SCREENING OF PHYTOCHEMICALS AND BIOACTIVE COMPOUNDS IN PUNICA GRANATUM PEEL TO EVALUATE ITS HEMATOLOGICAL POTENTIAL

Background: Pomegranate is the most significant tree which has been domesticated for incalculable human benefits and serves as a food medicine of great importance. Pomegranate has been found to have ethnomedical hi phytochemicals. These are found to possess several chemicals a value. Its various parts are exploited by pharmaceuticals and are considered as a powerhouse of energy due to its nutritive value. Methods: The present study was carried out for phytochemical analysis in the hydroalcoholic pomegranate peel extracts. The phytochemical analysis and analysis of bioactive compounds are done, the pomegranate peel extracts are found to have carbohydrates, alkaloids, flavonoids, steroids, tannic acids and phenolic compounds. The antioxidant activity, DPPH free radical scavenging activity, nitric oxide radical scavenging activity was analyzed. Results: For good health, the quality of blood should be maintained and for may be the key so as to protect the blood from oxidative stress. The results of various tests performed are compiled in various graphs and tables. Conclusions: Thus, from the present study it is concluded that pomegranate peel is a rich source of phytochemicals and bioactive compounds such as antioxidants and so can be used for various pharmaceutical and therapeutic purposes like increase in hemoglobin count.


INTRODUCTION
Phytochemicals are found in plants and are non nutritive in nature but have certain disease preventing properties. They offer protection against pathogens and are not required by human beings for perpetuating life [Kokate CK Phytochemicals work in many ways such as antioxidants which protect the cells from damage. These can stimulate certain enzymes and can reduce the risk of various ailments. These can also act as anti-bacterial and hormonal There are various pharmacological and epidemiological evidences which prove that plants contain biologically active components which offer various health benefits and also protects against degenerative ailments [Mutahar S, Shiban al, 2012]. The health benefits are often associated with plant phytochemicals as of consumption of higher levels of fruits and vegetables.
The phenolic compounds found in plants are found to exihibit various properties such as anti-allergic, anti-inflammatory, Phytochemicals are found in plants and are non nutritive in nature but have certain disease preventing properties. They offer protection against pathogens and are not required by Kokate CK et al 2006]. Phytochemicals work in many ways such as antioxidants which protect the cells from damage. These can stimulate certain enzymes and can reduce the risk of various ailments.
bacterial and hormonal stimulant.
There are various pharmacological and epidemiological evidences which prove that plants contain biologically active components which offer various health benefits and also Mutahar

SCREENING OF PHYTOCHEMICALS AND BIOACTIVE COMPOUNDS IN PUNICA GRANATUM PEEL TO EVALUATE ITS HEMATOLOGICAL POTENTIAL
Pomegranate is the most significant tree which has been domesticated for incalculable human benefits and serves as a food medicine of great importance. Pomegranate has been found to have ethnomedical history and is a rich reservoir of phytochemicals. These are found to possess several chemicals and have immense economic Its various parts are exploited by pharmaceuticals and are considered as a The present study was carried out for phytochemical analysis in the hydroalcoholic pomegranate peel extracts. The phytochemical analysis and analysis of bioactive compounds are done, the pomegranate peel extracts are found to have flavonoids, steroids, tannic acids and phenolic compounds. The antioxidant activity, DPPH free radical scavenging activity, nitric oxide radical scavenging For good health, the quality of blood should be maintained and for this antioxidant may be the key so as to protect the blood from oxidative stress. The results of various tests Thus, from the present study it is concluded that pomegranate peel is a rich source of phytochemicals and bioactive compounds such as antioxidants and so can be used for various pharmaceutical and therapeutic purposes like increase in hemoglobin count.  [Olaleye MT.2007]. Glycosides are naturally cardio active drug and is used to treat heart failures [Brain FH et al 1985]. Thus the purpose for the present investigation was to evaluate the phytochemicals present in pomegranate peel using in vitro methods. The screening results are tabulated.

