Xanthone from Garcinia eugenifolia (Clusiaceae)

*1,2Vivien Jong Yi Mian, 2Gwendoline Ee Cheng Lian, 1Khong Heng Yen

1Faculty of Applied Sciences, Universiti Teknologi MARA,94300 Jalan Meranek, Kota Samarahan , Sarawak, Malaysia. 2Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia.

*Corresponding Author: vivien@sarawak.uitm.edu.my

Abstract— Studies on the twigs of Garcinia eugenifolia have led to the isolation of one xanthone 5,9-dihydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-enyl)pyrano[3,2-b]xanthen-6(2H)-one (1) and one triterpenoid, sitosterol (2). Meanwhile, studies on the leaves of Garcinia eugenifolia provided another triterpenoid, friedelin (3). The structures of these compounds were determined by spectroscopic methods such as 1H NMR, 13C NMR, mass spectrometry (MS) and by comparison with previous studies. The hexane and ethyl acetate extracts of Garcinia eugenifolia indicated strong cytotoxicity towards the HL-60 cell line with IC50 values of 1.9 μg/ml and 2.5 μg/ml respectively. However, the methanol extract gave moderate inhibitory activities (IC50<10 μg/ml) towards HL-60 cell line.

Keywords- Garcinia eugenifolia; Xanthone; Cytotoxicity

I. INTRODUCTION Garcinia (Clusiaceae) is distributed in Thailand, India, Sri Lanka, Myanmar, Indonesia, Malaysia, Philippines and China. People in these countries often use Garcinia as the traditional medicines to treat abdominal pain, dysentery, diarrhea, suppuration, infected wound, leucorrhoea and chronic ulcer, and gonorrhea [1]. Furthermore, Garcinia exhibits anti-inflammatory [2], antitumor and antioxidant abilities [3], and antibacterial activity against Staphylococcus aureus [4] and Helicobacter pylori [5]. It was reported that Garcinia was a source of mangostin, tannin, xanthone, isoflavone, flavone and other bioactive substances [6][7]. Extensive research has shown that some bio-compounds from Garcinia species exhibited a wide range of biological and pharmacological activities such as cytotoxicity and antioxidant abilities. Detailed studies on the roots of Garcinia eugenifolia have provided 5,9-dihydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-enyl)pyrano[3,2-b]xanthen-6(2H)-one (1) and sitosterol (2).

II. EXPERIMENTAL

A. Plant Material The twigs (1.3 kg) and leaves (1.2 kg) of G. eugenifolia

was collected from Sarawak Forestry Department, Semengok, Malaysia.

B. Extraction and Isolation The fine ground twigs of G. eugenifolia were extracted with

hexane, ethyl acetate and methanol, twice for each to yield 7g, 28.9g and 19.5g of crude extracts respectively. Meanwhile, the fine ground leaves of G. eugenifolia were extracted with methanol, twice to yield 30.6g of crude extract. The dry crude extracts were purified by column chromatography using hexane, hexane/chloroform, chloroform/ ethyl acetate and chloroform/ methanol as eluting solvents. Besides, the crude extracts also were purified by preparative thin layer chromatography and sephadex LH-20.

C. 5,9-dihydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-enyl)pyrano[3,2-b]xanthen-6(2H)-one

(1) Yellow gum. IR: 3384 (broad OH), 2934 (C-H stretching), 1604 (C=O). 1H NMR (400 MHz, CDCl3): δ 13.7 (s, 1H, OH-5), δ 6.84 (s, 1H, H-10), δ 6.74 (d, J = 10.1 Hz, 1H, H-4), δ 6.25 (s, 1H, H-12), δ 5.56 (d, J = 10.1 Hz, 1H, H-3), δ 5.26 (t, J = 6.4 Hz, 1H, H-2'), δ 4.10 (d, J = 6.4 Hz, 2H, H-1'), δ 3.81 (s, 3H, 8-OMe), δ 1.83 (s, 3H, H-4'), δ 1.69 (s, 3H, H-5'), δ 1.46 (s, 6H, 2CH3). 13C NMR (100 MHz, CDCl3): δ 181.9 (C-6), δ 159.9 (C-12a), δ 157.9 (C-5), δ 156.3 (C-11a), δ 155.7 (C-10a), δ 154.6 (C-9), δ 142.6 (C-8), δ 136.9 (C-7), δ 132.2 (C-3'), δ 127.2 (C-3), δ 123.1 (C-2'), δ 115.7 (C-4), δ 112.2 (C-6a), δ 104.5 (C-4a), δ 103.7 (C-5a), δ 101.7 (C-10), δ 94.1 (C-12), δ 77.9 (C-2), δ 62.0 (8-OMe), δ 28.3 (2-CH3), δ 26.5 (C-1'), δ 25.8 (C-4'), δ 18.2 (C-5')

D. Cytotoxic Activity The cytotoxic assay against the HL-60 cell line (Human

Promyelocytic Leukemia) were carried out on the methanol extracts of G. eugenifolia. Briefly, HL-60 cells were cultured in RPMI 1640 media supplemented with 10% Fetal Bovine Serum. The cells were maintained at 37°C in 5% CO2 atmosphere. Further maintenance and subculturing of cells were done according to supplier’s protocol.

