CN107064480B

CN107064480B - Chinese medicinal ingredients promote evaluation method to azithromycin effect baseline and evaluate the application of gynaecology's prescriptions worth thousand gold

Info

Publication number: CN107064480B
Application number: CN201610663460.1A
Authority: CN
Inventors: 龚云; 李伏君; 张鹏; 邹亮; 彭开锋
Original assignee: Zhuzhou Qianjin Pharmaceutical Co Ltd
Current assignee: Zhuzhou Qianjin Pharmaceutical Co Ltd
Priority date: 2016-08-12
Filing date: 2016-08-12
Publication date: 2019-05-03
Anticipated expiration: 2036-08-12
Also published as: CN107064480A

Abstract

Evaluation method is promoted to azithromycin effect baseline the invention discloses a kind of Chinese medicinal ingredients and evaluates the application of gynaecology's prescriptions worth thousand gold.The present invention is using additions such as baselines, the exposure level for having proven to clearly effective therapeutic agent azithromycin is measured first, azithromycin is given to experimental model parallel with for examination Chinese medicine composition, the baseline level of drug combination and the curative effect trend of drug combination are measured, population pharmacokinetics-Population pharmacedynamics conjunctive model is established and is verified.Scientific design of the present invention can dynamics-pharmacodynamics systematic variation dynamic system for participating in of quantitative assessment priming, the drawbacks of changing the research method that weak efficiency curative effect of medication index point method measurement mean value compares.Apply the inventive method to the promotion evaluation of gynaecology's prescriptions worth thousand gold, specify that gynaecology's prescriptions worth thousand gold composition influences the synergy of each pharmacokinetic parameters of azithromycin, summarize the key agents group for obtaining playing anti-inflammatory and promoting blood circulation effect in gynaecology's prescriptions worth thousand gold composition, in terms of the method for the present invention can be advantageously applied to evaluation Chinese medicine composition and antibiotic combination safety and validity, it is particularly applied to the effect aspect that disappears in Radix Angelicae Sinensis ingredient priming in evaluation gynaecology's prescriptions worth thousand gold.

Description

Chinese medicinal ingredients promote evaluation method and evaluation gynaecology's a thousand pieces of gold to azithromycin effect baseline The application of side

Technical field

The present invention relates to drug evaluation technical fields, more particularly, to active constituent in Chinese medicine composition to Zitromax The evaluation method of element effect baseline winding level, active constituent makees azithromycin especially in Chinese medicine composition gynaecology prescriptions worth thousand gold With the evaluation method of baseline winding level.

Technical background

According to the literature, traditional Chinese medicine think chronic pelvic inflammatory disease (chronicpelvicinflammatorydisease, CPID) dominant mechanism is that married woman's menstrual period, postpartum blood room are just opened, and remaining blood is not easily the six external factors which cause diseases to the greatest extent, seven feelings, diet, overstrain and sexual strain institute Wound influences punching and appoints qi and blood to suffer from the stasis of blood.

In recent years, as TCM Modernization process accelerates, combined Chinese & western medicine is increasingly extensive.Clinic is to chronic pelvic Scorching treatment is also frequently with the combined therapy of Chinese and Western medicine or Chinese medicine and Antibiotic combination application.Currently, numerous studies report gynaecology thousand Gold plaque and the raising significant in efficacy of Antibiotic combination application for the treatment of chronic pelvic inflammatory disease, and adverse reaction rate is substantially reduced.Gynaecology A thousand pieces of gold piece combines azithromycin to the curative effect of chronic gynaecological imflammation better than azithromycin is applied alone, and the two is often clinically shared treatment Chronic gynaecological inflammation disease.

In practice, chronic pelvic inflammatory disease is treated, Chinese medicine application activating blood and removing stasis Method can obviously improve clinical symptoms, and western medicine is then recognized For the aseptic inflammation that it is after acute pelvitis of pelvic cavity, the abnormal changes such as hemorheology, immunology, pathology are shown as.Therefore, Treatment uses drug local action in anti-infective, anti-inflammatory and uterine cavity also to have clinical literature to think that uterus is promoted to receive for the basic rules for the treatment of Contracting is discharged infectious uterine secretions or has more significant curative effect using uterine cavity is cleared up in the case where a large amount of antibiotic.This is controlled The book on Chinese herbal medicine understanding of the same Radix Angelicae Sinensis of thought " disappearing in priming " is treated with the resemblance in thinking.

Quantify the overall efficacy that different pharmaceutical group applies compound and compound joint azithromycin in Chinese medicine composition It is furtherd investigate in terms of contribution rate, to probing into Chinese medicine composition the key agents group for playing anti-inflammatory and promoting blood circulation effect, comprehensively Improve the product quality of Chinese medicine composition, it is ensured that clinical application is quality controllable, uniform, safety provides theoretical foundation, guidance is clinical The rational use of medicines has great importance.

FUKE QIANJIN PIAN (capsule) is a kind of good typical Chinese medicine composition of application, by philippine flemingia root, single side needle, Jin Ying Root, Herba Andrographitis, leatherleaf mahonia, Radix Codonopsis, Radix Angelicae Sinensis, eight taste active constituent of Caulis Spatholobi composition, removing damp-heat, benefiting qi and removing blood stasis.It is mainly used for Vaginal discharge diseases caused by dampness and heat stasis, abdominal pain symptoms include profuse leukorrhea, color Huang matter are thick, smelly dirty, and underbelly pain, waist sacrum is ached, and mind is tired Power；Chronic pelvic inflammatory disease, endometritis, chronic cervicitis are shown in above-mentioned patient.It is obtained in terms for the treatment of chronic pelvic inflammatory disease significant Effect.Meanwhile wherein the active constituent Radix Angelicae Sinensis of a herb is " can draw the warp that all blood respectively returns its Radix Angelicae Sinensis, therefore named Radix Angelicae Sinensis "； " married woman is with blood for this ", Radix Angelicae Sinensis is blood man key medicine.Mainly there are replenishing and activating blood, the function of regulating menstruation and relieving pain, moistening intestines and relaxing bowels, in gynecological disease Middle application is extremely wide." book on Chinese herbal medicine newly organized " cloud " with when be attributed in Toxin-Vanquishing detoxification medicine, just taking its property dynamic, then disappear in priming, directly become to defecating and Out, it yields result real mind.Therefore the person that burst it is disconnected it is preferably big use, be allowed to promoting blood circulation with myogenic, i.e., the outstanding preferably urgent need of person of not bursting, be allowed to detoxification and by dirty ?."

It is rarely seen to be directed to gynaecology but in the research of FUKE QIANJIN PIAN and Antibiotic combination application for the treatment of chronic pelvic inflammatory disease Radix Angelicae Sinensis " disappearing in priming " effect whether there is and the research of inherent mechanism of action and effect in prescriptions worth thousand gold, also have no that Radix Angelicae Sinensis priming is made Be with its in which kind of active constituent group of Toxin-Vanquishing detoxification effect basis on, active constituent and activity in FUKE QIANJIN PIAN The mechanism that the effect baseline level to azithromycin how is promoted at grouping also lacks correlative study and guide.

Summary of the invention

The technical problem to be solved by the present invention is to fill up existing Chinese medicine composition joint azithromycin treatment chronic pelvic inflammatory disease Effect assessment technical deficiency, provide in a kind of Chinese medicine composition active constituent to the amount of azithromycin effect baseline winding level Change evaluation method.

The purpose of the present invention is achieved by the following technical programs:

A kind of Chinese medicinal ingredients are provided, evaluation method is promoted to azithromycin effect baseline, is using additions such as baselines, first Measurement has proven to the exposure level of clearly effective therapeutic agent azithromycin, by azithromycin with it is parallel for trying Chinese medicine composition Experimental model is given, the baseline level of drug combination and the curative effect trend of drug combination are measured, passes through population pharmacokinetics-group's medicine Effect is learned conjunctive model and is verified.

Scientific design technology path of the present invention, the Chinese medicinal ingredients provided promote evaluation side to azithromycin effect baseline Method, specifically includes the following steps:

S1. using chronic pelvic inflammatory disease rat as experimental model；

S2. the exposure level of azithromycin is measured；

S3. Chinese medicine composition PPK-PPD connection model is constructed；Being introduced into each component part in Chinese medicine composition is covariant, Quantitatively evaluating azithromycin acts on baseline winding level.

Wherein, experimental model described in step S1 can use chronic pelvic inflammatory disease rat model, preferably prepare the side of model Method is that rat uterine cavity and slight mechanical damage method completion are mechanically injected using mixed bacteria liquid.The mixed bacteria liquid is with golden grape Coccus and escherichia coli are tested bacterium.Preferably, in mixed bacteria liquid tested bacterium best training method are as follows: choose 3~4 respectively Single bacterium is fallen in 40 milliliters M-H (B) culture medium, and Zengjing Granule overnight takes out the multi-functional reading of original bacteria liquid all-wave length after 16 hours Number view surveys OD value (the bacterial growth period of saturation)；20mL is taken, is sub-packed in centrifuge tube, every pipe 2mL is centrifuged 10000~15000rpm/ Min 10 minutes, abandons or adopts the M-H (B) among supernatant, after being mixed with 2mL physiological saline, then is centrifuged 15000rpm/min, and 10 points Clock abandons or adopts the physiological saline in supernatant, collects bacterial cell thallus 2mL.By the gold-coloured staphylococci of same procedure culture and Escherichia coli obtains 4mL mixed bacteria liquid by 1:1 mixing.It is 100 bacterial concentrations by the bacterium solution, carries out 10 times with physiological saline Dilution, is diluted to 0.1 × 10⁰, 0.01 × 10⁰, spare (totally 3 dosage).It is further preferred that with 0.01 × 10⁰Most preferably to feel Microbiological contamination amount.

