Effect of Muntingia calabura L. Leaf Extract on Blood Glucose Levels and Body Weight of Alloxan-Induced Diabetic Mice

Diabetes Mellitus (DM) is a metabolic disease characterized by hyperglycemia caused by insufficiency in the number and function of insulin which led to an abnormality in the metabolism of carbohydmicees, lipid, and protein1,2. DM is a noncontagious disease in Indonesia with increasing prevalence and may lead to fatal complications3. Factors related to the increased prevalence of DM in Indonesia include changes in dietary patterns, obesity, urbanization, and lack of exercise4. Several acute and chronic complications can affect DM patients due to oxidative stress caused by hyperglycemia5.


INTRODUCTION
Diabetes Mellitus (DM) is a metabolic disease characterized by hyperglycemia caused by insufficiency in the number and function of insulin which led to an abnormality in the metabolism of carbohydmicees, lipid, and protein 1,2 . DM is a noncontagious disease in Indonesia with increasing prevalence and may lead to fatal complications 3 . Factors related to the increased prevalence of DM in Indonesia include changes in dietary patterns, obesity, urbanization, and lack of exercise 4 . Several acute and chronic complications can affect DM patients due to oxidative stress caused by hyperglycemia 5 .
The insulin hormone has the main function of regulating blood glucose levels. Lack of insulin or inadequate activity of the hormone due to reduced sensitivity of the insulin receptor can cause an abnormality in blood glucose homeostasis 6 . Exposure to the diabetogenic substance of alloxan reduces the level of insulin and disrupts blood glucose homeostasis. Alloxan is a diabetogenic substance that is cytotoxic to the pancreatic islets 7 .
The current oral medicines for diabetes mellitus are in the sulfonylurea, biguanide, and acarbose groups 8 . One of the medicines widely used in Indonesia is glibenclamide. Synthetic drugs, aside from expensive, often cause side effects 9 . On the other hand, natural drugs from selected herbal medicines cost cheaper and have a relatively easy method of application 10-12 .
One of the herbal medicines in Indonesia that can be used for diabetes mellitus is M. calabura, especially its leaves. M. calabura is a fast-growing wild plant that is easy to obtain. The leaves of M. calabura can be utilized as an antidiabetic drug. M. calabura leaf contains high antioxidants. Substance screening from M. calabura leaves showed alkaloid, flavonoid, tannin, saponin, triterpenoid, and steroid 13,14 Several previous studies stated that M. calabura leaf has potential as an antidiabetic drug, including a study by Zulham, Hendrarti, and Wahyuni (2019) 15 who showed that ethanol extract from M. calabura leaves with 50, 100, and 150 mg/kg obtained from macemiceion could reduce blood glucose level in alloxan-induced diabetic mice. The results from Herlina, Amriani, Solihah, and Sintya (2018) 16 also showed that ethanol extract from M. calabura leaves with 65, 130, and 260 mg/kg could reduce blood glucose levels to 28.90%, 32.16%, and 35.66% in male albino mice induced by alloxan.
Based on the statements above, the author is interested in conducting a further study to determine the effect of graded dose of M. calabura leaf extract on blood glucose level and body weight of alloxaninduced diabetic mice. This study is expected to be a new source of information for the public regarding the benefit of M. calabura leaves as an optimal antidiabetic herbal medicine.

Prepamiceion of animals
Male mice (Mus musculus L.), aged ± 3 months, weighing between 25-35 grams were used in this study. The cage used was a plastic caged covered with wire mesh. Every other day, the cage's bottom was coated in 1 cm high husks. The scientific range for light, moisture, and room temperature were adjusted. Ethical code, institutional, and national regulation on live animals were strictly observed. The Health Research Ethical Clearance Commission, Faculty of Dental Medicine, Airlangga University granted ethical permission for the animals used in this investigation, with the number 507/HRECC. FODM/XI/2020.

Prepamiceion of extract
M. calabura leaves were rinsed from dust and heated in an oven at 60 o C. The leaves were then ground with a leaf grinder with a miceio of 1:7 of M. calabura leaf powder immersed in 96% ethanol. They were mixed and let sit still at room tempemiceure for 2 hours. Afterward, the leaves were filtered, and macemicees were sepamiceed and remacemiceed with a miceio of 1:4 with an addition of 96% of ethanol. All macemicees were evapomiceed at 60 o C after filtered to obtain the thick extract. The solvent used was Carboxyl Methyl Cellulose (CMC) with 0.5% concentmiceion to obtain the desired extract.

