CN107056868A - Epigynum Auritum pregnane glucoside compound and its application - Google Patents
Epigynum Auritum pregnane glucoside compound and its application Download PDFInfo
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- CN107056868A CN107056868A CN201710180951.5A CN201710180951A CN107056868A CN 107056868 A CN107056868 A CN 107056868A CN 201710180951 A CN201710180951 A CN 201710180951A CN 107056868 A CN107056868 A CN 107056868A
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- -1 pregnane glucoside compound Chemical class 0.000 title claims abstract description 17
- 229930182478 glucoside Natural products 0.000 title claims abstract description 13
- 239000003814 drug Substances 0.000 claims abstract description 18
- 239000003018 immunosuppressive agent Substances 0.000 claims abstract description 16
- 229940124589 immunosuppressive drug Drugs 0.000 claims abstract description 8
- 229930182470 glycoside Natural products 0.000 claims description 32
- 208000023275 Autoimmune disease Diseases 0.000 claims description 6
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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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a class Epigynum Auritum pregnane glucoside compound and its application in immunosuppressive drug is prepared, experimental result is shown, such compound has the activity of very strong suppression mice spleen lymphocytes proliferation；The present invention provides lead compound to develop new immunodepressant, is conducive to developing plant medicine resource.
Description
Technical field
The present invention relates to a class Epigynum Auritum pregnane glucoside compound and its application in immunosuppressive drug is prepared,
Application particularly in preparing treating organs to transplant the immune drug of anti-rejection and autoimmune disease.
Background technology
In recent years, stepping up with industrialization degree, autoimmune disease, hypersensitivity disease etc. are by being immunized
Disease caused by overreact is also being continuously increased, and find the immunodepressant of high-efficiency low-toxicity has turned into when previous research heat
Point
Immune drug is the new medicine of the class grown up after 1970s.Immunization therapy is by adjusting machine
Body immunity function, corrects immunoreaction abnormity, reaches purpose of curing the disease.The medicine that majority acts on immune system has positive and negative two side
The effect in face, i.e. Immune-enhancing effect and immunosupress.Immunodepressant
(immunosuppressant) provided effectively for the rejection after autoimmunity disease and organ transplant in clinic
Medicine, widely paid attention to.
Immune response is originally body self-protection, a kind of defense reaction of homeostasis, suppresses this reaction for a long time
Some serious consequences such as infection and induced tumor can be brought, this is also some problems that current immunodepressant is run into.Exempt from
Epidemic disease inhibitor provides effective medicine in clinic for the rejection after autoimmunity disease and organ transplant, is commonly used to
Suppress the graft versus host disease(GVH disease) occurred after the rejection occurred after organ transplant, treatment bone-marrow transplantation, or treatment rheumatoid
The autoimmune diseases such as property arthritis, disease, typically can carry out immunosupress by medicine.Autoimmune disease is big
Mostly chronic or progressive disease needs long-term prescription, and existing glucocorticoid (such as dexamethasone) and immunodepressant are long
Phase medication generally existing toxic side effect is big, in-convenience in use the shortcomings of, such as dexamethasone glucocorticoid medicine, chloramphenicol
Class medicine, tetracycline medication and sulfa drugs etc. can often cause immunity of organism to suppress.Therefore, it is high in the urgent need to developing a class
Imitate the neotype immunosuppressant of low toxicity, compared to chemotherapy, Chinese medicine as a kind of natural products, with people's histocompatbility
Matter is good, Small side effects the advantages of, be increasingly valued by people in the research of immunodepressant.
Epigynum Auritum (Epigynum auritum) is a kind of Apocynaceae Epigynum Auritum race Simao Calamus, originates in Yun Nannan
Portion, according to the result of study of our early stages, contains the novel androstane of more structure, C in Epigynum Auritum21Steroidal and its glycoside chemical combination
Thing.In the case where finding the interest drive of new drug lead compound, we have carried out substantial amounts of bioactivity sieve to these noval chemical compounds
Choosing experiment, pregnane glucoside compound and its have not yet to see report as immunosuppressive drug that the present invention is provided.
