Antioxidant and Alpha Glucosidase Inhibitor Screening of Merremia peltata L. as Potential Traditional Treatment for Diabetes Mellitus

Diabetes mellitus is a metabolic disorder that is marked by the rise in blood sugar due to a decrease in insulin secretion by pancreatic beta cells and insulin function or disorder1. World Health Organization (WHO) estimates 422 million adults over the age of 18 lived with diabetes globally in 2014 and caused 1.5 million deaths in 20122. The whole world diabetes mellitus prevalence increases continuously and predicted in 2030 people with diabetes mellitus will reach 550 million people, this is equivalent to about three new cases every 10 seconds, or nearly 10 million per year3.


INTRODUCTION
Diabetes mellitus is a metabolic disorder that is marked by the rise in blood sugar due to a decrease in insulin secretion by pancreatic beta cells and insulin function or disorder 1 .World Health Organization (WHO) estimates 422 million adults over the age of 18 lived with diabetes globally in 2014 and caused 1.5 million deaths in 2012 2 .The whole world diabetes mellitus prevalence increases continuously and predicted in 2030 people with diabetes mellitus will reach 550 million people, this is equivalent to about three new cases every 10 seconds, or nearly 10 million per year 3 .
One of the therapies used in the treatment of diabetes is alpha glucosidase inhibitor (eg: Acarbose, Miglitor, and Voglibose) 1 .Acarbose is Food and Drug Administration (FDA) approved for the treatment of adults with type 2 diabetes mellitus.Acarbose is a complex oligosaccharide that acts as a competitive, reversible inhibitor of pancreatic alpha amylase and membrane-bound intestinal alpha glucosidase enzymes.The enzyme breaks down oligosaccharides, trisaccharides, and disaccharides (sucrose, maltose) to monosaccharides (glucose, fructose) in the brush border of the small intestine, so the absorption of monosaccharides after a meal is delayed and transport through the mucosal surfaces into the circulation is interrupted 4 .Unfortunately, there are some side effects of acarbose therapy, like flatulence, diarrhea, and abdominal pain causing uncomfortable to patients 5 .
Indonesia has so many biodiversity consisting of more than 40.000 endemic plants, and 7000 among them reported as medical plants 6 .More than 400 plants have reported as hypoglycemic agent with various mechanism.the plant has some bioactive constituent such as components, phenolics, glycosides, alkaloids, terpenoids, flavonoids 7 .Traditional medicines have been used for a long time and play an important role as alternative medicines.Using herbal medicine prove has less side effects and more acceptable in the Indonesian society also in diabetic therapy.Since ancient times, the ancestors of the Indonesian people have used plants to maintain health and treat various diseases, called "Jamu".Jamu is a traditional herb that can be made from plants, animals, minerals, preparations (galenic) or a mixture of these ingredients, which have been used from generation to generation for treatment based on experience 8 .
Merremia peltata (L.) Merr.) is perennial herbaceous vine from Convolvulaceae family, which often wrapped around the other plants.M. peltata known as "Aka Lambuang" is an ethnobotanical plant used as traditional medicine in Sulawesi, Sumatra, Maluku and Papua.Phytochemical screening showed that leaves extract of M. peltata contains terpenoid, steroid, saponin, and phenolic 9 .Traditionally, M. peltata is used as a potion for inflammation, abdominal pain, wound healing, infectious disease, diabetic therapy 10 .Despite of M. peltata is used traditionally as diabetic therapy, antidiabetic activity of M. peltata has not been investigated.
Previous in vitro and in vivo studies showed that some plants from merremia genus have antidiabetic activity.Plants in the merremia genus are reported to contain phenolic compounds, flavonoids, sulfates, aliphatic pyrrolidine amides, tropane, and alkaloid 11 .In vitro study showed that leaves ethanol extract of Merremia hederacea and hexane fraction of Merremia mammosa had inhibitor activity of enzyme alpha amylase and alpha glucosidase 6,11 .In vivo study using streptozotocin induced diabetic rats showed extract etanol of Merremia tridentata, Merremia mammosa, Merremia emarginata, and Merremia hederacea have potential antidiabetic activity 11,12,13,&14 .Based on the chemotaxonomy of chemical compounds in the same genus, M. peltata has the possibility of having the same pharmacological activity as alpha glucosidase inhibitor for potential antidiabetic agent, but such tests have not been conducted.This research purpose is to investigate in vitro antioxidant activity and alpha glucosidase enzym inhibitor of leaves and stem extract of M. peltata.

Plant material
Samples used in this study were the bark and leaves of M. peltata which obtained from Ratatotok district, North Sulawesi and identified by Herbarium Bogoriensis, Biological Research Centre, Indonesian Institute of Science.

Sample preparation
Fresh Stem and leaves of M. peltata were washed using fresh water and dried in the oven, then crushed with a grinder to get powder simplisia.

