CN104193845B - A kind of Nostoc commune antitumor polysaccharide and its preparation method and application - Google Patents
A kind of Nostoc commune antitumor polysaccharide and its preparation method and application Download PDFInfo
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- CN104193845B CN104193845B CN201410459857.XA CN201410459857A CN104193845B CN 104193845 B CN104193845 B CN 104193845B CN 201410459857 A CN201410459857 A CN 201410459857A CN 104193845 B CN104193845 B CN 104193845B
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Abstract
The invention provides a kind of Nostoc commune antitumor polysaccharide and preparation method thereof and the application that this polysaccharide is in preparing antitumor drug and health food。Its preparation method is by pure natural Nostoc commune crushed after being dried, weighs appropriate, adds distilled water backflow lixiviate, united extraction liquid；Said extracted liquid rotary evaporation is concentrated in right amount, adds 10.6% potassium ferrocyanide solution and 21.9% acetic acid zinc solution protein precipitation with volume ratio 1:1, Aspirate supernatant after centrifugal；The rotary evaporation that reduces pressure again is concentrated into certain volume, after being dialysed by concentrated solution distilled water, adds dehydrated alcohol and precipitates out White Flocculus, vacuum lyophilization again after centrifugal, namely obtain Nostoc commune polysaccharide。Anticancer experiment in vitro shows, this polysaccharide can substantially suppress human breast cancer cell to breed and no cytotoxicity, has good anti-tumor activity, provides possibility for developing new type anticancer health product or medicine。
Description
Technical field
The present invention relates to the preparation of blue green algae vegetable polysaccharides and application, particularly belong to a kind of Nostoc commune antitumor polysaccharide 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 (NostoccommuneVauch.), it is commonly called as land for building dish, another name lichens, dish mushroom, soft youngster, dish skin, ear, for the lamellar algae of algae Cyanophyceae nostocaceae (nostocaceae) nostoc nostoc (nostoccommunevauch)。Generally it is grown in the sandy soil surface of humidity, often mixes between the base portion and the liquor-saturated flora of tongue set between the stem and leaf of weeds base portion, greatly。Nostoc commune almost all has distribution in the whole nation, and there is wild resource distribution widely especially Changbaishan area, area northeastward, also has significantly high yield in Shanxi。
Compendium of Material Medica describes Nostoc commune: " abnormal smells from the patient: sweet, flat, nontoxic；Cure mainly: improving eyesight QI invigorating, the kidney invigorating。" " main improving eyesight, QI invigorating make us there is son in " Mingyi Bielu " middle description。" " food is examined " is thought: " clear god is antipyretic, and phlegm-fire can be treated " " of a specified duration clothes is prolonged life, the clear dirty hot person of Gai Yineng。Existing bibliographical information at present: it contains rich in protein, saccharide, various trace elements and certain crude fat and crude fibre, has higher edible and nutritive value, is the green hill wholefood of typical case；Simultaneously often edible have heat-clearing and toxic substances removing, cooling blood for improving eyesight, effect of enhancing metabolism, is good medical-care health food。Therefore, Nostoc commune can as the health food of a kind of food and medicament dual-purpose。
Polysaccharose substance refers to the natural polymer that a class is polymerized by more than 10 monosaccharide molecule, is one of macromolecular substances maintaining normal activities。There are some researches show, Nostoc commune polysaccharide material has antioxidation and bacteriostatic activity, also has the functions such as resisting fatigue, antiviral, blood sugar lowering, blood fat reducing, antiinflammatory, but not yet sees the research of Nostoc commune anti-tumor activity of polysaccharides。This experiment, with Nostoc commune for material, extracts a kind of polysaccharide with anti-tumor activity, and this polysaccharide can significantly inhibit the propagation of human breast cancer cell。Therefore, the present invention can not only 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 offer theoretical foundation。
