1. INTRODUCTION:

The persistent metabolic condition of diabetes is primarily distinguished by the loss of homeostasis of carbohydrates with protein and fat metabolism disruptions that result from outcome from either insulin discharge or insulin activity deserts or both. The protein (hormone) is insulin, synthesized in pancreatic beta cells respond to multiple stimuli, such as glucose, sulphonylurea, arginine, but the key determinant is glucose¹.

It is recognized that the most common factors donating to the pathophysiology of type II diabetes, a continuum of disease arising from tissue insulin blocking and ultimately progressing to a disorder characterized by complete loss of secretary function of the pancreatic beta cells, are insulin secretion deficiency, resistance to insulin tissue acts, or a mix of the two¹.

At present available medications for type II diabetes are include: biguanides , sulfonylurea, thiazolidinediones or glitazones, glucagon-like peptide-1 (GLP-1) agonists, dipeptidyl peptidase four (DPP-4) inhibitors, alpha-glucosidase inhibitors, glinides or meglitinides, sodium-glucose cotransporter- 2 inhibitors and insulin¹.

Biguanides increases insulin sensitivity, decreases fasting plasma sugar and insulin concentrations; in the lack of insulin, it is not effective. The glucose-reducing effect is primarily due to reduced development of hepatic glucose and increased peripheral glucose absorption in NIDDM patients. Type of diabetes is type 1, depending on insulin. Pancreatic islet beta cells are destroyed, with most cases being autoimmune antibodies (type IA) that destroy beta cells in the blood, but some are also idiopathic (type IB). Type II is a type of diabetes that is not insulin dependent. GLP-1 is an essential insulin-inducing incretin from pancreatic beta cells. This will inhibit the release of glucagon. The GLP-1 receptors are activated where appetite is suppressed².

Type II diabetes mellitus requires multiple processes and is a chronic progressive condition. Metformin is a derivative of biguanide that decreases the production of hepatic glucose as well as intestinal glucose absorption and increases insulin sensitivity by rising the absorption and use of peripheral glucose³.

Type II diabetes commonly occurs in adults, but children are often affected by obesity as well as any other physiological and pathological disorders causing it. Because of its effect on small blood vessels that supply the kidney, nerves and eyes, diabetes is responsible for many complications, thereby causing nephropathy, neuropathy and retinopathy. Larger blood vessels are also impaired, resulting in angina pectoris, myocardial infarction, acute ischaemic attacks, stroke, and peripheral arterial diseases⁴. Diabetics have dramatically raised oxidative stress levels and this is a major contributor to the majority of neurological, cardiovascular, retinal, and renal diabetic complications⁵.

Chemically, metformin is an oral active medication for type II diabetes mellitus, N-dimethylimidodicarbonimidic diamide, belonging to the biguanide class, which works essentially by stimulating the enzyme AMPK, which further controls the metabolism of carbohydrates by falling intestinal glucose uptake, hepatic glucose making and increasing insuccess⁶. And it is commonly used by nearly 120 million people worldwide for the treatment of type II diabetes. Biguanides is the active compound of the Galega officinalis. In the 1920, they were first accepted and used as therapeutic agents in the 1950. Metformin was approved as an antihyperglycemic agent in the United Kingdom in 1958, Canada in 1972 and the United States in 1995 and is suggested as a first-line treatment for type II diabetes by the European Association.⁶

The key effect of Biguanides is to reduce the output of hepatic glucose as an antihyperglycemic agent. In addition, it rises the usage of insulin-mediated glucose in peripheral tissues (e.g. liver and muscle), reduces the absorption of glucose in the small intestine and decreases plasma-free fatty acid concentrations, thus reducing the availability of gluconeogenesis substrates. As a consequence, it lowers the blood sugar levels in type II diabetes and does not cause excessive hypoglycemia. Metformin has recently been found to be effectual in treating multiple cancers, especially cancers of the prostate, colon, and breast cancer. Its biological half-life (t1/2) is in the 0.9-2.6 h range. For effective treatment, regular use of large doses of metformin (500 mg two or three times a day, or 850 mg once or twice a day with or after meals) is also essential⁶. With its approximate absolute bioavailability of about 50 to 60 percent, it is absorbed steadily and incompletely from the gastrointestinal tract, liberally soluble in water⁷.

