CN102764253A - Application of benzopyran alkaloid - Google Patents
Application of benzopyran alkaloid Download PDFInfo
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- CN102764253A CN102764253A CN2012102448117A CN201210244811A CN102764253A CN 102764253 A CN102764253 A CN 102764253A CN 2012102448117 A CN2012102448117 A CN 2012102448117A CN 201210244811 A CN201210244811 A CN 201210244811A CN 102764253 A CN102764253 A CN 102764253A
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- benzopyran
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Abstract
The invention relates to the technical field of medicines, in particular to application of a benzopyran alkaloid. The application of the benzopyran alkaloid to the preparation of medicines for restraining formation of hypertrophic scars is involved; the benzopyran alkaloid has the structural formula which is shown in specifications; and the benzopyran alkaloid can restrain proliferation and viability of normal human skin fibroblast, block the adhesion of normal human skin fibroblast (NHSF) and human mononuclear/macrophage (THP-1), and weaken contractility of the normal human skin fibroblast (NHSF).
Description
Technical field
The present invention relates to medical technical field, the alkaloidal application of particularly a kind of .alpha.-5:6-benzopyran.
Background technology
At present, about the more existing bibliographical informations of .alpha.-5:6-benzopyran alkaloid benzopyran compounds, like Japanese patent [Kawamura, N.; Tsuji, E.; Watanabe, Y.; Tsuchihashi, K.; Takako; T.Benzopyran derivatives; Their manufacture with Streptomyces species, and their use for treatment of asthma and rheumatoid arthritis.Daiichi Seiyaku Co., Ltd.; Mercian Corp.:Kyoto, Japan, 7March2000.] and document [Min-Juan Xu; Xiao-Jin Liu, Yi-Lei Zhao, Dong Liu; Zhen-Hao Xu, Xiao-Meng Lang, Ping Ao; Wen-Han Lin, Song-Lin Yang, Zhi-Gang Zhang and Jun Xu; Identification and Characterization of an Anti-Fibrotic Benzopyran Compound Isolated from Mangrove-Derived Streptomyces xiamenensis, Marine Drugs10:639-654,2012.] in; Once reported that the .alpha.-5:6-benzopyran alkaloid extraction was separated and structure is identified, and reported that it has antiinflammatory and fibrosis is active, but this compounds of Shang Weijian has the active report of anti-hypertrophic cicatrix.
Hypertrophic cicatrix is a kind of of pathologic scar; Often occur in surgical operation, outward injure burn after, be out of control because of fibroblast (NHSF) propagation, growth, the collagen over-deposit causes corium fabricization; Hypertrophy often continues several months or several years, degeneration just takes place gradually change.Because the viscoelasticity of scar tissue reduces, and the normal anatomical structures of human body surface skin histology is destroyed, not only influence attractive in appearancely, and symptoms such as pruritus, pain can be occurred, even serious dysfunction can be caused.Simultaneously, because hypertrophic cicatrix and inflammation are closely related, therefore, and inflammatory cell, (THP-1) has important function in the cicatrization process like people's Monocytes.The fibroblast (NHSF) in blocking-up people normal skin corium source and the adhesion of people's Monocytes (THP-1) have become one of target spot of treatment hypertrophic cicatrix.
Summary of the invention
The object of the invention is to provide a kind of .alpha.-5:6-benzopyran alkaloidal application.
The object of the invention realizes through following technical scheme:
The application of .alpha.-5:6-benzopyran alkaloid in the medicine that preparation inhibition hypertrophic cicatrix forms, the alkaloidal structural formula of said .alpha.-5:6-benzopyran is following:
Preferably, described medicine is the hypertrophic cicatrix inhibitor.
.alpha.-5:6-benzopyran alkaloid of the present invention can suppress normal human skin fibroblasts proliferation and vigor; Can also block the fibroblast (NHSF) in people's normal skin corium source and the adhesion of people's Monocytes (THP-1); Can also weaken the contractility of the fibroblast (NHSF) in people's normal skin corium source.
