CN103724447A - Extraction and classification method of lentinan - Google Patents
Extraction and classification method of lentinan Download PDFInfo
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Abstract
The invention provides an extraction and classification method of lentinan. The extraction and classification method comprises the steps: sequentially carrying out drying, crushing, soaking and water extraction on Shitake mushroom to obtain crude lentinan extract firstly; then deproteinize the obtained crude lentinan extract sequentially by using chlorobenzene, calcium chloride, ammonium oxalate, trypsin and a Sevage reagent; and carrying out an alcohol precipitation method finally to prepare the lentinan. When the lentinan is extracted and prepared by using the method provide by the invention, the yield is high, the damage to the lentinan, caused by use of acid of alkaline is also avoided, the lentinan after purification has higher purity, the lentinan with different weight-average molecular weight can be obtained by virtue of classification, the molecular weight distribution range is narrower, and the lentinan has wide application prospect in the fields of organism immune regulation and tumor adjuvant therapy.
Description
Technical field
The present invention relates to extracts active ingredients classification technique field, relate in particular to a kind of extraction and stage division of lentinan.
Background technology
Mushroom belongs to Basidiomycetes umbrella shape fungi, is edible mushrooms all the fashion in the world.Main component in mushroom is lentinan, mainly with the form of mixing polysaccharide, exists, and comprises 4 kinds of mixed polysaccharide and 2 kinds of dextran, wherein, having bioactive is dextran, and its structure is take β-(1 → 3) Glucopyranose glycosidic bond as main chain, take β-(1 → 6) Glucopyranose glycosidic bond as branch.
The functions such as modern study shows that lentinan has adjusting immunizing power, antitumor, hypoglycemic, antiviral and anti-oxidant.Lentinan has immunoregulation effect to body, mainly because it belongs to typical t cell activation agent, can promote in vivo delayed hypersensitive reaction, improve antibody-dependent cytotoxicity cytoactive, can also induce the factors such as producing acute protein inducible factor, thereby produce related immune, reply simultaneously.There is report to show that lentinan can suppress the growth of mouse S-180 solid tumor, for treatment colorectal carcinoma, mammary cancer, lung cancer, cancer of the stomach etc., there is good curative effect simultaneously, and can extend the survival time of tumour patient, also can greatly alleviate the side effect that chemotherapy is brought; Clinical application also finds, lentinan can improve the common symptons such as weak, nauseating, the abdominal distension of hepatitis B patient, promotes transaminase and bilirubin to recover normal; Lentinan is also to Abelson virus, A2(H2N2) virus, 12 type adenovirus and influenza virus etc. all has restraining effect.
Although lentinan is more and more subject to people's attention in clinical application in recent years, its mechanism of action is also very not clear and definite, the particularly mechanism of anti-tumor activity, wherein a very important complicacy that reason is exactly lentinan itself.
Therefore, lentinan is extracted to classification, for the mechanism of action of research lentinan, have very important meaning.For the extracting method of lentinan, mainly comprise at present: diluted alkaline extraction method is extraction agent mainly with sodium hydroxide, and extraction can obtain more alkali-soluble polysaccharide, the polysaccharide impurity that still this method obtains is more, and polysaccharide is also easily destroyed; Also having Thinner acid method, is extraction agent mainly with trichoroacetic acid(TCA), can obtain more solubility in acid polysaccharide, but polysaccharide is also easily destroyed; Also having rare salt extraction method, is extraction agent mainly with zinc chloride, but this method extraction yield is lower, and raw material is caused to larger waste; In addition, also have microwave, supersonic method, take deionized water as extraction agent, but thermo-sensitivity polysaccharide is easily destroyed.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of extraction and stage division of lentinan, can not damage polysaccharide, and higher to the extraction yield of polysaccharide, can carry out good classification to polysaccharide simultaneously.
The extracting method that the invention provides a kind of lentinan, comprising:
A) mushroom is dried successively, pulverizes, immersion, water extraction obtain lentinan crude extract;
B) by steps A) the lentinan crude extract that obtains removes albumen with chlorobenzene, calcium chloride, ammonium oxalate, trypsinase, Sevage reagent successively, finally adopts the method for alcohol precipitation to obtain lentinan.
Preferably, the granularity of described pulverizing is 50 order～300 orders.
Preferably, the time of described immersion is 12h～72h; The temperature of described water extraction is 60 ℃～98 ℃.
Preferably, 10wt%～40wt% that the consumption of described calcium chloride is described mushroom; The consumption of described ammonium oxalate is the 1wt%～5wt% of described mushroom.
Preferably, the alcohol that the method for described alcohol precipitation adopts is ethanol.
Preferably, described step B) in, with ammonium oxalate, trypsinase, lentinan crude extract being removed in the middle of the step of albumen successively, also comprise: add sodium-chlor.
Preferably, described step B) in, with Sevage reagent, lentinan crude extract being removed after albumen, also comprise: add gac.
The present invention also provides the method for the lentinan classification that a kind of said extracted method obtains, and comprising:
Lentinan is carried out to fractionation precipitation with acetone, obtain the lentinan after classification.
