CN105543328A - Anti-rheumatism traditional Chinese medicine quality evaluation method based on bioavailability - Google Patents
Anti-rheumatism traditional Chinese medicine quality evaluation method based on bioavailability Download PDFInfo
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Abstract
The present invention relates to a traditional Chinese medicine quality evaluation method based on anti-rheumatism bioavailability. According to the quality evaluation method, bioavailability is adopted as an evaluation index, and particularly bioavailability for chondrocyte protection, bioavailability for COX-2 activity inhibition, and bioavailability for inflammatory factor secretion inhibition are adopted as the quality evaluation indices, wherein the bioavailability for chondrocyte protection is the bioavailability for chondrocyte NO secretion inhibition, and the inflammatory factor is TNF-alpha or IL-1. According to the present invention, the quality evaluation method can be adopted as the effective complement of the traditional chemical detection method, directly reflects the clinical effectiveness, and is the quality control and detection method which truly meets the traditional Chinese medicine theory, reflects the clinical application characteristics of the traditional Chinese medicine, and ensures the safety and the effectiveness of the traditional Chinese medicine.
Description
Technical field
The invention belongs to traditional Chinese medicine quality to control and evaluation technique field, particularly a kind of medicine quality evaluated method based on estimation of biological potency, more particularly, relate to the antirheumatic activity detection method of Chinese medicine and the biological quality evaluation method of antirheumatism traditional Chinese medicine.
Background technology
Rheumatic arthritis (RA) is one of difficult and complicated illness, is 0.3% at China's average attack rate.Main clinical manifestation is arthroncus, pain, limitation of activity, morning stiff phenomenon.Later stage causes ankylosis, deformity, afunction even maimed.Because its pathology is extensive, sickness rate and disability rate high, and the cause of disease, pathogenesis are not yet illustrated, and not yet solve so far to the treatment problem of RA, are clinical and the focus of drug research and difficult point always.This sick short term effect of western medicine still can, but side effect is larger.Treatment by Chinese herbs RA is based upon on long-term clinical practice basis, lot of experiments is carried out to Chinese medicine compound prescription and the anti-RA effect of single medicinal material both at home and abroad, develop many clinical efficacies good, the Traditional Chinese Medicine Anti rheumatic medicine that side effect is little, but, lower to the Quality Control level of such Chinese medicine at present, mainly utilize the means such as chemical analysis method or microscopical identification to carry out quality examination, control or evaluation to its starting material, intermediate, Chinese patent medicine.
Compare chemical drugs, traditional Chinese medicine ingredients is very complicated, and the modernization of its quality standard and internationalization are perplex the core that Chinese medicine develops always, and the key of the quality standard of Erecting and improving sets up science, reliable quality control and evaluation method.Present stage traditional Chinese medicine quality control with evaluate with the qualitative and quantitative analysis of chemical composition for Main Means.Along with deepening continuously of traditional Chinese medicine research, find that this pattern controls at traditional Chinese medicine quality and shown very large limitation gradually in appraisement system.Such as single component triptolide content height, can not represent the quality of this taste quality of medicinal material of trypterygine, more overall reflection can not contain the antirheumatic activity of the compound preparation of tripterygium wilfordii or its extract just.Using the content of indivedual or part index number composition as an index of quality standard, although can control the amount of index composition, but be only the amount of a certain or some composition controlled wherein, be difficult to the stability and the consistence that ensure all the components, more be difficult to the overall biological effect embodying Chinese medicine, ensure the consistence of its clinical efficacy.
But, current estimation of biological potency method is little at the achievement in research report of field of Chinese herbal, and the measuring method of biological value for Traditional Chinese Medicine Anti rheumatic medicine, or the quality evalution of the antirheumatic Chinese medicine recorded based on biological value or detection method have not yet to see report.
Therefore, be necessary the quality evaluating method building a kind of antirheumatic Chinese medicine based on estimation of biological potency, its evaluation index can the antiheumatic clinical efficacy of direct correlation, this evaluation method can comprehensive evaluation Traditional Chinese Medicine Anti rheumatism group effect or its total quality, and this quality evaluating method also can be used for feature and the power of evaluating rheumatism pharmacologically active between different medicine.
Summary of the invention
Existing antirheumatic quality control and evaluation method are main mainly with the qualitative and quantitative analysis of chemical index composition, but this chemical detection means cannot be associated with clinical efficacy, therefore, really effectively can not evaluate the quality of medicine.For solving the defect existed in existing antirheumatic quality control and evaluation method, the invention provides a kind of antirheumatic quality evaluating method based on estimation of biological potency.
Rheumatism general reference affects one group of disease of bone, joint and surrounding soft tissue thereof.The arthritis normally caused by immune system obstacle, arthralgia, discomfort, swelling and stiff, and with Progressive symmetric erythrokeratodermia bone and cartilage erosion, finally cause degenerative joint, dysfunction.Cartilage degeneration is one of its major pathologic features.The NO of articular chondrocytes middle and high concentration can promote articular chondrocyte apoptosis; repair of cartilage ability is made to decline and can cartilage destruction be caused; in chondrocyte, NO content raises is one of cause of disease of cartilage degeneration; therefore, the provide protection of medicine to chondrocyte can be reflected by measuring chondrocyte NO secretion inhibiting rate.Inflammatory factor plays an important role in various immunological disease pathogenesis, IL-1 causes rheumatisant's systemic symptoms such as underground heat, weak, acute phase protein to synthesize the major cytokine increased, it is the principal element causing c reactive protein and erythrocyte sedimentation rate to raise, therefore, by measure IL-1 secrete inhibiting rate can drug characterization rheumatism biological effect; In addition, other inflammatory factors such as TNF-α secretes and increases, and impels synovial membrane to be in chronic inflammatory state, and TNF-α destroys cartilage further, causing joint deformity, therefore, also can reflect the restraining effect of medicine to rheumatism by measuring TNF-α secretion inhibiting rate; Inflammatory factor stimulates or can produce COX-2 under stressed condition, and COX-2 is the key enzyme that conversion of arachidonic acid is converted into prostaglandin(PG), by suppressing COX-2 active, blocking arachidonic acid and being converted into prostaglandin(PG), thus playing anti-inflammatory and analgesic effect.Therefore, by measuring medicine to the inhibiting rate of COX-2 activity, this medicine rheumatism biological effect can be characterized.
