In vitro and In vivo Evaluation of Potential Anti Diabetic Efficacy On Cassia Auriculata Flowers

Diabetes mellitus (DM) is most significant health problem in various developed and developing countries due to alteration of various clinical and pathological factors. Current work was intended for examine in vitro and in vivo anti diabetic competence of Cassia Auriculata flowers and its phytochemical analysis. The inhibitory effects on carbohydrate digestive enzyme áamylase interaction with various extracts of Cassia Auriculata were contrast with acarbose. Hypoglycemic activity was executed with standard as a glibenclamide. Study indicated ethanolic extract showed higher action on áamylase inhibition with assessment of IC50 value; 43.6%. Based on the above results of in vitro studies were used to selected ethanolic extract of Cassia Auriculata flower used for further study. Results of animal studies indicated that the ethanolic extract of C. Auriculata has shown dose dependent action (200 mg/ kg (1.20±0.91“!) and 400 mg / kg (4 ± 0.01“!) when compared to control and standard drug treated groups. Our study confirmed to ethanolic extract work through the áamylase inhibition mechanism. Our view bioactive constituents confirm anti diabetic capacity and afford methodical source for validation of Cassia Auriculata flowers in ayurvedic formulations on diminution of DM prevalence.

Free radicals were extremely hasty chemical species frequently produced in the human system by usual organic reactions on various exogenous systems 1 .A number of radicals were involved in the process of biological functions.Consequently, their effects on the organism are plaid by protection system that includes various enzymes which are excoriated oxidative stress and dent cells.Amplified oxidative stress directed diabetes mellitus and complications.Accordingly, endorsement on curative natural agents requires methodical exploration evaluation of effectiveness with variety of Ex vivo systems, besides properties involving whole animal preparation. 2entified plant has with splendid alluring brilliant golden yellow blossoms yellow dispersed in dry parts of India and Asia.A dissimilar fraction of plant has been reported on the survey articulate practice of plant against constipation, skin disorder and various other disorders.and 5 Traditional medicine flower part of Cassia Auriculata incited investigated in features, avert oxidation efficiency, anti diabetic action, plummeting clout, shifting ferrous ions, anti-peroxidation and ROS amend capabilities were in vitro conditions.Survey report of C. Auriculata has been illustrated assorted limitation were determined whole animal preparation but no proper scientific validation.In current research required to be finding as a plant based new entity for the treatment against diabetes mellitus and also perform complete scientific validation of above plant. 6and7

Identification and authentication
Cassia Auriculata blossoms were collected at Coimbatore District.Plant matters recognized and genuinely checkered at Botanical survey of India, Coimbatore No: BSI / SRC / 5 / 23 / 2012-13 Tech / 496.

Preparation of extract
The blossoms were isolated, washed altogether with water and shade dried for 6 days.1000 grams of powdered blossoms was subjected to extraction with methanol, ethanol, chloroform, petroleum ether, ethyl acetate (2000ml) in a round bottom flask at room temperature for 15days 8 .After fifteen days decanted and press the mark up to collect the fluidized product which were resolute utilizing rotating vacuum evaporator under lessened weight for collected extract 17.8%.

Phytochemical Test
Screening and identification of phytochemical constituents observational study was done in standard methodology.

Mayer's reagent
Required quantity of mercuric chloride + 60 ml of refine water + 5.0 g of potassium iodide were mix 20 ml of refine water water.Both solutions were mixed and volume was raised to 100 ml with distilled water 9 .

Dragendorff's reagent
First preparation: 1.7 grams of basic bismuth nitrate and 20 g of tartaric acid added 80 ml of refine water in a 100 ml standard flask.Second preparation: 16 grams of potassium iodide + 40 ml of refine water.First + Second mixed equal ratio 9 .

Test for alkaloids
About 0.6 g of plant extract + 8 milliliter of 1% Hydrochloric acid temperate and filtered.Required quantity of filtrate mixed both reagents such as Mayer's and Dragendorff's Test for steroids 0.7g gram of plant extract was diverse with two milliliter of acetic anhydride chase by two milliliter of sulphuric acid 9 .

