Chemical Constituents of Hoya buotii Kloppenb .

1 Chemistry Department, De La Salle University, 2401 Taft Avenue, Manila 1004, Philippines. 2 School of Applied Sciences (Discipline of Chemistry), Health Innovations Research Institute (HIRi) RMIT University, GPO Box 2476V Melbourne, Victoria 3001, Australia. 3 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 Hoyas found in the Philippines, 88 of these are endemic to the country (Aurigue, 2013).Hoya buotii Kloppenb.is an endemic Philippine ornamental plant which was first collected from Mt. Banahaw, Quezon Province and was also found in the Sierra Madre Mountain Range of Luzon and Mt. Halcon in Mindoro (Aurigue, 2013).

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).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
The sample was collected from a garden in Caloocan City, Philippines in March 2014.The sample was authenticated at the Philippine National Herbarium, Botany Division, National Museum of the Philippines.It was identified as Hoya buotii Kloppenb.by Danilo N. Tandang.

General isolation procedure
A glass column 18 inches in height and 1.0 inch internal diameter was packed with silica gel.The crude extracts were fractionated by silica gel chromatography using increasing proportions of acetone in CH 2 Cl 2 (10% increment) as eluents.Fifty milliliter fractions were collected.All fractions were monitored by thin layer chromatography.Fractions with spots of the same R f values were combined and rechromatographed in appropriate solvent systems until TLC pure isolates were obtained.A glass column 12 inches in height and 0.5 inch internal diameter was used for the rechromatography.Two milliliter fractions were collected.Final purifications were conducted using Pasteur pipettes as columns.One milliliter fractions were collected.

Isolation of the chemical constituents of the stems
The air-dried stems of H. buotii (71.5 g) were ground in a blender, soaked in CH 2 Cl 2 for 3 days and then filtered.The solvent was evaporated under vacuum to afford a crude extract (2.3 g) which was chromatographed using increasing proportions of acetone in CH 2 Cl 2 at 10% increment.The 10% acetone in CH 2 Cl 2 fraction was rechromatographed (4 ×) using 5% EtOAc in petroleum ether to afford a mixture of 4a and 4b (3 mg) after washing with petroleum ether.The 20% acetone in CH 2 Cl 2 fraction was rechromatographed (3 ×) using 10% EtOAc in petroleum ether to afford 1 (6 mg) after washing with petroleum ether.The 30% acetone in CH 2 Cl 2 fraction was rechromatographed (3 ×) using 20% EtOAc in petroleum ether to afford 2 (12 mg) after washing with petroleum ether.The 40% acetone in CH 2 Cl 2 fraction was rechromatographed (2 ×) using CH 3 CN:Et 2 O:CH 2 Cl 2 (0.5:0.5:9, v/v) to yield a mixture of 3a and 3b (8 mg) after washing with petroleum ether.

Isolation of the chemical constituents of the roots
The air-dried roots of H. buotii (7.2 g) were ground in a .
. blender, soaked in CH 2 Cl 2 for 3 days and then filtered.The solvent was evaporated under vacuum to afford a crude extract (0.1 g) which was chromatographed using increasing proportions of acetone in CH 2 Cl 2 at 10% increment.The 20% acetone in CH 2 Cl 2 fraction was rechromatographed using 10% EtOAc in petroleum ether to afford 1 (4 mg) after washing with petroleum ether.
The 40% acetone in CH 2 Cl 2 fraction was rechromatographed using 15% EtOAc in petroleum ether.The less polar fractions were combined and rechromatographed using the same solvent (2 ×) to afford 2 (5 mg) after washing with petroleum ether.The more polar fractions were combined and rechromatographed using 20% EtOAc in petroleum ether to yield 3a (3 mg) after washing with petroleum ether.

Isolation of the chemical constituents of the flowers
The air-dried flowers of H. buotii (6.0 g) were ground in a blender, soaked in CH 2 Cl 2 for 3 days and then filtered.The solvent was evaporated under vacuum to afford a crude extract (0.1 g) which was chromatographed using increasing proportions of acetone in CH 2 Cl 2 at 10% increment.The CH 2 Cl 2 fraction was rechromatographed (3 ×) using 5% EtOAc in petroleum ether to afford a mixture of 4a and 4b (5 mg) after washing with petroleum ether.

Isolation of the chemical constituents of the leaves
The air-dried leaves of H. buotii (71.5 g) were ground in a blender, soaked in CH 2 Cl 2 for 3 days and then filtered.The solvent was evaporated under vacuum to afford a crude extract (6.5 g) which was chromatographed using increasing proportions of acetone in CH 2 Cl 2 at 10% increment.
The CH 2 Cl 2 was rechromatographed in petroleum ether.The less polar fractions were combined and rechromatographed in petroleum ether to yield saturated hydrocarbons (25 mg) after washing with petroleum ether.