The antidiabetic activity test through a mechanism of inhibition of α-glucosidase enzyme was studied against ethanol, n-hexane, ethyl acetate and n-butanol fractions of ethanol extract of Artocarpus altilis (Parkinson) Fosberg (Moraceae) leaves and four flavonoid compounds isolated from ethyl acetate extracts of A. altilis. Ethyl acetate fraction has strongest antidiabetic activity compared to ethanol, n-hexane, and n-butanol fractions with IC50values5.98,6.79, 440.18and14.42μg/mL, respectively. Four flavonoid compounds (1-(2,4-dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-5-yl]-1-propanone (AC-31), 2-geranyl-2',3,4,4'-tetrahydroxy dihydrochalcone (AC-51), 8-geranyl-4',5,7-trihydroxyflavone (AC-33) andcyclocommunol (AA-3), have been isolated from ethylacetate fraction. AC-31 was the strongest antidiabetic compound compared to AC-51, AC-33 and cyclocommunolwithIC50values are 15.73, 24.41,49.49,and72.20μg/mL. Kineticstudies of AC-31 using Lineweaver-Burk method showed that inhibition mechanism of enzymeα-glucosidase was anon-competitivetype

Akhmad Darmawan

Chandra Risdian

Puspa Dewi Narrij Lotulung

Tjandrawati Mozef

Indonesian Journal of Chemistry

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Indo. J. Chem., 2014, 14 (1), 7 - 11Puspa Dewi Narrij Lotulung et al.7* Corresponding author. Tel/Fax : +62-22-2503051/2503240Email address : tjandrawm@yahoo.comIN VITRO ANTIDIABETIC ACTIVITIES OF EXTRACT AND ISOLATED FLAVONOIDCOMPOUNDS FROM Artocarpus altilis (Parkinson) FOSBERGPuspa Dewi Narrij Lotulung, Tjandrawati Mozef*, Chandra Risdian, and Akhmad DarmawanResearch Center for Chemistry, Indonesian Institute of SciencesKampus LIPI Gd. 50, Jl. Sangkuriang Bandung, IndonesiaReceived November 4, 2013; Accepted December 13, 2013ABSTRACTThe antidiabetic activity test through a mechanism of inhibition of α-glucosidase enzyme was studied against ethanol, n-hexane, ethyl acetate and n-butanol fractions of ethanol extract of Artocarpus altilis (Parkinson) Fosberg(Moraceae) leaves and four flavonoid compounds isolated from ethyl acetate extracts of A. altilis. Ethyl acetatefraction has strongest antidiabetic activity compared to ethanol, n-hexane, and n-butanol fractions with IC50 values5.98, 6.79, 440.18 and 14.42 μg/mL, respectively. Four flavonoid compounds (1-(2,4-dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-5-yl]-1-propanone (AC-31), 2-geranyl-2',3,4,4'-tetrahydroxydihydrochalcone (AC-51), 8-geranyl-4',5,7-trihydroxyflavone (AC-33) and cyclocommunol (AA-3), have been isolatedfrom ethyl acetate fraction. AC-31 was the strongest antidiabetic compound compared to AC-51, AC-33 andcyclocommunol with IC50 values are 15.73, 24.41, 49.49, and 72.20 μg/mL. Kinetic studies of AC-31 using Lineweaver-Burk method showed that inhibition mechanism of enzyme α-glucosidase was a non-competitive type. Keywords: Artocarpus altilis; antidiabetic; α-glucosidase; flavonoid ABSTRAKUji aktivitas antidiabetes melalui mekanisme penghambatan aktivitas enzim α-glukosidase telah dilakukan terhadap fraksi etanol, n-heksana, etil asetat, dan butanol dari ekstrak etanol daun Artocarpus altilis (Parkinson)Fosberg (Moraceae) dan empat senyawa flavonoid hasil isolasi dari ekstrak etil asetat tanaman yang sama. Fraksietil asetat mempunyai aktivitas antidiabetes paling kuat dibanding dengan fraksi etanol, n-heksana dan butanoldengan nilai IC50 berturut-turut 5,98, 6,79, 440,18 dan 14,42 