Study On Purification And Neuroprotective Effect Of Alkaloids From Dendrobium

Alkaloid is one of the main active components in Dendrobium.Modern pharmacological studies have shown that alkaloids isolated from Dendrobium have activities of improving memory and cognitive impairment.Dendrobium huoshanense,as a genuine medicinal material in Anhui Province,is kown as a product of protection of geographical indications of Dendrobium in China.At present,the study of alkaloids from Dendrobium huoshanense mainly focuses on the detection of total alkaloids.It is of great necessary to study the alkaloid composition and neuroprotective effects of Dendrobium huoshanense for the development of its health care efficacy.Therefore,three kinds of alkaloids(DHALE,DHALSE-2,DHALSE-3)were obtained from Dendrobium huoshanense fresh stems by heating extraction,liquid-liquid extraction and solid phase extraction.The structure of alkaloids from Dendrobium huoshanense were analyzed by means of HPLC-DAD,GC-MS and LC-QTOF-MS/MS.Finally,the protective effect on oxidative damage of nerve cells of alkaloids from Dendrobium huoshanense was studied The main research results are as follows:(1)The optimum solvent for extracting total alkaloids from Dendrobium huoshanense is acid ethanol(70% EtOH,pH=3).When the ratio of lyophilized powder of Dendrobium huoshanense to acid ethanol was 1:25 and extracted in water bath at 80℃for 2 h each time,the total alkaloid content was 0.0371%(in terms of lyophilized powder),which is 41.06% higher than that of 95% ethanol extractor,and 508.19% higher than that of trichloromethane extractor.(2)The alkaloids in Dendrobium huoshanense extract can be effectively enriched and purified by liquid-liquid extraction method.The total alkaloids in obtained by three extraction solvents are similar.The results of HPLC-DAD analysis showed that the matrix interference peaks of Dendrobium alkaloid extracts in liquid chromatography at 0-7 minutes could be significantly reduced after liquid-liquid extraction.GC-MS analysis showed that liquid-liquid extraction could remove carbohydrates,ethers and alcohols from Dendrobium extract.HPLC-DAD and GC-MS analysis confirmed that the alkaloid composition of Dendrobium huoshanense extracted by three solvents(acidic ethanol,95% ethanol and trichloromethane)was similar.(3)Compared with that of Dendrobium officinale or Dendrobium nobile,the alkaloid composition of Dendrobium huoshanense is specific.The total alkaloid extracts from Dendrobium huoshanense(DHALE),Dendrobium.nobile(DNALE)and Dendrobium officinale(DOALE)were detected by HPLC-DAD.The chromatographic fingerprint similarity evaluation system of traditional Chinese medicine was used to analyze the HPLC images of DHALE,DNALE and DOALE.The results showed that Dendrobium officinale and Dendrobium nobile samples exhibited rather low similarity with the correlation coefficients 0.435 and 0.564,respectively,which indicated that the alkaloids composition of Dendrobium huoshanense had its own unique alkaloid composition system.(4)DHALSE-2 and DHALSE-3 were obtained from the extracts from Dendrobium huoshanense by and liquid-liquid exaction and solid-phase extraction.LC-QTOFMS/MS qualitative analysis showed that the extracts mainly contained six alkaloid compounds.DHALE was separated and purified by solid phase extraction column with PCX as adsorbent.The alkaloid could be completely eluted by using 1:1 methanol/acetonitrile solution containing 3% ammonia water as eluent.Alkaloids in DHALSE-3 were 6-hydroxynobiline,2-hydroxydendrobine,findlayine C,dendrobine,nobilonine and trans-dendrochrysanine,and 6-hydroxynobiline,dendrobine and trans-dendrochrysanine,in DHALSE-2 espectively.(5)DHALE,DHALSE-2 and DHALSE-3 all have protective effects on PC12 cells induced by hydrogen peroxide.The pre-protection effect of DHALSE-3 is better than that of DHALE and DHALSE-2.