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Asian Transactions on Basic and Applied Sciences (ATBAS ISSN: 2221-4291) Volume 02 Issue 01 Development of Antidiabetic Active Compounds from Ethyl Acetate Extract of Acorus calamus L. Sri Hartati, Rizna T. Dewi, A. Darmawan and Megawati diabetes mellitus in the World. In the year 2000, there are Abstract— In research development of herbal medicine from
around 5.6 million diabetics in Indonesia. However, in 2006 selected plants for antidiabetic, ethyl acetate extract of Acorus
the estimated numbers of diabetics in Indonesia increased calamus L. plays on biological role in differentiation of
sharply up to 14 million people.
preadiposites and posses powerful in diabetes mellitus (DM type
Acorus calamus L. (AC) also know as Calamus or 2). Two active compounds are already known are 3-,22--
Sweet Flag have been used in the Indian and Chinese system of medicine for hundreds years. The radix AC widely used for and -sitosterol-3-O--D-glucosidase. The proposed of this
the therapy of diabetes in traditionally folk medicine in research is to isolate antidiabetic active compound with activity
America and Indonesia [4][5]. AC useful in cought, brochitiss, as inhibitor -glucosidase from ethyl acetate extract and modify
gout, inflammation, skin diseases, numbness, general debility, the active compounds to obtained compound with higher activity
and lower side effect based on structure activity relationship
emetic and stomatic, and to treat dispepsia, colic pain, (SAR). From the ethyl acetate extracts obtained four semi-polar
bronchitis, remittent fever and dysentery in children. Ethanol fractions that has similar TLC spot (fraction 4, 5, 6 and 7) that
exract of AC demonstrated significant hypolipidemic activiy was active as -glucosidase inhibitor with IC
[5][6][7]. Previous study by Wu, et al., [8]-[9] showed that 50 values are 22.86,
13.54, 13.34, and 18.92 μg/mL, respectively, and three polar
ethyl acetate fraction of AC was found to enhance adipocytes fractions (fractions 20, 21, and 22) were active as -glucosidase
differentiation as did by roglitazone. AC has potential to be inhibitor with IC50 values 13.55, 3.08, and 6.85 μg/mL,
useful for the treatment of diabetes and cardiovascular respectively. SAR studies showed that -sitosterol-3-O--D-
complication without body weight gain. Purpose of this study glucosidase active compound have similarity with acarbose as
was to isolate the antidiabetic active compound from ethyl positive standard of antidiabetic drug.
acetate extract of AC that acts as -glucosidase inhibitor and Keywords— Antidiabetes, -glucosidase, diabetes mellitus,
Acorus calamus L
sitosterol-3-O--D-glucosidase based on structure activity relationship (SAR). iabetes mellitus (DM) is a disease in which levels of glucose (simple sugar) in the blood is high because the D body can not release or use insulin normally. Insulin is a hormone secreted by the pancreas, which is responsible in A. Isolation maintaining normal blood sugar levels. Insulin incorporate Acorus calamus L. Rhizome material dried at 50oC and sugar into cells so that it can produce energy or stored as made into powder. macerated with methanol for 2 x 24 hours energy reserves. The number of diabetics worldwide currently three times and concentrated using rotary evaporator to aford is estimated at 150 million people, numbers will increase up to methanol extract. Methanol extract partitioned with solvent 220 million by 2010 and 300 million by 2025 with 90% of mixture n-hexane:water (1:1). Water fraction further them were DM type 2 diabetics[1][2][3]. According to WHO partitioned with ethyl acetate and butanol to obtained ethyl data, Indonesia ranks 4th largest in the number of patients with acetate, butanol and water extracts. Ethyl acetate extracts washed with n-hexane to reduce - and -asarone contents. Manuscript received February 12, 2012. Ethyl acetate extract further isolated using gravitation column Sri Hartati is with the Research Center for Chemistry, Indonesian Institute chromatography method with G of Science, Kawasan PUSPIPTEK Serpong, Tangerang Selatan, Banten, 60 silica gel as stationary phase Indonesia. 15314. (corresponding author to provide phone: +62-21-7560929; and n-hexane, ethyl acetate, and methanol as mobile phase is fax: +62-21-7560549; e-mail: [email protected]). eluted in a gradient. The fraction obtained was evaporated, Rizna T. Dewi is with the Research Center for Chemistry, Indonesian collected and analyzed by SiGF254 thin layer chromatography Institute of Science, Kawasan PUSPIPTEK Serpong, Tangerang Selatan, (TLC) aluminum plates using appropriate eluent. Fractions are Banten, Indonesia. 15314. (e-mail: [email protected]). A. Darmawan is with the Research Center for Chemistry, Indonesian grouped according to the TLC spot patterns. Fractions were Institute of Science, Kawasan PUSPIPTEK Serpong, Tangerang Selatan, tested for α-glucosidase activity and compared with Banten, Indonesia. 15314. (e-mail: [email protected]). nojirimicyn and quercetin as positive standard. Extract is Megawati is with the Research Center for Chemistry, Indonesian Institute considered active when the IC of Science, Kawasan PUSPIPTEK Serpong, Tangerang Selatan, Banten, 50 value close to or smaller than Indonesia. 15314. IC50 value of the standards.

