CN109456418B - Active eurycoma longifolia polysaccharide and preparation method thereof - Google Patents
Active eurycoma longifolia polysaccharide and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an active eurycoma longifolia polysaccharide and a preparation method thereof, belonging to the technical field of extraction and separation of natural plant active polysaccharides. The method comprises the following steps: the Eurycoma longifolia skin branches are subjected to pretreatment, degreasing, polysaccharide extraction, polysaccharide purification and other steps to obtain Eurycoma longifolia polysaccharide with the molecular weight of more than 100KDa, and the polysaccharide can effectively promote the proliferation of probiotics in fermented milk. The extraction rate of the Eurycoma longifolia polysaccharide prepared by the method is high and can reach 4.52%, and compared with alcohol precipitation polysaccharide prepared by a traditional method and decolorized polysaccharide or deproteinized polysaccharide obtained after decolorization or deproteinization purification of the alcohol precipitation polysaccharide, the Eurycoma longifolia polysaccharide has a better effect of promoting proliferation of probiotics in fermented milk; compared with polysaccharide with molecular weight more than 10KDa, the probiotic proliferation promoting agent also has better probiotic proliferation promoting effect in fermented milk. Has wide development potential and wide market prospect.
Description
Technical Field
The invention relates to an active eurycoma longifolia polysaccharide and a preparation method thereof, belonging to the technical field of extraction and separation of natural plant active polysaccharides.
Background
Eurycoma longifolia (Eurycoma longifolia, and Eurycoma longifolia)Eurycoma longifolia) Is of Eurycoma of Simaroubaceae (Simaroubaceae) ((R))Eurycoma) Plants, wild shrubs, are broadly dividedCloth is distributed in southeast Asia countries, Vietnam, Indonesia and Malaysia, and is still in the stage of introduction and cultivation in China at present. Eurycoma longifolia is called as 'Malaysia ginseng', and the whole plant can be used as a medicine, wherein the root part is the medicinal main body of the medicine, and the medicine has obvious curative effect on diseases such as malaria, fever, male sexual dysfunction and the like. In addition, the extract has the effects of improving physical strength, relieving fatigue, sterilizing, resisting ulcer, resisting hypertension, resisting diabetes and the like. The research on the active ingredients of Eurycoma longifolia is mainly focused on compounds such as terpenoids and alkaloids, and the research on polysaccharide as an active ingredient which is widely recognized is very few at present, and the active ingredient is mostly separated from the root of Eurycoma longifolia, and the active ingredient is less involved in other parts.
The polysaccharide compound is one of the main active compounds at present, and researches show that the polysaccharide has the effects of protecting liver, reducing blood sugar, regulating immunity, resisting bacteria, resisting tumors, promoting urination, clearing heat and the like. According to the invention, the Eurycoma longifolia polysaccharide with the function of promoting the proliferation of probiotics is extracted and separated from Eurycoma longifolia serving as a raw material, the activity expression of the polysaccharide is determined, and meanwhile, the preparation method of the polysaccharide is established, so that a good foundation is laid for industrialization, and therefore, the Eurycoma longifolia polysaccharide has a good development prospect.
Disclosure of Invention
The invention provides an active Eurycoma longifolia polysaccharide and a preparation method thereof, wherein the molecular weight of the polysaccharide is larger than 100KDa, and the polysaccharide has an excellent effect of promoting proliferation of probiotics in fermented milk.
The technical scheme adopted by the invention for solving the technical problems is as follows: the invention takes Eurycoma longifolia as a raw material, extracts and separates an active Eurycoma longifolia polysaccharide, and also provides a preparation method thereof.