Step S3 is used exist including HPLC-UV or EC (Electrochemical Detection) method in advance by constantly analysis and design Interior a variety of detection methods carry out, complete selectivity in two kinds of bio-matrixes of rat model blood and uterine tissue, working curve, Stability test, the final method for measurement of concentration for determining azithromycin in preferably two kinds of bio-matrixes.The measurement Zitromax The method of the exposure level of element is: HPLC-ECD method or HPLC-ESI-MS method.

The sample-pretreating method of the HPLC-ECD method or HPLC-ESI-MS method is to take 50 μ L of blood plasma, is added 10M NH₄OH solution and internal standard roxithromycin methanol solution (160ng/mL)；Diethyl ether is added, stands after vortex, is inhaled after centrifugation Upper organic phase is taken, is dried up under air stream, flowing phased soln is added in residue, is vortexed, and centrifugation takes supernatant to be analyzed.

The chromatographic condition of the HPLC-ECD method are as follows: chromatographic column Aglient poroshell120EC-C18 (2.7 μ ms 4.6 × 100mm) 695975-902, online filter: SSI35-0149；Mobile phase: acetonitrile-pH7.0 phosphate buffer solution (45: 55, V/V)；Flow velocity 0.8mL/min；30 DEG C of column temperature.Glass carbon working electrode potential+1.2V, reference electrode Ag/AgCl.

The chromatographic condition of the HPLC-ESI-MS method are as follows: chromatographic column Aglientporoshell120EC-C18 (2.7um × 4.6 × 100mm) 695975-902, online filter: SSI35-0149；Mobile phase: acetonitrile -10mMpH5.2 ammonium acetate (65: 35), flow velocity 0.2mL/min, 5 μ L of sample volume；Mass Spectrometer Method condition are as follows: electrospray ionisation source (ESI), selective cation inspection It surveys (SIM), azithromycin m/z 749.5 [M+H]⁺With internal standard roxithromycin m/z 837.5 [M+H]⁺, ionization source voltage 4.5kV, Spraying gas (N₂) flow velocity be 1.5L/min, desolventizing temperature be 250 DEG C, detector voltage 1.5kv.

Step S3 is to quantify each active constituent in gynaecology's prescriptions worth thousand gold composition and side with group mode for crucial covariant The influence mode and related coefficient of the population model parameter for the PK and PD parameter that group acts on azithromycin.Basic Ways include with Lower step:

S31. the PPK model of AZM is established；

S32. it determines the linear space of Integrative expression totality drug effect or selects each prescription association in each pharmacodynamics index The obvious index of same-action is as PD output valve；

S33. it determines suitable PD quantification of targets model, establishes the PPD model of truncated data；

S34. analyze Tissue distribution, antibacterial type, infection and inflammation relationship quantization factor, determine suitable PPK-PPD Connecting mode；

S35. each active constituent group in Chinese medicine composition and composition based on mechanism of action is established to assist azithromycin The drug combination PPK-PPD collective model of same-action calculates corresponding model parameter；

S36. related hypothesis testing and interval estimation are carried out to obtained model parameter, completes evaluation.

Wherein, the method that PPK-PPD collective model is established described in step S35 and calculates corresponding model parameter is: reference The foundation of Kinettica and monolix software progress overall model.After each node adjustment relevant parameter, to front and back calculated result It carries out AIC and Log likelihood value to compare, corresponding calculating parameter appropriate is set.Use nonlinear mixed-effect model point Analyse all pharmaco-kinetic processes, can estimated driving force parameter population distribution and due to physiology and pathology (fixed effect and association Variable) contribution between making a variation individual.Parser is mainly EM method, and E is conditional expectation Bayesian Estimation, and M is maximum likelihood Estimation.Pharmacokinetic parameter has known prior distribution (mean ± side in assumed group when conditional expectation Bayesian Estimation Difference), at the same also assume residual error distribution (mean value ± variance) it is known that under the described conditions to individual drugs kinetic parameter into Row estimation；Maximal possibility estimation asks calculation using the individual parameter of conditional expectation Bayesian Estimation as initial value, with maximum likelihood estimate Group's mean and variance.Evaluation criterion is changed into the model conspicuousness of front and back Modifying model twice when EM algorithm iteration carries out, Without conspicuousness, then operation terminates computation model parameter change twice for front and back.

Evaluation method of the present invention can be advantageously applied to evaluation Chinese medicine composition and antibiotic combination safety and Application in terms of validity.

Preferably, present invention simultaneously provides the method in gynaecology's prescriptions worth thousand gold composition and its active component to azithromycin The application in terms of the evaluation of baseline winding level is acted on, answering for the effect aspect that disappears in wherein Radix Angelicae Sinensis ingredient priming is especially evaluated With.

The applicant combines the composition of gynaecology's prescriptions worth thousand gold, it is believed that the effect of Radix Angelicae Sinensis priming is exactly with it in Toxin-Vanquishing detoxification Philippine flemingia root, cherokee rose root, Herba Andrographitis, leatherleaf mahonia, in single side needle, and receive the function of the priming of " detoxification and by dirty "；Gynaecology's prescriptions worth thousand gold Middle philippine flemingia root, cherokee rose root, Herba Andrographitis, leatherleaf mahonia, single side needle removing damp-heat ingredient most probably play anti-infective anti-inflammatory solution exopathogen Effect acts on similar modern antimicrobial, and the overlap action of Radix Angelicae Sinensis, Caulis Spatholobi, Radix Codonopsis is similar to " uterine secretions row There is similarity out ".Evaluation method of the present invention is established accordingly and is tested by population pharmacokinetics-Population pharmacedynamics conjunctive model science Card.

The beneficial effects of the present invention are:

The present invention is directed to chronic pelvic inflammatory disease disease, and using PPK-PPD model-fitting technique, tested Chinese medicine is quantitatively calculated in realization Each active constituent group is to the winding level for confirming clearly effective therapeutic agent azithromycin effect baseline in composition and side.

The present invention is measured as " draw in poison and disappear " key index to body fluid intestinal endotoxin migration under corresponding pathological state, " living Blood is with myogenic, i.e., the outstanding preferably urgent need of person of not bursting, be allowed to detoxification and by dirty " surveyed with uterus pathology rank scores, the tissue inflammation factor Fixed, blood coagulation correlation factor is key index, establishes the key index system and evaluation supporting point of chronic pelvic inflammatory disease treatment evaluation, And by the scientific accuracy of population pharmacokinetics-Population pharmacedynamics conjunctive model verifying index system.

The present invention verifies in model, and PPK uses the method by the full course for the treatment of of large sample, with index components in the full course for the treatment of in body The exposed amount of liquid and target area is modeling critical data, has comprehensively considered the statistical distribution of individual of sample difference, scientific design Can dynamics-pharmacodynamics systematic variation dynamic system for participating in of quantitative assessment priming, make a variation weak efficiency curative effect of medication index The drawbacks of research method that point method measurement mean value compares.

The present invention specifies that gynaecology's prescriptions worth thousand gold composition influences the synergy of each pharmacokinetic parameters of azithromycin, closes to be clinical It manages medication and guidance is provided；By PPK-PPD model, summarizes and obtain playing anti-inflammatory and promoting blood circulation effect in gynaecology's prescriptions worth thousand gold composition Key agents group, for the product quality for improving FUKE QIANJIN PIAN comprehensively, it is ensured that clinical application is quality controllable, uniform, safety provides Theoretical foundation；By the foundation of evaluation method and the PPK/PPD model of foundation science, quantify in gynaecology's prescriptions worth thousand gold composition not With drug group to the contribution rate of compound overall efficacy, it is provided with for the secondary development and industry of gynaecology's prescriptions worth thousand gold composition The Technical Reference and foundation of power.

