CN103724447B - The extraction of a kind of lentinan and stage division - Google Patents
The extraction of a kind of lentinan and stage division Download PDFInfo
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Abstract
The invention provides extraction and the stage division of a kind of lentinan, first Lentinus Edodes is dried successively, pulverizes, soaks, water raises lentinan crude extract；Then the lentinan crude extract obtained is carried out removing protein with chlorobenzene, calcium chloride, ammonium oxalate, trypsin, Sevage reagent successively, finally use the method for precipitate with ethanol can prepare lentinan.The method is when lentinan is prepared in extraction, there is higher productivity, and avoid the destruction using acid or alkali that lentinan is caused, the lentinan obtained after purification has higher purity, the lentinan of different weight average molecular weight can be obtained by classification, and range of molecular weight distributions is narrower, have broad application prospects in Organism immunoregulation and tumor aid treatment.
Description
Technical field
The present invention relates to extracts active ingredients classification technique field, particularly relate to the extraction of a kind of lentinan
And stage division.
Background technology
Lentinus Edodes belongs to Basidiomycetes umbrella shape fungus, is edible fungi the most all the fashion.Master in Lentinus Edodes
Wanting composition is lentinan, how presented in mixing polysaccharide, gathers including 4 kinds of heteropolysaccharide and 2 kinds of Portugals
Sugar, wherein, having bioactive is glucosan, and its structure is with β-(1 → 3) Glucopyranose. glycosidic bond
For main chain, with β-(1 → 6) Glucopyranose. glycosidic bond as branch.
Modern study shows that lentinan has regulation immunity, antitumor, blood sugar lowering, antiviral and anti-
The functions such as oxidation.Lentinan has immunoregulation effect to body, belongs to typical T mainly due to it
Cell activator, can promote delayed hypersensitive reaction in vivo, improves antibody-dependent cytotoxicity cytoactive,
The factors such as generation acute protein inducible factor can also be induced simultaneously, thus produce related immune response.There is report
Road shows that lentinan can suppress the growth of mice S-180 solid tumor, simultaneously for treatment colon cancer, breast
Adenocarcinoma, pulmonary carcinoma, gastric cancer etc. have a good curative effect, and can extend the time-to-live of tumor patient, also can
Enough alleviate the side effect that chemotherapy is brought greatly；Clinical practice is it was also found that lentinan can improve hepatitis B trouble
The common symptons such as weak, nauseating, the abdominal distention of person, promote that transaminase and bilirubin recover normal；Lentinus Edodes is many
Sugar is also to Abelson virus, A2(H2N2) virus, 12 type adenoviruss and influenza virus etc. all have suppression
Effect.
Although lentinan is increasingly subject to people's attention in clinical practice in recent years, but its effect
Mechanism is the clearest and the most definite, and the particularly mechanism of anti-tumor activity, a most critically important reason is exactly
The complexity of lentinan itself.
Therefore, carrying out lentinan extracting classification, the mechanism of action for research lentinan has very
Important meaning.Extracting method for lentinan specifically includes that diluted alkaline extraction method, how with hydrogen at present
Sodium oxide is extractant, extracts and can obtain more alkali-soluble polysaccharide, it is impossible to but this method obtains
Polysaccharide impurity more, polysaccharide is the most easily destroyed；Also has Thinner acid method, how with trichloroacetic acid for extracting
Agent, can obtain more acid-soluble polysaccharide, but polysaccharide is the most easily destroyed；Also have dilute salt extraction method,
Many with zinc chloride as extractant, but this method extraction ratio is relatively low, and raw material causes bigger waste；
Additionally, also microwave, supercritical ultrasonics technology, with deionized water as extractant, but thermal sensitivity polysaccharide is easily broken
Bad.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is provide the extraction of a kind of lentinan and divide
Level method, will not damage polysaccharide, and higher to the extraction ratio of polysaccharide, simultaneously can be to polysaccharide
Carry out preferable classification.
The invention provides the extracting method of a kind of lentinan, including:
A) be dried successively by Lentinus Edodes, pulverize, soak, water raises lentinan crude extract；
B) by step A) the lentinan crude extract that obtains is successively with chlorobenzene, calcium chloride, ammonium oxalate, pancreas
Protease, Sevage reagent carry out removing protein, finally use the method for precipitate with ethanol to obtain lentinan.
Preferably, the granularity of described pulverizing is 50 mesh～300 mesh.
Preferably, the time of described immersion is 12h～72h；The temperature that described water carries is 60 DEG C～98 DEG C.
Preferably, the consumption of described calcium chloride is 10wt%～40wt% of described Lentinus Edodes；Described ammonium oxalate
Consumption is 1wt%～5wt% of described Lentinus Edodes.
Preferably, the alcohol that the method for described precipitate with ethanol uses is ethanol.
Preferably, described step B) in, using ammonium oxalate, trypsin to lentinan crude extract successively
Carry out, in the middle of the step of removing protein, also including: add sodium chloride.
