76 CHAPTER 5 PHYTOCHEMICAL EVALUATION Phytochemistry is mainly concerned with enormous varieties of secondary plant metabolites which are biosynthesized by plants. The plant kingdom represents a treasure trove of structurally diverse bioactive molecules. Most of the best plant medicines are the sum of their constituents. The beneficial physiological and therapeutic effects of plant materials typically result from the combinations of these secondary products present in the plant. The information on the constituents of the plant clarifies the uses of the plants but only a small percentage have been investigated for their phytochemicals and only a fraction has undergone biological or pharmacological screening. As more phytoconstituents are being identified and tested, traditional uses of the plants are being verified [44]. In phytochemical evaluation the powdered leaves were subjected to phytochemical screening for the detection of various plant constituents, characterized for their possible bioactive compounds, which have been separated and subjected to detailed structural analysis. 5.1 MATERIALS AND METHODS After authentification, the fresh, healthy plant leaves of Symplocos cochinchinensis (Lour.) were properly dried in shade for 2-3weeks. It was pulverized in a blender, sieved and used for further studies.
77 5.1.1 Preparation of the Extracts About 2 kg of air-dried plant material was extracted in soxhlet assembly successively with n-hexane, chloroform, ethyl acetate and methanol (order of increasing polarity). Each time before extracting with the next solvent, the powdered material was dried. Each extract was concentrated by using rotary vacuum evaporator. The extract obtained with each solvent was weighed and the percentage yield was calculated in terms of dried weight of the plant material. The colour and consistency of the extract were also noted. All the solvents used for this entire work were of analytical reagent grade (Merck, Mumbai). 5.1.2 Qualitative Chemical Tests [45, 46] The n-hexane, chloroform, ethyl acetate, methanol extracts and the leaf powder were subjected to qualitative chemical analysis. Test for alkaloids A small portion of the extract was stirred separately with a few drops of dilute hydrochloric acid and filtered. The filtrate was carefully tested with various alkaloidal reagents such as Mayer s reagent, Dragondroff s reagent, Hager s reagent and Wagner s reagent. Test for carbohydrates The minimum amount of the extracts were dissolved in 5ml of distilled water and filtered. The filtrate was subjected to test for carbohydrates.
78 Molisch s test The filtrate was treated with 2-3 drops of 1% alcoholic alpha naphthol and 2ml of concentrated sulphuric acid was added along the sides of the test tube. Fehling s test The filtrate was treated with 1 ml of Fehling s A and B and heated in a boiling water bath for 5-10min. Appearance of reddish orange precipitate shows the presence of carbohydrates. Test for glycosides Cardiac glycoside Keller-Killani test- To 2 ml of extract, glacial acetic acid, one drop 5 % ferric chloride and concentrated sulphuric acid were added. Appearance of reddish brown colour at the junction of the two liquid layers indicates the presence of cardiac glycosides. Anthraquinone glycosides Borntrager s Test To 3 ml extract dilute sulphuric acid was added, boiled and filtered. To the cold filtrate equal volume benzene or chloroform was added. The organic layer was separated and ammonia was added. Ammonical layer turns pink or red. c) Saponin glycosides water. Foam test The extract and powder were mixed vigorously with
79 d) Coumarin glycosides fluorescence. Alcoholic extract when made alkaline, shows blue or green Test for phytosterol 1gm of the extract was dissolved in few drops of dry acetic acid, 3ml of acetic anhydride was added followed by few drops of concentrated sulphuric acid. Appearance of bluish green colour shows the presence of phytosterol. Test for fixed oils and fats (a) Small quantity of the various extracts was separately pressed between two filter papers. Appearance of oil stain on the paper indicates the presence of fixed oil. (b) Few drops of 0.5N alcoholic potassium hydroxide was added to a small quantity of various extracts along with a drop of phenolphthalein. The mixture was heated on a water bath for 1-2hrs. Formation of soap or partial neutralization of alkali indicates the presence of fixed oil and fats. Test for tannins and phenolic compounds Small quantity of various extracts were taken separately in water tested for the presence of phenolic compounds and tannins with (a) Dilute ferric chloride solution (5%) - violet colour (b) 1% solution of gelatin with 10%NaCl - white precipitate (c) 10% lead acetate solution - white precipitate
