CN1817899A - Polypeptide of ascidicea and preparation thereof - Google Patents
Polypeptide of ascidicea and preparation thereof Download PDFInfo
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- CN1817899A CN1817899A CNA2006100330613A CN200610033061A CN1817899A CN 1817899 A CN1817899 A CN 1817899A CN A2006100330613 A CNA2006100330613 A CN A2006100330613A CN 200610033061 A CN200610033061 A CN 200610033061A CN 1817899 A CN1817899 A CN 1817899A
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Abstract
Ascidian polypeptide and its production are disclosed. The procedure is carried out by immersion depressing at normal temperature by alcohol, recovering alcohol to obtain extract, extracting by organic solvent, concentrating, separation purifying by macroporous resin chromatography, silica-gel chromatography and Sephadex LH20 chromatography, gradient eluting by eluent in proportion of CHCl3: MeOH: H2O=7:3:0.5, and collecting concentrated eluent with thin-layer chromatographic ultraviolet verification Rf=0.5~0.6. The molecular weight of ascidian polypeptide is 711.4. It is simple, has less investment, convenient control for production and various raw materials.
Description
Invention field
The invention belongs to biochemistry and biomedicine field, be specifically related to Acidian polypeptide and preparation method thereof.
Background technology
Ascidian (Ascidia) is the urochordate of Urochordata (Uronordata) guiding principle, and the existing minority of the anticancer component that extracts from Ascidian enters clinical or preclinical phase.Discover that the ascidian biology contains tens of kinds of compounds such as alkaloids, peptide class, indoles, heavy metal chelant, polysulfide, macrolide, terpene, have stronger antitumor, antiviral, antibiotic, induce sarcoplasmic reticulum to release calcium, suppress multiple physiologically active such as calmodulin activity, especially so that more (Li Zhijun is reported in antineoplastic research, Xue Changhu, Wang Changhai. the anti-tumor biologically active substance in the Ascidian.The ocean circular, 2004,23 (5): 82-86; Ji Yubin, Chi Wenjie. the research of anti-tumor active substance in the Ascidian. Harbin University of Commerce's journal (natural science edition), 2005,21 (1): 6-10.).Chinese patent CN03111710.4 mentions and extract the Ascidian lipid acid with anticoagulant and blood coagulation resisting function from Ascidian, Chinese patent CN01812043.1 mentions extracting to have antitumor and the ecteinascidin antiviral activity effect from Ascidian, Chinese patent CN03807576.8 mentions ecteinascidin and preparation method thereof.The patent application of ascidian at present has report at aspects such as ecteinascidin and lipid acid, and extracts Acidian polypeptide and the application in the treatment hepatitis B does not appear in the newspapers as yet from Ascidian.
Summary of the invention
The object of the invention provides a kind of Acidian polypeptide with anti-HBV effect.
Another purpose of the present invention provides a kind of preparation method with Acidian polypeptide of anti-HBV effect.
Acidian polypeptide of the present invention is represented with following list type:
Lys-Gly-Lys-Ile-Glu-His(1)。
Lys is a Methionin in the formula, and Gly is a glycine, and Ile is an Isoleucine, and Glu is a L-glutamic acid, and His is a Histidine.
The Acidian polypeptide of formula of the present invention (1) expression is light yellow needle crystal, the ninhydrin reaction positive, and molecular weight is 711.4.
Acidian polypeptide preparation method of the present invention contains following steps: at first use the alcohol immersion Ascidian, reclaim ethanol and obtain medicinal extract; With the medicinal extract water dissolution, obtain the Ascidian crude extract with organic solvent extraction then; Using chromatography to separate at last purifies.
Ascidian crude extract preparation method is among the Acidian polypeptide preparation method of the present invention, soaks for 2 weeks with 70-90% (volumetric concentration v/v) alcohol at normal temperature of weight such as Ascidian, and decompression recycling ethanol gets medicinal extract; With the medicinal extract water dissolution, use organic solvent extraction then, concentrated aqueous solution obtains the Ascidian crude extract.
The consumption of water of the present invention is 2～3 times of medicinal extract.Wherein said multiple is meant that the volume (ml) of water is the multiple of medicinal extract quality (g).
Organic solvent of the present invention be ethyl acetate, chloroform, methylene dichloride, propyl carbinol, ether, sherwood oil, normal hexane, hexanaphthene wherein one or more.Organic solvent of the present invention better is that methylene dichloride or chloroform are wherein a kind of, preferably chloroform.Extraction times of the present invention is 2～4 times.The amount ratio of each used consumption of organic solvent of extraction of the present invention and water is volume ratio 1: 1.
