| Orally administration of water decoction is the representatinve form of the Traditional Chinese Medicine(TCM), so the pharmaceutical effective constituents of TCM may be the metabolites in vivo as well as the original substances. In order to investigate the in vivo effective components of Scutelariae Radix(SR) and Forsythiae Fructus(FF) which were most common heat-clearing herbs in TCM clinic, our study analyzed the gastrointestinal metabolites of SR and FF in vitro and in vivo, searched the anticomplementary, antibacterial and antiendotoxin metabolies. The results indicated that the metabolic samples exhibited better activities than the water extract of SR(WESR), water extract of FF(WEFF) and their main components(Norwogonin and hydroxytyrosol expressed the highest biological activities for SR and FF, respetively). In addition, the important role of intestinal bacteria in the metabolism and absorption process of the main constituents from SR and FF was investigated combined with means of metabonomics, serum pharmacochemistry as well as pharmacokinetics. The object of the study was to clarify that intestinal metabolites may be the effective constituets of SR and FF, and the pharmacological effects of them may be dependent on intestinal bacterial metabolism, which provides new research approaches for modernization of TCM.1. The establishment of the intestinal bacterial inubation method. The study compared two common intestinal bacterial incubation methods by ERIC-PCR, established a better method, which the intestinal bacterial structure was more similar with the fresh feces and more stable, as a basis for further intestinal bacteria incubation in vitro.2. Gastrointestinal metabolism of SR and activity screening of intestinal bacterial metabolites in vitro. The results indicated that the main components from SR were metabolized by human intestinal bacteria. The metabolites were demonstrated to have higher activities than those of WESB. Norwogonin was the most active compound among the metabolites and original compounds.Norwogonin-7-O-β-D-glucuronide was isolated from SR for the first time. A high performance liquid chromatography(HPLC) method was developed for the simultaneous quanification eight compounds namely baicalin, norwogonoside, oroxyloside, wogonoside, norwogonin, baicalein, wogonin and oroxylin A in WESR, which was determined to be 110.72±4.25、35.49±2.78、7.52±0.04、16.34±0.51、1.53±0.22、39.27±2.93、12.10±0.15、3.25±0.21 mg/g crude drug, respectively. WESR was found to be stable in simulated gastric juice and intestinal juice. After incubated with artificial juices, main constituents of WESR remained unchanged based on the comparison of peak areas of individual constituents during the course of incubation(RSD<3.0%). A UPLC-QTOF/MS method was developed for the determination of the metabolic process of main flavonoid glycosides incubated with human intestinal bacteria. Baicalin was metabolized to its aglycone baicalein and methylation product oroxylin A, wogonoside was metabolized to its aglycone wogonin and demethyl product norwogonin, oroxyloside was metabolized to its aglycone oroxylin A and demethyl product baicalein. When WESR was incubated with human intestinal bacteria, baicalin, wogonoside, oroxyloside and norwogonoside were metabolized into their respective aglycons, presented lower metabolic rate than the monomeric compound form.WESR showed no anticomplementary effect through the classical pathway or alternative pathway, whereas the metabolic samples at 24 h poessed the activity, suggested that anticomplementary metabolites may be generated during the metabolic process. The intestinal bacterial metabolites baicalein, oroxylin A and norwogonin did display an interesting anticomplementary activity, especially norwogonin(CP50=0.138 ± 0.004 mg/m L; AP50=0.147 ± 0.008 mg/m L). All three active compounds had no effect on coagulation system. Similarly, the metabolic samples exhibited better antibacterial and antiendotoxin activities than WESR. Baicalin, baicalein, wogonin, oroxylin A and norwogonin revealed inhibitory effects against the test bacteria. Norwogonin displayed an overall highest antimicrobial activity. The antiendotoxin effect assay showed that norwogonin and baicalein showed the highest activity among the active metabolites.Norwogonin expressed the highest biological activities among the compounds before and after intestinal bacterial metabolism, which may exert pharmacological activity of SF after the in vivo process.3. Gastrointestinal metabolism of FF and activity screening of intestinal bacterial metabolites in vitro. The results indicated that the main components from FF were metabolized when incubated with human intestinal bacteria. The metabolites were demonstrated to have higher activities than those of WEFF. Hydroxytyrosol was the most active compound among the metabolites and original compounds.Six compounds were isolated from FF, and their structures were elucidated based on NMR spectra data. The isolated copounds were(+)-phillyrin,(+)-pinoresinol-4-O-β-D-glucoside,(+)-pinoresinol monomethyl ether-O-β-D-glucoside,(-)-matairesinol4-O-β-D- glucoside,(-)-pinoresinol monomethyl ether-O-β-Dglucoside and forsythoside E. A HPLC method was developed for the simultaneous quanification three main compounds namely forsythoside A, phillyrin and(+)-pinoresinol-4-O-β-D-glucoside in WEFF, which was determined to be 28.37±0.03、3.74±0.02、3.512±0.01 mg/g crude drug, respectively. Main constituents from WEFF remained unchanged during the incubation with simulated gastric juice and intestinal juice by HPLC-DAD method. Forsythoside A, the highest content in WEFF, was metabolized to caffeic acid and hydroxytyrosol, and caffeic acid was further hydrogenated to form 3,4-dihydroxybenzenepropionic acid. Phillyrin was metabolized to its aglycone phillygenin, demethyl product lantibetin, bond cleavage product 1,2-Benzenediol,4-[(2S,3R,4R)-4-[(3,4-dimethoxyphenyl)methyl]tetrahydro-3-(hydroxymethyl)-2-furanyl] and enterolactone.