CN108558975B - 12 beta-hydroxy-androstane 4, 14-diene-16-ketone compound and application thereof - Google Patents
12 beta-hydroxy-androstane 4, 14-diene-16-ketone compound and application thereof Download PDFInfo
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Abstract
The invention discloses a 12 beta-hydroxy-androstane 4, 14-diene-16-ketone compound or pharmaceutically acceptable salt thereof, and application thereof in preparing immunosuppressive drugs and antitumor drugs; the compound is extracted and separated from the plant of the genus Sinomenium, and the structural formula of the compound is shown as the following formula:
wherein R is selected from hydrogen, methoxy, ethoxy, halogen, aliphatic alkyl and aliphatic amino; in the formula
Description
Technical Field
The invention relates to a 12 beta-hydroxy-androstane 4, 14-diene-16-ketone compound or a medicinal salt thereof and application thereof in preparing immunosuppressive and antitumor medicaments.
Background
The immune response is a defense reaction of self-protection and self-stabilization of the body, and long-term inhibition of the reaction can bring serious consequences such as infection, tumor induction and the like, which are also some problems encountered by the prior immunosuppressant. Immunosuppressive agents are generally used for immunosuppression through drugs, and are generally used for suppressing rejection after organ transplantation, treating graft-versus-host disease after bone marrow transplantation, or treating autoimmune diseases such as rheumatoid arthritis, crohn's disease and the like.
At present, more than 100 malignant tumors exist, the number of cancer deaths accounts for about 13% of the number of disease deaths worldwide, and the number of cancer deaths worldwide is expected to continue to increase; the occurrence and development of tumors are closely related to the decline of the immune function of the whole body, and once the tumors occur, the inhibition of the immune function of the body is deepened, thereby promoting the development of the tumors. The metastasis of tumor cells from the primary site to other sites continues to grow, which is a significant life-threatening cause of tumors. Among a plurality of factors of tumor pathogenesis, various types of immunosuppressive molecules play a key role in the formation and development process of tumors, can be used as a new target of antitumor drugs, and provide a new idea for the biological treatment of tumors.
The autoimmune diseases and cancers need to be taken for a long time, and the existing long-term administration of glucocorticoids (such as dexamethasone) and immunosuppressants generally has the defects of large toxic and side effects, inconvenient use and the like. As a natural product, the traditional Chinese medicine has the advantages of good compatibility with human tissues, small side effect and the like, and is increasingly paid more attention to the research of immunosuppressants and antitumor preparations. Therefore, the compound with the functions of immunosuppression and malignant tumor cell proliferation inhibition is searched from natural products, and the application value of the compound for developing novel immunosuppressive agents and antitumor preparations with high efficiency and low toxicity is achieved.
Sedum (Epigynum) is rich in androstane, pregnane and their glycosides. Under the drive of the interest of finding new medicine lead compounds, early stage biological activity screening finds that the polyene androsterone compound has obvious immunosuppressive activity; the polyene androsterone compound and the application thereof as an immunosuppressive drug have not been reported.
Disclosure of Invention
The invention extracts and separates a 12 beta-hydroxy-androstane 4, 14-diene-16-ketone compound or pharmaceutically acceptable salt thereof from a plant of the genus Sinomenium, and the chemical structural formula is shown as the formula I:
formula I;
wherein R is selected from hydrogen, methoxy, ethoxy, halogen, aliphatic alkyl and aliphatic amino;
in the formula
Represents a single bond or a double bond.
The aliphatic substituent in the above aliphatic hydrocarbon group and aliphatic amino group means a saturated or unsaturated aliphatic substituent, wherein the saturated aliphatic substituent means a straight-chain or branched alkyl group, a cycloalkyl group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, sec-butyl, cyclopropylcyclododecyl and the like, and the unsaturated aliphatic group means an alkenyl group such as allyl, isopentenyl, 1-dodecenyl, alkynyl group such as 1-octadecynyl, 2-octadecynyl, alkadienyl group such as geranyl, 1, 3-octadecenyl, 9-octadecenyl and the like, or an alkadienyl group.
The 12 beta-hydroxy-androstane 4, 14-diene-16-ketone compound is selected from one of the compounds shown in formula II, formula III and formula IV:
the formula II is 3 beta, 12 beta-dihydroxy androstane 4, 14-diene-16 ketone, the formula III is 3 beta-ethoxy-12 beta-hydroxy-androstane 4, 14-diene-16 ketone, and the formula IV is 3 beta-methoxy-12 beta-hydroxy-androstane 4, 14-diene-16 ketone.