80 Test for proteins Various extracts were dissolved in few ml of water and treated with (a) Millon s reagent: Appearance of red colour shows the presence of proteins and free amino acids. (b) Biuret test: Equal volume of 5% solution of sodium hydroxide and 1% copper sulphate were added. Appearance of pink or purple colour indicates the presence of proteins and free amino acids. Test for gums and mucilages About 10ml of various extracts were added separately to 25ml of absolute alcohol with constant stirring and filtered. The precipitate was dried in air and examined for its swelling properties and for the presence of carbohydrates. Test for flavanoids (a) (b) (c) With aqueous solution of sodium hydroxide blue to violet colour (Anthrocyanins), yellow colour (Flavones), yellow to orange (Flavonones). With concentrated sulphuric acid yellowish orange colour (Anthrocyanins), orange to crimson colour (Flavonones). Shinoda s test the extracts were dissolved in alcohol, to that a piece of magnesium and followed by concentrated hydrochloric acid was added drop wise and heated. Appearance of magenta colour shows the presence of flavonoids.
81 Test for lignin With alcoholic solution of phloroglucinol and concentrated hydrochloric acid appearance of red colour shows the presence of lignin. Test for terpenoids Noller s test: The substance was warmed with tin and thionyl chloride. Pink coloration indicates the presence of triterpenoids. Test for steroids Libermann Burchard Reaction: 2 ml extract was mixed with chloroform. To this 1-2 ml acetic anhydride and 2 drops concentrated sulphuric acid were added from the side of test tube. First red, then blue and finally green colour appears. 5.1.3 Thin Layer Chromatography [47,48] Of the various methods of separating and isolating plant constituents, thin layer chromatography (TLC) is one of the most powerful technique used for the separation, identification and estimation of single or mixture of components present in various extracts. Mechanism employed in this reliable technique is adsorption in which solute adsorbs on the stationary phase according to its affinity. Substances are separated by differential migration that occurs when a solvent flows along the thin layer of stationary phase. The substance which is having more affinity towards mobile phase moves faster when compared to the substance which has less affinity leading to the separation of the compounds.
82 TLC Plates phase. Precoated silica gel on aluminium plates were used as a stationary Sample application The extracts to be analysed were diluted with respective solvents and then spotted with help of capillary tube just 2 cm above its bottom. Selection of mobile phase Solvent mixture was selected on the basis of the phyto constituents present in each extract. Solvents were analysed as its order of increasing polarity. Several mobile phases were tried for the separation of maximum components. After several trials, the best solvent system was selected which showed good separation with maximum number of components. Solvent system Hexane extract - Methanol :Chloroform(9:1) Chloroform extract - Methanol :Chloroform(9:1) Ethylacetate extract - Methanol : Ethyl acetate: Hexane: Acetic acid (2:7:1: 0.5) Methanol extract - Ethyl acetate: Water ( 6:3:1) R f values were noted down for each selected extracts after elution by using different detecting agents such as Dragendroff s, Ninhydrin, Libermann Burchard, concentrated sulphuric acid and ferric chloride.
83 5.1.4 High Performance Thin Layer Chromatography[49] HPTLC method is a modern sophisticated and automated separation technique derived from TLC. Pre-coated HPTLC graded plates and auto sampler was used to achieve precision, sensitive, significant separation both qualitatively and quantitatively. High performance thin layer chromatography (HPTLC) is a valuable quality assessment tool for the evaluation of botanical materials efficiently and cost effectively. HPTLC method offers high degree of selectivity, sensitivity and rapidity combined with single-step sample preparation. In addition it is a reliable method for the quantitation of nanograms level of samples. Thus this method can be conveniently adopted for routine quality control analysis. It provides chromatographic fingerprint of phytochemicals which is suitable for confirming the identity and purity of medicinal plant raw materials. Basic steps involved in HPTLC Extracts used : n-hexane, chloroform, ethyl acetate and methanol Application mode : CAMAG Linomet IV. Development mode : CAMAG Twin Trough chamber. Sample application The samples were dissolved in same solvent and 10 µl quantity of sample was applied on the HPTLC silica merk 60F 254 graded plate sized 6cm x 10 cm as narrow bands using CAMAG Linomat 5 injector.
