| Objective1. To study the absorption, distribution, metabolism and excretion of cinnamic aldehyde in the body by detecting its concentration in blood of New Zealand rabbits administered with Shenqi-Pill extraction with the method of HPLC and analyzing its pharmacokinetic parameters.2. To study the influence of different proportions of Shenqi-Pill on the clinical symptoms, content of adrenocorticotropic hormone (ACTH) and cortisol (COR) in serum of Kidney-yang deficiency mouse models. To elucidate the mechanism of Shenqi-Pill and the relationship between its effects and dosage, to find out the optimal dosage of the drugs in Shenqi-Pill and to provide reliable reference for its effective applications in clinic.Methods1. Quality control method for Cortex Cinnamomi:Cortex Cinnamomi was made into powder and sieved by the No.2sieve.0.2gram of Cortex Cinnamomi powder was precisely weighed and placed in a100mL round bottom flask with54mL70%ethanol in it. The mixture was refluxed on heat for1hour after soaking for30min. The extraction solution was filtrated and its volume was set to100mL. HPLC procedure was performed to determine the content of cinnamic aldehyde on the chromatographic column of Diomonsil TM C18(5μm,4.6mm×250mm), and the mobile phase was methanol and water (65:35, v/v) with the flow velocity of1.0mL/min. The detection wavelength was275nm and the column temperature was RT. The injection volume was10μL.2. Preliminary study of the pharmacokinetics of the effective compositions in Shenqi-Pill:A mass of Shenqi-Pill with2.5grams of Cortex Cinnamomi (the dosage of Radix Aconiti Lateralis Preparata, Cortex Cinnamomi, Radix Rehmanniae, Rhizoma Dioscoreae, Fructus Corni, Cortex Moutan, Rhizoma Alismatis and Poria was1:1:8:4:4:3:3:3), with ten times the amount of70%ethanol, was refluxed for1hour. Solution was obtained by decompression and concentrated to lg/mL. New Zealand rabbits were administered by the solution above.1mL blood samples were collected at different times, and processed by matrix solid dispersion(MSPD). Cinnamic aldehyde in blood were determined by HPLC on the chromatographic column of Diomonsil TM C18(5μn,4.6mm×250mm), and the mobile phase was methanol and water (65:35, v/v) with the flow velocity of1.0mL/min. The detection wavelength was275nm and the column temperature was RT. The injection volume was50μL. The results was analysised by pharmacokinetic software DAS2.1.1.3. Study of the optimization of formulating prescriptions in Shenqi-Pill:190mouses were classificated into19groups (n=10) randomly according to response surface design,17groups for Chinese medicine treating groups,1group for blank control and1group for model. After one week's feed, kidney-yang deficiency mouse models were made by intraperitoneal injection of hydrocortisone injection with the dose of25mg· kg1· d-1for ten days, except the blank control group. At the same time, Chinese medicine treating groups was administered with Shenqi-Pill decoction, but blank control and model group were administered with the same volume saline for20days.0.3mL blood samples were collected from eyeball of mice, which were placed in tubes for1hour and centrifuged for10min at the speed of4000rpm on21st day. The supernatant was separated and detected the content of ACTH and COR by the method of ELISA. The results were analyzed by response surface software Design Expert V.8.0.5b.Results1. Quality control results:Content of cinnamic aldehyde in the six batches of was above2.4%, greater than1.5%stipulated in the Pharmacopoeia. So cinnamic aldehyde content was stable and the drugs of Cortex Cinnamomi can be used for Shenqi-Pill extraction preparation.2. Pharmacokinetic parameters results of cinnamic aldehyde:Standard curve equation for cinnamic aldehyde was established:Y=111298x+2397.6(r=0.9992), with good linear relationship for cinnamic aldehyde from0.063to6.3Mg/mL. The detection limit of cinnamic aldehyde was0.0315μg/mL and its retention time was6.7min. The content of cinnamic aldehyde in blood of New Zealand rabbits administered with Shenqi-pill reached to the maximum absorption peak after1.72hours, with the peak concentration (Cmax of6.26mg/L and the half-life period (t1/2) of0.446hour. The result indicated that the change process of cinnamic aldehyde was one-compartment model.3. Optimization results of formulating prescriptions in Shenqi-Pill. Multivariate quadratic response surface regression models was established for ACTH and COR in the mouse serum, with the equation of that: YACTH=14.32213+16.55723X1-6.84099X2-9.26621X3+6.94731X,X2-11.47842X1X3+0.11478X2X3-37.78926X.2+1.74038X22+7.04112X32YCOR=17.10794+14.39793X1-7.68334X2-10.22769X3+8.36777X1X2-11.01928X1X3-0.37305X2X:,-40.22727X,2+2.01543X22+8.00224X32Y stands for the effect value of content of ACTH or COR, X,, X2and X:, stand for independent variables. It was not a simple linear relationship between various factors and indicators, but a square relationship instead.4. The results analyzed by response surface software Design Expert V.8.0.5b, was that the best Shenqi-Pill dose for mouse was Cortex Cinnamomi0.15g, Radix Aconiti Lateralis Preparata0.15g, Radix Rehmanniae0.56g, Rhizoma Dioscoreae0.28g, Fructus Corni0.28g, Cortex Moutan0.42g, Rhizoma Alismatis0.42g and Poria0.42g. In the verification experiment under these conditions we found that content of ACTH was12.02pg/mL and content of COR was13.63pg/mL According to the experimental results, combined with the practical clinical application, if the dosages of Cortex Cinnamomi, Radix Aconiti Lateralis Preparata, Cortex Moutan, Rhizoma Alismatis and Poria were doubled, but those of Radix Rehmanniae, Rhizoma Dioscoreae, Fructus Corni kept unchanged, the effect of Shen-pill to tonify adrenal insufficiency would be more significant, and its effect to tonify kidney-yang is better.Conclusions1. The experiments confirmed that cinnamic aldehyde could be measured by the high performance liquid chromatography (HPLC) in the blood samples.A method for cinnamic aldehyde determined by HPLC was set up, which was conf i rmed to be stable, sensitive, accurate, but low recovery rate.2. The pharmacokinetic parameters showed that cinnamic aldehyde in vivo was metabolized and eliminated quickly, and the maintaining time of effective blood concentration was short.3. The dose of Shenqi-Pill prescription was relatively low. If the dosages of Cortex Cinnamomi, Radix Aconiti Lateral is Preparata, Cortex Moutan, Rhizoma Alismatis and Poria were doubled, but those of Radix Rehmanniae, Rhizorma Dioscoreae, Fructus Corni kept unchanged (the dosage of Radix Aconiti Lateralis Preparata, Cortex Cinnamomi, Radix Rehmanniae, Rhizoma Dioscoreae, Fructus Corni, Cortex Moutan, Rhizoma Alismatis and Poria was2:2:8:4:4:6:6:6), the effect of Shen-pill to tonify adrenal insufficiency would be more significant, and its effect to tonify kidney-yang is better. |
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