Cytotoxic compounds from the leaves of Garcinia cowa Roxb

Fatma Sri Wahyuni, Khozirah Shaari, Johnson Stanslas, Nordin Lajis, Dachriyanus Hamidi Faculty of Pharmacy, Andalas University, Kampus Limau Manis, Padang, West Sumatra, Indonesia. Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia. Faculty of Medicine and Health Sciences, Universiti Putra Malaysia 43400 UPM, Serdang, Selangor, Malaysia.Faculty of Nursing, Andalas University, Kampus Limau Manis, Padang, West Sumatra, Indonesia.


INTRODUCTION
The Genus Garcinia, belonging to the Family Clusiaceae have been widely investigated in terms of their bioactive ingredients.The plants are small to medium sized trees, which grow up to 30 m in height and are widely distributed in the tropical regions of the world (Kijjoa and Vieira, 2009).This genus has various biological activities such as antioxidant (Muharni et al., 2009 andDachriyanus et al., 2003), cytotoxic (Wahyuni et al., 2009) and antimicrobial activities (Dachriyanus et al., 2004).Garcinia cowa Roxb known as asam kandis in West Sumatera It is widely distributed throughout Indonesia and the Malay peninsula.The fruits are edible with a sour taste and used as spices in Indonesia especially in Minang tribes.(Dachriyanus et al., 2003).Many parts of G. cowa have been used in traditional folk medicine.The bark, latex and root have been used as an .antipyretic agent (Mahabusarakam, et al, 2005 andPathong et al, 2009) while the fruit and leaves have been used for indigestion and improvement of blood circulation, and as an expectorant (Pathong et al., 2009).Some pharmacological properties such as antitumorpromoting (Mukarami et al., 1995), inhibition of human lowdensity lipoprotein peroxidation and anti-platelet activities have been reported on the crude extract of leaves (Jantan et al., 2011).The chemical composition and biological activities of various parts of G. cowa have been investigated.Previous investigation on the fresh leaves, fruits and dried rinds of G. cowa has been investigated and found that (-)-hydroxycitric acid and its lactone constitute the major constituents (Jena et al., 2002).Previously, we reported the isolation of [2E,6E,10E]-(+)-4-hydroxy-3-methyl-5- (3,7,11,15tetramethyl-2,6,10,14-hexadecatetraenyl- 2) and rubraxanthone (3) from the stem bark of this plant.(Wahyuni et al., 2004) In continuation of our study on Garcinia cowa (Wahyuni et al., 2004), cytotoxic properties of isolated compounds from the leaves of Garcinia cowa against cancer cell-lines are reported.

General
Vacuum liquid chromatography (VLC) was conducted on silica gel (Merck 9385) and column chromatography (CC) was conducted on either silica gel (Merck 7734).The eluates were monitored by analytical thin layer chromatography (TLC) utilizing precoated silica gel (Merck 5554).The spots were visualized under ultraviolet lights at 254.Fractions containing similar profile were combined.Radial chromatography (RC) was carried out using Harrison Research Chromatotron model 7924T on plates coated with silica gel 60 PF 254 (Merck 7749) containing gypsum, at either 1 or 2 or 4 mm thickness.
UV (in absolute ethanol) and IR (KBr) spectra were recorded on a J ASCO V-560 spectrophotometer and a Perkin-Elmer 1650 FTIR spectrophotometer, respectively.Mass spectra were obtained on a J EOL J MS HX-110A spectrometer. 1 H and 13 C NMR spectra (CDCl 3 ) were recorded on a Varian 500 MHz NMR Spectrometer at 500 MHz ( 1 H)and 125 MHz ( 13 C), respectively and interpreted with the aid of the 1H-1H COSY, HMBC, and HMQC techniques.

Plant material
The leaves of Garcinia cowa Roxb were collected at Sarasah Bonta, Harau Valley, and West Sumatra at an altitude of 500 m.The voucher specimens (DR-181) were identified by Dr Rusdi Tamin and were deposited in the herbarium of Andalas University, Padang, Indonesia.Plant materials were air dried and ground to powder before extraction.

Extraction
Ground air-dried leaves (3.2 kg) were sequently defatted with hexane (3 x 2.5 L) and extracted with dichlorometahane (dichloromethane) (10 x 2.5 L).The dichloromethane extract was evaporated to dryness under reduced pressure to give the dark mass 153 g.

Cytotoxic asssay
Breast cancer MCF-7 and Lung cancer H-460 cell lines were used in this study.Cells were cultured in RPMI-1640 medium.Cells were cultured in RPMI with 10% FBS in 25 cm2culture flasks and incubated at 37oC, with 90% humidity level and 5% CO2 level.Varying concentrations of the pure isolated compounds were prepared from the stock solutions by serial dilution (100 μg/mL, 10 μg/mL, 1 μg/mL, 0.1 μg/mL) in RPMI-1640 to give the volume of 200 µl in each well of the microtiter plate.
Each concentration was tested in quadruplicate and the culture plate was incubated for 96 hours.After incubation, 50 μL of 2 mg/mL.MTT solution was added to each well and allowed to incubate.After 4 hours, all supernatant were discarded.100 μL DMSO was added to each well to dissolve the formazan crystals.Absorbance values at 550 nm was measured with a microplate reader.Cytotoxicity was stated as IC 50 (Mossman, 1983).
The position of prenyl unit and an aromatic proton were at C-2 and C-5, respectively, on the basis of HMBC correlations of the methylene protons (H-1', δ 3.33) of prenyl unit to C-2 (δ 162.2) and C-3 (δ 106.6) and the aromatic proton (H-5,δ 5.98, s) to C-1 (δ 93.91), C-3, (δ 106.6) and C-6 (δ 162.2) (Figure 4.8).The assignment of the protons and carbons of (1) are summarized in Table 1.The carbon chemical shifts of C-2, C-4 and C-6 established the attachment of hydroxyl groups at these carbons (Silverstein, 1987).Thus, the methyl ester moiety was linked at C-1.The EIMS data showed fragment ion at m/z 237 revealed the loss of CH 3 , while the fragment ion at m/z 220 and m/z 192, supporting this compound by losing OCH 3

Characterization of compound (3)
Compound 3 was obtained as red needless, mp 115-117 o C. The EIMS spectrum gave molecular ion at [m/z 457] + corresponding to molecular formula C 25 H 28 O 8 .The UV spectrum showed an absorption band at λ max 266.60 nm (4.86) and 226.40 nm (5.00).The IR spectrum exhibited absorption bands at 3201 cm -1 and 1655 cm -1 due to hydroxyl and carbonyl functionalities, respectively.The 13 C NMR spectrum also supported the presence three carbonyl groups by the signals at 184.16, δ182.06 and δ170.72 indicating the two p-benzoquinone carbonyls and an ester carbonyls, respectively (Permana, D., et al 2001).The 1 H NMR and HSQC spectra indicated the presence of two methoxyls from the signals at δ 4.01 (δ C 61.7) and δ 3.72 (δ C 52.9).The HMBC spectrum further suggested that the former is attached to an olefinic carbon (δ C 159.8), and the latter is located at a carbonyl carbon (δ C 170.7) (Permana et al., 2001).

Table 1 :
The 1 H-NMR , 13 C-NMR, and HMBC data of methyl