CN105111196A - Highly-esterified limonin compound and medical application thereof - Google Patents
Highly-esterified limonin compound and medical application thereof Download PDFInfo
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Abstract
The invention discloses a highly-esterified limonin compound and medical application thereof, and provides a compound structure, a pharmaceutical composition containing the compound, and a preparation method and application thereof. The compound is reported for the first time, is a highly-esterified limonin compound with novel structure, and can be extracted, separated and purified from the dry cluster of self-heal. The in-vitro test proves that the compound can inhibit multiple mammary cancer cells from proliferation and induce the apoptosis of the mammary cancer cells, and can be used for developing drugs for treating mammary cancer.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to the limonin compound being separated a kind of height esterification obtained from Spica Prunellae, containing its pharmaceutical composition and its preparation method and application.
Background technology
Spica Prunellae (Classification system: Prunellavulgaris), is commonly called as ox enemy, also known as doing in nine Paris polyphylla, iron look grass, large headdress flower, excellent column cap flower, sheep intestines dish, hammer grass, June, caput post etc.; Per nnial herb.Stolon, long on joint have fibrous root.Grow and to wet thick grass, wasteland, roadside at gully meadow bog or both sides, riverbank, be distributed widely in all over China, economize as the main place of production with Henan, Anhui, Jiangsu, Hunan etc.Mainly clinically be used as medicine with its drying and ripening fruit ear, improving eyesight of can relieving inflammation or internal heat, can control the effect such as conjunctival congestion with pain and swelling of the eye, headache.
Spica Prunellae has various active composition, mainly contains the compounds such as triterpenes, steroid, flavonoid, coumarins.At present, researchist has been separated and has identified numerous chemical composition from the Spica Prunellae of different genera.
Spica Prunellae decoction, water-leach liquor, alcohol-water leach liquor and alcohol leaching liquid all can obviously reduce laboratory animal blood pressure, and stem, leaf, fringe and herb all have hypotensive effect, but the effect of fringe is more obvious; Spica Prunellae water decoction-alcohol sedimentation liquid mouse peritoneal is injected, and has obvious anti-inflammatory action; Spica Prunellae decoction all has certain restraining effect to dysentery bacterium, Corynebacterium diphtheriae, vibrio cholerae, intestinal bacteria, Bacillus proteus, staphylococcus and Bacillus tuberculosis in vitro.
The speed of normal human cell's division is carried out within the specific limits, and when body is subject to the impact of externalities or the unbalance of internal environment, the mitotic cycle of tumour cell or apoptosis speed change, and tumour cell quantity continues to increase, and lump increases.Therefore, the proliferation function of inhibition tumor cell is an antineoplastic important channel.At present, research proves that Spica Prunellae extract has the effect of inhibition tumor cell propagation.
Summary of the invention
The object of this invention is to provide a kind of limonin compound being separated a kind of height esterification obtained from the fruit ear of Spica Prunellae drying, containing its pharmaceutical composition and its preparation method and application.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
There is the compound (I) of following structural formula,
The preparation method of described compound (I), comprise following operation steps: the Spica Prunellae fruit ear that (a) is dry is pulverized, extract with 80% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract macroporous resin removal of impurities in (b) step (a), use 20% ethanol and 75% ethanol elution successively, collect 75% ethanol eluate, concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract; C in () step (b), 75% ethanol elution medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 100:1,60:1,30:1,15:1 and 1:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, be the methanol aqueous solution isocratic elution of 60% by concentration expressed in percentage by volume, collect 9-12 column volume elutriant, elutriant concentrating under reduced pressure obtains pure compound (I).
Further, described macroporous resin is AB-8 type macroporous adsorbent resin.
Pharmaceutical composition, the described compound (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
The application of described compound (I) in the medicine of preparation treatment mammary cancer.
The application of described pharmaceutical composition in the medicine of preparation treatment mammary cancer.
When the compounds of this invention is used as medicine, directly can uses, or use with the form of pharmaceutical composition.
This pharmaceutical composition contains the compounds of this invention (I) for the treatment of significant quantity, and all the other are acceptable on pharmacology, nontoxic to humans and animals and pharmaceutically acceptable carrier of inertia and/or vehicle.
