CN100375755C - Longleaf Grateloupia acuminata polysaccharide extract and its prepn and use - Google Patents
Longleaf Grateloupia acuminata polysaccharide extract and its prepn and use Download PDFInfo
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- CN100375755C CN100375755C CNB2005100235510A CN200510023551A CN100375755C CN 100375755 C CN100375755 C CN 100375755C CN B2005100235510 A CNB2005100235510 A CN B2005100235510A CN 200510023551 A CN200510023551 A CN 200510023551A CN 100375755 C CN100375755 C CN 100375755C
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Abstract
The present invention relates to a long-leaf grateloupia acuminata polysaccharide extract, a preparation process thereof and application in preparing medicine for resisting vascularization and/or resisting tumors.
Description
Technical field
The present invention relates to a kind of plant milk extract, especially relate to 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.
Background technology
Biotherapy strategy at angiogenesis inhibitor, especially direct therapeutic strategy at tumor vessel self, having medicine is easy to arrive tumor locus, be difficult for produces resistance and is not subjected to advantage such as knurl kind restriction, so this strategy is considered to one of antitumor strategy of tool prospect, become the focus of tumor biotherapy research in recent years already.(Zetter, B.R.et al.Ann.Rev.Med. 1998 49:407-424.), were carrying out the I/II clinical trial phase to kind mostly surplus the anti-angiogenic medicaments of having developed at present reached 30.They are different on chemical structure, comprise steroid, polypeptide, microbiotic and electronegative polysaccharide such as heparin, sulfation heparitin etc.Discover that heparin, sulfation heparitin class very easily combine (Digabreiele with many somatomedins (as fibroblast growth factor FGF1, FGF2, blood vessel endothelial cell growth factor VEGF, pHGF etc.), A.D.et al.Nature, 1998,393:812-817.).In addition, the sulfation heparitin also has other indirect actions for tumor growth.
Although heparin, sulfation heparitin have these important function, be good angiogenesis inhibitor, its maximum shortcoming be often cause unmanageable hemorrhage, thereby limited its widespread use as clinical antineoplastic vascular formation inhibitor.Scientist is just attempting to solve by two approach at present: the one, obtain the small molecular weight hyparinoids from animal organs by chemical degradation or complete synthesis mode; The one, obtain to have high angiogenesis inhibiting activity and do not have or have only a polysaccharide of very low anticoagulant active from other source separation and purification.Scientists has found that some activity are higher than the heparin or the sulfation heparin of the small molecular weight of heparin in last approach, and complete synthesis active some oligosaccharides (Digabreiele, A.D.et al.Nature, 1998,393:812-817 far above heparin; Ram, S.et al.Nature Review:Cancer.2002,2:521-528.), but too high by the cost of this approach acquisition angiogenesis inhibitor inhibitor.
The Grateloupia filicina (Wulf.) polysaccharide of finding in the research of our polysaccharide component in to red algae to come into leaves not only has blood coagulation resisting function, but also has stronger blood vessel formation against function and antitumor action.About the structure of the Grateloupia filicina (Wulf.) polysaccharide that comes into leaves and anti-tumor activity thereof and anti-angiogenesis activity there is no bibliographical information.
Summary of the invention
The present invention aims to provide a kind of Grateloupia filicina (Wulf.) polyoses extract that comes into leaves with angiogenesis inhibitor and antitumor action.The method that the present invention also provides a kind of preparation to come into leaves the Grateloupia filicina (Wulf.) polyoses extract.The present invention also provides the purposes of Grateloupia filicina (Wulf.) polyoses extract in preparation angiogenesis inhibitor and/or antitumor drug that come into leaves.
Technical scheme of the present invention is:
A kind of Grateloupia filicina (Wulf.) polyoses extract that comes into leaves contains following weight ratio composition (weight unit is gram): 67～87% semi-lactosis, 1～20%3,6-Anhydrogalactose and 1～7%6-methylgalactose.This extract also contains 0.5～6.5% wood sugar and 0.5～6.5% glucose.Particularly, extract of the present invention contains following weight ratio composition: 77.2% semi-lactosi, 10.5%3,6-Anhydrogalactose, 3.9%6-methylgalactose, 3.5% wood sugar and 3.5% glucose.
