CN108558978A - Pregnane glucoside compound and its application - Google Patents
Pregnane glucoside compound and its application Download PDFInfo
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- CN108558978A CN108558978A CN201810408817.0A CN201810408817A CN108558978A CN 108558978 A CN108558978 A CN 108558978A CN 201810408817 A CN201810408817 A CN 201810408817A CN 108558978 A CN108558978 A CN 108558978A
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- pregnane
- epigycoside
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- -1 Pregnane glucoside compound Chemical class 0.000 title claims abstract description 19
- 229930182478 glucoside Natural products 0.000 title claims abstract description 11
- 150000001875 compounds Chemical class 0.000 claims abstract description 29
- 239000003018 immunosuppressive agent Substances 0.000 claims abstract description 13
- 239000002260 anti-inflammatory agent Substances 0.000 claims abstract description 8
- 229940124599 anti-inflammatory drug Drugs 0.000 claims abstract description 7
- 229940124589 immunosuppressive drug Drugs 0.000 claims abstract description 6
- 125000003147 glycosyl group Chemical group 0.000 claims abstract description 4
- 229930182470 glycoside Natural products 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 abstract description 16
- 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 8
- 241001408435 Epigynum auritum Species 0.000 abstract description 4
- 241000196324 Embryophyta Species 0.000 abstract description 2
- 241001529246 Platymiscium Species 0.000 abstract description 2
- 150000002611 lead compounds Chemical class 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 23
- 230000000694 effects Effects 0.000 description 19
- 229960003957 dexamethasone Drugs 0.000 description 15
- 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 15
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
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- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
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- LOGFVTREOLYCPF-KXNHARMFSA-N (2s,3r)-2-[[(2r)-1-[(2s)-2,6-diaminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxybutanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-KXNHARMFSA-N 0.000 description 1
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 1
- 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
- JWMFYGXQPXQEEM-NUNROCCHSA-N 5β-pregnane Chemical compound C([C@H]1CC2)CCC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H](CC)[C@@]2(C)CC1 JWMFYGXQPXQEEM-NUNROCCHSA-N 0.000 description 1
- 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
- 244000205574 Acorus calamus Species 0.000 description 1
- 241000209134 Arundinaria Species 0.000 description 1
- 108700031361 Brachyury Proteins 0.000 description 1
- 235000011996 Calamus deerratus Nutrition 0.000 description 1
- 241001269238 Data Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241001408451 Epigynum Species 0.000 description 1
- 241001408647 Epigynum cochinchinense Species 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 102100028999 High mobility group protein HMGI-C Human genes 0.000 description 1
- 101000986379 Homo sapiens High mobility group protein HMGI-C Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
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- 230000001070 adhesive effect Effects 0.000 description 1
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- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 230000003409 anti-rejection Effects 0.000 description 1
- 238000004500 asepsis Methods 0.000 description 1
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- 230000010261 cell growth Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
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- 230000009514 concussion Effects 0.000 description 1
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- 230000009977 dual effect Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000002024 ethyl acetate extract Substances 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
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- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry 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
- 230000001939 inductive effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 239000006166 lysate Substances 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
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 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
- 150000002894 organic compounds Chemical class 0.000 description 1
- 210000003024 peritoneal macrophage Anatomy 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000006187 pill 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
- 238000012797 qualification Methods 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
- C07J17/005—Glycosides
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Transplantation (AREA)
Abstract
The invention discloses a kind of pregnane glucoside compound and its applications in preparing immunosuppressive drug and anti-inflammatory drug；The compound is to be extracted from Epigynum Auritum platymiscium isolated, and structural formula is shown below：Wherein, R1、R2It is selected from glycosyl；Experiments have shown that the compound reported in the present invention has excellent immunosupress and anti-inflammatory effect；The compounds of this invention provides lead compound to develop immunosuppressor and anti-inflammatory preparation, is conducive to develop and use plant medicine resource.
Description
Technical field
The present invention relates to a kind of pregnane glycoside compounds and its answering in preparing immunosuppressive drug, anti-inflammatory drug
With.
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 provided by the invention and
It is not yet reported as immunosuppressive drug and anti-inflammatory drug.
Invention content
A kind of the purpose of the present invention is to provide structural formulas pregnane glucoside compound as shown in formula I：
R1,R2It is selected from glycosyl.
Above-mentioned glycosyl refer to 2,3- methoxyl group -6- deoxidation-β-D- idoses and bis- deoxidation-β-D- pyrans of 2,6- it is Arabic oneself
Sugar；It is located at R1And R2Position.
