INTRODUCTION:
The reactive oxygen species (ROS) inducing superoxide anionic radical (O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH) are implicated in oxidative damage to various cellular macromolecules. Increasing number of evidence suggested that oxidative stress induced biochemical changes are crucial etiological factors in several chronic human diseases such as diabetes mellitus, cancer, atherosclerosis, arthritis, inflammation and neurodegenerative diseases1. Although several synthetic antioxidants such as butylated hydroxyanisol (BHA) and butylated hydroxytoluene (BHT) are available which are quit unsafe2. Therefore, in recent years considerable attention has been directed to identify the natural antioxidants (plant derived) that may be used for human consumption.
In the present investigation we have attempted to investigate various active constituents present in methanolic extract of peel of Punica granatum which is responsible for their antioxidant potential.
Pomegranate is high prized fruit and its medicinal values have been known since ancient times. It’s all parts, root, stem bark, leaves, flower, fruit rind are used medicinally and botanically knowns as Punica granatum (Punicaceae). Within the country it is known under different names DADIMA (Sanskrit), ANAR (Hindi), Pomegranate (English).3,4
A deciduous tree or shrub, 1.5-5m tall with thorny branches, Leaves 2.5-5.5 cm long, oblong, obviate or elliptic- lanceolate, shining above. Flowers long solitary. Fruit- a hard, globose berry, 4-8 cm in diameter crowned with calyx and thick leathery rind. Seeds red or pink and juicy.5
It contains alkaloids chiefly pelletierine. It is rich source of tannin. Juice contain malic acid, citric acid, the seeds are the richest source of estrone, traditionally it is used in itching, pyorrhea, gum and teeth disorder. Stem and root bark is an effective anthelmintic and taeniacide. Seeds and pulp possess antibacterial activity. The food rind powder has appreciable immuno-stimulatory activity and hepato protective activity. It is also used as astringent6.
Pomegranate (Punica granatum Linn) has been traditionally used as antihelmintic, hepatoprotective, immunostimulant and antidiarrhoeal. The fruit rind of pomegranate shows significant reduction of the percent of diarrhoea. Pomegranate has been used for treatment of itching; Tooth and gum disorder juice makes delicious drink. A recent study found that Pomegranate juice exhibit three times greater antioxidant activity than other bioflavoniods such as red wine or green tea7.
MATERIAL AND METHODS:
The Plants part was collected from Bhopal (M.P) in month of Sept-Oct 2007. The plants were authenticated by Dr. A. K. Pathak HOD, Department of Pharmacy Barkatullah University, Bhopal. Herbarium no. BUPH-no.4022/B. The peel of P. granatum was dried under shade.
PREPARATION OF EXTRACT:
Approximately 70g of powder crude drug was extracted with methanol by soxhelation and the solvent was recovered by distillation. The extract was concentrated under reduced pressure and air dried.
Determination of active constituents:
(a)Total Phenolic content: The Folin–Ciocalteu reagent assay was used to determine the total phenolics content. The extract 1ml (10mg/ml) was mixed with 0.5 ml Folin–Ciocalteu reagent previously diluted with 7 ml deionized water. The solution was allowed to stand for 3 min at 25 şC before adding 0.2 ml of saturated sodium carbonate solution. The mixture was allowed to stand for another 120 min. The absorbance was measured at 725 nm. Gallic acid was used as standard for the calibration curve. The total phenolics content of the extract was calculated in terms of Gallic acid equivalent (GAE).8,9
C = c× V/m
Where, C= total content of phenolic compound in mg/gm of the extract.
c= concentration of gallic acid established via calibration curve.
V= volume of extract in ml.
m= wt. of extract in gm.
