INTRODUCTION:

It generally accepted that the sulphonyle urea produce hypoglycemia in normal animals by stimulating the pancreatic ß cells to release more insulin .These drugs, however, do not decrease blood glucose in alloxan diabetic animals .⁵In addition, the exogenous administration of insulin is well known to produce hypoglycaemia in both normal and alloxan induced subjects.⁹ This leads to increasing demand for natural products with potent antidiabetic and less side effect. Various parts of Pomegranate (Punica granatum L.) have been used for various medicinal purposes . Many studies have shown that the pomegranate peel extract has wound healing properties,² possesses antioxidant activity, 2002,¹immunomodulatory activity,⁶ antibacterial activity,¹⁹ gastroprotectective effect,⁴ larvicidal activity,¹²antifungal activity,³ antitumor action,11 antimicrobial effect,¹³ antiviral activity ²⁴ and hypoglycemia effect ^{15, 23.} It was found that Punica grantum peels contain tannins, anthocynins, flavonoids ,pectin’s,¹⁷ three estrogen compounds luteolin, quercetin and kaempferol.²² This investigation aims to evaluate the role of Punica grantum powder peels extract in its human therapeutic dose on beta cell number, blood glucose and plasma insulin levels in normal and alloxan diabetic rats for 4-weeks of treatment.

MATERIAL AND METHODS:

Plant material:

200ml boiling distilled water were added to3gm powder pomegranate peel, left it for 10 minutes and filtered .The filtrate was dried at 40-45C⁰ in the incubator .

Animals:

Male albino rats weighing 120±10gm were obtained from the observed first generation for a period of time .The animals were caged and provided with food and water ad libitum. Injection of alloxan monohydrate, in a dose of 120mg /kg Body Wt. dissolved in acetate buffer immediately prepared before usage. Seven days after injection of alloxan blood glucose levels of all surviving rats were determined. Only rats with glucose levels above 200mg/ml were considered diabetic and employed for the assay.

Experimental design:

24 adult male albino rats weighing (110-130gm) were divided into four groups 6 rats each. Group I, represented control, group II diabetic rats, group III diabetic rats treated with 0.43g of P. granatum peel aqueous extract and group IV treated with 0.43g of P. granatum peel extract, that dose equivalent to human therapeutic dose ¹⁸ daily for 4 weeks.

Blood sampling:

Blood samples were collected from retro-orbital vein in two separate tubes, one tube with EDTA for determination of hematological parameter. Red blood cells (RBCs) and (WBCs) were counted in Hemocytometer. The second tube containing potassium oxalate and sodium fluoride for estimation of glucose ²¹and insulin was estimated by kits.

Table (1): Showing the effect of treatment with 0.43g/kgBW Pomegranate (Punica granatum) peel aqueous extract for 4 weeks in body weights, some hematological parameters, blood glucose and insulin in normal and diabetic rats.

Groups / parameters	Control	Diabetic	Diabetic treated with pomegranate	Normal treated with pomegranate
Body weight (gm)	145.83± 4.92	*123.33±6.83**	130..5	140 ±3.5
RBCs x10⁶	5.35 ±0.66	2.25*±2	5.5±0.5	7.5x10⁶±1
WBCs x 10³	4.66 ±0.6	6.76 ±0.8	7±1	8±2
Hb (gm%)	16.33 ±1	12.5±0.8*	16.5±0.5	18±1
Glucose (mg/dl)	85±2	280±5	120±10	75±2.5
Insulin (µU/ml)	12.5v0.5	7.58± 0.8	12±0.5	15*±1

Fresh pancreas samples were collected in formal saline, stained in Hand E .Some section of pancreas were stained by modified aldehyde fuchsine.⁸

Statistical analysis:

All data obtained were analyzed using student ’t’ test.