Preferably, described step B) in, with Sevage reagent, lentinan crude extract is being carried out except egg
Bai Hou, also includes: add activated carbon.
The method that present invention also offers the lentinan classification that a kind of said extracted method obtains, including:
With acetone, lentinan is carried out fractional precipitation, obtain the lentinan after classification.
Preferably, described method particularly as follows:
A) lentinan obtained by the extracting method described in claim 1 is dissolved in distilled water,
Add acetone to precipitation occurs, centrifugal obtain supernatant and precipitation, described in be precipitated as first order lentinan；
B) taking the supernatant obtained in step a), addition acetone, to there is precipitation, is centrifuged and obtains supernatant
And precipitation, described in be precipitated as second level lentinan；
C) repeat said process, reach 90wt% to acetone concentration.
Present invention also offers a kind of lentinan answering in the medicine preparing enhancing immunity or food
With, the weight average molecular weight of described lentinan is 16200.
Compared with prior art, the invention provides the extracting method of a kind of lentinan, have employed except egg
White complex method, is first dried Lentinus Edodes successively, pulverizes, soaks, water raises lentinan
Crude extract；Then the lentinan crude extract obtained is used chlorobenzene, calcium chloride, ammonium oxalate, pancreas egg successively
White enzyme, Sevage reagent carry out removing protein, finally use the method for precipitate with ethanol can prepare lentinan.
The method, when lentinan is prepared in extraction, has higher productivity, and avoids employing acid or alkali pair
The destruction that lentinan causes, the lentinan obtained after purification has higher purity, it is possible to by dividing
Level obtains the lentinan of different number-average molecular weight.The lentinan purity that the present invention provides is the highest, almost
There is not impurity and harmful substance, therefore after sterilization treatment, i.e. can be used for external experiment in vivo carry out biology
Activity research, and the biological of research lentinan is lived by the lentinan of the different number-average molecular weights after classification
Property mechanism more significant, therefore the present invention provide extracting method be conducive to the further of lentinan
Research and exploitation.
Accompanying drawing explanation
Fig. 1 is the infared spectrum of the lentinan of the embodiment of the present invention 23 preparation；
Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram of the lentinan of the embodiment of the present invention 23 preparation；
Fig. 3 is the osmogels electrophoretogram of the lentinan that the embodiment of the present invention 24～42 classification obtains；
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 diagram；
Fig. 6 is the embodiment of the present invention 43 small mouse thymus index variation diagram；
Fig. 7 is the embodiment of the present invention 43 small mouse peripheral white blood cell variation diagram；
Fig. 8 is the embodiment of the present invention 43 small mouse bone marrow micronucleus number variation diagram.
Detailed description of the invention
The invention provides the extracting method of a kind of lentinan, including:
A) be dried successively by Lentinus Edodes, pulverize, soak, water raises lentinan crude extract；
B) by step A) the lentinan crude extract that obtains is successively with chlorobenzene, calcium chloride, ammonium oxalate, pancreas
Protease, Sevage reagent carry out removing protein, finally use the method for precipitate with ethanol to obtain lentinan.
Lentinan is extracted by the present invention by the complex method of removing protein, select chlorobenzene, calcium chloride,
Ammonium oxalate, trypsin, Sevage reagent etc. carry out removing protein, have higher productivity, and avoid
Using the destruction that lentinan is caused by acid or alkali, the lentinan that obtains after purification has higher pure
Degree, it is possible to obtained the lentinan of different number-average molecular weight by classification.The lentinan that the present invention provides
Purity is the highest, there's almost no impurity and harmful substance, therefore i.e. can be used for external body after sterilization treatment
Interior experiment carries out bioactivity research, and the lentinan of the different number-average molecular weights after classification is to research perfume
The biological activity mechanism of mushroom polysaccharide is more significant, and the extracting method that therefore present invention provides is conducive to perfume
The research further of mushroom polysaccharide and exploitation.
The extracting method of the lentinan that the present invention provides, comprises the following steps:
First be the preparation of lentinan crude extract, will Lentinus Edodes be dried successively, pulverize, soak,
Water raises lentinan crude extract, and concrete operation step is as follows:
Being cleaned by Lentinus Edodes and remove handle, 60 DEG C～80 DEG C dry, pulverize to 50 mesh～300 mesh, then at powder
Adding distilled water in sample after broken, the volume of described distilled water is preferably with the ratio of the quality of Lentinus Edodes
(10mL～200mL): 1g, more preferably (100mL～150mL): 1g, soak after adding distilled water
12h～72h, 60 DEG C～98 DEG C of high temperature bath 12h～48h carry out water and carry the most again, and solution is cooled down after carrying by water
To room temperature and with sodium bicarbonate adjust pH value to 5.0～8.0, more preferably adjust to 6～7, add compound egg
White protein hydrolysis, 35 DEG C～55 DEG C, more preferably 40 DEG C～50 DEG C of water-bath 1h～3h, then at stirrer
Stirring lower 80 DEG C～90 DEG C of water-bath 1h～3h makes enzyme inactivate, and then cools down, and filters, and residue distilled water redissolves,
Repeat 1～3 time, collect filtrate, obtain the thick product of lentinan water extract.The granularity of described pulverizing is more excellent
Elect 100 mesh～300 mesh as；The temperature that described water carries is more preferably 85 DEG C～95 DEG C, the time of high temperature bath
More preferably 20h～40h；The consumption of described compound protease is preferably 1wt%～5wt% of Lentinus Edodes, more excellent
Elect 2wt%～4wt% as.