(b)Total Flavonoid contents: The content of total flavanoids was determined by aluminium chloride colorimetric method. The content of flavanoid was determined as quercetin equivalent. 10 mg/ml of plant extract in respective solvent (stock solution SS) was mixed with 2 ml AlCl3 (2% w/v) in methanol and the solution was made up to 25ml with methanolic solution of acetic acid (0.5% v/v) (Probe solution PS). 1ml of SS was made up to 25ml with methanolic solution of acetic acid (contrast solution CS).The absorbance of PS and CS was measured at 420nm after 30 minutes. The result expressed as % of total flavanoid content.9
Absorbance at 420 x dilutin x 100
TFC =
E 1% 1 cm x wt. of extract in gms
Proanthocyanidin Content: Proanthocyanidin were determined by a butanol-HCl assay. In this method 1ml (10mg/ml) of plant extract was added with 4 ml of butanol-HCL reagent(95:5) and 0.1 ml of ferric reagent (2% ferric ammonium sulfate in 2.0M HCL). Then the mixture was kept in water bath for 60 min. After cooling, absorbance were recorded at 545nm against a blank containing solvent (0.5ml) instead of the extract10.
% Proanthocyanidin =
Absorbance at 545nm x dilution factor x 100
E1%1cm of leucocynanidine x wt.
of extract in gm
Estimation of Tannins (Volumetric method): To an aliquot of filtrate of extract(10 ml) in a porcelain dish, add 20 ml of indigo carmine solution and about 750 ml of water. Add KMno4 solution from a burette, 1 ml at a time with vigorous stirring, until the colour become light green. Then add drop wise until the colour changes to bright yellow or to a faint pink at the rim. Note the ml of KMno4 used (A).
To 50 ml of the clear filtered extract in a 250 ml flask, add 25 ml of gelatin solution and make up volume with acid Nacl solution. Transfer to a conical flask, add a little amount of filter aid (Kaolin), shake for 15 min. and filter. To 50ml. of filterate, add 20ml. of the indigo carmine solution, and about 750ml. of water, and titrate with KMno4 solution as before (B). The amount of ml. of KMno4 solution used in this case (B) subtracted from A gives the quantity of KMno4 solution required to oxidize the tannins11.
Where, A = Tannin like materials
B = Non- tannins materials
A-B = True tannins
[1ml of 0.1N KMno4 = 0.0041 g of tannin]
Titre(A-B) x N x 0.0041 x 100
%Tannins =
1 x 0.1 x wt. of sample
Evaluation of Antioxidant Activity:
(a) In-vitro antioxidant activity using DPPH:
DPPH Radical Scavenging Assay: (Chang et al 2003)
Different Concentration of methanolic extracts of rind of Punica granatum (25-250 ug/ml) in methanol were mixed with 400mM methanolic solution of 1,1, - Diphenyl -2-picryl hydrazine (DPPH) at a ratio of 1:3. The mixtures were left in the dark at room temperature for 90 minutes. Absorbance of resulting solution was measured by a spectrophotometer. The capability of scavenging DPPH radical was then calculated. The antioxidant activity is expressed as IC50. The IC50 value is defined as the concentration (in ug/ml) of extract that inhibits the formation of DPPH radicals by 50%.9,12
1 - Absorbance of sample at 517 nm X 100
Scavenging activity (%) =
Absorbance of Control at 517 nm
(b) Reducing power of herbal plants:
The reducing power of nutraceutical herbs was determined according to the method of Oyaizu (1986). Extracts were prepared in different concentrations ranging from 20µg/ml to 100µg/ml and 1 ml of each in distilled water were mixed with phosphate buffer (2.5 ml, 2 M, pH 6.6) and potassium ferricyanide (2.5 ml, 1%); the mixture was incubated at 50şC for 20 min. A portion (2.5 ml) of trichloroacetic acid (TCA, 10%) was added to the mixture which was then centrifuged at 1500 RPM for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%), and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. The Reducing Power was expressed as AAE means that reducing power of 1mg sample is equivalent to reducing power of 1nmol ascorbic acid.9
(c) Nitric oxide radical scavenging Assay:
Nitric oxide radical scavenging activity was determined according to the method reported by Garrat. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions, which can be determined by the use of the Griess Illusory reaction. 2 ml of 10 mM sodium nitroprusside in 0.5 ml phosphate buffer saline (pH 7.4) was mixed with 0.5 ml of extract at various concentrations and the mixture incubated at 250C for 150 min. From the incubated mixture 0.5 ml was taken out and added into 1.0 ml sulfanilic acid reagent (33% in 20% glacial acetic acid) and incubated at room temperature for 5 min. Finally, 1.0 ml Griess reagent (1%sulphanilamide in 2% phosphoric acid and 0.1%naphthylethylene diamine diamine dihydrochoride in water) was mixed and incubated at room temperature for 30 min. The absorbance at 546 nm was measured using a spectrophotometer. The nitric oxide radicals scavenging activity was calculated according to the equation13:
% Inhibition = ((A0-A1) / A0 × 100)
Where,
A0 = absorbance of the control (blank, without extract)
A1 = the absorbance in the presence of the extract
The IC50 of the extracts was compared with IC50 of Standard (curcumin)
Formulation of Oral suspension:
Tragacanth mucilage was prepared:-
Glycerin – 18ml
Tragacanth – 6g
Benzoic acid – 0.2g
Water(q.s.) – 100 ml .