Chromatography of the present invention can be macroporous resin chromatography, silica gel chromatography, Sephadex LH20 chromatography wherein one or more.Chromatography of the present invention is preferably used macroporous resin chromatography, silica gel chromatography, SephadexLH20 chromatography successively.
Macroporous resin chromatography of the present invention is this area macroporous resin chromatographic process commonly used.Its concrete steps are: behind the macroporous resin chromatography, colourless to post on the Ascidian crude extract with 50% ethanol elution, and concentrate drying.Macroporous resin of the present invention is non-polar macroporous resin preferably.Macroporous resin of the present invention is nonpolar or low-pole polystyrene macroporous resin, for example HPD-100, D-101, AB-8, HP-20, HPD-300.Macroporous resin of the present invention is the D-101 macroporous adsorbent resin preferably.
Silica gel column chromatogram method of the present invention is this area silica gel chromatography method commonly used.Those skilled in the art can obtain best parameter according to this area knowledge.Silica gel chromatography method concrete steps of the present invention are: behind silica gel chromatographic column on the Acidian polypeptide crude extract, use CHCl
3: MeOH: H
2The O ratio is 7: 3: 0.5 an eluent gradient elution, and flow velocity is 2～3ml/ minute, collects the inspection of thin-layer chromatography UV-light and knows R
fBe 0.5～0.6 elutriant, concentrate.Thin-layer chromatography condition wherein: developping agent is a propyl carbinol: water: acetic acid is 4: 5: 1; Developer is 5% sulfuric acid ethanol liquid.
Sephadex LH20 chromatography of the present invention, concrete steps are: with Sephadex LH20 gel chromatographic columns on the Acidian polypeptide crude extract, use CH
3OH: H
2O was with 1: 1 eluent wash-out, and flow velocity is 1～2ml/ minute, collected the inspection of thin-layer chromatography UV-light and knew R
fBe about 0.55 elutriant, concentrate.Thin-layer chromatography condition wherein: developping agent is a propyl carbinol: water: acetic acid is 4: 5: 1; Developer is 5% sulfuric acid ethanol liquid.
Sephadex LH20 of the present invention is the hydroxypropyl dextrane gel.
Ascidian of the present invention (Ascidian) can be the urochordate wrinkle knurl Ascidian [Styela plicata (lesueur)] of Urochordata (Uronordata) Ascidiacea.
Acidian polypeptide preparation method of the present invention can also carry out pre-treatment to Ascidian, concrete steps be with fresh Ascidian clean, drain, dry, pulverize; Drying temperature is lower than 80 degree; That drying comprises is air-dry, oven dry, vacuum-drying etc.
The preparation method of Acidian polypeptide of the present invention preferably 70～90% (volumetric concentration) alcohol at normal temperature of weight such as at first uses Ascidian to soak for 2 weeks, and decompression recycling ethanol gets medicinal extract; With the water dissolution of medicinal extract with 2～3 times, use the chloroform extraction identical then 2～4 times with volume of water, concentrate and obtain the Ascidian crude extract; Behind macroporous resin chromatography on the Ascidian crude extract, colourless to post with 50% ethanol elution, concentrate drying obtains the Acidian polypeptide crude extract; Behind silica gel chromatographic column on the above-mentioned Acidian polypeptide crude extract, use CHCl
3: MeOH: H
2The O ratio is 7: 3: 0.5 an eluent gradient elution, and flow velocity is 2～3ml/ minute, collects the inspection of thin-layer chromatography UV-light and knows R
fBe 0.5～0.6 elutriant, concentrate and obtain the Acidian polypeptide crude extract; Behind Sephadex LH20 gel chromatographic columns on the above-mentioned Acidian polypeptide crude extract, use CH
3OH: H
2O was with 1: 1 eluent wash-out, and flow velocity is 1～2ml/ minute, collected the inspection of thin-layer chromatography UV-light and knew R
fBe about 0.55 elutriant, concentrate and obtain Acidian polypeptide.
Acidian polypeptide of the present invention has anti-HBV effect.For a better understanding of the present invention, with relevant pharmacological testing of the present invention and result its purposes is described below.
