DEVELOPMENT OF ACTIVE COMPOUNDS FROM ETHYL ACETATE EXTRACT OF RHIZOME Acorus calamus L. AS ANTIDIABETIC

  

DEVELOPMENT OF ACTIVE COMPOUNDS FROM ETHYL ACETATE EXTRACT OF

RHIZOME Acorus calamus L. AS ANTIDIABETIC

Pengembangan senyawa aktif dari ekstrak etil asetat rimpang Acorus calamus L.

sebagai antidiabetes

  

Sri Hartati, Rizna T. Dewi, A. Darmawan and Megawati

  Research Center for Chemistry - LIPI Kawasan PUSPIPTEK Serpong Tangerang Selatan

  

ABSTRAK

Penelitian sebelumnya menunjukkan bahwa ekstrak etil asetat rimpang Acorus calamus L. memiliki aktivitas

biologis dalam diferensiasi pra-adiposit dan potensial dalam pengobatan diabetes tipe 2. Komponen aktif da-

lam ekstrak tersebut adalah 3-β, 22α-trihidroksiolean-30-metoksikarbonil-12 ene-22-O-α-L-rhamnosida dan

β-sitosterol-3-O-β-D-glukosidase. Penelitian ini bertujuan untuk mengisolasi komponen ekstrak etil asetat

rimpang Acorus calamus L yang memiliki aktivitas penghambatan enzim α-glukosidase serta melakukan modi-

fikasi struktur komponen aktif (SAR: Structure Activity Relationship). Hasil penelitian menunjukkan terdapat

4 fraksi semi polar dengan profil KLT yang mirip (fraksi 4, 5, 6, dan 7). Keempat fraksi tersebut menghambat

berturut-turut sebesar 13.54; 13.34; dan 18.92 µg/ml. Selain itu, aktivitas enzim α-glukosidase dengan IC ⁵⁰

terdapat pula 3 fraksi polar (fraksi 20, 21, dan 22) yang aktif dengan IC berturut-turut sebesar 13.55; 3.08;

₅₀

dan 6.85 µg/ml. Studi SAR menunjukkan bahwa β-sitosterol-3-O-β-D-glukosidase diduga memiliki struktur

yang mirip dengan acarbose, suatu obat antidiabetes. Penelitian ini sebaiknya dilanjutkan untuk memperoleh

komponen aktif yang dapat didesain sebagai obat antidiabetes.

  Kata kunci: antidiabetes, α-glukosidase, Acorus calamus L.

  

ABSTRACT

In search development herbal medicine of selected plant for antidiabetic, ethyl acetate extract of Acorus cala-

mus L. plays on biological role in differentiation of preadiposites and posses powerful in diabetes (type 2) and

have known active compounds are 3- β, 22 α-trihiroksiolean-30-metoksikarbonil–12 ene-22-O-α-L-rhamnoside

and β-sitosterol-3-O-β-D-glukosidase. The proposed of this search to isolate active compound which as inhibitor

α-glucosidase activity from ethyl acetate extract and modify of active compounds or (SAR = Structure Activity Re-

lationship). The results of fraction of ethyl acetate extracts were four semi-polar fractions which similar TLC spot

(4, 5, 6 and 7 fractions) were active inhibition of α-glucosidase activity which IC 22.86; 13.54; 13.34 and 18.92

  50

ug/ml respectively and three polar fractions (20, 21 and 22 fractions) were active inhibition of α-glucosidase

activity which IC 13.55; 3.08 and 6.85 ug/ml respectively. SAR studied showed β-sitosterol-3-O-β-D-glukosidase

  50

with suspicion active compound have similarity with acarbose antidiabetic drug. This research should be contin-

ued to establish of active compounds for further on antidiabetic drug design.

  Key words: antidiabetic, α-glucosidase, Acorus calamus L.

