It was an animal based experimental study conducted at Animal House of Faculty of Palamur Bioscience Private Limited for a period of one month (June 2018). The study was approved by Institutional Animal Ethics Committee (IAEC) Palamur Biosciences Private Limited (CPCSEA Registration Number -1312/PO/ReBiBt-S/09/CPCSEA). Animals were obtained from in house bred at Palamur Biosciences Pvt., Ltd.

Sample size: The sample size included total 30 albino Wistar rats weighing between 120-200 gm. These were divided in to five groups (T0, T1, T2, T3 and T4). Each group included six rats. Each group included three male and three female rats. All the animals were fed by standard rat pellet diet and were allowed for free access to water. The rats were housed in standard cages at a constant temperature (15^o–25^o C) with fixed 12:12 hour light-dark cycle.

Rats in all the groups were subjected to overnight fasting. Next day the rats in control group (T0) were subjected to intraperitoneal fresh normal saline (2 mL). Group T1, T2, T3 and T4 were the test group. Next day induction of diabetes mellitus was performed at different concentrations of STZ among T1,T2,T3 and T4 groups respectively with the doses of 45 mg/kg, 55 mg/kg, 65 mg/kg and 75 mg/kg intraperitoneally in one shot.

The STZ before use was dissolved in 0.1 M of freshly prepared sodium citrate buffer, pH 4.5, made isotonic by the addition of 0.25 M NaCl. Glucose water of 5% was given for two days to prevent drug induced hypoglycaemic shock. Body weight of the rats, blood glucose levels and C-peptide levels were noted after 7^th days, 14^th days, 21^st days and 28^th days of STZ injection. The blood sample was collected from retro-orbital plexus and centrifuged at 1000 RPM (Revolutions Per Minute) for 20 minutes and the serum (supernatant) was collected.

Blood glucose: It was estimated by semi-auto analyser using ERBA blood glucose kit. Method used was Trinder’s method [23]. Standard procedure as per the instruction manual was followed. Principle of this method was glucose in sample is oxidised to yield gluconic acid and hydrogen peroxide in the presence of glucose oxidase. The enzyme peroxidase catalyses the oxidative coupling of 4-aminoantipyrine with phenol to yield coloured quinonimine complex, with absorbance proportional to the concentration of glucose in sample.

C-peptide levels (C-P): It was estimated by Enzyme-linked Immunosorbent Assay (ELISA) test kit with catalogue number ITER0304 in an ELISA reader with the detection range of 0.07-5 ng/mL. Standard procedure as per the instruction manual was followed. Principle of this assay was based on sandwich ELISA technology [24]. The C-peptide antibody was precoated onto 96-well plates and the biotin conjugated anti C-peptide antibody.

The standards, test samples and biotin conjugated detection antibody was added to the wells subsequently, and washed with wash buffer. Horseradish peroxidase (HRP)-Streptavidin was added and unbound conjugates were washed away with wash buffer. The TMB substrates were added. 3,3′,5,5′-Tetramethylbenzidine or TMB was catalysed by HRP to produce a blue colour product that changed to yellow after adding acidic stop solution. The density of yellow was proportional to the C-peptide amount of sample captured in plate. Read the OD absorbance at 450 mm in a microplate reader, and then the concentration of C-peptide was measured.

The data has been entered in the excel sheet, column wise and row wise. After entering, the data has been analysed with Graph pad prism software version 6.3.1. The F statistics, one-way Analysis of Variance (ANOVA) was used to compare the different groups. Denny’s test was used to compare the control group versus different test groups. Statistical significance was tested for p-value <0.05.

The overall group analysis of body weights of all rats in different groups at variable time intervals were analysed with F statistics. The F-value of over group analysis of body weights of rats in different groups on 28^th day after STZ was 4.962 with p-value 0.0044. This was represented in [Table/Fig-1]. To conclude there was a significant difference (decrease) in the body weight between the control group T0 versus test group T3 and T4 on 28^th day after injection of STZ.

