Study On The Anti-inflammatory Activity And Chemical Constituents Of Tibetan Medicine Herba Oxytropis

The whole study was seperated into the following five parts:PartⅠ:Documental StudyThe systemic documental investigation on chemical constituents and pharmacological activities of Oxytropis D.C.was carried out.At present,the study on chemical constituents of this genus was focus on O.microphylla,O.ochrocephala,O.kansuensis,O.glabra,O. bicolor.However,only few reports about some others plants of Oxytropis D.C.,for instance,O.falcata,O.chiliophylla and O.psammocharis,could be found.In brief,the study on this genus is still being in primary status.PartⅡ:Pharmacognosical StudyThe four aspects including basic source,characters,microstructures and physical and chemical reactions of this drug were investigated in this part.The microstructures and characters of root and flower were observed and depicted for the first time.In addition,the physical and chemical reaction for identification of alkaloids and saponins in the drug were added.PartⅢ:Pharmacological Study1.Acute Toxicity(LD₅₀)Increasing doses of total extract（1.2,2.5,5.0,10.0,20.0 and 40.0 g/kg） were administered by lavage.The mice were kept under observation for a period of 7 days.None of mice were dead within 7 days,and both psychosis and change of weight were normal.2.Analgesic Effect of EOFOur findings showed that oral administration of EOF could significantly inhibit acetic acid-induced writhing in mice,maximum inhibition of writhing was observed at 1200 mg/kg dose（57.2%）.The extraction was remarkably active in the second phase of formalin-induced pain（1200 mg/kg dose,40.1%）,but not in the hot plate assay.O.falcata may have peripheral but not central analgesic properties.3.Anti-inflammatory Activity of EOFThe EOF also produced anti-inflammatory effects,as demonstrated by maximum inhibition of mouse ear edema（58.0%）,mouse peritonitis induced by carrageenan（59.4%）, and rat granuloma induced by cotton pellets（49.2%） at 1200 mg/kg dose.O.falcata may effect on both the acute and chronic phases of the inflammation.4.Screening for Anti-inflammatory Activity Fraction from O.falcataFlavonoid of O.falcata possessed the best anti-inflammatory effect among five fractions,and have no significant difference compared with indomethacin（10 mg/kg） as a reference drug.In addition,the alkaloid of O.falcata have evident anti-inflamatory activity, but was not stronger than flavonoid and indomethacin.5.Preliminary study on Mechanism of Anti-inflammatory Activity FractionThe content of MDA was increased greatly in both serum and right paw of model rats after the injection of carrageenan.However,the increased content of MDA in model rats could not be suppressed in 6 h after oral admistration of and indomethacin either.We suggested that the total flavonoid from O.falcata could exert its anti-inflammatory effect through cyclooxygenase suppression but not free radical production as indomethacin,a non-steroidal anti-inflammatory drug.PartⅣ:Study on Chemical Contituents1.Preliminary Chemical TestsAlkaloids,saponins,flavonoids,polysaccharides and glycosides,and volatile oil were present in this drug largely.2.Preparation of Five Fractions for Anti-inflammatory Activity ScreeningThe total flavonoids（The