The content of the invention
It is an object of the invention to provide a class Epigynum Auritum pregnane glucoside compound, its structural formula for example formula I, formula II,
Shown in formula III：
Formula I：(3β,17α,20S)pregn-5-ene-3,17,20-triol 2,3-dimethoxy-6-deoxy-β-D-
idopyranose；Epigynoside E, Chinese entitled 3 β, 17 α, 20 α-pregnane -5 (6)-alkene -3,17,20- triols 3-O-
2 ', 3 '-dimethoxy -6 '-deoxidation-β-D- idose glycosides, popular name is Epigynum Auritum glycosides penta.
Formula II：(3β,17α,20S)pregn-5-ene-3,15,17,20-tetraol 2,3-dimethoxy-6-
deoxy-β-D-idopyranose；Epigynoside F, Chinese entitled 3 β, 17 α, 20 α-pregnane -5 (6)-alkene -3,15,
17,20- tetrol 3-O-2 '-methoxyl group -6 '-deoxidation-β-D- idose glycosides, popular name be Epigynum Auritum glycosides oneself.
(3 β, 17 α, 20S) the pregn-5-ene-3,17,20-triol 2-methoxy-6-deoxy- β of formula III-D-
idopyranose；Epigynoside G, Chinese entitled 3 β, 17 α, 20 α-pregnane -5 (6)-alkene -3,17,20- triols 3-O-
2,6-2 ', 3 '-dimethoxy -6 '-deoxidation-β-D- idose glycosides, popular name are Epigynum Auritum glycosides heptan.
The preparation process of three pregnane glycoside compounds is specific as follows in the present invention：Take Epigynum Auritum plant sample leaf portion point
Dry powder 4.5kg, is extracted 3 times, each 48h with 75% alcohol reflux, is filtered to remove the filtrate for merging three extractions after filter residue, decompression
It is extracted with ethyl acetate again after concentration 3 times, concentrates ethyl acetate extract layer；By ethyl acetate layer extract macroporous absorbent resin
D101 carries out rough segmentation, is carried out eluting always respectively with the methanol aqueous solution of percent by volume 20%, 40%, 60%, 80%, 100%
5 parts (Fr.A, Fr.B, Fr.C, Fr.D, Fr.E) are obtained.Fr.B is by preparing liquid phase with percent by volume 30%-70%
Methanol aqueous solution carry out gradient elution obtain six part Fr.B1-B6.Fr.B2 therein parts are passed through into 200-300 mesh again
Silicagel column, with chloroform-acetone (volume ratio 10:1-3:1) eluted for eluent, four part Fr.B2a- are always obtained
Fr.B2d.By Fr.B2b parts, by gel column Sephadex LH-20, (mobile phase is chloroform：Methanol=1:1) formula is prepared
No. 2 compounds in II.By gel column Sephadex LH-20, (mobile phase is methanol for Fr.D parts：Water=1:1) separate
To four part Fr.D1-D4.Fr.D2 parts first pass through middle pressure C18 post (methanol-water, 40:60-80:20) gradient elution, then
Efficient liquid phase (acetonitrile-water, 50 are prepared by half:50) purifying obtains No. 1 compound in formula I.Fr.D3 parts are with chloroform-the third
Ketone (4:1) eluted for eluent by silicagel column, then by gel column Sephadex LH-20, (mobile phase is chlorine again
It is imitative：Methanol=1:1) elute, prepare No. 3 compounds in formula III.Identified, three compounds in formula I are newization
Compound.
It is another object of the present invention to apply above-mentioned Epigynum Auritum pregnane glucoside compound to prepare immunosuppressive drug
In thing.
Specifically immunosuppressive drug is applied in the medicine for preparing autoimmune disease, or is applied in preparing device
Official is transplanted in the medicine of anti-rejection.
One or more pharmaceutically acceptable auxiliary materials can also be added in application of the present invention, the auxiliary material includes medicine
Field conventional filler, diluent, adhesive, excipient, sorbefacient, filler, surfactant and stabilizer
Deng can also add flavouring agent, pigment and sweetener etc. if necessary.
Pill, pulvis, tablet, granula, oral liquid and injection can also be made in addition to capsule is made in application of the present invention
The diversified forms such as liquid.
Preparing splenocyte in the present invention with the Balb/c mouse of health is used to test.After the completion of prepared by splenocyte, pass through
ConA or LPS are induced, while after test group adds various concentrations test compound, culture 72h, being tried by CCK-8
Agent method distinguishes determination test group, induction control group and does not induce the light absorption value of control group, thus evaluate compound stimulate T cell or
The ability of person's B cell proliferation.Test result indicates that three pregnane glucoside compounds being related in the present invention, Epigynum Auritum glycosides
Pentath, the Balb/c mice spleens that oneself, Epigynum Auritum glycosides heptan (epigynoside E-G) of Epigynum Auritum glycosides are stimulated ConA (ConA)
Lymphopoiesis has obvious inhibitory action, there is significant difference (p compared with the control group for being not added with the compound<0.05).
