WO2019109779A1

WO2019109779A1 - Detection method for chinese medicine composition based on inhibition of cox-2 activity

Info

Publication number: WO2019109779A1
Application number: PCT/CN2018/114831
Authority: WO
Inventors: 王伽伯; 肖小河; 贾振华
Original assignee: 石家庄以岭药业股份有限公司
Priority date: 2017-12-05
Filing date: 2018-11-09
Publication date: 2019-06-13
Also published as: CN109870432A

Abstract

A detection method for a Chinese medicine composition. The method uses an inhibition ratio on COX-2 enzyme of the Chinese medicine composition as a determination index to detect anti-inflammatory activity thereof. The method comprises: A) preparing a Chinese medicine composition sample solution and/or a control solution; B) determining inhibition ratios on COX-2 enzyme activity of the sample solution and the control solution; and C) calculating potency and percentage fiducial limits using the inhibition ratios on COX-2 enzyme activity as indices.

Description

\¥0 2019/109779 ?€1^2018/114831

Method for detecting traditional Chinese medicine composition based on inhibition of <:0-2 activity

[0001] The present scheme relates to a biological detection method for a traditional Chinese medicine composition, and belongs to the field of medicine.

Background technique

[0002] At present, the quality evaluation of a traditional Chinese medicine compound is only carried out by measuring a single chemical component. First, whether the chemical component is a compound that exerts a pharmacodynamic effect. Second, whether the minimum content of the chemical component is controlled can effectively control the quality thereof. Effectiveness and consistency? From the current research, it is obviously impossible to evaluate the quality of traditional Chinese medicine compound by measuring only a single chemical component. Therefore, according to the specific conditions of traditional Chinese medicine compound, combined with modern science and technology, to explore the ability to meet the characteristics of traditional Chinese medicine, to a certain extent, not only related to clinical efficacy and related mechanisms, the operability of traditional Chinese medicine compound preparation quality assessment method, to ensure Chinese medicine The consistency and stability of compound clinical efficacy is one of the most important topics in the field of traditional Chinese medicine research.

[0003] The guiding principle of biological activity measurement has been included in the Chinese Pharmacopoeia, and the method of biological evaluation has been adopted in the quality control of medicinal materials such as leeches;

The "Botanical Drug Development Industry Guide" also clearly states that biological evaluation will be the main content of the registration review of botanical drugs. It can be seen that biological evaluation has become the development direction of the quality evaluation of traditional Chinese medicine and traditional Chinese medicine compound preparations. For traditional Chinese medicine, the vast majority of clinical applications are traditional Chinese medicine compounds, and there is rarely a single medicinal use. The complex composition and various pharmacological effects determine that the detection of one or several active ingredients does not reflect its overall efficacy. It is not feasible to evaluate Chinese medicine only through this standard. Introducing the biological evaluation method into the quality control system of traditional Chinese medicine can not only identify the variety, but also evaluate the quality. It can also be linked to its efficacy and observe its side effects. To some extent, the use of biological evaluation methods to detect the quality of traditional Chinese medicine has obvious advantages over traditional chemical methods. Among them, the analysis of the Chinese compound big compound can better reflect its advantages.

Summary of invention

technical problem

[0004] For traditional Chinese medicine, the vast majority of clinical applications are traditional Chinese medicine compounds, and there is rarely a single medicinal use. The complex composition and various pharmacological effects determine that the detection of one or several active ingredients does not reflect its Overall efficacy, it is not feasible to evaluate Chinese medicine only through this standard. \¥0 2019/109779 ?€1^2018/114831 Solution to the problem

Technical solution

[0005] In view of the deficiencies of the prior art, the present invention provides a biological detection method for a traditional Chinese medicine composition, and the detection method for detecting only one or several active ingredients relative to the prior art, the biological detection method of the present embodiment can be more Comprehensively characterize the overall efficacy of traditional Chinese medicine compositions to control their quality.

[0006] A method for detecting a traditional Chinese medicine composition, the method comprising: preparing a traditional Chinese medicine composition as a detectable sample

, in contact with cyclooxygenase ((:0), determine the inhibition rate of the sample on cyclooxygenase ((:0)).

In a specific implementation, the detecting method includes the following steps:

[0008] preparing a traditional Chinese medicine composition sample solution and / or a reference solution;

[0009] 3) determining the inhibition rate of the sample solution and the reference solution solution (: 0-2 enzyme activity;

[0010] 0 (: 0 - 2 enzyme activity inhibition rate index, calculate the titer and confidence rate;

[0011] wherein, the step of determining the inhibition rate of the sample solution and the reference solution solution (0-2 enzyme activity) can be determined by an enzyme immunoassay or the like.

[0012] As a preferred embodiment, the inhibition rate can be determined as follows:

[0013] adding (: 0 - 2 enzyme to the solution to be tested, cultured for a certain period of time;

[0014] 15) adding eight 0 and eight eight to the solution to be tested;

[0015] 显) color development, detecting fluorescence intensity;

[0016] (1) Calculate the inhibition rate according to the following formula,

[0017] % inhibition rate = (fluorescence intensity of initial active sample - fluorescence intensity of sample to be tested) / fluorescence intensity of initial active sample X 100%.

[0018] Preferably, the step & medium (: 0 - 2 enzyme dosage is 50011 / 1 ^ ~ 1000 11 / 1 ^;

[0019] Preferably, in step 15, eighty-eight is purified;

[0020] Preferably, the fluorescence intensity in the step 〇 is detected at an excitation light wavelength of 530-54011111 and an emission wavelength of 585-59511111.

