CN108403706A - A kind of new application of pregnane glucoside compound - Google Patents
A kind of new application of pregnane glucoside compound Download PDFInfo
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- -1 pregnane glucoside compound Chemical class 0.000 title claims abstract description 12
- 229930182478 glucoside Natural products 0.000 title claims abstract description 8
- 239000003018 immunosuppressive agent Substances 0.000 claims abstract description 11
- 239000002260 anti-inflammatory agent Substances 0.000 claims abstract description 9
- 229940124599 anti-inflammatory drug Drugs 0.000 claims abstract description 7
- 229940124589 immunosuppressive drug Drugs 0.000 claims abstract description 4
- 150000001875 compounds Chemical class 0.000 abstract description 26
- 238000002360 preparation method Methods 0.000 abstract description 18
- 238000002474 experimental method Methods 0.000 abstract description 13
- 239000003814 drug Substances 0.000 abstract description 10
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 7
- 241001408435 Epigynum auritum Species 0.000 abstract description 3
- 241001529246 Platymiscium Species 0.000 abstract description 2
- 241000196324 Embryophyta Species 0.000 abstract 1
- 150000002611 lead compounds Chemical class 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 23
- 229960003957 dexamethasone Drugs 0.000 description 17
- 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 17
- 230000000694 effects Effects 0.000 description 17
- 229930193348 epigynoside Natural products 0.000 description 15
- 239000013641 positive control Substances 0.000 description 15
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- QZLYKIGBANMMBK-UGCZWRCOSA-N 5α-Androstane Chemical compound C([C@@H]1CC2)CCC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CCC[C@@]2(C)CC1 QZLYKIGBANMMBK-UGCZWRCOSA-N 0.000 description 1
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- VHRSUDSXCMQTMA-PJHHCJLFSA-N 6alpha-methylprednisolone Chemical compound C([C@@]12C)=CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2[C@@H](O)C[C@]2(C)[C@@](O)(C(=O)CO)CC[C@H]21 VHRSUDSXCMQTMA-PJHHCJLFSA-N 0.000 description 1
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- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 1
- OAIVIYSBZFEOIU-UHFFFAOYSA-N chloroform;propan-2-one Chemical compound CC(C)=O.ClC(Cl)Cl OAIVIYSBZFEOIU-UHFFFAOYSA-N 0.000 description 1
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- HWJHWSBFPPPIPD-UHFFFAOYSA-N ethoxyethane;propan-2-one Chemical compound CC(C)=O.CCOCC HWJHWSBFPPPIPD-UHFFFAOYSA-N 0.000 description 1
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- 239000000796 flavoring agent Substances 0.000 description 1
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- 239000000499 gel Substances 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 229940125721 immunosuppressive agent Drugs 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
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- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000003526 lymphopoietic effect Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- 229960004584 methylprednisolone Drugs 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229940100688 oral solution Drugs 0.000 description 1
- 210000003024 peritoneal macrophage Anatomy 0.000 description 1
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- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 229960004618 prednisone Drugs 0.000 description 1
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 244000145591 rattan cane Species 0.000 description 1
- 235000012950 rattan cane Nutrition 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 210000002707 regulatory b cell Anatomy 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Rheumatology (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Pain & Pain Management (AREA)
- Transplantation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of new applications of pregnane glucoside compound, i.e. its application in preparing immunosuppressive drug or anti-inflammatory drug;The compound is to be extracted from Epigynum Auritum platymiscium isolated, which is shown below:
Description
Technical field
The present invention relates to a kind of new applications of pregnane glycoside compounds, i.e., it is preparing immunosuppressive drug, anti-inflammatory agent
Application in object.
Background technology
Immunosuppressor provides effective medicine in clinic for the rejection after autoimmunity disease and organ transplant
Object treats the graft versus host disease(GVH disease) occurred after bone-marrow transplantation commonly used to the rejection for inhibiting to occur after organ transplant, or
Treat the autoimmune diseases such as rheumatoid arthritis, disease.Anti-inflammatory agent is for treating institute after tissue is damaged
The drug of the reaction inflammation of generation;Steroidal drug is clinically used immunosuppressor and anti-inflammatory drug, as prednisone, fill in
Meter Song, methylprednisolone etc..It can effectively inhibit inflammatory factor, reduce various immunocytes.