Worldwide, T-II-DM predominance has achieved outbreak proportions. As diabetes induces the risk of cardiovascular diseases and early mortality, T-II-DM prevention and management has become a major public health iss ue around the world⁸.The risk of diabetes rises day by day and is primarily found in women rather than in men⁹. 150 million people have diabetes, according to epidemiological studies, and 300 million people worldwide are expected to be affected by 2025¹⁰.

As oral glucose-reducing agents for the treatment of non-insulin dependent mellitus diabetes (NIDDM), the two biguanides, metformin and phenformin, were introduced in 1957. With its association with lactic acidosis, phenformin has been discontinued in many countries, but metformin does not bear the same risk if properly administered. Buformin was introduced in 1958 only restricted use was given to Buformin, but phenformin was broadly introduced in the 1960 and early 1970^11,12. Contact with lactic acidosis has resulted in the removal of phenformin in some countries^13,14, but it is still used in some countries with caution, either as monotherapy or in combination with sulfonylurea. Special interest in this has been demonstrated by a fixed combo tablet containing phenformin and glyburide¹⁵. Metformin, however, took precedence among the biguanides.

Aim:

The aim of the present study is to evaluate the antidiabetic activity of methanolic leaf extract of Nephelium lappaceum L. against streptozotocin-nicotinamide induced type-II diabetes in Wistar albino rats.

2. MATERIALS AND METHODS:

2.1. Plant materials:

The fresh leaves of the plant Nephelium lappaceum L. were collected from the surrounding areas of Malappuram, Kerala during the month of February and authenticated by Botanical survey of India (BSI) southern circle, Coimbatore, Tamilnadu. The authentication certificate number is No:BSI/SRC/5/23/2020/Tech/730. 500g of plant materials were extracted with 100ml of different solvents (petroleum ether, chloroform, methanol (70%), and water (30%)) based on low polarity to high polarity by cold maceration method. The mixture was filtered by using muslin cloth and through Whatman No. 1 filter paper. The filtrate was concentrated using heating mantle at 40°C, and the resultant residue was kept in a refrigerator till further use. The extracts were analyzed their phytoconstituents by GC/MS and the extract stored in air tight glass container at 4^o C for biological studies.¹⁶

2.2. Chemicals:

Streptozotocin and Nicotinamide were obtained from Sigma Chemicals, Bangalore, India. Kits to estimate (SGOT), (SGPT)were purchased from Qualigens Diagnostics.

2.3. Experimental animals:

Healthy adult Wistar albino rats of 6-8 weeks old and 150-250g body weight were purchased from Biogen Laboratory, Bangalore. Protocols for the study were approved by the Institutional Animal Ethical Committee (IAEC) for Animal Care and were in accordance with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines, Government of India.(Approval No.: KMCRET/M.Pharm/8/2020-2021). All rats were housed and maintained under standard conditions of temperature (25⁰C ± 5⁰C), relative humidity (55 ± 10%), and 12/12 h light/dark cycle. Animals were fed with commercial pellet diet and water ad libitum freely throughout the study.

2.4. Glucose tolerance test (GTT) in normal rats¹⁷:

Normal rats were tested for the Oral glucose tolerance test (OGTT) of the various concentrations of MLEN and standard drug Metformin. Wistar albino rats weighing 150-250gm were divided into 5 groups consisting of 6 rats in each group. The oral glucose tolerance test (OGTT) was performed on overnight fasted normal rats. Distilled water, MLEN (400and 800mg/kg p.o) and Metformin (500mg/kg p.o) were administered to respective groups. Glucose (2g/kg) was fed 30 min after pre treatment with distilled water, MLEN and Metformin. Blood glucose levels were measured at 0, 30, 60, 90, and 120min after glucose load to access the effect of extract on blood glucose levels of the glucose loaded animals. The blood glucose was measured using blood glucose test strips and glucometer.

