Triterpenes and Sterols from Leaves of Hoya meliflua Merr

Introduction: Hoya plants are also called wax plants due to the waxy appearance of their leaves and flowers. Most species are cultivated as ornamental plants. In the Philippines, there are at least 109 species of Hoya; 88 of these are endemic to the country. One of the endemic species is Hoya meliflua often confused with H. diversifolia, which can also be found in other countries. This study is part of our research on the chemical constituents of Philippine native Hoyas. Methods: The compounds were isolated by silica gel chromatography and identified by NMR spectroscopy. Results: Chemical investigation of the dichloromethane extract from the leaves of Hoya meliflua afforded squalene and mixtures of β-amyrin (1a), α-amyrin (1b) and lupeol (1c) in about 1:1:0.25 ratio; oleanone (2a), ursenone (2b) and lupenone (2c) in about 1:1:0.3 ratio; β-amyrin cinnamate (3a), α-amyrin cinnamate (3b) and lupenyl cinnamate (3c) in about 0.5:0.3:1 ratio; and β-sitosterol and stigmasterol in about 5:1 ratio. Conclusion: The results of our study indicate that Hoya meliflua shares similar chemical characteristics with other members of the genus Hoya. The triterpenes and sterols obtained from H. meliflua were also identified from other Hoya species. It is interesting to note that although most Hoya plants have no known biological activity and medicinal property, the compounds isolated from H. meliflua possess diverse bioactivities.


INTRODUCTION
Hoya Br. is considered the largest genus of flowering plants in the family Apocynaceae.Commonly called wax plants due to the waxy appearance of their leaves and flowers, most species have ornamental value.A few have been reported to have medicinal properties or used in traditional medicine.In the Philippines, there are at least 109 species of Hoya; 88 of these are endemic to the country.One such endemic species is Hoya meliflua Merr.found in Apayao, Bataan, Laguna, La Union and Rizal in Luzon Island and in Leyte, Mindoro, Negros, Palawan and Panay islands in Central Philippines all at low altitudes. 1New records of occurrence in other provinces, such as Laguna, have not been published yet.The Plant List. 2 states that H. meliflua is an unresolved name because some data suggest that it is synonymous with H. diversifolia Blume which is also found in other countries.This may be attributed to the confusion on the identity of the two Philippine native species.This study is part of our research on the chemical constituents of Philippine native hoyas.We earlier reported the isolation of lupenone (I) and lupeol (II) from the roots; II, squalene (III) and β-sitosterol (IV) from the leaves; and betulin (V) from the stems of H. mindorensis Schlechter. 3 In another study, we reported the isolation of II, α-amyrin (VI), β-amyrin (VII), lupeol acetate (VIII), α-amyrin acetate (IX) and β-amyrin acetate (X) from the stems; and III, IV, VI, bauerenol (XI), lutein (XII) and stigmasterol (XIII) from the leaves of H. multiflora Blume. 4urthermore, the isolation of β-amyrin cinnamate (XIV) and taraxerol (XV) from the stems; and IV, XIII, XV, triglycerides (XVI) and chlorophyll a (XVII) from the leaves of H. wayetii Kloppenb has been reported. 5Moreover, the isolation of IV, XIII, XIV, XV, taraxerone (XVIII) and α-amyrin cinnamate (XIX) from the stems; IV, XV and XVIII from the roots; XIV and XIX from the flowers; and III, IV and saturated hydrocarbons (XX) from the leaves of H. buotii has been reported. 6e also reported the isolation of II, III, IV, VI, VII, XIV and XX from the leaves; and III, IV, XIII, XV and lupeol cinnamate (XXI) from the stems of H. diversifolia Blume. 7Chemical investigation of the dichloromethane extracts of H. cumingiana Decne.yielded II, IV, VI, VII, XIII and bauerenol (XXII) from the leaves; and XV from the stems. 8Recently, we reported the isolation of IV, XIII and XV from the stems 9 and I, IV, XIII, 2-hydroxyethylbenzoate (XXIII) and fatty acid methyl esters (XXIV) from the leaves 10 of Hoya pubicalyx.In another study, the dichloromethane extracts of H. paziae Kloppenb.yielded IX, X, XV and taraxeryl acetate (XXV) from the stems 11 and II, VI, VII, lupeol fatty acid esters (XXVI), α-amyrin fatty acid esters (XXVII) and β-amyrin fatty acid esters (XXVIII) from the leaves. 12Furthermore, the dichloromethane extracts of H. cagayanensis afforded I, II, IV, XIII, XX and dihydrocanaric acid (XXIX), from the stems; and 2-hydroxyethyl benzoate (XXX) from the leaves. 13Recently, the dichloromethane extracts of H. madulidii yielded II, IV, XIII, XIV, XIX, XX and XXI from the stems and III, IV, XIII, XVII and XX from the leaves. 14n this study, the dichloromethane extract of the leaves of Hoya meliflua yielded squalene, β-amyrin (1a), α-amyrin (1b), lupeol (1c), oleanone (2a), ursenone (2b), lupenone (2c), β-amyrin cinnamate (3a), α-amyrin cinnamate (3b), lupenyl cinnamate (3c), β-sitosterol and stigmasterol (Figure 1).

