| Huoxiang Zhengqi Tincture(HZT), a famous Chinese herbal formula in Tai Ping HuiMing He Ji Ju Fang, was researched in this artical. The formula was studied through theoptimizing strategy gradually and hierarchically. It’s the effective substances, havingantibacterial efficacy, which were researched gradually through the levels of chemicalgroups and chemicals. And first, it is studying from the effective parts to the serumchemistry, serum pharmacology and intestinal pharmacological and then to spectralefficiency-related laws; Besides, the fingerprints of HZT is investigated from all parties tothe overall quality control which is based on the efficacy of the muti-componentdetermination; Finally, it is researching from the HPLC method for the control ofnon-volatile and GC method of control. Some adopting suitable analysis means werechosen to establish its quality control method. Main research contents was included asfollows:We were determining the Staphylococcus aureus as the target bacteria throughscreening and comparing the inhibition of HZT, which has the largest inhibition zone valueand the lowest minimum inhibitory concentration. Since the bacteriostatic ring experimenthas the advantage of small dosage sample, simple, reproducible and so on, it was chosen tothe follow-up test.Following the above target bacteria and experimental methods, we compared thepharmacodynamic of different polar fractions of HZT extraction in order to determinewhich is the effective parts, and study the pharmacodynamic of monomer which wasextracted with separation method from traditional natural product. The results were showed that the petroleum ether layer has the strongest inhibitory effect, and then separated10compounds from it, which were identified as: patchouli alcohol (I), Lee honokiol (Ⅱ),iso-imperatorin (Ⅲ), honokiol (Ⅳ), imperatorin prime (V), atractylodes lactone III (Ⅵ),8-methoxy-5-hydroxy psoralen prime (VII), do imperatorin hormone (VIII), nobiletinprime (IX), orange peel the hormone (X). After study showed that honokiol has thestrongest inhibition on Staphylococcus aureus.Serum pharmacology and serum medicinal chemistry research are suitable for thetreatment effect of traditional Chinese medicine through blood. The results were showedthat it may not be applicable because the target organ of HZT is gastrointestinal tract.Experiment shows that the serum from HZT in all time points expressed no bacteriostasis,so we can’t use this method for further study. Professor Li Kang creatively proposesintestinal drug chemistry and intestinal pharmacology which is based on the theories ofchemical drugs and serum pharmacology. We extract intestinal contents after feeding with11batches of homemade Huoxiang Zhengqi Tincture to rats, detect the bacteriostatic effect,establish HPLC fingerprint of intestinal drug chemistry, and analyze the data of inhibitionzone diameter and the fingerprint with chemometric. There were10related peaks about theantibacterial activity which were introduced into the regression equation. The results werethe peak of No6,13,26and30having the positive correlation with the antibacterial activity,and the experiment was proved that the peak of No26(honokiol) has antibacterial effect.Those compounds of Huoxiang Zhengqi Tincture were having the reference value forquality control, in other words that the method was feasible.The Fingerprint is a kind of comprehensive and the quantification appraisal method,which was mainly used in appraising the traditional Chinese medicine as well as theChinese native medicine preparation half-finished product quality authenticity, the qualityand the stability. According to the earlier period experimental result, we were establishedthe HPLC fingerprint according to the chemical composition research, the spectrum effectcorrelation, metabolism research synthesis. Divided the HPLC fingerprint into high and lowpolar part, and identified32common peaks with separation and stable peak area, to be usedfor quality control of HZT, which can be regarded as a supplement to the method of quality control.We developed a liquid chromatography method for rapid determination of hesperidin,glycyrrhizin, magnolol, honokiol and other bacteriostatic components, which was accordingto the basis of the pharmacopoeia methods. An aliquot(20μL) was injected into a DiamonsilC18column(250mm×4.6mm,5μm), which was kept at35℃, the eluent was monitored by aUV detector at270nm. The mobile phase consisted of0.05%formic acid in water, methodand acetonitrile and the flow rate was1.0mL/min. The method was validated over theconcentration range0.006～0.380μg for hesperidin,0.08～5.28μg for glycyrrhizin,0.065～4.160μg magnolol and0.026～1.680μg honokiol, and had a good linearrelationship of peak area. The average recovery ranged from99.7%(RSD=1.4%),99.4%(RSD=0.6%),99.9%(RSD=0.2%) and100.9%(RSD=0.8%) respectively. Thismethod can be used as a supplement of quality controlling for the tincture.The HZT exists large numbers of volatile components. We had preliminary establishedGC fingerprint of HTZ with correlation analysis and regression analysis which were basedon the pharmacodynamic indicators of inhibition zone value, and finally found the materialbasis. We introduced multiple linear regression to the data analyze. The results shown thatthere were10chromatographic peaks related with antibacterial activity introduced into thisregression equation, which were positive correlation between the chromatographic peak ofNO1,2,3,4,9,12,13and14. The inhibition increased with the fingerprint ofchromagraphic peak of NO1,2,3,4,9,12,13and14peak intensity increases. Theexperiment proved that NO12peak for honokiol had inhibitory. Patchouli and perilla hadNO2and9peak, NO3,4and13peak which came from perilla, and there were unknownabout NO1and14peak. All results shown that this method can be used as a supplement tothe quality control method of volatile components of HTZ. |
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