European Journal of Scientific Research

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

© EuroJournals Publishing, Inc. 2008
http://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: tatisuhartati@unila.ac.id

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

Sutopo Hadi
Department of Chemistry, University of Lampung, Bandar Lampung 35145 Indonesia
E-mail: sutopohadi@unila.ac.id

Abstract

From the bark of Artocarpus 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-388
cell. In bioactivity test, artonin E was active against E. coli and B. subtilis, however it was
not active against Rizhopus. oryzae and Rizhopus. oligosporus.

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

1. Introduction
The research on Artocarpus plant growing in Lampung Province is continuously being carried out
especially 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 was
Artocarpus 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 of
these 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 of A. rigida. In this paper from the bark of A. rigida, we reported the isolation of artonin
E. 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 and B. Subtilis (Barry, 1980; Berghe and Vlientinck, 1991; Bauer et al., 1966) but did not show a
good activity against R. oryzae or R. Oligosporus (Berghe and Vlientinck, 1991).
The Bioactivity Test of Artonin E from the Bark of Artocarpus Rigida Blume 331

2. Materials and Methods

2.1. Plant Material
Samples of the bark of A. rigida Blume were collected from Keputran Village, Sukoharjo Tanggamus
Lampung in April 2007 and were identified by the staff at the Herbarium Bogoriense, Research Centre
for 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 (Merck
Kieselgel 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 8501
Scientific 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 of
compound, determination of the molecule structure with physical and UV-VIS, IR, and 1H-NMR
spectroscopies 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 of
ethylacetate (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 the
spot 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 8
fractions 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 0.49
respectively.

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; Bauer et al., 1966) and
antifungal activity test (Berghe and Vlientinck, 1991).

3. Results and Discussions

3.1. The Analysis of Spectrometry
The crystal (L1) obtained was analyzed by UV-VIS spectrophotometry and produced λ max. (MeOH),
nm (log ε): 203 (4.61); 268 (4.62); and 347 (3.96); λ max. (MeOH + AlCl3): 203 (4.62); 22 (4.54); 276
(4.65); and 425 (3.98); λ max. (MeOH + AlCl3 + HCl): 203 (4.62); 226 (4.52); 277 (4.65); and 403
(3.86); λ max. (MeOH + NaOAc): 203 (4.82); 267 (4.60); and 347 (3.85).
332 Tati Suhartati, Yandri and Sutopo Hadi

The yellow crystal obtained for compound L1 has a UV spectrum as shown in Figure 1 with
maximum 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 λ max. of 268 nm. On the addition of AlCl3, the batochromic shifting
occurred 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.
The Bioactivity Test of Artonin E from the Bark of Artocarpus Rigida Blume 333
Figure 2: IR spectrum of compound L1

Figure 3: 1H-NMR spectrum of compound L1 in acetone d-6

334 Tati Suhartati, Yandri and Sutopo Hadi
Table 1: The comparison of UV, IR, and MS spectrum data of artonin E (Hano et al., 1990) and the
compound L1

IR, υ maks cm
KBr −1
UV, λ max nm (log ε)
Artonin E L1 Artonin E L1
EtOH MeOH
211 (4.21) 203 (4.61) 3440 3434
266 (4.34) 268 (4,62). 3390 3379

300 (sh, 3.63) 2982

349 (3.51) 347 (3.96) 1662 1662
1650 (sh)
EtOH + AlCl3 MeOH +AlCl3 1645 1643
203 (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 1523
403 (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 of 1H-NMR spectrum of artonin E (Hano et al., 1990) and the compound L1
1
H-NMR, δ (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)

17 15 3'
HO 2' OH
14
4'
18
16
O O
1 1' B
5' OH
A 2 6'
6 9
4 3
5
12
OH O 10
11

13
The Bioactivity Test of Artonin E from the Bark of Artocarpus Rigida Blume 335

The 1H-NMR spectrum of L1 (Figure 3) (acetone d-6, 500 MHz) δ (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 the 1H-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 B
ring. 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 3.14
ppm (2 H, d, J = 14,7 Hz) and 5.13 ppm (1 H, m). While the 2,2-dimethylcromene ring of isoprenyl
substituent 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 by
Hano 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 and R. oligosporus, while the microbia used were E. coli and B. subtilis. The compound L1 with
the amount of 250 µg disk showed microbial activity against E. coli and B. subtilis produced clear zone
with 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 µg 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 and R. Oligosporus (Figure 7).
The cytotoxicity tests of compound L1 and cycloarthobiloxanthon have been previously been
done where artonin E showed higher cytotoxicity (IC50 0.06 μg/mL) than cycloarthobiloxanthone (IC50
4.6 μg/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 used
as antitumor and antibiotics.

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

L1 L1
Canamycin

Control Canamycin
Control
336 Tati Suhartati, Yandri and Sutopo Hadi
Figure 6: The microbial activity test of compound L1 against B. subtilis

L1 L1

Canamycin
Control
Canamycin
Control

Figure 7: The antifungal activity test of compound L1 against R. oryzae and R. oligosporus

L1
Clotrimazol
Clotrimazol L1

R. oryzae R. oligosporus

Conclusions
From the bark of A. 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 against
Leukemia murine P-388 cell. Artonin E also showed antimicrobial activity against both E. coli and B.
subtilis, the antimicrobial activity of artonin E is believed to be the first report available; however
artonin E did not show a good antifungal activity against R. oligosporus or R. oryzae.

Acknowledgment
The 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
The Bioactivity Test of Artonin E from the Bark of Artocarpus Rigida Blume 337

with contract number of 028/SP2H/PP/DP2M/III/2007. Thanks also go to Erlina Eka S., Iswanti, and
Hernawan 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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