2.5. Induction of diabetes:

Streptozotocin was dissolved in citrate buffer (pH 4.5) and nicotinamide was dissolved in normal saline. Non-insulin dependent diabetes mellitus was induced in overnight fasted rats by a single intraperitoneal injection of 60mg/kg streptozotocin 15min after the i.p administration of 120mg/kg of nicotinamide. Hyperglycemia was confirmed by the elevated levels of blood glucose were determined at 72h. The animals with blood glucose concentration more than 250mg/dl will be used for the study.¹⁷

2.6. Experimental Design:

In the experiment totally 30 rats (6 normal and 18 STZ-NIC induced diabetic surviving rats) were used. These rats were divided into five groups of 6 rats each. The NL methanol extract were dissolved in distilled water and administered orally for 28 days.

Group I- Distilled water

Group II- Receives Streptozotocin 60mg/kg/b.w.(i.p)and Nicotinamide 120mg/kg/b.w. (i.p) the day of induction

Group III - Diabetic rats to be treated with Metformin 250mg/kg (p.o) for 28days.

Group IV- Diabetic rats to be treated with MLEN extract (400mg/kg p.o) for 28days

Group V - Diabetic rats to be treated with MLEN extract (800mg/kg p.o) for 28days

2.7. Biochemical analysis:

Blood glucose estimation was done using a glucometer on 0, 7, 14, 21, 28 days after treatment with NL extract. At the end of 28 days, the animals were euthanized between 9:00-11:00 am to minimize diurnal variation. The urine volume analysed based on the Lipschitz et al method. The glycogen level of liver and skeletal muscles was measured by anthrone method. The activities of SGOT and SGPT were assayed in the serum using commercial kits. The haematological parameters measured by Sahli’s acid haematin method. And the Determination of antioxidant enzymes and lipid peroxidation also included for the study.

2.8. Histopathological examination of pancreas:

Pancreas was instantly dissected out, excised and rinsed in icecold saline solution. A portion of pancreas was fixed in 10% neutral formalin fixative solution, were fixed in 10% formalin, dehydrated in alcohol and then embedded in paraffin. Microtome sections of 4–5μm thickness were made by using a rotary microtome. The sections were stained with haematoxylin–eosin (H&E) dye to observe histopathological changes.

2.9. Statistical analysis:

One-way ANOVA followed by Dunnett’s test were carried out to compare the data with the level of significance set at P<0.05.

Table 2: Effect of MLEN on Urine volume

Groups	Water intake (ml)	Urine volume (ml)
Group I	14.01±0.25	9.66±0.42
Group II	29.35±0.34^**	26.06±0.29^**
Group III	17.03±0.23	13.03±0.23
Group IV	24.01±0.24	19.83±0.30
Group V	19.83±0.16	15.01±0.B25

Table 3: Effect of MLEN on Haematological parameters

Group	Haemoglobin (g/dL)	Glycosylated haemoglobin (%)
Group I	15.35±0.13	5.01±0.07
Group II	8.50±0.02***	8.13±0.27***
Group III	12.51±0.12***	5.8±0.32
Group IV	12.05±0.07***	7.15±0.22***
Group V	12.38 ±0.08***	6.91±0.31***

Table 4: Effect of MLEN on Liver and kidney functional parameters in serum

Groups

SGOT (U/L)

SGPT (U/L)

Blood Urea

(mg/dl)

Creatinine (mg/dl)

Group I

35.50±

0.76

43.18±

0.58

32.867± 0.61

0.423±

0.004

Group II

67.51±

0.75^***

75.50±

0.76^***

61.55 ±0.44^***

0.907±

0.006^***

Group III

44.53±

0.74^***

50.55±

0.77^***

39.51±

0.48^***

0.458±

0.005^***

Group IV

59.55±

0.73^***

64.58±

0.80^***

43.58 ±0.24^***

0.578±

0.003^***

Group V

49.56±

0.74^***

58.16±

0.60^***

41.26±

0.51^***

0.517±

0.004^***

Table 5: Effect of MLEN on Total protein and glycogen estimation in liver homogenate