Different antirheumatics affects one or more pathology link by different mechanism and plays its pharmacological action, and traditional Chinese medicine ingredients is complicated, plays Comprehensive harmonies, the pathophysiological change of refractory disease complexity of more hitting often through Mutiple Targets, multipath.Based on this characteristic of Chinese medicine, the invention provides a kind of quality evaluating method, can control from the group effect of many aspects to the complicated ingredient of antirheumatic Chinese medicine or evaluate, for medication combined exploitation is (such as with the antirheumatic Drug combination of other mechanism of action, play better antirheumatic effect by machine-processed complementation) or the medication of the clinical side of sending reference frame is provided, can also with the inner quality of comprehensive biological value metrics evaluation antirheumatic Chinese medicine or active height.From different approaches, this quality evaluating method reflects that described medicine rheumatism biological activity is strong and weak respectively, embody its group effect, with the validity direct correlation of clinical application, and more can also know which pathology link medicine mainly acts on, thus better for clinical application provides foundation.
For achieving the above object, the invention provides a kind of antirheumatic medicine quality evaluated method, it is using rheumatism biological value as quality evaluation index.
In some embodiments, described method protects the biological value of the biological value of chondrocyte, the biological value suppressing COX-2 activity and inflammation-inhibiting cytokine secretion as quality evaluation index.
Wherein, the biological value of described protection chondrocyte refers to the biological value suppressing chondrocyte NO secretion, evaluates the power of Chinese medicine protection cartilage cell activity with the height of measured biological value.
IL-1 and TNF-α is common inflammatory factor, also be the pro-inflammatory cytokine held the central place in rheumatic arthritis pathogeny, therefore, the present invention is using them as the representative Testing index evaluating rheumatism biological value, such as, but other inflammatory mediator, IL-6, IL-8, IL-15, IL-17 etc. all can be used as the detection of index for rheumatism biological value.
In the embodiment of the invention, supplementing as above-mentioned quality evaluating method, particularly when medicine itself not plays the activeconstituents of anti rheumatism action, but the product produced after bio-transformation is in vivo only active substance, this quality evaluating method also comprises makes described Chinese medicine after intestinal microflora fermentation, obtain tunning, experiment in vitro is adopted to detect the biological value of this tunning suppression chondrocyte NO secretion, suppress the biological value of COX-2 activity, suppress the biological value of TNF-α secretion and suppress the biological value of IL-1 secretion, quality evaluation index using measured biological value as antirheumatic Chinese medicine.
In another embodiment, supplementing as above-mentioned quality evaluating method, particularly when medicine itself not plays the activeconstituents of anti rheumatism action, but the product produced after metabolism is in vivo only active substance, this quality evaluating method can also comprise makes described Chinese medicine obtain meta-bolites under the effect of liver microsomes enzyme, experiment in vitro is adopted to detect the biological value of this meta-bolites suppression chondrocyte NO secretion, suppress the biological value of COX-2 activity, suppress the biological value of TNF-α secretion and suppress the biological value of IL-1 secretion, quality evaluation index using measured biological value as antirheumatic Chinese medicine.
Here cannot the exhaustive rheumatism biological value based on all Testing index, the present invention only exemplarily illustrates four kinds in order to characterize the biological value of antirheumatic activity power: suppress the biological value of chondrocyte NO secretion, suppress the biological value of IL-1 secretion, suppress the biological value of TNF-α secretion and suppress the biological value of COX-2 activity.It is to be understood that, should not limit the scope of the invention with this.The specific embodiment of specification sheets has been only the exemplary effect exemplified, and not does any type of restriction to the present invention, and therefore, the method for any introducing biological value metrics evaluation antirheumatic traditional Chinese medicine quality all should belong within protection scope of the present invention.
In the specific embodiment of the present invention, described biological value can be any one in above-mentioned 4 kinds of biological values, also can be the arbitrary combination of above-mentioned 4 kinds of biological values, the wherein combination of preferred above-mentioned 4 kinds of biological values, because of the biological value that it can be secreted by complete detection Drug inhibition chondrocyte NO, suppress the biological value of IL-1 secretion, suppress the biological value of TNF-α secretion and suppress the biological value of COX-2 activity, from its rheumatism biological activity of many aspects comprehensive evaluation, reflect the rheumatism effect of its entirety, and act on which pathology link by this quality evaluating method detection of drugs, which approach to play anti rheumatism action by, the rheumatism special efficacy of the different herbal species of comprehensive representation, quantize and characterize effect speciality of dissimilar antirheumatic, for the joint development or safer clinically of medicine, effectively, the side's of sending medication provides foundation exactly.
In some embodiment, Chinese medicine of the present invention can be Chinese medicine compound prescription, also can be single medicinal material material or medicine materical crude slice, can also be their extract.In a specific examples, described Chinese medicine is compound preparation QUFENG ZHITONG PIAN, and its prescription is made up of Herba Erodii 334g, mistletoe 167g, teasel root 167g, Root of Sixpetal Clematis 83g, levisticum 83g, Radix Aconiti Kusnezoffii Preparata 83g Flos Carthami 83g.In another specific examples, described Chinese medicine is folk prescription Chinese patent medicine Glucosidorum Tripterygll Totorum or peony root total glycosides capsules.
