CN106279458B - A kind of Lycium barbarum polysaccharide extract, extracting method and its preparing the application in blood lipid-lowering medicine - Google Patents
A kind of Lycium barbarum polysaccharide extract, extracting method and its preparing the application in blood lipid-lowering medicine Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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Abstract
The present invention relates to a kind of Lycium barbarum polysaccharide extract, extracting method and its application in blood lipid-lowering medicine is being prepared, is belonging to technical field of natural product extraction.Wherein purity of polysaccharide is 56~62%, and weight average molecular weight Mw range is 18000~32000.The problem that the active polysaccharide that can solve conventional wolfberry fruit extract is not high, purity is low, Lycium barbarum polysaccharide extract have preferable reducing blood lipid application effect.
Description
Technical field
The present invention relates to a kind of Lycium barbarum polysaccharide extract, extracting method and its application in blood lipid-lowering medicine is being prepared, is being belonged to
In technical field of natural product extraction.
Background technique
Fructus lycii is the traditional rare traditional Chinese medicine in China, is known as " red treasured " laudatory title, is rich in plant polyose, protein, vitamin
Etc. nutritional ingredients.The primary efficacy of fructus lycii is nourishing liver and kidney, benefiting shrewd head；It is lost for consumptive disease essence, soreness of waist and knee joint；Dizziness and tinnitus,
Nei Rexiao is thirsty, blood deficiency chlorosis, blurred vision.Polysaccharides is a kind of water-soluble polysaccharide obtained by extracting in fructus lycii.It has defined
The polysaccharide system proteoglycan, molecular weight is 22~25 kD, by arabinose, glucose, galactolipin, mannose, xylose, sandlwood
6 kinds of monosaccharide components compositions of sugar, detailed structural analysis are still in process.Research has shown that polysaccharides is that fructus lycii is adjusted
Main active that is immune, delaying senescence, can also play positive effect to malignant tumour, the prevention and treatment of AIDS.
In addition it can improve the symptoms such as the elderly's fatiguability, loss of appetite and eye-blurred, and have reducing blood lipid, anti-fatty liver, resist and swell
The effects of tumor, anti-aging.
CN104231097A discloses a kind of preparation method of plant polyose, comprising the following steps: (1) by fructus lycii screening and removing impurities
Heated-air drying dehydration is carried out afterwards, and three-level screening separates out pulp powder and fructus lycii seed after coarse granule powder crusher machine；(2) by pulp powder mistake
Charging carries out SCF-CO 2 after 20 meshes；(3) pure water heating extraction is added in extractor, temperature drops in the extraction later period
Pectase and cellulase heat preservation are added when to 45 DEG C, is centrifuged；(4) by the juice electrodialysis desalination after separation,
Then level Four hyperfiltration treatment, reverse osmosis concentration are carried out, then carries out drying bu sublimation, low-temperature grinding is finished product after being packaged
Polysaccharides.A kind of isolation and purification method of polysaccharides of CN104558229A, this method use novel temperature-sensitive block copolymer
Polyethylene oxide-polypropylene oxide-polyethylene oxide and inorganic salts form double-aqueous phase system, remove the albumen in polysaccharides
Matter, the small molecular weight impurities such as pigment.The inorganic salts in lower phase are removed using dialysis, ethanol precipitation obtains polysaccharide finished product.
But that there is recovery rates is not high for above-mentioned polysaccharide, the low problem of activity.
Summary of the invention
The purpose of the present invention is: the active polysaccharide for solving the problems, such as conventional wolfberry fruit extract is not high, purity is low, provides
A kind of new Lycium barbarum polysaccharide extract, also provide the extract extracting method and its preparing answering in blood lipid-lowering medicine
With.
