INTRODUCTION:

Oxidative stress (ost) depicts the existence of products called free radicals (molecules possessing an unpaired electron) and reactive oxygen species (ros), which are formed, in normal physiology but become deleterious when not being quenched by a cascade of antioxidants systems (aos). This can result either from an overproduction of ROS or from the inactivation of the AOS, thus shifting the OST/AOS balance in favour of stress. ROS oxidize various types of biomolecules, finally leading to cellular lesions by damaging DNA or stimulating apoptosis for cell death Some ROS are considered more important than others, such as superoxide (O2· ), hydroxyl radicals (·OH) or peroxides (ROO·). However not all oxygen-containing radicals have high oxidative potential ¹.

In the present study Free radical scavenging activity of Punica granatum Linn. (PG) was carried out by DPPH (1, 1-diphenyl-2-picryl hydrazyl) method, Reducing power assay, Scavenging activity against hydrogen peroxide, Total antioxidant activity, Estimation of total flavonoid content in vitro methods. In folk medicine pomegranate preparations of the dried pericarp and the juice of the fruits are employed as an oral medication in the treatment of colic, colitis leucorrhea, menorrhagia, oxyuriasis, paralysis and rectocele, and external application to caked breast ²and to the nape of the neck in mumps ³and headache ⁴. A number of therapeutic actions of these materials have been described including vermifugal, taenicidal, astringent, antispasmodic, antihysteric, diuretic, carminative, sudorific, galactogogue and emmenagogue ⁵Pomegranate peel is used for treating the infection of male or female sexual organs, mastitis, acne, folliculitis, piles, allergic dermatitis, tympanitis and for the treatment of oral diseases ⁶

METHODS:

In vitro antioxidant study

DPPH (1, 1-diphenyl-2-picryl hydrazyl) method ^{7, 8}

Assay principle:

Scavenging free radical potential was evaluated against ethanolic solution of DPPH, a stable free radical. Antioxidants react with DPPH and convert it to 1, 1-diphenyl-2-picryl hydrazine (non-radical). Degree of discoloration indicates the scavenging activity of drug. The change in absorbance produced at 517 nm, has been used as a measure of antioxidant activity.

Procedure:

DPPH (2.365 mg) was dissolved in 10 ml of 95% ethanol and the PJ extract was dissolved in 95% ethanol to make the stock solutions, which was diluted to give concentrations ranging from 50-800 ppm. The PG extract of varying concentration (1.5 ml) were added to both blank as well as in the test tubes. DPPH (1.5 ml) was added only in test and finally 95% ethanol (1.5ml) was added in both blank and test. The tubes were kept aside for 20 min allowing the reaction to take place. After 20 min the absorbance of the solutions was recorded against the respective blanks at 517 nm 8. Absorbance of control 􀌛 Absorbance of sample % Scavenging activity = × 100 Absorbance of control

Reducing power assay ^9,10

The reducing power of the dried PG extract was determined by the method reported by

Siddhuraju et al., (2002).

Assay principle:

The yellow color of the test solution changes to various shades of green and blue depending upon the reducing power of each extract. The presence of reductants (antioxidants) in the PJ extract causes the reduction of Fe3+/Ferric cyanide complex to Fe2+/ferrous form. Therefore, the Fe2+ complex can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm.

Procedure:

PG extracts (20–800 μg) in 1 ml of phosphate buffer with 5 ml of 0.2 M phosphate buffer (pH 6.6) and 5 ml of 1% potassium ferricyanide solution were incubated at 500C for 20 min. After the incubation, 5 ml of 10% TCA was added. The content was then centrifuged at 1000 rpm for 10 min. The upper layer of the supernatant (5 ml) was mixed with 5 ml of distilled water and 0.5 ml of 0.1% ferric chloride. Then the absorbance of the reaction mixture was read spectroscopically at 700 nm.

