Evaluation of Anti-diabetic and Anti-hyperlipidemic Activity of Rhododendron arboreum bark extract against Streptozocin Induced Diabetes in Rats

Rhododendron arboreum Sm. (R. arboreum) also known as “Laligurans” or simply “Gurans” national flower of Nepal, is a small evergreen tree with a bright red flower. It has habitat in the lap of Himalayas. The plant has the medicinal value and various parts are used for the treatment of the ailments. Flower of the plant is traditionally used in the treatment of diabetes by rural Nepalese people [1]. A flower is also used in the acute inflammation treatment [2] and also has the properties of anti-nociceptive and anti-inflammatory [3]. Hypolipidemic activities in induced hypercholesteremia rabbits [4] and hepatoprotective activity [5] has also been noted. The plant contains many flavonoids and triterpenoids/sterols which are known for their bioactive principles for anti-diabetic potential [6]. Flavinoids also is known for the regeneration of the damaged b-cells in diabetic mice [7,8].


INTRODUCTION
Rhododendron arboreum Sm. (R. arboreum) also known as "Laligurans" or simply "Gurans" national flower of Nepal, is a small evergreen tree with a bright red flower. It has habitat in the lap of Himalayas. The plant has the medicinal value and various parts are used for the treatment of the ailments. Flower of the plant is traditionally used in the treatment of diabetes by rural Nepalese people [1]. A flower is also used in the acute inflammation treatment [2] and also has the properties of anti-nociceptive and anti-inflammatory [3]. Hypolipidemic activities in induced hypercholesteremia rabbits [4] and hepatoprotective activity [5] has also been noted. The plant contains many flavonoids and triterpenoids/sterols which are known for their bioactive principles for anti-diabetic potential [6]. Flavinoids also is known for the regeneration of the damaged b-cells in diabetic mice [7,8].
Diabetes is a chronic disorder of carbohydrate, fat and protein metabolism characterized by complete or partial insufficiency of insulin action [9,10]. Diabetes mellitus (DM) is a chronic disorder caused by insufficient production of insulin and decreases in the absorption of glucose by the cells in the human system which leads to an increase in the concentration of glucose blood. It is also produced due to the hereditary Characters. Increase glucose level in blood leads to various deficiencies and alters the normal physiological effects of the human system like blood vessels and nerves system etc. It is projected that the Diabetic is the main disease which can increase the deaths retain next coming 25 years in Asian countries and Africans [11].
According to the World Health Organization (WHO), there are approximately 422 million diabetic's people world-wide; the global prevalence of diabetes has risen from 4.7% in 1980 to 8.5% in 2014. In 2016, an estimated 1.6 million deaths were directly caused by diabetics so it was listed as the 7 th leading cause of death in 2016 [12]. Diabetes is considered a major medical concern due to its high prevalence and potential deleterious effect on a patient physical and psychological state. The disease remains incurable and can only control with drugs [13].
The detailed survey of literature revealed that Rhododendron arboreum is an important plant of a hilly region with extensive medical and commercial uses. The plant exhibited antiinflammatory, hepato-protective, anti-diarrhoeal, anti-diabetic,

Evaluation of anti-diabetic and antihyperlipidemic activity of Rhododendron arboreum bark extract against streptozocin induced diabetes in rats
Suman Gautam 1 *, Kabiraj Chaudhary 2,3 2 Mallige College of Pharmacy, Bangalore, Karnataka, India, 3 Curex Pharmaceutical Pvt Ltd, Nepal, 1 Central Department of Biotechnology, Tribhuvan University, Nepal antioxidant properties due to the presence of flavonoids, saponins, tannins, and other phytochemicals [1]. So, the present study was conducted to find out the antidiabetic activity of the Rhododendron arboreum bark extract in streptozotocin-induced diabetic rats.

Collection and Authentication of Plant Material
The bark of Rhododendron arboreum was collected from the hilly region of Nepal. The plant was identified and authenticated by Dr. K. Ravi kumar, senior botanist at FRLHT (Foundation for Revitalisation of Local Health Traditions) Jarakabande Kaval, Post Attur, Yehalanka, Bengaluru (560106). The barks were shade dried at room temperature for 15 days and pulverized.

Preparation of Extract
The bark of Rhododendron arboreum was shade dried and then powdered with a mechanical grinder. The powder was stored in an airtight container and stored at 5°C. Powdered bark (500 g) was defatted with 1L petroleum ether (40-60°C) to remove lipid constituents by soxhlation for 24 hours not exceeding the boiling point of the solvent. It is then filtered with filter paper, and the residue was air-dried at 30°C and extracted with 95% ethanol (1L) by soxhlation for 48 h, not exceeding the boiling point of the solvent. Ethanol is evaporated in a rotary evaporator at 40-50°C under reduced pressure [14].