The Grateloupia filicina (Wulf.) polyoses extract that comes into leaves of the present invention can be made by following method: the Grateloupia filicina (Wulf.) that will come into leaves is pulverized the back and is used hot water extraction 1～3 hour, and temperature is controlled at 90～100 ℃, and pH value of solution is controlled between 5～7, gets an extracting solution;
Described extracting solution is stayed supernatant liquor after centrifugal, stir the ethanol that adds 1～3 times of amount down, recentrifuge stays throw out; With
With the water-soluble postlyophilization of the described throw out Grateloupia filicina (Wulf.) polyoses extract that obtains coming into leaves.
Preferably the pH with described solution is controlled between 6～7.
Another aspect of the present invention has provided the come into leaves method of Grateloupia filicina (Wulf.) polyoses extract of a kind of preparation, comprises the steps:
The Grateloupia filicina (Wulf.) that will come into leaves is pulverized the back and is used hot water extraction 1～3 hour, and temperature is controlled at 90～100 ℃, and pH value of solution is controlled between 5～7, gets an extracting solution;
Described extracting solution is stayed supernatant liquor after centrifugal, stir the ethanol that adds 1～3 times of amount down, recentrifuge stays throw out; With
With the water-soluble postlyophilization of the described throw out Grateloupia filicina (Wulf.) polyoses extract that obtains coming into leaves.
Preferably the pH with described solution is controlled between 6～7.
Another aspect of the present invention has provided the purposes of Grateloupia filicina (Wulf.) polyoses extract in preparation angiogenesis inhibitor and/or antitumor drug that come into leaves.Particularly the preparation leukemia, lung cancer or S-180 sarcoma medicine in purposes.
The details of all respects of the present invention will be able to detailed description in chapters and sections subsequently.By hereinafter and the description of claim, other characteristics of the present invention, purpose and advantage will be more obvious.
Description of drawings
Fig. 1 comes into leaves Grateloupia filicina (Wulf.) polyoses extract A (GLP) to angiogenesis restraining effect synoptic diagram
Not dosing of A control group; BGLP25 μ g/egg; CGLP50 μ g/egg
Fig. 2 comes into leaves Grateloupia filicina (Wulf.) polyoses extract A (GLP) to vascular endothelial cell tube chamber formation restraining effect synoptic diagram
Not dosing of A control group, endotheliocyte forms complete luminal structure;
BGLP0.313mg/ml, endotheliocyte aggregation variation, cell has pseudopodium to stretch out, but is not arranged in luminal structure;
CGLP1.25mg/ml, endotheliocyte are dispersed in and do not have distortion, do not have pseudopodium to stretch out, and do not have the sign that forms tube chamber;
Fig. 3 comes into leaves Grateloupia filicina (Wulf.) polyoses extract A (GLP) to migration of vascular endothelial cells restraining effect synoptic diagram
The A negative control; The B positive control; CGLP0.313mg/ml;
DGLP1.25mg/ml； EGLP2.5mg/ml； FGLP5.0mg/ml；
Embodiment
Appearance part of the present invention is based on such discovery: the Grateloupia filicina (Wulf.) polyoses extract that comes into leaves has angiogenesis inhibitor and antitumor action.
Can add pharmaceutically acceptable carrier to promote its administration in the Grateloupia filicina (Wulf.) polyoses extract that comes into leaves of the present invention.Preferable, pharmaceutical composition of the present invention contains the extract of 0.1-99.9% weight ratio." pharmaceutically acceptable carrier " can not destroy the pharmaceutical active of the Grateloupia filicina (Wulf.) polyoses extract that comes into leaves, its effective level simultaneously, and promptly can playing pharmaceutical carrier, to make the consumption of time spent nontoxic to human body.
