The Analisis Method Establishment Of Periplocae Cortex And Inhibitory Effect Of Ethanol Extract On Human Lung Cancer A549 Cell Proliferation

Periplocae Cortex is the dry root bark of Periploca plants of Asclepiadaceae and Periploca sepium Bunge, is commonly used in Chinese herbal medicine. Periplocae Cortex can dispel rheumatism, strengthen bones and waist, eliminate edema, be used for the treatment of rheumatism,lumbar bone pain, lumbar debility, shortness of breath, palpitation and symptoms of lower extremity edema. In recent years, the effect of the treatment of chronic congestive heart failure is better and clinical demand is greater. Periplocae Cortex chemical constituents are complex, mainly cardiac glycosides compounds, terpenoids and C21 steroidal compounds.At present, the content of active ingredient on determination of Periplocae cortex and pharmacologic research reports are more, Periplocae cortex quality control is rarely reported. This paper focused on 14 different areas of active ingredient content of Periplocae cortex which were the object of study, optimized its extraction process, established the method of HPLC-DAD fingerprint of Periplocae cortex, to establish a more perfect quality standard, to distinguish Periplocae cortex and Acanthopanacis cortex more systematically and completely, to ensure the safety of drug use.Lung adenocarcinoma is one of the most common types of non small cell lung cancer. It has high infection and metastasis, easy to produce drug resistance, therefore, it is urgent to find a new drug for the treatment of lung adenocarcinoma. This paper according to the characteristics of Periplocae cortex can induce apoptosis of cancer cells, chose the lung adenocarcinoma A549 cell as the research object to carry out the experiment in vitro, to study effect of Periplocae cortex on proliferation of human lung adenocarcinoma A549 cells. Using morphological observation,cell cycle distribution and so on, determined the Periplocae cortex can inhibit the proliferation of A549 cell line. Hope to provide a theoretical basis for the treatment of lung cancer at the cellular level, to seek new drugs for the treatment of lung adenocarcinoma.1.Optimization of extraction process of Periplocae cortex Used periplocin and 4- methoxy salicylaldehyde as index components,the extraction process was optimized by HPLC-DAD determination. First,by single factor method to examine the extraction solvent and method, and then used the L9(34) orthogonal experiment to further optimize the extraction process of Periplocae cortex, determined 4 factors of the extraction, such as solvent concentration, solvent ratio, extraction time and extraction times, 3 levels of each factor, designed orthogonal experiment.The final extraction process was 60% ethanol, 20 times of the solvent, 1.5 h,3 times of extraction. Periplocin and 4- methoxy salicylaldehyde were within the range of 10-60 ?g·ml-1(R2= 0.9996), 9.5~76 ?g ·ml-1(R2= 0.9997)with good linearity, the recoveries were between the 93%~105%(RSD3.77%), 94%~102%(RSD 3.23%). The component content vary significantly influenced by areas and climate, so this paper collected 14 samples from different areas of Periplocae cortex, according to the best process to extract and determine the content of periplocin and 4- methoxy salicylaldehyde.2.The establishment of HPLC fingerprint of Periplocae cortex This paper established the HPLC fingerprint of Periplocae cortex, and reviewed methodology, confirmed the 16 common peaks, established the control fingerprint of Periplocae cortex, and the similarity of 14 batches of Periplocae cortex fingerprints was analysed. Final chromatographic conditions were Hypersil BDS C18 column(250mm×4.6mm, 5?m), mobile phase: acetonitrile:0.05% Phosphoric acid solution(A:B),gradient elution program:0 min A:B 5:95, 5 min A:B 15:85, 15 min A:B 25:75, 20 min A:B 30:70, 30 min A:B 30:70, 35 min A:B 60:40, 60 min A:B60:40;wavelength:239 nm, flow rate: 1.0 ml·min-1, column temperature:30 ?, injection volume: 20 ?l. Extraction method was 10 times of methanol, ultrasonic extraction.Acanthopanacis Cortex as cortex Periplocae easily confusable herbs,respectively according to the Periplocae cortex extraction and chromatographic conditions to research 7 groups of cortex Acanthopanacis,established the HPLC charactereristic chromatogram of Acanthopanacis cortex and confirmed the 14 common peaks, and carried on the analysis of the similarity. The results showed that there were big differences with the fingerprint of Periplocae cortex and Acanthopanaci cortex, by comparing the fingerprint, the two kinds of medicines can be distinguished.3.Inhibitory action of ethanol extract from Periplocae cortex on lung cancer cell line A549 This paper used MTT assay to determin Periplocae cortex alcohol extract on A549 cell proliferation inhibition, the highest inhibition rate reached 73.94%. Fluorescence microscope to observe morphological changes of apoptotic cells, alcohol extract of cortex Periplocae A549 cells group appeared red cell apoptosis. Flow cytometry to detect the cell cycle distribution, the results showed that with the increase of drug concentration,the proportion of G0/G1 phase cells decreased, and the proportion of S cells increased. Experimental results showed that the cortex Periplocae alcohol extract on A549 lung cancer cells can inhibit the proliferation and to provide experimental basis for further study of Periplocae cortex anti-cancer effect.In summary, this paper optimized the extraction process of Periplocae cortex, can let the periplocin and 4- methoxy salicylaldehyde of Periplocae cortex content max, using HPLC determined the content of periplocin and4- methoxy salicylaldehyde of 14 different areas Periplocae cortex. The contents of periplocin from Periplocae cortex in Shanxi is highest, can reach 5.840mg·g-1, 4- methoxy salicylaldehyde in standard medicines is highest, can reach 3.959 mg·g-1. This paper also established the HPLC fingerprint of Periplocae cortex, and distinguished with Acanthopanaci cortex. This paper showed Periplocae cortex alcohol extract can inhibit the proliferation of A549 cells, and the inhibitory effect increased with the increase of the concentration of the drug and the time of the use of the drug,the highest inhibition rate can reach 73.94%.