Detailed description of the invention

Fig. 1 the method for the present invention technology path design diagram.Fig. 2 bare substrate SIM total ion current figure.Fig. 3 standard substance+ Bare substrate SIM total ion current figure.Fig. 4 typical case's low concentration sample SIM total ion current figure.Fig. 5 treated with combined medication Whole blood Undercut reduced viscosity situation of change.Fig. 6 treated with combined medication Whole blood height cuts reduced viscosity situation of change.Fig. 7 is medication combined Pretherapy and post-treatment high cut phase is to index variation situation.Undercut relative indices situation of change before and after Fig. 8 treated with combined medication.Fig. 9 drug Hematocrit situation of change before and after combination therapy.Figure 10 treated with combined medication Whole blood viscosity situation of change.Figure 11 is medication combined to be controlled Treat front and back erythrocyte mechanical fragility situation of change.Erythrocyte aggregation index situation of change before and after Figure 12 treated with combined medication.Figure 13 Deformable index situation of change before and after treated with combined medication.Red blood cell count(RBC) changes feelings before and after Figure 14 treated with combined medication Condition.Hematid specific volume change in count situation before and after Figure 15 treated with combined medication.Chromocytometer before and after Figure 16 treated with combined medication Number situation of change.Figure 17 treated with combined medication forward backward averaging erythrocyte volume situation of change.It is flat before and after Figure 18 treated with combined medication Equal content of hemoglobin situation of change.Figure 19 treated with combined medication forward backward averaging protein concentration situation of change.Figure 20 is medication combined Pretherapy and post-treatment red blood cell width coefficient of variation situation of change.Red blood cell width criteria difference changes feelings before and after Figure 21 treated with combined medication Condition.Figure 22 treated with combined medication Platelet number situation of change.Figure 23 treated with combined medication forward backward averaging volume of platelets becomes Change situation.Leukocyte count situation of change before and after Figure 24 treated with combined medication.Lymphocyte percentage before and after Figure 25 treated with combined medication Compare situation of change.Intermediate cell percentage situation of change before and after Figure 26 treated with combined medication.Grain before and after Figure 27 treated with combined medication Cell percentages situation of change.Figure 28 treated with combined medication Platelet volume change situation.Before Figure 29 treated with combined medication Platelet distribution width situation of change afterwards.Animal uterus pathological section figure after Figure 30 treated with combined medication.Wherein, divide from left to right It Wei not AZM, AZMQR, AZMDG, AZMFF treatment group animal uterus pathological section figure.Uterus is dirty before and after Figure 31 treated with combined medication Device index variation situation.Ovary organ coefficient situation of change before and after Figure 32 treated with combined medication.Before and after Figure 33 treated with combined medication Spleen organ coefficient situation of change.Thymus gland organ coefficient situation of change before and after Figure 34 treated with combined medication.Figure 35 is medication combined to be controlled Treat Plasma Before And After endotoxin situation of change.Figure 36 treated with combined medication Plasma Before And After changes of Nitric Oxide situation.Figure 37 drug connection Close pretherapy and post-treatment plasma TNF situation of change.Figure 38 pharmacokinetics PK tendency chart.Figure 39 pharmacokinetics is all Individual PK concentrates figure.The first schematic diagram of Figure 40 calculation method.The second schematic diagram of Figure 41 calculation method.Figure 42 calculation method third is shown It is intended to.The dimension in Figure 43 drug effect space selects figure.The recombination in Figure 44 drug effect space and each sample projection value are on sample time axis Distribution map.Weight distribution figure of the original pharmacodynamics index of Figure 45 in drug effect space 1,2 principal components.Figure 46 overall structure model knot Structure schematic diagram.Fitting result of each cell mean of Figure 47 to full model.Figure 48 each group mean concentration matched curve.Figure 49 each group is flat Equal concentration match value-measured value scatter plot is (left: population parameter fitting control；It is right: individual parameter fitting control).Figure 50 is based on The each group average effect value of calculation.Each group average effect curve after the fitting of Figure 51 full model.Figure 52 each group average effect match value- Measured value scatter plot is (left: population parameter fitting control；It is right: individual parameter fitting control).All sample of the Figure 53 for calculating Concentration value.12 bulk concentration matched curves before Figure 54 full model.Figure 55 concentration match value-measured value scatter plot is (left: group's ginseng Number fitting control；It is right: individual parameter fitting control).Figure 56 concentration measured value and match value accumulated probability confidence interval.Figure 57 is dense Spend measured value variation section and match value accumulated probability distributed area.Figure 58 concentration measured value variation section and match value are cumulative general Rate distributed area.All sample effect value of the Figure 59 for calculating.12 individual effects matched curves before Figure 60 full model.Figure 61 Effect match value-measured value scatter plot is (left: population parameter fitting control；It is right: individual parameter fitting control).The actual measurement of Figure 62 effect Value and match value accumulated probability confidence interval.Figure 63 effect measured value variation section and match value accumulated probability distributed area.Figure 64 effect measured values variation section and match value accumulated probability distributed area.The distribution of the boxlike of each covariant of Figure 65 and model parameter Figure.The boxlike figure of Figure 66 model parameter population parameter error distribution.Power of Figure 67 sample in drug effect space re-projection distance Credit cloth.The NaNO of Figure 68 detection NO₂The dilution step schematic diagram of titer.

Specific embodiment

The present invention is further illustrated combined with specific embodiments below.Following embodiments only for illustration, Bu Nengli Solution is limitation of the present invention.Unless stated otherwise, raw material used in following embodiments is that regular market purchase or commercial sources obtain The raw material obtained, unless stated otherwise, method and apparatus used in following embodiments are method commonly used in the art and set It is standby.

Embodiment 1

One, the principle analysis that the present embodiment experimental method is established is summarized

1. prescription analysis:

Composition: philippine flemingia root, cherokee rose root, Herba Andrographitis, leatherleaf mahonia, single side needle, Radix Angelicae Sinensis, Caulis Spatholobi, Radix Codonopsis composition.

Major function: removing damp-heat, benefiting qi and removing blood stasis.It is mainly used for vaginal discharge diseases caused by dampness and heat stasis, abdominal pain symptoms include leukorrhagia Amount is more, color Huang matter is thick, smelly dirty, and underbelly pain, waist sacrum is ached, spiritlessness and weakness；Chronic pelvic inflammatory disease, endometritis, chronic uterine neck Inflammation is shown in above-mentioned patient.

Prescription analysis: for composition, as seen from Table 1, the nature and flavor of drug, plant origin, chemical component can be by groups Recipe efficacious prescriptions are to can be divided into two effect groups.One be removing damp-heat group, containing philippine flemingia root, cherokee rose root, Herba Andrographitis, leatherleaf mahonia, Single side needle, section belongs to source and existing chemical component has antimicrobial antiphlogistic analgesia and immunoregulation effect more；Secondly for promoting blood circulation benefit Gas group contains Radix Angelicae Sinensis, Caulis Spatholobi, Radix Codonopsis, and the effect of this three tastes medicine is overlapped mutually, and modern pharmacological research has improvement lectin from hemolymph more, adjusts Save the functions such as smooth muscle function and blood generation.

1 composition of table and corresponding plants are learned and chemical component context analyzer abridged table

2. pathogenesis and principle of reatment preliminary analysis

Traditional Chinese medicine thinks that chronic pelvic inflammatory disease (chronicpelvicinflammatorydisease, CPID) dominant mechanism is Married woman's menstrual period, postpartum blood room are just opened, and remaining blood to the greatest extent, is not hurt by the six external factors which cause diseases, seven feelings, diet, overstrain and sexual strain easily, are influenced punching and are appointed qi and blood To be to suffer from the stasis of blood.It can obviously improve clinical symptoms using activating blood and removing stasis Method in practice.Western medicine then thinks it for acute pelvitis of pelvic cavity Aseptic inflammation afterwards shows as the abnormal changes such as hemorheology, immunology, pathology.Therefore, treatment using it is anti-infective, Drug local action is the basic rules for the treatment of in anti-inflammatory and uterine cavity, also has clinical literature to think that uterine contraction is promoted to make infectious uterine cavity point Clearing up uterine cavity in the case where secretion discharge or a large amount of antibiotic of application has more significant curative effect.The same Radix Angelicae Sinensis of this therapeutic thoughts " draws Disappear in medicine " book on Chinese herbal medicine understanding have thinking on resemblance.

" warp that all blood respectively returns its Radix Angelicae Sinensis, therefore named Radix Angelicae Sinensis can be drawn "." married woman is with blood for this ", Radix Angelicae Sinensis is blood man key medicine, main There are replenishing and activating blood, the function of regulating menstruation and relieving pain, moistening intestines and relaxing bowels, using extremely wide in gynecological disease." book on Chinese herbal medicine is newly organized " cloud " is attributed to working as It in Toxin-Vanquishing detoxification medicine, is just taking its property dynamic, is then disappearing in priming, directly becoming to defecating and go out, real mind of yielding result.Therefore the disconnected preferably big use of the person that burst, make Promoting blood circulation with myogenic, i.e., the outstanding preferably urgent need of person of not bursting, be allowed to detoxification and by dirty." in conjunction with the composition of gynaecology's prescriptions worth thousand gold, it can recognize It is the effect of Radix Angelicae Sinensis priming exactly with it in the philippine flemingia root of Toxin-Vanquishing detoxification, cherokee rose root, Herba Andrographitis, leatherleaf mahonia, single side needle, and receives The function of the priming of " detoxification and by dirty ".

Taking into account the above, removing damp-heat group should be and play anti-infective anti-inflammatory solution exopathogen effect in gynaecology's prescriptions worth thousand gold, act on class Like modern antimicrobial, and the overlap action of Radix Angelicae Sinensis, Caulis Spatholobi, Radix Codonopsis be similar to " uterine secretions discharge " have it is similar it Place.

3. experimental principle

Many experiments are analyzed and pass through by studying for a long period of time, the applicant's design is measured first using additions such as baselines The exposure level for confirming clearly effective therapeutic agent azithromycin is given azithromycin to rat with for examination concurrent, is surveyed Determine the baseline level of drug combination and the curative effect trend of drug combination.

Overall experimental program and process are as shown in Figure 1.

Two, major experimental process

1. experimental strain:

Staphylococcus aureus (ATCC25923), escherichia coli (ATCC25922) are bought in China national strain library. Strain subject freeze-dried powder is uniformly laid on sterile M-H (A) planar surface, 35 DEG C of overnight incubations after being suspended with sterile M-H (B) culture medium It bringing back to life, planar surface grows lawn after 18h, and the single cenobium wherein to grow fine is chosen, is applied to the inclined-plane M-H (A) again, It is incubated for 18h under the same terms, seals, it is stored refrigerated under the conditions of 4 DEG C；Test the previous day takes inclined-plane bacterium to be applied to M-H (A) plate, 35 DEG C of overnight incubations, that is, can be used.All operations are completed in superclean bench.

