Studies On The Water-soluble Chemical Constituents And Quality Control Of Anoectochilus Roxburghii(Wall.) Lindl

Objectives:This subject extracts,separates,purifies and identifies the water-soluble chemical constituents from the N-butanol part and water part of Anoectochilus formosanus,and establishes the determination method of the anoectochilus glucoside from the Anoectochilus formosanus by using the anoectochilus glucoside as a quantitative indicator.Meanwhile,the metabolism of the plant growth regulators added to the Anoectochilus formosanus is also determined for better quality control of Anoectochilus formosanus.It lays the foundation for the revision and completeness the standards of the Anoectochilus formosanus in the Chinese Herbal Medicine Standards in Fujian Province.Methods:1.Accelerated Solvent Extraction(ASE)method was employed for the extraction from the whole herb of the Anoectochilus formosanus,and different chromatographic columns(normal silica gel,macroporous adsorption resin,ODS,Sephadex LH-20)were selected,according to the polarities and phases of different solvents,to separate,purify and structurally identify the N-butanol extraction part and the water extraction part.2.High Performance Liquid Chromatography-Evaporative Light Scattering Detector(HPLC-ELSD)was used to determine the content of the anoectochilus glucoside from the Anoectochilus formosanus in different habitats,different cultivation methods,different harvest time and the wild Anoectochilus formosanus.Meanwhile,the content of the anoectochilus glucoside was also qualitatively analyzed by Ultra Performance Liquid Chromatography －Tandem Mass Spectrometry,and compared with the results of HPLC-ELSD method.3.A high performance Liquid Chromatography-Tandem Mass Spectrometry(LC-MS/MS)method was established for simultaneous determination of the residues of the 5 kinds of plant growth regulators,including Paclobutrazol(PP333),6-Benzyl Amino Purine(6-BA),2,4-Two Chloro Phenoxy Acetic Acid(2,4-D),a-Naphthoacetic Acid(NAA),and Indolobutyric Acid(IB A).And the residual quantities of the 5 kinds of plant growth regulators in different habitats,different cultivation methods and different harvest time were measured.Results:1.4 compounds were separated from the N-butanol and water extraction parts of the Anoectochilus formosanus.The structure was analyzed as:Glc-a(1→2)b-Fru-a(1→1)-Gal(C18H32016),9-β-D-Furribonyl Adenine(C10H13N5O4),Kinsenoside(C10H16OO8),Rutinose(C12H22O10).It is noted that the compound,Rutinose,was separated for the first time from Anoectochilus formosanus.2.A HPLC-ELSD method was established for the determination of the content of anoectochilus glucoside in different habitats,different cultivation methods,different harvest time and the samples of wild Anoectochilus formosanus.The results showed different contents of anoectochilus glucoside in different habitat samples:the content of anoectochilus glucoside from the tissue culture seedlings species was relatively low;the content of anoectochilus glucoside from the under forest species after six months and one year cultivation was relatively high;and the content of anoectochilus glucoside from the wild species was not higher than that from the under forest species.3.A method for the simultaneous determination of the residues of the 5 kinds of plant growth regulators(Paclobutrazol(PP333),6-Benzyl Amino Purine(6-BA),2,4-Two Chloro Phenoxy Acetic Acid(2,4-D),a-Naphthoacetic Acid(NAA),and Indolobutyric Acid(IBA))by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry was established.The residual quantities of plant growth regulators in different growth stages were measured.The results showed that there was a risk of residue in the tissue culture seedlings,however.there was no plant growth regulator residue left after transplanting to under forest for more than 1 year.Conclusions:1.Four compounds were separated from the n-butanol and water extract parts of Anoectochilus formosanus,and one of them was separated for the first time from Anoectochilus formosanus.2.The HPLC-ELSD method was established for the determination of the content of Anoectochilus glycosides in Anoectochilus formosanus.By comparison,the method is accurate,convenient and reliable and can be used for the quality control of Anoectochilus formosanus.3.The LC-MS/MS method was established for the determination of five plant growth regulator residues in Anoectochilus formosanus.The method is simple and sensitive,and can be used as a control method for plant growth regulators in Anoectochilus formosanus.