Phytochemical Analysis of Bruguiera Gymnorhiza Stem Bark As Antioxidant and Α- Glycosidase Inhibitors

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. The reaction was stopped by adding 100 μL of Na2CO3 (0.2M). Acarbose was used as a positive control with a concentration of 0.1; 0.5; 1; 5; 10 ppm. Solution absorption was measured using an Elisa reader at a wavelength of 410 nm

Analysis GCMS Identification of the active compound on the bark of B. gymnorrhisa was carried out on the sample with the best activity value against α-glucosidase inhibitor (lowest IC50). . A total of 300 mg of sample were dissolved with 3 mL of 99.9% ethanol and injected as much as 1 μL. GC-MS conditions used a capillary column HP-5 (Agilent19091J- 433: 0.25 mm x 30 m x 0.25 μm containing 5% diphenyl 95% dimethylpolysiloxane), the flow rate used was 1.0 mL/minute with an injection temperature of 270 ⁰C in splitless mode, and a pressure of 18, 21 psi. The carrier gas used is He (helium). The MS parameter used is to detect compounds with a mass of 40-800. MS conditions are MS

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

quad temperature 150-200 ⁰C and MS source temperature 250-300 ⁰C. The chromatogram results were analyzed using a database to determine the components of the compounds contained in the extract.

Analysis of data Data were analyzed descriptively by describing the results obtained in tables and figures and analyzing by comparing them with the literature

RESULTS

The yield of each solvent was different (Table 1). The yield of n-hexane extract is the smallest because it only contains wax, fat, and essential oil compounds, while Ethyl acetate extract contains the largest alkaloids, phenolic glycosides, steroids, terpenoids, tannins, and methanol because many compounds are dissolved. Table 1. The yield of B. gymnorrhiza stem bark extract in various solvents Name of Sample Solvent Yield (%) Color of Extract B. gymnorrhiza stem bark n-Hexane 0.90±0.01 Deep green Ethyl Acetate 4.27±0.53 Yellowish green Methanol 12.65±0.64 Deep green

Active compounds in plants are important elements in therapy. In tracing active compounds, the initial examination of active compounds is often carried out through phytochemical screening. In this study, the research is qualitative using this method. The extract contained flavonoids (phenol compounds), which showed a yellow color on TLC with Rf 0.84, 0.76, 0.55, and 0.44. After being evaporated, the ammonia turned brownish yellow and fluorescent blue at UV 366. In tannin identification, after being sprayed with reagent FeCl3, the color of the stain turned black. In the identification of steroids, it shows a green-blue color after spraying with Liberman-Buchard reagent, this is likely the extract contains steroid self-contained at Rf 0.96, 0.6, and 0.31 (figure 1-3 and table 2)

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

Table 2. Results of phytochemical screening with TLC on the bark of B. gymnorrhiza Rf after elution sprayed with reagent description Senyawa Flavonoid uap ammonia visual UV 254 nm UV 366 nm visual UV 254 nm UV 366 nm 0.93 yellow black orange greenish black orange negatif yellow o.87 green blue red green black blue red 0.84 yellow black light blue brownish black light blue positif fluorescence yellow fluorescence 0,76 yellow black light blue brownish black light blue fluorescence yellow fluorescence 0.55 yellow black light blue brownish black light blue fluorescence yellow fluorescence 0.44 yellow black light blue brownish black light blue fluorescence yellow fluorescence

Tannin FeCl3 Reagent 0,89 yellow black black black black black positif 0,80 yellow blue black black - 0.69 yellow red black black - - 0.017 yellow red black black - - Steroid Liberman Bourchad reagent 0.96 green black faint red bluish green black greenish yellow positif 0.83 yellow - yellow black red 0,78 - black red black orange 0.70 - black orange black red 0.60 green black red bluish green black dim red 0.43 - black red yellow black red 0.31 black red bluish green black dim red Antioxidant Activity of B gymnorrhiza Bark Crude Extracts. The results of the observation of antioxidant activity (Table 3) indicated that the value of the concentration of antioxidant compounds was different .

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

Table 3. IC50 value of antioxidant extract B. gymnorrhiza Material Test IC50 Value (Ppm) Antioxidant Category Comparison With Other Literature n-hexane extract ethyl 342,70 ± 68,37 very weak 177.051 acetate extract methanol 12.42 ±0,36 very strong 11.661 extract vitamin C 17,36 ±1,02 very strong 26.192 3,80 ±0.060 very strong - 1Herawati (2012) Soneratia alba hasil fraksi; 2Putri dan Hidajati 2015 (Xylocarpus moluccencis)

In this study, the activity of α-glucosidase enzyme from stem bark extract B gymnorrhiza all extracts was used in the α-glucosidase inhibitor test (Table 4). Table 4. IC50 Values Of The B. gymnorrhiza Extract Sample Values Ic50 (Ppm) n-hexane extract 1014,081±0,08 ethyl acetate extract 181,46 ± 0,12 methanol extract 156,52 ± 0,28 acarbose 151,026 ±0.39 GC-MS analysis was performed on extracts with the best α-glucosidase enzyme inhibitor activity. The compounds detected by this instrument were compounds that could change become a gas or a volatile one because the principle of this instrument is to vaporize compounds at high temperatures. Compound GC-MS results in the methanol extract of B gymnorrhiza stem bark (Table 5) Table 5. The result of compounds of of B. gymnorrhisa extract with GC-MS Run time Name compound Similarity (%) abundance (%) molecular formula 14.62 Hexadecanoic acid 95 18.52 C17H34O2 20.42 Phenol, 2,2 methylenebis 96 81.48 C23H32O2

