CN104098710A - Method for separating silkworm excrement total polysaccharides from water decoction of silkworm excrement - Google Patents
Method for separating silkworm excrement total polysaccharides from water decoction of silkworm excrement Download PDFInfo
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Abstract
The invention discloses a method for separating silkworm excrement total polysaccharides from water decoction of silkworm excrement, which belongs to the field of traditional Chinese medicine. According to the method, a macroporous adsorption resin adsorption process is adopted, process steps consisting of extraction, removal of protein, macroporous resin adsorption and decoloring are carried out, and high-purity silkworm excrement total polysaccharides are prepared from silkworm excrement; the method has improved production efficiency and obviously better effects than the prior art and is beneficial for further development of silkworm excrement into a drug used for treating diabetes so as to obtain greater economic values and social benefits.
Description
Technical field
The invention belongs to the field of Chinese medicines, be specifically related to a kind of method that separates silkworm excrement total polysaccharides from silkworm excrement decocting liquid.
Background technology
Silkworm excrement is traditional Chinese medicine simply, can dispel rheumatism, stimulate the circulation of the blood and cause the muscles and joints to relax, dampness elimination and in.Cure mainly rheumatic arthralgia, limbs are unsuccessful, rubella itch, the convulsion of vomiting and diarrhoea, women's amenorrhoea, uterine bleeding.
Silkworm is made a living with mulberry leaf, after edible fresh mulberry leaf, silkworm has has only digested and assimilated the nutritive substances such as the part carbohydrate in mulberry leaf, protein and fat, the nutritive substance that chlorophyll in mulberry leaf is not absorbed and used with other is discharged from ight soil with together with metabolic end product in silkworm body, so the nutritive ingredient of silkworm excrement is abundant.
After measured, in silkworm excrement, contain the polysaccharide component of 10% left and right.The document of report silkworm excrement polysaccharide is less, but silkworm excrement is the enrichment of mulberry leaf effective constituent, and its polysaccharide researches can be with reference to polysaccharides of Folium Mori correlative study.Chen Fujun (referring to: Chen Fujun, Lu Jun, Zhang Yongyu, in pharmacological research (I)-Sang of mulberry, blood-sugar-reducing effective components is on the glycometabolic impact of diabetic animal [J], Shenyang Pharmaceutical University's journal, 1996,13 (1): 24-27.) etc. reported that mulberry leaf total polysaccharides has significant hypoglycemic activity to alloxan diabetes mouse; Zhao Jun etc. (referring to: Zhao Jun, Gao Lan. the hypoglycemic lipid-lowering effect [J] of polysaccharides of Folium Mori, Tianjin Chinese materia medica, 2004,21 (6): 505-506.), by feeding diabetes B rat after the water-soluble polysaccharide separating-purifying in mulberry leaf, result shows that polysaccharides of Folium Mori has the effect that reduces blood sugar and suppress blood fat rising; The long brave a kind of polysaccharide peptide mixture that obtains separating from mulberry leaf that waits of Xue, the diabetic mice result of feeding STZ induction shows that mulberry leaf Polysaccharide-Peptide complex has the effect that reduces blood sugar and blood fat (referring to Xue Changyong, Teng Junying, Qiu Jihong, Deng. the hypoglycemic blood fat [J] of polysaccharides of Folium Mori one peptide complex, Journal of Nutrition, 2005,27 (2): 167-168.); In tradition application, this just treats for diabetes silkworm excrement, this may be relevant with the polysaccharide component in silkworm excrement, can consider silkworm excrement polysaccharide to further investigate, silkworm excrement further be developed to the medicine for the treatment of diabetes, to obtain larger economic worth and social benefit.
The object of the present invention is to provide a kind of mass-producing of silkworm excrement polysaccharide to extract process for purification.
Summary of the invention
The object of the present invention is to provide a kind of new method that separates silkworm excrement total polysaccharides taking silkworm excrement as raw material, overcome prior art weak point, improve production efficiency and the product purity of silkworm excrement total polysaccharides.
