Studies On The Chemical Constituents Of Achillea Alpine L.

Achillea alpine L., which is widely used by Uighur native people, is a plant of Compositae. The plant is widespread in Xin-jiang region of China especially in Ba-li-kun and Ta-cheng areas, as well as in Central Asia and Europe. As a popular folk medicine, Achillea alpine L. has a very long history of medical usage. It is recorded in"Supplement to the Compendium of Materia Medica"by Zhao Xue-min in Qing Dynasty. In ancient, a folk saying of"Where there is Achillea alpine L., where there no disease"is widespread in Xin-jiang. Achillea alpine L. grows in the region of Tian-shan mountains, distributing widely at an altitude of 1600-3000 m and grows on grassy areas of subalpine and in needle-leaved forests. In China, some clinical data indicated that Achillea alpine L. has pharmacological activities such as detoxication, strengthening the spleen to promote digestion, anti-anaphylaxis, anti-bacterial, anti-virus and liver-protective. Besides, the extracts of Achillea alpine L. exhibit biological activities including anti-cancer, antihistamine and antispasmodic. In folk, Achillea alpine L. is used as medicinal plant for relieving toxin of snakes, urticaria, indigestion, injury from falling down, feverish conditioned, common cold, swelling and pain in the throat, detoxicating and relieving sore throat and so on. It is reported that Achillea alpine L. contains chemical constituents such as flavonoids, keto-acid oxoacid, terpenoids, glycosides, polysaccharides volatile oil, polypeptide and alkaloids.Object: To develop the natural resources, search for its active components, extend the clinical effects and try to find out a new drug from Traditional Chinese Medicine, We conducted the extract of the air-dried aerial parts of Achillea alpine L. with Silica gel column chromatography to fractionate the crude extracts; with preparative TLC and HPLC to separate and to get compounds in pure form; with spectroscopic methods including UV, IR, HR-FAB-MS, 1H-NMR, 13C-NMR, 1H-1H-COSY, HMQC, HMBC and NOESY to establish the structures of the isolated pure compounds.Methods: The air-dried aerial parts of Achillea alpine L. (4.2 Kg) were chipped and extracted with 95% alcohol three times, filtered, and eliminated chlorophyll with activated carbon. The alcohol extract was concentrated to yield the total crud extract (368.6 g). The total crud extract was suspended in salt water (1:1) and re-extracted with petroleum ether, dichloromethane and ethyl acetate in order. At last three sections were obtained: the petroleum ether section (42.7 g), the dichloromethane section (38.8 g), and the ethyl acetate section (25.2 g). Dichloromethane section was applied to silica gel column chromatography for preliminary fractionation, each fraction was monitored with TLC, developed TLC were detected by UV light at 254 nm and followed by color reaction with spraying 10% H2SO4-ethanol reagent and heating at 140℃. Similar fractions were pooled and subjected to silica gel column chromatography. Combined some similar subfractions and applied to preparative TLC and/or reversed phase preparative HPLC for further purification at a flow rate of 3 mL/min. The extract of the ethyl acetate was subjected to polyamide column and eluted with mixtures of H2O-MeOH containing increasing amounts of MeOH, followed by silica gel and Sephadex LH-20 column chromatography. At last, pure compounds can be obtained and the spectroscopic methods including various of 1D and 2D NMR methods were used for their structural identification.Result: Separation of extract of the air-dried aerial parts of Achillea alpine L. by all kinds of methods including chromatography and chemistry yielded 16 compounds. Of them, the structures of 11 compounds were identified on the basis of chemical and spectral analysis. Compound B 5-Hydroxy-3,6,7,3',4'-pentamethoxy-flavone Compound C 3β,9β-Diacetoxy-l(l0),4(5),-germacra-dien-12,6α-olide (Sinten) Compound F1 (2S,3S,4R,8E)-2N [(2'R)-2'-hydroxyoctadec anoyl]-2-amino-8(E)- tetracosene-1,3,4-triol Compound F2 (2S,3S,4R,8E)-2N [(2'R)-2'-hydroxyoctadec anoyl]-2-amino-8(E)-pentacosene-1,3,4-triol Compound F3 (2S,3S,4R,8E) -2N [(2'R)-2'-hydroxyoctadec- anoyl]-2-amino-8(E)- hexacosene-1,3,4-triol Compound F4 (2S,3S,4R,8E)-2N [(2'R)-2'-hydroxyoctadec anoyl]-2-amino-8(E)- heptacosene-1,3,4-triol Compound F5 (2S,3S,4R,8E)-2N [(2'R)-2'-hydroxyoctadec anoyl]-2-amino-8(E)-octacosene-1,3,4-triol Compound G (±)-threo-N-isobutyl-4,5-dihydroxy-2E -decaenamide Compound H 6,7-cyclopropyl-5,18-dioleanene-3β-D-glucoside Compound K 5,7,3',4'-tetra-OH-flavone 6-C-glucoside (Iso-oriontin) Compound L 5,7,4'-tri-OH-flavone 6-C-glucoside (Isovitexin)Conclusion: The results of our experiment indicated that the solvents and methods of extraction used in this experiment are practicable. Column chromatography, preparative TLC and preparative HPLC were employed to isolate and purify the components in the ethanol extract of Achillea alpine L., and variety of spectroscopic methods were used to elucidate the structures of the compounds. Among them compound G is novel compound and compounds F1-F5 are reported for the first time from the genus of Achillea. Compound C is isolated for the first time from this plant.