Estimation of biological potency method of the present invention can be carry out according to " Chinese Pharmacopoeia " 2010 editions two annex XIV Bioassay-statistical methods, such as according to parallel line assay principle, carry out according to 22 methods in the parallel line analysis assay method specified under " Chinese Pharmacopoeia " 2010 editions two annex XIV Bioassay-statistical method items; Also can be adopt method in existing document, " research (II) of Motherwort Herb medicinal material biological assay method---oxytocin, Motherwort Herb dose-effect relationship and calibrating are suitable for the foundation of Effective pattern " " four-point method " shown in a literary composition that " biological assay of the biological detection method III-Herba Andrographis of the antipyretic and antidotal type Chinese medicine " people such as " one point method " shown in a literary composition or Yang Minghua that the people such as such as Wang Shengmin deliver delivers; Other science, the feasible estimation of biological potency method that can quantize biological effect can also be adopted.Any restriction is not done, as long as can realize the object of the invention for which kind of estimation of biological potency method of employing and the present invention of statistical procedures method.
Exemplarily illustrate described in order to characterize the measuring method of 4 kinds of biological values of antirheumatic activity power, but should can not limiting the scope of the invention with this below.
The biological value of suppression chondrocyte NO of the present invention secretion can measure as follows:
A. reference substance solution preparation: take dexamethasone as reference substance, be solvent, be made into the reference substance solution of respective concentration with the DMEM of 20% foetal calf serum, filters, for subsequent use;
B. need testing solution preparation: with medicine to be measured for trial-product, preferably, took the trial-product powder of 10-100 mesh sieve, with appropriate solvent extraction, extracting solution after filtration, according to the estimated value that trial-product is tired, obtains need testing solution with the DMEM dilution of 20% foetal calf serum;
C. need testing solution and reference substance solution are diluted to the dosage group of different concns with identical dose of distance, and agent distance is 0.5-1.0, such as, when being 0.6, and middle dosage=high dosage * 0.6; Low dosage=middle dosage * 0.6;
D. data processing calculates with tiring: the chondrocyte NO adopting nitrogen protoxide (NO) nitrate reductase method test kit to record trial-product and reference substance various dose group respectively secretes inhibiting rate; calculate the ratio that trial-product and reference substance reach dosage when substantially identical chondrocyte NO secretes inhibiting rate; biological value using this ratio as medicine to be measured relative to reference substance, the size of this ratio reflects the bioactive power of medicament protection chondrocyte to be measured.
The biological value of suppression IL-1 of the present invention secretion can measure as follows:
A. positive drug dexamethasone solution: with IL-1 inhibitor for reference substance, be dissolved in DMEM nutrient solution, be mixed with the reference substance solution of respective concentration, filter, for subsequent use, described IL-1 inhibitor can be dexamethasone or diacerein, in one embodiment, specification used is the Dexamethasone Injection of 5mg/ml, gets this injection liquid 2ml, add 8mlDMEM nutrient solution, piping and druming mixing, concentration 1mg/ml, solution takes on a red color transparent clarified liq, degerming through 0.22 μm of membrane filtration in super clean bench, 4 DEG C of preservations;
B. need testing solution preparation: with medicine to be measured for trial-product, preferably, took the trial-product powder of 10-100 mesh sieve, with appropriate solvent extraction, extracting solution after filtration, according to the estimated value that trial-product is tired, obtains need testing solution with DMEM dilution;
C. need testing solution and reference substance solution are diluted to the dosage group of different concns with identical dose of distance, and agent distance is 0.5-1.0, such as, when being 0.6, and middle dosage=high dosage * 0.6; Low dosage=middle dosage * 0.6;
D. data processing calculates with tiring: the IL-1 adopting mouse interleukin-11 (IL-1) ELISA kit to record trial-product and reference substance various dose group respectively secretes inhibiting rate, calculate the ratio that trial-product and reference substance reach dosage when substantially identical IL-1 secretes inhibiting rate, biological value using this ratio as medicine to be measured relative to reference substance, the size of this ratio reflects the bioactive power that Drug inhibition IL-1 to be measured secretes.
The biological value of suppression TNF-α of the present invention secretion can measure as follows:
A. reference substance solution preparation: with TNF-alpha inhibitor for reference substance, be dissolved in RPMI-1640 nutrient solution, be made into the reference substance solution of respective concentration, filters, for subsequent use, and it is general that described TNF-alpha inhibitor is preferably benefit match;
B. need testing solution preparation: with medicine to be measured for trial-product, preferably, took the trial-product powder of 10-100 mesh sieve, with appropriate solvent extraction, extracting solution after filtration, according to the estimated value that trial-product is tired, obtains need testing solution with the dilution of RPMI-1640 nutrient solution;
C. need testing solution and reference substance solution are diluted to the dosage group of different concns with identical dose of distance, and agent distance is 0.5-1.0, such as, when being 0.6, and middle dosage=high dosage * 0.6; Low dosage=middle dosage * 0.6;
D. data processing calculates with tiring: by putting the method for exempting from and measuring the method for L929 Cell viability, measure TNF-α secretory volume, the TNF-α recording trial-product and reference substance various dose group respectively secretes inhibiting rate, calculate the ratio that trial-product and reference substance reach dosage when substantially identical TNF-α secretes inhibiting rate, biological value using this ratio as medicine to be measured relative to reference substance, the size of this ratio reflects the bioactive power that Drug inhibition TNF-α to be measured secretes.