The present invention separates silkworm excrement total polysaccharides method taking silkworm excrement as raw material comprises the following steps:
A. silkworm excrement is got in right amount and decocted a drug wrapped, add the water of ten times of amounts, decoct a drug wrapped and decoct 2 times, each 30 minutes, merging filtrate.
B. after filtrate being concentrated, with 4800r/min, after centrifugal 10min, remove precipitation, obtain silkworm excrement polysaccharide crude extract.
C. add existing preparation 10% trichoroacetic acid(TCA), add-on and silkworm excrement polysaccharide crude extract volume ratio are 1: 10, and the refrigeration of silkworm excrement Crude polysaccharides extracting solution is left standstill to 24h, get supernatant liquor with 10000r/min, 17 DEG C, high speed centrifugation 20min, discards precipitation, and supernatant liquor merging is concentrated into after certain volume, add 95% ethanol precipitation to make to reach 80% containing alcohol amount, leave standstill after 48h in refrigerator cold-storage, leaching precipitation, obtains silkworm excrement Crude polysaccharides.
D. the above-mentioned a small amount of distilled water of silkworm excrement Crude polysaccharides removing after albumen is dissolved, upper AB-8 type macroporous adsorptive resins, with distilled water wash-out, is eluted to elutriant and adds 95% ethanol without till precipitating.Elutriant is concentrated into certain volume, obtains refining polysaccharide extraction liquid.
E. refining polysaccharide extraction liquid, add 60 DEG C of about 2h of decolouring of gac (solid-to-liquid ratio is 1: 10), filtered while hot, filtrate adds 95% ethanol to make to reach 80% containing alcohol amount after being concentrated into small volume, suction filtration after leaving standstill, for precipitation, repeatedly, 60 DEG C of following temperature are dry for dehydrated alcohol, acetone repetitive scrubbing, obtain refining silkworm excrement total polysaccharides.
In step D, said macroporous adsorbent resin is AB-8 type, D-101 type, HPD-400 type, NKA-9 type or H801 type macroporous adsorbent resin.
Absorption with macroporous adsorbent resin method is the one in solid-liquid pillar chromatographic separation technology, since the seventies in last century, be progressively applied in the clarifying process of Chinese medicine preparation, its adsorption is to reach by surface adsorption, surface electrical behavior or formation hydrogen bond, is a kind of very effective, method simple, reliable for effect, production cost is lower of regenerating.
Absorption with macroporous adsorbent resin method has a wide range of applications in the clarifying process of Chinese medicine preparation, especially in the separation of water soluble component, is showing more and more important effect.The inventor also once attempted macroporous adsorbent resin purification technique in the research process that improves silkworm excrement total polysaccharides extraction yield, that is: according to ordinary method silkworm excrement is decocted-centrifugation, then filtrate is carried out to purification with macroreticular resin technology and carry out purifying, found that the impurity extracting is very many, the separation costs of silkworm excrement total polysaccharides is high.Contriver has carried out test repeatedly, to finding better separation means, but always undesirable; After this in the time that the each composition of silkworm excrement is carried out to chromatographic analysis, contriver chances on, need to carry out the disengaging of protein, so contriver adopts trichoroacetic acid(TCA) deproteination matter, after test of many times, finally determine again the best approach using present method as refining silkworm excrement total polysaccharides.Highly purified silkworm excrement total polysaccharides, contains less acrobatics and organic solvent, and whole technological process is safe and simple, with low cost, is beneficial to suitability for industrialized production.
