CN118255663A - Extraction method and application of 3, 4-dihydroxymethyl benzoate - Google Patents
Extraction method and application of 3, 4-dihydroxymethyl benzoate Download PDFInfo
- Publication number
- CN118255663A CN118255663A CN202410352484.XA CN202410352484A CN118255663A CN 118255663 A CN118255663 A CN 118255663A CN 202410352484 A CN202410352484 A CN 202410352484A CN 118255663 A CN118255663 A CN 118255663A
- Authority
- CN
- China
- Prior art keywords
- ethyl acetate
- extracting
- ethanol
- gradients
- petroleum ether
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- CUFLZUDASVUNOE-UHFFFAOYSA-N methyl 3,4-dihydroxybenzoate Chemical compound COC(=O)C1=CC=C(O)C(O)=C1 CUFLZUDASVUNOE-UHFFFAOYSA-N 0.000 title claims abstract description 106
- 238000000605 extraction Methods 0.000 title claims abstract description 15
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 56
- 241000700159 Rattus Species 0.000 claims abstract description 37
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 33
- 206010039073 rheumatoid arthritis Diseases 0.000 claims abstract description 17
- 239000000469 ethanolic extract Substances 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 11
- 210000001503 joint Anatomy 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 11
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 11
- 210000000544 articulatio talocruralis Anatomy 0.000 claims abstract description 9
- 210000000845 cartilage Anatomy 0.000 claims abstract description 9
- 238000002791 soaking Methods 0.000 claims abstract description 8
- 230000008961 swelling Effects 0.000 claims abstract description 8
- 230000006378 damage Effects 0.000 claims abstract description 7
- 229940079593 drug Drugs 0.000 claims abstract description 7
- 206010061218 Inflammation Diseases 0.000 claims abstract description 6
- 230000004054 inflammatory process Effects 0.000 claims abstract description 6
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims abstract description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 36
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 32
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 28
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 239000003208 petroleum Substances 0.000 claims description 18
- 230000002757 inflammatory effect Effects 0.000 claims description 14
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 7
- 102100032367 C-C motif chemokine 5 Human genes 0.000 claims description 6
- 241000238631 Hexapoda Species 0.000 claims description 6
- 101000797762 Homo sapiens C-C motif chemokine 5 Proteins 0.000 claims description 6
- 102000013691 Interleukin-17 Human genes 0.000 claims description 6
- 108050003558 Interleukin-17 Proteins 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000000499 gel Substances 0.000 claims description 4
- 239000000741 silica gel Substances 0.000 claims description 4
- 229910002027 silica gel Inorganic materials 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 claims description 3
- 102000003777 Interleukin-1 beta Human genes 0.000 claims description 3
- 108090000193 Interleukin-1 beta Proteins 0.000 claims description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 3
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 3
- -1 compound methyl3,4-dihydroxybenzoate Chemical class 0.000 claims description 3
- 239000002024 ethyl acetate extract Substances 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 230000003110 anti-inflammatory effect Effects 0.000 claims description 2
- 230000008595 infiltration Effects 0.000 claims description 2
- 238000001764 infiltration Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 8
- 241000283690 Bos taurus Species 0.000 abstract description 4
- 102000000503 Collagen Type II Human genes 0.000 abstract description 4
- 108010041390 Collagen Type II Proteins 0.000 abstract description 4
- 238000010171 animal model Methods 0.000 abstract description 2
- 230000003356 anti-rheumatic effect Effects 0.000 abstract description 2
- 239000002038 ethyl acetate fraction Substances 0.000 abstract 1
- 238000005227 gel permeation chromatography Methods 0.000 abstract 1
- 238000004237 preparative chromatography Methods 0.000 abstract 1
- 238000010298 pulverizing process Methods 0.000 abstract 1
- 238000000465 moulding Methods 0.000 description 11
- 210000003371 toe Anatomy 0.000 description 10
- 206010003246 arthritis Diseases 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 6
- FPVRUILUEYSIMD-RPRRAYFGSA-N [(8s,9r,10s,11s,13s,14s,16r,17r)-9-fluoro-11-hydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-3-oxo-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-17-yl] acetate Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(OC(C)=O)[C@@]1(C)C[C@@H]2O FPVRUILUEYSIMD-RPRRAYFGSA-N 0.000 description 4
- 210000003423 ankle Anatomy 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 210000000988 bone and bone Anatomy 0.000 description 4
- 239000000839 emulsion Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000545405 Tripterygium Species 0.000 description 3
