CN102764253B - Application of benzopyran alkaloid - Google Patents
Application of benzopyran alkaloid Download PDFInfo
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- CN102764253B CN102764253B CN201210244811.7A CN201210244811A CN102764253B CN 102764253 B CN102764253 B CN 102764253B CN 201210244811 A CN201210244811 A CN 201210244811A CN 102764253 B CN102764253 B CN 102764253B
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- KYGFJSXICFBSLT-XBZLUXOVSA-N C[C@](CCC=C(C)C)([C@H](C1)O)Oc(cc2)c1cc2C(NC(C(C)=O)C(O)=O)=O Chemical compound C[C@](CCC=C(C)C)([C@H](C1)O)Oc(cc2)c1cc2C(NC(C(C)=O)C(O)=O)=O KYGFJSXICFBSLT-XBZLUXOVSA-N 0.000 description 1
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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 particularly alkaloidal application of 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, as 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 the alkaloidal extraction separation of .alpha.-5:6-benzopyran and Structural Identification had been reported, and reported that it has antiinflammatory and fibrosis is active, but there is not yet the report that this compounds has anti-hypertrophic cicatrix activity.
Hypertrophic cicatrix is a kind of of pathologic scar, often occurring in surgical operation, injure after burn outward, is to breed, grow out of control because of fibroblast (NHSF), and collagen over-deposit causes corium fabric, hypertrophy often continues several months or several years, degeneration just occurs gradually and change.Because the viscoelasticity of scar tissue reduces, the normal anatomical structures of human body surface skin histology is destroyed, not only affect attractive in appearancely, and can there is the symptoms such as pruritus, pain, even can cause serious dysfunction.Meanwhile, due to hypertrophic cicatrix and inflammation closely related, therefore, inflammatory cell, as people's Monocytes/Macrophages (THP-1) has important function in Process of Forming Scar.The fibroblast (NHSF) in blocking-up people normal skin corium source and the adhesion of people's Monocytes/Macrophages (THP-1) have become one of target spot for the treatment of 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 is achieved through the following technical solutions:
Application in the medicine that .alpha.-5:6-benzopyran alkaloid forms at preparation inhibition hypertrophic cicatrix, the alkaloidal structural formula of described .alpha.-5:6-benzopyran is as follows:
Preferably, described medicine is hypertrophic cicatrix inhibitor.
.alpha.-5:6-benzopyran alkaloid of the present invention can suppress the fibroblastic propagation of normal human skin and vigor; Can also block the fibroblast (NHSF) in people's normal skin corium source and the adhesion of people's Monocytes/Macrophages (THP-1); Can also weaken the contractility of the fibroblast (NHSF) in people's normal skin corium source.
Accompanying drawing explanation
Fig. 1 is the alkaloidal three-dimensional structure diagram of .alpha.-5:6-benzopyran of the present invention;
Fig. 2 suppresses the schematic diagram of the morphological observation in proliferation of human dermal fibroblasts experiment at .alpha.-5:6-benzopyran alkaloid;
Fig. 3 is the quantitative schematic diagram after .alpha.-5:6-benzopyran alkaloid suppresses the CCK-8 reagent dyeing in proliferation of human dermal fibroblasts experiment;
Fig. 4 is in .alpha.-5:6-benzopyran alkaloid blocking-up human skin fibroblast and Adherence of Monocytes experiment, the schematic diagram of the morphological observation after dyeing;
Fig. 5 is in .alpha.-5:6-benzopyran alkaloid blocking-up human skin fibroblast and Adherence of Monocytes experiment, the schematic diagram of adherent cell number quantitative result;
Fig. 6 is for to weaken in the experiment of the contractility of human skin fibroblast in 3D collagen gel at .alpha.-5:6-benzopyran alkaloid, the schematic diagram of morphological observation;
Fig. 7 is for to weaken in the experiment of the contractility of human skin fibroblast in 3D collagen gel at .alpha.-5:6-benzopyran alkaloid, measures the schematic diagram of quantitative result after glue area;
Fig. 8 is suppressing in the hypertrophy experiment of cicatrix in mouse model at .alpha.-5:6-benzopyran alkaloid, the quantitative result schematic diagram of hypertrophic cicatrix area and cicatrix index;
Fig. 9 is suppressing in the hypertrophy experiment of cicatrix in mouse model at .alpha.-5:6-benzopyran alkaloid, the section schematic diagram of hypertrophic cicatrix after Picro-Sirius red dyeing;
Figure 10 is suppressing in the hypertrophy experiment of cicatrix in mouse model at .alpha.-5:6-benzopyran alkaloid, the schematic diagram of cicatrix index (SEI=D/d).
