Studies On The Chemical Constituents Of The Aerial Parts Of Artemisia Frigida

Artemisia frigid, named xiaobaihao too, is a perennial herb and sometimes subshrub belonging to genus Artemisia. It is widespread in our country, middle Asia, Siberia, Europe and North America[1]. In our country, it is constructive species or companion species of phytobiocoenose growing in forest grassland, grassland, desert grassland and some arid regions [1]. The whole plant of A. frigid was used to treat icteric hepatitis, cholecystitis, difficulty in micturition, itch of skin, eczema, ascariasis and enterobiasis in traditional Chinese medicine. And in traditional Mongolia medicine, it is named"a'ge", used to remedy haematemesis, haemorrhagia nasalis, irregular menses, bleeding wound and"qi ha"disease. Sometimes, A. frigida was used as substitunte of A. capillaries and favourable nutritive value forage for livestocks in grazing areas. Plants of Artemisia were rich in sesquiterpenes, flavones, coumarins, monterpenes and polyacetylenes, particularly sesquiterpene compounds with variable pharmacological activities is noticeable. Especially, there is an increasing interest in species of the genus Artemisia following the discovery and successful clinical trials of the antimalarial sesquiterpene artemisinin obtained from the ancient Chinese medicinal plant A. annua[2]. By far, reports on the chemical constituents of A. frigida are rare in home or abroad and the existing literature shows that the main constituents of A. frigida are flavonoids, sesquiterpenes and acetylenic compounds[3,4,5]. So, it is of particular significance to look for new chemical composition from A. frigida, on the basis of this, to explore structure-activity relationship of similar compounds and as a guide to conduct in-depth research on the active ingredients. We systemically investigated the chemical constituents of the aerial parts of A. frigida, and established the structures of the isolated pure compounds on the basis of spectroscopic techniques including 1D and 2D NMR methods. In addition, we carried out a wide range of bioassay for part of the compounds obtained.Objective: In order to develop the natural resources, enrich the diversities of Traditional Chinese Medicine and find out new compounds from the aerial parts of Artemisia frigid, we use the technologies of Silica gel column chromatography, Sephadex-LH-20 gel column chromatography, preparative TLC, HPLC and recrystallization to systemically isolate and purify the compounds of this plant, and use the spectroscopic methods including MS, 1H-NMR, 13C-NMR, 1H-1H-COSY, HMQC and HMBC to characterize the structures of the isolated pure compounds. To check up the effect of the pure compounds isolated from I. japonica on human tumor cells.Methods: The pulverized dried aerial parts of Artemisia frigida (7.12 Kg) were macerated for 7 days at room temperature with 95% alcohol for three times. Heating the leachate and adding in absorbite, after filtered and evaporated of the alcohol in vacuum to yield the total crud extract of 2Kg. The crude extract was suspended in saturated brine and extracted successively with petroleum ether, dichloromethane and ethyl acetate in order to obtain three fractions: the petroleum ether fraction 118 g, the dichloromethane fraction 120 g and the ethyl acetate fraction 45 g. The dichloromethane fraction and the ethyl acetate fraction were applied to silica gel column chromatography for preliminary fractionation in turn; each fraction was monitored with TLC and combined the similar fractions. Combined subfractions were subjected to Silica gel column chromatography, Sephadex-LH-20 gel column chromatography, preparative TLC and/or reversed phase preparative HPLC for further separation and purification to get pure compounds. The spectroscopic methods including various of 1D and 2D NMR methods were used for the structural identification of these compounds.Results: Systemical separation of the aerial parts of A. frigida yielded 19 compounds of them, the structures of 7 compounds were identified on the basis of chemical and spectral analysis, including 4 sesquterpenes, a coumarin, a benzoate and an acetophenone. They are 11,13-dihydrozaluzanin-D(1), isoscopoletin(5), 1,15-Dioxoeudesm-12,6-olide (6), 8α-hydroxy-11βH-11,13-dihydrodehydrocostuslactone(7), 11,13-dihydrozaluzanin-C(11), terephthalic acid tricyclic ethylene ester (19), p-hydroxyacetophenone (20)。Conclusion: The results of our experiment indicated that the solvents and methods of extraction and isolation used in this experiment are practicable. Silica gel column chromatography, Sephadex-LH-20 gel column chromatography, preparative TLC and preparative HPLC were employed to isolate and purify the components of the aerial parts of A. frigida, and variety of spectroscopic methods were used to establish the structures of the compounds. We obtained 19 compounds in all, and the structures of 7 compounds were identified with the aid of spectroscopic methods. 11,13-Dihydrozaluzanin-D (1), 11,13-dihydrozaluzanin-C (11) and terephthalic acid tricyclic ethylene ester (19) were isolated as natural products from the plants for the first time. Isoscopoletin (5) and p-hydroxyacetophenone (20) were isolated from A. frigida for the first time, 8α-hydroxy-11βH-11,13-dihydrodehydrocostuslactone (7) was isolated from plants of Artemisia for the first time. 1,15-Dioxoeudesm-12,6-olide (6) is a new compound. Compound 5 showed notable inhibition on HLE at 0.125 mg/mL.