Experimental animal
Albino Wistar male rats (180-220 gm) were selected for experimental study. The animals were kept and maintained under laboratory conditions of temperature (24 ± 2°C), humidity (60 ± 1%), and 12-hour light/dark cycle. They were allowed free access to food (standard pellets) and water ad libitum. Experimental protocols and procedures used in the following ways:

Acclimatization
One week in the experimental room.

Selection of animals
After acclimatization, the animals were subjected to a gross observation to ensure that the selected rats were in good state of health. Rats were randomly selected for final allotment to the study.

Acute toxicity studies
Acute toxicity studies were performed according to OECD (Organization for Economic Co-operation and Development) guidelines 423. Rats are selected for the study and are fasted for 3-4 h with free access to water. Extracts of above plant parts at a dose of 5, 50, 300, 2000 mg/kg are given p.o. at 48 h intervals simultaneously to the respective groups. Animals are observed individually after dosing for signs of toxicity and mortality rates [15].

Induction of Diabetes
Rats were fasted overnight before inducing diabetes with streptozotocin. The rats were given an intraperitoneal injection of streptozotocin (60 mg/kg) freshly prepared in 0.1M sodium citrate buffer. The diabetic state was confirmed 48 h after streptozotocin injection. A threshold value of fasting blood glucose was taken as >200mg\dl.

Blood Sugar and Biochemical Estimation
Blood samples were obtained through puncture tail vein and collected blood samples were analyzed by Accu-Check Glucometer. Blood glucose levels were expressed in terms of mg/ dl and TC, TG, HDL, and LDH were also estimated.

Qualitative Phytochemical Analysis
The ethanolic extract was tested to know the different constituents present in it by the standard procedures. The extracts were tested for the alkaloids, carbohydrates, saponins, flavonoids, triterpenoids, sterols and protein.

Experimental Design
4 Groups of animals having six animals in each group were used.

Phytochemical Analysis
The ethanolic extract of Rhododendron arboreum was subjected to a qualitative test to identify the presence of phytoconstituents. The result of phytochemical analysis summarized in Table 1 confirmed the presence of alkaloids, flavonoids, saponins, carbohydrates, steroids, phenol, proteins, tannins and triterpenoids compounds.

Effect of Rhododendron arboreum Bark Extract on Blood Glucose Level of Glucose Overloaded Healthy Albino Rats
The result of the OGTT is summarized in Table 2 as well as in Figure 1. The standard drug glibenclamide 0.25 mg/kg, p.o. reduced blood glucose level 36.39 % at 1.5 th hr and 37.3% at 2 nd hr. The ethanolic extract of Rhododendron arboreum100 mg/kg, p.o. also reduced the blood glucose from 0.5 th to the 2 nd hour of treatment the highest being 22.05% at the 2 nd hour. The ethanolic extract of Rhododendron arboreum at 200 mg/kg reduced the blood glucose from 0.5 th hour to 2 nd hour and 28.34% was the highest reduction observed at the 2 nd hour.

Effect of Rhododendron Arboreum Bark Extract on Streptozocin Induced Diabetic Albino Rats
The Rhododendron arboreum ethanolic extract at 200 mg/kg exhibited a significant reduction of blood glucose level from 7 th day to 28 th day of the treatment 42.37 % of the reduction was found to be the highest and was shown on the 28 th day with 200 mg/kg of ethanolic extract. The ethanolic extract at a dose of 100 mg/kg also significantly reduced the blood glucose level in diabetic rats. The percentage of reduction was highest at 32.71% on the 28 th day. As compared to 200 mg/kg of ethanolic extract 100 mg/kg of ethanolic extract of Rhododendron arboreum showed nearly 9.66% less. And the results are presented in Table 3 and Figure 2.

Effect of Rhododendron arboreum Bark Extract on Biochemical Parameters in STZ Induced Diabetic Rats
The results of R. arboreum ethanolic bark extract on biochemical parameters in STZ induced diabetic rats are shown in Table 4 and Figure 3. The STZ induced diabetic rats showed significant hypercholesterolemia, hyper-triglyceridemia and an increase in levels of LDH and a decrease in the level of HDL when it is compared with the normal control rats.