After obtaining the thick product of lentinan water extract, it is carried out precipitate with ethanol, concrete, by filtrate with
The rotating speed of 3500rpm～4500rpm is centrifuged 10min～30min, collects supernatant, by supernatant 40 DEG C～50
DEG C rotary evaporation concentrates volume to the 1/15～1/3 of original volume, is subsequently adding 3～8 times (volume ratios) of concentrated solution
Dehydrated alcohol, stirring mixing 30min～60min, stand, be centrifuged with the rotating speed of 7000rpm～9000rpm
10min～30min also collects precipitation, precipitation adds distilled water and redissolves, repeat above-mentioned precipitate with ethanol step 3～5 times,
The precipitation vacuum lyophilization finally obtained, obtains the crude extract of lentinan.Described ethanol more preferably adds
Enter 5～7 times (volume ratios).
After obtaining lentinan crude extract, it is purified, concrete, in lentinan crude extract
Adding distilled water, the volume of described distilled water is preferably (2mL～10mL) with the ratio of the quality of Lentinus Edodes:
1g, is heated to fully dissolving to its agitation as appropriate after adding distilled water, is subsequently adding chlorobenzene and purifies,
Centrifugal after stirring 30min collecting supernatant, supernatant repeats step 5 that above-mentioned chlorobenzene processes～8 times, finally
Obtain the lentinan solution after chlorobenzene processes；The consumption of described chlorobenzene is preferably the 1/4～1/2 of liquor capacity,
More preferably 1/3.The purpose adding chlorobenzene is removing protein.
Then the lentinan solution obtained after above-mentioned chlorobenzene processes adds anhydrous calcium chloride, persistently boils
Rise 1min～3min, then 5000rpm～7000rpm be centrifuged 3～5min after collect supernatant, supernatant adds
Ammonium oxalate electric stirring 30min～60min, then 5000rpm～7000rpm is centrifuged in 3～5min collections
Clearly, the lentinan solution after ammonium oxalate processes is obtained；The consumption of described anhydrous calcium chloride is preferably Lentinus Edodes
10wt%～40wt%；The consumption of described ammonium oxalate is preferably 1wt%～5wt% of Lentinus Edodes.Preferably,
After addition calcium chloride, ammonium oxalate carry out removing protein, the supernatant obtained is additionally added sodium chloride satiety
With also electric stirring 0.5h～3h, then 7000rpm～9000rpm is centrifuged 8～10min collection supernatants, chlorination
Change the purpose of sodium for separating out protein.
After obtaining the lentinan solution after above-mentioned ammonium oxalate processes, adjust its pH value with sodium bicarbonate and arrive
7.5～8.5, it is subsequently adding trypsin, obtains the lentinan solution after trypsin hydrolyzing albumen, institute
State tryptic addition and be preferably 5wt%～15wt% of Lentinus Edodes.
After using trypsin hydrolyzing albumen, the lentinan solution obtained is added 0.5～1 times of volume
Sevage reagent (chloroform: n-butyl alcohol=4:1), electric stirring 10min～30min, then 1000rpm
Centrifugal 3min～5min, separatory is removed lower floor's liquid, is continued 5000rpm～7000rpm and be centrifuged 3min～5min
Collecting supernatant, supernatant repeats above-mentioned addition Sevage step 3～8 times, the supernatant rotation that will finally collect
Turn evaporation and concentration volume to the 1/5～1/4 of original volume, collect supernatant and add the anhydrous second of 3～8 times of volumes
Alcohol, stirring mixing 30min～60min, stand, then 3500rpm～4500rpm is centrifuged 10min～30min
Collect precipitation, precipitation is added distilled water redissolution, repeat the step 3 of above-mentioned precipitate with ethanol～5 times, will finally obtain
The precipitation arrived is successively with washes of absolute alcohol 2～4 times, and absolute ether cleans 2～4 times, and vacuum is drained and obtained
The lentinan of purification.Preferably, after lentinan crude extract being carried out removing protein with Sevage reagent,
Also include: adding activated carbon, the consumption of described activated carbon is preferably 1wt%～3wt% of Lentinus Edodes.Add and live
Property charcoal effect be decolouring.