Glycerin (18g) with water (75 ml.) in a tarred vessel, heated the mixture to boiling, discontinued the application of heat, added tragacanth (6g), benzoic acid(0.2g) and macerated the mixture during 24 hrs with occasional stirring. Then added enough purified water to make the mixture weigh 100g, stirred actively until of uniform consistency and strain forcibly through muslin14.
(a) In-vivo Evaluation of Antioxidant Activity (Lipid peroxidation):_
Chemicals:- Thiobarbituric acid (TBA), trichloroacetic acid (TCA), 5,5’dithiobis (2nitrobenzoic acid) (DTNB), phosphate buffer . All other reagents used were of analytical grade.
Animals: - The albino rats of both sex having average weight (150-200 g) were housed at controlled temperature (25±2OC) with proper food and water. The experimental group was subdivided in such a manner and formulation was given for four weeks; after that Lipid peroxidation of the blood was determined.
Without Ethanol Treated Groups:In this groups only drugs was given daily for 1 month.
GroupI (CONTROL) - received vehicle (Tragacanth Mucilage)
GroupII (STANDARD) - received vitamin C ( 100mg/kg, orally), Once daily
Group III (Test) - Suspension of methanolic extract of peel of Punica granatum (100mg/kg, orally) Once daily
Ethanol Treated Groups: In Ethanol treated Groups 1ml of 20%v/v ethanol was given daily for 1 month before drugs admistration.
GroupI (CONTROL) - received vehicle (Tragacanth Mucilage).
GroupII (STANDARD)- received vitamin C ( 100mg/kg, orally), Once daily.
Group III (Test) -Suspension of methanolic extract of peel of Punica granatum (100mg/kg, orally) Once daily
Lipid peroxidation (LPO):- To 2 ml, 5% suspension of separated RBC in 0.1 M phosphate buffered saline; 2 ml of 28% trichloroacetic acid was added and centrifuged. One ml of 1% thiobarbituric acid was added to 4 ml of supernatant, heated in boiling water for 60 min and cooled immediately. The absorbance was measured spectrophotometrically at 532 nm. The lipid peroxidation was calculated on the basis of the molar extinction coefficient of malondialdehyde (MDA) (1.56x 105), and expressed in terms of nanomoles of MDA/g Hb.15
HPLC Analysis:-HPLC procedure for analysis of Tannins in methanolic extract of P.granatum.
Mobile phase A:- Prepare 1000ml solution of water containing orthophosphoric acid [pH 2.4-2.6].Filter and degas.
Mobile Phase B:-Filtered and degassed acetonitrile.
Standard Solution:-Dissolve an accurately weighed quantity of tannic acid in water to obtain a known concentration of 0.03mg of Tannic acid/ml.
Test Solution:-P.granatum extract solution.
Chromatographic system:-The liquid chromatographic equipment is equipped with a 275nm detector and 0.5mm x15cm column that contains packing LI and is programmed to provide variable mixture of mobile phase A and B. Initially the system is held at a mixture consisting of 90% mobile A and10% B for 5 min.
Procedure:-Separately inject equal vol.(25ul)of standard and test solution to chromatograph to record the chromatogram and measure the response of all peaks. Calculate the area and percentage16.
