CN104897791A - Simulation and prediction method for pharmacokinetic curve of traditional Chinese medicine - Google Patents

Abstract

The invention provides a simulation and prediction method for a pharmacokinetic curve of traditional Chinese medicine. Through certain steps, the method discovers and confirms that components with similar chemical structures in a complex system of an oral traditional Chinese medicine prescription have similar pharmacokinetic changes, i.e., similar gamma<+> values; and under the condition that the gamma<+> values of a component A at each moment are known, AUC-time curves of other components with similar structures can be predicated, so a novel pharmacokinetic prediction idea and method are established. The method provided by the invention can be used for pharmacokinetic prediction of similar-structure compounds of a single medicine or compound traditional Chinese medicine and comprises the following concrete steps: 1, respectively acquiring plasma concentration-time curves of the component A in a single medicine and in a compound medicine; 2, acquiring the AUC-time changing curve of the component A; 3, calculating a ratio gamma<+> of the sum of AUC at each moment to the mean value of AUC at each moment; 4, carrying out cluster analysis on a component B based on chemical structure similarity; and 5, predicating the AUC-time changing curve of the component B if the component B and the component A are of a same kind according to cluster analysis results.

Description

A kind of Chinese medicines is for dynamic (dynamical) curve simulation and Forecasting Methodology

Technical field

The present invention relates to a kind of Chinese medicines for dynamic (dynamical) curve simulation and Forecasting Methodology and system thereof, specifically, relate to AUC (under time curve the area)-time curve of a kind of composition in simulation simple and compound, and predict the AUC-time curve of another kind of composition in simple and compound.

Background technology

Chinese medicine is complicated chemical system, and there is complicated drug-drug interactions, oral monomer component and oral traditional Chinese medicine compound preparation, even if its oral dose is just the same, can exist very large difference between the two.But the property prediction that ADME is relevant is all derive from high-throughout monomeric compound, be used for predicting the ADME character of monomeric compound with their modelings.But, because Chinese medicine compound prescription exists complicated drug-drug interactions, so these forecast models are inapplicable for traditional Chinese medicine complex system, need new Research Thinking, set up new forecast model and method.

The information being disclosed in this background technology part is only intended to deepen the understanding to general background technology of the present invention, and should not be regarded as admitting or imply in any form that this information structure has been prior art known in those skilled in the art.

Summary of the invention

In the present invention, propose a kind of new predicting strategy and forecast model, mainly as follows: Chinese medicine compound prescription compound, according to chemical constitution similarity score, be divided into different classifications, of a sort compound, is considered to similar.The compound that compound structure is similar, identical target may be acted on, show similar physicochemical property, pharmacokinetic property and pharmacologically active, for this reason, a hypothesis is proposed, that is: oral traditional Chinese medicine compound preparation, wherein, the compound of similar, metabolic risk that may be similar, so, for compound Chinese medicinal preparation, acquisition one or a few compound blood concentration-time curve in vivo can be passed through, thus predict other compounds blood concentration-time curve in vivo, thus obtain relevant pharmacokinetic parameter, for Chinese medicine compound prescription pharmacokinetics provides new approaches.

For this reason, the invention provides a kind of Chinese medicines for dynamic (dynamical) curve simulation and Forecasting Methodology, can be used for the prediction of the pharmacokinetics of the structurally similar compounds of simple or Chinese medicine compound prescription, it is by some steps, find that the composition similar with confirming chemical constitution has similar change of pharmacokinetics, namely similar Γ after the complex system of oral traditional Chinese medicine prescription ⁺value; Then, at the Γ of known composition A in each moment ⁺value, predicts the AUC-time curve of other similar compositions, thus sets up pharmacokinetics prediction new approaches and method.

Particularly, the method comprises:

Step 1. couple composition A obtains its blood concentration-time curve in simple and compound medicine respectively;

Step 2. calculating composition A is each blood concentration-time area under a curve AUC measuring the moment in simple and compound medicine, and then obtains its AUC-time changing curve;

Step 3. calculating composition A is the AUC sum in each moment and the ratio Γ of mean value in simple and compound medicine ⁺, its computing formula is as follows:

${\mathrm{\&Gamma;}}^{+}\left(t_i\right)=\frac{x_A(t_i+)y_A\left(t_i\right)}{\frac{1}{n}\underset{i=0}{\overset{n-1}{\mathrm{\&Sigma;}}}(x_A\left(t_i\right)+y_A\left(t_i\right))},i=\mathrm{0,1,2},...,n-1.---\left(1\right)$

Wherein, x _a(t _i), y _a(t _i) AUC in i-th moment that be respectively composition A in compound medicine and simple;

Step 4. couple composition B does the cluster analysis based on chemical constitution similarity;

If step 5. belongs to a class together according to cluster analysis result composition B and composition A, then can at the Γ of known composition A in each moment ⁺value, the AUC value of composition B in simple (or compound medicine) and the 3rd hour (i.e. t in compound medicine (or simple) thereof ₇moment) AUC value when, prediction composition B the 4th hour (i.e. t in compound medicine (or simple) ₈moment) and later AUC-time curve, that is

$x_B\left(t_i\right)=\frac{{\mathrm{\&Gamma;}}^{+}\left(t_i\right)}{{\mathrm{\&Gamma;}}^{+}\left(t_7\right)}[x_B\left(t_7\right)+y_B\left(t_7\right)]-y_B\left(t_i\right),i=\mathrm{8,9},...,n-1---\left(2\right)$

Or

$y_B\left(t_i\right)=\frac{{\mathrm{\&Gamma;}}^{+}\left(t_i\right)}{{\mathrm{\&Gamma;}}^{+}\left(t_7\right)}[x_B\left(t_7\right)+y_B\left(t_7\right)]-x_B\left(t_i\right),i=\mathrm{8,9},...,n-1---\left(3\right)$

Wherein, x _b(t _i), y _b(t _i) AUC in i-th moment that be respectively composition B in compound medicine and simple.

In described step 1, obtained the blood concentration-time curve of composition A in simple and compound by high performance liquid chromatography mass spectrum/mass spectrometry.

When testing blood concentration, select formic acid water-methanol as mobile phase, and adopt electro-spray ionization source ESI, positive ion mode detects, and many reaction detection MRM pattern measures medicine ion concentration.

When testing blood concentration, adopt protein precipitation method in sample pre-treatments.