Preferably, described method is specially:
A) lentinan obtaining by extracting method claimed in claim 1 is dissolved in distilled water, add acetone to occur precipitation, centrifugal supernatant liquor and the precipitation of obtaining, described in be precipitated as first step lentinan;
B) get the supernatant liquor obtaining in step a), add acetone to occur precipitation, centrifugal supernatant liquor and the precipitation of obtaining, described in be precipitated as second stage lentinan;
C) repeat said process, reach 90wt% to acetone concentration.
The present invention also provides the application of a kind of lentinan in medicine or the food of preparing strengthening immunity, and the weight-average molecular weight of described lentinan is 16200.
Compared with prior art, the invention provides a kind of extracting method of lentinan, adopted the complex method except albumen, first mushroom is dried successively, pulverizes, immersion, water extraction obtain lentinan crude extract; Then the lentinan crude extract obtaining is removed to albumen with chlorobenzene, calcium chloride, ammonium oxalate, trypsinase, Sevage reagent successively, finally adopt the method for alcohol precipitation can prepare lentinan.When this method is prepared lentinan in extraction, there is higher productive rate, and avoided the destruction that adopts acid or alkali to cause lentinan, the lentinan obtaining after purification has higher purity, can obtain by classification the lentinan of different number-average molecular weights.Lentinan purity provided by the invention is very high, there is hardly impurity and objectionable impurities, therefore can be used for experiment in external body after sterilising treatment, carry out bioactivity research, and the lentinan of different number-average molecular weights after classification is more significant to the biological activity mechanism of research lentinan, therefore extracting method provided by the invention is conducive to further research and the exploitation of lentinan.
Accompanying drawing explanation
Fig. 1 is the infared spectrum of the lentinan prepared of the embodiment of the present invention 23;
Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram of the lentinan prepared of the embodiment of the present invention 23;
Fig. 3 is the infiltration gel electrophoresis figure of the lentinan that obtains of the embodiment of the present invention 24～42 classifications;
Fig. 4 is the embodiment of the present invention 43 small mouse body weight change figure;
Fig. 5 is the embodiment of the present invention 43 small mouse index and spleen index variation diagrams;
Fig. 6 is the embodiment of the present invention 43 small mouse thymus index variation diagrams;
Fig. 7 is the embodiment of the present invention 43 small mouse peripheral white blood cell variation diagrams;
Fig. 8 is that the embodiment of the present invention 43 small mouse marrow micronucleus are counted variation diagram.
Embodiment
The extracting method that the invention provides a kind of lentinan, comprising:
A) mushroom is dried successively, pulverizes, immersion, water extraction obtain lentinan crude extract;
B) by steps A) the lentinan crude extract that obtains removes albumen with chlorobenzene, calcium chloride, ammonium oxalate, trypsinase, Sevage reagent successively, finally adopts the method for alcohol precipitation to obtain lentinan.
The present invention extracts lentinan by the complex method except albumen, select chlorobenzene, calcium chloride, ammonium oxalate, trypsinase, Sevage reagent etc. to remove albumen, there is higher productive rate, and avoided the destruction that adopts acid or alkali to cause lentinan, the lentinan obtaining after purification has higher purity, can obtain by classification the lentinan of different number-average molecular weights.Lentinan purity provided by the invention is very high, there is hardly impurity and objectionable impurities, therefore can be used for experiment in external body after sterilising treatment, carry out bioactivity research, and the lentinan of different number-average molecular weights after classification is more significant to the biological activity mechanism of research lentinan, therefore extracting method provided by the invention is conducive to further research and the exploitation of lentinan.
The extracting method of lentinan provided by the invention, comprises the following steps:
First be the preparation of lentinan crude extract, be about to that mushroom is dried successively, pulverizes, immersion, water extraction obtain lentinan crude extract, concrete operation step is as follows:
Mushroom is cleaned and removed handle, 60 ℃～80 ℃ air-dry dry, be crushed to 50 order～300 orders, then in the sample after pulverizing, add distilled water, the ratio of the volume of described distilled water and the quality of mushroom is preferably (10mL～200mL): 1g, more preferably (100mL～150mL): 1g, after adding distilled water, soak 12h～72h, and then 60 ℃～98 ℃ high temperature bath 12h～48h carry out water extraction, after water extraction, solution is cooled to room temperature and with sodium bicarbonate adjust pH value to 5.0～8.0, more preferably be adjusted to 6～7, add again composite protease hydrolysis albumen, 35 ℃～55 ℃, more preferably 40 ℃～50 ℃ water-bath 1h～3h, under stirrer stirs, 80 ℃～90 ℃ water-bath 1h～3h make enzyme deactivation again, then cooling, filter, residue redissolves with distilled water, repeat 1～3 time, collect filtrate, obtain the thick product of lentinan aqueous extract.The granularity of described pulverizing is 100 order～300 orders more preferably; More preferably 85 ℃～95 ℃ of the temperature of described water extraction, more preferably 20h～40h of the time of high temperature bath; The consumption of described compound protease is preferably the 1wt%～5wt% of mushroom, more preferably 2wt%～4wt%.