Embodiment
The present invention is described in further detail below by embodiment, but present disclosure is not limited thereto, this
Method operating according to a conventional method unless otherwise specified, the use conventional commercial of agents useful for same unless otherwise specified in embodiment
Reagent or the reagent configured according to a conventional method.
Embodiment 1：The preparation of pregnane glucoside compound
Epigynum Auritum leaf portion point dry powder 4.5kg is taken, is extracted 3 times with 75% alcohol reflux, each 48h is filtered to remove after filter residue
Merge the filtrate of three extractions, be extracted with ethyl acetate again after being concentrated under reduced pressure 3 times, concentration ethyl acetate extract layer is weighed to obtain 57g；
Ethyl acetate layer extract is subjected to rough segmentation with macroporous absorbent resin D101, respectively with percent by volume 20%, 40%, 60%,
80%th, 100% methanol-water elute that 5 parts (Fr.A-E) are always obtained.Fr.B (4.6g) is by preparing liquid phase with first
Alcohol water 30%-70% carries out gradient elution and obtains six part Fr.B1-B6.Fr.B2 therein (2.5g) is passed through into 200- again
The silicagel column of 300 mesh, with chloroform-acetone (10:1-3:1) eluted for eluent, four part Fr.B2a- are always obtained
Fr.B2d.By Fr.B2b (298mg), by gel column Sephadex LH-20, (mobile phase is chloroform：Methanol=1:1) it is prepared into
To the Epigynum Auritum glycosides in formula II oneself (5.5mg).By gel column Sephadex LH-20, (mobile phase is methanol to Fr.D (6.4g)：
Water=1:1) isolated four part Fr.D1-D4.Fr.D2 first passes through middle pressure C18 post (methanol-water, 40:60-80:20) it is terraced
Degree elution, then prepares efficient liquid phase (acetonitrile-water, 50 by half:50) purifying obtains the Epigynum Auritum glycosides penta (23mg) in formula I.
Fr.D3 is with chloroform-acetone (4:1) eluted for eluent by silicagel column, then pass through gel column Sephadex LH- again
20 (mobile phase is chloroform：Methanol=1:1) elute, prepare Epigynum Auritum glycosides heptan (5.7mg) in formula III.It is identified, in formula I
Three compounds be noval chemical compound.
Qualification result is as follows：
Epigynum Auritum glycosides penta (epigynoside E) is transparent crystal, is dissolved in chloroform methanol mixed liquor (chloroform：Methanol=1:
1), pyridine etc..-98.5(c 0.04,MeOH)；UV(MeOH)λmax(logε):203nm(3.5)；IR(KBr)νmax 3473
2902,2831,1733,1717,1458,1375,1329,1047,962cm–1；HR-ESI-MS m/z 531.3298[M+Na]+
(calcd.for C29H48O7Na+,531.3298).1HNMR(CDCl3, 500Mz) and13C NMR(CDCl3, 125Mz) and it is shown in Table 1；With
Upper data combination 2D NMR analyses confirm the chemical structural formula of Epigynum Auritum glycosides penta (epigynoside E) for shown in formula I.
Epigynum Auritum glycosides oneself (epigynoside F) is white powder, is dissolved in chloroform methanol mixed liquor (chloroform：Methanol=1:
1), pyridine etc..-205.9(c 0.01,MeOH)；UV(MeOH)λmax(logε):203nm(3.8)；IR(KBr)νmax
3429,2933,1632,1517,1379,1179,1097,1036cm–1；positive-ion HR-ESI-MS m/z
547.3245[M+Na]+(calcd.for C29H48O8Na+,547.3247)1H NMR(CDCl3, 500Mz) and13C NMR
(CDCl3, 125Mz) and it is shown in Table 1；Data above combination 2DNMR analyzes the chemistry for confirming Epigynum Auritum glycosides oneself (epigynoside F)
Structural formula is shown in formula II.