Using INFRARED ABSORPTION and nuclear magnetic resonance, NMR to characterize the lentinan structure of preparation, result shows,
The lentinan that the present invention is extracted has a structure shown in formula (I):
In formula (I), n is the degree of polymerization, it is preferred that 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 with β-(1 → 3) Glucopyranose. glycosidic bond
For main chain, with β-(1 → 6) Glucopyranose. glycosidic bond as branch.Its tertiary structure is as shown in the formula (II):
After preparing the lentinan of purification, with acetone, lentinan is carried out fractional precipitation, divided
Lentinan after Ji.
Concrete operation step is as follows:
A) being dissolved in distilled water by the lentinan of above-mentioned purification, addition acetone is to there is precipitation, centrifugal
Obtain supernatant and precipitation, described in be precipitated as first order lentinan；
B) taking the supernatant obtained in step a), addition acetone, to there is precipitation, is centrifuged and obtains supernatant
And precipitation, described in be precipitated as second level lentinan；
C) repeat said process, reach 90wt% to acetone concentration.
Concrete, the lentinan of the purification of above-mentioned preparation is placed in closed system, is added dropwise over acetone,
Stirring while adding, until acetone concentration reaches 5wt%, stand 60min～100min, repeat above operation
Improving constantly acetone concentration, until there being precipitation to produce, then 3500rpm～4500rpm is centrifuged
10min～30min collects precipitation, remains supernatant and continuously add acetone repeat the above steps after collecting precipitation,
Until acetone concentration reaches 90wt%, collect each precipitation, by gel permeation chromatography or Ubbelohde viscometer
Survey its molecular weight.
Test result indicate that, use the stage division that the present invention provides, it is possible to obtain different weight average molecular weight
Lentinan, grading effect is preferable, narrow molecular weight distribution.
Present invention also offers a kind of lentinan answering in the medicine preparing enhancing immunity or food
With, the weight average molecular weight of described lentinan is 16200.
The present invention passes through immunology confirmatory experiment, finds the lentinan of the different weight average molecular weight after classification
Immunologic competence the most different, by immunosuppressant mice is studied, eventually found raising exempt from
The optimum weight scope 16200 of epidemic disease power, for research further and the exploitation of lentinan medical value
It is made that contribution.
In the present invention, the medicine of described enhancing immunity preferably treats gastric cancer, colon cancer, the exempting from of pulmonary carcinoma
Epidemic disease ancillary drug, for strengthening the curative effect of chemotherapy, and alleviates the toxic action of chemotherapy, and reduces chemotherapy mistake
The phenomenon of leukopenia, gastrointestinal toxicity, liver function injury and vomiting that patient occurs in journey；Or treatment
The ancillary drug of chronic hepatitis B, for improving the effect of turning out cloudy of hepatitis b virus marker, reduces disease-resistant
The side effect of cytotoxic drug；Or treatment mycobacterium tuberculosis infection, the medicine of senile chronic bronchitis.
The food of described enhancing immunity is preferably the health food of flu-prevention, reduces cholesterol or blood glucose
Health food.
The invention provides the complex method of a kind of removing protein, first Lentinus Edodes is dried successively, pulverizes,
Soak, water raises lentinan crude extract；Then by the lentinan crude extract that obtains successively with chlorobenzene,
Calcium chloride, ammonium oxalate, trypsin, Sevage reagent carry out removing protein, the method finally using precipitate with ethanol
Lentinan can be prepared.The method, when lentinan is prepared in extraction, has higher productivity,
And avoiding the destruction using acid or alkali to cause lentinan, the lentinan obtained after purification has
Higher purity, it is possible to obtained the lentinan of different number-average molecular weight by classification.The present invention provides
Lentinan purity is the highest, there's almost no impurity and harmful substance, therefore the most available after sterilization treatment
Bioactivity research is carried out in external experiment in vivo, and the lentinan of the different number-average molecular weights after classification
More significant to the biological activity mechanism of research lentinan, that therefore the present invention provides extracting method
Be conducive to research further and the exploitation of lentinan.
In order to further illustrate the present invention, below in conjunction with embodiment carrying the lentinan that the present invention provides
Take agent stage division to be described in detail.
In following embodiment:
Quality × 100% of the product quality/raw material of reaction yield=actually obtain；
Quality × 100% of the purification quality/crude polysaccharides of purification yield=actually obtain；
The quality that classification product assay=classification obtains/purified polysaccharide quality × 100%.
