CN102070725B - Sulfated galactan, and preparation method and application thereof - Google Patents
Sulfated galactan, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses sulfated galactan, which has the molecular weight of 8.5*10<4>. The mass percentage of the sulfate group is between 25 percent and 27 percent. A chemical structure is irregularly formed by a repeated unit A, a repeated unit B, a repeated unit C and a repeated unit D, wherein the mol percentage of the repeated unit A is 10 to 13 percent, the mol percentage of the repeated unit B is 6 to 7 percent, the mol percentage of the repeated unit C is 63 to 65 percent, the mol percentage of the repeated unit D is 17 to 19 percent, and the total value of the mol percentage of the four repeated units is 98 to 100 percent. The sulfated galactan is prepared according to the following steps: firstly, extracting grateloupia filicina by water, and then, carrying out alcohol deposition, freeze drying and separation purification operation on extracted liquid. The sulfated galactan disclosed by the invention can be used for preparing anti-blood-vessel-generation and/or anti-tumor medicine and can be particularly used for preparing anti-S-180-sarcoma and liver cancer medicine.
Description
Technical field
The present invention relates to a kind of galactan sulfate, specifically, relate to a kind of from the polyoses extract of Grateloupia filicina (Wulf.) the galactan sulfate of isolated a kind of homogeneous, belong to natural medicine technical field.
Background technology
Sulfated polysaccharides in the marine alga is anti-oxidant because of having, antiviral, anticoagulation and multiple biological activity such as antitumor, receives extensive concern both domestic and external in recent years.Grateloupia filicina (Wulf.) is a kind of red algae sea film section centipede Trentepohlia algae; Grateloupia filicina (Wulf.) has clearing heat and detoxicating and effect expelling parasite since ancient times just as a kind of ocean Chinese medicine, and aboundresources; In Qingdao of China, ground such as Dalian coastal have distribute widely, and eaten by the locals.Polysaccharide is one of staple of Grateloupia filicina (Wulf.), and result of study in recent years shows: it has preferably anticoagulation, biological activity such as antiviral, has demonstrated it and has had the potential value that is developed to medicine.Though Chinese patent document ZL 200510023551.0 discloses a kind of Grateloupia filicina (Wulf.) polyoses extract that comes into leaves, its preparation method and the purposes in preparation angiogenesis inhibitor and/or antitumor drug thereof; And the patent No. is that the patent of ZL200610026830.7 discloses the purposes of a kind of Grateloupia filicina (Wulf.) polyoses extract in preparation anticoagulation, antithrombotic, anti-AIDS and/or antitumor drug.But disclosed Grateloupia filicina (Wulf.) polyoses extract in the above-mentioned patent documentation; It is a kind of Crude polysaccharides that is grouped into by multiple one-tenth; To be applied to the polyoses extract in Grateloupia filicina (Wulf.) source clinical; Produce a kind of effectively, safety, the clear and definite medicine of mechanism of action, also need separate the effective constituent in its polyoses extract, structural confirmation and drug effect thereof further go deep into clinical study.
Summary of the invention
The purpose of this invention is to provide a kind of from the polyoses extract of Grateloupia filicina (Wulf.) the galactan sulfate of isolated a kind of homogeneous, can be used widely for the Grateloupia filicina (Wulf.) polyoses extract lays the foundation.
In order to realize the foregoing invention purpose, the technical scheme that the present invention adopts is following:
Galactan sulfate provided by the invention is characterized in that:
Molecular weight is 8.5 * 10
4The quality percentage composition of sulfate group is 25～27%;
Chemical structure is by repeating unit A, repeating unit B, repeating unit C and repeating unit D is random forms; Wherein: the molar content of repeating unit A is 10～13%; The molar content of repeating unit B is 6～7%; The molar content of repeating unit C is 63～65%, and the molar content of repeating unit D is 17～19%;
The chemical structural formula of repeating unit A is following:
The chemical structural formula of repeating unit B is following:
The chemical structural formula of repeating unit C is following:
The chemical structural formula of repeating unit D is following:
The summation of the molar content of above-mentioned four kinds of repeating units is 98～100%.
Also contain molar content in the galactan sulfate of the present invention and be 0～2% the D-semi-lactosi that 1,3 of acetone acidic group connects that contains.