Description of drawings
Fig. 1 is the alkaloidal three-dimensional structure diagram of .alpha.-5:6-benzopyran of the present invention;
Fig. 2 is the sketch map that suppresses the morphological observation in HSF's proliferation experiment at the .alpha.-5:6-benzopyran alkaloid;
Fig. 3 is the quantitative sketch map after the .alpha.-5:6-benzopyran alkaloid suppresses the CCK-8 reagent dyeing in HSF's proliferation experiment;
Fig. 4 adheres in the experiment sketch map of the morphological observation after the dyeing .alpha.-5:6-benzopyran alkaloid blocking-up HSF and mononuclear cell;
Fig. 5 adheres in the experiment sketch map of adherent cell number quantitative result .alpha.-5:6-benzopyran alkaloid blocking-up HSF and mononuclear cell;
Fig. 6 is for to weaken in the experiment of the contractility of HSF in the 3D collagen gel at the .alpha.-5:6-benzopyran alkaloid, the sketch map of morphological observation;
Fig. 7 is for to weaken in the experiment of the contractility of HSF in the 3D collagen gel at the .alpha.-5:6-benzopyran alkaloid, measures the sketch map of quantitative result behind the glue area;
Fig. 8 is suppressing in the hypertrophy experiment of cicatrix in the mouse model the exponential quantitative result sketch map of hypertrophic cicatrix area and cicatrix at the .alpha.-5:6-benzopyran alkaloid;
Fig. 9 is suppressing in the hypertrophy experiment of cicatrix in the mouse model section sketch map of hypertrophic cicatrix behind Sirius red colouring at the .alpha.-5:6-benzopyran alkaloid;
Figure 10 is suppressing in the hypertrophy experiment of cicatrix in the mouse model sketch map of cicatrix index (SEI=D/d) at the .alpha.-5:6-benzopyran alkaloid.
The specific embodiment
Below illustrate the present invention, but and be not used in qualification protection scope of the present invention.
The separation and purification of embodiment 1 .alpha.-5:6-benzopyran alkaloid (chemical compound 1)
Get the S.xiamenisis bacterial strain and carry out flat board cultivation (30 liters), 7 days, after dull and stereotyped agar collection and chopping, use isopyknic ethyl acetate: methanol: the mixed solvent of formic acid=80:15:5 (volume ratio) extracted 3 times, each 12 hours.Extracting solution merges, and concentrates, and gets total extract 12 grams.Get total extract (6 gram), mix appearance, go up to being filled with on the 50 gram purification on normal-phase silica gel glass decompression posts with adding 10 gram 200-300 order silica gel G (Qingdao Haiyang Chemical Industry Group Corp.'s product) after an amount of chloroform-methanol mixed solvent dissolving; With chloroform-methanol (100:1,50:1,20:1; 10:1; 5:1, (volume ratio) is to methanol) gradient elution, the polarity of eluting solvent comes gradient to increase progressively through the consumption that improves methanol in the chloroform; Each eluting flows part and receives 200 milliliters respectively, detects according to thin layer chromatography to merge stream part.The component of getting the chloroform-methanol volume ratio and be 10:1 continues to separate with gel filtration chromatography, and adopting Sephadex LH-20 is filler, and methanol is eluant.Then all components is carried out efficient liquid phase chromatographic analysis, at 206nm, the uv absorption of 260nm is as the criterion with this chemical compound, merges the component with same absorbent.At last, adopt reversed phase column chromatography to carry out purification the component after merging, adopting the methanol/water solution of 75% (volume ratio) is eluant, obtains 15 milligrams of benzopyran compounds as shown in Figure 1 (chemical compound 1).
The physicochemical property of chemical compound 1: yellow unformed powder; [α]
22 D+ 39.5 ° (c0.044, MeOH); UV λ
Max(MeOH) 206,260nm; CD (MeOH) Δ ε
201+ 0.15, Δ ε
202+ 0.55, Δ ε
205+ 0.3, Δ ε
207.5+ 0.5, Δ ε
229-0.05, Δ ε
259+ 0.28;
1H and
13C NMR data, see Table1; HRESIMSm/z392.2069 [M+H]
+, (calcdfor C
21H
30NO
6, m/z392.2073), 390.1886 [M-H]-, (calcd for C
21H
29NO
6, m/z390.1917).
This table signal ownership based on DEPT,
1H-
1H COSY, HMQC and HMBC spectrum analysis result.The multiple degree of carbon signal utilizes the DEPT method to confirm and uses C (singlet), CH (doublet), CH respectively
2(triplet) and CH
3(quartet) expression.
Material is following:
Cell: normal human skin fibroblast (normal human skin fibroblasts, NHSF cell); Medicine: the chemical compound 1 that obtains by the foregoing description.
Method: be inoculated in 96 orifice plates, every hole 1 * 10 after will growing to NHSF cell 0.25% trypsinization of 70-80% fusion
3Individual cell.Change liquid and dosing after 24 hours, drug treating group chemical compound 1 final concentration is 30 μ g/ml, and solvent DMSO concentration is 1/10000, and matched group is DMSO, and final concentration is 1/10000.Changed culture medium and medicine in per two days once.0d after the dosing, 2d, 4d, 6d measure cell viability respectively.Measuring method does, the sucking-off culture medium, and every hole adds complete medium 100 μ l and CCK-8 reagent 10 μ l, and incubator is hatched ELIASA measure light absorption value after 60 minutes, and wavelength is 450nm.Experiment repetition 3 times.