2. bacterium solution preparation method

Method one: (preparation of high concentration bacterium solution)

It chooses in M-H (B) culture medium that 3~4 single bacteriums fall within 40 milliliters.Tested bacterium Zengjing Granule overnight 16 hours (37 DEG C, Shaking bath shakes 16 hours), next day takes out the multi-functional reading view of original bacteria liquid all-wave length and surveys OD value (the bacterial growth period of saturation)； 20mL is taken, is sub-packed in centrifuge tube, every pipe 2mL is centrifuged 10000~15000rpm/min, 10 minutes, abandons or adopts among supernatant M-H (B) after being mixed with 2mL physiological saline, then is centrifuged 15000rpm/min, 10 minutes, abandons or adopts the physiological saline in supernatant, Collect bacterial cell thallus 2mL.The gold-coloured staphylococci of same procedure culture and escherichia coli are mixed by 1:1 and obtained 4mL mixed bacteria liquid.It is 10 by the bacterium solution⁰Bacterial concentration carries out 10 times of dilutions with physiological saline, is diluted to 0.1 × 100,0.01 ×10⁰, spare (totally 3 dosage).

Method two: (preparation of low concentration bacterium solution)

Single bacterium colony (including gold-coloured staphylococci and escherichia coli) is chosen in 2 milliliters of physiological saline, is prepared respectively As 0.5 Maxwell concentration (about 1 × 10⁸CFU·mL^{- 1}) bacterial suspension, by gold-coloured staphylococci and escherichia coli suspension It mixes to get mixed bacteria liquid 4mL is arrived.The bacterium solution is set to 10A bacterial concentration, carries out two-fold dilution with physiological saline, it is dilute It releases to 0.5 × 10^A, 0.25 × 10^A, spare (totally 3 dosage).

3. drug and reagent: M-H (A) culture medium (OXOID company, product batch number: CM0337A)；M-H (B) culture medium (OXOID company, product batch number: CM0405B)；Urethane solution (20%)；Physiological saline (the limited public affairs of Sichuan Cologne medicine company share Department, product batch number: M12012004)；Other reagents and consumptive material are bought by Chengdu Ya Rong biochemical equipment business department to be provided.

4. instrument: electro-heating standing-temperature cultivator (the permanent Science and Technology Ltd. in Shanghai one, BPH-9162)；Excellent general serial ultrapure water machine (Chengdu Ultra Pure Science & Technology Co., Ltd, UpK-1-10T)；Micro sample adding appliance (EppendorfResearch, 10,100,100uL)； Air bath oscillator (Harbin City east joins electronic technology exploitation, HZQ-C)；Automatic electric heating pressure steam sterilization boiler (Shanghai Shen peace Medical apparatus and instruments factory, LDZX-40BI)；The multi-functional reading view (U.S. Thermo) of all-wave length；Bacterial colony counting instrument.

5. prepared by chronic pelvic inflammatory disease model

Rat weight label, abdomen routine disinfection, 20% urethane 0.5mL/100g intraperitoneal injection of anesthesia are injected with 1mL Device extracts previously prepared mixed bacteria liquid, and left hand holds rat-tail and lifts rat, grinds off needle tip, the bottom Yu Yindao with 10mL syringe Cervix crotch carefully enter in the uterine cavity of side, syringe needle in uterine cavity back and forth pull 2 times with mechanical injuries endometrium Tissue, then injects bacterium solution 0.1mL towards ovary direction, and 0.1mL bacterium solution is injected other side uterus, injected by same operating procedure Bi Hou is inverted rat 5min, to prevent bacterium solution leakage.Respectively at for 24 hours, 48h observes and records each group rat infection situation, with rat without Death, vagina scraping blade, progress bacterium colony culture select bacterium colony to count >=30 ,≤100 respectively for 24 hours, after 72h, 96h after modeling Infection dosage is as best infection dosage.Using the bacterium amount as the bacterium amount of In vivo infection.Tested rat infection bacterium amount is 2 institute of table Show:

Table 2

Animal group (n=4)	Infection dosage
		1	10⁰
2	0.1×10⁰
		3	0.01×100
4	10^A
		5	0.5×10^A
6	0.25×10^A

All experimental animals after modeling for 24 hours, 72h and 96h, anesthesia, routine disinfection abdomen protrudes into palace with aseptic cotton carrier Mouthful, back and forth after pull 2 times, cotton swab is put into the test tube of the physiological saline containing 2mL, obtains suspension containing test article, taking-up cotton swab, Mixed liquor is sufficiently dipped with new aseptic cotton carrier again and is uniformly applied to sterile M-H (A) planar surface, and 35 DEG C are incubated overnight, after 18h Carry out bacterium colony counting.240h sacrifices animal, observes uterine infection situation.Each group typical case mouse infects bacterium colony and counts, as a result such as following table 3:

The tested rat infection bacterium amount of table 3

Animal group (n=4)	Infection dosage
		1	10⁰
2	0.1×10⁰
		3	0.01×10⁰
4	10^A
		5	0.5×10^A
6	0.25×10^A

All experimental animals after modeling for 24 hours, 72h and 96h, anesthesia, routine disinfection abdomen is aobvious with no bacterium colony count results Show, 3 groups of mouse institute infection dosages meet requirement of experiment.Meanwhile the intuitive visible inflammatory congestion such as visible uterus and inflammatory water after dissection It is swollen.Experiment determines that method (one) is the best training method of tested bacterium；0.01×10⁰, it is best infection dosage；Experimental method can To replicate rat pelvic cavity bacterium chronic infection model.

6. the building of azithromycin measuring method in rat plasma

(1)HPLC-ECD

Instrument: LC-30AUPLC (Japanese Shimadzu), AntecDecadeIII electrochemical detector (safe and sound gram of Holland), chromatography Work station (labsolution5.5)；QL-901 vortex mixed instrument (Shanghai Hu Xi Analytical Instrument Co., Ltd)；MTN-2800W type Nitrogen evaporator (Tianjin Ao Tesaiensi Instrument Ltd.)；Ultra low temperature freezer (FIS13-990-16THERMOSCIENTIFIC)； H2050R tabletop refrigerated centrifuge (Xiang Yi centrifuge Instrument Ltd.)；Electronic balance (Beijing Sai Duolisi Co., Ltd) Deng.

Animal: SPF grades of SD rats, 220g ± 20g are bought by institute of lab animals, Sichuan Academy of Medical Sciences, experiment Animal productiong licensing number: SCXK (river) 2008-24.

Drug and reagent: Zithromax azithromycin tablet (Zithromax, pfizer inc)；Acetonitrile, methanol (color Compose pure FisherScientific)；Formic acid (chromatographically pure Chengdu Ke Long chemical reagent factory)；Water is Watson distilled water.Archie Mycin, roxithromycin standard items are purchased from Chinese food pharmaceutical biological product calibrating institute.

Administration and sampling: rat oral gavage gives AZM CMC-Na suspension solution (60mg/Kg), respectively at administration 15min, 30min, 1h, 1.5h, 2h, 4h, 8h, 12h, take blood 0.2ml in test tube of hepari centrifuge tube for 24 hours, 37 DEG C after water-bath 10-20 minutes, 3500r/min is centrifuged 10min, and separation supernatant is saved in -80 DEG C.

Liquid-liquid extraction: taking 50 μ L of blood plasma, and 10M NH is added₄Internal standard roxithromycin methanol solution is added in 10 μ L of OH solution (160ng/ml)10μL.2ml diethyl ether is added, vortex 1min stands 3min, and 13300 r/min are centrifuged 5min, and drawing upper layer has Machine phase dries up under 30 DEG C of air streams, and residue is added 150 μ L and flows phased soln, and vortex 1min, 14000r/min are centrifuged 3min, 30 μ L are taken to be analyzed.

Chromatographic condition: chromatographic column Aglient poroshell120EC-C18 (2.7 4.6 × 100mm of μ m) 695975- 902, online filter: SSI35-0149；Mobile phase: acetonitrile-pH7.0 phosphate buffer solution (45: 55, V/V)；Flow velocity 0.8mL/ min；30 DEG C of column temperature.Glass carbon working electrode potential+1.2V, reference electrode Ag/AgCl.

(2)HPLC-ESI-MS

Instrument: Shimadzu HPLC-MS2020 is included in line vacuum degasser, binary pump, autosampler, column oven, ultraviolet Detector, ESI, chromatographic work station (labsolution5.5)；Remaining is the same as instrument under HPLC-ECD method.

Chromatographic condition: chromatographic column Aglientporoshell120EC-C18 (2.7um × 4.6 × 100mm) 695975- 902, online filter: SSI35-0149；Mobile phase: acetonitrile -10mMpH5.2 ammonium acetate (65:35), flow velocity 0.2mL/min, into 5 μ L of sample amount.

Mass Spectrometer Method condition: electrospray ionisation source (ESI), selective positive ion detection (SIM), azithromycin m/z 749.5[M+H]⁺With internal standard roxithromycin m/z 837.5 [M+H]⁺, the flow velocity of ionization source voltage 4.5kV, spraying gas (N2) is 1.5L/min, desolventizing temperature are 250 DEG C, detector voltage 1.5kv.

Exemplary ion flow graph: AZM about 6min, IS about 8.4min.

Fig. 2 show bare substrate SIM total ion current figure, and Fig. 3 show standard substance+bare substrate SIM total ion current Figure.Fig. 4 show typical low concentration sample SIM total ion current figure.Experiment discovery, two methods are more sensitive with ESI detection, fixed Amount is limited up to 15ng/ml, and can be by improving flow velocity, to realize the shortening of analysis time.Consider that PPK is quasi- dense with stable state medicine simultaneously For main test object, ECD should can substantially meet quantitative demand.

7.IL-1 β, LPS, TNF-α, NO detection method

Referring to kit operation instructions, coherent detection is carried out.

