| Objectivel.To explor the source, the combination principle, the thought of compatibility of Banxia xiexin decoction, pursuing a better formation of Banxia xiexin decoction, consequently, improve the clinical efficacy.2.To develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for concentration measurement and pharmacokinetic analysis of baicalin in human saliva using monoclonal antibodies (MAb) against baicalin(anti-BAL), monoclonal antibodies (MAb) validating icELISA method to determine baicalin concentration in human saliva, providing an new method and an new idea of medicine concentration analysis and pharmacokinetic studies the Banxia xiexin decoction in human saliva.3.To study pharmacokinetic characteristics of baicalin and ginsenoside-Re in human saliva of the bitter and sweet medicine in Banxia xiexin decoction, observing dynamic changes of concentrations in saliva in different time, providing research foundation and scientific basis of saliva-based medicines monitoring of various clinical therapeutic substances and the research of prescription compatibility.Methods1.To solve these problems and restore the thought of Banxia xiexin decoction, from the origin of it, we use the point of the embryology and combine it with the medical science, philosophy and philology to study it.2.Indirect competition ELISA (ic-ELISA) methods were developed and validated for measurements and pharmacokinetic analysis of baicalin and its methodology including assay linearity and sensitivity of detection in human saliva, standard curve were prepared and its methodology including assay linearity and sensitivity of detection, precision and accuracy, recovery, stability and specificity anti-BAL MAb were validated.3.Fourteen healthy volunteers were randomly divided into two groups, the one group of eight subjects received baicalensis Georgi capsules, and the other group of six subjects received Banxia xiexin decoction capsules in the morning. The saliva from each subject was collected 30 min before drug administration, followed by30,45,60,120,180,240,300,315,330,360, 420,540,660 and 720 min after drug administration using Salivette(?) cotton swab tubes. After processing saliva samples, a standard curve comparing concentration of baicalin and ginsenoside-Re with its absorbance measured by ic-ELISA was plotted. These pharmacokinetic parameters were calculated in Kinetica version 5.0 and SPSS 20.0.Results1.We find the origin of Banxia xiexin decoction is Xiexin decoction from Tang ye jing Fa(record in Auxiliary Verse on Drugs and Methods for Zang-fu Organs, hereinafter referred to as Fu xing jue). Banxia xiexin decoction adding the pinellia ternate and the jujube to the xiexin decoction, which has the same attending disease. We can see some disappeared contents of Tang ye jing fa from Fu xing jue. Fu xing jue and Treatise on febrile and Miscellaneous Diseases has the same origin. Treatise on febrile and Miscellaneous Diseases is a book for treatment, it has rarely say formulate principle but has many symptoms and formulas, but the Fu xing jue is executable, it mainly expounds the compose and actions of the formulas, and the core principles of all the formulas was recorded in the "picture of enhancement or Inhibition by the five flavours".2.An indirect competition ELISA method was developed and validated for the measurements and pharmacokinetic analysis of baicalin in human saliva. We obtained a linear regression coefficient of 0.9958, and the linear regression equation, y=-0.2561n(x)+1.9224. A linear relationship was found within the range of 30ng/mL-1000ng/mL. The sensitivity (ICso) of the assay was determined as 228ng/mL. The limit of detection was 11.71ng/mL(mean±S.D× 3,n=20). Both intra-day and inter-day repeatability and precision was achieved, with relative standard deviation (RSD) lower than 15%. A mean recovery of 95-115% was obtained, with RSD lower than 15%. The RSD of stability of dissolving saliva are increased after stored for a long time.3.Pharmacokinetics of baicalin in human saliva of baicalensis Georgi capsules group after oral Administration:Our data show that baicalin in saliva reached maximum concentration first at 40±17.32min and second at 342±26.83min, with an average Cmax of 271.75± 167.24 ng/mL, An AUCO-t of 68763.15±58914.55 ng/mL-min and MRT of 578.2± 340.4min, was measured. Maximum concentration of baicalin was measured at two different time-points following administration, the first at about 30-60min and the second at about 330-360min.4.Pharmacokinetics of baicalin in human saliva of Banxia xiexin decoction capsules group after oral Administration:Our data show that baicalin in saliva reached maximum concentration first at 35±8.66min and second at 320±17.32min, with an average Cmax of 1057.41±801.88 ng/mL, An AUCO-t of 303955.3±259071.5 ng/mL·min and MRT of 976.24±515.61min, was measured. Maximum concentration of baicalin was measured at two different time-points following administration, the first at about 35min and the second at about 330min. Pharmacokinetics of ginsenoside-Re in human saliva:Our data show that ginsenoside-Re in saliva reached maximum concentration at 336±48.14min, with an average Cmax of 146.75±261.16 ng/mL, An AUCO-t of 30858.26±46828.19 ng/mL·min and MRT of 556.67±158.90min, was measured. Maximum concentration of ginsenoside-Re was measured at one time-points following administration, the concentration of ginsenoside-Re have reached a peak at 330min.Conclusions1.The principles of formulating prescription of five flavors in the book of Fu xing jue were relevant with the theory of excessive-surpass of six qi and the theory of tonification and purgation based on the conditions of five zang in the Yellow emperor’s internal canon of medicine. Inheriting from Zang qi fa shi lun, five flavors matching five xing in the book of Fu xing jue was distributed by the function of herbal medicines corresponding to season qi. In the book of Fu xing jue, Xiexin decoction was actually prescribed for purging "earth", which was associated with heart belonging to "earth" in the theory of five zang matching five xing. The compatibility of bitter and sweet played an important role in Xie xin decoction in the book of Fu xing jue, and the medicines are the key herbs for purging "earth".Banxia xiexin decoction is for purging "earth". Zhongjing Zhang combined his clinical practice to form the prescription, taking the prescription from Tang Ye Jing Fa, increasing some herbal medicines which is recorded in Shen nong ben cao jing. The thought of the prescription influenced by the theory of five flavor and closely related to the theory of tonification and purgation in the Yellow emperor’s internal canon of medicine and the pharmaceutic rule related to the thought of "formulating prescription according to the flavor of Chinese herbal" before Han Dynasty. The compatibility of bitter and sweet played an important role in Banxia xiexin decoction and the medicines are the key herbs for purging "earth"2.Quick, specific and sensitive indirect competitive ELISA assay methods for the measurement and pharmacokinetic analysis of baicalin in human saliva were developed for the first time. Method validations in human saliva were satisfactory and meet the criteria specified by the United States Food and Drug Administration (US FDA) for the analysis of biological samples. The validated ic-ELISA method can be successfully applied to the investigation of the pharmacokinetics of baicalin in human saliva, providing a new method of medicine concentration analysis and pharmacokinetic studies of traditional Chinese medicine in human saliva.3.The concentration and time line of baicalin was basically coincide in baicalensis capsule group and Banxia xiexin decoction capsule group, but the concentration and time line of Ginsenoside-Re was changed in Banxia xiexin decoction capsule group, which was no statistical difference with the second maximum concentration time of baicalin in baicalensis capsule group. We can say that after compatibility the pharmacokinetics characteristic of baicalin was changed, and the change were related to baicalensis. |
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