" pharmaceutically acceptable carrier " includes but not limited to: ion-exchange material, aluminum oxide, aluminum stearate, Yelkin TTS, self-emulsifying drug delivery system (SEDDS) is as d-a-vitamin E polyethylene glycol 1000 succinates, the surfactant that pharmaceutical preparations such as tween (Tweens) or other similar polymerisation mediums are used, serum protein such as human serum albumin, buffer substance such as phosphoric acid salt, Padil, Sorbic Acid, potassium sorbate, saturated vegetable fatty acid partial glycerol ester mixture, water, salt, ionogen such as vitriol protamine, phosphoric acid hydrogen two is received, potassium hydrogen phosphate, sodium-chlor, zinc salt, silica gel, Magnesium Silicate q-agent etc.Povidone, cellulosic material, polyvinyl alcohol, Xylo-Mucine, polypropylene acid esters, ethene-polyoxyethylene-block polymer and wool grease, cyclodextrin such as a-, β-and γ-Huan Hujing or its all can be used for promoting the useful for drug delivery of the Grateloupia filicina (Wulf.) polyoses extract that comes into leaves of the present invention through hydroxyalkyl cyclodextrin such as the derivative of chemically modified such as 2-and 3-hydroxypropyl-beta-cyclodextrin or other soluble derivatives etc.
Other pharmaceutically acceptable auxiliaries such as weighting agent (as lactose hydrous, starch, lactose bead and glucose), tackiness agent (as Microcrystalline Cellulose), disintegrating agent (as crosslinked carboxymethyl fecula sodium, croscarmellose sodium, low-substituted hydroxypropyl cellulose and cross-linked pvp), lubricant (as 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 composition of the present invention.
Above-mentioned Grateloupia filicina (Wulf.) polyoses extract and the pharmaceutical composition thereof of coming into leaves can pass through enteron aisle or parenteral route administration.The enterally administering preparation comprises pill, granule, capsule, suspension or solution.Non-intestinal drug delivery agent comprises injection, creme, paste, patch or sprays.That the parenterai administration approach comprises is subcutaneous, in the intracutaneous, artery, vein, muscle, joint, synovia, breastbone, sheath, intralesional, intracranial injection or instillation.Other route of administration can comprise part, rectum, intranasal, through cheek, vagina, hypogloeeis, mucous membrane, tracheae or urethra.Come into leaves Grateloupia filicina (Wulf.) polyoses extract and pharmaceutical composition thereof can also suck or implant and accumulate or mode administration such as acupuncture by aerosol.
The oral preparations of Grateloupia filicina (Wulf.) polyoses extract and pharmaceutical composition thereof of coming into leaves includes but not limited to: capsule, tablet, emulsion, water suspending agent, colloidal solution, solution, microcapsule, pill, lozenge, granule, pulvis.The pharmaceutically acceptable carrier that is usually used in tablet comprises lactose and W-Gum.Usually also can add lubricants such as Magnesium Stearate.The pharmaceutically acceptable carrier that is usually used in capsule comprises lactose and dried corn starch.When making oral water suspending agent and/or emulsion, the Grateloupia filicina (Wulf.) polyoses extract that comes into leaves can suspend or be dissolved in the oil phase and with emulsifying agent or suspension agent and combine.If desired, also can add some sweeting agents and/or flavouring agent and/or toner.