(1)IL-1β

1, it is loaded: setting gauge orifice, sample to be tested hole, blank well respectively.If 7 hole of gauge orifice, it is dense to sequentially add 100 μ L differences The standard items (preparing referring to kit operation instructions reagent) of degree.Blank well adds 100 μ L (referring to kit operation instructions), 100 μ L of Yu Kongjia sample to be tested, ELISA Plate add overlay film, and 37 DEG C incubate 2 hours.

2, liquid is discarded, is dried, washing is not had to.

3, every hole adds detection 100 μ L (prepared before use) of solution A working solution, and ELISA Plate adds overlay film, and 37 DEG C of incubations 1 are small When.

4, liquid in hole is discarded, every hole is washed with the cleaning solution of 350 μ L, is impregnated 1~2 minute, is sucked (untouchable plate Wall) or liquid in ELISA Plate is got rid of, the several layers of blotting papers of place mat on experimental bench, ELISA Plate is firmly clapped downward several times (can also be light Bat pats dry liquid in hole), it repeats board-washing 3 times.After last time is washed, the cleaning solution in hole is dried completely.Automatically it washes Trigger also may be used.

5, every hole adds detection solution B working solution (prepared before use) 100 μ L, in addition overlay film, 37 DEG C are incubated 30 minutes.

6, liquid in hole is discarded, is dried, board-washing 5 times, method is the same as step 4.

7, every hole adds 90 μ L of substrate solution, and ELISA Plate adds overlay film, and 37 DEG C are protected from light colour developing (reaction time, control was 15~25 Minute, it does not exceed 30 minutes.There is an apparent gradient blue in 3~4 holes before gauge orifice, when rear 3~4 gradient pores are unobvious, It can terminate).

8, every hole adds 50 μ L of stop bath, terminates reaction, and blue is vertical at this time turns yellow.The addition sequence of terminate liquid should be as far as possible It is identical as the addition sequence of substrate solution.Such as there is uneven color one, ELISA Plate please be shake gently so that solution is uniformly mixed.

9, after ensuring ELISA Plate bottom without bubble-free in water droplet and hole, immediately with microplate reader in each hole of 450nm wavelength measurement Optical density (O.D. value).

(2)LPS

1, it is loaded: setting gauge orifice, sample to be tested hole, blank well respectively.If 5 hole of gauge orifice, it is dense to sequentially add 50 μ L differences The standard items of degree (referring to kit operation instructions).Blank well adds 50 μ L (referring to kit operation instructions), and Yu Kongjia is waited for 50 μ L of sample, every hole adds detection 50 μ L of solution A working solution immediately after, gently vibrates, and mixes, has been careful not to bubble, enzyme Target adds overlay film, and 37 DEG C incubate 1 hour.

2, liquid in hole is discarded, every hole is washed with the cleaning solution of 350 μ L, is impregnated 1~2 minute, is sucked (untouchable plate Wall) or liquid in ELISA Plate is got rid of, the several layers of blotting papers of place mat on experimental bench, ELISA Plate is firmly clapped downward several times (can also be light Bat pats dry liquid in hole), it repeats board-washing 3 times.After last time is washed, the cleaning solution in hole is dried completely.Automatically it washes Trigger also may be used.

3, every hole adds detection solution B working solution (prepared before use) 100 μ L, in addition overlay film, 37 DEG C are incubated 30 minutes.

4, liquid in hole is discarded, is dried, board-washing 5 times, method is the same as step 2.

5, every hole adds 90 μ L of substrate solution, and ELISA Plate adds overlay film, and 37 DEG C are protected from light colour developing (reaction time, control was 15~25 Minute, it does not exceed 30 minutes.There is an apparent gradient blue in 3 holes behind gauge orifice, when preceding 3 gradient pores are unobvious It terminates).

6, every hole adds 50 μ L of stop bath, terminates reaction, and blue is vertical at this time turns yellow.The addition sequence of terminate liquid should be as far as possible It is identical as the addition sequence of substrate solution.Such as there is uneven color one, ELISA Plate please be shake gently so that solution is uniformly mixed.

7, after ensuring ELISA Plate bottom without bubble-free in water droplet and hole, immediately with microplate reader in each hole of 450nm wavelength measurement Optical density (O.D. value).

(3)TNF-α

1, it is loaded: setting gauge orifice, sample to be tested hole, blank well respectively.If 7 hole of gauge orifice, it is dense to sequentially add 100 μ L differences The standard items of degree (referring to kit operation instructions).Blank well adds 100 μ L (preparing last pipe of second step see reagent), Yu Kong Add 100 μ L of sample to be tested, ELISA Plate adds overlay film, and 37 DEG C incubate 2 hours.

2, liquid is discarded, is dried, washing is not had to.

4, liquid in hole is discarded, every hole is washed with the cleaning solution of 350 μ L, is impregnated 1-2 minutes, is sucked (untouchable wooden partition) Or get rid of liquid in ELISA Plate, the several layers of blotting papers of place mat on experimental bench, ELISA Plate firmly clap downward several times (can also pat by Liquid pats dry in hole), it repeats board-washing 3 times.After last time is washed, the cleaning solution in hole is dried completely.Automatic washer Also may be used.

7, every hole adds 90 μ L of substrate solution, and ELISA Plate adds overlay film, and 37 DEG C are protected from light colour developing (reaction time, control was 15~25 Minute, it does not exceed 30 minutes.There is an apparent gradient blue in 3~4 holes before gauge orifice, when rear 3-4 gradient pores are unobvious, i.e., It can terminate).

(4) NO is detected

1、NaNO₂The dilution of titer:

7, test tube are taken, successively label 1~7.Distilled water 1ml successively is added to 2~No. 7 test tubes with buret 1, with drop Determine pipe 2 and NaNO is added to No. 1 test tube₂Titer 2ml, then 1ml liquid is sucked out from No. 1 test tube with buret 2, No. 2 examinations are added Pipe mixes, then 1ml liquid is sucked out, No. 3 test tubes are added, and mixes.Repeat this step.It is abandoned when 1ml liquid is sucked out from No. 6 test tubes It goes.Dilution step is shown in Figure 68.

2,1% sulfanilamide (SN) 0.5ml successively is added to 1~No. 7 test tube with buret 3.

3,0.1%N-1- naphthyl ethylenediamine hydrochloric acid 0.5ml successively is added to 1~No. 7 test tube with buret 4.

4,1~No. 7 examination liquid in pipe is successively mixed, is incubated for 20 minutes under room temperature.

5, the suction of 1~No. 6 examination liquid in pipe as a control group with No. 7 examination liquid in pipe, is measured respectively with spectrophotometer Shading value simultaneously records.

6, it should be noted that each buret is only used for each reagent when liquid is added dropwise with buret, never use with.

8. test method

(female SPF SD rat, 220g ± 20g are tested 150 rats of model and grouping by Sichuan Academy of Medical Sciences Institute of Botany is bought, experimental animal production licence number: SCXK (river) 2008-24).16 are served only for blank, separately raise.Its Remaining be all made of after modeling method is anaesthetized injects mixed bacteria liquid.Observation 5 days, per daily randomly select 6 only reach blank group 2 anesthesia after It acquires vaginal fluid and is used for nacterial smear.Survival rats are randomly divided into 5 groups after 5 days, i.e. model, AZM, AZMQR, AZMDG, AZMFF (respectively indicates the model group not treated, azithromycin group, azithromycin heat-clearing merging group, azithromycin is used alone Lack Radix Angelicae Sinensis merging group, azithromycin square merging group entirely).

Model: the model group not treated；

AZM: azithromycin treatment model group.

AZMQR: azithromycin+with philippine flemingia root, Herba Andrographitis, cherokee rose root, leatherleaf mahonia, single side needle is according to FUKE QIANJIN PIAN formula In ratio and method be made composition combination therapy model group.

AZMDG: azithromycin+with philippine flemingia root, Herba Andrographitis, cherokee rose root, leatherleaf mahonia, single side needle, Caulis Spatholobi, Radix Codonopsis is according to woman Obtained composition combination therapy model group is made in ratio and method in section's a thousand pieces of gold slice prescription.

AZMFF: azithromycin+FUKE QIANJIN PIAN (including philippine flemingia root, Herba Andrographitis, cherokee rose root, leatherleaf mahonia, single side needle, when Return, Caulis Spatholobi, Radix Codonopsis ingredient, Zhuzhou Qianjin Pharmacy Co., Ltd produces) combination therapy model group.

1. study dosage

(1) AZM dosage: the present embodiment analyzed and determined to the optional study dosage of azithromycin (AZM), purpose For selection one study dosage that action intensity is moderate, duration is moderate after on probation, at the same it is clear with when Chinese medicine drug combination when Cheng Changdu.Prerun 60,30,20,10mg/kg.d tri- days find that former setting dosage is excessively high, finally use 10mg/kg.d.

(2) Test Drug Dosages:

Original sets the full side (AZMFF) of gynaecology's prescriptions worth thousand gold as 2.058g/kg.d, but prerun is found, this dosage is given using maximum What volume was prepared is thin pulp shape to medical fluid, can not pass through stomach-filling syringe needle.It is given with the final full side AZMFF dosage of adjustment for maximum Dose 1.66g/kg.d, adjustment are lacked when grouping dosage DG is 1.41g/kg.d, heat-clearing group dosage QR is 1.16g/kg.d.

2. administration time

In addition to blank and model, the 6th day after modeling starts to give relative medicine.AZM gives respectively with each Chinese medicine, difference Time about 2 hours.All administration groups first give the AZM of corresponding dosage at 8 points or so, start to give after two hours corresponding Chinese medicine physiological saline water mixes object.