A preparation method of active Eurycoma longifolia polysaccharide comprises the following steps:
1. pretreatment of Eurycoma longifolia
Crushing branches with skins of Eurycoma longifolia into 60 meshes, and drying and storing in a shade;
2. defatting Eurycoma longifolia
Adding 95% ethanol into the Eurycoma longifolia powder according to the mass-volume ratio of 1:10-1:20, soaking for 24 hours, filtering, repeatedly soaking filter residues for 1 time by the same method, naturally drying the Eurycoma longifolia powder in a shade after soaking, and storing the Eurycoma longifolia powder in a dry environment for later use;
3. polysaccharide extraction
3.1 extracting degreased Eurycoma longifolia by adding distilled water according to the mass volume ratio of 1:10-1:30, and extracting for 1-3 hours in an extraction tank at 80-100 ℃;
3.2 centrifuging the extract at 5000 r/min for 20min, separating the supernatant and the precipitate (Eurycoma longifolia particles);
3.3 re-extracting for 1 time according to the method of the steps 3.1 and 3.2, and combining the supernatant fluid extracted for two times;
3.4 removing impurities from the supernatant, filtering by decompression paper to remove micro particles, and preparing polysaccharide extraction clear solution;
3.5 concentrating the polysaccharide clear solution to 1/8 of the original volume, then adding absolute ethyl alcohol until the final concentration of the ethyl alcohol in the solution is 80%, precipitating at 4 ℃ for 24h, centrifuging at 5000 r/min for 15min, collecting the precipitate, washing the precipitate with 95% ethyl alcohol for 1 time, and drying to obtain the Eurycoma longifolia alcohol precipitated polysaccharide;
4. polysaccharide purification
4.1 adding distilled water into the ethanol precipitated polysaccharide of the eurycoma longifolia subjected to ultrafiltration to prepare a solution of 10-35mg/mL, and performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 100 KDa; adopting an optimized ultrafiltration process: the ultrafiltration pressure is 0.8-1.5 Mpa, when the volume of the polysaccharide solution is ultrafiltered and concentrated to 1/2 times of the volume of the original solution, 1/2 times of distilled water of the volume of the original solution is supplemented into the concentrated solution, ultrafiltration is continued until the volume of the concentrated solution is recovered to the volume before water supplement, water supplement is repeated in the same way until the total water supplement volume is 15-40 times of the volume of the original solution, and water is not supplemented any more;
4.2 after further concentration, freeze drying or spray drying, the Eurycoma longifolia polysaccharide with molecular weight more than 100KDa is obtained.
Preferably, the preparation method of the active eurycoma longifolia polysaccharide comprises the following steps:
1. pretreatment of Eurycoma longifolia
The branches with the skin of the Eurycoma longifolia Jack are crushed into 60 meshes, and dried and stored in a shade place.
2. Defatting Eurycoma longifolia
Adding 95% ethanol into the Eurycoma longifolia powder according to the mass-volume ratio of 1:10, soaking for 24 hours, filtering after soaking, repeatedly soaking filter residues for 1 time by the same method, naturally drying the Eurycoma longifolia powder in a shade after soaking, and storing the Eurycoma longifolia powder in a dry environment for later use.
3. Polysaccharide extraction
3.1 extracting degreased Eurycoma longifolia, adding distilled water according to the mass volume ratio of 1:20, and extracting for 2 hours in an extraction tank at 100 ℃;
3.2 centrifuging the extract at 5000 r/min for 20min, separating the supernatant and the precipitate (Eurycoma longifolia particles);
3.3 re-extracting the precipitate for 1 time by the same method (according to the 3.1 and 3.2 steps), and combining the supernatants obtained by the two extractions;
3.4 removing impurities from the supernatant, filtering by decompression paper to remove micro particles, and preparing polysaccharide extraction clear solution;
3.5 concentrating the polysaccharide clear solution to 1/8 of the original volume, then adding absolute ethyl alcohol until the final concentration of the ethyl alcohol in the solution is 80%, precipitating for 24h at 4 ℃, centrifuging for 15min at 5000 r/min, collecting the precipitate, washing the precipitate for 1 time by using 95% ethyl alcohol, and drying to obtain the Eurycoma longifolia alcohol precipitated polysaccharide.