III. RESULTS AND DISCUSSION 5,9-dihydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-

enyl)pyrano[3,2-b]xanthen-6(2H)-one (1) was isolated as a yellow gum. It reacted positively with FeCl3 reaction to reveal

2010 International Conference on Science and Social Research (CSSR 2010), December 5 - 7, 2010, Kuala Lumpur, Malaysia

978-1-4244-8986-2/10/$26.00 ©2010 IEEE 786

its phenolic nature. The [M+] at m/z 408 in the EI-MS spectrum corresponds to the molecular formula C24H24O6. The ultra violet absorptions at 243.5 and 316.5 nm show that (1) is an oxygenated xanthone.

The 1H NMR spectrum in CDCl3 showed the 5-OH signal at δ 13.7 (1H, s) The signals at δ 6.74 (d, 1H, J = 10.1 Hz, H-4), δ 5.56 (d, 1H, J = 10.1 Hz, H-3) and δ 1.46 (s, 6H, 2CH3) indicated the presence of a 2,2-dimethyl chromene ring substitution at the xanthone ring, with the chromene double bond ortho to the C-5 hydroxyl. In addition, 1H NMR signals at δ 5.26 (t, 1H, J = 6.4 Hz, H-2’), δ 4.10 (d, 2H, J = 6.4 Hz, H-1’), δ 1.83 (s, 3H, H-4’) and δ 1.69 (s, 3H, H-5’) were typical signals for a prenyl moiety which is ortho to the C-6 carbonyl. Two isolated aromatic protons that were observed at δ 6.84 (s, 1H) and δ 6.25 (s, 1H) were assigned to H-10 and H-12, respectively. Finally, a singlet signal that resonates at δ 3.81 was due to the methoxy group in compound (1).

The COSY spectrum showed correlations between H-1’ and H-2’ as well as between H-2’ and H-4’ and H-5’ which confirmed the presence of an isopentenyl side chain in (1). A correlation also occurred between H-3 and H-4 in the COSY analysis. The 13C NMR spectrum gave a total of twenty four carbons which validated the molecular formula of (1). The very downfield signal at δ 181.9 was assigned to the carbonyl group at C-6. Other protonated carbons of (1) were assigned by the HMBC experiment.

The 24 carbon signals in the 13C NMR spectrum were characterized by the DEPT spectrum, which indicated that (1) consists of five methyl, one methylene, five methine and thirteen quaternary carbons. In the HMBC analysis, the methylene proton (H-1’) gave cross peak to C-8 (δ 142.6) and C-7 (δ 136.9), thus suggesting the location of the prenyl moiety to be at C-7. Furthermore, the HMBC spectrum also showed that the methoxyl group was attached to C-8.

Comparison of these data with those reported earlier by Sen et al., (1982) [8] showed that (1) was identical to 5,9-dihydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-enyl)pyrano[3,2-b]xanthen-6(2H)-one (see Table I and Figure 1).

TABLE I. 1HNMR (400 MHZ, CDCL3) AND 13C NMR (100 MHZ, CDCL3), ASSIGNMENTS FOR 5,9-DIHYDROXY-8-METHOXY-2,2-DIMETHYL-

7-(3-METHYLBUT-2-ENYL)PYRANO[3,2-B]XANTHEN-6(2H)-ONE AND THEIR CONNECTIVITIES OBTAINED FROM COSY AND HMBC EXPERIMENT.

Sen et al., (1982)

Figure 1. 5,9-dihydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-enyl)pyrano[3,2-b]xanthen-6(2H)-one

Position δH δC 1H-1H COSY HMBC

2 77.9 3 5.56 (1H, d, J

= 10.1, Hz) 127.2 H-4 C-2, C-4a

4 6.84 (1H, d, J = 10.1, Hz)

115.7 H-3 C-2

4a 104.5 5 13.7 (1H, s,

OH) 157.9

5a 103.7 6 181.9 6a 112.2 7 136.9 8 142.6 9 154.6

10 6.84 (1H, s) 101.7 C-6a, C-8, C-9, C-10a

10a 155.7 11a 156.3 12 6.25 (1H, s) 94.1 C-4a, C-5a, C-

11a, C-12a 12a 159.9 1’ 4.09 (2H, bt, J

= 6.4 Hz) 26.5 H-4’, H-5’

2’ 5.26 (1H, bt, J = 6.4 Hz)

123.1 H-1’, H-4’,

H-5’

C-3’, C-7, C-8

3’ 132.2 4’ 1.69 (3H, s) 25.8 C-2’, C-3’, C-

5’ 5’ 1.83 (3H, s) 18.2 C-2’, C-3’, C-

4’ 2-Me 1.46 (3H, s)

1.46 (3H, s) 28.3 28.3

C-2, C-3, 2-Me C-2, C-3, 2-Me

8-OMe 3.80 (3H, s) 62.0 C-8

O O

OH

H3CO

HO

O

12

3

44a

55a66a

78

9

1010a

1111a

1212a

1'

2'3'

4' 5'

787

TABLE II. IC50 VALUES OF THE CRUDE EXTRACTS ON HL-60 CELL LINE

Extract IC50 (μg/ml)

Hexane 1.9

Ethyl Acetate 2.5

Methanol 9.0

Table 2 shows the IC50 values of the crude extracts for G. eugenifolia against the HL-60 (Human promyelocytic leukemia) cells line. The hexane and ethyl acetate extract of G. eugenifolia indicated strong cytotoxicity towards the HL-60 cells line with IC50 values of 1.9 μg/ml and 2.5 μg/ml respectively. However, the methanol extract gave good cytotoxic activities (IC50<10 μg/ml) towards HL-60 cells line.

