Antiplasmodial isoprenylated flavonoids from the stem bark of

Available online at www.scholarsresearchlibrary.com

Scholars Research Library Der Pharmacia Lettre, 2015, 7 (2):35-39 (http://scholarsresearchlibrary.com/archive.html) ISSN 0975-5071 USA CODEN: DPLEB4

Antiplasmodial isoprenylated flavonoids from the stem bark of Erythrina ovalifolia Roxb Tjitjik Srie Tjahjandarie*and Mulyadi Tanjung Natural Products Chemistry Research Group, Organic Chemistry Division, Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia _____________________________________________________________________________________________ ABSTRACK Fourisoprenylated flavonoid derivatives,erythrisenegalone (1), alpinumisoflavone (2), phaseollidin(3), and sandwicensin (4) were isolated from the stem bark of ErythrinaovalifoliaRoxbTheir structures were elucidated on the basis of spectroscopic analyses. Compounds 1–4 were evaluated for their antiplasmodial properties against Plasmodium falciparum, showing their IC50 were 1.69, 1.98, 1.66, and 1.83µg/mL, respectively. Keywords: Erythrinaovalifolia,Phenolic compound, Antiplasmodial _____________________________________________________________________________________________ INTRODUCTION Malaria is a major cause of death in the world than any parasitic infection, especially in tropical developing countries. This disease has been found endemic atall region in Indonesia. Recently, chloroquine and artemisinin have used as antimalarial drugs and showed resistance against Plasmodium parasites in Indonesia. Erythrinabelongs to the family Leguminosae, this plant has been shown to produce a number of pterocarpan, flavonoid, and alkaloid compounds which showed activity as anticancer, antioxidant, antiviral and antimalaria[1,2,3,4].Decoction of the stem bark or leaves of ErythrinaovalifoliaRoxbhas been used in the Indonesian people as a traditional medicine for malaria. In continuation of these chemical investigations, we have examinedErythrinaovalifoliaRoxband succeeded in isolating fourflavonoid compounds, with namely erythrisenegalone (1), alpinumisoflavone (2), phaseollidin(3), and sandwicensin (4). This paper discusses the structure elucidation of the fourisoprenylated flavonoids andantiplasmodial properties of compounds 1-4against Plasmodium falciparum. MATERIALS AND METHODS General UV and IR spectra were measured with a Shimadzu 1800and Perkin Elmer Spectrum One FTIR spectrometer, respectively. 1H and 13C NMR spectra were recorded with a JEOL ECA400 spectrometer operating at 400 (1H) and 100 (13C) MHz in CDCl3 using TMS as the internal standard. Mass spectra were obtained with a Waters LCT Premier XE. Vacuum liquid chromatography (VLC) and radial chromatography were carried out using Si gel 60 GF254 and Si gel 60 PF254, for TLC analysis, pre-coated silica gel plates (Merck Kieselgel 60 GF 254, 0,25 mm thickness) were used. Solvents used forextraction and preparative chromatography were of technical grade and distilled before use. Plant material The stem barkof E. ovalifoliaRoxb was collected from Kunir Kidul Village, District Lumajang, East Java, Indonesia. The plant was identified at the Herbarium Bogoriense, Bogor Botanical Garden, Bogor, Indonesia.

