Study On Structure Confirmation Of Polyacetylene And Induced Differentiative Effects On A549 Hepatocarcinoma Cells

Panax Ginseng is a valuable traditional Chinese medicine in China, ginsenoside is its main active ingredient. Recent studies have found that the ether soluble components of fibrous root which called polyacetylene also have a wider pharmacological activity. In particular, it has significant anti-tumor activity which attracted researchers’attention.Lung cancer, especially non-small cell lung cancer, is a common serious disease harmful to human health, and has become an important global public health problem.The fibrous root of ginseng was used as research object in our study. Extraction process of panaxynol, structure verification and standards research, content determination method, the inclusion process of total polyacetylene andβ-CD and anti-cancer activity were studied, the research results were as follow:1. Extraction process optimization of panaxynolBoth organic solvent extraction process and supercritical CO₂ extraction were adopted in our study. petroleum ether was used as extraction solvent, Orthogonal design was adopt to the heating reflux extraction process optimization.the result indicated that the optimized heating reflux extraction process was as follow: optimum extraction temperature was 80℃,15 times the amount of petroleum ether for once, the extraction time was 1h, according to the process, the extraction rate of panaxynol can be achieved to 0.42‰. Orthogonal design of supercritical CO₂ extraction was used to optimize process, The result showed that the optimum for the extraction temperature was 45℃, the concentration of ethanol concentration was 95% (the amount of crude drug was 1.2 L ? kg^-1), extraction pressure was 30 MPa, extraction time was 80min, according to the process, the extraction rate of panaxynol can be achieved to 0.45‰. Compare with the organic reagent extracton method, supercritical CO₂ extraction has a characteristic of environmental protectable, high efficiency and low cost.2. The structure verification of polyacetylene and standards researchAfter repeated studies, we optimized the silica gel G column chromatography to separate and purify compound A; combined Silica gel G column chromatography with LH-20 gel column chromatography to separate and purify compound B. The compound A and compound B were isolated and purified by using the above method, through the analysis of physical and chemical properties and spectral data, we draw a concrete conclusion that compound A is panaxynol, and compound B is panaxydol. Both of them were detected by TLC,and show single spot; and they were determined by HPLC normalized method, the content were no less than 98.0%.meanwhile, the quality standard of panaxynol and panaxydol were established on the basis of experimental result and literature.3. Quality control of polyacetylene componentsAfter repeated tests, the independent/simultaneous determination method of panaxynol and panaxydol were established; meanwhile, the total polyacetylene determination method was screened by using UV spectrophotometer. The HPLC conditions of panaxynol determination was as follow: column: Elite C18 column （150 mm×4.6mm, 5μm）; detection wavelength: 230nm; mobile phase: acetonitrile-0.025% phosphoric acid （70:30）; flow rate: 1.0mL?min^-1; column temperature: room temperature; the content of panaxynol in fibrous of Panax Ginseng was 0.42‰; The HPLC conditions of panaxydol determination was as follow: column: Elite C18 column （150mm×4.6mm,5μm）; detection wavelength: 230nm; mobile phase: acetonitrile-water（54:46）; flow rate: 1.0mL?min^-1; column temperature: room temperature; the content of panaxydol in fibrous of Panax Ginseng was 0.29‰; The HPLC conditions of simultaneous determine panaxynol and panaxydol was as follow: column: Elite C18 column （150mm×4.6mm, 5μm）; detection wavelength: 230 nm; mobile phase: acetonitrile–water,gradient elution; flow rate: 1.0mL?min^-1; column temperature: room temperature; the content of panaxynol and panaxydol in fibrous of Panax Ginseng were 0.42‰and 0.29‰respectively; The determination conditons of total polyacetylene was TU^-1901 UV spectrophotometer;wavelength: 230nm; the content of total polyacetylene in fibrous of Panax Ginseng was 0.84‰.4. The inclusion process of TPA-β-CDAfter stability test, the result indicated thatβ-CD has a stabilizing effect on polyacetylene. On this basis, orthogonal design was used to optimize the preparation of inclusion, the best TPA-β-CD inclusion process was as follow: TPA andβ-CD has a ratio of 1:8 （W:W）,inclusion temperature was 70℃, inclusion time was 60min. The process is simple, reproducible, and can be used for industrial production.5. Induced differentiative effect on A549 hepatocarcinoma cellsExperiments showed that TPA has a strong inhibitory effect on A549 cells in dose and time-dependent manners. 3.125¹2.5μg·ml^-1 of TPA performance of non-cytotoxic effects mediated growth inhibition, 25μg·ml^-1 and above of TPA showed cytotoxicity.