4. Polysaccharide purification
4.1 Ultrafiltration of Eurycoma longifolia precipitated polysaccharide adding distilled water to prepare 25mg/mL solution, and ultrafiltration with ultrafiltration membrane with molecular weight cutoff of 100 KDa. Adopting an optimized ultrafiltration process: the ultrafiltration pressure is 1.2 Mpa, when the volume of the polysaccharide solution is ultrafiltered and concentrated to 1/2 times of the volume of the original solution, 1/2 times of distilled water of the volume of the original solution is supplemented into the concentrated solution, ultrafiltration is continued until the volume of the concentrated solution is recovered to the volume before water supplement, water supplement is repeated in the same way until the total water supplement volume is 20 times of the volume of the original solution, and water supplement is not performed.
4.2 after further concentration, freeze drying or spray drying, the Eurycoma longifolia polysaccharide with molecular weight more than 100KDa is obtained.
The separation and purification of the polysaccharide are mainly carried out by adopting an ultrafiltration method. The ultrafiltration method is implemented by separating small molecular substances with molecular weight smaller than that of ultrafiltration membrane from extractive solution with permeate liquid by means of pressure and concentration differenceThe macromolecular substances with the molecular weight cut off by the ultrafiltration membrane are completely retained. The main factors influencing the ultrafiltration method include the cut-off molecular weight of the ultrafiltration membrane, the ultrafiltration pressure, the ultrafiltration water supplementing mode, the water supplementing amount and the like, and the factors finally determine the main properties of the polysaccharide, such as the molecular weight, the polysaccharide content, the activity and the like. The parameters of the technology are the parameters of the preparation method of the Eurycoma longifolia polysaccharide which can ensure higher extraction rate and has better biological activity by comparing the extraction rate and the biological activity through experimental research. The extraction rate can reach 4.52%, and the total amount of lactobacillus can reach 7.58 × 10 by adding 1% of the polysaccharide in the preparation of fermented milk9CFU/mL, much higher than the negative control without added sugar (8.13X 10)7CFU/mL) and sucrose addition control (1.70X 10)8CFU/mL）。
The invention has the beneficial effects that: the invention provides a Eurycoma longifolia polysaccharide with a molecular weight of more than 100KDa, which can effectively promote the proliferation of probiotics in fermented milk. The extraction rate of the Eurycoma longifolia polysaccharide prepared by the method is high and can reach 4.52%, and compared with alcohol precipitation polysaccharide prepared by a traditional method and decolorized polysaccharide or deproteinized polysaccharide obtained after decolorization or deproteinization purification of the alcohol precipitation polysaccharide, the Eurycoma longifolia polysaccharide has a better effect of promoting proliferation of probiotics in fermented milk; compared with polysaccharide with molecular weight more than 10KDa, the probiotic proliferation promoting agent also has better probiotic proliferation promoting effect in fermented milk. Therefore, the polysaccharide is a good beneficial bacteria proliferation promoter, can be used as a bioactive substance to be added into a fermented dairy product or be independently developed into a health food for regulating the intestinal flora function, and has wide development potential and wide market prospect.
Drawings
FIG. 1 shows the effect of different polysaccharides on promoting the proliferation of probiotics in fermented milk.
Detailed Description
The invention will now be further described with reference to the accompanying drawings and specific embodiments, which are included to provide a better understanding and understanding of the invention, and are not intended to limit the invention.
Example 1
A preparation method of active Eurycoma longifolia polysaccharide comprises the following steps:
1. pretreatment of Eurycoma longifolia
The branches with the skin of the Eurycoma longifolia Jack are crushed into 60 meshes, and dried and stored in a shade place.
2. Defatting Eurycoma longifolia
Adding 95% ethanol into the Eurycoma longifolia powder according to the mass-volume ratio of 1:10, soaking for 24 hours, filtering after soaking, repeatedly soaking filter residues for 1 time by the same method, naturally drying the Eurycoma longifolia powder in a shade after soaking, and storing the Eurycoma longifolia powder in a dry environment for later use.
