Protective role of Phyllanthusfraternus in alloxan-induced diabetes in rats

Abstract

Background: Phyllanthusfraternus is a pantropical weed of family phyllanthaceae, mainly found in northeast India. It has been used in the folklore medicine of Manipur tribe for treating type 2 diabetes.

Objective: The present study was commenced to evaluate the anti-diabetic and renoprotective potential of P.fraternus (aerial parts) in alloxan-induced diabetes in rats.

Materials and methods: Alloxan (130 mg/kg, ip) was used for the induction of diabetes in adult male wistar rats. Animals with blood glucose level greater than 280 mg/dL were treated once daily for 14 days with various test extracts. The biochemical parameters were measured from serum on the 15th day post-treatment. Necropsy samples harvested from pancreas and kidneys were examined for histopathological changes in these organs.

Results: Alloxan-induced diabetes not only caused significant increases in blood glucose, triglycerides, total cholesterol, creatinine and urea levels, but also provoked high oxidative stress in pancreas and kidneys. Profound morphological injuries were observed in islets of Langerhans and kidneys of diabetic animals. Administration of methanol extract (200 and 400 mg/kg) and mother liquor (200 and 400 mg/kg) ameliorate the elevated levels of blood glucose, triglycerides, total cholesterol as well as other biochemical parameters, but highest reduction in blood glucose concentration was observed with the largest dose of ethyl acetate fraction (400 mg/kg) of P.fraternus. Histopathological examination of pancreas and kidneys also exhibited greater protection by treatment with acetate fraction (400 mg/kg). The HPLC analysis showed the presence of four polyphenols such as catechin, gallic acid, caffeic acid and ellagic acid in ethyl acetate fraction of P. fraternus during HPLC analysis.

Conclusion: The results suggest that polyphenols present in P.fraternus may be responsible for the anti-diabetic and renoprotective activity in rats. Such protective effects of could be mediated through flavonol-induced anti-oxidant and anti-inflammatory activities in the pancreas and kidneys.

Keywords: Antidiabetic; Antioxidant; Phyllanthusfraternus; Polyphenols.

Graphical abstract

Fig. 1

(A) Showing effects of methanol extract, ethyl acetate fraction and mother liquor on the blood glucose levels. Values are expressed as mean ± S.E.M (N = 6). P^a < 0.05 Vs. normal control; p^b < 0.05 Vs. diabetic; p^c < 0.05 Vs. positive control; p^d < 0.05 Vs. MeOH (200 mg/kg); p^e < 0.05 Vs. MeOH (400 mg/kg); p^f < 0.05 Vs. ethyl acetate fraction (400 mg/kg); p^g < 0.05 Vs. mother liquor (200 mg/kg) (B): Showing effects of methanol extract, ethyl acetate fraction and mother liquor on the blood triglyceride levels. Values are expressed as mean ± S.E.M (N = 6). P^a < 0.05 Vs. normal control; p^b < 0.05 Vs diabetic; p^c < 0.05 Vs. positive control; p^d < 0.05 Vs. MeOH extract (200 mg/kg); p^e < 0.05 Vs. MeOH extract (400 mg/kg); p^f < 0.05 Vs. ethyl acetate fraction (100 mg/kg): p^g < ethyl acetate fraction (200 mg/kg) (C): Showing effects of methanol extract, ethyl acetate fraction and mother liquor on the blood cholesterol levels. Values are expressed as mean ± S.E.M (N = 6). P^a < 0.05 Vs. normal control; p^b < 0.05 Vs. diabetic; p^c < 0.05 Vs. positive control (D): Showing effects of methanol extract, ethyl acetate fraction and mother liquor on the blood urea levels. Values are expressed as mean ± S.E.M (N = 6). P^a < 0.05 Vs. normal control; p^b < 0.05 Vs. diabetic; p^c.<0.05 Vs. positive control; p^d < 0.05 Vs. MeOH extract (200 mg/kg); p^e < 0.05 Vs. MeOH extract (400 mg/kg) (E): Showing effects of methanol extract, ethyl acetate fraction and mother liquor on the creatinine levels. Values are expressed as mean ± S.E.M (N = 6). P^a < 0.05 Vs. normal control; p^b < 0.05 Vs. diabetic; p^c < 0.05 Vs. positive control; p^d < 0.05 Vs. MeOH extract (200 mg/kg); p^e < 0.05 Vs. MeOH extract (400 mg/kg).