The preparation of embodiment 1～4 lentinan crude extract is extracted
Mushroom stem removing retains sporophore, and 100～300 mesh sieves pulverized by 60 DEG C～80 DEG C of machines of dry, pulverize
Obtain Lentinus Edodes powder, then weigh four parts of 20.0000g Lentinus Edodes powder and be placed in 4 5L round-bottomed flasks, point
After not adding distilled water 2L, 2.4L, 2.6L, 3L immersion 24h, meet 90 DEG C of water-bath 36h of reflux, cold
But adjust pH value to 6.5 to standby sodium bicarbonate after room temperature, add compound protease 0.5g, 45 DEG C of water-baths
2h, then 90 DEG C of water-bath 2h under stirrer stirs, filter after being cooled to room temperature, and residue is multiple with distilled water
Molten, in triplicate.The solution 4000rpm obtained is centrifuged 20min, collects supernatant, supernatant 50 DEG C
Rotary evaporation concentration volume, to 200mL, is placed in 2L beaker, adds 1.6L dehydrated alcohol, stirring
Mix 40min under son stirring, stand, the solution 8000rpm obtained is centrifuged 15min and collects precipitation,
Precipitation adds 200mL distilled water and redissolves, and repeats the step 4 time of above-mentioned precipitate with ethanol, by cold for the precipitation finally given
Lyophilizing is dry, obtains the crude extract of lentinan, calculates productivity.The results detailed in Table 1, table 1 is that the present invention is real
Executing in example 1～4, soak volume affects data to reaction yield.
In table 1 embodiment of the present invention 1～4, soak volume affects data to reaction yield
The preparation of embodiment 5～8 lentinan crude extract is extracted
Mushroom stem removing retains sporophore, and 100～300 mesh sieves pulverized by 60 DEG C～80 DEG C of machines of dry, pulverize
Obtain Lentinus Edodes powder, then weigh four parts of 20.0000g Lentinus Edodes powder and be placed in 4 5L round-bottomed flasks, add
After distilled water 3L soaks 12h, 24h, 36h, 48h respectively, meet 90 DEG C of water-bath 36h of reflux, cold
But adjust system pH to 6.5 to standby sodium bicarbonate after room temperature, be subsequently adding 0.5g compound protease,
45 DEG C of water-bath 2h, then 90 DEG C of water-bath 2h under stirrer stirs, filter after being cooled to room temperature, used by residue
Distilled water redissolves, and in triplicate, obtains lentinan aqueous solution.The solution 4000rpm obtained is centrifuged
20min, collects supernatant, the 50 DEG C of rotary evaporations of supernatant obtained is concentrated volume and is placed in 200mL
In 2L beaker, add 1.6L dehydrated alcohol, mix 40min under stirrer stirring, stand, 8000rpm
Centrifugal 15min collects precipitation, adds 200mL distilled water and redissolves, repeat the step of above-mentioned precipitate with ethanol in precipitation
Rapid 4 times, the pellet frozen obtained is dried, obtains the crude extract of lentinan, calculate productivity.Result
Referring to 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 extract is extracted
Mushroom stem removing retains sporophore, and 100～300 mesh sieves pulverized by 60 DEG C～80 DEG C of machines of dry, pulverize
Obtain Lentinus Edodes powder, then weigh four parts of 20.0000g Lentinus Edodes powder and be placed in 4 5L round-bottomed flasks, add
After distilled water 3L soaks 24h respectively, connect reflux, respectively at 80 DEG C, 85 DEG C, 90 DEG C, 95 DEG C of water
Bath 36h, after being cooled to room temperature, adjustment pH value is to 6.5, adds compound protease 0.5g, 45 DEG C of water-bath 2h,
Then 90 DEG C of water-bath 2h under stirrer stirs, filter after being cooled to room temperature, are redissolved by residue distilled water,
In triplicate, lentinan aqueous solution is obtained.The solution 4000rpm obtained is centrifuged 20min, collects
Supernatant, 50 DEG C of rotary evaporations of supernatant concentrate volumes and are placed in 2L beaker to 200mL, add 1.6L without
Water-ethanol, mixes 40min under stirrer stirring, stands, and 8000rpm is centrifuged 15min and collects precipitation,
Precipitation adds 200mL distilled water redissolve, repeat above-mentioned precipitate with ethanol step 4 time, the pellet frozen that will obtain
It is dried, obtains the crude extract of lentinan, calculate productivity.The results detailed in Table 3, table 3 is that the present invention implements
In example 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 purification of embodiment 13～16 lentinan crude extract
Lentinan crude extract embodiment 1 obtained weighs four parts, and every part of 10.0000g is separately added into
20mL, 40mL, 80mL, 100mL distilled water, agitation as appropriate is heated to fully dissolving, then distinguishes
Add chlorobenzene 6.67mL, 13.33mL, 22.67mL, 33.33mL to purify, stirring reaction 30min
Rear centrifugal collection supernatant, supernatant repeats above-mentioned chlorobenzene and processes step 8 time；By gained lentinan solution
It is separately added into anhydrous calcium chloride 1g, 1.5g, 2.5g, 4g, constantly boiling 1min, then 5000rpm centrifugal
3min collects supernatant, and supernatant adds ammonium oxalate 0.3g electric stirring 30min, then 5000rpm from
Heart 3min collects supernatant, and the supernatant obtained continuously adds sodium chloride supersaturation electric stirring 1h, then
7000rpm is centrifuged 8min and collects supernatant；The supernatant pH value obtained after adjusting supersaturation with sodium bicarbonate
To 8.0, it is subsequently adding trypsin 1g, obtains lentinan solution.