Epigynum Auritum glycosides heptan (epigynoside G) is transparent crystal, is dissolved in chloroform methanol mixed liquor (chloroform：Methanol=1:
1), pyridine etc..-177.7(c 0.05,MeOH)；UV(MeOH)λmax(logε):203nm(3.6)；IR(KBr)νmax
3419,2931,1713,1633,1457,1376,1255,1104,1041,884cm–1；positive-ion HR-ESI-MS m/
z 517.3143[M+Na]+(calcd.for C28H46O7Na+,517.3141)1H NMR(methanol-d4, 600Mz) and13C
NMR(methanol-d4, 150Mz) and it is shown in Table 1；Data above combination 2D NMR analysis confirm Epigynum Auritum glycosides oneself
The chemical structural formula of (epigynoside F) is shown in formula III.
Three pregnane glucoside compounds described above are new natural organic-compound.
Table 1：Pregnane glucoside compound1H NMR and13C NMR datas
Embodiment 2：Immunosupress detection experiment
(1) preparation of splenic lymphocytes suspension
18~22g healthy BABL/c mouse sacrificed by exsanguination is taken, soaking disinfection 5 minutes in 75% alcohol are placed in, takes out, puts
In sterile tray, left side upward, in super-clean bench, picks up fur in the middle part of abdomen with the tweezers sterilized, makees a kerf, with addition
A set of apparatus cuts off each layer of stomach wall, is taken out spleen with the 3rd set of apparatus, removes fat and connective tissue, and being put into PBS, (phosphate delays
Fliud flushing) in, wash away floating blood.Then spleen tissue is moved in the plate for filling the endless full nutrient solutions of RPMI 1640, be cut into scissors
Fritter, is ground spleen in 200 mesh stainless steel mesh with asepsis injector core, is repeatedly rinsed on a small quantity with PBS, by suspension liquid relief
Device is transferred in 15mL centrifuge tubes.1000r/min rotating speeds centrifuge 5min.Supernatant is abandoned in suction, adds 3mL erythrocyte cracked liquids
(Tris-NH4Cl) mix, stand and 10mL PBS terminating reactions are added after 2min, centrifuge (1200rpm, 5min), remove supernatant, sink
Shallow lake is washed twice with 5mL PBS, is centrifuged under similarity condition.Precipitate and cultivated completely with RPMIs 1640 of the 5mL containing 10% hyclone
Liquid suspends；Expect that blue living cells is refused dye method and counted with 0.8%, viable count is no less than 95%, plus the complete culture solutions of RPMI 1640
Dilution, adjustment cell concentration to 1 × 106Individual/mL or so.
(2) preparation of test liquid
Precision weighs monomeric compound 2mg, adds and is diluted to required concentration before DMSO dissolvings, loading with PBS, and causes
DMSO final concentrations after sample are no more than 0.1%.
(3) experiment packet
Normal group：The μ LPBS of+10 1640 complete mediums of μ L RPMI of 100 μ L splenocyte suspensions+10
Model group：The μ LConA of 100 μ L splenocyte suspensions+10 (final concentration of 10 μ g/mL)+10 μ LPBS
Sample sets：The μ LConA of 100 μ L splenocyte suspensions+10 (final concentration of 10 μ g/mL)+10 μ L samples
In 96 orifice plates, lymphocyte suspension (1 × 10 is added per hole6Individual/mL) 100 μ L, ConA10 μ L (final concentration of 10 μ
G/mL), the μ L of various concentrations reagent chemical compound diluted liquid 10 (final concentration is respectively 12.5,25,50 μ g/mL), dexamethasone is also done
Three corresponding concentration groups, Normal group hole is respectively with 10 μ L 1640 complete culture solutions (containing 10% hyclone) and 10 μ L
PBS polishings, each concentration group set 4 it is parallel.
(4) cultivate：It is placed in 37 DEG C, 5%CO2Culture 72 hours in incubator.
(5) CCK-8 methods determine cell OD values
After culture 72 hours, 10 μ L CCK-8 reagents (the green skies) are added in every hole, 37 DEG C, 5%CO are placed in2Incubator
It is interior to continue after cultivating 4 hours, the light absorption value per hole is determined 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 represent that mathematical statistics and variance analysis work are used using " average ± standard deviation "
Origin softwares are completed.