Grateloupia filicina (Wulf.) polyoses extract and the pharmaceutical composition thereof of coming into leaves of the present invention can be made into aseptic injection, as aseptic water or oil phase suspension.This suspension can promptly use suitable dispersion agent or wetting agent (as Tween 80) and suspension agent etc. to make by the ordinary method of this area.But it can also be at the nontoxic thinner of enteron aisle external administration or the aqueous solution or the suspension in the solvent, as the solution in 1,3 butylene glycol.Relevant available support or solvent comprise N.F,USP MANNITOL, water, ringer's solution, isotonic sodium chloride etc.In addition, aseptic fixed oil often is used as the media of solvent or suspension agent, thereby comprises that the multiple soft fixed oil (bland fixedoil) of synthetic glycerine monoesters or triglyceride all is suitable for.Lipid acid can be used for preparing described injection as octadecenic acid and glyceride derivative thereof (as sweet oil or Viscotrol C, particularly its polyoxyethylene radical derivative) etc.Described oil solution or suspension also can comprise a kind of alcohol dilution agent of long-chain or dispersion agent or carboxymethyl cellulose or similar other dispersion agents, and this type of material is usually used in preparing pharmaceutical acceptable emulsion and/or suspension agent.Tensio-active agent that some other preparation is commonly used such as Tweens or Spans and/or other similar emulsifying agents or bioavailability promotor etc. can be used for preparing this preparation too.
Come into leaves Grateloupia filicina (Wulf.) polyoses extract and pharmaceutical composition thereof of the present invention can be made into suppository and pass through rectal administration, method is that the Grateloupia filicina (Wulf.) polyoses extract that comes into leaves is mixed with the non-irritating excipient that suits, the latter is liquid under rectal temperature for solid at room temperature, thereby this suppository can melt in rectum and discharges active ingredient.This type of vehicle includes but not limited to: theobroma oil, beeswax and polyethylene.The local administration preparation (as ointment) of come into leaves Grateloupia filicina (Wulf.) polyoses extract and pharmaceutical composition thereof of the present invention can be directly used in the affected part.This type of topical formulations contains active ingredient and pharmaceutically acceptable carrier, and the latter includes but not limited to: mineral oil, liquid petroleum, white oil, propylene glycol, polyoxyethylene or the polyoxy third desaturation compound, emulsification is cured or water.In addition, pharmaceutical composition of the present invention also can be made into lotion or finish.The carrier that is suitable for includes but not limited to: mineral oil, sorbic alcohol monostearate, polysorbate60, spermaceti ester, cetyl alcohol, 2-Stearyl alcohol, phenmethyl ethanol or water.The Grateloupia filicina (Wulf.) polyoses extract that comes into leaves of the present invention also can be made into enema etc. and is used for the rectum topical.The topical transdermal patch is also within protection scope of the present invention.But Grateloupia filicina (Wulf.) polyoses extract and pharmaceutical composition thereof also intranasal spraying or the inhalation of coming into leaves of the present invention, promptly, use phenmethyl ethanol or other sanitass, absorption enhancer, fluorocarbon and/or other solubilizing agent or dispersion agent to make salts solution by the ordinary method of this area.
Grateloupia filicina (Wulf.) polyoses extract and the pharmaceutical composition thereof of coming into leaves of the present invention also can pass through drug delivery implant.Adopt the drug delivery implant mode can reach in the administration subject and continue, regularly discharge the effect of come into leaves Grateloupia filicina (Wulf.) polyoses extract and pharmaceutical composition thereof of the present invention.In addition, drug delivery implant can also be at local organization and organ site-specific delivery of drugs (Negrinetal., Biomaterials22 (6): 563,2001) regularly release tech also can be used for the present invention and comes into leaves in the administration of Grateloupia filicina (Wulf.) polyoses extract and pharmaceutical composition thereof, as delayed release capsule, slow release method and the preparation technique for packing (as polymer and liposome) etc. based on the polymer technology.
Patch comprises within the scope of the present invention equally.It comprises basic unit's (as polymer, cloth, yarn and bandage) and pharmaceutical composition of the present invention.One side of basic unit can be provided with a protective layer to prevent the outflow of active ingredient.Described patch also can contain a tackiness agent that is used for fixing, and the latter can be a kind of natural or synthetic material, can temporarily adhere on the skin when it contacts with the administration subject's skin.Tackiness agent can be a waterproof.
