CN103073655B - The extracting method of a kind of Angelica Polysaccharide and application - Google Patents
The extracting method of a kind of Angelica Polysaccharide and application Download PDFInfo
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- CN103073655B CN103073655B CN201310047719.6A CN201310047719A CN103073655B CN 103073655 B CN103073655 B CN 103073655B CN 201310047719 A CN201310047719 A CN 201310047719A CN 103073655 B CN103073655 B CN 103073655B
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Abstract
The present invention discloses extracting method and the application of a kind of Angelica Polysaccharide.The present invention uses high-temperature organic acid method to extract and combines ultrafiltration membrane treatment and prepare Angelica Polysaccharide.After Radix Glycyrrhizae is pulverized, extracted by high-temperature organic acid, ultrafiltration is dammed, concentrate and ethanol precipitate and separate obtains Angelica Polysaccharide.The Angelica Polysaccharide present invention prepared joins in Medicated cigarette with tobacco shred weight 0.05% ~ 0.2%, and the Nicotiana tabacum L. agglutinator of gained Medicated cigarette is significantly higher than blank sample to human bronchial epithelial BEAS 2B cell survival rate, has positive effect.
Description
Technical field
The present invention relates to extracting method and the application of glycyrrhiza extract deep process technology, specifically Angelica Polysaccharide.
Background technology
Along with improving constantly of people's living standard and quality of life, cigarette consumption changes to brand consumption, especially high-grade cigarette market, consumer to health, the local flavor of Medicated cigarette it is also proposed that the highest requirement.Production low tar, the Medicated cigarette of less harmful have become one of important directions of China or even the development of world's cigarette industry.At present, the main technological route reducing Medicated cigarette harm employing has two: one is " fall Jiao ", i.e. the burst size by reducing coke tar in cigarette makes harmful components reduce simultaneously, i.e. concertedness reduces cigarette smoke harmful components；Another kind is " harm reduction ", i.e. uses special technique and adds functional component to reduce part harmful components in cigarette smoke, and harmful components in the purpose less to other composition effects, i.e. selectivity reducing cigarette fume.Nicotiana tabacum L. agglutinator (CSC) is an important indicator of evaluating cigarette safety to the cytotoxicity of human bronchial epithelial BEAS-2B cell.The most existing many documents report CSC induction human bronchial epithelial BEAS-2B apoptosis has also inquired into its action pathway and mechanism.Have not yet to see the Cytotoxic method reducing Nicotiana tabacum L. agglutinator (CSC) to human bronchial epithelial BEAS-2B cell.
Radix Glycyrrhizae is perennial herb, is distributed mainly on the ground such as Xinjiang, Qinghai, Gansu.Radix Glycyrrhizae has the effect of QI invigorating invigorating middle warmer, relieving spasm to stop pain, nourishing the lung to arrest cough, eliminating fire and detoxication and coordinating the actions of various ingredients in a prescription, is widely used in medicine and food service industry.China Radix Glycyrrhizae is mainly for the production of Radix Glycyrrhizae extract, glycyrrhizic acid and derivant thereof at present, and glycyrrhiza extract is the by-product of Radix Glycyrrhizae processing.For a long time, the glycyrrhizin (having another name called glycyrrhizic acid) in Radix Glycyrrhizae is only extracted by industrialized production as principle active component, extracts the glycyrrhiza extract after glycyrrhizic acid and is then taken as garbage to abandon, causes environmental pollution and the wasting of resources.Owing to process technology means limit, current China glycyrrhiza extract is not the most fully used, or for breeding feed, or soil is directly filled go back to as fertilizer.In recent years, the deep processing and utilization of glycyrrhiza extract becomes the focus and emphasis of studies in China.Granted patent (patent No. 201010242200.X) uses glycyrrhiza extract to be that reconstituted tobacoo prepared by raw material, and finds reconstituted tobacoo to add in Nicotiana tabacum L. to have without tar content, carbon monoxide release amount flavouring relatively low, obvious and the effect of minimizing Medicated cigarette excitement.Granted patent (patent No. 201010111656.2) discloses a kind of method extracting blood-sugar decreasing active from licorice slag.Invention (application number 200510119580.7) discloses a kind of method extracting glycyrrhizic acid from licorice slag.Invention (application number 02109474.8) discloses a kind of method extracting licoflavone from licorice slag.Having not yet to see with glycyrrhiza extract or Radix Glycyrrhizae as raw material, preparation has the report of the functional Angelica Polysaccharide of Nicotiana tabacum L. agglutinator (CSC) cytotoxicity reducing Medicated cigarette.
