The Effective Substances Study And Clinical Observation Of Jinhong Decoction In The Treatment Of Sepsis

Objective:Based on TCM characteristic theory“six hollow organs must keep its unobstructed”,Jinhong Decoction is developed by professor Gu Bo-hua,who is the successor of Gu’s surgery department in Longhua Hospital Shanghai University of TCM.The formula has the function of clearing heat and detoxifying,catharsis,activating blood,and removing wind and relieving pain.This study is put forward to achieve integral study on the medicinal material base and the clinical effect of Jinhong Decoction（JHD）in treatment of sepsis,and to provide references for clinical rational use of JHD.Methods:（1）Pharmacodynamics of JHD in the treatment of sepsis:All mice were administrated JHD orally for 7 consecutive days before operation.Cecal ligation and puncture（CLP）was performed at the 8^th day.Samples were harvested after 24 h.The plasma levels of blood urea nitrogen（BUN）,creatinine（CRE）,alanine transaminase（ALT）and aspartate aminotransferase（AST）and pathological changes were detected to assess the liver and kidney damage.Tumor necrosis factor-α（TNF-α）and Interleukin-6（IL-6）were detected by enzyme-linked immunsosrbent assay.TNF-α,IL-1β,IL-6 and interferon-γ（INF-γ）m RNA was determined by real-time PCR.The expression levels of inducible nitric oxide synthase（i NOS）,cyclooxygenase-2（COX-2）,TNF-αand Nuclear factorkappa B（NF-κB）p50 proteins were detected by Western bloting.（2）Plasma pharmacokinetics of JHD in septic rats after oral administration:CLP was performed to establish rat model of sepsis.After 24 h,sham and CLP rats were administrated JHD orally.The plasma concentrations of emodin-8-glucoside,rhein,aloe emodin,emodin,luteolin,rhein-8-glucosidem,physcion-8-O-β-D-monoglucoside,aloe-e Modin-8-O-β-D-glucopyranoside,4-hydroxycinnamic acid,ferulic acid,protocatechuic acid,epicatechin and salidroside were quantified by a sensitive liquid chromatography-tandem mass spectrometric（LC-MS/MS）method at 2,5,10,20,30,60,90,120,180,240,360 and 480 min after oral administration.（3）Chemical constituents of Sargentodoxa cuneata,as one of core prescription drugs of JHD:The chemical constituents of of Sargentodoxa cuneata was isolated by silica gel,open ODS column chromatography,Sephadex LH-20,pre-HPLC chromatography,etc.The structures of these compounds were determined by 1D and 2D NMR,HRMS,and quantum chemistry calculations.（4）The clinical effect of JHD in treatment of COVID-19 complicated with sepsis:77patients with COVID-19 complicated with sepsis were selected.The random number table was used to assigned all patients into two groups at a ratio of 1:1.The treatment group will be given standard western medicine therapy and JHD（oral,two sachets per day）for 14 days,while the control group only receives standard western medicine therapy for 14 days.The primary outcome is clinical improvement up after treatment.The mortality,SOFA score,APACHEⅡscore,lymphocyte count and D dimer were evaluated.Results:（1）Pharmacodynamics of JHD in the treatment of sepsis:JHD alleviated CLP-induced increase in serum levels of BUN,CRE,ALT,AST,TNF-αand IL-6,reduced the m RNA levels of TNF-α,IL-1β,IL-6 and INF-γin the lung of septic mice.Furthermore,JHD inhibited the expression of i NOS,COX-2,TNF-αand p50 in the lung of septic mice.（2）Plasma pharmacokinetics of JHD in septic rats after oral administration:Eleven constituents（emodin-8-glucoside,rhein,aloe emodin,emodin,luteolin,rhein-8-glucosidem,physcion-8-O-beta-D-monoglucoside,aloe-e Modin-8-O-β-D-glucopyranoside,protocatechuic acid,epicatechin and salidroside）in septic rats had higher C_max and AUC than those in normal rats,and 4-hydroxycinnamic acid and ferulic acid had lower C_max and AUC than those in normal rats.