Comparative Study On Pharmacokinetics-pharmacodynamics And Preliminary Study On Metabonomics Of Yinhuang Injection And Yinhuang Oral Solution In Rats

The mechanism study on traditional Chinese medicine (TCM) usually imitates the model of study on pharmaceutical chemicals, which involves the pharmacokinetic study of major active components and pharmacodynamic study on pathological model. Since most TCMs are compound recipes which contain complex components, the pharmacokinetic study on minor active components does not represent the pharmacokinetic characteristics of the whole TCM combination formula, and mismatches the "holism" of TCM. In addition, due to the very complex syndrome of TCM, it is difficult to choose one or several laboratory "golden standards" of high correlation and good specificity, which makes the study on pharmacodynamics of TCM truly difficult. The emergence of metabonomics will spell god-given chance element for the study on mechanism of action of TCM. Metabonomics, defined as the quantitative measurement of the multi-parametric metabolic response of living systems to pathophysiological stimuli or genetic modifications, is a branch of science concerned with the exploration on global metabolic mechanism of organism and its coinosite. It mainly investigates the change of endogenous metabolism response to drug, and reflects the biochemical process and condition modification and fits the global and dynamic principle of TCM. In the present study, a comparative study on classical pharmacokinetics-pharmacodynamics and metabonomics in Yinhuang injection and Yinhuang oral solution was undertaken to reveal the defects of pharmacokinetics and pharmacodynamics and to evaluate the potential of metabonomics on TCM combination formulas.Yinhuang injection and Yinhuang oral solution, prepared with extracts from Flos Lonicerae and Radix Scutellariae, are widely used to treat acute tonsillitis, upper respiratory infection, febris, tussis, pharyngalgia, etc., with such therapeutic effects as relieving fever, deintoxication and relieving sore-throat. Chlorogenic acid (ChA) is the major active component of Flos Lonicerae, having therapeutic effects of anti-inflammatory, conservancy of hepar, cholagogue, antibiosis and antivirus, etc.; Baicalin (Bc) is the major active component of Radix Scutellariae, having therapeutic effects of anti-inflammatory, antibiosis and antivirus, conservancy of ischemical reperfusion injury, conservancy of hepar, cholagogue, etc.1 The study on analytical method in vivo of Yinhuang injection and Yinhuang oral solution1.1 The study on HPLC analytical method for simultaneous determination of ChA and Bc in plasmaExtracted from the plasma samples with methanol-acetonitrile (3:1), the two compounds were successfully separated using a C₁₈ column with a gradient elution composed of 15% and 54% methanol-acetonitrile (1:1) in 0.2% phosphoric acid water solution. The flow-rate was set at 1 ml·min^-1 and the eluents were detected at 327 nm for ChA, 278 nm for Bc. Puerarin and rutin were used as the internal standards for chlorogenic acid and baicalin, respectively. The method was linear over the range of 0.388 - 12.4μg·ml^-1, 0.485 - 124μg·ml^-1 for chlorogenic acid and baicalin, respectively. The correlation coefficient for each analyte was above 0.998. The intra-day and inter-day precisions were better than 7% and 9%, with the relative error ranging from -9.5% to 7.3% and from -4.2% to 1.8%. The limit of detection and the limit of quantification for ChA and Bc in plasma were 0.194μg·ml^-1, 0.122μg·ml^-1, 0.388μg·ml^-1, and 0.485μg·ml^-1, respectively. 1.2 The study on capillary zone eleetrophoresis (CZE) analytical method for metabolite fingerprints in rat urineThe optimum metabolite fingerprints based on CZE was obtained under following conditions: 40 mM sodium borate as the run buffer (pH was adjusted to pH 8.2 with 0.5 M hydrochloric acid); 24 kV applied voltage, 25℃temperature; injection 3s×0.5 psi. Each CZE data was imported into Computer Aided Similarity Evaluation System as ASCâ…¡format and treated as a two-dimensional matrix (time, absorbance). After the peak alignments of the electropherograms, the congruence coefficient based on included angle cosine was calculated to evaluate the similar degree of intragroup fingerprints. The results showed that this method had good reproducibility and stability and the individual variation in fingerprints of rat urine was small.2 Comparative study on pharmaeokinetics and pharmaeodynamics of Yinhuang injection and Yinhuang oral solution2.1 Comparative study on pharmacokinetics of Yinhuang injection and Yinhuang oral solution10 male Wistar rats, randomly divided into two equal groups, were intravenously (i.v.) administrated with Yinhuang injection at a single dose of 0.5 ml·kg^-1 and intragastrically (i.g.) administrated with Yinhuang oral solution at a single dose of 20 ml·kg^-1, respectively. Collected blood salnples at different time points were centrifuged to obtain plasma and the concentration of ChA and Bc in plasma was determined simultaneously byHPLC described as above. Calculations of pharmacokinetic parameters and pharmacokinetic modeling were carried out using DAS 1.0 software. It was shown that the concentration of ChA and Bc in plasma could be determined simultaneously after