Chemical Constituents of Hoya cagayanensis C . M . Burton

1 Chemistry Department, De La Salle University, 2401 Taft Avenue, Manila 1004, Philippines. 2 Chemistry Department, De La Salle University Science & Technology Complex Leandro V. Locsin Campus, Biñan City, Laguna 4024, Philippines. 3 Food and Nutrition Research InstituteDepartment of Science and Technology, Bicutan, Taguig, Metro Manila, Philippines. 4 Agriculture Research Section, Atomic Research Division, Philippine Nuclear Research Institute-Department of Science and Technology, Commonwealth Avenue, Diliman, Quezon City 1101, Philippines. 5 School of Science (Discipline of Applied Chemistry and Environmental Science), RMIT University (City Campus), Melbourne 3001, Victoria, Australia.


INTRODUCTION
Hoya is a genus of flowering plants in the family Apocynaceae.Most are epiphytic vines with succulent leaves and attractive clusters of flowers.There are at least 109 species of Hoya found in the Philippines, 88 of these are endemic to the country (Aurigue, 2013).Among them is Hoya cagayanensis C. M. Burton, first discovered in Cagayan province but also collected from Laguna and Quezon provinces in Luzon island, Philippines.It has become popular as an ornamental plant among collectors in the country and also abroad due to its spice-scented flowers, lush foliage, and ease of culture.No report on their ethnobotany or chemical analysis have been found.This study is part of our research on the chemical constituents of Philippine endemic and indigenous hoyas.We earlier reported the isolation of lupenone and lupeol from the roots; lupeol, squalene and βsitosterol from the leaves; and betulin from the stems of H. mindorensis Schlechter (Ebajo et al., 2014).In another study, we reported the isolation of lupeol, α-amyrin, β-amyrin, lupeol acetate, α-amyrin acetate, and β-amyrin acetate from the stems; and α-amyrin, bauerenol, squalene, lutein, β-sitosterol, and stigmasterol from the leaves of H. multiflora Blume (Ebajo et al., 2015a).The isolation of β-amyrin cinnamate and taraxerol from the stems; and taraxerol, triglycerides, chlorophyll a, and a mixture of β-sitosterol and stigmasterol from the leaves of H. wayetii Kloppenb.has been reported (Ebajo et al., 2015b).Furthermore, the isolation of taraxerol, taraxerone, a mixture of β-sitosterol and stigmasterol, and a mixture of α-amyrin cinnamate and β-amyrin cinnamate from the stems; taraxerol, taraxerone, and β-sitosterol from the roots; a mixture of α-amyrin cinnamate and β-amyrin cinnamate from the flowers; and squalene, β-sitosterol, and saturated hydrocarbons from the leaves of H. buotii has been reported (Ebajo et al., 2015c).
We report herein the isolation of hydrocanaric acid (1), lupeol (2), lupenone (3), and saturated hydrocarbons from the stems; and 2-hydroxyethyl benzoate (4) and a mixture of βsitosterol (5a) and stigmasterol (5b) from the leaves of H. cagayanensis.To the best of our knowledge this is the first report on the isolation of 1-5 and hydrocarbons from H. cagayanensis.

General Experimental Procedure
1 H (500 MHz) and 13 C (125 MHz) NMR spectra were acquired in CDCl 3 on a 500 MHz Agilent DD2 NMR spectrometer with referencing to solvent signals (δ 7.26 and 77.0 ppm).
Two dimensional NMR experiments recorded included gCOSY, HSQCAD, and gHMBCAD NMR experiments.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
Stem cuttings were taken from two large specimen plants of

General Isolation Procedure
The air-dried stems (81.25 g) and leaves (61.95 g) of H. cagayanensis 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 stems (4.79 g) and leaves (4.00 g) which were separately chromatographed by gradient elution with CH 2 Cl 2 , followed by increasing amounts of acetone at 10% increments 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 millilitre 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 millilitre fractions were collected.