Flavonoids Test
A solution of hydrochloric alcohol from 2 mL of amyl alcohol was added with Mg powder and 5 mL of M. calabura leaf extract, then vigorously shaken and let to sepamicee. The formation of the yellow amyl alcohol layer showed a positive result.

Tannins Test
Steasny reagent was added to the solution of M. calabura leaf extract, then heated in a water bath to obtain a pink deposit. The result was filtered and the filtmicee from the solution was satumiceed with NaCl and added with gelatin. The formation of white deposits showed a positive result.

Saponins test
A reaction tube containing 10 mL of M. calabura leaf extract shaken for 10 seconds and let still for 10 minutes. The formation of bubbles or foam showed a positive result.

Polyphenol test
3 drops of FeCl reagent were added to 5 mL of M. calabura leaf extract. The formation of a blue-green color showed a positive result of polyphenol.

Quinone test
NaOH of 1 N was added to 5 mL of M. calabura leaf extract. The formation of a red color showed a positive result of quinone. Acute toxicity test M. calabura leaf extract with 5 mg/kg was given to 6 male mice orally using a probe and was observed every 6 hours for 48 hours. The toxic dose was confirmed if there are 3 or more mice died. However, if only one mice died, then the same dose was given to confirm non-toxicity. Repetition of the procedure with a higher dose of 50, 100, and 2000 mg/ kg was performed if there was no death.

Antidiabetic test
The mice were divided into 5 groups, each consisted of 6 mice. Alloxan with 150 mg/kg was dissolved in a NaCl 0.9%, induced peritoneally, and given to all groups of mice except the normal control group. After 5 days of alloxan injection, mice with blood glucose levels higher than 200 mg/dL were included for further treatment. The groups were divided into normal control group which was without alloxan, model control group group with only alloxan, extract treatment group with 100 mg/kg and 300 mg/kg of M. calabura leaf extract, and positive control group 600 µg/kg of glibenclamide. All treatments were applied orally using a probe. On days 1, 7, and 14, blood glucose level was checked on all groups using instant Accu check instan glucometer 17 .
Data analysis SPSS ver. 22 software was used to analyze all data in this study. Oneway ANOVA was used to determine the significance. Tukey was used to test the difference between groups. Differences were considered significant if P<0.05.

RESULTS AND DISCUSSION
Phytochemical screening of M. calabura leaf extracts Antidiabetic effect of M. calabura leaf extracts on blood glucose levels On days 1, 7, and 14, the results of this study revealed significant increase blood glucose levels in the model control group compared to the normal control group (P<0.05). Meanwhile, the M. calabura group and glibenclamide group showed a significant reduction in blood glucose level compared to the model control group (P < 0.05). Effects of M. calabura leaf extracts on blood glucose levels in mice after 14 days treatment can be seen in Table 2.

Antidiabetic effect of M. calabura leaf extracts on body weight
The statistical test results showed weight gain on day 7 and day 14 in the control group and the M. calabura leaf extract group. The glibenclamide group showed significant weight loss on day 7 compared to the model control group. Effect of M. calabura leaf extract on body weight in diabetic mice during 14 days can be seen in Table 3.

