Study On The Chemical Composition And Intestinal Absorption Characteristics Of 35% Ethanol Extract Of Spider Fragranc

Purpose:With the fierce social competition and increasing pressure on life,anxiety disorders has become common and frequently-occurring disease in modern diseases,which has serious impacts on people’s quality of life.Valeriana jatamansi Jones is a traditional Chinese herb widely used in Yunnan,Guizhou and Sichuan provinces of China,of which the 35%ethanol extract has a definite anti anxiety activity.In order to take a comprehensive understanding of its material basis of the clinical efficacy,and to further develop and use of medicinal resources of Valeriana jatamansi Jones,the 35%ethanol extract of Valeriana jatamansi Jones was taken as the research object.We established its HPLC specific chromatogram analysis method and the chemical composition was characterized.And related ingredients’ characteristics and preliminary absorption mechanism in vitro and in vivo intestinal absorption of rats were conducted in this study,which makes a preliminary exploration for the establish of a suitable Chinese medicinal material new method using the correlation of intestinal absorption spectrum and the effect for convenient and rapid optimizing the effective components and provides a new idea for the evaluation of the overall quality of Chinese medicine.Methods:1 Component Analysis1)Study of HPLC specific chromatogram15 batches of Valeriana jatamansi Jones from different countries across the country were collected.The analysis of the HPLC chromatogram of the 35%ethanol extract of Valeriana jatamansi Jones was performed using an Agilent Z0RBAX SB-C18(4.6×250 mm,5 μm)column,at a wavelength of 285 nm,with a flow rate of 1.0 mL/min,eluted with a mobile phase consisted of acetonitrile-0.1%the aqueous formic acid solution.This method was combined with the traditional Chinese medicine fingerprints similarity evaluation system to identify the spectrum from various regions as a whole.2)Quantitative analysis of 7 componentsA new HPLC method was established to simultaneously quantify neochlorogenic acid,chlorogenic acid,4-O-caffeoylquinic acid,3,4-O-dicaffeoylquinic acid,3,5-O-dicaffeoyl-quinic acid,hesperidin,4,5-O-dicaffeoylquinic acid in 15 batches 35%ethanol extracts of Valeriana jatamansi Jones from different regions.3)Study of compositional characterizationBased on the HPLC-LTQ-Orbitrap-MS technology,the chemical components of the 35%ethanol extract of Valeriana jatamansi Jones were characterized.2 Study of intestinal absorption1)Study of intestinal absorption with method of rat everted gut sacEstablished a method for the study of intestinal absorption of 35%ethanol extracts of Valeriana jatamansi Jones using the rat everted gut sac.After incubated in the 35%ethanol extracts with different concentrations of Valeriana jatamansi Jones,the content of neochlorogenic acid,chlorogenic acid,4-O-caffeoylquinic acid,3,4-O-dicaffeoylquinic acid,3,5-O-dicaffeoylquinic acid,hesperidin,4,5-O-dicaffeoylquinic acid were analyzed by means of HPLC methods established.The cumulative intestinal absorption(Q)of the seven components in the duodenum,jejunum,and ileum was calculated.2)Study of intestinal absorption with method of single-pass intestine perfusionEstablished a method for the intestinal absorption of 35%ethanol extracts of Valeriana jatamansi Jones using rat in situ single-pass intestine perfusion model.After perfusion with 35%ethanol extracts with different concentrations,the subsequent changes of the composition of neochlorogenic acid,chlorogenic acid,4-O-caffeoylquinic acid,3,4-O-di-caffeoylquinic acid,3,5-O-dicaffeoylquinic acid,hesperidin,4,5-O-dicaffeoylquinic acid were analyzed by means of HPLC methods established.The absorption rate constant(Ka)and apparent permeation coefficient(Papp)of the seven investigation components in the whole bowel segment of rats were calculated.In addition,the addition of Verapamil(Ver)the inhibitor of P-glycoprotein(P-gp)to the 35%ethanol extract was investigated.The subsequent changes of the composition of neochlorogenic acid,chlorogenic acid,4-O-caffeoylquinic acid,3,4-O-dicaffeoylquinic acid,3,5-O-dicaffeoylquinic acid,hesperidin,4,5-O-dicaffeoylquinic acid contained in the intestinal perfusion were observed.The absorption rate constant(Ka)and apparent permeation coefficient(Papp)of the seven investigation components in the whole bowel segment of rats were also calculated.Result1 Component Analysis1)Study of HPLC specific chromatogramA HPLC chromatogram method of the 35%ethanol extract of Valeriana jatamansi Jones was established.Using chlorogenic acid as the reference peak,10 common characteristic peaks were identified.Seven chromatographic peaks in the specific chromatogram were identified by comparison with reference substances,which identified as neochlorogenic acid,chlorogenic acid,4-O-caffeoylquinic acid,3,4-O-dicaffeoylquinic acid,3,5-O-dicaffeoyl-quinic acid,hesperidin,4,5-O-dicaffeoylquinic respectively.Similarity analysis was performed on the HPLC profiles of 15 batches of 35%ethanol extracts.The results showed that the HPLC fingerprints of 15 batches of 35%ethanol extracts of Valeriana jatamansi from different origins were between 0.900 and 0.984.The main chromatographic peaks,the overall peak appearance is basically same,and the chemical composition content is somewhat different.2)Quantitative analysis of 7 componentsThe linear ranges of neochlorogenic acid,chlorogenic acid,4-O-caffeoylquinic acid,3,4-O-dicaffeoylquinic acid,3,5-O-dicaffeoylquinic acid,hesperidin,4,5-O-dicaffeoylquinic were 24～121 μg/mL(R2=0.9997),62～835 μg/mL(R2=0.999),39～197 μg/ml(R2=0.9991),37～18μg/mL(R2=0.9994),and 13～66 μg/mL(R2=0.999)27～138 μg/mL(R2=0.9998)and 98～490 μg/mL(R2=0.9991)respectively.The results showed that there were differences in the content of each component of Valeriana jatamansi Jones from different origins.The contents of neochlorogenic