Laporan Eksekutif
UJI PREKLINIS TETRAPRENILTOLUQUINOL SENYAWA ANTIKANKER
BARU DARI TUMBUHAN KANDIS (GARCINIA COWA ROXB)(1)
Oleh
Fatma Sri Wahyuni, Nordin Hj Lajis dan Desi Eka Putri (2)
I.
PERMASALAHAN DAN TUJUAN PENELITIAN
Menurut Organisasi Kesehatan Dunia, WHO, setiap tahun jumlah penderita
kanker di dunia bertambah 6,25 juta orang. Dalam 10 tahun mendatang diperkirakan 9
juta orang akan meninggal setiap tahun akibat kanker. Dua pertiga dari penderita kanker
di dunia akan terdapat di negara-negara yang sedang berkembang (Anonimous, 2005). Di
Indonesia diperkirakan setiap tahunnya terdapat 100 penderita kanker baru dari setiap
100.000 penduduk (DepKes, 2002). Peningkatan jumlah penderita kanker di Indonesia
merupakan gejala yang sangat mengkhawatirkan, karena disamping membutuhkan biaya
pengobatan yang besar, obat-obat kanker yang ada pada saat ini juga memiliki efek
samping yang besar juga. Hal ini disebabkan karena belum adanya obat yang bekerja
specifik untuk pengobatan kanker. Oleh sebab itu upaya untuk menemukan obat alternatif
baru yang bisa digunakan mengobati penyakit kanker yang murah, memiliki toksisitas
yang rendah dan bekerja specifik sangat giat dilakukan.
Usaha untuk mendapatkan bahan yang bisa mengobati penyakit kanker yang
murah, mudah didapat dan memiliki kerja yang spesifik dimungkinkan dengan
melakukan pencarian sumber bahan obat baru yang berasal dari tanaman yang ada di
Indonesia. Dari hasil penelitian terdahulu terhadap beberapa species dari tumbuhan
Garcinia, diketahui bahwa ekstrak tumbuhan Garcinia cowa memiliki aktifitas yang
potensial serta spesifik menghambat pertumbuhan sel kanker paru-paru. Oleh sebab itu
kami berusaha untuk menemukan senyawa yang bertanggungjawab terhadap aktifitas ini.
______________________
1) Penelitian ini dibiayai melalui Hibah Bersaing, tahun anggaran 2006, Rp.
45.650.000,2) Dosen Sekolah Tinggi Farmasi Indonesia, Yayasan Perintis Padang dan Institute
of Biosciences, Univerisity Putra Malaysia
1
II.
INOVASI IPTEKS
Penemuan senyawa potensial yang memiliki aktifitas antikanker yang spesifik
dari tumbuhan Garcinia cowa ini merupakan suatu terobosan baru didalam dunia
pengobatan penyakit kanker, karena sampai saat sekarang belum ada obat kanker yang
bekerja secara spesifik.
III.
KONTRIBUSI PENELITIAN TERHADAP PEMBANGUNAN
Penemuan senyawa potensial yang memiliki aktifitas antikanker yang spesifik
dari tumbuhan Garcinia cowa ini merupakan suatu terobosan baru didalam dunia
pengobatan penyakit kanker. Senyawa yang akan didapat pada penelitian ini bisa
dijadikan model baru pada pengobatan kanker sehingga bisa dipublikasikan secara
internasional dan atau dipatenkan. Dengan ditemukannya senyawa antikanker yang
bekerja spesifik akan memberikan nilai tambah pada kegunaan tumbuhan Indonesia,
khususnya Garcinia cowa sehingga diharapkan bisa menjadi sumber pendapatan baru
bagi penduduk, khususnya yang berada dipedesaan dan negara pada umumnya.
IV.
MANFAAT BAGI INSTITUSI
Penelitian ini sangat berarti bagi perkembangan institusi kami dimana dengan
adanya kerjasama penelitian dengan pihak luar akan memberi cakrawala baru pada
institusi kami yang relatif baru berdiri.