[0021] In a specific embodiment, the reference substance in step VIII is selected from a conventional anti-inflammatory drug, such as celecoxib, rofecoxib.

, or can be used as a control for the traditional Chinese medicine composition. The preparation method of the reference solution comprises: taking a reference substance and extracting with an organic solvent, and the extract is a reference solution; preferably, the concentration of the reference solution is IX 10 ⁵ 11/011^2x10 ⁵ 11/011^ More preferably, the pair \¥0 2019/109779 卩(:17 \2018/114831 The concentration of the solution is 1.2x10 ⁵ 11/1^.

[0022] The preparation method of the sample solution of the traditional Chinese medicine composition in the step 8 includes: taking the traditional Chinese medicine composition, extracting with an organic solvent, and extracting the solution is a reference solution; preferably, the concentration of the sample solution is 1×10 ⁵

^111^2x10 ⁵ 11/1111^ More preferably, the sample solution concentration is 1.2x10 ⁵ 11/1111 _^ .

[0023] wherein the organic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), methanol, ethanol, etc.; and the extraction method is selected from any one of ultrasonic extraction, reflux extraction, and microwave extraction.

[0024] Step (: the "potency" and "credit limit rate" are calculated according to the "quality reaction parallel line method" in the "Bioassay of Drugs" (edited by Zhou Haijun); inhibition (: 0-2 enzyme activity titer) Not less than 500 \J-\ig ¹ , the average confidence rate is less than 25%.

[0025] The traditional Chinese medicine composition of the present scheme is composed of the following raw materials:

[0026] Forsythia 200-300 parts by weight, honeysuckle 200-300 parts by weight, ramie 50-100 parts by weight, fried almonds 50-100 parts by weight, gypsum 200-300 parts by weight, Radix is 200-300 parts by weight, 200-300 parts by weight of Mianma Guanzhong, 200-300 parts by weight of Houttuynia cordata, 50-100 parts by weight of patchouli, 30-80 parts by weight of rhubarb, 50-100 parts by weight of Rhodiola, 5-10 weight of menthol Parts, licorice 50-100 parts by weight.

[0027] Preferably, the traditional Chinese medicine composition is composed of the following raw materials:

[0028] Forsythia 210-270 parts by weight, honeysuckle 210-270 parts by weight, ramie 60-90 parts by weight, 60-90 parts by weight of fried almonds, 210-270 parts by weight of gypsum, 210-270 parts by weight of radix isatidis, cotton Ma Guanzhong 210-270 parts by weight, Houttuynia 210-270 parts by weight, patchouli 60-90 parts by weight, rhubarb 40-60 parts by weight, Rhodiola 60-9 0 parts by weight, menthol 6-8 weight Parts, licorice 60-90 parts by weight.

[0029] More preferably, the traditional Chinese medicine composition is composed of the following raw materials:

[0030] 255 parts by weight of forsythia, 255 parts by weight of honeysuckle, 85 parts by weight of ramie, 85 parts by weight of fried almonds, 255 parts by weight of gypsum, 255 parts by weight of radix isatidis, 255 parts by weight of mulberry, 255 weight of houttuynia A portion, 85 parts by weight of patchouli, 51 parts by weight of rhubarb, 85 parts by weight of Rhodiola, 7.5 parts by weight of menthol, and 85 parts by weight of licorice.

[0031] The preparation method of the traditional Chinese medicine composition: the above thirteen flavors, patchouli and water distilled extract volatile oil, collecting volatile oil, water extract filtered, spare; wing, ramie, houttuynia, rhubarb with 70 % ethanol extraction twice, the first 2 hours, the second 1.5 hours, the extract is filtered, combined, recovered ethanol, ready to use; honeysuckle, gypsum, Banlangen, Mianmaguanzhong, licorice, Rhodiola and water to cook until Boiling, adding fried almonds Decoction twice, the first 1.5 hours, the first 1 hour, the decoction filtered, the filtrate combined, add the aqueous solution after the patchouli oil, and concentrate to a relative concentration of U0~U5 (60 °C) , adding ethanol to make the alcohol content up to 70%, at 4. C is refrigerated for 24 hours, filtered, and the filtrate is recovered from ethanol. It is combined with the four kinds of alternate alcohol extracts such as forsythia, concentrated to a relative density of U0~U5 (60°〇, spray dried, mixed with appropriate amount of starch, made) Granules, drying, sieving, sieving out the appropriate amount of fine powder, dissolving the menthol and patchouli volatile oil with an appropriate amount of ethanol, spraying into the fine powder, mixing, mixing with the above particles, sealing for 30 minutes, and filling into capsules. Into 1000 capsules, that is.

[0032] The principle of the detection method of the present scheme is based on a fluorescence method, under the action of COX-2, arachidonic acid (AA) is converted into prostaglandin endoperoxide (: PGG2), catalyzed by: PGG2 and ADHP ( The reaction between 10-Acetyl-3,7-dihy droxyphenoxazine) produces a fluorinated substance, resorufin. Fluorescence is produced at a specific wavelength. By measuring the fluorescence intensity of resorufin, the COX-2 enzyme activity can be expressed, and the drug to be tested can be evaluated to inhibit the activity of COX-2 (see Fig. 1).

