CN104193845A - Nostoc commune antitumor polysaccharide as well as preparation method and application thereof - Google Patents
Nostoc commune antitumor polysaccharide as well as preparation method and application thereof Download PDFInfo
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- CN104193845A CN104193845A CN201410459857.XA CN201410459857A CN104193845A CN 104193845 A CN104193845 A CN 104193845A CN 201410459857 A CN201410459857 A CN 201410459857A CN 104193845 A CN104193845 A CN 104193845A
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- nostoc commune
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Abstract
The invention provides a nostoc commune antitumor polysaccharide and a preparation method thereof as well as application of the polysaccharide in preparing an antitumor drug and healthcare food. The preparation method for the nostoc commune antitumor polysaccharide comprises the following steps: drying and crushing pure natural nostoc commune, weighing proper amount of the dried and crushed nostoc commune, adding distilled water to reflux and extract the weighed nostoc commune, and combining extracting liquor; carrying out rotary evaporation and concentration on the extracting liquor to an appropriate amount, adding 10.6% potassium ferrocyanide liquor and 21.9% zinc acetate liquor in a volume ratio being 1:1 to precipitate proteins, absorbing liquid supernatant after centrifuging the mixture; carrying out rotary evaporation and concentration on the mixture under reduced pressure to a certain volume, adding absolute ethyl alcohol to separate out white flocculus after dialyzing concentrated liquor by virtue of distilled water, and freezing and drying in vacuum after centrifuging to obtain the nostoc commune polysaccharide. According to an in-vitro antitumor experiment, the polysaccharide can be used for obviously inhibiting multiplication of human breast cancer cells, is free of cell toxicity, has good antitumor activity, and provides possibility for developing and researching novel anticancer healthcare products or drugs.
Description
Technical field
The present invention relates to preparation and the application of blue green algae vegetable polysaccharides, specifically belong to antitumor polysaccharide of a kind of Nostoc commune and preparation method thereof, and the application in preparing antitumor drug and natural health care.
Background technology
Nostoc commune, formal name used at school Nostoc Commune (Nostoc commune Vauch.), be commonly called as land for building dish, another name lichens, dish mushroom, soft youngster, dish skin, ear, be the sheet algae of algae Cyanophyceae nostocaceae (nostocaceae) nostoc nostoc (nostoc commune vauch).Generally be grown in moist sandy soil surface, often mix between the cauline leaf of weeds base portion, between the large base portion and the liquor-saturated flora of tongue of setting.Nostoc commune almost all has distribution in the whole nation, area especially has wild resource widely to distribute in Changbaishan area northeastward, also has very high output in Shanxi.
< < Compendium of Materia Medica > > describes Nostoc commune: " smell: sweet, flat, nontoxic; Cure mainly: improving eyesight benefit gas, kidney tonifying." describe that " main improving eyesight, beneficial gas, has made us son in < < Mingyi Bielu > >." < < food examines in > > and thinks: " clear god is antipyretic, and convulsive seizure due to phlegm-fire can be treated " " take and prolong life for a long time, Gai Yineng is dirty hot person clearly.At present existing bibliographical information: it contains rich in protein, carbohydrate, various trace elements and certain crude fat and robust fibre, has higher edible and nutritive value, is typical green hill whole food; Simultaneously often the edible effect that has clearing heat and detoxicating, cooling blood for improving eyesight, enhances metabolism, is good medical-care health food.Therefore, Nostoc commune can be used as a kind of protective foods of food and medicament dual-purpose.
Polysaccharose substance refers to the natural high moleculer eompound that a class is formed by 10 above monose molecule aggregations, is one of macromolecular substance maintaining normal activities.There are some researches show, Nostoc commune polysaccharide material has anti-oxidant and bacteriostatic activity, also has the functions such as antifatigue, antiviral, hypoglycemic, reducing blood-fat, anti-inflammatory, but not yet sees the research of Nostoc commune anti-tumor activity of polysaccharides.This experiment be take Nostoc commune as material, extracts a kind of polysaccharide with anti-tumor activity, and this polysaccharide can significantly suppress the propagation of human breast cancer cell.Therefore, the present invention not only can be applied to the development of antitumor drug, natural new food and health drink, simultaneously for promoting the deep development of Nostoc commune resource and effectively utilizing theoretical foundation is provided.
Summary of the invention
The object of the present invention is to provide a kind of Nostoc commune polysaccharide with anti-tumor activity and preparation method thereof, with and application in preparing antitumor drug and protective foods.