RESULTS AND DISCUSSION:

Preliminary studies showed that P. granatum rind extract possessed significant blood sugar lowering activity.^14-16,23The antihyperglycaemic action of the peel extract of P. granatum, to the inhibitory intestinal absorption of glucose in rats This investigation aims to evaluate the role of Punica grantum powder peel aqueous extract in its human therapeutic dose on beta cell number, blood glucose and plasma insulin levels in alloxan diabetic rats for 4-weeks of treatment As well as to illustrate the mechanism of hypoglycaemic effect of the extract There was a significant weight loss in diabetic rats (table1), (fig.1), while in diabetic group treated with P. granatum aqueous peel extract ,an improvement in their body weights was observed. The ability of pomegranate rind extract to prevent body weight loss seems to be due to its antidiabetic activity. Pomegranate peel extract treated normal rats resulted in no significant increase in body weight after4- weeks.

The red blood cells (table1, fig.2) and haemoglobin decreased significantly in diabetic rats (table1, fig.4). WBCs showed non-significant change at 30 days by alloxan treatment (table1,fig.3) Diabetic rats treated with pomegranate peel extract displayed non-significant alternation in the red blood cells , haemoglobin and white blood cells from their corresponding control .In rats treated with pomegranate peel extract ,the red blood cells and haemoglobin increased significantly as the result of pomegranate peel haemostatic property¹⁰while WBCs displayed non-significant variation comparable to control for 4-weeks.

Fig 1: Showing the effect of treatment with 0.43g/kgBW Pomegranate (Punica granatum) peel aqueous extract for 4 weeks in body weights in normal and diabetic rats.

Fig 2: Showing the effect of treatment with 0.43g/kgBW Pomegranate (Punica granatum) peel aqueous extract for 4 weeks in R BCs x 106 in normal and diabetic rats.

Fig 3: Showing the effect of treatment with 0.43g/kgBW Pomegranate (Punica granatum) peel aqueous extract for 4 weeks in W B Csx 103 in normal and diabetic rats.

Fig 4: Showing the effect of treatment with 0.43g/kgBW Pomegranate (Punica granatum) peel aqueous extract for 4 weeks in Hb(gm %) in normal and diabetic rats.

Fig 5: Showing the effect of treatment with 0.43g/kgBW Pomegranate (Punica granatum) peel aqueous extract for 4 weeks in Glucose (mg/dl) in normal and diabetic rats.

Fig 6: Showing the effect of treatment with 0.43g/kgBW Pomegranate(Punica granatum) peel aqueous extract for 4 weeks in Insulin (μU/ml) in normal and diabetic rats

(Table1, fig.5) displayed significant elevation in plasma glucose and significant reduction in insulin level in diabetic rats (table1,fig.6). Pancreas showed reduction in ß cell numbers, ß cells necrosis and intracellular vacuolation (fig8) compared to control (fig.7). Diabetic rats treated with 0.43g/KgB.W. of Punica granatum aqueous peel extract for 4 weeks displayed significantly lowered blood sugar level (table1, fig 5) and augmentation in insulin level(table 1and fig.6) .The number of ß cells relatively increased, while vacuolation still appeared in some cells (fig.10) . Punica granatum aqueous peel extract significantly lowered blood sugar and increased insulin

level (table1 and figs. 8, 9) and the number of ß cells increased (fig.10) Alloxan has shown to induce free radical production and cause tissue energy⁷ The pancreas is especially susceptible to the action of alloxan –induced free radical damage. Punica grantum aqueous peel extract possesses strong antioxidant property¹can act as free radical scavenger and protect ß cells from damage. Plant extract treated normal rats’ revealed significant reduction in blood sugar and elevation in insulin level. The number of ß cells increased. The mechanism of the antidiabetic activity shown by the extract is protection of pancreas, the mechanism of the antiatialetic activity shown by pomegranate agueaous stimulation of ß cells, increase number of ß cells and subsequent release of insulin. Also it may increase insulin receptor. In conclusion pomegranate peel aqueous extract can reduce blood sugar through regeneration of ß cells.

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Received on 30.08.2009 Modified on 28.10.2009

Research J. Pharm. and Tech. 3(1): Jan. - Mar. 2010; Page 272-274