Estimation of total tannins, total phenolics and total flavonoids of different extracts of cedrela toona roxb. Fruits.

: 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. Total flavonoids content was measured with the ‘Aluminium Chloride Colorimetric Assay Method’. Total Phenolic content in the aqueous and methanol extracts of fruits of Cedrela toona Roxb. were determined using the Folin Denis Method and expressed as mg of tannic acid equivalents per gram dry weight of extract. 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.

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.
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.

Determination of total flavonoids content of leaves of Cedrela toona Roxb.
Aluminum chloride colorimetric assay method: 23, 24 Total flavonoids content was measured with the 'Aluminum chloride colorimetric assay method'. Aqueous and methanol extracts of leaves of Cedrela toona Roxb.that has been adjusted to come under the linearity rangei.e. (400μg/ml) and different dilution of standard solution of Quercetin and Rutin (10-100μg/ml) were added to 10ml volumetric flask containing 4ml of water. To the above mixture, 0.3ml of 5% NaNO 2 was added. After 5 minutes, 0.3ml of 10% AlCl 3 was added. After 6 min, 2ml of 1 M NaOH was added and the total volume was made up to 10ml with distill water. Then the solution was mixed well and the absorbance was measured against a freshly prepared reagent blank at 510 nm. Total flavonoid content of the extracts was expressed as percentage of Quercetin and Rutin equivalent per 100 g dry weight of sample.

Folin -Denis Method:
Total Phenolic content in the aqueous and methanol extracts of leaves of Cedrela toona Roxb. were determined using the Folin-Denis method and expressed as mg of tannic acid equivalents per gm dry weight of extract. The absorbance values of the test extracts after subtraction of control (y) were translated into total phenolic content (mg/gm of TAEs) using the tannic acid calibration plotwith the following formula: [Total phenolic content (mg/gm of TAEs) = y -0.0004 / 0.0012] Principle: The polyphenolic compounds are estimated by spectrophotometric method known as Folin-Denis method. The method is based on the oxidation of the molecules containing a phenolic hydroxyl group. The tannins and tannin like compounds reduce phospotungstomolybdic acid in alkaline solution to produce a highly blue coloured solution, the intensity of which is proportional to the amount of tannins and phenolic compounds and can be estimated against standard tannic acid solution.

Preparation of Reagents:
1) Folin-Denis Reagent: Sodium tungustate (10 gm) and phosphomolybdic acid (2 gm) were dissolved in distilled water (75 ml) along with phosphoric acid (5 ml). The mixture was refluxed for 2 hours and volume was made with water up to 100 ml.
2) Sodium carbonate solution: sodium carbonate (35 gm) was dissolved at 70-80°C in distilled water and volume was made up to 100 ml. It was filtered through glass wool after allowing it to stand over night.
3) Working standard solution of Tannic acid: Accurately weighed standard Tannic acid (100 mg) was dissolved in distilled water in volumetric flask. 5 ml of this solution was diluted with water to 100 ml in another volumetric flask to give 50 µg/ml Tannic acid solutions.

4) Preparation of solution:
A series of calibrated 10 ml volumetric flasks were taken and appropriate aliquots of standard Tannic acid solution ranging from 1.6, 2, 2.4, 2.8, 3.4, 4, 5, 6 ml were added. To these solutions Folin-Denis reagent (0.5 ml) and Sodium Carbonate solution (1 ml) and Distilled water (up to 10 ml) were added. And the absorbance was measured at 700 nm within 30 min of the reaction. The calibration curve was prepared and concentration of total phenolic compound was find out from methanol and water extract by taking their absorbance respectively.

5) Preparation of test solution:
a) Methanol Extract: Take 2 gm dried powder obtains from methanolic extract. Solubalize it into 50 ml methanol and take 0.5 ml of this solution and dilute up to 10 ml with methanol. Take 1 ml of this solution and dilute up to 10 ml with methanol and take this solution as a test solution. b) Aqueous Extract: Take 5 gm dried powder obtains from Water extract. Solubalize it into 20 ml water and take 0.1 ml of this solution and dilute up to 10 ml with methanol. Take 1 ml of this solution and dilute up to 10 ml with methanol and take this solution as a test solution.

Determination of total tannin content of fruit extract of Cedrela toona Roxb. 27,28
Take 0.1 gm of both extracts (aqueous and methanol) of Cedrela toona Roxb., dissolved in 10 ml of distilled water separately, add 10ml of indigo carmine dye, add 300 ml of distilled water, heat it at 60-70°C and titrate with 0.1 M KMnO 4 . Carry out same experiment by omitting substance.

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)

Concentration (µg/ml) Absorbance
Result indicated that Quercetin gave maximum absorbance at λmax 257 and linear relationship with concentration and absorbance between the concentration applied. (Y= 0.081X+0.1573; R 2 =0.995). Aqueoues extract of Cedrela toona Roxb. was found to contain total Flavonoid 0.91%w/w equivalent to Quercetin. Ethanol extract of Cedrela toona Roxb. contained total Flavonoid 1.56%w/w equivalent to Quercetin. Flavonoid content was higher in alcoholic extract of Cedrela toona Roxb. fruits extract than water extract. were determined using the 'Folin-Denis method' and expressed as mg of tannic acid equivalents per gm dry weight of extract (Table 5.12). The absorbance values of the test extracts after subtraction of control (y) were translated into total phenolic content (mg/gm of TAEs) using the tannic acid calibration plot with the following formula: Total phenolic content (mg/gm of TAEs) = y -0.0004 / 0.0012