Calculated in simple and compound by trapezoidal method in described step 2 that composition A is at the lower area of blood concentration-time curve of each time point, described trapezoidal method computing formula is as follows:

$\begin{array}{cc}A\left(t_i\right)=A\left(t_{i-1}\right)+\frac{[C\left(t_i\right)+C\left(t_{i-1}\right)](t_i-t_{i-1})}{2},& i=\mathrm{1,2},...,n-1---\left(4\right)\end{array}$

Wherein, A (t _i) for composition A in compound and simple is in the AUC value in the i-th moment, C (t _i) for composition A in compound and simple is in the blood concentration in the i-th moment, and t ₀=0, A (0)=0, C (0)=0.

Method and apparatus of the present invention has further feature and advantage, these further features and advantage are by apparent from being incorporated in this accompanying drawing and following embodiment, or state in detail in the drawings and specific embodiments, the drawings and specific embodiments are jointly for explaining some principle of the present invention.

Accompanying drawing explanation

The description undertaken by reference accompanying drawing is below manifested by other features and advantages of the present invention, in the drawing:

Figure 1A, Figure 1B and Fig. 1 C shows corydaline (I), α-allocryptopine (II), tetrahydropalmatine (III), N-1 (IV), Imperatorin (V), Isomperatorin (VI), byak-angelicin (VII), warfarin sodium (VIII respectively, IS) and the blank plasma samples chromatogram of promethazine hydrochloride (Ⅸ, IS), blank plasma standard add LLOQ chromatogram and the plasma sample chromatogram of administration after 2 hours;

Fig. 2 is the mean blood plasma concentration-time plot (n=6) of α-allocryptopine after the different extract of SD Oral Administration in Rats;

Fig. 3 is the mean blood plasma concentration-time plot (n=6) of tetrahydropalmatine after the different extract of SD Oral Administration in Rats;

Fig. 4 is the mean blood plasma concentration-time plot (n=6) of N-1 after the different extract of SD Oral Administration in Rats;

Fig. 5 is the mean blood plasma concentration-time plot (n=6) of corydaline after the different extract of SD Oral Administration in Rats;

Fig. 6 is the mean blood plasma concentration-time plot (n=6) of byak-angelicin after the different extract of SD Oral Administration in Rats;

Fig. 7 is the mean blood plasma concentration-time plot (n=6) of Imperatorin after the different extract of SD Oral Administration in Rats;

Fig. 8 is the mean blood plasma concentration-time plot (n=6) of Isomperatorin after the different extract of SD Oral Administration in Rats;

Fig. 9 display is based on 7 composition cluster analysis results in the Yuanhu Zhitong Prescription of chemical constitution similarity score between any two;

Figure 10 shows the AUC-time changing curve of 7 compositions in simple and compound;

Figure 11 display is based on the result of 7 composition cluster analyses in the Yuanhu Zhitong Prescription of AUC-time curve change;

Figure 12 shows the Γ of 7 compositions ⁺the scatter diagram of value.

Embodiment

Present will specifically with reference to each embodiment of the present invention, in the accompanying drawings with the example of these embodiments shown in following description.Although the present invention combines with exemplary and is described, should understand, this instructions not intended to be limits the invention to those exemplary.On the contrary, the present invention is intended to not only cover these exemplary, and covers and can be included in various replacements within the spirit and scope of the present invention that limited by claims, amendment, equivalents and other embodiment.

At Chinese medicines of the present invention in dynamic (dynamical) curve simulation and Forecasting Methodology, first set up 7 main active analysis in vivos in Yuanhu Zhitong Prescription.

Yuanhu Zhitong Prescription mainly contains corydalis tuber and the root of Dahurain angelica two taste medicine composition, in corydalis tuber, principal ingredient is alkaloid compound, and principal ingredient is coumarin kind compound in the root of Dahurain angelica, to the pharmacokinetic aspect of two medicines, domestic and foreign literature concentrates on the assay to tetrahydropalmatine mostly, but the feature of Chinese medicine is multicomponent to play a role jointly, investigate other compositions content in vivo and be also extremely necessary.In order to better understand Pharmacological Mechanism, the physiological disposition of Yuanhu Zhitong Prescription, be necessary to set up a kind of sensitive and analytical approach accurately, to measure the content of principal ingredient in biological sample (as blood plasma, tissue homogenate and urine).

The LC-MS-MS method of 7 kinds of principal ingredients such as α-allocryptopine, tetrahydropalmatine, N-1, corydaline, byak-angelicin, Imperatorin, Isomperatorin will set up in Simultaneously test rat plasma sample in the present invention.

Experimental apparatus used in the present invention:

Waters Acquity UPLC System, Waters Quattro Premier XE, water generation Science and Technology Ltd.); MIKRO 220R type hydro-extractor, German Hettich company;

MX-S type turbine mixer, SCILOGEX company of the U.S.;

XS205 type analysis balance, plum Teller-Tuo benefit Instrument Ltd. of Switzerland.

Medicine used in the present invention and reagent:

Acetonitrile (chromatographically pure, Merck company);

Methyl alcohol (chromatographically pure, Merck company);

α-allocryptopine (Mei He bio tech ltd of Shenzhen, lot number: 20100318, purity: 98%);

Tetrahydropalmatine (National Institute for Food and Drugs Control, lot number: 110726-201011, purity 99.9%);

Corydaline (sigma, lot number: S449121, purity: 98%);

N-1 (upper Hiroad standing grain medical sci-tech Development Co., Ltd, lot number: 100516, purity: 98%);

Byak-angelicin (Pusi Biological Science & Technology Co., Ltd., Chengdu, lot number: RFS-B-100610-04, purity 99.9%);

Isomperatorin (National Institute for Food and Drugs Control, lot number: 110827-200407, purity: 98%);

Imperatorin (National Institute for Food and Drugs Control, lot number: 110826-200712, purity: 99.9%).

Medicinal material used in the present invention:

The root of Dahurain angelica (Angelica Dahurica (Fisch.ex Hoffm.) Benth.et Hook.f.) originates from Hebei province;

Corydalis tuber (Corydalis yanhusuoW.T.Wang) originates from Hangzhou,

All be purchased from Anguo Chinese crude drug company limited of Hebei province, be accredited as medicinal material certified products by what uncommon flourish pharmacist of Institute Of Chinese Materia Medica Of China Academy of Chinese Medical Sciences.

