Streptozotocin and Nicotinamide was purchased from Himedia, Mumbai, Glibenclamide gift sample was obtained from Cipla, Mumbai, and ethanol employed to extract the samples was obtained from Pure Chemicals Co from Chennai. Other substances and reagents utilised in this work were of analytical grade, and the enzymatic kits were purchased commercially.

In May, the leaves of the C. rotang L plant were collected in Kerala's Malappuram District. Prof. P. Jayaraman, Director of the National Institute of Herbal Science in Chennai, recognised the plant, and the Pharmacognosy Department Herbarium retained a voucher specimen (Ref. No. PARC/2012/2178).

The leaf 500 gm was pulverised in a fireproof blender then extracted using ethanol in a soxhlet apparatus. The extract was concentrated and dried in a vacuum evaporator (S. K. Appliances, Haryana. Model: DRV60). The dried residue 18 gm was kept in desiccator and utilised in subsequent experiments.

The dried extract was examined for the presence of various phytoconstituents. 24

Wistar albino rats, either sex, weighing between 150 to 200 gm were used in the experiment. 25 Under standard laboratory conditions of temperature (22 ± 2˚C) and humidity (45 ± 5˚C), the animals were housed in a 12 hour day/12 hour night cycle. The animals were fed a conventional laboratory diet and had access to water at all times. All experimental protocols were approved (JKKMMRFCP/IACE/2012/007) by the Institutional Animal Ethics Committee (IAEC), and all animal research followed the principles and guidelines of India's CPCSEA (Committee for the Purpose of Control and Supervision of Experimentation on Animals).

The standards 425 of the Organisation for Economic Cooperation and Development (OECD) were used to perform oral toxicity (acute) research. 26

To investigate the hypoglycaemic potential of the ethanol extract of C. rotang L leaves (EECR) in normal rats, fasting blood glucose (FBG) and oral glucose tolerance test (OGTT) were done at doses of 100 and 200 mg/kg administered orally by gavage. In diabetic rats, the antihyperglycemic effect of the EECR was also tested at dosages of 100 and 200 mg/kg.

The experiment utilized 24 healthy rats that had fasted over night and were divided into four groups of six animals each.

Group I: Normal control + distilled water.

Group II: Normal rats +100 mg/kg EECR.

Group III: Normal rats + 200 mg/kg EECR.

Group IV: Normal rats + 5 mg/kg Glibenclamide.

A single dosage of extract was given to each of the experimental animal groups. After the extract was administered, blood samples were taken from the tail vein at 2, 4, 6, and 8 hours, followed by FBG.

A second group of 24 healthy rats was separated and treated similarly to the first group. After measuring the animal’s FBG levels, they were given oral glucose (2g/kg) 90 minutes later. Blood samples were taken from the tail vein just before (0h) and 1^st, 2^nd and 3^rd h subsequently following glucose administration and blood glucose levels were calculated. 27

The animal model for diabetes mellitus type 2 (NIDDM) was created by administering STZ 60 mg/kg  intraperitoneally, succeeded by NC 120 mg/kg 15 minutes later. Hyperglycemia was diagnosed when blood glucose levels were elevated 72 hours after the injection and again on day 7. In the anti-diabetic investigation, only rats with verified persistent NIDDM were employed. 28, 29

On severely diabetic rats, a 28-day treatment was done. Thirty rats were split into five groups, each having six rats.

Group I: Normal control + distilled water,

Group II: Diabetic control + distilled water,

Group III: Diabetic + EECR (100mg/kg),

Group IV: Diabetic + EECR (200mg/kg),

Group V: Diabetic + Glibenclamide (5mg/kg).

