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European Journal of Scientific Research

ISSN 1450-216X Vol.23No.2 (2008), pp.330-337

EuroJournals Publishing, Inc. 2008http://www.eurojournals.com/ejsr.htm

The Bioactivity Test of Artonin E from the Bark of

Artocarpus Rigida Blume

Tati Suhartati

Department of Chemistry, University of Lampung, Bandar Lampung 35145 Indonesia

E-mail: [email protected]

Yandri


Sutopo Hadi


E-mail: [email protected]

Abstract

From the bark ofArtocarpus rigida Blume which was collected from Tanggamus,Lampung Province Indonesia has successfully been isolated the cycloartobiloxanthone and

artonin E. The structure of these compounds has been identified by physical and

spectroscopies methods. These compounds have high cytotoxicity against leukemia P-388cell. In bioactivity test, artonin E was active against E. coli andB. subtilis, however it was

not active againstRizhopus. oryzae andRizhopus. oligosporus.

Keywords: A. rigida, cycloartobiloxanthone, artonin E, cytotoxicity, leukemia P-388 cell

1. IntroductionThe research on Artocarpus plant growing in Lampung Province is continuously being carried outespecially to that of the rare plant. Previous report by Suhartati and Yandri (2007), based on the result

of plant identification from Herbarium Bogoriense, Bogor stated that the plant used for this work wasArtocarpus dadah, but after the second and third identifications, the plant used indeed was A. rigida.Therefore, this paper is to clarify the plant used in our previous work. Both A. rigida and A. dadah

(Moraceae family) are rare plants and endemic in Indonesia in which the chemical content of these

plants are required to be determined. A variety of compounds has been isolated from some species ofthese plants and a few of them showed very interesting biological activities (Lemmens et al., 1995;

Nomura et al., 1998; Su et al., 2002; Han et al., 2006).

Suhartati and Yandri (2007) have previously reported the finding of cycloartobiloxanthone

from the bark ofA. rigida. In this paper from the bark ofA. rigida, we reported the isolation of artoninE. The structure of this compound has been identified physically and by UV-VIS, IR, and

1H-NMR

spectroscopies. In the bioactivity test of artonin E (Suhartati et al., 1999) showed activity against E.coli andB. Subtilis (Barry, 1980; Berghe and Vlientinck, 1991; Baueret al., 1966) but did not show agood activity againstR. oryzae orR. Oligosporus (Berghe and Vlientinck, 1991).

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The Bioactivity Test of Artonin E from the Bark of Artocarpus Rigida Blume 331

2. Materials and Methods2.1. Plant Material

Samples of the bark ofA. rigida Blume were collected from Keputran Village, Sukoharjo Tanggamus

Lampung in April 2007 and were identified by the staff at the Herbarium Bogoriense, Research Centrefor Biology, Indonesia Institute of Sciences Bogor, Indonesia and a voucher specimen has been

deposited at the herbarium.

2.2. General experimental procedure

VLC was carried out using Merck Si-gel 60, TLC analysis on pre-coated Si-gel plates (MerckKieselgel 60 F254, 0.25 mm).The UV lamp of Spectroline, Model ENF-240 C/F was used to see the

spot in TLC. Melting points were determined on a Fisher Johns micro-melting point apparatus and are

uncorrected. UV and IR spectra were measured with Beckman DU-7000 and Shimadzu FT-IR 8501Scientific spectrophotometers respectively.

1H NMR spectrum was recorded with a JEOL JNM A500

spectometer, operating at 500.00 MHz.

2.3. Research Methodology

The research stages carried out are in the following order: extraction, isolation, purification ofcompound, determination of the molecule structure with physical and UV-VIS, IR, and 1H-NMRspectroscopies and the bioactivity test on the pure compound.

2.3.1. Isolation and Purification of the compounds obtained

The milled, dried root bark (2.7 kg) was extracted exhaustively in turn with n-hexane and a mixture ofethylacetate (EtOAc)-methanol 1:1. On removal of the solvents under reduced pressure, gave residues

of 62.6 g and 119 g respectively. The EtOAc-methanol extract was then fractioned by Si-gel vacuum

liquid chromatography (VLC) three times. The fraction which has spot with the same Rf value with thespot on TLC for every VLC was then combined to give 6 major fractions. Based on the TLC spots, the

fractions which might contain flavonoid were fractions 5 (7.5 g) and 6 (37.9 g). The fraction 5 was