Groups	Total protein mg/dL	Glycogen mg/g
Group I	8.23±0.03	67.33±0.42
Group II	4.08±0.04^***	17.98±0.44 ^***
Group III	7.73±0.03^***	54.16±0.60 ^***
Group IV	6.33±0.06^***	40.16±0.30 ^***
Group V	6.98±0.06^***	47.83±0.47 ^***

Table 6: Estimation of In-vivo antioxidant (non enzymatic)

GROUPS	GSH (µg/mg Protein/mg of tissue extract)	VITAMIN C (mg/g)
Group I	9.65±0.07	1.083±0.017
Group II	2.53±0.08^***	0.707±0.027^***
Group III	8.08±0.06^***	1.303±0.009^***
Group IV	6.10 ±0.05^***	0.905±0.008^***
Group V	7.11±0.06^***	1.173±0.007^**

All the Values in Table 1-6 are expressed as mean± SEM

n=6, One way ANOVA followed by Dunnett’s test

ns = non-significant

* P < 0.05; ** P < 0.01; *** P < 0.001 denotes significant difference compared with diabetic control

Histological Analysis:

Group I: Pancreas shows normal pancreatic beta cells destruction

Group II: Islets are reduced in number and size with focal cytoplasmic vacuolation

Group III: Islets are normal in number few islets showing destruction

Group IV: Islets are normal in number and size

Group V: Regeneration of pancreatic beta cells

3. DISCUSSION:

The type II diabetes is induced by using Streptozotocin and Nicotinamide. The diabetogenic action of STZ is related to its selective destruction of pancreatic β-cells, which are the only source of insulin in body. Where the NIC signiﬁcantly decrease the cytotoxicity of STZ by reducing PARP-activation. Preliminary phytochemical tests of MLEN confirms the presence of alkaloids, flavonoids, glycosides, saponins, tannins, proteins and amino acids. Antidiabetic activity producing various constituents were identified in MLEN by using GC/MS technique. The present study was undertaken to assess the modification in serum glucose level and after systematic treatment with MLEN in STZ-NIC induced type II diabetes rats.¹⁸ After continuous treatment (28 days) with high and lower doses of MLEN, produced a significant decrease in serum glucose level in diabetic rats and the antidiabetic activity was produced MLEN which results in the increased glucose tolerance. Reduction of sugar absorption from the gut, increased insulin production from the pancreas, reduction of release of glucose from the liver, increasing glucose uptake by fat and muscle cells are probable mechanisms which may be involved.¹⁹

Diabetic rats (Type-II) decrease in body weight compared to control rats which confirms that diabetes has been induced, and it may be due to partial necrosis of pancreatic β-cell by STZ-NIC. Further the body weight of diabetic control rats have been decreased the possible mechanism behind reduction of body weight may be due to reduction of insulin level. Physiologically, insulin regulates protein synthesis and proteolysis in skeletal muscle.

Oral administration of MLEN (800mg/kg/bw dose) and metformin (250mg/kg) to the diabetic rats showed significant reduction of blood glucose level and increase in body weight than diabetic control rats. Therefore, MLEN mediated above effect may be due to its preventive effect on STZ- NIC mediated β-cell damage in diabetic rats and further this leads to increase insulin release and inhibits muscle proteolysis which causes improvement in body weight of MLEN-treated diabetic rats.

The glycosylated haemoglobin (HbA1C) is a major clinical marker in diabetes which helps to find the degree of protein glycation during diabetes.²⁰ In persistent hyperglycemic state, formation of HbA1c occurred by nonenzymatic reaction between glucose and free amino groups of haemoglobin. HbA1c level in diabetes helps to find long-term glycemic control, and it helps to predict the risk of the development or progression of diabetic complications.²¹ Published studies supported that reduction in HbA1c levels during the diabetes treatment considerably reduced microvascular complications.

