CN110105461A - A kind of adjunct antineoplastic compound sea lettuce extract and its technology of preparing - Google Patents
A kind of adjunct antineoplastic compound sea lettuce extract and its technology of preparing Download PDFInfo
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- CN110105461A CN110105461A CN201910471433.8A CN201910471433A CN110105461A CN 110105461 A CN110105461 A CN 110105461A CN 201910471433 A CN201910471433 A CN 201910471433A CN 110105461 A CN110105461 A CN 110105461A
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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Abstract
The technology of preparing of the invention discloses a kind of compound sea lettuce extract with adjunct antineoplastic effect.The process characteristic is to utilize ultrasonic wave assisted extraction, Thick many candies ingredient is isolated using water extraction and alcohol precipitation method, albumen and depigmentation are taken off again, the sea lettuce active polysaccharide of purity is high is obtained after purified, recycle hydrogen peroxide and Ascorbic Acid Oxidation degradation sea lettuce polysaccharide, the sea lettuce activated oligosaccharide of purity is high is obtained after purified, and sea lettuce polysaccharide and sea lettuce oligosaccharides are used in mixed way.It is confirmed through animal pharmacodynamics, the compound sea lettuce extract has exact killing ability to H22 liver cancer tumor in KM Mice Body, the growth of tumour can be inhibited to spread, and have certain repairing effect to pathological tissues, and do not have apparent side effect.Compound sea lettuce extract of the present invention is a kind of nontoxicity, Small side effects, foolproof bioactive substance, can be convenient the drug that various dosage forms are prepared into pharmaceutically acceptable carrier.
Description
Technical field
The invention belongs to pharmaceutical technology fields, particularly with regard to a kind of compound sea lettuce extract with adjunct antineoplastic effect
Technology of preparing.
Background technique
For tumour as a kind of malignant disease, lethality is higher, and patient is also deep by its subsidiary ailing puzzlement, and it is suffered from
Sick rate just rises year by year, and society goes up year by year for the demand of anti-tumor drug.Anti-tumor drug is conventionally used to be often accompanied by
Various side effects, or the price is very expensive, thus it is wide for high efficiency, low side effects of, inexpensive, applicability novel
Anti-tumor drug has sizable demand.Natural polymer such as polysaccharide in seaweed etc., wherein many have centainly
Antitumor action, but since polysaccharide cannot directly absorb, but act on intestinal flora, while oligosaccharides is as polysaccharide degradation product
But it can directly be absorbed by the body, and become new research emphasis.Polysaccharide acts on intestinal flora, and oligosaccharides is directly made after absorbing
For body interior, the two action pathway is different, but can tell on, and good cooperation effect will likely be generated by being used in mixed way
Fruit is the field for being worth exploring.
Sea lettuce is also known as sea lettuce, rock head blueness etc., widely distributed, cheap, and the application as food has longer history,
And traditional medicine record on have inducing diuresis for removing edema, softening and eliminating sputum, it is clearing heat and detoxicating the effect of, though about its medicinal research,
But it is deeply excavated and range exploitation then relatively lags behind, and then has more perfect research with the Enteromorpha of its equal category, therefore open
The pharmaceutical potential of hair sea lettuce is necessary.It is living that numerous studies data shows that polysaccharide and oligosaccharides all have anti-tumor biological
Property, therefore the preparation and pharmacological activity analysis of sea lettuce polysaccharide and oligosaccharides are systematically completed, it develops a kind of with anti-tumor activity
And the compound sea lettuce extract of Small side effects be quite it is necessary to.In disclosed at present algal polysaccharides and oligosaccharides related patents
In, the application there has been no compound sea lettuce extract in anti-tumor aspect, the compound sea lettuce extract for preparing and its anti-in the present invention
Tumor promotion is there is not yet other research reports or patent.
The scale application of oligosaccharides is limited to the deficiency of oligosaccharides Study on Preparation Technology, and is difficult to large-scale promotion.Oligosaccharides
Oxidizing process preparation is using glycosidic bond inside the oxidability of hydrogen peroxide and ascorbic acid dismantling polysaccharide, compared to traditional acid system
Preparation, the invention avoids strong acid is used, has the advantages that easy to operate, high-efficient, free of contamination.Heretofore described sea lettuce
Oligosaccharides is that the green technology of preparing decomposed using hydrogen peroxide and Ascorbic Acid Oxidation is made, and side reaction, item will not be occurred by having
The features such as part is mild, environmental-friendly.The side of green alga ulva polysaccharide is prepared in the present invention by hydrogen peroxide and Ascorbic Acid Oxidation
Method is there is not yet other research reports or patent.