Animal used as test used in the present invention:

SD rat, male, body weight 230 ± 20g, is provided by Laboratory Animal Science portion of Department Of Medicine, Peking University, conformity certification number: SCXK (capital) 2009-0017.At temperature 20-22 DEG C, relative humidity 45%-65%, raises under illumination/dark 12h/12h condition, free diet, drinking-water, and adaptability starts experiment after raising 3 days.

The preparation of the standard solution that Fa Benming uses:

Precision takes α-allocryptopine respectively, tetrahydropalmatine, N-1, corydaline, byak-angelicin, Imperatorin and Isomperatorin are dissolved in methyl alcohol, be made into Stock concentrations and be respectively 1mg/mL, storing solution methanol dilution is become the mixed solution of variable concentrations, respectively containing α-allocryptopine, N-1 0.5, 1, 2.5, 5, 25, 50, 100 and 250ng/ml, containing tetrahydropalmatine and corydaline 1, 2, 5, 10, 50, 100, 200 and 500ng/ml, containing byak-angelicin, Imperatorin and Isomperatorin 2, 4, 10, 20, 100, 200 and 400 and the standard solution of 1000ng/ml, for the preparation of typical curve.

Precision takes interior mark warfarin sodium and promethazine hydrochloride is dissolved in the storing solution that methyl alcohol is mixed with 100 μ g/mL, and dilution is 50ng/mL and 25ng/mL standard solution respectively.It is for subsequent use that all solution is placed in-20 DEG C of Refrigerator stores.

The preparation of plasma sample typical curve used in the present invention and Quality Control (QC) sample:

Get rat blank plasma 50 μ l, add hybrid standard serial solution 50 μ l, being mixed with the final concentration being equivalent to α-allocryptopine and N-1 is 0.5,1,2.5,5,25,50,100 and 250ng/ml, the final concentration of tetrahydropalmatine and corydaline is 1,2,5,10,50,100,200 and 500ng/ml, the final concentration of byak-angelicin, Imperatorin and Isomperatorin is the correcting sample of 2,4,10,20,100,200,400 and 1000ng/ml, except not adding except methyl alcohol, all the other operate by under " plasma sample pre-treatment " item.

QC sample is with method dilution preparation, concentration is respectively as follows: α-allocryptopine and N-1 be 1,100,200ng/ml, tetrahydropalmatine and corydaline be 2,200,400ng/ml, byak-angelicin, Imperatorin and Isomperatorin be 4,400,800ng/ml.

Chromatograph mass spectrum analysis condition:

Chromatographic column: ACQUITY UPLC BEH C ₁₈post (2.1mm × 50mm, 1.7 μm), mobile phase: 0.1% formic acid water-methanol, elution program in Table 3-1-1, flow velocity: 0.3ml/min, column temperature: 50 DEG C, sample size: 5 μ l.

Table 1 gradient elution

Adopt electro-spray ionization source (ESI), positive ion mode detects, desolventizing gas is nitrogen, desolventizing temperature degree: 350 DEG C, flow velocity: 650L/h, ion source temperature: 120 DEG C, adopts multiple-reaction monitoring (MRM) pattern to measure medicine ion concentration, and 7 kinds of analysis ingredients and the optimum MRM parameter of interior target are in table 2.

7 compositions and 2 interior target MS parameters in table 2 Yuanhu Zhitong Prescription

Plasma sample pre-treatment:

Get rat plasma 50 μ l, add 50 μ l methyl alcohol successively, 150 μ l are containing the methanol solution of interior mark (warfarin sodium 50ng/mL and promethazine hydrochloride 25ng/mL), and vortex shakes 3min, the centrifugal 10min of 15000r/min, get supernatant 200 μ l, nitrogen dries up, and redissolves with 100 μ l 50% methyl alcohol, vortex 1min, 15000 centrifugal 5min, get supernatant 5 μ l sample introduction, carry out LC-MS analysis.

The analysis in vivo checking that the present invention carries out is comprised:

(1) specificity of method is investigated:

The blank plasma of 6 Different Individual and standard are added blood plasma, and (α-allocryptopine and N-1 are 0.5ng/ml, tetrahydropalmatine and corydaline are 1ng/ml, byak-angelicin, Imperatorin and Isomperatorin are 2ng/ml), except not adding interior mark, other carry out UPLC-MS/MS analysis by after the method operation under " plasma sample pre-treatment " item, more different blank plasma and standard add the chromatogram of blood plasma, whether there is interference with the endogenous component investigated in blank plasma.

(2) range of linearity and lower limit of quantitation (LLOQ):

In three days of methodology confirmation, every day prepares 3 cover typical curves, and take testing concentration as horizontal ordinate, the peak area ratio of determinand and internal standard compound is ordinate, with weighting (W=1/x ²) least square method carries out regressing calculation, try to achieve equation of linear regression and be typical curve.

6 samples were prepared to typical curve minimum point concentration in the 3rd day and analyze in method confirmation, and calculate each sample mensuration concentration according to typical curve on the same day, LLOQ is the least concentration point on typical curve, its response should be more than 5 times (S/N >=5) of blank bio-matrix chaff interference response, and precision is expressed as relative standard deviation (relative standard deviation, RSD)≤20% is answered, accuracy is expressed as bias (bias), should in ± 20% scope.

(3) precision and accuracy:

Get blank rat plasma 50 μ L, operate by " preparation of plasma sample typical curve and Quality Control (QC) sample " Xiang Tongfa, prepare high, medium and low three concentration (α-allocryptopine and N-1 be 1,100,200ng/ml, tetrahydropalmatine and corydaline be 2,200,400ng/ml, byak-angelicin, Imperatorin and Isomperatorin be 4,400,800ng/ml) quality-control sample, each concentration carries out 6 sample analyses every day, METHOD FOR CONTINUOUS DETERMINATION three days, according to the same day typical curve calculate quality-control sample record concentration.According to quality-control sample measurement result calculate this law in a few days, day to day precision and accuracy.

