Chemical Composition And Fingerprint Tsugeki Study

Purpose:1.The study is started by the orthogonal test method,optimization the extraction technology of total flavonoids from Cudrania tricuspidata,and give reference for development and utilizeetion of it.Then isolation and identification the chemical composition from the EtoAc part and butanol part,and screeing experiment of vitro-anticancer active of the monomeric compounds, to find the material basis for anticancer of Cudrania tricuspidata.For the development of new anti-cancer drug and provide a theoretical basis,at the same time for the establishment of HPLC fingerprint of Cudrania tricuspidata to provide index parameter.2.Through determining the five flavonoids effective constituents in Cudrania tricuspidata and used Systematic Cluster Analysis Method with a qualitative assessment of the different com-modities medicine.For identification of the quality control offer reference;Determining the inorganic elements of Cudrania tricuspidata by ICP-MS.To provide some theoretical references for further study the anticancer mechanism.3.To establish HPLC fingerprint of Cudrania tricuspidata,which was expected to be standards of quality control and identification of the drugs.Material and method:1.Material:Cudrania tricuspidata(Carr.) Bur is the dried root and rhizome of Moraceae plants,which is collected from Guangxi.They are provided and identify by the teacher come from Guangxi College of Traditional Chinese Medicine.The kinds of commercial drugs is collected from Guangxi and Anguo.2.Method:(1) The process was studied by orthogonal design L₉(3⁴) with the extraction rate of total flavonoids and paste-forming rate as comprehensive index;Through many kinds of chromatographic methods such as silica gel,and Sephadex LH-20,isolation and purification monomeric compound from the EtoAc part and butanol part.(2) By MTT method,make 2 monomeric compounds to evaluate their antitumor activity in vitro.(3) Determining the five species content in flavone ingredients through HPLC and using systematic cluster analysis method classify 10 batches different of commodity of medicinal materials.Determining the inorganic elements by ICP-MS.(4)The HPLC method was used,chromatography conditions were Aglinet C18(150×4.6mm,5μm) column with gradient mobile phase of acetonitril-0.1% phosphoric acid.UV detection wavelength was at 230nm and the column temperature was at 30℃with the flow rate of 1.0 mL·min^-1.By referening of Dihydromorin establish the fingerprint spectrum.Results:1.Studies on chemical constituents:The optimum extraction condition of the total flavonoids obtained was:the crude drug was extracted three times,the 60%alcohol density,12 times alcohol was added and 1.5 hours every time.Ten monomers have been isolated form the EtoAc part and butanol part.They are:Ⅰβ-sitosterol,ⅡDaucosterine,ⅢQuercetin,ⅣKaempferol,Ⅴ5,7,4′-trihydroxyldihydroisoflavone,Ⅵ(13α,14β,17α,20R) -Lanosta-7, 24-diene-3β-ol,ⅦTaxifolin,Ⅷ(13α,14β,17α,20R)-Lanosta-7,24-diene-3β-O-actate,ⅨDihydromor- in,ⅩCycloartocarpesin.TheⅢ,Ⅳ,Ⅴ,Ⅶ,Ⅸ,Ⅹare all the flavonoids.For effective substance of anti-cancer provide reference,and for evaluation of the quality of medicines,then establish HPLC fingerprint provide more parameters.2.Anticancer experiment of components shows that:Dihydromorin and Cycloartocarpesin are all have the effect in vitro cancer-restraining.The inhibition effects of cultured HeLa cells is Cycloartocarpesin＞5-Fu＞Dihydromorin.,the inhibition effects of SGC-7901 cells is 5-Fu＞Cycloartocarpesin＞Dihydromorin.3.Studies on content determination:(1) The contents of the five flavonoids effective constituents of the ten commercial drugs have larger difference.Then that doesn′s evaluation the quality of commercial drugs is good or bad,so using systematic cluster analysis method make the five flavonoids as comprehensive index;Research quality evaluation method for the 10 batches different of commodity of medicinal.The samples are divided into 4 classes,the best, the good,the general and the poor.(2) Determining the 36 inorganic elements by ICP-MS. The results showed that:It riches in elements which is good for people in Cudrania tricuspidata,and the contents of harmful metal elements in low.4.Studies on fingerprint:Apply HPLC to set up the finger print of Cudrania tricuspidata, dihydromorin as the standard preparation.12 peaks in the chromatogram were common,4 peaks have been calibrated.All the similarities are above 0.9.Conclusion:1.Studies on chemical constituents:(1) The process was studied by orthogonal design L₉(3⁴) with the extraction rate of total flavonoids and paste-forming rate as comprehensive index; (2) Ten monomers have been isolated form the EtoAc part and butanol part.The Cycloartocarpesin(Ⅹ) is obtained from Cudrania tricuspidata for the first time.And monomers 13α,14β,17α,20R)-Lanosta-7,24-diene-3β-ol(Ⅵ) and(13α,14β,17α,20R) -Lanosta-7,24-diene-3β-O-actate(Ⅷ) are obtained from the genius for the first time.2.Anticancer experiment of components shows that:Dihydromorin and Cycloartocarpesin are all have the effect in vitro cancer-restraining.Antitumor mechanism shows that:the 2 monomeric compounds are playing a role through induce apoptosis.3.Studies on content determination:(1) Determining the five species content in flavone ingredients through HPLC and using systematic cluster analysis method classify 10 batches different of commodity of medicinal materials.Form that can preliminary evaluation the the quality of commercial drugs and for identification of the quality control offer reference.(2) First determining the 36 inorganic elements by ICP-MS,it can provide some theoretical references for further study the anticancer mechanicsm,and for the use of the security, non-toxic and provide a reliable basis.4.Studies on fingerprint:First used the HPLC establish the fingerprint spectrum for Cudrania tricuspidata,and 4 peaks have been calibrated.The method is convenient,quick and exact, and its reproducibility is good,it will be a scientific reference for qualitation identification and quality evaluation of Cudrania tricuspidata.