The method of anti-HBV cell experiment of the present invention: with 0.25% trypsinase the 2.2.15 cell is dispersed into the individual cells suspension, by 3 * 10
4The cells/well branch is planted in 96 orifice plates, uses the pastille nutrient solution instead after 2 days, and each concentration adds 4 holes.Sample with cell culture fluid be diluted to 16,8 respectively, 3 concentration of 4mg/ml, with cytosis after 12 days, draw cell conditioned medium liquid, measure HBsAg, HBeAg titre with the ELISA method.Remaining cell is measured the cytotoxicity of medicine with mtt assay.Experiment with lamifudin as the positive control medicine, with the cell that do not add any medicine as the cell control group.
1.1 the ELISA method is surveyed HBsAg, HBeAg titre.
1.1.1 every hole adds sample 50 μ l to be measured, establishes feminine gender, each 2 hole of positive control, and establishes blank 1 hole.
1.1.2 every hole adds enzyme conjugates 50 μ l (except that blank), fully shrouding behind the mixing is put 37 ℃ and was hatched 8 hours.
1.1.3 manual wash plate: discard the liquid hole in, full each hole of washings storage is left standstill after 5 seconds and is dried, and pats dry after repeating 5 times.
1.1.4 every hole adds developer A liquid, each 50 μ l of B liquid, abundant mixing, and shrouding is put 37 ℃ and was hatched 15 minutes;
1.1.5 every hole adds stop buffer 50 μ l, mixing;
1.1.6 measure with microplate reader.Get wavelength 450nm, with blank well school zero, read each hole OD value then earlier, negative control OD value is lower than 0.05 as 0.05 calculating, is higher than 0.05 according to actual OD calculating.
1.2 mtt assay is measured the half toxic concentration of medicine cell growth.
In cell, add 400 μ g/ml MTT liquid 0.1ml/ holes, 37 ℃ of 5%CO
2Hatch 4h, visible yellow black first a ceremonial jade-ladle, used in libation particle discards MTT liquid, add 100%DMSO 0.1ml/ hole, after treating that first a ceremonial jade-ladle, used in libation particle dissolves (about 10min) fully, measure the absorbance A value in wavelength 570nm place with ultraviolet spectrophotometer, the 100%DMSO that does not add cell with 4 holes is a blank.
1.3 the calculating of experimental result.
Half-inhibition concentration (IC
50) be that HBsAg or HBeAg inhibiting rate are 50% o'clock drug level, half toxic concentration (TC
50) be the concentration of experimental port survivaling cell when being cell control well 50%.
The anti-HBV effect of Acidian polypeptide is as shown in table 1, and the toxic action of Acidian polypeptide pair cell is as shown in table 2.According to calculating, Acidian polypeptide of the present invention suppresses the medium effective concentration difference 3.65mg/ml of HBsAg, and the medium effective concentration that suppresses HBeAg is respectively 4.40mg/ml.This polypeptide all shows certain toxicity when high density, but in survey concentration, cell-damaging rate does not all reach 50%, so its half toxic concentration TC
50Greater than 64mg/ml, it suppresses HBsAg therapeutic index TI greater than 17.53, suppresses HBeAg therapeutic index TI greater than 14.54.
The anti-HBV effect of table 1 Acidian polypeptide
| Sample | Dosage mg.mL -1 | HBsAg | HBeAg | ||
| OD±S | Inhibiting rate % | OD±S | Inhibiting rate % | ||
| The Acidian polypeptide lamifudin | 64 32 16 8 4 4 2 1 | 0.071±0.001 0.085±0.004 0.120±0.002 0.301±0.037 0.487±0.087 0.110±0.004 0.275±0.008 0.541±0.010 | 92.82 * 91.28 * 87.42 * 67.44 * 46.91 * 88.52 * 70.31 * 40.94 * | 0.103±0.014 0.164±0.012 0.309±0.097 0.492±0.035 0.676±0.074 0.299±0.067 0.464±0.041 0.793±0.123 | 92.42 * 87.76 * 76.66 * 62.66 * 48.58 * 77.43 * 64.80 * 39.63 * |
| Cell contrast blank | 0.912±0.007 0.006 | 1.311±0.007 0.004 | |||
* compare p＜0.05 with the cell contrast.