acid,chlorogenic acid,4-O-caffeoylquinic acid,3,4-O-dicaffeoylquinic acid,3,5-O-dicaffeoylquinic acid,hesperidin,4,5-O-dicaffeoylquinic in the 15 batches of Valeriana jatamansi Jones materials were 0.100～0.396 mg/g,1.942～6.273 mg/g,0.196～0.839 mg/g,0.042～0.483 mg/g,0.024～0.895 mg/g,0.209～1.245 mg/g,0.016～1.361 mg/g respective-ly.The quantitative analysis method is a simple,fast,accurate method of multi-component content.3)Study of compositional characterizationBased on the HPLC-LTQ-Orbitrap-MS technology,the chemical components of the 35%ethanol extract of Valeriana jatamansi Jones were characterized.A total of 50 components were identified,mainly including flavonoids and phenylpropanoic acids.2 Study of intestinal absorption1)Study of intestinal absorption with method of rat everted gut sacCompared with the HPLC chromatograms of the herbs,11 components chromatographic peaks detected in the intestine samples of in vitro rat everted gut sac which could pass through the intestinal sac and 7 were identified,namely neochlorogenic acid,chlorogenic acid,4-O-caffeoylquinic acid,3,4-O-dicaffeoylquinic acid,3,5-O-dicaffeoylquinic acid,hesperidin,4,5-O-dicaffeoylquinic.And these seven components were studied for intestinal absorption characteristics.The results showed that the 7 components except for 3,5-O-dicaffeoylquinic acid in the duodenum,jejunum absorption and hesperidin in the bowel absorption,the cumulative absorption of other components in each intestinal segment increased with the liquid concentration and time increased without high concentration saturation.But cumulative absorption of 3,5-O-dicaffeoylquinic acid in the duodenum and jejunum and cumulative absorption of hesperidin in various intestinal segments had a downward trend with the increasing concentration of drug solution,there is a clear high-level inhibition.There were differences in the cumulative absorption of the components in the examined intestinal segments.2)Study of intestinal absorption with method of single-pass intestine perfusionThe intestinal absorption parameters were calculated for the seven investigated components of the in situ single-pass intestine perfusion samples.The results showed that in addition to 3,5-O-dicaffeoylquinic acid and hesperidin among seven components identified the Ka values and Papp values of other components were not significantly different with different concentration of the drug solution,so there was no high concentration inhibition phenomenon.However,the absorption parameter Ka and Papp of 3,5-O-dicaffeoylquinic acid and hesperidin had a downward trend with increasing concentrations indicating that their absorption is inhibited by the concentration.The Papp of seven components were all between 0.18×10-3 cm·min-1 and 1.2×10-3 cm·min-1.It was speculated that the absorption was medium in the entire intestine.However the Papp value of the medium and high concentrations of hesperidin was only 0.2 cm·min-1,so the absorption of hesperidin was poor.3)reliminary study on intestinal absorption mechanismP-gp inhibition experiment showed that the Ka value and Papp of seven components increased after adding Ver in the extract with statistically significant difference.ConclusionBased on the anxiolytic activity,the HPLC specific chromatogram of the 35%ethanol extract of Valeriana jatamansi Jones and multi-component quantitative analysis were constructed.The overall chromatographic peaks of the main chromatogram peaks in the specific chromatogram are basically same,the established method is stable and reliable by which the whole characteristics of the sample can be fully reflected.The simultaneous determination of 7 active ingredients such as hesperidin and chlorogenic acid showed differences in the content of components of Valeriana jatamansi Jones from different habitats.Simultaneously,the chemical composition of 35%ethanol extracts was systematically characterized by HPLC-LTQ-Orbitrap-MS technology,which enriched its chemical information.The experimental results of the rat everted gut sac and in situ single-pass intestine perfusion experiments of 35%ethanol extracts showed that 7 investigational components could be absorbed into the intestine,suggesting that these 7 components may be the effective ingredient of Valeriana jatamansi Jones 35%ethanol extracts.The neochlorogenic acid,chlorogenic acid,4-O-caffeoylquinic acid,3,4-O-dicaffeoylquinic acid,4,5-O-dicaffeoyl-quinic may be absorbed by free-diffusion method.However there may exist active diffusion in addition to free diffusion in the absorption of 3,5-O-dicaffeoylquinic acid and hesperidin.The ileal and jejunal absorption of each component always be better.Under the experimental conditions,the 7 components are absorbed moderately in the entire intestinal segment.However,the medium and high concentration group of hesperidin are absorbed poorly.All of the 7 components were P-gp substrates,which were subjected to P-gp efflux pump action during the absorption process.Thus Ver was able to increase its absorption in the small intestine by inhibiting P-gp and the possible absorption mechanism of each component was preliminarily explored.In this study,focusing on the treatment needs of anxiety disorders,the chemical composition and intestinal absorption characteristics and possible mechanisms of 35%ethanol extracts of Valeriana jatamansi Jones were systematically analyzed.This study provides a scientific basis for material basis of anti-anxiety efficacy and the evaluation of anti-anxiety activity of medicinal materials from different origins.What’s more,this paper makes a preliminary exploration for a new method using the intestinal absorption spectrum-effect correlation method to select and optimize effective components quickly and rapidly which is suitable for the characteristics of Chinese medicinal materials in the later stage of the project and will provide a new idea for the quality evaluation of traditional Chinese medicine.