阅读说明：本技术 一种检测药物镇痛评价过程中cox-2酶活性的方法 (A method of COX-2 enzymatic activity in detection medicine analgesic evaluation procedure ) 是由 肖伟 孙郊 郑伟然 李猛 张新庄 李尔广 柯志鹏 陶晓倩 于 2019-08-01 设计创作，主要内容包括：本发明涉及一种检测药物镇痛评价过程中COX-2酶活性的方法,其特征在于,该方法包括：对药物进行抑制COX-2酶的活性检测；将所述COX-2酶的活性检测结果进行数据处理与效价计算以确定药物的镇痛效果。利用本发明所提供的基于生物效价中药质量评价方法可进行对具有相应靶点的中西镇痛药物或活性组分,如桂枝茯苓制剂或其提取物等进行质量评价,并更好地控制药物的质量,确保临床用药的有效性。(The present invention relates to a kind of methods of COX-2 enzymatic activity in detection medicine analgesic evaluation procedure, which is characterized in that this method comprises: inhibit to drug the Activity determination of COX-2 enzyme；The Activity determination result of the COX-2 enzyme is subjected to data processing and potency calculating to determine the analgesic effect of drug.It can be carried out based on biological value medicine quality evaluated method to Chinese and Western analgesic or active component with corresponding target spot using provided by the present invention, such as cassia twig and poria cocos preparation or its extract carry out quality evaluation, and preferably control the quality of drug, it is ensured that the validity of clinical application.)

1. a kind of method of COX-2 enzymatic activity in detection medicine analgesic evaluation procedure, which is characterized in that this method comprises: to medicine Object inhibit the Activity determination of COX-2 enzyme；The Activity determination result of the COX-2 enzyme is subjected to data processing and potency calculates To determine the analgesic effect of drug.

2. the method according to claim 1, wherein the drug is selected from GUIZHI FULING JIAONANG.

3. the method according to claim 1, wherein the activity of the COX-2 enzyme is to be measured using fluorescent method.

4. the method according to claim 1, wherein the analgesia is pain caused by treatment dysmenorrhea.

5. a kind of method for detecting medicine analgesic effect assessment benchmark, which is characterized in that this method comprises:

Using the solution that qualified drug is prepared as test solution；

Inhibit to the test solution active testing of COX-2 enzyme；

Data processing is carried out to the result of the active testing and potency calculates, obtains the potency of qualified drug as evaluation base It is quasi-.

6. according to the method described in claim 5, it is characterized in that, this method further include:

Reference substance solution is prepared using cox 2 inhibitor as reference substance；Wherein, the cox 2 inhibitor is selected from celecoxib；

Inhibit to the reference substance solution active testing of COX-2 enzyme.

7. according to the method described in claim 5, it is characterized in that, the drug is selected from GUIZHI FULING JIAONANG；The analgesia is Treat pain caused by dysmenorrhea.

8. application of the evaluation criteria that claim 5-7 either method obtains in evaluation drug analgesic effect.

9. the method for a kind of pair of medicine analgesic effect detection, which is characterized in that this method comprises:

Using the solution that qualified drug is prepared as test solution；

Inhibit to the test solution active testing of COX-2 enzyme；

Data processing is carried out to the result of the active testing and potency calculates, the potency for measuring qualified drug establishes evaluation base It is quasi-；

Medicine preparation to be measured inhibit at solution to be measured the active testing of COX-2 enzyme, and is carried out using the evaluation criteria It examines.

10. according to the method described in claim 9, it is characterized in that, this method comprises:

Using the solution that GUIZHI FULING JIAONANG commercially available product is prepared as test solution；

Reference substance solution is prepared using celecoxib as reference substance；

Inhibit to the test solution and reference substance solution the active testing of COX-2 enzyme；

Data processing is carried out to the result of the active testing and potency calculates, establishes evaluation criteria；

Medicine preparation to be measured inhibit at solution to be measured the active testing of COX-2 enzyme, and is treated using the evaluation criteria The effect for surveying drug therapy dysmenorrhea is tested.

