Anti-diabetic activity of crude polysaccharide and rhamnose-enriched polysaccharide from G. lithophila on Streptozotocin (STZ)-induced in Wistar rats

The aim of the present study was to elucidate the anti-diabetic effects of the crude polysaccharide and rhamnose-enriched polysaccharide derived from G. lithophila on streptozotocin (STZ)-induced diabetic Wistar rats. Treatment with crude polysaccharide and rhamnose-enriched polysaccharide showed increases in body weight and pancreatic insulin levels and a decrease in blood glucose levels compared with control diabetic rats. The blood concentrations of total cholesterol (TC), triglycerides (TGs), low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) decreased, and high-density lipoprotein (HDL) increased both in the crude polysaccharide- and rhamnose-enriched polysaccharide-treated rats. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels increased, and malondialdehyde (MDA) levels decreased in the livers, kidneys and pancreases of crude polysaccharide- and rhamnose-enriched polysaccharide-treated rats. Immunohistological examination further confirmed that restoration of the normal cellular size of the islets of Langerhans and the rebirth of β-cells were found to be greater in the body region than in the head and tail regions of the pancreas. The crude polysaccharide- and rhamnose-enriched polysaccharide-treated diabetic rats showed normal blood glucose levels and insulin production, and reversed cholesterol levels and enzymatic actions. Therefore, rhamnose-enriched polysaccharide from G. lithophila acts as a potent anti-diabetic agent to treat diabetes and can lead to the development of an alternative medicine for diabetes in the future.

Body weight changes. Once weekly, the body weights were recorded for all the groups of experimental rats.
Blood glucose estimation. The fasting blood glucose level was determined in all experimental rats initially to determine the diabetic status and once a week thereafter throughout the study period. Blood was obtained by snipping the tails of the rats with the help of a sharp razor, and blood glucose levels were determined using a glucometer (Ultra One Touch, Johnson and Johnson).

Blood insulin estimation. Blood insulin was measured using the double-antibody method of Bailey and
Ahmed-Sorour 23 .