When the Grateloupia filicina (Wulf.) polyoses extract and pharmaceutical composition and preparation thereof of coming into leaves of the present invention can mix with one or more additional medicines or prophylactic agent.Additional medicaments can be used as independently formulation and come into leaves Grateloupia filicina (Wulf.) polyoses extract and pharmaceutical composition administration respectively thereof of the present invention.Additional medicaments also can with Grateloupia filicina (Wulf.) polyoses extract and the pharmaceutical composition thereof of coming into leaves of the present invention together with single formulation administration.
For the ease of understanding the present invention, the spy enumerates following examples.Its effect should be understood that it is to annotation of the present invention and absolutely not to any type of restriction of the present invention.Each above listed reference is all introduced the application as a reference in full.
Embodiment 1 prepares the Grateloupia filicina (Wulf.) polyoses extract A (GLP) that comes into leaves
The Grateloupia filicina (Wulf.) 300g that comes into leaves pulverizes, add 90 ℃ of water then and extracted 1 hour for 6 liters, during add Glacial acetic acid control extracting solution the pH value be 6.2.Centrifugal 5 liters of the supernatant liquors that get add 15 liters of 95% ethanol under stirring, and are centrifugal, abandoning supernatant.Water-soluble 3 liters of throw out, lyophilize must come into leaves Grateloupia filicina (Wulf.) polysaccharide 30g (the A extract, GLP).
Embodiment 2 prepares the Grateloupia filicina (Wulf.) polyoses extract B that comes into leaves
The Grateloupia filicina (Wulf.) 1.5kg that comes into leaves pulverizes, add boiling water then and extracted 2 hours for 50 liters, during add Glacial acetic acid control extracting solution the pH value be 6.8.Centrifugal supernatant concentration to 30 liter stirs and adds 60 liters of 95% ethanol down, and is centrifugal, abandoning supernatant.Water-soluble 10 liters of throw out, the cryodesiccated Grateloupia filicina (Wulf.) polysaccharide 160g (B extract) that comes into leaves.
Embodiment 3 prepares the Grateloupia filicina (Wulf.) polyoses extract C that comes into leaves
The Grateloupia filicina (Wulf.) 1.0kg that comes into leaves pulverizes, add 70 ℃ of water then and extracted 3 hours for 30 liters, during add Glacial acetic acid control extracting solution the pH value be 5.5.Centrifugal supernatant concentration to 15 liter stirs and adds 15 liters of 95% ethanol down, and is centrifugal, abandoning supernatant.Water-soluble 10 liters of throw out, the cryodesiccated Grateloupia filicina (Wulf.) polysaccharide 105g (C extract) that comes into leaves.
Embodiment 4 come into leaves Grateloupia filicina (Wulf.) polyoses extract A (GLP) external/the extracorporeal suppression tumor cell growth experiment
Through experiment confirm: GLP the mouse leukemia growth of tumour cell is had the obvious suppression effect, inhibiting rate is 100% when 1mg/ml.GLP has the obvious suppression effect equally to the growth of human lung adenocarcinoma tumour cell, and inhibiting rate is 95.3% when 1mg/ml.
Experiment mice is transplanted after the S-180 sarcoma and to be pressed 200mg/kg, 100mg/kg, 50mg/kg intravenously administrable in second day, continuous 7 days, the average knurl of determination experiment group and control group is heavy, and GLP had obvious suppression effect (P＜0.01) (table 1) to mouse transplantability S-180 sarcoma when the result showed 200mg/kg.