The toxic action of table 2 Acidian polypeptide pair cell
| Sample | Dosage mg.mL -1 | OD±S | Destructive rate (%) |
| The Acidian polypeptide lamifudin | 64 32 16 8 4 4 2 1 | 0.712±0.035 * 0.875±0.074 * 0.981±0.099 1.121±0.100 1.103±0.103 1.090±0.114 1.241±0.201 1.115±0.123 | 35.83 21.12 11.55 - - 1.71 - - |
| The contrast of blank cell | 0.001 1.109±0.095 | ||
* compare p＜0.05 with the cell contrast.
In Acidian polypeptide of the present invention and preparation method thereof, Ascidian is distributed widely in marine site, the South Sea, and output is abundant, is convenient to gather, and extracting method technology is simple, and facility investment is few, and production process is convenient to control.
Embodiment
Following examples make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.
Embodiment 1
Fresh wrinkle knurl Ascidian 50Kg removes internal organ, cleans, and at room temperature soaks for 2 weeks with 75% ethanol 50kg, uses the rotatory evaporator recovered alcohol, gets extract 1000g.Get extract 200g 500ml dissolved in distilled water,, receive dried solvent and obtain water-soluble portion 825g with the dichloromethane extraction of 500ml; With the dissolving as far as possible of 500ml distilled water,, receive dried solvent and get water-soluble portion 712g again with the n-butanol extraction of equal volume; The dissolubility part of then fetching water 100g, last D-101 macroporous resin column chromatography, with 50% ethanol 500ml wash-out, flow velocity is 3ml/ minute, receives dried solvent and gets product 14g; Carry out silica gel column chromatography with this, use CHCl
3: MeOH: H
2The O ratio is 7: 3: 0.5 an eluent gradient elution, and flow velocity is 3ml/ minute, collects the inspection of thin-layer chromatography UV-light and knows R
fBe 0.5～0.6 elutriant, receive dried solvent and get the Acidian polypeptide crude extract, with Sephadex LH20 gel column chromatography on this, use CH at last
3OH: H
2O was with 1: 1 eluent wash-out, and flow velocity is 1ml/ minute, collected the inspection of thin-layer chromatography UV-light and knew R
fBe about 0.55 elutriant, receive dried solvent and obtain Acidian polypeptide 146mg.Above-mentioned thin layer is washed condition: developping agent is a propyl carbinol: water: acetic acid is 4: 5: 1; Developer is with 5% sulfuric acid ethanol liquid.
Embodiment 2
Fresh wrinkle knurl Ascidian 50Kg removes internal organ, cleans, and at room temperature soaks for 2 weeks with 75% ethanol 50kg, to tasteless, gets extract 1000g with 60 ℃ of recovered alcohols of rotatory evaporator.Get extract 200g with the dissolving as far as possible of 500ml distilled water,, receive dried solvent and obtain water-soluble portion 805g with the chloroform extraction of equal volume; With the dissolving as far as possible of 500ml distilled water,, receive dried solvent and get water-soluble portion 683g again with the n-butanol extraction of equal volume; The dissolubility part of then fetching water 100g, last D-101 macroporous resin column chromatography, with 50% ethanol 500ml wash-out, flow velocity is 2.5ml/ minute, receives dried solvent and gets product 10g; Carry out silica gel column chromatography with this, use CHCl
3: MeOH: H
2The O ratio is 7: 3: 0.5 an eluent gradient elution, and flow velocity is 2.5ml/ minute, collects the inspection of thin-layer chromatography UV-light and knows R
fBe 0.5～0.6 elutriant, receive dried solvent and get crude product, go up Sephadex LH20 gel column chromatography respectively with this at last, use CH
3OH: H
2O was with 1: 1 eluent wash-out, and flow velocity is 1.5ml/ minute, collected the inspection of thin-layer chromatography UV-light and knew R
fBe about 0.55 elutriant, receive dried solvent and obtain Acidian polypeptide 102mg.Above-mentioned thin-layer chromatography condition: developping agent is a propyl carbinol: water: acetic acid is 4: 5: 1; Developer is with 5% sulfuric acid ethanol liquid.