Lentinan solution derived above is added the Sevage reagent with lentinan solution same volume
(VChloroform: VN-butyl alcohol=4:1), electric stirring 10min, then 1000rpm is centrifuged 3min, and separatory goes
Except lower floor's liquid, continuing 5000rpm and be centrifuged 3min collection supernatant, supernatant repeats above-mentioned addition Sevage
Step 6 time, concentrates the supernatant rotary evaporation finally collected volume to the 1/5 of original volume, is subsequently adding
Activated carbon 0.2g, after stirring, 3500rpm is centrifuged 10min, and supernatant adds the dehydrated alcohol of 8 times of volumes,
Stirring mixing 30min, stands, and then 3500rpm is centrifuged 10min and collects precipitation, and precipitation is added water
Redissolve, repeat the step 4 time of above-mentioned precipitate with ethanol, the precipitation finally given is used washes of absolute alcohol 3 successively
Secondary, absolute ether cleans 3 times, and vacuum drains the lentinan obtaining purification, calculates purification yield.In detail
Being shown in Table 4, table 4 is in the embodiment of the present invention 13～16, and calcium chloride adds quality affects data to purification yield.
In table 4 embodiment of the present invention 13～16, calcium chloride adds quality affects data to purification yield
The purification of embodiment 17～20 lentinan crude extract
Lentinan crude extract embodiment 1 obtained weighs four parts, and every part of 10.0000g adds respectively
100mL distilled water, agitation as appropriate is heated to fully dissolving, and is subsequently adding chlorobenzene 30mL and purifies,
Centrifugal collection supernatant after stirring 30min, supernatant repeats above-mentioned chlorobenzene and processes step 8 time；Gained is fragrant
Mushroom polysaccharide solution add 2.5g anhydrous calcium chloride, constantly boiling 1min, then 5000rpm be centrifuged 3min receive
Collection supernatant, supernatant is separately added into 0.2g, 0.3g, 0.4g, 0.5g ammonium oxalate electric stirring 30min,
Then 5000rpm is centrifuged 3min and collects supernatant, and the supernatant obtained continuously adds sodium chloride supersaturation electricity
Dynamic stirring 1h, then 7000rpm is centrifuged 8min and collects supernatant；Obtain after adjusting supersaturation with sodium bicarbonate
The supernatant pH value arrived, to 8.0, is subsequently adding trypsin 1g, obtains lentinan solution.
Lentinan solution derived above is added the Sevage reagent with lentinan solution same volume
(VChloroform: VN-butyl alcohol=4:1), electric stirring 10min, then 1000rpm is centrifuged 3min, and separatory goes
Except lower floor's liquid, continuing 5000rpm and be centrifuged 3min collection supernatant, supernatant repeats above-mentioned addition Sevage
Step 6 time, concentrates the supernatant rotary evaporation finally collected volume to the 1/5 of original volume, is subsequently adding
Activated carbon 0.2g, after stirring, 3500rpm is centrifuged 10min, and supernatant adds the dehydrated alcohol of 8 times of volumes,
Stirring mixing 30min, stands, and then 3500rpm is centrifuged 10min and collects precipitation, and precipitation is added water
Redissolve, repeat the step 4 time of above-mentioned precipitate with ethanol, the precipitation finally given is used washes of absolute alcohol 3 successively
Secondary, absolute ether cleans 3 times, and vacuum drains the lentinan obtaining purification, calculates purification yield.In detail
Being shown in Table 5, table 5 is in the embodiment of the present invention 17～20, and ammonium oxalate adds quality affects data to purification yield.
In table 5 embodiment of the present invention 17～20, ammonium oxalate adds quality affects data to purification yield
The purification of embodiment 21～23 lentinan crude extract
Lentinan crude extract embodiment 1 obtained weighs three parts, and every part of 10.0000g adds respectively
100mL distilled water, agitation as appropriate is heated to fully dissolving, and is subsequently adding chlorobenzene 30mL and purifies,
Centrifugal collection supernatant after stirring 30min, supernatant repeats above-mentioned chlorobenzene and processes step 8 time；Gained is fragrant
Mushroom polysaccharide solution add 2.5g anhydrous calcium chloride, constantly boiling 1min, then 5000rpm be centrifuged 3min receive
Collection supernatant, supernatant adds 0.3g ammonium oxalate electric stirring 30min, and then 5000rpm is centrifuged 3min
Collecting supernatant, the supernatant obtained continuously adds sodium chloride supersaturation electric stirring 1h, then 7000rpm
Centrifugal 8min collects supernatant；With sodium bicarbonate adjust the supernatant pH value obtained after supersaturation respectively to 7.7,
8.0,8.2, it is subsequently adding trypsin 1g, obtains lentinan solution.