General Experimental Procedure
Sample spectra were obtained on a JEOL ECS 400 spectrometer with CDCl 3 as solvent.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 meliflua leaves and stems were collected from healthy vines of a clone that has been cultivated for more than 10 years.Samples labeled as PNRI-H.CA were provided under Material Transfer Agreement No. 2016-004 dated June 24, 2016.The original plant came from Quezon Province, Philippines and authenticated by one of the authors (FBA).

General Isolation Procedure
A glass column 12 inches in height with 0.5 inch internal diameter was used for the fractionation of the crude extract.Fractions of 10 mL volumes were collected and monitored by thin layer chromatography.Fractions containing spots with similar Rf values were combined and rechromatographed using the appropriate solvent.Final purification was carried out using Pasteur pipette as the column, collecting 1 mL fractions.TLC-pure isolates were combined and after evaporation of the solvent, were subjected to NMR analysis.

Isolation of Chemical Constituents of the Leaves
The air-dried leaves (252.8 g) were ground in a blender, soaked in CH 2 Cl 2 for three days and then filtered.The filtrate was concentrated under vacuum to afford a crude extract (7.8 g) which was chromatographed by gradient elution with CH 2 Cl 2 , followed by increasing amounts of acetone at 10% increment by volume as eluents.The CH 2 Cl 2 fraction was rechromatographed (2 ×) using petroleum ether to afford squalene (12.8 mg).The 10% acetone in CH 2 Cl 2 fraction was rechromatographed (3 ×) using 1% EtOAc in petroleum ether to yield a mixture of 3a, 3b and 3c (5.0 mg), after washing with petroleum ether.The 20% acetone in CH 2 Cl 2 fraction was rechromatographed (3 ×) using 2.5% EtOAc in petroleum ether to yield a mixture of 2a, 2b and 2c (10.3 mg), after washing with petroleum ether.The 30% acetone in CH 2 Cl 2 fraction was rechromatographed (4 ×) using 7.5% EtOAc in petroleum ether to afford a mixture of 1a, 1b and 1c (10.6 mg) after washing with petroleum ether.The 40% acetone in CH 2 Cl 2 fractions was rechromatographed (3 ×) using 10% EtOAc in petroleum ether to yield a mixture of β-sitosterol and stigmasterol (21.0 mg) after washing with petroleum ether.