Existing non-steroidal anti-inflammatory drug and immunosuppressor long-term administration generally existing such as glucocorticoid etc generates
The shortcomings of drug resistance, toxic side effect are big, inconvenient to use, therefore normally only used in the treatment of major disease.Chinese medicine conduct
A kind of natural products, has many advantages, such as and people's tissue compatible property is good, Small side effects, in grinding for immunosuppressor and anti-inflammatory preparation
It is increasingly valued by people in studying carefully.Thus, the compound with immunosupress and anti-inflammatory effect is found from natural products,
There is application value for the neotype immunosuppressant and anti-inflammatory drug of developing high-efficiency low-toxicity.
Simao Calamus (Epigynum) is one of Apocynaceae Epigynum Auritum race category, according to the result of study of our early periods,
Androstane, pregnane and its glucosides substance are rich in Epigynum Auritum platymiscium.Pregnane glycoside compounds of the present invention are as immune
Inhibit drug and anti-inflammatory drug, it is not yet reported.
Invention content
The purpose of the present invention is to provide a kind of new application of pregnane glucoside compound, i.e. the compound is exempted from preparation
Epidemic disease inhibits the application in drug or anti-inflammatory drug, the structural formula of compound to be shown below:
It is 17 α, and -3 β-O-2- methoxyl group -6- deoxidation-β-D- idose glycosides of 20S- dihydroxy-pregnant steroid -5 (6)-alkene leads to
Popular name epigynoside G.
Specific immunosuppressive drug is applied in the medicine for preparing autoimmune disease, or is applied and prepared organ
In the medicine for transplanting anti-rejection.
It can include that pharmaceutical field is conventional with pharmaceutically acceptable auxiliary material, the auxiliary material in application of the present invention to fill out
Agent, diluent, adhesive, excipient, sorbefacient, filler, surfactant and stabilizer etc. are filled, can also be added when necessary
Enter flavouring agent, pigment and sweetener etc..
Pill, pulvis, tablet, granula, oral solution and injection can also be made in addition to capsule is made in application of the present invention
The diversified forms such as liquid.
Splenocyte is prepared for testing with the Balb/c mouse of health in the present invention;After the completion of prepared by splenocyte, pass through
Concanavalin A (ConA) or lipopolysaccharides (LPS) are induced, while various concentration test compound is added in test group, with
The dexamethasone (Dexamethasone, abbreviation DXMS) of corresponding concentration is used as positive control, after cultivating 72h, passes through Cell
Counting Kit-8 (CCK-8) reagent methods measure test group, positive controls, induction control group and do not induce control group respectively
Light absorption value, to evaluate the ability of compound regulatory T-cell or B cell proliferation;The experimental results showed that pregnant steroid in the present invention
Alkane glucoside compound:Epigynoside G are to the ConA or LPS Balb/c mouse T splenic lymphocytes stimulated or bone-marrow-derived lymphocyte
Proliferation significantly inhibits, the significant difference (p compared with the control group for being not added with the compound<0.05).
The Turnover of Mouse Peritoneal Macrophages (RAW264.7) that LPS is induced is used to be used as inflammatory model, RAW264.7 in the present invention
After cell growth state is stablized, the test compound of various concentration is added in test group, is sun with the dexamethasone of corresponding concentration
Property control, continue to be incubated 2h LPS induction RAW264.7 cells are added are inflamed reaction, continue culture for 24 hours, on collection culture medium
It is clear to carry out using enzyme linked immunosorbent assay (ELISA) (ELISA) method detection tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-
1 β) and prostaglandin E2 (PGE2) three kinds of inflammatory factors expression;The extracorporeal anti-inflammatory activity of compound is evaluated with this;It is real
It tests the result shows that pregnane glucoside compound in the present invention:Epigynoside G secrete the RAW264.7 cells that LPS is induced
TNF-α, IL-1 tri- kinds of inflammatory factors of β and PGE2 expression all significantly inhibit, and be not added with the chemical combination
The control group of object has significant difference (p<0.05).