- 101150095962 aaaP gene Proteins 0.000 description 3
- 229960003657 dexamethasone acetate Drugs 0.000 description 3
- 239000003995 emulsifying agent Substances 0.000 description 3
- 230000003628 erosive effect Effects 0.000 description 3
- 150000002338 glycosides Chemical class 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 241000700157 Rattus norvegicus Species 0.000 description 2
- 208000025747 Rheumatic disease Diseases 0.000 description 2
- 230000003044 adaptive effect Effects 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000002683 foot Anatomy 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 210000000548 hind-foot Anatomy 0.000 description 2
- 238000000265 homogenisation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 208000018937 joint inflammation Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- MSWZFWKMSRAUBD-GASJEMHNSA-N 2-amino-2-deoxy-D-galactopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O MSWZFWKMSRAUBD-GASJEMHNSA-N 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 206010051728 Bone erosion Diseases 0.000 description 1
- 241000272201 Columbiformes Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 206010013954 Dysphoria Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101000588302 Homo sapiens Nuclear factor erythroid 2-related factor 2 Proteins 0.000 description 1
- 208000004044 Hypesthesia Diseases 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102100031701 Nuclear factor erythroid 2-related factor 2 Human genes 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 241000797328 Vespa velutina auraria Species 0.000 description 1
- 241000256856 Vespidae Species 0.000 description 1
- 241000212749 Zesius chrysomallus Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 231100000439 acute liver injury Toxicity 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 210000001188 articular cartilage Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 235000009569 green tea Nutrition 0.000 description 1
- 210000004247 hand Anatomy 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 208000034783 hypoesthesia Diseases 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 208000030175 lameness Diseases 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000010603 microCT Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000036651 mood Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000004112 neuroprotection Effects 0.000 description 1
- 230000000324 neuroprotective effect Effects 0.000 description 1
- 231100000862 numbness Toxicity 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000002040 relaxant effect Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 230000000552 rheumatic effect Effects 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an extraction method and application of 3, 4-dihydroxybenzoic acid methyl ester, and relates to the technical field of medicines, wherein the extraction method comprises the following steps: pulverizing air-dried wasp body, soaking in 95% ethanol to obtain ethanol extract, extracting with ethyl acetate to obtain ethyl acetate fraction, and purifying with silica gel column chromatography, sephadex LH-20 gel chromatography and semi-preparative chromatography to obtain methyl 3, 4-dihydroxybenzoate; the anti-rheumatoid arthritis efficacy is evaluated by establishing a bovine type II collagen-induced rat rheumatoid arthritis model (CIA) and administering methyl 3, 4-dihydroxybenzoate (MDHB) for treatment; the animal model selected by the method is simple and easy to operate, and experimental results show that the 3, 4-dihydroxybenzoic acid methyl ester can obviously relieve ankle joint and toe swelling of CIA rats, inhibit inflammatory reaction process, reduce damage to joints and cartilage, and achieve the effect of treating rheumatoid arthritis.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to an extraction method and application of 3, 4-dihydroxymethyl benzoate.
Background
Rheumatoid arthritis (Rheumatoidarthritis, RA) is a chronic autoimmune disease characterized by joint inflammation, fibroblast-like cell proliferation, cartilage destruction and bone erosion, manifested by joint redness, tenderness and restricted movement. RA repeatedly and continuously activates the innate and acquired immune systems, over time leading to a cascade of immune tolerance failures, autoantibody production and excessive production of inflammatory cytokines, leading to joint inflammation, ultimately leading to permanent and disabling cartilage and bone damage. RA can cause great damage to joints, if the joints cannot be effectively treated for a long time, the illness state can be further aggravated, the joints gradually lose the mobility, finally the joints are disabled, other complications can be caused after the illness state is aggravated, such as respiratory diseases, skin diseases, heart diseases, kidney diseases and the like, the mood of a patient is more dysphoria after the disability occurs, and psychological disorders are seriously caused.