The pharmaceutically acceptable salts of the present invention are salts with organic acids including but not limited to tartaric acid, citric acid, formic acid, acetic acid, oxalic acid, butyric acid, maleic acid, succinic acid, or salts with inorganic acids including but not limited to hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid.
The compound is prepared by solvent extraction, separation and purification from a plant raw material of the genus Sinomenium; if necessary salified with an appropriate acid.
The solvent extraction is preferably organic solvent reflux extraction, ultrasonic-assisted extraction or extraction, and the organic solvent is selected from aqueous solutions of methanol, ethanol, acetone and the like in different proportions.
The invention also aims to apply the 3, 16-androstenedione compound or the pharmaceutically acceptable salt thereof in preparing immunosuppressive drugs and antitumor drugs.
The specific immunosuppressive drug is applied to the preparation of a therapeutic drug for autoimmune diseases, or an anti-rejection therapeutic drug for organ transplantation, or an anti-tumor drug.
The medicine can also be added with one or more pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials comprise conventional filling agents, diluents, adhesives, excipients, absorption promoters, filling agents, surfactants, stabilizers and the like in the field of pharmacy, and flavoring agents, pigments, sweeteners and the like can also be added when necessary.
The medicine can be prepared into various forms such as pills, powder, tablets, granules, oral liquid, injection and the like besides capsules.
In the invention, healthy Balb/c mice are used for preparing splenocytes for experiment; after the preparation of splenocytes is finished, inducing by ConA or LPS, simultaneously adding test compounds with different concentrations into a test group, culturing for 72h, and respectively measuring the light absorption values of the test group, an induced control group and a non-induced control group by a CCK-8 reagent method, thereby evaluating the capacity of the compounds for stimulating the proliferation of T cells or B cells; the experimental result shows that the three androstadienone compounds related in the invention, namely the cistus androstadienone A, B, C, have obvious inhibition effect on the proliferation of Balb/c mouse spleen lymphocytes stimulated by ConA (concanavalin), and have significant difference (p is less than 0.05) compared with a control group without the compound.
Inoculating cell suspension with certain concentration adjusted to human tumor cells in logarithmic growth phase to 96-well culture plate at 90 μ l/well, culturing for 24 hr, adding compounds with different concentrations at 10 μ l/well after adherence3 multiple holes are arranged in the depth. The cells were counted and the viability of the cells, i.e. the number of unstained cells/total number of cells x 100%, determined by the disk blue staining method, was > 95% for the test, the cell suspension was adjusted to 1 x 106one/mL. Setting cell blank control group, positive control group (dexamethasone) and sample group (concentration is 25 μmol. L according to the result of preliminary test)-1All containing equal concentrations of DMSO). The negative control is equal volume of culture medium, and the corresponding DMSO concentration at 25. mu.g/ml of compound is used as vehicle control to eliminate the influence of DMSO on cell growth. Adding the medicine, continuously placing at 37 ℃ and 5% CO2Culturing in incubator, adding 20 μ L MTT (5mg/ml) per well after 1, 3, 6, 12, 18, and 24 hr of drug action, culturing for 4 hr, adding triple solution [ 10% SDS-5% isobutanol-0.012 mol/L HCl ] per well]Formazan is dissolved in 100 mu l. The inhibition rate of cell proliferation was estimated by measuring the OD value of each well at a wavelength of 570nm using a microplate reader.
The invention obtains 12 beta-hydroxy-androstane 4, 14-diene-16-ketone compounds from Sinomenium plants for the first time, experiments show that the compounds have significant immunosuppressive activity at the concentration of 25 mu M and 50 mu M, and have better inhibitory action on 24 human tumor cell lines, and in addition, the three new compounds have inhibitory action on other 15 tumor cells (SK-OV3, MDA _ MB-468, Hop92, EKVX, U0-31, OVCAR8, UACC-62, MALME-3M, CCRF-CEM, SK-MEL-2, SNB-19, U251, HT-29, T-47D, NIC-H322M) and have equivalent positive control activity; the three compounds provide lead compounds for developing immunosuppressant and antitumor preparation, and are favorable for developing and utilizing plant medicinal resources.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but the present invention is not limited thereto, and the method in the examples is carried out in a conventional manner unless otherwise specified, and reagents used therein are, for example, conventional commercially available reagents or reagents prepared in a conventional manner without specifically specified.