35 Scholar Research Library

Tjitjik Srie Tjahjandarie and Mulyadi Tanjung

Der Pharmacia Lettre, 2015, 7 (2):35-39

______________________________________________________________________________ Extraction and isolation The dried and powder of the stem barkE. ovalifoliaRoxb(4.0 kg) were macerated with methanol two times at room temperature, and then concentrated under reduced pressure. The residue was suspended in water (9:1) and partitioned sequentially with n-hexane and EtOAc (16 g). The crude EtOAc was then fractionated using vacuum liquid chromatography on silica gel eluting with mixtures n-hexane-EtOAc (9:1, 4:1, 1:1, and 3:7) to give four major fractions A-D.Mayor fraction B (250 mg) was separated with radial chromatography and eluting with mixtures ofnhexane-EtOAc (9:1, and 4:1) to give two subfractions B1-B2.Separation of subfraction B1using radial chromatography eluted with mixturesn-hexane-diisopropylether (9:1, and 7:3) gave erythrisenegalone (1;10 mg) and subfraction B2 with n-hexane-acetone (9:1, and 8:2)yielded sandwicensin (4;20 mg).Furthermore, fraction C purified using radial chromatography eluted with mixtures n-hexane-CHCl3 (7:3, to chloroform) yielded phaseollidin (3;8 mg) and alpinumisoflavone (2;6 mg). Structure elucidation Erythrisenegalone (1), yellow solid, UV (MeOH) λmaks nm (log ε) : 236 (3.95), 293 (4.14), and325 sh (3.60). IR (KBr) ѵmax(cm-1) 3425, 2948, 2864, 1632, 1546, 1448, 1170.HR-ESI-MS m/z407.1863)[M+H]+ (calcd for C25H27O5: 407.1858); 1H NMR (400 MHz, CDCl3) δH (ppm): 5.33 (1H, dd, J = 13,0, 3,0 Hz, H-2),3.04 (1H, dd, J = 17,0, 13,0 Hz, H-3ax), 2.79 (1H, dd, J = 17,0, 3,0 Hz, H-3eq), 7.32 (2H, d, J = 8.0 Hz, H-2’/6’), 6.87 (1H, d, J = 8.0 Hz, H3’/5’), 3.21 (1H, d, J = 7.0 Hz, H-1’’), 5.15 (1H, t like, J = 7.5 Hz, H-2’’), 1.81 (3H, s, H-4’’), 1.75 (3H, s, H-5’’), 5.49 (1H, d, J = 10.0 Hz, H-3’’’), 6.63 (1H, d, J = 10.0 Hz, H-4’’’), 1.70 (6H, s, H-5’’’/6’’’), 12.54 (1H, s, 5-OH); 13 C NMR (100 MHz, CDCl3) δH (ppm): 78.5 (C-2), 43.3 (C-3), 196.6 (C-4), 102.8 (C-4a), 159.3 (C-5), 107.3 (C-6), 162.3 (C-7), 106.4 (C-8), 157.8 (C-8a), 131.1 (C-1’), 127.7 (C-2’/6’), 115.5 (C-3’/5’), 155.9 (C-4’), 21.9 (C-1’’), 122.0 (C-2’’), 134.7 (C-3’’), 17.9 (C-4’’), 25.8 (C-5’’), 78.8 (C-2’’’), 121.8 (C-3’’’), 134.0 (C-4’’’), 25.7 (C5’’’/6’’’). Alpinumisoflavone (2), yellow solid, HR-ESI-MS m/z 337.1082[M+H]+ (calcd for C20H17O5: 337.1076); 1H NMR (400 MHz. DMSO-d6) δH (ppm): 8.17 (1H,s,H-2), 6.36 (1H,s,H-8), 7.45 (2H, d, J = 8.5, H-2’/6’), 6.90 (2H, d, J = 8.5, H-3’/5’), 5.76 (1H, d, J = 10.0, H-3’’), 6.67 (1H, d, J = 10.0, H-4’’), 1.46 (6H,s,H-5’/6’), 13.42 (1H,s,5-OH);13C NMR (100 MHz. DMSO-d6) δH (ppm): 154.4 (C-2), 124.1 (C-3), 181.8 (C-4), 106.0 (C-4a), 157.7 (C-5), 106.7 (C6), 160.2 (C-7), 95.4 (C-8), 158.1 (C-8a), 122.9 (C-1’), 131.1 (C-2’/6’), 116.0 (C-3’/5’), 158.5 (C-4’), 78.8 (C-2’’), 129.4 (C-3’’), 115.7 (C-4’’), 28.4 (C-5’’/6’’). Phaseollidin(3), yellow solid, UV (MeOH) λmaks nm (log ε) : 224 (4.86), 281 (3.93), and287(3.95).HRESI-MS: m/z [M+H]+calcd. for C20H21O4325.1472, found 325.1440. 1H NMR (400 MHz, CDCl3) δH (ppm): 7.38 (1H, d, J = 8.4 Hz, H-1),6.54 (1H, dd, J = 8.4, 2.5 Hz, H-2),6.40 (1H, d, J = 2.5 Hz, H-4),4.21 (1H, dd, J = 11.1, 4,8 Hz, H-6α), 3.61 (1H, t, J = 11.1 Hz, H-6β), 3.51 (1H, m, H-6a),6.94 (1H, d, J = 8.0 Hz, H-7),6.37 (1H, d, J = 8.0 Hz, H-8), 5.44 (1H, d, J = 6.7 Hz, H-11a),3.33 (1H, d, J = 7.4 Hz, H-1’), 5.27 (1H, t, J = 7.2 Hz, H-2’),1.78 (3H, s, H-4’), 1.72 (3H, s, H-5’), 5,39 (1H, s, 5-OH);13C NMR (100 MHz, CDCl3) δH (ppm): 132.4 (C-1), 109.8 (C-2), 157.1 (C-3), 103.7 (C-4), 156.7 (C-4a), 66.7 (C-6), 40.1 (C-6a), 118.7 (C-6b), 121.4 (C-7), 108.3 (C-8), 155.9 (C-9), 110.4 (C-10), 158.5 (C-10a), 78.2 (C-11a), 112.9 (C-11b), 23.3 (C-1’), 122.5 (C-2’), 135.4 (C-3’), 18.0 (C-4’), 25.9 (C-5’). Sandwicensin (4), yellow solid, UV (MeOH) λmaks nm (log ε) :223 (4.60), 285 (3.84), and287(3.89).HRESI-MS: m/z [M+H]+339.1642calcd.forC21H23O4, found 339.1596. 1H NMR (400 MHz, CDCl3) δH (ppm): 7.41 (1H, d, J = 8.4 Hz, H-1),6.58 (1H, dd, J = 8.4, 2.4 Hz, H-2),6.44 (1H, d, J = 2.4 Hz, H-4),4.23 (1H, dd, J = 11.0, 5,1 Hz, H-6α), 3.66 (1H, t, J = 11.0 Hz, H-6β), 3.52 (1H, m, H-6a),7.02 (1H, d, J = 8.1 Hz, H-7),6.43 (1H, d, J = 8.1 Hz, H-8), 5.46 (1H, d, J = 7.2 Hz, H-11a),3.31 (1H, d, J = 7.9 Hz, H-1’), 5.26 (1H, t, J = 7.2 Hz, H-2’),1.78 (3H, s, H-4’), 1.68 (3H, s, H-5’), 3.81 (3H, s, 9-OCH3);13C NMR (100 MHz, CDCl3) δH (ppm): 132.3 (C-1), 109.9 (C-2), 157.5 (C-3), 103.1 (C-4), 156.5 (C-4a), 66.5 (C-6), 40.0 (C-6a), 119.4 (C-6b), 121.6 (C-7), 103.5 (C-8), 158.5 (C-9), 112.6 (C-10), 158.4 (C-10a), 78.0 (C-11a), 113.3 (C-11b), 22.9 (C-1’), 122.3 (C-2’), 132.3 (C-3’), 17.8 (C-4’), 25.8 (C-5’), 56.0 (9-OCH3).