Mahasiswa S-2 yang terlibat dalam penelitian ini adalah :
1. Desi Eka Putri, NIM 05213002 Judul Skripsi adalah Kajian Farmakokinetik dari
Senyawa Tetrapreniltoluquinol, (Sedang berlangsung)
Mahasiswa S-3 yang terlibat adalah :
1. Fatma Sri Wahyuni, Matrik no GS12416, Judul Disertasi adalah Isolation and
Biological Activity Study of Garcinia spp (Sedang berlangsung)
V.
PUBLIKASI ILMIAH
Fatma Sri Wahyuni, Desi Eka Putri, Nordin H. Lajis, and Johnson
Stanslas, Tetraprenyltoluquinone, A New Selective compound from Garcinia
cowa, will be submitted to Journal of Pharmaceutical Biology.
2
Draft Artikel Ilmiah
Tetraprenyltoluquinone, A New Selective compound from Garcinia cowa
Fatma Sri Wahyuni,A,B Desi Eka PutriB, Nordin H. LajisA, and Johnson
StanslasA,C
A
Institute of Bioscience, University of Putra Malaysia, 43400 Serdang,
Selangor, Malaysia.
B
Sekolah Tinggi Farmasi Indonesia, Yayasan Perintis Padang, Simpang
Kalumpang Padang, West Sumatra, Indonesia.
C
Faculty of Biomedics, University of Putra Malaysia, 43400 Serdang,
Selangor, Malaysia.
Abstract
The hexane fraction of the methanolic extract of the stem bark of Garcinia
cowa Roxb. (Guttiferae) yielded [2E,6E,10E]-(+)-4-hydroxy-3-methyl-5(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl-2-cyclohexen-1-one
(1). The structural elucidation followed from spectroscopic evidence. This
extract and tetraprenyltoluquinone (1) showed active and selective activity
towards non small lung cancer cells (H-460) with IC50 of 16 ± 4 µg/mL.
Introduction
Garcinia cowa Roxb. (Guttiferae), known as kandis in West Sumatra, is a medium-sized
tree which attains a height of ca. 30 m and it is widely distributed throughout Indonesia
and the Malay peninsula. Previous workers have isolated a number of prenylated
xanthones from the stem bark.[1,2,3] In this work we report the isolation and structural
elucidation from the hexane soluble portion of the methanolic extract of a new ringreduced tetraprenyl-toluquinone.
Results and Discussion
Chromatography of the hexane soluble portion of the methanolic extract yielded a
dextrorotatory yellow oil which was assigned structure (1) on the following grounds. The
high-resolution mass spectrum (HRMS) indicated the molecular formula C27 H42 O2. The
1
H and
13
C nuclear magnetic resonance (NMR) spectra, shown in Table 1, were
interpreted by the assistance of the distortionless enhancement of polarization transfer
(DEPT), double-quantum filtered correlation spectroscopic (DQF-COSY), heteronuclear
3
single quantum coherence (HSQC) and heteronuclear bond correlation (HMBC)
techniques and clearly indicated the presence of a linear geranylgeranyl chain [4,5] which
must therefore be attached to a fragment C7 H9 O2 containing three units of unsaturation.