Obtain after the thick product of lentinan aqueous extract, it is carried out to alcohol precipitation, concrete, centrifugal 10min～the 30min of rotating speed by filtrate with 3500rpm～4500rpm, collect supernatant, 40 ℃～50 ℃ rotary evaporations of supernatant liquor are concentrated to volume to 1/15～1/3 of original volume, then add 3～8 times of (volume ratio) dehydrated alcohols of concentrated solution, be uniformly mixed 30min～60min, standing, with the centrifugal 10min～30min of rotating speed the collecting precipitation of 7000rpm～9000rpm, to precipitate adding distil water redissolves, repeat above-mentioned alcohol precipitation step 3～5 time, the precipitation vacuum lyophilization finally obtaining, obtain the crude extract of lentinan.Described ethanol more preferably adds 5～7 times (volume ratios).
Obtain after lentinan crude extract, it is carried out to purifying, concrete, in lentinan crude extract, add distilled water, the ratio of the volume of described distilled water and the quality of mushroom is preferably (2mL～10mL): 1g, its agitation as appropriate is heated to abundant dissolving after adding distilled water, then add chlorobenzene to purify, centrifugal collection supernatant after stirring 30min, supernatant liquor repeats step 5～8 time of above-mentioned chlorobenzene processing, finally obtains chlorobenzene lentinan solution after treatment; The consumption of described chlorobenzene is preferably 1/4～1/2 of liquor capacity, and more preferably 1/3.Add the object of chlorobenzene for removing albumen.
Then in the lentinan solution obtaining, add Calcium Chloride Powder Anhydrous after above-mentioned chlorobenzene is processed, constantly boiling 1min～3min, after the centrifugal 3～5min of 5000rpm～7000rpm, collect supernatant again, in supernatant liquor, add ammonium oxalate electric stirring 30min～60min, then the centrifugal 3～5min of 5000rpm～7000rpm collects supernatant, obtains ammonium oxalate lentinan solution after treatment; The consumption of described Calcium Chloride Powder Anhydrous is preferably the 10wt%～40wt% of mushroom; The consumption of described ammonium oxalate is preferably the 1wt%～5wt% of mushroom.Preferably, adding calcium chloride, ammonium oxalate to remove after albumen, in the supernatant liquor obtaining, also add sodium-chlor supersaturation electric stirring 0.5h～3h, then the centrifugal 8～10min of 7000rpm～9000rpm collects supernatant, adds the object of sodium-chlor for separating out protein.
Obtain after above-mentioned ammonium oxalate lentinan solution after treatment, adjust its pH value to 7.5～8.5 with sodium bicarbonate, then add trypsinase, obtain the lentinan solution after trypsin hydrolyzing albumen, described tryptic add-on is preferably the 5wt%～15wt% of mushroom.
Adopt after trypsin hydrolyzing albumen, the lentinan solution obtaining is added to the Sevage reagent (trichloromethane: propyl carbinol=4:1) of 0.5～1 times of volume, electric stirring 10min～30min, then the centrifugal 3min～5min of 1000rpm, separatory is removed lower floor's liquid, continue 5000rpm～7000rpm centrifugal 3min～5min and collect supernatant, supernatant liquor repeats the above-mentioned Sevage of adding step 3～8 time, the supernatant liquor rotary evaporation of finally collecting is concentrated to volume to 1/5～1/4 of original volume, collect supernatant liquor and add the dehydrated alcohol of 3～8 times of volumes, be uniformly mixed 30min～60min, standing, then the centrifugal 10min～30min of 3500rpm～4500rpm collecting precipitation, add distilled water to redissolve precipitation, repeat step 3～5 time of above-mentioned alcohol precipitation, the precipitation finally obtaining is cleaned 2～4 times with dehydrated alcohol successively, anhydrous diethyl ether cleans 2～4 times, vacuum is drained the lentinan that obtains purifying.Preferably, lentinan crude extract is removed after albumen with Sevage reagent, also comprise: add gac, the consumption of described gac is preferably the 1wt%～3wt% of mushroom.The effect that adds gac is decolouring.
Adopt infrared absorption and nucleus magnetic resonance to characterize the lentinan structure of preparation, result shows, the lentinan that the present invention extracts has structure shown in formula (I):
In formula (I), n is the polymerization degree, preferred, 1≤n≤3300.
The weight-average molecular weight of described lentinan is preferably 3900～33000.
The described lentinan with formula (I) structure, its structure is take β-(1 → 3) Glucopyranose glycosidic bond as main chain, take β-(1 → 6) Glucopyranose glycosidic bond as branch.Its tertiary structure is as shown in the formula (II):
Prepare after the lentinan of purifying, lentinan is carried out to fractionation precipitation with acetone, obtain the lentinan after classification.
Concrete operation step is as follows:
A) lentinan of above-mentioned purifying is dissolved in distilled water, add acetone to occur precipitation, centrifugal supernatant liquor and the precipitation of obtaining, described in be precipitated as first step lentinan;
B) get the supernatant liquor obtaining in step a), add acetone to occur precipitation, centrifugal supernatant liquor and the precipitation of obtaining, described in be precipitated as second stage lentinan;
C) repeat said process, reach 90wt% to acetone concentration.