______________________________________________________________________________

Fig. 1. Structures of flavonoid compounds

Antiplasmodial analysis Antiplasmodial properties of the isolated compounds 1-4against Plasmodium falciparumwas obtained from the Institute of Tropical Diseases, UniversitasAirlangga, Surabaya, Indonesia. In vitro antimalarial activity against Plasmodium falciparumwas carried out according to a modified method of Trager and Jensen using RPMI 1640medium with10% O+ serum [5]. The antimalarial activity of four flavonoid compounds and chloroquine (positive control) were measured in triplicate. Fresh red blood cells was used as a negative control. The active compound was dissolved in DMSO and diluted with RPMI 1640 medium to prepare a series of concentration. Parasitaemia was evaluated after 48 by Giemsa stain and the average percentage suppression of parasitaemia was calculated by following equation [6,7]: %

=

100 × %

%

− %

The influence of the active compound on the growth of parasites was expressed by the 50% inhibitory concentrations (IC50), which was determined using linier regression analysis. RESULTS AND DISCUSSION Extraction of the dried milled the tree bark of E. ovalifoliaRoxbwith methanolgave a fraction which was separated by vacuum liquid chromatography and radial chromatography to give namely erythrisenegalone (1), alpinumisoflavone (2), phaseollidin(3), and sandwicensin (4).The structures of fourisoprenylated flavonoids were identified by UV, HR-ESI-MS, 1D and 2D NMR spectra. Erythrisenegalone (1) was isolated as a yellow solid and the molecular formula C25H27O5 was deduced from its HRESI-MS data. The UVspectrum of 1 exhibited maxima typicalfor a flavanone structure (λmax236, 293and 325 sh nm[8]. TheIR spectrum indicated absorptions forhydroxyl (3425 cm-1), conjugated carbonyl(1632 cm-1), and aromatic (1632 and1546 cm-1) and C-O-C ether (1170cm-1) groups.The 1H NMR spectrum of 1showed three doublet-doublet proton signals at δH 5.33 (1H, dd, J = 13.0, 3.0 Hz, H-2), 3.04 (1H, dd, J = 17.0, 13.0 Hz, H-3ax), and 2.79 (1H, dd, J = 17.1, 3.0 Hz, H-3eq) confirmed for the flavanonestructure.The 1H NMR spectrum showed a pair of doublets (J=8.0 Hz), each integrating for two protons, at δ 6.87 and 7.32characteristic for aromatic in the ring B.In the 1H NMRspectrum of 1showed two isoprenyl groups, one is 3-methyl-2-buten-1-yl group at δH5.15 (1H, t, J = 7.5 Hz, H-2’’), 3.21 (2H, d, J = 7.0 Hz, H-1’’), 1.81 (3H, s, H-4’’), 1.75 (3H, s, H-5’’), and another is 2,2dimethylpyrano ringatδH5.49 (1H, d, J = 10.0 Hz, H-3’’’), 6.63 (1H, d, J = 10.0 Hz, H-4’’’), and 1.70 (6H, s, H5’’’/6’’’).The presence of a chelated hydroxyl group (δH 12.54) suggested that hydroxyl group attached at C-5.In the 13C NMR spectrum, the presence of four oxyaryl (δC162.3, 159.3, 157.8, 155.9) suggested that1 is naringenin structure.The placement 3-methyl-2-buten-1-yl and2,2-dimethylpyrano groupsat C-6 or C-8 were determined with HMQC and HMBC spectra.The presence of long-rangecorrelations in the HMBC spectrum of 1between the proton signal of a chelated 5-OH group at δH 12.54 with threequaternary carbon signals at δC 102.8 (C-4a), 159.3 (C-5), 107.3(C-6), andcorrelation proton singlet δH5.49(H-3’’’) with carbon signals at δC107.3(C-6), consequently these correlations correspond to the 2,2-dimethylpyrano ring was fused at C-6 and C-7.Therefore3-methyl-2-buten-1-yl was located at C-8. From the HR-ESI-MS, 1D and 2D NMR data, compound 1 was identified as erythrisenegalone [9]. Alpinumisoflavone (2) was obtained as a yellow solid and its HR-ESI-MS spectrum showed the molecular formula C20H17O5([M+H]+337.1082, calcd 337.1076). The 1H NMR spectrum of 2showed singlet proton signals at δH8.17