The DEPT technique indicated that this fragment contained a ketone (C 198.99), a
tertiary vinylic carbon (C 126.44), a quaternary vinylic carbon (C 161.14), a tertiary
carbon (C 73.09) presumably attached to a hydroxy group, a tertiary carbon (C 43.47), a
methylene group (C 41.06) and a methyl group (C 20.30). The infrared spectrum of the
new compound indicated the presence of a hydroxyl function (max 3435 cm-1) and an
-unsaturated ketone (max 1653 cm-1) in keeping with the
13
C NMR spectrum. These
data suggested that the fragment C7 H9 O2 was -methyl--unsaturated cyclohexenone
substituted with a geranylgeranyl side chain and a hydroxy group. The tertiary vinylic
carbon (C 126.44) was associated with a broad singlet at H 5.84 as shown by the HSQC
technique so that it can be assigned to C(). The quaternary vinylic carbon (C 161.14)
was deduced from the HMBC technique to be coupled (2 bonds) to the protons of a
methyl group (H 2.02, C 20.38) so that this carbon is C(). The tertiary carbon (C
73.09, H 4.09), by application of the HMBC technique, was deduced to be coupled (3
bonds) to the methyl group protons (H 2.02) and to the vinyl proton (3 bonds) at H 5.80
so that this carbon is C(). Carbon C() was also coupled (3 bonds) to a proton at H 2.35
which is attached (HSQC) to C(1') of the geranylgeranyl chain which must therefore be
attached to C(). This compound was elucidated as structure (1).[6]
Methanol extracts
and tetraprenyltoluquinol isolated from the stem bark of
Garcinia cowa extract showed active and selective activity towards non small lung
cancer cells (H-460) with IC50 of 16 ± 4 µg/mL. [7]
Experimental
General directions have been given previously.[8] The 600 MHz 1NMR spectrum was
recorded on a Bruker AV-600 instrument.
4
Extraction and Isolation of Compounds from the bark of Garcinia cowa
The stem bark of Garcinia cowa was collected in February 2006 at Sarasah
Bonta, Harau Valley, 50 Kota District, West Sumatra at an altitude of 500m. A voucher
specimen (DR-180) was identified by Dr Rusjdi Tamin and is deposited in the herbarium
at Universitas Andalas.
Ground air-dried stem bark (5 kg) was percolated with hexane (10 L) followed by
dichloromethane (10 L). The extracts were evaporated to dryness under reduced pressure
and the dark mass (17.12 g, hexane) and (212 g, dichloromethane)
Isolation of [2E,6E,10E]-(+)-4 -hydroxy-3-methyl-5 -(3,7,11,15-tetramethyl-2,6,10,14hexadecatetraenyl-2-cyclohexen-1-one 1
A portion (60.0 g) of the hexane extract was chromatographed over a column of silica gel
with increasing amounts of ethyl acetate in hexane as eluent. Further purification was
achieved using radial chromatography with the same eluent.
This process yielded
[2E,6E,10E]-(+)-4-hydroxy-3-methyl-5-(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl-2-cyclohexen-1-one 1 (4g) as a yellowish oil.
High-resolution FABMS
[M+H]+• found: 399.3263 C27 H43 O2 requires m/z 399.3359. []26
D +50.0° (c,10.0,
CHC13). EIMS: m/z 398 [M]+• (11%), 355(2), 329(2), 205(10), 161(10), 149(12),
138(11), 137(24), 136(18), 135(20), 133(12), 125(64), 124(16), 123(17), 122(11)
121(24), 108(32), 106(18), 104(13), 69(100). max/nm (MeOH) 230 ( 11,000). max/cm-1
(film) 3435, 3310, 2980, 2925, 2860, 1653, 1437, 1377, 1111, 1008, 886, 667.
Cytotoxic assay ( MTT Assay)
Cells will be attached by incubat suspensiocells (180 ul in each well axcept
blank) for over night. Varying concentrations of the plant extracts will be prepared from
the stock solutions by serial dilution (10-fold dilution start from 100 ug, 10 ug, 1 ug, 0.1
ug) in RPMI-1640 to give a volume of 200 ul in each well of microtiter plate (96-well).
The assay for each concentration of extract will be performed in quarted and the culture
plate will be kept at 37 0 C with 5 % (v/v) CO2 for 4 days. After 96 hours of incubation,
50 ul of 0.2 % w/v MTT will be dissolved in phosphate buffer saline, will be added to
each well and allowed to incubate for a further 4 hours. After 4 hours incubation, discard
all supernatant. Subsequently, 100 ul DMSO will be added to each well and vigorously
mixed to dissolve the formazan crystals. Absorbance values at 570 nm will be measured
5
with a microplate reader. Cytotoxicity will be expressed as IC50 , i.e. the concentration
to reduce the absorbance of treated cells by 50% with reference to the control (untreated
cells). [9]
Acknowledgements
We thank Dr Rusjdi Tamin for the identification of the plant material. Department of
Education, Republic of Indonesia is thanked for financial support under Hibah Bersaing
XIV.