then further subjected with VLC using Si-gel as the adsorbent and was eluted with solvents of EtOAc-n-hexane in a variety of concentration between 15 40 %. Based on the TLC chromatogram, three

fractions (a, b and c) are obtained in the amount of 0.2 g, 6.1 g, and 3.5 g respectively. The c fraction

was then further subjected with VLC, was eluted with solvents of EtOAc-n-hexane 10 40%, and 8fractions were obtained. On combining of fractions 2 4 and eluted with EtOAc-n-hexane 15%,

yellow crystalline (compound L1), 110 mg), mp 255-257 C was obtained. The TLC of this compound

showed a single spot when it was eluted with three different solvent systems, i.e. EtOAc-n-hexane

35%; acetone-n-hexane 35% and acetone-dichloromethane 5% with Rf of 0.51, 0.23, and 0.49respectively.

2.3.2. Bioactivity test on the pure compound

The bioactivity test done includes the cytotoxic test of compound L1 based on the method of Mayer et

al. (1982), antibacterial activity test (Barry, 1980; Berghe and Vlientinck, 1991; Baueret al., 1966) and

antifungal activity test (Berghe and Vlientinck, 1991).

3. Results and Discussions3.1. The Analysis of Spectrometry

The crystal (L1) obtained was analyzed by UV-VIS spectrophotometry and produced max. (MeOH),

nm (log ): 203 (4.61); 268 (4.62); and 347 (3.96); max. (MeOH + AlCl3): 203 (4.62); 22 (4.54); 276(4.65); and 425 (3.98); max. (MeOH + AlCl3 + HCl): 203 (4.62); 226 (4.52); 277 (4.65); and 403

(3.86); max. (MeOH + NaOAc): 203 (4.82); 267 (4.60); and 347 (3.85).

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332 Tati Suhartati, Yandri and Sutopo Hadi

The yellow crystal obtained for compound L1 has a UV spectrum as shown in Figure 1 withmaximum absorbances at 203, 268, 300, and 347 nm. This UV spectrum indicated a prenylated

flavonoid on C-3 on the flavon frame (Markham, 1973), in which the band I at 347 nm has a lower

intensity than the band II with a max. of 268 nm. On the addition of AlCl3, the batochromic shiftingoccurred on band I and band II were 78 nm and 8 nm respectively which indicated there is no prenyl

group on C-6 on the A ring. On the addition of HCl, the hypsochromic shifting on band I was 22 nm

indicated ortho dihydoxy present on the B ring. While on the addition of NaOAc, there is no shifting

on band II which indicated no free OH group on C-7 at the flavon frame.

Figure 1: UV-VIS spectrum of compound L1

The IR spectrum of compound L1 (Figure 2) indicated absorption in KBr (cm-1

): 3434, 3379,

2982, 1662, 1643, 1605, 1583, 1560, 1523, 1481, and 1462. The stretching at 3434 cm-1

indicated the

OH group, the present of conjugated carbonyl group shown by strong stretching at 1662 and 1643 cm-1

in compound L1 (Harborne, 1973). The present of aromatic system is shown by the stretching at 1605-

1426 cm-1

.

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Table 1: The comparison of UV, IR, and MS spectrum data of artonin E (Hano et al., 1990) and thecompound L1

UV, max nm (log ) IR,1

cmKBrmaks

Artonin E L1 Artonin E L1

EtOH MeOH211 (4.21) 203 (4.61) 3440 3434

266 (4.34) 268 (4,62). 3390 3379

300 (sh, 3.63) 2982349 (3.51) 347 (3.96) 1662 1662

1650 (sh)

EtOH + AlCl3 MeOH +AlCl3 1645 1643203 (4.62)

266 (4.17) 226 (4.54) 1605 1605

277 (4.28) 276 (4.65) 1585 1583

410 (3.55) 425 (3.98) 1560 1560

MeOH +AlCl3 + HCl 1525 1523403 (3.86) 1483 1481

277 (4.65) 1462 1462

226 (4.52)

203 (4.62)+NaOAc

203 (4.82)267 (4.60)

347 (3.85)

Table 2: The comparison of1H-NMR spectrum of artonin E (Hano et al., 1990) and the compound L1

1H-NMR, (ppm)

Artonin E (DMSO-d6) L1 (Acetone-d6)

1,42 (9H, s, C16-CH3 x 2 1,44 (6H, s)

dan C11-CH3) 1,46 (3H, s)