μg/mL. Empat senyawa flavonoid (1-(2,4-dihidroksifenil)-3-[8-hidroksi-2-metil-2-(4-metill-3-pentenil)-2H-1-benzopiran-5-yl]-1-propanon (AC-31), 2-geranil-2',3,4,4'-tetrahidroksidihidrochalkon (AC-51), 8-geranil-4',5,7-trihidroksiflavon (AC-33) dan siklokomunol (AA-3)merupakan senyawa-senyawa hasil isolasi dari fraksi etil asetat. AC-31 merupakan senyawa dengan aktivitasantidiabetes paling kuat dibanding dengan AC-51, AC-33, dan siklokomunol dengan nilai IC50 berturut-turut 15,73;24,41; 49,49 and 72,20 μg/mL. Studi kinetik yang dilakukan menggunakan metoda Lineweaver-Burk menunjukkan bahwa mekanisme penghambatan aktivitas enzim α-glukosidase oleh senyawa AC-31 mempunyai tipe non-kompetitif.Kata Kunci: Artocarpus altilis; antidiabetes; α-glukosidase; flavonoidINTRODUCTIONDiabetes mellitus (DM) is a chronic disease causedby inherited and/or acquired deficiency in production ofinsulin by the pancreas, or by the ineffectiveness of theinsulin produced [1]. DM can be classified into 2 types:DM type 1 (insulin-dependent diabetes mellitus or IDDM)and DM type 2 diabetes (non-insulin dependent diabetesmellitus or NIDDM). In DM type 2, insulin-resistance is acharacteristic feature and several drugs to increase theinsulin sensitivity are currently being used in clinic [2]. Atthe present time, it is estimated that 150 million peopleworldwide have diabetes and that this will increase to220 million by 2010 and 300 million by 2025 [3].Indonesian health research foundation in 2007showed that DM is a serious health problem forIndonesian peoples with 14.7% proportion of deathsdue to diabetes disease in the age 45-54 years.DM is a debilitating and often life-threateningdisorder with increasing incidence throughout the world[2]. Diabetic complications arise partly fromglycosylation damage to structural and functionalproteins and reflect chronic failure to maintain bloodglucose homeostasis. Other complications such asdiabetic nephropathy, diabetic retinopathy, diabeticneuropathy and diabetic cardiomyopathy prevail as aresult of hyperglycemia. The pathophysiology involvesan interaction between metabolic and hemodynamicIndo. J. Chem., 2014, 14 (1), 7 - 11Puspa Dewi Narrij Lotulung et al.8factors. Metabolic factors include advanced glycation,increased formation of polyols and activation of proteinkinase-C. Hemodynamic factors include systemichypertension, intraglomerular hypertension and the roleof vasoactive hormones, such as anglotensin II. Clinicalcourse progresses from microalbuminuria to overtproteinuria and then to renal failure [5].Dietary intake is related to blood glucose levels,and it is considered to be important to suppresshyperglycemia induced by overeating in order to preventworsening of the symptoms in the hyperglycemicindividuals with uncertain diabetic status. Inhibition of α-glucosidase is one way that could do in order tosuppressing postprandial hyperglycemia. By inhibitionthe activity of enzyme will affect to the delaying glucoseabsorption, controlling the hyperglycemia [6-7] andreducing chronic vascular complications [8]. Therefore,by using α-glucosidase inhibitors is a cost-effective means to preventing the progression of diabetes [9-10].Several inhibitors of this enzyme have beenreported such as acarbose, voglibose, miglitol [11],nojirimycin and 1-deoxynojirimycin from plants [12].Plants as sources of medicinal compounds have longbeen used by people. With a wealth of biodiversity,Indonesia