CN104262443A - Preparation method of polyhydroxy sterol monomeric compound and application - Google Patents
Preparation method of polyhydroxy sterol monomeric compound and application Download PDFInfo
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- CN104262443A CN104262443A CN201410442533.5A CN201410442533A CN104262443A CN 104262443 A CN104262443 A CN 104262443A CN 201410442533 A CN201410442533 A CN 201410442533A CN 104262443 A CN104262443 A CN 104262443A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N45/00—Biocides, pest repellants or attractants, or plant growth regulators, containing compounds having three or more carbocyclic rings condensed among themselves, at least one ring not being a six-membered ring
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Abstract
The invention discloses a preparation method of a polyhydroxy sterol monomeric compound and an application. The preparation method comprises the following steps: triturating and uniformly homogenizing fresh styela plicata, carrying out ultrasonic extraction by 95% ethanol, decompressing and concentrating to obtain an extract, dispersing the extract by using water, extracting by virtue of ethyl acetate, and carrying out concentration under reduced pressure on the extraction liquid to obtain an ethyl acetate liquid extract; fully dissolving the ethyl acetate liquid extract by methanol, carrying out SephadaxLH-20 gel column chromatography and RP-18 reverse silica-gel column chromatography; and finally, preparing by using efficient high performance liquid chromatography to respectively obtain a compound 1, dendronesterol A and a compound 2 (20S, 22E, 24R)-ergosta-7, 22-dien-3beta, 5alpha, 6beta-triol, wherein the artemia resistant activity of the compounds 1 and 2 is detected, and the fatality rates to artemia are respectively 87.4% and 82.9% when the concentration is 50 microgramme/mL and rise along with the concentration, showing concentration dependence. The compound can be applied to preparing artemia-resisting drugs. The compound which can be applied to preparing artemia-resisting drugs has easily available raw materials and is wide in prospect.
Description
Technical field
The present invention relates to the preparation method of polyhydroxy sterol class monomeric compound, also relate to the application of polyhydroxy sterol class monomeric compound, described polyhydroxy sterol class monomeric compound refers to the monomer constituents of extraction and isolation from wrinkle knurl Ascidian, belongs to marine natural product chemistry field.
Background technology
Since the eighties in 20th century, some Natural products researchs personnel have found to have the compound of antitumor, antiviral activity and have much entered clinical study from Ascidian in the world, make Ascidian become except sponge and coral, the mankind obtain the important marine Biological resources having remarkable pharmacological active substance.Research both at home and abroad about Ascidian nutritive ingredient is a lot, as amino acid, mineral element, lipid acid and polysaccharide etc., has sizable benefit, be called as " northeast rare delicacies " human body.Wrinkle knurl Ascidian (Styela plicata) belongs to Chordata (phylum Chordata), Urochordata (Tunicata), Ascidiacea (Ascidiacea).Few about the Research Literature of its biologically active substance at present, foreign study personnel find to have the diterpene of pharmacologically active and the compound of polypeptide forms in early days from this kind of Ascidian, the domestic research about coastal area of china wrinkle knurl Ascidian is also at the early-stage, the people such as Su Jing joy study discovery sterol and ceramide secondarily in level meta-bolites proportion larger.There is not yet the report of its chemical composition anti-halogen worm activity so far.
Summary of the invention
The object of this invention is to provide the methods and applications of extraction and isolation polyhydroxy sterol class monomeric compound from wrinkle knurl Ascidian, to overcome the deficiencies in the prior art.