Concrete, the lentinan of the purifying of above-mentioned preparation is placed in to closed system, dropwise add acetone, limit edged stirs, until acetone concentration reaches 5wt%, standing 60min～100min, repeat above operation and improve constantly acetone concentration, until there is precipitation to produce, then the centrifugal 10min～30min of 3500rpm～4500rpm collecting precipitation, remains supernatant liquor and continues to add acetone to repeat above-mentioned steps, until acetone concentration reaches 90wt% after collecting precipitation, collect each precipitation, by gel permeation chromatography or Ubbelohde viscometer, survey its molecular weight.
Experimental result shows, adopts stage division provided by the invention, can obtain the lentinan of different weight-average molecular weight, and grading effect is better, narrow molecular weight distribution.
The present invention also provides the application of a kind of lentinan in medicine or the food of preparing strengthening immunity, and the weight-average molecular weight of described lentinan is 16200.
The present invention is by immunology confirmatory experiment, find the also difference of immunologic competence of the lentinan of the different weight-average molecular weight after classification, by immunosuppressant mouse is studied, finally found the optimum weight scope that improves immunizing power---16200, for contribution has been made in further research and the exploitation of lentinan pharmaceutical use.
In the present invention, the medicine of described strengthening immunity is preferably the immune ancillary drug for the treatment of cancer of the stomach, colorectal carcinoma, lung cancer, for strengthening the curative effect of chemotherapy, and alleviate the toxic action of chemotherapy, and reduce the phenomenon of leukopenia, gastrointestinal toxicity, hepatic disorder and vomiting that in chemotherapy process, patient occurs; Or the ancillary drug for the treatment of chronic hepatitis B, for improving the effect of turning out cloudy of hepatitis b virus marker, reduce the side effect of antiviral; Or the medicine for the treatment of mycobacterium tuberculosis infection, senile chronic bronchitis.
The food of described strengthening immunity is preferably the protective foods of flu-prevention, reduces the protective foods of cholesterol or blood sugar.
The invention provides a kind of complex method except albumen, first mushroom is dried successively, pulverizes, immersion, water extraction obtain lentinan crude extract; Then the lentinan crude extract obtaining is removed to albumen with chlorobenzene, calcium chloride, ammonium oxalate, trypsinase, Sevage reagent successively, finally adopt the method for alcohol precipitation can prepare lentinan.When this method is prepared lentinan in extraction, there is higher productive rate, and avoided the destruction that adopts acid or alkali to cause lentinan, the lentinan obtaining after purification has higher purity, can obtain by classification the lentinan of different number-average molecular weights.Lentinan purity provided by the invention is very high, there is hardly impurity and objectionable impurities, therefore can be used for experiment in external body after sterilising treatment, carry out bioactivity research, and the lentinan of different number-average molecular weights after classification is more significant to the biological activity mechanism of research lentinan, therefore extracting method provided by the invention is conducive to further research and the exploitation of lentinan.
In order to further illustrate the present invention, below in conjunction with embodiment, the extraction agent stage division of lentinan provided by the invention is described in detail.
In following embodiment:
Quality × 100% of reaction yield=actual product quality/raw material obtaining;
Quality × 100% of purifying yield=actual purifying quality/Crude polysaccharides obtaining;
Quality/purified polysaccharide quality × 100% that the content=classification of classification product obtains.
The preparation of embodiment 1～4 lentinan crude extract is extracted
Mushroom stem removing retains sporophore, 60 ℃～80 ℃ air-dry dry, pulverizer was pulverized 100～300 mesh sieves and was obtained mushroom powder, then take four parts of 20.0000g mushroom powder and be placed in 4 5L round-bottomed flasks, adding distil water 2L, 2.4L, 2.6L, 3L soak after 24h respectively, meet 90 ℃ of water-bath 36h of reflux, after being cooled to room temperature, standby sodium bicarbonate is adjusted pH value to 6.5, add compound protease 0.5g, 45 ℃ of water-bath 2h, then 90 ℃ of water-bath 2h under stirrer stirs, filter after being cooled to room temperature, residue redissolves with distilled water, in triplicate.By the centrifugal 20min of solution 4000rpm obtaining, collect supernatant, the concentrated volume of 50 ℃ of rotary evaporations of supernatant liquor is to 200mL, and be placed in 2L beaker, and adding 1.6L dehydrated alcohol, stirrer stirs the lower 40min of mixing, standing, by centrifugal the solution 8000rpm obtaining 15min collecting precipitation
Precipitation adds 200mL distilled water redissolves, and repeats the step 4 time of above-mentioned alcohol precipitation, by the precipitation lyophilize finally obtaining, obtains the crude extract of lentinan, calculates productive rate.The results detailed in Table 1, table 1 is in the embodiment of the present invention 1～4, and soak solution volume affects data to reaction yield.
