CN104163844B - Steroidal ketene compounds caulerpa ketenes and preparation thereof and purposes - Google Patents
Steroidal ketene compounds caulerpa ketenes and preparation thereof and purposes Download PDFInfo
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Abstract
The present invention relates to pharmaceutical technology field, relate to steroid compound caulerpa ketenes that the side chain that obtains of extracting and developing is novel from China's Caulerpa racemosa (Forssk) Web V. Bos and preparation method thereof and at diabetes, obesity or protein tyrosine esterase 1B(PTP1B) application in inhibitor.The caulerpa ketenes external PTP1B suppression test of the present invention shows, this compound for protein TYR esterase 1B(PTP1B) there is remarkable inhibiting activity, therefore can be used to prepare PTP1B inhibitor it can also be used to diabetes, obesity and the medicine of complication thereof are treated in preparation.
Description
Technical field
The present invention relates to pharmaceutical technology field, relate in particular to a kind of steroid compound with special side chain of isolated from China's Caulerpa racemosa (Forssk) Web V. Bos.
Background technology
Caulerpa racemosa (Forssk) Web V. Bos Caulerpa
racemosa (Forsskål)
J. Agardh system Chlorophyta (Chlorophyta) Chlorophyceae (Chlorophyceae) Siphonales (Siphonales) Caulerpaceae (Caulerpaceae) caulerpa belong to (Caulerpa) sea-plant, be distributed mainly on Perenniporia martius marine site, be grown on the rock of below intertidal zone, coral reef or in, on the sand ground of low tide band.Also have widely distributed in China marine site, focus primarily upon Dongshan, Fujian, Taiwan, Hainan, Xisha, Coast of Guangdong Province.
Diabetes (diabetes mellitus) are one group of clinical syndromes caused by h and E factor interaction.At present, typically diabetes are divided into two classes, I-patients with type Ⅰ DM (insulin-dependent diabetes mellitus, insulin-dependent
Diabetes mellitus, IDDM) and II-patients with type Ⅰ DM (Non-Insulin Dependent Diabetes Mellitus, non-insulin-dependent diabetes mellitus, NIDDM).In diabetes, 90% is above II-patients with type Ⅰ DM.
The feature of II-patients with type Ⅰ DM is insulin sensitive tissues such as skeletal muscle, liver, the adipose tissue opposing to insulin action.Protein-tyrosine-phosphatase (PTPases) in statocyte in insulin action path the effect in GAP-associated protein GAP tyrosine phosphorylation level increasingly come into one's own, become treatment II-patients with type Ⅰ DM new way.PTPase includes big nation's cross-film (receptor type) and intracellular (non-receptor type) enzyme, participates in a series of important life processes.At present, PTPase acceptor or acceptor metasomite in insulin path are affected the research of Normal insulin effect, is concentrated mainly on LAR-PTPase, SHPTP-2, PTP1B.
PTP1B is first certified protein-tyrosine-phosphatase (protein tyrosine phosphatase); the experiment on mice rejected by PTP1B is shown; PTP1B is by being acylated the dephosphorization of insulin receptor, and then plays very important effect in regulation insulin sensitivity and fat metabolic process.Thus, PTP1B inhibitor selective, highly active has important value in the treatment of II-patients with type Ⅰ DM, obesity and complication thereof.
Summary of the invention
The present invention be from China Caulerpa racemosa (Forssk) Web V. Bos (C. racemesaSteroid compound caulerpa ketenes (Caulerpenone) with special side chain of isolated is extracted in).Show through pharmacological testing research, this compound for protein TYR phosphate 1B(PTP1B) there is remarkable inhibiting activity.
Therefore, it is an object of the present invention to provide new steroid compound caulerpa ketenes.
It is a further object to provide the preparation method of described caulerpa ketenes.
A further object of the present invention is to provide the purposes of described caulerpa ketenes.Specifically, described caulerpa ketenes is preparing protein-tyrosine-phosphatase 1B (PTP1B)
Application in the medicine of inhibitor, the further application in the medicine of preparation treatment diabetes, obesity and complication thereof.
First purpose according to the present invention, the present invention is found that the steroid compound caulerpa ketenes that a side chain is special first from Caulerpa racemosa (Forssk) Web V. Bos, and its chemical constitution is as follows:
。
According to second object of the present invention, the present invention provides the preparation method of described caulerpa ketenes, and it is isolated from Caulerpa racemosa (Forssk) Web V. Bos, specifically comprises the following steps that
1) extract medicinal extract is prepared
By freezing Caulerpa racemosa (Forssk) Web V. Bos (C. racemosa) with ethanol seepage pressure effects routinely, obtain extract, extract reduced pressure concentration is reclaimed ethanol, obtains CE；
2) isolated and purified
(1) said extracted thing is dispersed in water into suspension, suspension is used petroleum ether, ethyl acetate and extracting n-butyl alcohol, gained extract concentrate successively and respectively obtains petroleum ether extraction medicinal extract, ethyl acetate extraction medicinal extract and n-butanol extraction medicinal extract；
(2) ethyl acetate extract is carried out silica gel column chromatography, with petroleum ether/acetone gradient elution, merge similar stream part according to TLC colour developing and obtain 5 components (A-E)；Wherein component B(petroleum ether/acetone volume ratio 85:15 elution fraction) through Sephadex LH-20 gel filtration chromatography, elute with methylene chloride/methanol volume ratio 1:1, again through silica gel column chromatography, with petroleum ether/acetone 85:15 volume ratio wash-out, through preparation HPLC after, elute with methanol/water 90:10 volume ratio, obtain the compounds of this invention caulerpa ketenes.