The wasp (Vespa velutina auraria Smith) is one of the raw materials for preparing the wasp wine, which is an proved recipe of the Yunnan Jingbao nationality, and the wasp wine has the functions of treating rheumatic diseases, relaxing tendons and activating collaterals, and can improve the symptoms of numbness of hands and feet, joint ache and the like of patients caused by rheumatic blockage through external application or oral administration. Through systematic component separation and identification of wasps, more than 30 monomer components are obtained, wherein the monomer components comprise 3, 4-dihydroxybenzoic acid Methyl ester (Methyl 3,4-dihydroxybenzoate, MDHB).
Methyl3,4-dihydroxybenzoate (Methyl 3,4-dihydroxybenzoate, MDHB) was originally the major metabolite of antioxidant polyphenols found in green tea plants, and has good antioxidant and anti-inflammatory effects. Studies have shown that MDHB can provide neuroprotection against TBHP-induced oxidative damage to SH-SY5Y cells in vitro, demonstrating that MDHB is an effective neuroprotective compound that can relieve oxidative stress of cells. MDHB can also prevent D-galactosamine/lipopolysaccharide induced acute liver injury by inhibiting mouse inflammation, and MDHB can up-regulate Nrf2 and inhibit excessive osteoclast activity in mice to treat osteoporosis.
Therefore, an extraction method of the 3, 4-dihydroxybenzoic acid methyl ester and application thereof are provided.
Disclosure of Invention
The invention aims to provide a method for separating 3, 4-dihydroxybenzoic acid methyl ester from a wasp body for the first time, soaking the wasp body in 95% ethanol to obtain an ethanol extract, extracting the ethanol extract with ethyl acetate to obtain an ethyl acetate part, and continuously purifying the ethyl acetate part with silica gel column chromatography and Sephadex LH-20 gel to obtain the 3, 4-dihydroxybenzoic acid methyl ester.
In order to achieve the technical effects, the invention is realized by the following technical scheme: the extraction method of the 3, 4-dihydroxybenzoic acid methyl ester is characterized by comprising the following steps of:
S1, extracting: respectively taking crushed wasp air-dried insect bodies and ethanol according to the ratio of 1:3-5 of the mass to the volume, then soaking the crushed wasp air-dried insect bodies in the ethanol for 48 hours, and filtering to obtain a first ethanol extract; repeatedly soaking and extracting the residues with the same volume of ethanol for 3-4 times, each time for 24 hours, filtering, combining with the first extract, and recovering ethanol under reduced pressure to obtain the wasp ethanol extract;
s2, extraction: mixing the ethanol extract of the wasp with pure water according to the volume ratio of 1:1, stirring and uniformly mixing, sequentially extracting the mixed solution with solvents with different polarities for 3-5 times, combining ethyl acetate extracts, and recovering ethyl acetate to obtain an ethyl acetate part.
S3, separating: separating ethyl acetate part by silica gel column chromatography, gradient eluting with petroleum ether, chloroform and methanol at different volume ratios, and combining TLC results to obtain components C1-C11.
S4, separating the component C3 in the components C1-C11 obtained in the S2 through silica gel column chromatography, carrying out gradient elution by petroleum ether and acetone with different volume ratios, combining TLC results to obtain 9 components C3-1-C3-9, carrying out gradient elution on the component C3-5 in the components C3-1-C3-9 through ethyl acetate and acetone with different volume ratios, combining TLC results to obtain 10 components C3-5-1-C3-5-10; continuously purifying component C3-5-6 in the components C3-5-1 to C3-5-10 by silica gel column chromatography and Sephadex LH-20 gel to obtain the compound methyl3,4-dihydroxybenzoate, the structure of which is as follows:
In the S1, the concentration of the ethanol is 90-98%.