Table 1 GLP is to the restraining effect of mouse S-180 sarcoma
| Group | Dosage (mg/kg) | Average knurl weight ± SD | Tumour inhibiting rate (%) | The P value |
| Control group (NS) | 1.86±0.40 | ----- | ----- | |
| GLP | 50 | 1.50±0.61 | 19.4 | >0.05 |
| GLP | 100 | 1.39±0.34 | 25.3 | >0.05 |
| GLP | 200 | 0.56±0.21 | 52.1 | <0.01 |
| 5-FU | 75 | 0.53±0.25 | 71.5 | <0.01 |
Embodiment 5 comes into leaves Grateloupia filicina (Wulf.) polyoses extract A (GLP) to angiogenesis inhibition experiment (CAM)
It is 6 days (the air chamber end upwards) of incubator hatching of 95% that some pieces of fertilization chicken embryos place 38 ℃, humidity, leave standstill after 15 minutes leave standstill the chicken embryo above the position of windowing of standardized 2 * 2cm size, cut window with the scissors mill then, blow away the dust that mill is cut on the eggshell, throw off the eggshell at the place of windowing.Cut the broken shell film with corectome, expose chorioallantoic membrane.GLP5ul is added on the Whatman filter paper of 0.5 * 0.5cm of autoclave sterilization size, then filter paper is placed on the less position of chorioallantoic membrane blood vessel, corresponding solvent and not dosing contrast is set.Put into incubator hatching two days behind the sterile transparent adhesive tape envelope window.Open scotch tape, press from both sides out Whatman filter paper gently with tweezers.Dissecting microscope is observed the angiogenesis situation down, and shooting.Increase along with administration concentration, vessel branch new life has tangible minimizing trend (arrow is depicted as newborn little blood vessel), when administration concentration reaches 50 μ g/egg, great vessels right side dosing place is compared with the left side, angiogenesis quantity has obvious minimizing, blood vessel inhibition ability is ++, no angiogenesis district is greater than 6mm (accompanying drawing 1).
Embodiment 6 comes into leaves Grateloupia filicina (Wulf.) polyoses extract A (GLP) to vascular endothelial cell tube chamber formation experiment
The Matrigel60 μ L/ hole of 4 ℃ of precoolings adds 96 orifice plates, and 37 ℃ of cell culture incubators are stablized and treated that it solidified in 40 minutes.HMEC is with the complete culture solution dilution and plant 96 orifice plates (5 * 10
8Cells.L
-1, 5 * 10
4Cells.well
-1), the GLP physiological saline solution is established 5 concentration (5,2.5,1.25,0.625 and 0.313mg/ml), and each concentration is established two multiple holes, and the blank of not dosing accordingly is set.Hatching took pictures under phase microscope after 8 hours observes tube chamber network formation situation.The result shows that GLP does not have the tube chamber sign (accompanying drawing 2) of formation when 1.25mg/ml.
The embodiment 7 Grateloupia filicina (Wulf.) polyoses extract A (GLP) that comes into leaves tests endothelial cell migration
Balance is after 1 hour in incubator through the MCDB131 of serum-free training liquid for the transwell that 1% gelatin is handled, and last chamber adds 100 μ l 5 * 10 of serum-free MCDB131 dilution
4The HMEC in/hole and the GLP of different concns, MCDB131 training liquid and 20%FCS that following chamber adds 0.6ml serum-free stimulate migration, and corresponding feminine gender and positive control are set simultaneously, place CO
2Effect is 8 hours in the incubator, discard Kong Zhongpei liquid then, fix 30 minutes with 90% alcohol normal temperature, 0.1% Viola crystallina normal temperature dyeing 10 minutes, clear water rinses, dab off not migrating cell of upper strata with cotton swab, microscope (Olympus, DP50, Japan). observe down and take pictures, use 10% acetate, 100 μ l/ hole extractings 10 minutes at last, measure the OD value in the 600nm place.
The inhibiting rate calculation formula is:
Inhibiting rate (%)=[(the OD value administration group-non-stimulated negative control of OD value)/(not dosing of the OD value positive control-non-stimulated negative control of OD value)] * 100%
Along with the increase of GLP activity, the cell count of chamber is minimizing trend under the Transwell that moves into.When concentration reached 2.5mg/ml, inhibiting rate reached 60%, and the 5mg/mlGLP inhibiting rate is 81.1%, with batch experiment 10
-5M Suramin inhibiting rate is 25.9%, enlargement ratio * 100 (accompanying drawing 3).