3. Polysaccharide extraction
3.1 extracting degreased Eurycoma longifolia, adding distilled water according to the mass volume ratio of 1:20, and extracting for 2 hours in an extraction tank at 100 ℃;
3.2 centrifuging the extract at 5000 r/min for 20min, separating the supernatant and the precipitate (Eurycoma longifolia particles);
3.3 extracting the precipitate again for 1 time by the same method, and combining the supernatants obtained by the two extractions;
3.4 removing impurities from the supernatant, filtering by decompression paper to remove micro particles, and preparing polysaccharide extraction clear solution;
3.5 concentrating the polysaccharide clear solution to 1/8 of the original volume, then adding absolute ethyl alcohol until the final concentration of the ethyl alcohol in the solution is 80%, precipitating for 24h at 4 ℃, centrifuging for 15min at 5000 r/min, collecting the precipitate, washing the precipitate for 1 time by using 95% ethyl alcohol, and drying to obtain the Eurycoma longifolia alcohol precipitated polysaccharide.
4. Polysaccharide purification
4.1 Ultrafiltration of Eurycoma longifolia precipitated polysaccharide adding distilled water to prepare 25mg/mL solution, and ultrafiltration with ultrafiltration membrane with molecular weight cutoff of 100 KDa. Adopting an optimized ultrafiltration process: the ultrafiltration pressure is 1.2 Mpa, when the volume of the polysaccharide solution is ultrafiltered and concentrated to 1/2 times of the volume of the original solution, 1/2 times of distilled water of the volume of the original solution is supplemented into the concentrated solution, ultrafiltration is continued until the volume of the concentrated solution is recovered to the volume before water supplement, water supplement is repeated in the same way until the total water supplement volume is 20 times of the volume of the original solution, and water supplement is not performed.
4.2 after further concentration, freeze drying or spray drying, the Eurycoma longifolia polysaccharide with molecular weight more than 100KDa is obtained. The extraction rate of the polysaccharide is 4.52%, and the total sugar content in the polysaccharide is 48.6% (total sugar is determined by adopting a phenol-sulfuric acid method).
Example 2
A preparation method of active Eurycoma longifolia polysaccharide comprises the following steps:
1. pretreatment of Eurycoma longifolia
The branches with the skin of the Eurycoma longifolia Jack are crushed into 60 meshes, and dried and stored in a shade place.
2. Defatting Eurycoma longifolia
Adding 95% ethanol into the Eurycoma longifolia powder according to the mass-volume ratio of 1:20, soaking for 24 hours, filtering after soaking, repeatedly soaking filter residues for 1 time by the same method, naturally drying the Eurycoma longifolia powder in a shade after soaking, and storing the Eurycoma longifolia powder in a dry environment for later use.
3. Polysaccharide extraction
3.1 extracting degreased Eurycoma longifolia with distilled water according to the mass volume ratio of 1:30, and extracting for 3 hours in an extraction tank at 95 ℃;
3.2 centrifuging the extract at 5000 r/min for 20min, separating the supernatant and the precipitate (Eurycoma longifolia particles);
3.3 extracting the precipitate again for 1 time by the same method, and combining the supernatants obtained by the two extractions;
3.4 removing impurities from the supernatant, filtering by decompression paper to remove micro particles, and preparing polysaccharide extraction clear solution;
3.5 concentrating the polysaccharide clear solution to 1/8 of the original volume, then adding absolute ethyl alcohol until the final concentration of the ethyl alcohol in the solution is 80%, precipitating for 24h at 4 ℃, centrifuging for 15min at 5000 r/min, collecting the precipitate, washing the precipitate for 1 time by using 95% ethyl alcohol, and drying to obtain the Eurycoma longifolia alcohol precipitated polysaccharide.