Test for terpenoids
Required quantity of plant extract was adding two milliliter of chloroform in a test tube and added three milliliter of concentrated sulphuric acid. 8

Test for flavonoids
Substance treated alcohol, a couple of magnesium turnings and few drops of concentrated HCL were added and bubbled for five minutes. 9Test for tannins 0.5 gram of sample boiled in twenty milliliter of refine water in test tube and filtered. 9

Test for Phytosterol
Sample was liquefying in Two milliliter of acetic anhydride, animated for sweltering, cooled and one milliliter of concentrated sulfuric acid was added. 9.Foam Test: 5 milliliter test solution taken single test tube was disturbed well for 5 mins.2. Olive oil test: -Additional a couple of olive oil to required quantity in test tube congaing sample. 9

Test for glycosides
Keller -Killiani test: Additional required quantity of glacial acetic acid + few drops of 5 % ferric chloride solution to a little of dry extract.Further 0.5 ml of concentrated sulfuric acid was added along the side of the test tube carefully. 9n vitro á-Amylase Inhibition activity [10][11] Five hundred micro liter of samples in a test tube and additional to Five hundred micro liter of 0.20 mM buffer of phosphate containing á-amylase solution and incubate at 25°C for 10 minutes.Five hundred micro liter of one percentage starch solution in 0.02 M sodium phosphate buffer additional each tube. Reactn combination was incubate at 25°C for 10 minutes and mix with 3, 5 dinitro salicylic acid colour reagent which incubate boiling water bath for five minutes and cooled to room temperature then make up 10 ml refine water which absorbance estimated at 540 nm.Proportion inhibition in each examine was intended formulae

Experimental animals Mice for acute toxicity study
The Adult female Swiss mice weighing between (20-30 grams) were used to calculate LD 50 .Housed animals in clean cages and kept up under standard states of light (12 hours amid elective day/night cycles), relative humidity (60-70%) and temperature (26 ± 1 °C).][14][15] Preliminary In vivo activity The Adult rats weight were measured range between 200-230 grams and used to perform the hypoglycemic action and Institutional Animal Ethical Committee (1164/ac/08/CPCSEA). 16 Animals were fasted for 48 hours and weigh the animals.Divided animals above mentioned format.Collected first drop of blood by tail nipping method and glucose level monitored by using Glucometer, which considered as a 0 hour reading.All the extracts and drug administer orally by using oral feeding needle.After the administration glucose level were constantly measured different time intervals ½ and 1 hours.

Statistical Analysis
Results were articulated as mean± SD assesses by one way analysis of variance pursued by Dunnett's technique of several assessment.

Appearance and percentage yield of Extract
AEECA (Aqueous Ethanolic Extract of Cassia Auriculata ) were a semisolid brownish color extract and the percentage yield was found to be 17.8%.

Phyochemical Analysis
The phytochemical screening results revealed that the alkaloids were present due to turbidity formation.Changed from violet to blue was showed steroids.Reddish russet was formed and positive result for presence of terpenoid.Red color observed and present flavonoids.A colour change was observed in the test tube, which point out tannins present.A darker ring was development at the intersection and the turning of the upper layer to dim green which indicated the test for phytosterols present.Below two observations indicated presence of saponins due to formation of stable foam confirmed the test and formation emulsion.Formation of blue and red color.Above two color changes indicated presence of glycosides.

Acute toxicity
Plant a dose of 2000 mg/kg has rejection unfavorable consequences tested with mice up to    Obtained R f values were confirmed by standard R f values.R f values obtained for my extract ranges from 0.56 to 0.88, So my extract may contain compounds like flavanoids, glycosides and alkaloids.Quantity determination of total phenolics carried out with respect standard curve of gallic acid (r 2 = 0.99) showed 115.8 mg of extract and also quercetin standard curve (r 2 = 0.994) helps concentration of flavonoids observed114.2mg of extract.

Hypoglycemic Test
In clinically oral hypoglymic drugs are evaluated by using above preclinical method which help to find the hypoglycemic property for the development of new chemical entity present in the ethanolic extract of C. AuriculataThe hypoglycemic study demonstrated that the ethanolic extract of C. Auriculata flowers two dose levels of glucose level milder changes were

CONCLUSION
The results of the present study provides scientific evidence for anti diabetic activity of flowers by the evaluation of various in vitro and in vivo models and hence supports the therapeutic usage of flowers in traditional medicines for treating DM and its associated complications.This work will be useful for diabetic research workers to

Table 3 .
α-Amylase Inhibition of Ethyl acetate Extract of C.

Table 6 .
Results of Ethanolic extract of C. Auriculata