In table 1 embodiment of the present invention 1～4, soak solution volume affects data to reaction yield
The preparation of embodiment 5～8 lentinan crude extracts is extracted
Mushroom stem removing retains sporophore, 60 ℃～80 ℃ air-dry dry, pulverizer was pulverized 100～300 mesh sieves and was obtained mushroom powder, then take four parts of 20.0000g mushroom powder and be placed in 4 5L round-bottomed flasks, adding distil water 3L soaks respectively 12h, 24h, 36h, after 48h, meet 90 ℃ of water-bath 36h of reflux, after being cooled to room temperature, standby sodium bicarbonate is adjusted system pH to 6.5, then add 0.5g compound protease, 45 ℃ of water-bath 2h, 90 ℃ of water-bath 2h under stirrer stirs again, after being cooled to room temperature, filter, residue is redissolved with distilled water, in triplicate, obtain the lentinan aqueous solution.By the centrifugal 20min of solution 4000rpm obtaining, collect supernatant, 50 ℃ of rotary evaporations of the supernatant liquor obtaining are concentrated to volume to 200mL and are placed in 2L beaker, add 1.6L dehydrated alcohol, stirrer stirs the lower 40min of mixing, standing, the centrifugal 15min collecting precipitation of 8000rpm adds 200mL distilled water to redissolve in precipitation, repeat the step 4 time of above-mentioned alcohol precipitation, by the precipitation lyophilize obtaining, obtain the crude extract of lentinan, calculate productive rate.The results detailed in Table 2, table 2 is that in the embodiment of the present invention 5～8, soak time affects data to reaction yield.
In table 2 embodiment of the present invention 5～8, soak time affects data to reaction yield
The preparation of embodiment 9～12 lentinan crude extracts is extracted
Mushroom stem removing retains sporophore, 60 ℃～80 ℃ air-dry dry, pulverizer was pulverized 100～300 mesh sieves and was obtained mushroom powder, then take four parts of 20.0000g mushroom powder and be placed in 4 5L round-bottomed flasks, adding distil water 3L soaks respectively after 24h, connect reflux, respectively at 80 ℃, 85 ℃, 90 ℃, 95 ℃ of water-bath 36h, after being cooled to room temperature, adjust pH value to 6.5, add compound protease 0.5g, 45 ℃ of water-bath 2h, then 90 ℃ of water-bath 2h under stirrer stirs, after being cooled to room temperature, filter, residue is redissolved with distilled water, in triplicate, obtain the lentinan aqueous solution.By the centrifugal 20min of solution 4000rpm obtaining, collect supernatant, the concentrated volume of 50 ℃ of rotary evaporations of supernatant liquor is placed in 2L beaker to 200mL, adds 1.6L dehydrated alcohol, stirrer stirs the lower 40min of mixing, standing, the centrifugal 15min collecting precipitation of 8000rpm adds 200mL distilled water to redissolve in precipitation, repeat above-mentioned alcohol precipitation step 4 time, by the precipitation lyophilize obtaining, obtain the crude extract of lentinan, calculate productive rate.The results detailed in Table 3, table 3 is that in the embodiment of the present invention 9～12, bath temperature affects data to reaction yield.
In table 3 embodiment of the present invention 9～12, bath temperature affects data to reaction yield
The purifying of embodiment 13～16 lentinan crude extracts
The lentinan crude extract that embodiment 1 is obtained weighs four parts, every part of 10.0000g, add respectively 20mL, 40mL, 80mL, 100mL distilled water, agitation as appropriate is heated to abundant dissolving, then add respectively chlorobenzene 6.67mL, 13.33mL, 22.67mL, 33.33mL to purify, centrifugal collection supernatant after stirring reaction 30min, supernatant liquor repeats above-mentioned chlorobenzene treatment step 8 times; Gained lentinan solution is added respectively to Calcium Chloride Powder Anhydrous 1g, 1.5g, 2.5g, 4g, constantly boiling 1min, the centrifugal 3min of 5000rpm collects supernatant again, supernatant liquor adds ammonium oxalate 0.3g electric stirring 30min, then the centrifugal 3min of 5000rpm collects supernatant, the supernatant liquor obtaining continues to add sodium-chlor supersaturation electric stirring 1h, and then the centrifugal 8min of 7000rpm collects supernatant; With sodium bicarbonate, adjust the supernatant liquor pH value to 8.0 obtaining after supersaturation, then add trypsinase 1g, obtain lentinan solution.
The lentinan solution obtaining is above added and the Sevage reagent (V of lentinan solution phase same volume
trichloromethane: V
propyl carbinol=4:1), electric stirring 10min, then the centrifugal 3min of 1000rpm, separatory is removed lower floor's liquid, continue the centrifugal 3min of 5000rpm and collect supernatant, supernatant liquor repeats the above-mentioned Sevage of adding step 6 time, the supernatant liquor rotary evaporation of finally collecting is concentrated to volume to 1/5 of original volume, then add gac 0.2g, the centrifugal 10min of 3500rpm after stirring, supernatant liquor adds the dehydrated alcohol of 8 times of volumes, be uniformly mixed 30min, standing, then the centrifugal 10min collecting precipitation of 3500rpm, add water to redissolve precipitation, repeat the step 4 time of above-mentioned alcohol precipitation, the precipitation finally obtaining is cleaned 3 times with dehydrated alcohol successively, anhydrous diethyl ether cleans 3 times, vacuum is drained the lentinan that obtains purifying, calculate purifying yield.Refer to table 4, table 4 is in the embodiment of the present invention 13～16, and calcium chloride adds quality to affect data to purifying yield.