MATERIALS AND METHODS
The fresh pomegranate fruits were collected from the fruit market, Bittan Market, Bhopal. The fruits were washed and then disinfected. The peel was carefully removed from the seeds. These peels were then shade dried for 10 days .Then the pomegranate peels were grinded in the grinder. Then these are taken for extraction process

Preparation of hydro alcoholic extract
The powdered material is extracted with the help of Soxhlet apparatus. 200ml of the solvent is used in the ratio of 70:30 (methanol/water). A Soxhlet extraction is used when the desired compound has a limited solubility in a solvent, and the impurity is insoluble in that solvent. It allows for unmonitored and unmanaged operation while efficiently recycling a small amount of solvent to dissolve a larger amount of material.

Phytochemical analysis
The hydroalcoholic extract of Punicagranatum fruit peel were studied for their phytoconstituents using different phytochemical tests [Ayoola GA et.al 2008].

Qualitative Phytochemical analysis
The crucial starting point for assessing the pharmacological aspect of pomegranate is the i.dentification of the phytochemicals. The pomegranate peel extract was screened to identify the key families of phytochemicals. Following tests were performed for identification.

Molisch test
This test is performed to identify carbohydrates. In this 2.3 ml of the extract was taken and 4 drops of naphthol in alcohol is added. This is then shaked well and conc. H 2 SO 4 is then poured from the sides of the test tube.

Test for Steroids
This test is known as the Salkowiski test. In this 2 ml of chloroform and 2 ml of conc.H 2 SO 4 are added to 2ml of the extract. This is then shaken well.

Test for Flavanoids
Take 2 ml of test solution then add Small quantity of reduced lead acetate solution (10%).

Tannic acid and Phenolic tests
Take 2-3ml. of aqueous or alcoholic extract add few drops for following reagent.

Test for Sulphates
With lead acetate reagent gives white ppt. will form that soluble in NaOH solution.

Test for Chloride
Take 5-7 ml. filterate plant extract solution and add 3-5 ml. lead acetate solution.

Test for saponins
Foam test -The extract and powder were mixed vigorously with water.

Total Flavonoid content
A clean test tube was taken and 0.5 ml of the sample (Extract) is added, containing 1.25 ml of distilled water. Then 0.075 ml of 5 % sodium nitrite solution is added and allowed to stand for 5 min. 0.15 ml of 10 % aluminium chloride is added, after 6 min 0.5 ml of 1.0 M sodium hydroxide was added and the mixture were diluted with another 0.275 ml of distilled water. The absorbance of the mixture at 510 nm was measured immediately. The flavonoid content was expressed as mg querecitin equivalents /g sample.

DPPH Free Radical Scavenging Activity
The free-radical scavenging activities were tested by their ability to bleach the stable radical DPPH. The antioxidant activity using the DPPH (1, 1-diphenyl-2-Picrylhydrazyl) assay was assessed by this method. The sample extracts and standards (BHA and ascorbic acid) were prepared at various concentrations (200-1000 ppm) and mixed with ethanolic solution of DPPH with a concentration of 0.04 mg/ml. After stand for 20 min in the dark, the mixtures were measured at 517 nm against ethanol as blank using UV-Vis Spectrophotometer.
The result obtained was calculated using formula:

Nitric oxide radical scavenging activity
The procedure is based on the principle that, sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent. Scavengers of nitric oxide complete with oxygen, leading to reduced production of nitrite ions. Plant extract was dissolved in distilled water for this quantification. Sodium Nitroprusside (5 mM) in standard phosphate buffer saline (0.025m, pH 7.4) was incubated with different concentration (100-400μg/ml) of methanol extract and tubes were incubated at 29 o C for 3 hours. Control experiment without the test compounds but with equivalent amount of buffer was conducted in an identical manner. After 3 hours incubated samples were diluted with 1 ml of Griess reagents. The absorbance of the colour developed during diazotization of Nitrite with sulphanilamide and its subsequent coupling with Napthylethylenediamine hydro-chloride was observed at 550nm on spectrophotometer. Same procedure was done with ascorbic acid which was standard in comparison to methanol extract. Calculated the % inhibition by formula and plot graph in compared to standard.
Where, A control = Absorbance of control reaction A test = Absorbance in the presence of the samples of extract.