4. Polysaccharide purification
4.1 Ultrafiltration of Eurycoma longifolia precipitated polysaccharide adding distilled water to prepare 10mg/mL solution, and ultrafiltration with ultrafiltration membrane with molecular weight cutoff of 100 KDa. Adopting an optimized ultrafiltration process: the ultrafiltration pressure is 1.5 Mpa, when the volume of the polysaccharide solution is ultrafiltered and concentrated to 1/2 times of the volume of the original solution, 1/2 times of distilled water of the volume of the original solution is supplemented into the concentrated solution, ultrafiltration is continued until the volume of the concentrated solution is recovered to the volume before water supplement, water supplement is repeated in the same way until the total water supplement volume is 40 times of the volume of the original solution, and water supplement is not performed.
4.2 after further concentration, freeze drying or spray drying, the Eurycoma longifolia polysaccharide with molecular weight more than 100KDa is obtained. The extraction rate of the polysaccharide is 4.26%, and the total sugar content in the polysaccharide is 50.3% (total sugar is determined by adopting a phenol-sulfuric acid method).
Example 3
A preparation method of active Eurycoma longifolia polysaccharide comprises the following steps:
1. pretreatment of Eurycoma longifolia
The branches with the skin of the Eurycoma longifolia Jack are crushed into 60 meshes, and dried and stored in a shade place.
2. Defatting Eurycoma longifolia
Adding 95% ethanol into the Eurycoma longifolia powder according to the mass-volume ratio of 1:15, soaking for 24 hours, filtering after soaking, repeatedly soaking filter residues for 1 time by the same method, naturally drying the Eurycoma longifolia powder in a shade after soaking, and storing the Eurycoma longifolia powder in a dry environment for later use.
3. Polysaccharide extraction
3.1 extracting degreased Eurycoma longifolia, adding distilled water according to the mass volume ratio of 1:10, and extracting for 1 hour in an extraction tank at the temperature of 80 ℃;
3.2 centrifuging the extract at 5000 r/min for 20min, separating the supernatant and the precipitate (Eurycoma longifolia particles);
3.3 extracting the precipitate again for 1 time by the same method, and combining the supernatants obtained by the two extractions;
3.4 removing impurities from the supernatant, filtering by decompression paper to remove micro particles, and preparing polysaccharide extraction clear solution;
3.5 concentrating the polysaccharide clear solution to 1/8 of the original volume, then adding absolute ethyl alcohol until the final concentration of the ethyl alcohol in the solution is 80%, precipitating for 24h at 4 ℃, centrifuging for 15min at 5000 r/min, collecting the precipitate, washing the precipitate for 1 time by using 95% ethyl alcohol, and drying to obtain the Eurycoma longifolia alcohol precipitated polysaccharide.
4. Polysaccharide purification
4.1 Ultrafiltration of Eurycoma longifolia precipitated polysaccharide adding distilled water to prepare 35mg/mL solution, and ultrafiltration with ultrafiltration membrane with molecular weight cut-off of 100 KDa. Adopting an optimized ultrafiltration process: and (3) ultrafiltration pressure is 0.8 Mpa, when the volume of the polysaccharide solution is ultrafiltered and concentrated to 1/2 times of the volume of the original solution, 1/2 times of distilled water in the volume of the original solution is supplemented into the concentrated solution, ultrafiltration is continued until the volume of the concentrated solution is recovered to the volume before water supplement, and water supplement is repeated in the same way until the total water supplement volume is 15 times of the volume of the original solution and water supplement is not performed.
4.2 after further concentration, freeze drying or spray drying, the Eurycoma longifolia polysaccharide with molecular weight more than 100KDa is obtained. The extraction rate of the polysaccharide is 3.89%, and the total sugar content in the polysaccharide is 46.9% (total sugar is determined by adopting a phenol-sulfuric acid method).
Example 4
Promoting intestinal probiotic proliferation
1 method of experiment
1.1 preparation of fermented milk
Strictly according to aseptic operating specifications
1. Raw milk (commercial sterile milk, not fat milk solid ≧ 8.5%), is preheated to about 40 deg.C in water bath.
2. The package is opened in a sterile operation. Packaging with plastic or paper box (bag), sterilizing the box cover or bag opening with 75% alcohol cotton ball, cutting with sterilizing scissors, and slowly adding the raw milk into a sterile beaker.