Technical field

The invention belongs to traditional Chinese medicine quality control and assessment technique field, in particular to a kind of detection medicine analgesic evaluation procedure The method of middle COX-2 enzymatic activity.

Background technique

The so-called pain of modern medicine is a kind of Physiological Psychology activity of complexity, is clinically one of most common symptom. It includes pain reaction (the body fortune that noxious stimulation acts on the feel of pain caused by body and body to noxious stimulation Dynamic property reaction and/or internal organ vegetative reaction, are often accompanied by strong mood color).Dysmenorrhea belongs to one kind of pain, wherein Primary dysmenorrhea (primary dysmenorrhea) i.e. functional dysmenorrhea, refers to painful menstrual periods, often in spastic, concentrates on Lower abdomen, other symptoms include have a headache out of strength, dizzy, nausea and vomiting, diarrhea, pain in waist and lower extremities, very common in young woman, shadow The quality of life of determining female physiological periodicity is rung, epidemiological study shows that primary dysmenorrhea is the most common disease of current gynaecology, state The ratio 43-90% of outer report primary dysmenorrhea, country's report dysmenorrhea disease incidence 33.1%, wherein primary dysmenorrhea accounts for 53.2%.

Doctor trained in Western medicine has certain understanding to dysmenorrhea mechanism at present, but and it is indefinite, Western medicine mostly use non-steroidal anti-inflammatory drugs or Contraceptive come alleviate dysmenorrhea generation pain, therapeutic effect is poor, and have stronger side effect.Compared to Western medicine, Chinese medicine is controlled Treat dysmenorrhea it is more advantageous-therapeutic effect is good and side effect is smaller.However, it is lower for the quality control level of Chinese medicine at present, It is main that quality control and evaluation is carried out to its medicinal material, bulk pharmaceutical chemicals, finished product using chemical analysis method or microscopical characters method.

Compared with Western medicine, since traditional Chinese medicine ingredients are extremely complex, criteria of quality evaluation be always perplex Chinese medicine changes now with The core of Basic national topographic map, and the key for establishing the criteria of quality evaluation of comparatively perfect is science of establishing, the control of reliable quality and comments Valence method.At this stage traditional Chinese medicine quality control with evaluation using the qualitative and quantitative analysis method of chemical component as main means, with Deepening continuously for traditional Chinese medicine research, it is found that it is very big this mode has gradually been shown in traditional Chinese medicine quality control and appraisement system Limitation.For example, certain single reference compound component content is high, it is good to represent its whole drug quality, can not be anti- It is more effective to reflect its whole drug.By in control herbal mixture certain single index compounds as medicine quality standard One Xiang Zhibiao, although to the accurate control that the amount of index ingredient carries out, it can be difficult to guaranteeing the steady of all the components in Chinese medicine Qualitative and homogeneity can not reflect the biological effect of Chinese medicine entirety, guarantee the consistency of its clinical efficacy.

But estimation of biological potency method is seldom in the research achievement report of field of Chinese herbal at present.Therefore, it is necessary to construct A kind of quality evaluating method based on biological value, evaluation index can be directly linked the clinical efficacy of the pain such as treatment dysmenorrhea.

Summary of the invention

In view of this, the present invention provides a kind of quality evaluating method based on biological value treatment pain class disease.This hair It is bright to propose a kind of method for detecting COX-2 enzymatic activity in medicine analgesic evaluation procedure, which is characterized in that this method comprises:

Inhibit to drug the Activity determination of COX-2 enzyme；

The Activity determination result of the COX-2 enzyme is subjected to data processing and potency is calculated and imitated with the analgesia for determining drug Fruit.

Specifically, the drug can be GUIZHI FULING JIAONANG.

Preferably, the activity of the COX-2 enzyme is to be measured using fluorescent method.

Preferably, the analgesia is pain caused by treatment dysmenorrhea especially primary dysmenorrhea.

The invention also provides a kind of methods for detecting medicine analgesic effect assessment benchmark, which is characterized in that this method packet It includes:

Using the solution that qualified drug (being optionally the commercially available product for having passed through inspection) is prepared as test solution；

Inhibit to the test solution active testing of COX-2 enzyme；

Data processing is carried out to the result of the active testing and potency calculates, obtains the potency of qualified drug as evaluation Benchmark.