Summary of the invention
It is answered present invention aims to solve the deficiencies of the prior art, and provides a kind of a kind of anti-tumor activity is strong, adverse reaction is low
Ashlar water shield extract, and the technology of preparing of the compound sea lettuce extract is provided.
In order to achieve the above object, the present invention adopts the following technical scheme:
A kind of sea lettuce polysaccharide with anti-tumor activity, it is characterised in that: the number-average molecular weight of the polysaccharide is 8.10 × 104Da,
Weight average molecular weight is 1.45 × 105Da, by rhamnose, fucose, arabinose, xylose, mannose, glucose, grape alditol
Acid, galactolipin, galacturonic acid composition, molar ratio 39.44:0.65:0.48:15.68:1.61:1.94:0.26:0.73:
0.89。
The technology of preparing of sea lettuce polysaccharide with anti-tumor activity provided by the invention, comprising the following steps:
(1) it dries and crushes after sea lettuce being impregnated desalination using conventional method, 60-80%v/v ethyl alcohol is added into sea lettuce dry powder and returns
Stream extracts 1-2 hours, and dry powder weight g and ethyl alcohol volume mL ratio are 1:20-30, and drying obtains the stone of drying defatted after reflux
Water shield powder, recycling ethyl alcohol are recycled；
(2) distilled water is added into degreasing sea lettuce powder made from step (1), wherein sea lettuce quality g and distilled water volume mL ratio
For 1:30-40, and with 350-400W ultrasonic wave assisted extraction 120 minutes under 60-70 DEG C of extraction temperature, then in 80-90
It extracts 2-3 hours in DEG C hot water, after extraction, is filtered, filter residue is repeated into 2-3 extraction, merging filtrate,
4000-5000rpm is centrifuged 10-20 minutes, obtains supernatant is concentrated under reduced pressure into original volume at 65-70 DEG C 1/10, and concentration is added
Make in system concentration of alcohol up to 80% v/v for 95%v/v ethyl alcohol, then 4 DEG C standing 8-12 hours, leaching precipitating, precipitating is successively
It is washed, is dried under reduced pressure with dehydrated alcohol, acetone, ether, obtain sea lettuce Thick many candies；
(3) the sea lettuce Thick many candies for taking step (2) to be prepared, are dissolved completely in distilled water, the dissolution body of polysaccharide and distilled water
Product ratio is 1:100-1:120, and the neutral proteinase of 0.1%-0.2% weight is then added, after standing 2-3 hours at 40 DEG C, in
100 DEG C are boiled 20-30 minutes, and solution is centrifuged 10-15 minutes in 4000-5000rpm, precipitating is removed, obtains the crude of removing protein
Polysaccharide solution；
(4) step (3) obtained solution is taken, by HPD-826 macroporous absorbent resin packed column, eluent is inhaled by nonpolarity
Attached dose of active carbon removes depigmentation, and active carbon usage amount g and effluent volume mL are according to w/v 1:35-50, with 60-
80rpm and shaking table shake 1-2 hours, and then 6000-8000rpm is centrifuged 20-30 minutes, collect supernatant；
(5) bag filter for utilizing molecular cut off 8000-14000 or more removes small molecule and color in step (4) acquired solution
Trapped fluid is concentrated under reduced pressure element, is concentrated into paste, and carry out vacuum freeze drying, and purification sea lettuce polysaccharide is obtained after crushing
Powder；
(6) the resulting purification sea lettuce polysaccharide powder of step (5) is taken, is dissolved completely in distilled water, the dissolution of polysaccharide and distilled water
Volume ratio is 1:80-1:100, and the hydrogen peroxide of solution weight 0.05% and 0.026% ascorbic acid or different anti-bad is then added
Hematic acid reacts 3-4 hours at 65-75 DEG C, and is aided with the stirring of 100-200rpm, obtains sea lettuce polysaccharide degradation solution；
(7) the resulting sea lettuce polysaccharide degradation solution of step (6) is taken, it is that 95%v/v ethyl alcohol reaches concentration of alcohol in system that concentration, which is added,
75% v/v, then 4 DEG C standing 8-12 hours, by solution in 4000-5000rpm be centrifuged 10-15 minute, remove precipitating, collection
Supernatant is concentrated under reduced pressure, and the 1/5-1/4 of original volume is concentrated into, and obtains crude sea lettuce oligosaccharide solution；
(8) it by sea lettuce oligosaccharide solution crude in step (7), is handled using molecular cut off 2000D bag filter, collects dialyzate；
(9) dialyzate in step (8) is handled using molecular cut off 500D bag filter, trapped fluid is freeze-dried, is prepared into
To sea lettuce activated oligosaccharide；
(10) by sea lettuce oligosaccharides powder mixed in equal amounts obtained by sea lettuce polysaccharide powder obtained by step (5) and step (9) to get compound stone
Water shield extract.