Estimation of the biochemical parameters. Estimation of triglycerides (TG).
The estimation was performed using a GPO kit (Sigma chemical, Maharastra, India) according to the following procedure 24 . The enzyme reagent (1 ml) was added to 10 µl of the sample. The mixture was shaken well and incubated at 37 °C for 10 min. The OD reading was taken at 546 nm using a spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). Estimation of total cholesterol (TC). The total cholesterol estimation was performed using a GPO kit according to the following procedure 24 . One millilitre of the enzyme reagent was added to 10 µl of sample. The mixture was shaken well and incubated at 47 °C for 5 min. The OD reading was noted at 505 nm using a spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan).
= × Concentration of Cholesterol (mg/dl) OD of the sample OD of the standard 200 (2) Estimation of HDL Cholesterol. The estimation was performed using a GPO kit according to the following procedure 24 . One millilitre of enzyme reagent was added to 10 µl of sample. The mixture was shaken well and incubated at 37 °C for 5 min. The OD reading was noted at 505 nm using a spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). Estimation of antioxidant markers. Estimation of superoxide dismutase (SOD). The activity of SOD in the liver and kidney tissues was determined according to the procedure of Kakkar 25 . Superoxide radicals react with nitroblue tetrazolium (NBT) in the presence of NADH to produce formazan blue. SOD removes the superoxide radicals and inhibits the formation of formazan blue. The intensity of the colour is inversely proportional to the activity of the enzyme. One unit of enzymatic activity is defined as the enzyme reaction that gives 50% inhibition of NBT reduction in one minute under the assay conditions and is expressed as the specific activity in units/mg protein.
Method for histology. The pancreas was removed soon after the dissection of the experimental rats and promptly fixed in 10% formalin for histological investigation. The formalin-fixed pancreases were minced to 5 mm to randomize the perception zone and inserted in paraffin. Staining was performed over the paraffin areas with haematoxylin and eosin. Immunohistochemistry was executed according to the method of Kakimoto et al. 29 . Briefly, pancreas paraffin areas were deparaffinized. For insulin immunostaining, the sections were incubated at 4 °C with a primary rabbit polyclonal anti-insulin antibody (Elabscience, India) followed by an HRP-conjugated secondary antibody. Stanning was performed on the segments with 3,3′-diaminobenzidine. Then, images of the areas were captured with an Olympus DP73 advanced camera framework and cellSens Software furnished with an Olympus BX51 magnifying instrument.
Quantification of the beta-cell area in the intact pancreas. As per the procedure of Kakimoto et al. 29 .
The entire pancreas was cut into serial 5 μm thick sections spaced 150 μm apart. For analysis, every 30 th section was stained. Image processing is a reliable and reproducible method of analysing pancreatic beta cells in islet biology and is equivalent to conventional morphometric analysis. This method is cost effective for analysing the immunohistochemical index. As per the procedure given for the quantification of beta cells by Golson et al. 30 , a similar method of quantification was used in our study to quantify the accumulation of beta cell mass and size using Fiji/ImageJ with Java Plugin, the open access software developed by the NIH, USA, and the obtained data were interpreted with MATLAB. The detailed steps involved in image processing were adapted from Golson et al. 30 . The longitudinal section of rat pancreas with head, body and tail regions was examined in multiple fields of focus to avoid any error in the surface measurement, and the total mass of the beta cells was extracted for the analysis.
Statistical analysis. All experimental data were subjected to one-way analysis of variance (ANOVA), and Dunnett's multiple comparison test (GraphPad Software, USA) was also used to determine the difference among means at the level of p < 0.05. The data obtained from the beta cells of the pancreas were evaluated using MATLAB (version R2017a, Mathworks, MA, USA).
Ethics approval. All procedures performed in this research work were carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India and approved by the Animal Ethical Committee of Annamalai University (No.343; dated 7.9.12). There were no human samples involved in this study.

Results
Acute toxicity study. Oral administration of the crude polysaccharide and rhamnose-enriched polysaccharide of G. lithophila was found to be safe up to the p.o. dose level 2000 mg/kg, which showed no signs of toxicity. Furthermore, doses up to 2000 mg/kg crude and rhamnose-enriched polysaccharides did not show any negative behavioural changes to motor activity. No mortality was observed up to 14 days. Hence, the median lethal dose (LD 50 ) of crude polysaccharide and rhamnose-enriched polysaccharide from G. lithophila was determined to be more than 2000 mg/kg body weight ( www.nature.com/scientificreports www.nature.com/scientificreports/ control (group II) rats expanded less body weight than the normal control (group I) rats (p < 0.01). Both the crude polysaccharide-and rhamnose-enriched polysaccharide-treated groups (IV and V) rat body weights significantly increased, (p < 0.01) as did the xiaoke pill-treated group (III) rats compared to the diabetic control rats (group II) ( Table 2).
Changes in the blood glucose levels in diabetic rats after crude polysaccharide and rhamnose-enriched polysaccharide treatment. On the first day of the experiment, the diabetes-induced groups of rats (II, III, IV and V) showed increased blood glucose levels compared to the control rats (group I) (see Table 3). After 30 days of the experiment, diabetic STZ (group II; 427 ± 4.33 mg/dl) rats exhibited increased blood glucose levels compared with normal control rats (group I; 97 ± 0.59 mg/dl). The crude polysaccharide-and rhamnose-enriched polysaccharide-treated rats (group IV and V; 124 ± 1.05 and 117 ± 0.94 mg/dl, respectively) showed reduced blood glucose concentrations compared to the diabetic control (group II) rats. Moreover, in the standard drug xiaoke pill-treated (group III; 109 ± 1.83 mg/dl) rats, the blood glucose concentration decreased significantly (p < 0.01) after 30 days of the experiment, which was similar between the crude polysaccharide (group IV; 124 ± 1.05 mg/dl) and rhamnose-enriched polysaccharide (group V; 117 ± 0.94 mg/dl)-treated groups of rats.