There is a significant decrease in Hb and increase in HbA₁C levels in STZ-NIC induced diabetic rats. The treatment with MLEN (800mg/kg dose) showed reduction of HbA1c and improvement in Hb levels nearby equivalent to standard group, and it may be possibly due to blood glucose lowering effect of MLEN through reversal of insulin resistance or increasing insulin secretion by regeneration of pancreatic 𝛽-cells.

One of the key enzymes for carbohydrate metabolism in the small intestine is pancreatic 𝛼-amylase which converts consumed polysaccharides to monosaccharides. This enzyme action causes postprandial blood glucose level elevation due to absorption of formed glucose from polysaccharides in the small intestine. Drugs having an inhibitory action on this enzyme possess an ability to control of postprandial blood glucose level specifically in type II diabetes groups. The present study results was clearly demonstrated that MLEN possesses potent pancreatic 𝛼-amylase inhibition which confirms that in vivo antidiabetic action of high dose MLEN may be due to the inhibition of alpha amylase.

In type II diabetes, peripheral insulin resistance and impaired insulin secretion from pancreatic B-cells are two important features. Drug which diminishes insulin resistance will effectively control hyperglycemia, normalize lipid metabolism in type II diabetes, and hence it will prevent the diabetes-mediated cardiovascular complications.

The drugs like metformin and pioglitazone will ameliorate insulin resistance and control the hyperglycemia and abnormal lipid metabolism. This class of drugs has adverse effects such as lactic acidosis, gastrointestinal disturbance, liver toxicity, and cardiovascular risk.²¹ Thus, drugs which improve insulin sensitivity without adverse effects were reported to be useful for the long-term treatment in type II diabetes.

Lipid peroxide mediated tissue damage has been observed in the development in type II diabetes mellitus. Insulin secretion is impaired during diabetes and this may evoke lipid peroxidation in biological systems. Present study shows that administration of high dose MLEN and metformin inhibits production of liver peroxides.

LPO and GSH are the two major scavenging enzymes that remove toxic free radicals in vivo and are thought to play important role in protecting the cell against the potentially deleterious effects of reactive oxygen species.

Reduced activity of CAT and GSH may result in a number of deleterious effects due to the accumulation of superoxide radicals (O₂-) and hydrogen peroxide.²²Administration of high dose of MLEN and metformin results in the activation of CAT and GSH to near normal levels in diabetic rats. The result of the CAT and GSH activity clearly shows that high dose of MLEN has free radical scavenging activity, which could exert a beneficial action against pathological alterations caused by the presence of O₂- and OH.

CONCLUSION:

From the present study, it was concluded that MLEN may be useful in treating Diabetes mellitus with no visible signs or symptoms of toxicity. MLEN exhibited anti-diabetic activity comparable to that of a standard drug metformin. The traditional use of MLEN to treat diabetes is supported by laboratory results from this study, suggesting a need to isolate and evaluate active constituents responsible for the exhibited biological activity for the further studies.

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Received on 22.12.2022 Modified on 20.09.2023

Research J. Pharm. and Tech. 2024; 17(3):1376-1381.

DOI: 10.52711/0974-360X.2024.00217

Groups	Blood glucose levels (mg/dL)
Groups	0 minute	30 minutes	60 minutes	120 minutes	180 minutes
Group I	77.66±0.80	103.00±0.85	95.16±0.60	80.66±0.49	72.50±0.67
Group II	121.83±0.94^***	144.83±0.79^***	133.33±0.88^***	126.00±0.51^***	124.66±0.80^***
Group III	80.33±0.49	103.00±0.85	97.16±0.65	80.66±0.49	72.50±0.67
Group IV	96.83±0.37^*	123.50±0.61^*	114.83±1.35^*	106.83±0.40^*	98.50±0.42^*
Group V	87.50±0.61	116.80±0.583	104.16±0.87	98.66±0.21	80.50±2.32