Triterpenes and sterols from Hoya diversifolia Blume

1 Chemistry Department, De La Salle University, 2401 Taft Avenue, Manila 1004, Philippines. 2 Chemistry Department, Central Luzon State University, Munoz, Nueva Ecija 3121, Philippines. 3 Agriculture Research Section, Atomic Research Division, Philippine Nuclear Research Institute-Department of Science and Technology, Commonwealth Avenue, Diliman, Quezon City 1101, Philippines. 4 National Research Institute of Chinese Medicine, Ministry of Health and Welfare, 155-1, Li-Nong St., Sec. 2, Taipei 112, Taiwan. 5 Chemistry Department, De La Salle University Science & Technology Complex Leandro V. Locsin Campus, Biñan City, Laguna 4024, Philippines.


INTRODUCTION
Hoya plants are also called wax plants due to the waxy appearance of their leaves or flowers.There are at least 109 species of Hoya found in the Philippines, 88 of these are endemic to the country.Hoya diversifolia Blume is an indigenous Philippine ornamental plant found in Palawan and Luzon, specifically Quezon Province (Aurigue, 2013).It is distributed in Myanmar, Indo-China, peninsular Thailand, Peninsular Malaysia, Singapore, Sumatra and Java (Mansur, 2003).An earlier study reported that H. diversifolia afforded apigenin-O-glycoside, apigenin-di-C-glycoside, vitexin and isovitexin.The extract of this plant exhibited strong antinematodal activity against the pine wood nematode Bursaphelenchus xylophilus.In Malaysia and Vietnam, a decoction of the leaves of H. diversifolia is used as a bath to treat rheumatism (Mansur, 2003).Another study reported that H. diversifolia contains 11.1% free terpenoids and 81.7% triterpene cinnamates (Warnaar, 1984).This study is part of our research on the chemical constituents of Philippine native hoyas.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 H. mindorensis Schlechter (Ebajo et al., 2014).In another study, we reported the isolation of lupeol, α-amyrin, βamyrin, lupeol acetate, α-amyrin acetate, and β-amyrin acetate from the stems; and α-amyrin, bauerenol, squalene, lutein, βsitosterol, and stigmasterol from the leaves of H. multiflora Blume (Ebajo et al., 2015a).Recently, 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 in about 2:1 ratio, and a mixture of α-amyrin cinnamate and β-amyrin cinnamate in about 1:2 ratio from the stems; taraxerol, taraxerone, and β-sitosterol from the roots; a mixture of α-amyrin cinnamate and β-amyrin cinnamate in about 3:2 ratio from the flowers; and squalene, βsitosterol, and saturated hydrocarbons from the leaves of H. buotii has been reported (Ebajo et al., 2015c).We report herein the isolation of β-amyrin cinnamate (1), squalene (2), β-sitosterol (3), and a mixture of β-amyrin (4a), α-amyrin (4b) and lupeol (4c) in a 4:2:1 ratio and saturated hydrocarbons from the leaves; and 2, taraxerol (5), lupeol cinnamate ( 6), and a mixture of 3 and stigmasterol (7) in a 2:1 ratio from the stems of H. diversifolia.The chemical structures of 1-7 are presented in Fig. 1.

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
Healthy

General Isolation Procedure
The air-dried leaves (157.2 g), and stems (383.9 g) of H. diversifolia 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 (9.1 g), and stems (11.75 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 18 inches in height and 1 inch internal diameter was used for the fractionation of the crude extracts.Eleven 20 mL fractions were collected.A glass column 12 inches in height and 0.5 inch internal diameter was used for the rechromatography of fractions from the crude extracts.2 mL 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. 1 mL fractions were collected.

Isolation of Chemical Constituents of the Leaves
The 10% acetone in CH 2 Cl 2 fraction from the chromatography of the crude extract was rechromatographed using 1% EtOAc in petroleum ether. 1 mL fractions were collected.Fractions 7-12 yielded 2 (5 mg), while fractions 1-5 afforded saturated hydrocarbons (6 mg).The 20% acetone in CH 2 Cl 2 fraction was rechromatographed using 2.5% EtOAc in petroleum ether and portions of 2 mL fractions were collected.Fractions 17-21 were combined to yield 1 (3 mg) after washing with petroleum ether.The 40% acetone in CH 2 Cl 2 fraction was rechromatographed using 10% EtOAc in petroleum ether, followed by 15% EtOAc in petroleum ether. 1 mL fractions were collected.Fractions 5-6 eluted with 10% EtOAc in petroleum ether afforded a mixture of 4a, 4b and 4c (4 mg) after washing with petroleum ether.The 10-13 fractions eluted with 15% EtOAc in petroleum ether afforded 3 (5 mg) after washing with petroleum ether.

CONCLUSION
Hoya diversifolia Blume is an ornamental plant native to the Philippines and other Asian countries with chemical constituents from dichloromethane extracts identified as follows: β-amyrin cinnamate, squalene, β-sitosterol, a mixture of β-amyrin, α-amyrin and lupeol in a 4:2:1 ratio, and saturated hydrocarbons from the leaves; and taraxerol, lupeol cinnamate, and a mixture of β-sitosterol and stigmasterol in a 2:1 ratio from the stems.