Lentinan solution derived above is added the Sevage reagent with lentinan solution same volume
(VChloroform: VN-butyl alcohol=4:1), electric stirring 10min, then 1000rpm is centrifuged 3min, and separatory goes
Except lower floor's liquid, continuing 5000rpm and be centrifuged 3min collection supernatant, supernatant repeats above-mentioned addition Sevage
Step 6 time, concentrates the supernatant rotary evaporation finally collected volume to the 1/5 of original volume, is subsequently adding
Activated carbon 0.2g, after stirring, 3500rpm is centrifuged 10min, and supernatant adds the dehydrated alcohol of 8 times of volumes,
Stirring mixing 30min, stands, and then 3500rpm is centrifuged 10min and collects precipitation, and precipitation is added water
Redissolve, repeat the step 4 time of above-mentioned precipitate with ethanol, the precipitation finally given is used washes of absolute alcohol 3 successively
Secondary, absolute ether cleans 3 times, and vacuum drains the lentinan obtaining purification, calculates purification yield.In detail
Being shown in Table 6, table 6 is that in the embodiment of the present invention 21～23, pH value affects data to purification yield.
The lentinan of the purification of embodiment 23 preparation is carried out infrared and nuclear magnetic resonance, NMR sign, experimental result
Seeing Fig. 1 and Fig. 2, wherein, Fig. 1 is the infared spectrum of the lentinan of the embodiment of the present invention 23 preparation,
Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram of the lentinan of the embodiment of the present invention 23 preparation.As seen from Figure 1,
At 3600cm-1～3200cm-1Between 3400cm-1Left and right shows-OH stretching vibration absworption peak, and explanation is deposited
At intermolecular hydrogen bond；3000cm-1～2800cm-1Between 2900cm-1Left and right shows the weak suction of C-H
The characteristic peak received；At 1400cm-1～1200cm-1Between characteristic peak be saccharide C-H angle vibration absorption peak,
Features above peak shows that this compound belongs to saccharide compound.At 1630cm-1Left and right is the carbonyl of amide
(C=O) stretching vibration；At 1030cm-1Left and right absworption peak is pyranose ring (C-O-C) and hydroxyl
Absworption peak, and 890cm-1Left and right absworption peak is β type C-H angle vibration performance absworption peak, features above peak
Show this compound be callose be main lentinan.Show further according to nuclear-magnetism, may certify that this
Invention has prepared the lentinan of purification.
In table 6 embodiment of the present invention 21～23, pH value affects data to purification yield
The classification of embodiment 24～42 lentinan
Weigh the lentinan after purification that 5.0000g embodiment 20 obtains, dropwise add in closed system
Enter acetone, stirring while adding, until acetone concentration reaches 5wt%, stand 90min, repeat above operation
Improving constantly acetone concentration, until there being precipitation to produce, then 3500rpm is centrifuged 20min and collects precipitation,
Remain supernatant after collecting precipitation and continuously add acetone repeat the above steps, until concentration reaches 90%.Collect
Each precipitation, surveys its molecular weight by gel permeation chromatography or Ubbelohde viscometer, refers to table 7, and table 7 is this
In inventive embodiments 24～42, lentinan acetone classification afterproduct content and molecular weight thereof collect.
The gel infiltration electrophoretogram of classification afterproduct is shown in Fig. 3, and wherein curve A is weight average molecular weight 32900
Lentinan gel infiltration electrophoretogram；Curve B is that the lentinan gel of weight average molecular weight 16200 oozes
Electrophoretogram thoroughly；Curve C is the lentinan gel infiltration electrophoretogram of weight average molecular weight 16000；Curve D
Lentinan gel infiltration electrophoretogram for weight average molecular weight 14800；Curve E is weight average molecular weight 10500
Lentinan gel infiltration electrophoretogram；Curve F is the lentinan gel infiltration of weight average molecular weight 9100
Electrophoretogram；Curve G is the lentinan gel infiltration electrophoretogram of weight average molecular weight 7800；Curve H is
The lentinan gel infiltration electrophoretogram of weight average molecular weight 5100；Curve I is the perfume (or spice) of weight average molecular weight 4500
Mushroom polysaccharide gel infiltration electrophoretogram；Curve J is the lentinan gel infiltration electrophoresis of weight average molecular weight 3900
Figure.Meanwhile, gel infiltration electrophoretogram does not has impurity peaks, also indicates that the lentinan tool that the present invention extracts
There is higher purity.
Table 7 lentinan acetone classification afterproduct content and molecular weight thereof collect
Embodiment 43
Lentinan after the classification that embodiment 27,29,32,35,37,38 and 40 obtains is as reality
Test material, carry out immunology checking.
Laboratory animal is H22 mice, by intraperitoneal injection of cyclophosphamide (CTX) modeling, obtains immunity
The low mouse model of power, is divided into experimental group, cyclophosphamide group (CTX), levamisole group (LMS)
With blank group (CON), experimental group is divided into again 7 groups: respectively corresponding embodiment 27,29,32,
35,37,38 and 40 lentinan obtained.Specifically it is grouped as follows: A embodiment 27；B——
Embodiment 29；C embodiment 32；D embodiment 35；E embodiment 37；F implements
Example 38；G embodiment 40；H blank group (CON)；I cyclophosphamide group (CTX)；
J levamisole group (LMS).