Further, the technology of preparing of above-mentioned compound sea lettuce extract with anti-tumor activity is applied to preparation prevention and treatment
In the drug of tumour.
The tumour is H22 liver cancer tumor.
The dosage form of the drug includes tablet, granule, capsule.
The present invention has the advantages that there has been no the reports of the anti-tumor activity about compound sea lettuce extract for research at present.
The present invention utilizes ultrasonic wave assisted extraction, Thick many candies ingredient, then de- albumen and depigmentation is isolated using water extraction and alcohol precipitation method, through pure
The sea lettuce active polysaccharide of purity is high is obtained after change, recycles hydrogen peroxide and Ascorbic Acid Oxidation degradation sea lettuce polysaccharide, it is purified
The sea lettuce activated oligosaccharide of purity is high is obtained afterwards, and sea lettuce polysaccharide and sea lettuce oligosaccharides are used in mixed way.It is confirmed through animal pharmacodynamics, institute
Stating compound sea lettuce extract has exact killing ability to H22 liver cancer tumor in KM Mice Body, and the growth of tumour can be inhibited to expand
It dissipates, and has certain repairing effect to pathological tissues, and not there is no apparent side effect.Compound sea lettuce extract of the present invention
It is a kind of nontoxicity, Small side effects, foolproof bioactive substance, is further benefit of the sea lettuce as marine pharmaceutical resource
With new approach is provided, new selection also is provided to treat or prevent the drug development of tumour, can be convenient and pharmaceutically
Acceptable carrier is prepared into the drug of various dosage forms.
Detailed description of the invention
Fig. 1 is the functional compound sea lettuce extract of the present invention to H22 liver cancer tumor model mice serum tumor index of correlation
Influence.
Fig. 2 is the paraffin section HE dyeing microscopic examination image (enlargement ratio 1:400) of administration group tumor tissues.
Specific embodiment
Embodiment 1
(1) it dries and crushes after sea lettuce being impregnated desalination using conventional method, 60%v/v alcohol reflux is added into sea lettuce dry powder
It extracting 1 hour, dry powder weight g and ethyl alcohol volume mL ratio are 1:20, and drying obtains the sea lettuce powder of drying defatted after reflux,
Ethyl alcohol is recycled to be recycled；
(2) distilled water is added into degreasing sea lettuce powder made from step (1), wherein sea lettuce quality g and distilled water volume mL ratio
For 1:30, and 2 then were extracted in 80 DEG C of hot water with 350W ultrasonic wave assisted extraction 120 minutes under 60 DEG C of extraction temperature
Hour, it after extraction, being filtered, filter residue is repeated into 2 extractions, merging filtrate is centrifuged 10 minutes in 4000rpm,
It obtains supernatant and is concentrated under reduced pressure into the 1/10 of original volume at 65 DEG C, it is that 95%v/v ethyl alcohol reaches concentration of alcohol in system that concentration, which is added,
Then 80%v/v stands 8 hours at 4 DEG C, leaching precipitating, precipitating is successively washed with dehydrated alcohol, acetone, ether, is dried under reduced pressure,
Obtain sea lettuce Thick many candies；
(3) the sea lettuce Thick many candies for taking step (2) to be prepared, are dissolved completely in distilled water, the dissolution body of polysaccharide and distilled water
Then product is added the neutral proteinase of 0.1% weight, after standing 2 hours at 40 DEG C, boils 20 points in 100 DEG C than being 1:100
Solution is centrifuged 10 minutes in 4000rpm, removes precipitating, obtain the crude polysaccharide solution of removing protein by clock；
(4) step (3) obtained solution is taken, by HPD-826 macroporous absorbent resin packed column, eluent is inhaled by nonpolarity
Attached dose of active carbon removes depigmentation, and active carbon usage amount g and effluent volume mL are according to w/v 1:35, with 60rpm in shaking
Bed concussion 1 hour, then 6000rpm is centrifuged 20 minutes, collects supernatant；
(5) utilize molecular cut off 8000-14000 or more bag filter, remove step (4) acquired solution in small molecule and
Trapped fluid is concentrated under reduced pressure pigment, is concentrated into paste, and carry out vacuum freeze drying, and it is more that purification sea lettuce is obtained after crushing
