Terpenoids and Sterols from Hoya multiflora Blume

Article history: Received on: 30/01/2015 Revised on: 12/02/2015 Accepted on: 07/03/2015 Available online: 27/04/2015 Chemical investigation of the dichloromethane extracts of Hoya multiflora Blume led to the isolation of lupeol (1a), α-amyrin (1b), β-amyrin (1c), lupeol acetate (2a), α-amyrin acetate (2b), and β-amyrin acetate (2c) from the stems; and 1b, bauerenol (3), squalene (4), lutein (5), β-sitosterol (6a), and stigmasterol (6b)from the leaves. The structures of 1-6 were identified by comparison of their H and/orC NMR data with those reported in the literature.

This study was conducted as part of our research on the chemical constituents of the genus Hoya.We earlier, reported the isolation of lupenone and lupeol from the roots; lupeol, squalene and β-sitosterol from the leaves; and betulin from, the stems of Hoya mindorensis Schlechter (Ebajo et al., 2014).

General Experimental Procedure
NMR spectra were recorded on a Varian VNMRS spectrometer in CDCl 3 at 600 MHz for 1 H NMR and 150 MHz for 13 C NMR spectra.Column chromatography was performed, with silica gel 60 (70-230 mesh).Thin layer chromatography, was .performed with plastic backed plates coated with silica gel F 254 and the plates were visualized by spraying with vanillin/H 2 SO 4 solution followed by warming.

Sample Collection
Hoya multiflora Blume was collected from a garden in Pangasinan, Philippines in September 2013.Voucher specimens were authenticated at the Botany Division of the Philippine National Museum.

General Isolation Procedure
The air-dried leaves (23.0 g), and stems (86.5 g) of H. multiflora were ground in a blender, soaked in CH 2 Cl 2 for three days and then filtered.The filtrates were concentrated under vacuum to afford crude extracts of leaves (2.0 g), and stems (1.0 g) which were each chromatographed by gradient elution with CH 2 Cl 2 , followed by increasing amounts of acetone at 10% increment by volume as eluents.A glass column 12 inches in height and 0.5 inch internal diameter was used for the fractionation of crude extracts.Two milliliter fractions were collected.Fractions with spots of the same R f values were combined and rechromatographed in appropriate solvent systems until TLC pure isolates were obtained.Rechromatography and final purifications were conducted using Pasteur pipettes as columns.One milliliter fractions were collected.