Further, in S2, the solvents with different polarities are specifically petroleum ether, chloroform, ethyl acetate and n-butanol; the extraction sequence is petroleum ether, chloroform, ethyl acetate and n-butanol.
In the step S3, the silica gel column is a 200-300 mesh silica gel column.
Further, in S3, the petroleum ether and the chloroform are divided into three gradients according to different volume ratios, wherein the volume ratio of the petroleum ether to the chloroform in the three gradients is 1:1, 3:7 and 0:1 respectively; the chloroform and the methanol are divided into five gradients according to different volume ratios, and the volume ratio of the chloroform to the methanol in the five gradients is 25:1, 10:1, 5:1, 3:2 and 0:1 respectively.
Further, in S4, the petroleum ether and the acetone are divided into five gradients according to different volume ratios, and the volume ratios of the petroleum ether and the acetone in the five gradients are respectively 10:1, 5:1, 7: 3. 6: 4. 1:1; the ethyl acetate and the acetone are divided into seven gradients according to different volume ratios, and the volume ratio of the ethyl acetate to the acetone in the seven gradients is 1: 0. 100:1, 50:1, 25:1, 10:1, 5:1, 3:7.
Another object of the present invention is to provide an application of methyl 3, 4-dihydroxybenzoate, which is characterized in that the application of methyl 3, 4-dihydroxybenzoate in preparing a medicament for treating rheumatoid arthritis.
Further, the application of the medicine for treating rheumatoid arthritis specifically comprises the following steps: the anti-inflammatory agent can be used for treating rheumatoid arthritis by relieving ankle joint and toe swelling of CIA rats, reducing inflammatory factors such as TNF-alpha, IL-17, IL-1 beta and CCL5, inhibiting inflammatory reaction process, reducing inflammatory infiltration, and reducing damage to joints and cartilage.
The beneficial effects of the invention are as follows:
The invention separates 3, 4-dihydroxybenzoic acid methyl ester from the wasp body with rheumatoid arthritis resistance for the first time, and the specific steps of extraction and separation are easy to operate; the anti-rheumatoid arthritis effect of the 3, 4-dihydroxybenzoic acid methyl ester is evaluated by establishing a bovine type II collagen-induced rat rheumatoid arthritis model (CIA), and the result shows that MDHB can obviously relieve ankle joint and toe swelling of the CIA rat, inhibit inflammatory reaction process, reduce damage to joints and cartilage and achieve the effect of treating the rheumatoid arthritis. Methyl 3, 4-dihydroxybenzoate can be used for preventing and treating rheumatoid arthritis; and the obtained 3, 4-dihydroxybenzoic acid methyl ester has not been reported to have therapeutic effects on rheumatoid arthritis.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is an image taken of the hind legs of each group of rats at day 35 of the experiment showing the condition of each group of rats after administration;
FIG. 2 shows the daily body weight change of rats in each group;
FIG. 3 is a graph showing toe thickness variation after molding administration for each group of rats;
FIG. 4 is a graph showing the change in ankle circumference after model administration in each group of rats;
FIG. 5 is a graph showing the effect of methyl 3, 4-dihydroxybenzoate on inflammatory factors in serum of rats in each group;
Fig. 6 is a micro-CT of ankle joints of rats of each group.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The extraction method of the 3, 4-dihydroxybenzoic acid methyl ester is characterized by comprising the following steps of:
S1, extracting: respectively taking 10g of crushed wasp air-dried insect bodies and 30ml of ethanol according to the ratio of 1:3 of the mass to the volume, then soaking the crushed wasp air-dried insect bodies in the ethanol for 48 hours, and filtering to obtain a first ethanol extract; repeatedly soaking and extracting the residues with the same volume of ethanol for 3-4 times, each time for 24 hours, filtering, combining with the first extract, and recovering ethanol under reduced pressure to obtain the wasp ethanol extract;
s2, extraction: mixing the ethanol extract of the wasp with pure water according to the volume ratio of 1:1, stirring and uniformly mixing, sequentially extracting the mixed solution with solvents with different polarities for 3-5 times, combining ethyl acetate extracts, and recovering ethyl acetate to obtain an ethyl acetate part.