Specific implementation mode
Below by embodiment, invention is further described in detail, but present disclosure is not limited thereto, this
Method operating according to a conventional method unless otherwise specified in embodiment, agents useful for same unless otherwise specified use conventional commercial
Reagent or the reagent configured according to a conventional method.
Embodiment 1:The preparation of Epigynoide G
The Simaos the 15kg Laos little Hua rattan sample after air-drying is taken, is extracted 3 times with methanol eddy after crushing, after reduced pressure
It is water-soluble, then be extracted with ethyl acetate 3 times, ethyl acetate layer is concentrated, weigh to obtain 1.3kg;By ethyl acetate layer with macroporous absorption tree
Fat D101 carries out rough segmentation, elute always there are with the methanol aqueous solution of percent by volume 40%, 60%, 80%, 100% respectively
To 4 parts (Fr.A, Fr.B, Fr.C, Fr.D).Fr.D (200g) admixes the silica gel of 1.5 times of amounts, mixes sample silica gel and carries out conventional do
It is dry, column is filled, with chloroform-acetone (0:100-90:10) two-phase system is that mobile phase carries out gradient elution, and merging is inspected according to TLC
Obtain three part Fr.D1-D3;Fr.D2 C18Filler mixes reverse phase C on sample18Column methanol-water gradient elution obtains two parts,
Fr.D2a-D2b;Fr.D2a is by silicagel column with petroleum ether-acetone (10:1-1:1) gradient elution is carried out, is then passed through
Sephadex LH-20 gel columns use chloroform methanol (1:1, V/V) mixed solvent elutes, is pure by half preparation efficient liquid phase again
Compound epigynoide G (7mg) are obtained after change.
Embodiment 2:Immunosupress detection experiment
(1) preparation of splenic lymphocytes suspension
Neck is taken off after taking the healthy Balb/c mouse of 18~22g to extract eyeball bloodletting to put to death, and is placed in 75% alcohol of volumetric concentration
Middle soaking disinfection 3-5 minutes, taking-up was placed in sterile tray, on the left of abdomen upward, in safety cabinet, was picked up with the tweezers sterilized
Fur in the middle part of abdomen begins to make a kerf from lower end, each layer of stomach wall is cut off with other set instrument, is taken out spleen with third set instrument,
Removal fat and connective tissue, are put into PBS (phosphate buffer), wash away floating blood.Then spleen tissue is moved to and fills RPMI
It in the plate of 1640 endless full nutrient solutions, is cut into small pieces with scissors, with asepsis injector core by spleen in 200 mesh stainless steel mesh
It gently grinds, is repeatedly rinsed on a small quantity with PBS, suspension is transferred to pipettor in 15mL centrifuge tubes;1000r/min rotating speeds centrifuge
5min;Supernatant is abandoned, 3mL erythrocyte cracked liquids (Tris-NH is added4Cl 9mL PBS terminations are added instead after standing 2min in) mixing
It answers, centrifuges (1000rpm, 5min), supernatant, precipitation is gone to be washed twice with 5mL PBS, centrifuged under similarity condition;Finally precipitation is used
RPMI 1640 complete culture solutions of the 5mL containing 10% fetal calf serum suspends;Expect that blue living cells is refused dye method and counted with 0.8%, it is living thin
Born of the same parents' number is no less than 95%, and 1640 complete culture solutions of RPMI is added to dilute, adjustment cell concentration to 1 × 106A/mL or so.
(2) preparation of test liquid
Precision weighs monomeric compound and each 2mg of dexamethasone, and DMSO dissolvings are added, are diluted with RPMI-1640 culture mediums
To required concentration;Precision weighs each 2mg of ConA and LPS and uses PBS and DMSO to dissolve respectively, is diluted with RPMI1640 culture mediums
To required concentration.It controls each additive dissolving DMSO final concentrations used and is no more than 0.1%.
