CYTOTOXIC ACTIVITY OF APORPHINE ALKALOIDS FROM ALSEODAPHNE PEDUNCULARIS (WALL. EX. NESS) MEISSN AGAINST HELA CELL LINE

Genus of Alseodaphne has been known as a source of apoprhine alkaloid that displayed potent cytotoxic activities against selected cell lines. Two aporphine alkaloids, Corydine 1 and norlirioferine 2 were isolated from leaves of Alseodaphne peduncularis (Wall. Ex. Ness) Meissn. (Lauraceae). Phytochemical study involved extraction, separation and purification by using various chromatography methods and structural elucidation by using spectoscopic techniques such as UV, IR LC-MS and 1D and 2D NMR. These compounds were then assayed for cytotoxicity against human uteric cervical tumor (heLa) and compare for safety with normal mouse fibroblast (NIH/3T3) cell lines by using the MTT assay. Compound 2 displayed potent cytotoxic activities against heLa cell, but compound 1 showed as inactive. Both compounds have no significant effect against NIH/3T3 cell lines indicated good for cancer agent treatment.


INTRODUCTION
showed specific emphases on their potential development as anticancer agents [1] .Plant secondary metabolites and their semi-synthetic derivatives have been used as anticancer drugs therapy, such as vinblastine, vincristine, the camptothecin derivatives, topotecan and irinotecan, etoposide, etoposide derived from epipodophyllotoxin and paclitaxel (taxol).
Looking for new secondary metabolites is still necessary for the development of novel pharmaceuticals [1,2] .
Alseodaphne peduncularis is a genus of Lauraceae, which is widely distributed in peninsular Malaysia and Sumatra.Most of this genus has been known as a source of aporphine alkaloids [3] .More than 500 aporphine alkaloids, such proaporphines, oxoaporphines and aporphines have been isolated from various plant families and many of these compounds also displayed potent cytotoxic activities which may be exploited for the design of anticancer agents [4,5] .This work aimed to identify the active compounds by assessing the cytotoxic activity of aporphinoids isolated from leaves of Alseodaphne peduncularis (Wall.Ex.Ness) Meissn.on HeLa cell lines and the structural evidence related to cytotoxicity is also discussed.

Cell Culture and MTT Cytotoxicity Assay
Cytotoxic activity in this study was treated against HeLa and NIH/3T3 cell lines.Both cells were recognized from the American Type Cell Collection (ATCC).
Medium without compound was used as negative control and positive control was used vincristine sulfate as comparation.
The cells were cultured using RPMI 1640 culturing media and maintained at 37°C in 5% CO2 atmosphere and counted using hemocytometer.
The MTT assay was carried out in the 96-wells plate.Briefly, a volume of

DISCUSSIONS Compound Characterization
Corydine 1 [7] was isolated from the leaves of A. peduncularis with a suggested by UV spectrum that showed absorptions at 230, 265 and 305 nm [9] .
The IR spectrum showed OH absorption band at 3278 cm -1 .Absorption peaks at 1425 and 1634 cm -1 indicated the presence of aromatic system [10] .
The specific 1 H NMR spectrum ( Compound 2 isolated from leaves of A.

In-vitro cytotoxic
In this study, the compounds were evaluated for cytotoxicities against NIH/3T3 and HeLa Cell lines.The cytotoxicities were assayed at various concentrations under continuous exposure for 72 hours, are expressed in CD50 values (μg/mL), and were summarized in Table 2.
Result expressed as CD50 that represent the compound concentration doses that reduced the mean absorbance at 570 nm to 50% of those in the untreated control wells.The CD50 value was obtained from the plot between the concentrations of compound versus percent of cell viability.
The value was used to describe the degree of cytotoxicity of the compounds towards cell lines.
Compounds which demonstrated the CD50 value of less than 5.0μg/mL were considered very active, while compounds with the CD50 value between 5.0 and 10.0 μg/mL were classified as moderately active.Those compounds that have CD50 value of 10-25 μg/mL were considered to be weak in cytotoxicity [9] .
Positive control, vincristine sulfate, showed a very good anticancer agents where gave a strongly effect against heLa and HL-60 cell lines, but have no cytotoxic against a normal cell NIH/3T3.
In case of cytotoxicity against heLa cell line, compound 2 showed very strong activity with CD50 values of 2 μg/mL while compound 1 displayed as inactive.
Both compounds showed no significant cytotoxicity effect against NIH/3T3.This result indicated as good for anticancer agent that indicated as save for normal cell.
In addition, cytotoxicities were also enhanced by presence of methoxyl and N-methyl groups.Compound 2 which have no N-methyl group gave better cytotoxic activity against HeLa cell line.
In case of compound 1, cytotoxic activity also affected by substituent arrangement.
Previous research reported that aporphine alkaloid with has 10-CH3 and 11-OH substitution had no activity [7] .This type of structure made 1 being inactive in cytotoxic.On the other side, the low activity of this compound also caused of the presence of N-methyl group.

Conclusions
Plants are the most important sources of biologically active natural products that have differences in the structure and biological properties.Active compounds from plant resulted by secondary metabolite pathway.Plants have long history of use in the treatment of cancer.About 60% of anticancer agents used nowadays are obtained from natural sources.The present review presents that most of alkaloids isolated from large number of plant families dichloromethane crude extract from A. peduncularis was subjected to CC over silica gel and eluted with increasing polarity solvent system of hexane, dichloromethane and methanol.Fractions which have the same pattern shown in TLC were grouped into a series of fractions.Isolation and purification (44 g) of sample yielded 14 fractions after grouped.Fraction 5 (100 mg) was then purified by PTLC to gave compound 1 (5 mg) and fraction 11 (2 g) gave 2 (100 mg) after purified by CC and PTLC.The structure of compounds were elucidated by using 1D-NMR ( 1 H, 13 C and DEPT) and 2D-NMR (COSY,

100. 0
μL of complete growth medium was added into each well of 96-wells flat bottom microtiter plate (Nunclon, USA).The compounds or vincristine sulphate solution (95.0-105.0%purity by HPLC, Sigma, USA) at 60.0 μg/mL was aliquoted into wells in triplicate and serially diluted.A volume of 100.0 μL of 1x10 5 cells/mL heLa or NIH/3T3 cells were seeded into 96-wells flat microtiter plates and incubated for 72 hours in CO2 incubator.After 72 hours incubation, a volume of 20.0 μL of MTT solution (5.0 mg/mL) was added into each well and incubated for 4 hours.The culture medium was removed and 100.0 μL of 100% DMSO solution were added to each well to solubilise the formazan formed.The plates were read using the plate reader at 570nm wavelength (Infinite M200, Tecan, Switzerland).A dose response curve of the percentage of cell viable versus extract concentration was plotted.
from the leaves of Alseodaphne peduncularis yielded two known aporphine alkaloids, corydine 1 and norlirioferine 5. Compound 2 displayed potent cytotoxic activity against heLa cell, but compound showed as inactive.Both compounds have no significant effect against NIH/3T3 cell lines.

Table 1 1
H and 13 CNMR Data of Compound 1 and 2

Table 2
Cytotoxic Activity of Compound 1 and 2 against NIH/3T3 and HeLa Cell lines