Further, the method for the detection medicine analgesic effect assessment benchmark further include:

Reference substance solution is prepared using cox 2 inhibitor as reference substance；Wherein, it is next preferably to be selected from plug for the cox 2 inhibitor Former times cloth；

Inhibit to the reference substance solution active testing of COX-2 enzyme.

Specifically, the drug is preferably GUIZHI FULING JIAONANG；The analgesia preferably treatment dysmenorrhea especially primary Pain caused by dysmenorrhea.

The invention also provides the evaluation criteria the answering in evaluation drug analgesic effect that such as preceding either method obtains With.

The invention also provides the methods of a kind of pair of medicine analgesic effect detection, which is characterized in that this method comprises:

Using the solution that qualified drug (being optionally the commercially available product for having passed through inspection) is prepared as test solution；

Inhibit to the test solution active testing of COX-2 enzyme；

Data processing is carried out to the result of the active testing and potency calculates, the potency for measuring qualified drug establishes evaluation Benchmark；

Medicine preparation to be measured inhibit at solution to be measured the active testing of COX-2 enzyme, and utilizes the evaluation criteria It tests.

Preferably, the method to medicine analgesic effect detection includes:

Using the solution that GUIZHI FULING JIAONANG commercially available product is prepared as test solution；

Reference substance solution is prepared using celecoxib as reference substance；

Inhibit to the test solution and reference substance solution the active testing of COX-2 enzyme；

Data processing is carried out to the result of the active testing and potency calculates, establishes evaluation criteria；

Medicine preparation to be measured inhibit at solution to be measured the active testing of COX-2 enzyme, and utilizes the evaluation criteria It tests to the effect of drug therapy dysmenorrhea to be measured.

Beneficial effects of the present invention include at least:

1, it can be carried out based on biological value medicine quality evaluated method to corresponding target spot using provided by the present invention Chinese and Western analgesic or active component, such as cassia twig and poria cocos preparation or its extract carry out quality control and evaluation；

2, using the quality of the Chinese medicines analgesics such as preferably control cassia twig and poria cocos preparation of the invention, and can be inhibited COX-2 bioactivity controls in the reasonable scope, it is ensured that the validity of clinical application.

Detailed description of the invention

Fig. 1 is GUIZHI FULING JIAONANG sample in the present invention to the amount-result relation figure of COX2 inhibiting effect；

Fig. 2 is GUIZHI FULING JIAONANG sample concentration and inhibiting rate linear relationship chart in the present invention in the present invention.

Specific embodiment

The present invention mainly establishes the drug biological assessment method of the COX-2 enzyme activity based on the recombination of fluorescence detection source of people.For Preferably it is easy to understand the present invention, will illustrate the present invention so that cassia twig tuckahoe is to the treatment of dysmenorrhea as an example below, these implementations Example only serves illustrative, does not limit to and application range of the invention.

The quality control of existing cassia twig and poria cocos preparation and evaluation method are mostly with the qualitative and quantitative analysis of chemical index ingredient Based on, however, this chemical detection method can not be associated with clinical efficacy, therefore cannot really face effective evaluation drug Bed mass.In order to solve the above problem, it is necessary to provide a kind of based on estimation of biological potency cassia twig and poria cocos preparation treatment dysmenorrhea class disease The quality evaluating method of disease.The pain especially generation of intermenstrual period dysmenorrhea and prostaglandin (PGs) amount correlation are very strong, big absolutely Some women dysmenorrhea is that PGs dependence endogenous PGE2 and PGF2 α is increased and stimulated uterus strong contraction, vasopasm, Jin Erzao Cause intrauterine pressure temporarily to increase at uterus ischemic, anaerobic condition and generates spasmic pain indirectly.And the synthesis of prostaglandin There is a critical oxidation enzyme-cyclooxygenase 2 (i.e. COX-2) in the process, cassia twig and poria cocos preparation can be by inhibiting COX-2 activity, resistance The synthesis of disconnected prostaglandin, to reduce, intrauterine Prostaglandin is horizontal, and performance analgesic acts on.COX-2 enzyme activity and pain Through clinical evaluation index pain, there are very strong correlations, therefore can be active to COX-2 by measurement cassia twig and poria cocos preparation Inhibiting rate characterizes the biological effect of the drug.

