CN103254325A - Preparation method of abrus cantoniensis hance polysaccharide - Google Patents
Preparation method of abrus cantoniensis hance polysaccharide Download PDFInfo
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Abstract
The invention discloses a preparation method of abrus cantoniensis hance polysaccharide. The method comprises the following steps of weighing the dry abrus cantoniensis hance, and pulverizing the dry abrus cantoniensis after being segmented, adding deionized water, enabling the material-liquid ratio to be 1:20 to 1:40, extracting 2 to 3 times through backflow, ensuring the extraction temperature to be 60 to 95 DEG C, extracting one time per 1.5 to 3 hours, merging the extracted liquid, filtering the extracted liquid, removing insoluble impurities, treating the filtered liquid by utilizing neutral protease, dialyzing the filtered liquid through a dialysis band, decompressing, distilling and concentrating the dialyzed liquid, adding absolute ethyl alcohol while stirring the concentrated liquid, enabling the alcohol content to reach 75 to 80 percent, standing the liquid, filtering the liquid, collecting the precipitate, and drying the precipitate to obtain the abrus cantoniensis hance polysaccharide. The abrus cantoniensis hance has a function of clearing away the heat and toxic materials and dispersing the depressed liver-qi and alleviating the pain, is a conventional clinical traditional Chinese medicine and has a good development prospect. According to the extraction method of the abrus cantoniensis hance polysaccharide, the technological condition is easy to control, and the purity of the polysaccharide is high. The preparation method has the advantages of moderate reaction condition, no toxicity and no side effect of the reaction products, and the like.
Description
Technical field
The present invention relates to a kind of production method of polysaccharide, particularly relate to the extracting method of Herba Abri polysaccharide, belong to the health food technology field.
Background technology
Herba Abri is the dry complete stool of leguminous plants Semen Abri Precatorii Abrus cantoniensis, the Chinese medicine Herba Abri is pulse family abrus plant, is used as medicine with complete stool, has the dampness removing removing jaundice, clearing heat and detoxicating, the effect of dispersing the depressed liver-QI for alleviating pain is clinical conventional Chinese medicines, is used for jaundice, side of body rib is bad, the gastral cavilty distending pain, acute, chronic hepatitis, mazoitis also are one of China's product herbal varieties.Herb contain abrine (Abrine, namely N ?methyl tryptophan), choline (choline), adenosine class, sterols, triterpene saponin, flavonoid, amino acid, carbohydrate.
In recent years, prove through the science medical research that Herba Abri has tangible liver protection effect.Herba Abri and hair Herba Abri decocting liquid all can reduce CCL4 and bacille Calmette-Guerin vaccine and lipopolysaccharide-induced immunological liver injury mice serum ALT activity, and the rising of immunological liver injury mice serum gpt, glutamic-oxal(o)acetic transaminase is had tangible reduction effect.Though Herba Abri is applied to protect the liver removing jaundice and mazoitis history is longer, the basic substance of its pharmacological action is which chemical position and which pharmacological action are associated not fully aware of.The domestic exploitation of also not seeing its efficient part.
Polysaccharide is a class natural high moleculer eompound, is present in all membrane structures, participates in the various physiological activities of cell, is one of the base substance of activity normal operation that earns a bare living.Polysaccharide also is one of active ingredient of Chinese herbs, has many pharmacologically actives and pharmacodynamic feature.At present, both at home and abroad be the research to this natural resources of Chinese medicinal materials of Herba Abri, concentrate on the aspects such as assay, pharmacologically active of composition evaluation, alkaloids and flavonoid, yet do not see both at home and abroad that for STUDY ON POLYSACHAROSE in the Herba Abri report is arranged.
Summary of the invention
The object of the invention is, overcomes existing defective to Herba Abri research, and a kind of production method of Herba Abri polysaccharide is provided.Herba Abri extraction method of polysaccharides of the present invention, easy control of process conditions, reaction conditions gentleness, product extraction yield height.
