Study On Metabolism,Anti-COPD Mechanism And Drug Combination Of S-allylmercapto-L-cysteine

Garlic(Allium sativum)has been widely used for the prevention and treatment of various diseases,such as inhibition of carcinogen activation,detoxification,anti-inflammation,DNA impairment and so on.However,the irritation and unpleasant smell of garlic limits its consumption and use.Aged garlic,the high temperature fermentation of garlic,is safer comparing with ordinary raw garlic,and can significantly reduce the original irritating smell of raw garlic.The main sulfur compounds of aged garlic include S-allyl cysteine(SAC),S-allyl mercapto cysteine(SAMC)and allicin(SACS),and the pharmacological activity of SAMC is higher.A large number of previous literatures found that SAMC has significant therapeutic effects in anti-cancer,liver protection and anti-chronic obstructive pulmonary disease.Most of the current researches are limited to the evaluation of activity.However,its synthesis,metabolism,active mechanism of anti-COPD and combination of medication have not been reported.Therefore,this subject studied on the above aspects of SAMC.The contents of this subject include the following six chapters.(Chapter Ⅰ)The synthesis,characterization and preliminary pharmaceutical evaluation of SAMC were studied;(Chapter Ⅱ&Ⅲ)The metabolic mechanism of SAMC in vitro was studied by using several models;(Chapter Ⅳ)The absorption,distribution,metabolism and excretion of SAMC and established methods for the determination of SAMC and its metabolite determination method were researched;(Chapter Ⅴ)According to the results of metabolic studies,the active mechanism of SAMC on anti-chronic obstructive pulmonary disease was studied from the aspects of cell signal pathway,anti-inflammatory,anti-oxidation,and protein modification.(Chapter Ⅵ)The alleviation of SAMC on the adverse reactions caused by antifungal agents was evaluated and studied.The preliminary pharmaceutical evaluation of SAMC(Chapter 1)included studies on synthesis,purity analysis,structural confirmation,physicochemical properties and stability of SAMC.The content of SAMC in the solid garlic is very low,the extraction is time-consuming and laborious,and the purification process is complex and inefficient.In order to obtain a stable and reliable source of SAMC,this subject has carried on the thorough investigation to the synthesis methods of SAMC and prepared a high purity SAMC by a two-step synthesis way.Subsequently,the structure of SAMC was confirmed by UV,IR,HRMS,1H-NMR and 13C-NMR,and the related spectra data were established.The melting point,solubility in different solvents,oil and water partition coefficient,dissociation constant,stability of SAMC were measured.The results showed that SAMC is white or almost white flaky crystals,soluble in water,and almost insoluble ethanol,acetone,dichloromethane and other organic solvents.The melting point of SAMC is 179.21 ℃(also the decomposition point).The oil-water partition coefficient logP is below zero.It means SAMC is a hydrophilic compound.The dissociation constants are 2.7 and 9.1,respectively.At the same time,HPLC method for the determination of SAMC in vitro was established,and the analytical method was verified.The results of method validation showed that the method had good specificity and linear relationship,and the recovery and precision satisfied the requirements.The results of stability study showed that SAMC was stable in acidic environment,but it was very unstable and easy to degrade in alkaline environment.Accelerated test results showed that SAMC can be stored in aluminum and plastic packaging.The study on the metabolic mechanism of SAMC in vitro(Chapter II)includes the metabolism in hepatocytes and liver microsomes.The metabolites of SAMC in hepatocytes and liver microsomes were identified.The main CYP450 isoforms involved in SAMC metabolism and the inhibitory effect of SAMC on CYP450 isoforms were investigated.The enzymatic kinetic parameters were determined.The results showed that SAMC had two metabolites in hepatocytes and four metabolites in liver microsomes.The optimal incubation time and protein concentration were 15 min and 1 mg/mL.respectively.The main subtypes were CYP3A4 and CYP1A1,and SAMC had some inhibitory effect on CYP2E1.The enzymatic kinetic results showed that Vmax and Km were 24.29 μM/min/mg liver microsomes and 339.7 μM,respectively.The study on the metabolic mechanism of SAMC in vitro(Chapter Ⅲ)focused on the metabolism of SAMC in blood.First,the metabolites of SAMC in blood were identified.The metabolic sites of SAMC were determined,and the effects of endogenous thiol compounds on SAMC were studied.Secondly,the metabolic mechanism and metabolic kinetics of SAMC were studied.At last,the plasma protein binding and protein modification of SAMC were researched.The results showed that six metabolites of SAMC were detected in the blood,and its main metabolic site was>30kD proteins.The metabolic rate of SAMC in the red cells was fast,its half-life was short and some containing mercapto-endogenous substances had a greater impact on the metabolism of SAMC.Protein binding and modification results showed that the plasma protein binding rate was about 50%,the 93 and 125 cysteine residues in hemoglobin β chain could be S-allyl thiolation and the same effect could be detected in rat blood after multiple injections.The metabolic study of SAMC in vivo(Chapter IV)included the establishment and validation of biological sample analysis methods,metabolites identification,pharmacokinetics,tissue distribution and other research content.Firstly the in vivo LC-MS and GC-MS analysis methods of SAMC were established and verified.The metabolism,distribution and excretion of SAMC after oral administration were studied.The results suggested that the two methods had good specificity,high precision,and their accuracy,stability