Experimental result can be learnt by accompanying drawing like Fig. 2, shown in 3: chemical compound 1 can suppress HSF's propagation.When dosage is 30 μ g/ml, administration four days to six days, the administration group is compared the difference with significance with matched group.
The fibroblast in embodiment 3 .alpha.-5:6-benzopyran alkaloids blocking-up people normal skin corium source and the adhesion experiment of people's Monocytes
Material is following:
Cell: normal human skin fibroblast (normal human skin fibroblasts, NHSF cell); People's Monocytes (Human acute monocytic leukemia cell line, THP-1); Medicine: the chemical compound 1 that obtains by the foregoing description.
Method: with NHSF cell inoculation to 24 orifice plate, 500 μ l cell suspension are inoculated in every hole, and density is 1 * 10
5/ ml cultivates after 3 days and merges fully.Cultivate before the 24h adding 10%FBS and 1% pair with DMEM behind the THP-1 cell centrifugation anti-resuspendedly altogether, with calcein dyeing, centrifugal and to add 10%FBS and 1% pair with DMEM anti-resuspended when cultivating altogether, making the THP-1 cell density is 5 * 10 before cultivating altogether
4/ ml.With the sucking-off of NHSF cell conditioned medium, PBS cleans once the every hole adding in back THP-1 suspension 500 μ l, and it is 30 μ g/ml that drug treating group every hole adding 318 makes final concentration, and solvent DMSO concentration is 1/10000, and matched group is DMSO, and final concentration is 1/10000.Put into and take out after incubator is hatched 5h, clean 3 times with PBS after the sucking-off culture medium, flush away does not adhere to the THP-1 cell.Through observing the calcein fluorescence staining, 9 visual field statistics adherent cell numbers are chosen in every hole immediately under inverted microscope 200 * visual field.Experiment repetition 3 times.
Experimental result can be learnt by accompanying drawing like Fig. 4, shown in 5: chemical compound 1 can be blocked HSF and monocytic adhesion, and has concentration dependent.When dosage was 10 μ g/ml and 30 μ g/ml, the administration group is compared with matched group had significant difference.
Material is following: cell: normal human skin fibroblast (normal human skin fibroblasts, NHSF cell); Medicine: the chemical compound 1 that obtains by the foregoing description.
Method: add and grow to NHSF cell that 70-80% merges and add the DMEM complete medium after with 0.25% trypsinization and end digestion, centrifugal back supernatant discarded adds the DMEM complete medium, and making cell density is 3 * 10
5/ ml.In each 35mm culture dish, add 0.92 milliliter of 1.76 * DMEM culture medium, 0.1MNaOH0.04ml, FBS0.19ml, 3mg/ml collagen 0.20ml adds cell suspension 0.67ml at last.It is 30 μ g/ml that the every ware adding of drug treating group compound1 makes final concentration, and solvent DMSO concentration is 1/10000, and matched group adds DMSO, and final concentration is 1/10000.Put into incubator behind the mixing, take out behind the 1h and shake gently, make the 3D collagen gel become flexible from culture dish.Respectively at 6h, 12h, 24h takes a picture behind the 36h and use Photoshop software measurement glue area.Experiment repetition 3 times.
Experimental result can be learnt by accompanying drawing like Fig. 6, shown in 7: when dosage was 30 μ g/ml, chemical compound 1 can weaken the contractility of HSF in three-dimensional collagen gel, and in 6 hours obvious difference of administration.
Material is following: eight ages in week, female C57BL/6 mice was 10, was divided into experimental group and matched group, and buying is from Chinese Academy of Sciences's Shanghai Experimental Animal Center; Medicine: the chemical compound 1 that obtains by the foregoing description.
Method: with the mouse back skin depilatory, sew up after after 24 hours the back being hit exactly 2 centimetres of skin holostrome longitudinal incisions with sodium sulfide.Take out stitches after 4 days and sew up the installation pulling device, retracted 2mm the same day, later on every other day tractive once, each tractive 4mm, tractive to postoperative is 10 days altogether.Be administered once every day behind the beginning tractive, and experimental group is pressed benzopyran compounds 110mg/kg lumbar injection, and the DMSO of matched group injection equivalent quantity of solvent in experimental group was injected to the tractive postoperative 10 days.Removed pulling device in 10 days behind the tractive, statistics cicatrix area, and get the fixedly embedding of cicatrix BIAO and BEN, section back Sirius red colouring, statistics scar hyperplasia index (Scar Elevation Index, SEI).
