CN112574326A - Rhizoma gastrodiae macromolecule linear straight-chain glucan and preparation method and application thereof - Google Patents
Rhizoma gastrodiae macromolecule linear straight-chain glucan and preparation method and application thereof Download PDFInfo
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- CN112574326A CN112574326A CN202011469544.4A CN202011469544A CN112574326A CN 112574326 A CN112574326 A CN 112574326A CN 202011469544 A CN202011469544 A CN 202011469544A CN 112574326 A CN112574326 A CN 112574326A
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- Prior art keywords
- gastrodia elata
- glucan
- polysaccharide
- rhizoma gastrodiae
- linear
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/716—Glucans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- 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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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Polymers & Plastics (AREA)
- Immunology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Materials Engineering (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- General Chemical & Material Sciences (AREA)
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- Food Science & Technology (AREA)
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Abstract
The invention discloses a gastrodia elata macromolecule linear straight-chain glucan and a preparation method and application thereof, and belongs to the field of traditional Chinese pharmacology. The invention dries and crushes the tuber of the gastrodia tuber and degreases the tuber by ethanol. Extracting at room temperature, centrifuging the extractive solution, concentrating, precipitating with ethanol, dissolving precipitate, dialyzing with dialysis bag with molecular weight cutoff of 8000-14000Da, concentrating the dialysate, and separating and purifying with ion exchange resin and gel exclusion chromatography to obtain rhizoma Gastrodiae linear dextran. The polysaccharide is composed of glucose only, is alpha-D-1, 4 connected homopolyglucan, and is a novel glucan. When the concentration is 400 mu g/mL, the compound can obviously promote the proliferation of macrophages, enhance the phagocytic capacity of the macrophages, promote the macrophages to release NO and cytokines TNF-alpha and IL-6, and enhance the immunity of organisms.
Description
Technical Field
The invention relates to the field of traditional Chinese pharmacology, in particular to a gastrodia elata macromolecule linear straight-chain glucan and a preparation method and application thereof.
Background
Gastrodia elata (Gastrodia elata Blume) is a heterotrophic perennial herb of Gastrodia of Orchidaceae, and is sweet in taste, neutral in nature and enters liver meridian. The medicinal dried tubers are mainly produced in Yun, Gui, Chuan and Tibetan provinces in China. The gastrodia elata is mainly used for treating hypertension, dizziness, headache, facial distortion, limb numbness, infantile convulsion, epilepsy and tetanus, and is one of the traditional and rare Chinese medicines in China. Modern pharmacological research finds that the gastrodia elata also has the effects of improving intelligence, strengthening brain, removing free radicals and delaying aging, and has a certain curative effect on senile dementia. The polysaccharide is used as an important component substance in the tuber of the gastrodia elata, and has important theoretical and application values when being researched.
According to literature reports, the gastrodia elata polysaccharide contains monosaccharides such as xylopyranose and glucopyranose, is a heterogeneous polysaccharide, and has a main structure of alpha-D-glucopyranose (structural representation of gastrodia elata polysaccharide, food research and development, 2010, 31-52.). Analyzing the structure of rhizoma Gastrodiae polysaccharide produced from Sichuan province by Chongxiaxia, etc., and finding that the molecular weight of rhizoma Gastrodiae polysaccharide is 4.97 × 105u, which mainly comprises glucose and also contains a small amount of rhamnose and mannose, and the main chain of the u is alpha-1, 4-pyran-type-D-glucose (structural representation of gastrodia elata polysaccharide, food research and development, 31(9), 52-56 and 2010) discovered by nuclear magnetic resonance spectrum analysis. Liuming science and