References
[1]
[2]
[3]
[4]
[5]
[6]
H.-H. Lee, H.-K. Lee, Phytochemistry, 1976, 15, 2038.
K. Likhitwitayawuid, T. Phandungchareon, C. Mahidol, S. Ruchirawat,
Phytochemistry, 1997, 45, 1299.
K. Likhitwitayawuid, T. Phandungchareon, J. Krungkrai, Planta Medica, 1998, 64,
70.
V. Amico, F. Cunsolo, M. Piatelli, G. Ruberto, Phytochemistry, 1985, 24, 1047.
M. D’Ambrosio, A. Guerriero, D. Fabbri, F. Pietra, Helv. Chim. Acta, 1986, 69,
1581.
Fatma Sri Wahyuni, N.H.Lajis, J.Stanslas, D.A.I.Ali, K.Shaari and Dachriyanus,
14th Indonesian National Symposium on Natural Products Chemistry, Bandung,1617th December 2004.
[7]
Fatma Sri Wahyuni, Lindsay T. Byrne, Dachriyanus, Roza Dianita, Junuarty
Jubahar, Nordin H. Lajis and Melvyn V. Sargent, Aust. J. Chem., 2004, 57, 223226.
[8] R. W. Baker, Z. Brkic, M. V. Sargent, B. W. Skelton, A. H. White, Aust. J. Chem.,
2000, 53, 925.
[9] Shahan, T.A., P.D. Siegel, W.G. Sorenson, W.G. Kuschner, and D.M. Lewis,
Journal of Immunological Methods, 1994,175, 181-187.
H
3
2
OH
4
5
1'
16'
5'
2'
3'
4'
9'
6'
7'
8'
13'
10'
11'
12'
14'
15'
1
O
(1)
6
Table 1. NMR data for cyclohexenone (1) in CDC13
Atoms
C(125MHz
)
H(600MHz,Eu(fod)3)
H (500 MHz)
mult., coupling constants
1
2
3
3-Me
4
5
6
1
2
3
4,8,12
5,9,13
6,10,14
7,11
15
16
15-Me
7-Me, 11Me
3-Me
OH
198.99
126.44
164.14
20.38
73.07
43.47
41.06
30.55
120.52
138.16
39.75 
39.60 
39.55 
26.64 
26.49 
26 .41 
124 .28 
124 .06 
123 .76 
135 .20 
134 .78 
6.34, brs, Wh/2 7.0 Hz
5.84
2.23
4.50, brt, J ca. 5.5 Hz, Wh/2 18.0 Hz
2.45, m, Wh/2 27.0 Hz
2.68, dd, J6ax,6eq 16.0, J6ax,5ax 12.5 Hz
3.07, dd, J6eq,6ax 16.0, J6eq,5ax 2.5 Hz
2.30, ddd, J1,1 14.0, J1,2 7.3, J1,5 6.5
Hz
2.54, ddd, J1,1 14.0, J1,2 7.3, J1,5 5.5
Hz
5.30, brt, J2,1 7.3 Hz
2.04
4.14
2.10
2.10
2.51
2.10
2.35
5.20
1.96 – 2.14, m
5.10 m
5.09
131.07





17.55
25.56
15.93 
15.87 
16.14
2.84, brs, Wh/2 19.5 Hz
1.64, d, J 1.1 Hz
1.60, s, 6H, Wh/2 3.5
Hz
1.59, d, J 1.1 Hz
1.67,d, J 1.1 Hz
3.11, brs, Wh/2 14.0
Hz
7
8