Separate suspensions of EECR and glibenclamide were made in 2 % gum acacia. Each dose was given orally through oral gavage on a daily basis for 28 days, based on body weight. In overnight starved animals, body weights and blood glucose levels were assessed weekly using blood glucose test strips and an ACCU CHEK glucometer. The animals were starved overnight after the experiment ended, and blood was obtained for several biochemical tests. Cervical decapitation was used to kill the animals. The pancreas was removed and quickly washed in ice cold saline before being refrigerated for biochemical analysis. 30, 31

The serum was analysed for haemoglobin (Hb), glycosylated haemoglobin (HbA₁C), total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL), AST (Aspartate Amino Transaminase) or SGOT (Serum Glutamate Pyruvate Transaminase), ALT (Alanine Amino Transaminase) or SGPT (Serum Glutamate Oxaloacetate Transaminase) ALP (Alkaline Phosphatase). Very low density lipoprotein (VLDL) and low density lipoprotein (LDL). Values were calculated using the formula given below. 32, 33, 34

VLDL = TG/5, LDL = TC – (HDL + VLDL).

The pancreas was separated, washed in normal saline, then treated in 10% formalin solution for 24 hours and dehydrated with alcohol. Paraffin was used to clean and embed all of the tissues. 5µ thick slices were cut and stained with haematoxylin and eosin for histological examination. 35

All of the data was expressed using the mean ± standard error mean (SEM). Graphpad Instat version 3.06 computer software was used to run one-way analysis of variance (ANOVA) and Dunnett's post hoc test to find significant differences. At p<0.05, differences between the groups was considered significant.

In the EECR's phytochemical analysis, carbohydrates, alkaloids, saponins, flavonoids, tannins, and phenolic compounds were discovered.

In oral toxicity (acute) testing, a sole dose of 2000 mg/kg EECR exhibited no effect on behaviour. There were no fatalities or poisoning symptoms detected over the 24-hour and 14-day monitoring period. The oral LD₅₀ of EECR must be more than 2000 mg/kg.

Figure 1 depicts the impact of EECR at 100 and 200 mg/kg on fasting blood glucose levels in normal healthy rats. After 6 hours of oral treatment, rats given 200 mg/kg EECR, exhibited a marked reduction of 14 % in FBG, whereas rats given 100 mg/kg had a maximum reductin of 8.6 %. On the OGTT of normal rats, Figure 2 depicts the hypoglycaemic impact of oral therapy at doses of 100 and 200 mg/kg. Three hours after glucose administration, the dose of 200 mg/kg caused a maximum decline of 15.2%, whereas the dose of 100 mg/kg caused a reduction of 6.7%.

Figure 2

Table 1 shows the antihyperglycemic impact of repeated oral EECR treatment at various dosages on fasting blood glucose levels in STZ-NC diabetic rats. When diabetic rats were given STZ-NC, their blood glucose levels jumped much more than normal controls. When STZ-NC diabetic rats were given different dosages of EECR and glibenclamide, their blood glucose levels dropped considerably (p<0.01) when compared to normal control rats, and this was related to the dose and duration of therapy. Blood glucose levels for the 200 mg/kg and 100 mg/kg EECR doses were 66.9% and 63.9%, respectively, at the end of the study (the 28th day). In diabetic rats, the EECR at 200mg/kg was more efficacious than the EECR at 100mg/kg (p<0.01).

STZ-NC animals lost a lot of weight during the trial as compared to normal animals. Although different dosages of EECR and glibenclamide exhibited significant improvements in body weight (p<0.01) when compared to diabetic control, which lost 9.51%, 14.63%, and 16.11% respectively, diabetic control lost weight until the conclusion of the trial.

HbA₁C levels in STZ-NC diabetic rats were substantially higher (p<0.01) than in normal control rats, whereas Hb levels were lower. In STZ-NC diabetic rats, different dosages of EECR and glibenclamide resulted in substantial (p<0.01) reductions in HbA₁C levels of 56.38 % and 65.37 %, respectively. When compared to diabetic control rats, the Hb level increased by 26.22 % and 29.80 %, respectively. Glibenclamide, a common drug, led in a 63.64 % decline in HbA₁C and a 29.99 % increase in Hb, which would have been identical to the treatment with 200mg/kg of EECR.