In above-mentioned preparation method, in preparing extract medicinal extract step, the described ethanol used that extracts is 95% ethanol.
In above-mentioned preparation method, in separating step, the concentration of petroleum ether/acetone gradient elution is followed successively by volume ratio 100:0,95:5,85:15,70:30 and 50:50.
In above-mentioned preparation method, in separating step, the filler of the chromatographic column of described preparation HPLC is RP-18.
According to third object of the present invention, the invention provides described caulerpa ketenes in the purposes preparing PTP1B inhibitor, diabetes medicament, obesity drug.
The present invention has carried out external PTP1B suppression test to gained caulerpa ketenes, and result shows that this compound has obvious inhibitory activity to PTP1B.Therefore, can be used to prepare PTP1B inhibitor, be used for treating diabetes, obesity and complication thereof.
The caulerpa ketenes of the present invention can be obtained by isolated and purified in marine alga；Can also obtain through chemical modification method well known to those skilled in the art synthesis.
Accompanying drawing explanation
Fig. 1 PTP1B inhibitory activity test philosophy.
Detailed description of the invention
The chemical structural formula (Arabic numerals in structural formula are the marks of carbon atom in chemical constitution) of the caulerpa ketenes of indication in examples below:
。
The preparation of embodiment 1 caulerpa ketenes
1. prepare Caulerpa racemosa (Forssk) Web V. Bos extract medicinal extract
(1) extract is prepared
By freezing Chinese Caulerpa racemosa (Forssk) Web V. Bos (C. racemosa) (picking up from Zhanjiang coastal) 5 kg(dry weight) extract three times with 30L 95% ethanol percolation respectively, each diacolation 5 days, merge extract；
(2) extract medicinal extract is prepared
Said extracted liquid is reclaimed ethanol at temperature≤45 DEG C reduced pressure concentration, obtains CE 350g；
The most isolated and purified
1) said extracted thing medicinal extract is dispersed in water into suspension, being extracted three times respectively with petroleum ether (1.5L), ethyl acetate (1.5L) and n-butanol (1L) successively by suspension, gained extract reduced pressure concentration respectively obtains petroleum ether and extracts medicinal extract (38g), ethyl acetate extraction medicinal extract (160g) and n-butanol extraction medicinal extract (120g)；
2) ethyl acetate extract is carried out silica gel column chromatography, with petroleum ether/acetone gradient elution；The solubility of gradient elution, as volume ratio 100:0,95:5,85:15,70:30 and 50:50, merges similar stream part according to TLC colour developing and obtains 5 components (A-E)；
3) component B(85:15) through Sephadex LH-20 gel filtration chromatography, with petroleum ether/methylene chloride/methanol volume ratio 2:1:1 wash-out, merge similar stream part according to TLC colour developing and obtain 5 components (B1-B7)；
4) component B5 is again through silica gel column chromatography, with petroleum ether/acetone volume ratio 85:15 wash-out, merges similar stream part according to TLC colour developing and obtains 3 components (B5-1-B5-3)；
5) component B5-2 is through preparation HPLC (filler of chromatographic column is RP-18), elutes with methanol/water volume ratio 90:10, and flow velocity is 3 mL/min, and retention time is 15min, obtains the compounds of this invention caulerpa ketenes, is identified as noval chemical compound.
Structural Identification
Routinely through the various modern spectral technique such as NMR, HRESIMS, UV, IR and optically-active, it is determined that the chemical constitution of compound caulerpa ketenes, its physicochemical property is as follows:
White powder, molecular formula is C29H46O2；
Specific rotatory power [α] 20
D -10.0 (c 1.0, CHCl3)；
Ultraviolet spectra UV (MeOH)λ max(log ε): 233 (4.42)nm；
Infrared spectrum IR (KBr)ν max: 3447, 2932, 1689, 1457, 1307,
1223, 1188, 917, 742 cm– 1；
High resolution mass spectrum HRESIMSm/z 449.3409 [M+Na]+ (calcd for C29H46O2Na,
449.3396)；
Proton nmr spectra1H NMR
(600 MHz) and carbon-13 nmr spectra13C NMR (150 MHz) data are shown in Table one
Caulerpa ketenes described in table one1H and13C NMR(ppm in CDCl3)
The test of embodiment 2 PTP1B inhibitory activity:
Test philosophy: see Fig. 1.Molecular biology method is utilized to express people source protein TYR phosphate 1B(hPTP1B at E. coli system) catalyst structure domain, hPTP1B recombinant protein energy hydrolysis substrate p-nitrophenyl phosphoric acid (p-Nitrophenyl phosphate after purified, pNPP) phosphatide key, obtain yellow soluble product p-nitrophenol (p-Nitrophenol), this product has the strongest light to absorb at 410nm, therefore can directly detect change and the activity change of observation enzyme and the compound suppression situation to enzymatic activity that at 410nm, light absorbs.