The biological value of suppression COX-2 activity of the present invention can measure as follows:
A. reference substance solution preparation: take cox 2 inhibitor as reference substance, by reaction system as dissolution with solvents, obtain reference substance solution, described reaction system is 10 μ lCOX-2 (final concentration is 0.48U/ μ l)+100 μMs of AA+2.1mMTMPD; Described cox 2 inhibitor can select celecoxib;
B. need testing solution preparation: with medicine to be measured for trial-product, preferably, took the trial-product powder of 10-100 mesh sieve, with appropriate solvent extraction, extracting solution after filtration, according to the estimated value that trial-product is tired, obtains need testing solution with described reaction system dilution;
C. need testing solution and reference substance solution are diluted to the dosage group of different concns with identical dose of distance, and agent distance is 0.5-1.0, such as, when being 0.6, and middle dosage=high dosage * 0.6; Low dosage=middle dosage * 0.6;
D. data processing calculates with tiring: adopt microplate reader method to record the COX-2 maximum inhibition of trial-product and reference substance various dose group respectively, calculate the ratio of dosage when trial-product and reference substance reach substantially identical COX-2 maximum inhibition, biological value using this ratio as medicine to be measured relative to reference substance, the size of this ratio reflects the power of Drug inhibition COX-2 activity to be measured.
Beneficial effect of the present invention at least comprises:
1, antirheumatic medicine quality evaluated method provided by the present invention is utilized to carry out traditional Chinese medicinal materials assortment qualification and quality evaluation, quality control and evaluation can also be carried out to Chinese medicine compound prescription or Chinese medicinal materials or its extract, with suppressing the biological value of chondrocyte NO secretion, suppress the biological value of IL-1 secretion, suppress the biological value of TNF-α secretion or suppress the biological value of COX-2 activity to characterize the inner quality of antirheumatic Chinese medicine and activity strong and weak;
2, estimation of biological potency method susceptibility provided by the invention is high and reliable and stable, selected biological value index and antirheumatic activity direct correlation, can Efficient Characterization rheumatism biological activity, the present invention efficiently solves the quality evalution of the antirheumatic based on estimation of biological potency, ensure that the qualitative, quantitative of antirheumatic uses, as supplementary and raising that is conventional and chemical detection, jointly can to check on its inner quality from multiple angles, improve existing Quality Management Mode, and thinking and Technical Reference can be provided for the research of other medicine quality evaluated and control, economic and social benefit is huge,
3, the present invention passes through from its rheumatism biological activity of many aspects comprehensive evaluation, reflect the rheumatism effect of its entirety, and detection of drugs which approach can play anti rheumatism action by, for medicine joint development or clinically safer, the side's of sending medication efficiently and accurately foundation is provided;
4, the positive control drug used in rheumatism estimation of biological potency method of the present invention is all that drug effect is clear and definite on this point of application, be widely used clinically, the medicine of " mark post " property effect can be played, medicine to be measured is tired relative to the Relative biological of positive control drug can characterize its activity intensity in this similar drug, therefore, quality evaluating method of the present invention also can be used for evaluating the bioactive feature of rheumatism and intensity between different medicine;
5, the present invention not only may be used for the quality evaluating finished product or intermediate, and also can be used for the evaluation index of process optimization, the monitor and managment for production process is significant;
6, the method for the invention can be applied to TCM mechanism research, quality control, therapeutic evaluation or clinical application etc.
Embodiment
For making the present invention easier to understand, describe the present invention in detail below in conjunction with embodiment, these embodiments only play illustrative effect, are not limited to range of application of the present invention, NM specific experiment method in the following example, conveniently experimental technique carries out usually.
For the ease of understanding the present invention better, provide the implication that relevant letter abbreviations refers to below.
DMEM is the abbreviation of dulbecco'smodifiedeaglemedium, is a kind of improved culture medium, and the DMEM substratum used in the present invention is purchased from Hyclone company;
NO represents nitrogen protoxide;
IL-1 represents interleukin 1;
TNF-α represents tumor necrosis factor-alpha;
COX-2 represents Transitional cell carcinomas;
RPMI is the abbreviation of RoswellParkMemorialInstitute, and 1640 is code names of substratum, and RPMI-1640 nutrient solution used in the present invention is purchased from Invitrogen company;
TMPD is the abbreviation of neopentyl glycol, purchased from CAYMANCHEMICAL company;
AA represents arachidonic acid, purchased from Sigma Co., USA;
DMSO refers to dimethyl sulfoxide (DMSO), is purchased from Tianjin Chemical Reagents Factory No.1.
Technological thought of the present invention is: choose the medicine that experiment in vivo result shows to have anti-inflammatory pain-stopping effect, first carry out biological activity determination experiment, namely the biological activity that detection of drugs suppresses chondrocyte NO secretion, suppression IL-1 secretes, suppresses TNF-α secretion, suppresses COX-2 activity, choose that experimental result is positive and the point of application that dose-effect is dependency measures biological value further, described experimental result is that the positive refers to that trial-product group is compared with negative control group, has significant difference statistically; Test positives control group result for positive for biological activity determination, and trial-product group result is negative, then the biological activity assay result of this point of application is judged to be feminine gender, and the point of application of negative findings no longer carries out estimation of biological potency.Choose one or more biological activity determination experimental result for positive and dose-effect be dependency point of application measured by biological value as quality evaluation index, consider the cost that mass analysis detects and efficiency, preferably the biological value of 1 or 2 point of application is as quality evaluation index, for the quality evalution of the quality control in traditional Chinese medicine quality evaluation, Chinese Traditional Medicine or the finished product.If the rheumatism special efficacy in order to fully understand medicine, for medication combined exploitation, such as with the antirheumatic Drug combination of other mechanism of action, play better antirheumatic effect by machine-processed complementation and scientific basis is provided, then preferably measure the positive and biological value of point of application in dose-effect dependency of each biological activity determination experimental result, by this can the rheumatism special efficacy of the different herbal species of comprehensive representation, quantize and characterize effect speciality of dissimilar antirheumatic, for the joint development of medicine provides foundation.Biological value of the present invention refer to compare under the same conditions trial-product and reference substance (or standard substance) produce identical or substantially identical biological effect time the ratio of dosage.