Technical solution is:
A kind of Lycium barbarum polysaccharide extract, wherein purity of polysaccharide is 56~62%, weight average molecular weight Mw range is 18000~
32000。
The extracting method of above-mentioned Lycium barbarum polysaccharide extract, includes the following steps:
Step 1 takes 200~220 parts of fructus lyciis dried by weight, is added into 100~120 parts of petroleum ether, stirs
After mixing uniformly, 10~20min is stood, is filtered, solids is dried into 1~3h in being dried under reduced pressure case, obtains the first fructus lycii powder；
First fructus lycii powder is carried out under 20~40 DEG C of range HIGH PRESSURE TREATMENT, obtains the second fructus lycii powder by step 2；
Step 3, the second fructus lycii powder are added in distilled water, and control solid-liquid ratio is 1g/30~35ml, then is added in feed liquid
0.1~0.2% enzyme is digested and is extracted, extracting solution is collected after suction filtration；
Ethyl alcohol is added in extracting solution in step 4, and ethyl alcohol volumetric concentration is made to be greater than 80%, stands 2~3h, collects precipitating, then use
The mixed solution of ethyl alcohol and acetone washing precipitating, obtains crude extract；
Crude extract is dissolved in water by step 5, and feed liquid is concentrated with nanofiltration membrane, after obtained concentrate spray drying,
Obtain Lycium barbarum polysaccharide extract.
In the step 1, it is dried under reduced pressure 40~60 DEG C of temperature range.
In the step 2, high pressure range is 0.5~1Mpa of finger pressure.
In the step 3, digests and the temperature range extracted is 60~70 DEG C, the time is 2~4h.
In the step 3, the enzyme is selected from alkaline pectate lyase, alkali cellulose enzyme, alkalescent xylanase
With the mixture according to weight ratio 2:3:2:0.5 of laccase.
In the step 5, adding water weight is 20~35 times of crude extract weight, the molecular cut off of the nanofiltration membrane
Range is 500~800Da.
Application of the Lycium barbarum polysaccharide extract in preparation treatment high blood cholesterol drug.
Lycium barbarum polysaccharide extract total cholesterol, triglycerides or high density rouge in preparation degradation mammalian serum
Application in protein cholesterol drug.
Beneficial effect
The present invention provides a kind of new Lycium barbarum polysaccharide extracts, can solve the active polysaccharide of conventional wolfberry fruit extract
Problem not high, purity is low, Lycium barbarum polysaccharide extract have preferable reducing blood lipid application effect.
Specific embodiment
So-called percentage refers to mass percent in no special instruction in the present invention.
Embodiment 1
The extracting method of Lycium barbarum polysaccharide extract, includes the following steps:
Step 1 takes 200 parts of fructus lyciis dried by weight, is added into 100 parts of petroleum ether, after mixing evenly,
10min is stood, is filtered, solids is dried under reduced pressure to dry 1h in case at 40 DEG C, obtains the first fructus lycii powder；
First fructus lycii powder is carried out under 20 DEG C of range 0.5Mpa HIGH PRESSURE TREATMENT, obtains the second wolfberry fruit powder by step 2
End；
Step 3, the second fructus lycii powder are added in distilled water, and control solid-liquid ratio is 1g/30ml, then 0.1% is added in feed liquid
Enzyme, digested and extracted, the enzyme be selected from alkaline pectate lyase, alkali cellulose enzyme, alkalescent xylanase and
The mixture according to weight ratio 2:3:2:0.5 of laccase, digests and the temperature ranges of 60 DEG C of extractions are, the time is 2h, after suction filtration
Collect extracting solution；
Ethyl alcohol is added in extracting solution in step 4, makes ethyl alcohol volumetric concentration 80%, stands 2h, collects precipitating, then with ethyl alcohol and third
The mixed solution of ketone washs precipitating, obtains crude extract；
Crude extract is dissolved in water by step 5, and adding water weight is 20 times of crude extract weight, by feed liquid molecular cut off
500Da nanofiltration membrane is concentrated, and after obtained concentrate spray drying, obtains Lycium barbarum polysaccharide extract.
Lycium barbarum polysaccharide extract obtained above, wherein purity of polysaccharide is 58.2%, and weight average molecular weight Mw is 22125.
