CN109400742B

Movatterモバイル変換

Info

Publication number: CN109400742B
Application number: CN201811330972.1A
Authority: CN
Inventors: 吴月国; 陈峰阳; 赵铮蓉; 吴蓓丽; 刘骅; 张萍
Original assignee: Zhejiang Academy of Medical Sciences
Current assignee: Yunnan Pinhutang Biotechnology Co ltd
Priority date: 2018-11-09
Filing date: 2018-11-09
Publication date: 2021-10-29
Anticipated expiration: 2038-11-09
Also published as: CN109400742A

Abstract

Translated fromChinese

本发明公开了一种齿瓣石斛精制多糖及其制备方法和应用，齿瓣石斛精制多糖的制备方法包括：齿瓣石斛先用水提取，提取液浓缩后添加乙醇醇沉，沉淀用水溶解后脱蛋白，干燥得粗多糖，粗多糖经离子交换柱纯化得到半精制多糖，半精制多糖再经凝胶柱纯化后得到齿瓣石斛精制多糖。该齿瓣石斛精制多糖具有增强免疫，缓解体力疲劳的作用，安全有效，可用于制备增强免疫力和缓解体力疲劳的药物和功能性食品中，还可作为TLR4激动剂在制备添加到疫苗中的免疫佐剂，和TLR4相关的抗肿瘤药物的应用。

The invention discloses a refined polysaccharide of Dendrobium dentobium, a preparation method and application thereof. The preparation method of the refined polysaccharide of Dendrobium denticola includes: firstly extracting Dendrobium denticola with water, concentrating the extract, adding ethanol for precipitation, dissolving the precipitate with water and then deproteinizing , drying to obtain crude polysaccharide, crude polysaccharide is purified by ion exchange column to obtain semi-refined polysaccharide, and semi-refined polysaccharide is purified by gel column to obtain Dendrobium dendrobii refined polysaccharide. The refined polysaccharide of Dendrobium candidum has the functions of enhancing immunity and relieving physical fatigue, is safe and effective, can be used in the preparation of medicines and functional foods for enhancing immunity and relieving physical fatigue, and can also be used as a TLR4 agonist in the preparation of vaccines. Immune adjuvants, and the application of TLR4-related antitumor drugs.

Description

Dendrobium devonianum refined polysaccharide and preparation method and application thereof

Technical Field

The invention relates to the technical field of dendrobium devonianum, and particularly relates to dendrobium devonianum refined polysaccharide and a preparation method and application thereof.

Background

TLR4 is a key transmembrane pattern recognition receptor in the Toll-like receptor (TLR) family, expressed primarily in immune cells, as well as in normal epithelial and cancer cells. TLR4 responds to a variety of invading foreign pathogens, including viruses, bacteria, protozoa and fungi, by pathogen-associated molecular patterns (PAMPs). In addition, it recognizes endogenous substances by risk (or injury) associated molecular patterns (DAMPs). These endogenous substances are typically released from cells that are inflamed, oxidized, damaged, or necrotic, and include beta-defensins, beta-amyloid, peroxidase, high mobility group box 1 protein (HMGB1), Heat Shock Proteins (HSPs), hyaluronic acid, heparin sulfate, substance P, and the like. These capabilities make TLR 4a key regulator of innate immunity and adaptive immune response and are involved in many immune and other diseases. Thus, TLR4 has become an important drug target. TLR4 antagonists are being developed for the treatment of inflammatory diseases, including asthma, arteriosclerosis,type 2 diabetes, autoimmune and neuroinflammatory diseases, while TLR4 agonists are used for the treatment of cancer and infectious diseases (e.g. fungi and parasites), either directly or as immunoadjuvants in vaccines.

Dendrobium devonianum (Dendrobium devonianum Paxton.) is dry stem of Dendrobium devonianum (Dendrobium devonianum Paxton.) belonging to Orchidaceae, also called Dendrobium devonianum, Violet, herba Clausenae Lansii and radix et rhizoma Rhei Paxton, and is adopted in the Chinese medicinal material Standard of Yunnan province, and has the effects of benefiting stomach, promoting fluid production, nourishing yin, clearing heat and the like. Meanwhile, dendrobium devonianum is the only dendrobium variety which is published by administrative departments and has food safety standards at present, and the research and development of dendrobium devonianum are more and more emphasized.