Scavenging activity against hydrogen peroxide ¹¹

The scavenging capacity of PG extract on hydrogen peroxide was determined according to the method of Ruch et al., (1989).

Procedure: Test tubes were prepared with 2.0 ml of various concentrations (50-800 ppm) of PJ extract and a solution of H2O2 (1.2 ml, 40 mM) in phosphate buffer (pH

7.4). A blank solution was prepared the same way but without H2O2. After incubation of the mixture during 10 min, the absorbance was recorded at 230 nm. The Scavenging activity was calculated using the following formula

Total antioxidant activity ¹²

The total antioxidant activity of PG extract was determined using the thiocyanate method Various concentrations (50, 100, 250, and 500 ppm) of PG extract were prepared in methanol and added to a linoleic acid emulsion (2.5 ml, 40 mM, pH 7.0) and phosphate buffer (2ml, 40mM, pH 7.0). The linoleic acid emulsion was prepared by mixing 0.2804 g linoleic acid with 0.2804 g Tween-20 as emulsifier in 50 ml phosphate buffer (40 mM). The mixture was then homogenized. The final volume was adjusted to 5 ml with 40 mM phosphate buffer, and pH 7.0. The mixed samples were then incubated at 370C in glass flask for 60 h, to accelerate the oxidation process (Yen and Chen, 1995). One milliliter of the incubated sample was removed at 12 h interval and 0.1 ml 20 mM FeCl2 and 0.1 ml 30% ammonium thiocyanate was added. The absorbance of this was measured at 500 nm, using a spectrophotometer (Shimadzu, 2450). Alpha Tocopherol was used as a reference compound. To eliminate the solvent effect, the control sample, which contained the same amount of solvent added to linoleic acid emulsion in the test sample and the reference compound, was used. All data reported are the average of triplicate analysis. Percent inhibition of lipid peroxide generation was calculated using formula.

Estimation of total flavonoid content ¹³

The aluminium chloride colorimetric method was used for flavonoids determination in PJ extract Rutin was used to make the calibration curve. Ten milligrams of rutin was dissolved in 80% ethanol and then diluted to 25, 50 and 100 μg/ml. The diluted standard solutions (0.5 ml) were separately mixed with 1.5 ml of 95% ethanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. The same concentrations of PJ extract were also prepared. After incubation at room temperature for 30 min, the absorbance of reaction mixture was measured at 415 nm with Schimadzu UV-2450 spectrophotometer. The total flavonoid content of PG was calculated from standard calibration curve equation.

Estimation of total phenolic content ¹⁴

Total soluble phenolics compounds present in PG extract were determined with the Folin-Ciocalteu reagent. The calibration curve was prepared by preparing gallic acid (GA) solutions at concentrations 0, 50, 100, 150, 250, 500 μg/ml in ethanol. To 0.1 mL of PJ extract (1mg/ml in distilled water) 1 ml of Folin-Ciocalteu reagent was added. After 3 minutes, 3 ml 2 % Na2CO3 was added. Subsequently, the mixture was shaken for 2 h at room temperature and the absorbance was measured using Schimadzu UV-2450 spectrophotometer at 760 nm. All tests were performed in triplicate. Total phenol values were expressed in terms of gallic acid equivalent (mg/g of dry mass).

RESULTS:

DPPH (1, 1-diphenyl-2-picryl hydrazyl) method

A dose dependent increase in scavenging activity was observed with varying concentration of PG extract. The EC50 value was found to be 450 ppm.

Reducing power assay

A dose dependent increase in optical density was observed with varying concentration of PG extract.

Scavenging activity against hydrogen peroxide

A dose dependent increase in scavenging activity was observed with varying concentration of PG extract. The EC50 value was found to be 190 ppm.

Total antioxidant activity

A dose dependant increase in percent inhibition was observed with varying concentration of PG extract. The IC50 value was found to be 210 ppm.