Summary of the invention
It is an object of the invention to provide a kind of Nostoc commune polysaccharide with anti-tumor activity and preparation method thereof and its application in preparing antitumor drug and health food。
The method of a kind of Nostoc commune antitumor polysaccharide provided by the invention, comprises the steps:
The first step: take Nostoc commune, pulverizes after dry 2 hours, adds distilled water and be heated to reflux 3 hours, stand for 60 DEG C, and double gauze filters, and takes supernatant, extraction repeated as above 1 time, merging filtrate, be evaporated under reduced pressure be concentrated into thick；
Second step: the extracting solution after concentration adds the potassium ferrocyanide (K of 10.6% while stirring4[Fe(CN)6]·3H2O), shake up, add the zinc acetate (C of 21.9%4H8O4Zn), jolting is uniform, and with protein precipitation, ambient temperatare puts 30 minutes, and 5000rpm takes supernatant after centrifugal 20 minutes, reduction vaporization is concentrated into thick；Described potassium ferrocyanide, zinc acetate and extracting liquid volume ratio are 11 8-15；
3rd step: be the bag filter of 3000 with interception, the concentrated solution obtained by second step is dialysed in distilled water, within every 12 hours, changes water once, until conductivity of dialysate is constant, with the small molecular weight impurity such as deionizing and monosaccharide；
4th step: adding dehydrated alcohol, vortex in the extracting solution that the 3rd step obtains, room temperature is placed 0.5-1 hour, with precipitate polysaccharides, then centrifugal 15-20 minute of 5000rpm, collect precipitation；Described extracting solution and dehydrated alcohol volume ratio are 3-2 7-8；
5th step: add appropriate distilled water in above-mentioned precipitation, stirring and dissolving at 50-60 DEG C, until becoming homogeneous liquid, then vacuum lyophilization becomes floccule namely to obtain crude polysaccharides sample, and called after NVPS, in 4 DEG C of preservations。
10.6% potassium ferrocyanide solution described in second step, 21.9% acetic acid zinc solution and extracting liquid volume are than being preferably 11 10。
Extracting solution described in 4th step and dehydrated alcohol volume ratio are preferably 37。
Dissolution precipitation heating-up temperature described in 5th step is preferably 55 DEG C。
The Nostoc commune polysaccharide that above-mentioned preparation method prepares can be applied in preparing new type anticancer medicine, it is possible to applies in preparing health product。
Through anti tumor activity in vitro detection display, the Nostoc commune polysaccharide that above-mentioned preparation method prepares can substantially be bred by anticancer, has good active anticancer。Result show with this polysaccharide of variable concentrations process human breast cancer cell (MCF-7) 24 hours, concentration be 300ug/ml time, fatality rate reaches about 25%；Process 36 hours, concentration is when being 200ug/ml, and fatality rate is high to about 60%, so cancerous cell (MCF-7) is had good inhibition by this polysaccharide。
Polysaccharide prepared by the method, because it derives from pure natural algae, purifying process is simple in addition, therefore has bigger DEVELOPMENT PROSPECT and using value。As: in medicine, can have the cancer ancillary drug of anti-tumor activity as the adjuvant of other drug or preparation；In health product, the health care oral liquid with antitumor and anti-oxidation function can be prepared；Furthermore it is also possible to be applied in the minds of beverage and point as natural additive for foodstuff, it is alternatively arranged as emulsifying agent, wetting agent is added in cosmetics。