3. sample collection

After 1st gives AZM two hours, each group rat systemic vein blood is acquired through tail vein.From each administration of second day beginning Group rat is twice daily taken a blood sample, and is respectively acquired once through tail vein before giving Archie and before giving Chinese medicine, is acquired blood volume about every time 0.3mL, sample time error about 30min.After administration gives AZM two hours on the 2nd, 4,6,8, about 2 are sacrificed after each group anesthesia ~4 rats, acquire cavity of resorption artery anticoagulated whole blood, and be collected simultaneously sacrifice rat target organ (nest, thymus gland, spleen are incubated in uterus It is dirty).

4. sample presentation is tested

All time point 0.3mL and last whole blood divide and about 0.2mL blood plasma are taken to detect AZM concentration for UPLC-MS.Last Anticoagulated whole blood inspection blood count, lectin from hemolymph.Last blood plasma is for detecting LPS, TNF-α, NO.Nest, thymus gland, spleen are incubated in uterus Dirty weighing simultaneously calculates organ coefficient.Uterus is from cutting in the middle part of corpus uteri, for pathology inspection after taking 1/2 formalin fixed.

Experimental result: the result shows that, after experimental animal modeling, whole blood undercut reduced viscosity, whole blood are viscous shown in Fig. 5 to Figure 13 Degree and erythrocyte aggregation index increase compared with normal group, and after giving different combinational drug therapies, each index is all reduced, It is intended to level back to normal.Therapeutic effect between each treatment group compares, whole blood undercut reduced viscosity: AZMFF > AZM > AZMDG>AZMQR；Whole blood viscosity: AZMFF > AZMDG > AZMQR > AZM；Erythrocyte aggregation index: AZMDG > AZM > AZMFF > AZMQR.The above result shows that showing that drug for invigorating blood circulation and eliminating stasis plays a major role in the change of hemorheology state, " blood can be improved This pathological state of the stasis of blood ".

Experimental result is shown in shown in Figure 14 to Figure 29 for Blood cell counting and classification.

Organ coefficient is also known as dirty body ratio, is the weight of experimental animal internal organs and the ratio of its weight.Each internal organs when normal It is constant compared with the ratio of weight.After animal contaminated, impaired organ weights can change, therefore organ coefficient also changes therewith Become.Organ coefficient increases, and indicates congested internal organs, oedema or hypertrophy etc.；Organ coefficient reduce, indicate internal organs atrophy and other Degenerative change.The result shows that, after experimental animal modeling, the organ coefficient of 4 kinds of internal organs organizes ratio with normal shown in Figure 30 to Figure 34 It more increases, after giving different combinational drug therapies, each coefficient is all reduced, and is intended to level back to normal.Each treatment group it Between therapeutic effect compare, uterus organ coefficient: AZMDG > AZMQR > AZMFF > AZM；Ovary organ coefficient: AZM is better than other, Spleen organ coefficient: being difficult to compare, thymus gland organ coefficient: AZMFF > AZMDG > AZM > AZMQR.The above result shows that showing slow Property pelvic inflammatory disease rat model uterus, ovary, four kinds of internal organs of spleen and thymus gland due to inflammatory reaction, lead to that internal organs are congested, oedema, it is dirty Device coefficient increases；After giving antibiosis anti-inflammatory drug, organ coefficient tends to restore normal level, but the effect between four kinds of drugs Intensity is difficult to compare.

The result shows that, after experimental animal modeling, 3 kinds of inflammatory factor levels are bright compared with normal group shown in Figure 35 to Figure 37 Aobvious to increase, after giving different combinational drug therapies, each coefficient is all reduced, and is intended to level back to normal.Between each treatment group Therapeutic effect compare, plasma endotoxin: AZMQR > AZMDG > AZM > AZMFF；Plasma Nitric Oxide: AZMDG, AZMQR > AZMFF > AZM, plasma TNF: AZMFF > AZM > AZMDG > AZMQR.The above result shows that showing chronic pelvic inflammatory disease Rat model plasma endotoxin, Plasma Nitric Oxide, plasma TNF due to infection and inflammation generation, cause this three Kind inflammatory factor largely generates, its level is made to be higher than normal value；After giving drug therapy, inflammatory factor level tends to restore Normal level makees in intensity substantially between four kinds of drugs are as follows: AZMQR > AZMDG > AZMFF > AZM.

9. pharmacokinetic studies

The Drug-time curve and range of distribution of related double administrations are calculated using Kinetica5, experimental result is shown in Figure 38 and figure 39.The result shows that giving Chinese medicine each group again after giving daily AZM two hours has substantially no effect on associated biomolecule availability.Heat-clearing group Later period slightly reduces its bioavilability trend, but this effect can be introduced by the promoting blood circulation group drug in compound and be offset, and be shown Compound compatibility may have unknown some internal influences.

10. the modeling of population pharmacokinetics-Population pharmacedynamics conjunctive model

1. Basic Ways the following steps are included:

S31. the PPK model of AZM is established；

S34. analyze azithromycin Tissue distribution, antibacterial type, infection and inflammation relationship quantization factor, it is suitable to determine PPK-PPD connecting mode；

S35. each active constituent group is established in gynaecology's prescriptions worth thousand gold composition and side based on mechanism of action to azithromycin The drug combination PPK-PPD collective model of synergistic effect calculates corresponding model parameter；

2. calculation method and process are as shown in Figure 40 to 42.

The foundation of 3.PPK/PPK model

The projection setting in 3.1 overall drug effect spaces

Pharmacodynamics index after drug effect for evaluation is various, how to carry out comprehensive evaluation and selects which kind of pharmacodynamics index Carrying out quantitatively evaluating has natural difficulty and biased property.Therefore, to realize that the synthesis that nobody is subjective selection and preference is commented Sample in all periods for carrying out evaluating drug effect is based on by valence, the present invention using PCA-DA, PLS-DA and OPLS-DA The physiology of all measurements and the multivariable classification identification of pharmacology index, and by all drug effects of all samples according to non-supervisory point Space-like carries out reprojection, to form the sample space that can be used for PPD that can reflect comprehensive drug, and to composition sample space High weight group indication distinguish.

Analysis method: data analysis is referring to R version 3.1.2 (R Core Team；2014R:A Language and Environment for Statistical Computing；R Foundation for Statistical Computing, Vienna,Austria；URL:https://www.r-project.org),PCA-DA,PLS-DA,and OPLS-DA in package MUMA(Metabolomics Univariate and Multivariate Analysis,Ver 1.4； Edoardo G, Francesca C, Dimitrios S, Silvia M, Andrea S, and Michela G, 2012), R package version 1.4.https://cran.r-project.org/web/packages/muma/index.html).

(2) initial data is constituted: every one sample of behavior of analysis matrix, and column are formed by each time point weight, sampled into internal organs Respectively respectively value, inflammation index are respectively worth composition, matrix size 84*44 for value, lectin from hemolymph for weight, organ coefficient, blood count.

(3) analyze result: using PCA-DA, PLS-DA, and OPLS-DA analysis shows, it is 4 main that pharmacodynamics index is compressible Ingredient (see shown in Figure 43) can reflect that the information percentage of overall pharmacodynamics index is about 80%, can reflect the overwhelming majority after compression Drug action makes a variation between group.

(4) recombination in drug effect space and the calculating of each sample projection value: each sample is respectively tieed up in above four principal component space Spend coordinate value be used as position vector X=(X1, X2, X3, X4), using each dimension reflection raw medicine imitate space information content as weight to It measures W=(w1, w2, w3, w4) and each sample projection value is defined with the average out to symbolic vector N of each sample point X of blank control group are as follows: Sgn ((X-N) .W) * (X.W) (wherein sgn is sign function).Draw power credit of the gained projection value on sample time axis Butut is as shown in figure 44, the results showed that gained distance value can reflect that whole drug effect is empty after drug effect spatial recomposition and each sample re-projection Between dynamic variation at any time, there is specific regularity.The drug effect that this projector distance can be used for concentrated expression drug effect represents Value.

(5) high-resolution pharmacodynamics index: (as shown in figure 45) is analyzed to the composition ratio of 1,2 principal component, hence it is evident that can See drug effect makes a variation between each group main body index be inflammation (lower right corner: LPS, NO, TNF) and lectin from hemolymph index (lower right corner: whole blood is low Cut reduced viscosity；The upper left corner: whole blood height cuts relative viscosity, erythrocyte mechanical fragility, erythrodegeneration index).Other haemocytes Counting, weight, organ index are minimum in each between-group variation, do not constitute the evaluation index for reflecting final curative effect.

(6) determination of PD index: in conclusion the projector distance using each sample in above four principal component space is formed most Each sample PD index (as shown in table 4 below) for calculating eventually.

The projector distance (information content 79.29%) in the drug effect space of the online property variation of each sample of table 4

The building of 3.2PPK/PPD model

(1) modeling software of data analysis: ADAPT 5.1, Kinetica5.5, Monolix4.3.3within Matlab2014A and R version 3.1.2with package saemix 1.2 (Comets E, Lavenu A, Lavielle M.SAEMIX,an R version of the SAEM algorithm.20th meeting of the Population Approach Group in Europe,Athens,Greece(2011),Abstr 2173.http:// Www.page-meeting.org/default.asp? abstract=2173).

(2) PK model: summarizing this Drug-time curve of bulk sample, carries out one, two, three Room compartment models and reversed Gauss model respectively Fitting is carried out the selection of PPK basic model by model goodness and PPK-PPD Estimating The Model Coefficients totality goodness.