1,57 (3H, br s, C11-CH3) 1,57 (3H, s)

3,06 (2H, br d, J = 7Hz, C9-Hx2) 3,14 (2H, d, J = 14,7 Hz)5,06 (1H, m, C10-H) 5,13 (1H, m)

5,70 (1H, d, J = 10 Hz,C15-H) 5,66 (1H, d, J = 19,6 Hz)

6,22 (1H, d, J = 0,5 Hz, C6-H) 6,15 (1H, s)

6,48 (1H, s, C3-H) 6,59 (1H, s)

6,54 (1H, dd, J = 0,5; 6,60 (1H, d, J = 20,8 Hz)10 Hz, C14-H)

6,70 (1H, s, C6-H) 6,88 (1H, s)

13,21 (1H, s, C5-OH) 7,80 (1H, br s)

8.28 (1H, s)8.34 (1H, br s)

13,25 (1H, s)

Figure 4: The molecule structure of compound L1 (artonin E)

OO

O

OHOH

OH

OH

A

B1

2

345

6 9

1011

12

13

14

15

16

17

18 1'

2'

3'

4'

5'

6'

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The1H-NMR spectrum of L1 (Figure 3) (acetone d-6, 500 MHz) (ppm): 1,44 (6H, s); 1.46

(3H, s); 1.57 (3H, br s); 3.14 (2H, br d, J= 14,7 Hz); 5.13 (1H, m); 5.66 (1H, d,J= 19.6 Hz); 6.15 (1H,s); 6.40 (1H, s); 6.62 (1H, d,J= 10 Hz); and 6.68 (1H, s); 7.80 (1H, br s); 8.28 (1H, s); 8.34 (1H, br s);dan 13.25 (1H, s).

Based on the1H-NMR spectrum of compound L1 (acetone-d6) that the compound L1 has 3

singlet aromatic protons with chemical shift of 6.15 ppm on the A ring; 6.59 and 6.88 ppm on the Bring. The isoprenyl substituents on C-3 are shown by protons of 2 CH3 groups with chemical shifts of

1.46 ppm (3 H, s) and 1.57 ppm (3 H, s) and 3 protons in ABX system with chemical shifts of 3.14ppm (2 H, d,J= 14,7 Hz) and 5.13 ppm (1 H, m). While the 2,2-dimethylcromene ring of isoprenylsubstituent on C-8 is shown by proton from 2 CH3 groups with chemical shift of 1.14 ppm (6 H, s) and

2 protons from vinyl groups directly attached on the A ring with chemical shift of 6.60 ppm (1 H, d, J= 20.8 Hz) and 5.66 ppm (1 H, d,J= 19.6 Hz).

Based on the spectroscopies data above, the values obtained are similar to artonin E reported byHano et al. (1990), therefore it was suggested that the compound L1 is artonin E (Table 1 and Table 2)with the molecule structure as shown in Figure 4.

3.2. Bioactivity test

The antifungal and antimicrobial bioactivity tests were done on compound L1. The fungi used were R.oryzae andR. oligosporus, while the microbia used wereE. coli andB. subtilis. The compound L1 withthe amount of 250 g diskshowed microbial activity againstE. coli andB. subtilis produced clear zonewith a diameter of 1.2 cm and 0.9 cm respectively (Figure 5), while the standard used the canamycin

sulphate with the amount of 240 g disk produced clear zone with a diameter of 2.2 (Figure 6).However, in the antifungal activity test this compound did not have a good activity against both R.oryzae andR. Oligosporus (Figure 7).

The cytotoxicity tests of compound L1 and cycloarthobiloxanthon have been previously beendone where artonin E showed higher cytotoxicity (IC50 0.06 g/mL) than cycloarthobiloxanthone (IC50

4.6 g/mL) against leukemia P-388 cell (Suhartati et al., 2001).Based on these data, compound L1 (artonin E) not only has high cytotoxicity against leukemia

P-388 cell, but it also has antimicrobial effect which make this compound has a possibility to be usedas antitumor and antibiotics.