(4) extraction recovery and matrix effect:

Investigate the impact of component to be measured matrix effect under variable concentrations, and the blood plasma extraction recovery under this condition.The standard preparing component to be measured adds plasma sample (basic, normal, high QC concentration, operation with method under " preparation of plasma sample typical curve and Quality Control (QC) sample " item), measure concentration (set 3 sample) according to after the operation of " plasma sample pre-treatment " method; Standard after blank plasma albumen precipitation is added compound mensuration concentration to be measured (set 2 sample); By mobile phase preparation respective concentration not containing the pure sample product measured in solution concentration (set 1 sample) of matrix.The calculated value of matrix effect is set 2 sample and the chromatographic peak area ratio of set 1 sample; The calculated value of extraction recovery is set 3 sample and the chromatographic peak area ratio of set 2 sample.Mark matrix effect and the extraction recovery of warfarin sodium (50ng/mL) and promethazine hydrochloride (25ng/mL) in investigating simultaneously.

(5) sample stability:

In Method validation process, the stability analyzing sample is investigated, comprise the stability after 1 time, 3 times Frozen-thawed cycled, preserve the long-time stability of 2 weeks under short-term stability – 20 DEG C of freezing conditions of the lower 25 DEG C of placement 4h of room temperature, and the plasma sample after process (4 DEG C) in automatic sampler places the stability of 12h.During study on the stability, get blank plasma 50 μ L, according to legal system below " preparation of plasma sample typical curve and Quality Control (QC) sample " item for Quality Control (QC) sample of basic, normal, high three concentration, add in blank plasma by 1:10, each concentration carries out 3 sample analyses, with measured value compared with sign concentration, represent with relative deviation (RE).

Analysis in vivo the result is as follows:

(1) specificity:

Blank rat plasma, after the additional standard solution of blank plasma and rat administration, plasma sample chromatogram is shown in Figure 1A to Fig. 1 C.Component to be measured and interior target retention time specific as follows: α-allocryptopine is 3.55min, tetrahydropalmatine is 3.54min, N-1 is 3.64min, corydaline is 3.68min, byak-angelicin is 4.63min, Imperatorin is 5.76min and Isomperatorin is 6.12min, N-1 is 6.85min, warfarin sodium (IS) is 4.49min, promethazine hydrochloride (IS) is 5.67min, and the endogenous component in blank plasma does not cause interference to mensuration.

(2) typical curve and lower limit of quantitation:

Article 3, typical curve all shows good linear relationship in the following concentration range investigated, α-allocryptopine and N-1: 0.5-500ng/mL, tetrahydropalmatine and corydaline: 1-1000ng/mL, byak-angelicin, Imperatorin and Isomperatorin: 2-2000ng/mL, each ingredient standard curve correlation coefficient (R ²) be all greater than 0.99.Lower limit of quantitation experimental result shows, under mensuration concentration, the withinday precision (RSD) of 7 kinds of compositions to be measured is respectively 2.4%, 2.8%, 1.9%, 1.9%, 8.1%, 3.9%, 1.7%, and accuracy (RE) is respectively 4.0%, 3.0%, 4.0%, 4.0% ,-9.0% ,-3.0%, 5.0%.The equation of linear regression of composition to be measured is in table 3.

The equation of linear regression of table 37 kinds of compositions

(3) precision and accuracy:

As shown in table 4, under high, medium and low three concentration, in a few days be respectively 1.0% ~ 5.3% and 1.5% ~ 6.1% with day to day precision (RSD), in a few days be respectively-3.2% ~ 7.1% and-7.0% ~ 7.5% with accuracy in the daytime (RE), data show that the preci-sion and accuracy of component to be measured in rat plasma all meets the analysis requirement of biological sample.

Table 4 α-allocryptopine, tetrahydropalmatine, N-1, corydaline, byak-angelicin, Imperatorin and the preci-sion and accuracy of Isomperatorin in rat plasma (n=6)

(4) recovery and matrix effect:

Table 5 lists component to be measured and interior mark (the warfarin sodium 50ng/mL of basic, normal, high 3 concentration; Promethazine hydrochloride 25ng/mL) matrix effect and extraction recovery investigate result.Extraction recovery result shows, under this blood plasma pre-treating method, precipitation of protein to the extraction recovery of 7 compositions to be measured all more than 88.8%.Matrix effect investigates result display, and the ion of the endogenous material under this method condition determination in blood plasma suppresses or humidification can be ignored, on the mass spectrum response not impact of 7 kinds of compositions to be measured.

Extraction recovery in rat plasma of table 5 α-allocryptopine, tetrahydropalmatine, N-1, corydaline, byak-angelicin, Imperatorin and Isomperatorin and matrix effect (n=6)

(5) stability:

Table 6 α-allocryptopine, tetrahydropalmatine, N-1, corydaline, byak-angelicin, Imperatorin and the stability of Isomperatorin in rat plasma (n=6)

Study on the stability result shows that 7 kinds of component blood slurry samples are through three Frozen-thawed cycled (RE:-10.4%-10.9%), lower 25 DEG C of room temperature places 4h (RE:-5.5%-13.0%), all stable after preserving 2 weeks (RE:-10.3%-10.5%) under-20 DEG C of freezing conditions, the plasma sample after process (4 DEG C) in automatic sampler places stable (RE:-9.2%-12.1%) concrete outcome of 12h in table 6.

From above table, 7 kinds of compositions all keep stable in storage, process and mensuration process.

Through checking, this analytical approach meets the requirement of Determination of Biological Samples.

Next, carry out UPLC-MS/MS condition optimizing, interior target is selected and the optimization of plasma sample pre-treating method.

First investigate mobile phase, methyl alcohol and acetonitrile all relatively good as mobile phase chromatographic peak profile, but acetonitrile as mobile phase time, the response of byak-angelicin be methyl alcohol as 1/10th of mobile phase, so, select methyl alcohol as mobile phase.In mass spectrophotometry, in mobile phase, add the acid ingredients such as formic acid, chromatographic peak profile can be improved significantly.Investigate the addition of formic acid in aqueous phase herein, find along with formic acid addition increases, from 0.05%, 0.1% and 0.2%, but the response of alkaloids composition significantly strengthens, and its peak shape also makes moderate progress, therefore we have selected chromatographic peak profile and the mass spectrum response that a suitable formic acid concn (0.1%) takes into account all components to be measured.