Isolation of Chemical Constituents of the Stems
The CH 2 Cl 2 fraction from the chromatography of the crude extract was rechromatographed using 1% EtOAc in petroleum ether to afford a mixture of 2a-2c (9 mg) after washing with petroleum ether.The 20% acetone in CH 2 Cl 2 fraction was rechromatographed using 5% EtOAc in petroleum ether, to afford a mixture of 1a-1c (4 mg) after washing with petroleum ether.
Although no biological activity tests were conducted on the isolated compounds (1-6), literature search revealed that these have diverse bioactivities as follows., Lupeol (1a) exhibited antiurolithiatic and diuretic activity (Vidya et al., 2002).It prevented the formation ofvesical calculi and reduced the size of the preformed stones in rats (Anand et al., 1994).It also showed antifungal activity against Fusarium oxysporum and Penicillium notatum (Manzano et al., 2013).Lupeol significantly reduced the 451Lu tumor growth in athymic nude mice (Saleemet al., 2008), inhibited the proliferation of MDA-MB-231 human breast cancer cells in a dose dependent manner (Lambertini et al., 2005), and induced growth inhibition and apoptosis in hepatocellular carcinoma SMMC7721 cells by down-regulation of the death receptor 3 (DR3) expression (Zhang et al., 2009).Lupeol and lupeol acetate (2a) have shown hypotensive activity (Saleem et al., 2003), while 1a also exhibited antidyslipidemic activity in hamster at 100 mg/Kg body weight (Reddy et al., 2009).It exhibited potent anti-inflammatory activity in an allergic airway inflammation model by a significant reduction in eosinophils infiltration and in Th2-associated cytokines levels that trigger the immune responses in asthma (Vasconcelos et al., 2008).A review on the biological activities of lupeol has been provided (Gallo and Sarachine, 2009).β-Amyrin (1c) and α-amyrin (1b) were reported to possess antiinflammatory (Recio et al., 1995;Madeiros et al., 2007;Okoye et al., 2014) and analgesic (Otuki et al., 2005;Soldi et al., 2008) properties.β-Amyrin showed antifungal activity against A. rabiei with an MIC value of 0.0156 mg/mL (Jabeen et al., 2011)., α-Amyrin was proposed as a possible biomarker for the fungal resistance of grape-vine leaves (Vitis vinifera) (Batovska et al., 2008).The mixture of 1b and 1c effectively reduced the elevated plasma glucose levels during the oral glucose tolerance test (OGTT).Furthermore, the mixture of 1b and 1c at 100 mg/kg significantly decreased the VLDL and LDL cholesterol and increased the HDL cholesterol (Santos et al., 2012).A review on the sources and biological activities of 1b and 1c has been provided (Vasquez et al., 2012).The anti-inflammatory effect of lupeol acetate (2a) involves the opioid system, as indicated by the complete blockade of the opioid antagonist naloxone (Lucetti et al., 2010).α-Amyrin acetate (2b) at 100 mg/kg showed significant (p < 0.05) inhibition of egg albumen-induced paw edema with 40 % inhibition at the 5th hour.β-Amyrin acetate (2c) and 2b isolated from the Alstonia boonei stem bark exhibited profound antiinflammatory activity (Okoye et al., 2014).Triterpenes 2b and 2c were also reported to exhibit sedative, anxiolytic and anticonvulsant properties (Aragao et al., 2009).A mixture of bauerenol (3), 1b and 1c obtained from Ardisia species exhibited angio-suppressive effects on duck chorioallantoic membrane (CAM) (Raga et al., 2013b); restricted inter-capillary length and reduced branch point with 100% CAM viability and embryo survivability and promoted intense expression of the von Willebrand factor (F8) (Raga et al., 2013c); was found toxic to A. salina nauplii after 48h of exposure and showed teratologic manisfestations on Danio rerio embryos (Raga et al., 2014a); and exhibited analgesic property in the acetic acid writhing test and hot plate assay (Raga et al., 2014b).Another study reported that a mixture of bauerenol, α-amyrin and β-amyrin from Carmona retusa exhibited 51% analgesic activity and showed 20% antiinflammatory activity at dosage of 100 mg/kg mouse, while of 250 mg/kg mouse showed a 29% anti-diarrheal activity (Villasenor et al., 2004).Squalene (4) was reported to significantly suppress colonic ACF formation and crypt multiplicity**y which strengthened the hypothesis that it possesses chemopreventive activity against colon carcinogenesis (Rao et al., 1998).
It showed cardioprotective effect which is related to inhibition of lipid accumulation by its hypolipidemic properties and/or its antioxidant properties (Farvin et al., 2006).A recent study reported that tocotrienols, carotenoids, squalene and coenzyme Q10 have anti-proliferative effects on breast cancer cells (Loganathan et al., 2013).The preventive and therapeutic potential of squalene containing compounds on tumor promotion and regression have been reported (Desai et al., 1996).A recent review on the bioactivities of squalene has been provided (Ronco and De Stéfani, 2013).
Dietary lutein ( 5), especially at 0.002%, inhibited tumor growth by selectively modulating apoptosis, and by inhibiting angiogenesis (Chew et al., 2003).Another study reported that the chemopreventive properties of all-trans retinoic acid and lutein may be attributed to their differential effects on apoptosis pathways in normal versus transformed mammary cells (Sumantran et al., 2000).Moreover, very low amounts of dietary lutein (0.002%) can efficiently decrease mammary tumor development and growth in mice (Park et al., 1998).Another study reported that lutein and zeaxanthine reduces the risk of age related macular degeneration (SanGiovanni et al., 2007).
β-Sitosterol (6a) was observed to have growth inhibitory effects on human breast MCF-7 and MDA-MB-231 adenocarcinoma cells (Awad et al., 2007).It was shown to be effective for the treatment of benign prostatic hyperplasia (Jayaprakasha et al., 2007).It was also reported to attenuate βcatenin and PCNA expression, as well as quench radical making it a potential anticancer drug for colon carcinogenesis (Baskar et al., 2010).It can inhibit the expression of NPC1L1 in the enterocytes to reduce intestinal cholesterol uptake (Jesch et al., 2009).It was reported to induce apoptosis mediated by the activation of ERK and the down regulation of Akt in MCA-102 murine fibrosarcoma cells (Moon et al., 2007).

CONCLUSION
Hoya multiflora is a Philippine indigenous ornamental plant with no reported chemical studies and biological activities.This study reports on the terpenoids and sterols with known diverse biological activities which were isolated from the leaves and stems of the plant.Most of these compounds (1a-1c, 3-6) were reported to exhibit cytotoxic and anticancer properties, while 2a-2c were reported to possess anti-inflammatory activity.