CN114133461A - Extraction method and application of gastrodia elata oligosaccharide GEP-2 - Google Patents
Extraction method and application of gastrodia elata oligosaccharide GEP-2 Download PDFInfo
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
The invention belongs to the technical field of medicines and biology, and particularly relates to an extraction method and application of gastrodia elata oligosaccharide GEP-2. The invention provides a method for extracting gastrodia elata oligosaccharide GEP-2. The gastrodia elata oligosaccharide GEP-2 provided by the invention has a network-like structure to prepare a dissolving-assisting platform, so that the dissolving state of an extract can be obviously improved. Meanwhile, the gastrodia elata oligosaccharide GEP-2 has excellent Nerve Growth Factor (NGF) similar activity, can induce nerve cell differentiation and promote the differentiation of pheochromocytoma (PC 12). Experiments prove that the oligosaccharide GEP-2 of gastrodia elata provided by the invention can effectively improve the differentiation rate of nerve cells.
Description
Technical Field
The invention belongs to the technical field of medicines and biology, and particularly relates to an extraction method and application of gastrodia elata oligosaccharide GEP-2.
Background
The Chinese medicine Gastrodia elata (Gastrodia elata Bl.) is a dry tuber of Gastrodia elata (Gastrodia R.Br.) Bl. of Gastrodia of Orhidaceae, and has the functions of calming wind and relieving spasm, calming liver yang, dispelling wind and dredging collaterals. The traditional Chinese medicine gastrodia elata is mainly used for treating liver wind stirring, fright epilepsy and convulsion, dizziness, headache, limb numbness, paralysis of hands and feet, rheumatism and arthralgia and the like; is a traditional and rare traditional Chinese medicine in China. The research on the chemical components of gastrodia elata goes back to the 19 th century. At present, the main chemical components of gastrodia are phenolic components and derivatives thereof, polysaccharides, polypeptides and the like, and the total number of the chemical components is more than 100 according to literature reports. The above components show various biological activities in research, such as tranquilizing, reducing resistance of peripheral blood vessel, cerebral blood vessel and coronary blood vessel, lowering blood pressure, slowing down heart rate, relieving pain and resisting inflammation, and rhizoma Gastrodiae polysaccharide shows immunological activity in research.
Gastrodia elata is rich in polysaccharide, the skeleton type of the polysaccharide of the gastrodia elata reported at present is mainly alpha- (1-4) -pyran-type-D-glucose, and the molecular weight of the polysaccharide of the gastrodia elata is found to be 1.54 multiplied by 10 by previous research3kDa or 28.8kDa, has a defect of large molecular weight which is difficult to utilize, and has a molecular weight of 1.54X 103kDa polysaccharide is a starch component, and an aqueous solution is easily suspended and is difficult to dissolve in water for use. Therefore, there is a need in the art for a gastrodia elata extract that is easy to use.
Disclosure of Invention
In order to solve the problems, the invention provides an extraction method and application of gastrodia elata oligosaccharide GEP-2. The gastrodia elata oligosaccharide GEP-2 provided by the invention has a net-like structure, and can remarkably improve the dissolution state of an extract. Meanwhile, further pharmacological activity research shows that the gastrodia elata oligosaccharide also has activity similar to Nerve Growth Factor (NGF), can induce nerve cell differentiation and promote differentiation of pheochromocytoma (PC12), and has important significance for treating neurodegenerative diseases.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a method for extracting gastrodia elata oligosaccharide GEP-2, which comprises the following steps:
carrying out water extraction, filtration and concentration on the degreased gastrodia elata to obtain a crude concentrated extract;
carrying out primary alcohol extraction on the crude extract concentrate to obtain a crude polysaccharide mixture A; the ethanol concentration in the first ethanol extraction system is 35 percent;
standing, filtering and concentrating the crude polysaccharide mixture A in sequence to obtain a crude polysaccharide extract concentrated solution;
carrying out secondary alcohol extraction on the crude polysaccharide extract concentrated solution to obtain a crude polysaccharide extract B; the concentration of ethanol in the system of the second ethanol extraction is 90 percent;
standing and centrifuging the crude polysaccharide extract B to obtain crude polysaccharide extract precipitate;
redissolving the crude polysaccharide extract precipitate to obtain a complex solution;
removing protein in the complex solution to obtain gastrodia elata oligosaccharide GEP-2.