  DEVELOPMENT OF ACTIVE COMPOUNDS FROM ETHYL ACETATE EXTRACT OF RHIZOME Acorus calamus L. AS ANTIDIABETIC Pengembangan senyawa aktif dari ekstrak etil asetat rimpang Acorus calamus L. sebagai antidiabetes

  INTRODUCTION

  Diabetes mellitus is a disease in which lev- els of glucose (simple sugar) in the blood is high because the body can not release or use insulin normally. Insulin is a hormone secreted by the pancreas, which is responsible in maintaining normal blood sugar levels. Insulin incorporate sugar into cells so that it can produce energy or stored as energy reserves. The number of diabet- ics worldwide currently is estimated at 150 mil- lion people and this figure is expected to increase to reach 220 million by 2010 and with the current rate of increase as in 2025 will be 300 million. Di- abetes mellitus of these type 2 are 90% (Collene

  et al., 2005; So et al., 2000). According to WHO

  data, Indonesia ranks 4th largest in the number of people with diabetes mellitus in the World. In the year 2000, there are around 5.6 million peo- ple in Indonesia have diabetes. However, in 2006 the estimated number of diabetics in Indonesia increase sharply to 14 million people.

  Acorus calamus L. have been used in the

  Indian and Chinese system of medicine for hundreds years. The radix A. calamus widely used in the therapy of diabetes in traditionally folk medicine of America and Indonesia (Wu et

  al., 2009). Previous study by Wu et al., (2007;

  2009). The ethyl acetate fraction of A. calamus was found to enhance adipocyte differentiation as did roglitazone. A. calamus has potentially to be useful for the treatment of diabetes and car- diovascular complication without body weight gain. The proposed of this search to isolate ac- tive compound which as inhibitor

  α-glucosidase activity from ethyl acetate extract and modify of active compounds 3-

  β, 22 α-trihiroksiolean-30- metoksikarbonil-12ene-22-O- α-L-rhamnoside and

  β-sitosterol-3-O-β-D-glukosidase or (SAR = Structure Activity Relationship).

  materials and metHods Methods of isolation

  Rhizome material dried in oven at a tem- perature of 50 ^o

  C, dry sampel then ground using a grinder with a certain subtlety, then macerated with technical methanol for 2 x 24 hours as much as 3x. Concentrated and the residue was separat- ed and then the concentrate was concentrated by rotary vacuum evaporator to aford methanol ex- tract. Methanol extract partitioned with solvent mixture n-hexane: water (1:1) as much as 3x. The results of partition obtained hexane and water fractions. On the water fraction partitioned with ethyl acetate: water (1:1) obtained ethyl acetate extracts and water fractions. In the same way it is partitioned with butanol, to afford butanol ex- tract and water fraction was dried to obtained water extract. Ethyl acetate extracts was washed again with n-hexane to reduce contained of

  α and β asasron. Ethyl acetate extract was subjected to silica gel G ₆₀ of gravity column chromatography and using gradient mixture of n-hexane–ethyl acetate and methanol as mobile phases. Frac- tions obtained is evaporated then collected and analyzed by thin layer chromatography (TLC) aluminum plates SiGF ₂₅₄ sheet with eluent ad- justed. Results are grouped by suitability TLC spots. The fractions were tested to the activity of α-glucosidase, result of these samples compared with a standard against koji or quersetrin. Ex- tract is considered active when the IC ₅₀ close to or smaller than IC ₅₀ of the standard. Sri Hartati, Rizna T. Dewi, A. Darmawan and Megawati

  α-glucosidase test methods α-Glucosidase reaction mechanism is to catalyze the reaction of substrate to p- nitrofenol and glucose. p-Nitrophenyl-

  C. The reaction was stopped by the addition of 1000 µl solution of 0.2

  SAR (Structure Activyti Relationships) Materials required

  From the linear graph by entering the 50% inhi- bition (Y) will be calculated concentration from the regression formula. Y = ax + b

  IC ₅₀ values (concentration that inhibits 50% of the working enzyme) obtained from the curve equation between% inhibition (Y) as the ordinate axis with a concentration ug / mL (x) as ordinate.