Summary of the invention
It is an object of the invention to fill up and reduce Medicated cigarette Nicotiana tabacum L. agglutinator (CSC) blank to the cytotoxicity method of human bronchial epithelial BEAS-2B cell about application Angelica Polysaccharide at present, it is provided that the extracting method of a kind of Angelica Polysaccharide and application.The Angelica Polysaccharide present invention prepared joins in Medicated cigarette, and the Nicotiana tabacum L. agglutinator of gained Medicated cigarette is significantly higher than blank sample to human bronchial epithelial BEAS-2B cell survival rate, has positive effect.
Invention also provides in Medicated cigarette, add Angelica Polysaccharide reduction Medicated cigarette Nicotiana tabacum L. agglutinator (CSC) the Cytotoxic application to human bronchial epithelial BEAS-2B cell.
The purpose of the present invention is achieved by following technical solution:
The extracting method of a kind of Angelica Polysaccharide, it comprises the steps:
(1) glycyrrhiza extract removes sand clean, pulverizes to obtain glycyrrhiza extract raw material after drying；
(2) in glycyrrhiza extract raw material, add organic acid soln, extract 2-5 hour at 110-120 DEG C, filter, separate glycyrrhiza extract extracting solution, with potassium hydroxide regulation extracting liquid pH value to 6.5-7.0；
(3) the daltonian film of extracting solution molecular cut off 5000 of step (2) gained is carried out ultrafiltration, collect liquid part of damming；
(4) ultrafiltration permeate is concentrated in vacuo, to the 1/7 ~ 1/3 of original volume, obtains concentrated solution；
(5) in concentrated solution, add ethanol, after mix homogeneously, at 0-4 DEG C, stand 1-3 hour, filter, remove supernatant part, obtain polysaccharide precipitation；
(6) precipitate, lyophilization, it is thus achieved that Angelica Polysaccharide are collected.
Improving further, step (1) described glycyrrhiza extract is crushed to 40-100 mesh.
Improve further, step (2) described organic acid soln be in citric acid or malic acid any one, and the pH value of solution is 1.0-3.0.
Improving further, the consumption of step (2) described organic acid soln adds by 20-40 times of glycyrrhiza extract raw material weight.
Improving further, after step (5) described ethanol addition meets mix homogeneously, final ethanol concentration is 75-90%(v/v).
Improving further, step (5) described polysaccharide precipitation ethanol embathes 2-3 time.
Improving further, step (6) gained precipitate carries out lyophilization, obtains light yellow Angelica Polysaccharide.
The Angelica Polysaccharide that said method obtains application in Medicated cigarette, specifically according to the ratio of 0.05-0.2% of tobacco quality, is added in tobacco shred or filter plug by spray pattern.
Present invention employs above technical scheme, have such advantages as and beneficial effect:
1, the invention provides the extraction separation method of a kind of Angelica Polysaccharide, the Angelica Polysaccharide present invention prepared joins in Medicated cigarette with tobacco shred weight 0.05% ~ 0.2%, the Nicotiana tabacum L. agglutinator of gained Medicated cigarette is significantly higher than blank sample to human bronchial epithelial BEAS-2B cell survival rate, has positive effect.Additionally, the Angelica Polysaccharide prepared of the present invention on the sucking quality of Medicated cigarette without impact.