Preferably, the method for redissolving the crude polysaccharide extract precipitate comprises: mixing the crude polysaccharide extract precipitate with water; the temperature of the water is 90-100 ℃.
Preferably, the method for removing the double solution comprises a Sevage method; the frequency of removing the protein in the complex solution is more than or equal to 3.
The invention also provides the gastrodia elata oligosaccharide GEP-2 extracted by the extraction method, wherein the molecular weight of the gastrodia elata oligosaccharide GEP-2 is 4.0-10.0 KDa.
The invention also provides the gastrodia elata oligosaccharide GEP-2 extracted by the extraction method or the application of the gastrodia elata oligosaccharide GEP-2 in extracting a medicine for protecting a nervous system.
The invention also provides the gastrodia elata oligosaccharide GEP-2 extracted by the extraction method or the application of the gastrodia elata oligosaccharide GEP-2 in extracting nervous system protection food.
The invention also provides the gastrodia elata oligosaccharide GEP-2 extracted by the extraction method or the application of the gastrodia elata oligosaccharide GEP-2 in extracting biological agents for protecting nervous systems.
Preferably, the content of the gastrodia elata oligosaccharide GEP-2 in the medicine, food or biological agent is more than or equal to 50 percent in percentage by mass.
The invention provides a method for extracting gastrodia elata oligosaccharide GEP-2. The gastrodia elata oligosaccharide GEP-2 extracted by the extraction method provided by the invention is used for preparing a dissolving-assisting platform through a net-like structure, so that the dissolving state of the extract can be obviously improved. Meanwhile, the gastrodia elata oligosaccharide GEP-2 has excellent NGF-like activity, can induce substances of nerve cell differentiation and promote the differentiation of pheochromocytoma (PC 12). The embodiment proves that the gastrodia elata oligosaccharide GEP-2 provided by the invention can effectively improve the differentiation rate of nerve cells.
Drawings
FIG. 1 is a flow chart of the extraction of the oligosaccharide GEP-2 and GEP-1 from Gastrodia elata of the present invention;
FIG. 2 is an HPLC chromatogram of GEP-1;
FIG. 3 is an HPLC chromatogram of GEP-2;
FIG. 4 is a graph showing the difference in solubility between GEP-1 and GEP-2;
FIG. 5 is an electron micrograph of GEP-1;
FIG. 6 is an electron micrograph of GEP-2;
FIG. 7 is a Blank control group (Blank) in the neuroprotective activity experiment;
FIG. 8 shows a Negative control group (Negative) in the neuroprotective activity experiment;
FIG. 9 is a Positive control group (Positive) in the neuroprotective activity experiment;
FIG. 10 shows Compound B (GEP-1) in the neuroprotective Activity assay;
FIG. 11 shows Compound A (GEP-2) in the neuroprotective activity assay.
Detailed Description
The invention provides a method for extracting gastrodia elata oligosaccharide GEP-2, which comprises the following steps:
carrying out water extraction, filtration and concentration on the degreased gastrodia elata to obtain a crude concentrated extract;
carrying out primary alcohol extraction on the crude extract concentrate to obtain a crude polysaccharide mixture A; the ethanol concentration in the first ethanol extraction system is 35 percent;
standing, filtering and concentrating the crude polysaccharide mixture A in sequence to obtain a crude polysaccharide extract concentrated solution;
carrying out secondary alcohol extraction on the crude polysaccharide extract concentrated solution to obtain a crude polysaccharide extract B; the concentration of ethanol in the system of the second ethanol extraction is 90 percent;
standing and centrifuging the crude polysaccharide extract B to obtain crude polysaccharide extract precipitate;
redissolving the crude polysaccharide extract precipitate to obtain a complex solution;
and removing protein in the complex solution to obtain the gastrodia elata oligosaccharide.