3. Pure sucrose or experimental samples (addition amount of 1%) are added, and the mixture is stirred by a sterile glass rod and dissolved uniformly.
4. Adding probiotic bacteria (mixed strains of Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium lactis, and Lactobacillus acidophilus, with total viable count of 1 × 106CFU/mL) was added at 16.7%.
5. Filling, subpackaging in aseptic beakers, sealing and binding.
6. Putting the mixture into a constant-temperature incubator to be cultured for 8 hours at the culture temperature of 45 ℃.
1.2 lactic acid bacteria count
According to the estimation of the total number of viable bacteria of a sample to be detected, 2-3 continuous proper dilutions are selected, each dilution uses a 1mL pipette to suck 0.1mL sample uniform solution to be respectively placed on 3 MRS agar plates, and a coating rod is used for surface coating. Counting all colonies on the plate after anaerobic culture for 48 h +/-2 h at 36 +/-1 ℃. Dilution from sample to plate coating requires completion within 15 min.
1.3 presentation of the results
The total number of colonies was counted by selecting plates with a colony count of 30 CFU to 300 CFU. If only one dilution is within the appropriate count range, the total number of colonies can be averaged and multiplied by the corresponding dilution factor. If both dilutions are in this range, then the formula is calculated:
in the formula: n-number of colonies in the sample;
ΣC-the sum of the colony counts of the plates (plates containing the appropriate range of colony counts);
n1the number of plates at the first dilution (low dilution factor);
n2-number of plates at second dilution (high dilution factor);
d-dilution factor (first dilution).
2 results of the experiment
As can be seen from FIG. 1, the total number of lactic acid bacteria in the fermented milk prepared by adding the polysaccharide of the present invention was 7.58X 109CFU/mL (logarithm is 9.88), adding polysaccharide with molecular weight more than 10kDa, and the total number of lactobacillus reaches 3.09 × 109CFU/mL (logarithm is 9.49), and the effect of the CFU/mL is obviously superior to that of the alcohol precipitated polysaccharide (5.13 multiplied by 10) prepared by the traditional method in the aspect of promoting the proliferation effect of probiotics in the fermented milk8CFU/mL, logarithm 8.71), is also obviously superior to the decolourized polysaccharide (5.75 multiplied by 10) obtained by decolourizing and deproteinizing the alcohol precipitated polysaccharide8CFU/mL, log 8.76) and deproteinized polysaccharide (7.24X 10)8CFU/mL, log 8.86). Compared with the polysaccharide with the molecular weight of more than 10kDa, the polysaccharide of the invention also has better probiotic proliferation promoting effect in fermented milk, and has obvious difference. Showing the parameters of the technology established by our experimental study,can ensure to obtain the Eurycoma longifolia polysaccharide which has higher extraction rate and excellent biological activity.