(3) PD model: small, the guarantor from the dynamics distribution map of sample PD index as it can be seen that blank group PD index makes a variation at any time It holds nearly pseudo-linear normality to stablize, meets the homeostasis features such as no modeling attack；Have after model group modeling attack certain spontaneous extensive Recurrent images carry out a variety of curve model fittings to its dynamics, the results showed that Methods of Linear Growth Model AIC and BIC value is smaller, can For describing associated dynamic process.The dynamic variation of model group PD index can be considered from pathological change degree and physiology normal value Degree of change.Each pharmaceutical intervention group PD curve is the macroreaction using pharmaceutical intervention model group PD space curve.Modelling is retouched State this phenomenon i.e.: model group PD=physiology PD function-pathology PD function；Each treatment group PD=physiology PD function-pathology PD letter Number+drug effect function.Drug effect function investigates differential and integral variation.

(4) connection of model: AZM belongs to typical Time dependence antibiotic, i.e. bactericidal activity is contacted with it with bacterium Duration it is directly proportional, i.e., the antibacterial curative effect of drug depend on drug in the tissue concentration maintain MIC or more it is lasting when Between.Therefore the cumulative model of the site of action for being directly coupled and being coupled with effect compartment is investigated, bind mode is selected according to the goodness of fit.

(5) normal state, lognormal, the distribution group of logit of all model parameters the probability distribution of model parameter: are investigated It closes, selection preferably parameter probability-distribution function；

(6) covariant model: after the probability distribution for determining structural model and model parameter, calculate individual parameter and weight, The figure distribution of drug combination group classification, selects a variety of covariant models fittings, the evaluation goodness of fit and hypothesis testing as a result, And covariant parameter distribution figure is combined to select more significant covariant model.

(7) error model: after the probability distribution for determining structural model and model parameter, the fitting knot of concentration and drug effect output Fruit carries out the inspection of error map shape to measured value, selects appropriate model.

(8) the stochastic effects error covariance model of model parameter: to simplify workflow, fixed covariance matrix is pair Angular moment battle array, without selection and optimization.

3.3 calculation method

(1) it data: using only four groups of mean concentration-effect datas for giving AZM when structural model determines, can significantly reduce When machine；When determining parameter distribution probabilistic model, using all samples, contain blank and model group；Determine covariant model and error When model, using four groups of entirety samples for giving AZM.

(2) initial value: the convergence of program iteration is significantly decided by the setting of initial value, each piecemeal difference of model when calculating Calculate value in average group, the initial value as full model.

(3) iterative algorithm: SAME, ML simultaneously combine MCMC and BOOTSTRAP verifying (sample number random sampling with replacement, composition 500 sample of calculated example).

3.4 overall models used

(1) shown in overall structure model specification such as Figure 46 (structural model block diagram).

(2) structural model:

Structure Mode1

Pysocolog y (t)=pysoA+pysoBt

Hazards (t)=hazaA+hazaBt

Dose (0)=fDose

Ac (0)=0

Ad (0)=0

Effect=physocolog y (t)-hazrads (t)+Drug (t)

Output={ Cc, Effect }

(3) covariant model:

Covariate Model:

(4) probability distribution of non-covariant model parameter:

Other parameters distribution:

(5) error model:

Residual error Model:

Y1=Cc

Y2=Effect

(6) covariance model: diagonal matrix, i.e., non-correlation between each model parameter stochastic effects are independently distributed.

(7) biological significance: medicine movable model is preferable with InverseGaussin models fitting, but is coupled model convergence after PD Property it is poor, therefore final determine two compartment models.Drug effect is decomposed into three as previously shown, reflects physiological status, pathological state respectively With the cumulative improvement of drug.Wherein physiology and pathology function are selected as linear model, and fitting result preferably reflects blank pair According to the temporal behavior of group and model control group, its model population parameter is fixed in calculating thereafter, is set its probability distribution and is positive State distribution.Time according to AZM feature and drug effect space projection is additive, selects directly to be coupled after more a variety of connection models Active component drug accumulation amount model, the i.e. drug action of AZM are the function of the richness product dose value of drug action target position, function shape Formula is Emax model.Regression variable is time t and target position weight (uterus, when being sampled by sample in terms of uterus weight), and weight is Continuity covariant, group be classification covariant (wherein AZM group number is 2, QR 3, DG 4, FF 5, blank 0, 1) model group is.Group covariant is to organize 2 as reference group, i.e. the population value typical value that is AZM group.

3.5 Estimating The Model Coefficients and verifying

(1) repeatedly calculated using the two-way comparative approach of one-in and one-out, and act on entirely with covariant and The models fitting figure not acted on entirely compares and compare relevant parameter, and (remaining fitted figure is shown in corresponding document packet, reduces in this text Piece spoke is not listed), determine the covariant model in final mask.Fitting result such as Figure 47 institute of each cell mean to full model Show, it is seen that model carries out more sufficient structure to each sample group mean concentration and effect value and action character is explained.

(2) model described above carries out parameter fitting to all samples, and uses BOOTSTRAP method, carries out model Verifying.As a result as shown in Figure 53 to Figure 66.By Figure 53 to Figure 66 as it can be seen that models fitting result is good, steady, to each sample and PK-PD feature has carried out more accurate quantity characterization.The error map of covariant and model parameter also indicates that, the covariant of setting Amount model and error distribution can reflect the final medicine of influence for the variation relation of feature and pharmacodynamic profile, can on the basis of this to merging Medication and physiologic factor influence final medicine generation and drug action carries out quantization parsing.

4.6 Estimating The Model Coefficients results

The parameter fitting of model as described above, the final iterative value reflection of the parameter of conditional probability distribution are carried out to all samples AIC the and BIC value of model information amount is respectively less than -10^{^4}, population parameter fitting result is shown in Table 5.

The parameter value of 5 models fitting of table

It is above-mentioned the results showed that

1. the preparation of experimental animal model: chronic pelvic inflammatory disease animal model established by the present invention, after modeling For 24 hours, vagina scraping blade after 72h, 96h, carries out bacterium colony culture, and bacterium colony count results show that each group rat institute infection dosage meets reality Test requirement.The intuitive visible obvious inflammatory congestion swelling of model rat uterus, most tortuous expansions of uterine cavity, end are expanded after dissection, Show that rat chronic pelvic inflammatory disease model can be successfully, reproduced using this method, provides reliable guarantee for subsequent experimental development.

2. azithromycin measuring method in rat plasma: in HPLC-MS method measurement rat plasma of the present invention AZM concentration, this method is selectively good, has good linear, precision, accuracy, and sample analysis time is short, detects sensitive Degree is high, and quantitative limit provides effective ways in the intracorporal pharmacokinetic of rat up to 15ng/ml, for subsequent AZM.

3. influence of the drug combination to azithromycin pharmacokinetics: compared with AZM group is used alone, drug combination each group Pharmacokinetic parameters CLt, V1, CLd, V2 and MIT of azithromycin do not make significant difference (P > 0.05), and main pharmacokinetic parameters are shown in Table Shown in 6.This result of study explanation, FUKE QIANJIN PIAN perfect square and the respectively side's of tearing open combination not appreciably affect azithromycin in chronic pelvic inflammatory disease The intracorporal pharmacokinetic characteristic of rat, it was demonstrated that FUKE QIANJIN PIAN clinically can be united and applied in chronic pelvic with azithromycin The treatment of the diseases such as inflammation.

Table 6 be used alone azithromycin main pharmacokinetic parameters after AZM group and drug combination group multiple dosing (N= 10)

4. different pharmaceutical group drug effect overall merit

The present invention is illustrated difference of each pharmacodynamics index each group, due to the medicine after drug effect for evaluation Effect index is various, and the improvement of fuselage state cannot be only illustrated by a certain or certain several index variation, and the present invention is further led to The statistical method for crossing science selects reasonable index to carry out comprehensive quantitatively evaluating to overall drug effect from numerous indexs. Using PCA-DA, PLS-DA, and OPLS-DA, analysis shows, compressible pharmacodynamics index is 4 principal components, can be reflected after compression total The information percentage of body pharmacodynamics index is about 80%, and drug action makes a variation between can reflecting most groups.By each sample above Each latitude coordinates value in four principal component spaces is as position vector X=(X1, X2, X3, X4), with each dimension reflection raw medicine effect space Information content is fixed with the average out to symbolic vector N of each sample point X of blank control group as weight vectors W=(w1, w2, w3, w4) Each sample projection value of justice are as follows: sgn ((X-N) .W) * (X.W) (wherein sgn is sign function).Gained projection value is drawn in sampling Between distribution map on axis, i.e. the dynamics distribution map of PD index, as shown in Figure 67.

The result shows that gained distance value can reflect whole drug effect space at any time after drug effect spatial recomposition and each sample re-projection Between dynamic variation, there is specific regularity.This projector distance can be used for the drug effect typical value of concentrated expression drug effect.From sample The dynamics distribution map of this PD index is kept nearly pseudo-linear normality to stablize, met as it can be seen that blank group PD index makes a variation small at any time The homeostasis features such as no modeling attack；There is certain spontaneous recovery phenomenon after model group modeling attack, its dynamics is carried out more Kind curve model fitting, the results showed that Methods of Linear Growth Model AIC and BIC value is smaller, can be used for describing associated dynamic process, mould The dynamic variation of type group PD index can be considered the degree of change from pathological change degree and physiology normal value；Each pharmaceutical intervention group PD is bent Line is the macroreaction to pharmaceutical intervention model group PD space curve.The description of modelling this phenomenon is i.e.: model group PD=is raw Manage PD function-pathology PD function；Each treatment group PD=physiology PD function-pathology PD function+drug effect function.The results show that After experimental animal modeling, body comprehensive characteristics are substantially change compared with normal group, after giving different combinational drug therapies, Comprehensive drug with the continuity for the treatment of time, is intended to level back to normal.Therapeutic effect between each medicine group compares, each group Overall drug effect is on close level, no significant difference.