For achieving the above object, this invention takes such technical measures: the preparation method of polyhydroxy sterol class monomeric compound, concrete technological step is as follows:
(1) fresh wrinkle knurl Ascidian is got, homogenate in tissue mashing machine, 4 times are extracted through 95% EtOH Sonicate ripple, each 30min, united extraction liquid, obtains medicinal extract when heat survey relative density is 1.1-1.4 when Rotary Evaporators concentrating under reduced pressure extracting solution to 35 DEG C, with the ratio that the mass ratio of medicinal extract and water is 1:1, use water-dispersion medicinal extract, use equivalent extraction into ethyl acetate after dispersion, when extraction liquid is evaporated to 35 DEG C, heat surveys relative density obtains ethyl acetate phase medicinal extract when being 1.1-1.4;
(2) ethyl acetate phase medicinal extract methyl alcohol is dissolved completely, with Sephadax LH-20 gel filtration chromatography, methanol-eluted fractions, then RP-18 reversed-phase silica gel column chromatography is used, methanol/water concentration is 30% respectively, 40%, 50%, 60%, 70%, 80%, 90%, 100% carries out gradient elution, finally use preparative high-performance liquid chromatographic, 80% methanol-water wash-out, obtain 2 pure compounds, be respectively compound 1 and compound 2, the physico-chemical property of compound and spectral data are analyzed, and contrast with document, compound 1 is accredited as: dendronesterol A, compound 2 is accredited as: (20S, 22E, 24R)-ergosta-7, 22-dien-3 β, 5 α, 6 β-triol, its structural formula is respectively:
The anti-halogen worm activity test method that the invention provides 2 polyhydroxy sterol monomeric compounds is: according to Solis improved method, get 24 well culture plates, every hole adds the solution (every hole 20 halogen worm) of 1mL containing artemia larvae, each compound arranges 50 μ g/mL, 25 μ g/mL, 10 μ g/mL tri-final concentrations, blank group (adding the seawater 1mL processed) and DMSO control group (DMSO containing with the suitable concentration of test sample) are set simultaneously, three Duplicate Samples established by the sample of each concentration, make test cultures plate, after cultivating 24h, record halogen worm dead individuals number.
Result evaluation: corrected mortality %=[(DMSO control group survival rate-sample sets survival rate)/DMSO control group survival rate] × 100%.
Anti-halogen worm according to aforesaid method detection compound 1 and compound 2 is active, when concentration is 50 μ g/mL, 87.4% and 82.9% are respectively to the lethality rate of halogen worm, and along with the rising killing rate of concentration also raises, show concentration dependent, can be used for the application preparing pest-resistant medicine.
Take the present invention of above-mentioned measure, preparation method is simple, and cost is low, little to the pollution of environment; The research and development that the monomeric compound obtained is marine drug provides basic data.The compound of separation and purification of the present invention, dendronesterol A and (20S, 22E, 24R)-ergosta-7,22-dien-3 β, 5 α, 6 β-triol are all good to the activity of halogen worm.Halogen worm has the advantages such as wide material sources, amount that is simple to operate, required compound be less as the model organism of bioactivity screening, the screening that application halogen worm carries out insecticide active substance is a kind of method that cost is low, efficiency is high, application of the present invention can shorten the cycle of new drug initiative greatly, significant to the research and development of new drug.
Embodiment
Embodiment 1
Preparation method:
(1) fresh wrinkle knurl Ascidian weight in wet base 1.5kg is got, homogenate in tissue mashing machine, 4 times are extracted through 95% EtOH Sonicate ripple, each 30min, united extraction liquid, obtains medicinal extract 1.5g, with 1500g water-dispersion medicinal extract when heat survey relative density is 1.1-1.4 when Rotary Evaporators concentrating under reduced pressure extracting solution to 35 DEG C, use equivalent extraction into ethyl acetate after dispersion, when extraction liquid is evaporated to 35 DEG C, heat surveys relative density obtains ethyl acetate phase medicinal extract 700.0mg when being 1.1-1.4;
(2) ethyl acetate phase medicinal extract methyl alcohol is dissolved completely, with Sephadax LH-20 gel filtration chromatography, methanol-eluted fractions, then RP-18 reversed-phase silica gel column chromatography is used, methanol/water concentration is 30% respectively, 40%, 50%, 60%, 70%, 80%, 90%, 100% carries out gradient elution, finally use preparative high-performance liquid chromatographic, 80% methanol-water wash-out, obtain 2 pure compounds, be respectively compound 1 and compound 2, the physico-chemical property of compound and spectral data are analyzed, and contrast with document, compound 1 is accredited as: dendronesterol A, compound 2 is accredited as: (20S, 22E, 24R)-ergosta-7, 22-dien-3 β, 5 α, 6 β-triol, its structural formula is respectively:
The physical constant of compound and spectral data are: compound dendronesterol A: white, needle-shaped crystals, ESI-MS m/z:461 [M-H
2o+H]
+.