2, the present invention is with the glycyrrhiza extract of low value as raw material, and preparation has the Angelica Polysaccharide of functional activity, it is achieved that the higher value application of glycyrrhiza extract resource.Disclosed Angelica Polysaccharide extracting method has the advantages that technological process is simple, be prone to industrialization.
Detailed description of the invention
Nicotiana tabacum L. agglutinator cell toxicity test: collecting cigarette smoke by standard smoking conditions (GB/T 16450-1996, the standard conditions that conventional analysis defines with smoking machine), prepare flue gas agglutinator, be configured to the solution of 1 cigarette/ml with cell culture fluid, liquid nitrogen storage is standby.During experiment, it is diluted to experimental design concentration.Human bronchial epithelial BEAS-2B cell uses the LHC-8 culture medium of serum-free, cultivates under 37 degree, 5% carbon dioxide and 95% damp condition.
By the cell of exponential phase of growth, being inoculated in 96 orifice plates with suitable density, add the Nicotiana tabacum L. agglutinator of variable concentrations after 24h, every kind of condition sets 6 parallel oceans, culture medium cumulative volume is 200ul, is continuing cultivation 72h under 37 degree, 5% carbon dioxide and 95% damp condition.Cultivation terminates front 4h, and every hole adds 5mg/ml MTT solution 20ul.After cultivation terminates, take supernatant, add 150ulDMSO, piping and druming mixing, after purple crystal all dissolves, measure the OD value of 490nm wavelength by microplate reader, calculate cell survival fraction.
It is embodied as being described further to the present invention below in conjunction with embodiment and subordinate list.
Embodiment
1
(1) glycyrrhiza extract removes sand clean, pulverizes after drying, and crosses 40 mesh sieves and obtains glycyrrhiza extract raw material；
(2) press 35 times of addition water of glycyrrhiza extract raw material weight, extract 5 hours at 110 DEG C, filter, separate glycyrrhiza extract, obtain extracting solution, with potassium hydroxide regulation extracting liquid pH value to 7.0；
(3) the daltonian film of extracting solution molecular cut off 5000 of step (2) gained is carried out ultrafiltration, collect liquid part of damming；
(4) extracting solution is concentrated in vacuo, to the 1/5 of original volume, obtains concentrated solution；
(5) adding ethanol in concentrated solution, after mix homogeneously, final ethanol concentration is 70%(v/v), at 2 DEG C, stand 3 hours, filter, remove supernatant part, obtain polysaccharide precipitation；
(5) precipitate, lyophilization, it is thus achieved that light yellow glycyrrhiza extract polysaccharide 1# are collected.
Embodiment
2
(1) glycyrrhiza extract removes sand clean, pulverizes after drying, and crosses 40 mesh sieves and obtains glycyrrhiza extract raw material；
(2) adding pH value by 35 times of glycyrrhiza extract raw material weight is the citric acid solution of 2, extracts 5 hours, filters, separate glycyrrhiza extract, obtain extracting solution at 110 DEG C, with potassium hydroxide regulation extracting liquid pH value to 7.0；
(3) the daltonian film of extracting solution molecular cut off 5000 of step (2) gained is carried out ultrafiltration, collect liquid part of damming；
(4) liquid that will dam is concentrated in vacuo, and to the 1/5 of original volume, obtains concentrated solution；
(5) adding ethanol in concentrated solution, after mix homogeneously, final ethanol concentration is 70%(v/v), at 2 DEG C, stand 3 hours, filter, remove supernatant part, obtain polysaccharide precipitation；
(5) precipitate, lyophilization, it is thus achieved that light yellow glycyrrhiza extract polysaccharide 2# are collected.
