Contents Of Some Micromolecular Primary Metabolites In Several Traditional Chinese Medicine Extracts And Their Enhancive Effects On Intestinal Absorption

Aim Loss of activity often occurs after purification of traditional Chinese medicine(TCM)extracts.It may be related to the loss of synergistic effect of each constituent at pharmaceutical,pharmacokinetic or pharmacodynamic levels after purification.The solubility of the drug significantly affects its intestinal absorption.It is worth noting that the solubility of some TCM constituents in the extract is significantly higher than that in the monomer form,which may be related to the synergistic effect between the constituents in the extract.There are micromolecular primary metabolites in TCM extract,such as organic acids,sugars,amino acids,urea and choline.Natural deep eutectic solvent(NADES)with significant solubilizing effects can be obtained by artificially mixing certain micromolecular primary metabolites in a certain proportion.However,the contents of each primary metabolite and their proportions in TCM extracts are not clear.In addition,it is not clear whether the formation of a solubilizing NADES based on this proportion is possible.Therefore,this paper intends to establish quantitative analysis methods for a variety of micromolecular primary metabolites.Then the actual content and proportion of each primary metabolite in various TCM extracts will be determined.Then whether NADES can be obtained according to the composition and proportion will be determined.After that,the effects of the prepared NADES on the solubility and intestinal absorption of some TCM constituents will be studied.The mechanism by which TCM constituents are significantly more soluble in extracts than their monomer forms will finally be proposed.Methods LC-MS/MS methods for the determination of some plant produced primary metabolites including organic acids(malic acid,chlorogenic acid,neochlorogenic acid,cryptochlorogenic acid,caffeic acid,ferulic acid),sugars(xylitol,glucose,fructose,sucrose),amino acids(proline,alanine,glycine,proline,histidine),urea,and choline were established.Then methodological validation was performed based on indexes such as quantitative lower limit linear range,precision and accuracy,recovery,stability and repeatability.Based on the internal standard method,the established LC-MS/MS methods were used to determine the contents of the above primary metabolites in the extracts of several TCM(Coptidis Rhizoma,Puerariae Lobatae Radix,Scutellariae Radix,Ginseng Radix et Rhizoma,Ginkgo Folium,Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle,Salviae Miltiorrhizae Radix et Rhizoma)extracts.Heating based method was used to determine whether NADES could be formed by the main primary metabolites in Coptidis Rhizoma extract according to their proportions.The effect of the prepared NADES on the in vitro intestinal absorption of berberine was studied.After that,the effect of the prepared NADES on the intestinal absorption and in vivo exposure of berberine was studied based on a pharmacokinetic experiment in mice.Results The established LC-MS/MS methods were fast,sensitive,and accurate.It could be used to quantitatively determine the contents of primary metabolites such as sugars,organic acids,amino acids,choline and urea in TCM extracts.By using the LC-MS /MS method,it was found that sucrose,malate and choline were the main primary metabolites in the tested TCM extract.According to the proportion of primary metabolites in Coptidis Rhizoma extract,a NADES was successfully prepared and the solubility of berberine could be increased to 20 mg/m L by the prepared NADES.The solubility of berberine in the diluted NADES(50%,25%,12.5%,6.25%,3.125%)decreased significantly with the increase of the dilution ratio,but still significantly higher than that of pure water.The NADES had significant concentration-dependent promoting effect on the in vitro intestinal absorption of berberine.The results of the pharmacokinetic experiment also showed that the NADES significantly improved the exposure levels of berberine in the portal vein blood and liver in mice.Conclusion Three fast,sensitive,and accurate LC-MS/MS quantitative analysis methods for some micromolecular primary metabolites in several TCM extracts were established.Sucrose,malate and choline were the major primary metabolites in the tested TCM extracts.A NADES was successfully prepared according to the composition and proportion of the primary metabolites in Coptidis Rhizoma extract.The prepared NADES significantly promoted the intestinal absorption and in vivo exposure level of berberine.