S3, separating: separating the ethyl acetate part by 200-300 mesh silica gel column chromatography, carrying out gradient elution by petroleum ether and chloroform with different gradients in volume ratios of 1:1, 3:7 and 0:1 and chloroform with different gradients in volume ratios of 25:1, 10:1, 5:1, 3:2 and 0:1 respectively, and combining TLC results to obtain components C1-C11.
S4, separating the component C3 in the components C1-C11 obtained in the S2 through silica gel column chromatography, wherein the volume ratio is respectively 10:1, 5:1 and 7: 3. 6: 4. gradient elution is carried out on petroleum ether and acetone with different gradients in a ratio of 1:1, the TLC results are combined into 9 components, components C3-1 to C3-9 are obtained, and the volume ratio of components C3-5 in the components C3-1 to C3-9 is respectively 1: 0. gradient elution is carried out on ethyl acetate and acetone with different gradients of 100:1, 50:1, 25:1, 10:1, 5:1 and 3:7, and the combined TLC results are combined into 10 components to obtain C3-5-1 to C3-5-10 components; continuously purifying component C3-5-6 in the components C3-5-1 to C3-5-10 by silica gel column chromatography and Sephadex LH-20 gel to obtain the compound methyl 3, 4-dihydroxybenzoate.
Example 2
In this example, preliminary efficacy studies on CIA rats using methyl 3, 4-dihydroxybenzoate (MDHB) were performed as follows:
Step one, material preparation:
(1) Test article: methyl 3, 4-dihydroxybenzoate (MDHB), dexamethasone acetate tablet (DXMS, positive drug), tripterygium glycosides tablet (GTW, positive drug);
(2) Experimental animals: wistar rats, male, weight 180-220 g, spf grade, supplied by beijing Bei Fu biotechnology limited, animal license number: SCXK (Beijing) 2019-0010.
(3) Instrument and apparatus: stomach needle, shanghai Boli pigeon industry and trade company, specification: 12#; vernier calipers Gui Linan land digital measurement and control stock company;
Step two, experimental flow:
(1) Preparing bovine type II collagen (BC II) emulsifier: 5ml of complete Freund's adjuvant was removed by a 1ml pipette into 15ml of EP tube, 5ml of bovine type II collagen solution (2 mg/ml) was removed by a 1ml pipette into EP tube, and the solution was mixed uniformly by a homogenizer, and after white homogenization had occurred, homogenized again for 1min to be uniform, taking care of the whole procedure on ice. And (3) finishing homogenization, dripping the emulsion into an aqueous beaker, and if the emulsion appears in a granular form on the water surface, keeping the emulsion not to spread for a long time, so that the emulsion has a good emulsifying effect.
(2) Grouping of the test objects: after 7d of adaptive feeding, wistar rats were randomly divided into 10 groups according to body weight, namely a normal group, a model group, a dexamethasone acetate group (0.5 mg/kg), a tripterygium glycosides group (10 mg/kg) and 6 groups of different doses (160, 80, 40, 20, 10, 5 mg/kg) of methyl 3, 4-dihydroxybenzoate (MDHB) with 6 doses each.
(3) And (3) molding: selecting 54 rats of other nine groups except the normal group after the adaptive feeding, sterilizing the rats by alcohol, and injecting 0.1ml of BC II emulsifier in the left rear toe and the third of the left side, which are far from the tail root of the rats, so as to cause inflammation, wherein the same position of the normal group is injected with 0.1mol/L acetic acid solution; after 7 days, 0.1ml of emulsifier was injected at the right third of the tail root to boost the immunity, and the molding was performed for 14 days. Generally, the heel starts to red and swollen about 10 days, and the peak period is 14-28 days. Rats with successful modeling were selected for group drug administration.