Asian Transactions on Basic and Applied Sciences (ATBAS ISSN: 2221-4291) Volume 02 Issue 01 semi-polar fractions (fraction 4, 5, 6, 7) and 3 polar fractions B. -Glucosidase Test Methods (fraction 22, 23, 24) with IC50 values are 22.86, 13.54, 13.34, 18.92, 13.55, 3.08 and 6,85 μg/mL, respectively. All fractions α-glucosidase reaction mechanism is to catalyze the have α-glucosidase inhibitor activity higher than quercetin breakdown reaction of p-nitrophenyl-D-glucopyranosyde (38.49 μg/mL) and only fraction 4 and 7 have α-glucosidase (PNP) substrate to p-nitrophenol and glucose at 37oC inhibitor activity lower than nojirimicyn (14.15 μg/mL) (Table temperature (Fig. 1). The enzyme activity was measured by uptake of p-nitrophenol generated. If the sample has the ability From the TLC profile spots of the active fractions showed to inhibit the activity of the -glucosidase, p-nitrophenol that fraction 4, 5, 6 and 7 (semi-polar fractions), as well as generated will be reduced [10]. -glucosidase enzymatic fraction 22, 23 and 24 (polar fractions) (Fig. 2). method is a cheaper and faster alternatives in vitro method that use as an initial screening test to determine the -glucosidase inhibitor ability of a compound [10]. -glucosidase activity inhibition test performed according to Kim Yong-Mu, et al. (2005) [11] (kit Waco Chemical Ltd.) p-nitrophenyl- Fig. 2. Thin layer chromatography (TLC) results, (a) semi-polar active -glucosidase enzyme fractions, eluted with n-hexane:ethyl acetate (9:1), (b) polar active fractions, eluted with 5% methanol in chloroform, (c) polar active fractions, eluted with α-GLUCOSIDASE INHIBITION ACTIVITY TEST RESULTS p-nitrophenol α-D-glucose IC50 (ug/mL) -glucosidase reaction mechanism C. SAR (Structure Activity Relationships) To determine the relationship between structure and activity (SAR) required several chemical computational facilities such as virtual molecular docker (using Molegro Virtual Docker/MVD), ChemDraw Ultra 10.0, Chem3D Ultra 10.0, α-glucosidase enzyme proteins (1LWJ) as receptors taken from http://www.pdb.org and ligand (active compound Based on literature study, majority antidiabetic active or synthesis target compound). compounds contained in A. calamus are 3β,22α,23- Data processing and analysis using a computer program, by first making the chemical structure of the ligand compound noside and -sitosterol-3-O-b-D-glucopyranoside (Fig. 3) using ChemDraw Ultra 10.0, followed by the the most stable conformation structures of the ligand using Chem-3D Ultra 10.0. α-glucosidase enzymes as a receptor docked with ligand compound using MVD in order to obtain bond energy value between them and compared with bond energy value from acarbose as positive standard. III. RESULT AND DISCUSSIONS A. Fractionation Results and α-glucosidase inhibition activity test Fig. 3. Estimation of the chemical structure of the active compounds from A. calamus plants. (a) 3β,22α,23-trihydroxyolean-30-methoxycarbonyl-12-ene- From 311 g of ethyl acetate extract of A. calamus obtained (rhamnoside-acorus), 26 fractions. All fraction of ethyl acetate was tested its glucopyranoside (-sitosterol-acorus) antidiabetic activity using in vitro -glucosidase inhibition test B. Structure Activity Relationship (SAR) Study method and obtained 7 antidiabetic active fractions, there are 4