The overall group analysis of blood glucose levels of all rats in different groups at variable time intervals were analysed with F statistics. The F-value of over group analysis of blood glucose levels of rats in different groups on 7^th day after STZ was 4.369 with p-value=0.0081. The F-value of over group analysis of blood glucose of rats in different groups on 14^th day after STZ was 0.7327 with p-value=0.5783. The F-value of over group analysis of blood glucose of rats in different groups on 21^st day after STZ was 0.8841 with p-value=0.4877. The F-value of over group analysis of blood glucose of rats in different groups on 28^th day after STZ was 0.4064 with p-value=0.8023. This was represented in [Table/Fig-2]. To conclude there was a significant difference (increase) in the blood glucose levels between the control group T0 versus test group T3 and T4 on 7^th day after injection of STZ.

The overall group analysis of C-peptide levels of all rats in different groups at variable time intervals were analysed with F-statistics. The F-value of over group analysis of C-peptide levels of rats in different groups on 7^th day after STZ was 67.54 with p-value <0.0001. The F-value of over group analysis of C-peptide levels of rats in different groups on 14^th day after STZ was 59.62 with p-value <0.0001. The F-value of over group analysis of C-peptide levels of rats in different groups on 21^st day after STZ was 63.06 with p-value <0.0001. The F-value of over group analysis of body weights of rats in different groups on 28^th day after STZ was 86.31 with p-value <0. 0001. This was represented in [Table/Fig-3]. To conclude there was a significant difference (increase) in the C-peptide levels between the control group T0 versus test group T1, T2, T3 and T4 on 7^th, 14^th, 21^st and 28^th day after injection of STZ.

The present study showed that, there was a significant difference (decrease) in the body weight between the control group T0 versus test group T3 and T4 on 28^th day after injection of STZ. Studies have shown an association between increased glucose levels and decreased body weight of diabetic animals [25-28]. Streptozotocin was used in a dose of 45 mg/kg body weight while Mozaffari et al. (1997) [25] used 90 mg/kg body weight intraperitoneally in rats, Kang N et al., used 70 mg/kg body weight intravenously in rats and Oscika TM et al., used STZ in a dose of 50 mg/kg body weight intravenously in rats for producing hyperglycaemia [25-27]. The selection of lower dose was adopted as present strain of rats could not tolerate and survive with the dose used by previous investigators. The observations and results of the present study demonstrated that STZ was effective in producing persistent hyperglycaemia in experimental animals with a dose of 55 mg/kg body weight which co related with the finding of Greg H and Terri J in 2007 [28]. The animals treated with STZ in groups T3 and T4 appeared ill-looking with loss of their body weights because of injurious effects of STZ which caused alkylation of DNA and produced hyperglycaemia and necrotic lesions. Present observations are in agreement with the findings of Piyachaturawat P et al., Habibuddin M et al., concluded that the animals treated with STZ appeared very week with loss of their body weights because of injurious effects of STZ which caused alkylation of DNA and produced hyperglycaemia and necrotic lesions [29,30].