[0033] Experimental Example 1 Protective effect of traditional Chinese medicine composition on H1N1 influenza virus-infected mice and verification of biological evaluation indexes

[0034] 1. Experimental materials

[0035] 1.1 Experimental Instrument

[0036] LEICA DM3000 inverted microscope (Leica, Germany); D3024R desktop high speed frozen microcentrifuge (DragonLab); SIGMAJ-26 refrigerated centrifuge (SIGMA, Germany); AL201 electronic balance (METTLER TOLEDO instrument (Shanghai) ))) RM2245 manual rotary slicing machine for electronically controlled injection, EG1150H split paraffin embedding machine (Leica, Germany); S1000 Thermal Cycler (BIO-RAD); JY-1000M electric thermostat blast drying Box (Wujiang Zhisheng Oven Equipment Factory); Stepone plus Xenon Quantitative PCR Instrument (ABI Company, USA); SW-CJ-1FD Ultra-clean Workbench (Sujing Antai Company); NanoDrop2000 Ultra-micro Spectrophotometer (American Thermo Company) ).

1.2 drugs and reagents

[0038] Chinese medicine composition capsule (prepared in Example 1); PBS purchased from Gibco; 4% paraformaldehyde solution purchased from GBCBIO company; xylene purchased from Guangzhou Chemical Reagent Factory; hematoxylin dye, eosin dye purchased in Zhuhai Besso Biotechnology Co., Ltd.; IL-6, COX-2, GAPDH primers purchased from Life Company, USA; TRizol \¥0 2019/109779 ?€1^2018/114831

Reagent purchased from life company (batch No. 15596-026), DEPC treated water purchased from Solarbio (batch R1600)

cDNA Synthesis SuperMix was purchased from Novoprotein (batch E044-01B); trichloromethane, isopropanol and absolute ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (batch numbers 10006818, 8010921 8, 10009218); first strand cDNA synthesis The kit was purchased from Thermo Company of the United States (batch #K1622)

; FastStart Universal SYBR Green

Master (Rox) was purchased from Roche, USA (batch number ⁴ 9139U001).

[0039] 1.3 animals

[0040] BALB/c mice, female, 6-8 weeks old, clean grade, weight 18-20 g, Guangdong Provincial Experimental Animal Center, Certificate No.: [SCXK (Guangdong) 2003-0002]. IVC cage feeding, free drinking water, room temperature 25±2 °C, humidity 60-70%, set 12 hours of indoor light/dark cycle, feeding with standard food and water, keeping cages, tableware and environment clean Keep the room quiet. All animals were pre-adapted for 7 days prior to the experiment and all mice were carefully cared for in the experiment. Approved by the Animal Experimental Ethics Committee.

[0041] 1.4 virus and cell strain

[0042] Influenza A virus PR8 strain (A/PR/8/34, H1N1) was purchased from the American Classic Culture Collection (ATCC). When inoculated, the 9-11 day old chicken embryo allantoic cavity was inoculated, cultured at 37 ° C for 3 days, and the allantoic fluid was collected for use (hemagglutination titer). LD _{5Q was} measured in mice according to the method of Reed-Muench

(50% lethal dose). MDCK (Madin Darby Canine Kidney, dog kidney cells) cells were purchased from the Concord Cell Bank of the Chinese Academy of Sciences.

[0043] 2. Experimental method

[0044] 2.1 Determination of the median lethal dose of mice infected with virus (LD ₅ 〇)

[0045] 50 BALB/c mice, 6-8 weeks old, female, randomly divided into 5 groups, 10 times serial dilution of virus (A/PR/8/34, H1N1) with sterile PBS was 10-2 , 10-3, 10-4, 10-5, 10-6 total 5 dilutions

. After mild anesthesia with diethyl ether, the mice were infected by intranasal route. Each mouse was infected with 5 (VL, observed for 15 days, and the survival and death of mice infected with different dilutions of the virus solution were recorded. According to Reed-Muench The method calculates the median lethal dose LD _{5Q of the} virus in mice.

[0046] 2.2 Animal experiment grouping and administration

[0047] Mice were randomly divided into 5 groups of 15 each. Normal group (Normal), model group (Model)

, oseltamivir (Oseltamivir) (60 Mg/kg/day), Chinese medicine composition was administered in high dose group (1300 mg/kg/day) and low dose group (650 mg/kg/day). The mice underwent mild respiratory anesthesia. The model group was infected with two LD _5Q HlN1 virus dilutions, and each mouse was infected with 5 (VL; blank group was instilled into sterile PBS. The day of infection was 2 hours before infection. Dosing, continuous administration for 5 days. 0.2 mg of each of the high- and low-dose groups in the oseltamivir group and the traditional Chinese medicine composition, administered twice a day, 8 hours apart, and the blank group and the model group were administered in the same manner. Saline.

[0048] 2.3 Determination of IL-6 and COX-2 mRNA expression in lung tissue

[0049] 2.3.1 Extraction of total RNA from lung tissue

[0050] 100 mg of lung tissue of each group of mice was weighed, and Trizol lmL was added thereto at 15,000 rpm for three times for 10 s. The homogenate was completed and the RNA was extracted by the Trizor method. RNA purity OD260/OD280 is between 1.8 and 2.0. Used in subsequent experiments.