The method comprises the steps of crushing fresh or dried gastrodia elata, then carrying out alcohol extraction treatment to obtain filter residues, and placing the filter residues to obtain the degreased gastrodia elata. In the present invention, the alcohol extraction method preferably comprises: leaching the crushed gastrodia elata by using an ethanol water solution to obtain filter residue; the mass percentage content of the ethanol in the ethanol water solution is preferably 90-95%, and more preferably 95%; the leaching frequency is preferably 1-3 times, and more preferably 3 times.
After the defatted gastrodia elata is obtained, the defatted gastrodia elata is subjected to water extraction, filtration and concentration to obtain a crude concentrated extract. In the invention, the water extraction method of the defatted gastrodia elata preferably comprises boiling water extraction; the boiling water extraction method preferably comprises: mixing the degreased gastrodia elata with water and then boiling; the water is preferably distilled water; the boiling time is preferably 1.5-3 h, and more preferably 2 h; the number of times of extraction with boiling water is preferably 1-3 times, and more preferably 2 times; filtering and concentrating after the boiling treatment is finished; the temperature of the concentration is preferably 50 to 80 ℃, and more preferably 65 ℃.
After the crude concentrated extract is obtained, the crude concentrated solution is subjected to primary alcohol extraction to obtain a crude polysaccharide mixture A; when the first alcohol extraction is carried out, the final volume percentage of the ethanol in the extraction system of the first alcohol extraction is 35%. In the present invention, the first alcohol extraction method preferably comprises: and adding 95 mass percent of ethanol aqueous solution into the crude extract concentrated solution until the mass percent of ethanol in the ethanol extraction system reaches 35 percent, and stopping adding the ethanol aqueous solution. The invention is beneficial to more purifying and extracting the GEP-2 by controlling the content of ethanol in the crude polysaccharide extract.
After obtaining the crude polysaccharide mixture a, the application sequentially stands, filters and concentrates the crude polysaccharide mixture a to obtain a crude polysaccharide extract concentrate. In the invention, the standing time is preferably more than or equal to 12 hours; during the filtration, double-layer gauze is preferably used for filtration;
after the crude polysaccharide extract concentrated solution is obtained, carrying out secondary alcohol extraction on the crude polysaccharide extract concentrated solution to obtain a crude polysaccharide extract B; and during the second alcohol extraction, the final volume percentage of the ethanol in the extraction system of the second alcohol extraction is 90%. In the present invention, the second alcohol extraction method preferably comprises: and adding absolute ethyl alcohol into the crude polysaccharide extract concentrated solution until the mass percentage of the ethyl alcohol in the alcohol extraction system reaches 90%, and stopping adding the absolute ethyl alcohol. According to the invention, the extract containing GEP-2 can be accurately obtained by controlling the content of ethanol in the crude polysaccharide extract B.
After obtaining the crude polysaccharide extract B, the application performs standing and centrifugation on the crude polysaccharide extract B to obtain a crude polysaccharide extract precipitate. In the invention, the standing time is preferably more than or equal to 12 hours; the rotation speed of the centrifugation is preferably 3500rpm, and the time of the centrifugation is preferably 20 min.
After the crude polysaccharide extract precipitate is obtained, the application redissolves the crude polysaccharide extract precipitate to obtain a reconstituted solution. In the present invention, the solution used for reconstitution includes water; the method for redissolving the crude polysaccharide extract precipitate preferably comprises: mixing the crude polysaccharide extract precipitate with water; the temperature of the water preferably comprises 90 ℃ to 100 ℃. The invention can fully remove GEP-1 by re-dissolving with hot water, improve the purity of GEP-2 and improve the solubility.
After obtaining the compound solution, the protein in the compound solution is removed to obtain GEP-2. In the present invention, the method for removing proteins in the supernatant preferably comprises the Sevage method; the number of times of protein removal is preferably 3 or more, preferably 3; after removal of the protein, the present invention preferably further comprises concentrating and freeze-drying the protein-removed reconstituted solution; the concentration temperature is preferably 50-60 ℃, and the pressure is preferably-1.0 Pa; the freeze drying temperature is preferably-42 ℃, and the freeze drying time is preferably 1-2L/24-72 h.