Example 1: preparation of 12 beta-hydroxy-androstane 4, 14-diene-16-ones
Taking 6kg of dry powder of Szechwan sturgeon, and concentrating by volumeExtracting with 75% ethanol under reflux for 3 times, each for 48 hr, filtering to remove residue, mixing the filtrates, concentrating under reduced pressure, extracting with ethyl acetate for 3 times, and weighing the concentrated ethyl acetate extraction layer to obtain 72 g; roughly separating ethyl acetate layer extract with macroporous adsorbent resin D101, and eluting with 20%, 40%, 60%, 80%, and 100% methanol water solution respectively to obtain 5 fractions (Fr.A, Fr.B, Fr.C, Fr.D, Fr.E); the Fr.D part is subjected to gradient elution by preparing a liquid phase and using a methanol water solution with the volume concentration of 60-100% to obtain five parts Fr.D 1-D5; passing Fr.D2 through 40-60 mesh silica gel column, eluting with chloroform-acetone (volume ratio of 5:1-1:1) to obtain total two parts Fr.D2a-Fr.D2 b; d2b is separated and purified by high performance liquid chromatography HPLC (mobile phase is acetonitrile: water: 3:2) to obtain compound ii; then, enabling Fr.D3 to pass through an MCI gel column, and carrying out gradient elution by taking a methanol-water two-phase system as a mobile phase to obtain two parts Fr.D3a-Fr.D3b, enabling the Fr.D3a part to pass through the gel column, and finally passing through a 100-mesh 200-mesh silica gel column, and eluting by taking chloroform-acetone (the volume ratio is 8:1-1:1) as an eluent to obtain a compound III; part of Fr.D3b warp C18Passing through a 100-mesh 200-mesh silica gel column, eluting with chloroform-acetone (volume ratio of 10:1-1:1) as eluent, and separating and purifying by High Performance Liquid Chromatography (HPLC) (mobile phase is acetonitrile: water: 3:2) to obtain compound IV; three compounds in the formulas II, III and IV are all new compounds through identification.
The identification results are as follows:
the cissampelos andradienone A is white powder, and is dissolved in chloroform-methanol mixture (chloroform: methanol: 1), pyridine, etc.
(c 0.1,MeOH)；UV(MeOH)λmax(logε):234(3.8),202(3.4)nm；IR(KBr)νmax3423,2928,1661,1608,1403,1254,1036,855cm–1；HRESIMS m/z 303.1958[M+H]+(calcd.for C19H27O3,303.1960).1H (600MHz) and13C NMR(150MHz)(CDCl3) NMR is shown in Table 1; the above data are combined with 2D NMR analysis evidenceThe cissampelos hance androstadienone A is 3 beta, 12 beta-dihydroxy-androstane 4, 14-diene-16-ketone, and the chemical structural formula is shown in a formula II.
The cissampelos androstadienone B is white powder, and is dissolved in chloroform-methanol mixture (chloroform: methanol: 1), pyridine, etc.
110.5(c 0.1,C5H5N)；UV(MeOH)λmax(logε):234(3.1),202(2.9)；IR(KBr)vmax3416,2936,1680,1609,1443,1410,1373,1234,1180,1078,1040,885cm-1；HR-ESI-MS m/z 331.2270[M+H]+(calcd.for C21H31O3,331.2273)1-H NMR(CDCl3500Mz) and13C NMR(CDCl3125Mz) is shown in Table 1; the data are combined with 2D NMR analysis to confirm that the chemical structural formula of the cissampelos arundinacea androstadienone B is shown as a formula III in the specification, and the chemical structural formula is 3 beta-ethoxy-12 beta-hydroxy-androstane 4, 14-diene-16-ketone.
The Sinomenium acutangula androstadienone C is white powder, and is dissolved in chloroform-methanol mixture (chloroform: methanol 1:1), pyridine, etc.;
(c 0.05,C5H5N)；UV(MeOH)λmax(logε):234(3.2),202(3.0)nm；IR(KBr)vmax3421,2936,2864,1680,1609,1445,1374,1246,1080,958,886cm-1；HR-ESI-MS m/z 317.2115[M+H]+(calcd.for C20H29O3,317.2117)1h NMR (MeOD,500MHz,) and13C NMR(methanol-d4150Mz) are shown in Table 1; the data are combined with 2D NMR analysis to confirm that the chemical structural formula of the cissampelos arundinacea androstadienone is shown in a formula IV as 3 beta-methoxy-12 beta-hydroxy-androstane 4, 14-diene-16-ketone.