Accompanying drawing explanation
Fig. 1 Nostoc commune antitumor polysaccharide (NVPS) sample
Fig. 2 Nostoc commune antitumor polysaccharide (NVPS) affects bar diagram to Cytotoxic
Fig. 3 Nostoc commune antitumor polysaccharide (NVPS) induces MCF-7 apoptosis to have dose dependent bar diagram
Detailed description of the invention
The preparation method of embodiment 1 Nostoc commune antitumor polysaccharide (NVPS)
(1) will dry 2 hours of 60 DEG C of Auricularia, pulverizing natively。
(2) taking Nostoc commune powder 100g, add 5L distilled water and be heated to reflux 3 hours, stand, double gauze filters, and takes supernatant, extraction repeated as above 1 time, and 2 times filtrate merges, and is evaporated under reduced pressure and is concentrated into 1000ml；
(3) the land for building dish extracting solution obtained in step (2) is added 100ml potassium ferrocyanide (K while stirring4[Fe(CN)6]·3H2O) (10.6%), shakes up, and adds 100ml zinc acetate (C4H8O4Zn) (21.9%), jolting is uniform, and with protein precipitation, ambient temperatare puts 30 minutes, and 5000rpm takes supernatant after centrifugal 20 minutes, be evaporated under reduced pressure and be concentrated into 100ml；
(4) bag filter that concentrated solution interception is 3000 that step (3) obtains is dialysed in distilled water, within every 12 hours, change water once, until conductivity of dialysate is almost constant, the dialysis solution 120ml of the small molecular weight impurity such as ion and monosaccharide that is removed；
(5) adding 280ml dehydrated alcohol, vortex in the extracting solution that step (4) obtains, room temperature is placed 1 hour, with precipitate polysaccharides, and then centrifugal 20 minutes of 5000rpm, collect precipitation；
(6) adding 50ml distilled water heated and stirred in the precipitation that step (5) obtains to dissolve, until becoming homogeneous liquid, then vacuum lyophilization becomes floccule namely to obtain crude polysaccharides sample, and called after NVPS, in 4 DEG C of preservations；
Adopting phend-sulphuric acid, with glucose as a standard product measure Nostoc commune polyoses content。Found that polyoses content is about 8.734g Nostoc commune polysaccharide sample (as shown in Figure 1) in the Nostoc commune powder of 100g。
Embodiment 2: choose the MCF-7 cell of exponential phase, cell is made cell suspension, by cell counting, every hole 4 × 104Individual it is inoculated in 96 orifice plates, 37 DEG C, 5%CO2Incubator cultivates 24 hours after cell attachment, discard old culture medium, add not homopolysaccharide final concentration (0,25,50,100,200,300ug/ml) culture medium, if 5 parallel holes, continue to cultivate 24 hours by culture plate, every hole adds 20 μ L5.0mg/mLMTT, then hatches 4 hours。Removing culture fluid in plate, every hole adds the DMSO of 150 μ L and dissolves the first a ceremonial jade-ladle, used in libation crystallization that living cells produces, and puts vibration 10min on shaking table, in microplate reader in mensuration each hole, wavelength 570nm place optical absorbance。If the minimizing of the hole absorbance containing polyoses extract compared with control wells is more than 10% (namely suppression ratio is more than 10%), then it is assumed that under this concentration, cell is toxic。
Cytotoxicity experiment result shows: with variable concentrations (25,50,100,200,300ug/ml) Nostoc commune polysaccharide (NVPS) processes MCF-7 cell 24 hours, suppression ratio respectively may be about 7.62%, 9.25%, 9.51%, 5.25%, 8.64% compared with the control, suppression ratio is respectively less than 10%, so cell is not had toxicity (as shown in Figure 2) by NVPS at these concentrations。
Embodiment 3:
