PHYTOCHEMICAL SCREENING AND DEVELOPMENT OF VARIOUS THIN LAYER CHROMATOGRAPHIC METHODS FOR DIFFERENT CEDRELA TOONA ROXB. FRUIT EXTRACTS

Air dried powdered material of the fruits of cedrela toona Roxb. was successively extracted with petroleum ether, hexane, acetone, methanol and water extract by soxhlet extraction and subjected to various qualitative chemical tests to determine presence of various phytoconstituents like alkaloids, glycosides, carbohydrates, phenolics and tannins, phtosterols, fixed oils and fats, proteins, amino acids, flavonoids, saponins etc. The various extracts of fruits of cedrela toona Roxb. were than subjected to thin layer chromatographic studies to identify the number and nature of the chemical constituents present. This study helps researchers for developmentof isolation method of active ingredient having vast pharmacological effects.


Authentication and Collection of Fresh Plant
The fresh parts of Cedrela toona Roxb.were collected in March 2010, from botanical garden of Dang, Gujarat, India. Dried Samples of Bark and fruit of Cedrela toona Roxb. were collected from Paritosh Herbals, Dehradun. The plant was identified by comparing its morphological and microscopical with description given in different standard texts, floras and Ayurvedic Pharmacopoeia of India 1 . Besides these, the plant was then identified and authenticated by Dr. M. S. Jangid, Botany Department, Sir P. T. Science College, Modasa, Gujarat, India and a voucher specimen was deposited. For further confirmation, the microscopic characters of this plant was studied and compared with available literature as mentioned above. The leaves were dried in shade and stored at 27 o C. It was powdered, passed through 40# and stored in air tight containers. Phytochemical Studies Preliminary phytochemical screening 258,259 Successive solvent extraction: 100g of each of air-dried powdered material of leaves, stems and fruits of Cedrela toona Roxb. was successively extracted with the following solvents of increasing polarity in a soxhlet apparatus.  petroleum ether (60º -80ºc)  hexane  chloroform/acetone  ethanol/methanol  distilled water All the extracts were concentrated by distilling the solvents and the extracts were dried in an oven at 50 0 c. Each time before extracting with the next solvent, the marc was dried in an air oven below at 50 0 c.The marc was finally macerated with water for 24 hours to obtain the aqueous extract. The completion of the extraction was confirmed by evaporating a few drops of extract from the thimble on watch glass to observe that no residue remained after evaporation of the solvent. The liquid extracts obtained with different solvents were collected. The consistency, odour, colour, appearance of the extracts and their percentage yield were noted. The extracts were then subjected to various qualitative tests using reported methods, to determine the presence of various phytoconstituents such as alkaloids, glycosides, flavonoids, carbohydrates, amino acids, saponins, sterols and terpenoids, anthraquinone glycosides, coumarins, carotenoids, tannins, phenolic compounds, fixed oils, fats etc. Qualitative chemical identification ofCedrela toona Roxb. [7][8][9][10][11][12][13][14][15][16][17][18][19] The extracts were subjected to various qualitative chemical tests to determine the presence of various phytoconstituents like alkaloids, glycosides, carbohydrates, phenolics and tannins, phytosterols, fixed oils and fats, proteins amino acids, flavonoids, saponins, etc. using reported methods. Alkaloids: Extracts were dissolved individually in dilute hydrochloric acid and filtered. The filtrates were tested carefully & treated with alkaloid reagents.  Mayer's Test: Filtrates were treated with Mayer's reagent (potassium mercuric iodide). The formation of a yellow cream precipitate indicated the presence of alkaloids.  Wagner's Test: Filtrates were treated with Wagner's reagent (iodine in potassium iodide) and observed. Formation of brown or reddish brown precipitate indicated the presence of alkaloids.  Dragendorff's Test: Filtrates were treated with Dragendorff's reagent (solution of potassium bismuth iodide). Formation of red precipitate indicated the presence of alkaloids.  Hager's Test: Filtrates were treated with Hager's reagent (saturated picric acid solution). Formation of yellow colored precipitate indicated the presence of alkaloids. Proteins and Amino acids:  Millon's Test: The extracts were treated with 2 ml of Millon's reagent. The formation of white precipitate, which turned to red upon heating, indicated the presence of proteins and amino acids.  Biuret Test: The extracts were treated with 1ml of 10% sodium hydroxide solution and heated. A drop of 0.7% copper sulphate solution to the above mixtures was added. The formation of purplish violet color indicated the presence of proteins.
 Ninhydrin Test: To the extracts, 0.25% ninhydrin reagent was added and boiled for few minutes. Formation of blue color indicated presence of amino acid.