The three 12 beta-hydroxy-androstane 4, 14-diene-16-ketone compounds are all novel natural organic compounds.
Table 1: process for preparing androstadienone compounds from cistema sinensis1H NMR and13c NMR data
Example 2: immunosuppressive detection assay
(1) Preparation of spleen lymphocyte suspension
Taking 18-22 g of healthy BABL/c mice, exsanguinating, killing, soaking and disinfecting in 75% alcohol for 5 minutes, taking out, placing in a sterile tray with the left side facing upwards, clamping the skin hair in the middle of the abdomen with disinfected forceps in an ultra-clean bench to form an incision, shearing each layer of the abdominal wall with another set of instruments, taking out the spleen with a third set of instruments, removing fat and connective tissues, placing in PBS (phosphate buffered saline), and washing away floating blood; then, the spleen tissue is transferred to a plate containing RPMI 1640 incomplete culture solution, cut into small pieces by scissors, the spleen is ground in a 200-mesh stainless steel screen mesh by using a sterile syringe core, washed by PBS for a small amount of times, the suspension is transferred to a 15mL centrifuge tube by using a pipettor, centrifuged for 5min at the rotating speed of 1000r/min, supernatant is sucked off, and 3mL erythrocyte lysate (Tris-NH) is added4Cl), standing for 2min, adding 10mL PBS to terminate the reaction, centrifuging (1200rpm,5min), removing supernatant, washing the precipitate twice with 5mL PBS, centrifuging under the same condition, and suspending the precipitate with 5mL RPMI 1640 complete culture solution containing 10% fetal calf serum; counting by 0.8% trypan blue viable cell dye exclusion method, wherein the viable cell number is not less than 95%, diluting with RPMI 1640 complete culture solution, and adjusting cell concentration to 1 × 106About one/mL.
(2) Preparation of test solutions
2mg of monomeric compound is precisely weighed, dissolved by adding DMSO, and diluted to the required concentration by PBS before loading, and the final concentration of DMSO after loading is not more than 0.1%.
(3) Experiment grouping
Normal control group: 100 μ L spleen cell suspension +10 μ L RPMI 1640 complete Medium +10 μ L LPBS
Model group: mu.L spleen cell suspension + 10. mu.L of LCoA (final concentration 10. mu.g/mL) + 10. mu.L of LPBS
Sample group: 100 μ L spleen cell suspension +10 μ L of LCoA (final concentration 10 μ g/mL) +10 μ L sample;
in 96-well plates, lymphocyte suspensions (1X 10) were added to each well6one/mL) 100. mu.L, ConA 10. mu.L (final concentration of 10. mu.g/mL), different concentrations of reagent compound dilutions 10. mu.L (final concentrations of 12.5, 25, 50. mu.g/mL, respectively), dexamethasone were also prepared in three corresponding concentration groups, and wells of the normal control group were filled with 10. mu.L of 1640 complete medium (containing 10% fetal bovine serum) and 10. mu.L of PBS, 4 replicates per concentration group, respectively.
(4) Culturing: standing at 37 deg.C for 5% CO2Culturing in an incubator for 72 hours.
(5) CCK-8 method for determining OD value of cells
After 72 hours of incubation, 10. mu.L of CCK-8 reagent (Biyunyan) was added to each well, and the mixture was incubated at 37 ℃ with 5% CO2After further culturing in the incubator for 4 hours, the absorbance at 450nm was measured per well to calculate the cell proliferation, and the Stimulation Index (SI) was calculated as follows:
SI (stimulation index) ═ OD value of mitogen-added cultures/OD value of non-mitogen-added cultures.
(6) Data processing
The OD value of the experimental data is expressed by means of 'mean +/-standard deviation', and the mathematical statistics and the analysis of variance work are completed by Origin software.