Icing Sugar end；
(6) the resulting purification sea lettuce polysaccharide powder of step (5) is taken, is dissolved completely in distilled water, the dissolution of polysaccharide and distilled water
Volume ratio is 1:80, and the hydrogen peroxide of solution weight 0.05% and 0.026% ascorbic acid is then added, reacts 3 at 65 DEG C
Hour, and it is aided with the stirring of 100rpm, obtain sea lettuce polysaccharide degradation solution；
(7) the resulting sea lettuce polysaccharide degradation solution of step (6) is taken, it is that 95%v/v ethyl alcohol reaches concentration of alcohol in system that concentration, which is added,
Then 75% v/v stands 8 hours at 4 DEG C, solution is centrifuged 10 minutes in 4000rpm, precipitating is removed, collects supernatant, carry out
It is concentrated under reduced pressure, is concentrated into the 1/4 of original volume, obtains crude sea lettuce oligosaccharide solution；
(8) it by sea lettuce oligosaccharide solution crude in step (7), is handled using molecular cut off 2000D bag filter, collects dialyzate；
(9) dialyzate in step (8) is handled using molecular cut off 500D bag filter, trapped fluid is freeze-dried, is prepared into
To sea lettuce activated oligosaccharide；
(10) by sea lettuce oligosaccharides powder mixed in equal amounts obtained by sea lettuce polysaccharide powder obtained by step (5) and step (9) to get compound stone
Water shield extract.
Embodiment 2
(1) it dries and crushes after sea lettuce being impregnated desalination using conventional method, 70%v/v alcohol reflux is added into sea lettuce dry powder
It extracting 2 hours, dry powder weight g and ethyl alcohol volume mL ratio are 1:25, and drying obtains the sea lettuce powder of drying defatted after reflux,
Ethyl alcohol is recycled to be recycled；
(2) distilled water is added into degreasing sea lettuce powder made from step (1), wherein sea lettuce quality g and distilled water volume mL ratio
For 1:35, and 3 then were extracted in 85 DEG C of hot water with 400W ultrasonic wave assisted extraction 120 minutes under 65 DEG C of extraction temperature
Hour, it after extraction, being filtered, filter residue is repeated into 3 extractions, merging filtrate is centrifuged 15 minutes in 4500rpm,
It obtains supernatant and is concentrated under reduced pressure into the 1/10 of original volume at 65 DEG C, it is that 95%v/v ethyl alcohol reaches concentration of alcohol in system that concentration, which is added,
Then 80% v/v stands 12 hours at 4 DEG C, leaching precipitating, precipitating is successively washed with dehydrated alcohol, acetone, ether, and decompression is dry
It is dry, obtain sea lettuce Thick many candies；
(3) the sea lettuce Thick many candies for taking step (2) to be prepared, are dissolved completely in distilled water, the dissolution body of polysaccharide and distilled water
Then product is added the neutral proteinase of 0.15% weight, after standing 2-3 hours at 40 DEG C, boils 25 in 100 DEG C than being 1:120
Minute, solution is centrifuged 15 minutes in 4500rpm, precipitating is removed, obtains the crude polysaccharide solution of removing protein；
(4) step (3) obtained solution is taken, by HPD-826 macroporous absorbent resin packed column, eluent is inhaled by nonpolarity
Attached dose of active carbon removes depigmentation, and active carbon usage amount g and effluent volume mL are according to w/v 1:45, with 70rpm in shaking
Bed concussion 2 hours, then 7000rpm is centrifuged 25 minutes, collects supernatant；
(5) bag filter for utilizing molecular cut off 8000-14000 or more removes small molecule and color in step (4) acquired solution
Trapped fluid is concentrated under reduced pressure element, is concentrated into paste, and carry out vacuum freeze drying, and purification sea lettuce polysaccharide is obtained after crushing
Powder；
(6) the resulting purification sea lettuce polysaccharide powder of step (5) is taken, is dissolved completely in distilled water, the dissolution of polysaccharide and distilled water
Volume ratio is 1:90, and the hydrogen peroxide of solution weight 0.05% and 0.026% arabo-ascorbic acid is then added, reacts at 70 DEG C
3 hours, and it is aided with the stirring of 150rpm, obtain sea lettuce polysaccharide degradation solution；
(7) the resulting sea lettuce polysaccharide degradation solution of step (6) is taken, it is that 95%v/v ethyl alcohol reaches concentration of alcohol in system that concentration, which is added,
