Bioassay-guided Isolation of Triterpene Compounds from Dillenia suffruticosa and Their Cytotoxic Activities against Cancer Cells

Sains Malaysiana 51(5)(2022): 1487-1504 http://doi.org/10.17576/jsm-2022-5105-18

ABSTRACT Previous studies showed the ability of Dillenia suffruticosa to inhibit the growth of cancer cells by means of cell cycle arrest and apoptosis, thus validating the traditional use of the plant in treating cancer. Therefore, the present study was designed to isolate and elucidate the bioactive compounds responsible for the anticancer properties of D. suffruticosa extract. Bioassay-guided fractionation of the most potent fractions of DCM and EtOAc extract of D. suffruticosa was performed via column chromatography followed by purification using preparative HPLC. The structures of the isolated compounds were elucidated by NMR spectroscopy. Biological experiment by MTT assay and a series of column chromatography resulted in the isolation of three triterpene compounds. However, only the structure of compound (3) was confirmed as 1-isopropenyl-4α, 4β, 8, 10, 14-pentamethyl-icosahydro-cyclopenta[a]chrysene-3, 17-diol. Another two compounds were labelled as (1) and (2), which currently are unidentified due to unsuccessfulness in the full analysis of the spectroscopic data that enable the assignment of all protons, carbons, and confirmation of the structure. Compound (2) isolated from DCM extract of D. suffruticosa was most cytotoxic towards the selected cancer cells compared to the other two compounds and selected chemotherapeutic drugs, tamoxifen, and cisplatin. The ability of the isolated compounds to inhibit the growth of cancer cells indicates that these compounds are the bioactive constituents in D. suffruticosa that is mainly responsible for the cytotoxic activities. For this reason, these isolated compounds could be used as a means for the standardisation of herbal product from D. suffruticosa.

INTRODUCTION Recent global cancer statistics estimated to have risen to 18.1 million new cases and 9.6 million deaths in 2018, with breast and lung cancer, were the most frequently diagnosed among women and men, respectively (Bray et al. 2018). In Malaysia, a total of 103,507 new cases were identified from 2007 until 2011, with breast and colorectal cancer being the highest detected among females and males, respectively (Manan et al. 2016). Many chemotherapeutic drugs have been used intensively in the clinic as a standard treatment for the management of cancer, such as tamoxifen (for estrogen receptor (ER)-dependent growth breast cancer) (El Saghir et al. 2011), trastuzumab (for human epidermal growth factor receptor 2 (HER-2) positive breast cancer) (Perez 2011), gefitinib (for non-small lung carcinoma in epidermal growth factor receptor mutated patients) (de Marinis et al. 2011) and cisplatin (for cervical cancer) (Randal-Whitis & Monk 2007). The chemotherapeutic drugs are used either to alleviate advanced unresectable cancer or in an attempt to reduce the risk of recurrence after radical surgery (Aschele et al. 2009). Despite the success of these chemotherapeutic drugs, they may cause adverse side effects such as increased incidence of endometrial cancer associated with tamoxifen (Wysowski et al. 2002), cardiac dysfunction related to trastuzumab (Chen et al. 2011; Shaffer et al. 2009), arthralgia linked with aromatase inhibitors (Din et al. 2011), and hematologic toxicity and myelosuppression resulting from topotecan (Lee et al. 2010). Consequently, developments of new therapeutic medicine from natural product-derived medications are highly in demand and become a promising candidate for the drug lead.

Dillenia suffruticosa (Family: Dilleniaceae) is one of the medium-sized trees found in the perennial forests of Malaysia. Locally known as ‘Simpoh air’ (Malay), D. suffruticosa has been used ethno-medically to treat cancerous growth (Ahmad & Holdsworth 1995), managerheumatism (Hanum & Hamzah 1999), promote wound healing and to relieve fever (Mat-Salleh & Latiff 2002). D. suffruticosa also demonstrates antimicrobial (Wiart et al. 2004) and antivirus properties against dengue (Muliawan 2008). Findings from our previous in vitro studies shown that the dichloromethane (DCM) and ethyl acetate (EtOAc) extract from the roots of D. suffruticosa exhibited significant cytotoxicity towards various human cancer cell lines, including cervical adenocarcinoma cell (HeLa), breast adenocarcinoma cell (MCF-7 and MDAMB-231), lung carcinoma cell (A549), colon carcinoma (HT29) and ovarian adenocarcinoma (CaOV3) cell lines. Phytochemical analysis of DCM and EtOAc extract showed the presence of saponins, triterpenes, and sterols along with polyphenolic compounds, which are believed to contribute to their cytotoxic properties (Armania et al. 2013a). Further study shows that the cytotoxicity of the active fractions derived from chromatographic fractionation of DCM and EtOAc of D. suffruticosa was due to G2 /M cell cycle arrest and induction of apoptosis (Armania et al. 2013b). All the above results suggest the potential use of D. suffruticosa extract for the treatment of cancer.

The utilisation of crude extract from the medicinal plant for therapeutic purposes has major drawbacks concerning discrepancy due to variation in the amount of the active constituents owing to ecological conditions, geographical areas, variability in the collection, storage, and preparations of the raw materials (batch-to-batch variation) (Colegate & Molyneux 2008). As such, the uniformity of a natural-based product is crucial. One of the essential keys towards the standardisation of naturalbased product is by identification and quantification of the target active compound(s) that reflects the efficacy of the herbal product. Recognition of the active compound(s) could be achieved through bioassay-guided fractionation (Alsayari et al. 2018; Chen et al. 2007; Colegate & Molyneux 2008; Mfotie Njoya et al. 2014). Considered as a valuable approach in drug discovery (Pieters & Vlietinck 2005), the bioassay-guided fractionation technique is one of the methods used in isolating the bioactive components from medicinal herbs which elicit particular bioactive characteristics, including compounds with anticancer properties (Alsayari et al. 2018; Lai et al. 2010). Isolation and purification of the bioactive compounds that induce a particular pharmacological response required a simple, specific and rapid in vitro test such as 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) assay to screen the source material and monitor the chromatographic fractionation (such as column chromatography, preparative thin-layer chromatography (Prep TLC) and preparative HPLC) (Colegate & Molyneux 2008). Therefore, the present study aims to elucidate the bioactive compound(s) responsible for the cytotoxicity of D. suffruticosa by using bioassay-guided fractionation techniques.

Sains Malaysiana 51(5)(2022): 1487-1504 http://doi.org/10.17576/jsm-2022-5105-18