(7) Results of the experiment
Table 2: stimulation index of Marsdenia tenacissima androstadienone A on ConA-stimulated splenic lymphocyte proliferation of Balb/c mice
| Mean value of | SD | Significant analysis | |||||
| Model set | 3.26028 | 3.17562 | 2.97889 | 3.30698 | 3.180442 | 0.14495 | |
| yp low concentration | 2.69635 | 2.58213 | 2.39673 | 2.42662 | 2.52545 | 0.13994 | 0.0097609 |
| yp middle-concentration | 1.90216 | 1.96399 | 2.03898 | 1.88999 | 1.94878 | 0.06830 | 8.0436E-10 |
| yp high concentration | 1.68732 | 1.71988 | 1.68213 | 1.63991 | 1.68231 | 0.03283 | 7.5839E-11 |
The irritation indexes of the cispunica arundinacea androstadienone A at the concentrations of 12.5, 25 and 50 mu M are respectively 2.52, 1.94 and 1.63; the significance analysis shows that the medium concentration and high concentration groups have significant difference (P < 0.05) compared with the model group.
Table 3: stimulation index of cismus arundinacea androstadienone B on ConA-stimulated splenic lymphocyte proliferation of Balb/c mice
| Mean value of | SD | Significant analysis | |||||
| Model set | 3.13973 | 3.49012 | 3.36782 | 3.32117 | 3.32971 | 0.14531 | |
| yp low concentration | 2.48526 | 2.24749 | 2.67548 | 2.56724 | 2.49386 | 0.18178 | 0.00213824 |
| yp middle-concentration | 1.87385 | 2.01879 | 2.00194 | 1.97765 | 1.96805 | 0.06503 | 2.78108E-9 |
| yp high concentration | 1.73933 | 1.61787 | 1.82276 | 1.71787 | 1.72445 | 0.08423 | 3.11143E-10 |
The irritation indexes of the cispunica arundinacea androstadienone B at the concentrations of 12.5, 25 and 50 mu M are respectively 2.49, 1.96 and 1.72; the significance analysis shows that the high concentration group has significant difference (P < 0.05) compared with the model group.
Table 4: stimulation index of cismus arundinacea androstadienone C on ConA-stimulated splenic lymphocyte proliferation of Balb/C mice
| Mean value of | SD | Significant analysis | |||||
| Model set | 3.21314 | 3.38673 | 3.21782 | 3.09178 | 3.22736 | 0.12120 | |
| yp low concentration | 2.68143 | 2.79125 | 2.47818 | 2.59916 | 2.63750 | 0.13218 | 0.0025835 |
| yp middle-concentration | 1.99868 | 1.97814 | 2.00123 | 2.02165 | 1.99992 | 0.01779 | 5.1291E-10 |
| yp high concentration | 1.88283 | 1.77286 | 1.87687 | 1.69668 | 1.80731 | 0.08938 | 5.1556E-11 |
The irritation indexes of the cispunica arundinacea androstadienone C at the concentrations of 12.5, 25 and 50 mu M are respectively 2.63, 1.99 and 1.80; the significance analysis shows that the medium concentration and high concentration groups have significant difference (P < 0.05) compared with the model group.
The positive control results were:
table 5: stimulation index of dexamethasone to ConA-stimulated splenic lymphocyte proliferation in Balb/c mice
| Mean value of | SD | Significant analysis | |||||
| Model set | 3.19048 | 3.14881 | 3.20988 | 3.09912 | 3.16207 | 0.04909 | |
| yp low concentration | 1.48621 | 1.54397 | 1.68853 | 1.59691 | 1.57890 | 0.08593 | 3.0950E-11 |
| yp middle-concentration | 1.38769 | 1.34693 | 1.45138 | 1.38393 | 1.39248 | 0.04335 | 1.7449E-14 |
| yp high concentration | 1.25774 | 1.14981 | 1.19167 | 1.16877 | 1.19199 | 0.04705 | 6.7812E-15 |
The stimulation indexes of dexamethasone at the concentrations of 12.5, 25 and 50 mu M are respectively 1.57, 1.39 and 1.19; the significance analysis shows that each concentration group has significant difference (P < 0.05) compared with the model group.
The results of the experiment show that the cissampelos androstadienone A, B, C has immunosuppressive activity at concentrations of 25. mu.M and 50. mu.M.
Example 3: in vitro antitumor Activity test
(1) Material
DMSO (Sigma, USA), fetal bovine serum (HyClone, USA), RPMI-1640 culture medium (HyClone, USA), phosphate buffer (Beyotime, Shanghai), double antibody (HyClone, USA), CCK-8 (Token chemical technology, Inc.), human tumor cells (CCRF-CEM, MOLT-4, K-562, MALME-3M, UACC-62, SNB-75, OVCAR8, EKVX, U0-31, SF-295, NCI-H226, SK-OV3, MDA _ MB-468, Hop92), the compound of the invention and dexamethasone were all formulated with DMSO.