Administration/ Treatment
Changes in the histopathology of the pancreas. The control rats (group I) showed no pathological changes in the pancreatic cellular architecture. The diabetic control (group II) rats showed moderate to marked diffused necrotic changes in the pancreas as a result of the significant reduction in the number of β-cells in the islets of Langerhans. The positive control, the xiaoke pill-treated (group III) rats, showed a slight development of islets of  Table 6. Effects of crude polysaccharide and rhamnose enriched polysaccharide on blood, liver and kidney (MDA, GPx and SOD) level in STZ diabetic rats. a,b,c,d&e comparisons were made between Groups I, II, III, IV and V.  www.nature.com/scientificreports www.nature.com/scientificreports/ Langerhans along with β-cells. The crude polysaccharide-treated rats (group IV) showed an improvement in islets of Langerhans and specific improvement in the β-cells, and rhamnose-enriched polysaccharide-treated (group V) rats showed significant regeneration of β-cells when compared to the diabetic control (Fig. 6).

Quantification of beta cells.
To measure the in situ beta cell zone of the rat pancreas for all experimental groups of rats, an entire pancreas was cut as a virtual cut with high magnification to evaluate the whole dissemination of beta cells in situ for analysis. Macro-plugin was used in Fiji/ImageJ for the size distribution from the pancreatic images. The images with beta cells were segmented for the surface measurement, total area, circularity and Feret's diameter and then evaluated. The images were physically checked the region of interest (ROI) produced via the programmed threshold was revised to ensure ROI displays of only the beta cells region. Then, the examinations were undertaken, and each islet area was traced manually to calculate the surface measurement.
Dynamic changes of cell arrangement throughout the islet measurements were observed for each experimental group. The immunostaining image of the rat pancreas and proliferation of the beta cells in the experimental group are shown in Fig. 7a-o. As per Olehink et al. 19 60% of beta cells in the total endocrine surface are <100 μm in diameter. Similarly, most of the islet areas covered <100 to 150 μm, as shown in Fig. 7a-e (group I) shows the exception, where the healthy pancreas beta cells are distributed for up to 600 μm of the pancreatic body region. Three-dimensional scatter plots show the islet area, circular shape and Feret's diameter. There were significant changes found in group III (positive control), with the accumulation of beta cells found in the body region (Fig. 7a-e) compared with the head (Fig. 7f-j) and tail regions (Fig. 7k-o) of the pancreas. Groups IV and V also showed high proliferation rates but were not equivalent to group III. There were few beta cells observed in the STZ-treated rat pancreas, i.e., group II, compared to all the diabetic rats. The control group (group I) rat pancreas consisted of a high number of beta cells with intact islet areas. Overall, the distribution of beta cells from the islet mass shows that the greater accumulation in the body region gradually decreases to the tail region, although there is some fluctuation.