2.3.2 PCR Reaction System and Procedure

[0052] PCR reaction system: Primer l _[ xL, 10xdNTP 2 _[ xL, Ri l _[ xL, Rt l _[ xL, Reaction Buffer 4]

\ih; Adjust to the same concentration of Template RNA and DEPC water for a total of 11 ^ L. Total 20^. Reverse transcription into cDNA, reaction conditions: 25. C 5min, 42. C 60 min, 70. C 5 min.

[0053] PCR reaction procedure: After reverse transcription was completed, it was diluted 5 times with DEPC water. Add sample to each well, premix

9^1 (SYBR Green 4^, F l^iL, R l^iL, DEPC water 3^L), film, centrifuged and tested on the machine. Test procedure: Pre-denaturation 95°C 10min, cycle 40 times 95°C 15s®60°C 60s, dissolution curve 75°C ® 95°C, 1°C per 20s.

[0054] The PCR primer sequences are shown in Table 1.

Table 1 PCR primer sequences

[0056]

\¥02019/109779 卩(:17 \2018/114831

[Table 1]

[0057] 2.4 statistical analysis

[0058] 〇1^& ^]^1116.0 and 〇3^11^] ^* 〇 9.2 software processing, 1 test for statistical analysis, ? <0.05 was considered statistically significant.

For the internal reference, 2 - ^{△△ was} used for relative gene expression analysis.

[0059] 3. Experimental results

[0060] The lung tissue samples of each group of mice were collected on the 5th day after the administration of each infection, and 11 _^ -6, (: 0 X-2 RNA in the lung tissue was extracted for real-time quantification.

Detection. The results are shown in Table 2. The high- and low-dose groups of the traditional Chinese medicine composition had significant inhibitory effects on the expression of 11 _^ -6 and (: 0 - 21111 八 ^ < 0.01, ? < 0.05) and had a dose-effect relationship.

Table 2 Effect of Chinese Herbal Medicine Composition on Expression of 11 _^ -6 and COX-2 mRNA in Mouse Lung Tissue

[Table 2]

[0063] *^<0.05, **p<0·01 compared with Model [0064] The above results indicate that the normal group of mice has a good mental state, agile action, a lighter hair, a more uniform breathing, and a weight gain. After the second day of infection, the mice in the model group had uneven breathing, contracture, and dullness, and the diet was significantly reduced and eliminated. This H1N1 influenza virus mouse model was shown to be successful. After the virus infection, the body weight of the mice began to decrease, and the administration of the oseltamivir-positive drug and the traditional Chinese medicine composition all reduced the weight loss of the mice. The lung viral load, lung index data and lung histopathological sections of the mice also showed that the traditional Chinese medicine composition can effectively inhibit the replication of influenza virus in mice, and has obvious anti-influenza-induced pneumonia.

[0065] The mRNA expression of IL-6 and COX-2 in mouse lung tissue was determined by real-time quantitative PCR, and it was found that the traditional Chinese medicine composition can significantly reduce the expression of two genes in lung tissue in a dose-dependent manner. The overall animal level verified the reliability of the selection of IL-6 and COX-2 for the anti-inflammatory biological evaluation indicators of traditional Chinese medicine compositions.

Experimental Example 2 Bioassay method for traditional Chinese medicine composition based on inhibition of COX-2 activity

Based on the results of the experimental example 1, it was confirmed that COX-2 is closely related to the anti-inflammatory mechanism of the traditional Chinese medicine composition, and therefore, it is used as an indicator for biological evaluation of the traditional Chinese medicine composition, and a biological evaluation method for the traditional Chinese medicine composition is established.

[0068] 1. Experimental materials

[0069] 1.1 Instruments and Materials

[0070] SynergyH2 full-function microplate detector (BioTek, USA); desktop high-speed centrifuge (Thermo, USA); micropipette, multi-channel pipette (Eppendorf, Germany); one-tenth electronic analysis Balance (METTLER TOLEDO Instrument Shanghai Co., Ltd.); DK-600S three-use constant temperature water tank (Shanghai Jinghong Experimental Equipment Co., Ltd.); Vortex-Genie® 2 vortex instrument (Shenzhen An Bisheng Technology Co., Ltd.); Centrifuge tube (Corning, USA).

1.2 Drugs and Reagents

[0072] DMSO was purchased from AMRESCO, USA (batch No. 3304C252); K547-100 COX-2 inhibitor screening kit (F) was purchased from Biovision, USA (batch number)

2F280547); Chinese medicine composition prepared according to Example 1, a total of 40 batches (No. S01-S40); capsule contents were placed under high temperature (60 ° C), high humidity (RH95%), light (45001x) conditions 20d samples Al, A2, A3 (No. S41-S43); Celecoxib was purchased from aladdin (batch #E15281 16).

2. Experimental method [0074] 2.1 Detection Step

[0075] Step 1: In the 96-well plate, the drug to be tested is added; COX-2 is added and incubated for a certain period of time to inhibit the reaction of the drug with C OX-2;

[0076] Step 2: Adding ADHP and AA respectively, using catalytic generation: PGG2 combined with ADHP to produce resorufin

[0077] Step 3: color development of the enzyme standard pore, the excitation light wavelength is 530-540 nm, and the emission intensity is detected at 585-595 nm, compared with the blank and 100% active pores, and the drug is calculated according to the formula for COX-2. Inhibition rate

[0078] Step 4: After the coordinate conversion of the inhibition rate of the drug to COX-2, the titer and the confidence limit are calculated.