the like extract alpha-1, 4-D-glucan with 1, 6-connecting branched chains from gastrodia elata, and the alpha-1, 4-D-glucan has certain scavenging capacity on oxygen free radicals (gastrodia elata polysaccharide separation, structure analysis and free radical scavenging action research, food science, 30(3), 29-32). Li super et al found that the polysaccharides GBP-I and GBP-II of Gastrodia elata Blume both consist of rhamnose, galactose, glucose, xylose and mannose (separation of Gastrodia elata Blume. polysaccharide and analysis of monosaccharide composition thereof, Chinese agronomy report, 24(7), 89-92, 2008). Gastrodia elata dextran (Glc: Gal ═ 96:4)Has better protective effect on mice acute liver injury induced by CCl4 (the influence of gastrodiae glucan on mice acute liver injury induced by CCl4, Shanghai J.Med., 43(12), 2009). Chenjuncheng finds that the polysaccharides RGP-1a and RGP-1b of gastrodia elata have obvious capacity of eliminating DPPH and hydroxyl free radicals and can also stimulate macrophages to release NO and enhance the phagocytic capacity of the macrophages (separation and purification of the two plant polysaccharides of gastrodia elata and elaeagnus angustifolia and biological activity research thereof, northwest university of agriculture and forestry, Master thesis, 2016). The gastrodia elata polysaccharide can inhibit the proliferation of liver cancer H22 cells, and can reduce the damage of cyclophosphamide to an immune system when being used together with cyclophosphamide (the gastrodia elata polysaccharide can inhibit the tumor growth by influencing the immune system of a mouse, J.Immunity, 2014, 30(6), 566-568). The polysaccharide WPGB-A-H is extracted from rhizoma Gastrodiae by Sunyaman, and has a molecular weight of 28.84kDa, and monosaccharide composition analysis shows that WPGB-A-H comprises glucose and mannose. (Gastrodia elata polysaccharide separation and purification and physicochemical property research, university in southwest, Master thesis, 2007). Sulfated gastrodia elata polysaccharide can obviously inhibit the growth activity of glioma cells and can effectively inhibit the migration of glioma cells (research on the anti-glioma activity of sulfated gastrodia elata polysaccharide, China pharmacist, 227 one-material 231, 23(2) and 2020). Researches show that the content of water-insoluble gastrodia elata glucan is 92.1%, the content of immunoglobulin in immunosuppressive mice can be increased, and the thymus index of the immunosuppressive mice can be increased (the influence of gastrodia elata polysaccharide on the immune function of the mice, China national folk medicine journal, (85), 112-114, 2007). The gastrodia elata polysaccharide can relieve the inhibition effect of cyclophosphamide on the humoral immune function of mice (the influence of the gastrodia elata polysaccharide on the humoral immune function of mice with low immune function caused by cyclophosphamide, China journal of senior schools, 36, 1027-. Research on Wanghem and the like finds that the gastrodia elata polysaccharide has better antioxidant activity in vitro (influence of different extraction methods on the antioxidant activity of the gastrodia elata polysaccharide, food technology 40(3), 208-; the molecular weight of the gastrodia elata polysaccharide PGEB-3-H extracted from the Yunnan gastrodia elata is 28.8kDa, and the gastrodia elata polysaccharide has a structure that alpha-1, 4 is connected to glucan with a small amount of alpha-1, 6 branches on the main chain. The activity research shows that PGEB-3-H can reduce the triglyceride level of rat with hyperlipemia and can increase B in brainThe content of the acetylcholine improves the memory of the mice (research on specific edible and medicinal fungi active polysaccharide in Yunnan, university in southwest, doctor's paper, 2008). Zhu et al extracted polysaccharide PGE from Gastrodia elata Blume by hot water extraction method, its molecular weight is 1.54 × 103kDa, Structural study found that PGE consists of 1,4 linked glucose to form linear skeleton, and 1,3 and 1,46 linked glucose branches exist, activity study found that half inhibitory concentration of PGE to angiotensin converting enzyme is 0.66mg/mL (Structural characterization and ACE inhibitory activities of polysaccharides from Gastrodia elata Blume, Natural Product Research,33, 1721-.