Then 75% v/v stands 10 hours at 4 DEG C, solution is centrifuged 15 minutes in 4500rpm, precipitating is removed, collects supernatant, into
Row is concentrated under reduced pressure, and is concentrated into the 1/5 of original volume, obtains crude sea lettuce oligosaccharide solution；
(8) it by sea lettuce oligosaccharide solution crude in step (7), is handled using molecular cut off 2000D bag filter, collects dialyzate；
(9) dialyzate in step (8) is handled using molecular cut off 500D bag filter, trapped fluid is freeze-dried, is prepared into
To sea lettuce activated oligosaccharide；
(10) by sea lettuce oligosaccharides powder mixed in equal amounts obtained by sea lettuce polysaccharide powder obtained by step (5) and step (9) to get compound stone
Water shield extract.
Embodiment 3
(1) it dries and crushes after sea lettuce being impregnated desalination using conventional method, 80%v/v alcohol reflux is added into sea lettuce dry powder
It extracting 2 hours, dry powder weight g and ethyl alcohol volume mL ratio are 1:30, and drying obtains the sea lettuce powder of drying defatted after reflux,
Ethyl alcohol is recycled to be recycled；
(2) distilled water is added into degreasing sea lettuce powder made from step (1), wherein sea lettuce quality g and distilled water volume mL ratio
For 1:40, and 3 then were extracted in 90 DEG C of hot water with 400W ultrasonic wave assisted extraction 120 minutes under 70 DEG C of extraction temperature
Hour, it after extraction, being filtered, filter residue is repeated into 3 extractions, merging filtrate is centrifuged 20 minutes in 5000rpm,
It obtains supernatant and is concentrated under reduced pressure into the 1/10 of original volume at 70 DEG C, it is that 95%v/v ethyl alcohol reaches concentration of alcohol in system that concentration, which is added,
Then 80% v/v stands 12 hours at 4 DEG C, leaching precipitating, precipitating is successively washed with dehydrated alcohol, acetone, ether, and decompression is dry
It is dry, obtain sea lettuce Thick many candies；
(3) the sea lettuce Thick many candies for taking step (2) to be prepared, are dissolved completely in distilled water, the dissolution body of polysaccharide and distilled water
Then product is added the neutral proteinase of 0.2% weight, after standing 3 hours at 40 DEG C, boils 30 points in 100 DEG C than being 1:120
Solution is centrifuged 15 minutes in 5000rpm, removes precipitating, obtain the crude polysaccharide solution of removing protein by clock；
(4) step (3) obtained solution is taken, by HPD-826 macroporous absorbent resin packed column, eluent is by nonpolarity
Activated carbon of sorbent removes depigmentation, active carbon usage amount g and effluent volume mL according to w/v 1:50, with 80rpm in
Shaking table shakes 2 hours, and then 8000rpm is centrifuged 30 minutes, collects supernatant；
(5) bag filter for utilizing molecular cut off 8000-14000 or more removes small molecule and color in step (4) acquired solution
Trapped fluid is concentrated under reduced pressure element, is concentrated into paste, and carry out vacuum freeze drying, and purification sea lettuce polysaccharide is obtained after crushing
Powder；
(6) the resulting purification sea lettuce polysaccharide powder of step (5) is taken, is dissolved completely in distilled water, the dissolution of polysaccharide and distilled water
Volume ratio is 1:100, and the hydrogen peroxide of solution weight 0.05% and 0.026% ascorbic acid is then added, reacts 4 at 75 DEG C
Hour, and it is aided with the stirring of 200rpm, obtain sea lettuce polysaccharide degradation solution；
(7) the resulting sea lettuce polysaccharide degradation solution of step (6) is taken, it is that 95%v/v ethyl alcohol reaches concentration of alcohol in system that concentration, which is added,
Then 75% v/v stands 12 hours at 4 DEG C, solution is centrifuged 15 minutes in 5000rpm, precipitating is removed, collects supernatant, into
Row is concentrated under reduced pressure, and is concentrated into the 1/5 of original volume, obtains crude sea lettuce oligosaccharide solution；
(8) it by sea lettuce oligosaccharide solution crude in step (7), is handled using molecular cut off 2000D bag filter, collects dialyzate；
(9) dialyzate in step (8) is handled using molecular cut off 500D bag filter, trapped fluid is freeze-dried, is prepared into
To sea lettuce activated oligosaccharide；
(10) by sea lettuce oligosaccharides powder mixed in equal amounts obtained by sea lettuce polysaccharide powder obtained by step (5) and step (9) to get compound
Sea lettuce extract.