The experiment of embodiment 8 vascular endothelial cell proliferations
The use srb assay is measured, and is about to HMEC or HUVEC cell (1 * 10
8Cells.L
-1, 1 * 10
4Cells.Well
-1) insert 96 orifice plates and cultivate and to make it adherent back dosing in 24 hours, the GLP physiological saline solution is established 5 concentration (5,2.5,1.25,0.625 and 0.313 mg/ml), and each concentration is established three and is answered holes, and establishes corresponding zeroing hole and blank.Behind the drug effect 72 hours, outwell nutrient solution, add 100 μ L10% Tricholroacetic Acid (TCA)/holes and fix 1 hour in 4 ℃, outwell stationary liquid, with distillation washing 5 times, seasoning.Add 100 μ L4mg/ml SRB (Sigma)/holes again, room temperature dyeing 15 minutes is abandoned it, washes 5 times seasoning with 1% Glacial acetic acid (HAC).Add 150 μ LTris base buffer (10mM)/holes at last, shake up.Microplate reader (VERSAmax) 540nm wavelength is measured the OD value down.
Inhibiting rate (%)=(OD value control wells-OD value dosing holes)/OD value control wells * 100%
According to each concentration inhibiting rate, adopt LOGIT method calculation of half inhibitory concentration IC
50More than each experiment repeat 3 times, obtain the average IC of 3 experiments
50Value is as the final index of the ability of inhibition.Calculating GLP according to experimental result is 0.86mg/ml (table 2) to the IC50 mean value that influences that capillary endothelium HMEC-1 breeds.
Table 2GLP is to the influence of capillary endothelium HMEC-1 propagation
| Concentration (mg/ml) | 5 | 2.5 | 1.25 | 0.625 | 0.3125 | IC50 (mg/ml) |
| Inhibiting rate (%) | 85.113 | 68.878 | 72.536 | 47.814 | 29.763 | 0.605 |
| 85.691 | 76.949 | 77.930 | 39.220 | 22.519 | 0.727 | |
| 80.303 | 58.873 | 53.424 | 41.207 | 35.031 | 1.247 |
GLP is 0.63mg/ml (table 3) to the IC50 mean value that influences of huve cell HUVEC propagation.
Table 3 GLPA is to the influence of huve cell HUVEC propagation
| Concentration (mg/ml) | 5 | 2.5 | 1.25 | 0.625 | 0.3125 | IC50 (mg/ml) |
| Inhibiting rate (%) | 86.866 | 85.957 | 65.748 | 54.911 | 41.901 | 0.481 |
| 88.123 | 87.554 | 80.546 | 69.163 | 53.763 | 0.253 | |
| 53.474 | 64.013 | 46.328 | 41.337 | 27.844 | 1.17 |
Experimental result shows: there is dose-effect relationship preferably in GLP to the inhibiting rate of vascular endothelial cell growth.
Many aspects involved in the present invention have been done as above and have been set forth.Yet, it should be understood that before not departing from spirit of the present invention to put that those skilled in the art can carry out equivalents and modification to it.Their scope is also included within the appended claim.
Claims (11)
1. the Grateloupia filicina (Wulf.) polyoses extract that comes into leaves contains following weight ratio composition: semi-lactosi 67-87%, 3,6-Anhydrogalactose 1-20% and 6-methylgalactose 1-7%, and this Grateloupia filicina (Wulf.) polyoses extract that comes into leaves is made by following method:
The Grateloupia filicina (Wulf.) that will come into leaves is pulverized the back and is used hot water extraction 1-3 hour, and temperature is controlled at 90-100 ℃, and pH value of solution is controlled between the 5-7, gets an extracting solution;
Described extracting solution is stayed supernatant liquor after centrifugal, add the ethanol that 1-3 doubly measures under stirring, recentrifuge stays throw out; With
With the water-soluble postlyophilization of the described throw out Grateloupia filicina (Wulf.) polyoses extract that obtains coming into leaves.
2. the extract of claim 1, wherein said pH value of solution is controlled between the 6-7.
