Asian Transactions on Basic and Applied Sciences (ATBAS ISSN: 2221-4291) Volume 02 Issue 06

Isolation of Prenylated Flavonoid from Ethyl Acetate Fraction of Artocarpus altilis Leaves using Counter-Current Chromatography
Sofa Fajriah, Tjandrawati Mozef, Nina Artanti, Puspa D.N. Lotulung, and Jamilah Abbas
Abstract A counter-current chromatographic system (CCC) for isolation of prenylated flavonoid from Artocarpus altilis had been done. The two phase of solvent system composed of n-hexane ethyl acetatemethanolwater (5:5:7:3, v/v/v/v) had been selected by HPLC. About 100 mg amount of ethyl acetate fraction of A. altilis was separated, yielding 5 mg of compound 1. The purity of compound 1 was 98%, determined by HPLC analysis. The 1 chemical structure of compound 1 was identified by ESI-MS, Hand 13C-NMR as prenylated flavonoid group, [1-(2,4dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3pentenyl)-2H-1- benzopyran-5-yl]-1-propanone].

Artocarpus communis J. R. and G. Forster (synonym A. altilis (Parkinson) Fosberg) or breadfruit is locally known as Sukun. It is a large evergreen tree, up to 30-m tall. A. communis is a native of tropical Asia and the Pacific, and the center of genetic diversity extends from Indonesia to Papua New Guinea. It has long been an important staple food in Polynesia. Now it is widely distributed throughout the humid tropics, and commonly grown in home gardens for its edible fruit [13]. Counter-current chromatography (CCC) is a liquidliquid chromatography technique that separates components of a mixture based on their distribution between two immiscible liquid phases. One of these phases is used as the mobile phase Keywords Counter-current chromatography, Artocarpus the other one as the stationary phase. Solutes elute from the altilis, prenylated flavonoid CCC column according to their partition ratio (KD), which is GHILQHG DV WKH UDWLR RI WKH VROXWHV FRQFHQWUDWLRQ LQ WKH I. INTRODUCTION stationary phase to its concentration in the mobile phase. Compounds, which have a higher affinity to the mobile phase ne of medicinal plants as focus on collaborative research (low KD), elute earlier, while compounds that have a higher between Zhejiang University and Indonesian Institute of affinity to the stationary phase (high KD) elute later [14]. CCC Sciences is Artocarpus altilis. The genus Artocarpus benefits from great advantages when compared with the (Moraceae) comprises approximately 50 species and is widely traditional liquidsolid separation methods: it eliminates the distributed in tropical and subtropical regions, including complications resulting from the solid support matrix, such as Indonesia. Some members of this genus have been used irreversible adsorptive sample loss and deactivation, tailing of medicinally to treat various diseases [1]. These plants are solute peaks, and contamination. In addition, CCC has the known to produce a variety of isoprenylated flavonoids containing the unique feature of an isoprenyl side chain at C-3, unique features of high recovery, high efficiency and ease to scale up. Regarding the recent numerous literature about CCC DV ZHOO DV ,-GLR[\JHQDWLRQ RU ,,-trioxigenation development and applications, CCC has been widely used in patterns in ring B of the flavone skeleton [2];[3]. Many of the separation and purification of various natural products. these compounds exhibit interesting biological properties However, since the crude natural product extract is a complex including cytotoxic [3], anti-inflammatory [3]; [4], antiplatelet [5], antioxidant [6], antibacterial [7], and anticomplementary mixture which contains extremely large number of molecules with a wide range of polarity and different molecular weight, effects [8]. They also show activity as inhibitor of TNF-. it is a significant challenge for conventional CCC technique formation [3]; [4], tyrosinase [9];[10];[11], .-reductase [12]. due to the narrow hydrophobicity window of any single Manuscript received January 4, 2013. biphasic solvent system in the isocratic elution mode [15]. Sofa Fajriah is with the Research Center for Chemistry, Indonesian Because of the advantages of counter-current Institute of Sciences, Kawasan PUSPIPTEK, Serpong, Tangerang Selatan, chromatography and broadened our knowledge about Banten, INDONESIA, 15314 (corresponding author to provide phone: +62chromatography techniques, the research focused on isolation 21-7560929; fax: +62-21-7560549; e-mail: syahfa03@yahoo.com). Tjandrawati Mozef is with the Research Center for Chemistry, Indonesian of lead compound from Artocarpus altilis.