The preparation method of galactan sulfate provided by the invention comprises following concrete operations step:
A) water extraction: shred after the Grateloupia filicina (Wulf.) of fresh collection rinsed out salt surfactant with tap water, add zero(ppm) water, transfer to pH=5～7, extracted 0.5～1.5 hour at 85～95 ℃ with Glacial acetic acid min. 99.5; Residue extracts 1 time by said process again; Merge 2 times extracting solution;
B) alcohol precipitation: under agitation add 2～4 times of volume of ethanol of said extracted liquid, leave standstill centrifugal collecting precipitation;
C) freeze-drying: gained is precipitated water-soluble, dialysed lyophilize then 24～72 hours;
D) separation and purification: after above-mentioned freeze dried polysaccharide used pH=7.2, the concentration phosphate buffer soln dissolving as 20mmol/L; With DEAE-Sepharose Fast Flow gel chromatography column chromatography; Sodium chloride solution and the pH=7.2, the concentration that earlier with concentration are 0.5mol/L are 2 times of column volumes of phosphate buffer soln wash-out of 20mmol/L; Use concentration to be the sodium chloride solution of 0.8mol/L and pH=7.2, concentration phosphate buffer soln wash-out again as 20mmol/L; The phenolsulfuric acid method detects collects, flowing water dialysis 24～72 hours, lyophilize; Use dissolved in distilled water again, with Sepharose CL-6B column purification, with the zero(ppm) water wash-out, the sulfuric acid phynol method detects to be collected, and lyophilize promptly gets galactan sulfate of the present invention.
Water extraction condition in the step a) preferentially is recommended as: transfer to pH=6 with Glacial acetic acid min. 99.5, extracted 1 hour at 90 ℃.
Alcohol precipitation condition in the step b) preferentially is recommended as: the volumn concentration that under agitation adds 3 times of volumes of said extracted liquid is 95% ethanol.
Dialysis time in the step c) preferentially is recommended as 48 hours.
Flowing water dialysis time in the step d) preferentially is recommended as 48 hours.
The present invention also provides the application of described galactan sulfate in preparation angiogenesis inhibitor and/or antitumor drug, the particularly application in the medicine of anti-S-180 sarcoma of preparation and liver cancer.
Compared with prior art, beneficial effect of the present invention is following:
The present invention first from the polyoses extract of Grateloupia filicina (Wulf.) separation and purification obtained a kind of galactan sulfate of homogeneous; And it has been carried out drug efficacy study; For the Grateloupia filicina (Wulf.) polyoses extract can prepare a kind of effectively, safety, the clear and definite medicine of mechanism of action provides valuable approach, the Grateloupia filicina (Wulf.) polyoses extract can be used widely becomes possibility.
Description of drawings
Fig. 1 is the high performance size exclusion chromatography figure (HPGPC figure) of the GFP08 sample that makes of embodiment 1.
Fig. 2 is the infared spectrum of the GFP08 sample that makes of embodiment 1.
Fig. 3 is the infared spectrum of GFP08 sample after base-modified processing that embodiment 1 makes.
Fig. 4 is the GFP08 sample that embodiment 1 makes
13The C-NMR collection of illustrative plates.
Fig. 5 is the inversion Photomicrograph of Human umbilical vein endothelial cells HUVEC tube chamber under the GFP08 of different concns effect, and wherein: a is the blank group, and b is 10mg/L GFP08 group, and c is 50mg/L GFP08 group, and d is 100mg/L GFP08 group.
Fig. 6 is the inhibiting mathematical statistics figure of the GFP08 of different concns to Human umbilical vein endothelial cells HUVEC tube chamber, and among the figure: * * representes to compare p＜0.01 with control group, and * * * representes to compare p＜0.001 with control group.
Fig. 7 induces group, 20%FBS to induce the inversion Photomicrograph of the Human umbilical vein endothelial cells HUVEC under group, positive drug control group and the drug group in nothing; Wherein: a does not induce group for having; B is that 20%FBS induces group; The positive medicine control group of c (20%FBS+10 μ M Suramin group), d is drug group (20%FBS+100mg.L
-1The GFP08 group).
Fig. 8 is the mathematical statistics figure that GFP08 carries out with the OD value Human umbilical vein endothelial cells HUVEC migration restraining effect, and among the figure: the positive medicine control group of FBS1 (20%FBS+10 μ M Suramin group), FBS2 is drug group (20%FBS+100mg.L
-1The GFP08 group); * expression is compared p＜0.05 with control group, and * * representes to compare p＜0.01 with control group.