The survey live body system of standard: 10mM Tris.Cl tri-(methylol) aminomethane hydrochloride), pH 7.6,10mM pNPP, 2%DMSO, 100nM hPTP1B.
Observation index: dynamic measurement wavelength is that the light at 410 nm absorbs, and the time is 3 minutes, and the slope of its kinetic curve first order reaction is as the activity index of enzyme.
Test method: be from expression in escherichia coli the gst fusion protein that purifies for the protein-tyrosine-phosphatase PTP1B of screening.Use ultraviolet suitable substrates p-nitrophenyl phosphoric acid (pNPP), observe the variable concentrations inhibitory action to the activity of recombinase, with the medicinal effects of preliminary assessment compound.Before use sample is dissolved in DMSO and is made into debita spissitudo, 3 times of dilutions, 7 dilution factors, three wells is set, takes 2 μ L sample solutions and add 96 orifice plates, be subsequently adding 88 μ L assay mix (assay buffer, pNPP, H2O), 10 μ L PTP1B are added.It is detection absorbance value at 410nm that 96 orifice plates are placed on VERSAmax dynamically detection wavelength, and the time is 3 minutes.
The judge of experimental result and explanation:
The selection result is the compound concentration percent inhibition to enzymatic activity when being 20 μ g/ml, when inhibiting rate is higher than 50%, the inhibiting rate detected diluted chemical compound higher than 50% (is become different concentration by screening routinely, reacting according to above-mentioned method of testing, all tests are respectively provided with multiple hole) draw IC50, the IC of positive control sodium vanadate50It it is 2 μMs.
Experimental result: the compounds of this invention caulerpa ketenes IC to PTP1B enzyme inhibition activity50It it is 3.8 μMs.
Experiment conclusion: pass through molecular biology test, it can be seen that compound caulerpa ketenes is to protein tyrosine esterase 1B(PTP1B) there is preferable inhibitory activity.Therefore, during the caulerpa ketenes of the present invention can be used for the medicine of preparation diabetes, obesity and complication thereof.
Claims (2)
1. a steroid compound caulerpa ketenes, chemical structural formula is as follows:
。
The steroid compound caulerpa ketenes the most according to claim 1 application in preparing protein tyrosine phosphate 1B inhibitor, diabetes medicament, obesity drug.
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| CN104497090B (en) * | 2014-11-03 | 2016-03-02 | 南昌大学 | Caulerpa ketenes and its production and use |
| CN107501362B (en) * | 2017-07-18 | 2019-10-15 | 南阳师范学院 | A kind of naphthoquinones glycosides compound and its preparation method and application |
| CN107652347B (en) * | 2017-10-11 | 2020-09-18 | 南昌大学 | Compound Dictyopterissini I and application thereof in preparation of diabetes or obesity medicines |
| CN107674108B (en) * | 2017-10-11 | 2020-09-22 | 南昌大学 | Application of stigmastane steroid compound in preparing medicine for treating diabetes or obesity |
| CN107802626B (en) * | 2017-10-11 | 2020-01-21 | 南昌大学 | Hypoglycemic composition and preparation method and application thereof |
Citations (1)
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| CN103054131A (en) * | 2011-09-08 | 2013-04-24 | 浙江海洋学院 | Preparation method of high-purity Caulerpa racemesa polyphenol and application thereof to fresh-keeping of shrimp |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103054131A (en) * | 2011-09-08 | 2013-04-24 | 浙江海洋学院 | Preparation method of high-purity Caulerpa racemesa polyphenol and application thereof to fresh-keeping of shrimp |
Non-Patent Citations (3)
| Title |
|---|
| "A new polyacetylenic fatty acid and other secondary metabolites from the Chinese green alga Caulerpa racemosa (Caulerpaceae) and their chemotaxonomic significance";Shui-Chun Mao et al.;《Biochemical Systematics and Ecology》;20110918;第39卷;第253-257页 * |
| "Caulerchlorin, a novel chlorinated bisindole alkaloid with antifungal activity from the chinese green alga Caulerpa racemosa";Ding-Quan Liu et al.;《Heterocycles》;20120106;第85卷(第3期);第661-666页 * |
| "中国南海总状蕨藻的脂肪酸类化学成分和生物活性研究";刘定权 等;《中国海洋药物》;20131231;第32卷(第6期);第13-20页 * |
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Effective date of registration: 20201023 Address after: 226000 No. 266 Century Avenue, hi tech Zone, Jiangsu, Nantong Patentee after: Nantong Yaoxiang Technology Co., Ltd Address before: 999 No. 330031 Jiangxi province Nanchang Honggutan University Avenue Patentee before: Nanchang University |
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