Institute of Sciences, Kawasan PUSPIPTEK, Serpong, Tangerang Selatan, Banten, INDONESIA, 15314 (e-mail: tjandrawm@yahoo.com). Nina Artanti is with the Research Center for Chemistry, Indonesian Institute of Sciences, Kawasan PUSPIPTEK, Serpong, Tangerang Selatan, Banten, INDONESIA, 15314 (e-mail: n i naa rt an ti @yahoo . com ). Puspa D.N. Lotulung is with the Research Center for Chemistry, Indonesian Institute of Sciences, Kawasan PUSPIPTEK, Serpong, Tangerang Selatan, Banten, INDONESIA, 15314 (email: puspade@yahoo . com ). Jamilah Abbas is with the Research Center for Chemistry, Indonesian Institute of Sciences, Kawasan PUSPIPTEK, Serpong, Tangerang Selatan, Banten, INDONESIA, 15314 (email: jamilahabbas@yahoo.com).

II. EXPERIMENTAL METHODS A. Preparation of Sample The dried leaves of A. altilis collected from Serpong area (Indonesia) were extracted exhaustively using macerator with ethanol. The ethanol extracts were concentrated using vacuum

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ATBAS-30216065Asian Transactions

Asian Transactions on Basic and Applied Sciences (ATBAS ISSN: 2221-4291) Volume 02 Issue 06 rotary evaporator and then partitioned with hexane-water (1:1), then ethyl acetate-water (1:1). Extraction and partition process to obtained ethyl acetate fraction of A. altilis leaves were conducted in Research Centre for Chemistry, Indonesian Institute of Sciences. The A. altilis leaves ethyl acetate fraction then separated further using counter-current chromatography (CCC) system. This CCC experiments were conducted in Department of Chemistry, Zhejiang University, China. B. Selection of the two-phase solvent system for CCC by HPLC The preliminary studies were carried out with this twophase solvent system at various volume ratios (5:5:6.5:3.5, 5:5:6.8:3.2, 5:5:7:3, v/v/v/v (n-hexane ethyl acetate methanolwater) to get coefficient distribution (K) near 1. For calculating K, can be counted as follows: K = % Area of upper phase peak / % Area of stationary phase peak The condition of HPLC to obtain result for solvent system selection is: Time (min) % Methanol % Water Flow (mL/min) 0 20 80 0.8 5 40 60 0.8 10 70 30 0.8 15 90 10 0.8 25 90 10 0.8 C. Preparation of the two-phase solvent systems and sample solution The two-phase solvent system used was composed of nhexaneethyl acetatemethanolwater at volume ratio 5-5-7-3 that selected from preliminary study. The solvent mixture was thoroughly equilibrated in a separatory funnel at room temperature and the two phases were separated shortly before use. The sample solution was prepared by dissolving the crude sample in a solvent mixture consisting of equal volumes of both upper and lower phases at suitable concentration according to the preparative scale of CCC separation. D. CCC separation procedure CCC separation was performed as follows: the coiled column of CCC was first entirely filled with the upper stationary phase using pump about 15 minutes. Then the sample solution (100 mg crude sample in 1.5 ml upper phase and 2.5 ml lower phase) was injected through the sample port and the lower mobile phase was pumped into the head end of the inlet column at a flow rate of 5 ml/min while the column was rotated at 500 rpm. The effluent from CCC columns was monitored on-line at 254 nm and automatically collected in fraction collectors. The fractions were identified with HPLC. E. HPLC analysis and identification of CCC peak fractions The high-performance liquid chromatography (HPLC) system used was an Agilent 1100 system including a G1311A QuatPump, a G1322 Degasser, a G1314A variable-wavelength detector, a model 7725 injection valve with a 20 _l loop, a PT100 column oven and an Agilent ShemStation for LC. HPLC analysis of the crude sample and CCC peak fractions was performed with a CAPCELL PAK-C18 column, type MG 5 m, size 4.6mm l.D. x 250 mm. The mobile phase was methanol (solvent A) and water (solvent B) at the gradient: A from 50 to 90% and B from 50 to 10% for 20 min. The flow rate was 0.8 ml/min, and the effluent was monitored at 254 nm. Identification of the CCC peak fraction was performed by ESI-MS, 1H NMR and 13C NMR. NMR experiments were carried out using a Bruker Advanced DMX 500 NMR spectrometer with acetone-d 6 as solvent and TMS as internal standard. III. RESULTS AND DISCUSSIONS From preliminary study and calculating of K value with HPLC, the volume ratio of two phase of solvent system is 5:5:7:3 (n-hexane ethyl acetatemethanolwater) with K value 1.1. This solvent system used as solvent in CCC experiment. Based on the result of CCC chromatogram (Fig. 1), there have three peaks at retention time 45, 70 and 120 minutes, but only peak at retention time 70 minutes shows the pure compound, so this peak identified with HPLC and the result can be seen at Figure 2.

Fig. 1. The CCC Chromatogram of ethyl acetate fraction from A. altilis leaves.

Fig. 2. HPLC Chromatogram from CCC experiment peak at retention time 70 minutes of ethyl acetate fraction of A. altilis.