  % Inhibition = C = absorbance of blank (DMSO) S = absorbance of sample (difference ab- sorbance with and without enzyme)

  (C - S) x 100 C

  Presentation inhibitory activity was measured by using the equation:

  µM Na ₂ CO ₃ . The number of p-nitrofenol re- leased measured with spectrophotometer at λ 400 nm.

  C. 250 µl of α-glucosidase enzyme solution was added and incubation was continued for 15 minutes at 37 ^o

  α-D-glukopiranosida was used as substrate, at temperature of 37 ^o C.

  µl test so- lution added 250 µl PNP and 495 µl of phosphate buffer solution, then pre incubation for 5 minutes at the temperature 37 ^o

  α-glucosidase activity inhibitor performed according to Kim Yong-Mu et al. (2005) (kit Waco Chemical Ltd.). In test tube containing 5

  test inhibition of α-glucosidase activity (in vitro)

  Enzymatic α-glucosidase activity is an in vitro method for testing the ability of alternative that is cheaper and faster for initial screening.

  Figure 1. Reaction mechanism decomposition of the substrate p-nitrophenyl α-D- glukopiranosida.

  et al., 2002).

  α-glucosidase, p-nitrofenol generated will be reduced (Artanti

  The enzyme activity was measured by uptake of p-nitrofenol generated. If the sample has the ability to inhibit the activity of the

  In the search process with the relation- ship between structure or activity is better known as SAR (structure activity relationship), the necessary ingredients of computational pro- gram (in this study, the program is used for MVD (virtual molecule Docker), Chem-Office 2-D and

  DEVELOPMENT OF ACTIVE COMPOUNDS FROM ETHYL ACETATE EXTRACT OF RHIZOME Acorus calamus L. AS ANTIDIABETIC Pengembangan senyawa aktif dari ekstrak etil asetat rimpang Acorus calamus L. sebagai antidiabetes

  3D, proteins or enzymes that act as receptors comparison quercetin with 38.49 ug/ml. Where test results ethyl acetate fraction obtained four (α-glucosidase enzyme), and ligand (active com- pound or compounds isolated and target synthe- active fractions that have similar spots semipo- sis). lar TLC are fraction number 4, 5, 6, 7 and three

  Method polar fractions of fractions number 22, 23, 24.

  The method used the of data processing

  IC value of each fraction to 4, 5, 6, 7 are 22.86, ₅₀ 13.54, 13.34 and 18.92 ug/ml, while the IC frac- and analysis using a computer program, by first ₅₀ tions no 22. 23, 24 is 13.55, 3.08 and 6.85 ug/mL. describing ligand compounds using Chem-Office

  2D program, to further sought the position of the Those fraction 5, 6, 22, 23 and 24 showed more most stable structure of the ligand using Chem- actively inhibit the activity of α-glucosidase than the standard koji and quercetin, because these

  3D Office. 3D structure of α-glucosidase enzyme obtained from http://www.pdb.org. Value log P factions have IC values less than blank. While ₅₀ fractions 4 and 7 activity slightly below the stan- (lipofilisitas) obtained through the calculation of ligand 3D structures using the program Hy- dard koji but more active than quercetin blank. perChem. Then the enzyme α-glucosidase as a receptor ligand compound using docking with

  Active fraction

  MVD program to learn the value of the bond en- ergy between them for later comparison with the value of the docking was conducted on a positive standard (acarbose), so based on that value can be known correspondence between receptor and ligand with whether the alleged activities of li- gands will have better or equivalent to a positive Figure 1. Thin layer chromatography (TLC),

  the result of semi polar active fractions eluted with standar. n-hexane and ethyl acetate (9: 1).