Antirheumatic Chinese medicine of the present invention refers to that experiment in vivo proves to have the Chinese medicine of rheumatism drug effect, includes but not limited to Chinese medicine compound prescription, medicinal material and their extract or their coliform tunning or hepatomicrosome meta-bolites.
Specific embodiments of the invention have chosen Chinese patent medicine QUFENG ZHITONG PIAN, Glucosidorum Tripterygll Totorum and peony total glucosides tablet.But method of the present invention is not limited to carry out quality evalution to these kinds, only exemplarily exemplify herein, not any type of restriction is done to the present invention, in fact, the medicine that any experiment in vivo shows to have antirheumatic activity all can adopt ill vitro test method of the present invention to measure its biological value relative to positive control medicine to carry out quality evalution, evaluate the bioactive direction of its rheumatism and potency, the bioactive direction of described rheumatism mainly refers to it plays anti rheumatism action by which approach, such as by suppressing chondrocyte NO secretion, suppress IL-1 secretion, suppress TNF-α secretion or suppress COX-2 active, described rheumatism potency is exactly the biological value of corresponding positive contrast medicine on each point of application.
Chinese patent medicine QUFENG ZHITONG PIAN used in the present invention (the accurate word Z34020421 of traditional Chinese medicines, Anhui Province Xuefeng Medicine Co., Ltd), Glucosidorum Tripterygll Totorum (the accurate word Z37020344 of traditional Chinese medicines, Lunan Pharmaceutical Co., Ltd.), peony root total glycosides capsules (the accurate word H20055058 of traditional Chinese medicines, the limited public affairs of magnificent pharmacy are found in Ningbo) all purchased from commercially available prod, three kinds of Chinese patent medicines use suitable dissolution with solvents, filtration respectively, filtrate is concentrated into 0.5g crude drug/ml, for subsequent use respectively as need testing solution.
Positive drug benefit match selected by the present invention is general purchased from Shanghai CP Guojian Pharmaceutical Co., Ltd; Celecoxib purchased from American Pfizer; Dexamethasone is purchased from Tianjin Pharmaceutical Jiaozuo Co., Ltd..
We have carried out associated biomolecule titration test respectively to compound Chinese patent medicine QUFENG ZHITONG PIAN, folk prescription Chinese patent medicine Glucosidorum Tripterygll Totorum and peony root total glycosides capsules, now test-results are reported as follows:
Embodiment 1 QUFENG ZHITONG PIAN, Glucosidorum Tripterygll Totorum and peony root total glycosides capsules relevant biological activity measure
1, nitrogen protoxide (NO) RNA isolation kit measures the biological activity of Drug inhibition chondrocyte NO secretion
By chondrocyte's method, to cultivate based on Primary chondrocyte, interleukin-(IL)-1 β stimulated in vitro method is adopted to cause chondrocyte matrix to be degraded as pharmacological model, detect NO content with nitrogen protoxide (NO) nitrate reductase method test kit, secrete inhibiting rate for index with chondrocyte NO.
Specific experiment method: the chondrocyte that vitro culture is good be inoculated in 96 orifice plates, add the IL-1 β that concentration is 100ng/ml after cultivating 24h, each concentration establishes 3 repetitions, and be arranged in parallel blank group, is placed in 37 DEG C, 5%CO
224h cultivated by incubator, for subsequent use.After positive drug Dexamethasone group and trial-product (QUFENG ZHITONG PIAN, Glucosidorum Tripterygll Totorum and peony root total glycosides capsules) each dosage group act on above-mentioned chondrocyte 48h, collecting cell supernatant liquor, by NO content in cell conditioned medium after nitrogen protoxide (NO) nitrate reductase method test kit detection of drugs (comprising positive drug and trial-product) effect.Mensuration positive drug and each dosage group of trial-product are to the concentration of NO in chondrocyte's supernatant liquor after induced damage, and more each acute drug effect group, model group, normal control intergroup relation, result is all used
represent, compare between group and adopt t to detect.Secrete inhibiting rate by following formulae discovery chondrocyte NO, with this drug characterization, chondrocyte is secreted to the restraining effect of NO.Concrete outcome is in table 1.
The restraining effect that table 1 QUFENG ZHITONG PIAN, Glucosidorum Tripterygll Totorum and peony root total glycosides capsules are secreted chondrocyte NO
Note: * * represents compared with model group, P < 0.01; * represent compared with model group, P < 0.05.
Above-mentioned experimental result shows, positive drug dexamethasone has obvious restraining effect to chondrocyte NO secretion, and Glucosidorum Tripterygll Totorum also has obvious restraining effect to chondrocyte NO secretion, and in dose-dependently.And QUFENG ZHITONG PIAN and peony root total glycosides capsules are without the effect obviously suppressing chondrocyte NO secretion.
2, mouse interleukin-11 (IL-1) ELISA kit method measures the biological activity of Drug inhibition IL-1 secretion
Based on peritoneal macrophage, adopt the IL-1 synthesis of LPS stimulated in vitro method induction and discharge as pharmacological model, detecting IL secretory volume with mouse interleukin-11 (IL-1) ELISA kit, secreting inhibiting rate for index with IL-1.