The extraction method provided by the invention can accurately extract and obtain the GEP-2.
The invention also provides the gastrodia elata oligosaccharide GEP-2 extracted by the extraction method, wherein the molecular weight of the gastrodia elata oligosaccharide GEP-2 is 4.0-10.0 KDa. The gastrodin GEP-2 provided by the invention has a net-like structure, and a dissolving-assisting platform can be established based on the physicochemical property to improve the solubility problem; gastrodia elata GEP-2 also has the function similar to Nerve Growth Factor (NGF), has the activity of inducing nerve cell differentiation, can promote the differentiation of pheochromocytoma (PC12), and has important significance for treating neurodegenerative diseases.
The invention also provides the gastrodia elata oligosaccharide GEP-2 extracted by the extraction method or the application of the gastrodia elata oligosaccharide GEP-2 in extracting a medicine for protecting a nervous system. In the invention, the content of the gastrodia elata oligosaccharide GEP-2 in the medicament is preferably more than or equal to 50% in percentage by mass. The application provided by the invention can effectively promote the differentiation of PC 12.
The invention also provides the gastrodia elata oligosaccharide GEP-2 extracted by the extraction method or the application of the gastrodia elata oligosaccharide GEP-2 in extracting nervous system protection food. In the invention, the content of the gastrodia elata oligosaccharide GEP-2 in the food is preferably more than or equal to 50% in percentage by mass. The application provided by the invention can effectively improve the differentiation rate of PC 12.
The invention also provides the gastrodia elata oligosaccharide GEP-2 extracted by the extraction method or the application of the gastrodia elata oligosaccharide GEP-2 in extracting biological agents for protecting nervous systems. In the invention, the content of the gastrodia elata oligosaccharide GEP-2 in the biological preparation is preferably more than or equal to 50% in percentage by mass. The application provided by the invention can effectively improve the differentiation activity of PC 12.
For further illustration of the present invention, the following detailed description will be made of the extraction method and application of gastrodia elata oligosaccharide GEP-2 provided by the present invention with reference to the drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Extraction, separation and purification and structure identification of GEP-1 and GEP-2
1. Extraction, separation and purification of GEP-1 and GEP-2
Taking 7.2kg of fresh rhizoma gastrodiae tubers, cutting into small pieces, adding 95% ethanol water solution by mass for leaching for three times, filtering to obtain filter residues, placing the filter residues in a ventilation position at room temperature, and ventilating until the filter residues have no alcohol smell, thus obtaining the degreased rhizoma gastrodiae;
extracting defatted rhizoma Gastrodiae with boiling water, mixing defatted rhizoma Gastrodiae with 20L distilled water, boiling and extracting for 2 hr, and extracting for 2 times. And filtering after extraction is finished, combining the two filtrates, and concentrating the combined filtrate under reduced pressure at 65 ℃ to 7-10L to obtain a crude concentrated extract.
And adding an ethanol water solution with the mass percentage of 95% into the crude concentrated extract, and slowly and quickly stirring until the final concentration of the ethanol reaches 35% to obtain a crude polysaccharide mixture A.
Standing the crude polysaccharide mixture A overnight, filtering with double-layer gauze to obtain precipitate and 35% ethanol water solution, mixing the precipitate with 10L hot water (90-100 deg.C), and re-dissolving to obtain aqueous solution containing GEP-1.
Concentrating 35% ethanol water solution to about 500ml, adding anhydrous ethanol, stirring, extracting with ethanol for the second time, and stopping adding anhydrous ethanol when ethanol concentration in the ethanol extraction system reaches 90% to obtain crude polysaccharide extract B.
The crude polysaccharide extract B was left overnight and centrifuged (5000rpm, 20min) to obtain a crude polysaccharide extract precipitate.
Mixing the crude polysaccharide extract precipitate with 2L hot water (90-100 deg.C) for redissolution to obtain a composite solution, i.e. aqueous solution containing GEP-2.