Total Antioxidant Activity
Determination of total antioxidant capacity was evaluated by the Phosphomolybdenum method. 0.3 ml of extract and subfraction in ethanol, ascorbic acid used as standard (5 to 200g/ml) and blank (ethanol) were combined with 3 ml of reagent mixture separately and incubated at 95°C for 90 min. After cooling to room temperature, the absorbance of each sample was measured at 695 nm against the blank. Ascorbic acid was used as standard and the total antioxidant capacity is expressed as equivalents of ascorbic acid or Gallic acid. The antioxidant activity is expressed as the number of equivalents of ascorbic acid and was calculated by the following equation: Where,
Solution will form white ppt. Gelatin solution (10%)Solution Solution will form white ppt. Acetic acid solution.
Solution will appear red colour Dilute iodine solution.
Solution will appear red colour Dilute potassium permagnate solution Solution will decolourizes its colour.  Table 1.

DPPH Free Radical Scavenging Activity
The DPPH radical scavenging activity of Pomegranate peel extract is depicted by the figure 1.Pomegranate fruit peel extract potently scavenged DPPH radicals similar to catechin, it is likely that peelextract possessed proton and in association with a number of hydroxyl groups to stabilize free radicals   Nitric oxide radical generated from sodium nitroprusside at physiological pH was found to be inhibited by the peel extracts. The Nitric Oxide scavenging activity is shown in figure 2. The scavenging activity is higher in pomegranate peels as compared to the standard ascorbic acid. From the figure it is clear that with increasing concentration the activity also increases.

Total phenolic content
The Total Phenolic Content was found to be 273.26 catechol equivalents/g. The results are in agreement to 2002]who studied this process for extraction from pomegranate peel extracts but this result differs slightly due to environmental and experimental conditions. The best solvent for the exraction of the phenolic compound is methanol as it has the ability to inhibit the reaction of PPO which leads to the oxidation of phenolics [ 1994]. The results are shown in figure 2 and

Screening of phytochemicals and bioactive compounds in punica granatum
The respective values for pomegranate peel are also shown in Nitric oxide radical generated from sodium nitroprusside at physiological pH was found to be inhibited by the peel Oxide scavenging activity is shown in figure 2. The scavenging activity is higher in pomegranate peels as compared to the standard ascorbic acid. From the figure it is clear that with increasing concentration the activity The Total Phenolic Content was found to be 273.26 catechol equivalents/g. The results are in agreement to [Singh RP et al who studied this process for extraction of antioxidants from pomegranate peel extracts but this result differs slightly due to environmental and experimental conditions. The best solvent for the exraction of the phenolic compound is methanol as it has the ability to inhibit the reaction of PPO hich leads to the oxidation of phenolics [Yen GC et.al ]. The results are shown in figure 2

Nitrous Oxide Scavenging Activity
The total flavanoid content in pomegranate peel extracts were found to be 87.122 mg of quercetin equivalent /g. The regression coefficient taken here is R 2 = 0.990 and the equation for standard curve is taken as y= 0.244x +0.697 (figure 4) (table 4).
The antioxidant activity of samples was evaluated by the green phosphomolybdenum complex formation according to the method of Prieto (1999).This method is based on the reduction of phosphomolybdic acid to phosphomolybdenum blue complex by sodium sulfide. The obtained phosphomolybdenum blue complex is oxidized by the addition of nitrite and this causes a reduction in intensity of the blue colour. The results shows that at lower concentration the percentage inhibition of pomegranate peel is more as compared to standard ascorbic acid. This is shown in figure 5. P. Granatumpeel extracts can be used as a natural source of antioxidants.

CONCLUSIONS
Pomegranate has an important role in folk medicine. It is known as a rich source of pharmacological properties. The present study showed interesting preliminary phytochemical constituents in hydroalcoholic peel extracts of Punica granatum. As the pomegranate peel extracts have promising antioxidant properties so these can act as a powerful ingredient in increasing the hemoglobin count. Further characterization and quantitative assay may be carried out to test the peel extracts for various therapeutic and pharmacological activity.