has potential resources of various plants withvarious active compounds content which could inhibit theactivity of enzyme α-glucosidase to overcome the disease diabetes [3-4]. Artocarpus altilis (Parkinson)Fosberg is a tropical plant, widely distributed in thetropics regions including Indonesia. Various parts of theplant, from leaves, latex, bark, roots were used astraditional medicine for various diseases ranging frominfectious diseases (skin, urinary tract, and ear),diarrhea, dysentery, oral inflammation, fever, liver todiabetes and hypertension [13]. In vivo experimentsshowed that ethanol extract of A. altilis leaves (10 and20 mg/kg) can reduced blood glucose levels significantly[16]. In this study, we investigated the inhibitory effect ofA. altilis leaves extracts and four isolated flavonoidcompounds on α-glucosidase activity. We also investigated kinetics inhibition reaction of thecompounds.EXPERIMENTAL SECTIONMaterialsArtocarpus altilis leaves was collected fromParung, Bogor, Indonesia in March 2006 and determinedat Herbarium Bogoriense, Research Center for Biology,Indonesian Institute of Sciences.Four flavonoid compounds isolated from A. altilis(collection of Research Center for Chemistry, IndonesianInstitute of Sciences) AC-31 (1-(2,4-dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-5-yl]-1-propanone), AC-51 (2-geranyl-2',3,4,4'-tetrahydroxydihydrochalcone), AC-33 (8-geranyl-4',5,7-trihydroxyflavone) and cyclocommunol (AA-3)[13-14].Ethanol, n-hexane, ethyl acetate, n-butanol,enzyme α-glucosidase, p-nitrofenil-α-D-glukopirano sida (p-NPG), bovine serum albumin and quercetinfrom Sigma. Solution of sodium bicarbonate andDimethylsulfoxide (DMSO) from Merck.InstrumentationIn this study, we used various instruments tosupport our research such as: macerator, rotaryevaporator, and ELISA reader.ProcedureExtractionPowdered and dried of A. altilis leaves (4.95 Kg)were extracted exhaustively with ethanol 70%. Theethanol extracts (250 g) were concentrated usingvacuum rotary evaporator and then partitioned withn-hexane, ethyl acetate and n-butanol.α-Glucosidase activity. Xα-glucosidase enzyme activity was analyzed using Sancheti method (Sanchetiet al., 2009) with modifications. 0.001% enzyme stocksolution dissolved in phosphate buffer solution (pH 7)contained 0.2% bovine serum albumin. Enzymaticreactions by mixing 25 μL 20 mM p-NPG as the substrate, 45 μL phosphate buffer solution and 5 μL samples were then added to the homogeneousα-glucosidase enzyme was diluted 50 times in phosphate buffer solution. The reaction mixture thenwater-bath incubated at 37 °C for 30 min. The reactionwas stopped by the addition of 100 μL 0.2 M of Na2CO3solution. The resulted p-nitrophenol was measured atλ 400 nm using ELISA reader. The experiments were performed with 3 replicates and % inhibition wascalculated using the following formula:C S% x100%CInhibitionC absorbance without sampleS absorbance with the sampleKinetics inhibition against α-glucosidase enzymeKinetics inhibition against α-glucosidase enzyme was analyzed by measuring the formation of p-NP onthe time interval 0 to 20 min with a variety of p-NPGsubstrate concentrations between 1 to 20 mM with orwithout samples. Linewaver-Burk method used todetermine the type of inhibition to create a graph plot1/v (speed of reaction) against 1/[S] (substrateconcentration changes are directly proportional to theproduct formation).Indo. J. Chem., 