CN109846870A - Phenolic acid compound is preparing the purposes in resisting rheumatoid arthritis drug - Google Patents
Phenolic acid compound is preparing the purposes in resisting rheumatoid arthritis drug Download PDFInfo
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- CN109846870A CN109846870A CN201910330178.5A CN201910330178A CN109846870A CN 109846870 A CN109846870 A CN 109846870A CN 201910330178 A CN201910330178 A CN 201910330178A CN 109846870 A CN109846870 A CN 109846870A
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- rheumatoid arthritis
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Abstract
The invention discloses a kind of phenolic acid compound 3,4- dihydroxy -5- methoxy benzoic acids to prepare the purposes in resisting rheumatoid arthritis drug.Present invention firstly discovers that 3,4- dihydroxy -5- methoxy benzoic acids can obviously inhibit the proliferation of synoviocytes in rheumatoid arthritis, the apoptosis of synoviocytes in rheumatoid arthritis is induced.Therefore, which can be used for preparing prevention and/or treats the drug of rheumatoid arthritis, clinical value with higher and development prospect.
Description
Technical field:
The present invention relates to pharmaceutical technology field, in particular to a kind of phenolic acid compound is preparing resisting rheumatoid arthritis
Purposes in drug.
Background technique:
Rheumatoid arthritis (rheumatoid arthritis, RA) is a kind of chronic systemic based on arthropathy
Property autoimmune disease, its pathogenesis remains unknown at present, clinic think RA morbidity mainly due to patient itself
Immune dysfunction leads to allergic reaction, makes the rotten denaturation of anti-inflammatory cells, be deposited on joint part and make joint tissue deformity,
Disable.According to investigations, the disease incidence of RA is about 1-2% in world wide, and China RA patient populations fall ill more than 4,000,000
Rate is still in rising trend.
Currently, mainly using non-steroidal anti-inflammatory drugs (diclofenac sodium, aspirin etc.) for rheumatoid arthritis and being immunized
Inhibitor (Ismipur etc.) is treated, but these drugs have apparent adverse reaction, as the former damages gastrointestinal tract
Mucous membrane leads to peptic ulcer, and the latter's selective action is poor, is easy to induce infection etc..Therefore, safe and effective control is researched and developed
Treating rheumatoid arthritis drug is a current urgent task.
In recent years, domestic and foreign scholars have made extensive and intensive studies in terms of botanical medicine treats RA, find many days
Right drug such as tripterygium glycosides, total glucoside, malicious rattan alkali etc. has preferable RA therapeutic effect.Therefore, it is developed from botanical medicine
Safe and efficient RA therapeutic agent has broad prospects out.
Summary of the invention:
In view of this, it is necessary to provide a kind of phenolic acid compounds to prepare the use in resisting rheumatoid arthritis drug
On the way.
The purpose of the present invention is to provide a kind of phenolic acid compound 3,4- dihydroxy -5- methoxy benzoic acids to inhibit class
Application in rheumathritis synovial cell proliferation and prevention and/or treatment rheumatoid arthritis.
Compound 3,4- dihydroxy -5- methoxy benzoic acid structural formula according to the present invention is shown in formula I:
The application of 3,4- dihydroxy -5- methoxy benzoic acid provided by the present invention includes two aspects: 1) preparing for it
Application in the product of prevention and/or treatment rheumatoid arthritis；2) inhibit synoviocytes in rheumatoid arthritis in preparation for it
Application in the product of proliferation.
It is in particular in that 3,4- dihydroxy -5- methoxy benzoic acid can obviously inhibit synoviocytes in rheumatoid arthritis
Proliferation, induces the apoptosis of synoviocytes in rheumatoid arthritis, can be used for preparing prevention and/or treats the medicine of rheumatoid arthritis
Object.
In addition, with 3,4- dihydroxy -5- methoxy benzoic acid is following products of effective component preparation: 1) prevention and/or
The product for treating rheumatoid arthritis；2) product for inhibiting synoviocytes in rheumatoid arthritis proliferation, also belongs to guarantor of the invention
Protect range.