Histopathological Investigation
Histopathology of the pancreas is present in Figure 4-7. The pancreas of albino rats of the control group showed normal histology, the normal appearance of the islet of Langerhans containing α, ß and δ cells. The ß-cells are the most abundant cells.
Diabetic rat pancreas showed a reduction in the pancreatic ß-cell numbers and necrosis along with few surviving ß-cells. Severe infiltration of inflammatory cells was also observed. It  In the reference group, i.e., diabetic rats treated with Glibenclamide, pancreas architecture was similar to that observed in the control rat and it also showed slight regeneration of the beta cell, less damage to beta cells as compared with the diabetic rat.
Histopathological study of the pancreas of diabetic rats treated with alcoholic extract of R. arboreum at the dose of 100 mg/kg p.o. showed a significant improvement in the number of b-cells.
It was observed that it exhibited less damage to beta cells, improved beta cell regeneration and showed slight necrosis as compared to diabetic rats.
The pancreas of the rats treated with the alcoholic extract of R. arboreum at the dose of 200 mg/kg p.o. showed a reduction in the extent of necrosis and inflammation where as increased in the number of islet cells of the pancreas and less deposition of the fatty material as compared with diabetic control.

DISCUSSION
In this study, the anti-diabetic action of ethanolic extract of Rhododendron arboreum bark extract has been evaluated and its efficacy has been compared with that of standard oral hypoglycemic drug Glibenclamide owing to its traditional uses and proved its claimed uses. The ethanolic extract of Rhododendron arboreum found to contain wide varieties of chemical constituents such as carbohydrate, saponins, flavonoids, triterpenoid, and sterols.
The ethanolic extract of Rhododendron arboreum was evaluated for its hypoglycemic effect in healthy rats and STZ induced diabetes in albino rats. The ethanolic extract of Rhododendron arboreum significantly reduced blood glucose in normal healthy rats. The ethanolic extract of Rhododendron arboreum also reduced the blood glucose in glucose overloaded healthy albino rats. This effect may be due to the reduction of glucose absorption or due to increased insulin secretion and alteration in carbohydrate and lipid metabolism.
The number of b-cell improved significantly by extract may be the mechanism of anti-diabetic action of R. arboreum extract. The leaves extract of R. arboreum also reported for its potent antioxidant activity. Treatment with both extracts elevated enzymatic and non-enzymatic antioxidant levels. This property may also play a role in the reduction of inflammation, and necrosis leading to the regeneration of damaged beta cells in the pancreas.
The subsequent increase in uptake of blood glucose and its utilization may be another mechanism of action of the extract. Other possible mechanism includes the stimulation of b-cells and subsequent release of insulin and activation of the insulin receptors. Estimation of insulin level and insulin receptor may give more insight into the mechanism of the anti-diabetic activity exhibited by the extract.
A plant may act on blood glucose through different mechanisms, some of them may have insulin-like substances and some may inhibit insulinase activity. The extract might possess an insulinlike effect on peripheral tissues either by promoting glucose uptake and metabolism or inhibiting hepatic gluconeogenesis. The phytochemical studies on ethanolic extract of R. arboreum revealed the presence of tannins and flavonoids. Flavonoid and tannins isolated from other anti-diabetic medicinal plants has been found to stimulate the secretion or possess an insulin-like effect.
The results of the present study indicate the Rhododendron arboreum bark extract exhibited a dose-dependent reduction in blood glucose levels in healthy and diabetic rats. The results from the present study also indicate that the Rhododendron arboreum bark extract can reduce the levels of serum cholesterol, serum triglyceride, lactate dehydrogenase and increase the level of HDL, confirms the possibility that the major function of the leaves extract are on the protection of vital tissues including the pancreas, thereby reducing the causation of diabetes in the experimental animals.
Flavonoids like quercetin and its derivatives were reported to potentiate insulin secretion and protect INS-1 pancreatic b-cells against oxidative damage via phosphorylation pathways. The presence of such active components might be responsible for the anti-diabetic as well as anti-hyperlipidemia activity by the R. arboreum bark extract supported by the various research work [1,2,[16][17][18][19][20][21].
The aqueous extract of Rhododendron arboreum at 200 mg/kg dose significantly reduced blood glucose levels in diabetic rats. The extract at the dose of 100 and 200 mg/kg reduced 31.25% and 44.78 % of blood glucose respectively on the 28 th day of treatment. The results of histopathological studies revealed that the aqueous extract of Rhododendron arboreum 100 mg/kg and 200 mg/kg have increased the number of b-cells and also decrease necrosis and inflammation in the pancreas as seen in various work [22,23].
From the above discussion, we have observed that the ethanolic extract of R. arboreum bark has anti-diabetic activity as well as major role in the reduction of TG, TC, LDL and increase HDL.