Described pharmaceutically acceptable carrier or vehicle are that one or more are selected from solid, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent.Pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention is applied to by form that is oral or injection the patient needing treatment.For time oral, tablet, slow releasing tablet, controlled release tablet, capsule, dripping pill, micropill, suspensoid, emulsion, powder or granule, oral liquid etc. can be made into; During for injecting, can be made into water-based or oily solution, aseptic powder injection, liposome or the emulsion etc. of sterilizing.
figure of description
Fig. 1 is compound (I) structural formula;
Fig. 2 is compound (I) two-dimentional hsqc spectrum;
Fig. 3 is the two-dimentional HMBC spectrum of compound (I);
Fig. 4 is the two-dimentional ROESY spectrum of compound (I);
Fig. 5 is that various dose compound (I) is on the impact of Cells Proliferation of Human Breast Cancer vigor;
Fig. 6 is that various dose compound (I) is on the impact of Apoptosis of Breast Cancer.
Embodiment
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.
Main raw, reagent source and instrument type:
Ethanol, sherwood oil, ethyl acetate, propyl carbinol, methylene dichloride are analytical pure, and purchased from Shanghai Ling Feng chemical reagent company limited, methyl alcohol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
DMEM/F12 is high, and sugared nutrient solution is purchased from Beijing Hyclone company.Modified form RPMI-l640 substratum is purchased from Beijing Hyclone company.Standard foetal calf serum is purchased from Tianjin TBD company.CCK-8 cell viability detection reagent is purchased from Amada Co., Ltd. colleague chemistry institute.AnnexinV-FITC/PI apoptosis detection kit is purchased from Shanghai Yan Bin Chemical Industry Science Co., Ltd.Dimethyl alum (DMSO) and phosphate buffer soln (PBS) are purchased from Sigma Products.
-70 DEG C of cryogenic refrigerators: FormaScientificInc. company, the U.S.; Electronic balance: Shimadzu AEL-200 type, Japan; Constant temperature oscillator: THZ-C type, domestic); Disinfection with high pressure steam pot: YX-400B type, domestic; CO
2cell culture incubator: FormaSeries II, Thermalelectroncorp., the U.S.; Thermostat water bath: BHW2-I type, domestic; Inverted phase contrast microscope: CK40 type, Olympus, Japan; Microscope: (BX51 type, Olympus, Japan; Micro-digit collecting system: DP71 type, Olympus, Japan; Multi-functional microplate reader: InfiniteM200 type, TECAN, Switzerland; Desk-top room temperature supercentrifuge: TGL-16G type, Beijing; Desk-top low-temperature and high-speed whizzer: 2K15 type, sigma company, the U.S.; BL60 electronic balance: Beijing Sai Duolisi instrument system company limited; LD4-40 Large Copacity whizzer: system in Beijing Jing founds whizzer company limited; 2K15 high temperature low speed refrigerated centrifuge: (sigma company; UV765 ultraviolet spectrophotometer: Shanghai Precision Scientific Apparatus Co., Ltd; Laser confocal scanning microscope: MRC-1024 type, Bio-Rad, Britain.
Embodiment 1: compound (I) is separated preparation and structural identification
A Spica Prunellae fruit ear (8kg) pulverizing that () is dry, (25L × 3 time) are extracted with 80% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste (6L), use sherwood oil (6L × 3 time), ethyl acetate (6L × 3 time) and water saturated propyl carbinol (6L × 3 time) to extract successively, obtain petroleum ether extract, acetic acid ethyl ester extract (312g) and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract AB-8 type macroporous resin removal of impurities in (b) step (a), use 20% ethanol (8L) and 75%(12L) ethanol elution successively, collect 75% ethanol eluate, concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract (152g); C in () step (b), 75% ethanol elution medicinal extract purification on normal-phase silica gel is separated, be 100:1(8 column volume successively by volume ratio), a 60:1(8 column volume), a 30:1(6 column volume), a 15:1(8 column volume) and 1:1(5 column volume) methylene chloride-methanol gradient elution obtain 5 components; Component 4(32g in (d) step (c)) be separated further by purification on normal-phase silica gel, be 20:1(8 column volume by volume ratio successively), a 12:1(10 column volume) and 5:1(6 column volume) methylene chloride-methanol gradient elution obtain 3 components; Component 2(9g in (e) step (d)) be separated with the reverse phase silica gel of octadecylsilane bonding, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 60%, collect 9-12 column volume elutriant, elutriant concentrating under reduced pressure obtains pure compound (I) (28mg).
