Anti-Tumor Activities of Analogues Derived from the Bioactive Compound of Zingiber zerumbet

RESULTS

IC₅₀ obtained from MTT assay was used to determine the viability of HeLa cells. The higher IC₅₀ value, the more viable cells remaining after treatment, which indicates the lesser effective the compound was, for cytotoxicity. The synthetic analogues of ZER were screened at concentration ranged from 10 to 100 μM. Based on the Cytotoxicity Screening Index from the National Cancer Institute Chemotherapeutic Standard (Geran et al., 1972), the IC₅₀ value less than 18 μM is considered very significant whilst between 18 and 137.6 μM is considered significant. The finding (Fig. 2) indicated that IC₅₀ of the analogues felt between 18 and 137.6 μM, which is considered significant.

From Fig. 2, the IC₅₀ for the analogues obtained were 70.0±2.5 μM in n-Butylbenzene, 79.4±4.1 μM 1, 1`-(4-Chlorobutylidene) bis(4-fluorobenzene), 87.6±4.6 μM in alpha,alpha-Diphenyl-gamma-butyrolactone and 80.0±7.8 μMin (1,4`-Bipiperidine)-4`-carboxaminde. The finding indicated significant different (p<0.05) in IC₅₀ among analogues. In comparison within the analogues as seen in the Fig. 2, n-Butylbenzene shown significant lower value of IC₅₀, while alpha, alpha-Diphenyl-gamma-butyrolactone shown significant higher value of IC₅₀. Among the screened synthetic compounds, n-Butylbenzene considered most cytotoxic compound as its lowest IC₅₀.

Parent compound, ZER and commercial drug, cisplatin were previously established their IC₅₀ which were 20.7±0.9 and 5.3±1.3 μM. This study found that the analogues were incompetent to neither ZER nor cisplatin for cytotoxicity in HeLa cells as their IC₅₀ were higher (Fig. 2). Thus, ZER was the most active natural compound, comparing to its synthetic analogues and comparable to cisplatin as well.

Fig. 2:	The concentration-response curve of human cervical cancer cell (HeLa) derived from MTT cytotoxicity assay performed after 72 h exposures with compound 5, 6, 7, 8 ZER and cisplatin are presented as Means±SD (n = 3)

DISCUSSION

Natural products possess a pedigree to justify quality and appreciation in drug discovery and development (Nielsen, 2002; Bogevik et al., 2008). Currently, there is rapid increase in application of natural products in combinatorial chemistry and vice versa (Koehn, 2008). The therapeutic areas of oncology are emerging (Saif, 2008). Natural products or intermediates have served as building blocks or scaffolds in the synthesis of complex natural products, bioactive analogues or designed hybrid molecules (Ortholand and Ganesan, 2004). Finally, structural motifs from the biologically active parent molecule have been identified and have served for design of natural product mimicry, which improves the knowledge and research in this area (Ertl et al., 2008).

ZER is a crystalline sesquiterpene derived from the wild ginger, Z. zerumbet. This bioactive component has its unique structure, with a cross-conjugated ketone in an 11-membered ring, as well as remarkable biological activity (Kitayama et al., 2003). Antiproliferative activity of Z. zerumbet is mainly modulated by the ZER component which is the main cytotoxic compound that constitutes about 37% of the whole Z. zerumbet content (Murakami et al., 2002). Terpenoids, including mono-, sesqui-, di- and triterpenoids, are biosynthesized by tandem reactions of the phosphorylated isoprene unit consisting of five carbons and are ubiquitously found in the plant kingdom. Some of these dietary terpenoids have anti-carcinogenic activities in a variety of rodent experiments and clinical trial results also demonstrated that their potential of treating cancers without major toxicity (Damodaran and Dev, 1968; Crowell, 1999; Rabi and Gupta, 2008). The further understanding of their biological and physiological mechanisms may lead to the identification of more effective compounds in this category for the prevention and treatment of targeted cancer types.

Studies demonstrated that ZER inhibited the proliferation of human colonic adenocarcinoma cell lines in a dose-dependent manner, while the growth of normal human dermal and colon fibroblasts was less affected. Intriguingly, a-humulene (HUM), a structural analogue lacking only the carbonyl group in ZER, was virtually inactive in all experiments conducted, indicating that the a; b-unsaturated carbonyl group in ZER may play some pivotal roles in interactions with unidentified target molecule(s) (Murakami et al., 2002). Thus, second-generation chemicals may be synthesized to mimic naturally occurring compounds, ZER but with greater specificity and less toxicity.

The MTT tetrazolium salt colorimetric assay previously described by Mosmann (1983); to measure cytotoxicity and cell proliferation. The level of MTT cleavage by viable cells of various origins was found to be directly proportional to the number of cells (Gerlier and Thomasset, 1986).

The synthetic analogues of ZER screened in this study, were n-Butylbenzene, 1, 1`-(4-Chlorobutylidene) bis(4-fluorobenzene), alpha,alpha-Diphenyl-gamma-butyrolactone and (1,4`-Bipiperidine)-4`-carboxaminde. The synthetic analogues were shown in the result, to have cytotoxic effect towards cervical cancer cells. Yet, these analogues were not as effective as ZER and Cisplatin, if compare to their MTT findings established previously in the laboratory. Since IC₅₀ of the analogues were high, we suggest that it may be used for anti-proliferative studies in order to investigate it preventive potential in cancer development. Nevertheless, the screening stage of this study concluded that ZER remains the most active cytotoxic natural compound on cervical cancer cells. A future chemical synthesis is required to produce more analogues, however, this approach must be followed by preserving the most functions biological active groups in ZER.

ACKNOWLEDGMENTS

The authors wish to thank gratefully Universiti Putra Malaysia (RUGS 91143) and the Malaysian National Council for Cancer for financial support and The Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Malaysia for providing analogues of zerumbone.