Phytochemical Analysis of Bruguiera Gymnorhiza Stem Bark As Antioxidant and Α- Glycosidase Inhibitors
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Warsinah & Diastuti (2020): Phytochemical analysis of B gymnorhiza stem bark Feb 2021 Vol. 24 Issue 3 Phytochemical Analysis of Bruguiera Gymnorhiza Stem Bark as Antioxidant and Α- Glycosidase Inhibitors Warsinah1 and Hartiwi Diastuti2 1 Department of Pharmacy, Faculty of Health Science, University of Jenderal Soedirman, Purwokerto, Central Of Java, Indonesia 2 Department of Chemistry ’Faculty of Mathematics and Natural Sciences, University of Jenderal Soedirman, Purwokerto, Central Of Java, Indonesia Correspondent author: Warsinah, [email protected] Abstract Objective: Bruguiera gymnorrhizza is a mangrove plant used for traditional medicine. This plant contains alkaloid compounds, flavonoids, saponins, tannins, and steroids so that it has potential activity as a source of antioxidants and α-glucosidase inhibitors. Antioxidants are substrate oxidation inhibitors that are easily oxidized, whereas α - glucosidase inhibitors can limit the action of α-glucosidase that digests carbohydrates in the intestine. This study aims to determine the class of active compounds that function as antioxidants and α-glucosidase enzymes in vitro in B. gymnorrhiza stem bark extract. Methods: This research includes extraction using n-hexane, ethyl acetate, and methanol solvent, phytochemical tests with the TLC method, antioxidant activity test with the DPPH method, α-glycosidase inhibitor activity, and active extracts are fractionated with preparative TLC, then active fractions are identified by GCMS. Results: The results showed that the yield of n-hexane extract was 0.90%, ethyl acetate extract was 4.27% and methanol bark extract was 12.65%. Ethyl acetate and methanol extracts are a group of compounds that provide very strong antioxidant activity while the n-Hexane extract is very weak. Ethyl acetate and methanol extracts provide the inhibitory activity of 66.03% and 68.80%, at a concentration of 250 ppm while at acarbose at 90.89%. Thin layer chromatography analysis produced the best eluent, methanol: DCM: n-hexane (2: 3: 1) and showed 5 fractions. Fraction 1 has α-glucosidase inhibiting activity with an IC50 value of 167.5 ppm. Infraction 1 hexadecanoic acid and phenol, 2,2 methylenebis were identified. Conclusion: methanol extract has the highest antioxidant activity and α-glucosidase inhibitors and the extract contains hexadecanoic acid and phenol, 2.2 methylenebis. Keyword: Bruguiera gymnorrhiza, active compounds, inhibitors, antioxidants, α-glucosidase How to cite this article: Warsinah, DIastuti H (2021): Phytochemical analysis of Bruguiera gmynorhiza stem bark as antioxidant and A-glycosidase inhibitors, Ann Trop Med & Public Health; 24(S03): SP24369 DOI: http://doi.org/10.36295/ASRO.2021.24369 INTRODUCTION The development of natural-based medicines is increasing because traditional medicines are proven to be safer than synthetic drugs. this encourages researchers to continue to explore the medical benefits of natural ingredients such as plants. Medicinal plants have been used for many years to prevent and treat various diseases. There are an estimated 750,000 species of plants on Earth, 10% of which are used as food and medicine (1)(2). Medicinal plants have various pharmacological effects such as antioxidants (3), anti-diabetes (4), the benefits of plants as medicine lie in Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2021.24369 Warsinah & Diastuti (2020): Phytochemical analysis of B gymnorhiza stem bark Feb 2021 Vol. 24 Issue 3 the bioactive compound groups such as alkaloids, flavonoids, steroids, triterpenoids, saponins, and tannins. Each in a different amount and quality in plants (5). A shift in diet by consuming fast food and foods/drinks with high sugar content results in an increase in free radicals and sugar in the body. These conditions cause degenerative diseases such as Diabetes Mellitus (DM) with signs of hyperglycemia. Type I diabetes mellitus occurs because pancreatic beta cells do not produce insulin, while diabetes mellitus type 2 is due to a lack of insulin receptors on the surface of the intestinal cells (6). It is currently estimated that 150 million people worldwide suffer from diabetes and it will increase to 300 million by 2025. Globally, the percentage of type 2 diabetes mellitus is greater than 90% (7). α - glucosidase inhibitor (AGI) is an antidiabetic agent with the mechanism of inhibiting the action of the alpha- glucosidase enzyme. A therapeutic approach by reducing the absorption of carbohydrates by the intestine. The use of synthetic inhibitors, such as acarbose, in the treatment of type II diabetes patients, can cause side effects in the form of flatulence (8). So it is necessary to find AGI from natural sources to treat diabetes. Mangroves are a new alternative because of their uniqueness. Several studies have shown that mangroves are effective and potential to inhibit the α-glucosidase enzyme (9). Mangrove plants such as