The gastrodia elata polysaccharide extracted in the research is composed of various monosaccharides such as glucose and the like, is heteropolymeric polysaccharide with a complex structure, brings certain difficulty for development and utilization of the heteropolymeric polysaccharide, and is small in molecular weight, so that the separation and extraction process of the heteropolymeric polysaccharide is influenced, and impurities are easy to appear during separation. At present, no report of high molecular weight gastrodia elata linear glucan exists.
Disclosure of Invention
The object of the present invention is to provide a high molecular weight linear glucan which solves the above-mentioned problems of the prior art.
In order to achieve the purpose, the invention provides the following scheme:
in a first aspect, a tall gastrodia tuber macromolecular linear straight-chain glucan is provided, and the tall gastrodia tuber macromolecular linear straight-chain glucan has the following structure:
[→4)-α-D-Glcp-(1→]n
wherein n is a positive integer.
Preferably, the molecular weight range of the tall gastrodia tuber macromolecular linear straight-chain glucan is 1000-4000kDa, and the average molecular weight is 2000 kDa.
In a second aspect, a method for preparing the gastrodia elata macromolecular linear straight-chain glucan is provided, which comprises the following steps:
1) polysaccharide extraction: drying rhizoma Gastrodiae tuber, pulverizing, and defatting with ethanol. Extracting at room temperature, centrifuging the extractive solution, concentrating, precipitating with ethanol, redissolving the precipitate, and freeze drying to obtain crude polysaccharide extracted from rhizoma Gastrodiae by cold water extraction;
2) polysaccharide purification: dialyzing the crude polysaccharide of rhizoma Gastrodiae with dialysis bag with cut-off molecular weight of 8000-.
Preferably, in step 1), the polysaccharide extraction specifically comprises: drying and crushing rhizoma Gastrodiae tuber, heating and refluxing with 80% ethanol for defatting, and drying at room temperature to obtain defatted powder of rhizoma Gastrodiae tuber; taking degreased gastrodia elata powder, adding deionized water to enable the material-liquid ratio to be 1: 20-60 (g/v), standing for 1-6 hours at room temperature, stirring and extracting, centrifuging an extracting solution, repeatedly extracting precipitates after centrifugation for 2-3 times, combining supernate after centrifugation, concentrating under reduced pressure, adding 2-6 times of volume of absolute ethyl alcohol to carry out alcohol precipitation, standing overnight in a refrigerator at 4 ℃, carrying out vacuum filtration on alcohol precipitation precipitates, re-dissolving the precipitates in water, and carrying out freeze drying to obtain crude gastrodia elata polysaccharide.
Preferably, the mass volume ratio of the rhizoma gastrodiae tuber degreased powder to the deionized water is 1 g: 40 ml.
Preferably, in step 2), the specific steps of purifying the polysaccharide comprise: dissolving crude rhizoma Gastrodiae polysaccharide in water, dialyzing with dialysis bag with molecular weight cutoff of 8000-14000Da, separating and purifying with Q-Sepharose fast flow anion exchange resin and Sephacryl S-400 gel filtration chromatography, detecting with sulfuric acid-phenol, collecting polysaccharide component, concentrating, and lyophilizing to obtain rhizoma Gastrodiae linear dextran.
Preferably, the rhizoma gastrodiae tuber is a Yunnan Zhaotong rhizoma gastrodiae tuber.
In the third aspect, the application of the gastrodia elata macromolecular linear glucan in preparing a medicine or food or health-care product for enhancing the immune function is provided.
In a fourth aspect, a pharmaceutical composition is provided, which comprises the tall gastrodia tuber macromolecular linear glucan and pharmaceutically acceptable auxiliary materials.
In a fifth aspect, a health food composition is provided, which comprises the gastrodia elata macromolecular linear straight-chain glucan and auxiliary materials acceptable in food.