(3) experiment packet
Negative control group:+ 10 μ LRPMI-1640 culture solutions of+10 μ LConA solution of 100 μ L splenocyte suspensions
Positive controls:+ 10 μ L dexamethasone solutions of+10 μ LConA solution of 100 μ L splenocyte suspensions
Test sample group:+ 10 μ L given the test agent of+10 μ LConA solution of 100uL splenocyte suspensions
Normal group:+ 20 μ LRPMI-1640 culture solutions of 100uL splenocyte suspensions
Blank control group:120 μ LRPMI-1640 culture solutions;
In 96 orifice plates, lymphocyte suspension (1 × 10 is added per hole6A/mL) 100 10 μ L (final concentration of 10 μ of μ L, ConA
G/mL), 10 μ L of various concentration reagent chemical compound diluted liquid (final concentration is respectively 12.5,25,50 μ g/mL), dexamethasone is also done
Three corresponding concentration groups, each concentration group set 4 it is parallel.
(4) it cultivates:37 DEG C are placed in, 5%CO2Culture 72 hours in incubator.
(5) CCK-8 methods measure cell OD values
After culture 72 hours, the CCK-8 reagents (the green skies) of 10 μ L are added in every hole, are placed in 37 DEG C, 5%CO2Incubator
After inside continuing culture 4 hours, the light absorption value per hole is measured at 450nm to calculate cell proliferative conditions, and thorn is calculated as follows
Swash index (SI):
SI (stimulus index)=plus OD values of mitogen culture/is not added with the OD values of mitosis stock culture.
(6) data processing
Experimental data OD values indicate that mathematical statistics and variance analysis work are used using " average ± standard deviation "
Origin softwares are completed.
(7) experimental result
Table 1:Epigynoide G intervene lower Balb/c mice spleen T lymphopoiesis stimulus indexes
Compound epigynoside G at a concentration of 12.5,25,50 μM, test group stimulus index is respectively 2.76,
1.98、1.71;Significance analysis shows each concentration function and effect and the poor anisotropic all extremely significantly (P of negative control group<
0.01)。
At a concentration of 12.5,25,50 μM, test group stimulus index is respectively positive control dexamethasone (DXMS)
1.43、1.24、1.14;Each concentration group function and effect and negative control group more all have extremely significant difference (P<0.01).
Inhibit T lymphopoietic the experimental results showed that the compound epigynoside G have at 12.5 μM
Activity increases the enhancing of its inhibition within the scope of 50 μM with concentration.
Table 2:EpigycosideG intervenes the stimulus index of lower Balb/c mice spleen B lymphocyte proliferations
Epigynoside G at a concentration of 12.5,25,50 μM, test group stimulus index is respectively 5.92,4.80,
2.64;Significance analysis shows each concentration function and effect and the poor anisotropic all extremely significantly (P of negative control group<0.01).
Positive control dexamethasone (DXM) at a concentration of 12.5,25,50 μM, test group stimulus index is respectively 4.11,
3.46、1.89;Each concentration group function and effect and negative control group more all have extremely significant difference (P<0.01).
The experimental results showed that work of the compound epigynoside G at 12.5 μM with inhibition B lymphocyte proliferation
Property, within the scope of 50 μM, increase with concentration, stimulus index reduces, inhibition enhancing.
In summary experimental result, heretofore described compound epigynoside G can significantly inhibit ConA or LPS
The proliferation of the Balb/c mouse spleen T lymphocytes and bone-marrow-derived lymphocyte that induce respectively.Heretofore described compound
Epigynoside G have outstanding immunosuppressive activity, can be used for the preparation of immunosuppressive agents.
Embodiment 3:Extracorporeal anti-inflammatory activity test
(1) preparation of related reagent is tested
Sample dilutes the preparation of working solution:Concentrating sample dilution takes out from -4 DEG C of refrigerators, and room temperature is slowly thawed, according to
Experiment institute expense takes part using distilled water according to 1:5 dilution proportion.