Embodiment 2
The extracting method of Lycium barbarum polysaccharide extract, includes the following steps:
Step 1 takes 220 parts of fructus lyciis dried by weight, is added into 120 parts of petroleum ether, after mixing evenly,
20min is stood, is filtered, solids is dried under reduced pressure to dry 3h in case at 60 DEG C, obtains the first fructus lycii powder；
First fructus lycii powder is carried out under 40 DEG C of range 1Mpa HIGH PRESSURE TREATMENT, obtains the second fructus lycii powder by step 2；
Step 3, the second fructus lycii powder are added in distilled water, and control solid-liquid ratio is 1g/35ml, then 0.2% is added in feed liquid
Enzyme, digested and extracted, the enzyme be selected from alkaline pectate lyase, alkali cellulose enzyme, alkalescent xylanase and
The mixture according to weight ratio 2:3:2:0.5 of laccase, digests and the temperature ranges of 70 DEG C of extractions are, the time is 4h, after suction filtration
Collect extracting solution；
Ethyl alcohol is added in extracting solution in step 4, makes ethyl alcohol volumetric concentration 80%, stands 3h, collects precipitating, then with ethyl alcohol and third
The mixed solution of ketone washs precipitating, obtains crude extract；
Crude extract is dissolved in water by step 5, and adding water weight is 35 times of crude extract weight, by feed liquid molecular cut off
800Da nanofiltration membrane is concentrated, and after obtained concentrate spray drying, obtains Lycium barbarum polysaccharide extract.
Lycium barbarum polysaccharide extract obtained above, wherein purity of polysaccharide is 61.2%, and weight average molecular weight Mw is 28479.
Embodiment 3
The extracting method of Lycium barbarum polysaccharide extract, includes the following steps:
Step 1 takes 210 parts of fructus lyciis dried by weight, is added into 110 parts of petroleum ether, after mixing evenly,
15min is stood, is filtered, solids is dried under reduced pressure to dry 2h in case at 45 DEG C, obtains the first fructus lycii powder；
First fructus lycii powder is carried out under 30 DEG C of range 0.8Mpa HIGH PRESSURE TREATMENT, obtains the second wolfberry fruit powder by step 2
End；
Step 3, the second fructus lycii powder are added in distilled water, and control solid-liquid ratio is 1g/32ml, then is added in feed liquid
0.15% enzyme is digested and is extracted, and the enzyme is selected from alkaline pectate lyase, alkali cellulose enzyme, alkaline xylan
The mixture according to weight ratio 2:3:2:0.5 of enzyme and laccase, digests and the temperature range of 65 DEG C of extractions is that the time is 3h, takes out
Extracting solution is collected after filter；
Ethyl alcohol is added in extracting solution in step 4, makes ethyl alcohol volumetric concentration 80%, stands 3h, collects precipitating, then with ethyl alcohol and third
The mixed solution of ketone washs precipitating, obtains crude extract；
Crude extract is dissolved in water by step 5, and adding water weight is 30 times of crude extract weight, by feed liquid molecular cut off
700Da nanofiltration membrane is concentrated, and after obtained concentrate spray drying, obtains Lycium barbarum polysaccharide extract.
Lycium barbarum polysaccharide extract obtained above, wherein purity of polysaccharide is 58.4%, and weight average molecular weight Mw is 29647.
Reference examples 1
Difference with embodiment 3 is: the petroleum ether extraction for not carrying out step 1 to fructus lycii operates.
The extracting method of Lycium barbarum polysaccharide extract, includes the following steps:
Step 1 takes 210 parts of fructus lyciis dried by weight, and dry 2h is dried under reduced pressure in case at 45 DEG C, obtains the
One fructus lycii powder；
First fructus lycii powder is carried out under 30 DEG C of range 0.8Mpa HIGH PRESSURE TREATMENT, obtains the second wolfberry fruit powder by step 2
End；
Step 3, the second fructus lycii powder are added in distilled water, and control solid-liquid ratio is 1g/32ml, then is added in feed liquid
0.15% enzyme is digested and is extracted, and the enzyme is selected from alkaline pectate lyase, alkali cellulose enzyme, alkaline xylan
The mixture according to weight ratio 2:3:2:0.5 of enzyme and laccase, digests and the temperature range of 65 DEG C of extractions is that the time is 3h, takes out
Extracting solution is collected after filter；
Ethyl alcohol is added in extracting solution in step 4, makes ethyl alcohol volumetric concentration 80%, stands 3h, collects precipitating, then with ethyl alcohol and third
The mixed solution of ketone washs precipitating, obtains crude extract；
Crude extract is dissolved in water by step 5, and adding water weight is 30 times of crude extract weight, by feed liquid molecular cut off
700Da nanofiltration membrane is concentrated, and after obtained concentrate spray drying, obtains Lycium barbarum polysaccharide extract.