(+)-Pinoresinol-4-O-β-D-glucoside was metabolized to its aglycone pinoresinol, secoisolariciresinol, lariciresinol, enterolactone,(2R,3R)-2,3-Bis(4-hydroxy-3-methoxybenzyl)butyrolactone and(-)-(2R,3R)-3-(3′′-hydroxybenzyl)-2-(4′-hydroxy-3′-methoxybenzyl)butyrolactone. Hydrolysis, demethylation, reduction, condensation, oxidation and dehydroxylation were the main reaction types. When WEFF was incubated with human intestinal bacteria, it presented lower metabolic rate than the monomeric compound form.WEFF showed no anticomplementary effect through the classical pathway or alternative pathway, neither. However, its metabolic samples at 24 h showed the anticomplementary activity. The lignans and their main intestinal bacterial metabolites had no anticomplementary effect through the classical pathway or alternative pathway. However, hydroxytyrosol and 3,4-dihydroxybenzenepropionic acid, as intestinal bacterial metabolites of forsythoside A, demonstrated the anticomplementary activity, especially hydroxytyrosol(CP50=0.096 ± 0.007 mg/m L; AP50=0.121 ± 0.012 mg/m L). Similarly, the metabolic samples exhibited better antibacterial and antiendotoxin activities than WEFF. The lignans and their main intestinal bacterial metabolites showed no antibacterial and antiendotoxin effects. Forsythoside A, its intestinal metabolites hydroxytyrosol and 3, 4-dihydroxy benzenepropionic acid demonstrated inhibitory effects against the bacteria. Hydroxytyrosol displayed the highest antimicrobial activity. Hydroxytyrosol and 3, 4-dihydroxybenzenepropionic acid showed antiendotoxin effect in vitro, especially hydroxytyrosol.Hydroxytyrosol expressed the highest biological activities among the compounds before and after intestinal bacterial metabolism, which may exert pharmacological activity after the in vivo process. The pharmacological effects of FF may be dependent on intestinal bacterial metabolism and the real active ingredients may be the intestinal bacterial metabolites instead of the original substances.4. The effect of the intestinal bacteria on the metabolism and absorption of the main components from SR and FF. The results indicated that glucuronidation, methylation, demethylation and hydrolysis metabolites of TCM ingredient were present in rat in vivo. The intestinal bacteria could influence the metabolism and asorbtion of TCM ingredient in vivo by means of hepatoenteral circulation and hydrolytic actionPseudo-germfree rat model was obtained by the orally administration of broad-spectrum antibiotics. The study compared the metabolic process of main components from SR and FF incubated with the rat intestinal bacteria between the pseudo-germfree and normal rats. The results indicated that the metabolic rate and species of the metabolites may be influenced by the disturbance of intestinal bacteria. In addition, the metablic species of the metabolites were also different when main components incubated with human and rat intestinal bacteria because of the difference between different species in gut.The metabolites in the caecum and urine of pseudo-germfree and normal rat were analyzed by UPLC-QTOF/MS. Glucuronide conjugates, methylation, demethylation and hydrolysis products were determined in the normal rats, whereas no metabolite was found in pseudo-germfree rat. The urine samples of pseudo-germfree and normal rat were analyzed by the multivariate statistical analysis of metabonomics, which screened the different metablic markers between the two groups. The results indicated that the metablic markers included urinary amino acid metabolites and the in vivo metabolism of TCM, suggested the important significance of intestinal bacteria in the maintenance of host health and normal metabolism.The pharmacokinetics of main components from SR and FF and their main intestinal bacterial metabolites in pseudo-germfree and normal rat plasma were compared, which proved that the intestinal bacteria influence the metabolism and asorbtion of TCM ingredient in vivo by means of hepatoenteral circulation and hydrolytic action, then affect the pharmacological effects.5. Study on constituents aborbed into blood of SR and FF in compound preparation, which proved that active intestinal bacterial metabolites may be the pharmaceutical effective constituents of SR and FF in Shuanghuanglian.We analyzed the rat serum samples after oral administration of water extract and oral liquid of SHL(including SR, FF and Lonicera japonica), verified the active intestinal bacterial metabolites existed in the medicated serum, which may be the pharmaceutical effective constituents in vivo, proved the critical role of intestinal bacterial metabolism in treating diseases and playing pharmaceutical efficacy. |
PDF Full Text Request