Purpose of the present invention is achieved through the following technical solutions:
A kind of extracting method of Herba Abri polysaccharide comprises the steps:
(1) drying and crushing: behind the Herba Abri raw material drying, pulverize, sieve, get the Herba Abri powder;
(2) lixiviate: add deionized water extraction in step (1) gained Herba Abri powder, the mass ratio of deionized water and Herba Abri powder is 1:20～1:40, and extracting temperature is 60～95 ℃, and extraction time is 1.5～3h, extracts 1 time or repeatedly;
(3) except insoluble impurities: with filtration or centrifugal after the extracting solution cooling of extracting solution or merging, remove insoluble impurities;
(4) except albumen: add neutral protease and handle 18～25min for 40～50 ℃ in step (3) gained filtrate, the mass concentration of neutral protease in filtrate is 0.4～0.6%, and be centrifugal, and filtrate drives attitude dialysis 24h～48h through dialysis;
(5) alcohol precipitation: trapped fluid underpressure distillation in the dialysis band in the step (4) is concentrated into 1/6～1/4 of original volume, and concentrated solution adds ethanol while stirring, makes the ethanol mass concentration reach 75%～80%, leaves standstill, and is centrifugal, collecting precipitation;
(6) drying: precipitation obtains sheet Herba Abri polysaccharide after drying in the step (5).
Cross the order number of net solarization and be preferably 100 mesh sieves for further realizing the object of the invention, described step (1) Herba Abri powder.
The mass ratio of the described deionized water of step (2) and Herba Abri powder is preferably 1:25～1:35, extracts temperature and is preferably 60～80 ℃, and extraction time is preferably 2～3h.
The mode of the described filtration of step (3) is preferably vacuum filtration; Described centrifugal rotation speed is preferably 4500r/min.
The aperture of described dialysis band is preferably 3000～4000Da, and the mode of described dynamic dialysis is flowing water dialysis or magnetic agitation dialysis.
Described neutral protease is preferably subtilis neutral protease (bacillus subtilis neutral protease, unikneutral S), bacillus subtilis neutral proteinase (SUKAPro NE) or aspergillus oryzae neutral proteinase (aspergillus oryae F ?81neutral protease).
It is to adopt Rotary Evaporators that described underpressure distillation concentrates, and vacuum degree control is between 0.08～0.09MPa, and temperature is controlled at 55 ± 3 ℃.
Described ethanol is preferably dehydrated alcohol.
Described drying is lyophilize or spraying drying; Described spray-dired inlet temperature control is at 170 ± 3 ℃, and air outlet temperature is controlled at 60 ± 3 ℃; Pre-freeze 3h in the described lyophilize, vacuum-drying 12h.
The present invention compared with prior art has following advantage and beneficial effect:
1, the present invention extracts polyose component in the Herba Abri first, and extraction yield can reach 6～10%.。
2, the present invention extracts the Herba Abri polysaccharide from the modern thinking of Chinese medicine, for further investigation and the application of main chemical position and corresponding medicine efficacy relation in the Herba Abri provides new thinking.
3, the present invention has improved traditional extraction technique, takes hot water backflow lixiviate, neutral protease deproteinated, and reaction process is easy to control, sanitation and hygiene, and is safe and harmless, significantly improves purity of polysaccharide, and under the limiting case, purity of polysaccharide can reach 78%.
4, raw material of the present invention is easy to get, and technology is simple, and is practical, can deep processing produce high performance health medicine, and production cost is low, also enriched the research field of vegetable polysaccharides science simultaneously.
Description of drawings
Fig. 1 is the uv scan graphic representation of embodiment 1 product;
Fig. 2 is the infrared spectrogram of embodiment 1 product;
Fig. 3 is the GPC spectrogram of embodiment 1 product.
Embodiment
For understanding the present invention better; the present invention is described further below in conjunction with embodiment; the contriver is to passing through further investigation and test; many successful embodiment have been arranged; enumerate four specific embodiments below with explanation the present invention, but the scope of protection of present invention is not limited to the scope of embodiment statement.
Embodiment 1
The first step with Herba Abri raw material (the dry complete stool of leguminous plants Guangzhou plant Semen Abri Precatorii Abrus cantoniensis Hance) drying after, with the plant pulverizer pulverize the Herba Abri powder, cross 100 mesh sieves, and take by weighing 200g.Add deionized water, make the feed liquid mass ratio reach 1:20,60 ℃ of refluxing extraction 3 times, each 1.5h, united extraction liquid;
The gained extracting solution is removed insoluble impurities through the 4500r/min high speed centrifugation in the second step previous step;
Subtilis neutral protease (unikneutral S) the reaction 18min that adds 0.5wt% in the 3rd step step 2 gained clear liquid, 45 ℃ of temperature of reaction, the cooling back is centrifugal, and supernatant liquor is collected trapped fluid through dialysis band flowing water dialysis 24h; The aperture of dialysis band is 3500Da.