Structural identification: pale yellow needles crystallization, is soluble in chloroform, ethyl acetate, acetone and methyl alcohol; HR-ESIMS shows [M+Na]
+for m/z689.2822, can obtain molecular formula in conjunction with nuclear-magnetism feature is C
33h
46o
14, degree of unsaturation is 11.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, methanol-
d 4, 400MHz): H-1(7.00, br, s), H-2(2.85, d,
j=13.4), H-2(2.42, dd,
j=13.4,9.9), H-5(2.16, d,
j=11.8), H-6(5.13, dd,
j=11.8,2.0), H-7(4.80, d,
j=2.0), H-9(2.63, m), H-11(2.08, m), H-11(1.93, m), H-12(1.50, m), H-12(1.75, m), H-15(3.59, s), H-17(5.62, s), H-18(1.25, 3H, s), H-19(1.31, 3H, s), H-21(7.51, s), H-22(6.43, s), H-23(7.52, s), H-28(1.43, 3H, s), H-29(1.19, 3H, s), H-30(1.25, 3H, s), OAc-1(1.96, 3H, s), OAc-6(2.00, 3H, s), OAc-7(2.15, 3H, s), OMe-3(3.64, 3H, s), carbon-13 nmr spectra data δ
c(ppm, methanol-
d 4, 100MHz): 78.4(CH, 1-C), 37.2(CH
2, 2-C), 174.1(C, 3-C), 74.1(C, 4-C) and, 48.0(CH, 5-C), 73.9(CH, 6-C) and, 72.0(CH, 7-C), 43.5(C, 8-C) and, 36.9(CH, 9-C), 48.3(C, 10-C) and, 18.3(CH
2, 11-C), 27.4(CH
2, 12-C), 39.6(C, 13-C), 71.0(C, 14-C) and, 57.1(CH, 15-C), 169.6(C, 16-C) and, 79.9(CH, 17-C), 18.0(CH
3, 18-C), 16.1(CH
3, 19-C), 121.9(C, 20-C), 142.8(CH, 21-C) and, 111.0(CH, 22-C), 144.5(CH, 23-C) and, 36.9(CH
3, 28-C), 28.1(CH
3, 29-C), 18.4(CH
3, 30-C), 171.7(C, OAc-1), 21.3(CH
3, OAc-1), 172.0(C, OAc-6), 21.0(CH
3, OAc-6), 172.0(C, OAc-7), 21.1(CH
3, OAc-7), 52.6(CH
3, OMe-3), carbon atom mark is see Fig. 1.Total score liberation of hydrogen spectrum and carbon spectrum nuclear magnetic resonance data, find compound (I) and (Phytochemistry2008 in document, 69,1782 1787) compound odoralide structure comparison is close, the OAc-11 group of the latter is instead of, epoxy construction hydrocracking unlike the OAc-6 group 4 in compound (I).HMBC spectrum in, H-6(δ H5.13) with C-5(δ C48.0), C7(δ C72.0), and in acetyl group carbonyl carbon (δ C172.0) be correlated with, confirm this structure.The relative configuration of compound (I) is determined by ROESY spectrum.In ROESY spectrum, H-6 and H-7, H
3-19, H
3the intersection peak of-30 shows that OAc-6 group is α configuration.14 and 16 hydroxyl configurations are according to ROESY spectrum and reference compound odoralide nuclear magnetic signal is defined as beta comfiguration.Therefore, this compound structure can be determined as shown in Figure 1.