Embodiment
Embodiment 1:
Get 100g silkworm excrement, add 1000g water, decoct a drug wrapped and decoct 2 times, each 30 minutes, merging filtrate; After concentrated, with 4800r/min, after centrifugal 10min, remove precipitation, obtain silkworm excrement polysaccharide crude extract 460g.Add existing preparation 10% trichoroacetic acid(TCA), add-on and silkworm excrement polysaccharide crude extract volume ratio are 1: 10, and the refrigeration of silkworm excrement Crude polysaccharides extracting solution is left standstill to 24h, get supernatant liquor with 10000r/min, 17 DEG C, high speed centrifugation 20min, discards precipitation, and supernatant liquor merging is concentrated into after certain volume, add 95% ethanol precipitation to make to reach 80% containing alcohol amount, leave standstill after 48h in refrigerator cold-storage, leaching precipitation, obtains silkworm excrement Crude polysaccharides.The above-mentioned a small amount of distilled water of silkworm excrement Crude polysaccharides removing after albumen is dissolved, and upper AB-8 type macroporous adsorptive resins, with distilled water wash-out, is eluted to elutriant and adds 95% ethanol without till precipitating.Elutriant is concentrated into 170ml, obtains refining polysaccharide extraction liquid.Refining polysaccharide extraction liquid, add 60 DEG C of about 2h of decolouring of gac (solid-to-liquid ratio is 1: 10), filtered while hot, filtrate adds 95% ethanol to make to reach 80% containing alcohol amount after being concentrated into small volume, suction filtration after leaving standstill, for precipitation, repeatedly, 60 DEG C of following temperature are dry for dehydrated alcohol, acetone repetitive scrubbing, obtain the refining total polysaccharides 13g of silkworm excrement.
Embodiment 2:
Get 500g silkworm excrement, add 5000g water, decoct a drug wrapped and decoct 2 times, each 30 minutes, merging filtrate; After concentrated, with 4800r/min, after centrifugal 10min, remove precipitation, obtain silkworm excrement polysaccharide crude extract 1980g.Add existing preparation 10% trichoroacetic acid(TCA), add-on and silkworm excrement polysaccharide crude extract volume ratio are 1: 10, and the refrigeration of silkworm excrement Crude polysaccharides extracting solution is left standstill to 24h, get supernatant liquor with 10000r/min, 17 DEG C, high speed centrifugation 20min, discards precipitation, and supernatant liquor merging is concentrated into after certain volume, add 95% ethanol precipitation to make to reach 80% containing alcohol amount, leave standstill after 48h in refrigerator cold-storage, leaching precipitation, obtains silkworm excrement Crude polysaccharides.The above-mentioned a small amount of distilled water of silkworm excrement Crude polysaccharides removing after albumen is dissolved, and upper AB-8 type macroporous adsorptive resins, with distilled water wash-out, is eluted to elutriant and adds 95% ethanol without till precipitating.Elutriant is concentrated into 850ml, obtains refining polysaccharide extraction liquid.Refining polysaccharide extraction liquid, add 60 DEG C of about 2h of decolouring of gac (solid-to-liquid ratio is 1: 10), filtered while hot, filtrate adds 95% ethanol to make to reach 80% containing alcohol amount after being concentrated into small volume, suction filtration after leaving standstill, for precipitation, repeatedly, 60 DEG C of following temperature are dry for dehydrated alcohol, acetone repetitive scrubbing, obtain the refining total polysaccharides 64g of silkworm excrement.
Embodiment 3:
Get 1000g silkworm excrement, add 10000g water, decoct a drug wrapped and decoct 2 times, each 30 minutes, merging filtrate; After concentrated, with 4800r/min, after centrifugal 10min, remove precipitation, obtain silkworm excrement polysaccharide crude extract 5640g.Add existing preparation 10% trichoroacetic acid(TCA), add-on and silkworm excrement polysaccharide crude extract volume ratio are 1: 10, and the refrigeration of silkworm excrement Crude polysaccharides extracting solution is left standstill to 24h, get supernatant liquor with 10000r/min, 17 DEG C, high speed centrifugation 20min, discards precipitation, and supernatant liquor merging is concentrated into after certain volume, add 95% ethanol precipitation to make to reach 80% containing alcohol amount, leave standstill after 48h in refrigerator cold-storage, leaching precipitation, obtains silkworm excrement Crude polysaccharides.The above-mentioned a small amount of distilled water of silkworm excrement Crude polysaccharides removing after albumen is dissolved, and upper AB-8 type macroporous adsorptive resins, with distilled water wash-out, is eluted to elutriant and adds 95% ethanol without till precipitating.Elutriant is concentrated into 1750ml, obtains refining polysaccharide extraction liquid.Refining polysaccharide extraction liquid, add 60 DEG C of about 2h of decolouring of gac (solid-to-liquid ratio is 1: 10), filtered while hot, filtrate adds 95% ethanol to make to reach 80% containing alcohol amount after being concentrated into small volume, suction filtration after leaving standstill, for precipitation, repeatedly, 60 DEG C of following temperature are dry for dehydrated alcohol, acetone repetitive scrubbing, obtain the refining total polysaccharides 125g of silkworm excrement.