The invention discloses the following technical effects:
the invention extracts linear straight-chain glucan with high molecular weight from gastrodia elata, wherein the polysaccharide only consists of glucose and is alpha-D-1, 4 connected homopolyglucan, and the glucan is novel glucan. The polysaccharide provided by the invention has a simple structure, is beneficial to development and utilization, has a large molecular weight and is convenient to separate. When the concentration is 400 mu g/mL, the compound can obviously promote the proliferation of macrophages, enhance the phagocytic capacity of the macrophages, promote the macrophages to release NO and cytokines TNF-alpha and IL-6, and enhance the immunity of organisms. The gastrodia elata alpha-D-1, 4-glucan provided by the invention is homopolysaccharide, and the gastrodia elata alpha-D-1, 4-glucan prepared by the preparation method provided by the invention has good structural integrity of sugar chains and shows better immune enhancement activity in vitro.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a diagram showing the purity of alpha-D-1, 4-glucan from Gastrodia elata Blume;
FIG. 2 is the nuclear magnetic resonance spectrum of rhizoma Gastrodiae alpha-D-1, 4-dextran;
FIG. 3 is a carbon spectrum of alpha-D-1, 4-glucan of Gastrodia elata;
FIG. 4 shows the preparation of alpha-D-1, 4-glucan from Gastrodia elata1H-13C HMQC spectrogram;
FIG. 5 is a view showing the monosaccharide composition of alpha-D-1, 4-glucan of Gastrodia elata Blume;
FIG. 6 is a graph of the survival rate of macrophages from mice incubated with Gastrodia elata alpha-D-1, 4-glucan;
FIG. 7 is a graph of macrophage phagocytosis index of rhizoma Gastrodiae alpha-D-1, 4-glucan;
FIG. 8 is a graph showing TNF-. alpha.production by cultured cells of Gastrodia elata.alpha. -D-1, 4-glucan;
FIG. 9 is a graph showing the IL-6 production amount of cultured cells of Gastrodia elata alpha-D-1, 4-glucan.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The materials and reagents used in the present invention are commercially available, unless otherwise specified.
Example 1
1. Extraction of gastrodia tuber alpha-D-1, 4-glucan
A. Drying and crushing rhizoma gastrodiae tubers, degreasing the obtained powder for 24h by using 80% ethanol, drying at room temperature, adding 3000mL of distilled water into 100g of rhizoma gastrodiae powder, soaking at room temperature for 2h, stirring and extracting for 3h, centrifuging, repeatedly extracting residues for 2 times, mixing supernate, concentrating, adding 95% ethanol with 3 times of volume, precipitating with ethanol, precipitating, and freeze-drying to obtain crude polysaccharide of the rhizoma gastrodiae cold water extract.
B. Preparing 1% solution from crude gastrodia elata polysaccharide, adding dichloromethane/n-butanol mixed solution (chloroform: n-butanol is 4:1 in volume ratio) with the same volume, stirring to remove protein, repeating protein removal for 2 times, concentrating the polysaccharide solution after protein removal, dialyzing by using a dialysis bag with the molecular weight cut-off of 8000 14000Da, concentrating the dialysate, purifying the polysaccharide by using Q-Sepharose Fast Flow anion exchange resin, eluting by using 0-1 mol/L sodium chloride solution, detecting by using a sulfuric acid-phenol method, collecting water elution components, concentrating, further purifying by using gel column chromatography Sephacryl S-400, detecting by using a sulfuric acid-phenol method, and collecting polysaccharide components to obtain the gastrodia elata alpha-D-1, 4-glucan.
Example 2
1. Extraction of gastrodia tuber alpha-D-1, 4-glucan
A. Drying and crushing rhizoma gastrodiae tubers, degreasing the obtained powder for 24h by using 80% ethanol, drying at room temperature, adding 100g of rhizoma gastrodiae powder into 2000mL of distilled water, soaking at room temperature for 1h, stirring and extracting for 3h, centrifuging, repeatedly extracting residues for 3 times, mixing supernate, concentrating, adding 2 times volume of 95% ethanol for alcohol precipitation, precipitating, and freeze-drying to obtain the crude polysaccharide of the rhizoma gastrodiae cold water extract.