The preparation of reagent dilutions working solution:Concentrated reagent liquid takes out from -4 DEG C of refrigerators, and room temperature slows down slow defrosting, according to experiment
Institute's expense takes part using distilled water according to 1:5 dilution proportion.
The preparation of cell cracking working solution:Concentrating cells lysate takes out from -4 DEG C of refrigerators, and room temperature is slowly thawed, according to examination
Testing institute's expense takes part using distilled water according to 1:2 dilution proportion.
The preparation of wash operating solution:Concentrated cleaning solution takes out from -4 DEG C of refrigerators, is placed at room temperature for half an hour, jog makes it dissolve
Uniformly, take part using distilled water according to 1 according to experiment institute expense:20 dilution proportion.
The preparation of biotinylated antibody working solution:Concentrated biological element antibody takes out from -20 DEG C of refrigerators, and it is small to be placed at room temperature for half
When, brief centrifugation makes it dissolve and is deposited to bottom of the tube, 100 μ l 1 × reagent dilutions working solutions is added, with pipette tips blowing gently
It beats for several times, it is made fully to dissolve.Take part using distilled water according to 1 according to experiment institute expense:80 dilution proportion.
The preparation of horseradish peroxidase working solution:It concentrates horseradish peroxidase to take out from -20 DEG C of refrigerators, be placed at room temperature for
Half an hour, after brief centrifugation liquid-transfering gun piping and druming make fully to dissolve.Take part using distilled water according to 1 according to experiment institute expense:
200 dilution proportion.
(2) preparation of sample
Sample is before use, must at least need to dilute 5-10 times with 1 × sample dilution working solution.
(3) preparation of standard items
The use of standard items must comply with now with the current principle;
A. standard items must preserve in -20 DEG C or -80 DEG C of refrigerators;
B. standard items are taken out for -20 DEG C from refrigerator, is placed at room temperature for half an hour, brief centrifugation makes its whole be deposited to pipe
Bottom is added 1x samples and dilutes working solution;
C. concussion gently is for several times, it is ensured that standard items powder all, fully dissolving, a concentration of 10ng/mL, is labeled as at this time
Mother liquor;
D. based on mother liquor, 6 gradient concentrations are diluted to according to 5 times successively;
E. 1x samples dilution working solution (a concentration of 0) as a control group is used;
F. the final dosage of each concentration is prepared according to actual experiment institute expense.
(4) cell culture and experiment packet
RAW264.7 cells using DMEM complete culture solutions (addition 10% fetal calf serum, 1% is dual anti-) in 37 DEG C, 5%CO2
Culture to logarithmic phase uses in environment incubator.After logarithmic growth phase RAW264.7 cells add DMEM culture mediums to adjust concentration,
With 2 × 104In 96 orifice plate of Standard entertion in a/hole.After continuing culture for 24 hours, each sample of various concentration is added;This experiment is tested
Compound test concentration is respectively set to 0.4 μ g/mL, 1 μ g/mL, 5 μ g/mL.Made using dexamethasone (10 μ g/mL of final concentration)
For positive control, PBS does negative control.Continue to be incubated after 2h plus LPS makes its final concentration of 5 μ g/ml, continue culture for 24 hours, collects
Culture medium supernatant is detected.
Specific experiment is grouped as follows:
Negative control group:Cell suspension+PBS+LPS
Positive controls:Cell suspension+LPS+ dexamethasone
Blank control group:Cell suspension
Test sample group:Cell suspension+each concentration of test sample+LPS
Each experimental group does three groups of parallel tests.