ACKNOWLEDGMENT The authors wish to thank the Research Management Institute, Universiti Teknologi MARA for the Excellent Fund (DANA Cemerlangan) and MOSTI for FRGs grant for financial support. The authors also wish to thank the staffs of Forest Research Centre, Kuching, Sarawak for their assistance in the collection and identification of the plant.

REFERENCES

[1] G. K. Jayaprakasha, P.S. Negi, and B.S. Jena, “Antioxidative and antimutagenic activities of the extracts from the rinds of Garcinia pedunculata,” Innovative Food Science & Emerging Technologies, vol. 7, pp. 246–250, 2006.

[2] C. Gopalakrishnan, D. Banumathi, and S. K. Suresh, “Effect of mangostin, a xanthone from Garcinia mangostana Linn. in immunopathological and inflammatory reactions,” Indian Journal of Experimental Biology, vol. 19, pp. 843–846, 1997.

[3] P. Williams, M. Ongsakul, J. Proudfoot, K. Croft, and L. Bellin, “Mangostin inhibits the oxidative modification of human low density lipoprotein,” Free Radical Research, vol. 23, pp. 175–184, 1995.

[4] Y. Sakagami, M. Iinuma, K. G. N. P. Piyasena, and H. R. W. Dharmaratne, “Antibacterial activity of α-mangostin against vancomycin resistant Enterococci (VRE) and synergism with antibiotics,” Phytomedicine, vol. 12, pp. 203–208, 2005.

[5] W. Mahabusarakum, S. Phongpaichit, C. Jansakul, and P. Wiriyachitra, “Screening of antibacterial activity of chemicals from Garcinia mangostana Songklanakarin,” Journal of Science and Technology, vol. 5, pp. 337–339, 1983.

[6] S. Deachathai, W. Mahabusarakam, S. Phongpaichit, and W. C. Taylor, “Phenolic compounds from the fruit of Garcinia dulcis,” Phytochemistry, vol. 66, pp. 2368–2375, 2005.

[7] H. A. Jung, B. N. Su, W.J. Keller, R. G. Mehta, and A. D. Kinghorn, “Antioxidant xanthones from the pericarp of Garcinia mangostana (Mangosteen),” Journal of Agricultural and Food Chemistry, vol. 54, pp. 2077–2082, 2006.

[8] A. K. Sen, K. S. Kalyan, P.C. Mazumder, B. Nilima, R. Uusvuori, and T. A. Hase, “A Xanthone from Garcinia mangostana,” Phytochemistry, vol. 19, pp. 2223-2225, 1980.

[9] O. A. Adaramoye, E. O. Farombi, E.O. Adeyemi, and G.O. Emerole, “Comparative Study on the Antioxidant Properties of Flavonoids of Garcinia Kola Seeds,” Journal of Medicinal Science, vol. 21, pp. 331-339, 2005.

[10] C. H. Chang, C. C. Lin, M. Hattori, and T. Namba, “Four prenylated xanthones from Cudrsnis cochinchinensis,” Phytochemsitry, vol. 28, pp. 595-598, 1989.

[11] P. Dharma, H. L. Nordin, M. M. Mackeen, M. A. Abdul, A. Norio, K. Mariko, and T. Hiromitsu, “Isolation and Bioactivities of Constitutents of the Roots of Garcinia atroviridis,” Journal of Natural Product, vol. 64, pp. 976 -979, 2001.

[12] A. S. Khalid, S. Khozirah, A. Faridah, A. I. Daud, S. H. Ahmad, S. Normawati, S. Khoushik and H. L. Nordin, “Cytotoxic caged-polyprenylated xanthonoids and a xanthone from Garcinia cantleyana,” Phytochemistry vol. 68, pp. 2537-2544, 2007.

[13] M. M. Mackeen, A. M. Ali, N. H. Lajis, K. Kawazu, Z. Hassan, M. Amran, M. Habsah, L. Y. Mooi, and S. M. Mohamed, “Antimicrobial, antioxidant, antitumour-promoting and cytotoxic activities of different plant part extracts of Garcinia atroviridis,” Journal of Ethnopharmacology, vol. 72, pp. 395-402, 2000.

[14] W. Mahabusarakam, J. Proudfoot, W. Taylor, and K. Croft, “Inhibition of lipoprotein oxidation by prenylated xanthones derived from mangostin,” Free Radic Res, vol. 33, pp. 643-659, 2000.

788