Embodiment 3
Fresh wrinkle knurl Ascidian 50Kg removes internal organ, cleans, and at room temperature soaks for 2 weeks with 90% ethanol 50kg, to tasteless, gets extract 1300g with 60 ℃ of recovered alcohols of rotatory evaporator.Get extract 200g 600ml dissolved in distilled water,, receive dried solvent and obtain water-soluble portion 830g with the chloroform extraction of equal volume; Above-mentioned extracting process repeats 3 times, receives dried solvent and gets water-soluble portion 730g; The dissolubility part of then fetching water 100g, last AB-8 macroporous resin column chromatography, with 50% ethanol 500ml wash-out, flow velocity is 2ml/ minute, receives dried solvent and gets product 15g; Carry out silica gel column chromatography with this, use CHCl
3: MeOH: H
2The O ratio is 7: 3: 0.5 an eluent gradient elution, and flow velocity is 3ml/ minute, collects the inspection of thin-layer chromatography UV-light and knows R
fBe 0.5～0.6 elutriant, receive dried solvent and get the Acidian polypeptide crude extract, with Sephadex LH20 gel column chromatography on this, use CH3OH at last: H2O was with 1: 1 eluent wash-out, and flow velocity is 2ml/ minute, collects the inspection of thin-layer chromatography UV-light and knows R
fBe about 0.55 elutriant, receive dried solvent and obtain Acidian polypeptide 126mg.Above-mentioned thin-layer chromatography condition: developping agent is a propyl carbinol: water: acetic acid is 4: 5: 1; Developer is with 5% sulfuric acid ethanol liquid.
Embodiment 4
Fresh wrinkle knurl Ascidian 50Kg removes internal organ, cleans, and at room temperature soaks for 2 weeks with 80% ethanol 50kg, to tasteless, gets extract 1300g with 60 ℃ of recovered alcohols of rotatory evaporator.Get extract 200g 400ml dissolved in distilled water,, receive dried solvent and obtain water-soluble portion 825g with the chloroform extraction of equal volume; Use the 600ml dissolved in distilled water,, receive dried solvent and obtain water-soluble portion 725g with the ethyl acetate extraction of equal volume; Use the 600ml dissolved in distilled water,, receive dried solvent and obtain water-soluble portion 605g, receive dried solvent and get water-soluble portion 512g with the n-hexane extraction of equal volume; The dissolubility part of then fetching water 100g, last D-101 macroporous resin column chromatography, with 50% ethanol 500ml wash-out, flow velocity is 2ml/ minute, receives dried solvent and gets product 13g; Carry out silica gel column chromatography with this, use CHCl
3: MeOH: H
2The O ratio is 7: 3: 0.5 an eluent gradient elution, and flow velocity is 3ml/ minute, collects the inspection of thin-layer chromatography UV-light and knows R
fBe the elutriant of 0.5-0.6, receive dried solvent and get the Acidian polypeptide crude extract, with Sephadex LH20 gel column chromatography on this, use CH3OH at last: H2O was with 1: 1 eluent wash-out, and flow velocity is 2ml/ minute, collected the inspection of thin-layer chromatography UV-light and knew R
fBe about 0.55 elutriant, receive dried solvent and obtain Acidian polypeptide 146mg.The thin-layer chromatography condition: developping agent is a propyl carbinol: water: acetic acid is 4: 5: 1; Developer is with 5% sulfuric acid ethanol liquid.
Embodiment 5
Fresh wrinkle knurl Ascidian 50Kg removes internal organ, cleans, and at room temperature soaks for 2 weeks with 75% ethanol 50kg, to tasteless, gets extract 1000g with 60 ℃ of recovered alcohols of rotatory evaporator.Get extract 200g 500ml dissolved in distilled water,, use the ethyl acetate extraction of equal volume again, receive dried solvent and get water-soluble portion 712g with the dichloromethane extraction of equal volume; The dissolubility part of then fetching water 100g, last HDP-100 macroporous resin column chromatography, with 50% ethanol 500ml wash-out, flow velocity is 3ml/ minute, receives dried solvent and gets product 14g; Carry out silica gel column chromatography with this, use CHCl
3: MeOH: H
2The O ratio is 7: 3: 0.5 an eluent gradient elution, and flow velocity is 3ml/ minute, collects the inspection of thin-layer chromatography UV-light and knows R
fBe the elutriant of 0.5-0.6, receive dried solvent and get the Acidian polypeptide crude extract, with Sephadex LH20 gel column chromatography on this, use CH3OH at last: H2O was with 1: 1 eluent wash-out, and flow velocity is 2ml/ minute, collected the inspection of thin-layer chromatography UV-light and knew R
fBe about 0.55 elutriant, receive dried solvent and obtain Acidian polypeptide 146mg.The thin-layer chromatography condition: developping agent is a propyl carbinol: water: acetic acid is 4: 5: 1; Developer is with 5% sulfuric acid ethanol liquid.