(7) experimental result
Table 2：Stimulus index of the Epigynum Auritum glycosides penta to the ConA Balb/c mice spleen lymphocytes proliferations stimulated
In formula I Epigynum Auritum glycosides penta (epigynoside E) concentration be 12.5,25,50 μM when stimulus index
Respectively 2.56,1.96,1.69；Significance analysis shows that middle concentration and high concentration group has significant difference compared with model group
(P<0.05)。
Table 3：The stimulus index of oneself the Balb/c mice spleen lymphocytes proliferations to ConA stimulations of Epigynum Auritum glycosides
In formula II Epigynum Auritum glycosides oneself (epigynoside F) concentration be 12.5,25,50 μM when stimulus index
Respectively 3.37,2.97,1.92；Significance analysis shows that high concentration group has significant difference (P compared with model group<0.05).
Table 4：The stimulus index for the Balb/c mice spleen lymphocytes proliferations that Epigynum Auritum glycosides heptan is stimulated ConA
In formula III Epigynum Auritum glycosides heptan (epigynoside G) concentration be 12.5,25,50 μM when stimulus index
Respectively 3.23,3.01,1.94；Significance analysis shows that high concentration group has significant difference (P compared with model group<0.05).
Positive control result is：
Table 5：The stimulus index for the Balb/c mice spleen lymphocytes proliferations that dexamethasone is stimulated ConA
Dexamethasone concentration be 12.5,25,50 μM when stimulus index be respectively 1.62,1.41,1.21；Conspicuousness point
Analysis shows that each concentration group has significant difference (P compared with model group<0.05).
Test result indicates that, Epigynum Auritum glycosides penta (epigynoside E) has immunosuppressive activity when concentration is 25 μM,
Epigynum Auritum glycosides oneself (epigynosides F), Epigynum Auritum glycosides heptan (epigynosides G) there is immune suppression when concentration is 50 μM
System activity.
Claims (4)
1. Epigynum Auritum pregnane glucoside compound of the structural formula as shown in formula I, formula II, formula III：
Formula I：3 β, 17 α, 20 α-pregnane -5 (6)-alkene -3,17,20- triol 3-O-2 ', 3 '-dimethoxy -6 '-deoxidation-β-D-
Idose glycosides；
Formula II：3 β, 17 α, 20 α-pregnane -5 (6)-alkene -3,15,17,20- tetrols 3-O-2 '-methoxyl group -6 '-deoxidation-β-D-
Idose glycosides；
Formula III：3 β, 17 α, 20 α-pregnane -5 (6)-alkene -3,17,20- triol 3-O-2,6-2 ', 3 '-dimethoxy -6 '-are gone
Oxygen-β-D- idose glycosides.
2. application of the Epigynum Auritum pregnane glucoside compound in immunosuppressive drug is prepared described in claim 1.
3. application according to claim 2, it is characterised in that：Immunosuppressive drug is the curative of autoimmune disease
Thing.
4. application according to claim 2, it is characterised in that：Immunosuppressive drug is controlled for the anti-rejection of organ transplant
Treat medicine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108403706A (en) * | 2018-04-26 | 2018-08-17 | 昆明理工大学 | A kind of new application of pregnane glucoside compound |
| CN108558978A (en) * | 2018-04-26 | 2018-09-21 | 昆明理工大学 | Pregnane glucoside compound and its application |
| CN108558975A (en) * | 2018-04-26 | 2018-09-21 | 昆明理工大学 | 12 beta-hydroxies-androstane-14,14- diene -16- ketone compounds and its application |
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| CN103073607A (en) * | 2013-02-26 | 2013-05-01 | 昆明理工大学 | 12[beta]-hydroxyandrostane-4,6,8(9),13(14)-tetraene-3,11,16-triketone and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108403706A (en) * | 2018-04-26 | 2018-08-17 | 昆明理工大学 | A kind of new application of pregnane glucoside compound |
| CN108558978A (en) * | 2018-04-26 | 2018-09-21 | 昆明理工大学 | Pregnane glucoside compound and its application |
| CN108558975A (en) * | 2018-04-26 | 2018-09-21 | 昆明理工大学 | 12 beta-hydroxies-androstane-14,14- diene -16- ketone compounds and its application |
| CN108558978B (en) * | 2018-04-26 | 2020-11-17 | 昆明理工大学 | Pregnane glycoside compounds and application thereof |
| CN108558975B (en) * | 2018-04-26 | 2021-03-02 | 昆明理工大学 | 12 beta-hydroxy-androstane 4, 14-diene-16-ketone compound and application thereof |
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