RESULTS AND DISCUSSION

  Results of fractination of 311 g of ethyl ac- etate extract of A. calamus are afforded 26 frac- tions is the result of combining fractions base on similarity spotting TLC results. From the results of fractionation was carried out testing of barriers work activities

  α-glucosidase enzyme that can be seen in Table 1. Where in Table 1 these data show

  Figure 2. Thin layer chromatography (TLC) results of

  that the active fractions compared with standard

  the polar active fraction F (f - -f ), F (f _{21 175 178 22 179}

  positive blank koji fraction MTC (methylene

  f - ) and, F (f ). a. eluted with CHCl : _{191 22 192 203 2} chloride) with IC was 14.15 ug/ml and IC is Methanol 5%, b. eluted with CHCl . _{50 50 2} Sri Hartati, Rizna T. Dewi, A. Darmawan and Megawati

  the assumption, that in general compounds that

  Tabel 1. α-glucosidase inhibition activity Test Results

  have similar chemical structure would have or

  Sampel

  IC (ug/ml) ₅₀

  show similar biological activity the same time, it

  Standard Koji (nejorimi-

  14.15 sin) is a basic principle of the SAR.Compounds guide Quercetin

  38.49 F4

  22.86

  is not intended specifically as a clinical agent, but

  F5

  13.54

  it is a starting point to develop compounds that

  F6

  13.34 F7

  18.92

  function in the clinic. Thus, to increase its activ-

  F22

  13.55 F23 3.08 ity then studied the relationship of structure F24

  6.85

  and biological activity clusters by changing the _{COOCH 3} substituents in lead compounds. To predict the activity of synthesized compounds that would _{O Rha} be predicted by using software Molegro Virtual Docking (MVD), HyperChem Pro-6.0, or compare the lead with a compound/ drug (native ligand) _HO glucosidase enzyme, (acarbose, deoxynojirimi- cin, and miglitol) of drugs such as MIMS Catalog _OH Indonesia 2006 (http://www/mims.com) (Fig- ure 3) also with a few isolated compounds of

  3 β,22α,23-trihydroxyolean-30-

  natural materials that have proved active as an inhibitor of-glucosidase, accessible base on line of site (http://pubchem.ncbi.nlm.nih.gov) (Fig- _{HO HO HO HO O O} ure 4). _{HO HO OH}

  OH H C _{HN HO 3 O OH} β-sistosterol-3-O-b-D-glucopyranoside _{(1) HO OH O O OH OH O} Figure 3. Estimated active structure antidiabetes OH _{HO HO OH H C N 3 OH O HO OH OH}

  Study Results SAR (structure Modification O _{(2) HO NH HO HO OH} Study Results) ^OH _{OCH 3 (3)}

  Modification of the structure is the de- velopment of the markers compounds of known

  Figure 3. Some of the structure of α-glucosidase

  biological activity to produce derivatives/ana-

  inhibitor. (1) akarbosa, (2) methyl, β-

  logues. It aims to produce new compounds that

  acarviosinida, (3) 1-deoksinojirimisin

  more effectively and safely used. This is based on

  DEVELOPMENT OF ACTIVE COMPOUNDS FROM ETHYL ACETATE EXTRACT OF RHIZOME Acorus calamus L. AS ANTIDIABETIC Pengembangan senyawa aktif dari ekstrak etil asetat rimpang Acorus calamus L. sebagai antidiabetes

  N ^N _{OH N} ^N _OH ^HO _{Vasicine Vasicinol S} O ^HO ₃ ^{S O} _HO ^{OH OH} _Salacinol Figure 4. Some of the structure of α-glucosidase inhibitors isolated from plants.

  .

  With the software we can determine phar- macopore of compounds that have been known active against α-glucosidase enzyme. The result of calculation parameters can be known whe- ther the compound or our compound has some similarities (similarity) with comparison com- pound (deoxynojirimisin/ gliset) and likelihood (posibility) to synthesize an analog or derivative compound of the isolates. In phase I of this ac- tivity, we have performed calculations using the software to look for similarities sulochrin MVD with the active compounds are α-Gis deoxynoji- rimisin or miglitol (mimic sugar), vasacine and vasacinol (isolated from Adthoda vasica Ness), and salasinol (results isolated from Salacia ob- longa). Doxynojirimisin used as reference com- pounds because these compounds are active as compounds Glyset (oral drug α-glucosidase inhi- bitor) with the mechanism of action as a compe- titive inhibitor. From the calculation of similarity of data obtained as in Table 2.