Isolation of the Chemical Constituents of the Stems
The crude leaves extract (4.00 g) was chromatographed using increasing proportions of acetone in CH 2 Cl 2 (10% increment) as eluents.The CH 2 Cl 2 fraction was rechromatographed (2 ×) using petroleum ether to afford hydrocarbons (5 mg) after washing with petroleum ether.The 20% acetone in CH 2 Cl 2 fraction was rechromatographed (3 ×) using 5% EtOAc in petroleum ether to yield 3 (10 mg) after washing with petroleum ether.

Isolation of the Chemical Constituents of the Leaves
The crude stems extract (4.79 g) was chromatographed using increasing proportions of acetone in CH 2 Cl 2 (10% increment) as eluents.The CH 2 Cl 2 fraction was rechromatographed (3 ×) using 10% EtOAc in petroleum ether to yield a mixture of 5a and 5b (12 mg) after washing with petroleum ether.The 70% acetone in CH 2 Cl 2 fraction was rechromatographed using 15% EtOAc in petroleum ether.Fractions collected from this column were combined and rechromatographed using CH 3 CN:Et 2 O: CH 2 Cl 2 (0.5:0.5:9, v/v) to afford 4 (3 mg) after washing with petroleum ether.

RESULTS AND DISCUSSION
Silica gel chromatography of the dichloromethane extract of H. cagayanensis yielded 1-5 and hydrocarbons.The structures of 1 and 4 were elucidated by extensive 1D and 2D NMR spectroscopy.The NMR data of 1 are similar to those of canaric acid (Lopes et al., 1999), except at C-4 and C-24 where their structures differ.In canaric acid, a double bond is found between C-4 and C-24, while a single bond is present in 1.There are also differences in the chemical shifts of the protons and carbons close to C-4 and C-24.The NMR spectra of 2 are in accordance with data reported in the literature for lupeol (Ebajo et al., 2015a), 3 for lupenone (Prakash et al., 2012); 4 for 2-hydroxyethyl benzoate (Sharghi and Sarvari, 2003); 5a for β-sitosterol (Ebajo et al., 2015a), 5b for stigmasterol (Ebajo et al., 2015a) and hydrocarbons (Ebajo et al., 2015c).
Although there is no reported biological acitivity for H. cagayanensis, the compounds (1-3 and 5a-5b) isolated from the plant were reported to possess diverse activities.
An earlier study reported that dihydrocanaric acid (1) exhibited growth inhibitory activity against both HeLa and SW480 cells (Sadhu et al., 2008).It was also reported as a powerful antioxidant (Ghosh et al., 2010).
Lupeol (2) exhibited antiurolithiatic and diuretic activity (Vidya et al., 2002).It prevented the formation of vesical 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 (Saleem et 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).
Lupenone (3) inhibits adipocyte differentiation by suppressing PPARγ and C/EBPα protein levels (Ahn and Oh, 2013).It also increases the tyrosinase enzyme expression via mitogen-activated protein kinase phosphorylated extracellular signal regulated kinases 1 and 2 phosphorylation inhibition which results to stimulation of melanogenesis.This suggests that lupenone could be a possible treatment for hypopigmentation (Villareal et al., 2013).β-Sitosterol (5a) has been reported 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 (Jayaprakasya et al., 2007).It was observed to attenuate β-catenin and PCNA expression, as well as quench free radical in vitro, making it a potential anticancer drug for colon carcinogenesis (Baskar, et al., 2010), and inhibit the expression of NPC1L1 in the enterocytes to reduce intestinal cholesterol uptake (Jesch et al., 2009).It was also reported to induce apoptosis mediated by the activation of ERK and the downregulation of Akt in MCA-102 murine fibrosarcoma cells (Moon et al., 2007).
H. cagayanensis cultivated at the Philippine Nuclear Research Institute Hoya Germplasm Collection under MTA No. 2015-06 dated August 13, 2015 and MTA No. 2016-04 dated June 24, 2016.Accession No. PNRI-H.23 is grown from a cutting that originally came from Quezon province and was subsequently propagated since 1997.It was identified and authenticated by Dr. Simeona V. Siar of the Institute of Plant Breeding, University of the Philippines-Los Baños on February 26, 2008.