The 4 antitumor test of H22 liver cancer tumor model mice of embodiment
H22 liver cancer tumor model is established with healthy adult male KM mouse.Before modeling by the H22 liver cancer oncocyte of culture in
1000rpm is centrifuged 10 minutes, is resuspended with the standard 1x PBS solution of pH7.4, and cell concentration is adjusted after counting to 108/ mL, as
It is inoculated with tumor liquid.Tumor liquid is injected into the left front oxter of mouse with subcutaneous injection, inoculum concentration is 2 × 107/ mouse is inoculated with volume
0.2mL.Mice Inoculated is randomly divided into normal group, model group, positive group, four groups of administration group, every group 5, normal group
(normal) and model group (model) gives 0.3mL physiological saline stomach-filling, and positive group (control) is given 0.3mL 20mg/kg
Cyclophosphamide solution intraperitoneal injection, administration group (ULPL) are given 150 mg/kg compound sea lettuce extract solution of 0.3mL, are continuously given
It medicine 12 days, removes tumour with mouse and weighs, contrast model group knurl weight calculates tumour inhibiting rate, observes compound sea lettuce extract pair
The influence of H22 liver cancer tumor model mice anti-tumor capacity, the tumour stripped carry out paraffin section and HE dyeing, observe tissue shape
State.Meanwhile the 0th day in zoopery takes blood, and takes blood after the administration is complete, separates serum, test serum tumor necrosis
The level of factor TNF-α and blood vessel endothelium growth factor VEGF appraises and decides its antitumor property.
Functionality compound sea lettuce extract of the invention analyzes H22 liver cancer tumor model mice tumor suppression capacity
Influence of the compound sea lettuce extract of the present invention to H22 liver cancer tumor model mice anti-tumor capacity: normal group does not find tumour,
On the basis of the knurl weight of model group, and when its tumour inhibiting rate (%) is set as 0, the tumour inhibiting rate (%) of cyclophosphamide positive drug group is
70.08 ± 10.65, the tumour inhibiting rate (%) of administration group is 78.99 ± 2.27, has the significance difference opposite sex (p < 0.01) compared to model group,
Illustrate that compound sea lettuce extract has significant antitumor action to H22 liver cancer tumor model mice.To TNF-α in serum and VEGF
Analysis shows that, Fig. 1 show compound sea lettuce extract TNF-α and the content of VEGF can be significantly reduced, it is evident that inhibit tumour
Growth is the important component of its tumor-inhibiting action, can effectively inhibit the growth and diffusion of tumour.In slice HE dyeing microscopic examination
There is the necrosis cavity of large area in middle discovery (Fig. 2), tumor tissues nucleus, are the performances that tumour cell is largely killed,
Meanwhile the cell chromosome disorder of Partial tumors area is inhibited, nucleus molding, form is approached toward normal cell, and in progressive
Variation tendency illustrates that drug can activate immunity, repairs lesion region caused by tumour, and pathological tissues is promoted to restore normal,
The imaging of multiple spot microscopy shows this trend.Through comprehensive analysis, the tumor suppression ability of compound sea lettuce extract is higher than traditional antineoplastic
Object cyclophosphamide, and the growth of tumour can be inhibited to spread, while having certain repairing effect to pathological tissues, and its drug pair is made
With low, do not find that mouse has organ lesion or other significant discomfort phenomenons.
Influence of the functional compound sea lettuce extract of 1 present invention of table to H22 liver cancer tumor model mice tumour inhibiting rate
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with repair
Decorations, are all covered by the present invention.