i.v. administration of Yinhuang injection; the AUC change of ChA followed the three open - compartment model and that of Bc followed the two open - compartment model. It was shown that the ChA in plasma could not be determined via i.g. administration of Yinhuang oral solution and the AUC change of BC presented multiple - peak phenomenon, and the absolute bioavailability of Bc was about 15.2%.2.2 Comparative study on pharmacodynamics of Yinhuang injection and Yinhuang oral solution30 Wistar rats, 15 male and 15 female, were randomly assigned to 3 equal groups: the oral solution group, the injection group and the control group. Each rat in the injection group and the oral solution group was intravenously administrated with Yinhuang injection at 1 ml·kg^-1 and intragastrically administrated with Yinhuang oral solution at 5 ml·kg^-1, respectively, twice a day, for 4 days successively; the rats in the control group were allowed access to water ad libitum without any other treatment. On the third day, 0.1ml of the 1% carrageen normal saline solution was hypodermically injected into left and right back voix pedis of each rat in the three groups. The bulk of right back voix pedis at different time after administration of carrageen and the normal voix pedis were measured and the degree of tumefaction was calculated. It was shown that the tumefaction values of two dosage groups were significantly lower than that in the control group (P＜0.05) and the degree of tumefaction in the oral solution was equivalent to that in the injection group (P＞0.2), which indicated that both of the two dosage groups have equivalent anti-inflammatory effect.3 Preliminary study on metabonomics of Yinhuang injection and Yinhuang oral solution30 Wistar rats, 15 male and 15 female, acclimated in individual metabolism cages, were randomly assigned to 3 equal groups: the oral solution group, the injection group and the control group. Each rat in the injection group and the oral solution group i.v. administrated with normal saline solution at 1 ml·kg^-1 and i.g. administrated with normal saline solution at 5 ml·kg^-1, respectively, twice a day, for 2 days successively; then, rats were i.v. administrated with Yinhuang injection at 1 ml·kg^-1 and i.g. administrated with Yinhuang oral solution 5 ml·kg^-1, respectively, twice a day, for 4 days successively. The rats in the control group were allowed access to water ad libitum without any other treatment. On the fifth day, 0.1ml of the 1% carrageen normal saline solution was hypodermically injected into left and right back voix pedis of each rat in the three groups. At selected time intervals, urine samples from each rat were collected and 150 metabolite fingerprints were obtained by CZE method described as above.After peak alignments and data autoscaling, 150 fingerprints were subjected to Principal Components Analysis (PCA) to obtain PCA general scores plot, which showed that most of the scores were mapped in four different clusters except for several scores. The grouping and typing fingerprints were subjected to PCA and variance weight analysis to be shown as follows: The performance of PCA of fingerprints of normal urine sample revealed that the sexual and diurnal variation was not significant. The variation of PCA induced by the stimuli of i.g. was not significant while that induced by the stimuli of i.v. was significant. The variation of PCA was not significant in the group via i.g. administration of Yinhuang oral liquid and normal saline solution while significant in the i.v. group administrated with Yinhuang injection and normal saline solution. The variation of PCA was significant in i.g. administration of Yinhuang and i.v. administration of Yinhuang injection. The performance of PCA of fingerprints in three groups revealed significant discrimination where samples with inflammation lay apart from samples preinflammation, and the variance weight analysis was shown that multitude characteristic peaks varied significantly; while the variation of PCA of fingerprints in three groups was not significant during 0 - 36 h after causing inflammation. The variation of PCA during 0 - 36 h after causing inflammation was significant between two dosage groups and control group, while that was not significant between oral solution group and injection group, which indicated that both of the two dosage groups have equivalent therapeutic effect of anti-inflammation.ConclusionThe study on pharmacokinetics :indicated that pharmacokinetic characteristics of ChA and Bc in Yinhuang injection and Yinhuang oral solution varied greatly, while the study on pharmacodynamics indicated that the curative effect of Yinhuang oral solution equaled to that of Yinhuang injection. It is evident that the findings of pharmacokinetics and pharmacodynamics are inconsistent, which demonstrates that it is unreliable to study the mechanism of TCM combination formulas by traditional pharmacokinetic and pharmacodynamic methods.Application of CZE-based urine metabolite fingerprints could successfully discriminate the metabolite phenotypes of different treatment, overlook physiological variations and obtain characteristic peaks of biomarkers, which indicates that metabonomics is an all-round,, fast and reliable science branch on study of the mechanism of action of TCM combination formulas.