Therapeutic Ef icacy ofMimosa Pudica -Aegle Marmelos formulation on Diabetes Mellitus

Anitha V1, Rajarajeswari1, Bupesh G*2,3, Vasanth S2, Tirumalai Vasan P4, Sahoo U5, Pitambar Humane6 1Department of Noinadal, Sri Sairam Siddha Medical College & Research Center, Chennai, Tamilnadu, India 2Sree Balaji Medical College and Hospital, R&DWing, BIHER, Chennai, Tamilnadu, India 3Department of Forest Science, School of Science, Nagaland University (Central), Lumami, Zunheboto, Nagaland-798627, India 4Department of Biotechnology, Srimad Andavar Arts and Science College, Tiruvanaikaval,Tiruchirappalli, Tamilnadu, India 5Department of Forestry, Mizoram University, Aizawl, Mizoram -796004 6Department of Botany, Dharampeth M P Deo Memorial Science College, Nagpur (MS), India


INTRODUCTION
Diabetes is a group of metabolic disorder characterized via a high blood sugar level which results from defects in insulin secretion. Type II Diabetes mellitus is the commonest metaoblic disorder in India. Heritable element, obesity, deskbound existence fashion and aging had been proven to raise the risk for diabetes. Changes in life style plays a major role in Diabetes. The proper medical care and an ordinary tracking of diabetes are crucial now not best to maintain the disorder under control; however, additionally to prevent Diabetes associated Troubles (Ananthan, 2003).
Diabetes mellitus affected nearly 7% of the world population and predicted that it would be the 7 th leading cause of death by the year of 2030. Glucose-6-phosphatase is one of the key enzymes of gluconeogenesis which is involved in dephosphorylation of Glucose-6-phosphate to glucose, as the inal step in gluconeogenesis and glycogenolysis. Fructose1,6bis phosphatase is another enzyme that catalyzes the dephosphorylation of fructose1, 6-bisphosphate to fructose-6-phosphate. Both of these enzymes will be elevated in diabetes, which is mainly due to insulin resistance. During the diabetic condition, the gluconeogenic enzymes are activated or increased, so it leads to the production of more glucose (Viswanathan et al., 2013).
The agents which are most commonly used for the treatment of diabetes mellitus include synthetic drugs and these are identi ied to be coupled with severe adverse effects such as hypoglycemia, weight gain, drug resistance (Selvam et al., 2020). Hence, in recent times medicinal plants have paid more attention for the treatment of diabetes mellitus because of low cost with less damaging consequences. The phytoconstituents present within the medicinal plants or natural method is known to make contributions towards the hypoglycemic assets of that unique plant thereby supporting in the management of diabetes mellitus (Sahariah et al., 2016). In Siddha System, diabetes is correlated as "Madhumegam" -Madhu meaning honey and megam meaning urine -which directly translates to increased sugar in the urine. It is also known as Pramegham, Neerizhivu noi, Thithippu neer. Mathumegam is one among the 20 types of Mega mentioned by the great Sage Yugi. Madhumegam comes under subclass pitha. Madhumegam deteriorates all the seven body constitutions, thus lead to emaciation (Sivaraj et al., 2011).
Aegle marmelos is commonly known as bael. It contains furocoumarins, including xanthotoxol and the methyl ester of alloimperatorin as well as lavonoids, rutin and marmesin a number of essential oils; and, among its alkaloids, a-fargarine(=allocryptopine), Oisopentenylhalfordinol, O-methylhafordinol. Aegeline (N-[2-hydroxy-2(4-methoxyphenyl) ethyl]-3-phenyl-2-propenamide) is a constituent that can be extracted from bael leaves. Leaf juice 10 to 15 ml can be given daily for treating Diabetes. Leaves may be soaked overnight time in the water then ground properly next morning and given with the remaining water to Diabetes patients. Powdered leaves of Aegle and Turmeric powder may be given inside the dose of half teaspoon two times or thrice an afternoon to deal with Diabetic ulcers. Siddha Medicine, Vilvam tablet is ideal to treat Hyperglycemia.
Mimosa pudica Linn is a common herb which grows in the course of in India. The stems are branched, with bristly hairs. The leaves are small lea lets on the stalk, and on touch, they fold collectively. Mimosa pudica Linn is traditionally utilized in Indian system of medication for the treatment of various sicknesses. This short-lived evergreen subshrub is typically handled as an annual. It is grown for its interest value-the fern-like leaves near up and hunch when touched, typically re-beginning inside minutes. It has prickly stems and small, luffy, ball formed purple vegetation in the summertime. It grows to a height of ive ft and spreads around 3 ft-a perennial plants, it grows to a top of 0.5m with a selection of 0.3m. In a few areas, this plant is becoming a noxious weed (Sundaresan and Radhiga, 2015). Mimosa owns the following movements Astringent, Styptic, Alterative and Aphrodisiac. The juice of the whole plant at the dose of 25-30ml in the early morning keeps the blood glucose stages within regular limits. In Siddha system, the leaves and the roots are dried and powdered and given in the dose of 2-5 gm for diabetes. M. Pudica is wealthy in medicinally critical secondary metabolites, consisting of carbohydrates, proteins, amino acids, tannins, phenolics, steroids, lavonoids, saponins, mucilage, and alkaloids. In the present study, the M. Pudica and A.marmelos were evaluated for the antioxidant activity and antidiabetic activity in in-vitro and healthy volunteer trial.