purity is 83.7%,and the yield rate is 2.56%）,saponins（The purity is 68.3%,and the yield rate is 0.89%）,polysaccharides（The purity is 85.5%,and the yield rate is 2.26%）,volatile oil（The yield rate is 1.34%） and alkaloids（The yield rate is 1.45%） were seperated from the Herba Oxytropis.3.Isolation and Identification of CompoundsSilica gel column chromatographic and semi-preparative high performance liquid chromatographic method were used to isolate constituents from the petroleum ether, chloroform and ethyl acetate extract,and 33 compounds were obtained.Subsequently,the structures of 30 compounds among these were elucidated by UV,EI-MS,NMR spectral data and physical and chemical properties as A₁（n-nonacosane）,A₂（eicosanoic acid）,A₃ （β-sitosterol）,B₄（pinostrobin）,B₅（2’,4’-dihydroxy dihydrochalcone）,B₆（2’,4’-dihydroxy chalcone）,B₇（pinocembrin）,B₈（stigmasterol）,B₉（2’-hydroxy-4’-methoxy chalcone）, B₁₀/C₁₉（7-hydroxy flavonone）,B₁₂（Ψ-baptigenin）,B₁₃（formononetin）,B₁₄（liquiritigenin）, B₁₅（2’-methoxy-4’-hydroxy chalcone）,C₁₆（chrysin）,C₁₇（genistein）,C₁₈（apigenin）,C₂₀ （2’,4-dihydroxy-4’-methoxy chalcone）,C₂₁（luteolin）,C₂₂（phenethyl cinnamide）,C₂₃ （3’,7-dihydroxy-2’,4’-dimethoxy isoflavane）,C₂₄（7-hydroxy flavone）,C₂₆（2’-hydroxy phenethyl cinnamide）,C₂₈（isoliquiritigenin）,C₂₉（isorhamnetin）,C₃₀（quercetin）,C₃₁ （myricetin）,C₃₂（kaempferol）,C₃₃（diisobutyl phthalate）,C₃₄（β-daucosterol）.A₁,A₂,B₄,B₇, B₈,B₁₀/C₁₉,B₁₂,B₁₃,B₁₄,C₁₆,C₁₈,C₂₀,C₂₁,C₂₂,C₂₄,C₂₆,C₃₀,C₃₁ and C₃₃ were isolated from Herba Oxytropis for the first time.A₂,B₄,B₇,B₁₀/C₁₉,B₁₂,B₁₃,B₁₄,C₂₀,C₂₄ and C₃₃ were isolated from Oxytropis D.C.for the first time.4.Determination of Flavonoid Aglycones in Herba OxytropisThe rapid and simple HPLC-DAD analytical method of five flavonoid aglycones including B₄,B₅,B₆,B₇ and B₁₀ in Herba Oxytropis were established.The samples were separated on an Agilent Zorbax Eclipse XDB-C₈ column（150 mm×4.6 mm,5μm） coupled with an Agilent Zorbax Eclipse XDB-C₈ guard column（12.5 mm×4.6 mm,5μm）. The mobile phase was composed of ACN（A） and water containing 0.1%FA water solution （B）,which were applied in a gradient elution of 0-50min,linear change from A-B（25:75, v/v） to A-B（50:50,v/v）;50-60 min,A-B（50:50,v/v） to A-B（60:40,v/v）;60-65 min,A-B （60:40,v/v） to A-B（25:75,v/v）.And the instrument was then eluted with the beginning gradient for 5 min.The mobile phase flow rate was 1.0 ml/min and monitored at 280 nm and 365 nm with the column temperature at 20℃.The injectin volume was 5μL.The raw data were collected and analyzed by HP Chemsation 10.02 software.The linear ranges of B₄,B₅,B₆,B₇ and B₁₀ were 0.101-0.707μg,0.029-0.203μg,0.332-2.324μg,0.134-0.938μg and 0.122-0.854μg,and r=0.9996,0.9998,0.9998,0.9996 and 0.9998,respectively.The accuracy of these five compounds was between 0.61%-1.64%,and the average recovery is 96.9%-101.2%.The method was successfully applied to quantify the five components in samples from three different areas（Qinghai,Tibet and Gansu）.The content of Compound B₄ is 0.16%,0.17%and 0.09%;B₅ is 0.04%,0.05%and 0.02%;B₆ is 0.64%,0.73%and 0.45%;B₇ is 0.32%,0.48%and 0.30%;B₁₀ is 0.20%,0.16%and 0.10%.This simple and reliable method provided a new basis for overall assessment on quality of Herba Oxytropis and should be considered as a suitable quality control method.