Embodiment
3
(1) glycyrrhiza extract removes sand clean, pulverizes after drying, and crosses 80 mesh sieves and obtains glycyrrhiza extract raw material；
(2) adding pH value by 20 times of glycyrrhiza extract dry powder weight is the citric acid solution of 1, extracts 2 hours, filter at 120 DEG C, separates glycyrrhiza extract slag and obtains extracting solution, with potassium hydroxide regulation extracting liquid pH value to 6.5；
(3) the daltonian film of extracting solution molecular cut off 5000 of step (2) gained is carried out ultrafiltration, collect liquid part of damming；
(4) ultrafiltration permeate is concentrated in vacuo, to the 1/5 of original volume, obtains concentrated solution；
(5) adding ethanol in concentrated solution, after mix homogeneously, final ethanol concentration is 85%(v/v), at 4 DEG C, stand 12 hours, filter, remove supernatant part, obtain polysaccharide precipitation, and embathe 2-3 time with ethanol and acetone；
(6) precipitate, lyophilization, it is thus achieved that light yellow glycyrrhiza extract polysaccharide 3# are collected.
Embodiment
4
(1) glycyrrhiza extract removes sand clean, pulverizes after drying, and crosses 100 mesh sieves and obtains glycyrrhiza extract raw material；
(2) adding pH value by 25 times of glycyrrhiza extract dry powder weight is the malic acid solution of 2.5, extracts 4 hours, filter at 115 DEG C, separates glycyrrhiza extract slag and obtains extracting solution, with potassium hydroxide regulation extracting liquid pH value to 6.7；
(3) the daltonian film of extracting solution molecular cut off 5000 of step (2) gained is carried out ultrafiltration, collect liquid part of damming；
(4) ultrafiltration permeate is concentrated in vacuo, to the 1/7 of original volume, obtains concentrated solution；
(5) adding ethanol in concentrated solution, after mix homogeneously, final ethanol concentration is 80%(v/v), at 3 DEG C, stand 10 hours, filter, remove supernatant part, obtain polysaccharide precipitation, embathe 2-3 time with ethanol；
(6) precipitate, lyophilization, it is thus achieved that light yellow glycyrrhiza extract polysaccharide 4# are collected.
Embodiment
5
(1) glycyrrhiza extract removes sand clean, pulverizes after drying, and crosses 80 mesh sieves and obtains glycyrrhiza extract raw material；
(2) adding pH value by 25 times of glycyrrhiza extract dry powder weight is the citric acid solution of 3.0, extracts 5 hours, filter at 110 DEG C, separates glycyrrhiza extract slag and obtains extracting solution, with potassium hydroxide regulation extracting liquid pH value to 6.8；
(3) the daltonian film of extracting solution molecular cut off 5000 of step (2) gained is carried out ultrafiltration, collect liquid part of damming；
(4) ultrafiltration permeate is concentrated in vacuo, to the 1/3 of original volume, obtains concentrated solution；
(5) adding ethanol in concentrated solution, after mix homogeneously, final ethanol concentration is 90%(v/v), at 4 DEG C, stand 3 hours, filter, remove supernatant part, obtain polysaccharide precipitation, embathe 3 times with ethanol；
(6) precipitate is collected, lyophilization, it is thus achieved that lurid glycyrrhiza extract polysaccharide 5#,.
The Medicated cigarette CSC impact on human bronchial epithelial BEAS-2B cell proliferation
| Product | Addition | IC50 |
| Angelica Polysaccharide 1# | 0.1% | 0.00299 cigarette/ml |
| Angelica Polysaccharide 2# | 0.1% | 0.00485 cigarette/ml |
| Angelica Polysaccharide 3# | 0.1% | 0.00496 cigarette/ml |
| Angelica Polysaccharide 4# | 0.1% | 0.00499 cigarette/ml |
| Angelica Polysaccharide 5# | 0.1% | VC |
| Blank | 0.00295 cigarette/ml |
From table 1, using deionized water dissolving Angelica Polysaccharide, added the Angelica Polysaccharide of 0.1% by spray pattern in Medicated cigarette, its Medicated cigarette CSC is to 1.74 times that the IC50 value of human bronchial epithelial BEAS-2B cell proliferation is blank sample.Angelica Polysaccharide its Medicated cigarette CSC using conventional hot water extraction to obtain is suitable with blank sample to the IC50 value of human bronchial epithelial BEAS-2B cell proliferation.This shows, the Angelica Polysaccharide prepared by the present invention can significantly improve the Medicated cigarette CSC IC50 value to human bronchial epithelial BEAS-2B cell proliferation, and the safety cigarette that i.e. with the addition of Angelica Polysaccharide is higher.