Extraction
The dried powder of stem and leaves M. peltata were extracted sequentially using Ultrasonic Assisted Extraction (UAE).The sample (100 g) extracted using N-Heksan (NH) (500 ml) for 30 minutes three times, then filtered to obtain filtrate and residue.The residue was dried and then re-extracted using Ethyl Acetate (EA) then Methanol (ME) using the same method.The filtrate was evaporated using a rotary evaporator to obtain crude extract.

Microscopic observation by light and SEM microscope
The microscopic part of dried powder of M. peltata leaves and stem were observed using light and Scanning Electron Microscope (SEM) microscope.

Antioxidant assay by DPPH inhibition
The antioxidant ability of extract was determined using (1.1-diphenyl-2-picrylhidrazyl) DPPH radical scavenging activity method described by Burda & Oleszek (2001) with slight modification.Ascorbic acid was used for positive control.Five milligram extracts were dissolved in 10 ml methanol solution, and diluted to get sample concentrations 20, 40, 60, 80, and 100 ppm.500 μL sample solution were added into 1.5 mL DPPH solution, mixed for 2 minutes, and incubated in dark room for 30 minutes.After incubation, the sample absorbance was measured using Spectrophotometer UV-Vis in 517 nm.The color change from purple to yellow means free radical scavenging efficiency 15.Free radical scavenging activity was calculated as the percentages of color decreasing of DPPH solution using the following equation: Extract sample (100 μg/ml) was mixed with 3 mL FRAP reagent then the absorbance was measured after 10 min.Ascorbate acid was used as positive control.The calibration curve prepared using FeSO4.7H2Omethanol solution in concentration 100 to 2000 mM and measured concentration of Fe2+ at 593 nm using Spectrophotometer UV-Vis.The amount of Fe2+ produced from the reduction of Fe3+ by the extract was calculated from the standart curve prepared from ferrous sulphate solution and results were expressed as mg Fe2+ / 100 g dry sample.All determinations were performed in triplicate 16 .

Alpha glucosidase inhibitor activity assay
The alpha glucosidase inhibitor was determined using adapted published protocol from Elya et al (2015).Acarbose used as a positive control.Extract of stem and leaves M. peltata were dissolved in maximum 10% DMSO and phosphate buffer solution pH 6.8.Five various concentrations of sample and acarbose were made to determine IC50 of alpha glucosidase inhibitor .Thirty microliter of sample, 36 μL of phosphate buffer solution, and 17 μL PNPG substrate at concentration of 4 mM were put in 96 well microplate.The mixture was incubated at 37°C for 5 min.After incubation, 17 μL of alpha glucosidase enzyme solution 0.025 U/ml was added into each well.The mixture was incubated at 37°C for 15 min.The reaction was stopped by adding 100 μL of Sodium Carbonate (Na2CO3) 200 mM.Absorbance was measured at 405 nm using a microplate reader 17 .Each test was repeated three times.
Percent inhibition of the sample was determined using following equation:

Extraction
Five hundred gram dried powder of stem and leaves of M. peltata were extracted using UAE method.The ultrasonic extraction method was faster and more effective to extract secondary metabolites from plants than conventional methods like maceration or soxhletation 18 .The high power ultrasound (20 t0 25 kHz) could degrade the cell wall and increase the penetration of solvent through the plant cells, enhancing the solubility of phytochemicals..
Extraction was carried out sequentially with different polarity solvent to maximize solubility of phytochemical from the sample, with increasing polarity solvent starting from N-Hexane (NH), Ethyl Acetate (EA), then Methanol (ME).Solvent polarity would affect the type and amount of chemical compounds to be extracted, the antioxidant capacity, and biological activity of the extract 19 .The yield of UAE sequential extraction was displayed in the following table 1. Table 1 showed that NH extract from stem and leaves had the highest amount indicating that the sample contained more non polar than polar substituents.

Antioxidant assay by DPPH inhibition
Antioxidant assay using (DPPH) free radical scavenging method was the first approach for evaluating the antioxidant potential of a compound, developed by Blois (1958) 20 .A stable free radical α, α-diphenyl-β-picrylhydrazyl (DPPH; C18H12N5O6, M=394.33) had odd electron of nitrogen atom.An antioxidant compound donated a hydrogen atom formed hydrogen bond with nitrogen atom in DPPH, marked with decolorization of violet DPPH solution turned to yellow.The absorbance of DPPH at 517 nm was interrupted by light, oxygen, pH, and type of solvent in addition to the antioxidant.Polar solvent may decrease the odd electron density of nitrogen atoms in DPPH and increase the reactivity of DPPH.DPPH in methanol solution had good stability under the light.Incubation under dark room was needed to prevent photochemical decomposition of DPPH that positively correlated with the absorbed light energy 21 Ascorbic acid (AA) was used as a positive control because of its strong antioxidant properties.AA acts primarily as a donor of single hydrogen atoms, and the radical anion monodehydroascorbate reacts mainly with radicals.Together these properties account for the remarkable antioxidant actions of ascorbic acid 22 .
IC50 is the concentration of compounds that have 50% inhibition of DPPH.The compound with higher antioxidant activity will have the lower value of IC50.According to Phongpaichit et al ( 2007), a compound stated as free antiradical very strong when the IC50 value <10 μg / mL, strong if the IC50 value is between 10-50 μg / mL, moderate if the IC50 value ranges from 50-100 μg /mL, weak when the IC50 value is between 100-250 μg / mL and is inactive when IC50 above 250 μg / mL.DPPH inhibition of stem and leaves M. peltata were showed in picture 2. IC50 obtained from a regression linear equation with plotting sample concentration in x axis and % inhibition in y axis of graph.Regression equation and IC50 of the sample were presented in table 3.
AA as a positive control showed very strong antioxidant activity with IC50 value 10.49 μg/mL.