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ATBAS-30216065Asian Transactions

Asian Transactions on Basic and Applied Sciences (ATBAS ISSN: 2221-4291) Volume 02 Issue 06 From HPLC chromatogram, it shows that the compound almost pure (compound 1) and identified using NMR and MS. From MS data, compound 1 has molecular weight 408. The analysis of its NMR data and comparison with reference showed in Table 1. From the 13 C-NMR spectrum, compound 1 2 indicated 25 carbons, including 8 sp methine carbons, 4 methylene carbons, 3 methyl groups, and 10 quartenery carbons.
Table 1. Comparison of and reference
13 1 C and H NMR assignment between compound 1

From NMR spectrum, MS data, and comparison with previous spectral data [16];[17];[18], compound 1 was identified as prenylated flavonoid, named 1-(2,4dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3pentenyl)-2H-1-benzopyran-5-yl]-1-propanone (Figure 3). In [18], this compound was isolated by general gravitation column chromatography that needs various solid and mobile phase system for separations and purifications before obtaining the pure compound 1. Therefore this suggests the advantage of using CCC system for isolation of compound.
OH O
7 6

13

C and H NMR assignment from compound 1 C No. /C (ppm) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 165.20 103.62 163.48 108.27 132.45 113.66 204.14 39.77 26.66 128.08 121.29 114.71 143.11 139.72 119.22 119.49 130.30 78.9 40.89 22.94 123.99 132.10 25.80 17.78 26.20 /H (ppm) ( H, multiplicity, J in Hz) 6.38 (1H, d, 2.45) 6.37 (1H, dd, 2.45; 8.5) 7.54 (1H, d, 8.5) 3.1 (2H, q) 2.9 (2H, m) 6.60 (1H, d, 8.5) 6.72 (1H, d, 8.5 )

13

1 C and H NMR assignment from reference [16]

/C (ppm) 165.15 103.51 163.14 107.97 132.27 113.59 203.89 39.65 26.49 127.91 121.14 114.53 143.02 139.56 119.03 119.36 130.12 78.76 40.77

/H (ppm) (mult., J in Hz) 6.37 (2.0) 6.34 (8.8; 2.0) 7.55 (8.8)

HO

10

15 16 23

B
13

C
18

14

OH

O
25

22 24

19

3.08 (m) 2.97 (m) 6.61 (8.2) 6.73 (8.2)

Fig. 3. Molecular Structure Compound Isolate A [1-(2,4dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3pentenyl)-2H-1- benzopyran-5-yl]-1-propanone]. IV. CONCLUSIONS Counter-current chromatography technique is more efficient for compound isolation from ethyl acetate fraction of A. altilis leaves compare to column chromatography because it GRHVQW QHHG D ORW RI SXULILFDWLRQ VWHS. Based on NMR spectra, MS data, and compared with previous published spectral data, compound 1 from ethyl acetate extract of A. altilis leaves was identified as a prenylated flavonoid, [1-(2,4-dihydroxyphenyl)-3-[8-hydroxy2-methyl-2-(4-methyl-3-pentenyl)-2H-1- benzopyran-5-yl]-1propanone]. ACKNOWLEDGMENT

6.54 (1H, d, 9.8) 5.63 (1H, d, 9.8)

6.54 (10.2) 5.63 (10.2) 1.70 (m)

20 22.80 2.08 (m) 21 123.84 5.08 (7.0) 22 131.95 23 1.66 (3H, s) 25.66 1.65 (bs) 24 1.57 (3H, s) 17.64 1.56 (bs) 25 1.38 (3H, s) 26.07 1.38 (s) 1-OH 12.85 (1H, s) 12.81 (s) 3-OH 7.20 (bs) 13-OH 5.58 (bs) * Spectra compound 1 recorded at 500 MHz for H 1spectrum and 125 MHz 13 for C spectrum in CDCl 3 DQG UHIHUHQFHV VSHFWUD UHFRUGHG DW 00 0+]. 7KH values are in ppm and J values (Hz) in parentheses. Abbreviations for NMR signal are as follows: s = singlet, d = doublet, and t = triplet.

1.7 (2H, m) 2.09 (2H, m) 5.08 (1H, t)

The H-NMR spectra suggested the existence of two aromatic rings, ring A and B. At ring A have three proton aromatics, revealed a clear AMX system as indicated by the proton resonances at 6.38 (H-, d, J 2.45 Hz) and 6.37 ppm (H-, dd, J 2.45 Hz and 8.5 Hz) and at 7.54 ppm (H-, d, J 8.5 Hz) and 6.37 ppm (1 H, dd, J 2.45 Hz and 8.5 Hz). The down field chemical shift at 12.85 ppm (s) indicated the presence of hydroxyl group formed a hydrogen bond with a carbonyl group (C-1) at 204.14 ppm. The multiplicities of carbons were assigned by the DEPT-135 experiment.