2014, 14 (1), 7 - 11Puspa Dewi Narrij Lotulung et al.9Fig 1. Chemical structure of flavonoid compounds isolated from A. AltilisTable 1. Enzyme α-glucosidase inhibition activity of  A. altilis leaves ethanol extract and its fractions.IC50 (μg/mL)  Quercetin 11.60Ethanol 6.79n-Hexane 440.18Ethylacetate 5.98n-Butanol 14.42RESULT AND DISCUSSIONα-Glucosidase inhibition activities of each sample was compared by IC50 value resulted in the experimentalcondition. Smaller IC50 values mean stronger inhibitionactivity. Ethanol extract showed stronger inhibitionactivity against α-glucosidase compared with quercetin as positive control with IC50 6.79 μg/mL. Further fractionation of the ethanol extract showed that the ethylacetate fraction was the strongest fraction with IC50 5.98μg/mL (Table 1). This result suggests that the active compounds from the ethanol extract concentrated onsemi polar fraction, and flavonoids were the active majorcompounds contained in A. altilis plant.AC-31 (1-(2,4-dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-5-yl]-1-propanone), yellow crystal,1H-NMR (in CDCl3, ppm)δH 6.38 (1H, d, J= 2.45 Hz,. H-2); 6.37 (1H, dd, J= 2.45;8.5 Hz, H-4), 7.54 (1H, d, J= 8.5 Hz, H-5); 3.1 (2H, q, H-8); 2.9 (2H, M, H-9); 6.60 (1H, d, J= 8.5 Hz, H-11); 6.72(1H, d, J= 8.5 Hz, H-12); 6.54 (1H, d, 9.8 Hz, H-16); 5.63(1H, d, 9.8 Hz, H-17); 1.70 (2H, m, H-19); 2.09 (2H, m,H-20); 5.08 (1h, t, H-21); 1.66 (3H, s, H-23); 1.57 (3H,s, H-24); 1.28 (3H, s, H-25);13C-NMR (in CDCl3, ppm)δC 165.2 (C-1), 103.6 (C-2), 163.48 (C-3), 108.3 (C-4),132.45 (C-5), 113.6 (C-6), 204.1 (C-7), 39.8 (C-8), 26.7(C-9), 128.8 (C-10), 121.29 (C-11), 114.7 (C-12), 143.1(C-13), 139.7 (C-14), 119.2 (C-15), 119.5 (C-16), 130.3(C-17), 78.9 (C-18), 40.9 (C-19), 22.9 (C-20), 123.9(C-21), 132.1 (C-22), 25.8 (C-23), 17.8 (C-24), 26.2(C-25). Molecular weight with [M+] 409.5460 [17].AC-51 (2-geranyl-2',3,4,4'-tetrahydroxydihydrochalcone), yellow gum,1H-NMR (in CDCl3, ppm)δH 6.71 (1H, d, 8.2 Hz, H-5); 6.65 (1H, d, J= 8.2 Hz,H-6); 3.11 (2H, t, J= 7.8 Hz, H-α); 2.97 (2H, t,  J= 7.8 Hz, H-β), 6.37 (1H, d, J= 2.4 Hz, H-3’); 6.35 (1H,dd, J= 8.7, 2.4 Hz, H-5’); 7.58 (1H, d, J= 8.7 Hz, H-6’);3.40, (2H, d, J= 6.7 Hz, H-1”); 5.17 (1H, brt, J= 6.7 Hz,H-2”); 2.06 (2H, t, J= 6.2 Hz, H-4”); 2.08 (2H, q,J= 6.2 Hz, H-5”); 5.02 (1H, brt, J= 6.2 Hz, H-6”); 1.79(brs, 3H, H-8”); 1.57 (brs, 3H, H-9”); 1.66 (brs, 3H,H-10”).13C-NMR (in CDCl3, ppm) δC 131.1 (C-1), 126.1(C-2), 142.4 (C-3), 142.6 (C-4), 112.9 (C-5), 121.3(C-6), 113.5 (C-1’), 164.9 (C-2’), 103.5 (C-3’), 163.0(C-4’), 108.0 (C-5’), 132.2 (C-6’), 204.0 (C=O), 39.6 (α), 27.7 (β), 25.8 (C-1”), 121.7 (C-2”), 138.6 (C-3”), 39.6 (C-4”), 26.3 (C-5”), 123.7 (C-6”), 132.1 (C-7”), 25.7(C-8”), 17.7 (C-9”), 16.2 C-10”) [19].AC-33 (8-geranyl-4',5,7-trihydroxyflavone), whitecrystal,1H-NMR (in CD3OD, ppm) δH 5.3 (1H, dd, H-2);2.70 (1H, dd H-3a); 3.06 (1H, dd, H-3b); 5.93 (1H, s,H-6); 3.18 (2H, dd, H-9); 5.14 (1H, t, H-10); 1.91 (2H, t,H-12); 2.02 (2H, t, H-13); 5.04 (1H, t, H-14); 1.55 (3H,Indo. J. Chem., 2014, 14 (1), 7 - 11Puspa Dewi Narrij Lotulung et al.10Table 2. Enzyme α-glucosidase inhibition activity of the isolated flavonoid compounds from A. altilis leavesIC50 (µM)Quercetin 38.40AC-51 24.41AC-31 15.73AC-33 49.49AA-3 72.20Table 3. Vm and Km values of the enzymatic reactionsof α-glucosidase with and without inhibitor (AC31) Vm (mM/mnt) Km (mM)Without Inhibitor 0.033± 0.009 3.246± 