3. the extract of claim 1, it also contains wood sugar 0.5-6.5% and glucose 0.5-6.5%.
4. claim 1 or 2 extract, it contains following weight ratio composition: semi-lactosi 77.2%, 3,6-Anhydrogalactose 10.5%, 6-methylgalactose 3.9%, wood sugar 3.5% and glucose 3.5%.
5. the extract of claim 4 is characterized in that, it is made by following method:
The Grateloupia filicina (Wulf.) that will come into leaves is pulverized the back and is used hot water extraction 1-3 hour, and temperature is controlled at 90-100 ℃, and pH value of solution is controlled between the 5-7, gets an extracting solution;
Described extracting solution is stayed supernatant liquor after centrifugal, add the ethanol that 1-3 doubly measures under stirring, recentrifuge stays throw out; With
With the water-soluble postlyophilization of the described throw out Grateloupia filicina (Wulf.) polyoses extract that obtains coming into leaves.
6. the extract of claim 5, wherein said pH value of solution is controlled between the 6-7.
7. a method for preparing the Grateloupia filicina (Wulf.) polyoses extract that comes into leaves comprises the steps:
The Grateloupia filicina (Wulf.) that will come into leaves is pulverized the back and is used hot water extraction 1-3 hour, and temperature is controlled at 90-100 ℃, and pH value of solution is controlled between the 5-7, gets an extracting solution;
Described extracting solution is stayed supernatant liquor after centrifugal, add the ethanol that 1-3 doubly measures under stirring, recentrifuge stays throw out; With
With the water-soluble postlyophilization of the described throw out Grateloupia filicina (Wulf.) polyoses extract that obtains coming into leaves.
8. the method for claim 7, wherein said pH value of solution is controlled between the 6-7.
9. the Grateloupia filicina (Wulf.) polyoses extract that comes into leaves of claim 1 is in the purposes of preparation in the anti-angiogenic medicaments.
10. the Grateloupia filicina (Wulf.) polyoses extract that comes into leaves of claim 1 is in the purposes of preparation in the antitumor drug.
11. the purposes of claim 10, wherein said tumour are leukemia, lung cancer or S-180 sarcoma.
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| CN102349863A (en) * | 2011-11-01 | 2012-02-15 | 广东海洋大学 | Preparation method of algal polysaccharides toner |
| CN103329733B (en) * | 2013-06-21 | 2015-03-18 | 南阳医学高等专科学校 | Methods for preparing entomogenous fungus conjugate and extracting polysaccharides of entomogenous fungus conjugate |
| CN103951760B (en) * | 2014-04-29 | 2016-01-13 | 上海中医药大学 | A kind of Grateloupia filicina (Wulf.) oligosaccharides and its preparation method and application |
| CN104045735B (en) * | 2014-06-21 | 2016-03-30 | 广东绍河珍珠有限公司 | The method of a kind of double-enzyme method high efficiency extraction full composition Grateloupia filicina (Wulf.) extracting solution |
| CN109608561A (en) * | 2019-02-01 | 2019-04-12 | 中国水产科学研究院南海水产研究所 | A kind of extracting method of centipede polysaccharides |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1108572A (en) * | 1993-09-24 | 1995-09-20 | 株式会社雅库路特本社 | Antiulcer agent and adhesion inhibitor for helicobacter pyroli |
| CN1392160A (en) * | 2001-06-15 | 2003-01-22 | 中国科学院化工冶金研究所 | Flocculating extraction process of seawweed polysaccharide |
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2005
- 2005-01-25 CN CNB2005100235510A patent/CN100375755C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1108572A (en) * | 1993-09-24 | 1995-09-20 | 株式会社雅库路特本社 | Antiulcer agent and adhesion inhibitor for helicobacter pyroli |
| CN1392160A (en) * | 2001-06-15 | 2003-01-22 | 中国科学院化工冶金研究所 | Flocculating extraction process of seawweed polysaccharide |
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| CN1810842A (en) | 2006-08-02 |
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