Compound Chinese herbal medicinal ingredients are more complicated, play integrally-regulated effect often through multiple target point, multipath, can more hit The change of disease complexity pathology, so as to improve or cure effect.For this feature of Chinese medicine, biology effect proposed by the present invention The method of quality control of valence evaluation can carry out quality control and evaluation based on the whole of Chinese medicine complicated ingredient, for opening for drug Hair and the control of clinical application quality and curative effect embodiment provide important reference.The method of quality control can be from different approaches Reflect that cassia twig and poria cocos preparation for improving the power of dysmenorrhea bioactivity, embodies the group effect that plays a role of drug, and faces The validity association of bed curative effect, can clearly reflect pharmaceutically-active pathology link, provide reliable basis for clinical application.

In the present invention, except particularly pointing out, drug used, reagent, instrument can be by normal in technical solution provided by the invention Rule channel or market are bought.The specific experiment method that do not mention in the following example, usually carries out according to conventional methods.

To facilitate the understanding of the present invention, the meaning that related letter abbreviations are referred to is provided below:

COX-2: Transitional cell carcinomas

DMSO: dimethyl sulfoxide

The biological assessment method of the COX-2 enzyme activity of fluorescence detection source of people recombination

Test apparatus: fluorescence microplate reader

Test material:

GUIZHI FULING JIAONANG, Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov

Transitional cell carcinomas (COX-2) inhibitor screening kit, the green skies, the examination that Item No.S0168 company newly purchases Agent box

COX-2 Assay Buffer (reagent caching liquid), Cayman, Item No.S0168-1

COX-2 Probe (probe), the green skies, Item No.S0168-2,600ul

COX-2 Cofactor (co-factor working solution) (50 ×), the green skies, Item No.S0168-3,20ul

COX-2 Substrate (substrate) (50 ×), the green skies, Item No.S0168-4

Substrate Buffer (substrate caching liquid), the green skies, Item No.S0168-5

RhCOX-2 (25 ×) (people recombinant C OX-2), the green skies, Item No.S0168-6,22ul

Celecoxib (100 μM) (celecoxib), the green skies, Item No.S0168-7

96 porocyte plates (Corning 3925black plate, black bottom)

Celecoxib: green skies Bioisystech Co., Ltd

Sample configuration:

Reagent prepares: enzymatic hydrolysis freezes spare to room temperature centrifugation mixing；Probe,Cofactor(50×),Substrate(50 ×) DMSO thaw (can 37 DEG C of water-bath 0.5-2min) to room temperature, spare (long-term -20 DEG C are kept in dark place) is kept in dark place.

Cofactor working solution: Cofactor (50 ×) dilutes 50 times in right amount, with assaybuffer, and 4 DEG C of preservation same day are standby With.

COX-2 working solution: rhCOX-2 (25 ×) dilutes 25 times in right amount, with assay buffer, and ice bath saves standby in 1h With.

Substrate working solution: Substrate (50 ×) in right amount, adds isometric Substrate buffer to dilute, whirlpool Rotation mixes, and mixture dilutes 125 times with ultrapure water again, and ice bath saves spare in 1h.

Positive drug solns are prepared: taking cox 2 inhibitor is positive reference substance, with DMSO be solvent carry out dissolution be configured to it is dense Degree is the solution of 0.3429ug/ml, and the final concentration of 0.019 μ g/ml (50nM) in reaction system, the cox 2 inhibitor can Select celecoxib.

Test solution is prepared: being taken GUIZHI FULING JIAONANG content appropriate, is mentioned with the solvent (DMSO) of appropriate ratio It takes, seals, mix, 37 DEG C of water-baths 30min, 7000g are centrifuged 2min, and supernatant crosses 0.22 μm of filter membrane, and filtrate is as mother liquor, concentration note It is 1.