Asian Transactions on Basic and Applied Sciences (ATBAS ISSN: 2221-4291) Volume 02 Issue 01 The main purpose of doing chemical structure modification For the first phase, we have to performed calculations using by develop marker compounds that known have biological MVD software to look for similarities between sulochrin and activity is to produce new compounds that more effectively α-glucosidase active compounds such as deoxynojirimicyn, and safely used. This is based on the general assumption that a miglitol, vasacine and vasacinol (isolated from Adthoda vasica compound that have similar chemical structure backbone with Ness), and salasinol (results isolated from Salacia oblonga). the similar pharmacophore groups would have or show similar Deoxynojirimicyn used as reference compounds because these biological activity. Lead compounds guide is not intended compounds are active as Glyset compound (oral drug α- specifically as a clinical agent, but it is a starting point to glucosidase inhibitor) with the competitive inhibitor action develop a new compounds that have clinical function. Study about structure and biological activity relationship of the lead compound undertaken through a change or addition of substituents [12] [13]. The biological activity of synthesize LIGAND SIMILARITY CALCULATION SCORE RESULTS USING MVD SOFTWARE target compounds would be predicted by Molegro Virtual Docking (MVD), HyperChem Pro-6.0, or compared with the Similarity score other lead compounds/drug (native ligand) or drugs such as acarbose, deoxynojirimicyn, and miglitol with α-glucosidase enzyme (Fig. 4), also with some natural product isolated deoxinojirimicin compounds that approved active as an α-glucosidase inhibitor Rhamnosida -acorus http://pubchem.ncbi.nlm.nih.gov) (Fig. 5). Based on the results above, acarbose has the lowest similarity value and close to the value of β-sitosterol-acorus, or it can be said instead that β-sitosterol-acorus compound with the acarbose (Fig. 6). The next step is to place (docking) ligand on the target enzyme (1LWJ) to determine whether the ligand has affinity towards the target (Fig.7). Fig. 4. Some of chemical structure of α-glucosidase inhibitor active compounds. (1) acarbosa, (2) methyl, β-acarviocynida, (3) 1-deoxy-nojirimicyn Fig. 6. Similarity alignment result of the lead compounds with comparable Fig. 5. Some of chemical structure of α-glucosidase inhibitors isolated from plants. (4) vasicine, (5) vasicinol, (6) salacinol. With the software we can determine pharmacophore crystallographic groups of the compounds that have been known active against (http://www.pdb.org) α-glucosidase enzyme. From the calculation results can be known a few parameters that describe whether the compounds After predicting the α-glucosidase enzyme binding site, the have some similarities with standard active compounds next step is to calculate the docking score of the ligand (acarbose, deoxynojirimicyn, miglitol) (Table II) and compound with deoxynojirimicyn and miglitol as a reference, possibility to synthesize an analog or derivative compound.

Asian Transactions on Basic and Applied Sciences (ATBAS ISSN: 2221-4291) Volume 02 Issue 01 salacinol as comparator and synthesize target compounds as [5] K. Heyri, T – H Han, S – G Lee, ―Anti-Inflammatory Activity of water listed in Table III. extract of Acorus calamus L. Leaves on Keratinocyte‖, HaCaT Cells‖, Journal of Ethnopharmcology, 122, 2009, pp. 149 –156. [6] R. S. Parab, Sushma A. Mengi, ― Hypolipidemic activity of Acorus calamus L. in rats‖, Fitoterapia, 73, 2002, pp. 451-455. [7] S. Manikanda, R. Srikumar, and N. Jeva Parthasarathy, ―Protective effect of Acorus calamus L. in free radical scavenger and lipid DOCKING CALCULATION RESULTS SCORE peroxidase in discrete regions of brain against noise stress‖. Biol. USING MVD SOFTWARE Pharm. Bul, (28), 2, 2005, pp. 2327-2330. [8] H-S Wu, Y-Y Li, L-J Weng, C-X Zhou, Q-J He and Y-J Lou, ―A fraction of Acorus calamus