In table 4 embodiment of the present invention 13～16, calcium chloride adds quality to affect data to purifying yield
The purifying of embodiment 17～20 lentinan crude extracts
The lentinan crude extract that embodiment 1 is obtained weighs four parts, and every part of 10.0000g, adds respectively 100mL distilled water, agitation as appropriate is heated to abundant dissolving, then add chlorobenzene 30mL to purify, centrifugal collection supernatant after stirring 30min, supernatant liquor repeats above-mentioned chlorobenzene treatment step 8 times; Gained lentinan solution is added to 2.5g Calcium Chloride Powder Anhydrous, constantly boiling 1min, the centrifugal 3min of 5000rpm collects supernatant again, supernatant liquor adds respectively 0.2g, 0.3g, 0.4g, 0.5g ammonium oxalate electric stirring 30min, then the centrifugal 3min of 5000rpm collects supernatant, the supernatant liquor obtaining continues to add sodium-chlor supersaturation electric stirring 1h, and then the centrifugal 8min of 7000rpm collects supernatant; With sodium bicarbonate, adjust the supernatant liquor pH value to 8.0 obtaining after supersaturation, then add trypsinase 1g, obtain lentinan solution.
The lentinan solution obtaining is above added and the Sevage reagent (V of lentinan solution phase same volume
trichloromethane: V
propyl carbinol=4:1), electric stirring 10min, then the centrifugal 3min of 1000rpm, separatory is removed lower floor's liquid, continue the centrifugal 3min of 5000rpm and collect supernatant, supernatant liquor repeats the above-mentioned Sevage of adding step 6 time, the supernatant liquor rotary evaporation of finally collecting is concentrated to volume to 1/5 of original volume, then add gac 0.2g, the centrifugal 10min of 3500rpm after stirring, supernatant liquor adds the dehydrated alcohol of 8 times of volumes, be uniformly mixed 30min, standing, then the centrifugal 10min collecting precipitation of 3500rpm, add water to redissolve precipitation, repeat the step 4 time of above-mentioned alcohol precipitation, the precipitation finally obtaining is cleaned 3 times with dehydrated alcohol successively, anhydrous diethyl ether cleans 3 times, vacuum is drained the lentinan that obtains purifying, calculate purifying yield.Refer to table 5, table 5 is in the embodiment of the present invention 17～20, and ammonium oxalate adds quality to affect data to purifying yield.
In table 5 embodiment of the present invention 17～20, ammonium oxalate adds quality to affect data to purifying yield
The purifying of embodiment 21～23 lentinan crude extracts
The lentinan crude extract that embodiment 1 is obtained weighs three parts, and every part of 10.0000g, adds respectively 100mL distilled water, agitation as appropriate is heated to abundant dissolving, then add chlorobenzene 30mL to purify, centrifugal collection supernatant after stirring 30min, supernatant liquor repeats above-mentioned chlorobenzene treatment step 8 times; Gained lentinan solution is added to 2.5g Calcium Chloride Powder Anhydrous, constantly boiling 1min, the centrifugal 3min of 5000rpm collects supernatant again, supernatant liquor adds 0.3g ammonium oxalate electric stirring 30min, then the centrifugal 3min of 5000rpm collects supernatant, the supernatant liquor obtaining continues to add sodium-chlor supersaturation electric stirring 1h, and then the centrifugal 8min of 7000rpm collects supernatant; With sodium bicarbonate, adjust supernatant liquor pH respectively to 7.7 of value, 8.0,8.2 obtaining after supersaturation, then add trypsinase 1g, obtain lentinan solution.
The lentinan solution obtaining is above added and the Sevage reagent (V of lentinan solution phase same volume
trichloromethane: V
propyl carbinol=4:1), electric stirring 10min, then the centrifugal 3min of 1000rpm, separatory is removed lower floor's liquid, continue the centrifugal 3min of 5000rpm and collect supernatant, supernatant liquor repeats the above-mentioned Sevage of adding step 6 time, the supernatant liquor rotary evaporation of finally collecting is concentrated to volume to 1/5 of original volume, then add gac 0.2g, the centrifugal 10min of 3500rpm after stirring, supernatant liquor adds the dehydrated alcohol of 8 times of volumes, be uniformly mixed 30min, standing, then the centrifugal 10min collecting precipitation of 3500rpm, add water to redissolve precipitation, repeat the step 4 time of above-mentioned alcohol precipitation, the precipitation finally obtaining is cleaned 3 times with dehydrated alcohol successively, anhydrous diethyl ether cleans 3 times, vacuum is drained the lentinan that obtains purifying, calculate purifying yield.Refer to table 6, table 6 is in the embodiment of the present invention 21～23, and pH value affects data to purifying yield.