Collection and Formulation of the Antidiabetes drug Mimosa pudica and Aegle marmelos.
The Fresh Leaves and roots of Mimosa pudica and leaves of Aegle marmelos were collected from Sri Sairam Siddha Medical College Herbal Garden, Tambaram. Mimosa pudica and Aegle marmelos were shade dried as a powder. Then it is formulated in the combination (1:0.5) and were dissolved in 200 ml of drinking water and heated up to half (100ml) then it was iltered as a concoction.

In vitro antioxidant assay (DPPH)
2, 2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay: A volume of 1.0 mL of 0.3 mM DPPH in methanol was added to 1.0 mL of formulated drug in different concentrations 50, 100, 150, 250 and 500 µg /mL in test tubes. Ascorbic acid was used as a reference. The contents were mixed and incubated in the dark for 30 min and after that absorbance was read at 517 nm against a reagent blank (Selvam et al., 2020;Vasanth et al., 2018). The inhibition of alpha-glucosidase enzyme activity was determined (Krishnaveni et al., 1984). Incubating a solution to starch substrate (2% w/v maltose) 1 ml with 0.2 M Tris buffer pH 8.0 and different concentrations (20-100 µl) of the formulated drug were added incubation for 5 min at 37 • C. The reaction was initiated by adding 1 ml of the alpha-glucosidase enzyme (1 U/ml) to it followed by incubation for 40 min at 35 • C. Then, the reaction was terminated by the addition of 2 ml of 6 N HCl. Then, the colour development was measured at 540 nm. Acarbose were used as a positive control for amylase inhibitor.

In vitro Pancreatic Lipase Inhibitory Activity
The inhibitory activity against pancreatic lipase was measured using p-nitrophenyl butyrate (p-NPB) as a substrate with a modi ied method from (Zhang et al., 2008). 10 mL of the formulated drug (prepared at concentrations of 20, 40, 60, 80 and 100 µg/mL), positive control (Orlistat, 100 mM) and were pipetted into respective wells of a 96 well plate. Freshly prepared porcine pancreatic lipase was added at fourfold the amount of the test samples, positive and negative controls (40 mL). The plates were initially incubated at 37 C for 15 minutes. Thereafter 170 mL of the substrate solution was added to the wells. The plate was then incubated at 37 was read at 405 nm.

A Pilot Clinical study
A 50-year-old man who was diagnosed with type 2 diabetes two weeks before admission to the hospital, Sri Sairam Siddha Medical College and Research Center. He had a strong family history of type 2 diabetes. He smoked heavily (> 20 cigarettes/day) and had no habit of alcohol consumption. His treatment started following the irst line of treatment. His fasting blood glucose level was 181 and posted prandial 246mg/dl. His blood pressure was 140/80 mmHg, pulse was 85bpm, and the temperature was 98.7 • F, hemoglobin of 15.1 g/dl. His Liver Functions and Renal functions were normal. He was prescribed with the formulated Mimosa pudica and Aegle marmelos for 30 days.