Chinese patent application No. 201510040088.4 (grant No. CN104628798B) discloses a method for simultaneously preparing anthocyanin and polysaccharide from dendrobium devoninum paxt raw material. The method comprises the following steps: 1) preparing water extract: extracting fresh dendrobium devoninum paxt stems in water, and carrying out solid-liquid separation to obtain a liquid as a water extract; 2) concentrating the water extract: evaporating the water extract to obtain a concentrated water extract; 3) and (3) extracting polysaccharide: adding ethanol into the concentrated water extract, standing, performing solid-liquid separation, and drying the obtained solid to obtain polysaccharide; 4) extracting anthocyanin: removing ethanol from the liquid obtained by the solid-liquid separation in the step 3), and drying the solution to obtain the anthocyanin. The method can simply and conveniently extract anthocyanin pigment and polysaccharide with functional activity in the dendrobium devonianum at the same time, and the obtained anthocyanin pigment and polysaccharide have higher purity; the energy consumption in the extraction process is low; the method can improve the time efficiency, can also fully utilize resources, and has better application prospect.

Chinese patent application with application number 201610121973.X (application publication number CN106632708A) discloses a method for separating and purifying Dendrobium devonianum homopolysaccharide, which comprises separating by fractional alcohol precipitation method, and purifying with gel column to obtain pure Dendrobium devonianum homopolysaccharide. In addition, the invention also adopts multispectral analysis to identify the framework structure of the dendrobium devonianum homogeneous polysaccharide, can accurately identify the primary structure of the polysaccharide, and determines that the main polysaccharide in the dendrobium devonianum is acetyl glucomannan.

Chinese invention patent application with application number 201710214362.4 (application publication number CN107033253A) discloses a dendrobium devonianum polysaccharide with immune promoting and hypoglycemic activities and a preparation method thereof, the dendrobium devonianum polysaccharide is prepared by the steps of water extraction, alcohol precipitation, redissolution, drying or water extraction, ultrafiltration, drying and the like, and the dendrobium devonianum polysaccharide is characterized in that: iodine-potassium iodide reagent has no color development and molecular weight of 3 × 10⁵－5×10⁵Da, monosaccharide composition is mainly mannose, acetylated mannose (Acetyl-Man) and contains a small amount of glucose (Glc), the composition ratio of the mannose to the glucose is more than 20:1, and glycosidic bonds are mainly beta-1, 4-Manp and beta-1, 4-Glcp. The polysaccharide has effects of promoting immunity and lowering blood sugar, and can be used for development of related products for treating hypoimmunity and hyperglycemia.

Disclosure of Invention

The invention aims to provide a dendrobium devonianum refined polysaccharide and a preparation method and application thereof, wherein the dendrobium devonianum refined polysaccharide has the function of enhancing immunity, and also has the application of the polysaccharide as a TLR4 agonist in preparation of an immunologic adjuvant added into a vaccine and an anti-tumor drug related to TLR 4.

In order to achieve the purpose, the invention adopts the following technical scheme: extracting herba Dendrobii with water, concentrating the extractive solution, adding ethanol to make ethanol content reach about 80%, precipitating with ethanol, dissolving the precipitate with water, deproteinizing, drying to obtain crude polysaccharide, purifying the crude polysaccharide with ion exchange column to obtain semi-refined polysaccharide, and purifying with gel column to obtain herba Dendrobii refined polysaccharide.

A preparation method of dendrobium devonianum refined polysaccharide comprises the following steps:

1) pretreating the dendrobium devonianum raw material into powder, granules, sheets or strips to obtain pretreated dendrobium devonianum;

2) placing the pretreated dendrobium devonianum in a container, adding water, extracting, and filtering to obtain an extracting solution;

3) concentrating the extractive solution under reduced pressure to obtain concentrated solution;

4) adding ethanol into the concentrated solution to ensure that the ethanol content of the liquid medicine reaches 50-90 percent, and precipitating with ethanol to obtain a precipitate;

5) dissolving the precipitate with water to obtain precipitate solution, extracting with chloroform-n-butanol mixture, collecting the uppermost layer liquid, concentrating under reduced pressure, and freeze drying to obtain crude polysaccharide of herba Dendrobii;

6) dissolving the dendrobium devonianum crude polysaccharide with water, loading the dendrobium devonianum crude polysaccharide to an ion exchange column, eluting with ultrapure water, collecting eluent, measuring absorbance by a phenol-sulfuric acid method, washing each gradient until sugar is detected, combining same fractions according to absorption peaks, concentrating under reduced pressure, dialyzing, and freeze-drying to obtain semi-refined polysaccharide of dendrobium devonianum;

7) dissolving semi-refined polysaccharide of Dendrobium devonianum with water, loading onto gel column, eluting with ultrapure water, collecting eluate, measuring absorbance by phenol-sulfuric acid method, washing each gradient until no sugar is detected, mixing same fractions according to absorption peak, concentrating under reduced pressure, dialyzing, and freeze-drying to obtain refined polysaccharide of Dendrobium devonianum.