Total flavonoid content and total phenolic content of the PG extract

The total flavonoid content of PG extract in terms of rutin equivalent (The standard curve equation: y = 0.00241x + 0.00426) was found to be 415.7 ± 3. 29 mg/g of dry weight. The phenolic content of PJ extract in terms of Gallic acid equivalent (The standard curve equation: y = 0.001162x + 0.10266) was found to be and 84.15 ± 6.365 mg/g of dry weight.

DISCUSSION:

It is known that reactive oxygen species (ROS) contribute to the pathogenesis of numerous cardiovascular diseases including hypertension, atherosclerosis, cardiac hypertrophy, heart failure and restenosis, being NAD (P)H oxidase the predominant source of ROS^15. Activation of this enzyme leads to a variety of intracellular signaling events that ultimately promote endothelium dysfunction, vascular smooth muscle cells proliferation, pro-inflammatory genes expression and reconstruction of the extracellular matrix¹⁶ Assessment of antioxidant activities and lipid peroxidation byproducts in hypertensive subjects indicates an excessive amount of ROS and a reduction of antioxidant mechanism activity in both blood as well as in several other cellular systems¹⁷

As reactive oxygen species contribute to endothelium-dependent contraction and to increased vascular resistance, antioxidants can possibly restore endothelial function and hence decrease blood pressure^18.Polyphenols are the most abundant antioxidants in our diets. The main classes of polyphenols are phenolic acids (mainly caffeic acid) and flavonoids the most abundant in the diet are flavanols (catechins plus proanthocyanidins), anthocyanins, and their oxidation products], which account for one- and two-thirds of polyphenols, respectively Polyphenols are reducing agents that may protect the body’s tissues against oxidative stress and associated pathologies such as cancer, chronic heart disease, vascular diseases, and inflammation The preliminary phytochemical investigation of PG extract in the present study reveals the presence of flavonoids, anthocynins, tannins and phenolic compounds. PG is documented to possess impressive antioxidant property due to its ployphenolics, tannins and anthocynins. The antioxidant level in PG was found to be higher than in other natural juices such as blueberry, cranberry, and orange, as well as in red wine.

In the present study we found the excellent antioxidant activity of PGextract as measured by various methods such as DPPH radical scavenging activity, reducing power assay, H2O2 scavenging activity and total antioxidant activity. Antioxidants such as vitamin C, vitamin E, 􀈕-caroteone and coenzyme Q were recently shown to possess hypotensive properties ¹⁹. Catalase, glutathione peroxidase and SOD are the three primary antioxidant enzymes among the endogenous systems for removal of reactive oxygen species. The present study reveals that there is significant decrease in antioxidant enzymes SOD, CAT, GSH and increase in the level of TBARS in kidneys and pancreas of diabetic and diabetic hypertensive rats. Endothelial dysfunction is attributed to increased oxidative stress and decreased NO production and action ²⁰ Administration of antioxidants in experimental models has been shown to reduce the Severity of atherosclerosis by reducing oxidative stress and increasing NO production and action ²¹ Finally, a recent study shows that PG consumption for 3 years by patients with carotid artery stenosis reduced common carotid intima-media thickness, blood pressure, and LDL oxidation . Similarly, the ingestion of polyphenols contained in purple grape juice had beneficial effects on endothelial function in patients with coronary heart disease. Taken together, these data suggest that polyphenols can protect arteries from vascular damage via antioxidant effects and NO restoration. In the present study, pretreatment with PG extract restored the antioxidant enzymes level and decreased TBARS level, which in turn indicate the protective effect of PG against oxidative stress as PG possess polyphenolic compounds.

ACKNOWLEDGEMENT:

Authors are thankful to Principal and Management of KCT’s R.G. Sapkal College of Pharmacy, Anjaneri, Nashik for providing all necessary facility for research project.

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Received on 18.04.2014 Accepted on 30.04.2014

Asian J. Res. Pharm. Sci. 4(2): April-June 2014; Page 51-54