Embodiment 2: compound (I) pharmacological action is tested
One, test method
1, cell strain and cultivation
Select MCF-7 Breast Cancer Cell (estrogen receptor positive ER
+, the negative HERZ/neu of human epidermal growth factor acceptor-2
-), mammary cancer MDA-MB-231 cell (ER
-, HERZ/neu
-) and mammary cancer MDA-MB-453(ER
-, HERZ/neu
+) cell strain, three kinds of cell strains all come from Chinese Sciences Academy Biochemistry And Cell Biology Institute.MCF-7 cell and MDA-MB-231 cell cultures are in the high sugared nutrient solution of the DMEM/F12 containing 10% standard foetal calf serum, and MDA-MB-453 cell cultures is in the RPMI-1640 nutrient solution containing 10% standard foetal calf serum, and above cell is all in 37 DEG C, 5%CO
2under condition, cellar culture goes down to posterity.Three kinds of breast cancer cells are attached cell, going down to posterity of attached cell: when cell attachment growth 2 ~ 3d to 80% merges, discard old nutrient solution, 0.1mol/LPBS(pH7.4 with appropriate) add the 0.1mol/LPBS preparation of the pancreatin (pH7.4) of appropriate 0.25%, filtration sterilization in rinsing cell 2 backward bottles.Digestion, be advisable just to cover bottom Tissue Culture Flask, light Microscopic observation, treats that tenuigenin bounces back, and after intercellular substance increases, adds fresh nutrient solution immediately and stops digestion, and repeatedly blow and beat gently with suction pipe, makes to form cell suspension at the bottom of cell detachment bottle; Adjustment cell concn, draws appropriate cell suspension and adds in new culturing bottle, add 6mL fresh culture, basis of microscopic observation, be positioned over cell culture incubator.
2, cell proliferation viability examination
Utilize CCK-8 method detection compound (I) (by the preparation of embodiment 1 method, HPLC purity the is greater than 98%) impact on three kinds of Cells Proliferation of Human Breast Cancer vigor.CCK-8 test kit (CellCountingKit-8) is the test kit of the detection cell proliferation of being developed by Japanese colleague's chemistry institute, containing WST-8 [2-(2-methoxyl group-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2 in CCK-8 reagent, 4-disulfonic acid benzene)-2H-tetrazolium monosodium salt], it is reduced to the yellow formazan product with high water soluble under the effect of electron carrier 1-methoxyl group-5-toluphenazine methyl-sulfate (1-MethoxyPMS) by the desaturase in cell mitochondrial, the quantity generating formazan thing is directly proportional to the quantity of viable cell.Measure its absorbance value by microplate reader at 450nm wavelength place, can indirectly reflect viable cell quantity.
Concrete operations are: by breast cancer cell MCF-7, MDA-MB-231, MDA-MB-453 routine passage of logarithmic phase, cell counting, with 2.5 × 10
5/ mL cell concn is inoculated in 96 porocyte culture plates, and every hole 200 μ L, overnight incubation, treats cell attachment and well-grown.Experiment divides 5 groups, often organize work 6 parallel holes, add the compound (I) of different amount respectively, make its final concentration be respectively 200,150,100,50mg/mL, control group adds equivalent distilled water, cultivate 24h, discard nutrient solution, wash 3 times with PBS, add fresh medium every hole 200 μ L, then every hole adds 10 μ LCCK-8, cultivate 4h harvested cell, detect 450nm place optical density value [D (450)] with microplate reader, with the zeroing of distilled water blank, calculate the proliferation inhibition rate that different concns compound (I) acts on.Cell proliferation inhibition rate=[contrast D (450) experimental group D (450)]/contrast D (450) × 100%
3, apoptotic detection
Utilize AnnexinV/PI two dye Fluorescein activated cell sorter detection compound (I) on the impact of breast cancer cell MCF-7, MDA-MB-231, MDA-MB-453 apoptosis.Experiment is carried out in strict accordance with AnnexinV apoptosis detection kit operation instructions.Concrete operations are: by breast cancer cell MCF-7, MDA-MB-231, MDA-MB-453 routine passage of logarithmic phase, cell counting, with 10
5/ mL cell concn is inoculated in 6 porocyte culture plates, every hole 5mL, and overnight incubation, treats cell attachment and well-grown.Experiment divides 5 groups, often organizes work 3 parallel holes, adds the compound (I) of various dose respectively, make its final concentration be respectively 150,100,50mg/mL, control group adds equivalent distilled water, cultivates 24h, discards nutrient solution, PBS(pH7.2 with 4 DEG C of precoolings) wash cell 3 times, conventional trysinization, makes single cell suspension, centrifugal, with the binding damping fluid Eddy diffusion cell that 250 μ L dilute, and its concentration is made to be 1 × 10
6/ mL, the cell suspension getting 100 μ L, in 5mL streaming pipe, adds 5 μ LAnnexinV/FITC solution, leave standstill 5min, add the PI solution 10 μ L of 20 μ g/mL, after mixing, place 10min in room temperature lucifuge, in reaction tubes, add 400 μ LPBS, utilize machine testing on flow cytometer respectively to organize fluorescence situation.Cell divides to 4 phases according to AnnexinV and PI staining conditions respectively, AnnexinV and the PI equal negative cells that dyes is survive cell, PI stained positive, AnnexinV are negative staining is physical abuse or ruptured cell, AnnexinV and the PI dyeing all positive is downright bad or non-viable apoptotic cell, and AnnexinV stained positive, PI are negative staining is judged to be viable apoptotic cell.