and others met requirements.Although,SAMC were not detected,its two major metabolites,AMSO and AMSO2,were detected instead of.The maximal plasma concentration of AMSO was 134.933±32.041 μg/mL,and the highest plasma concentration of AMSO2 was 122.27±21.937 μg/mL.The peak time of AMSO and AMSO2 were 24 h and 36 h respectively.The half-life of AMSO was 12 hours or so,and the half-life of AMSO2 was up to 26 h or more.Tissue distribution studies showed that the two metabolites of SAMC were distributed in the main organs of rat,including heart,liver,spleen,lung and kidney.The concentration of two metabolites in liver tissue was significantly higher than that in other tissues.Excretion studies showed that the two metabolites of SAMC could be excreted in the urine,feces and bile.To further speculate the metabolic dynamics of SAMC in vivo,a test of SAMC by intravenous injection via tail vein was studied.In the research,Tween-80 was added in and high-pressure homogenizer was used in the preparation of SAMC dosage form.Although,the dosage belonged to the micro-particle dispersion system and had a certain sustained-release effect,the results showed that the half-life of SAMC in vivo were less than 5 min.The research on pharmacological activity of SAMC for the treatment of chronic obstructive pulmonary disease(Chapter V)was composed of the discovery of signal pathway in the lung adenocarcinoma cell line SPC-Al,the mechanism of anti-inflammatory and anti-oxidative protection and the target of drug.(1)A COPD model was established to evaluate the inhibition of SAMC on MUC-5AC and the activation of SAMC on membrane protein AQP-5.The results of ELISA showed that SAMC had a significant inhibitory effect on LPS-induced MUC5AC expression in SPC-Al cells.RT-PCR results further demonstrated the inhibitory effect of SAMC on LPS-induced MUC5AC expression from RNA level.Western blotting and immunofluorescence showed that SAMC could significantly inhibit the degradation of IκBα protein and the transfer of NF-κB P65 protein in SPC-Al cells.Based on the above results,it can be speculated that the alleviate effect on COPD of SAMC can inhibit the secretion of MUC5AC in SPC-Al by regulating NF-κB signal pathway.(2)In the way of anti-inflammatory and anti-oxidative effects,the protective mechanism of SAMC on COPD inflammation and oxidative damage induced by LPS was studied.The model of inflammation and oxidative damage of NR8383 and SD rat was established by administering a certain dose of LPS.The results showed that SAMC could significantly inhibit the expression levels of TNF-α,IL-1β and IL.-8 in macrophages NR8383 and rat BALF which were increased by LPS.The results of oxidative stress showed that SAMC could regulate ROS,SOD and MDA levels in macrophages.At the same time,SAMC could reduce the wet weight and dry weight ratio which were increased by LPS,and decrease LPS-induced MPO and NO levels in rat lungs.Pathological and immunohistochemical observation showed that SAMC could not only inhibit LPS-induced pulmonary inflammation,but also could repair damaged lung tissue.Immunohistochemistry analysis showed that anti-inflammatory effects of SAMC may be achieved by modulating NF-κB signal pathway,which was consistent with the results in vitro.(3)The active sit of SAMC in LTQ experiments showed that SAMC could directly modify the MUC5AC protein.SAMC could induce No.5564 CYS of the MUC5AC protein S-cysteineization,masking the surface of the protein exposed cysteine residues.Prevent the formation of disulfide bonds between proteins,alter the three-dimensional conformation of mucin,reduce the mucus viscosity,make the respiratory sputum easily cough up,and alleviate COPD symptoms caused by airway obstruction.The study on SAMC and antifungal agents(posaconazole,POS)(Chapter VI)included the evaluation of inhibition and mechanism of SAMC on anti-fungal drugs-induced adverse reactions.First,POS enteric microspheres were prepared by spray drying technique and characterized.The properties of the polymer feeding solution for the preparation of POS microspheres were also studied.And then,KunMing mice model were used to study the protective effect of SAMC on the adverse reactions of mice induced by POS which was orally administrated for one week.The results showed that POS group showed reduced diet,lazy,burnout,vertical hair and the incidence of diarrhea was up to 40%.The combination therapy groups could significantly improve the mental state and the incidence of diarrhea reduced to 20%.The anti-inflammatory mechanism study showed that the combination therapy groups significantly reduced the expression levels of TNF-a,IL-1β and IL-6 induced by POS(p<0.05).The results of antioxidant mechanism showed that the levels of MDA in Co-L and Co-H groups were decreased by about 20%and 30%comparing with POS group,respectively.The histopathological results showed that the colonic tissue of the combined administration group had a certain repair in inflammatory infiltration and mucosal adhesions versus the POS group,and the colon tissue structure was more clearly discernible.In a word,SAMC could inhibit POS-induced adverse reactions by anti-oxidation and anti-inflammatory ways.In summary,the pharmaceutical properties of SAMC,such as synthesis,structure identification,physical and chemical properties,purity and stability,were studied and evaluated in this subject.The identification of the metabolites,tissue distribution,drug absorption and excretion of SAMC and the pharmacokinetic parameters were determinated in metabolic studies in vivo and in vivo.The pharmacological activity of anti-COPD was researched from cell signal pathway,anti-inflammatory,anti-oxidation and protein modification aspects.Finally,the mechanism of SAMC on the adverse reaction of antifungal agents was analyzed.The results showed that SAMC has better druggability and advantages in combination therapy.