Lycium barbarum polysaccharide extract obtained above, wherein purity of polysaccharide is 51.9%, and weight average molecular weight Mw is 45771.
Reference examples 2
Difference with embodiment 3 is: not being added in the enzymatic hydrolysis neutral and alkali transelminase of step 3, weight is by alkali
Property zytase is substituted.
The extracting method of Lycium barbarum polysaccharide extract, includes the following steps:
Step 1 takes 210 parts of fructus lyciis dried by weight, is added into 110 parts of petroleum ether, after mixing evenly,
15min is stood, is filtered, solids is dried under reduced pressure to dry 2h in case at 45 DEG C, obtains the first fructus lycii powder；
First fructus lycii powder is carried out under 30 DEG C of range 0.8Mpa HIGH PRESSURE TREATMENT, obtains the second wolfberry fruit powder by step 2
End；
Step 3, the second fructus lycii powder are added in distilled water, and control solid-liquid ratio is 1g/32ml, then is added in feed liquid
0.15% enzyme is digested and is extracted, and the enzyme is selected from alkali cellulose enzyme, alkalescent xylanase and laccase according to weight
The mixture than 2:3:4:0.5 is measured, digests and the temperature range of 65 DEG C of extractions is that the time is 3h, collect extracting solution after suction filtration；
Ethyl alcohol is added in extracting solution in step 4, makes ethyl alcohol volumetric concentration 80%, stands 3h, collects precipitating, then with ethyl alcohol and third
The mixed solution of ketone washs precipitating, obtains crude extract；
Crude extract is dissolved in water by step 5, and adding water weight is 30 times of crude extract weight, by feed liquid molecular cut off
700Da nanofiltration membrane is concentrated, and after obtained concentrate spray drying, obtains Lycium barbarum polysaccharide extract.
Lycium barbarum polysaccharide extract obtained above, wherein purity of polysaccharide is 51.5%, and weight average molecular weight Mw is 56227.
With high cholesterol and lipid forage feed animal and be aided with Fat Emulsion stomach-filling can fast and stable formation lipid metaboli it is disorderly
Random animal model gives the Lycium barbarum polysaccharide extract of animal above embodiments 1~3 of the present invention, reference examples 1~2, to detect fructus lycii
Influence of the polyoses extract to hyperlipemia.
Experimental material
Drug: the Lycium barbarum polysaccharide extract of Examples 1 to 3, reference examples 1~2.
Experimental animal
Germline: SD rat.
Rank: SPF grades.
Gender and quantity: 80, half male and half female.
The weight of animals: 50~80g.
Rearing conditions: the daily amount of drinking water of animal and food-intake in order to facilitate observation of, the raising of experimental animal single cage, room temperature 20~25
DEG C, relative humidity be 40~70%, lighting hours 12 hours.Feed is added at regular time and quantity, eats the grains dedicated feed of mouse, drink is certainly
Water replaces padding daily.
Other reagents and drug
Positive drug: Simvastatin Tablets.
High lipid food: 10% yolk powder, 10% lard, 1% cholesterol, 0.2% cholate and 78.8% basal feed.
Fat emulsion: 10% cholesterol, 20% lard, 2% sodium taurocholate, 1% methylthiouracil.
Laboratory apparatus: automatic clinical chemistry analyzer
Experimental method
The preparation of fat emulsion
Reference literature takes lard 20g to be placed in 500ml beaker, the heating and melting on electric furnace, and 10g cholesterol is added and dissolves,
2g sodium taurocholate and 1g methylthiouracil are added, is sufficiently stirred evenly；20ml Tween 80,20ml propylene glycol and 30ml is then placed in steam
Distilled water is stirred continuously.After methylthiouracil dissolution, be cooled to room temperature, then plus distilled water to 100ml, and mix well, i.e.,
At 10% cholesterol, 20% lard, 2% sodium taurocholate, 1% methylthiouracil fat emulsion.Refrigerator saves, when use prior to
Melt in 37 DEG C of water-baths.
Experimental group and modeling method
It SD rat 80, adapts to raising and weighs after a week, under experimental situation, normal group is given the feeding of normal rat feed.