Claims (4)
1. a kind of technology of preparing of compound sea lettuce extract with anti-tumor activity, which comprises the following steps:
(1) it dries and crushes after sea lettuce being impregnated desalination using conventional method, 60-80%v/v ethyl alcohol is added into sea lettuce dry powder and returns
Stream extracts 1-2 hours, and dry powder weight g and ethyl alcohol volume mL ratio are 1:20-30, and drying obtains the stone of drying defatted after reflux
Water shield powder, recycling ethyl alcohol are recycled；
(2) distilled water is added into degreasing sea lettuce powder made from step (1), wherein sea lettuce quality g and distilled water volume mL ratio
For 1:30-40, and with 350-400W ultrasonic wave assisted extraction 120 minutes under 60-70 DEG C of extraction temperature, then in 80-90
It extracts 2-3 hours in DEG C hot water, after extraction, is filtered, filter residue is repeated into 2-3 extraction, merging filtrate,
4000-5000rpm is centrifuged 10-20 minutes, obtains supernatant is concentrated under reduced pressure into original volume at 65-70 DEG C 1/10, and concentration is added
So that concentration of alcohol is reached 80%v/v for 95%v/v ethyl alcohol, then 4 DEG C standings 8-12 hour, leaching precipitates, and precipitates successively
It is washed, is dried under reduced pressure with dehydrated alcohol, acetone, ether, obtain sea lettuce Thick many candies；
(3) the sea lettuce Thick many candies for taking step (2) to be prepared, are dissolved completely in distilled water, the dissolution body of polysaccharide and distilled water
Product ratio is 1:100-1:120, and the neutral proteinase of 0.1%-0.2% weight is then added, after standing 2-3 hours at 40 DEG C, in
100 DEG C are boiled 20-30 minutes, and solution is centrifuged 10-15 minutes in 4000-5000rpm, precipitating is removed, obtains the crude of removing protein
Polysaccharide solution；
(4) step (3) obtained solution is taken, by HPD-826 macroporous absorbent resin packed column, eluent is inhaled by nonpolarity
Attached dose of active carbon removes depigmentation, and active carbon usage amount g and effluent volume mL are according to w/v 1:35-50, with 60-
80rpm and shaking table shake 1-2 hours, and then 6000-8000rpm is centrifuged 20-30 minutes, collect supernatant；
(5) utilize molecular cut off 8000-14000D or more bag filter, remove step (4) acquired solution in small molecule and
Trapped fluid is concentrated under reduced pressure pigment, is concentrated into paste, and carry out vacuum freeze drying, and it is more that purification sea lettuce is obtained after crushing
Icing Sugar end；
(6) the resulting purification sea lettuce polysaccharide powder of step (5) is taken, is dissolved completely in distilled water, the dissolution of polysaccharide and distilled water
Volume ratio is 1:80-1:100, and the hydrogen peroxide of solution weight 0.05% and 0.026% ascorbic acid or different anti-bad is then added
Hematic acid reacts 3-4 hours at 65-75 DEG C, and is aided with the stirring of 100-200rpm, obtains sea lettuce polysaccharide degradation solution；
(7) the resulting sea lettuce polysaccharide degradation solution of step (6) is taken, it is that 95%v/v ethyl alcohol reaches concentration of alcohol in system that concentration, which is added,
75%v/v, then 4 DEG C standing 8-12 hours, by solution in 4000-5000rpm be centrifuged 10-15 minute, remove precipitating, collection
Supernatant is concentrated under reduced pressure, and the 1/5-1/4 of original volume is concentrated into, and obtains crude sea lettuce oligosaccharide solution；
(8) it by sea lettuce oligosaccharide solution crude in step (7), is handled using molecular cut off 2000D bag filter, collects dialyzate；
(9) dialyzate in step (8) is handled using molecular cut off 500D bag filter, trapped fluid is freeze-dried, is prepared into
To sea lettuce activated oligosaccharide；
(10) by sea lettuce oligosaccharides powder mixed in equal amounts obtained by sea lettuce polysaccharide powder obtained by step (5) and step (9) to get compound stone
Water shield extract.
2. a kind of technology of preparing of compound sea lettuce extract with anti-tumor activity as described in claim 1 is prevented and treated in preparation
Application in tumour medicine.
3. application according to claim 2, which is characterized in that the tumour is H22 liver cancer tumor.
4. application according to claim 2, which is characterized in that the dosage form of the drug includes tablet, granule, capsule
Agent.
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