When analyzing biological sample, issuable error when being marked with correcting sample pre-service in usually adding and analyzing, and facilitate sample pretreatment, improve processing speed.For using the instrument of this highly sensitive low stability of mass spectrum as detecting device, interior target adds especially necessary.What consider that we analyze is cumarin and alkaloid two compounds, first by analyzing the structural similarity of similarity software calculated candidate compound and testing compound, the compound that a large amount of screening structure is similar, candidate compound is screened further by Determination of oil-water partition coefficient, every element of the first species selects 3 candidate compounds as interior mark, then according to chromatogram and mass spectrographic testing result, select best interior mark, finally we have selected warfarin sodium (for Coumarins composition) and promethazine hydrochloride (for biological alkali components) two medicines and mark as in this two constituents, respectively it is corrected.

In test, we attempt comparing albumen precipitation (comprising methanol extraction, acetonitrile precipitation and methyl alcohol: acetonitrile=1:1 precipitation), liquid-liquid extraction and Solid-Phase Extraction.In albumen precipitation, the recovery of methanol extraction method is higher than acetonitrile precipitation and methyl alcohol: acetonitrile=1:1 precipitation, and matrix effect is smaller; Liquid-liquid extraction has been investigated ethyl acetate respectively and has been added 12.5% ammoniacal liquor and 0.2M NaOH, the recovery adding 12.5% ammoniacal liquor is higher than and adds 0.2M NaOH, the method matrix effect adding alkali due to liquid-liquid extraction is larger, and the recovery of albumen precipitation method is higher than liquid-liquid extraction, consider the sample that subsequent experimental is a large amount of, costly and comparatively take, therefore, this experiment have finally chosen the pre-treating method of methanol extraction albumen to SPE.Find because the organic phase ratio in sample is higher than the organic phase ratio in mobile phase, take two kinds of methods: one is redissolve with 50% methyl alcohol after supernatant nitrogen dries up, and two is that supernatant dilutes with equal-volume water.Find that supernatant nitrogen dries up through investigation and be better than supernatant with water-reducible method, we have also investigated 3 simultaneously, 4, mass spectrum response after the methanol extraction plasma proteins of 5 times of volumes, find the methyl alcohol process of 3 times of volumes, component to be measured can be extracted comparatively completely and dilute less, thus obtain maximum mass spectrum response.

Therefore, establish simple, sensitive and LC-ESI-MS analytical approach accurately in the present invention, for α-allocryptopine, tetrahydropalmatine, N-1, corydaline, byak-angelicin, Imperatorin and Isomperatorin in Simultaneous Determination rat plasma.Sample pre-treatments adopts protein precipitation method, simple and fast.This method specificity is good, highly sensitive, its lower limit of quantitation is respectively 0.5ng/mL (corydaline and tetrahydropalmatine), 1ng/mL (N-1 and α-allocryptopine) and 2ng/mL (byak-angelicin, Imperatorin and Isomperatorin), for follow-up pharmacokinetic provides reliable analytical approach.

Hereinafter, the pharmacokinetics of Yuanhu Zhitong Prescription 7 main active in rat plasma is studied.

In order to compare simple and whether Yuanhu Zhitong Prescription changes to the pharmacokinetics of main active after Oral Administration in Rats administration, we by Corydalis P.E, Angelica Dahurica extract and corydalis tuber analgesic formula extraction (corydalis tuber: the root of Dahurain angelica=2:1) respectively to rat oral gavage, compared by the pharmacokinetics behavior of simple and Yuanhu Zhitong Prescription, interaction trend after both suppositions drug combination, for the application of Yuanhu Zhitong Prescription compatibility provides rational basis and guidance.

Get angelica root 1kg, pulverize No. 6 sieves.Soak 1h, extract with 4 times amount 70% alcohol refluxs, extraction time is 2h, extracts 2 times successively, and merge filtered fluid, 70 DEG C of drying under reduced pressure are to powder, and extraction ratio is 9.52%; Get corydalis tuber medicinal material 1kg, pulverize No. 6 sieves.Soak 1h, extract with 4 times amount 70% alcohol refluxs, extraction time is 2h, extracts 2 times successively, and merge filtered fluid, 70 DEG C of drying under reduced pressure are to powder, and extraction ratio is 8.15%.

Measure the content of main active in corydalis tuber and Angelica Dahurica extract respectively, result shows that in Corydalis P.E, the content of main active is as follows: α-allocryptopine is 3.20mg/kg, corydaline is 11.59mg/kg, tetrahydropalmatine is 4.19mg/kg, N-1 is 1.13mg/kg, in Angelica Dahurica extract, the content of main active is as follows: byak-angelicin is 0.69mg/kg, and Imperatorin is 3.78mg/kg, and Isomperatorin is 1.90mg/kg.

Get a heparin, often prop up 1g, be placed in the volumetric flask of 100mL, be settled to scale with physiological saline, be made into the heparin solution of 1%, prepare 500ml altogether, be placed in 4 DEG C and store for future use.

Take Corydalis P.E 6.52g respectively, Angelica Dahurica extract 3.81g, Yuanhu Zhitong Prescription (Corydalis P.E 6.52g, Angelica Dahurica extract 3.81g), the aqueous solution adding 50ml grinds to form suspension solution, for rat oral gavage.Liquid prepared before use.

18 SD rats, body weight 230 ± 20g, is divided into 3 groups at random, and often organize 6, male and female half and half, before administration, 12h fasting, freely drinks water, and gavage gives corydalis tuber analgesic formula extraction, Angelica Dahurica extract and Corydalis P.E respectively, feeds with feed after administration 4h.Before administration and after administration 0.17,0.33,0.67,1,1.5,2,3,4,6,8,12,24,36,48h gets blood by orbital venous plexus and is about 0.5mL, anticoagulant heparin, gets blood plasma after the centrifugal 10min of 3500 × g, to be measured in-20 DEG C of preservations.

The non-compartment model method (statistics moments method) of DAS2.0 data processing software (quantitative pharmacology Professional Committee of Chinese Pharmacological Society) is adopted to carry out pharmacokinetic parameter C _max, T _max, t _1/2, AUC, MRT, CL/F etc. calculating.

In experiment after the oral Corydalis P.E of female rats, plasma sample drug concentration is far away higher than male rat blood concentration, to cause in indivedual plasma sample corydalis paper mill wastewater beyond the range of linearity of typical curve, concentration is exceeded to the plasma sample of the range of linearity, redeterminate after diluting with blank plasma, investigated the dilution effect of sample, experimental result shows, adopt blank plasma diluting high-concentration sample post analysis, the recovery, blood concentration are had no significant effect.