  Table 2. Calculation results with MVD ligand simila- rity scores No. Ligand Similarity score

  1 Acarbose -398.456 2 β-sitosterol-acorus -362.157 3 deoxinojirimicin -118.83 4 miglitol -133.648

  5 Rhamnosida -acorus -493.623

  6 Salacinol -180.87

  7 Vasicine -178.314

  8 Vasicinol -184.239

  Based on these results compound acar- bose, has the lowest similarity value and close to the value of β-sitosterol-acorus, or it can be said instead that the compound β-sitosterol- acorus has some similarities with the compound / drug acarbose (Figure 5). The next step is to place (docking), compound ligand on the target enzyme (α-glucosidase crystallographic results that can be downloaded on line: http// www. rbcs.pdb), this stage is to determine whether the compound has ligand affinity towards the target (Figure 7). After predicting the binding sites in the enzyme α-glucosidase, the next stage is to calculate the docking score good ligand drug compounds (de- oxynojirimisin and miglitol as reference), com- pounds the comparison (salacinol), and com- pounds the sample as listed in Table 3. Sri Hartati, Rizna T. Dewi, A. Darmawan and Megawati Figure 5. The result of alignment of the lead Figure 6: X-ray crystallographic α-glucosidase compounds with comparable compounds enzyme

  Table 3. Docking calculation results with the MVD score Ligand MolDock Score Rerank Score ACG_989 [Acarbose] -181.76 -165.093 b-sitosterol acorus.MOL -134.266 -115.66 deoksinojirimisin.MOL -60.0821 -58.3225 rahamnosida acorus .MOL -110.567 -46.8888 Pose compounds (ligands) in the binding site can be viewed as shown in Figure 7.

  1

  3

  2 Figure 7. Pose ligand binding sites on α-glucosidase enzyme (MVD docking). 1, Amino acid residues on the side α-glucosidase enzyme bond; 2, native ligandacarbose); 3, didocking ligands (b-sitosterol, rham- nosida, and acarbose).

  DEVELOPMENT OF ACTIVE COMPOUNDS FROM ETHYL ACETATE EXTRACT OF RHIZOME Acorus calamus L. AS ANTIDIABETIC Pengembangan senyawa aktif dari ekstrak etil asetat rimpang Acorus calamus L. sebagai antidiabetes

  CONCLUSIONS

  α-Glucosidase Activity and Postprandial The results activity of (F4, F5, F6 and F7) are Hyperglikemia, Nutrition, 21: 756-761. inhibited 22.86; So WY., Ng MC, Lee Sc., Sanke T., Lee HK., Chan JC.

  α-glucosidase activity which IC ₅₀ 13.54; 13.34 and 18.92 ug/ml respectively and 2000. Genetics of type 2 diebetes mellitus. three polar fractions (F20, F21 and F22) are in- Hongkong Med. J., 6:69-76 hibited 13.55; Wu HS., Li YY., Weng LJ., Zhou CX., He QJ., and Lou

  α-glucosidase activity which IC ₅₀ 3.08 and 6.85 ug/ml respectively. Those fractions YJ. 2007. A Fraction of Acorus calamus L.

  F5, F6, F22; F23 and F24 showed more actively Extract Devoid of β-asaron Enhances Adi- inhibit the activity of α-glucosidase than the stan- pocyte Differentiation in 3T4-Ll Cells, Phy- dard koji and quercetin, because these factions totherapy Research, 21: 262-264. have IC values less than blank. While F4 and F7 Wu SH., Zhu DF., Zhou CH., Feng CR., Lou YJ., Bo Y. ₅₀ activity slightly below the standard koji but more and He QJ. 2009. Insulin Sensitizing activity active than quercetin blank. SAR studied showed of Ethyl Acetate fraction of Acorus calamus

  L. In Vitro and In Vivo, J. of Ethnopharma- β-sitosterol-3-O-β-D-glukosidase with suspicion active compound have similarity with acarbose cology, 123, 288 -292. antidiabetic drug. This research should be con- tinued to establish of active compounds for fur- ther on antidiabetic drug design.

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