| Qiong-Yu-Gao(QYG),derived from Rehmanniae Radix(RR),Poriae(PO)and Ginseng Radix(GR),is a famous traditional Chinese medicine formulae that firstly was documented in song dynasty "Hong-Shi-Ji-Yan-Fang".QYG has the effects of nourishing yin to moisturize the lung and replenishing qi to invigorate the spleen,and was traditionally applied to treat tuberculosis caused by lung and kidney yin deficiency syndrome and spleen qi deficiency syndrome.It is now used for anti-aging,regulating the spleen and stomach and the treatment of chronic wasting diseases,such as cancer,lung tuberculosis,Alzheimer’s disease and so on based on a series of clinical researches.Although QYG has obvious curative effects,it is lack of overall systematic investigation.In this study,optimization of preparation,quality control methods and gut microbiota activity were systematically investigated so as to provide scientific data for inheritation and modernigation of QYG.1.The literature reviewThe research progress of QYG,including preparation,bioactive components,pharmacological activities and clinical application were summarized.The shortcomings of the current research and the prospections on QYG exploration were discussed.2.Quality inspection of raw materialsThe quality of the commercial materials that were used for this study were inspected according to the monographs conditions of these herbs documented in the"Chinese Pharmacopoeia".3.The scientific connotation of traditional long-time extraction and concentration procedurePrevious study revealed that iridoid glycosides,phenethylalcohol glycosides,furfural derivatives,ginsenosides and triterpenoid acids were the major components of QYG However,the traditional extraction time was up to 7 h,so,study on extraction and concentration time was implemented to explore the influence of extraction and concentration with long duration on the quality consistency of QYG The contents of 5-HMF,catalpol,melittoside,acetoside,ginsenoside Re,ginsenoside Rbi,ginsenoside 20(S)-Rg3,ginsenoside Rgi,ginsenoside Ro and pachymic acid and the standard deviation(SD)accumulation values thereof in repeatedly prepared samples were used as the evaluation index,and HPLC-TQ-MS was used to determine the contents.It was found that the total contents of 10 bioactive components had peaks when extracting 1 h and 5 h respectively.The SD accumulation values decreased with extending of the extraction duration and decreased first and then increased with extending of the concentration duration,indicating that long-time extraction can improve the quality consistency of QYG.4.Optimization of preparation conditionsa)The extraction procedureThree factors three levels orthogonal experimental design was used to optimize the main factors,such as ratio of solvent,times and time of sample extraction that influence water extraction procedure,with the contents of 10 bioactive components and polysaccharides and the yield of extract as the investigating index.The optimized long-time extraction procedure was as follows:the herbs were extracted 3 times,adding 8 volumes of water and 5 h for each time.The optimized short-time extraction procedure was as follows:the herbs were extracted 3 times,adding 10 volumes of water and 1 h for each time.Comparing total contents of the main active components and feasibility in two extraction procedures,the final optimal procedure for QYG was that the herbs were extracted 3 times,adding 10 volumes of water and 1h for each time.b)The concentration procedureEffects of different temperature on marker components in the decompression condition were investigated.Optimal concentration procedure was that the water extract was vacuum concentrated to relative density of 1.20-1.30 under the temperature of 70-80℃.5.The overall characterization of carbohydratesFirstly,QYG was divided into alcohol supernatant and polysaccharides by alcohol precipitation.Then,the free monosaccharides and oligosaccharides in the alcohol supernatant were determined by direct HPLC-ELSD and pre-column derivatization HPLC-UV method.The free monosaccharides included Xly,Fru,Man,Glc and Gal,with the average contents of 0.54%,3.93%,0.06%,0.92%and 2.13%respectively.The free oligosaccharides included sucrose,maltose,melibiose,raffinose,mannotriose and stachyose,with the average contents of 4.68%,0.32%,0.68%,4.28%,2.66%,25.25%respectively.A high performance gel permeation chromatography(HPGPC)coupled with HPLC-UV method was developed to characterize the molecular weight distribution and monosaccharide composition of polysaccharides of QYG.The molecular weight range was from 3.5×105 to 2.1×106 Da,and monosaccharide composition is mainly consisted of six monosaccharides,including Man,GalA,Glc,Gal,Ara and Fuc,with the mole ratios of 2:5:66:43:6:15.6.Practical quality control method of QYGIn order to practically control the quality of QYG product,the quantitative and qualitative methods were established.Firstly,qualitative inspection of RR and GR in QYG were established by TLC.Secondly,quantification of acetoside and ginsenoside Rb1 were developed by HPLC.And the content of total polysaccharides was determined by phenol-sulfuric acid method.7.Improvement of the structure of gut microbiotaSixty male mice were randomly divided into five groups:blank group,fructo-oligosaccharides(OF)group,low-dose QYG group,median-dose QYG group and high-dose QYG group.The drugs were administered intragastrically for 14 days.The selective culture method was established to analyze the intestinal microbial communities in mice feces.Significant increase of Bifidobacterium spp.and reduction of Enterobacter spp.,Enterococcus spp.and Clostridium spp.were observed in QYG treated groups,and to some extent,increase of Lactobacillus spp..Comparing to OF,the effects of QYG to improve the structure of gut microbiota were more obvious. |
PDF Full Text Request