Discussion
Streptozotocin is one of the most commonly used substances to induce diabetes in rats. This toxin causes the death of pancreatic cells by alkylation of DNA, resulting in reduced DNA synthesis and release of insulin 29 . STZ administration of more than 40 mg/kg to rats is capable of destroying pancreatic β-cells and causing the development of constant hyperglycaemia, a situation parallel to diabetes in humans 29,30 . In the present study, administration of STZ at 55 mg/kg to rats was found to be capable of increasing blood glucose levels on the 3 rd day of the experimental period, which may be due to the destruction of pancreatic islets and the death of β-cells. In    diabetes mellitus, body cells are unable to utilize glucose as a source of energy and proteins are instead used as energy sources. This leads to a decrease in protein storage and reduces body weight 31 . In the present study, streptozotocin-induced diabetic rats showed a decrease in body weight throughout the experimental period. Oral treatment with crude polysaccharide and rhamnose-enriched polysaccharide from G. lithophila to diabetic rats for 30 days resulted in a significant improvement in body weight compared to the control rats.
Diabetic mellitus is mainly associated with multiple metabolic disorders, amongst which lipid metabolism is relatively affected by high concentrations of triglycerides and total cholesterol and low levels of HDL cholesterol, as found in many cases 32 . The concentrations of TC, TGs and LDL cholesterol rise, and HDL cholesterol levels decrease in the blood, contributing to secondary complications of diabetes 32 . In the present study, the lipid profile of diabetic rats changed to show increased levels of TC, TGs, LDL cholesterol and VLDL cholesterol and decreased HDL cholesterol levels. Treatment with crude polysaccharide and rhamnose-enriched polysaccharide resulted in an elevated lipid profile, such as total cholesterol (TC), triglycerides (TGs) and LDL-cholesterol, similar to the reference level, and significantly increased the concentration of HDL-cholesterol. This finding was desirable in this study. These results may be associated with the possible protective effect of the polysaccharides to pancreatic β-cells. In addition, these effects may be due to the low activity of cholesterol biosynthetic enzymes or the insignificant level of lipolysis under the control of insulin 33 . Therefore, glycol metabolism and lipid metabolism may improve the renovation of β-cells and increase insulin secretion 34 .
In diabetes, hyperglycaemia results in the generation of free radicals, which weakens the body's defence mechanisms, thus leading to the disruption of cellular functions, oxidative damage to membranes and an enhanced susceptibility to lipid peroxidation 35 . Increased glycated Cu-and Zn-SOD activity have been reported in severe diabetes 36 . SOD and GPx are important enzymatic antioxidants that could be helpful in preventing lipid peroxidation and are involved in cellular defence mechanisms, thus protecting tissues against oxidative damage 37 . Under hyperglycaemic conditions, antioxidants are thought to regenerate the damaged extracellular matrix proteins and cell growth 35 . In addition, an increase in pancreatic MDA content has been reported previously in hyperglycaemic rats 37 .
Further, additional stimulation of MDA is associated with increased serum glucose levels and the destruction of ROS. Glucose possibly increases the formation of ROS through the non-enzymatic glycation of proteins, as well as through auto-oxidation 38 . The oxidative degradation of fructosamines may also contribute to oxidative stress in hyperglycaemia 36 . In our study, a significant increase in MDA levels and a reduction in SOD and GPx in the serum, livers and kidneys of STZ-induced diabetic rats was seen. However, the treatment of rats with crude polysaccharide and rhamnose-enriched polysaccharide lowered MDA levels and restored SOD and GPx activities to near normal levels. Similar results were observed with Achyranthes bidentata polysaccharides, which increased MDA concentrations and reduced other antioxidant enzymes, such as SOD and GPx, in the blood, liver and kidney tissues of STZ-induced diabetic rats 34 .
The enhanced activity of SOD and GPx in the present study may contribute to the potential effect of rhamnose-enriched polysaccharides on the scavenging of oxygen free radicals. Pancreatic β-cells may be protected from the oxidative damage induced by STZ. Therefore, it could be assumed that rhamnose-enriched polysaccharide has a direct protective effect against diabetes by decreasing the oxidative stress and preserving pancreatic β-cell damage, and this may be due to the reaction of rhamnose-enriched polysaccharide, which improves glycometabolism. In the present study, the pancreases of STZ-induced rats showed a significantly reduced size and number of β-cells in the islets of Langerhans after 30 days. Furthermore, the pancreases of rhamnose-enriched polysaccharide-treated rats showed the restoration of normal cell size of the islets of Langerhans and regenerated β-cells.