Remaining each group rat feeds high lipid food, while pressing 1ml/100g dosage stomach-filling fat emulsion, fasting after continuous 15 days daily
16 hours, blood is taken, measurement serum total cholesterol (TC), triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C) are horizontal,
According to the successful experimental animal of serum total cholesterol (TC) horizontally selected modeling, be randomly divided into 8 groups: Normal group, model group,
Positive controls, Examples 1 to 3 group, 1~2 group of reference examples, every group each 10.Continuing to give high lipid food and fat emulsion
While, different tested materials are given respectively.After administration starts after fasting 16 hours the 15th day, blood sampling, survey serum TC, TG,
HDL-C is horizontal.
Observation index and detection method:
Ordinary circumstance observation
Every morning observes and records general performance, behavior, poisoning manifestations and the death condition of an animal.According to surveying weekly
Fixed weight and food intake dose adjusts groups of animals dosage according to calculated result, to guarantee groups of animals tested material needs
Amount.Group is drunk for Pu'er tea, according to the amount of drinking water mean value measured weekly, calculates Average administration amount weekly.When occurring abnormal, often
It is at least observed twice.To the animal close observation record being seriously poisoned.
Biochemical indicator detection
Serum total cholesterol (TC), triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C) are horizontal, full-automatic raw
Change analyzer detection.
Data statistics:
It is for statistical analysis that each group of data is all made of SPSS variance analysis, and heterogeneity of variance person uses data conversion, such as converts
It is still uneven afterwards then to use nonparametric statistics.
Extract is freely drunk and stomach-filling effect for reducing fat serum total cholesterol (TC), triglycerides (TG) and high density rouge
Protein cholesterol (HDL-C) testing result see the table below.
Influence (unit mmol/L) of the Lycium barbarum polysaccharide extract to hyperlipemia animal serum total cholesterol (TC)
| Group pharmaceutical quantities | Before administration | 15th day | 30th day | |
| Normal group | ---- | 2.58±0.68 | 2.51±0.38 | 2.26±0.25 |
| Model group | ---- | 23.58±1.51 | 23.11±1.52 | 23.36±1.58 |
| Positive drug group | 20.5mg/kg/ days | 22.20±1.58 | 18.22±1.32 | 14.52±1.14** |
| Example 1 group | 0.50g/kg/ days | 22.12±1.25 | 17.25±1.25* | 12.33±1.15* |
| 2 groups of embodiment | 0.50g/kg/ days | 22.25±1.82 | 18.25±1.62 | 14.52±1.18* |
| 3 groups of embodiment | 0.50g/kg/ days | 22.05±1.58 | 17.17±1.25* | 13.15±1.02* |
| 1 group of reference examples | 0.50g/kg/ days | 22.58±1.54 | 19.01±1.58*▲ | 16.59±1.42*▲ |
| 2 groups of reference examples | 0.50g/kg/ days | 22.58±1.61 | 21.28±1.58* | 15.92±1.57* |
* compared with model group, P < 0.05, * * is compared with model group, P < 0.01；▲ compared with 3 groups of embodiment, p < 0.05；
Influence (unit mmol/L) to hyperlipemia animal serum triglyceride (TG)
| Group pharmaceutical quantities | Before administration | 15th day | 30th day | |
| Normal group | ---- | 0.55±0.19 | 0.52±0.17 | 0.52±0.44 |
| Model group | ---- | 0.67±0.45 | 1.15±0.57 | 1.54±0.95 |
| Positive drug group | 20.5mg/kg/ days | 0.62±0.64 | 1.14±0.94* | 1.08±0.64** |
| Example 1 group | 0.50g/kg/ days | 0.63±0.63 | 1.17±0.51* | 1.11±0.52* |
| 2 groups of embodiment | 0.50g/kg/ days | 0.63±0.75 | 1.15±0.74* | 1.08±0.47* |
| 3 groups of embodiment | 0.50g/kg/ days | 0.64±0.25 | 1.14±0.51* | 1.11±0.42* |
| 1 group of reference examples | 0.50g/kg/ days | 0.65±0.56 | 1.45±0.63* | 1.65±0.67* |
| 2 groups of reference examples | 0.50g/kg/ days | 0.66±0.46 | 1.24±0.37*▲ | 1.54±0.85*▲ |
* compared with model group, P < 0.05, * * is compared with model group, P < 0.01；▲ compared with 3 groups of embodiment, p < 0.05；