1H NMR(C
5D
5N,600MHz)δ:2.20(1H,m,H-1a),1.56(1H,m,H-1b),2.27(1H,m,H-2a),2.06(1H,m,H-2b),4.86(1H,m,H-3),2.94(1H,dd,J＝12.7,11.4Hz,H-4a),2.33(1H,dd,J＝12.7,4.9Hz,H-4b),4.16(1H,br s,H-6),2.22(1H,m,H-7a),1.72(1H,m,H-7b),2.24(1H,m,H-8),2.06(1H,m,H-9),1.66(1H,m,H-11a),1.58(1H,m,H-11b),2.53(1H,dt,J＝13.2,3.3Hz,H-12a),1.66(1H,m,H-12b),1.35(1H,m,H-14),1.66(1H,m,H-15a),1.04(1H,m,H-15b),1.86(1H,m,H-16a),1.50(1H,m,H-16b),1.25(1H,m,H-17),4.54(1H,d,J＝11.8Hz,H-18a),4.09(1H,d,J＝11.8Hz,H-18b),1.64(3H,s,H-19),1.57(1H,m,H-20),1.14(3H,d,J＝6.6,H-21),1.38(1H,m,H-22a),1.06(1H,m,H-22b),1.38(1H,m,H-23a),1.18(1H,m,H-23b),1.35(2H,m,H-24),1.14(1H,m,H-25),0.88(3H,d,J＝6.6Hz,H-26),0.88(3H,d,J＝6.6Hz,H-27),Ac2.08(3H,s)；
13C NMR(C
5D
5N,150MHz)δ:33.6(CH
2,C-1),32.8(CH
2,C-2),68.2(CH,C-3),43.1(CH
2,C-4),76.7(C,C-5),77.1(CH,C-6),42.5(CH
2,C-7),31.8(CH,C-8),46.2(CH,C-9),39.4(C,C-10),22.1(CH
2,C-11),36.2(CH
2,C-12),46.2(C,C-13),55.9(CH,C-14),24.6(CH
2,C-15),28.5(CH
2,C-16),55.7(CH,C-17),64.0(CH
3,C-18),17.5(CH
2,C-19),36.6(CH,C-20),19.6(CH
3,C-21),36.8(CH
2,C-22),24.2(CH
2,C-23),28.5(CH
2,C-24),40.1(CH,C-25),23.0(CH
3,C-26),23.3(CH
3,C-27),Ac21.3,170.9。
Compound (20S, 22E, 24R)-ergosta-7,22-dien-3 β, 5 α, 6 β-triol: white, needle-shaped crystals, ESI-MS m/z413 [M-H
2o+H]
+.
1H NMR(C
5D
5N,600MHz)δ:4.83(1H,m,H-3),4.30(1H,br s,H-6),5.72(1H,br s,H-7),0.53(3H,s,H-18),1.50(3H,s,H-19),1.03(3H,d,J＝6.7Hz,H-21),5.24(1H,dd,J＝15.2,7.2Hz,H-22),5.17(1H,dd,J＝15.2,7.2Hz,H-23),0.83(6H,d,J＝4.9Hz,H-26,H-27),0.92(3H,d,J＝6.7Hz,H-28)；
13C NMR(C
5D
5N,150MHz)δ:32.6(CH
2,C-1),33.8(CH
2,C-2),67.5(CH,C-3),41.9(CH
2,C-4),76.1(C,C-5),74.2(CH,C-6),120.4(CH,C-7),141.5(C,C-8),43.7(C,C-9),38.0(C,C-10),22.4(CH
2,C-11),39.8(CH
2,C-12),43.7(C,C-13),55.2(CH,C-14),23.4(CH
2,C-15),28.4(CH
2,C-16),56.1(CH,C-17),12.5(CH
3,C-18),18.8(CH
3,C-19),40.8(CH,C-20),39.8(CH
3,C-21),136.1(CH,C-22),132.0(CH,C-23),43.1(CH,C-24),33.3(CH,C-25),20.1(CH
3,C-26),21.3(CH
3,C-27),117.8(CH
3,C-28)。
Respectively to the anti-halogen worm determination of activity of above-mentioned 2 polyhydroxy sterol compounds:
According to Solis improved method, get 24 well culture plates, every hole adds the solution (every hole 20 halogen worm) of 1mL containing artemia larvae, and each compound arranges 50 μ g/mL, 25 μ g/mL, 10 μ g/mL tri-final concentrations, arrange blank group (adding the seawater 1mL processed) and DMSO control group (DMSO containing with the suitable concentration of test sample), three Duplicate Samples established by the sample of each concentration, make test cultures plate simultaneously, after cultivating 24h, record halogen worm dead individuals number.