DISCUSSION

In this study, the yield was different for each extract. The yield is influenced by the polarity of the solvent, temperature, and extraction time (14). In phytochemical fuses, the extract which is the largest in quantity is used. The quantitative test was selected based on the highest yield value (10), and the active compounds in plants were qualitatively very diverse (15). The test was carried out by the phytochemical screening method with the reagent of the appearance of stains on TLC. This screening test is for the initial investigation of the active compounds contained in B. gymnorrhiza, the appearance of stains is intended as a guide for the presence of active compounds with different distances from the predetermined eluent. The identification with UV showed the existing compound is fluorescent or not. This sign is an early guide to the existence of active compounds. Research is continuing on antioxidant activity and α-glucosidase inhibition. The IC50 value in the two activities is different, this is because the level of polarity provides a significant difference in antioxidant activity, there by stopping the radical chain reaction (16). The inhibitory power of the extract is caused by the content and variations of the compounds of each medicinal plant. Following the results of phytochemical screening, the compounds that play an active role as enzyme inhibitors of α-glucosidase are thought to be flavonoids and phenols. Alkaloid and flavonoid compounds in Makassar fruit extract (Brucea javanica (L.) Merr) inhibit the action of the α-Glucosidase enzyme (9). Whereas luteolin, myricetin, quercetin, and kaempferol compounds also inhibit the α-glucosidase enzyme (17)(18). The ability of the extract activity is due to the synergistic effect of the compound that mimics the transition position of the pyranosidic unit of

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 natural glucosidase substrate (17). The analysis GC-MS showed that the components of the methanol extract of the stem bark B. gymnorrhiza consisted of fatty acids and phenols. The compound detected has a low peak. Methanol extract has a similarity (> 90%) is hexadecanoic acid or palmitic acid (BM 270.26 g / mol) and phenol (BM 340 g / mol). These phenolic compounds are thought to have antidiabetogenic activity. The phenol compound, 2,2 methylenebis has the IUPAC name 2-tert-buthyl-6 - [(3-tert-butyl-2-hydroxy-5-methyl phenyl) methyl] -4- methyl phenol. This compound also has another name, namely bis (2-hydroxy-3-tert-butyl-5-methyl phenyl) methane; 2,2'-methylenebis (6-tert-butyl-4-cresol); and GERI-BP002-A. Amorati et al. (2003) stated that the compound 2,2'-methylenebis (6-tert-butyl-4-methyl phenol) had a higher antioxidant activity than 4,4'-methylenebis (2,6-di-tert-butyl phenol) (19). High phenol content is associated with antioxidant activity and there is a correlation to α-glucosidase inhibition (20), polyphenols have an effect on glucose absorption regulation and control of oxidative stress (21), methanol extract on the bark of Sandoricum koetjape contains Palmitic acid detected using GC-MS with Rt 22,651 minutes and abundance (area) 4.06 (13) Palmitic acid enhances lipid profile replacement which contributes to activity hypolipidemic by inactivating the hepatic enzyme HMG-Coa reductase on time of cholesterol synthesis (22).

CONCLUSION

Phytochemical screening on the methanol extract of B gymnorrhiza stem bark showed the presence of flavonoids, tannins, and steroids. Methanol extract has the highest antioxidant activity and α-glucosidase inhibitors and the extract contains hexadecanoic acid and phenol, 2.2 methylenebis

ACKNOWLEDGMENTS

I would like to thank the rector of the Jenderal Soedirman University, who has funded this research through the Project with the Institutional Research grant scheme. REFFERENCES 1. Qasi. M.A, Molvi.K. Herbal medicine: a comprehensive review. Int J Pharm Res. 2016;8:1–5. 2. Partha P.A, Bhusan .P . History of Indian traditional medicine: a medical inheritance. Asian J Pharm Clin Res. 2018;11:421. 3. Ashraf K, Halim H, Lim SM, Ramasamy K, Sultan S. In vitro antioxidant, antimicrobial and antiproliferative studies of four different extracts of Orthosiphon stamineus, Gynura procumbens and Ficus deltoidea. Saudi J Biol Sci. 2020;27:417–32. 4. Rasouli H, Yarani R, Pociot F, Popovic-Djordjevic J. Anti-diabetic potential of plant alkaloids: revisiting current findings and future perspectives. Pharmacol Res. 2020;155. 5. Bernhoft A, Brief A. Review on bioactive compounds in plants. Bioactive compounds in plants -benefits and risks for man and animals. Nor Acad Sci Lett. 2010;50:11–7. 6. Pulungan A, Herqutanto. Diabetes melitus tipe 1: penyakit baru yang akan makin akrab dengan kita. Majalah Kedokteran Indonesia. Maj Kedokt Indones. 2009;3:455–9. 7. Chan EWC, Tangah J, Kezuka M, Hoan HD, Binh CH. Botany, uses, chemistry and bioactivities of mangrove plants II: Ceriops tagal. JournalInternational Soc Mangrove Ecosyst. 2015;13(6):39–43. 8. Sancheti S, Sandesh S, Seo SY. Chaenomeles Sinensis: A Potent α-and β-Glucosidase Inhibitor. Am J Pharmacol Toxicol. 2009;4(1):8–11. 9. Srivastava AK, Tiwari P, Srivasta P , Srivasta R, Mishra A, N Rahuja, et al. Antihyperglycaemic and antidyslipidemic activities in ethyl acetate fraction of fruits of marine mangrove Xylocarpus moluccensis. J Pharm Pharm Sci. 2014;6(1):809–26. 10. Sudirman S,Nurjanah, Jacoeb AM. Proximate compositions, bioactive compounds and antioxidant activity

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Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2021.24369