Influence (unit mmol/L) to hyperlipemia animal serum High Density Lipoprotein Cholesterol (HDL-C)
| Group pharmaceutical quantities | Before administration | 15th day | 30th day | |
| Normal group | ---- | 0.63±0.29 | 0.68±0.35 | 0.63±0.62 |
| Model group | ---- | 3.63±0.52 | 3.22±0.75 | 5.02±1.58 |
| Positive drug group | 20.5mg/kg/ days | 3.33±0.47 | 2.33±0.64* | 2.33±1.25** |
| Example 1 group | 0.50g/kg/ days | 3.22±0.46 | 2.54±0.64* | 2.51±1.08* |
| 2 groups of embodiment | 0.50g/kg/ days | 3.23±0.59 | 2.59±0.74* | 2.42±1.18* |
| 3 groups of embodiment | 0.50g/kg/ days | 3.18±0.62 | 2.39±0.58* | 2.32±1.22* |
| 1 group of reference examples | 0.50g/kg/ days | 3.21±0.47 | 2.92±0.54* | 2.64±1.51* |
| 2 groups of reference examples | 0.50g/kg/ days | 3.11±0.75 | 2.68±0.81*▲ | 2.72±1.33*▲ |
* compared with model group, P < 0.05, * * is compared with model group, P < 0.01；▲ compared with 3 groups of embodiment, p < 0.05；
As can be seen from the above table, Lycium barbarum polysaccharide extract provided by the invention has preferable reduction serum total cholesterol
(TC), the effect of triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C), embodiment 3 for reference examples 1,
Impurity can be effectively removed by petroleum ether extraction operation, improve product purity, improve the Lowering cholesterol effect of drug；It is real
Example 3 is applied relative to reference examples 2, polysaccharide can be effectively facilitated by using alkaline pectate lyase and decomposed, it is sweet to improve reduction
The action activity of oily three esters.
Claims (5)
1. a kind of Lycium barbarum polysaccharide extract reduces the application in mammalian serum in triglycerides drug in preparation, feature exists
In the extracting method of the Lycium barbarum polysaccharide extract includes the following steps: that step 1 takes 200~220 parts by weight
The fructus lycii dried is added into 100~120 parts of petroleum ether, after mixing evenly, stands 10~20min, filters, by solids
Dry 1~3h, obtains the first fructus lycii powder in being dried under reduced pressure case；Step 2, the range by the first fructus lycii powder at 20~40 DEG C
Lower carry out HIGH PRESSURE TREATMENT, obtains the second fructus lycii powder；Step 3, the second fructus lycii powder are added in distilled water, and control solid-liquid ratio is
1g/30~35ml, then in feed liquid be added 0.1~0.2% enzyme, digested and extracted, extracting solution is collected after suction filtration；4th
Ethyl alcohol is added in extracting solution in step, and ethyl alcohol volumetric concentration is made to be greater than 80%, stands 2~3h, collects precipitating, then with ethyl alcohol and acetone
Mixed solution washing precipitating, obtains crude extract；Crude extract is dissolved in water by step 5, and feed liquid is concentrated with nanofiltration membrane, is obtained
After the concentrate spray drying arrived, Lycium barbarum polysaccharide extract is obtained；
In the step 3, the enzyme is alkaline pectate lyase, alkali cellulose enzyme, alkalescent xylanase and laccase
The mixture formed according to weight ratio 2:3:2:0.5.
2. application according to claim 1, which is characterized in that in the step 1, be dried under reduced pressure temperature range 40~60
℃。
3. application according to claim 1, which is characterized in that in the step 2, high pressure range be finger pressure 0.5~
1Mpa。
4. application according to claim 1, which is characterized in that in the step 3, digest and the temperature range extracted is
60~70 DEG C, the time is 2~4h.
5. application according to claim 1, which is characterized in that in the step 5, adding water weight is crude extract weight
20~35 times, the molecular cut off range of the nanofiltration membrane is 500~800Da.
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