Fig. 9 is the mathematical statistics figure that GFP08 carries out with the cell migration number Human umbilical vein endothelial cells HUVEC migration restraining effect, and among the figure: the positive medicine control group of FBS1 (20%FBS+10 μ M Suramin group), FBS2 is drug group (20%FBS+100mg.L
-1The GFP08 group); * representes to compare p＜0.01 with control group, and * * * representes to compare p＜0.001 with control group.
Figure 10 is that the GFP08 of 100mg/L is to the proteic expression figure of the TF of Human umbilical vein endothelial cells HUVEC.
Embodiment
Below in conjunction with embodiment the present invention is done further detailed, complete explanation:
One, preparation galactan sulfate
Shred after the Grateloupia filicina (Wulf.) of fresh collection rinsed out salt surfactant with tap water, add zero(ppm) water, transfer to pH=6, extracted 1 hour at 90 ℃ with Glacial acetic acid min. 99.5; Residue extracts 1 time by said process again; Merge 2 times extracting solution;
The volumn concentration that under agitation adds 3 times of volumes of said extracted liquid is 95% ethanol, leaves standstill centrifugal collecting precipitation;
Gained is precipitated water-soluble, dialysed lyophilize then 48 hours;
Getting the above-mentioned freeze dried polysaccharide of 3g uses 100mL pH=7.2, concentration to dissolve as the phosphate buffer soln of 20mmol/L; Use the DEAE-Sepharose Fast Flow gel chromatographic columns (chromatography of 2.6cm * 60cm) then; Sodium chloride solution and the pH=7.2, the concentration that earlier with concentration are 0.5mol/L are 2 times of column volumes of phosphate buffer soln wash-out of 20mmol/L; Use concentration to be the sodium chloride solution of 0.8mol/L and pH=7.2, concentration phosphate buffer soln wash-out again as 20mmol/L; The phenolsulfuric acid method detects collects, flowing water dialysis 48 hours, and lyophilize gets sample 500mg; This 500mg sample is used the 5mL dissolved in distilled water again; With the Sepharose CL-6B post (purifying of 2.6cm * 100cm); With the zero(ppm) water wash-out, the sulfuric acid phynol method detects, and collects 43～55 pipes (every pipe 5ml); Lyophilize promptly gets galactan sulfate of the present invention (being labeled as GFP08) sample 250mg.
Two, purity and molecular-weight determination
Detect with HPGPC; Detector is the differential detector, and chromatographic column is Shodex KS-805 and KS-804 series connection, the sodium chloride aqueous solution wash-out of 0.2mol/L; The VISOSE of different molecular weight is standard substance, calculates its molecular weight with AgilentGPC software according to elution time.
Prepared GFP08 sample detects to single symmetrical peak (seeing shown in Figure 1) through HPGPC, shows that galactan sulfate of the present invention is a homogeneous polysaccharide, and molecular weight is 8.5 * 10
4
Three, structure is identified
1, sugared compositional analysis
The monose that adopts the reductive water solution to measure the GFP08 sample is formed; Barium sulfate turbidimetry is measured sulfate content; The 2,4 dinitrophenyl hydrazine method is measured its acetone acidic group content, and D-and L-galactose content use chiral reagent (S)-(+)-Yi Bingchunan; According to Navarro, two one-step hydrolysis reductive ammonifications of report such as D.A. are measured.Measuring the result sees shown in the table 1.
The sugar of table 1GFP08 is formed and molecular weight
Annotate: 3, the absolute configuration of 6-Anhydrogalactose and 6-methylgalactose adopts
13C NMR confirms.
Can be found out by table 1: GFP08 is a galactan sulfate, and the ratio of D-semi-lactosi (D-semi-lactosi+6-methyl D-semi-lactosi) and L-semi-lactosi (L-semi-lactosi+L-3,6 Anhydrogalactoses) shows that near 1: 1 this polysaccharide is the agar-agar type structure.