[0079] 2.2 Preparation of liquid medicine

[0080] Dilute Assay buffer R (10x) 10-fold with sterile ultrapure water as a buffer after dilution; dilute heme (Heme), COX-2 with 1:10 diluted buffer (on ice) Operation), ADHP (pre-formulated); Dilute AA 1:1 with KOH, vortex and then dilute 1:5 with sterile ultrapure water to 2 mM.

[0081] Precision weighed a certain amount of celecoxib, force PDMSO dissolved ultrasonic extraction lOmin, 0.45 filter filter, dilute DMSO into O^pg / mL mother liquor, stored in a refrigerator at 4 ° C, diluted To the desired concentration

[0082] Weigh a certain amount of traditional Chinese medicine composition accurately, and dissolve the DMSO into a mother liquor of lmg/mL, ultrasonically extract for 10 min, centrifuge at 12000 rpm/min for 10 min, and take the supernatant for use. Dilute to the desired concentration when used.

2.3 Determination of COX-2 Inhibition Rate

[0084] Experiment Setting Background Group (Background), COX-2 100% Initial Activity Group (100% Initial)

Activity) Inhibitor group. In a 96-well plate, enter 15 (VL reaction buffer, HVL heme, HVL COX-2, and HVL solvent at 100% Initial Activity; add 16 (VL reaction buffer, IO^IL heme and HVL solvent; Inhibitor was added 150 reaction buffer, HVL heme, HVL COX-2 and HVL inhibitors at different concentrations. Incubate for 10 min at room temperature; add ADVL to HVL in each well; quickly add arachidonic acid HVL to each well to start The reaction was incubated for 5 minutes at room temperature, placed in a microplate reader, and the intensity of the excitation light was detected at a wavelength of 530-540 nm and an emission wavelength of 585-595 nm.

[0085] The fluorescence intensity of the 100% initial active sample and the inhibitor sample needs to be subtracted from the background fluorescence intensity. Buckle \¥0 2019/109779 ?€1^2018/114831 In addition to the background of the 100% initial active sample fluorescence intensity (Initial Activity) minus the inhibitor intensity of each inhibitor sample (Sample Activity), and then divided by Initial

Activity, multiplied by 100, to obtain the inhibition rate of COX-2 (Inhibition%), the calculation formula is as follows

[0086] Inhibition rate % = (Initial Activity-Sample Activity) / Initial ActivityxlOO%

[0087] 2.4 Determination of Biopotency of Traditional Chinese Medicine Composition

[0088] The celecoxib was used as the reference control substance and the reference composition of the traditional Chinese medicine composition (S40), and the concentration was reduced by 1:0.5 times, and the inhibition rate of COX-2 was determined by the three wells, and the reference of the traditional Chinese medicine composition was defined. The original valence value of the object (S40) is WOOU/pg. The other batches of samples were tested for the test group, and the potency (PT) and confidence limit (FL%) for inhibiting COX-2 activity were calculated according to the principle of the simple probability unit method.

[0089] 3. Experimental results

[0090] 3.1 reagent AA before and after purification on the drug inhibition of COX-2 activity

The fluorescence intensity of each concentration of celecoxib was measured according to the above experimental method, and the influence of the AA before and after the purification on the measurement results was examined.

[0092] The results showed that the fluorescence background value detected before AA purification was 317, and the inhibition rate exceeded 100%; while the background value after purification (purity >95%) decreased significantly to 50 or less. The IC50 of celecoxib after purification was 2.5. Therefore, the purified AA should be selected as the detection reagent during the experiment.

3.2 Effect of COX-2 enzyme dosage on drug inhibition of COX-2 activity

According to the above experimental method, a COX-2 enzyme amount of 500 U/mL and 1000 U/mL was selected for the reaction, and the bioavailability of the TCM composition for COX-2 inhibitory activity was determined. The results showed that the amount of different enzymes had little effect on the bioavailability of the drug to inhibit COX-2 activity. From the cost point of view, the enzyme dosage was chosen to be 500 U/mL.

3.3 Inhibition of COX-2 Enzyme Activity by Traditional Chinese Medicine Composition

[0096] The prepared Chinese medicine composition was subjected to a test solution, and the fluorescence intensity of each group was measured according to the method of 2.2.4. The inhibitory effect of the traditional Chinese medicine composition on the activity of COX-2 enzyme was examined, and the results are shown in FIG.

[0097] The results show that the composition of the traditional Chinese medicine composition is 1

<〇.〇1), indicating that the traditional Chinese medicine composition has a significant inhibitory effect on COX-2 enzyme activity and has a concentration-dependent relationship.

[0098] 3.4 Methodology findings [0099] 3.4.1 Precision inspection

[0100] Take the same Chinese medicine composition solution of the same concentration (60 ·!^ - ¹ ), determined according to the above method, and determine its inhibition effect on COX-2 6 times, and calculate the traditional Chinese medicine composition (: 0 - 2 activity average) The inhibition rate was 67.23%, and the 11 SD was 5.14%. The precision of the method was good within days.

[0101] Take the same batch of the same concentration of the traditional Chinese medicine composition solution, a total of 6 parts, on the first day to the sixth day, the inhibition of COX-2 was determined according to the above method, and the average inhibition of the activity of the traditional Chinese medicine composition COX-2 was calculated. The rate was 64.73 % and the RSD was 8.56%. The method was excellent in daytime precision.