The present study explained the variations of blood glucose levels in with the doses of STZ at, a varied time intervals (7^th, 14^th, 21^st and 28^th day). The route of drug administration, right dose and proper technique are key points to produce a diabetic animal model as stated by Jain DK and Arya RK who compared the different doses and routes of administration for alloxan to achieve a perfect diabetic animal model [31]. Authors used the STZ diluted in buffer solutions with pH range 4.0-5 as per many studies, administered it intraperitoneally and glycaemic levels were checked after 7^th day [32,33]. It was observed in the present study that animals who received an intraperitoneal injection of STZ at 55 mg/kg dose developed a sustained hyperglycaemic profile as reported by Masashi K and Jerrold M [34]. Similar to the present study, Greg H and Terri J have also highlighted that the same dose of STZ i.e., 55 mg/kg produced hyperglycaemia without causing any harm to animals [28]. We found mortality rates in two groups T3 and T4 who received STZ 65 mg/kg and 75mg/kg, respectively. Contrary to the present results few studies have documented that single intraperitoneal injection of STZ at a dose of 100 mg/kg will produce immediate hyperglycaemia in animals without harming them [35,36]. In our study, we found high mortality rate in one group (T4) who received STZ 75 mg/kg. Zhang M et al., concluded their study by stating that 45 mg/kg dose of STZ will give a persistent diabetic profile [37], Whereas in the present study, author observed that few rats from group T1 which were intervened by 45 mg/kg dose of STZ become euglycaemic by 7^th day of intervention. This can be explained as the young animals have an increased threshold of sensitivity to the potentially toxic effects of STZ. In addition, it is likely that the aggressive fluid administration after STZ injection contributed to the low morbidity and absent mortality. The present study revealed that intraperitoneal injection of STZ induced changes including hyperglycaemia and deceased C-peptide levels with an obvious evidence of diabetogenesis The possible mechanisms for β-cells destruction by STZ was reported to induce generation of some types of oxygen free radicals and alteration of endogenous scavengers of these reactive species, fragmentation of DNA and the subsequent increase in the activity of Polymeric Adenosine Diphosphate Ribose (poly-ADP) ribose synthase (an enzyme known to deplete nicotinamide adenine dinucleotide in β-cells), inhibition of Adenosine Triphosphate (ATP) synthesis and islet mitochondrial respiratory enzymes [38]. Authors found 55 mg/kg weight of STZ was ideal for inducing diabetes in albino wistar rats suitable to the experimental surroundings.

The present study explains the variations in C-peptide levels with the doses of STZ at a varied time interval (7^th, 14^th, 21^st and 28^th day). The C-peptide is a product of pro-insulin cleavage; numerous studies have demonstrated that C-peptide, although not influencing blood glucose control, may play a role in preventing and potentially reversing some of the chronic complications of diabetes [38]. Increasing evidence suggests that declining ß-cell function in diabetes and the lack of C-peptide secretion might play a putative role in the development of microvascular abnormalities, which go beyond the effects of declining insulin secretion or increased blood glucose levels in diabetes [39]. In this study, authors found that C-peptide level was reduced significantly in STZ diabetic compared with normal rats group. These findings coincide with the results of Frost T et al., and Cong L and Chen J [40,41]. The STZ diabetic rats showed a highly significant decrease in serum C-peptide level as compared to normal rats. The diabetogenic agent STZ selectively destructs β-cells of the islets of Langerhans in the pancreas result in inhibition of insulin synthesis. This can be explained as C-Peptide is produced in β-cells in the pancreas, and secreted into the blood stream. So, when there is destruction of β-cells by STZ, the secretion of C-peptide decreases.

This study was conducted for a short duration. The follow-up after giving STZ was done for 28 days only. After administration of streptozotocin authors observed the hyperglycaemic profile of animals for 28 days. Future studies with longer duration of follow-up of more than 28 days was recommended to find out the exact time of insulin reversal after administration of STZ at 55 mg/kg dose. Furthermore, histopathologic examinations should be performed immediately after STZ administration and it should be repeated after the insulin reversal to analyse the recovery of β islets from STZ toxicity.

This study would help in deciding the accurate dose of STZ for induction of diabetes in wistar albino rats in our scenario. Administration of 55 mg/kg intraperitoneal dose of STZ created diabetic animal model with persistent hyperglycaemia. Dose of 75 mg/kg was found to be lethal to animals indicated by high mortality rate. STZ at 45 mg/kg dose was insufficient to create persistent diabetic profile in animal model. It may be concluded that the STZ through its direct alkylating action can cause selective destruction of the β-cells producing hyperglycaemia at a dose of 55 mg/kg body weight. It may also be stated that STZ by producing diabetes (hyperglycaemia) and decrease in C-peptide levels causes reduction in the body weight of diabetic animals. The present study has been designed to observe the effects of STZ-induced diabetes and its association between the reduction in the weights of animal.