Carbohydrates:
Extracts were dissolved individually in 5ml of distilled water and filtered. The filtrates were used to test the presence of carbohydrates.  Benedict's test: Filtrates were treated with Benedict's reagent and heated on water bath. Formation of an orange red precipitate indicated the presence of reducing sugars.  Molisch's Test: Filtrates were treated with 2 drops of alcoholic α-naphthol solution in a test tube and 2 ml concentrated sulphuric acid was added carefully along the sides of the test tube. Formation of violet ring at the junction indicated the presence of carbohydrates.  Fehling's Test: Filtrates were hydrolyzed with dilute hydrochloric acid, neutralized with alkali and heated with Fehling's A and B solutions. A red precipitate was formed which indicated the presence of carbohydrates.  Barfoed's Test: Filtrates were treated with Barfoed's reagent and heated on water bath. Formation of an orange red precipitate indicated the presence of reducing sugars. Flavonoids:  Shinoda test: 1-5mg of dried extract was extracted with 10ml of ethanol (95%v/v) for 15 min on a boiling water bath and filtered. To the filtrate, added a small piece of magnesium ribbon and 3 to 4 drops of concentrated hydrochloric acid. Formation of red colour was observed.  Fluorescence test: 1-2 mg of dried extract was extracted with 15 ml methanol for 2 min., on a boiling water bath, filtered while hot and evaporated to dryness. To the residue 0.3 ml boric acid solution (3% w/v) and 1 ml oxalic acid solution (10% w/v) were added. The mixture was evaporated to dryness and the residue was dissolved in 10

Glycosides:
Extracts were hydrolyzed with dilute hydrochloric acid and the hydrolysate was subjected to glycosides tests.  Modified Borntrager's Test: The extracts were treated with ferric chloride solution and heated on boiling water bath for about 5 mins. The mixture was cooled and shaken with equal volume of benzene. The benzene layer was separated and treated with half of its volume of ammonia solution. The formation of rose pink or cherry red color in the ammonical layer indicated the presence of anthranol glycoside.  Legal's Test: The extracts were treated with sodium nitroprusside in pyridine and methanolic alkali. The formation of pink to red color indicated the presence of cardiac glycosides.  Balget Test: The extract of drug was treated with sodium picrate and the formation of a yellowish orange color confirmed the presence of cardiac glycosides.  Boiled the test solution with dilute sulphuric acid, filtered and added chloroform to the filtrate. Shook well and collected the organic layer . A few drops of strong ammonia solution was added , and shaken slightly and the test tube kept aside for a few minutes. The colour of lower ammonical layer was observed.

 Modified borntrager's test:
To the test solution of drug ferric chloride and dilute HCl were added; heated it, cooled it and filtered. Filtrate was shaken with ether or any other organic solvent. The organic layer was shaken with strong ammonia solution and the test tube kept aside. The colour of lower ammonical layer was observed.

Chromatographic Examination of Various Extracts 20,21,22
The various extracts of fruits of Cedrela toona Roxb prepared by successive solvent extraction procedure were subjected to thin layer chromatographic studies to identify the number and nature of chemical constituents present. The R f values of different phytoconstituents present in various extracts of leaves and fruits of Cedrela toona Roxbwere recorded. Preparation of TLC plate: Absorbent used : Silica gel G. Vehicle used for preparation of slurry: Distilled water Method of preparation: Pour plate method Activation of plate: In oven at 110˙c for 30 minutes Application of sample: About 10 to 15 l of sample was applied with thehelp of glass capillary.
Mobile phase: Take required quantity of solvents in TLC chamber, shake well and utilized for chamber saturation. Chamber saturation: 30 minutes Parameters observed: Color of the spot, R f value

RESULT AND DISCUSSION: Preliminary phytoprofiles
The presence of different chemical constituents in the crude drug can be detected by subjecting them to successive extraction using solvents in the order of increasing polarity. The extracts obtained were then dried completely and kept in vacuum desiccator. They were then subjected to qualitative chemical tests in order to detect the various chemical constituents present in them. The colour, consistency and percentage yield of extracts were determined which are shown in (Table 1)