[0102] 3.4.2 Repeatability

[0103] 6 parts of the same batch of Chinese medicine composition sample (S40) were taken to prepare a test solution, and the average inhibition rate of COX-2 activity of the traditional Chinese medicine composition was calculated to be 70.48%, and the RSD was 9.32%.

[0104] In summary, the precision is high, the repeatability is good, and the requirements of the biological evaluation method are met, and can be used for biological evaluation.

3.5 Biopharmaceutical Standardization of Reference Materials of Traditional Chinese Medicine Compositions

Using the method established above, celecoxib was used as a control substance, and the value of inhibition of COX-2 enzyme activity of the reference substance of the traditional Chinese medicine composition was standardized according to the "mass reaction parallel line method". The bioavailability of the reference of the traditional Chinese medicine composition was defined as 1000 Uyg ^-1 , and the titer of celecoxib was measured. The results are shown in Table 3.

Table 3: Conversion of the reference value of the Chinese medicine composition reference substance to celecoxib

[0109] After the inhibition rate and the corresponding administration concentration were coordinate-converted, the titer calculation software was used to calculate the titer, and the result was that the reference of the I II Chinese medicine composition was equivalent to 3.80513⁄4 of celecoxib.

3.6 Traditional Chinese Medicine Composition Inhibition (: Bioactivity Determination of 0-2 Activity)

According to the above detection method, the test composition of the traditional Chinese medicine composition is inhibited (the 0-2 enzyme activity value is measured, and the inhibition rate of each batch of the Chinese medicine composition (: 0-2 enzyme activity is determined, and 340 is used as a standard). The reference substance, after the inhibition rate and the corresponding administration concentration were coordinate-converted, the input titer calculation software was used to obtain the inhibition of each batch of the traditional Chinese medicine composition (: 0-2 enzyme activity), and the measurement results are shown in Table 4.

Table 4 Chinese medicine composition inhibition (: 0-2 titer determination result \¥0 2019/109779 ?€1^2018/114831

[table 3]

\¥0 2019/109779 卩(:17 \2018/114831

[0114] The measurement results show that different batches of traditional Chinese medicine compositions are inhibited (: 0-2 active titer is 51011/

~14131143⁄4, the average biopotency is 85611/. Different batches of traditional Chinese medicine composition inhibition (: 0 - 2 titer is different, 526 sample has the highest titer, sample 505 has the lowest titer, abnormal test sample and normal sample comparison titer Decreased, with significant differences, where inhibition (: 0 - 2 activity) \¥0 2019/109779 卩 (: 17 \2018/114831 The order of price drop is high humidity > high temperature illumination.

[0115] Experimental Example 3 Correlation analysis between quality chemical evaluation and biological evaluation of traditional Chinese medicine composition

[0116] 9 kinds of chemical components in the traditional Chinese medicine composition (chlorogenic acid, rutin, forsythiaside VIII, forsythiaside, isochlorogenic acid (:, isochlorogenic acid: 8, caffeic acid, menthol) , Baiqiu Li alcohol) The content index is the independent variable, the biological titer is the dependent variable, and the 40 batches of the traditional Chinese medicine composition is the statistical analysis sample, and the inhibition is observed (: 0 -2 biopotency, inhibition of macrophage secretion 11 _^ - Correlation analysis between the bio-potency and the determination results of the nine chemical components was calculated using multivariate statistical 8?88 statistical software.

[0117] The results are shown in Table 5.

Table 5 Correlation analysis between biological activity and chemical content

From the viewpoint of inhibition (: 0-2 active anti-inflammatory biopotency, the relevant components are chlorogenic acid, isochlorogenic acid isochlorogenic acid (: an acid component, and are positively correlated ( _? <0.05), suggesting that the higher the content of these three components, the inhibition (: 0-2 enzyme activity level also has a tendency to increase. Therefore, in the chemical component allegations should be considered to increase the detection of the indicator activity of such associated activity To improve the consistency of the quality of traditional Chinese medicine compositions to a certain extent.

From the anti-inflammatory biopotency of inhibiting the secretion of -6 activity by macrophages, the related component is forsythin, and it is positively correlated (/? <0.05), indicating that the content of forsythin is higher. The level of inhibition of cell secretion of 11 _^ -6 also increased. It is suggested that forsythin is an effective component for exerting anti-inflammatory effects, and its content has a certain influence on the quality of traditional Chinese medicine compositions.

Advantageous effects of the invention

Beneficial effect \¥0 2019/109779 ?€1^2018/114831

[0122] The beneficial effects of the solution:

[0123] 1. Based on the prediction of network pharmacology, the verification of transcriptomics and the analysis of whole animal metabolomics, this protocol established (: 0-2 is the anti-inflammatory bioactivity index to establish the quality bioactivity evaluation of traditional Chinese medicine composition). Method

[0124] 2. The protocol used 03⁄4-2 enzyme activity as an indicator to evaluate the inhibitory effect of the traditional Chinese medicine composition on the activity of 03⁄4-2 enzyme, and established an anti-inflammatory biological evaluation method combining the enzyme level of clinical indications. Methodological investigation, the biological evaluation method has high precision and good repeatability.