Claims (7)
1. Acidian polypeptide is characterized in that having following sequence:
Lys-Gly-Lys-Ile-Glu-His, Lys is a Methionin in the formula, and Gly is a glycine, and Ile is an Isoleucine, and Glu is a L-glutamic acid, His is a Histidine.
2. the described Acidian polypeptide of claim 1 is characterized in that molecular weight is 711.4.
3. the preparation method of claim 1 or 2 described Acidian polypeptides, at first the alcohol at normal temperature with 70～90% volumetric concentrations of weight such as Ascidian soaked for 2 weeks, and decompression recycling ethanol gets medicinal extract; With medicinal extract with 2～3 times of water dissolution, use then with water with the organic solvent extraction of volume 2～4 times, concentrated aqueous solution obtains the Ascidian crude extract; Separate to purify obtaining at last successively with macroporous resin chromatography, silica gel chromatography, Sephadex LH20 chromatography, wherein silica gel chromatography is with behind the silica gel chromatographic column on the Acidian polypeptide crude extract, uses CHCl
3: MeOH: H
2The O ratio is 7: 3: 0.5 an eluent gradient elution, and flow velocity is 2～3ml/ minute, and collection thin-layer chromatography UV-light inspection knowledge Rf is 0.5～0.6 elutriant, and concentrate, wherein the thin-layer chromatography condition: developping agent is a propyl carbinol: water: acetic acid is 4: 5: 1; Developer is 5% sulfuric acid ethanol liquid.
4. the described Acidian polypeptide preparation method of claim 3, it is characterized in that organic solvent be ethyl acetate, chloroform, methylene dichloride, propyl carbinol, ether, sherwood oil, normal hexane, hexanaphthene wherein one or more.
5. the described Acidian polypeptide preparation method of claim 4 is characterized in that organic solvent is a chloroform.
6. the described Acidian polypeptide preparation method of claim 3 is characterized in that macroporous resin chromatography concrete steps are: behind the D-101 macroporous resin chromatography, colourless to post on the Ascidian crude extract with 50% ethanol elution, and concentrate drying.
7. the described Acidian polypeptide preparation method of claim 3 is characterized in that described Sephadex LH20 chromatography, and concrete steps are: with Sephadex LH20 gel chromatographic columns on the Acidian polypeptide crude extract, use CH
3OH: H
2O was with 1: 1 eluent wash-out, and flow velocity is 1～2ml/ minute, collected the inspection of thin-layer chromatography UV-light and knew R
fBe about 0.55 elutriant, concentrate, wherein the thin-layer chromatography condition: developping agent is a propyl carbinol: water: acetic acid is 4: 5: 1; Developer is 5% sulfuric acid ethanol liquid.
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|---|---|---|---|
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174096A (en) * | 2010-12-24 | 2011-09-07 | 中国科学院海洋研究所 | Ciona intestinalis polypeptide and preparation method thereof |
| CN102172394A (en) * | 2010-12-24 | 2011-09-07 | 中国科学院海洋研究所 | Application of ciona intestinalis linnaeus peptides |
| CN103421870A (en) * | 2012-05-14 | 2013-12-04 | 宁波博丰生物科技有限公司 | Preparation method of sea squirt low molecular polypeptides |
| 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 |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103314973B (en) * | 2013-06-13 | 2014-06-18 | 广西壮族自治区海洋研究所 | Method for extracting anti-artemia active ingredients from styela canopus |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1067933B1 (en) * | 1998-04-06 | 2007-08-15 | The Board Of Trustees Of The University Of Illinois | Semi-synthetic ecteinascidins |
-
2006
- 2006-01-20 CN CNB2006100330613A patent/CN100355775C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174096A (en) * | 2010-12-24 | 2011-09-07 | 中国科学院海洋研究所 | Ciona intestinalis polypeptide and preparation method thereof |
| CN102172394A (en) * | 2010-12-24 | 2011-09-07 | 中国科学院海洋研究所 | Application of ciona intestinalis linnaeus peptides |
| CN102172394B (en) * | 2010-12-24 | 2013-05-15 | 中国科学院海洋研究所 | Application of ciona intestinalis linnaeus peptides |
| CN103421870A (en) * | 2012-05-14 | 2013-12-04 | 宁波博丰生物科技有限公司 | Preparation method of sea squirt low molecular polypeptides |
| 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 |
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|---|---|
| CN100355775C (en) | 2007-12-19 |
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