In the step 1), the dendrobium devonianum is crushed into powder and sieved by a sieve with 20-50 meshes to obtain powdery dendrobium devonianum; or cutting the dendrobium devonianum into slices, wherein the thickness of each slice is 2-5 mm to obtain the flaky dendrobium devonianum, and the slices are cut into the thicknesses, so that the extraction of the effective ingredients of the medicine is facilitated.

In the step 2), the weight ratio of the pretreated dendrobium devonianum to water is 1:10 to 100, and more preferably 1:20 to 40.

The extraction conditions are as follows: heating to 95-105 ℃, and extracting for 2-4 hours under the condition of heat preservation. The effective components of the medicine can be extracted by the extraction under the conditions.

The extraction times are 2-4 times, and the method specifically comprises the following steps: adding water, extracting, filtering to obtain an extracting solution, adding the same water into filter residues, extracting in the same way, repeating the steps in the same way for 2-4 times to obtain the extracting solution, and finally combining the extracting solutions.

In the step 3), the reduced pressure concentration condition is as follows: concentrating under reduced pressure at 50-70 deg.C, preferably 60-70 deg.C.

In the step 4), the alcohol content of the liquid medicine refers to the volume ratio of the ethanol to the volume of the concentrated solution plus the ethanol.

In the step 5), the volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixed solution is 3-5: 1, more preferably 4: 1.

the volume ratio of the mixed solution of chloroform-n-butanol (volume ratio 4: 1) to the precipitation dissolving solution is 1: 4. The reduced pressure concentration conditions are as follows: concentrating under reduced pressure at 50-60 deg.C.

In the step 6) and the step 7), the reduced pressure concentration condition is as follows: concentrating under reduced pressure at 50-60 deg.C. The dialysis conditions are as follows: dialyzing (dialysis membrane cut-off 7000) with flowing distilled water for 24 h.

The prepared herba Dendrobii refined polysaccharide has molecular weight of 8 × 10 determined by high performance liquid chromatography⁴-5×10⁶；

The prepared dendrobium devonianum refined polysaccharide is analyzed by gas chromatography, and monosaccharide components of the dendrobium devonianum refined polysaccharide comprise mannose and glucose, wherein the molar ratio of the glucose to the mannose is 1:10-1: 20.

The prepared dendrobium devonianum polysaccharide has the following structure after nuclear magnetic resonance analysis:

wherein n is the number of repeating structural units.

The prepared dendrobium devonianum polysaccharide can be used for preparing medicines and functional foods for enhancing immunity and relieving physical fatigue. The weight percentage content of the dendrobium nobile refined polysaccharide in the medicine and the functional food for enhancing the immunity and relieving the physical fatigue is 0.1-99.5 percent.

The medicine and functional food for enhancing immunity and relieving physical fatigue comprise the effective components of dendrobium devonianum refined polysaccharide and ginsenoside Rg1 in a mass ratio of 1: 1; or the effective components are dendrobium devonianum refined polysaccharide, ginsenoside Rg1, arabinose and galactose, and the mass ratio is 1: 1: 0.4: 0.2.

the results of the present invention are set forth below: firstly, through research, a method for purifying dendrobium devonianum polysaccharide by combining an ion exchange column with a gel column is created, and a new dendrobium devonianum refined polysaccharide with the molecular weight of 9.5 multiplied by 10 is separated from dendrobium devonianum⁴Da, the molar ratio is 15.85:1, researches show that the dendrobium devonianum refined polysaccharide is a TLR4 agonist, can directly stimulate the activation of macrophages in vitro and in zebra fish bodies, and indicate that the dendrobium devonianum refined polysaccharide can be used as a TLR4 agonist, is used for preparing an immunologic adjuvant added into a vaccine and an anti-tumor drug related to TLR4, and is used as a regulator of an immunologic function for health-care food.

Compared with the prior art, the invention has the following advantages:

in the invention, the dendrobium devonianum is a rare traditional Chinese medicine with the effects of tonifying stomach, promoting the secretion of saliva or body fluid, nourishing yin and clearing heat, and is the only dendrobium varieties with the quality of medicine and food at present. The invention discloses the preparation of the dendrobium devonianum polysaccharide by adopting the method of purifying by combining an ion exchange column with a gel column for the first time, and the prepared dendrobium devonianum polysaccharide has definite composition and structure, has obvious immunity enhancing effect, is suitable for developing functional foods and medicines with the immunity enhancing effect and has wide application prospect.