______________________________________________________________________________ characteristic for H-2 of the isoflavone structure.The 1H NMR spectrumalso exhibited the presence of a 2,2dimethylpyrano ring [δH 1.46 (6H, s), 5.76 (lH, d, J= 10.0 Hz), 6.67 (lH, d, J= 10.0 Hz)] and a pair of doublets (J=8.5 Hz), each integrating for two protons, at δ 6.90 and 7.45 assignable to the signals of apara-hydroxyphenyl group in the ring B.The remaining two signals at δ 13.42 and 6.36 (each 1H) could be attributed to the chelated hydroxyl group proton characteristic for 5-OH and H-6 or H-8 in the ring A of isoflavonoid structure. The 13C NMR spectrum, the presence of four oxyaryl (δC160.2, 158.5, 158.1 and 157.7) and a carbonyl carbon signal at δC181.8 suggested that the oxygenates functionalities are at C-5, C-7 and C-4’ of the isoflavonoid structure.Based on 1H and 13 C NMR spectra, the placement 2,2-dimethylpyrano ring were fused at C-6 and C-7 or C-7 and C-8. The location of the 2,2-dimethylpyrano unit at C-6 and C-7 determined on the basis of the HMBC correlations observed. The presence of long-range correlations in the HMBC spectrum of 2 between the proton signal of a chelated 5-OH group at δH 13.42 and three quaternary carbon signals at δC106.0 (C-4a), 157.7 (C-5), 106.7 (C-6) unambiguously placed the 2,2-dimethylpyrano fused at C-6 and C-7. The structure of 2 was, then, characterized as alpinumisoflavone [10]. Phaseollidin (3) had the molecular formula C20H17O4([M+H]+325.1440, calcd 325.1472) by the HR-ESI MS spectra. The UV and 1H NMR spectra (δH3.51 (1H, m, H-6a),3.61 (1H, t, J = 11.1 Hz, H-6β), 4.21 (1H, dd, J = 11.1, 4,8 Hz, H-6α), 5.44 (1H, d, J = 6.7 Hz, H-11a) suggested that 3 was a pterocarpan derivative.The 1H NMR spectrum of 3 showed two aromatic regions, one is ABX system at δH7.38 (1H, d, J = 8.4 Hz, H-1),6.54 (1H, dd, J = 8.4, 2.5 Hz, H-2), and 6.40 (1H, d, J = 2.5 Hz, H-4), and another is δH6.94 (1H, d, J = 8.0 Hz, H-7), and 6.37 (1H, d, J = 8.0 Hz, H-8) on rings A and D.Furthermore, in the 1H NMR spectra showed one isoprenyl group assignable to a 3-methyl-2buten-1-yl group at δH5.27 (1H, t, J = 7.2 Hz, H-2’),3.33 (1H, d, J = 7.4 Hz, H-1’), 1.78 (3H, s, H-4’), 1.72 (3H, s, H-5’). The 13C NMRspectra of 3 showed 20 carbon signals consistent for isoprenylatedpterocarpan.Based on 1H and 13 C NMR spectra, the placement 3-methyl-2-buten-1-ylonpterocarpan structure at C-4or C-10.The placement 3methyl-2-buten-1-yl group at C-4 or C-10 was determined with HMQC and HMBC spectra. In the HMBC spectrum showed correlations between a proton signal at δH3.33 (H-1’’)with two oxyaril at δC 155.5 (C-9), and 155.8 (C10a)unambiguously placed the 3-methyl-2-buten-1-yl group at C-10.From the above UV, HR-ESI-MS, 1D and 2D NMR data, compound 3 was identified as asphaseollidin [11]. Sandwicensin (4)had the molecular formula C21H23O4([M+H]+339.1642, calcd 339.1596) by the HR-ESI MS spectra. From HR-ESI-mass spectra indicatingof 4 is methylated from compound 3.The 1H and 13C NMRspectra of 4very similar with compound 3, but there is one methoxyl groups attached to the aromatic core from compound 4that proton signal at δH3.81and the carbon signal at 56.0. Based on 1H and 13C NMR spectra, the placement methoxyl group at C-3or C-9.The placement of a methoxy group in the C-3or C-9 confirmed by the HMQC and HMBC spectra. The HMBC spectrum showed a correlation between the proton signal at δH3.81with carbonoxyaril signal at δC 155.8. Furthermore, the correlation between aromatic proton signals at δH7.02 (d, 8.1, H-7)with oxyaril carbon signal at δC 155.8 indicated that the methoxyl group attached at C-9.From the above UV, HR-ESI-MS, 1D and 2D NMR data, compound 4 was identified as (6aR, 11aR)-3-hydroxy-9-methoxy-10-isoprenyl pterocarpan named as sandwicensin [11]. Compounds1-4isolated from the tree bark of E. ovalifoliaRoxbwere assessed for their antimalarial activity against Plasmodium falciparum. The results are presented in Table 1.Compounds 1–4 were evaluated for their antiplasmodialproperties against Plasmodium falciparum, showing their IC50 were 1.69, 1.98, 1.66, and 1.83µg/mL, respectively (chloroquine as a positive control, IC501.02 µg/mL). These antiplasmodial data suggested that the compound 1-4showed moderate activity against Plasmodium falciparum.The structure-activity relationship of compounds 3–4against Plasmodium falciparum suggested that the presence of hydroxyl group at C-9 on phaseollidin (3) increasing activity than the methoxyl group at C-9 on sandwicensin (4). Table 1. Antiplasmodialassay of 1-4 Compound Erythrisenegalone Alpinumisoflavone Phaseollidin Sandwicensin Chloroquine