The method of the antitumor polysaccharide of a kind of Nostoc commune provided by the invention, comprises the steps:
The first step: get Nostoc commune, 60 ℃ of dry pulverizing after 2 hours, adding distil water reflux 3 hours, standing, double gauze filters, and gets supernatant liquor, as above repeat to extract 1 time, merging filtrate, reduction vaporization is concentrated into thick;
Second step: the extracting solution after concentrated adds 10.6% yellow prussiate of potash (K while stirring
4[Fe (CN)
6] 3H
2o), shake up, then add 21.9% zinc acetate (C
4h
8o
4zn), jolting is even, with protein precipitation, places 30 minutes under room temperature, and 5000rpm gets supernatant liquor after centrifugal 20 minutes, and reduction vaporization is concentrated into thick; Described yellow prussiate of potash, zinc acetate and extracting liquid volume ratio are 1 ︰ 1 ︰ 8-15;
The 3rd step: the dialysis tubing that is 3000 with interception, the concentrated solution that second step is obtained is dialysed in distilled water, within every 12 hours, changes water once, until conductivity of dialysate is constant, with small molecular weight impurities such as deionizing and monose;
The 4th step: in the extracting solution obtaining in the 3rd step, add dehydrated alcohol, vortex, room temperature is placed 0.5-1 hour, to precipitate polysaccharide, the centrifugal 15-20 minute of 5000rpm then, collecting precipitation; Described extracting solution and dehydrated alcohol volume ratio are 3-2 ︰ 7-8;
The 5th step: in above-mentioned precipitation, add appropriate distilled water, stirring and dissolving at 50-60 ℃, until become homogeneous liquid, then vacuum lyophilization becomes floss to obtain Crude polysaccharides sample, called after NVPS, in 4 ℃ of preservations.
10.6% potassium ferrocyanide solution described in second step, 21.9% acetic acid zinc solution and extracting liquid volume ratio are preferably 1 ︰ 1 ︰ 10.
Extracting solution described in the 4th step and dehydrated alcohol volume ratio are preferably 3 ︰ 7.
Dissolution precipitation Heating temperature described in the 5th step is preferably 55 ℃.
The Nostoc commune polysaccharide that above-mentioned preparation method makes can be applied in preparing new type anticancer medicine, also can in preparing healthcare products, apply.
Through anti tumor activity in vitro detection display, the Nostoc commune polysaccharide that above-mentioned preparation method makes is anticancer propagation obviously, has good antitumour activity.Result shows that while being 300ug/ml by this polysaccharide of different concns handler's breast cancer cell (MCF-7) 24 hours, concentration, lethality rate reaches approximately 25%; Process 36 hours, concentration while being 200ug/ml, lethality rate is high to approximately 60%, so this polysaccharide has good inhibition to cancer cells (MCF-7).
Polysaccharide prepared by the method, because it derives from pure natural algae, purifying process is simple in addition, therefore has larger DEVELOPMENT PROSPECT and using value.As: aspect medical, can be used as the auxiliary material of other drug or the cancer ancillary drug that preparation has anti-tumor activity; Aspect healthcare products, can prepare and there is antitumor and health care oral liquid anti-oxidant function; In addition, can also be applied in beverage and point in the heart as natural additive for foodstuff, also can be used as emulsifying agent, wetting Agent for Printing Inks is added in makeup.
Accompanying drawing explanation
The antitumor polysaccharide of Fig. 1 Nostoc commune (NVPS) sample
The antitumor polysaccharide of Fig. 2 Nostoc commune (NVPS) is on the Cytotoxic column diagram that affects
The antitumor polysaccharide of Fig. 3 Nostoc commune (NVPS) induction MCF-7 apoptosis has dose-dependently column diagram
Embodiment
The preparation method of the embodiment antitumor polysaccharide of 1 Nostoc commune (NVPS)
(1) will be natively dry 2 hours of 60 ℃, auricularia auriculajudae, pulverize.