RESULTS AND DISCUSSION
Silica gel chromatography of the dichloromethane extract from the leaves of Hoya meliflua afforded squalene and mixtures of β-amyrin (1a), α-amyrin (1b) and lupeol (1c) in about 1:1:0.25 ratio; oleanone (2a), ursenone (2b) and lupenone (2c) in about 1:1:0.3ratio; β-amyrin cinnamate (3a), α-amyrin cinnamate (3b) and lupenyl cinnamate (3c) in about 0.5:0.3:1ratio; and β-sitosterol and stigmasterol in about 5:1 ratio.The structures of these compounds were identified by comparison of their NMR data with those reported in the literature.The NMR spectra of 1a are in accordance with data reported in the literature for β-amyrin, 4 1b for α-amyrin, 4 1c for lupeol, 4 2a for oleanone, 15 2b for ursenone, 15 2c for lupenone, 16 3a for β-amyrin cinnamate, 6,17 3b for α-amyrin cinnamate, 6,17 3c for lupeol cinnamate, 17 squalene, 18 β-sitosterol 18 and stigmasterol. 18he ratio of about 1:1:0.25 for the mixture of β-amyrin (1a), α-amyrin (1b) and lupeol (1c) was deduced from the intensities and integrations of the 1 H NMR resonances for the olefinic protons of 1a at δ 5.16 (brs), 7,12 1b at δ 5.11 (brs) 7,12 and 1c at δ 4.68 (brs) and 4.56 (brs). 7,12,18The ratio of about 1:1:0.30for the mixture of oleanone (2a), ursenone (2b) and lupenone (2c) was deduced from the intensities and integrations of the 1 H NMR resonances for the olefinic protons of 2a at δ 5.19 (t, J = 3.2 Hz), 15 2b at δ 5.14 (t, J = 3.2 Hz) 15 and 2c at δ 4.67 (brs) and 4.55 (brs). 19he ratio of about 0.5:0.3:1for the mixture of β-amyrin cinnamate (3a), α-amyrin cinnamate (3b) and lupenyl cinnamate (3c) was deduced from the intensities and integrations of the 1 H NMR resonances for the olefinic protons of 3a δ 5.18 (brs), 6,17 3b at δ 5.13 (brs); 6,17 and 3c at δ 4.68 (br s) and 4.56 (br s). 16The ratio of about 5:1 for the mixture of β-sitosterol and stigmasterol was deduced from the intensities and integrations of the 1 H NMR resonances for olefinic protons at δ 5.33 (brs, H-5) and methyl protons at δ 0.66 (s) for β-sitoterol and olefinic protons at δ 5.33 (brs, H-5), 5.13 (dd, J = 8.4, 15.2 Hz) and 5.00 (dd, J = 8.4, 15.2 Hz) and the methyl protons at δ 0.68 (s) for stigmasterol. 18H.meliflua is cultivated as an ornamental plant in the Philippines.There is no reported biological activity and medicinal property for the plant, but Blanco 20 mentioned as early as 1845 a record that H. meliflua causes "stoppage of birth, " or perhaps as an abortifacient.However, literature search revealed that the compounds isolated from the plant possess diverse activities.β-Amyrin (1a) and α-amyrin (1b) were reported to possess anti-inflammatory. 21-23and analgesic [24][25] properties Triterpene 1a showed antifungal activity against A. rabiei with an MIC value of 0.0156 mg/mL, 26 while 1b was proposed as a possible biomarker for the fungal resistance of grape-vine leaves (Vitis vinifera). 27The mixture of 1a and 1b effectively reduced the elevated plasma glucose levels during the oral glucose tolerance test (OGTT).Furthermore, the mixture of 1a and 1b at 100 mg/kg significantly decreased the VLDL and LDL cholesterol and increased the HDL cholesterol. 28A review on the sources and biological activities of 1a and 1b has been provided. 29On the other hand, lupeol (1c) exhibited antiurolithiatic and diuretic activity. 30It prevented the formation of vesical calculi and reduced the size of the preformed stones in rats. 31It also showed antifungal activity against Fusarium oxysporum and Penicillium notatum. 32Triterpene 1c significantly reduced the 451Lu tumor growth in athymic nude mice, 33 inhibited the proliferation of MDA-MB-231 human breast cancer cells in a dose dependent manner 34 and induced growth inhibition and apoptosis in hepatocellular carcinoma SMMC7721 cells by down-regulation of the death receptor 3 (DR3) expression. 35It 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. 36Another study reported that 1c and lupeol acetate have shown hypotensive activity. 37 review on the biological activities of lupeol has been provided. 38upenone (2c) inhibited adipocyte differentiation by suppressing PPARγ and C/EBPα protein levels. 