Windmill grass [Clinopodium chinensis O. Kuntze var. Grandiflorum (Maxim. Hara], belonging to wind round vegetable which are Labiatae species is perennial herbs. There are many kinds of chemical constituents in Windmill grassincluding flavonoids, saponins, volatile oil, fatty acids, triterpenes, steroids and other biologically active compounds. Pharmacological studies have shown that windmill grass has hemostasis, anti-tumor, anti-inflammatory, immunomodulatory, antibacterial and other effects.In this study, the chemical constituents of the windmill grass were studied systemically. Ten compounds were Isolated from the windmill grass. Using IR,UV, MS, NMR and other spectroscopic analysis of the data, including physical and chemical properties , the chemical structures of the 10 compounds were identified as buddlejasaponinâ…£b(A), buddlejasaponinâ…£(B), Clinoposaponinâ…ª(3β,28-epoxy-16β-hydroxyolean-11-en-3β-yl-β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-fucopyranoside)(C), didymin(D),3β,16β,28 trihydroxyoleana -11 ,13 (18)-dien -3-yl- [β-D- glucopyranosyl -(1→2) ] -[β-D-glucopyranosyl-(1→3) ]-β- D- fucopyranoside(G),quercetin-3-O-β-D-glucopyranosyl-(1→6)-O-β-D- glucopyanoside(H),Clinoposaponinâ…¤(I),Clinoposaponinâ…¢(J),Clinoposaponinâ…¤b(K) and Clinoposaponinâ…¢b(L). Among them, co-pounds C, H, I, J, K, L are the first time isolated from the windmill grass.Windmill grass as a widely used Chinese herbal medicines has been rcorded in"Chinese Pharmacopoeia"of 1995, 2000, 2005 Editions, but it still lack of quality standards in the determination of indicators of Terpenoids. To fill this gap, this paper established Determination method to analysis the main buddlejasaponinâ…£in the this plant using HPLC-ELSD method. Chemical composition of the extraction, isolation, structure elucidation5Kg dried windmill grass powders were extracted in 85% ethanol at reflux condition for three times, the volume of 85% ethanol were 35, 25, 20 liters and each reflux time were 2 hours, 1.5 hours and 1 hour, respectively. Filtrated solution was concentrated and diluted with water, and then was absorpted by macroporous resin column, eluted with ethanol. Extract 273g was gained after ethanol was evaporated in vavumm. The extraction was subjected to silica gel column and ODS column repeatedly to afford compounds A,B,C,D,G,H,I,J,K,L. The mobile phase were Ethyl acetate,Chloroform,Methanol and water with several proportions.Being identified by physical, chemical properties and IR, UV, MS, NMR spectral analysis, comparison with the literature, The results showed that compound A is buddlejasaponinâ…£b,compound B is buddlejasaponinâ…£,compound C is Clinoposaponinâ…ª(3β,28-epoxy-16β-hydroxyolean-11-en-3β-yl-β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-fucopyranoside),compound D is didymin, compound G is 3β,16β,28 trihydroxyoleana -11 ,13 (18)-dien -3-yl- [β-D- glucopyranosyl -(1→2) ] -[β-D-glucopyranosyl-(1→3) ]-β- D- fucopyranoside,compound H is quercetin-3-O-β-D-glucopyranosyl-(1→6)-O-β-D- glucopyanoside,compound I is Clinoposaponinâ…¤, compound J is Clinoposaponinâ…¢,compound K is Clinoposaponinâ…¤b and compound L is Clinoposaponinâ…¢b.HPLC determination of buddlejasaponinâ…£1.Chromatographic and detector conditionsThe mobile phase was methanol-water (70:30), flow rate was 1.0mL.min-1, the column temperature was 25℃. ELSD drift tube temperature degree was set at 80℃; Carrier gas flow rate was adjusted to 1.66 L·min-1; Amplification factor was and impactor was closed.2. Methodology Studyâ… Standard curveBuddlejasaponinâ…£sampling in the range of 0.5 ~ 20.0μg, into the amount of its value and are commonly used on the common logarithm of peak area showed good linearity. Regression equation and value are as follows. lgS=1.4157 lgm +5.159 r = 0.9997â…¡Precision experimentsThe same control solution 6 parallel determinations, Buddlejasaponinâ…£peak area RSD value was 1.24%.â…¢Repeatability testsThe RSD of Buddlejasaponinâ…£conten in the 6 copies test sample solution was 1.59%, the results show that the method has a good reproducibility.â…£Stability testIn the test solution, the RSD of peak area of buddlejasaponâ…£in sample solution during 12 hours was1.63%, indicating that the sample solution stability in 12 hours.â…¤Recovery testThe average recovery of buddlejasaponinâ…£98.8% (RSD 1.87%).The results of experiments showed that the above method has high accuracy, high precision, selectivity . After determination , the content of buddlejasaponinâ…£in Windmill grass ws 1.065 percent in average.
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