2, methylation analysis
Can find out from the result that methylates (seeing shown in the table 2) of GFP08DS: GFP08 is mainly by 1; The 3 D-semi-lactosi, 2 the Sulfated D-semi-lactosis and 1 that connect; The Sulfated L semi-lactosi of prosposition of 4 connections, 3,6 dehydration L semi-lactosis, 6 Sulfated L semi-lactosis are formed.
The methylation analysis result of table 2GFP08
3, IR analyzes
(see shown in Figure 2) in the infared spectrum of GFP08: sulfate group (1260cm is arranged
-1) and 3,6-Anhydrogalactose (935cm
-1) charateristic avsorption band, 842cm
-1About absorption peak show that this polysaccharide sulfate the position of substitution is at 2,3 or 4, at 820cm
-1The place has acromion explanation to have 6 sulfates to replace; Through base-modified processing back (seeing shown in Figure 3), 820cm
-1The acromion at place weakens many, and this shows has 6 sulfates to be substituted on 1,4 semi-lactosi that connects, and after alkaline purification, 6 sulfates are sloughed and formed 3,6 Anhydrogalactoses with 3 hydroxyl condensations.
4,
13C NMR analyzes
GFP08's
13Anomeric carbon fignal center position is mainly four among the C NMR (seeing shown in Figure 4): 104.4,103.0,102.1,99.2; Ratio 99.2 more High-Field place does not have absorption peak; Show that this polysaccharide does not contain 1; α-L-the semi-lactosi of 4 connections, and the existence of 3,6 dehydration α-L-semi-lactosis (99.2) has confirmed that further its structure is the agar-agar type; Ownership is seen shown in the table 3 in detail.
Table 3GFP08
13The ownership of C NMR signal
Annotate: D represents the D-semi-lactosi of 1,3 connection, and D2S represents 2 Sulfated 1, the 3 D-semi-lactosis that connect; The DP representative contains the D-semi-lactosi that 1,3 of acetone acidic group connects, the D-semi-lactosi that on behalf of 6 methyl 1,3, D6M connect; The L-semi-lactosi that on behalf of prosposition two Sulfated 1,4, L2S3S connect, L6S represents 6 sulfations 1; The L-semi-lactosi that on behalf of 3,6 dehydrations 1,4, the L-semi-lactosi of 4 connections, LA connect.
aBecause content is low, poor TV signal Chu only makes the part ownership.
To sum up analyze and can know by inference: galactan sulfate of the present invention is by repeating unit A, repeating unit B, repeating unit C and repeating unit D is random forms; Wherein: the molar content of repeating unit A is 10～13%; The molar content of repeating unit B is 6～7%; The molar content of repeating unit C is 63～65%, and the molar content of repeating unit D is that the summation of the molar content of 17～19%, four kinds of repeating units is 98～100%; Possibly also contain molar content and be 0～2% the D-semi-lactosi that 1,3 of acetone acidic group connects that contains.
Embodiment 2
Get well-grown 7～11 days S-180 sarcoma knurl kind, knurl liquid is processed 1 * 10
7/ ml cell suspension, the right armpit subcutaneous vaccination of mouse 0.1ml/ only.Inoculate and divide cage at random after 24 hours, the GFP08 of different concns, successive administration 12 days are given in posterior vein injections in 24 hours.Positive drug 5-FU was administered once behind tumor inoculation in per two days.Put to death animal in 24 hours after the drug withdrawal, weigh, knurl is heavy, calculate that respectively to organize average knurl heavy, obtain tumor control rate and carry out the t check by following formula.
Tumor control rate (%)=(the average knurl weight-treatment of blank group group average tumor)/blank group average tumor * 100%.
The therapeutic evaluation standard: tumor control rate＜40% is invalid; Tumor control rate >=40%, and be effective through statistical procedures p＜0.05.Concrete data are referring to shown in the table 4.
Table 4GFP08 is to the influence of mouse S-180 sarcoma growth
Visible by table 4: mouse behind tumor inoculation continuously vein give the galactan sulfate of the present invention (GFP08) 12 days of 25mg/kg, 50mg/kg, 100mg/kg; 50mg/kg, 100mg/kg group all can significantly suppress the growth of S-180 sarcoma; Tumour inhibiting rate is respectively 54.7% and 68.9%; Effect is superior to disclosed antitumous effect in the one Chinese patent application 200610026830.7, shows that the Grateloupia filicina (Wulf.) polysaccharide has improved its antitumous effect after purified.Positive control drug 5-FU 25mg/kg per two days intravenously administrables behind tumor inoculation once significantly suppress the growth of S-180 sarcoma, and tumour inhibiting rate is 90.5%.