Prevent and/or treat the drug of rheumatoid arthritis in the present invention and synoviocytes in rheumatoid arthritis is inhibited to increase
The drug grown can by injection, injection, infiltration, absorption, physically or chemically the method that mediates import body for example muscle, it is intradermal,
Subcutaneously, vein, mucosal tissue；Or body is imported after other material mixings or package.
When needs, one or more pharmaceutically acceptable carriers can also be added in said medicine.The load
Body includes diluent, excipient, filler, adhesive, wetting agent, disintegrating agent, the sorbefacient, surface of pharmaceutical field routine
Activating agent, absorption carrier, lubricant etc..
A variety of shapes such as injection, tablet, pulvis, granule, capsule, oral solution, paste, creme can be made in said medicine
Formula.The drug of the above various dosage forms can be prepared according to the conventional method of pharmaceutical field.
It is significant that the present inventor's first passage pharmacological evaluation proves that 3,4- dihydroxy -5- methoxy benzoic acid has
The activity for inhibiting synoviocytes in rheumatoid arthritis proliferation and induction synoviocytes in rheumatoid arthritis apoptosis, can be used for preparing pre-
Anti- and/or treatment rheumatoid arthritis drug, therefore its clinical value with higher and development prospect.
Detailed description of the invention:
Fig. 1 is the 3,4- dihydroxy -5- methoxy benzoic acid of various concentration in the embodiment of the present invention 1 to rheumatoid arthritis
The statistical results chart of synovial cell proliferation inhibiting effect.
Fig. 2 is the 3,4- dihydroxy -5- methoxy benzoic acid of various concentration in the embodiment of the present invention 2 to rheumatoid arthritis
The statistical results chart of apoptosis of synoviocytes facilitation.
In figure: " * " indicates the significance of difference in statistics.
Specific embodiment:
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The inhibiting effect that embodiment 1:3,4- dihydroxy -5- methoxy benzoic acid is proliferated synoviocytes in rheumatoid arthritis
Cell: human rheumatoid arthritis synovial cell (RA-FLS)；
Drug: 3,4- dihydroxy -5- methoxy benzoic acid；
Method: the recovery of 1.RA-FLS
RA-FLS cryopreservation tube is taken out rapidly from liquid nitrogen filling, puts the thermostat water bath that temperature is 37 DEG C into, it is continual to shake
It shakes, accelerates cell and thaw.After thawing completely, the RA-FLS suspension in cryopreservation tube is moved to pipettor in super-clean bench
In 10mL sterile centrifugation tube, it is centrifuged 5min, revolving speed 1000r/min.Supernatant is outwelled, 5mL complete culture solution is added, uses pipettor
It is lasting to blow and beat to uniform cell suspension, RA-FLS is inoculated in 25mL Tissue Culture Flask, is put into cell incubator and cultivates,
Incubator condition setting is 37 DEG C of temperature, CO2Concentration 5%.The growing state for observing RA-FLS after for 24 hours under the microscope, works as hair
Existing cell is adherent, triangular in shape and irregular shuttle shape when, show that cell growth status is good.
The passage of 2.RA-FLS
It observes under the microscope, when RA-FLS adherent growth is to account for entire Tissue Culture Flask bottom 80% or so, i.e.,
It can carry out cell passage.The original fluid in culture bottle is outwelled, is rinsed 2 times with PBS, it is therefore an objective to original fluid be cleaned, avoided
Influence cell dissociation；0.25% tryptic digestive juice is added to be digested, and digests situation in microscopically observation RA-FLS,
Suitable fetal calf serum (FBS) is added when seeing that most cells are rounded under mirror and terminates digestion, is then added a certain amount of complete
Full nutrient solution blows and beats culture bottle inner wall with aseptic straw in culture bottle repeatedly, is in uniform cell suspension；Use cell counting board
Cell is counted, after counting, RA-FLS is inoculated in 2 new culture bottles, and suitable complete culture is added
Liquid is put into cell incubator and cultivates, and incubator condition setting is 37 DEG C of temperature, CO2Concentration 5%, i.e., one mode for passing two pass
It is commissioned to train feeding.