Table 2 represents the levels of TC, TG, HDL, LDL, and VLDL in the control group and experimental groups. The serum concentrations for TC, TG, LDL, and VLDL were considerably greater (p<0.01) in STZ-NC diabetic rats while comparing to a normal control group, although serum HDL levels were significantly lower (p<0.01). The alteration in lipid metabolism was partially attenuated that after administration of different doses of EECR and glibenclamide, as evidenced by significant (p<0.01) declines in serum TC (43.72 %, 51.35 %, and 52.65 %), TG (47.11 %, 56.94 %, and 58.81 %), LDL (59.09 %, 70.18 %, and 72.24 %), and VLDL (47.10 %, 56.93 %, and 58.81% ) levels and by elevation in HDL level (40.14 %, 46.41%, and 48.70%), respectively while comparing with diabetic control rats. When comparing to other dosages of EECR 100 mg/kg and glibenclamide 5 mg/kg, EECR 200 mg/kg was more effective in enhancing the level of lipid parameter.

When comparing the diabetes control group to the normal control group, the diabetes control group had substantially (p<0.01) higher hepatic marker enzyme activity, such as AST, ALT, and ALP. While comparing to the diabetic control group, treatment with various doses of EECR and glibenclamide resulted in significant (p<0.01) reductions in AST (72.66%, 78.44%, and 78.00%), ALT (71.60%, 74.90%, and 74.48 %), and ALP (58.83%, 61.51%, and 63.14%) (Table 3). In STZ-NC diabetic rats, the EECR 200 mg/kg dose, rather than the 100 mg/kg dose, caused the highest reduction in liver enzymes such as AST, ALT, and ALP.

Photomicrographs of the islets of Langerhans in the pancreas of normal control groups revealed normal acini and cellular populations (Figure 3 A). Glibenclamide restored the normal cellular population size of islets having hyperplasia (Figure 3B), as well as the significant loss of pancreatic β-cell mass associated with Atrophic acini and the islets of diabetes control groups (Figure 3C). At the dosage level of 200 mg/kg (Figure 3D), EECR revealed a pancreas with acini and normal islets, β-cell regeneration, and notably normal regenerated and preserved cells, as well as noticeable proliferating and regenerated β-cells in comparison to the lower dose level of 100 mg/kg (Figure 3E).

Figure 3

Use of STZ for inducing diabetes in rodent models is well recognized. STZ-induced diabetes has been shown to match human DM 36 with glycosuria, hyperglycemia, polyphagia, polydipsia, and weight loss. 37 STZ has a cytotoxic impact on pancreatic beta cells, reducing endogenous insulin synthesis and raising blood glucose levels as a result. 38 NC is an antioxidant that protects type 2 DM, which produces pancreatic β-cell mass, from the cytotoxic effects of STZ by scavenging free radicals and causing very mild damage. 28

EECR had a dose-dependent hypoglycemic impact in normoglycemic rats after 3 hours in our investigation. EECR 200 mg/kg demonstrated a significant enhancement in glucose tolerance in the oral glucose tolerance test. In diabetic rats, EECR treatment reduced blood glucose levels in a dose-dependent manner, with a dosage of 200 mg/kg having a similar impact as glibenclamide, which is a common antidiabetic drug used to compare the anti-diabetic properties of a variety of bioactive substances in STZ-induced mild diabetes. 39, 40 The effect of glibenclamide is to increase insulin production in the Langerhans islets β-cell. Two suggested mechanisms of action for the EECR are insulin potentiation from the β-cell and increased peripheral glucose absorption. 41 Previous findings suggest that EECR works in a similar way as glibenclamide. In this situation, researchers have already demonstrated antidiabetic effect with an increase in insulin release from β-cells. 42 According to a recent research, diabetic rats treated with C. rotang L seed methanol extract demonstrated a substantial (p<0.01) drop in fasting blood glucose after 7 days of therapy compared to diabetic control rats who were not treated. 43

