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Clinopodium polycephalum (Vaniot.) C.Y.Wu et Hsuan ex Hsuan and Clinopodiumchinensis (Benth.) O.Kfze, belonging to Labiatae plant which is perennial herbs, andtheir dry part on the ground are named Duanxueliu. Clinopodium chinensis was firstrecorded in the grass part of JiuHuangBenCao(1406), which was called Fengluncaiand the leaves can edible. Clinopodium polycephalum was first recorded in thefragrant grass class of ZhiWuMingShiTuKao(1848), and called Dayexiangru. Theywere no medicinal records.“Duanxueliu”, including the two original plants, has beenrecorded in the People’s Republic of China Pharmacopoeia of2005edition. Theywere used for the treatment of various of bleeding symptoms in clinic, like uterinebleeding, hematuria, epistaxis, gingival bleeding, traumatic bleeding, fibroidsbleeding.In this study,18compounds were isolated from herba clinopodii extracts.15compounds were identified by IR UV MS NMR spectroscopic data analysis and thephysical and the chemical properties, which were Linarin(compound A), Apigenin-7-rutinoside (compound B), Naringenin(compound C), Apigenin(compound D),Homoeriodictyol(compound E), Luteolin glycosides(compound F), Naringenin-7–rutinoside (compound G), Didymin(compound H), Clinopodiside II(compound I),Clinoposaponin Ga(compound J), Buddlejasaponin IV b(compound K),3β,16β,24aldehyde,23,28-tetrahydroxyoleana-9(11),12(13)-diene-3-yl-[β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→3)]-β-D-fucopyranoside(compound L)、3β,16β,11ketone,23,28-tetrahydroxyoleana-12(13)–diene-3-yl -[β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→3)]-β-D-fucopyranoside(compound M),3β,16ethanoyl,23,28-tetrahy-droxyoleana-11(12),13(18)-diene-3-yl-[β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→3)]-β-D-fucopyranoside(compound N),5-methyl-8-(3-methoxy-4-hydroxy phenyl)-5,7-diene octoate(compound O), respectively in addition, the compounds L、M、N、O were new compounds.This experiment also established HPLC-ELSD determination of the triterpenoidsaponins about Clinopodiside II and Buddlejasaponin IVb in tabellae clinopodii.1Chemical composition of the separation, structure elucidation1.1SeparationTake5kg extracts of clinopodii for macroporous resin D101column chromatography,gradient eluted with water-ethanol (water,15%ethanol,70%ethanol,90%ethanol).70%ethanol elution was carried on polyamide column chromatography, usingwater-ethanol gradient elution(water,15%ethanol,30%ethanol,50%ethanol,70%ethanol,90%ethanol). The30%ethanol elution of polyamide column was carriedon silica gel column chromatography, obtained12constituents by chloroform-methanol-acetic acid gradient elution. The seventh part was dissolved bywater-saturated n-butanol, then extracted by0.01mol·L-1NaOH, which adjust pH to2with concentrated HCl. The results are divided into two layers: water saturatedn-butanol part and lye part. The water saturated n-butanol layer is flavonoid portionand lye layer is saponins portion. Compounds I-O were purified from the saponinsportion by Sephadex LH-20and preparative high performance liquid, and compoundsA-H were purified from flavonoid portion by polyamide column chromatography andpreparative high performance liquid. 1.2Structure identificationThese compounds were identified by physicochemical properties and IR, UV, MS,NMR spectral analysis and literature. They were linarin (compound A),apigenin-7-rutinoside (compound B), Naringenin(compound C), apigenin-(compoundD), homoeriodictyol(compound E), luteolin glycosides (compound F), naringenin-7-rutinoside(compound G), didymin(compound H), Clinopodiside II(compound I),Clinoposaponin Ga(compound J), Buddlejasaponin IV b(compound K),3β,16β,24aldehyde,23,8-tetrahydroxyoleana-9(11),12(13)-diene-3-yl-[β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→3)]-β-D–fucopyranoside(compound L),3β,16β,11ketone,23,28-tetrahydroxyoleana-12(13)–diene-3-yl-[β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→3)]-β-D-fuco–pyranoside(compound M),3β,16ethanoyl,23,28–tetrahy-droxyoleana-11(12),13(18)-diene-3-yl-[β-D-glucopyran-osyl-(1→2)]-[β-D-glucopyranosyl-(1→3)]-β-D-fucopyranoside(compound N),5-methyl-8-(3-methoxy-4-hydroxy)-5,7-diene octoate(compound O).2Content determination of ClinopolisideII、buddlejasaponin IVb in extractsclinopodii2.1The conditions of chromatographic and detectorChromatographic conditions: AichromaBond-AQ C18(4.6×250mm5μm), themobile phase was methanol-water(78:22), the flow rate was1.0mL·min-1and thecolumn temperature was30℃.Detector conditions: the atomization temperatures was27.0℃, the drift tubetemperature was60.0℃, the pressure of the carrier gas was30psi and the gain valuewas set to20.2.2Methodology studyI Standard curveThe linear regression equation of buddlejasaponin IVb is lg S=1.506lg m+4.469,and the correlation coefficient r is0.9980. The results show that the referencesubstance of buddlejasapnin IVb in0.496μg～9.92μg has a good relationship of the linear range. The linear regression equation of Clinopoliside II is lg S=1.460lg m+4.602, the correlation coeff icient r is0.9970. The results show that the referencesubstance of Clinopoliside II has a good relationship in the linear range from0.200μgto20.0μg.II Precision expeditionThe intraday peak area precisions of Clinopoliside II and buddlejasaponin IVb were2.26%and2.40%. the day precision were3.98%and4.45%. The days retention timeprecision were0.53%and0.69%. but the day retention time precision were0.69%and0.49%.III Repetition investigationThe RSD of content of Clinopoliside II and buddlejasaponin IVb in the6copies of thetest solution is4.48%and3.39%, the results show that this method has a goodrepeatability.IV Stability investigationThe RSD of peak area of Clinopoliside II and buddlejasaponin IVb were3.38%and2.42%after precise drawing the same test solution (first sample), and then injectingby prepared for0,2,4,8,12and24hours, respectively.Visible Clinopoliside II,buddlejasaponin IVb were stable within24hours, the good preparation for the testsolution can be measured within24hours.V Coefficient of recovery investigationThe mean coefficients of recovery of Clinopoliside II and buddlejasaponin IVb are99.7%and97.7%, the RSD are1.16%and1.46%, and the results demonstrate thatthe coefficient of recovery of Clinopoliside II and buddlejasaponin IVb are well.The methodology experiment shows that this method has higher accuracy, sensitivity,precision and selectivity. Assaying consequence indicate that the average contents of Clinopoliside II and buddlejasaponin IVb in extracts of clinopodii are0.35%and0.98%. The component of extracts of clinopodii was investigated deeply, and themethod of assaying of Clinopoliside II and buddlejasaponin IVb were constructed.The results provide scientific basis for the construction of multiindex components ofextracts of clinopodii, improve the quality standard of clinopodii preparations, andprovide scientific basis for new medicinal value of “duanxueliu”.
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