4, the statistical study of data
All data all represent with mean number scholar standard deviation (`X scholar s), and adopt SPSS12.0 statistical software, carry out one-way analysis of variance to experimental result, P<0.05 thinks to there is significant difference between the two.
Two, result and conclusion
1, cell proliferation viability examination result
CCK-8 method is utilized to detect the impact of different concns compound (I) effect on three kinds of Cells Proliferation of Human Breast Cancer vigor, result shows, compound (I) effect can make three kinds of Cells Proliferation of Human Breast Cancer activity all reduce, and reduce degree increase with the increase of activity, demonstrate dose-effect relationship, 100, 150 all there is significant difference (P<0.05 with 200mg/mL group compared with control group, P<0.01), compound (I) effect can significantly suppress breast cancer cell MCF-7, MDA-MB-231, the propagation of MDA-MB-453, demonstrate stronger vitro cytotoxicity, show that compound (I) has stronger In Vitro Anti mammary cancer effect.The results are shown in Figure 5 and table 1.
2, apoptotic detected result
Utilize AnnexinV/PI two dye Fluorescein activated cell sorter detection compound (I) on the impact of breast cancer cell MCF-7, MDA-MB-231, MDA-MB-453 apoptosis, apoptosis analytical results shows, compound (I) effect 24h can make the apoptosis rate of three kinds of breast cancer cells all raise, demonstrate short apoptosis effect, and there is dose-effect relationship, 100 have significant difference (P<0.05) with 150mg/mL group compared with control group shows that compound (I) can remarkable inducing mammary cancer cell-apoptosis, plays antitumor action.The results are shown in Figure 6.
Sum up, this research, by chemical compounds I anti-breast cancer Effect study, finds that chemical compounds I can make three kinds of Cells Proliferation of Human Breast Cancer activity reduce, significantly suppresses its propagation, demonstrate stronger vitro cytotoxicity, have In Vitro Anti mammary cancer effect.
Table 1 various dose compound (I) is to the inhibiting rate (%) of Cells Proliferation of Human Breast Cancer
| 50mg/mL | 100mg/mL | 150mg/mL | 200mg/mL | |
| MDA-MB-453 cell | 14.80 | 29.80 | 33.10 | 44.30 |
| MDA-MB-231 cell | 11.40 | 24.00 | 30.60 | 37.50 |
| MCF-7 cell | 10.30 | 20.20 | 26.20 | 35.10 |
Embodiment 3
The preparation of tablet: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, vehicle is added, pelletizing press sheet than the ratio for 1:9 in itself and excipient weight.
Embodiment 4
The preparation of oral liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, oral liquid method for making makes oral liquid routinely.
Embodiment 5
The preparation of capsule or granule: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, add vehicle in itself and excipient weight than the ratio for 1:9, make capsule or granule.
Embodiment 6
The preparation of injection liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, inject with water routinely, essence filter, injection liquid is made in embedding sterilizing.
Embodiment 7
The preparation of aseptic powder injection: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, be dissolved in sterile water for injection, stirring makes molten, filter with aseptic suction funnel, aseptic essence filter again, be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains powder injection.