Specific experiment method: adopt male KM mouse, in-vitro separation prepares peritoneal macrophage, and the suitableeest inoculation quantity that scavenger cell inoculates 24 orifice plates is 1 × 10
6/ hole, adding LPS in this, as target cell stimulates, the inflammatory pathology that in-vitro simulated cytokine IL-1 infiltrates.Blank group (acellular), cell controls group, standard control group, model group (adding the LPS effect 12-15h of concentration as 5 μ g/ml in scavenger cell), positive drug Dexamethasone group and each dosage group of trial-product is set in above-mentioned 24 orifice plates, positive drug and trial-product group are the need testing solutions adding dexamethasone and each concentration adding 5 μ g/mlLPS while, effect 12-15h.Detected the change of IL-1 content in each group of supernatant by ELISA kit, calculate inhibiting rate, detect the effect whether trial-product has IL-1 synthesis and the release suppressing LPS to induce, evaluate it with this and whether there is the biological activity suppressing IL-1 secretion.Each group of OD value is all used
represent, compare between group and adopt t inspection.In this experiment, standard concentration is 1000pg/ml.Concrete outcome is in table 2.
IL-1 secretes inhibiting rate %=(IL-1 model group-IL-1 administration group)/(IL-1 model group-IL-1 cell controls group) × 100%
The restraining effect that table 2 QUFENG ZHITONG PIAN, Glucosidorum Tripterygll Totorum and peony root total glycosides capsules are secreted IL-1
Note: * * represents compared with model group, P < 0.01; * represent compared with model group, P < 0.05.
Above-mentioned experimental result shows, positive drug dexamethasone has significant restraining effect to IL-1 secretion, and Glucosidorum Tripterygll Totorum and peony root total glycosides capsules also have obvious restraining effect to IL-1 secretion, and dose-effect relationship is better.And QUFENG ZHITONG PIAN is without the effect obviously suppressing IL-1 secretion.
3, by putting the method for exempting from and measuring the biological activity that L929 Cell viability measures Drug inhibition TNF-α secretion
The U937 cell after differentiation is stimulated by inducing U937 cytodifferentiation and LPS with VitD3, cells and supernatant is got by after U937 cell cultures 24h, join in L929 cell, add dactinomycin and strengthen L929 cell to the susceptibility of TNF α, the output of TNF α is judged roughly by the survival rate measuring L929 cell, add tested material (comprising positive control medicine and trial-product) after fixing experiment condition, observe tested material and whether there is the biological activity suppressing TNF-α secretion.
Model group: in 96 porocyte culture plates, 20000 U937 cells are inoculated in every hole, add VitD310nM, 37 DEG C, cultivate 24h in 5%CO2 incubator after, after adding LPS1000ng/ml stimulation 60min, collecting cell culture supernatant, join in the L929 cell in 10000/ hole being inoculated in 96 porocyte culture plates, 24h cultivated by 37 DEG C ﹑ 5%CO2 incubators, surveys cell survival rate with mtt assay.
Positive drug group and trial-product group: other conditions are the same, just exist; Be loaded with while adding U937 cell culture supernatant in 96 porocyte culture plates of 10000/ hole L929 cell and add test medicine (comprising positive drug and each dosage group of trial-product), preferably, in L929 Tissue Culture Plate, add dactinomycin makes its final concentration be 1 μ g/ml, and dactinomycin can improve the susceptibility of L929 cell to TNF α.The OD value of each hole at 570nm wavelength place is read by microplate reader.By following formulae discovery L929 cell inhibitory rate, represent that different pharmaceutical suppresses the effect of TNF-α secretion with this inhibiting rate.Concrete outcome is in table 3.
The inhibiting rate that table 3 QUFENG ZHITONG PIAN, Glucosidorum Tripterygll Totorum and peony root total glycosides capsules are secreted TNF-α
Note: * * represents compared with model group, P < 0.01; * represent compared with model group, P < 0.05.
Above-mentioned experimental result display, do not add only adding the culture supernatant of U937 cell benefit match general time, the amount of the TNF-α of generation is 46.21% to the inhibiting rate that TNF-α secretes, when to match general concentration be 125mg/l to benefit, have remarkable reduction to the inhibiting rate that TNF-α secretes, inhibiting rate is only 6%.QUFENG ZHITONG PIAN and Glucosidorum Tripterygll Totorum have no significant effect the inhibiting rate that TNF-α secretes, and this two preparation are described to the secretion of TNF-α without obvious restraining effect.Peony root total glycosides capsules has obvious effect to the inhibiting rate that TNF-α secretes, and amount effect relationship.
4, microplate reader method measures the biological activity of Drug inhibition COX-2 activity
With 96 orifice plates for reaction carriers, end reaction system is 210 μ l.140 μ L damping fluids are added in advance in plate hole, 10 μ LHeme, 10 μ LCOX-2,10 μ L test medicine, 25 DEG C, insulation 5min, add 20 μ LTMPD again, 20 μ L arachidonic acids, 25 DEG C of insulation 20min, 570nm place mensuration absorbancy in microplate reader, if enzyme blank group and positive drug group and trial-product group, positive drug group and trial-product group are reacted within 20 minutes, terminate reaction, 570nm place mensuration absorbancy in microplate reader respectively at being added positive drug celecoxib and each dosage trial-product in above-mentioned reaction system.By following formulae discovery medicine, different pharmaceutical is detected on the impact of cyclooxygenase-2 activity to the inhibiting rate of COX-2 enzymic activity.Concrete outcome is in table 4.
The calculation formula of COX-2 maximum inhibition is:
Table 4 QUFENG ZHITONG PIAN, Glucosidorum Tripterygll Totorum and peony root total glycosides capsules are on the impact of COX-2 enzymic activity
Note: * * represents compared with blank group, P < 0.01; * represent compared with blank group, P < 0.05.
Above-mentioned experimental result shows, when celecoxib concentration reaches 1mg/ml, can reach 41.71% to the inhibiting rate of COX-2 enzymic activity, compared with blank group, have obvious restraining effect to COX-2 enzymic activity.Glucosidorum Tripterygll Totorum, peony root total glycosides capsules are then without the effect obviously suppressing COX-2 enzymic activity.High, medium and low three the dosage groups of QUFENG ZHITONG PIAN all show the effect obviously suppressing COX-2 enzymic activity, and there is good dose-effect relationship.
