Anti-Type I Diabetic Activity of the Methanolic Extract of Aegle marmelos on Streptozotocin Induced Rat Model

Aegle marmelos, generally acknowledged as Bael, is being ancient in Ayurveda for the therapy of a number of disorders. All the components on it tree along with stem, bark, root, leaves, fruit and seeds at all stages of maturity have medicinal virtues and have been recorded in Ethno-medicine. Aims: The present investigation study the Anti-Type I diabetic activity of the methanolic extract of Aegle marmelos on STZ induced rat model. Study Design: In-vivo study in rat model Place and Duration of Study: Department of Pharmacology, Karnataka college of Pharmacy, Bangalore, India, between Jan 2021 to Dec 2021. Methodology: Extracted Aegle marmelos was to be evaluated the toxicity as per the OECD guidelines and biochemical, hematological and gross pathological analysis has been assessed. Type I Diabetes has been induced in Wistar rats through STZ 65mg/kg/b.w. I.P. During the experiment, Rat’s BW and FBS level were monitored. At the end of study, animals among all groups namely Group I: Normal control, Group II: STZ 65mg/kg, Group III: STZ + Insulin 4Ukg/b.w., Group IV: Aegle marmelos 250mg/kg and Group V: Aegle marmelos 500mg/kg were sacrificed and biochemical parameters like Lipid profile, C-Peptide, HbA1c, Serum insulin, pancreatic insulin, and histology of pancreas had been observed. Aegle marmelos was also screened for pro-inflammatory Original Research Article Haimed et al.; JPRI, 34(17B): 1-14, 2022; Article no.JPRI.83761 2 cytokines viz., IL-1β, IL-6 and TNF-α were assessed by ELISA test. Furthermore, Antioxidant Enzyme like SOD, CAT, LPO and GSH were performed. Results: The observed extract Aegle marmelos was shown safe in the toxicity data; B.W, Lipid Profile, RFT, LFT, hematological parameters were shown in the limit range and the vital organ tissue histology were not shown any anatomical and structural abnormalities. The findings of the other parameters have been shown significant impact in vivo to manage the diabetic markers like weight gain, blood glucose, lipid profile, C-Peptide, HbA1c, secretion of insulin, and pancreatic insulin. Diabetic pancreas of rats confirmed fall of beta cell density and disruption of normal architecture. But treated group were found to restore the mass of beta cells. Mediator of inflammatory cytokines like increased in STZ group and was inhibited by test chemicals. Elevated oxidative enzymes also have been seen to control upon the treatment with Aegle marmelos. Conclusion: All this findings and phytoconstituents present within the extract should be the possible chemical substances concerned in the prevention of diabetes.


INTRODUCTION
Diabetes mellitus is the most common metabolic disease characterized by way of continual hyperglycemia, which is due in accordance with carbohydrate, protein and lipid metabolism disturbance triggered through a relative or utmost deficiency in secretion of insulin and/or insulin action within the peripheral tissues [1]. DM has significantly higher risk of death after cancer and cardio, cerebrovascular diseases [2]. It is estimated as 5% on demise in the world is caused by diabetes, and it will be increased by 50% within the next 10 years [3]. There is thriving proof as the excess production of ROS into diabetes, as reason oxidative stress, may thoroughly and of part make a contribution towards the progression of problems of a variety over tissues [4,5]. The control of DM without any consequences is a challenge, medicine derived from plants may additionally lead an essential function between the remedy for DM [6]. Natural merchandise isolated from medicinal plant sources have been ancient for the siege and therapy on a number of diseases pathologies, consisting of cancers, heart disease, diabetes mellitus or high blood pressure [7,8]. Up to 2014, More than 800 kinds have been investigated and theirs hypoglycemic results have been reported [9].
Aegle marmelos is a medicinal plant of family Rutaceae which is typically acknowledged as like Bael, Bengal-quince, golden apple or wood/stone apple tree. It is a medium-sized deciduous tree, up to 12-15 m tall with a short trunk, thick, soft, flaking bark and spreading, occasionally spiny branches [10]. This plant is provincial to Northern India but extensively located throughout the Indian Peninsula and in Ceylon, Burma, Bangladesh, Thailand and China. It is also grown in partial Egyptian gardens, within Surinam and Trinidad [11,12]. A. marmelos crop plants are globose with a smooth, hard and aromatic shell that is grey-green when raw and yellowish when ripe. The fruit pulp is faded orange, sweet, resinous and noticeably aromatic [13,14]. This fruit is broadly used into folks remedy for the treatment of diabetes mellitus [15]. so properly it is used in the treatment over chronic diarrhea, dysentery and peptic ulcers, as a laxative and in conformity with get better out of respiratory affections [16]. A. marmelos crop plants has been acknowledged in imitation of possess antioxidant [17], radioprotective [18], gastroprotective [19], anti-ulcerative colitis [20], hepatoprotective [21], cardioprotective [22] and antidiabetic [23] activities. A. marmelos fruit possesses excessive nutritional value. The crop is aged to redact juice, jam, syrup, jelly, toffee and other products. The pulp is observed to contain water, sugars, protein, fiber, fat, calcium, phosphorus, potassium, iron, minerals yet nutritional vitamins (Vitamin A, B1, C then Riboflavin) [14,24] as like well as bioactive compounds, kind of carotenoids, phenolics, alkaloids, pectens, tannins, coumarins, flavonoids and terpenoids [25,26]. Therefore, this study was aimed to evaluate the anti-diabetic activity of A. marmelos fruits of methanolic extract against STZ induced diabetes in rats.