The building and evaluation of 5.PPK/PPD model

Medicine movable model is preferable with InverseGaussin models fitting, but is coupled model poor astringency after PD, therefore final Two compartment models have been determined.Drug effect is decomposed into three as previously shown, reflects physiological status respectively, pathological state and drug it is cumulative Improve.Wherein physiology and pathology function are selected as linear model according to Figure 46, and fitting result preferably reflects blank control group With the temporal behavior of model control group, its model population parameter is fixed in calculating thereafter, and setting its probability distribution is normal state point Cloth.Time according to AZM feature and drug effect space projection is additive, and the drug effect being directly coupled is selected after more a variety of connection models Fraction medicine accumulation amount model, the i.e. drug action of AZM are the functions of the richness product dose value of drug action target position, and functional form is Emax model.

7 model of fit population parameter calculated result of table

(1) influence of the drug combination to AZM pharmacokinetics

By the structure and covariant model that finally determine as it can be seen that merge QR, DG and FF group will not absorption, distribution to AZM And excretion parameter generates the larger impact for having conspicuousness that can be quantified.

(2) influence of the drug combination to AZM pharmacodynamics

By the structure and covariant model that finally determine as it can be seen that merging not to AZM EC₅₀Clearly measurable influence is generated, But each group can enhance final E_maxValue, E_max_ pop=5.96, QR contributive rate are e^{^0.209}=1.23, DG contributive rate are e^{^0.416} =1.51, FF contributive rate are e^{^0.3}The apparent ceiling effect of AZM enhances 1.23 times after=1.17, i.e. merging QR, remaining group effect Similar, multiplying power is also similar.

Pharmacokinetic parameters of the azithromycin in each group experimental animal body are fitted with InverseGaussin model, As a result drug combination each group has no significant effect azithromycin in the intracorporal pharmacokinetic parameters of chronic pelvic inflammatory disease rat；In conjunction with PPK- PPD the model calculation, three drug combination groups have humidification, and this action intensity to the Tissue distribution of azithromycin Multiplying power in tri- groups of AMZQR, AMZDG, AMZFF is suitable.This result of study explanation, FUKE QIANJIN PIAN perfect square and the respectively side's of tearing open combination Azithromycin is not appreciably affected in the intracorporal pharmacokinetic characteristic of chronic pelvic inflammatory disease rat, prompts FUKE QIANJIN PIAN clinically The treatment of the diseases such as chronic pelvic inflammatory disease can be united and applied in azithromycin.Drug combination is not to azithromycin EC₅₀It generates clear Measurable influence, i.e., but each group can enhance final E_maxValue, three drug combination groups are to azithromycin E_maxEnhancing effect It answers and multiplying power is similar.In conjunction with drug combination group and difference of the azithromycin group on pharmacokinetics and overall pharmacodynamics is used alone Property, three drug combination groups are on pharmacokinetics and pharmacodynamics compared with being used alone azithromycin group, and three is to pharmacokinetic parameters Improvement and the enhancing overall function of drug effect be on close level.Drug combination each group has certain increasing to azithromycin totality drug effect Pretend use.

The present invention is by PPK/PPD model quantitative study gynaecology prescriptions worth thousand gold different pharmaceutical group to azithromycin pharmacokinetics and medicine The influence learned is imitated, the PPK/PPD model of clinical application will be instructed, improved by specific aim and is optimized, Chinese medicine is organically applied to In the prescription research of compound, can quantitative study difference factor (such as: different prescription medicine groups) it is dynamic to group's medicine/shadow of pharmacodynamic parameter It rings, provides guidance for clinical rational drug use；The key agents group for playing anti-inflammatory efficacy in gynaecology's prescriptions worth thousand gold is specified simultaneously, is right The secondary development of Chinese medicine innovation preparation research and gynaecology's prescriptions worth thousand gold is of great significance, and provides one for Chinese prescription research New uses for reference method.

Claims

1. method for quantitatively evaluating of the active constituent to azithromycin effect baseline winding level, feature in a kind of Chinese medicine composition It is, comprising the following steps:

S1. using chronic pelvic inflammatory disease rat as experimental model；

S2. the exposure level of azithromycin is measured；

S3. Chinese medicine composition PPK-PPD connection model is constructed；Introducing each component part of Chinese medicine composition is covariant, and quantization is commented Valence azithromycin acts on baseline winding level；

Wherein, described in step S3 construct Chinese medicine composition PPK-PPD connection model Basic Ways the following steps are included:

S31. the PPK model of azithromycin is established；

S32. it determines the linear space of Integrative expression totality drug effect or each prescription collaboration is selected to make in each pharmacodynamics index Use obvious index as PD output valve；

S34. analyze Tissue distribution, antibacterial type, infection and inflammation relationship quantization factor, determine suitable PPK-PPD connection Mode；

S35. it establishes each active constituent group in Chinese medicine composition and composition based on mechanism of action and azithromycin is cooperateed with and make Drug combination PPK-PPD collective model calculates corresponding model parameter；

2. method for quantitatively evaluating according to claim 1, which is characterized in that in the preparation of experimental model described in step S1, It is that tested bacterium prepares mixed bacteria liquid with gold-coloured staphylococci and escherichia coli.

3. method for quantitatively evaluating according to claim 2, which is characterized in that in the preparation of experimental model described in step S1, It is to mix the gold-coloured staphylococci liquid of same procedure culture and escherichia coli liquid by 1:1 to obtain mixed bacteria liquid.

4. method for quantitatively evaluating according to claim 2, which is characterized in that the use of the mixed bacteria liquid is with 0.01 × 10⁰ For best infection dosage.

5. method for quantitatively evaluating according to claim 1, which is characterized in that measure the effect of azithromycin described in step S2 Horizontal method is using HPLC-ECD method or HPLC-ESI-MS method；

The sample-pretreating method of the HPLC-ECD method or HPLC-ESI-MS method is to take 50 μ L of blood plasma, and 10M is added NH₄OH solution and internal standard roxithromycin methanol solution；Diethyl ether is added, is stood after vortex, upper organic phase is drawn after centrifugation, it is empty It is dried up under air-flow, flowing phased soln is added in residue, is vortexed, and centrifugation takes supernatant to be analyzed；

The chromatographic condition of the HPLC-ECD method are as follows: specification is the chromatographic column Aglient of 2.7 μ m, 4.6 × 100 mm Poroshell 120EC-C18, part number 695975-902, online filter: SSI35-0149；Mobile phase: volume ratio is 7.0 phosphate buffer solution of acetonitrile-pH of 45:55；Flow velocity 0.8mL/min；30 DEG C of column temperature, glass carbon working electrode potential+1.2V, Reference electrode Ag/AgCl；

The chromatographic condition of the HPLC-ESI-MS method are as follows: specification is the chromatographic column of 2.7 μ m, 4.6 × 100 mm Aglientporoshell120EC-C18, part number 695975-902, online filter: SSI35-0149；Mobile phase: body Product is than 5.2 ammonium acetate of -10 mM pH of acetonitrile for 65:35, flow velocity 0.2mL/min, 5 μ L of sample volume；Mass Spectrometer Method condition Are as follows: electrospray ionisation source (ESI), selective positive ion detection (SIM), azithromycin m/z 749.5 [M+H]⁺With internal standard Luo Hong Mycin m/z 837.5 [M+H]⁺, ionization source voltage 4.5kV, spraying gas N₂Flow velocity be 1.5L/min, desolventizing temperature 250 DEG C, detector voltage 1.5kv.

6. method for quantitatively evaluating according to claim 1, which is characterized in that the step S3 is with group mode for key Covariant quantifies the group for the PK and PD parameter that each active constituent group acts on azithromycin in Chinese medicine composition and composition The influence mode and related coefficient of model parameter.

7. method for quantitatively evaluating according to claim 1, which is characterized in that establish the comprehensive mould of PPK-PPD described in step S35 Type and the method for calculating corresponding model parameter are: the foundation of overall model are carried out referring to Kinettica and monolix software, respectively After node adjusts relevant parameter, AIC and Log likelihood value is carried out to front and back calculated result and is compared, setting is corresponding appropriate Calculating parameter；Analyze all pharmaco-kinetic processes using nonlinear mixed-effect model, can estimated driving force parameter group Distribution and the contribution due to physiology and pathology to making a variation between individual；Parser is mainly EM method, and E is that conditional expectation Bayes estimates Meter, M is maximal possibility estimation；Pharmacokinetic parameter has known priori in assumed group when conditional expectation Bayesian Estimation Distribution, while also assuming residual error distribution it is known that estimating under the described conditions individual drugs kinetic parameter；It is maximum Possibility predication is using the individual parameter of conditional expectation Bayesian Estimation as initial value, with maximum likelihood estimate Qiu Suan group's mean value and side Difference；Evaluation criterion is changed into the model conspicuousness of front and back Modifying model twice when EM algorithm iteration carries out, front and back calculates twice Model parameter changes without conspicuousness then operation termination.

8. according to claim 1 to any one of 7 method for quantitatively evaluating in evaluation Chinese medicine composition and antibiotic combination safety Application in terms of property and validity.

9. according to claim 1 to 7 any one method for quantitatively evaluating each active constituents in evaluation gynaecology's prescriptions worth thousand gold and side The application in terms of safety and validity is combined with antibiotic.

10. method for quantitatively evaluating disappears in Radix Angelicae Sinensis ingredient priming the side of acting in evaluation gynaecology's prescriptions worth thousand gold according to claim 1 The application in face.