Antioxidant assay by FRAP
The FRAP assay is a relatively simple, quick, and inexpensive method for measuring total antioxidant activity of plant samples.The assay uses the reduction of ferric ions (Fe3+) to ferrous ions (Fe2+), indicated by a colour change from pale yellow color to intensive blue, and absorbance at 598 nm 23 .Calibration curve from ferrous sulfate heptahydrate (FSH) was made as a standart and obtained regression equation y = 0.00257x + 0.04715.The total antioxidant activity of the sample shown in following table 4.
Based on the FRAP assay, stem ME also had the highest total antioxidant power of the plant extract with value 207.08 μmol/g after ascorbic acid as a positive control antioxidant with value 340.04 μmol/g.

Alpha glucosidase inhibitor activity assay
The principle of this test is that a substance that acts as an inhibitor will bind to the α-glucosidase enzyme so that the enzyme activity in hydrolyzing the pNPG substrate (p-nitrophenyl-α-D-glucopyranoside) becomes p-nitrophenol which is yellow in color will be inhibited.The absorbance is measured at 405 nm based on the amount of p-pyrophenol formed.
The in vitro alpha glucosidase inhibitor assay of the M. peltata stem and leaves extract used five variations of sample concentration to get a graph which x axis as concentration and y axis as percent inhibition.Previous phytochemical study by Perez et al (2015), ethanol extract of M. peltata contained flavonoid, alkaloid, and tannin 9 .Many bioactive compounds from different plants have been reported to have hypoglycemic effect, in that mostly phenolics, resin glycosides, and flavonoids have a positive correlation as antidiabetic agents 11 .The ethanol extract shows greater inhibition activity in both stem or leaves of M. peltata compared to other extracts.The presence of flavonoids and phenolic compounds in ethanol extract of M. peltata may act against diabetes mellitus either through their capacity to avoid glucose absorption.

CONCLUSION
M. peltata has potential antioxidant and alpha glucosidase inhibitor activity for diabetic therapy.Antioxidant power of M. peltata extract had positive correlation in alpha glucosidase inhibitor activity.Stem ME has the best antioxidant and better alpha glucosidase inhibitor activity than acarbose as positive control.Phytochemical content of phenolics, resin glycosides, and flavonoids of the M. peltata extract had positive correlation of hypoglycemic activity and had proven as antidiabetic agent in previous study.
blanko (B*) -control of blanko (KB**) A2 : Absorbance of sample (S) -control of sample absorbance (KS***) * : Blanko contains substrate + enzyme, without extract ** : Control of blanko contains substrate and buffer, without enzyme and extract *** : Control of sample contains substrate + extract with the addition of the enzyme after incubation Percent inhibition obtained in each sample was processed in the form of a graph, which x as concentration and y as percent inhibition of sample to get linear regression equation Y= ax + b.The Inhibition Concentration (IC50) was determined using following equation: Af-idah, et al.: Antioxidant and Alpha Glucosidase Inhibitor Screening of Merremia peltata L. as Potential Traditional Treatment for Diabetes Mellitus IC50 =

Figure 1 :
Figure 1: Microscopic observation of stem and leaf M. peltata using light microscope and SEM.Stomata of M. peltata leaf observed using SEM (A and B) and light microscope ©.Vascular system in M.peltata stem observed using SEM (D) and light microscope (E).Calcium oxalate (CaO) found in M. peltata stem observed using light microscope (F).
The stem ME showed the best antioxidant activity of all sample extracts with the lowest IC50 values 47.37 μg/mL.The leaves ME, leaves EA, stem EA, and stem NH were categorized as moderate antioxidant activity with IC50 value respectively 70.15 μg/ mL, 74.20 μg/mL, 81.94 μg/mL, and 99.53 μg/mL.The Leaves NH were categorized as a weak antioxidant activity with IC50 value 108.64 μg/ mL.

Table 1 : The yield of M. peltata extraction using UAE.
Regression equation from the graph used to determine IC50 of the extract.The result showed that Stem ME had the best activity with IC50 value 47.44 μg/mL, almost two times better than acarbose as a positive