Embodiment 3
Bel7402 liver cancer cell liquid 90 μ l are implanted in the every hole of 96 orifice plates, and about 6000 cells were cultivated 24 hours.The GFP08 sample ligand is processed 1.0mg/ml, three kinds of different concentration of 0.1mg/ml and 0.01mg/ml, every then hole adds 100 μ l.Three holes of each concentration kind.Making a circle in week adds 200 μ l nutrient solutions, prevents fringing effect.Cultivated 3 days after the dosing.Whole liquid in the sucking-off hole, and wash one time with PBS.(preventing sample and MTT reaction) every then hole adds fresh MTT solution 20 μ l (5mg/ml), and vibration is 5 minutes on the shaking table.37 ℃ are continued to cultivate 4h behind the application of sample.Every hole adds 100 μ l DMSO, vibrates 5 minutes.Measure each hole absorbancy with enzyme-labeled immunity determinator 570nm place.Formula according to following calculates inhibiting rate, and the result sees shown in the table 5.
Inhibiting rate=(1-OD experimental group/OD control group) * 100%
Table 5GFP08 is to the influence of Bel-7402 liver cancer cell growth
| Concentration (mg/ml) | 1.0 | 0.1 | 0.01 |
| Inhibiting rate (%) | 98.4 | 70.0 | 20.5 |
Visible by table 5: the GFP08 of 1.0mg/ml has remarkable restraining effect to the BEL-7402 liver cancer cell growth, and inhibiting rate reaches 98.4%.
Embodiment 4
The every hole of 96 orifice plates is coated with the liquid ECMatix glue of 30 μ l, solidifies 45 minutes down at 37 ℃.Every hole adds the HUVEC cell suspension (3 * 10 of 100 μ l
4/ hole), add the medicine or the solvent of different concns, 37 ℃, 5%CO
2Cultivated 8 hours.With being inverted difference, calculate complete tube chamber immediately and form several N as document image under 250 times of mirrors of microscope.Tube chamber rate of formation=(N
Experimental group-N
Control group)/N
Control group* 100%.All experimental datas represent with mean ± standard deviation that all many groups are relatively adopted one-way analysis of variance, carry out statistical study with SPSS16.0 software.
Fig. 5 is the inversion Photomicrograph of Human umbilical vein endothelial cells HUVEC tube chamber under the GFP08 of different concns effect, and wherein: a is the blank group, and b is 10mg/L GFP08 group, and c is 50mg/L GFP08 group, and d is 100mg/L GFP08 group.
Fig. 6 is the inhibiting mathematical statistics figure of the GFP08 of different concns to Human umbilical vein endothelial cells HUVEC tube chamber, and among the figure: * * representes to compare p＜0.01 with control group, and * * * representes to compare p＜0.001 with control group.
Visible in conjunction with Fig. 5 and Fig. 6: the tube chamber that GFP08 all can significance ground 10,50, during 100mg/L suppresses the HUVEC cell forms and is dose-dependently.
Embodiment 5
The Millicell bottom film is immersed in 0.1% gelatin solution, and 37 ℃ of constant temperature 2h take out, and cell is inverted, and dries.Get one 24 orifice plate, establish respectively not have and induce group, 20%FBS to induce group, positive drug control group and drug group, does not wherein have to induce and add the M199 that 500 μ l contain 0.1%BSA in the group hole and train liquid, all the other are respectively organized the M199 that adding 500 μ l in the hole contain 20%FBS and train liquid.The Millicell that dries well is put into 24 orifice plates, in cell, add 100 μ l cell suspensions (3 * 10
5/ hole), add solvent or medicine successively, 37 ℃, 5%CO
2Cultivate 24h, take out, fixedly 2h is immersed in 4% Paraformaldehyde 96 in the Millicell bottom, wipe ventricular cell gently with cotton swab, following ventricular cell is counted (at random 5 visuals field average) to the 100X microscopically with 0.1% violet staining 30min after drying.Viola crystallina on the staining cell is dissolved in 300 μ l and contains in the solution of 30% acetate then, and the 600nm place measures absorbance value, cell migration rate=(OD
Experimental group/ OD
Control group)) * 100%.All experimental datas represent with mean ± standard deviation that all many groups are relatively adopted one-way analysis of variance, carry out statistical study with SPSS16.0 software.
