Journal of Applied Pharmaceutical Science Vol. 8(08), pp 012-020, August, 2018 Available online at http://www.japsonline.com DOI: 10.7324/JAPS.2018.8802 ISSN 2231-3354 Demethylbelamcandaquinone B Isolated from Labisia pumila Pumila Enhanced Proliferation and Differentiation of Osteoblast Cells Haryati Ahmad Hairi1, Jamia Azdina Jamal2, Nor Ashila Aladdin2, Khairana Husin2, Noor Suhaili Mohd Sofi1, Norazlina Mohamed1, Isa Naina Mohamed1, Ahmad Nazrun Shuid1* 1Department of Pharmacology, Faculty of Medicine, Preclinical Building, Universiti Kebangsaan Malaysia, Jalan Yaacob Latiff, Bandar Tun Razak, Cheras, 56000 Kuala Lumpur, Malaysia. 2Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Aziz, 50300 Kuala Lumpur, Malaysia. ARTICLE INFO ABSTRACT Article history: Labisia pumila (LP) or more commonly known as Kacip Fatimah in Malaysia has received much attention due to its Received on: 19/05/2018 estrogenic effects, including its role in the treatment of osteoporosis. This study was designed to explore the active Accepted on: 19/07/2018 compound of LP that may be responsible for its anti-osteoporotic effects. Crude aqueous extract of Labisia pumila Available online: 31/08/2018 var alata (LPva) was fractionated into hexane (Hex), dichloromethane (DCM) and methanol (Met) solvents and their proliferative effects on mouse osteoblastic cell line (MC3T3-E1) were evaluated with MTS bioassay. The DCM fraction significantly promoted cell proliferation in a dose-dependent manner. Thin layer chromatography (TLC) was Key words: performed on the DCM fraction of LPva to separate the constituents and the potential active compound was identified. active compound, Labisia Further isolation was achieved by column chromatography (CC) and Sephadex LH-20 column chromatography. The pumila, osteoporosis, bioactivity of the isolated compound was confirmed by its ability to replicate MC3T3-E1 accelerated proliferation osteoblasts. and differentiation. Mass spectrometry and nuclear magnetic resonance (NMR) identified the active compound as demethylbelamcandaquinone B. Further studies are required to determine the potential of this active compound of LPva in treating osteoporosis. INTRODUCTION therapy (HRT). However, HRT was linked to breast carcinoma Osteoporosis, a worldwide prevalence of disease, can be (Kotsopoulos et al., 2016), endometrial cancer (Chlebowski defined as a systemic skeletal disease characterized by weak bones et al., 2016) and cardiovascular disease (Bassuk and Manson, and high risk of fractures (Kanis and Johnell, 2006). This disease 2016). Bisphosphonate is a synthetic drug used to reduce the risk generally affects the elderly population and is associated with of vertebral and hip fractures, but daily bisphosphonate therapy postmenopausal estrogen deficiency and progressive bone loss may cause esophagitis and osteonecrosis of the jaw (Xu et al., (McCann et al., 2008; Pisani et al., 2016). Low estrogen level leads 2013). Selective estrogen receptor modulator or SERM is another to excessive bone resorption and reduced bone formation (Bord synthetic drug that binds to the intracellular estrogen receptors in and Beavan, 2003; Sorensen et al., 2006). The imbalance of bone target organs as agonists or antagonists. Unfortunately, it may also remodeling resulted in a bone loss and eventually osteoporosis cause serious side effects such as thromboembolic disorders and (Feng and Mcdonald, 2011). The main option for prevention and hot flushes (Maximov et al., 2013). treatment of postmenopausal osteoporosis is hormone replacement Recently, phytoestrogen became a favorable alternative therapeutic agent for treatment of osteoporosis. Phytoestrogens, such as isoflavones (Deyhima et al., 2003; Singh et al., 2015), *Corresponding Author lignans (Zhu et al., 2017) and coumestans (Ueda et al., 2011) acted Ahmad Nazrun Shuid, Department of Pharmacology, Faculty of as SERMs to exert therapeutic effects against postmenopausal Medicine, Pre clinical Building, Universiti Kebangsaan, Malaysia, osteoporosis. These phytoestrogens could also promote calcium Jalan Yaacob Latiff, Bandar Tun Razak, Cheras, 56000 Kuala Lumpur. absorption or regulate bone remodeling through estrogen receptor E-mail: anazrun @ yahoo.com © 2018 Haryati Ahmad Hairi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial- ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). Hairi et al. / Journal of Applied Pharmaceutical Science 8 (08); 2018: 012-020 013 pathway (Guo et al., 2012; Singh et al., 2015; Zhu et al., 2017). Extraction and isolation of compounds They have fewer side effects and suitable for long-term use The voucher specimen of Labisia pumila var. alata compared to synthesized drugs (Atteritano et al., 