39It also increased 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 2c could be a possible treatment for hypopigmentation. 40-Amyrin cinnamate (3a) was reported to inhibit the TPAS-induced inflammation (ID 50 0.27 μmol/ear; CI 95% 0.23-0.33μmol/ear) which is more inhibitory than the positive control, indomethacin (ID 50 0.91 μmol/ ear ;CI 95% 0.23-0.33μmol/ear). 19α-Amyrin cinnamate (3b), lupeol cinnamate (3c) and 3a exhibited marked anti-inflammatory activity against TPA-induced inflammation (ID 50 = 0.15-0.75µmol/ear).Triterpene 3c showed the highest activity with ID 50 of 0.15 µmol/ear. 12This triterpene at a dose of 10 mg/kg also exhibited anti-inflammatory activity on rat hind paw edema induced by carrageenan. 12Furthermore, 3c exhibited inhibitory effect on skin tumor promotion in an in vivo two-stage carcinogenesis test using DMBA as an initiator and TPA as a promoter. 12Triterpenes 3a-3c showed moderate inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) in Raji cells as a primary screening test for inhibitors of tumor promoters. 12qualene was reported to significantly suppress colonic ACF formation and crypt multiplicity which strengthened the hypothesis that it possesses chemopreventive activity against colon carcinogenesis. 41It showed cardioprotective effect which is related to inhibition of lipid accumulation by its hypolipidemic properties and/or its antioxidant properties. 42A recent study reported that tocotrienols, carotenoids, squalene and coenzyme Q10 have anti-proliferative effects on breast cancer cells. 43The preventive and therapeutic potential of squalene containing compounds on tumor promotion and regression have been reported. 44A recent review on the bioactivities of squalene has been provided. 45-Sitosterol was observed to have growth inhibitory effects on human breast MCF-7 and MDA-MB-231 adenocarcinoma cells. 46It was shown to be effective for the treatment of benign prostatic hyperplasia. 47It was also reported to attenuate β-catenin and PCNA expression, as well as quench radical in-vitro, making it a potential anticancer drug for colon carcinogenesis. 48It can inhibit the expression of NPC1L1 in the enterocytes to reduce intestinal cholesterol uptake. 49It was reported to induce apoptosis mediated by the activation of ERK and the downregulation of Akt in MCA-t102 murine fibrosarcoma cells. 50tigmasterol showed therapeutic efficacy against Ehrlich ascites carcinoma bearing mice while conferring protection against cancer induced altered physiological conditions. 51It lowered plasma cholesterol levels, inhibited intestinal cholesterol and plant sterol absorption and suppressed hepatic cholesterol and classic bile acid synthesis in Winstar as well as WKY rats. 52Other studies reported that stigmasterol showed cytostatic activity against Hep-2 and McCoy cells, 53 markedly inhibited tumour promotion in two stage carcinogenesis experiments, 54 exhibited antimutagenic, 55 topical anti-inflammatory, 56 antiosteoarthritic 57 and antioxidant 58 activities.

CONCLUSION
The results of our study indicate that Hoya meliflua shares similar chemical characteristics with other members of the genus Hoya: H. multiflora, 4 H. diversifolia, 7 H. cumingiana, 8 7 and H. madulidii 14 which contained squalene.The most common constituents of the eleven native Hoya species studied are β-sitosterol and stigmasterol which are found in ten species.Of the twelve compounds isolated from H. meliflua, lupeol is found in eight species, squalene, β-amyrin and α-amyrin in five species, lupenone and β-amyrin cinnamate in four species, α-amyrin cinnamate and lupenyl cinnamate in three species.Oleanone and ursenone are reported for the first time from Philippine Hoya species.It is interesting to note that although most Hoya plants have no known biological activity and medicinal property, the compounds isolated from H. meliflua possess diverse bioactivities.Lastly, the difference in results of phytochemical analysis of H. meliflua is a proof that it is an entirely different and distinct species from H. diversifolia.