Fig. 7 induces group, 20%FBS to induce the inversion Photomicrograph of the Human umbilical vein endothelial cells HUVEC under group, positive drug control group and the drug group in nothing; Wherein: a does not induce group for having; B is that 20%FBS induces group; The positive medicine control group of c (20%FBS+10 μ M Suramin group), d is drug group (20%FBS+100mg.L
-1The GFP08 group).
Fig. 8 is the mathematical statistics figure that GFP08 carries out with the OD value Human umbilical vein endothelial cells HUVEC migration restraining effect, and among the figure: the positive medicine control group of FBS1 (20%FBS+10 μ M Suramin group), FBS2 is drug group (20%FBS+100mg.L
-1The GFP08 group); * expression is compared p＜0.05 with control group, and * * representes to compare p＜0.01 with control group.
Fig. 9 is the mathematical statistics figure that GFP08 carries out with number of cells Human umbilical vein endothelial cells HUVEC migration restraining effect, and among the figure: the positive medicine control group of FBS1 (20%FBS+10 μ M Suramin group), FBS2 is drug group (20%FBS+100mg.L
-1The GFP08 group); * representes to compare p＜0.01 with control group, and * * * representes to compare p＜0.001 with control group.
Visible in conjunction with Fig. 7 to Fig. 9: 100mg/L GFP08 can suppress to significance the migration of external 20%FBS inductive HUVEC cell.
Embodiment 6
According to following treatment process, cell is with lysate (50mM Tris (pH7.5), 1mM EDTA; 150mM NaCl, 20mM NaF, 0.5%NP-40; 10% glycerine (glycerol), 1mM phenylmethylsulfonyl fluoride, 10 μ g/ml Trypsin inhibitor,Trasylols (aprotinin); 10 μ g/ml leupeptins (leupeptin), 10 μ g/ml pepstatin (pepstatin) A carry out cracking, are adjusted to the isopyknic sample of isoconcentration behind the determination of protein concentration.The sample for preparing is carried out electrophoresis with SDS-PAGE glue.Change and to go up behind the film to be detected one anti-ly, after 4 ℃ of shaking tables spend the night, go up corresponding two anti-.After the ECL colour developing, with X-ray sheet exposure image.In order to reuse protein blot, (62.5mM Tris (pH6.7), 20%SDS and 0.1M 2 mercapto ethanol (2-mercaptoethanol) be in 50 ℃ of water-baths 30 minutes, with anti-getting rid of of last mistake, goes up new one more again and resist with Strip buffer for we.
Figure 10 be the GFP08 of 100mg/L to the proteic expression figure of the TF of HUVEC, visible by Figure 10: GFP08 antitumor, blood vessel formation against function maybe be relevant with the TF signal path.
Be noted that in addition: galactan sulfate of the present invention can be prepared into various medicinal prepnss with pharmaceutically acceptable carrier.Said " pharmaceutically acceptable carrier " would not destroy the pharmaceutical active of galactan sulfate; Its effective level; Promptly can play pharmaceutical carrier and make the consumption reply human non-toxic of time spent, it includes but not limited to: ion-exchange material, aluminum oxide, StAl, Yelkin TTS, self-emulsifying drug delivery system.
Other pharmaceutically acceptable auxiliaries such as weighting agent are (like lactose hydrous; Starch; Lactose bead and glucose); Tackiness agent (like Microcrystalline Cellulose); Disintegrating agent is (like crosslinked carboxymethyl fecula sodium; Sodium Croscarmellose; Low-substituted hydroxypropyl cellulose and cross-linked pvp); Lubricant (like magnesium stearate); Absorption enhancer; Flavouring agent; Sweeting agent; Thinner; Vehicle; Wetting agent; Solvent; Solubilizing agent and tinting material etc. also can add in the pharmaceutical prepn by galactan sulfate preparation of the present invention.
Especially, can be prepared into intravenous administration formulation, certainly, also can be prepared into, aerosol sucks or implant and accumulate or the pharmaceutical prepn of mode administration such as acupuncture through enteron aisle or parenteral route administration by galactan sulfate of the present invention.