84 Chromatogram Development It was carried out in CAMAG Twin Trough chambers. Sample elution was carried out according to the adsorption capability of the component to be analysed. After elution, plates were taken out of the chamber and dried. Scanning Plates were scanned under UV at 254nm. The data s obtained from scanning were brought into integration through CAMAG software. Chromatographic finger print was developed for the detection of phytoconstituents present in each extract and R f values were tabulated. Mobile Phase Hexane extract - Hexane:Chloroform: Acetic acid (7:3:0.5) Chloroform extract - Hexane:Chloroform: Acetic acid (7:3:0.5) Ethyl acetate extract - Butanol:Water:Acetic acid ( 4:5:1) Methanol extract - Butanol:Water:Acetic acid ( 4:5:1)
85 5.2 RESULTS 5.2.1 Extraction The percentage yield of successive extractive values for leaves of Symplocos cochinchinensis (Lour.) is tabulated in Table 5.1 Table 5.1 The percentage yield of successive extracts of the leaves of Symplocos cochinchinensis (Lour.) S.No Extract Method of extraction Colour Physical nature Yield (%w/w) 1 n-hexane 2 Chloroform 3 Ethyl acetate 4 Methanol Successive solvent extraction in soxhlet apparatus Green/ Sticky mass Greenish brown/ Sticky mass Yellowish green/ Thick solid mass Brownish green/ Thick solid Waxy greasy semisolid 2.0 Semisolid 2.0 Solid 5.7 Solid 8.8 mass
86 5.2.2 Qualitative Chemical Analysis Qualitative chemical analysis of phytoconstituents of the leaf powder and various extracts of Symplocos cochinchinensis (Lour.) is tabulated in Table 5.2. Table 5.2 Qualitative chemical analysis of phytoconstituents of the leaf powder and various extracts of Symplocos cochinchinensis (Lour.) S. No. Test Powder n-hexane Chloroform Ethyl acetate Methanol 1. Alkaloids + - - + + 2. Glycosides + - - + + 3. Terpenoids - - - - - 4. Carbohydrate + - - + + 5. Proteins + - - + + 6. Steroids + + + - - 7. Flavonoids + - - + + 8. Phenols + - - - + 9. Tannins - - - - - 10. Iridoid glycoside - - - - - 11. Quinones - - - - - 12. Anthraquinone - - - - - 13. Saponins + - - + + Note: + ve indicates positive result, whereas ve indicates negative result
87 5.2.3 Thin Layer Chromatography The TLC studies of the n-hexane, chloroform, ethyl acetate and methanol extract are shown in Table.5.3 Table 5.3 The TLC studies of various extracts of Symplocos cochinchinensis (Lour.) S.No Extract Solvent system 1 n-hexane No. of spots 2 R f values 0.62 0.66 Methanol : Chloroform(9:1) 2 Chloroform 3 0.37 0.43 0.48 3 Ethyl acetate Methanol : Ethyl acetate: Hexane : Acetic acid (2:7:1: 0.5) 3 0.27 0.33 0.88 0.20 4 Methanol Methanol : Ethyl acetate: Water (6:3:1) 4 0.45 0.52 0.72
88 5.2.4 High Performance Thin Layer Chromatography The HPTLC fingerprints of the n-hexane, chloroform, ethylacetate and methanol extract are shown in Table 5.4 and Figures 5.1, 5.2, 5.3, and 5.4 Table. 5.4 The HPTLC fingerprints of various extracts of cochinchinensis (Lour.) Symplocos S.No Extract Detection Wavelength (nm) No. of spots R f values 1 n-hexane 280 6 0.07, 0.49, 0.60, 0.75, 0.82, 0.90 2 Chloroform 280 11 0.07, 0.28, 0.32, 0.42, 0.46, 0.50, 0.58, 0.76, 0.82, 0.88, 0.91 3 Ethyl acetate 280 6 0.12, 0.29, 0.36, 0.49, 0.57, 0.81 4 Methanol 280 10 0.06, 0.15, 0.18, 0.24, 0.33, 0.39, 0.51, 0.58, 0.65, 0.71