Claims (2)
1. A preparation method of active Eurycoma longifolia polysaccharide is characterized by comprising the following steps: the method comprises the following steps:
the first step is as follows: pretreatment of Eurycoma longifolia
Crushing branches with skins of Eurycoma longifolia into 60 meshes, and drying and storing in a shade;
the second step is that: defatting Eurycoma longifolia
Adding 95% ethanol into the Eurycoma longifolia powder according to the mass-volume ratio of 1:10-1:20, soaking for 24 hours, filtering, repeatedly soaking filter residues for 1 time by the same method, naturally drying the Eurycoma longifolia powder in a shade after soaking, and storing the Eurycoma longifolia powder in a dry environment for later use;
the third step: polysaccharide extraction
3.1 extracting degreased Eurycoma longifolia by adding distilled water according to the mass volume ratio of 1:10-1:30, and extracting for 1-3 hours in an extraction tank at 80-100 ℃;
3.2 centrifuging the extract at 5000 r/min for 20min, separating the supernatant and the precipitate;
3.3 re-extracting for 1 time according to the method of the steps 3.1 and 3.2, and combining the supernatant fluid extracted for two times;
3.4 removing impurities from the supernatant, filtering under reduced pressure to remove micro particles, and preparing polysaccharide extract;
3.5 concentrating the polysaccharide clear solution to 1/8 of the original volume, then adding absolute ethyl alcohol until the final concentration of the ethyl alcohol in the solution is 80%, precipitating at 4 ℃ for 24h, centrifuging at 5000 r/min for 15min, collecting the precipitate, washing the precipitate with 95% ethyl alcohol for 1 time, and drying to obtain the Eurycoma longifolia alcohol precipitated polysaccharide;
the fourth step: polysaccharide purification
4.1 adding distilled water into the ethanol precipitated polysaccharide of the eurycoma longifolia subjected to ultrafiltration to prepare a solution of 10-35mg/mL, and performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 100 KDa; adopting an optimized ultrafiltration process: the ultrafiltration pressure is 0.8-1.5 Mpa, when the volume of the polysaccharide solution is ultrafiltered and concentrated to 1/2 times of the volume of the original solution, 1/2 times of distilled water of the volume of the original solution is supplemented into the concentrated solution, ultrafiltration is continued until the volume of the concentrated solution is recovered to the volume before water supplement, water supplement is repeated in the same way until the total water supplement volume is 15-40 times of the volume of the original solution, and water is not supplemented any more;
4.2 after further concentration, freeze drying or spray drying, the Eurycoma longifolia polysaccharide with molecular weight more than 100KDa is obtained.
2. A preparation method of active Eurycoma longifolia polysaccharide is characterized by comprising the following steps: the method comprises the following steps:
the first step is as follows: pretreatment of Eurycoma longifolia
Crushing branches with skins of Eurycoma longifolia into 60 meshes, and drying and storing in a shade;
the second step is that: defatting Eurycoma longifolia
Adding 95% ethanol into the Eurycoma longifolia powder according to the mass-volume ratio of 1:10, soaking for 24 hours, filtering after soaking, repeatedly soaking filter residues for 1 time by the same method, naturally drying the Eurycoma longifolia powder in a shade after soaking, and storing the Eurycoma longifolia powder in a dry environment for later use;
the third step: polysaccharide extraction
3.1 extracting degreased Eurycoma longifolia, adding distilled water according to the mass volume ratio of 1:20, and extracting for 2 hours in an extraction tank at 100 ℃;
3.2 centrifuging the extract at 5000 r/min for 20min, separating the supernatant and the precipitate;
3.3 re-extracting for 1 time according to the method of the steps 3.1 and 3.2, and combining the supernatant fluid extracted for two times;
3.4 removing impurities from the supernatant, filtering under reduced pressure to remove micro particles, and preparing polysaccharide extract;
3.5 concentrating the polysaccharide clear solution to 1/8 of the original volume, then adding absolute ethyl alcohol until the final concentration of the ethyl alcohol in the solution is 80%, precipitating at 4 ℃ for 24h, centrifuging at 5000 r/min for 15min, collecting the precipitate, washing the precipitate with 95% ethyl alcohol for 1 time, and drying to obtain the Eurycoma longifolia alcohol precipitated polysaccharide;
the fourth step: polysaccharide purification
4.1 adding distilled water into the ethanol precipitated polysaccharide of the eurycoma longifolia subjected to ultrafiltration to prepare a solution of 25mg/mL, and performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 100 KDa; adopting an optimized ultrafiltration process: the ultrafiltration pressure is 1.2 Mpa, when the volume of the polysaccharide solution is ultrafiltered and concentrated to 1/2 times of the volume of the original solution, 1/2 times of distilled water of the volume of the original solution is supplemented into the concentrated solution, ultrafiltration is continued until the volume of the concentrated solution is recovered to the volume before water supplement, water supplement is repeated in the same way until the total water supplement volume is 20 times of the volume of the original solution, and water supplement is not performed;
4.2 after further concentration, freeze drying or spray drying, the Eurycoma longifolia polysaccharide with molecular weight more than 100KDa is obtained.
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