Another object of the present invention is to provide structural formula pregnane glucoside compound as shown in formula II：
Formula II：- 20 α-O-2 of -3 β-O-2,3- methoxyl group -6- deoxidation-β-D- idoses of 17 Alpha-hydroxies-pregnant steroid -5 (6)-alkene,
Bis- deoxidation-β-D- pyrans arabino-hexos glycosides of 6-, popular name epigycoside B.
It is another object of the present invention to apply above-mentioned pregnane glycoside compounds in preparing immunosuppressive drug.
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.
The present invention is another object is that applying above-mentioned pregnane glucoside compound in preparing anti-inflammatory drug.
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
ConA or LPS are induced, while various concentration test compound is added in test group, with the dexamethasone of corresponding concentration
(Dexamethasone, abbreviation DXMS) is used as positive control, after cultivating 72h, passes through Cell Counting Kit-8 (CCK-8)
Reagent method measures test group, positive controls, induction control group and the light absorption value for not inducing control group respectively, to evaluate chemical combination
The ability of object regulatory T-cell or B cell proliferation；The experimental results showed that pregnane glucoside compound in the present invention
Epigycoside B have the ConA or LPS Balb/c mouse T splenic lymphocytes stimulated or B lymphocyte proliferation apparent
Inhibiting effect, 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 epigycoside B secrete the RAW264.7 cells that LPS is induced in the present invention
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 Epigycoside B
By Burma's Epigynum Auritum (Epigynum cochinchinensis) branches and leaves go-no-go, gained leaf 5kg is at dry and ventilated
After natural drying, it is broken into pieces using pulverizer；Burma's Simao cane leaves fragment is immersed in 75% methanol solution of 10L volumetric concentrations
Soakage extraction, 12 hours every time, continuous extraction was three times；It collects and merges leaching liquor, be concentrated under reduced pressure using Rotary Evaporators and remove liquid
Body obtains medicinal extract 500g；Medicinal extract adds 2L water dissolutions to be placed on separatory funnel, then isometric ethyl acetate is added to be extracted repeatedly；
It is almost colourless to be repeatedly extracted to ethyl acetate layer, collects after combined ethyl acetate layer is evaporated and obtains crude extract medicinal extract 120g；It uses
Crude extract is crossed MCI columns by middle pressure chromatographic fractionation system, and rinsing 5 column volumes through 90% methanol-water of volumetric concentration obtains without plant
The sample of the impurity such as pigment, 100% methanol rinse the impurity such as the pigment of gained and discard；It is final to obtain subsequently separable gross sample
60g。
Gross sample 60g, 100g C are obtained after ethyl acetate extracts and removes depigmentaton18After filler mixes sample, color is pressed in use
Spectrum is detached, and uses 20% methanol-water of volumetric concentration, 40% methanol-water, 60% methanol-water, 80% methanol-water and 100% first successively
Alcohol water gradient washes, each gradient washes 10L mobile phases.Each concentration flushing gained sample, which is kept separately, to be respectively labeled as
Fr.A(10g)、Fr.B(10g)、Fr.C(14g)、Fr.D(12g)、Fr.E(12g)。
Fr.D (12g) partly uses C18Middle compression leg is with methanol-water (40:60-80:20) every 10% transformation of scale carries out gradient
Elution, every part of eluent 300mL are collected, and obtain 5 parts such as FrD1, D2, D3, D4, D5.
The parts Fr.D1 (2.1g) carry out silica gel column chromatography separation, with chloroform：Acetone (10:1-2:1) system gradient rushes column,
It is collected respectively per 150mL eluents, finally retains 3 after TLC detection and analysis in conjunction with the collection liquid of TLC combining data detection same compositions
A part, Fr.D1-1, Fr.D1-2, Fr.D1-3；Wherein Fr.D1-1 carries out being purified by flash processing using methanol by gel column,
It is final to obtain monomeric compound epigycoside B (3.5mg)；
Identified pregnane glycoside compounds epigycoside B of the present invention are noval chemical compound；Qualification result is as follows：
II compound epigycoside B of formula are white indefinite form powder, are soluble in chloroform, methanol equal solvent.[α](c 0.1,CH3OH)；UV(CH3OH)λmax(logε):203nm(3.5)；IR(KBr)vmax:3450,2922,
2853,1638,1535,1401,1384,1202,1034cm–1；(+)HR-ESI-MS m/z661.3920[M+Na]+
(calcd.for C35H58O10Na+,661.3922).
