King Formosanus Sterols Triterpenoids Extraction And Analysis Of Inhibition Of Chronic Leukemia, Myeloid K562 Activity

OBJECTIVE: In this study, we applied supercritical fluid extraction (SFE) to extract sterols and triterpene from Anoectochilus roxburghii, then the qualitation and quantitation were undertaken; to establish and perfect the quality standard system of Anoectochilus roxburghii and to provide the reference data to the Chinese Pharmacopeoia;To study the k562 hyperplasy inhibit activity of CO₂ supercritical fluid extraction (SFE0) products, and explored the mechanism of antineoplasmic activity of Anoectochilus roxburghii initially.METHODS: We apply supercritical fluid extraction (SFE) to extract sterols and triterpene from Anoectochilus roxburghii. Orthogonal design was used to optimize the SFE conditions. The effects of pressure, temperature and modifiers on the extraction efficiencies were studied by a three-level orthogonal array design with an L₉(3)³ orthogonal experiment table.Then liquid chromatography–atmospheric pressure chemical ionization-ion trap mass spectrometry (HPLC/APCI/MS) is proposed, for the purpose of qualitative analysis of sterols and triterpene, and the selected ion monitoring (SIM) to quantitative analyse. We take terpenoids oleanolic acid (OA) and ursolic acid (UA) as index ingredient, and the minimum content of these two components was regulated. Edematous amaenorrhea, total ashes and erhanol-soluble extracts of the 12 Anoectochilus roxburghii samples were investigated with the first edematous amenorrhea determination method in Appendix IX H, ashes determination method in Appendix IX K and 70% ethanol extract determination method in Appendix X A in Chinese Pharmacopeoia published in 2005. Through MTT dyeing method we detected the K562 cell toxic action, then got the IC50 through inhibition ratio curve. Apoptosis morphological transformation of K562 cell induced by SFE₀ was detected by the transmission electron microscope and AO-EB staining method. Alteration of cell cycle was analyzed by flow cytometer.RESULT: We got the optimal condition, i.e., a pressure of 25 MPa, a temperature of 45℃and ethanol as modifier. The concentration ofβ-sitosterol , stigmasterol and ergosterol significantly increased by 4.71, 3.97 and 3.95 times when the SEF was used compared with the Soxhlet extraction. The protonated molecules of the first full mass spectra of the analytes were recorded by APCI (+)-MS spectra, the diagnostic ions were selected for screening and quantification. Based on the m/z value of the fragment ion, retention time, published data and the comparison with standard compounds, these analytes in each sample were identified. For the first time we report ergosterol, oleanolic acid and ursolic acid exist in Anoectochilus roxburghii. And the concentration of ergosterol, stigmasterol,β-sitosterol,oleanolic acid and ursolic acid is 0.968mg/g,1.19 mg/g,1.06 mg/g,0.161 mg/g and 0.130 mg/g respectively.The minimum concentration of OA and UA in arvensis Anoectochilus roxburghii is 0.065% and 0.059% respectively. The edematous amaenorrhea were 10.47%～7.32%; the total ashes were 11.049%～7.444%; the ethanol soluble extracts in 70% ethanol were 18.127%～10.274%. SFE0 induced significant growth arrest of K562 cultured in vitro. After treatment with SFE0 showed typical of apoptosis, the diploid cell peak appeared.CONCLUSION: The concentration of the three sterols is higher compared with the two triterpene components, over 1‰. For the high selectivity of the extraction, much cleaner extracts obtained and less time used, SFE is fit to extract sterols and triterpene from Anoectochilus roxburghii; the concentration of the two triterpene components can not be lower than 0.124% when calculated as dry medical material; SFE₀ induced significant growth arrest of K562.