B. Preparing 1% solution from crude gastrodia elata polysaccharide, adding dichloromethane/n-butanol mixed solution (chloroform: n-butanol is 4:1 in volume ratio) with the same volume, stirring to remove protein, repeating protein removal for 2 times, concentrating the polysaccharide solution after protein removal, dialyzing by using a dialysis bag with the molecular weight cut-off of 8000 14000Da, concentrating the dialysate, purifying the polysaccharide by using Q-Sepharose Fast Flow anion exchange resin, eluting by using 0-1 mol/L sodium chloride solution, detecting by using a sulfuric acid-phenol method, collecting water elution components, concentrating, further purifying by using gel column chromatography Sephacryl S-400, detecting by using a sulfuric acid-phenol method, and collecting polysaccharide components to obtain the gastrodia elata alpha-D-1, 4-glucan.
Example 3
1. Extraction of gastrodia tuber alpha-D-1, 4-glucan
A. Drying and crushing tuber of gastrodia elata, degreasing the powder for 24h by using 80% ethanol, drying at room temperature, adding 6000mL of distilled water into 100g of gastrodia elata powder, soaking at room temperature for 6h, stirring and extracting for 3h, centrifuging, repeatedly extracting residues for 3 times, mixing supernate, concentrating, adding 95% ethanol with 6 times of volume of the supernate for alcohol precipitation, precipitating, freeze-drying, and obtaining crude polysaccharide of gastrodia elata cold water extract.
B. Preparing 1% solution from crude gastrodia elata polysaccharide, adding dichloromethane/n-butanol mixed solution (chloroform: n-butanol is 4:1 in volume ratio) with the same volume, stirring to remove protein, repeating protein removal for 2 times, concentrating the polysaccharide solution after protein removal, dialyzing by using a dialysis bag with the molecular weight cut-off of 8000 14000Da, concentrating the dialysate, purifying the polysaccharide by using Q-Sepharose Fast Flow anion exchange resin, eluting by using 0-1 mol/L sodium chloride solution, detecting by using a sulfuric acid-phenol method, collecting water elution components, concentrating, further purifying by using gel column chromatography Sephacryl S-400, detecting by using a sulfuric acid-phenol method, and collecting polysaccharide components to obtain the gastrodia elata alpha-D-1, 4-glucan.
Example 4
Polysaccharide structure characterization:
(1) the relative molecular weight and purity of the polysaccharide were determined by High Performance Gel Permeation Chromatography (HPGPC), and the purity identification result is shown in FIG. 1, which is a single symmetrical chromatographic peak, indicating that the obtained polysaccharide is a polysaccharide with uniform structure and relative weight average molecular weight of 2000 kDa. The instrument used was an Agilent 1260 high performance liquid chromatograph, the chromatographic column was Shodex Ohpak SB804, the mobile phase was 0.1% sodium azide, the flow rate was 1.0mL/min, and the column temperature was 35 ℃.
(2) Nuclear magnetic resonance hydrogen spectrum (figure 2), carbon spectrum (figure 3) and1H-13the C HMQC (figure 4) spectrogram can obtain the correlation between hydrogen signals and carbon signals, namely 5.39-100.5(H1/C1), 3.96-74.1(H3/C3), 3.84-72.2(H5/C5), 3.77,3.83-61.2(H6/C6) and 3.65-77.8(H4/C4) and 3.65-72.0 (H2/C2). The monosaccharide composition diagram of the polysaccharide is shown in FIG. 5.
The analysis result shows that the gastrodia elata polysaccharide structure is alpha-D-1, 4-glucan.
Example 5
Experiment for enhancing immunity
1. Experimental Material
The test sample is Gastrodia elata alpha-D-1, 4-dextran, the negative control is phosphate buffer solution, the positive control is Lipopolysaccharide (LPS), the cell strain is mouse macrophage RAW264.7, 0.25% trypsin, MTT, and an enzyme labeling instrument.