From table 1 to table 4, QUFENG ZHITONG PIAN suppresses COX-2 activity display obvious biological activity, and the increase of bioactive intensity dosage and increasing, present good dose-effect relationship.Its biological value can be detected further, using biological value as quality evaluation index.
Glucosidorum Tripterygll Totorum suppresses IL-1 secretion and suppresses chondrocyte NO secretion display obvious biological activity, and the increase of bioactive intensity dosage and increasing, present good dose-effect relationship.The biological value suppressing IL-1 secretion and/or suppress chondrocyte NO secretion can be detected further, using biological value as quality evaluation index.
Peony root total glycosides capsules suppresses TNF-α secretion and suppresses IL-1 secretion all to show obvious biological activity, and the increase of bioactive intensity dosage and increasing, present good dose-effect relationship.The biological value suppressing TNF-α secretion and/or suppress IL-1 secretion can be detected further, using biological value as quality evaluation index.
Embodiment 2 QUFENG ZHITONG PIAN suppresses the mensuration of the biological value of COX-2 activity
A. reference substance solution preparation: be reference substance with celecoxib, be dissolved in the reaction system of 10 μ lCOX-2 (final concentration is 0.48U/ μ l)+100 μMs of AA+2.1mMTMPD, filters;
B. need testing solution preparation: prepare QUFENG ZHITONG PIAN need testing solution by method as previously mentioned;
C. need testing solution and reference substance solution are diluted to the dosage group of different concns with identical dose of distance, and agent distance is 0.5; Diluting solvent used is 10 μ lCOX-2 (final concentration is 0.48U/ μ l)+100 μMs of AA+2.1mMTMPD;
D. data processing calculates with tiring: adopt the microplate reader method described in embodiment 1 to record the COX-2 maximum inhibition of trial-product and reference substance various dose group respectively, calculate the ratio of dosage when trial-product and reference substance reach substantially identical COX-2 maximum inhibition, using this ratio as QUFENG ZHITONG PIAN relative to the biological value of celecoxib, the size reflection QUFENG ZHITONG PIAN of this ratio suppresses the power of COX-2 activity.Specific experiment the results are shown in Table 5.
Table 5 QUFENG ZHITONG PIAN is to the restraining effect of COX-2 activity
Embodiment 3 Glucosidorum Tripterygll Totorum suppresses the mensuration of the biological value of chondrocyte NO secretion
A. reference substance solution preparation: take dexamethasone as reference substance, be solvent, be made into the reference substance solution of respective concentration with the DMEM nutrient solution of 20% foetal calf serum, filters, for subsequent use;
B. need testing solution preparation: prepare Glucosidorum Tripterygll Totorum need testing solution by method as previously mentioned;
C. need testing solution and reference substance solution are diluted to the dosage group of different concns with identical dose of distance, and agent, apart from being 0.5, is diluted with the DMEM nutrient solution of 20% foetal calf serum;
D. data processing calculates with tiring: the chondrocyte NO adopting the nitrogen protoxide described in embodiment 1 (NO) RNA isolation kit to record trial-product and reference substance various dose group gained respectively secretes inhibiting rate, relatively trial-product and reference substance reach the ratio of dosage when substantially identical chondrocyte NO secretes inhibiting rate, using this ratio as the biological value relative to dexamethasone, the size reflection Glucosidorum Tripterygll Totorum of this ratio suppresses the bioactive power of chondrocyte NO secretion.Specific experiment the results are shown in Table 6.
The restraining effect that table 6 Glucosidorum Tripterygll Totorum is secreted chondrocyte NO
Embodiment 4 Glucosidorum Tripterygll Totorum suppresses the mensuration of the biological value of IL-1 secretion
A. reference substance solution preparation: take dexamethasone as reference substance, dissolves with DMEM substratum, degerming through 0.22 μm of membrane filtration in super clean bench, 4 DEG C of preservations;
B. need testing solution preparation: prepare Glucosidorum Tripterygll Totorum need testing solution by method as previously mentioned;
C. need testing solution and reference substance solution are diluted to the dosage group of different concns with identical dose of distance, and agent is apart from being 0.5, and diluting solvent used is DMEM substratum;
D. data processing calculates with tiring: the IL-1 adopting mouse interleukin-11 (IL-1) the ELISA kit method described in embodiment 1 to record trial-product and reference substance various dose group respectively secretes inhibiting rate, calculate the ratio that trial-product and reference substance reach dosage when substantially identical IL-1 secretes inhibiting rate, using this ratio as Glucosidorum Tripterygll Totorum relative to the biological value of dexamethasone, the size reflection Glucosidorum Tripterygll Totorum of this ratio suppresses the bioactive power of IL-1 secretion.Specific experiment the results are shown in Table 7.
The restraining effect that table 7 different concns Glucosidorum Tripterygll Totorum is secreted IL-1
Embodiment 5 peony root total glycosides capsules suppresses the mensuration of the biological value of TNF-α secretion
A. reference substance solution preparation: general for reference substance with benefit match, is dissolved in RPMI-1640 nutrient solution, filters;
B. need testing solution preparation: prepare peony root total glycosides capsules need testing solution by method as previously mentioned;
C. need testing solution and reference substance solution are diluted to the dosage group of different concns with identical dose of distance, and agent distance is 0.5; Diluting solvent used is RPMI-1640 nutrient solution;
D. data processing calculates with tiring: adopt described in embodiment 1 by putting the method for exempting from and measuring the method for L929 Cell viability, measure TNF-α secretory volume, the TNF-α recording trial-product and reference substance various dose group respectively secretes inhibiting rate, calculate the ratio that trial-product and reference substance reach dosage when substantially identical TNF-α secretes inhibiting rate, match general biological value using this ratio as peony root total glycosides capsules relative to benefit, the size reflection peony root total glycosides capsules of this ratio suppresses the bioactive power of TNF-α secretion.Specific experiment the results are shown in Table 8.