DISCUSSION
The active substances in M. calabura include alkaloid, flavonoid, saponin, tannin, polyphenol, quinone, and steroids. The flavonoid content in M. calabura leaf can reduce blood glucose levels. Flavonoid is one of the most found secondary metabolic substances in plant tissues 18 . Flavonoid is known to capture free radicals or function as a natural antioxidant 19,20 . This activity enables flavonoids to capture or neutralized free radicals (such as ROS or RNS) related to the phenolic OH group to repair damaged tissues after induced by alloxan 21 . Flavonoids can also protect the lipid membrane from oxidative damage. Therefore, lipid peroxidation can be inhibited and an increased level of malondialdehyde (MDA) can be prevented 22,23 .
Alkaloids can regenemicee damaged pancreatic β cells. The antioxidant activity from M. calabura leaf is also high and could repair damaged pancreatic β cells. Antioxidant activity can capture free radicals that result in the repair of damaged pancreatic β cells causing DM 1. This leads to increased insulin in the body thus reduced blood glucose in the body 24,25 .
Saponin is a substance containing the isoprene structure of CH 2 =C(CH 3 )-CH=CH 2 26 . This saponin can be detected based on the ability to form foam because saponin acts like a soap. Saponin is an active surface substance grouped as triterpene glycosides. Saponin contained in M. calabura leaf supports the potential of the plant as a diabetic drug. Physiologically, saponin is an active substance that inhibits the absorption of glucose and prevents increased glucose in the blood, thus can be used to treat diabetes 27 . Tannin can stimulate glucose and fat metabolism, preventing these two energy sources from accumulating in the bloodstream. This substance also has hypoglycemic activity by increasing glycogenesis 28 .
Polyphenol can alleviate oxidative stress by contributing hydrogen atoms from the hydroxyl aromatic group of polyphenol to bind free radicals and release them through the excretion system, preventing a chain reaction from superoxide to hydrogen superoxide. Polyphenol has been shown to protect pancreatic cells against the harmful effects of free radicals generated under chronic hyperglycemia conditions. Antioxidant administration can enhance pancreatic β cell mass and keep insulin levels stable 29,30 . Steroids lowered blood glucose levels through altering insulin activity at the cellular level, distal insulin receptors,and reduced glucose production in the liver 31 .
In this study, the mice used were male mice because they have a more stable biological condition compared to female mice, which are not affected by the estrous cycle. Other than that, male mice also have faster drug metabolism. Mice were adapted for 2 weeks to the lifestyle in their new environment and to avoid stress during treatment.
Diabetes mellitus can be caused by several factors. These factors include genetic, nutrition, diabetogenic substance, and free radicals (oxidative stress) 32 . Alloxan is a toxic diabetogenic substance, especially on pancreatic β cells. Alloxan given to animal models such as mice can cause diabetes. The entrance of alloxan into pancreatic cells initiates the cytotoxic mechanism of alloxan. The diabetogenic property of alloxan is determined by the speed with which it is collected. Damage to cells is caused by a combination of mechanisms, including the oxidation of the sulfhydryl group and the production of free radicals. Alloxan damages pancreatic β cells by destroying biological components that contain the sulfhydryl group, cysteine amino acids, and proteins that attach to the SH group (including enzymes containing the SH group) 33 . Alloxan reacts with two SH groups that bind the sides of protein or amino acid and create a disulfide bond, which inactivates the protein and leads to the damaged function of the protein 34  The data were express in mean ± SD (n= 6 of each group). * P < 0.05 (compared with normal control group); *** P < 0.001 (compared with normal control group); # P < 0.05 (compared with model control group); ### P < 0.001 (compared with model control group).  The data were express in mean ± SD (n= 6 of each group). * P < 0.05 (compared with normal control group); # P < 0.05 (compared with model control group).

Groups
that M. calabura leaf with 100 and 300 mg/kg provided a significant effect in reducing blood glucose levels because they provide similar effects to glibenclamide as a positive control.
The positive control group that was given glibenclamide showed a significant decrease in blood glucose compared to the model control group. Glibenclamide is an oral hypoglycemic drug in the group of sulfonylurea that has a therapeutic effect in reducing blood glucose levels. Therefore, it is chosen as a comparison in this study. This is because glibenclamide works by increasing insulin secretion. Glibenclamide works by inducing the secretion of insulin hormone from the granules of the β cells in pancreatic Langerhans islets. Its interaction with ATP -sensitive K channel in β cells membrane cause membrane depolarization, which will open the Ca channel. After the Ca channel opens, Ca 2+ ions will go inside β cells and induce granules with insulin that results in insulin secretion 36,37 .
The results of this study showed that weight loss occurred on day 7 in the model control group because diabetes mellitus patients often had sudden significant weight loss due to insufficient insulin. Therefore, glucose provides a very low energy source in the body and the body took energy from fat and muscles which causes to weight loss. The administmiceion of M. calabura leaf extract in this study helped normalized the weight of mice because the ethanol extract of M. calabura leaf contains antioxidant substances such as flavonoid and saponin that can normalize weight.

CONCLUSION
The administmiceion of M. calabura leaf extract in this study effective in decrease blood glucose level and can normalized the weight of diabetic mice. However, further studies are needed to determine the microscopic effect on pancreatic and liver cells of diabetic mice after administmiceion of M. calabura leaf extract.