Studies On Chemical Constituents From Leaves Of Ilex Comuta Lindl.ex Paxt

Ilex cornuta Lindl.ex Paxt, as the evergreen plant- holly Ilex, distributes in Zhejiang, Jiangsu, Anhui, Jiangxi, Hubei, Hunan, Henan and Guangxi provinces. Holly leaves of this plant can cure headache, fever etc. Ilex cornuta Lindl.ex Paxt in the clinical application is still in the research stage. In addition to bactericidal anti-inflammatory effect, the more excessive suppression of the immune cell activation, anti-inflammatory, inhibit the function of the immune cell activation phase, together with its abundant source and cheap price, all the charaters makes it easy to develope new drug。Documents showed that the Ilex cornuta Lindl.ex Paxt,mainly contains flavonoids, triterpenes, saponins, sterols and polyphenols and other biologically active compounds.In this study, the extracts of the holly leaves with 95% ethanol as extractant was gained, then silica gel, polyamide and the gel chromatography, seven purified compounds were obtained. Using IR, UV, nuclear magnetic resonance spectroscopic data and the analysis of physical and chemical properties, identified their chemical structures as IlexsideⅡ, Ilexsaponin B, Ursolic acid, Daucosterol,β-sitosterol, Quercetin 3-O-β-D-xylopyranosyl(1→2)-O-3-D-glucopyranoside, Zigu-glucosideⅡ. Among them, quercetin-3-O-β-D-xylopyranosyl (1→2)-O-β-D-glucopyranoside for the first time isolated from this plant.This experiment also established HPLC determination method to analysis flavonoid-quercetin-3-O-β-D-xylopyranosyl (1→2)-O-β-D-glucopyranoside in the leaves of this plant. This method is consistent with the 2005 edition of Chinese Pharmacopoeia (1) AppendixⅧA method of Chinese medicine quality standards and Appendix VI D on High Performance Liquid Chromatographic Analysis of requirements. Extraction and SeparationThe leaves of Ilex cornuta Lindl.ex Paxt, (2kg) was grinder to crush and extracted by haet reflux with 95% ethanol for three times as 12L for 60min,10L for 45min and 10L for 30min repectively, combined ethanol extracting solution, concentrated in vacuum to gave ethanol extract 29g.Get Ilex cornuta Lindl.ex Paxt, ethanol extract 29g, using ethyl acetate methanol-water (1:5:0.1) as eluent over silica gel column chromatography (0.5kg), collected the elution solvent, through thin layer chromatography, collected the same part of the fraction, obtained four parts. After eluted with methanol-ethyl acetate, a portion of colorless crystals, compound Yl (88.7mg) was provided. From mixed solvent recrystallization, part B was subjected to silica gel and ODS column chromatography to obtain compound Y2 (15.5mg). From C part, we collected Y3 (367.1mg) by repeatedly perpormanced on silica gel, ODS column chromatography. As above operating manner, compound Y4 (72mg) and compound Y5 (42 mg) were obtained from part C, compound Y6 (150mg) and compound Y7 (270mg) were purified from Part D.Structure identificationInfrared spectrometry, Ultraviolet spectrometry and nuclear magnetic resonance spectroscopy data and the physical and chemical properties were analysized, then referenced to relevant literature, elucidated the chemical structures of these seven components. Y1 is the IlexsideⅡ; compound Y2 is Ilexsaponin B [3-O-β-D-glucopyranosyl (1→2)-β-D-xylopyranosyl Ilexgenin B ligand 28-O-β-D-glucopyranosyl ester glycosides], compound Y3 is Ursolic acid, compounds Y4 is Daucosterol, compound Y5 isβ-sitosterol, compound Y6 is quercetin-3-O-β-D-xylopyranosyl (1→2)-O-β-D-glucopyranoside, Y7 is Zigu-glucosideⅡ. The Compounds Y6 (quercetin-3-O-β-D-xylopyranosyl (1→2)-O-β-D-glucopyranoside) for the first time isolated from this plant.HPLC determination of triterpenoid Y6(Quercetin 3-O-β-D-xylopyranosyl(1→2)-O-β-D-glucopyranoside) in Ilex cornuta Lindl.ex Paxt,In this study, chromatographic conditions see Table 2 Table 2 The Chromatographic conditions of experiment Project condition chromatographic column Agilent extendC-18 column (250 mm×4.6 mm,5μm) mobile phase Acetonitrile-0.1％acetic acid solution (10:90) The detection wavelength detector 255nm Oven temperature 35℃Velocity 1.0mL·min-1In this study,The experimental content see Table 3Table 3 The content of the experimentstudy Experimential content Experimential Result Evaluate ProjectStandard compond Y6 was 0.1μg-Y=1.676*103X-85.44 Good curve 10.0μg. R=0.9999Precision Carries on compond Y6 Peak area RSD=1.63% Good parallel determinations Retention time RSD to 0.2 mg.mL-1 standard 1.62%. solution.Reprodurity The reprodurity tests of Sample content RSD=Good compond Y6 for six 1.19% samples Retention time RSD: 1.18%.Detectabilitie Take three 0.5 mg.mL-1 Use of signal to noise The detectabilities standard solution and ratio(S/N)≥3 was 2.Ong diluted 10,50 and500 criteria to determine the times, Into the detection limit chromatograph, record the peak height,and calculate the signal to noise ratio.Stability The solution fo compound The solution of compound Good Y6 was stable in ten hours Y6 was stable in ten hours RSD<2%Recovery the solution were RSD=1.85%. Good injected into me chromatograph instrument respectively Measured peak areaThe compounds of Ilex cornuta Lindl.ex Paxt were determined.The average content of the compound Y6is 0.0231%.This paper studied the major chemical constituents of the Ilex cornuta Lindl.ex Paxt,.The results can offer a new index on content determination for the establishment of quality standards of Ilex cornuta Lindl.ex Paxt,and also can provide a new scientific evidence for the development and research of this medicin herb..