Drawings

FIG. 1 is a gas chromatogram of the refined polysaccharide of Dendrobii nobile Lindl prepared in example 1 of the present invention;

FIG. 2 shows the polysaccharide prepared from Dendrobium devonianum of example 1 of the present invention¹H-NMR spectrum (500 MHz);

FIG. 3 shows the polysaccharide prepared from Dendrobium devonianum according to example 1 of the present invention¹³C-NMR spectrum (125 MHz);

FIG. 4 is a graph showing the results of experiments on the in vitro macrophage activation induced by dendrobium devonianum refined polysaccharide prepared in example 1 as TLR-4 agonist;

wherein a in figure 4 is a direct action diagram of refined dendrobium devonianum polysaccharide on TLR4, the abscissa is the concentration of the refined polysaccharide, and the ordinate is the relative stimulation intensity of TLR 4; in FIG. 4, b is a graph showing the effect of the dendrobium devonianum refined polysaccharide on the secretion of TNF-alpha, the abscissa is the concentration of the refined polysaccharide, and the ordinate is the content of TNF-alpha.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more clear, the present invention is further described with reference to the following examples, which should not be construed as limiting the scope of the present invention.

Example 1

The dendrobium devonianum refined polysaccharide of the embodiment is prepared according to the following specific mode:

1) crushing the dendrobium devonianum raw material, and screening the crushed dendrobium devonianum raw material through a 40-mesh screen to obtain powdery dendrobium devonianum for later use;

2) placing powdery dendrobium devonianum into a container, adding water with the weight of 40 times of that of the dendrobium devonianum, preserving heat at 100-105 ℃ for 3 hours for extraction, filtering to obtain filtrate, repeatedly extracting filter residues once again, filtering to obtain filtrate, and combining the two extracting solutions;

3) concentrating the extracting solution at 60-70 deg.c and decompression concentration to obtain concentrated solution;

4) adding 4 times of anhydrous ethanol into the concentrated solution to make the ethanol content of the medicinal liquid reach 80%, and precipitating with ethanol to obtain precipitate;

5) the precipitate was dissolved in water to form a precipitate dissolved solution, and the precipitate dissolved solution was dissolved in chloroform-n-butanol (volume ratio 4: 1) the mixture of (a) was extracted, and chloroform-n-butanol (volume ratio 4: 1) the volume ratio of the mixed solution to the precipitation solution is 1:4, the uppermost layer liquid is added with chloroform-n-butanol mixed solution with the same volume, the operation is repeated for 6 times, and finally, the uppermost layer liquid is taken to be decompressed and concentrated at the temperature of 50-60 ℃, and freeze-dried to obtain the dendrobium devonianum crude polysaccharide;

6) dissolving the crude polysaccharide of Dendrobium devonianum with water, loading a DEAE-Sepharose Fast Flow column, eluting with ultrapure water, collecting by an automatic collector, controlling the Flow rate of a peristaltic pump to be 1mL/min, and collecting one tube at 8 min. Measuring absorbance by phenol-sulfuric acid method, washing each gradient until no sugar is detected, combining same fractions according to absorption peak, concentrating under reduced pressure at 50-60 deg.C, dialyzing with flowing distilled water (dialysis membrane molecular weight cut-off of 7000) for 24h, and freeze drying to obtain semi-refined polysaccharide of herba Dendrobii;

7) dissolving semi-refined polysaccharide of Dendrobium devonianum with water, loading on Sephadex G-200 column, eluting with ultrapure water, collecting with automatic collector, peristaltic pump at flow rate of 1mL/min, and collecting in a tube for 11 min. Measuring absorbance by phenol-sulfuric acid method, washing each gradient until no sugar is detected, combining same fractions according to absorption peak, concentrating under reduced pressure at 50-60 deg.C, dialyzing with flowing distilled water (dialysis membrane molecular weight cut-off of 7000) for 24h, and freeze drying to obtain herba Dendrobii refined polysaccharide;

8) the molecular weight of the refined polysaccharide is 9.5 × 10 by high performance liquid chromatography⁴。

9) Analyzing the prepared dendrobium devonianum refined polysaccharide by gas chromatography, wherein a gas chromatogram is shown in figure 1, monosaccharide components of the dendrobium devonianum refined polysaccharide comprise mannose and glucose, and the molar ratio of the glucose to the mannose is 1: 15.85;