2. Experimental methods
2.1 cell viability assay
Mouse macrophage is digested by 0.25% trypsin after cells are fully adhered in an incubator containing 5% carbon dioxide at 37 ℃ by using a high-glucose DMEM culture medium containing 10% fetal calf serum, the cells are inoculated in a 96-well cell culture plate (the cell density is 5000/well), gastrodia elata alpha-D-1, 4-glucan sample solutions with different concentrations are added after the cells are adhered to the wall to ensure that the final concentrations are 25, 50, 100, 200 and 400 mu g/mL, a positive control is a lipopolysaccharide solution with the concentration of 5 mu g/mL, a negative control is a phosphate buffer solution, 3 multiple wells are arranged, after the cells are incubated for 24 hours, MTT is added to incubate the cells for 4 hours, supernatant is absorbed, DMSO is added, the OD value of each well is measured at the wavelength of 570nm by using an enzyme labeling instrument, and the cell survival rate (%) [ ODMedicine adding device-ODBlank group]/[ODNegative control group-ODBlank group]. The results are shown in fig. 6, and there is no significant difference in cell viability among the groups.
2.2 cell phagocytic Activity assay
Inoculating RAW264.7 cells in logarithmic growth phase to a 96-well plate, adding gastrodia elata polysaccharide sample solutions with different concentrations after the cells adhere to the wall to enable the final concentrations to be 25, 50, 100, 200 and 400 mu g/mL, adding a lipopolysaccharide solution with a positive control of 5 mu g/mL and a phosphate buffer solution as a negative control, setting 3 multiple wells, incubating the cells for 24 hours, discarding a culture medium PBS (phosphate buffer solution) for cleaning for 2 times, adding a neutral red solution, continuously culturing the cells for 1 hour, removing a supernatant, cleaning the cells with precooled PBS for 2 times, adding a cell lysate, and performing room-temperature cell lysisLysing the cells overnight, measuring the absorbance at 540nm, and calculating the phagocytic index of the macrophages, the phagocytic index being ODSample set/ODBlank group. The results are shown in fig. 7, where the phagocytic index was significantly increased for each group compared to the negative control.
2.3 cytokine TNF-. alpha.and IL-6 Release assay
Inoculating RAW264.7 cells in a logarithmic growth phase to a 6-well plate, adding gastrodia elata polysaccharide sample solutions with different concentrations after the cells adhere to the wall to enable the final concentrations to be 25, 50, 100, 200 and 400 mu g/mL, setting 3 multiple wells with a lipopolysaccharide solution with a positive control of 5 mu g/mL and a negative control of a phosphate buffer solution, incubating the cells for 24h, collecting cell supernatants, and detecting the contents of TNF-alpha and IL-6 generated by the cells by an ELISA method. As shown in FIGS. 8 and 9, the content of TNF-alpha and IL-6 in each group was increased relative to the negative control, wherein the content of TNF-alpha increased with the increase of the polysaccharide concentration of Gastrodia elata Blume, and the content of IL-6 decreased with the increase of the polysaccharide concentration of Gastrodia elata Blume.
The invention extracts a high molecular weight linear glucan from gastrodia elata, the polysaccharide only consists of glucose and is alpha-D-1, 4 connected homopolyglucan, and the glucan is a novel glucan. When the concentration is 400 mu g/mL, the compound can obviously promote the proliferation of macrophages, enhance the phagocytic capacity of the macrophages, promote the macrophages to release NO and cytokines TNF-alpha and IL-6, and enhance the immunity of organisms.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. The gastrodia elata macromolecule linear straight-chain glucan is characterized in that the gastrodia elata macromolecule linear straight-chain glucan is gastrodia elata alpha-D-1, 4-glucan and has the following structure:
[→4)-α-D-Glcp-(1→]n
wherein n is a positive integer.
2. The linear dextran of gastrodia elata macromolecule according to claim 1, wherein the molecular weight range of the linear dextran of gastrodia elata macromolecule is 1000-4000kDa, and the average molecular weight is 2000 kDa.
