Studies On The Substance Basis Of Anti-tumor In Vitro And Preparative Technology Of Xinjiang Artemisia Rupestris L.

Artemisia rupestris L., which is widely used by Uyghur native people, belongs to the genus Artemisia of family Compositae. It only grows in Xinjiang region of China, especially in the regions of Tian-shan mountains, A-ertai mountains and Kun-lun mountains, distributing widely at an altitude of 1600~4500 m and grows on grassy areas of subalpine and in needle-leaved forests, so it was named Xinjiang Artemisia rupestris. In ancient, a folk saying of"Where there is Artemisia rupestris L., Where there is no toxin of sankes."and"Where there is Artemisia rupestris L., Where there is no disease."is widespread in Xinjiang. It was recorded in"The Uyghur Traditional Medicine Recordsâ… "and some clinical data indicated that Artemisia rupestris L. has pharmacological activities such as dispelling wind and promoting blood flow, removing blood stasis and relieving swelling, strengthening the stomach to promote digestion, clearing away heat and toxins, anti-anaphylaxis and relieving toxins of snakes. It was reported that Artemisia rupestris L. contains mainly chemical constituents such as flavonoids, keto-acid oxoacid, terpenoids, polysaccharides, volatile oil, polypeptide and alkaloids. Nowadays, researching of Artemisia rupestris L. was focused on anti- influenza and anti-hepatitis B virus by scientists in Xinjiang. Lately, it was reported that flavonoids of Artemisia rupestris L. could exert their anti-proliferative effects on human liver tumor cell through inducing apoptosis and/or toxicity. In order to clarify the anti-tumor components, extend the clinical effects and further develop the traditional Uyghur resources, we carried out systematical investigation of active parts, active components of anti-tumor in vitro, preparative technology and the methods of quality control of the active part of Artemisia rupestris L.Objectives: 1) To screen the active parts and active constituents of anti-tumor in vitro of Artemisia rupestris L. 2) To optimize the preparative technology of the active part of Artemisia rupestris L. 3) To establish the methods of quality control of the active part of Artemisia rupestris L..Methods: 1) On the basis of systematical consulting the researching literatures of chemical constituents and biological activities study on the genus Artemisia plants, by means of bioactivity-guiding, anti-tumor activity screening assay of four-tumor cell lines in vitro were made on water extract, 95% ethanol extract, chloroform extract of Artemisia rupestris L. and water eluant, 30% ethanol eluant, 50% ethanol eluant, 70% ethanol eluant, 90% ethanol eluant by HPD-450 macroporous absorption resin, and the chemical constituents of the active part of Artemisia rupestris L. were researched for the first time. Several purification and separation methods (column chromatography on macroporous absorption resin, silical gel, polyamide, Sephadex LH-20) and many kinds of structure elucidation and identification technologies (UV, IR, EI-MS, 1H- NMR, 13C-NMR, DEPT, HMQC and HMBC) were used. And structure and MS/MS fragment pathway of casticin obtained from active part of Artemisia rupestris L. were elucidated with electron spray ionization mass spectrometry for the first time. The characteristic ions and fragment mechanism were explained. 2) According to the mono factor design and multifactors design, we used the contents of the mainly active constituents as index to select the extraction technology and purification technology of the active part of Artemisia rupestris L. for the first time. 3) The methods of quality control of the active part were established after the methodology validation.Results: 1) 70% ethanol eluant from water extract showed promising inhibitory tumor effects on the tested tumor cell lines and suggest that 70% ethanol eluant might be active part of anti-tumor in vitro, and 16 compounds were isolated from the 70% ethanol eluant of Artemisia rupestris L. and 13 compounds were elucidated on the basis of the physico-chemical properties and spectra analysis. Among them, 8 compounds were isolated from the plant for the first time and 2 compounds was isolated from the genus Artemisia for the first time. And among them, 8 compounds belong to flavonoids, 2 sesquiterpenes, 2 sterols and 1 other. And the results also showed that casticin mainly fragmented on the C ring and A ring. The characteristic ions of casticin were m/z 375, 342, 314, 299, 285 or 271 and m/z 375 fragment to m/z 342, ion m/z 342 fragment to m/z 314, ion m/z 314 fragment to m/z 299, ion m/z 299 fragment to m/z 285 or 271. And it was preliminaryly deduced that ESI-MS fragment pathway of flavonoids is similar to EI-MS fragment pathway of flavonoids. 2) After optimizing of the extraction technology and purification technology of the active part and verified through 3 batches of samples, the extraction technology of the active part was as following: the extracting solvent was pure water, extracting 3 times, first added 14 times extracting solvent to the materials, second added 14 times extracting solvent to the materials and third added 14 times extracting solvent to the materials and the extracting lasted 30 minutes each time. The purification technology of the active part was as following: water extract of Artemisia rupestris L. (the concentration of original solution of the extract was 30 mg/ml, pH=6) was subjected to HPD-450 resin column (diameter/high=1:9) with the adsorbing current velocity 2 ml/min, the ratio of loading capacity to resin volume was 1:15. After eluted with water (twice of resin volumn), the resin was eluted by 70% ethanol with 6 times of resin volumn under the flow rate 2 ml/min. The resin could be used for 7 times repeatedly after regenerated with 95% ethanol and 1 mol/L NaOH. 3) For the first time, the quantitative methods of mainly active constituents, such as quercetin, luteolin, kaempferol and casticin in Artemisia rupestris L. extract and Artemisia rupestris L. were established by HPLC.Conclusions: 1) The study of active parts and active constituents provided a substance basis of actions for further developing Artemisia rupestris L. And providing important information for flavonoids metabolism and structural modification researching on MS/MS fragment pathway of casticin. 2) The extraction and purification technology of Artemisia rupestris L. was reasonable, steady and repetitive. The yield ratio of Artemisia rupestris L. extract was 10.36~11.98%. The contents of total flavonoids was over 50% (51.29~53.95%) which met the demands. The mainly active constituents of luteolin and casticin were 9.77~9.84% and 23.77~24.16%, respectively. These results were steady and satisfied. The results of principle pharmacodynamics in vitro showed that the active part was as same as the 70% ethanol eluant screened assay in vitro in the first part, so it indicated that optimizing the preparative technology of the active part obtained anticipated result. 3) 3 batches of samples showed that established methods were easy and accurate, had nice recovery rates and good repetition, and could be used to control the quality of Artemisia rupestris L. extract and Artemisia rupestris L. which provided a basis for developing a novel drug and establishing its scientific, reasonable and strict quality control system in the future.