2.242AC-31 1.5 mM 0.012±0.001 4.262±0.413AC-31 3 mM 0.009±0.002 2.934±0.170 Fig 2. Kinetics inhibition of enzyme α-glucosidase by AC-31 compounds, H-16); 1.61 (3H, s, H-17), 1.56 (3H, s, H-18); 7.31(2H, d, J= 8.56 Hz, H-2’/6’); 6.81 (2H, d, J= 8.56 Hz,H-3’/5’).13C-NMR (in CD3OD, ppm) δC 80.4 (C-2), 44.1(C-3), 198.2 (C-4), 103.4 (C-4a), 163.2 (C-5), 96.5 (C-6),161.7 (C-7), 109.2 (C-8), 166.2 (C-8a), 22.5 (C-9), 124.1(C-10), 135.3 (C-11), 40.9 (C-12), 27.8 (C-13), 125.6(C-14), 132.1 (C-15), 17.9 (C-16), 25.9 (C-17), 16.3(C-18), 131.5 (C-1’), 129.0 (C2’/6’), 116.3 (C-3’/5’), 159.0(C-4’). Molecular weight [M+] 409.0832 [5].Cyclocommunol, yellow amorphous powder,1H-NMR (in CD3OD, ppm) δH 1.69 (3H, d, J= 1.2 HzH-14); 1.95 (3H, d, J= 1.4 Hz, H-15); 5.47 (1H, d,J= 9.5 Hz, H-12); 6.20 (1H, d, J= 9.5 Hz, H-11); 6.43(1H, d, J= 2.0 Hz, H-3’); 6.63 (1H, dd, J= 2.0, 8.5 Hz,H-5’); 7.70 (1H, d, J= 8.4 Hz, H-6’); 6.26 (1H, d,J= 1.8 Hz, H-6); 6.52 (1H, d, J= 1.8 Hz, H-8); 12.88 (1H,s).13C-NMR (in CD3OD, ppm) δC 160.5 (C-2), 109.4(C-3), 180.7 (C-4), 159.5 (C-5), 100.7 (C-6), 166.2 (C-7),95.7 (C-8), 164.8 (C-9), 106.4 (C-10), 71.0 (C-11), 123.3(C-12), 132.8 (C-13), 18.8 (C-14), 26.1 (C-15), 110.9(C-1’), 140.0 (C-2’), 105.8 (C-3’), 165.5 (C-4’), 111.8(C-5’), 127.4 (C-6’). Molecular weight [M] 352.0949 [18].AC-31, AC-51, AC-33, and cyclocommunol wereflavonoid compounds isolated previously from A. altilisleaves [5,17-19]. AC-31 showed the lowest IC50compared to quercetin, AC-51, AC-33 andcyclocommunol with IC50 value 15.73. Its means thatAC-31 has the strongest activity against enzyme α-glucosidase, followed by AC-51 which also has a betterIC50 value compared with quercetin as a positive control(Table 2).From the chemical structures of the isolatedflavonoid compounds, correlated with α-glucosidase inhibition activity, alleged that a hydroxyl functionalgroup in a particular place plays important role ininhibiting the enzyme.Kinetics study of the enzyme α-glucosidase inhibition by AC-31 compound based of Linewaver-Burkmethods showed that the Michaelis constant (Km)value of the reaction without and with inhibitor (AC31)at a concentration of 1.5 mM or 3 mM was notsignificantly different (Fig. 2 and Table 3). The resultsindicated that the type of enzyme α-glucosidase resistance by compound AC-31 is a non-competitivewhere the affinity of the enzyme to the substrate doesnot change despite the compound AC-31, it alsoshowed that compound AC-31 occupy a different locusof the p-NPG substrate in the enzyme α-glucosidase, and AC-31 compound only affects to the reaction rate(Table 3).CONCLUSIONEnzyme α-glucosidase inhibition assay of Artocarpus altilis leaf extract shown that ethanolextract, ethyl acetate fraction and two flavonoidcompounds (AC-31 and AC-51) have antidiabeticactivity stronger than quercetin as positive control withIC50 value 6.79 μg/mL, 5.98 μg/mL, 15.73 μM, and 24.41 μM, respectively. From the kinetic inhibition of enzyme α-glucosidase mechanism can be concluded that the type of inhibition of AC-31 compound is a non-competitive.ACKNOWLEDGEMENTWe are grateful to Indonesian Institute ofSciences for supported our research by ThematicProgram Research Grant.REFERENCES1. 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In Vitro Antidiabetic Activities of Extract and Isolated Flavonoid Compounds from <i>Artocarpus altilis</i> (Parkinson) Fosberg

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