3.MTT method measures cell inhibitory rate
Suitable 3,4- dihydroxy -5- methoxy benzoic acid is taken to be made into 0.01,0.1,1,5,10 μM of training respectively with DMEM
Nutrient solution.The RA-FLS in logarithmic growth phase is taken, the original fluid in culture bottle is outwelled, is rinsed 2 times with PBS, is added 0.25%
Tryptic digestive juice is digested, and digests situation in microscopically observation RA-FLS, to see that cell largely becomes under mirror
Bowlder is added suitable FBS and terminates digestion, and a certain amount of culture solution is then added in culture bottle, is ceaselessly blown with aseptic straw
Beat culture bottle inner wall, be in uniform cell suspension, counted with cell counting board, with culture solution adjust cell concentration be 1 ×
105RA-FLS is inoculated in 96 hole steril cell culture plates by a/mL, and every 100 μ L of hole is put into cell incubator and cultivates, training
Supporting case condition setting is 37 DEG C of temperature, CO2Concentration 5%.Be incubated for for 24 hours after, by 96 orifice plates be divided into 6 groups (i.e. blank control group,
0.01,0.1,1,5,10 μM of medicine group, every group of 6 multiple holes), sop up the original fluid in hole, be separately added into equivalent DMEM and
The pastille culture medium of various concentration, is put into cell incubator and intervenes.Intervene for 24 hours and after 48h, 20 μ L are added in each hole
MTS, 37 DEG C of incubation 1.5h, measures absorbance of each hole at 540nm wavelength with enzyme-linked immunosorbent assay instrument, calculates group of cells
Inhibiting rate.
Calculation formula is as follows:
Cell inhibitory rate/%=(1- blank control group OD value/each acute drug group OD value) × 100%
As a result: please refer to Fig. 1, concentration is that 0.1~10 μM of 3,4- dihydroxy -5- methoxy benzoic acid can be obvious
Inhibit the proliferation of RA-FLS, and inhibiting effect enhances with the increase of drug concentration.
Facilitation of the embodiment 2:3,4- dihydroxy -5- methoxy benzoic acid to synoviocytes in rheumatoid arthritis apoptosis
Cell: human rheumatoid arthritis synovial cell (RA-FLS)；
Drug: 3,4- dihydroxy -5- methoxy benzoic acid；
Method: the recovery of 1.RA-FLS
It is same as Example 1.
The passage of 2.RA-FLS
It is same as Example 1.
3.TUNEL method measures Apoptosis
The fracture of chromosomal DNA is a progressive process stage by stage in Apoptosis, and chromosomal DNA is first in endogenous
Hydrolase nucleic acid under the action of be degraded to the large fragment of 50-300kb.Then about 30% chromosomal DNA is in Ca2+And Mg2+
It under the endonuclease enzyme effect of dependence, is cut off at random between nucleosomal units, forms 180~200bp nucleosome DNA poly
Body.As long as a series of 3 ' ends-OH for the DNA for notch occur on DNA double chain fracture or a chain and generating can be in deoxyribose
Under the action of nucleotide terminal enzyme (DNA) (TdT), by deoxyribonucleotide and fluorescein, peroxidase, alkaline phosphatase
The derivative that enzyme or biotin are formed is tagged to the 3 '-ends of DNA, so as to carry out the detection of apoptotic cell, such methods one
As be known as deoxyribonucleotide terminal enzyme (DNA) mediate Nick End labelling method (TUNEL).
Suitable 3,4- dihydroxy -5- methoxy benzoic acid is taken to be made into 0.1 and 1 μM of culture solution, RA- respectively with DMEM
FLS cell is incubated for 48h in blank and drug containing culture solution, at room temperature using the fixed 30min of 4% paraformaldehyde.It is slow with phosphate
Fliud flushing (PBS) rinses 3 times, and cell membrane penetration liquid (0.1% sodium citrate, 0.1%Triton X-100) is added and acts on 2min.37
Deoxyribonucleotide terminal enzyme (DNA) (TdT) and biotin -11- deoxyuridine triphosphate (biotin-11- are used at DEG C
DUTP reaction 1h) is marked.Using fluorescence microscope cellular morphology and calculate apoptotic index.