(4) Dose and mode of administration: each group of rats is administrated by a gastric lavage mode, and the gastric lavage volume is 1ml/100g;
The general state and the weight change of the rat are observed and recorded every day, the toe thickness and the ankle circumference change of the rat are measured before molding, 14 days of molding and 21 days of administration, and meanwhile, the arthritis index score is carried out, and the molding success is indicated when the arthritis index score is greater than 4 minutes, wherein the standard is as follows: 0 point: no red swelling; 1, the method comprises the following steps: red spots or mild redness of joints appear; 2, the method comprises the following steps: moderate redness and swelling of joints; 3, the method comprises the following steps: severe swelling of the joints; 4, the following steps: all paw swelling, stiffness or rigidity including ankle joint.
Step three, experimental results:
the above experimental data were collected and summarized to obtain the data shown in table 1 below:
TABLE 1 arthritis index score for each group of rats
Note that: aa P <0.01 compared to normal group; bP<0.05,bb P <0.01 compared to model set
The results are shown in Table 1, and the rat arthritis index score after modeling is significantly increased (P < 0.01) compared with the normal group, indicating that the modeling was successful in this experiment. 21 days after dosing, each dosing group reduced the arthritis index score (P <0.01 or P < 0.05) of the rats to a different extent than the model group.
Figure 2 shows that the body weight of each group of rats is changed, the positive drug dexamethasone acetate has a larger influence on the body weight of rats, and the body weight of rats is reduced more.
Fig. 3 and 4 show that: the toe thickness and ankle circumference of the model group are obviously increased compared with those of the normal control group, and the common lameness of rats seriously indicates that the model is successful; the positive drug groups dexamethasone acetate and tripterygium glycosides can reduce the toe thickness and ankle circumference level of the rat, MDHB also show the same trend and are dose dependent, and the high concentration inhibition effect is better than the low concentration.
FIG. 3A is a secondary toe thickness variation; b is the thickness change of the inflammatory toe. Compared with the prior molding, aaaP is less than 0.001, aaP is less than 0.01, and aP is less than 0.05; compared with 14 days of molding, bbbP is less than 0.001, bbP is less than 0.01, and aP is less than 0.05.
FIG. 4A is a secondary ankle joint circumference change; b is the perimeter change of the inflammatory ankle joint. Compared with the prior molding, aaaP is less than 0.001, aaP is less than 0.01, and aP is less than 0.05; compared with 14 days of molding, bbbP is less than 0.001, bbP is less than 0.01, and bP is less than 0.05.
Example 3
The present example uses enzyme-linked immunosorbent assay (ELISA) to detect the serum inflammatory factor index of rats; inflammatory factors in the serum of each group of rats in example 2 were determined using an ELISA kit: TNF- α, IL-17, IL-1β and CCL5; the results are shown in Table 2:
TABLE 2 influence of MDHB on serum inflammatory factors of CIA rats
As shown in Table 2 and FIG. 5, the amounts of inflammatory factors TNF-alpha, IL-17, IL-1β and CCL5 in the serum of rats in the Model group (Model) were significantly increased as compared with the normal Control group (Control), and the inflammatory factors in the Model group were significantly different as compared with the normal Control group, indicating that the molding of rats was successful. Each MDHB group reduced the expression of inflammatory factors compared to the model group, and TNF- α, IL-17, IL-1β and CCL5 were significantly reduced in rat serum and exhibited a trend.
FIG. 5A is TNF- α; b is IL-17; c is IL-1 beta; d is CCL5. aaaP < 0.001 compared with the blank control group; bbbP < 0.001, bbP < 0.01, bP < 0.05, compared to model set.
Example 4
The effect of this example on methyl 3, 4-dihydroxybenzoate (MDHB) on CIA rat articular cartilage is as follows:
Taking the rat hind foot of example 2, stripping off the muscle tissue and ligaments attached to the toes, and fixing the hind foot bone tissue by adopting 4% paraformaldehyde; the fixed bone tissue is scanned and projected on the layer surface of each part of the sample by adopting a micro-focus X-ray bulb tube, and as can be seen from fig. 6, the bone tissue of the normal control group has no erosion of cartilage, while the ankle joint of the model group shows obvious erosion of cartilage and deformation of phalanges, and after the positive medicine and MDHB are given, the erosion of cartilage and the deformation degree of phalanges can be reduced.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are also considered to be within the scope of the present invention.