1H NMR(methanol-d4, 600Mz) and13C NMR(methanol-d4, 150Mz) and it is shown in Table 1；Data above combines
2D NMR analyses confirm that the chemical structural formula of the compound is shown in formula II, are a new natural organic-compound, life
Entitled epigycoside B.
Table 1：Epigycoside B1H NMR and13C NMR datas
aov:overlap。
Embodiment 2：Immunosupress detection experiment
(1) preparation of splenic lymphocytes suspension
Neck is taken off after taking the healthy BABL/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 2：Balb/c mice spleens T lymphopoiesis stimulus indexes under II compound intervention of formula
Compound epigycoside B are when activity is 12.5,25,50 μM in formula II, test group stimulus index point
It Wei 2.82,2.06,1.56；Significance analysis shows each concentration function and effect and the poor opposite sex of negative control group all extremely
Significantly (P<0.01).
Positive control dexamethasone (DXM) at a concentration of 12.5,25,50 μM, test group stimulus index is respectively 1.43,
1.24、1.14；Each concentration group function and effect and negative control group more all have and its significant difference (P<0.01).
Inhibit T lymphs thin the experimental results showed that heretofore described compound epigycoside B have at 12.5 μM
The activity of born of the same parents' proliferation increases the enhancing of its inhibition within the scope of 50 μM with concentration.
Table 3：The stimulus index of Balb/c mice spleen B lymphocyte proliferations under II compound intervention of formula
Epigycoside B when activity is 12.5,25,50 μM, test group stimulus index is respectively 6.02,
5.19、2.80；Significance analysis shows under 12.5 μM of concentration function and effect compared with negative control group with significant difference
(P<0.05), increase with concentration, stimulus index reduces, and enhances B lymphocyte proliferation inhibiting effect.Concentration is in 25 μM and 50 μM
When compared with negative control group difference extremely significantly (P<0.01).
At a concentration of 12.5,25,50 μM, test group stimulus index is respectively positive control dexamethasone (DXMS)
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 epigycoside B 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 epigycoside B 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
Epigycoside B have outstanding immunosuppressive activity, can be used for the preparation of immunosuppressive agents.
Embodiment 4：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/m L at this time, label
For 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 4：Influences of the compound epigcosideB to RAW264.7 cellular inflammations factor expression level
Positive control medicine dexamethasone is at the activity for 10 μ g/mL, inflammation RAW264.7 cells TNF-α, IL-
The expression of 1 β, PGE2 is respectively 14.29ng/L, 4.61ng/L, 11.32ng/L.
Compound epigycoside B 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 17.10ng/L, 16.77ng/L, 16.27ng/L, the table with negative control group 19.75ng/L
It is compared up to amount, all there is significant difference (P<0.05).
Compound epigycoside B 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 5.27ng/L, 4.77ng/L, 3.88ng/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, epigycoside B are in 1.0 μ g/mL concentration
Under, the expression of IL-1 β is suitable with after positive control effect after effect, and IL-1 β express water under 5.0 μ g/mL activities
It is flat then already below positive control exercising result.
Compound epigycoside B are when activity is 0.4 μ g/mL, 1.0 μ g/mL, 5.0 μ g/mL, test group
The expression contents of PGE2 are respectively 14.37ng/L, 13.69ng/L, 12.11ng/L, the expression with negative control group 16.72ng/L
Amount is compared, and all has significant difference (P<0.05).
The experimental results showed that the compound epigycoside B as shown in Formula II are in 0.4 μ g/mL-5.0 μ g/mL in the present invention
Activity under have and adjust to the expression of the TNF-α of inflammation RAW264.7 cells of LPS inductions, IL-1 β, the PGE2 factors
Section acts on, and can inhibit its expression, weakens inflammatory reaction, and under the activity of 5.0 μ g/mL, and effect is with positive drug
Sai meter Song is suitable.Heretofore described compound epigycoside B have significant extracorporeal anti-inflammatory activity, can be used for anti-inflammatory
The preparation of drug.
Claims (4)
1. the pregnane glucoside compound that structural formula is shown below：
Wherein, R1、R2It is selected from glycosyl.
2. pregnane glucoside compound according to claim 1, which is characterized in that structural formula of compound such as II institute of formula
Show：
3. application of the pregnane glycoside compounds as claimed in claim 1 or 2 in preparing immunosuppressive drug.
4. application of the pregnane glycoside compounds as claimed in claim 1 or 2 in preparing anti-inflammatory drug.
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