Chronic hyperglycaemia has deleterious effects on pancreatic β-cells by inducing oxidative stress 39 , which has been found to be more problematic than in other cells because β-cells possess the lowest intrinsic antioxidant defences 40 . In the present study, treatment with crude polysaccharide and rhamnose-enriched polysaccharide from G. lithophila to diabetic rats may prevent the development of diabetes by reducing glucose concentration. However, no significant differences were observed between the crude polysaccharide-and rhamnose-enriched polysaccharide-treated groups. The possible reasons may be that crude and rhamnose-enriched polysaccharides increased insulin release through antioxidant activity or the regeneration of β-cells 38 . Pancreatic β-cells are highly specialized cells responsible for producing the insulin required by an organism to maintain glucose homeostasis. Defects in the development, maintenance or expansion of β-cell mass can result in the impairment of glucose metabolism and diabetes 41 .
In the present study, STZ-induced rats showed a reduction in insulin levels in the serum compared to the control group. Treatment with crude polysaccharide and rhamnose-enriched polysaccharide significantly increased insulin levels in STZ rats compared to the diabetic control. The xiaoke pill-treated group rats also showed similar insulin levels that were on par with the rhamnose-enriched polysaccharide-treated group of rats. This strengthens the insulin-mimicking effect of rhamnose-enriched polysaccharides by regenerating insulin-producing cells.
The area of the beta cells was found in a limited number in the negative control (group II) rats with a depleted number of beta cells; all these areas were manually quantified in threshold images for their particle size. Furthermore, manual tracing of these regions was labour-intensive due to the irregularly shaped islets and beta cells, as has been observed in previous studies as well 41 . The present study shows that the expansion in beta cell mass is mainly due to the increase in beta cell replication, irrespective of the progenitors of beta cells that exist in the mature pancreas. In this study, mechanical methods were used for evaluating the islet region and insulin-positive region with beta cells in the insulin immunostained pancreas using Fiji/ImageJ software (Fig. 7). MATLAB simulation has been developed to ideally quantify the islet region from pancreatic areas (head, body and tail). The role of image investigation is to convert the subjective histological observations to quantitative features. The improvement of computerized vision has allowed the entire slide access with high resolution and has allowed for more tissues to be examined. This method of evaluation provides reliable information and is (2020) 10:556 | https://doi.org/10.1038/s41598-020-57486-w www.nature.com/scientificreports www.nature.com/scientificreports/ widely used in biomedical image analysis 19,41 . Overall, the distribution of beta cells from the islets of Langerhans shows greater accumulation in the body region and gradually decreases in the tail region. However, other studies conducted in the human pancreas have shown that more beta cells were present in the tail region of diabetes patients 41 .
The insulinogenic effect of various medicinal plant extracts resulting in the activation of β-cells has been reported 42,43 . The possible mechanism through which they exert their anti-hyperglycaemic effects might have be due to the increased release of insulin from β-cells or through the regeneration of β-cells. Under these circumstances, a number of other plants have also been reported to have anti-hyperglycaemic activity with a stimulatory effect on insulin release 37 .

conclusions
The present study suggested that rhamnose-enriched polysaccharides from G. lithophila could be used as an anti-diabetic agent due to the insulin-mimetic effects to reverse the blood glucose concentration, restore insulin production, improve cholesterolemic and enzymatic action and regenerate β-cells. Therefore, the rhamnose-enriched polysaccharide from G. lithophila can act as a potent anti-diabetic agent to treat diabetic patients, which will help future research working on the formulation of an alternative medicine for diabetes.
Limitations of the study. The effects of rhamnose-enriched polysaccharides in STZ-induced diabetic rats were studied in this work, but the mechanism of action at the molecular level is required to examine their role. A molecular mechanism study is therefore required to determine the improvement of dyslipidaemic conditions in diabetes for further drug discovery. Furthermore, beta cell accumulation was found to be high in the body region of the pancreas in our study. Compared to this result, beta cells originated more in the body and tail region of the human pancreas (Freeby et al. 44 ); however, Rahier et al. 45 reported a greater accumulation of beta cells in the posterior head region of the human pancreas. Therefore, the role of rhamnose-enriched polysaccharides in the increment of beta cells in the body region of the rat pancreas needs to be studied further.

Data availability
All data generated or analyzed during this study are included in this published article.