Claims (3)
1. the extracting method of an Angelica Polysaccharide, it is characterised in that comprise the steps:
(1) glycyrrhiza extract removes sand clean, pulverizes to obtain glycyrrhiza extract raw material after drying；Described glycyrrhiza extract is crushed to 40-100 mesh；
(2) in glycyrrhiza extract raw material, add organic acid soln, extract 2-5 hour at 110-120 DEG C, filter, separate glycyrrhiza extract extracting solution, with potassium hydroxide regulation extracting liquid pH value to 6.5-7.0；Described organic acid soln be in citric acid or malic acid any one, and the pH value of solution is 1.0-3.0；Described organic acid soln
Consumption add by 20-40 times of glycyrrhiza extract raw material weight；
(3) the daltonian film of extracting solution molecular cut off 5000 of step (2) gained is carried out ultrafiltration, collect liquid part of damming；
(4) ultrafiltration permeate is concentrated in vacuo, to the 1/7 ~ 1/3 of original volume, obtains concentrated solution；
(5) in concentrated solution, add ethanol, after mix homogeneously, at 0-4 DEG C, stand 1-3 hour, filter, remove supernatant part, obtain polysaccharide precipitation；After described ethanol addition meets mix homogeneously, final ethanol concentration is 75-90%(v/v)；
(6) precipitate, lyophilization, it is thus achieved that Angelica Polysaccharide are collected；Described Angelica Polysaccharide has the activity improving human bronchial epithelial BEAS-2B cell survival rate.
Extraction separation method the most according to claim 1, it is characterised in that step (5) described polysaccharide precipitation ethanol embathes 2-3 time.
Extraction separation method the most according to claim 1, it is characterised in that step (6) gained precipitate carries out lyophilization, obtains light yellow Angelica Polysaccharide.
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| CN104644570A (en) * | 2014-06-26 | 2015-05-27 | 黄心诚 | Preparation method of licorice root polysaccharide powder injection |
| CN106962194A (en) * | 2017-04-11 | 2017-07-21 | 乌鲁木齐天格尔农业科技开发有限公司 | The method for tissue culture of Chinese bush cherry |
| CN109957038A (en) * | 2019-03-16 | 2019-07-02 | 莎车县富源甘草制品有限公司 | A kind of preparation method of Radix Glycyrrhizae Thick many candies |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101525391A (en) * | 2009-03-31 | 2009-09-09 | 武汉大学 | Method for extracting amylase from liquorice and application thereof |
| CN101766680A (en) * | 2008-12-31 | 2010-07-07 | 李志方 | Comprehensive extraction method of glycyrrhiza |
| CN101921656A (en) * | 2009-06-11 | 2010-12-22 | 湖北中烟工业有限责任公司 | Method for extracting liquorice flavoring extract for cigarette additive from glycyrrhizic-acid produced waste liquid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101766680A (en) * | 2008-12-31 | 2010-07-07 | 李志方 | Comprehensive extraction method of glycyrrhiza |
| CN101525391A (en) * | 2009-03-31 | 2009-09-09 | 武汉大学 | Method for extracting amylase from liquorice and application thereof |
| CN101921656A (en) * | 2009-06-11 | 2010-12-22 | 湖北中烟工业有限责任公司 | Method for extracting liquorice flavoring extract for cigarette additive from glycyrrhizic-acid produced waste liquid |
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