Antimalarial IC50 (µg/mL) 1.69 1.98 1.66 1.83 1.02

Acknowledgements We would like to thank to Prof. Dr. Yana M.Syahfrom Department of Chemistry, ITB Bandung, Indonesia for HRESIMS spectrum measurements. We would like to thank to Mr. Ismail Rachmanfrom the Herbarium Bogoriense, Botanical Garden, Bogor, Indonesia for identifying the species.




______________________________________________________________________________ REFERENCES [1] PH Nguyen, TTDao, J Kim,DT Phong, DT Ndinteh, JT Mbafor, and WK Oh,Bioorg. Med. Chem. 2011; 19: 3378-3383. [2] PHNguyen, TNANguyen, KW Kang, DT Ndinteh, JT Mbafor, YR Kim, and WK Oh,Bioorg. Med. Chem. 2010; 18: 3335-3344. [3] P Innok, T Rukachaisirikul, Suksamrarn, Chem.Pharm. Bull.,2009; 57(9): 993-996. [4] M Ozawa, S Kawamata, T Etoh,M Hayashi, K Komiyama, AKishida, C Kuroda, andAOhsaki, A,Chem. Pharm.Bull.,2010; 58(8): 1119-1122. [5] I Zakaria, NAhmad, FM Jaafar, andA Widyawaruyanti, Fitoterapia.2012, 83, 968-972. [6] I Zakaria, NAhmad, FM Jaafar, andA Widyawaruyanti, Fitoterapia.2012, 83, 968-972. [7]Fidock,A., David, Rosenthal,P.J., Croft, S.L., Brun, R., and Solomon, N., Nature Reviews, 2004, 3, 509-520 [8]M. Tanjung.,E.H. Hakim., Elfahmi J. Latief., andY.M. Syah., J Nat. Prod. Comn.,2012, 7(10), 1309-1310. [9] P Khaomek, E Chikara, I Aki, S Hitomi, N Miyuki, R Nijsiri, S Ekarin, K Hiroaki, O Kazuhiko, O Sathosi and Y Haruki, J.Nat.Med., 2008; 62:217-220. [10] CBMNde, DNjamen, ST Fomum, J Wandji, E Simpson, C Clyne and GVollmer, European J Pharm., 2012;674:87–94. [11] SA Adesanya, MJ O’Neil, IR Pantry, and MF Robert,J. Pharm.Pharmacol.,2011,37: 512-517.
