The Isolation And Identification Of Chemical Constituents From The Culture Of Dendrobium Officinale

Dendrobium officinale Kimuraet Migo is a kind of traditional Chinese medicine which belongs to Orchidaceae Dendrobium,whose main medicinal part is fresh or dry stems.Dendrobium officinale have the function of nourishing kidney and clearing away heat-evil,benefiting the stomach and promoting the production of body fluid,moistening the lung and relieving cough,resisting cancer and prolonging life.The great demand has led to a severe shortage of resources and high price.However,the emergence of Dendrobium officinale protocorm can effectively alleviate the lack of Dendrobium officinale in the society.By studying the composition of Dendrobium officinale protocorm,we can get a better understanding of the differences between Dendrobium officinale protocorm and wild Dendrobium officinale,so as to make it better for use.In this paper,the main conclusions are as follows:1.The sinapic acid was separated and purified from Derdrobium officinale protocorm by High-Speed Countercurrent Chromatography,which was isolated for the first time from Dendrobium.Ether-ethyl acetate-methanol-water(4:6:3:7,v/v)was used as the two-phase solvent system.The flow rate ofstationary phase was set at 12.0 m L/min,and the flow rate ofmobile phasewas 2.0 m L/min.The sample size was 200 mg and the revolution speed was 850 rpm.The detection wavelength was 280 nm and the retention of stationary phase was 46.67 %.Finally,sinapic acid with a purity of 93.11 % was obtained in a one-step separation.The results showed that sinapic acid had a strong DPPH· radical scavenging ability in vitro and the IC50 is 0.046 mg/m L.2.The eluting fraction was successfully separated and purified by high-speed counter-current chromatography with a two-phase solvent system consisting of MTBE-methanol-acetonitrile-water(5:1:2:6,v/v)in one-step separation.The flow rate of stationary phase was set at 15.0 m L/min,and the flow rate of mobile phase was 1.0 m L/min.Thesample size was 150 mg and the revolution speed was 850 rpm.The detection wavelength was 280 nm and the retention of stationary phase was 41.67 %.At the same time,we can know that the compound I was ?-D-glucopyranose1-[(E)-3-(4-hydroxyphenyl)-2-propenoat] at 97.8 % purity,and thecompound II was ?-D-glucopyranose 1-[(E)-3-(3,4-dihydroxyphenyl)-2-propenoat] at 98.6 % purity.Thecompound III was 1-O-sinapoyl glucopyranoside at 98.4 % purity.The purity of the three compounds was determined by HPLC,and the structures of the three compounds were identified by ESI-MS,1H NMR and 13 C NMR.The results showed that the three compounds had a DPPH· radical scavenging ability in vitro.When the concentration was 0.01 mg/m L-0.2 mg/m L,the removal rate increased with the increase of the concentration.At the same time,the compound I had the more bad effect,and the compound II had the best effect.In addition,the IC50 is 0.6219 mg/m L,0.0497 mg/m L,0.1111 mg/m L,respectively.3.The extraction technology of polysaccharide from Dendrobium officinale protocorm was optimized with orthogonal test design in extraction temperature,time and solid/liguid ratio.When extraction temperature,time and solid/liguid ratio was 90 ?,60 min and 1:40(g/m L),the extraction efficiency of polysaccharide reached 78.51 % at best.The deproteinized methods were compared through Sevage,hydrochloricacid,trichloroacetic acid and papain.The experimental results showed thatthe effect of papain method to take off the protein was the best,the enzymolysis time of was 90 min,temperature was 70 ? and enzyme content was 30 U,the polysaccharide content was 85.01 % and the protein content was 5.89 %.In addition,the polysaccharide content decreased with increasing elution times by Sevage method.On the one hand,when the HCL concentration was 25 %,the polysaccharide content and protein content was 52.17 % and 4.82 % respectively.On the other hand,when the TCA concentration was 0.03 g/m L,polysaccharide content was 35.50 % and protein content was 7.34 %.4.The crude polysaccharide of Dendrobium officinale protocorm was deproteinized through the Sevage method,was purified by DEAE-52 cellulose column chromatography.We can get four components,they were called DOKM-A?DOKM-B?DOKM-C?DOKM-D,respectively.In addition,the highest content component DOKM-A was purified by Sephadex G-75 column chromatography for further separation and purification.At last,we can get two components,they were called DOKM-A-1,DOKM-A-2,respectively.And the two fractions were identified by HPGPC.The results showed that DOKM-A-1 was a more pure component than DOKM-A-2.Itsmolecular weight was 12 KDa by calculation.The monosaccharide composition is mannose,rhamnose,galacturonic acid,glucose,galactose,xylose,arabinose,and the monosaccharide molar ratio is 1: 1.821: 0.367: 1.269: 0.355: 0.323: 1.024.At the same time,the experiment result shows they all had a DPPH· radical scavenging ability in vitro,and DOKM-A>DOKM-D>DOKM-B>DOKM-C.The DOKM-A-1 was badder than DOKM-A in the scavenging effect of DPPH· radical.