Result evaluation: corrected mortality %=[(DMSO control group survival rate-sample sets survival rate)/DMSO control group survival rate] × 100%
Activity Results shows, as shown in table 1:
Table 1, the halogen worm killing activity of compound
Table1 Lethal effects on brine shrimp A.salina of the compounds from S.plicata
Table 1 result is pointed out, and 2 polyhydroxy sterol compounds show stronger halogen worm killing activity, and in concentration dependent, compound 1 and 2 is respectively 87.4% and 82.9% when 50 μ g/mL to the lethality rate of halogen worm, and insecticidal effect is good.
Claims (2)
1. the preparation method of polyhydroxy sterol class monomeric compound, is characterized in that the concrete technological step of described method is:
(1) fresh wrinkle knurl Ascidian is got, homogenate in tissue mashing machine, 4 times are extracted through 95% EtOH Sonicate ripple, each 30min, united extraction liquid, obtains medicinal extract when heat survey relative density is 1.1-1.4 when Rotary Evaporators concentrating under reduced pressure extracting solution to 35 DEG C, with the ratio that the mass ratio of medicinal extract and water is 1:1, use water-dispersion medicinal extract, use equivalent extraction into ethyl acetate after dispersion, when extraction liquid is evaporated to 35 DEG C, heat surveys relative density obtains ethyl acetate phase medicinal extract when being 1.1-1.4;
(2) ethyl acetate phase medicinal extract methyl alcohol is dissolved completely, with Sephadax LH-20 gel filtration chromatography, methanol-eluted fractions, then RP-18 reversed-phase silica gel column chromatography is used, methanol/water concentration is 30% respectively, 40%, 50%, 60%, 70%, 80%, 90%, 100% carries out gradient elution, finally use preparative high-performance liquid chromatographic, 80% methanol-water wash-out, obtain 2 pure compounds, be respectively compound 1 and compound 2, the physico-chemical property of compound and spectral data are analyzed, and contrast with document, compound 1 is accredited as: dendronesterol A, compound 2 is accredited as: (20S, 22E, 24R)-ergosta-7, 22-dien-3 β, 5 α, 6 β-triol, its structural formula is respectively:
2. the application of polyhydroxy sterol class monomeric compound as claimed in claim 1 in the anti-halogen worm medicine of preparation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105315192A (en) * | 2015-06-17 | 2016-02-10 | 厦门医学高等专科学校 | Method for separating 1-hydrogen-3-formylindole from styela plicata |
| CN108409537A (en) * | 2018-02-07 | 2018-08-17 | 厦门医学院 | A kind of method and its decadinene alcohol compound detaching decadinene alcohol compound from the tumor ascidian that wrinkles |
| CN112889833A (en) * | 2021-02-01 | 2021-06-04 | 苏州推动者生物科技有限公司 | Method for preventing and treating plant virus diseases |
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| CN1803831A (en) * | 2006-01-23 | 2006-07-19 | 南方医科大学 | Acidian polypeptide and preparation method thereof |
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| CN1803831A (en) * | 2006-01-23 | 2006-07-19 | 南方医科大学 | Acidian polypeptide and preparation method thereof |
| KR20100127601A (en) * | 2009-05-26 | 2010-12-06 | 공해순 | Compositions and powder making methods for the anti-inflammatory utilizing styela clava, styela plicata or a sea squirt |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105315192A (en) * | 2015-06-17 | 2016-02-10 | 厦门医学高等专科学校 | Method for separating 1-hydrogen-3-formylindole from styela plicata |
| CN105315192B (en) * | 2015-06-17 | 2017-11-10 | 厦门医学院 | A kind of method that the indolecarboxaldehyde of 1 hydrogen 3 is separated in the knurl ascidian from wrinkle |
| CN108409537A (en) * | 2018-02-07 | 2018-08-17 | 厦门医学院 | A kind of method and its decadinene alcohol compound detaching decadinene alcohol compound from the tumor ascidian that wrinkles |
| CN108409537B (en) * | 2018-02-07 | 2021-01-05 | 厦门医学院 | Method for separating decadienol compound from ascidians plicata and decadienol compound thereof |
| CN112889833A (en) * | 2021-02-01 | 2021-06-04 | 苏州推动者生物科技有限公司 | Method for preventing and treating plant virus diseases |
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