The lentinan of purifying prepared by embodiment 23 carries out infrared and nucleus magnetic resonance sign, experimental result is shown in Fig. 1 and Fig. 2, wherein, Fig. 1 is the infared spectrum of the lentinan prepared of the embodiment of the present invention 23, and Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram of the lentinan prepared of the embodiment of the present invention 23.As seen from Figure 1, at 3600cm
-1～3200cm
-1between 3400cm
-1left and right show-OH stretching vibration absorption peak, illustrates and has intermolecular hydrogen bond; 3000cm
-1～2800cm
-1between 2900cm
-1left and right shows the characteristic peak of the weak absorption of C-H; At 1400cm
-1～1200cm
-1between characteristic peak be carbohydrate C-H angle vibration absorption peak, above characteristic peak shows that this compound belongs to saccharide compound.At 1630cm
-1left and right is the stretching vibration of the carbonyl (C=O) of acid amides; At 1030cm
-1left and right absorption peak is the absorption peak of pyranose ring (C-O-C) and hydroxyl, and 890cm
-1left and right absorption peak is β type C-H angle vibration performance absorption peak, and above characteristic peak shows that this compound is that callose is main lentinan.According to nuclear-magnetism, show again, can prove that the present invention has prepared the lentinan of purifying.
In table 6 embodiment of the present invention 21～23, pH value affects data to purifying yield
The classification of embodiment 24～42 lentinans
Take the lentinan after the purifying that 5.0000g embodiment 20 obtains, in closed system, dropwise add acetone, limit edged stirs, until acetone concentration reaches 5wt%, standing 90min, repeats above operation and improves constantly acetone concentration, until there is precipitation to produce, then the centrifugal 20min collecting precipitation of 3500rpm, remains supernatant and continues to add acetone to repeat above-mentioned steps, until concentration reaches 90% after collecting precipitation.Collect each precipitation, by gel permeation chromatography or Ubbelohde viscometer, survey its molecular weight, refer to table 7, table 7 is that in the embodiment of the present invention 24～42, lentinan acetone classification after product content and molecular weight thereof gather.
The gel infiltration electrophorogram of classification after product is shown in Fig. 3, and wherein curve A is the lentinan gel infiltration electrophorogram of weight-average molecular weight 32900; Curve B is the lentinan gel infiltration electrophorogram of weight-average molecular weight 16200; Curve C is the lentinan gel infiltration electrophorogram of weight-average molecular weight 16000; Curve D is the lentinan gel infiltration electrophorogram of weight-average molecular weight 14800; Curve E is the lentinan gel infiltration electrophorogram of weight-average molecular weight 10500; Curve F is the lentinan gel infiltration electrophorogram of weight-average molecular weight 9100; Curve G is the lentinan gel infiltration electrophorogram of weight-average molecular weight 7800; Curve H is the lentinan gel infiltration electrophorogram of weight-average molecular weight 5100; Curve I is the lentinan gel infiltration electrophorogram of weight-average molecular weight 4500; Curve J is the lentinan gel infiltration electrophorogram of weight-average molecular weight 3900.Meanwhile, gel infiltration electrophorogram does not have impurity peaks, shows that the lentinan that the present invention extracts has higher purity yet.
Table 7 lentinan acetone classification after product content and molecular weight thereof gather
Embodiment 43
Lentinan after the classification obtaining take embodiment 27,29,32,35,37,38 and 40, as experiment material, carries out immunology checking.
Laboratory animal is H22 mouse, by intraperitoneal injection of cyclophosphamide (CTX) modeling, obtain hypoimmunity mouse model, be divided into experimental group, endoxan group (CTX), LEVAMISOLE HCL group (LMS) and blank group (CON), experimental group is divided into again 7 groups: the lentinan that corresponding embodiment 27,29,32,35,37,38 and 40 obtains respectively.Specifically be grouped as follows: A---embodiment 27; B---embodiment 29; C---embodiment 32; D---embodiment 35; E---embodiment 37; F---embodiment 38; G---embodiment 40; H---blank group (CON); I---endoxan group (CTX); J---LEVAMISOLE HCL group (LMS).
By tail vein injection, treat, dosage is 0.1mg/kg body weight, and administration volume is 0.1mL/kg body weight, within every 4 days, be administered once, and administration 5 times altogether, blank is organized not administration.Experiment finishes the rear following index that detects respectively: body weight gain situation, organ index (thymus index, index and spleen index, cardiac index, liver exponential sum renal index), peripheral white blood cell and marrow micronucleus number.
Experimental result is shown in Fig. 4～Fig. 8, Fig. 4 is the embodiment of the present invention 43 small mouse body weight change figure, can see that each group all occurs weight loss phenomenon after modeling, modeling success is described, in each group body weight after Lentinan for Treatment, there is obvious rising, best results is that weight-average molecular weight is 16200 lentinan, organizes B.Table 8 is that data corresponding to Fig. 4 small mouse body weight change curve gather, and by table 8, also can be found out, the body weight of group B small mouse is the heaviest.
Fig. 5 is the embodiment of the present invention 43 small mouse index and spleen index variation diagrams, after can seeing modeling, each group index and spleen index obviously reduces, after Lentinan for Treatment, each group mouse spleen index has to a certain degree rising, and best results is the lentinan of weight-average molecular weight 16200.
Fig. 6 is the embodiment of the present invention 43 small mouse thymus index variation diagrams, after can seeing modeling, each group thymus index obviously reduces, after Lentinan for Treatment, each group mouse thymus index has to a certain degree rising, and best results is the lentinan of weight-average molecular weight 16200.