Claims (8)
1. The extraction method of the 3, 4-dihydroxybenzoic acid methyl ester is characterized by comprising the following steps of:
S1, extracting: respectively taking crushed wasp air-dried insect bodies and ethanol according to the ratio of 1:3-5 of the mass to the volume, then soaking the crushed wasp air-dried insect bodies in the ethanol for 48 hours, and filtering to obtain a first ethanol extract; repeatedly soaking and extracting the residues with the same volume of ethanol for 3-4 times, each time for 24 hours, filtering, combining with the first extract, and recovering ethanol under reduced pressure to obtain the wasp ethanol extract;
s2, extraction: mixing the ethanol extract of the wasp with pure water according to the volume ratio of 1:1, stirring and uniformly mixing, sequentially extracting the mixed solution with solvents with different polarities for 3-5 times, combining ethyl acetate extracts, and recovering ethyl acetate to obtain an ethyl acetate part.
S3, separating: separating ethyl acetate part by silica gel column chromatography, gradient eluting with petroleum ether, chloroform and methanol at different volume ratios, and combining TLC results to obtain components C1-C11.
S4, separating the component C3 in the components C1-C11 obtained in the S2 through silica gel column chromatography, carrying out gradient elution by petroleum ether and acetone with different volume ratios, combining TLC results to obtain 9 components C3-1-C3-9, carrying out gradient elution on the component C3-5 in the components C3-1-C3-9 through ethyl acetate and acetone with different volume ratios, combining TLC results to obtain 10 components C3-5-1-C3-5-10; continuously purifying component C3-5-6 in the components C3-5-1 to C3-5-10 by silica gel column chromatography and Sephadex LH-20 gel to obtain the compound methyl3,4-dihydroxybenzoate, the structure of which is as follows:
2. The method for extracting methyl 3, 4-dihydroxybenzoate according to claim 1, wherein in S1, the concentration of ethanol is 90% -98%.
3. The method for extracting methyl 3, 4-dihydroxybenzoate according to claim 1, wherein in S2, the solvents with different polarities are petroleum ether, chloroform, ethyl acetate and n-butanol; the extraction sequence is petroleum ether, chloroform, ethyl acetate and n-butanol.
4. The method for extracting methyl 3, 4-dihydroxybenzoate according to claim 1, wherein in S3, the silica gel column is a 200-300 mesh silica gel column.
5. The method for extracting 3, 4-dihydroxybenzoic acid methyl ester according to claim 1, wherein in S3, the petroleum ether and chloroform are divided into three gradients according to different volume ratios, and the volume ratio of the petroleum ether to the chloroform in the three gradients is 1:1, 3:7 and 0:1 respectively; the chloroform and the methanol are divided into five gradients according to different volume ratios, and the volume ratio of the chloroform to the methanol in the five gradients is 25:1, 10:1, 5:1, 3:2 and 0:1 respectively.
6. The method for extracting 3, 4-dihydroxybenzoic acid methyl ester according to claim 1, wherein in S4, the petroleum ether and the acetone are divided into five gradients according to different volume ratios, and the volume ratio of the petroleum ether to the acetone in the five gradients is 10:1, 5:1, 7 respectively: 3. 6: 4. 1:1; the ethyl acetate and the acetone are divided into seven gradients according to different volume ratios, and the volume ratio of the ethyl acetate to the acetone in the seven gradients is 1: 0. 100:1, 50:1, 25:1, 10:1, 5:1, 3:7.
7. The application of the 3, 4-dihydroxybenzoic acid methyl ester is characterized in that the 3, 4-dihydroxybenzoic acid methyl ester is applied to the preparation of medicines with anti-inflammatory effect for treating rheumatoid arthritis.