The value is controlled within 10%, which is more stable than the anti-inflammatory biopotency method at the cellular level;

[0125] 3. Using the method to evaluate the biological activity of the traditional Chinese medicine composition, it was found that the batch-to-batch inhibition (the biological titer of 0-2 also has a certain difference, from the perspective of 40 batches of commercially available samples, The bioavailability ranged from 510 11/ to 14131143⁄4, with an average biopotency of 85,611/. Laboratory-made destructive sample inhibition (: 0-2 bioavailability was significantly lower than normal qualified samples, suggesting that the method It is also possible to distinguish between samples of different qualities;

[0126] 4. The method of the present invention makes up for the deficiency of the quality evaluation method of the chemical single index, and provides a more evidence-based detection method for the preparation of the traditional Chinese medicine composition;

[0127] 5. The method of the present invention can be applied not only to the quality evaluation of the traditional Chinese medicine composition described in the present scheme, but also to the quality evaluation of other traditional Chinese medicines and traditional Chinese medicine compounds having anti-inflammatory activity, and has universal applicability.

Brief description of the drawing

DRAWINGS

1 is (: 0-2 active detection principle;

2 is a graph showing the inhibitory effect of different concentrations of traditional Chinese medicine compositions on the enzyme activity of 03⁄4-2.

[0130] Embodiments of the present solution

Example 1

[0132] Group: Forsythia 255 _§ , Honeysuckle 255 _§ , Castor Yellow 85 _§ , Fried Bitter Almond 85 _§ , Gypsum 255 _§ , Banlangen 255 _§ . Mian Ma Guanzhong 255, Houttuynia 255, Patchouli 85, Rhubarb 51, Rhodiola 85, menthol 7. 5 _§ . Licorice 85 _{§ ;}

[0133] Preparation method: the above thirteen flavors, patchouli and water distillation to extract volatile oil, collect volatile oil, water extract filtered, spare; forsythia, ricin, houttuynia, rhubarb with 70% ethanol extracted twice, 2 hours at a time \¥0 2019/109779 卩(:17 \2018/114831

, the first 1.5 hours, the extract is filtered, combined, recovered ethanol, spare; honeysuckle, gypsum, Banlangen, Mianma Guanzhong, licorice, Rhodiola and water to boil, add fried bitter almond, decoction twice The first time is 1.5 hours, the first time is 1 hour, the decoction is filtered, the filtrate is combined, and the aqueous solution of the patchouli oil is added, and the solution is concentrated to a relative density of 1. 10-1.15 (60 ° 〇), and ethanol is added. 70% alcohol content, at 4 ° (: 24 hours of refrigeration, filtered, the filtrate recovered ethanol, combined with the above-mentioned forsythia and other four kinds of spare alcohol extract, concentrated to a relatively dense 1.15 ~ 1.20 (60 ° 〇, Spray drying, mixing with appropriate amount of starch, making granules, drying, sieving, sieving out appropriate amount of fine powder, dissolving menthol and patchouli volatile oil with appropriate amount of ethanol, spraying into fine powder, mixing, mixing with the above particles Evenly, sealed for 30 minutes, filled into capsules, made into 1000 capsules, that is.

[0134] Biological detection method:

[0135] Preparation of reference substance: Precision weighing Chinese medicine composition working reference material (standardized titer of celecoxib chemical pure product 1000 11·^ -〇, grinding fine, adding DMSO ultrasonic extraction 3011^11, 10000] ^* /11 Centrifuge 1011^11, take the supernatant to make a solution containing 1.2x10 ⁵ 11 per 11111.

[0136] Preparation of the test article: Take the appropriate amount of the content of the traditional Chinese medicine composition, grind fine, force PDMSO ultrasonic extraction 3011^11, 1 0000]·/!!^!! Centrifugal 10111111, take the supernatant, use the estimated effect The price is made into a solution containing 1.2\10 ⁵ II per 11111.

[0137] Determination method: Take the reference solution and the test solution according to the dilution ratio at 1:0.8 ~

Between 1:0.5, the inhibition rate was determined according to the "2.1 detection step" of Experimental Example 2, and the inhibition rate of the enzyme activity of 03⁄4-2 was used as an index. According to the "Biochemical Parallel Method" in the "Bioassay of Medicines" (edited by Zhou Haijun) "Calculating potency and confidence limits. Inhibition (: 0-2 enzyme activity titer must not be less than 500 11·^ - ¹

The average confidence rate is less than 25%.

Example 2

[0139] Biological detection method:

[0140] Preparation of reference substance: Precision weighing Chinese medicine composition working reference material (Ruoficob chemical pure product standardization titer

1000 11·^ ') , grinding fine, adding methanol solution ultrasonic extraction 30111111, 1000〇17111111 centrifugation 10111111, taking the supernatant to make a solution containing 11.x10 ⁵ 11 per 11111, that is.

[0141] Preparation of the test article: taking the appropriate amount of the content of the traditional Chinese medicine composition, grinding fine, force PDMSO ultrasonic extraction 3011^11, 1

0000]·/!!^!! Centrifuge 10111111, take the supernatant, and use a solution of 1.2\10 ⁵ II per 11111 at the estimated potency. \¥0 2019/109779 卩(:17 \2018/114831

[0142] Determination method: taking the reference solution and the test solution according to the dilution ratio at 1:0.8 ~

Between 1:0.5, according to the instructions of the commercially available (: 0 -2 Fluorescence Detection Kit (Shanghai Biyuntian Biotechnology Co., Ltd.), the inhibition rate is determined, and the inhibition rate of 〇3⁄4-2 enzyme activity is used as an indicator. "Bioassay" (edited by Zhou Haijun) in the "quality reaction parallel line method" to calculate the potency and confidence rate. Inhibition (: 0-2 enzyme activity titer must not be less than 500 1] · ^ average confidence rate is less than 25 %.