The inhibiting rate that table 8 peony root total glycosides capsules is secreted TNF-α
Embodiment 6 peony root total glycosides capsules suppresses the mensuration of the biological value of IL-1 secretion
A. reference substance solution preparation: take dexamethasone as reference substance, dissolves with DMEM substratum, degerming through 0.22 μm of membrane filtration in super clean bench, 4 DEG C of preservations;
B. need testing solution preparation: prepare peony root total glycosides capsules need testing solution by method as previously mentioned;
C. need testing solution and reference substance solution are diluted to the dosage group of different concns with identical dose of distance, and agent is apart from being 0.5, and diluting solvent used is DMEM substratum;
D. data processing calculates with tiring: the IL-1 adopting mouse interleukin-11 (IL-1) the ELISA kit method described in embodiment 1 to record trial-product and reference substance various dose group respectively secretes inhibiting rate, calculate the ratio that trial-product and reference substance reach dosage when substantially identical IL-1 secretes inhibiting rate, using this ratio as peony root total glycosides capsules relative to the biological value of dexamethasone, the size reflection peony root total glycosides capsules of this ratio suppresses the bioactive power of IL-1 secretion.Specific experiment the results are shown in Table 9.
The restraining effect that table 9 different concns peony root total glycosides capsules is secreted IL-1
Innovative point of the present invention is the quality determining method providing a kind of antirheumatic Chinese medicine, and it is using biological value as evaluation index; Particularly, be that the biological value of the biological value suppressing chondrocyte NO to secrete, the biological value suppressing IL-1 secretion, the biological value suppressing TNF-α secretion and/or suppression COX-2 activity is as evaluation index.Can be that the combination of above-mentioned 4 kinds of biological values is as the overall target evaluating drug quality, also can optionally wherein 1 or 2 kind of biological value as quality evaluation index, in addition, which kind of means is adopted to detect biological value, the present invention is not also restricted, as long as can stablize, accurately and easy handling, meet the requirement that pharmaceutical analysis detects.
Innovative point of the present invention is also the quality standard providing a kind of antirheumatic Chinese medicine, it is differentiated at conventional physics and chemistry, on the basis of assay, set up rheumatism estimation of biological potency project, with the quality of chemistry and biology index comprehensive evaluation antirheumatic Chinese medicine, the inner quality of medicine of jointly checking on.Described rheumatism biological value be protection chondrocyte biological value, suppress the biological value of the biological value of COX-2 activity and/or the biological value of inflammation-inhibiting cytokine secretion.The biological value of described protection chondrocyte secretes inhibiting rate for Testing index with chondrocyte NO; Described inflammatory factor refers to TNF-α or IL-1.
The above is only the preferred embodiments of the present invention, not any pro forma restriction is done to the present invention, although the present invention discloses as above with preferred embodiment, but and be not used to limit the present invention, any those skilled in the art, not departing from the scope of technical solution of the present invention, make a little change when the technology contents of above-mentioned announcement can be utilized or be modified to the Equivalent embodiments of equivalent variations, in every case be the content not departing from technical solution of the present invention, according to any simple modification that technical spirit of the present invention is done above embodiment, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.
Claims (6)
1. an antirheumatic medicine quality evaluated method, is characterized in that, using rheumatism biological value as quality evaluation index.
2. method according to claim 1, is characterized in that, described method protects the biological value of the biological value of chondrocyte, the biological value suppressing COX-2 activity and inflammation-inhibiting cytokine secretion as quality evaluation index.
3. method according to claim 2, is characterized in that, the biological value of described protection chondrocyte refers to the biological value suppressing chondrocyte NO secretion.
4. method according to claim 2, is characterized in that, described inflammatory factor refers to TNF-α or IL-1.
5. method according to claim 1, it is characterized in that, also comprise and make described Chinese medicine after intestinal microflora fermentation, obtain tunning, adopt experiment in vitro to detect this tunning and suppress the biological value of chondrocyte NO secretion, suppress the biological value of COX-2 activity, suppress the biological value of TNF-α secretion and suppress the biological value of IL-1 secretion.
6. method according to claim 1, it is characterized in that, also comprise and make described Chinese medicine obtain meta-bolites under the effect of liver microsomes enzyme, adopt experiment in vitro to detect this meta-bolites and suppress the biological value of chondrocyte NO secretion, suppress the biological value of COX-2 activity, suppress the biological value of TNF-α secretion and suppress the biological value of IL-1 secretion.
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| CN106591414A (en) * | 2016-11-29 | 2017-04-26 | 广州中医药大学 | Biological detection method used for quality evaluation on and quality control over heat-clearing and detoxifying traditional Chinese medicines |
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| CN109870432A (en) * | 2017-12-05 | 2019-06-11 | 石家庄以岭药业股份有限公司 | One kind is based on the inhibition active detection method of Chinese medicine composition of COX-2 |
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| CN110531076A (en) * | 2019-07-19 | 2019-12-03 | 江苏康缘药业股份有限公司 | A method of COX-2 enzymatic activity in detection medicine analgesic evaluation procedure |
| CN110702809A (en) * | 2019-10-12 | 2020-01-17 | 辽宁中医药大学 | Compound chicken granule quality control and evaluation method based on anti-hepatic fibrosis bioactivity |
| CN111500667A (en) * | 2020-05-22 | 2020-08-07 | 天津中新药业集团股份有限公司 | Evaluation method of anti-inflammatory activity of Chinese patent medicine and application thereof |
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