（3）Chemical constituents of Sargentodoxa cuneata,as one of core prescription drugs of JHD:Fifty-two compounds were isolated from S.cuneata,including two new compounds（1-2）.Compound 20,24,26,28,35,39,41,45,47 and 50 are isolated from the genus Sargentodoxa for the first time.They were identified as sargentodoxoside A（1）,sargentodoxoside B（2）,4-O-β-D-glucopyranosylvanillic acid（3）,p-hydroxy-phenethyl-O-β-D-glucfopyranoside（4）,（-）-catechin（5）,2-（3,4-dihydroxyphenyl）-ethyl（6-O-caffeoyl）-β-D-glucopyranoside（6）,3’,4’-dihydroxyphenethylalcohol1-O-β-D-glucopyranoside（7）,4-O-（β-D-glucopyranosyl）-benzoic acid（8）,aegineoside（9）,epicatechin（10）,secoisolariciresinol9-O-β-glucopyranoside（11）,1-（3’,4’-dihydroxycinnamoyl）cyclopentane-2,3-diol（12）,lirioresinol A（13）,syringaresinol 4’-O-β-D-glucopyranoside（14）,syringaresinol（15）、5-O-caffeoylquinic acid methyl ester（16）,cinchonain Ia（17）,1-O-（vanillic acid）-6-O-（3’’,5’’-dimethoxy-galloyl）-β-D-glycoside（18）,4-O-β-D-（6-O-vanilloylglucopyranosyl）vanillic acid（19）,fomannoxin acid（20）,protocatechuic acid ethyl ester（21）,（4S）-（2E）-4-hydroxy-2-nonenoic acid（22）,tyrosol（23）,syringaldehyde（24）,vanillic acid（25）,uridine（26）,trypacidin（27）,dimethyl 2,3’-dimethoxyosoate（28）,（7R,8S）-dihydrodehydrodiconiferyl alcohol 9-O-β-D-glucopyranoside（29）,1-（α-L-rhamnosyl（1-6）-β-D-glucopyranosyloxy）-3,4,5-trimethoxybenzene（30）,（+）-lyoniresinol3α-O-β-D-glucopyranoside（31）,（-）-lyoniresinol3α-O-β-D-glucopyranoside（32）,sargentodoside B（33）,tachioside（34）,4-hydroxy-3,5-dimethoxy-phenol（35）,cuneataside A（36）,（-）-8’-epilyoniresin-4-ylβ-glucopyranoside（37）,arjunglucosideⅡ（38）,isotachioside（39）,glucosyringic acid（40）,3,4,5-trimethoxyphenyl-β-D-glucopyranoside（41）,β-hydroxypropiovanillone-3-O-β-D-glucopyranoside（42）,3-O-（β-D-glucopyranosyl）-1-（3’,5’-dimethoxy-4’-hydroxyphenyl）-1-propanone（43）,（-）-lyoniresinol 2α-O-β-D-glucopyranoside（44）,vanillyl alcohol-4-O-β-D-glucopyranoside（45）,sargentodoside C（46）,4-hydroxy-1-（2-nitroethyl）benzene4-O-（6’-O-β-D-xylopyranosyl）-β-D-glucopyranoside（47）,androsin（48）,（+）-5’-methoxyisolariciresinol 3α-O-β-D-glucopyranoside（49）,staphylionoside D（50）,syringicacid（51）,rel-（7R,7’E,8S）-4,9-Dihydroxy-3,3’,5-trimethoxy-4’,7-epoxy-8,5’-neolign-7’-en-9’-oic acid（52）.（4）The clinical effect of JHD in treatment of COVID-19 complicated with sepsis:We included in this study 77 patients,including 38 received stabdard care plus Jinhong Decoction and 39 received standard care alone.There was no satatistically significant difference in age,gender,SOFA score and APACHEⅡscore between the test group and the control group（p>0.05）.After treatment,the overall improvement rate was 23.1%（9/39）in the control group,compared to 44.7%（16/38）in the treatment group（p<0.05）.At end point of this study,compared with control group,SOFA score,APACHEⅡscore was lower in treatment group（p<0.05）.Mortality was significantly lower in treatment group compared with control group（55.3%vs 76.9%;p<0.05）.After administration of JHD,an increased lymphocyte count and a decreased total D dimer were observed.Conclusion:We clarify that JHD can alleviate the inflammatory respond by reducing the release of inflammatory factors in the process of septic mice.Emodin-8-glucoside,rhein,aloeemodin,emodin,luteolin,rhein-8-glucosidem,physcion-8-O-beta-D-monoglucoside,aloe-emodin-8-O-β-D-glucopyranoside,protocatechuic acid,epicatechin and salidroside in septic rats had higher C_max and AUC than those in normal rats.Fifty-two compounds were isolated from Sargentodoxa cuneata.Twelve of them are isolated from the plant for the first time and compound 1and 2 are new natural compounds.JHD is a promising integrative therapy for COVID-19 complicated with sepsis,which provides a scientific basis for the treatment of sepsis and the development of anti-sepsis drugs.