2009; Singh et (LPva) is UKMB 30006/SM 2622 which were deposited in the al., 2015). Herbarium of Universiti Kebangsaan Malaysia. The extraction of Labisia pumila (Blume) Fern-Vill (synonym: LPva and isolation of the compound were prepared according to Marantodes pumilum (Blume) Kuntze) from Primulaceae family (Nor Ashila et al., 2016; Avula et al., 2011) previously with slight is locally known in Malaysia as Kacip Fatimah. Other common modification. The air-dried leaves of LPva (8720 g) with a ratio names are kunci Fatimah, selusoh Fatimah, rumput Fatimah of dried leaves:water of 1:30 were extracted at the temperature and akar Fatimah. There are three types of Labisia pumila (LP), of 60°C for 2 hours followed by freeze-drying to obtain a crude which are var alata, var pumila and var lanceolata (Stone, aqueous extract with the net yield of 700 g. Part of the crude 1988). A decoction of the whole plant of LP is often used in aqueous extract (200 g) was successively partitioned with an equal Southeast Asia folk medicine to treat menstrual irregularities, volume of hexane (Hex), dichloromethane (DCM) and methanol improve fertility, facilitate childbirth and as postpartum medicine (Met) under reflux for 4 hours. The net yield of Hex fraction was (Muhammad and Mustafa, 1994). Several bioactive compounds 1.5 g; DCM fraction was 1.3 g; Met fraction was 39.83 g and the in aqueous extract of LP have been reported including flavonoids, balanced residue was 157.39 g. A portion of DCM fraction (1.3 ascorbic acid, beta-carotene, anthocyanin and phenols (Mohamad g) was subjected to Si-gel column chromatography and separated et al., 2009). Recently, LP extract was found to exhibit a number with hexane-ethyl acetate (EtOAc) of increasing percentage of of biological activities including antioxidant, anti-inflammatory EtOAc to afford 15 sub-fractions (1−15). Sub-fraction 8 (81.9 (Sanusi et al., 2013), antimicrobial (Karimi et al., 2011), mg) was further separated using Sephadex LH-20 and eluted antifungal (Karimi et al., 2013) and antinociceptive (Samuagam with 1% MeOH in chloroform (CHCl3) to yield 10 sub-fractions. et al., 2011). To date, LP received considerable attention since Sub-fractions I and J (53.6 mg) were combined based on TLC it was proven to have phytoestrogenic properties. LP has the analysis. The combined fraction was purified using Si-gel column ability to displace estradiol and bind to antibodies raised against chromatography and eluted isocratically with CHCl3-EtOAc estradiol (Wahab et al., 2011). LP was also shown to increase (90:10) to yield pure interest compound (35 mg) (Figure 1). estrogen and testosterone levels and suppress follicle stimulating hormone (FSH) and luteinizing hormone (LH) in ovariectomised Cell culture rats (Wahab et al., 2011). These actions emulate endogenous Mouse pre-osteoblast cells (MC3T3-E1 cells) (ATCC, estrogen. The phytoestrogenic effects of LP meant that it could Rockville, MD, USA) were grown in modified alpha minimal counteract various estrogen-deficiency related diseases such as essential medium (α-MEM) containing 10% FBS and 1% insulin resistance, cardiovascular diseases, and osteoporosis. The antibiotic-antimycotic (Gibco Life Technologies, Inc., Grand phytoestrogenic properties of LP were proven by its uterotropic Island, NY, USA). Culture media were changed to differentiation effects and body weight regulation by modulating secretions of medium, α-MEM supplemented with 50 μg/ml ascorbic acid and leptin and resistin, and expression of adipokines in adipose tissue 10 mM β-glycerophosphate (Sigma, St. Louis, MO, USA) to (Fazliana et al., 2009). induce osteoblast differentiation (Yan et al., 2014). However, few studies were conducted aiming at the identification and characterization of the active compound that Cell viability increases bone formation have not been carried out. Hence, in For cell viability assay, two-time points, 24 hours and the present study, we aimed for the separation and isolation of 72 hours, were selected for proliferation phase (rapid increase the distinct active compound of Labisia pumila var. alata (LPva) in cell number until confluence) and the cells were initiated for which is relevant for anti-osteoporosis treatment using bio-guided differentiation when proliferation had arrested. Cell viability fractionation and purification method. This method is a procedure study was performed using the MTS assay (Promega, Madison, whereby an extract is fractionated and chromatographically WI, USA) (Lina Wati et al., 2017). Crude aqueous extract of re-fractionated until a pure bioactive is isolated. LPva, hexane, DCM and methanol fractions of LPva extract, demethylbelamcandaquinone B and 17-beta-estradiol (positive