8. The use of methyl 3, 4-dihydroxybenzoate according to claim 7, wherein said use for the preparation of a medicament for the treatment of rheumatoid arthritis comprises in particular: obviously relieves the ankle joint and toe swelling of CIA rats, reduces inflammatory factors such as TNF-alpha, IL-17, IL-1 beta, CCL5 and the like to inhibit inflammatory reaction process, reduces inflammatory infiltration, and reduces damage to joints and cartilage to achieve the medical application for treating rheumatoid arthritis.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202410352484.XA CN118255663A (en) | 2024-03-26 | 2024-03-26 | Extraction method and application of 3, 4-dihydroxymethyl benzoate |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202410352484.XA CN118255663A (en) | 2024-03-26 | 2024-03-26 | Extraction method and application of 3, 4-dihydroxymethyl benzoate |
Publications (1)
Publication Number | Publication Date |
---|---|
CN118255663A true CN118255663A (en) | 2024-06-28 |
Family
ID=91610567
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202410352484.XA Pending CN118255663A (en) | 2024-03-26 | 2024-03-26 | Extraction method and application of 3, 4-dihydroxymethyl benzoate |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN118255663A (en) |
-
2024
- 2024-03-26 CN CN202410352484.XA patent/CN118255663A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1893191B1 (en) | Medicinal acidic cannabinoids | |
Huang et al. | Anti-inflammatory effects of Brucea javanica oil emulsion by suppressing NF-κB activation on dextran sulfate sodium-induced ulcerative colitis in mice | |
Xie et al. | Eucommia ulmoides Oliv. bark aqueous extract inhibits osteoarthritis in a rat model of osteoarthritis | |
EP3037090B1 (en) | Composition containing monoacetyldiglyceride compound as active ingredient for preventing or treating rheumatoid arthritis | |
JP5478486B2 (en) | Plant extract and its therapeutic use | |
Zhou et al. | Hyperbaric oxygen improves functional recovery of the injured spinal cord by inhibiting inflammation and glial scar formation | |
Aulia et al. | A Powerful ELISA Technique to Test The Potential of Extra Virgin Olive Oil in Reducing TNF-α Level and Edema Volume in Male Rattus norvegicus Exposed to Carrageenan | |
Li et al. | Fengshi Qutong capsule ameliorates bone destruction of experimental rheumatoid arthritis by inhibiting osteoclastogenesis | |
Hong et al. | Total flavonoids of bidens pilosa ameliorates bone destruction in collagen-induced arthritis | |
TW200812605A (en) | Plant extracts for the treatment of rheumatoid arthritis | |
CN107951980B (en) | Application of hosta plantaginea flower and extract thereof in preparing medicine for treating chronic prostatitis | |
CN118255663A (en) | Extraction method and application of 3, 4-dihydroxymethyl benzoate | |
TWI435727B (en) | Use of modulating secretion of cytokines | |
EP3639835B1 (en) | Pharmaceutical composition consisting of actein and deoyxactein as active compounds | |
EP3639836B1 (en) | Composition comprising isoferulic acid for use in the treatment of autoimmune hepatitis | |
CN109568419B (en) | Morinda officinalis total iridoid glycoside and preparation method and application thereof | |
Dikwa et al. | Aqueous extract of Hybanthus enneaspermus exhibited aphrodisiac potentials in fluoxetine-induced sexually-impaired female rats | |
CN104435293A (en) | Application of heterogenous kadsura longepedunculata total triterpenes ethanol extract in preparation of anti-arthritis drug | |
EP2025340A1 (en) | Plant extracts for the treatment of rheumatoid arthritis | |
JP2729340B2 (en) | Dementia treatment | |
CN114159435B (en) | Application of Fuziling in preparing medicine for treating arthritis | |
THIS | Aqueous extract of Hybanthus enneaspermus exhibited aphrodisiac potentials in fluoxetine-induced sexually-impaired female rats | |
TWI763214B (en) | Preparation of moutan radices cortex extract and uses for preventing or treating of inflammatory diseases thereof | |
AU2009101279A4 (en) | Medicinal formulation containing selected cytokines | |
CN115252626B (en) | Pharmaceutical composition containing glucocorticoid and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination |