Characterization of the isolated fraction
To isolate the final fraction, the extract from M. charantia fruit was filtered and chromatographed according to our previous method . The FT-IR spectrum of the polysaccharide is shown in Figure 1A. The spectrum of the polysaccharide indicated the absorption peaks of C-H symmetric and asymmetric stretching vibrations at 2926 and 2854 cm−1, respectively. The peak at 1745 cm−1 was characteristic to the C=O stretching vibration of the ester and the peaks at 1143 and 1051 cm−1 were related to C-O stretching vibration of the ester. Also, the peak at 1665 cm−1 was a feature of C=O stretching vibration and the peaks at 1103, 1051 and 1018 cm−1 were a feature of C-O stretching vibration of the acid carboxylic. The broad peak at about 2500-3600 cm−1 has corresponded to OH stretching vibration. The presence of these peaks in the FT-IR spectrum confirmed the stretchers of polysaccharide. As indicated in the Fig. 1B, the mass spectrum of isolated fraction shows that the percentage of fragments was about 936.0 (3.5), 772.3 (7.1), 745.3 (16.6), 710.7 (8.2), 597.1 (7.1), 555.0 (11.7), 460.3 (11.7), 408.1 (11.7), 366.1 (47.1), 275.0 (29.4), 255.1 (30.5), 193.0 (17.6), 175.1 (25.9), 171.1 (100.0), 113.0 (97.6). As shown in Fig. 1C, the isolated fraction has a carbohydrate pattern which is very similar to the structure of an isolated polysaccharide from Rosa canina.
Isolated fraction improved the function of pancreas
As indicated in table 2, the level of blood glucose was increased in diabetic rats compared to healthy ones and fraction treatment significantly decreased the blood sugar in diabetic rats (P≤0.05). In addition, loss of weight was obvious in diabetic rats (P≤0.05) compared to normal animals. However, diabetic rats treated with fraction gained weight over the course of 6 weeks (P≤0.05). Owing to the causative role of insulin in normal function of the islets, the protein level of insulin was evaluated in the pancreas tissue. In animals treated with fraction, the expression level of insulin was remarkably increased compared to untreated diabetic ones (Fig. 2A). In addition, measurement of mRNA expression showed that insulin and Pdx-1 gene expressions were upregulated in fraction treated rats with respect to diabetic ones (P≤0.05) (Fig. 2B).
Effect of isolated polysaccharide on the mRNA expression of Notch signaling pathway
The mRNA expression of Notch 1 was increased to about 2.24 fold in untreated and metformin-treated diabetic rats and increased to above more than 1.64 fold in MCP-treated diabetic rats compared to normal group (Fig. 3A). However, the expression of hes1 as a downstream signaling factor of notch signaling pathway was not altered in diabetic group (1.07) while increased about 1.22 and 1.38 fold in metformin and MCP-diabetic rats. Considering the disconcertion between Notch1 and hes1, the expression of two ligands, DLL4 and Jagged1, were studied. As shown in Fig. 3A, the mRNA expression of Jagged1 was increased to about 2.05, 3.7 and 2.4, respectively, in non-treated, MCP- treated and metformin-treated diabetic rats compared to normal rats. Albeit, the expression of DLL4 was increased to nearly 1.32 fold in diabetic group while it was decreased to 0.72 and 0.88 fold, respectively, in MCP and metformin groups in comparison with healthy rats. In follow, the protein expression of hes1 and cyclin d1 was examined in the pancreas of rats. The expression levels of insulin, Hes1, cyclin d1 were evaluated by IHC in the pancreas tissues of treated and untreated diabetic rats. As shown in Fig. 3B, the expression of cyclin d1 was about 20%, 15%, 30% and 55%, respectively, in normal, diabetic, metformin and MC-treated diabetic rats. The expression of Hes1 was cytoplasmic in acinar cells of diabetic rats while it was positive in the cytoplasm and nucleus of acinar and islet cells from metformin and MCP-treated diabetic and normal rats. The ratio of Bax/Bcl2 is considered as the indicator of cell death. Owing to the significant change in the expression of cyclin d1, it was prompted to measure the expression of Bax and Bcl2 as two factors involved in cellular survival and death. As shown in Fig. 3C, the levels of Bax and also Bax/Bcl2 ratio was enhanced in diabetic rats compared to the healthy group. However, MC and metformin treatment reduced the expression level of Bax and also the ration of Bax/Bcl2 relative to diabetic rats.
Effect of isolated polysaccharide on the mRNA expression of VEGF, CD31 and CD34
Owing to the prominent role of angiogenesis and vascular system in diabetes, the expression of VEGF, CD31 and CD34 were assessed in the pancreas from diabetic rats. The expression of CD31 was negative in normal and diabetic rats and was positive in the vascular walls in metformin and MC-treated diabetic ones. The CD34 in vascular walls was strongly stained in normal, metformin and MC-treated diabetic rats and was poorly stained in diabetic rats. The VEGF expression was 3+ in acinar cells of normal rats. In untreated, and metformin-treated diabetic rats, the level of VEGF was 2+ and 3+ in islet and acinar cells, respectively. The expression of VEGF in MC-treated diabetic rats was 2+, 1+ and 3+ in islet, stromal and acinar cells, respectively (Fig. 4).