After 3 groups of each extracts of SD rat oral gavage, adopt the method for above-mentioned foundation to analyze each time point blood plasma, blood concentration-time data are in table 7 to 20, and mean blood plasma concentration-time data is in table 21.Adopt non-compartment model method to calculate pharmacokinetic parameter, the results are shown in Table 22 and 23.

Table 7 rat oral gavage gives the blood concentration-time data (ng/ml, n=6) of α-allocryptopine after first Corydalis P.E

N.d. do not detect, at below LLOQ

Table 8 rat oral gavage gives the blood concentration-time data (ng/ml, n=6) of α-allocryptopine after corydalis tuber analgesic formula extraction

Table 9 rat oral gavage gives the blood concentration-time data (ng/ml, n=6) of tetrahydropalmatine after Corydalis P.E

N.d. do not detect, at below LLOQ

The blood concentration-time data (ng/ml, n=6) of tetrahydropalmatine after table 10 Oral Administration in Rats corydalis tuber analgesic formula extraction

Table 11 rat oral gavage gives the blood concentration-time data (ng/ml, n=6) of N-1 after Corydalis P.E

Table 12 rat oral gavage gives the blood concentration-time data (ng/ml, n=6) of N-1 after corydalis tuber analgesic formula extraction

Table 13 rat oral gavage gives the blood concentration-time data (ng/ml, n=6) of corydaline after Corydalis P.E

N.d. do not detect, at below LLOQ

Table 14 rat oral gavage gives the blood concentration-time data (ng/ml, n=6) of corydaline after corydalis tuber analgesic formula extraction

Table 15 rat oral gavage gives the blood concentration-time data (ng/ml, n=6) of byak-angelicin after Angelica Dahurica extract

Table 16 rat oral gavage gives the blood concentration-time data (ng/ml, n=6) of byak-angelicin after corydalis tuber analgesic formula extraction

Table 17 rat oral gavage gives the blood concentration-time data (ng/ml, n=6) of Imperatorin after Angelica Dahurica extract

Table 18 rat oral gavage gives the blood concentration-time data (ng/ml, n=6) of Imperatorin after corydalis tuber analgesic formula extraction

Table 19 rat oral gavage gives the blood concentration-time data (ng/ml, n=6) of Isomperatorin after Angelica Dahurica extract

Table 20 rat oral gavage gives the blood concentration-time data (ng/ml, n=6) of Isomperatorin after corydalis tuber analgesic formula extraction

Table 21 mean blood plasma concentration-time data

Main pharmacokinetic parameter (n=6) after the alone and Yuanhu Zhitong Prescription oral administration of table 22 corydalis tuber

^*p<0.01, ^*p<0.05 is for corydalis tuber

Main pharmacokinetic parameter (n=6) after the alone and Yuanhu Zhitong Prescription oral administration of table 23 root of Dahurain angelica

^*p<0.01, ^*p<0.05 is for the root of Dahurain angelica

Pharmacokinetic parameter result of calculation shows: as shown in Figure 2, and SD rat oral gavage Yuanhu Zhitong Prescription group is compared with corydalis tuber group, and the peak time of α-allocryptopine extends to some extent, in body, average residence time extends 0.8 times, there is significant difference, P<0.01, AUC _{0 → t}and AUC _{0 → ∞}increase all to some extent by comparison, but not there is statistical significance.

As shown in Figure 3, Yuanhu Zhitong Prescription group is compared with corydalis tuber group, the AUC of tetrahydropalmatine _{0 → t}, AUC _{0 → ∞}and MRT _(0-t)have conspicuousness to improve, P<0.01, peak time extends 3.8 times, P<0.05.

As shown in Figure 4, Yuanhu Zhitong Prescription group is compared with corydalis tuber group, and N-1 Cmax improves 2 times, and peak time extends 4 times, and in body, average residence time extends 0.8 times, P<0.01, AUC _{0 → t}and AUC _{0 → ∞}all enlarge markedly, there is statistical significance.

As shown in Figure 5, Yuanhu Zhitong Prescription group is compared with corydalis tuber group, and corydaline Cmax increases, and peak time extends 2 times, has statistical significance, and in body, average residence time extends 0.65 times, has statistical significance, P<0.05.

Fig. 6 to Fig. 8, is respectively the blood concentration-time curve map deriving from composition byak-angelicin in the root of Dahurain angelica, Imperatorin, Isomperatorin 3 compositions.As can be seen from the figure, relative to simple, the AUC of these 3 compositions in compound _{0 → t}and AUC _{0 → ∞}all obviously reduce.Meanwhile, chemically compositional classification, angelicin, Imperatorin, Isomperatorin 3 one-tenth belong to coumarinoids, different with 4 alkaloids compositions, these 3 composition AUC _{0 → t}and AUC _{0 → ∞}all obviously reduce, bioavilability reduces, and expression is not the AUC of same class composition, chemical composition _{0 → t}and AUC _{0 → ∞}change different, the AUC of same class composition _{0 → t}and AUC _{0 → ∞}variation tendency has consistance.And according to document, Coumarins composition has hepatic injury effect, the above results provides foundation for compatibility attenuation.

Alkaloid component in Yuanhu Zhitong Prescription in corydalis tuber is the principal ingredient of analgesic activity, the corydalis tuber compatibility root of Dahurain angelica, and the root of Dahurain angelica can extend the peak time of principal alkaloid constituents in corydalis tuber, the time lengthening of blood concentration of remaining valid, simultaneously AUC _{0 → t}and AUC _{0 → ∞}enlarge markedly, make the blood concentration longer time be in Valid concentration, better play drug effect, from the angle of pharmacokinetics, explain the principle of corydalis tuber and the root of Dahurain angelica 5.Simultaneously, we find from experimental result, and after root of Dahurain angelica compatibility corydalis tuber, corydalis tuber significantly reduces the blood concentration of main coumarinoids in the root of Dahurain angelica, show that corydalis tuber and the root of Dahurain angelica two taste Chinese medicine exist drug-drug interactions, for the physiological disposition research of Yuanhu Zhitong Prescription is laid a good foundation.

Next, the AUC-time curve of calculating and matching 7 principal ingredients.

The AUC-time curve of 7 compositions in Yuanhu Zhitong Prescription is calculated according to following trapezoidal method computing formula:

$\begin{array}{cc}A\left(t_i\right)=A\left(t_{i-1}\right)+\frac{[C\left(t_i\right)+C\left(t_{i-1}\right)](t_i-t_{i-1})}{2},& i=\mathrm{1,2},...,13---\left(5\right)\end{array}$

Wherein, t ₀=0, A (0)=0, C (0)=0.

