On the basis of the hypoglycemic activity reported by plants belonging to the Asclepiadaceae family, the anti-diabetic activity of the aqueous extract of P. tomentosa aerial parts in rats affected by streptozotocin-induced diabetes has been evaluated. Diabetes mellitus is a complex and progressive metabolic disease characterized by chronic hyperglycemia. Pathogenesis of diabetes mellitus involves the generation of free radicals especially reactive oxygen species (ROS), glucose oxidation and lipid peroxidation. MDA is the well-known indicator of lipid peroxidation and oxidative stress. Moreover, it is well known that high levels of water intake, fasting blood glucose, with loss of body weights and polyuria are important indicators of diabetes [23].
Influence of an aqueous extract of P. tomentosa aerial parts on body weights and blood glucose level
Diabetic rats treated with an aqueous extract of P. tomentosa aerial parts showed improvement of body weight if compared to diabetic untreated control. Probably, the improvement of body weight could be associated to positive modification of blood sugar, which enhanced weight gain through successful glucose utilization [24, 25]. In addition, in diabetic rats, oral administration of P. tomentosa (Table 1) caused significant declines of the fasting blood glucose compared to untreated diabetic rats. The effects of P. tomentosa extract and water intake in diabetic rats on body weight and on blood glucose level are shown in Table 1. A significant difference between diabetic rats and diabetic rats treated with P. tomentosa has been observed. In particular, after injection of STZ, a significant loss (P < 0.001) in the final body weight if compared with initial body weight was observed. In diabetic group treated with P. tomentosa the final body weight was higher than the initial body weight. Moreover, the administration of P. tomentosa, at dose of 200 mg/kg to diabetic rats, caused significant (P < 0.01) reduction of blood glucose levels and water intake.
Influence of P. tomentosa aqueous extract on lipids blood level
Hypertriglyceridemia has been identified as a major risk factor for cardiovascular complications. In the present study an increase of triglycerides level after injection of STZ, was observed. The obtained results are in agreement with literature in fact the insulin action on lipoprotein metabolism is exerted mainly through the lipolysis increase of triglyceride rich lipoproteins by stimulating lipase and lipolysis prevention of fats stored in tissues by inhibition of hormones sensitive lipase [26]. Diabetic rats treated with the aqueous extract of P. tomentosa aerial parts displayed triglycerides levels not significantly different from the normal control. Furthermore a decrease of ALT and AST levels in serum of the diabetic rats treated with P. tomentosa extract, probably due to the compounds exerting free radical scavenging activity protecting liver cells against lipid-peroxidation, was observed (Table 2).
Triglyceride level of the diabetic group was significantly higher than the normal control, while the administration of P. tomentosa to STZ-treated diabetic rats, caused a significant reduction of blood triglyceride levels. HDL and LDL levels did not show evident significant differences between tested groups. Moreover, the MDA level was increased (P < 0.001) in the diabetic group and in diabetic rats treated with P. tomentosa if compared to control group (Table 2).
Histological assessment of pancreas, kidney and liver by Eaematoxylin and Eosin staining
To evaluate the effects of P. tomentosa aqueous extract on pancreas, kidney and liver of diabetic and control rats, the histological analysis of the above mentioned organs has been evaluated.
The pancreas cells of control rats showed the normal acinar cells which stained strongly and arranged in lobules with prominent nuclei. As shown in Fig. 1-IA, in control rats, the islet cells were embedded within the acinar cells and surrounded by capsule. The pancreas of the diabetic untreated group revealed a high level of cellular damage; in particular, diabetic rats revealed pathological changes of both exocrine and endocrine components. Islet β-cells were almost entirely lost in STZ-treated rats. Islets of Langerhans showed hyaline and necrotic changes (black arrow).
Wider interlobular and intralobular ducts were observed (Fig. 1-IB). P. tomentosa administration to diabetic rat demonstrated marked improvement of the cell injure, as evident from the partial restoration of Islets of Langerhans and exocrine components. (Fig. 1-1D). These observations showed that the extract could confer some protective effect on the pancreas, consequently improving glucose metabolism. This could be attributed to the antioxidant effect of some of the phytochemicals in P. tomentosa which prevent streptozotocin-induced free radical destruction of the pancreatic islets. The pancreas of control rats treated with P. tomentosa showed normal architecture of the pancreas (Fig. 1-IC).