Claims (2)
1. a method that separates silkworm excrement total polysaccharides from silkworm excrement decocting liquid, is characterized in that:
A. silkworm excrement is got in right amount and decocted a drug wrapped, add the water of ten times of amounts, decoct a drug wrapped and decoct 2 times, each 30 minutes, merging filtrate.
B. after filtrate being concentrated, with 4800r/min, after centrifugal 10min, remove precipitation, obtain silkworm excrement polysaccharide crude extract.
C. add existing preparation 10% trichoroacetic acid(TCA), add-on and silkworm excrement polysaccharide crude extract volume ratio are 1: 10, and the refrigeration of silkworm excrement Crude polysaccharides extracting solution is left standstill to 24h, get supernatant liquor with 10000r/min, 17 DEG C, high speed centrifugation 20min, discards precipitation, and supernatant liquor merging is concentrated into after certain volume, add 95% ethanol precipitation to make to reach 80% containing alcohol amount, leave standstill after 48h in refrigerator cold-storage, leaching precipitation, obtains silkworm excrement Crude polysaccharides.
D. the above-mentioned a small amount of distilled water of silkworm excrement Crude polysaccharides removing after albumen is dissolved, upper AB-8 type macroporous adsorptive resins, with distilled water wash-out, is eluted to elutriant and adds 95% ethanol without till precipitating.Elutriant is concentrated into certain volume, obtains refining polysaccharide extraction liquid.
E. refining polysaccharide extraction liquid, add 60 DEG C of about 2h of decolouring of gac (solid-to-liquid ratio is 1: 10), filtered while hot, filtrate adds 95% ethanol to make to reach 80% containing alcohol amount after being concentrated into small volume, suction filtration after leaving standstill, for precipitation, repeatedly, 60 DEG C of following temperature are dry for dehydrated alcohol, acetone repetitive scrubbing, obtain refining silkworm excrement total polysaccharides.
2. a kind of method that separates silkworm excrement total polysaccharides from silkworm excrement decocting liquid according to claim 1, is characterized in that: in step D, said macroporous adsorbent resin is AB-8 type, D-101 type, HPD-400 type, NKA-9 type or H801 type macroporous adsorbent resin.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10245389A (en) * | 1997-03-03 | 1998-09-14 | Zaiman Kin | New antiviral active substance |
| CN101654428A (en) * | 2009-09-11 | 2010-02-24 | 成都市金医生科技健康产业有限公司 | Method for extracting and separating 1-deoxynojirimycin with high purity from natural products |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10245389A (en) * | 1997-03-03 | 1998-09-14 | Zaiman Kin | New antiviral active substance |
| CN101654428A (en) * | 2009-09-11 | 2010-02-24 | 成都市金医生科技健康产业有限公司 | Method for extracting and separating 1-deoxynojirimycin with high purity from natural products |
Non-Patent Citations (2)
| Title |
|---|
| 张保国等: "《动物药》", 30 November 2003 * |
| 李奇豫等: ""桂蚕沙多糖组分的制备与GC-MS分析"", 《中国实验方剂学杂志》 * |
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Inventor after: Li Suli Inventor after: Zhang Guijun Inventor after: Li Qiyu Inventor after: Chen Xiao Inventor before: Zhang Guijun Inventor before: Li Suli Inventor before: Li Qiyu Inventor before: Chen Xiao |
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Effective date of registration: 20160519 Address after: 100044 Beijing city Xicheng District Debao Park No. 11 room 511 Applicant after: Beijing Hongtai Kangda Pharmaceutical Technology Co., Ltd. Applicant after: Guangxi University Address before: 100044 Beijing city Xicheng District Debao Park No. 11 room 511 Applicant before: Beijing Hongtai Kangda Pharmaceutical Technology Co., Ltd. |
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Application publication date: 20141015 |