Figure 5: The microbial activity test of compound L1 againstE. Coli

L1L1Canamycin

CanamycinControl

Control

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Figure 6: The microbial activity test of compound L1 againstB. subtilis

L1L1

Canamycin

CanamycinControl

Control

Figure 7: The antifungal activity test of compound L1 againstR. oryzae andR. oligosporus

R. oryzae R. oligosporus

L1

L1Clotrimazol

Clotrimazol

ConclusionsFrom the bark ofA. rigida has been isolated cycloartobiloxanthone dan artonin E, the derivative of prenylated flavonoid compound on C-3 which has been known to have high bioactivity againstLeukemia murine P-388 cell. Artonin E also showed antimicrobial activity against both E. coli andB.subtilis, the antimicrobial activity of artonin E is believed to be the first report available; howeverartonin E did not show a good antifungal activity againstR. oligosporus orR. oryzae.

AcknowledgmentThe authors would like to thank to The Directorate of Research and Community Services, Directorate

General of Higher Education, The Ministry of National Education of Republic of Indonesia that provides fund for this project to be undertaken through Fundamental Research Grant Scheme 2007

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with contract number of 028/SP2H/PP/DP2M/III/2007. Thanks also go to Erlina Eka S., Iswanti, andHernawan who have helped us in the preparation of the samples. We are also grateful to the Herbarium

Bogoriense Bogor, Indonesia, for the assistance in identification of the plant specimen.

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Laboratory Medicine (V. Lorian Ed.). Williams and Wilkin Co. Baltimore. USA. 1-23.[2] Bauer, A.W., W.M.M. Kirby, J.C. Sherris, and M. Truck. 1966. Antibiotic susceptibility testing

by a standard disc.Am. J. Clin. Pathol. 45: 493-496.[3] Berghe, DAV and A.J. Vlientinck. 1991. Screening methods for antibacterial and antiviral

agents from higher plants, In: Assays for Bioactivity (Hostettmann K. Ed) Vol 6, Methods inplant Biochemistry (Dey P.M. and J.B. Harborne Ed). Academic press. London. 47.

[4] Han, A.-R., You-J. Kang, T. Wibowo, S.K. Lee, and Eun-K. Seo. 2006. Prenylated flavonoidsfrom the heartwood of Artocarpus communis with inhibitory activity on lipopolysaccharide-induced nitric oxide production.J. Nat. Prod. 69: 719-721.

[5] Hano, Y., Yamagami, Y., Kobayashi, M., Isohata, R., and Nomura, T. 1990. Artonins E and F,two new prenylflavones from the bark of Artocarpus communis Forst, Heterocycles. 31(5):

877-882.[6] Harborne, J.B. 1973.Phytochemical Methods. London. Chapman and Hall, Ltd. 49-188.[7] Markham, K.R. 1988. Cara Mengidentifikasi Flavonoid. Penerbit ITB. Bandung. Hlm 1- 173[8] Lemmens, R.H.M.J., I. Soerianegara, and W.C. Wong (Ed). 1995. Plant Resources of South-

East Asia 5, (2) Timber Trees: Minor Commercial Timbers. Bogor. 59-70.[9] Mayer, N., N.R. Ferrigni, J.E. Putnam, D.E. Nichols, and J.L. McLaughlin. 1982. Brine

Shrimp: A Convenient General Bioassay for Active Plant Constituents. Plant. Medica. 45: 31-34.

[10] Nomura, T., S. Hano, and M. Aida. 1998. Isoprenoid-Substituted Flavonoid fromArtocarpusPlants (Moraceae).Heterocycles. 47(2): 1179-1205.

[11] Su, B.-N., M. Cuendet, M.E. Hawthorne, L.B. S. Kardono, S. Riswan, H.H.S. Fong, R.G.

Mehta, J.M. Pezzuto, and A.D. Kinghorn. 2002. Constituents of the bark and twigs ofArtocarpus dadah with cyclooxygenase inhibitory activity.J. Nat. Prod., 65(2): 163 169.

[12] Suhartati, T., S.A. Achmad, N. Aimi, dan E.H. Hakim. 1999. Artonin E, suatu senyawaflavonoid dariArtocarpus rotunda. J. Mat. & Sains. 4(2): 178-184.

[13] Suhartati, T., S.A Achmad, N. Aimi, E.H. Hakim, M. Kitajima, H. Takayama, and K. Takeya.

2001. Artoindonesianin L, a new prenylated flavone with cytotoxic activity from Artocarpusrotunda. Fitoterapia. 72: 912-918.

[14] Suhartati, T. dan Yandri A.S. 2007. Sikloartobilosanton dari kulit batang dan flavonoid dalam

beberapa bagian tumbuhanArtocarpus dadah yang tumbuh di Lampung.J. Sains MIPA. EdisiKhusus. 13(2): 82-86 (Indonesian).

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