(5) the expression ELISA double antibody sandwich methods of ELISA method detection culture medium supernatant IL-1 β, PGE2, TNF-α,
Concrete operation step is as follows:
1) reagent needed for ELIAS strip and experiment is taken out, is restored to room temperature, 100 μ L couple are added according to experimental design per hole
Standard items after the dilution answered and sample.After sealing plate film sealing plate, it is incubated at room temperature 2.5 hours;
2) each boreliquid is sucked, 300 μ L wash operating solutions are added per hole, is washed 4 times, is firmly patted on clean paper, really
Guarantor thoroughly removes liquid in hole;
3) the biotinylated antibody working solution after 100 μ L dilutions is added per hole, jiggles, after sealing plate film sealing plate, room temperature
It is incubated 1 hour, is washed 4 times after removing liquid;
4) the horseradish peroxidase working solution after 100 μ L dilutions is added per hole, jiggles, after sealing plate film sealing plate, room
Temperature is incubated 45 minutes, is washed 4 times after removing liquid;
5) the horseradish peroxidase working solution after 100 μ L dilutions is added per hole, jiggles, after sealing plate film sealing plate, room
Temperature is incubated 45 minutes;
6) 100 μ L color developing agents are added per hole, after sealing plate film sealing plate, are incubated at room temperature 30 minutes;
7) 50 μ L terminate liquids are added per hole, absorbance at 450nm is detected in microplate reader;
8) according to the standard items OD values read in microplate reader, standard curve and regression equation are drawn, and according to the OD of sample
Value calculates the expression contents of each factor.
(6) data processing
17.0 statistical softwares of SPSS analyze, and numerical value is indicated with equal X ± SE, it is poor with t inspections verification for the variation between different groups
It is anisotropic;Use the drawing of Origin 8.0 (* p<0.05 indicates that there are significant differences with negative control group).
(7) experimental result
Table 3:Influences of the compound epigcoside G to RAW264.7 cellular inflammations factor expression level
Compound epigycoside G are when activity is 0.4 μ g/mL, 1.0 μ g/mL, 5.0 μ g/mL, test group
The expression of TNF-α is respectively 15.47ng/L, 14.04ng/L, 12.67ng/L, the table with negative control group 19.75ng/L
It is compared up to amount, all there is significant difference (P<0.05), and with 10 μ g/mL exercising results of positive controls dexamethasone
14.29ng/L compares, and for epigycoside G under the activity of 1.0 μ g/mL, the expression of TNF-α is right with the positive
Photograph is worked as, and has then been less than positive control result under 5.0 μ g/mL activities.
Compound epigynoside G are when activity is 0.4 μ g/mL, 1.0 μ g/mL, 5.0 μ g/mL, test group IL-
The expression of 1 β is respectively 5.24ng/L, 4.44ng/L, 3.47ng/L, has significant difference compared with negative control group,
And compared with 10 μ g/mL exercising results 4.61ng/L of positive controls dexamethasone, works of the epigynoside G in 1.0 μ g/mL
With under concentration, the expression of IL-1 β is with less than positive control, and under the activity of 5 μ g/mL, IL-1 β expression quantity is
3.47ng/L, expression is far below positive controls, and makes the IL-1 β secretory volumes of inflammatory cell substantially close to normal level
3.18ng/L。
Compound epigynoside G are when activity is 0.4 μ g/mL, 1.0 μ g/mL, 5.0 μ g/mL, test group
The expression of PGE2 is respectively 13.47ng/L, 12.08ng/L, 10.78ng/L, has conspicuousness compared with negative control group
Difference, and compared with 10 μ g/mL exercising results 11.32ng/L of positive controls dexamethasone, epigynoside G are in 1.0 μ g/
Under the activity of mL, the expression of PGE2 is suitable with positive control, then low under 5.0 μ g/mL activities
In positive controls.
The experimental results showed that compound epigycoside G under the activity of 0.4 μ g/mL-5.0 μ g/mL to LPS
The TNF-α of the inflammation RAW264.7 cells of induction, IL-1 β, the PGE2 factors expression there is adjustment effect, it can be inhibited
Expression weakens inflammatory reaction, and wherein epigynoside G antiphlogistic effects are better than positive drug dexamethasone.It is heretofore described
Compound epigynoside G have significant extracorporeal anti-inflammatory activity, can be used for the preparation of anti-inflammatory drug.
Claims (1)
1. pregnane glucoside compound the answering in preparing immunosuppressive drug or anti-inflammatory drug that structural formula is shown below
With:
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CN110123855A (en) * | 2019-06-14 | 2019-08-16 | 昆明理工大学 | The purposes of Simao boisiana extract |
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