3. A method for preparing macromolecular linear glucan of gastrodia elata according to any one of claims 1-2, wherein the method comprises the following steps:
1) polysaccharide extraction: drying rhizoma Gastrodiae tuber, pulverizing, and defatting with ethanol; extracting at room temperature, centrifuging the extractive solution, concentrating, precipitating with ethanol, redissolving the precipitate, and freeze drying to obtain crude rhizoma Gastrodiae polysaccharide;
2) polysaccharide purification: dissolving crude polysaccharide of rhizoma Gastrodiae, adding organic solvent, stirring to remove protein, dialyzing with dialysis bag with cut-off molecular weight of 8000-.
4. The method for preparing macromolecular linear glucan of gastrodia elata according to claim 3, wherein in the step 1), the specific steps of polysaccharide extraction comprise: drying and crushing rhizoma Gastrodiae tuber, heating and refluxing with 80% ethanol for defatting, and drying at room temperature to obtain defatted powder of rhizoma Gastrodiae tuber; taking rhizoma gastrodiae defatted powder, adding deionized water to enable the volume mass ratio of feed liquid to be 1: 20-60, standing for 1-6 h at room temperature, stirring and extracting, centrifuging the extracting solution, repeatedly extracting the centrifuged precipitate for 2-3 times, combining the centrifuged supernatant, concentrating under reduced pressure, adding 2-6 times of volume of absolute ethyl alcohol to carry out alcohol precipitation, standing overnight in a refrigerator at 4 ℃, carrying out reduced pressure suction filtration on the alcohol precipitation precipitate, re-dissolving the precipitate in water, and freeze-drying to obtain the crude rhizoma gastrodiae polysaccharide.
5. The method for preparing tall gastrodia tuber macromolecular linear straight-chain glucan according to claim 4, wherein the mass-to-volume ratio of tall gastrodia tuber degreased powder to deionized water is 1 g: 40 ml.
6. The method for preparing macromolecular linear glucan of gastrodia elata according to claim 3, wherein in the step 2), the specific steps of purifying the polysaccharide comprise: dissolving crude polysaccharide of rhizoma Gastrodiae in water, dialyzing with dialysis bag with molecular weight cutoff of 8000-14000Da, separating and purifying with Q-Sepharose fast flow anion exchange resin and Sephacryl S-400 gel filtration chromatography, concentrating, and lyophilizing to obtain rhizoma Gastrodiae macromolecule linear dextran.
7. The method for preparing macromolecular linear glucan of gastrodia elata according to any one of claims 3-6, wherein gastrodia elata tubers are Yunnan Zhaotong gastrodia elata tubers.
8. Use of a macromolecular linear glucan of gastrodia elata as claimed in any one of claims 1-2 in the preparation of a medicament or food or health product for enhancing immune function.
9. A pharmaceutical composition comprising the macromolecular linear glucan of gastrodia elata as claimed in any one of claims 1-2 and pharmaceutically acceptable excipients.
10. A health food composition comprising the macromolecular linear glucan of gastrodia elata as claimed in any one of claims 1-2 and one or more food-acceptable excipients.
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| CN115572334A (en) * | 2022-09-01 | 2023-01-06 | 澳门科技大学 | Alpha- (1, 4) (1, 6) -glucan and preparation method and application thereof |
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| CN114133461A (en) * | 2021-12-06 | 2022-03-04 | 中国科学院昆明植物研究所 | Extraction method and application of gastrodia elata oligosaccharide GEP-2 |
| CN115572334A (en) * | 2022-09-01 | 2023-01-06 | 澳门科技大学 | Alpha- (1, 4) (1, 6) -glucan and preparation method and application thereof |
| CN115572334B (en) * | 2022-09-01 | 2024-04-02 | 澳门科技大学 | Alpha- (1, 4) (1, 6) -glucan and preparation method and application thereof |
| CN115558035A (en) * | 2022-10-12 | 2023-01-03 | 昆明理工大学 | A rhizoma Gastrodiae polysaccharide with immunoregulatory activity |
| CN115558035B (en) * | 2022-10-12 | 2023-10-13 | 昆明理工大学 | Gastrodia elata polysaccharide with immunoregulatory activity |
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