STZ-NC induced diabetes results in weight loss in rats because of protein wastage in the absence of glucose as just a source of energy. 44 After 28 days of EECR therapy, glycemic control improved considerably, avoiding weight loss. Improved glucose control or structural protein creation might explain the increase in body weight. 45 Hyperglycemia is a significant contributor to the onset of cardiovascular disease (CVD). Hyperglycemia causes protein glycation and peroxidation, which damages arterial walls, according to animal studies. 46 Diabetics have a 2-8 times higher risk of all forms of cardiovascular disease than non-diabetics. Accelerated coronary heart disease (CHD) has emerged as a leading cause of morbidity and death in diabetics across the world. 47 Diabetic hyperlipidemia is defined by elevated TG, TC, LDL, and VLDL cholesterol levels, and also reduced HDL cholesterol levels. As a result of these changes, diabetic patients are more likely to develop coronary heart disease. LDL-C has a positive relationship with the risk of cardiovascular disease, whereas HDL-C has a negative relationship. 48, 49 When compared to normal control rats, STZ-NC therapy resulted in alterations in normal lipid profiles, including greater TC, TG, LDL, and VLDL levels, along with lower HDL levels. The abnormal lipid profile in STZ-NC diabetic rats was recovered following treatment with both EECR and glibenclamide. This cholesterol-lowering action might be attributable to a good stabilisation of glucose levels following EECR therapy, which could help to restore diabetic rats impaired lipid metabolism. As a consequence, EECR's capacity to prevent CVD disorders associated with diabetes is supported by its hypolipidemic action in diabetic rats.

Liver is the primary organ for metabolism, detoxification, storage, and excretion of xenobiotics and their metabolites. The liver enzymes AST, ALT, and ALP give excellent idea of how well the liver is functioning. 50 The liver of STZ-induced diabetic rats was necrotized. Increased AST, ALT, and ALP activity in plasma may be due to the leakage of these enzymes from the liver cytosol into circulation, indicating that STZ has a hepatotoxic impact. 51 The effect of such enzymes in plasma was lowered in diabetic rats that were given EECR, lowering the liver damage caused by STZ-NC induced diabetes. The activity of these enzymes was significantly reduced in diabetic rats administered with EECR, indicating that it had a hepatoprotective impact in reducing diabetes complications.

Damage to pancreatic islets, in addition to insulin resistance, is another essential factor in the onset and development of diabetes. 52 The damage to diabetic rats' pancreatic islets produced by STZ-NC was a major contributor in the animals' hyperglycemia. In our study, histopathological examinations demonstrated that EECR therapy expanded the amount of pancreatic islets in a dose-dependent manner. These findings suggested that EECR may be able to preserve or promote the regeneration of pancreatic β-cells, both are beneficial to DM.

The EECR demonstrated good antihyperglycemic and antihyperlipidemic effects in the STZ-NC generated diabetes model, according to the above findings. Moreover, this study has offered scientific support for the safety and usefulness of CR leaves by traditional practitioners in diabetes treatment. However, further research is needed to isolate, purify and characterise the beneficial compounds in EECR, as well as to determine their mechanism of action at molecular level in diabetes and diabetic complications.

We reveal that we have not received any funding from any source for this research.

We declare that we have no conflicts of interest.

STZ: Streptozotocin; NC: Nicotinamide; HbA₁C: Glycosylated haemoglobin; Hb: Haemoglobin; TC: Total cholesterol; TG: Triglycerides; HDL: High density lipoprotein; LDL: Low density lipoprotein; VLDL: Very low density lipoprotein; AST: Aspartate Amino Transaminase; ALT: Alanine Amino Transaminase; ALP: Alkaline Phosphatase; CNS: Central nervous system, GC-MS: Gas chromatography - Mass spectrometry, IAEC: Institutional Animal Ethics Committee; CPCSEA: Committee for the Purpose of Control and Supervision of Experimentation on Animals; OECD: organization for economic cooperation and development; EECR: ethanol extract of Calamus rotang L leaves; FBG: Fasting blood glucose; OGTT: Oral glucose tolerance test; NIDDM Non-insulin dependent diabetes mellitus; SEM: Standard error mean; LD₅₀: Lethal dose 50; DM: Diabetes mellitus, CVD: Cardiovascular disease; CHD: Coronary heart disease.