This work is collaboration research between Zhejiang University and Indonesian Institute of Sciences (LIPI). We also thanks to Prof. Yuanjiang Pan and his group member for facilitating this research.

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ATBAS-30216065Asian Transactions

(LIPI) since 2005

Asian Transactions on Basic and Applied Sciences (ATBAS ISSN: 2221-4291) Volume 02 Issue 06 REFERENCES

Eisai PT., Medical Herb Index in Indonesia.1986. T. Nomura, Y. Hano, Isoprenoid substituted phenolic compounds of moraceous plants, Nat. Prod. Rep., 11, 205-218, 1994. [3] T. Nomura, Y. Hano, M. Aida, Isoprenoid substituted flavonoids from Artocarpus plants (Moraceae), Heterocycles, 47, 1179-1204, 1998. [4] M-I Chung, H-H Ko, M-H Yen, C-N Lin, Artocarpol A, a novel constituent with potent anti-inflammatory effect, isolated from Artocarpusrigida, Helv Chim Acta, 83: 1200-1204, 2000. [5] C-N Lin, C-M Lu, H-C Lin, S-C Fang, B-J Shieh, M-F Hsu, J-P Wang, F-N Ko, C-M Teng, Novel antiplatelet constituents from formosanMoraceous plants, J. Nat Prod., 59 (9): 834-838, 1996. [6] F.N Ko, Z.J. Cheng, C.N Lin, C.M. Teng, Scavenger and antioxidant properties of prenylflavones isolated from Artocarpus heterophyllus, Free Radical Biol Med, 25:160-168, 1998. [7] M. Sato, S. Fujiwara, H. Tsuchiya, T. Fujii, M. Linuma, H. Tosa, Y. Ohkawa, Flavones with antibacterial activity against cariogenic bacteria, J. Ethnopharmacol, 54: 171-176, 1996. [8] M.S.J Nascimento, H. Cidade, M. Pinto. A. Kijjoa, Anticomplementary activity of prenylated flavones from Artocarpus elasticus, Pharm. Pharmacol. Lett,. 7: 135, 1997. [9] K. Shimizu, R. Kondo, K. Sakai, S.H. Lee, H. Sato, The inhibitory components from Artocarpus incisus on melanin biosynthesis, Planta Med. 64, 408-412, 1998. [10] K. Shimizu, M. Fukuda, R. Kondo, K. Sakai, The 5 alpha-reductase inhibitory components from heartwood of Artocarpusincisus: structureactivity investigation, Planta Med., 66 (1): 16-9, 2000. [11] K. Likhitwitayawuid, B. Sritularak, A new dimericstilbene with tyrosinase inhibitory activity from Artocarpus gomezianus, J. Nat. Prod., 64, 1457-1459, 2001. [12] K. Shimizu, R. Kondo, K. Sakai, Inhibition of tyrosinase by flavonoids, stilbenes and related 4-substituted recorcinols: structure-activity investigations, Planta Med., 66 (1): 11-5, 2000 [13] E.W.M. Verheij, R.E. Coronel (Eds.), Plant resources of South-East Asia, No.2, edible fruit and nuts. PROSEA, Bogor, Indonesia, pp 79-96, 1992. [14] H. Guzlek, I.I.R. Baptista, P.L.Wood, and Livingston, A. 2010.A novel approach to modelling counter-current chromatography.J. Chromatography A, 1217, p 6230-6240. [15] McLean, S., Reynolds, W.F., Tinto, W.F., Chan, W.R., Shepherd, V., 1996. Complete 13C and 1H spectral assignments of prenylated flavonoids and a hydroxy fatty acid from the leaves of Caribbean Artocarpus communis. Magn.Reson. Chem. 34, 719-722. [16] Koshihara, Y., Fujimoto, Y., and Inoue, H., 1988. A new 5-lipoxygenase selective inhibitor derived from Artocarpus communis strongly inhibits arachidonic acid-induced ear edema. BiochemPharmacol. 37(11), 21612165. [17] Wang, Y., K. Xu, L. Lin, Y. Pan and X Zheng. 2007. Geranyl flavonoids from the leaves of Artocarpus altilis. Phytochemistry 68: 1300-1306. [1] [2]

S. Fajriah, was born in Serang, Banten, Indonesia, February 5, 1981, post graduated of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Bandung, West Java, Indonesia, graduated in 2003. Master degree of Chemistry, Faculty of Mathematics and Natural Sciences, Indonesia University, graduated in 2011. Position as reseacher at Natural Product, Food, and Pharmaceutical Division of Research Center for Chemistry, Indonesian Institue of Sciences

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ATBAS-30216065Asian Transactions