The aqueous solution containing GEP-1 and the aqueous solution containing GEP-2 were each deproteinized by the Sevage method (chloroform: n-butanol 4:1) three times, and after removing the proteins, concentrated and freeze-dried to give GEP-1(380g) and GEP-2(113.5 g). The flow chart of the above extraction method is shown in fig. 1.
2. Structural identification of GEP-1 and GEP-2
(1) GEP-1 is detected by HPLC-ELSD, and the HPLC spectrum of GEP-1 is shown in FIG. 2, wherein GEP-1 is a homogeneous polysaccharide molecule with a peak of 5.4 and a molecular weight of about 1.60X 103kDa。
(2) The GEP-2 is detected by HPLC-ELSD, the HPLC spectrum of the GEP-2 is shown in figure 3, and the molecular weight of the GEP-2 is about 4.0-10.0 KDa.
Example 2
100mg of each of GEP-1 and GEP-2 prepared in example 1 was dissolved in 10ml of pure water, and the dissolved state was observed and recorded. The solubilities of GEP-1 and GEP-2 are shown in FIG. 4. The aqueous GEP-2 solution on the left in FIG. 4 is clearly a clear aqueous solution, while the aqueous GEP-1 solution on the right is clearly turbid and in suspension, indicating that GEP-2 has good solubility.
The forms of polysaccharides having a large difference in molecular weight between GEP-1 and GEP-2 were observed by Transmission Electron Microscopy (TEM) using 10. mu.L each. The morphology of GEP-1 is shown in FIG. 5, and exhibits irregular round-like or triangular shapes similar to starch granules; the morphology of GEP-2 is shown in FIG. 6, and it is a net-like morphology, indicating that it has a function of improving the dissolution state of the extract.
Example 3
Neuroprotective Activity test
1. PC12 is cultured in a culture medium of 1640+ 10% HS + 5% FBS +100U/mL double antibody at 37 deg.C and 5% CO2；
2. When the PC12 cells grow to a proper amount, taking the PC12 cells, and carrying out trypsinization to prepare a cell suspension;
3. sucking the PC12 cell suspension into a 15mL centrifuge tube for centrifugation (1000rpm, 5 min);
4. after centrifugation is finished, disinfecting the centrifugal tube with alcohol, taking the centrifugal tube into a super clean bench, and pouring the supernatant into a waste liquid tank to leave a precipitate;
5. adding 5mL of new complete culture medium into the precipitate, blowing and beating for ten times by using a pipette, blowing the cells as much as possible, but not blowing too much force to obtain dispersed cell suspension;
6. taking 0.2ml of dispersed cell suspension, adding the dispersed cell suspension into a cell counting tube, adding 0.8ml of PBS, uniformly mixing, and counting;
7. the concentration of PC12 cells was adjusted to 5X 104Adding cells/mL into a 48-well plate coated with Polylysine (PLL) in advance, wherein each well is 0.2mL, and placing the plate into a cell culture box for culture;
8. the next day of culture, the original medium was aspirated, and then medium (48 wells, 0.24 mL/well) containing 1640+ 2.5% Fetal Bovine Serum (FBS), and 5ng/mL of Nerve Growth Factor (NGF) and test compound were added;
9. experiment design: divided into 5 groups of 3 replicates each.
Blank control (Blank): without NGF, only cells and final concentration of 0.1% dimethyl sulfoxide (DMSO);
negative control group (Negative): containing NGF at a final concentration of 5ng/mL and DMSO at a final concentration of 0.1%;
positive control group (Positive): containing NGF at a final concentration of 50ng/mL and DMSO at a final concentration of 0.1%;
compound group A (GEP-1): the final concentration of GEP-1 is 20 mug/mL, the final concentration of DMSO is 0.1%, and the final concentration of NGF is 5 ng/mL;
compound group B (GEP-2): the final concentration of GEP-2 was 0.1% at 20. mu.g/mL DMSO and 5ng/mL of NGF.