The kidney section of control untreated group and P. tomentosa treated group showed normal architecture of glomerular capillary (blue arrow), glomerular tubule and urinary space (black arrow), with normal basement membrane and capillaries (Fig. 1-IIA and IIC). In diabetic rats (Fig. 1-IIB), kidney sections showed mild thickening of the basement membrane, atrophy of glomerular capillaries (blue arrow), with increased Bowman’s space (urinary space, black arrow). Diabetic rats treated with P. tomentosa showed features of healing, i.e., normal structure of basal membrane and glomerulus. Moreover, Bowman’s space was improved towards normal condition after treatment with P. tomentosa (Fig. 1-IID). Therefore, the kidneys of diabetic rats treated with P. tomentosa showed an improvement if compared to those of the diabetic untreated group. In detail, the extract slowed down the renal impairment associated with diabetes mellitus.
From the histopathology of the liver an improvement of P. tomentosa treated diabetic rats was observed. The liver of the diabetic untreated group showed evidence of congestion, inflammation and necrosis. These observations suggest steatosis of the liver and are evident of the toxic effects of STZ on the liver of rats [27]. In detail, treatment with P. tomentosa extract improved the hepatic architecture; moreover, as evidenced by the liver biochemical parameters, a hepatoprotective effect at P. tomentosa extract could be inferred. Liver of control rats (Fig. 1-IIIA) showed a preserved architecture with central vine (black arrow), hepatocytes and sinusoids (blue arrow). Moreover, normal sinusoids with flatted endothelial cells were seen (Fig. 1-IIIA and IIIC). The histology of liver sections obtained from diabetic rats showed loss of the normal architecture with congested central vein (black arrow), disarranged sinusoids (blue arrow), binucleated hepatocytes (red arrow) and more Kupffer cells (green arrow) (Fig. 1-IIIB). Liver sections of diabetic rats treated with P. tomentosa (Fig. 3D) showed almost normal liver histology with slight dilated in sinusoids, lesser degree of inflammation
LC-MS qualitative profile of aqueous extract of P. tomentosa aerial parts
In order to correlate the hypoglycemic activity to the chemical composition, the aqueous extract of P. tomentosa aerial parts, has been investigated by an analytical approach based on LC-ESI/LTQOrbitrap/MS/MSn, operating in the same conditions reported previously [14]. The analysis of LC-HRMS spectra allowed to assign both accurate molecular mass and molecular formula to the [M-H]− pseudomolecular ions occurring in the LC-MS profile. Identification of compounds has been performed on the basis of the retention times, the accurate masses and characteristic fragmentation patterns, and by comparison with literature data on P. tomentosa.
By this way, 23 metabolites could be identified. In particular, the LC-MS analysis of P. tomentosa extract suggested the occurrence of cardenolides (4, 8, 16–17, 21) duble-linked cardenolides (1–3, 5, 7, 12–15, 18–20, 22–23), and flavone glycosides (6, 9–11) (Fig. 2 and Table 3).
LC-MS analysis showed for some compounds the same pseudomolecular ions; this is the case of compounds 2, 7, 12 and 13 with a pseudomolecular ion [(M + HCOOH)-H]− at m/z 593, compounds 5 and 14 with a pseudomolecular ion [(M + HCOOH)-H]− at m/z 595, as well as compounds 19 and 20 with a pseudomolecular ion [(M + HCOOH)-H]− at m/z 577 (Table 3).
In order to unambiguously establish the molecular structure of compounds 1–23, and to discriminate among structural isomers or stereoisomers, a LC/MS analysis of naturally occurring standards, previously isolated from the aerial parts and roots of P. tomentosa, has been carried out.
LC-MS analysis highlighted the occurrence of different classes of compounds, mainly cardiac glycosides. These compounds represent a group of secondary metabolites that share the capacity to bind the extracellular surface of the main ion transport protein in the cell, the membrane inserted sodium pump (Na+/K+-ATPase) [13]. Earlier studies showed the hypoglycemic effect exerted by ouabain, a cardiac glycoside present in plants belonging to the Asclepiadaceae family; in particular this compound displayed a significant decrease in glucose and glycerol concentrations [28, 29].