The 3rd step trapped fluid is evaporated to the vacuum degree control of 1/5(Rotary Evaporators of original volume between 0.08～0.09MPa through Rotary Evaporators, temperature control is at 55 ± 3 ℃), add dehydrated alcohol in the concentrated solution while stirring, make the ethanol mass content reach 75%, leave standstill 30min, the centrifugal 10min of 4500r/min, collecting precipitation.Throw out behind the vacuum-drying 12h, namely gets sheet Herba Abri polysaccharide crude through pre-freeze 3h.The Herba Abri polysaccharide extract rate is 6.32%.
Wherein Herba Abri polysaccharide extract rate calculation formula is:
Total sugar content use Ben Fen ?sulfuric acid process measure, reducing sugar content adopt 3,5 ?dinitrosalicylic acid (DNS) method measure; In the present embodiment, Herba Abri raw materials quality 200g, total sugar content are 13.09g, and reducing sugar content is: 0.4507g.
The evaluation of Herba Abri polysaccharide:
(1) physico-chemical property:
After testing, present embodiment sheet Herba Abri polysaccharide crude is water-soluble, is insoluble to organic solvents such as ethanol, acetone, ether, propyl carbinol, does not show blue with the potassiumiodide reaction, and the non-starch based of this polysaccharide product is described.
(2) uv scan:
As shown in Figure 1, this polysaccharide product does not have uv-absorbing at 260nm, 280nm wavelength place, illustrates not contain impurity such as protein and nucleic acid in this product.
(3) Infrared spectroscopy:
As shown in Figure 2, in infrared spectra (IR) spectrum, polysaccharide product 3500 ?3200cm
?1Between wide strong absorption peak is arranged, this peak be O ?the stretching vibration of H cause, 3000 ?2800cm
?1Between weak absorption peak is all arranged, this peak be C ?the stretching vibration of H cause, 1400 ?1200cm
?1Between all have not too the absorption peak of point, this peak be C ?the angle vibration peak of H, can judge tentatively that according to above absorption peak product is the polyose compound.
(4) mensuration of Herba Abri polysaccharide molecular weight
As shown in Figure 3, present embodiment gained Crude polysaccharides through DEAE Cellulose ?52 ion exchange columns, the NaCl eluant solution obtains the polysaccharide product that different molecular weight distributes.The molecular weight of polysaccharide mainly is distributed in 8～100kDa.
Embodiment 2
The first step with Herba Abri raw material (the dry complete stool of leguminous plants Guangzhou plant Semen Abri Precatorii Abrus cantoniensis Hance) drying after, with the plant pulverizer pulverize the Herba Abri powder, cross 100 mesh sieves, and take by weighing 200g.Add deionized water, make the feed liquid mass ratio reach 1:40,80 ℃ of refluxing extraction 2 times, each 2h;
The gained extracting solution is removed insoluble impurities through vacuum filtration in the second step previous step;
Bacillus subtilis neutral proteinase (SUKAPro NE) the reaction 22min that adds 0.4wt% in the 3rd step step 2 gained clear liquid, 50 ℃ of temperature of reaction, the cooling back is centrifugal, and supernatant liquor is collected trapped fluid through dialysis band magnetic agitation (300r/min) dialysis 36h; The aperture of dialysis band is 3000Da.
The 3rd step trapped fluid is evaporated to the vacuum degree control of 1/4(Rotary Evaporators of original volume between 0.08～0.09MPa through Rotary Evaporators, temperature control is at 55 ± 3 ℃), add dehydrated alcohol in the concentrated solution while stirring, make the ethanol mass content reach 75%, leave standstill 30min, the centrifugal 10min of 4500r/min, collecting precipitation.Throw out behind the vacuum-drying 12h, namely gets sheet Herba Abri polysaccharide product through pre-freeze 3h.The Herba Abri polysaccharide extract rate is 8.91%.The polysaccharide extract rate method of calculation are with embodiment 1, total sugar content 18.19g wherein, and reducing sugar content is 0.3721g.
After testing, present embodiment sheet Herba Abri polysaccharide crude is water-soluble, is insoluble to organic solvents such as ethanol, acetone, ether, propyl carbinol, does not show blue with the potassiumiodide reaction, and the non-starch based of this polysaccharide product is described.The uv scan graphic representation of present embodiment product, infrared spectrogram and GPC spectrogram are substantially with embodiment 1.