Calculation formula is as follows:
Apoptotic index/%=(apoptosis cell/1000 in 1000 cells) × 100%.
As a result: please refer to Fig. 2, concentration is that 0.1~10 μM of 3,4- dihydroxy -5- methoxy benzoic acid can be obvious
Promote the apoptosis of synoviocytes in rheumatoid arthritis, and facilitation enhances with the increase of drug concentration.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (6)
1. application of the phenolic acid compound in the product of preparation prevention and/or treatment rheumatoid arthritis.
2. application of the phenolic acid compound in the product that preparation inhibits synoviocytes in rheumatoid arthritis proliferation.
3. phenolic acid compound as described in claim 1 is in the product of preparation prevention and/or treatment rheumatoid arthritis
Using, it is characterised in that: it is drug or health care product that preparation, which inhibits the product of neuroinflamation,.
4. phenolic acid compound as claimed in claim 2 is in the product that preparation inhibits synoviocytes in rheumatoid arthritis proliferation
Application, it is characterised in that: inhibit synoviocytes in rheumatoid arthritis proliferation product be drug or health care product.
5. phenolic acid compound as described in claim 1 is in the product of preparation prevention and/or treatment rheumatoid arthritis
Using, it is characterised in that: phenolic acid compound 3,4- dihydroxy -5- methoxy benzoic acid.
6. phenolic acid compound as claimed in claim 2 is in the product that preparation inhibits synoviocytes in rheumatoid arthritis proliferation
Application, it is characterised in that: phenolic acid compound 3,4- dihydroxy -5- methoxy benzoic acid.
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| CN201910330178.5A CN109846870A (en) | 2019-04-23 | 2019-04-23 | Phenolic acid compound is preparing the purposes in resisting rheumatoid arthritis drug |
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| CN201910330178.5A CN109846870A (en) | 2019-04-23 | 2019-04-23 | Phenolic acid compound is preparing the purposes in resisting rheumatoid arthritis drug |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113521171A (en) * | 2021-08-10 | 2021-10-22 | 福建香缘生物科技发展有限公司 | Essential oil for relieving rheumatoid arthritis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102631434A (en) * | 2012-04-25 | 2012-08-15 | 中国人民解放军第二军医大学 | Application of pawpaw total phenolic acid extract in preparation of arthritis prevention and treatment medicaments or food |
| CN102641278A (en) * | 2012-04-06 | 2012-08-22 | 王青 | Application of ginkgolic acid in treating arthritis |
| CN103893161A (en) * | 2013-10-25 | 2014-07-02 | 南通瑞普埃尔生物工程有限公司 | Application of syringic acid-(4-hydroxyl-3,5-dimethoxybenzoic acid) in preparation of medicine for preventing and treating rheumatoid arthritis |
-
2019
- 2019-04-23 CN CN201910330178.5A patent/CN109846870A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102641278A (en) * | 2012-04-06 | 2012-08-22 | 王青 | Application of ginkgolic acid in treating arthritis |
| CN102631434A (en) * | 2012-04-25 | 2012-08-15 | 中国人民解放军第二军医大学 | Application of pawpaw total phenolic acid extract in preparation of arthritis prevention and treatment medicaments or food |
| CN103893161A (en) * | 2013-10-25 | 2014-07-02 | 南通瑞普埃尔生物工程有限公司 | Application of syringic acid-(4-hydroxyl-3,5-dimethoxybenzoic acid) in preparation of medicine for preventing and treating rheumatoid arthritis |
Non-Patent Citations (8)
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113521171A (en) * | 2021-08-10 | 2021-10-22 | 福建香缘生物科技发展有限公司 | Essential oil for relieving rheumatoid arthritis |
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Application publication date: 20190607 |