L. extract devoid of -asaron enhances adipocyte differentiation in 3T4-Ll Cells‖, Phytotherapy Research, 21, ACG_989 [Acarbose] 2007, pp.262 -264. β-sitosterol acorus [9] H-S Wu, D-F Zhu, C-H Zhou, C-R Feng, Y-J Lou, Yang Bo and Q-J He, ―Insulin sensitizing activity of ethyl acetate fraction of Acorus deoxynojirimicyn calamus L. in vitro and in vivo", J. of Ethnopharmacology,123, 2009, rhamnoside acorus [10] N. Artanti, M. Hanafi and L.B.S. Kardono, ―Inhibition of α-glucosidase Pose of the ligand compound in the binding site can be viewed enzyme activity of Uncaria gambir Roxb. And Taxus sumatrana (Miquel) De Launbenfels. (Published Conference Proceedings style)‖, in as shown in Fig. 8 Proc. 5th National Seminar of Chemistry. (in Bahasa Indonesia), Yogyakarta, Indonesia, 2002, pp.483 -488. [11] Y-M Kim, Y-K Jeong, M-H Wang, W-Y Lee, H-I Rhee, ―Inhibitory Effect of Fine Extract on α-Glucosidase Activity and Postprandial Hyperglikemia‖, Nutrition, 21, 2005, pp.756-761. [12] A. S. Cristoph, F. Wolfgang, H. W. Rudolf, M. R. Bernd, R. L. Klaus and M. v. Janos, ― Automated Docking to Antibodies : Method and Aplications‖ Methode, 20, 2000, p. 280-291. [13] S. Gisbert and B. Hans-Joachim, ― Vistual Screening and Fast automated Docking Methods‖ DDT (Drug Discovery Today), vol 7, (1) Januaray 2002. S. Hartati, Was born in Cilacap (Central Java) Indonesia, March 11,
1956, post graduated of Chemistry, Faculty of Mathematic and Sciences. Padjadajaran University Bandung Indonesia, graduated in 1984. Master Fig. 7. Pose of the ligand on α degree Chemistry of Natural Product, faculty of mathematic and sciences -glucosidase enzyme binding sites (MVD docking). (1) amino acid residues on the side α Indonesia University, graduated in 2000. Doctor Chemistry of Natural -glucosidase enzyme bond, (2) Product, faculty of mathematic and sciences Indonesia University, acarbose native ligand, (3) docked ligand compounds (β-sitosterol, graduated in 2007 . rhamnosida, and acarbose). Position Senior researcher at Natural Product Food and Farmaceutical Division of Research Center for Chenistry LIPI. Since 2001 member of The Indonesian Society of Natural Products Chemistry. Since 2009 member of Indonesian Society for Cancer Chemoprevention. -glucosidase activity test results showed that both semi-polar and polar fractions can inhibit the action of the enzyme α-glucosidase with IC50 22.86, 13.54, 13.34, 18.92, 13.55, 3.08, and 6.85 μg/mL, compared with nojirimicyn (IC50 14.15 μg/mL) and quercetin (38.49 μg/mL). SAR study indicates that the -sitosterol-3-O--D-glucosidase compound is similar with acarbose as the antidiabetic drug with molecular docking score (MVD molecular docking score) -134.266 and -181.76, respectively. [1] L. A. Collene, R. H. Steven, J. A Williams, and W. B. Wolf , ―Effects of a Nutritional Supplement Containing Salacia oblonga Extract and Insulinogenic Amino Acids on Postprandial Glycemia, Insulinemia, and Breath Hydrogen Responses in Healthy Adults‖, Nutrition, 21, 2005, pp. 848-854. [2] D-S Lee, and S-H Lee, ―Genistein, a Soy Isoflavon, is a Potent α- Glucosidase Inhibitor‖, FEBS Letters, 501, 2001, pp. 84-86. [3] Z. Dadan, F Isao, L. Changzheng, I, Kaori, G. Huashi, Z. Jien, ―The Stimulatory Activities of Polysaccharide Compounds Derived from Algae Extracts on Insulin Secretion in vitro‖, Biol. Pharm. Bull., 31 (5), 2008, pp. 921-924. [4] S.R. Yende, U.N. Harle, D. T. Raygure, T. A. Tuse and N. S. Vywahare, ―Pharmacological Profile of Acorus calamus : An Overview‖, Pharmacognosy Reviews [Phcog-Rev]-Suplement, 2 (4), pp. 22 – 26, July – Dec, 2008.

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