89 Figure 5.1 The HPTLC fingerprint of n-hexane extract of Symplocos cochinchinensis (Lour.)
90 Figure 5.2 HPTLC fingerprint of chloroform extract of Symplocos cochinchinensis (Lour.)
91 Figure 5.3 HPTLC fingerprint of Ethylacetate extract of Symplocos cochinchinensis (Lour.)
92 Figure 5.4 HPTLC fingerprint of methanol extract of cochinchinensis (Lour.) Symplocos
93 5.3 DISCUSSION Most of the traditional knowledge about medicinal plant was in the form of oral knowledge. There is no uniform or standard procedure for maintaining the inventory of these plants and the knowledge about their medicinal properties. Therefore, it is necessary that such procedures to be documented and studied for systematic regulation and widespread application. The leads for a significant number of modern synthetic drugs have originated from isolated plant ingredients since the search for new entities begins from either derivatizing the existing drug or from traditional medicinal system. It is very important to undertake phytochemical investigations along with biological screening to understand therapeutic dynamics of medicinal plants and also to develop quality parameters. In the phytochemical analysis different polarity of phytoconstituents were sorted out from the coarsely powdered leaves of Symplocos cochinchinensis (Lour.) by using solvents like n-hexane, chloroform, ethyl acetate and methanol by successive extraction using soxhlet apparatus. Successive extractive values revealed the solubility and polarity particulars of the metabolites in the plant. Methanolic extract showed high extractive yield 8.8 %w/w when compared to other extracts. Qualitative preliminary phytochemical analysis was performed initially with different chemical reagents to detect the nature of phytoconstituents and their presence in each extract and powder. Hexane and chloroform extracts showed the presence of steroids. Ethyl acetate extract was found to contain flavanoids, glycosides, proteins, saponins, alkaloids and carbohydrates. Methanolic extract showed the presence of carbohydrates, flavanoids, phenols, saponins, proteins, glycosides and alkaloids.
94 Qualitative chromatographic analysis of these extracts using thin layer chromatography was performed to separate and identify the single or mixture of constituents in each extract. The hexane extract showed 2 spots (R f values 0.62, 0.66), chloroform extract showed one spot (0.37), where as 3 spots were found with ethyl acetate extract (0.27, 0.33, 0.88) and 4 spots were found in methanol extract (R f values 0.20, 0.45, 0.52, 0.72). TLC was performed for the identification of different components in the extracts qualitatively. HPTLC was scanned at 280nm with the best solvent to detect the maximum number of components and peak abundance qualitatively. The finger print of hexane extract showed 6 spots in the solvent system and the R f values were 0.07, 0.49, 0.60, 0.75, 0.82, 0.90. Chloroform extract showed 11 spots in the solvent system and the R f values were 0.07, 0.28, 0.32, 0.42, 0.46, 0.50, 0.58, 0.76, 0.82, 0.88, 0.91. The ethyl acetate extract showed 6 spots in the solvent system and the R f values were 0.12, 0.29, 0.36, 0.49, 0.57, 0.81. The finger print of methanol extract showed 10 spots and the R f values were 0.06, 0.15, 0.18, 0.24, 0.33, 0.39, 0.51, 0.58, 0.65, 0.71. HPTLC fingerprint is one of the versatile tool for qualitative and quantitative analysis of active constituents. It is also a diagnostic method to find out the adulterants and to check the purity. Since, secondary metabolites are responsible for biological activity, this study would be the leading path way of information for selection of the extract for pharmacological activity and isolation of constituents responsible for the activity.