The specific embodiment
Below illustrate the present invention, but the protection domain being not intended to limit the present invention.
The separation and purification of embodiment 1 .alpha.-5:6-benzopyran alkaloid (compound 1)
Get S.xiamenisis bacterial strain and carry out flat board cultivation (30 liters), 7 days, after dull and stereotyped agar is collected and shredded, use isopyknic ethyl acetate: methanol: mixed solvent formic acid=80:15:5(volume ratio) extracts 3 times, each 12 hours.Extracting solution merges, concentrated, obtains 12 grams of total extracts.Get total extract (6 grams), after dissolving with appropriate chloroform-methanol mixed solvent, add 10 grams of 200-300 order silica gel G (Qingdao Haiyang Chemical Industry Group Corp.'s product) and mix sample, upper to being filled with on 50 grams of purification on normal-phase silica gel glass decompression posts, 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 by improving the consumption of methanol in chloroform, and each eluting stream part receives respectively 200 milliliters, according to thin layer chromatography combining data detection stream part.The component of getting chloroform-methanol volume ratio and be 10:1 continues to carry out separation with gel filtration chromatography, and adopting Sephadex LH-20 is filler, and methanol is eluant.Then all components is carried out to efficient liquid phase chromatographic analysis, with this compound, at 206nm, the uv absorption of 260nm is as the criterion, and merges the component with same absorbent.Finally, adopt reversed phase column chromatography to carry out purification the component after merging, adopt 75%(volume ratio) methanol/water solution be eluant, obtain 15 milligrams of benzopyran compounds (compound 1) as shown in Figure 1.
The physicochemical property of 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 DEPT method to determine and uses respectively C (singlet), CH (doublet), CH
2(triplet) and CH
3(quartet) represents.
Material is as follows:
Cell: normal human skin fibroblast (normal human skin fibroblasts, NHSF cell); Medicine: the compound 1 being obtained by above-described embodiment.
Method: be inoculated in 96 orifice plates, every hole 1 * 10 after growing to NHSF cell 0.25% trypsinization of 70-80% fusion
3individual cell.After 24 hours, change liquid dosing, drug treating group 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.Within every two days, change culture medium and medicine once.0d after dosing, 2d, 4d, 6d measures respectively cell viability.Measuring method is, sucking-off culture medium, and every hole adds complete medium 100 μ l and CCK-8 reagent 10 μ l, and incubator is hatched microplate reader measure light absorption value after 60 minutes, and wavelength is 450nm.Experiment repeats 3 times.
Experimental result as shown in Figure 2,3, can be learnt by accompanying drawing: compound 1 can suppress the propagation of human skin fibroblast.When dosage is 30 μ g/ml, administration four days to six days, administration group is compared the difference with significance with matched group.
The fibroblast in embodiment 3 .alpha.-5:6-benzopyran alkaloid blocking-up people normal skin corium sources and the adhesion experiment of people's Monocytes/Macrophages
Material is as follows:
Cell: normal human skin fibroblast (normal human skin fibroblasts, NHSF cell); People's Monocytes/Macrophages (Human acute monocytic leukemia cell line, THP-1); Medicine: the compound 1 being obtained by above-described embodiment.