(2) get Nostoc commune powder 100g, add 5L distilled water reflux 3 hours, standing, double gauze filters, and gets supernatant liquor, as above repeats to extract 1 time, and 2 times filtrate merges, and reduction vaporization is concentrated into 1000ml;
(3) the land for building dish extracting solution obtaining in step (2) is added to 100ml yellow prussiate of potash (K while stirring
4[Fe (CN)
6] 3H
2o) (10.6%), shakes up, then adds 100ml zinc acetate (C
4h
8o
4zn) (21.9%), jolting is even, with protein precipitation, places 30 minutes under room temperature, and 5000rpm gets supernatant liquor after centrifugal 20 minutes, and reduction vaporization is concentrated into 100ml;
(4) concentrated solution step (3) being obtained is dialysed in distilled water with the dialysis tubing that interception is 3000, within every 12 hours, changes water once, until conductivity of dialysate is almost constant, and the dialyzate 120ml of the small molecular weight impurities such as ion and monose that are removed;
(5) in the extracting solution obtaining in step (4), add 280ml dehydrated alcohol, vortex, room temperature is placed 1 hour, and to precipitate polysaccharide, then 5000rpm is centrifugal 20 minutes, collecting precipitation;
(6) in the precipitation obtaining in step (5), add 50ml distilled water heated and stirred to dissolve, until become homogeneous liquid, then vacuum lyophilization becomes floss to obtain Crude polysaccharides sample, and called after NVPS, in 4 ℃ of preservations;
Adopt phenolsulfuric acid method, with glucose as a standard product are measured Nostoc commune polysaccharide content.Found that in the Nostoc commune powder of 100g, polysaccharide content is about 8.734g Nostoc commune polysaccharide sample (as shown in Figure 1).
Embodiment 2: chooses the MCF-7 cell of logarithmic phase, cell made to cell suspension, and by cell counting, every hole 4 * 10
4individual being inoculated in 96 orifice plates, 37 ℃, 5%CO
2incubator is cultivated 24 hours after cell attachment, discard old substratum, the substratum that adds homopolysaccharide final concentration not (0,25,50,100,200,300ug/ml), if 5 parallel holes, culture plate is continued to cultivate 24 hours, every hole adds 20 μ L5.0mg/mL MTT, then hatches 4 hours.Remove nutrient solution in plate, every hole adds the DMSO of 150 μ L to dissolve the first a ceremonial jade-ladle, used in libation crystallization that viable cell produces, and puts the 10min that vibrates on shaking table, in microplate reader in wavelength 570nm place each hole optical absorbance of mensuration.If the minimizing of comparing with control wells containing the hole absorbancy of polyoses extract is greater than 10% (namely inhibiting rate is greater than 10%), think toxic to cell under this concentration.
Cytotoxicity experiment result shows: with different concns (25,50,100,200,300ug/ml) Nostoc commune polysaccharide (NVPS), process MCF-7 cell 24 hours, inhibiting rate is about respectively 7.62%, 9.25%, 9.51%, 5.25%, 8.64% compared with the control, inhibiting rate is all less than 10%, so NVPS does not have toxicity (as shown in Figure 2) to cell under these concentration.
Embodiment 3:
Choose the MCF-7 cell of logarithmic phase, adjusting cell concn is every hole 8 * 10
3individual cell/100 μ l nutrient solution, is added in 96 well culture plates.37 ℃, 5%CO
2incubated overnight in incubator.After cell attachment, take out culture plate, change the substratum containing different concns (0,25,50,100,200,300ug/ml) Nostoc commune polysaccharide, establish 5 parallel holes.Culture plate is put back in incubator and is continued to cultivate 24 hours and 36 hours, and every hole adds 20 μ L5.0mg/mL MTT, continues to hatch 4 hours.Suck nutrient solution in plate, every hole adds the DMSO of 150 μ l to dissolve the first a ceremonial jade-ladle, used in libation crystallization that viable cell produces, and puts the 10min that vibrates on shaking table, in microplate reader in wavelength 570nm place each hole optical absorbance of mensuration.
Gained Nostoc commune polysaccharide of the present invention (NVPS), carries out above-mentioned anti tumor activity in vitro detection to human breast carcinoma JEG-3.Result shows: with the NVPS of different concns (0,25,50,100,200,300ug/ml), process MCF-7 cell 24 hours, and process 36 hours, 25,50, the NVPS of 100ug/ml to MCF-7 inhibiting rate all in 20% left and right and following, and process 36 hour concentrations 200, during 300ug/ml, NVPS is respectively 57.6%, 73.3% to MCF-7 inhibiting rate, compares have utmost point significant difference (P<0.01) (as shown in Figure 3) with control group.