Should be noted that at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although with reference to preferred embodiment the present invention is specified, those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement the technical scheme of invention; And not breaking away from the spirit and the scope of technical scheme of the present invention, it all should be encompassed in the claim scope of the present invention.
Claims (10)
1. galactan sulfate is characterized in that:
Molecular weight is 8.5 * 10
4The quality percentage composition of sulfate group is 25～27%;
Chemical structure is by repeating unit A, repeating unit B, repeating unit C and repeating unit D is random forms; Wherein: the molar content of repeating unit A is 10～13%; The molar content of repeating unit B is 6～7%; The molar content of repeating unit C is 63～65%, and the molar content of repeating unit D is 17～19%;
The chemical structural formula of repeating unit A is following:
The chemical structural formula of repeating unit B is following:
The chemical structural formula of repeating unit C is following:
The chemical structural formula of repeating unit D is following:
The summation of the molar content of above-mentioned four kinds of repeating units is 98～100%.
2. galactan sulfate according to claim 1 is characterized in that: also contain molar content in the described galactan sulfate and be 0～2% the D-semi-lactosi that 1,3 of acetone acidic group connects that contains.
3. the preparation method of the described galactan sulfate of claim 1 is characterized in that, comprises following concrete operations step:
A) water extraction: shred after the Grateloupia filicina (Wulf.) of fresh collection rinsed out salt surfactant with tap water, add zero(ppm) water, transfer to pH=5～7, extracted 0.5～1.5 hour at 85～95 ℃ with Glacial acetic acid min. 99.5; Residue extracts 1 time by said process again; Merge 2 times extracting solution;
B) alcohol precipitation: under agitation add 2～4 times of volume of ethanol of said extracted liquid, leave standstill centrifugal collecting precipitation;
C) freeze-drying: gained is precipitated water-soluble, dialysed lyophilize then 24～72 hours;
D) separation and purification: after above-mentioned freeze dried polysaccharide used pH=7.2, the concentration phosphate buffer soln dissolving as 20mmol/L; With DEAE-Sepharose Fast Flow gel chromatography column chromatography; Sodium chloride solution and the pH=7.2, the concentration that earlier with concentration are 0.5mol/L are 2 times of column volumes of phosphate buffer soln wash-out of 20mmol/L; Use concentration to be the sodium chloride solution of 0.8mol/L and pH=7.2, concentration phosphate buffer soln wash-out again as 20mmol/L; The phenolsulfuric acid method detects collects, flowing water dialysis 24～72 hours, lyophilize; Use dissolved in distilled water again, with Sepharose CL-6B column purification, with the zero(ppm) water wash-out, the sulfuric acid phynol method detects to be collected, and lyophilize promptly gets described galactan sulfate.
4. the preparation method of galactan sulfate according to claim 3 is characterized in that, the water extraction condition in the step a) is: transfer to pH=6 with Glacial acetic acid min. 99.5, extracted 1 hour at 90 ℃.
5. the preparation method of galactan sulfate according to claim 3 is characterized in that, the alcohol precipitation condition in the step b) is: the volumn concentration that under agitation adds 3 times of volumes of said extracted liquid is 95% ethanol.
6. the preparation method of galactan sulfate according to claim 3 is characterized in that, the dialysis time in the step c) is 48 hours.
7. the preparation method of galactan sulfate according to claim 3 is characterized in that, the flowing water dialysis time in the step d) is 48 hours.
8. the application of the described galactan sulfate of claim 1 in the preparation angiogenesis inhibitor.
9. the application of the described galactan sulfate of claim 1 in the preparation antitumor drug.
10. the application of the described galactan sulfate of claim 1 in anti-S-180 sarcoma of preparation or liver-cancer medicine.
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| CN103467625B (en) * | 2013-09-10 | 2015-08-19 | 南京海兰琪生物科技有限公司 | A kind of preparation method of agar-agar Polygalactan sulfuric ester |
| CN109400743B (en) * | 2018-11-14 | 2021-06-18 | 浙江海洋大学 | Method for extracting sulfated galactan from sea grapes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1341126A (en) * | 1999-02-23 | 2002-03-20 | 宝酒造株式会社 | Sulfated fucogalactan |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1341126A (en) * | 1999-02-23 | 2002-03-20 | 宝酒造株式会社 | Sulfated fucogalactan |
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