Can following table be obtained by above-mentioned formula:

Table 24 medicine AUC-time data

At the AUC-time curve of simple and compound, correlation analysis between compound structure is changed to 7 principal ingredients:

MedChem Studio software, as a visual Chemoinformatics research platform, with Tanimoto coefficient, carries out structural similarity evaluation to 7 compounds.Compound similarity score between any two utilizes SPSS software in table 23., is analyzed, the results are shown in Figure 9 by cluster analysis (PCA) to chemical constitution similarity score between any two.

As can be seen from result, α-other recessive alkali, corydaline, tetrahydropalmatine and N-1 belong to alkaloid compound, carry out cluster analysis result display, from being divided into same class with structural similarity marking; Imperatorin, Isomperatorin and byak-angelicin belong to coumarin kind compound, carry out cluster analysis result display with structural similarity marking, another kind of from being divided into.

Table 25 7 compositions chemical constitution similarity score between any two

7 principal ingredients are as follows at the AUC-time changing curve computing function of simple and compound:

${\mathrm{AUC}}_{v_i}=\frac{{\mathrm{AUC}}_{d_i}-{\mathrm{AUC}}_{\mathrm{fi}}}{C_{\max }},i=\mathrm{1,2},...,13---\left(6\right)$

Wherein, AUC _vifor in different time points, the AUC change of composition I between simple and compound; AUC _difor the AUC of the simple in different time points; AUC _fithe AUC of the compound of different time points, C _maxfor concentration maximum between simple and compound.

In Yuanhu Zhitong Prescription and simple, 7 composition AUC-time changing curves are shown in Figure 10, as seen from the figure, along with the prolongation of time, and Coumarins composition (Imperatorin, Isomperatorin and byak-angelicin) AUC _vicontinuous increase, but, alkaloids composition (α-other recessive alkali, corydaline, tetrahydropalmatine and N-1) AUC _vicontinuous minimizing.Further cluster analysis is carried out to change curve, result shows, 3 Coumarins compositions (Imperatorin, Isomperatorin and byak-angelicin) are gathered same class, 4 alkaloids compositions (α-other recessive alkali, corydaline, tetrahydropalmatine and N-1), illustrate to there is the composition that chemical constitution is similar similar AUC _vichange curve.

By cluster analysis, chemical constitution similarity score is between any two analyzed, the results are shown in Figure 11.

Next, 1 compound is utilized to predict other compounds AUC-time curve in compound at the AUC-time curve Changing Pattern of simple and compound.

Find 7 kinds of compositions to be divided into two classes by the ratio calculating the AUC sum of each composition in Yuanhu Zhitong Prescription and corydalis tuber (or root of Dahurain angelica) and its mean value, and propose a kind of new Forecasting Methodology thus.

First, the AUC sum of each composition in compound and simple and the ratio Γ of its mean value is calculated ⁺, this ratio Γ ⁺computing formula as follows:

${\mathrm{\&Gamma;}}^{+}\left(t_i\right)=\frac{x\left(t_i\right)+b\left(t_i\right)}{\frac{1}{n}\underset{i=0}{\overset{n-1}{\mathrm{\&Sigma;}}}(x\left(t_i\right)+b\left(t_i\right))},i=\mathrm{1,2},...,13---\left(7\right)$

Wherein, x (t _i) be in compound composition I at the AUC in i moment, b (t _i) for become the AUC of I in the i moment in simple.

Each composition is at the ratio Γ in each moment ⁺in table 24.

The each composition of table 26 is at the ratio Γ in each moment ⁺

Scatter diagram is as shown in figure 12 made according to upper table.

As seen from Figure 12, above-mentioned seven curves can be divided into two classes by degree of closeness, namely the recessive alkali of α-other, tetrahydropalmatine-S, N-1-S are a class with corydaline, and byak-angelicin, Imperatorin and Isomperatorin-S are another kind of.

Following table is tetrahydropalmatine-S, N-1-S, corydaline and the recessive alkali of α-other and Imperatorin, Isomperatorin-S Γ corresponding to byak-angelicin ⁺ratio between value.

Table 27 tetrahydropalmatine-S, N-1-S, corydaline and the recessive alkali of α-other and Imperatorin, Isomperatorin-S Γ corresponding to byak-angelicin ⁺ratio between value

Can be found out by upper table, corresponding Γ after 3 hours ⁺ratio between value is all distributed in interval (0.84,1.11).

Infer based on above-mentioned theory, we can adopt following method at the Γ of known composition A in each moment ⁺value the AUC value of composition B in simple (compound) and in compound (simple) the AUC value x (t of the 3rd hour ₇) when, prediction composition B 4 hours and later AUC value in compound (simple)

Below, for sake of convenience, note by abridging i=0,1,2 ..., n-1.Following linear equation group can be obtained by (1)-(3).

$\left\{\begin{array}{c}{x}_7+b_7=\frac{a_7}{n}\underset{j=0}{\overset{n-1}{\mathrm{\&Sigma;}}}(x_j+b_j)\\ x_8+b_8=\frac{a_8}{n}\underset{j=0}{\overset{n-1}{\mathrm{\&Sigma;}}}(x_j+b_j)\\ ......\\ x_{n-2}+b_{n-2}=\frac{a_{n-2}}{n}\underset{j=0}{\overset{n-1}{\mathrm{\&Sigma;}}}(x_j+b_j)\\ {(x_j+b_j)}_{n-1}=\frac{a_{n-1}}{n}\underset{j=0}{\overset{n-1}{\mathrm{\&Sigma;}}}(x_j+b_j)\end{array}\right.---\left(8\right)$

Separate this system of equations, can obtain and separate as follows:

$x_i=\frac{a_i}{a_7}(x_7+b_7)-b_i,i=\mathrm{8,9},...,n-1---\left(9\right)$

That is

$x\left(t_i\right)=\frac{a\left(t_i\right)}{a\left(t_7\right)}[x\left(t_7\right)+b\left(t_7\right)]-b\left(t_i\right),i=\mathrm{8,9},...,n-1---\left(10\right)$

Embodiment 1 is by the Γ of the recessive alkali of composition α-other ⁺the AUC value of value prediction composition tetrahydropalmatine-S in Yuanhu Zhitong Prescription after 4 hours

Now the recessive alkali of composition α-other corresponds to moment t _iΓ ⁺value a (t _i) and tetrahydropalmatine-S in corydalis tuber correspond to moment t _iaUC value b (t _i) (i=7,8 ..., 13) and as following table.