Fig. 7 is the embodiment of the present invention 43 small mouse peripheral white blood cell variation diagrams, after can seeing modeling, each group peripheral white blood cell obviously reduces, after Lentinan for Treatment, each group murine interleukin number has to a certain degree rising, and best results is that weight-average molecular weight is 16200 lentinan.
Fig. 8 is that the embodiment of the present invention 43 small mouse marrow micronucleus are counted variation diagram, after can seeing modeling, each group marrow micronucleus number is all increased significantly, after Lentinan for Treatment, each group micronucleus number has to a certain degree decline, and best results is that weight-average molecular weight is 16200 lentinan.
Table 8 Fig. 4 small mouse body weight change curve corresponding data gathers
From above-described embodiment, the present invention extracts lentinan by the complex method except albumen, select chlorobenzene, calcium chloride, ammonium oxalate, trypsinase, Sevage reagent etc. to remove albumen, there is higher productive rate, and avoided the destruction that adopts acid or alkali to cause lentinan, the lentinan obtaining after purification has higher purity, can obtain by classification the lentinan of different number-average molecular weights.And by immunosuppressant mouse is studied, finally found the optimum weight scope that improves immunizing power---162000, for contribution has been made in further research and the exploitation of lentinan pharmaceutical use.
The explanation of above embodiment is just for helping to understand method of the present invention and core concept thereof.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of the claims in the present invention.
Claims (10)
1. an extracting method for lentinan, comprising:
A) mushroom is dried successively, pulverizes, immersion, water extraction obtain lentinan crude extract;
B) by steps A) the lentinan crude extract that obtains removes albumen with chlorobenzene, calcium chloride, ammonium oxalate, trypsinase, Sevage reagent successively, finally adopts the method for alcohol precipitation to obtain lentinan.
2. extracting method according to claim 1, is characterized in that, the granularity of described pulverizing is 50 order～300 orders.
3. extracting method according to claim 1, is characterized in that, the time of described immersion is 12h～72h; The temperature of described water extraction is 60 ℃～98 ℃.
4. extracting method according to claim 1, is characterized in that, 10wt%～40wt% that the consumption of described calcium chloride is described mushroom; The consumption of described ammonium oxalate is the 1wt%～5wt% of described mushroom.
5. extracting method according to claim 1, is characterized in that, the alcohol that the method for described alcohol precipitation adopts is ethanol.
6. extracting method according to claim 1, is characterized in that, described step B) in, with ammonium oxalate, trypsinase, lentinan crude extract being removed in the middle of the step of albumen successively, also comprise: add sodium-chlor.
7. extracting method according to claim 1, is characterized in that, described step B) in, with Sevage reagent, lentinan crude extract being removed after albumen, also comprise: add gac.
8. a method for the lentinan classification obtaining through extracting method claimed in claim 1, comprising:
Lentinan is carried out to fractionation precipitation with acetone, obtain the lentinan after classification.
9. stage division according to claim 8, is characterized in that, described method is specially:
A) lentinan obtaining by extracting method claimed in claim 1 is dissolved in distilled water, add acetone to occur precipitation, centrifugal supernatant liquor and the precipitation of obtaining, described in be precipitated as first step lentinan;
B) get the supernatant liquor obtaining in step a), add acetone to occur precipitation, centrifugal supernatant liquor and the precipitation of obtaining, described in be precipitated as second stage lentinan;
C) repeat said process, reach 90wt% to acetone concentration.
10. the application of lentinan in medicine or the food of preparing strengthening immunity, the weight-average molecular weight of described lentinan is 16200.
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| CN105330759A (en) * | 2015-11-23 | 2016-02-17 | 福建裕兴果蔬食品开发有限公司 | Method for extracting mushroom polysaccharides |
| CN107312108A (en) * | 2017-07-27 | 2017-11-03 | 福建农林大学 | A kind of preparation method and applications of lentinan chromic compound |
| CN109081876A (en) * | 2017-06-13 | 2018-12-25 | 陶宁 | The lentinan powder and its extraction process of prevention and/or adjuvant therapy of tumors |
| CN110256598A (en) * | 2019-07-17 | 2019-09-20 | 武汉工程大学 | A kind of preparation method of difference bioactivity lentinan |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104829734A (en) * | 2014-10-10 | 2015-08-12 | 沈阳市粮油食品科学研究所 | Method of producing pigment, protein, polysaccharide and dietary fiber from dry lentinula edodes |
| CN104829734B (en) * | 2014-10-10 | 2017-06-13 | 沈阳市粮油食品科学研究所 | A kind of method of utilization dried thin mushroom production pigment, albumen, polysaccharide and dietary fiber |
| CN105330759A (en) * | 2015-11-23 | 2016-02-17 | 福建裕兴果蔬食品开发有限公司 | Method for extracting mushroom polysaccharides |
| CN109081876A (en) * | 2017-06-13 | 2018-12-25 | 陶宁 | The lentinan powder and its extraction process of prevention and/or adjuvant therapy of tumors |
| CN107312108A (en) * | 2017-07-27 | 2017-11-03 | 福建农林大学 | A kind of preparation method and applications of lentinan chromic compound |
| CN110256598A (en) * | 2019-07-17 | 2019-09-20 | 武汉工程大学 | A kind of preparation method of difference bioactivity lentinan |
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