Method: NHSF cell is seeded to 24 orifice plates, and 500 μ l cell suspension are inoculated in every hole, and density is 1 * 10
5/ ml, cultivates after 3 days and merges completely.Cultivate altogether before 24h and will after THP-1 cell centrifugation, with DMEM, add 10%FBS and 1% dual anti-resuspended, before cultivating altogether, with calcein dyeing, centrifugal and add 10%FBS and 1% dual anti-resuspended with DMEM while cultivating altogether, making THP-1 cell density is 5 * 10
4/ ml.By the sucking-off of NHSF cell conditioned medium, after PBS cleans once, every hole adds THP-1 suspension 500 μ l, and the every hole of drug treating group adds 318, and to make final concentration be 30 μ g/ml, and solvent DMSO concentration is 1/10000, and matched group is DMSO, and final concentration is 1/10000.Put into after incubator is hatched 5h and take out, after sucking-off culture medium, with PBS, clean 3 times, wash away and do not adhere to THP-1 cell.By observing calcein fluorescence staining, in Xia Mei hole, inverted microscope 200 * visual field, choose immediately 9 visual field statistics adherent cell numbers.Experiment repeats 3 times.
Experimental result as shown in Figure 4,5, can be learnt by accompanying drawing: compound 1 can be blocked human skin fibroblast and monocytic adhesion, and has concentration dependent.When dosage is 10 μ g/ml and 30 μ g/ml, administration group is compared and is had significant difference with matched group.
Material is as follows: cell: normal human skin fibroblast (normal human skin fibroblasts, NHSF cell); Medicine: the compound 1 being obtained by above-described embodiment.
Method: add and grow to NHSF cell that 70-80% merges and add DMEM complete medium to end digestion after with 0.25% trypsinization, centrifugal rear supernatant discarded, adds 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, finally adds cell suspension 0.67ml.It is 30 μ g/ml that the every ware of drug treating group adds compound1 to make final concentration, and solvent DMSO concentration is 1/10000, and matched group adds DMSO, and final concentration is 1/10000.After mixing, put into incubator, after 1h, take out and shake gently, make 3D collagen gel become flexible from culture dish.Respectively at 6h, 12h, 24h, takes a picture after 36h and uses Photoshop software measurement glue area.Experiment repeats 3 times.
Experimental result as shown in Figure 6,7, can be learnt by accompanying drawing: when dosage is 30 μ g/ml, compound 1 can weaken the contractility of human skin fibroblast in three-dimensional collagen gel, and within 6 hours, occurs significant difference in administration.
Material is as follows: eight week age, 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 compound 1 being obtained by above-described embodiment.
Method: by mouse back skin depilatory, sew up after back being hit exactly after 24 hours to 2 centimetres of skin holostrome longitudinal incisions with sodium sulfide.After 4 days, take out stitches and sew up installation pulling device, retracting 2mm the same day, later every other day tractive once, each tractive 4mm, tractive was to postoperative 10 days altogether.After starting tractive, be administered once every day, 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, is injected to tractive postoperative 10 days.Within after tractive 10 days, remove pulling device, statistics cicatrix area, and get cicatrix specimen and be fixed embedding, Sirius red colouring after section, statistics scar hyperplasia index (Scar Elevation Index, SEI).
Experimental result as shown in Figure 8,9, 10, can be learnt by accompanying drawing: 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 the fibroblastic propagation of normal human skin and vigor; Can also block the fibroblast (NHSF) in people's normal skin corium source and the adhesion of people's Monocytes/Macrophages (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 preparation and suppresses the medicine that hypertrophic cicatrix forms.
Disclosed is above only several specific embodiments of the application, but the application is not limited thereto, and the changes that any person skilled in the art can think of, all should drop in the application's protection domain.
Claims (2)
1. .alpha.-5:6-benzopyran alkaloid suppresses the application in the synulotic medicine of skin hyperplasia in preparation, and the alkaloidal structural formula of described .alpha.-5:6-benzopyran is as follows:
2. application as claimed in claim 1, is characterized in that, described medicine is skin hyperplasia cicatrix inhibitor.
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| 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 |
| 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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| US20050245492A1 (en) * | 2004-04-28 | 2005-11-03 | Lephart Edwin D | Use of equol for treating skin diseases |
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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 |
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