Bioactive Goniothalamin from Goniothalamus tapis with Cytotoxic Potential

1Department of Chemistry, Faculty of Science and Technology, Phranakhon Rajabhat University, Bangkok 10220, Thailand 2Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50300, Thailand 3The Forest Herbarium, Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok 10220, Thailand Laboratory of Natural Products, Faculty of Science and Center for Innovation in Chemistry, Lampang Rajabhat University, Lampang 52100, Thailand


General Procedures
IR spectrum was recorded on Shimadzu 8900 FTIR spectrophotometer by using KBr pellets technique. The 1 H (400 MHz), 13 C (100 MHz) and 2D NMR (COSY, DEPT, HMBC, HMQC) spectra were reccord by using a DPX on a BrÜker DPX 400 spectrometer in CDCl 3 as an internal standard. The EIMS was obtained by using a Finnigan LC-Q. Silica gel (Merck grade 7734, 70-230 mesh, 60 A) was used for column chromatography and TLC analysis manage with silica gel GF 254 precoated plates with detection using UV detector.

Plant Material
Leaves and twigs of G. tapis were collected from Trang province, Thailand in January 2011. The plant was identified by Mr. Narong Nutasaen. A voucher specimen (BKF no. 130978) has been deposited at the Forest Herbarium, Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok, Thailand.

Extraction and Isolation
Leaves and twigs air-dried powdered of G. tapis (2.115 kg) was fermented with hexane 7 litres for 5 days. Filtration was used for separated hexane extracted solution and residue. The residue was reextracted for 6 times with this solvent. Hexane extracted solution was combined and evaporated to dryness under reduced pressure to receive 25.33 g. The residue of hexane extraction was sequential extracted with ethyl acetate (4 litres x 5 days x 6 times) with same process of hexane to give 79.22 g, then acetone (3.8 litres x 5 days x 4 times) to obtain 54.22 g and methanol (3.63 litres x 5 days x 4 times) to gain 59.25 g of extracted, respectively. The ethyl acetate extracted (79.22 g) of G. tapis was separated on the silica gel column and eluted with gradient elution system such as hexane:EtOAc (0:100%) to EtOAc:MeOH (0:100%) to afford seven fractions (F 1 -F 7 ). Fraction F 4 (11.59 g) was rechromatographed (silica gel) followed by crystallization with ethanol to obtain a goniothalamin (1.80 g).

Evaluation of Cytotoxic Activity
The cytotoxic activities of goniothalamin from G. tapis were performed by using the standard in vitro sulforhodamine B (SRB) assay. The cancer cell lines were grown in a 96-well plate (Vichai and Kirtikara, 2006). Ellipticine was used as a positive control. The cancer cell lines used were P-388 (murine lymphocytic leukemia), KB (human oralnasopharyngal), Col-2 (human colon cancer), MCF-7 (human breast cancer), Lu-l (human lung cancer), A549 (adenocarcinomic human alveolar basal epithelial cells), T24 (human urinary bladder cancer cells) and ASK (rat glioma cell). The normal cell line employed was HEK-293 (human embryonic kidney). The cytotoxic activity is expressed as 50% effective dose (ED 50 ).

Results
In the present work, the defatted ethyl acetate was subjected to phytochemical investigation leading to the isolation of goniothalamin (Fig. 1). The compound was performed on the basis of spectral and chemical evidence from spectroscopic techniques data ( 1 H, 13 C and 2D NMR) in Table 1 and also by comparison with closely related literature (Ahmad et al., 1991). Additionally, goniothalamin was assessed for cytotoxicities against nine cell lines (Table 2).     (100), corresponding to C 13 H 12 O 2 . The 1 H NMR displayed two olefinic protons at δ 6.08 and 6.73 for H-3 and H-7, which also showed that they are in a trans configuration. It also showed aromatic protons at δ 7. 43-7.24 (m, 5H). The 13 C NMR spectrum showed a carbonyl group signal at δ 163.83. Goniothalamin was first isolated from Goniothalamus species and found several times from the same genus (Ahmad et al., 1991;Hasan et al., 1994;Jewers et al., 1972;Jiang et al., 1997).

Conclusion
This study focused on the phytochemical of Thai medicinal plant together with biochemical evaluation.
The results presented herein reveal the goniothalamin from the G. tapis and its cytotoxic activity. Therefore, further intensive studies on the structure-anticancer activity relationships of this class of compound is highly recommended.