The Γ of the recessive alkali of table 28 α-other ⁺value a (t _i) and the AUC value of tetrahydropalmatine-S in corydalis tuber

X (t in addition ₇)=971.4730917.Can be regarded as tetrahydropalmatine-S corresponds to moment t in Yuanhu Zhitong Prescription according to (10) (n=14) _iaUC value x (t _i) (i=8,9 ..., 13).Now correlated results is listed in the table below.

The AUC experiment value of table 29 tetrahydropalmatine-S in Yuanhu Zhitong Prescription and predicted value comparable situation

Embodiment 2 is by the Γ of the recessive alkali of composition α-other ⁺the AUC value of value prediction composition N-1-S in Yuanhu Zhitong Prescription after 4 hours

According to method as above, following result can be obtained:

The AUC experiment value of table 30 N-1-S in Yuanhu Zhitong Prescription and predicted value comparable situation

Embodiment 3 is by the Γ of the recessive alkali of composition α-other ⁺the AUC value of value prediction composition corydaline in Yuanhu Zhitong Prescription after 4 hours

According to method as above, following result can be obtained:

The AUC experiment value of table 31 corydaline in Yuanhu Zhitong Prescription and predicted value comparable situation

Embodiment 4 is by the Γ of composition byak-angelicin ⁺the AUC value of value prediction composition Imperatorin in Yuanhu Zhitong Prescription after 4 hours

According to method as above, following result can be obtained:

The AUC experiment value of table 32 Imperatorin in Yuanhu Zhitong Prescription and predicted value comparable situation

Embodiment 5 is by the Γ of composition byak-angelicin ⁺the AUC value of value prediction composition Isomperatorin-S in Yuanhu Zhitong Prescription after 4 hours

According to method as above, following result can be obtained:

The AUC experiment value of table 33 Imperatorin in Yuanhu Zhitong Prescription and predicted value comparable situation

Claims (5)

1. a Chinese medicines is for dynamic (dynamical) curve simulation and Forecasting Methodology, it is after the complex system of oral traditional Chinese medicine prescription, find that the composition similar with confirming chemical constitution has similar change of pharmacokinetics, thus predicted the pharmacokinetic parameter of other similar compositions at the pharmacokinetic parameter in each moment by principal component, described method comprises:

Step 1. couple composition A obtains its blood concentration-time curve in simple and compound medicine respectively;

Step 2. calculating composition A is each blood concentration-time area under a curve AUC measuring the moment in simple and compound medicine, and then obtains its AUC-time changing curve;

Step 3. calculating composition A is the AUC sum in each moment and the ratio Γ of mean value in simple and compound medicine ⁺, its computing formula is as follows:

${\mathrm{\&Gamma;}}^{+}\left(t_i\right)=\frac{x_A\left(t_i\right)+y_A\left(t_i\right)}{\frac{1}{n}\underset{i=0}{\overset{n-1}{\mathrm{\&Sigma;}}}[x_A\left(t_i\right)+y_A\left(t_i\right)]},i=\mathrm{0,1,2},...,n-1.---\left(1\right)$

Wherein, x _a(t _i), y _a(t _i) AUC in i-th moment that be respectively composition A in compound medicine and simple;

Step 4. couple composition B does the cluster analysis based on chemical constitution similarity.

If step 5. belongs to a class together according to cluster analysis result composition B and composition A, then can at the Γ of known composition A in each moment ⁺value, the AUC value of composition B in simple or compound medicine and in compound medicine or simple when the AUC value of the 3rd hour, predicting composition B the 4th hour and later AUC-time curve in compound medicine or simple, the 3rd hour is wherein t ₇moment the 4th hour is t ₈moment, that is

$x_B\left(t_i\right)=\frac{{\mathrm{\&Gamma;}}^{+}\left(t_i\right)}{{\mathrm{\&Gamma;}}^{+}\left(t_7\right)}[x_B\left(t_7\right)+y_B\left(t_7\right)]-y_B\left(t_i\right),i=\mathrm{8,9},...,n-1.---\left(2\right)$

Or

$y_B\left(t_i\right)=\frac{{\mathrm{\&Gamma;}}^{+}\left(t_i\right)}{{\mathrm{\&Gamma;}}^{+}\left(t_7\right)}[x_B\left(t_7\right)+y_B\left(t_7\right)]-x_B\left(t_i\right),i=\mathrm{8,9},...,n-1.---\left(3\right)$

Wherein, x _b(t _i), y _b(t _i) AUC in i-th moment that be respectively composition B in compound medicine and simple.

2. Chinese medicines according to claim 1 is for dynamic (dynamical) curve simulation and Forecasting Methodology, is obtained the blood concentration-time curve of composition A in simple and compound medicine in wherein said step 1 by high performance liquid chromatography mass spectrum/mass spectrometry.

3. Chinese medicines according to claim 2 is for dynamic (dynamical) curve simulation and Forecasting Methodology, wherein select formic acid water-methanol as mobile phase, and adopt electro-spray ionization source ESI, positive ion mode detects, and many reaction detection MRM pattern measures medicine ion concentration.

4. Chinese medicines, for dynamic (dynamical) curve simulation and Forecasting Methodology, wherein adopts protein precipitation method in sample pre-treatments according to claim 2.

5. Chinese medicines according to claim 1 is for dynamic (dynamical) curve simulation and Forecasting Methodology, calculated in simple and compound medicine by trapezoidal method in wherein said step 2 that composition A is at the lower area of blood concentration-time curve of each time point, described trapezoidal method computing formula is as follows:

$A\left(t_i\right)=A\left(t_{i-1}\right)+\frac{[C\left(t_i\right)+C\left(t_{i-1}\right)](t_i-t_{i-1})}{2},i=\mathrm{1,2},...,n-1.---\left(4\right)$

Wherein, A (t _i) for composition A in compound and simple is in the AUC value in the i-th moment, C (t _i) for composition A in compound and simple is in the blood concentration in the i-th moment, and t ₀=0, A (0)=0, C (0)=0.