Collection of Plant Material
The fruits of Aegle marmelos were brought from Bangalore, Karnataka, India. The plant specimen has been identified and authenticated by department of botany, University of Rajasthan, Jaipur and specimens were kept for the reference. And reference number was RUBL 211761.

Preparation of extract
The fruits of Aegle marmelos were chopped into small pieces and dried under shade at room temperature for seven days. The dried fruits were powdered and passed through the sieve (Coarse 10/40). The powder was used for the preparation of methanolic extract.

Method of extraction
The100gm powder was subjected to extraction with 1000ml methanol in a reflux condenser for 3 cycles of 7hrs each till the volume reduced to half. Extract was filtered through Whattman filter paper No.1 and evaporated to dryness to get constant weight.

Experimental Animals
Female Albino mice (5-6 weeks old) weighing between 25-35gm were taken for toxicity studies and Wistar male rats (8-10 weeks old) weighing 150-200gm was used for the main experiment.  [32] Wistar male rats (150-200g) were considered for this analysis and diabetes induced through I.P., dose of STZ 65mg/kg/b.w. STZ was made freshly before administration and dissolved in the buffer of 0.1 M cold sodium citrate and pH 4.5. In order to avoid hypoglycaemia, STZ-Rats were fed 5% w/v glucose solution for 24 hours. After 72h, rats were recorded Fasting Blood Sugar (FBS) >180 mg/dL and chosen for the analysis. All the animals were given free access to have the tap water and pellet diet and held in polyethylene cages at room temperature. Rat's body weight, FBS levels of rats were taken with one-touch glucometer prior to and after the end of the test, i.e. 0 and 30 days.

Histology of pancreas tissue -H&E staining
The animals were euthanized using high dose of Pentobarbital and then sacrificed and the pancreas of each animal was isolated and was cut into small pieces, preserved and fixed in 10% formalin for two days., dehydrated with alcohol, embedded in paraffin, cut into 4-5 m thick sections, and stained with Haematoxylin-Eosin dye for photomicroscopic observation. The microscopic features of the organs of rats were compared with the control group.

STATISTICAL ANALYSIS
The results are expressed as Mean ± SEM from N=6 rats in each group. Data were analysed using statistical software Microsoft Excel worksheet. The significance of difference among the groups was assessed using Student t-test compared between Normal control (Untreated) vs. all groups p<0.05 were considered significant.

RESULTS
The yield of methanolic extract of fruits of Aegle marmelos was calculated and the % Yield was 27.5.
Mortality was not seen in the acute toxicity up to a dose of 5000mg/kg/b.w. p.o. No signs of illness, gastrointestinal intolerance or abnormal behavior were observed. Hence, noted the dose of 5000 mg/kg was safe and well tolerated. ( Dose: Selection of dose was done on the basis of acute toxicity OCED guideline 425. 5000 mg/kg body weight was tolerated dose and no signs of toxicity have been found, after performing the acute oral toxicity studies. 1/20th and 1/10th of the same dose was selected; 250mg/kg and 500mg/kg respectively and the further study were carried out.

Fig. 6. Effect of Lipid Profile with the treatment of Aegle marmelos (AM) in Diabetic Rats
Values are expressed as Mean ± S.E.M; (n =6/group).     allergy, diabetes, healing wounds, and swollen joints etc [45]. In screening of the toxic concerning of an herbal extract found to be safe and no impact on the test in rats after 14 days of observation. This study presents data on the treatment of diabetic markers, which were shown to be comparable efficacy then the standard one as Insulin, Aegle marmelos has shown marked decrease in the serum glucose level , Total cholesterol, triglycerides, LDL, VLDL, glycosylated hemoglobin, were also found to be a limited range. The HDL cholesterol, serum insulin and pancreatic insulin increased with test drug, increase in islet area was quite considerable. Similarly, mediator of inflammation was assessed and analysis showed Aegle marmelos inhibited moderately in STZ stimulated rats. Free radical concentrations were screened in terms of SOD, CAT, MDA, & GSH. And data revealed that there were significantly changes in the treated groups as compared with STZ rats. The data suggesting, it has the potential alternative and sustainable source for Ayurveda drugs.

CONCLUSION
In drawing the conclusion of the research carried out, the analysis is mainly focused on the toxicity and diabetic markers. Aegle marmelos has significant anti-diabetic activity executed of the present investigation should remain outcome of lower blood glucose levels, enhanced body mass, improvised lipid profile, and notable occurrence of beta cell mass in histopathology studies. The treated diabetic group confirmed notably lowered within the HbA1c levels. Similarly the increase in serum insulin and pancreatic insulin, controlled pro-inflammatory cytokines, anti-oxidant enzyme may additionally facilitate in conformity with prevent diabetic complication.

CONSENT
It is not applicable.

ETHICAL APPROVAL
All the experiments conducted on the animals were in accordance with the standards set for the use of the laboratory animal use and the experimental protocols were duly approved by the IAEC of Karnataka College of pharmacy, Bangalore (Reg. Number: 1564/PO/Re/S/11/CPCSEA).