Dilution process: taking GUIZHI FULING JIAONANG mother liquor, and DMSO dilutes 256 times and obtains GUIZHI FULING JIAONANG-a, mother liquor dilution 312 times obtain GUIZHI FULING JIAONANG-b, and mother liquor dilutes 624 times and obtains GUIZHI FULING JIAONANG-c, and mother liquor dilutes 1204 times and obtains osmanthus Branch Indian buead capsule-d.

It is grouped situation:

1 COX inhibitor sifting reaction system (90 μ l) of table

Experimental method:

It using 96 porocyte plates, is arranged by each hole, assaybuffer, Cofactor working solution (concussion), COX-2 is added Working solution (concussion), COX-2 working solution (concussion), sample solution, the volley of rifle fire mix, 25 DEG C of incubation 5min；Place into (4 DEG C on ice Refrigerator) it is incubated for 10min, cooling；Each hole adds 5 μ l of Probe working solution, and the volley of rifle fire mixes；Each hole adds 5 μ of low temperature Substrate working solution L is operated at low ambient temperatures as far as possible, and the volley of rifle fire mixes；

Add after working solution that 37 DEG C of measurement fluorescence, excitation wavelength 560nm, launch wavelength are that (every 30s surveys 1 to 590nm immediately It is secondary, continuous 15min).

Using " dose response-inhibition " model of GraphPad Prism and drug is calculated to COX-2 using following formula The inhibiting rate of enzymatic activity draws amount-effect curve, predicts IC50.

Inhibiting rate (%)=(RFU of COX-2_Sample-RFU_{Blank control})/(RFU_{Initial enzyme activity}-RFU_{Blank control}) * 100% interpretation of result:

Inhibiting rate of each dosage cassia twig tuckahoe of table 2 to COX enzyme

As shown in Fig. 1 and table 2, amount-effect of the GUIZHI FULING JIAONANG sample based on DMSO extraction to COX2 inhibiting effect Relationship investigates the middle section as a result, selection S type curve, i.e., 1/128 (252.4 μ g/ml) -1/1024 (31.55 μ g/ml) concentration range Concentration and inhibiting rate carry out linear dependence analysis, repeatedly test result shows GUIZHI FULING JIAONANG 1/128 three times In (252.4 μ g/ml) -1/1024 (31.55 μ g/ml) concentration range, good linearly related (such as figure is presented in concentration and inhibiting rate 2)。

The present invention uses COX-2 to pass through as the biological action target spot of dysmenorrhea pain by taking GUIZHI FULING JIAONANG as an example It is compareed with positive drug, makes GUIZHI FULING JIAONANG and inhibit the active dose-effect relationship of COX-2, and by the way that GUIZHI FULING JIAONANG is dense It spends in the control of the concentration limits of COX-2 inhibiting rate dose-effect relationship within the scope of reasonable and the similitude of amount effect curve, IC50 concentration etc., the quality for carrying out biological value to GUIZHI FULING JIAONANG control.

Chinese patent drug GUIZHI FULING JIAONANG (Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov) used in the present invention is originated from our company Self-made products, wherein specific embodiment chooses cassia twig tuckahoe glue capsule and carries out quality evaluation, but the present invention is only exemplary example It lifts, however it is not limited to which capsule formulation is also not necessarily limited to GUIZHI FULING JIAONANG.

Therefore since Chinese medicine treatment dysmenorrhea is effective, pain measurement is mostly subjective index, lacks objective indicator.But this Invention overcomes drawbacks described above well, and used biological value checking method can accurately and rapidly detect cassia twig tuckahoe system Agent (treatment dysmenorrhea drug) carries out unified biological value to drug and indicates to the inhibiting rate of COX2 enzymatic activity.In pharmaceutical production Intermediate product in the process, finished product, links, the minor biological cycling such as the quality of medicinal material and the place of production, collecting season can become One of the means of quality control.Quality evaluating method of the present invention can be used as effective benefit of traditional chemical detection means It fills, directly reaction Clinical efficacy, is that one kind really meets traditional Chinese medicine traditional theory, embodies Chinese medicine clinical application feature, guarantees Chinese medicine safely and effectively quality control and evaluation method.

The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as Protection scope of the present invention.