B. gymnorrhiza contain flavonols, flavones, and glycosyl flavones (10), with functions as antioxidants, anti-diarrhea, anti-tumor, antiviral and anti-hyperglycemia (11). Ethanol extract of bark can reduce total cholesterol, Low-Density Lipoprotein (LDL), and triglycerides (12), and reduce blood glucose levels in mice (12). Based on this information, B. gymnorrhiza has the potential as a source of antioxidant compounds and α-glucosidase enzyme inhibitors, so it is necessary to comprehensively study the responsible compounds with scientific evidence METHODS Experimental design This research is laboratory research, descriptive by exploring active compounds. This research was conducted qualitatively by extraction method, phytochemical screening with TLC, and GS-MS profile. The sampling technique was purposive sampling. The sample used was the bark of B. gymnorrhiza. Plant Material The stem bark of Bruguera gymnorrhiza was collected in February 2020 from Arboretum Mangrove Tourism Area "Kolak Sekancil" (Conservation of the Laguna in the Segara Anakan Cilacap Area), and was determined by the Faculty of Biology, Jenderal Soedirman University Chemicals and reagents The chemicals and reagents used were 75% ethanol, aqua dest, 2N HCl, dragendorff reagent, Mayer reagent, 10% NaOH, Liberman-Buchard reagent, Pb acetate, AlCl3 solution, 10% KOH solution, 5% FeCl3, ethyl acetate, chloroform, methanol, n-hexane, butanol, glacial acetic acid, H2SO4 10%. Experimental design this research is a laboratory descriptive study which was conducted qualitatively with the phytochemical s creening method with TLC and GS-MS profiles. The sampling technique was purposive sampling. The sample used was the bark of B gymnorhiza. Preparation of plant extract The B. gymnorrhiza bark was washed under running tap water to remove dirt and foreign materials. The stem was then dried in an oven with a temperature of 40 C for 3 days. After the stem sample had been dried completely, the sample was then crushed into a coarse powder using a heavy-duty blender. Next, the extraction of powder B. gymnorrhiza stem sample was prepared using n-hexane, ethyl acetate, and methanol following (13). Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2021.24369 Warsinah & Diastuti (2020): Phytochemical analysis of B gymnorhiza stem bark Feb 2021 Vol. 24 Issue 3 Phytochemical screening Identification of the constituents present in the B. gymnorrhiza stem, the extract obtained was subjected to three qualitative tests which are flavonoids, tannins, and steroids were using TLC and according to methods reported (13). Silica gel G60 F254 stationary phase preparation / TLC plate with a length of 8 cm and a width of 2 cm, then washed with methanol, then activated in an oven at a temperature of 100oC for 10 minutes, 10 mg of the extract was dissolved in 1 ml of ethanol then bottled in the stationary phase. The mobile phase used methanol: DCM: n -hexane (2: 3: 1) 1. Identification of Flavonoid Compounds The extract was bottled in the stationary phase and then eluted with a solvent mixture of methanol: DCM: n-hexane (2: 3: 1). Then evaporated with ammonia and then viewed in visible light, UV 366 nm. 2. Identification of Steroid Compounds In this identification, the stationary and mobile phases used were the same as in the identification of flavonoids with Liberman-Bouchard reagent stains and heating at 105oC for 5 minutes, then observed in visible light, UV 366 nm 3. Tannin Compound Identification In this study, the stationary phase and the mobile phase were the same as in the identification of flavonoids with the appearance of 5% FeCl3 reagent stains. then observed in visible light, UV 366 nm Antioxidant Activity Test A total of 50 μL of the ethanolic extract of the bark of B. gymnorrhiza was put into a measuring flask, as well as quercetin with various concentrations, then added with 1.0 ml of DPPH 0.4 mM. Furthermore, methanol is added to a volume of 5.0 mL (homogenized by vortex for 1 minute). The absorption was read at the maximum wavelength o f DPPH (517 nm) with methanol blank and control solution. Α-Glucosidase Inhibitor Activity Test (8) The testing of α-glucosidase inhibitor activity was carried out in vitro using the α-glucosidase inhibition method. The methanol extract was dissolved in dimethyl sulfoxide (DMSO) with various concentrations. 1 mg α-glucosidase enzyme was dissolved in 100 mL of phosphate buffer pH 7.0 then added 200 mg of bovine serum albumin (BSA) which had been dissolved in 100 mM phosphate buffer (pH 7.0). The 1 mL enzyme solution had previously been diluted 25 times with 100 mM phosphate buffer (pH 7.0). The reagent mixture used in this test contained 50 μL 0.1M phosphate buffer (pH 7.0), 25 μL 4-nitrophenyl α-D-glucopyranoside 0.5 mM (dissolved in 0.1 M phosphate buffer pH 7.0), 10 μL test samples with various concentrations ( 50, 100, 250, 500, 1000 ppm) and 25 μL of α- glucosidase solution (0.04 units mL-1). The reaction was initiated by adding 25 µL of enzyme solution and 25 µL of phosphate buffer followed by incubation for 30 minutes at 37 ° C.