Experimental result can be learnt by accompanying drawing like Fig. 8,9, shown in 10: chemical compound 1 can obviously reduce the generation of mice cicatrix, and laboratory animal does not find that body weight changes simultaneously.
Through experiment confirm .alpha.-5:6-benzopyran alkaloid of the present invention can suppress normal human skin fibroblasts proliferation and vigor; Can also block the fibroblast (NHSF) in people's normal skin corium source and the adhesion of people's Monocytes (THP-1); Can also weaken the contractility of the fibroblast (NHSF) in people's normal skin corium source.Therefore .alpha.-5:6-benzopyran alkaloid of the present invention can be used for preparing the medicine that suppresses hypertrophic cicatrix formation.
More than the disclosed several specific embodiments that are merely the application, but the application is not limited thereto, any those skilled in the art can think variation, all should drop in the application's the protection domain.
Claims (2)
1. the application of .alpha.-5:6-benzopyran alkaloid in the medicine that preparation inhibition hypertrophic cicatrix forms, the alkaloidal structural formula of said .alpha.-5:6-benzopyran is following:
2. application as claimed in claim 1 is characterized in that, described medicine is the hypertrophic cicatrix inhibitor.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103508993A (en) * | 2013-09-13 | 2014-01-15 | 上海交通大学 | Preparation method of benzopyran compound and application of protecting against pulmonary fibrosis |
| CN104407084A (en) * | 2014-11-28 | 2015-03-11 | 上海交通大学 | Quantitative detection method of streptomyces xiamenensis in plasma and qualitative detection method of metabolite of streptomyces xiamenensis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000072766A (en) * | 1998-08-27 | 2000-03-07 | Dai Ichi Seiyaku Co Ltd | Benzopyran derivative |
| US20050245492A1 (en) * | 2004-04-28 | 2005-11-03 | Lephart Edwin D | Use of equol for treating skin diseases |
-
2012
- 2012-07-16 CN CN201210244811.7A patent/CN102764253B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000072766A (en) * | 1998-08-27 | 2000-03-07 | Dai Ichi Seiyaku Co Ltd | Benzopyran derivative |
| US20050245492A1 (en) * | 2004-04-28 | 2005-11-03 | Lephart Edwin D | Use of equol for treating skin diseases |
Non-Patent Citations (1)
| Title |
|---|
| MIN-JUAN XU ET AL.: "Identification and Characterization of an Anti-Fibrotic Benzopyran Compound Isolated from Mangrove-Derived Streptomyces xiamenensis", 《MARINE DRUGS》, vol. 10, 15 March 2012 (2012-03-15), pages 639 - 654 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103508993A (en) * | 2013-09-13 | 2014-01-15 | 上海交通大学 | Preparation method of benzopyran compound and application of protecting against pulmonary fibrosis |
| WO2015035778A1 (en) * | 2013-09-13 | 2015-03-19 | 上海交通大学 | Method for preparing benzopyran compound and uses in protecting against pulmonary fibrosis |
| CN103508993B (en) * | 2013-09-13 | 2015-10-14 | 上海交通大学 | The preparation method of benzopyran compounds and the purposes of pulmonary fibrosis resistant |
| CN104407084A (en) * | 2014-11-28 | 2015-03-11 | 上海交通大学 | Quantitative detection method of streptomyces xiamenensis in plasma and qualitative detection method of metabolite of streptomyces xiamenensis |
| CN104407084B (en) * | 2014-11-28 | 2016-03-02 | 上海交通大学 | The quantitative detection of Xiamen mycin and the qualitative checking method of its metabolin in blood plasma |
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| CN102764253B (en) | 2014-03-05 |
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Effective date of registration: 20220629 Address after: 100000 Department of medicine, Peking University, 38 Xueyuan Road, Haidian District, Beijing Patentee after: Xu Minjuan Address before: 200240 No. 800, Dongchuan Road, Shanghai, Minhang District Patentee before: SHANGHAI JIAO TONG University |
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Effective date of registration: 20221125 Address after: 201109 Building 1, No. 600, Jianchuan Road, Minhang District, Shanghai Patentee after: Shanghai Shengtong Kangwei Pharmaceutical Co.,Ltd. Address before: 100000 Department of medicine, Peking University, 38 Xueyuan Road, Haidian District, Beijing Patentee before: Xu Minjuan |