Example 3

[0144] Biological detection method:

[0145] Preparation of reference substance: Precision weighing Chinese medicine composition working reference material (Selecidab chemical pure product standardization titer

1000 11·^ ') , grinding fine, adding ethanol solution ultrasonic extraction 3011^11, 1000〇17111111 centrifugation 1011^11, taking the supernatant to make a solution containing 11.x10 ⁵ 11 per 11111, that is.

[0146] Preparation of the test article: Take the appropriate amount of the content of the traditional Chinese medicine composition, grind fine, force PDMSO ultrasonic extraction 3011^11, 1 0000]·/!!^!! Centrifugal 10111111, take the supernatant, use the estimated effect The price is made into a solution containing 1.2\10 ⁵ II per 11111.

[0147] Determination method: Take the reference solution and the test solution according to the dilution ratio at 1:0.8 ~

The average confidence rate is less than 25%.

Example 4

[0149] Biological detection method:

[0150] Preparation of reference substance: Precision weighing Chinese medicine composition working reference material (Rofecoxib chemical pure product standardization titer

1000 ^^-1) , finely ground, add DMSO ultrasonic extraction 3011^11, 10000] ^* /11 Centrifuge 1011^11, take the supernatant to make a solution of 11111^1.2x105 II, that is.

[0151] Preparation of the test article: taking the appropriate amount of the content of the traditional Chinese medicine composition, grinding fine, force PDMSO ultrasonic extraction 3011^11, 1

0000]·/!!^!! Centrifuge 10111111, take the supernatant, and use a solution of 1.2\10 ⁵ II per 11111 at the estimated potency.

[0152] Assay method: Take the reference solution and the test solution according to the dilution ratio between 1:0.8 ~ 1:0.5, according to the "radiozyme immunoassay" inhibition rate, with the inhibition rate of 03⁄4-2 enzyme activity as Indicators, according to the 2015 edition of the Chinese Pharmacopoeia 卩 (: 17 52018/1148310 2019/109779

(3) The biometric verification statistical method calculates the titer and the confidence limit rate by the parallel line method of mass reaction. Inhibition (: 0 - 2 enzyme activity titer should not be less than 500 11. ¹ , the average confidence rate is less than 25%.

Claims

\¥0 2019/109779 卩(:17 \2018/114831 Claims

[Claim 1] A method for detecting a traditional Chinese medicine composition, comprising: preparing a traditional Chinese medicine composition as a detectable sample, contacting with a cyclooxygenase ((:0), measuring a sample to the ring Oxidase ((:0) inhibition rate;

Wherein, the traditional Chinese medicine composition is made of the following raw materials:

Forsythia 200-300 parts by weight, honeysuckle 200-300 parts by weight, ricin yellow 50-100 parts by weight, fried bitter almond 50-100 parts by weight, gypsum 200-300 parts by weight, radix is 200-300 parts by weight, Mian Ma Guanzhong 200-300 parts by weight, 200-300 parts by weight of Houttuynia cordata, 50-100 parts by weight of patchouli, 30-80 parts by weight of rhubarb, 50-100 parts by weight of Rhodiola, 5-10 parts by weight of menthol, licorice 50 - 100 parts by weight.

[Claim 2] The detecting method according to claim 1, wherein the detecting method comprises the following steps:

VIII) preparing a sample solution and/or a reference solution of the traditional Chinese medicine composition;

B) Determine the inhibition rate of the sample solution and the reference solution solution (: 0-2 enzyme activity).

[Claim 3] The detecting method according to claim 2, wherein the method for measuring the inhibition rate of the enzyme activity comprises the following steps:

Adding 03⁄4-2 enzyme to the solution to be tested, and culturing for a certain period of time;

15) adding 80 and arachidonic acid to the solution to be tested;

〇) color development, detecting fluorescence intensity;

计算 Calculate the inhibition rate as follows:

Inhibition rate % = (fluorescence intensity of initial active sample - fluorescence intensity of sample to be tested) / fluorescence intensity of initial active sample 100%.

[Claim 4] The detecting method according to claim 3, wherein the amount of the enzyme in the step _& medium is

50011 ~ 1000 11 ;

[Claim 5] The detecting method according to claim 3, wherein in step 15, eighty-eight is purified;

[Claim 6] The detecting method according to claim 3, wherein the fluorescence intensity in the step is detected at an excitation light wavelength of 530-54011111 and an emission wavelength of 585-59511111.

[Claim 7] The detecting method according to claim 2, wherein the reference item in the step VIII is selected \¥0 2019/109779 卩 (: 17 \2018/114831 from conventional anti-inflammatory drugs or the traditional Chinese medicine composition that can be used as a control.

[Claim 8] The detecting method according to claim 7, wherein the reference substance is celecoxib or rofecoxib.

[Claim 9] The detecting method according to claim 8, wherein the reference solution concentration is 1

10 ⁵ 11/1111^2x10 ⁵ 11/1111^ Preferably, the reference solution concentration is 1.2× ^{10 5} 11 .

[Claim 10] The detecting method according to claim 2, wherein the method comprises the step (:: to (:0)

-2 Enzyme activity inhibition rate index, calculation of potency and confidence rate; Chinese medicine composition inhibition (: 0-2 enzyme activity titer should not be less than 500 11·^ ^ The average confidence rate is less than 25%. Request 1.