Being treated by tail vein injection, dosage is 0.1mg/kg body weight, is administered volume and is
0.1mL/kg body weight, is administered once for every 4 days, is administered 5 times altogether, and blank group is not administered.After experiment terminates
Detect following index respectively: body weight growth pattern, organ index (thymus index, index and spleen index, heart
Index, liver index and renal index), peripheral white blood cell and bone marrow micronucleus number.
Experimental result is shown in that Fig. 4～Fig. 8, Fig. 4 are the embodiment of the present invention 43 small mouse body weight change figures, permissible
After seeing modeling, each group all there is weight loss phenomenon, modeling success is described, through Lentinan for Treatment
Rear each group body weight has substantially rising, best results be weight average molecular weight be the lentinan of 16200, i.e.
Group B.Table 8 is the data summarization that Fig. 4 small mouse body weight change curve is corresponding, by table 8 it can also be seen that
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 diagram, it can be seen that respectively organize spleen after modeling
Dirty index substantially reduces, and after Lentinan for Treatment, each group mouse spleen index has and to a certain degree rises,
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 diagram, it can be seen that respectively organize breast after modeling
Gland index substantially reduces, and after Lentinan for Treatment, each group mouse thymus index has and to a certain degree rises,
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 diagram, it can be seen that after modeling
Each group peripheral white blood cell substantially reduces, and after Lentinan for Treatment, each group murine interleukin number has
To a certain degree rise, best results be weight average molecular weight be the lentinan of 16200.
Fig. 8 is the embodiment of the present invention 43 small mouse bone marrow micronucleus number variation diagram, it can be seen that after modeling each group
Bone marrow micronucleus number is all increased significantly, under after Lentinan for Treatment, each group micronucleus number has to a certain degree
Fall, best results be weight average molecular weight be the lentinan of 16200.
Table 8 Fig. 4 small mouse body weight change curve corresponding data collects
From above-described embodiment, lentinan is extracted by the present invention by the complex method of removing protein,
Select chlorobenzene, calcium chloride, ammonium oxalate, trypsin, Sevage reagent etc. to carry out removing protein, have relatively
High productivity, and avoid the destruction using acid or alkali that lentinan is caused, the perfume (or spice) obtained after purification
Mushroom polysaccharide has higher purity, it is possible to obtained the lentinan of different number-average molecular weight by classification.And
And by immunosuppressant mice is studied, eventually found the optimum weight model improving immunity
Enclosing 162000, research further and exploitation for lentinan medical value are made that contribution.
The explanation of above example is only intended to help to understand method and the core concept thereof of the present invention.Should
Point out, for those skilled in the art, under the premise without departing from the principles of the invention,
The present invention can also be carried out some improvement and modification, these improve and modification also falls into right of the present invention and wants
In the protection domain asked.
Claims (8)
1. lentinan application in the medicine preparing enhancing immunity or food, described lentinan
Weight average molecular weight is 16200；
The extracting method of described lentinan includes:
A) be dried successively by Lentinus Edodes, pulverize, soak, water raises lentinan crude extract；
B) by step A) the lentinan crude extract that obtains is successively with chlorobenzene, calcium chloride, ammonium oxalate, pancreas
Protease, Sevage reagent carry out removing protein, then precipitate with ethanol, finally use acetone to carry out fractional precipitation,
Obtain described lentinan.
Application the most according to claim 1, it is characterised in that the granularity of described pulverizing is 50 mesh～300
Mesh.
Application the most according to claim 1, it is characterised in that the time of described immersion is 12h～72h；
The temperature that described water carries is 60 DEG C～98 DEG C.
Application the most according to claim 1, it is characterised in that the consumption of described calcium chloride is described
10wt%～40wt% of Lentinus Edodes；The consumption of described ammonium oxalate is 1wt%～5wt% of described Lentinus Edodes.
Application the most according to claim 1, it is characterised in that the alcohol that the method for described precipitate with ethanol uses
For ethanol.
Application the most according to claim 1, it is characterised in that described step B) in, successively
Carry out, in the middle of the step of removing protein, also including to lentinan crude extract with ammonium oxalate, trypsin: add
Sodium chloride.
Application the most according to claim 1, it is characterised in that described step B) in, with Sevage
After reagent carries out removing protein to lentinan crude extract, also include: add activated carbon.
Application the most according to claim 1, it is characterised in that described fractional precipitation particularly as follows:
A) lentinan that described precipitate with ethanol obtains is dissolved in distilled water, add acetone to occur precipitation,
Centrifugal obtain supernatant and precipitation, described in be precipitated as first order lentinan；
B) taking the supernatant obtained in step a), addition acetone, to there is precipitation, is centrifuged and obtains supernatant
And precipitation, described in be precipitated as second level lentinan；
C) repeat said process, reach 90wt% to acetone concentration.
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