Embodiment 3
The first step with Herba Abri raw material (the dry complete stool of leguminous plants Guangzhou plant Semen Abri Precatorii Abrus cantoniensis Hance) drying after, with the plant pulverizer pulverize the Herba Abri powder, cross 100 mesh sieves, and take by weighing 200g.Add deionized water, make the feed liquid mass ratio reach 1:30,95 ℃ of refluxing extraction 3 times, each 1.5h;
The gained extracting solution is removed insoluble impurities through the 4500r/min high speed centrifugation in the second step previous step;
Aspergillus oryzae neutral proteinase (aspergillus oryae F ?81neutral protease) the reaction 25min that adds 0.6wt% in the 3rd step step 2 gained clear liquid, 50 ℃ of temperature of reaction, the cooling back is centrifugal, and supernatant liquor is collected trapped fluid through dialysis band flowing water dialysis 24h; The aperture of dialysis band is 4000Da.
The 3rd step trapped fluid is evaporated to the vacuum degree control of 1/5(Rotary Evaporators of original volume between 0.08～0.09MPa through Rotary Evaporators, temperature control is at 55 ± 3 ℃), add dehydrated alcohol in the concentrated solution while stirring, the ethanol mass content reaches 80%, leave standstill 30min, the centrifugal 10min of 4500r/min, collecting precipitation.Throw out behind the vacuum-drying 12h, namely gets sheet Herba Abri polysaccharide product through pre-freeze 3h.The Herba Abri polysaccharide extract rate is 7.57%.The polysaccharide extract rate method of calculation are with embodiment 1, total sugar content 16.20g wherein, reducing sugar content 1.071g.
After testing, present embodiment sheet Herba Abri polysaccharide crude is water-soluble, is insoluble to organic solvents such as ethanol, acetone, ether, propyl carbinol, does not show blue with the potassiumiodide reaction, and the non-starch based of this polysaccharide product is described.
The uv scan graphic representation of present embodiment product, infrared spectrogram and GPC spectrogram are substantially with embodiment 1.
Embodiment 4
The first step with Herba Abri raw material (the dry complete stool of leguminous plants Guangzhou plant Semen Abri Precatorii Abrus cantoniensis Hance) drying after, with the plant pulverizer pulverize the Herba Abri powder, cross 100 mesh sieves, and take by weighing 200g.Add deionized water, make the feed liquid mass ratio reach 1:40,90 ℃ of refluxing extraction 2 times, each 3h;
The gained extracting solution is removed insoluble impurities through the 4500r/min high speed centrifugation in the second step previous step;
Subtilis neutral protease (unikneutral S) the reaction 22min that adds 0.5wt% in the 3rd step step 2 gained clear liquid, 45 ℃ of temperature of reaction, the cooling back is centrifugal, and supernatant liquor is collected trapped fluid through dialysis band magnetic agitation (300r/min) dialysis 48h; The aperture of dialysis band is 3500Da.
The 3rd step trapped fluid is evaporated to the vacuum degree control of 1/6(Rotary Evaporators of original volume between 0.08～0.09MPa through Rotary Evaporators, temperature control is at 55 ± 3 ℃), add dehydrated alcohol in the concentrated solution while stirring, the ethanol mass content reaches 80%, leave standstill 30min, the centrifugal 10min of 4500r/min, collecting precipitation.Throw out behind the vacuum-drying 12h, namely gets sheet Herba Abri polysaccharide product through pre-freeze 3h.The Herba Abri polysaccharide extract rate is 9.61%.The polysaccharide extract rate method of calculation are with embodiment 1, total sugar content 20.02g wherein, reducing sugar content 0.7974g.
After testing, present embodiment sheet Herba Abri polysaccharide crude is water-soluble, is insoluble to organic solvents such as ethanol, acetone, ether, propyl carbinol, does not show blue with the potassiumiodide reaction, and the non-starch based of this polysaccharide product is described.The uv scan graphic representation of present embodiment product, infrared spectrogram and GPC spectrogram are substantially with embodiment 1.