Fig. 2

(A) Showing effects of methanol extract, ethyl acetate fraction and mother liquor on the TBARS levels in pancreatic tissue. Values are expressed as mean ± S.E.M (N = 6). P^a < 0.05 Vs. normal control; p^b < 0.05 Vs diabetic; p^c < 0.05 Vs. positive control; p^d < 0.05 Vs. MeOH (200 mg/kg); p^e < 0.05 Vs. MeOH (400 mg/kg); p^f < 0.05 Vs. ethyl acetate fraction (100 mg/kg); p^g < ethyl acetate fraction (200 mg/kg); p^h < 0.05 Vs. ethyl acetate fraction (400 mg/kg) (B): Showing effects of methanol extract, ethyl acetate fraction and mother liquor on the TBARS levels in kidney tissues. Values are expressed as mean ± S.E.M (N = 6). P^a<0.05 Vs normal; p^b < 0.05 Vs diabetic; p^c < 0.05 Vs. positive control; p^d < 0.05 Vs MeOH (200 mg/kg); p^e < 0.05 Vs. MeOH (400 mg/kg); p^f < 0.05 Vs. ethyl acetate fraction (100 mg/kg); p^g < ethyl acetate fraction (200 mg/kg) (C): Showing effects of methanol extract, ethyl acetate fraction and mother liquor on the GSH levels in pancreatic tissue. Values are expressed as mean ± S.E.M (N = 6). P^a < 0.05 Vs normal control; p^b < 0.05 Vs diabetic; p^c < 0.05 Vs. positive control; p^d < 0.05 Vs. MeOH (200 mg/kg); p^e < 0.05 Vs. MeOH (400 mg/kg); p^f < 0.05 Vs. ethyl acetate fraction (100 mg/kg) (D): Showing effects of methanol extract, ethyl acetate fraction and mother liquor on the GSH levels in kidney tissue. Values are expressed as mean ± S.E.M (N = 6). P^a < 0.05 Vs. diabetic; p^b < 0.05 Vs. diabetic; p^c < 0.05 Vs. positive control; p^d < 0.05 Vs. MeOH (200 mg/kg); p^e < 0.05 Vs. MeOH (400 mg/kg); p^f < 0.05 Vs. ethyl acetate fraction (100 mg/kg) (E): Showing effects of methanol extract, ethyl acetate fraction and mother liquor on SAG levels. Values are expressed as mean ± S.E.M (N = 6). P^a < 0.05 Vs normal; p^b < 0.05 Vs diabetic; p^c < 0.05 Vs. positive control; p^d < 0.05 Vs. MeOH (200 mg/kg); p^e < 0.05 Vs MeOH (400 mg/kg); p^f < 0.05 Vs. ethyl acetate fraction (100 mg/kg); p^g < ethyl acetate fraction (200 mg/kg); p^h < 0.05 Vs ethyl acetate fraction (400 mg/kg); pⁱ < 0.05 Vs. mother liquor (200 mg/kg).

Fig. 3

(A) Pancreatic tissue section is unremarkable and shows normal islets of Langerhans in normal control group (H&E; 100X) (B) Photomicrograph shows degenerated islets of Langerhans replaced by fibrosis in diabetic control group (H&E; 100X) (C) Photomicrograph shows normal islets of Langerhans and features like binucleation and eosinophilia in positive control (H&E; 100X) (D) Section showing fibrotic islets of Langerhans in MeOH extract (200 mg/kg) treated group (E) Section showing mildly preserved islets of Langerhans in MeOH extract (400 mg/kg) treated group (H&E; 100X) (F and G) Photomicrographs showing degeneration of islets of Langerhans and lymphocytic infiltration in ethyl acetate fraction (100 and 200 mg/kg) treated groups (H&E; 100X) (H) Section showing preserved integrity of islets of Langerhans and hyperplasia of islet cells, exhibiting protective response to large dose of ethyl acetate fraction (400 mg/kg) (H&E; 100X) (I and J) Photomicrographs showing degenerated islets of Langerhans in mother liquor (200 and 400 mg/kg) treated groups (H&E; 100X).

Fig. 4

(A) Photomicrograph of kidney section showing normal glomeruli, tubules and vessels in normal control group (H&E; 100X) (B) Kidney section showing tubular changes, thickening of glomerular membrane and mesangial hypercellularity in diabetic control group (H&E; 100X) (C) Kidney section showing normal glomeruli in positive control group (H&E; 100X) (D) Kidney section showing mild inflammation in MeOH extract (200 mg/kg) treated group (H&E; 100X) (E) Kidney section showing interstitial inflammation and fibrosis in MeOH extract (400 mg/kg) treated group (H&E; 100X) (F and G) Kidney sections showing mild inflammation in ethyl acetate fraction (100 and 200 mg/kg) treated groups (H&E; 100X) (H) Photomicrograph showing kidney protection (i.e. normal glomerulus, tubules and blood vessels). in diabetic rats treated with ethyl acetate fraction (400 mg/kg) of Phyllanthusfraternus (H&E; 100X) (I and J) Kidney sections showing mild inflammatory changes in mother liquor (200 and 400 mg/kg) treated groups (H&E; 100X).