Claims (6)
1. there is the compound (I) of following structural formula,
。
2. the preparation method of compound according to claim 1 (I), it is characterized in that comprising following operation steps: the Spica Prunellae fruit ear that (a) is dry is pulverized, extract with 80% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract macroporous resin removal of impurities in (b) step (a), use 20% ethanol and 75% ethanol elution successively, collect 75% ethanol eluate, concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract; C in () step (b), 75% ethanol elution medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 100:1,60:1,30:1,15:1 and 1:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, be the methanol aqueous solution isocratic elution of 60% by concentration expressed in percentage by volume, collect 9-12 column volume elutriant, elutriant concentrating under reduced pressure obtains pure compound (I).
3. preparation method according to claim 2, is characterized in that: described macroporous resin is AB-8 type macroporous adsorbent resin.
4. pharmaceutical composition, the compound according to claim 1 (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
5. the application of compound according to claim 1 (I) in the medicine of preparation treatment mammary cancer.
6. the application of pharmaceutical composition according to claim 4 in the medicine of preparation treatment mammary cancer.
Priority Applications (1)
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105294819A (en) * | 2015-10-20 | 2016-02-03 | 淄博夸克医药技术有限公司 | Novel limonin compound for treating breast cancer |
| CN105294723A (en) * | 2015-12-10 | 2016-02-03 | 缪琼华 | Diterpenoid compound for treating breast cancer and preparation method thereof |
| CN105418728A (en) * | 2015-12-01 | 2016-03-23 | 杨飞杰 | New limonin compound as well as preparation method and pharmaceutical application in breast cancer treatment |
| CN106146315A (en) * | 2016-07-14 | 2016-11-23 | 朱正直 | A kind of medicinal compound and preparation method thereof |
| CN106187777A (en) * | 2016-07-14 | 2016-12-07 | 朱正直 | A kind of pharmaceutical composition of cefoperazone |
| CN106265623A (en) * | 2016-07-14 | 2017-01-04 | 朱正直 | Compound, cefoperazone pharmaceutical composition preparation treatment periodontitis medicine in application |
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Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102018759A (en) * | 2009-09-17 | 2011-04-20 | 上海中医药大学附属曙光医院 | Rosmarinic acid, rosmarinic acid-containing common selfheal fruit-spike active ingredient and preparation methods and application thereof to prevention and treatment of cancer postoperative metastasis |
| CN105153267A (en) * | 2015-08-28 | 2015-12-16 | 林天样 | Novel withanolides compound, novel withanolides compound preparation method and medical application of novel withanolides compound |
| CN105294723A (en) * | 2015-12-10 | 2016-02-03 | 缪琼华 | Diterpenoid compound for treating breast cancer and preparation method thereof |
| CN105294819A (en) * | 2015-10-20 | 2016-02-03 | 淄博夸克医药技术有限公司 | Novel limonin compound for treating breast cancer |
| CN105330636A (en) * | 2015-12-07 | 2016-02-17 | 西宁意格知识产权咨询服务有限公司 | New flavonoid compound and preparation method and medical application thereof |
| CN105418728A (en) * | 2015-12-01 | 2016-03-23 | 杨飞杰 | New limonin compound as well as preparation method and pharmaceutical application in breast cancer treatment |
| CN105439989A (en) * | 2015-12-31 | 2016-03-30 | 胡文杰 | Novel diterpenoid compounds as well as preparation method and medicine application thereof |
| CN105461672A (en) * | 2015-10-06 | 2016-04-06 | 温州高新技术产业开发区聚智汇科技信息咨询服务中心 | New diterpene compound, and preparation method and medicinal use thereof |
| CN105481802A (en) * | 2015-12-29 | 2016-04-13 | 吴金凤 | Clerodane diterpenoid compounds, and preparation method and medical application thereof |
| CN105503871A (en) * | 2015-12-30 | 2016-04-20 | 吴金凤 | Novel indole alkaloid compound and preparation method and medical application thereof |
| CN105503991A (en) * | 2015-12-31 | 2016-04-20 | 吴金凤 | Limonin compound for treating breast cancer and preparation method of limonin compound |