Table 1
|
S. No. |
Active Constituents |
Inference |
|
1. |
Total Phenolic content |
15.064mg gallic acid equivalent /g extract |
|
2. |
Total Flavonoid content |
8.32 % |
|
3. |
% Proanthocyanidine |
14.08% |
|
4. |
% Tannin |
14.48 % |
RESULTS AND DISCUSSION:
The Preliminary Phytochemical investigation of methanolic extract of P. granatum showed the presences of Tannins, sterol, and Flavonoids. The total phenolic (graph 1), Proanthocynidine, %Tannin and Flavonoid contents was determined (Table 1) .The percent inhibition showed decrease in concern traction of DPPH radical due to the scavenging ability of extract and ascorbic acid (standard). IC50 value of methanolic extract of P. granatum and Ascorbic acid were found to be 24.03µg/ml and 25.8 µg/ml respectively. Standard curve of ascorbic acid are showed (graph 2)
Graph.1-Standard curve of Gallic acid
Graph.2-Standard curve of ascorbic acid
Table – 2: DPPH scavenging assay
|
Extract |
Inhibitory concentration ( IC 50 µg/ml) |
|
Punica ganatum |
24.03 |
|
Ascorbic acid |
25.8 |
The In-vitro antioxidant activity by Reducing Power indicated that increases in absorbance with concentration showed that methanolic extract of P. granatum have reducing power (graph 3).
Graph.3- Reducing Power of P. granatum and Ascorbic acid (standard)
Table-3: Reducing Power of methanolic extract of P.granatum
|
Concentration(µg/ml) |
Absorbance (700nm) |
|
|
Ascorbic acid |
P.granatum |
|
|
20 |
0.33 |
0.571 |
|
40 |
0.47 |
0.723 |
|
60 |
0.62 |
0.751 |
|
80 |
0.74 |
1.153 |
|
100 |
0.82 |
1.782 |
In Nitric oxide assays the IC50 of methanolic extract of P. granatum and Standard (Curcumin) were found to be 39.06µg/ml and 20.16µg/ml respectively. .
In-vivo antioxidant activity revealed that the concurrent treatment of ethanol-administered rats with vitamin C prevented the above ethanol-induced changes in the markers of oxidative stress. Influence of methanolic extract of P. granatum suspension on ethanol-induced changes were more or less similar and comparable with the vitamin C treatment (table 5).
Table – 4: Nitric oxide scavenging assay
|
Extract |
Inhibitory concentration ( IC 50 µg/ml) |
|
P.granatum |
39.0.6 |
|
Curcumin |
20.16 |
Table – 5: Effect of Methanolic extract of Punica granatum peel on oxidative stress in rats(mean±SEM, n=6)
|
Groups |
Treatment |
Lipid Peroxidation (nmMDA/g Hb) |
|
Without Ethanol treated Control
Test1 Standard |
Vehicle(Tragacanth mucilage) P.granatum Vit. C |
72.75±1.5
68.58±0.71 69.025±0.79 |
|
Ethanol treated Control Test1 Standard |
Vehicle P.granatum Vit. C |
154.54±0.69 93.15±0.66* 99.99±0.90* |
Values are expressed as the mean ± SEM, n = 6 in each group P< 0.001 significance Vs control
Graph 4 Chromatogram of standard ellagic acid
Graph 5 Chromatogram of P. granatum extract
The methanolic extract of P. granatum were analyzed by HPLC using H2O:CH3CN (9:1).The Phenolic were found to be ellagic acid having Rt= 10.32(For standard R=11.55) which is responsible for plant antioxidant activity (graph 4 and 5).
ACKNOWELEGEMENT:
The Author thanks the Dr. A. K. Pathak H. O. D. Dept. of Pharmacy B. U. Bhopal (M.P) and Dr. A. K. Singhai, for giving the necessary permission to conduct the animal study and providing the necessary research facility.
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Received on 19.05.2010 Modified on 24.05.2010
Accepted on 13.06.2010 © RJPT All right reserved
Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page1170-1174
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