Claims (9)
1. the extracting method of a Herba Abri polysaccharide is characterized in that comprising the steps:
(1) drying and crushing: behind the Herba Abri raw material drying, pulverize, sieve, get the Herba Abri powder;
(2) lixiviate: add deionized water extraction in step (1) gained Herba Abri powder, the mass ratio of deionized water and Herba Abri powder is 1:20～1:40, and extracting temperature is 60～95 ℃, and extraction time is 1.5～3h, extracts 1 time or repeatedly;
(3) except insoluble impurities: with filtration or centrifugal after the extracting solution cooling of extracting solution or merging, remove insoluble impurities;
(4) except albumen: add neutral protease and handle 18～25min for 40～50 ℃ in step (3) gained filtrate, the mass concentration of neutral protease in filtrate is 0.4～0.6%, and be centrifugal, and filtrate drives attitude dialysis 24h～48h through dialysis;
(5) alcohol precipitation: trapped fluid underpressure distillation in the dialysis band in the step (4) is concentrated into 1/6～1/4 of original volume, and concentrated solution adds ethanol while stirring, makes the ethanol mass concentration reach 75%～80%, leaves standstill, and is centrifugal, collecting precipitation;
(6) drying: precipitation obtains sheet Herba Abri polysaccharide after drying in the step (5).
2. the extracting method of a kind of Herba Abri polysaccharide according to claim 1 is characterized in that: it is 100 mesh sieves that described step (1) Herba Abri powder is crossed the order number that net shines.
3. the extracting method of a kind of Herba Abri polysaccharide according to claim 1, it is characterized in that: the mass ratio of the described deionized water of step (2) and Herba Abri powder is 1:25～1:35, extracting temperature is 60～80 ℃, extraction time 2～3h.
4. the extracting method of a kind of Herba Abri polysaccharide according to claim 1, it is characterized in that: the mode of the described filtration of step (3) is vacuum filtration; Described centrifugal rotation speed is 4500r/min.
5. the extracting method of a kind of Herba Abri polysaccharide according to claim 1, it is characterized in that: the aperture of described dialysis band is 3000～4000Da, the mode of described dynamic dialysis is flowing water dialysis or magnetic agitation dialysis.
6. the extracting method of a kind of Herba Abri polysaccharide according to claim 1, it is characterized in that: described neutral protease is subtilis neutral protease (bacillus subtilis neutral protease, unikneutral S), bacillus subtilis neutral proteinase (SUKAPro NE) or aspergillus oryzae neutral proteinase (aspergillus oryae F ?81neutral protease).
7. the extracting method of a kind of Herba Abri polysaccharide according to claim 1 is characterized in that: it is to adopt Rotary Evaporators that described underpressure distillation concentrates, and vacuum degree control is between 0.08～0.09MPa, and temperature is controlled at 55 ± 3 ℃.
8. the extracting method of a kind of Herba Abri polysaccharide according to claim 1, it is characterized in that: described ethanol is dehydrated alcohol.
9. the extracting method of a kind of Herba Abri polysaccharide according to claim 1, it is characterized in that: described drying is lyophilize or spraying drying; Described spray-dired inlet temperature control is at 170 ± 3 ℃, and air outlet temperature is controlled at 60 ± 3 ℃; Pre-freeze 3h in the described lyophilize, vacuum-drying 12h.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107915786A (en) * | 2017-12-29 | 2018-04-17 | 广西汇智生产力促进中心有限公司 | The method for extracting abrus cantoniensis hance polysaccharide |
| CN110283258A (en) * | 2019-07-22 | 2019-09-27 | 玉林师范学院 | A kind of preparation method with liver-protecting function abrus cantoniensis hance polysaccharide |
| WO2022205518A1 (en) * | 2021-03-30 | 2022-10-06 | 海南云皓生物科技有限公司 | Method for preparing pandanus tectorius polysaccharide |
| CN115746159A (en) * | 2022-11-29 | 2023-03-07 | 广西大学 | Preparation process and application of carboxymethylated abrus cantoniensis hance polysaccharide |
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| CN101147537A (en) * | 2007-10-26 | 2008-03-26 | 华南理工大学 | Method for high efficiency extracting momordica polysaccharide from balsam pear |
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| CN101147537A (en) * | 2007-10-26 | 2008-03-26 | 华南理工大学 | Method for high efficiency extracting momordica polysaccharide from balsam pear |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107915786A (en) * | 2017-12-29 | 2018-04-17 | 广西汇智生产力促进中心有限公司 | The method for extracting abrus cantoniensis hance polysaccharide |
| CN110283258A (en) * | 2019-07-22 | 2019-09-27 | 玉林师范学院 | A kind of preparation method with liver-protecting function abrus cantoniensis hance polysaccharide |
| WO2022205518A1 (en) * | 2021-03-30 | 2022-10-06 | 海南云皓生物科技有限公司 | Method for preparing pandanus tectorius polysaccharide |
| CN115746159A (en) * | 2022-11-29 | 2023-03-07 | 广西大学 | Preparation process and application of carboxymethylated abrus cantoniensis hance polysaccharide |
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