10. Putting each group of cells into a cell culture box for continuous culture;
11. cell differentiation was observed every day, and the cell differentiation rate was counted after adding the compounds (GEP-1 and GEP-2) and inducing for 72 hours, as follows:
(1) and (4) judging the standard: cells with a projection length greater than the cell diameter are considered differentiated cells;
(2) compared with a negative control, if the length and the number of the protrusions of the compound group are not obviously improved, the compound group is considered to have no obvious differentiation activity, and the differentiation rate is not counted;
(3) if the number and length of the protrusions in the compound group are obviously larger than those of the negative control, the compound is considered to have differentiation activity, and the number of differentiated cells is required to be counted, wherein each group counts not less than 5 fields. The results of the differentiation-promoting activity of PC12 are shown in table 1 and fig. 7 to 11, in which fig. 7 is a Blank control group (Blank) in the experiment of neuroprotective activity, fig. 8 is a Negative control group (Negative) in the experiment of neuroprotective activity, fig. 9 is a Positive control group (Positive) in the experiment of neuroprotective activity, fig. 10 is a compound B group (GEP-1) in the experiment of neuroprotective activity, and fig. 11 is a compound a group (GEP-2) in the experiment of neuroprotective activity.
TABLE 1 Effect of different groups on the differentiation Rate of PC12
| Group of | Blank | Negative | Positive | GEP-1 | GEP-2 |
| 72h differentiation Rate (%) | Substantially without differentiation | 3.97％ | 19.83％ | 4.91％ | 12.32％ |
As is clear from table 1 and fig. 7 to 11, the number and length of projections of PC12 cells in the compound a group were significantly larger than those in the negative control group, and it was thus determined that GEP-2 had a significant differentiation activity and promoted the differentiation of PC 12. As can be seen by comparison with the positive control group, GEP-2 has an effect similar to NGF.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. The extraction method of gastrodia elata oligosaccharide GEP-2 is characterized by comprising the following steps of:
carrying out water extraction, filtration and concentration on the degreased gastrodia elata to obtain a crude concentrated extract;
carrying out primary alcohol extraction on the crude extract concentrate to obtain a crude polysaccharide mixture A; the ethanol concentration in the first ethanol extraction system is 35 percent;
standing, filtering and concentrating the crude polysaccharide mixture A in sequence to obtain a crude polysaccharide extract concentrated solution;
carrying out secondary alcohol extraction on the crude polysaccharide extract concentrated solution to obtain a crude polysaccharide extract B; the concentration of ethanol in the system of the second ethanol extraction is 90 percent;
standing and centrifuging the crude polysaccharide extract B to obtain crude polysaccharide extract precipitate;
redissolving the crude polysaccharide extract precipitate to obtain a complex solution;
removing protein in the complex solution to obtain gastrodia elata oligosaccharide GEP-2.
2. The extraction process according to claim 1, characterized in that the reconstitution process of the crude polysaccharide extract precipitate comprises: mixing the crude polysaccharide extract precipitate with water; the temperature of the water is 90-100 ℃.
3. The extraction method according to claim 1, wherein the method for removing the protein in the double solution comprises a Sevage method; the frequency of removing the protein in the complex solution is more than or equal to 3.
4. The gastrodia elata oligosaccharide GEP-2 extracted by the extraction method of any one of claims 1 to 3, wherein the molecular weight of the gastrodia elata oligosaccharide GEP-2 is 4.0 to 10.0 KDa.
5. Use of the oligosaccharide GEP-2 of Gastrodia elata extracted by the extraction method of any one of claims 1-3 or the oligosaccharide GEP-2 of Gastrodia elata of claim 4 in the extraction of a medicament for protecting the nervous system.
6. Use of the gastrodia elata oligosaccharide GEP-2 extracted by the extraction method of any one of claims 1 to 3 or the gastrodia elata oligosaccharide GEP-2 of claim 4 in extraction of food for protecting nervous system.
7. Use of the gastrodia elata oligosaccharide GEP-2 extracted by the extraction method of any one of claims 1 to 3 or the gastrodia elata oligosaccharide GEP-2 of claim 4 in extraction of biological agents for protecting nervous systems.
8. The use according to any one of claims 5 to 7, wherein the content of the gastrodia elata oligosaccharide GEP-2 in the medicament, the food or the biological agent is more than or equal to 50% by mass.
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