| CN105520928A (en) * | 2015-12-28 | 2016-04-27 | 吴芊葭 | Application of diterpene compound in preparation of breast cancer inhibition drugs |
| CN105585574A (en) * | 2015-12-28 | 2016-05-18 | 吴芊葭 | Diterpene compound and preparation method thereof |
| CN106366155A (en) * | 2016-08-28 | 2017-02-01 | 成都宝科生物科技有限公司 | Novel limonins compound as well as preparation method and medical application thereof |
-
2015
- 2015-09-06 CN CN201510558951.5A patent/CN105111196A/en active Pending
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102018759A (en) * | 2009-09-17 | 2011-04-20 | 上海中医药大学附属曙光医院 | Rosmarinic acid, rosmarinic acid-containing common selfheal fruit-spike active ingredient and preparation methods and application thereof to prevention and treatment of cancer postoperative metastasis |
| CN105153267A (en) * | 2015-08-28 | 2015-12-16 | 林天样 | Novel withanolides compound, novel withanolides compound preparation method and medical application of novel withanolides compound |
| CN105461672A (en) * | 2015-10-06 | 2016-04-06 | 温州高新技术产业开发区聚智汇科技信息咨询服务中心 | New diterpene compound, and preparation method and medicinal use thereof |
| CN105294819A (en) * | 2015-10-20 | 2016-02-03 | 淄博夸克医药技术有限公司 | Novel limonin compound for treating breast cancer |
| CN105418728A (en) * | 2015-12-01 | 2016-03-23 | 杨飞杰 | New limonin compound as well as preparation method and pharmaceutical application in breast cancer treatment |
| CN105330636A (en) * | 2015-12-07 | 2016-02-17 | 西宁意格知识产权咨询服务有限公司 | New flavonoid compound and preparation method and medical application thereof |
| CN105294723A (en) * | 2015-12-10 | 2016-02-03 | 缪琼华 | Diterpenoid compound for treating breast cancer and preparation method thereof |
| CN105520928A (en) * | 2015-12-28 | 2016-04-27 | 吴芊葭 | Application of diterpene compound in preparation of breast cancer inhibition drugs |
| CN105585574A (en) * | 2015-12-28 | 2016-05-18 | 吴芊葭 | Diterpene compound and preparation method thereof |
| CN105481802A (en) * | 2015-12-29 | 2016-04-13 | 吴金凤 | Clerodane diterpenoid compounds, and preparation method and medical application thereof |
| CN105503871A (en) * | 2015-12-30 | 2016-04-20 | 吴金凤 | Novel indole alkaloid compound and preparation method and medical application thereof |
| CN105503991A (en) * | 2015-12-31 | 2016-04-20 | 吴金凤 | Limonin compound for treating breast cancer and preparation method of limonin compound |
| CN105439989A (en) * | 2015-12-31 | 2016-03-30 | 胡文杰 | Novel diterpenoid compounds as well as preparation method and medicine application thereof |
| CN106366155A (en) * | 2016-08-28 | 2017-02-01 | 成都宝科生物科技有限公司 | Novel limonins compound as well as preparation method and medical application thereof |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105294819A (en) * | 2015-10-20 | 2016-02-03 | 淄博夸克医药技术有限公司 | Novel limonin compound for treating breast cancer |
| CN105418728A (en) * | 2015-12-01 | 2016-03-23 | 杨飞杰 | New limonin compound as well as preparation method and pharmaceutical application in breast cancer treatment |
| CN105294723A (en) * | 2015-12-10 | 2016-02-03 | 缪琼华 | Diterpenoid compound for treating breast cancer and preparation method thereof |
| CN106146315A (en) * | 2016-07-14 | 2016-11-23 | 朱正直 | A kind of medicinal compound and preparation method thereof |
| CN106187777A (en) * | 2016-07-14 | 2016-12-07 | 朱正直 | A kind of pharmaceutical composition of cefoperazone |
| CN106265623A (en) * | 2016-07-14 | 2017-01-04 | 朱正直 | Compound, cefoperazone pharmaceutical composition preparation treatment periodontitis medicine in application |
| CN107778346A (en) * | 2016-08-28 | 2018-03-09 | 成都宝科生物科技有限公司 | A kind of highly oxidized noval chemical compound and its medical usage |
| CN114105964A (en) * | 2021-12-16 | 2022-03-01 | 贵州医科大学 | Limonin compound, preparation method and application thereof as tobacco mosaic virus resistant medicament |
| CN114105964B (en) * | 2021-12-16 | 2023-08-01 | 贵州医科大学 | Limonin compound, preparation method and application thereof as tobacco mosaic virus resistant drug |
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