transitions occur in the wave region of the spectrum. Radio waves are considered to be the lowest form of electromagnetic radiation. The amount of energy is not sufficient to excite or vibrate or rotate an atom or molecule. The amount of energy available in radiofrequency region is just sufficient to affect the nuclear spin of the atom in a molecule and hence constitute the most fundamental part of NMR spectroscopy. The spectra given by all forms of spectroscopy may be described in the terms of the three important factors. 1. Frequency of spectral lines or bands 2. Intensity of spectral lines or bands 3. Shapes of spectral lines or bands All the above properties depend on the molecular parameters of the system. In case of 1 H NMR, these molecular parameters are found to be 1. shielding constant of nuclei 2. coupling constant of nuclei 3. lifetime of energy levels 132 Solvents used for 1 H NMR- A substance free of proton should be used as a solvent i.e., which does not give absorption of its own in NMR spectrum. Moreover the solvent should be capable of dissolving at least 10% of the substance under investigation. Commonly used solvents are 1. Carbon tetrachloride-ccl4 2. Carbon disulphide-cs2 3. Deuterochloroform-CDCl3

21 4. Hexachloro acetone-(ccl3)2c=o, etc. These solvents differ considerably as regards their polarity clearly, NMR spectrum of a compound measured in another solvent of different polarity. Hence, it is important to mention the solvent used in NMR spectrum 133. c) Mass spectroscopy Mass spectroscopy is the analytical technique in which a mixture of gaseous ions is separated according to their m/z (mass-charge) ratios. A mass spectrum is a plot of relative pressure or concentration of the gaseous components as a function of the mass-charge ratios of the components. Both positive and negative ions can be studied. Mass spectrum can only be used to separate gaseous ions, non-gaseous samples are first converted to a gas and then to ions prior, to being converted to a gas is accomplished in the inlet system of the mass spectrometer. A portion of the gaseous sample is admitted to the ionic source where the gaseous molecules are converted to gaseous ions. Only these samples which can be converted to gas can be studied using mass spectrometry. The majority of the gaseous ions that are created in the mass spectrometer are singly charged. The m/z ratio of those is equal to the mass of the ions. A doubly charged ions has a mass spectral peak at a mass charge ratio that is one half the value for a singly charged ion with the same mass 134. Organic chemist use mass spectrometry in three principal ways 1. To measure relative molecular masses with very high accuracy, from these can be deduced exact molecular formulae.

22 2. To detect within a molecule the places at which it prefers to fragment; from this can be deduced the presence of recognizable groupings within the molecule. 3. As a method for identifying analytes by comparison of their mass spectra with libraries of digitized mass spectra of known compounds 135. Principles of operation of mass spectrometer Mass spectrometer is an instrument that is used for the determination of constitution of materials. The mass spectrometers possess components to perform following functions 1. Ionisation of the sample. 2. Acceleration of ions by an electric field. 3. Dispersion of ions according to their mass and charge ratio. 4. Detection of the ions and production of a corresponding electric signal 136.

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