Claims (7)
1. a preparation method for the antitumor polysaccharide of Nostoc commune, is characterized in that, comprises the steps:
The first step: get Nostoc commune, 60 ℃ of dry pulverizing after 2 hours, adding distil water reflux 3 hours, standing, double gauze filters, and gets supernatant liquor, as above repeat to extract 1 time, merging filtrate, reduction vaporization is concentrated into thick;
Second step: the extracting solution after concentrated adds 10.6% yellow prussiate of potash while stirring, shakes up, then adds 21.9% zinc acetate, and jolting is even, places 30 minutes under room temperature, and 5000rpm gets supernatant liquor after centrifugal 20 minutes, and reduction vaporization is concentrated into thick; Described yellow prussiate of potash, zinc acetate and extracting liquid volume ratio are 1 ︰ 1 ︰ 8-15;
The 3rd step: the dialysis tubing that is 3000 with interception, the concentrated solution that second step is obtained is dialysed in distilled water, within every 12 hours, changes water once, until conductivity of dialysate is constant;
The 4th step: in the extracting solution obtaining in the 3rd step, add dehydrated alcohol, vortex, room temperature is placed 0.5-1 hour, the centrifugal 15-20 minute of 5000rpm then, collecting precipitation; Described extracting solution and dehydrated alcohol volume ratio are 3-2 ︰ 7-8;
The 5th step: in above-mentioned precipitation, add appropriate distilled water, stirring and dissolving at 50-60 ℃, until become homogeneous liquid, then vacuum lyophilization becomes floss to obtain Crude polysaccharides sample, in 4 ℃ of preservations.
2. the preparation method of the antitumor polysaccharide of a kind of Nostoc commune as claimed in claim 1, is characterized in that, 10.6% potassium ferrocyanide solution described in second step, 21.9% acetic acid zinc solution and extracting liquid volume ratio are 1 ︰ 1 ︰ 10.
3. the preparation method of the antitumor polysaccharide of a kind of Nostoc commune as claimed in claim 1, is characterized in that, the extracting solution described in the 4th step and dehydrated alcohol volume ratio are 3 ︰ 7.
4. the preparation method of the antitumor polysaccharide of a kind of Nostoc commune as claimed in claim 1, is characterized in that, the dissolution precipitation Heating temperature described in the 5th step is 55 ℃.
5. the antitumor polysaccharide of Nostoc commune that preparation method makes as described in claim 1,2,3 or 4.
6. the application of the antitumor polysaccharide of Nostoc commune as claimed in claim 5 in preparing new type anticancer medicine.
7. the application of the antitumor polysaccharide of Nostoc commune as claimed in claim 5 in preparing healthcare products.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106317234A (en) * | 2015-06-29 | 2017-01-11 | 甘肃凯源生物技术开发中心 | Nostoc commune polysaccharide extraction method |
| CN106615103A (en) * | 2016-11-28 | 2017-05-10 | 山东坤泰生物科技有限公司 | Fermentation stirring type goat yogurt and preparation method thereof |
| CN110420142A (en) * | 2019-08-27 | 2019-11-08 | 上海澄穆化妆品有限公司 | A kind of extracting method of Aplotaxis auriculata extract |
| CN116903760A (en) * | 2023-05-25 | 2023-10-20 | 浙江大学 | Auricularia auricula polysaccharide, preparation method thereof and application thereof in inhibiting obesity |
Citations (1)
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|---|---|---|---|---|
| CN1220999A (en) * | 1997-12-25 | 1999-06-30 | 中国科学院水生生物研究所 | Method for extracting and separating filiform blue-green algae water-soluble polyose and ecto-polyose |
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2014
- 2014-09-11 CN CN201410459857.XA patent/CN104193845B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1220999A (en) * | 1997-12-25 | 1999-06-30 | 中国科学院水生生物研究所 | Method for extracting and separating filiform blue-green algae water-soluble polyose and ecto-polyose |
Non-Patent Citations (5)
| Title |
|---|
| HAIFENG LI,ET AL.: "Antioxidant and moisture-retention activities of the polysaccharide from Nostoc commune", 《CARBOHYDRATE POLYMERS》 * |
| 张唐伟等: "地木耳多糖的抗氧化性与抑菌作用", 《食品与生物技术学报》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106317234A (en) * | 2015-06-29 | 2017-01-11 | 甘肃凯源生物技术开发中心 | Nostoc commune polysaccharide extraction method |
| CN106317234B (en) * | 2015-06-29 | 2018-10-26 | 河西学院 | A kind of extracting process of Nostoc commune polysaccharide |
| CN106615103A (en) * | 2016-11-28 | 2017-05-10 | 山东坤泰生物科技有限公司 | Fermentation stirring type goat yogurt and preparation method thereof |
| CN110420142A (en) * | 2019-08-27 | 2019-11-08 | 上海澄穆化妆品有限公司 | A kind of extracting method of Aplotaxis auriculata extract |
| CN116903760A (en) * | 2023-05-25 | 2023-10-20 | 浙江大学 | Auricularia auricula polysaccharide, preparation method thereof and application thereof in inhibiting obesity |
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| CN104193845B (en) | 2016-06-22 |
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