Study Metabolism Of Protopanaxatriol By Isolated Intestinal Flora

Objective:Based on the novel coronavirus pneumonia,it has reported that in treatment of novel coronary pneumonia（COVID-19）patients combined with cardiovascular disease,"Yiqi Fumai Lyophilized Injection"as the therapeutic drugs whose mainly medicine is red ginseng,were found in anti-inflammation,immunity enhancement,and fibrosis symptoms improvement,has the ability to treat pulmonary and chronic obstructive pulmonary disease.After fresh ginseng is processed into red ginseng,the small secondary ginsenosides,which generated by hydrolyzing glycosyl of ginsenoside,shows stronger pharmacological activity in anti-tumor and anti-inflammation.Therefore,in this study,the protopanaxatriol were isolated and purified from red ginseng.The pathway and metabolism of protopanaxatriol ginsenosides in human intestinal flora and the interaction between intestinal flora and protopanaxatriol after oral ginseng were evaluated.The results would provide scientific supports for the clinic application in ginseng.Methods:1.Extraction and separation of protopanaxatriol:The method of extraction of ginsenosides was followed the protocols of“Pharmacopoeia of the People’s Republic of China”（Version 2015）.The separation and purification of protopanaxatriol were performed to D101C macroporous resin coloum.Three factors,sample concentration,adsorption time,and ethanol concentration,influenced by extraction were optimized.2.The incubation of human intestinal flora with protopanaxatriol treatment in vitro:Fresh feces from volunteers with good health condition were collected,and incubated in an anaerobic workstation to obtain the bacterial solution of human intestinal flora.The protopanaxatriol standard and extracted protopanaxatriol were incubated with the bacterial solution of human intestinal flora,respectively.The samples were harvested and measured at 0,1,2,4,6,8,12,24,48,and 60 h.3.Identification of samples:The rapid resolution liquid chromatography quadrupole-time-of-flight mass spectrometry（RRLC-Q-TOF MS）was used to analyze the samples,which was metabolized by in intestinal flora.The Electrospray ionization（ESI）and negative ionization scanning mode was performed in RRLC-Q-TOF MS.4.Measurement of conversion rate:Ultra high performance liquid chromatography-mass spectrometry（UPLC-QQQ MS）was used for quantitation of samples.5.High-throughput 16S rDNA genes sequencing:DNA was extracted by commercial kit,and the purified samples were conducted with high-throughput sequencing using Illumina Hiseq 2500 platform.The productions of intestinal microorganisms at the level of phylum and genus were analyzed to investigate the effect of protopanaxatriol on intestinal microorganisms.Results:1.The extraction and separation of protopanaxatriol:Ginsenosides were extracted from red ginseng by the previous reports.The protopanaxatriol,isolated from red ginseng,mainly contains ginsenoside Re and Rg₁.2.The incubation of human intestinal flora with protopanaxatriol treatment in vitro:The protopanaxatriol standards（Re,Rg₁,Rg₂,Rh₁,F₁,Rf）and the extracted protopanaxatriol compounds were incubated in human intestinal flora,respectively.Ginsenoside Re has five kinds of transformation products:ginsenoside Rg₁,Rg₂,Rh₁,F₁ and PPT,with a total conversion rate of 91%.Ginsenoside Rg₁ has three kinds of transformation products:ginsenoside Rh₁,F₁ and PPT,with a total conversion rate of 80%.Ginsenoside Rg₂ has two kinds of transformation products:ginsenoside Rh₁ and PPT,with a total conversion rate of 73%.Ginsenoside Rh₁ has one kind of transformation product which is ginsenoside PPT,with a total conversion rate of 82%.Ginsenoside F₁ has one kind of transformation product which is ginsenoside PPT,with a total conversion rate of 81%.Additionally,ginsenoside Rf has two kinds of transformation products:ginsenoside Rh₁ and PPT,with a total conversion rate of 89%.The ginsenoside Re and Rg₁ in the protopanaxatriol group,after incubated in the human intestinal flora,has transformed products which are Rg₂,Rh₁,F₁ and PPT.At the period of 60 h incubation,the production rates of products Rg₂,Rh₁,F₁ and PPT were about 6%,12%,11%and 56%.3.Analysis of effect of protopanaxatriol in human intestinal flora:Ginsenoside Re,Rh₁,F₁ and protopanaxatriol group were incubated in human intestinal flora in vitro,respectively.According to the high-throughput 16S rDNA gene sequencing analysis technology,Firmicutes,Bacteroidetes,Proteobacteria and Actinobacteria were found at the species structure of the phylum level in intestinal flora.Compared with the control group,protopanaxatriol may change the relative abundance of intestinal flora,in which Firmicutes increased significantly（p<0.01）and Bacteroidetes decreased significantly（p<0.01）.The observation of in the genus level,the relative abundances of Escherichia-Shigella,Dorea,and Prevotella₉ were increased,while that of Sutterella was decreased in the extracted protopanaxatriol.At the main time,the relative abundance of Faecalibacterium and Dialister were significantly increased,while that of Lachnoclostridium was decreased.Conclusion:In this study,the protopanaxatriol,isolated from red ginseng,mainly contains ginsenoside Re and Rg₁.The protopanaxatriol（Re,Rg₁,Rg₂,Rh₁,F₁,Rf）and the extracted protopanaxatriol compounds were incubated in human intestinal flora,the protopanaxatriol ginsenosides were transformed or converted in the intestinal flora mainly by losing the C-6 and C-20 glucose.At the incubation time of 60 h,ginsenoside Re has five transformation products,and the transformation pathway was Re→Rg₁/Rg₂→Rh₁/F₁→PPT,conversion rate was 91%;Ginsenoside Rg₁ has three transformation products,and the transformation pathway was Rg₁→Rh₁/F₁→PPT,conversion rate was 80%;Ginsenoside Rg₂ has two transformation products,pathway was Rg₂→Rh₁→PPT,conversion rate was73%;Ginsenoside Rh₁ has one transformation product,and pathway was Rh₁→PPT,conversion rate was 82%;Ginsenoside F₁ has one transformation product,and pathway was F₁→PPT,conversion rate was 81%;Ginsenoside Rf has two transformation products,and pathway was Rf→Rh₁→PPT,conversion rate was 89%.The ginsenoside Re and Rg₁ in the protopanaxatriol group,the transformation pathway was same as protopanaxatriol standards,at the period of 60 h incubation,the production rates of products Rg₂,Rh₁,F₁ and PPT were about 6%,12%,11%and 56%.Ginsenoside Re,Rh₁,F₁ and protopanaxatriol group were incubated in human intestinal flora,respectively.According to the high-throughput 16S rDNA gene sequencing analysis technology,it was found that the protopanaxatriol acting on intestinal flora could regulate the phylum level and genes level of abundance of intestinal bacteria and microorganisms,from which it can affect the related metabolic pathways by regulating the abundance of microorganisms,and may play the role of anti-inflammatory and improving human immunity.