RESULTS AND DISCUSSION
After 30 days Fasting Blood Glucose reduced to 130mg/dl and Post parandial back to 170 mg/dl. He did not have any adverse effects during the course.

In-vitro Alpha Glucosidase Inhibitory Activity
The formulated drug revealed a considerable inhibitory action of the alpha-glucosidase enzyme. The percentage inhibition at a 20-100µl dose of the formulated drug showed a dose dependent increase in percentage inhibition. The percentage inhibition varied from 41.4% -12.42% for the highest concentration on the lowest concentration ( Figure 2).
There was a dosage-dependent increase in percentage inhibitory activity against the pancreatic enzyme. It showed 50% inhibition at a concentration of 100µg mL-1 (Figure 3). In the in vitro experiment, the ability to extract had condensed tannins as an inhibitor of pancreatic lipase.
The lectin extract of Aegel marmelos has antibacterial and anti-diabetic activity (18). The ethanolic extract of Aegel marmelos cause a reduction in blood sugar from the seventh day after continuous administration of the extract and on 28th day, the level of the sugars was found to be reduced by 54% (Avula et al., 2016).
The beta cells may be regenerated in Type 2 diabetic patients on GS 4 of Gymnema and lectin of aegle supplementation. GS 4 supplementation for 18 -20 months raised insulin levels in the serum of patients (Baskaran et al., 1990). Preliminary human research reports that Gymnema and its extract GS4 may be bene icial in patients with Type 1 or Type 2 Diabetes when it is added to diabetes drugs being taken by mouth or to insulin (Bhavani, 2014). The main cause of Type 2 diabetes is insulin resistence and /or de iciency of this receptor for a hormone which causes hyperglycemia. The key strategy in treating the diabetic patient should be the maintenance of Normoglycemia (Chatham-Stephens et al., 2017).
From the results of alpha-glucosidase enzyme inhibition assay, it was observed that the formulation drugs reveals highest percentage inhibition ranges from 41.4% -12.42% at the concentration of 10 µg/ml to 100µg/ml. The antidiabetic effect of Mimosa pudica is mainly due to the presence of phenolic and lavonoid compounds present in the plant Mimosa pudica (Lakshmibai et al., 2015;Rajendiran et al., 2017). Mimosa pudica alters the insulin and glucose levels and thus maintain the Glycated haemoglobin levels under control. Mimosa pudica treatment improves insulin sensitivity and attenuates fat accumulation in the liver (Muhammad et al., 2016).
Pancreatic lipase inhibition is one of such attempts and many researchers focused on the potential ef icacy of natural products as antiobesity agents (Yun, 2010). According to Siddha literature evidences as for available now, it is mentioned that Diabetes occurs as a result of impairment in day to day activities or Lifestyle modi ications (Khan et al., 2012;Mathew et al., 2008). These changes affect the body metabolism. Finally results in glucose intolerance.
Medicinal plant compounds are still the most accessible resource of pancreatic lipase inhibitors. Therefore, we investigated biologically active compounds from Mimosa pudica using different extraction ratios of petroleum ether to water.
Extracts under different concentrations of Mimosa pudica were tested for α-glucosidase inhibitory activity. The methanolic extract showed higher pancreatic lipase activity than aqueous extract, whereas petroleum ether and chloroform extract did show moderate inhibit pancreatic lipase at all. Notably, neat alcoholic (methanolic) extracts exhibited stronger inhibitory effects than their corresponding aqueous mixtures.Methanolic extract of Mimosa pudica showed signi icant antidiabetic and antihyperlipidemic activities on streptozotocin-induced diabetes mellitus in rats (Parasuraman et al., 2019).

CONCLUSIONS
The formulated drug Mimosa pudica and Aegle marmelos administered 100 mg concentration can signi icantly reduce the blood glucose level. Further, the in-vitro assays of antidiabetic and antioxidant of the formulated drug (herbal combination) demonstrate greater ef icacy against anti-diabetes Mellitus and demonstrated property. Moreover, they are a low cost-effective when compared to the current medications used as hypoglycemic drugs.