Choosing the MCF-7 cell of exponential phase, adjusting cell concentration is every hole 8 × 103Individual cell/100 μ l culture fluid, is added in 96 well culture plates。37 DEG C, 5%CO2Incubated overnight in incubator。After cell attachment, take out culture plate, change containing variable concentrations (0,25,50,100,200,300ug/ml) culture medium of Nostoc commune polysaccharide, if 5 parallel holes。Culture plate is put back to and is continued in incubator to cultivate 24 hours and 36 hours, and every hole adds 20 μ L5.0mg/mLMTT, continues to hatch 4 hours。Sucking culture fluid in plate, every hole adds the DMSO of 150 μ l and dissolves the first a ceremonial jade-ladle, used in libation crystallization that living cells produces, and puts vibration 10min on shaking table, in microplate reader in mensuration each hole, wavelength 570nm place optical absorbance。
Gained Nostoc commune polysaccharide (NVPS) of the present invention, carries out above-mentioned anti tumor activity in vitro detection to human breast carcinoma JEG-3。Result shows: with the NVPS of variable concentrations (0,25,50,100,200,300ug/ml) process MCF-7 cell 24 hours, and process 36 hours, 25,50, the NVPS of 100ug/ml is to MCF-7 suppression ratio all about 20% and below, and process 36 hour concentrations 200,300ug/ml time, NVPS, to MCF-7 suppression ratio respectively 57.6%, 73.3%, has pole significant difference (P < 0.01) (as shown in Figure 3) compared with matched group。
Claims (4)
1. Nostoc commune polysaccharide application in preparing anti-human breast cancer medicines；Described Nostoc commune polysaccharide is prepared by the method comprised the steps:
The first step: take Nostoc commune, pulverizes after dry 2 hours, adds distilled water and be heated to reflux 3 hours, stand for 60 DEG C, and double gauze filters, and takes supernatant, extraction repeated as above 1 time, merging filtrate, be evaporated under reduced pressure be concentrated into thick；
Second step: the extracting solution after concentration adds the potassium ferrocyanide solution of 10.6% while stirring, shakes up, adds the acetic acid zinc solution of 21.9%, jolting is uniform, ambient temperatare puts 30 minutes, and 5000rpm takes supernatant after centrifugal 20 minutes, reduction vaporization is concentrated into thick；Described potassium ferrocyanide solution, acetic acid zinc solution and extracting liquid volume ratio are 11 8-15；
3rd step: be the bag filter of 3000 with interception, the concentrated solution obtained by second step is dialysed in distilled water, within every 12 hours, changes water once, until conductivity of dialysate is constant；
4th step: adding dehydrated alcohol, vortex in the extracting solution that the 3rd step obtains, room temperature is placed 0.5-1 hour, then centrifugal 15-20 minute of 5000rpm, collects precipitation；Described extracting solution and dehydrated alcohol volume ratio are 3-2 7-8；
5th step: add appropriate distilled water in above-mentioned precipitation, stirring and dissolving at 50-60 DEG C, until becoming homogeneous liquid, then vacuum lyophilization becomes floccule namely to obtain crude polysaccharides sample, in 4 DEG C of preservations。
2. Nostoc commune polysaccharide application in preparing anti-human breast cancer medicines as claimed in claim 1, it is characterised in that 10.6% potassium ferrocyanide solution described in second step, 21.9% acetic acid zinc solution and extracting liquid volume ratio are 11 10。
3. Nostoc commune polysaccharide application in preparing anti-human breast cancer medicines as claimed in claim 1, it is characterised in that the extracting solution described in the 4th step and dehydrated alcohol volume ratio are 37。
4. Nostoc commune polysaccharide application in preparing anti-human breast cancer medicines as claimed in claim 1, it is characterised in that the dissolution precipitation heating-up temperature described in the 5th step is 55 DEG C。
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| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 (4)
| Title |
|---|
| Antioxidant and moisture-retention activities of the polysaccharide from Nostoc commune;Haifeng Li,et al.;《Carbohydrate Polymers》;20101027;第83卷;第1821-1827页 * |
| 地木耳多糖的抗氧化性与抑菌作用;张唐伟等;《食品与生物技术学报》;20111130;第30卷(第6期);第868-873页 * |
| 地木耳多糖的提取与纯化研究;张唐伟等;《中国野生植物资源》;20110531;第39卷(第3期);第34-37页 * |
| 苯酚-硫酸法测定西宁地区地木耳中多糖含量;张唐伟等;《分析实验室》;20100531;第29卷;第219-220页 * |
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