(2) Method of producing a composite material
5 kinds of cell suspension liquid which is adjusted to a certain concentration by human tumor cells in logarithmic growth phase is inoculated on a 96-hole culture plate, 90 mul/hole, compounds with different concentrations are added after the cells are cultured for 24 hours and adhere to the wall, 10 mul/hole, and each concentration is provided with 3 compound holes. The cells were counted and the viability of the cells, i.e. the number of unstained cells/total number of cells x 100%, determined by the disk blue staining method, was > 95% for the test, the cell suspension was adjusted to 1 x 106one/mL. Setting cell blank control group, positive control group (dexamethasone) and sample group (concentration is 25 μmol. L according to the result of preliminary test)-1All containing equal concentrations of DMSO). The negative control is equal volume of culture medium, and the corresponding DMSO concentration of compound 25. mu.g/mL is used as vehicle control to eliminate the influence of DMSO on cell growth.
(3) Culturing
Adding the medicine, continuously placing at 37 ℃ and 5% CO2Culturing in an incubator.
(4) Determination of OD value by MTT method
After the drugs act for 1, 3, 6, 12, 18 and 24 hours respectively, 20 mu L of MTT (5mg/ml) is added into each hole, the culture is continued for 4 hours, 100 mu L of triple liquid [ 10% SDS-5% isobutanol-0.012 mol/L HCl (W/V/V) ] is added into each hole to dissolve formazan, and the formazan generation amount is in direct proportion to the number of viable cells under normal conditions, so that the number of the viable cells can be presumed according to the OD value of the optical density. And the cell inhibition rate was calculated as follows:
inhibition ratio (%) - (OD value)Control wellOD valueSample adding hole) OD valueControl well×100％。
(5) Data processing
The OD value of the experimental data is expressed by means of 'mean +/-standard deviation', and the mathematical statistics and the analysis of variance work are completed by Origin software.
(6) Results of the experiment
The three new compounds in the formulas II, III and IV have better inhibition on 24 human tumor cell strains; the results showed that 9 of the human tumor cells (MOLT-4, K-562, SNB-75, SF-539, SF-295, NCI-H226, NCI-H522, SR, HS578T) showed resistance to dexamethasone; in addition, the three novel compounds had inhibitory effects on other 15 tumor cell lines (SK-OV3, MDA _ MB-468, Hop92, EKVX, U0-31, OVCAR8, UACC-62, MALME-3M, CCRF-CEM, SK-MEL-2, SNB-19, U251, HT-29, T-47D, NIC-H322M) and comparable positive control activity (Table 4).
Table 4: effect of Compounds on human tumor cell survival
Claims (2)
1. The application of 12 beta-hydroxy-androstane 4, 14-diene-16-ketone compounds with the structural formula shown as formula II or pharmaceutically acceptable salts thereof in preparing antitumor drugs:
2. use according to claim 1, characterized in that: the pharmaceutically acceptable salt is formed with organic acid selected from tartaric acid, citric acid, formic acid, acetic acid, oxalic acid, butyric acid, maleic acid and succinic acid, or inorganic acid selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61178920A (en) * | 1975-03-21 | 1986-08-11 | ビ−チヤム・グル−プ・リミテツド | Medicinal composition containing 4-ene-16, 16-disubstituted-3-one steroid |
| CN107056868A (en) * | 2017-03-24 | 2017-08-18 | 昆明理工大学 | Epigynum Auritum pregnane glucoside compound and its application |
| CN107365345A (en) * | 2017-06-21 | 2017-11-21 | 昆明理工大学 | A kind of D rings insert oxygen pregnane glycoside compounds and its application |
-
2018
- 2018-04-26 CN CN201810384371.2A patent/CN108558975B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61178920A (en) * | 1975-03-21 | 1986-08-11 | ビ−チヤム・グル−プ・リミテツド | Medicinal composition containing 4-ene-16, 16-disubstituted-3-one steroid |
| CN107056868A (en) * | 2017-03-24 | 2017-08-18 | 昆明理工大学 | Epigynum Auritum pregnane glucoside compound and its application |
| CN107365345A (en) * | 2017-06-21 | 2017-11-21 | 昆明理工大学 | A kind of D rings insert oxygen pregnane glycoside compounds and its application |
Non-Patent Citations (1)
| Title |
|---|
| 思茅藤C21甾体免疫抑制活性研究;姚元成;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20150415;E057-115 * |
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