10) the dendrobium devonianum refined polysaccharide is analyzed by nuclear magnetic resonance, and the monosaccharide component of the polysaccharide is mainly mannose, and the polysaccharide contains an acetyl absorption peak.¹The H-NMR spectrum (500MHz) is shown in FIG. 2,¹³the C-NMR spectrum (125MHz) is shown in FIG. 3, and the structural units of the polysaccharide are shown below:

example 2

1) slicing the dendrobium devonianum raw material to obtain flaky dendrobium devonianum with the thickness of 2-5 mm;

2) placing the flaky dendrobium devonianum into a container, adding water with the weight of 30 times of that of the dendrobium devonianum, preserving the heat at 100-105 ℃ for 3 hours, filtering to obtain filtrate, repeatedly extracting the filter residue once again, filtering to obtain filtrate, and combining the two extracting solutions;

5) the precipitate was dissolved in water, and the mixture was dissolved in chloroform-n-butanol (volume ratio 4: 1) the mixture of (a) was extracted, and chloroform-n-butanol (volume ratio 4: 1) the volume ratio of the mixed solution to the precipitation solution is 1:4, the uppermost layer liquid is added with chloroform-n-butanol mixed solution with the same volume, the operation is repeated for 6 times, and finally, the uppermost layer liquid is taken to be decompressed and concentrated at the temperature of 50-60 ℃, and freeze-dried to obtain the dendrobium devonianum crude polysaccharide;

6) dissolving the crude polysaccharide of Dendrobium devonianum with water, loading a DEAE-Sepharose Fast Flow column, eluting with ultrapure water, collecting by an automatic collector, controlling the Flow rate of a peristaltic pump to be 1mL/min, and collecting one tube at 8 min. Measuring absorbance by phenol-sulfuric acid method, washing each gradient until no sugar is detected, combining same fractions according to absorption peak, concentrating under reduced pressure at 50-60 deg.C, dialyzing with flowing distilled water (dialysis membrane molecular weight cut-off of 7000) for 24h, and freeze drying to obtain herba Dendrobii refined polysaccharide.

Comparative example 1

The preparation is carried out according to the following specific mode:

dendrobium devonianum homopolysaccharide prepared according to example 1 of the Chinese patent application with application number 201610121973.X (application publication number CN 106632708A).

Comparative example 2

The preparation is carried out according to the following specific mode:

dendrobium devonianum polysaccharide was prepared according to example 1 of the chinese patent application publication No. CN107033253A with application No. 201710214362.4.

Safety test

Animal experiments prove that the dendrobium devonianum refined polysaccharide has high safety.

1 materials of the experiment

1.1 sample: the dendrobium devonianum refined polysaccharide prepared in the example 1.

1.2 Experimental animals: the melanin allele mutant Albino strain zebrafish was bred in a natural pair mating breeding manner. 240 tails in total, the age is 2 days (2dpf) after fertilization, the fish is raised in water for fish culture at the temperature of 28 ℃ (water quality: 200mg of instant sea salt is added into per 1L of reverse osmosis water, the conductivity is 480-510 mu S/cm, the pH is 6.9-7.2, and the hardness is 53.7-71.6 mg/L CaCO₃) The license number for experimental animals is as follows: SYXK (Zhe) 2012-0171. The feeding management meets the requirements of international AAALAC certification.

1.3 Main reagents: vinorelbine, colorless solution, batch No. 140501, and sealed storage at 4 ℃ in dark place; the solution is diluted with physiological saline to a mother solution with a concentration of 0.05mg/mL for use.

1.4 Main instruments: dissecting microscopes (SZX7, OLYMPUS, Japan); a camera (VertA1, China) connected to the microscope; 6 well plates (Fisher Scientific, China); precision electronic balances (CP214, OHAUS, America); microinjection apparatus (IM-300, Narishige); needle puller (PC-10, Narishige, Japan).

2 method of experiment

Randomly selecting 240 black pigment allele mutant Albinostrain zebra fish 2 days (2dpf) after fertilization to be placed in a six-hole plate, treating 30 zebra fish in each hole (experimental group), and giving a dose of 0.25 ng/tail of vinorelbine by intravenous injection to establish a zebra fish macrophage reduction model. The model zebra fish is respectively dissolved in water to be provided with the concentration of 125, 250, 500, 1000, 1500 and 2000 mug/mL of dendrobium devoninum, meanwhile, a normal control group (zebra fish treated by water for fish culture) and a model control group are arranged, and the volume of each hole (experimental group) is 3 mL. During the experiment, the death of zebrafish was observed and recorded daily and the dead zebrafish was removed. After the dendrobium devonianum refined polysaccharide is treated for 3 days, the death number and the toxicity of the zebra fish of each experimental group are counted, and the Maximum Tolerance Concentration (MTC) of the dendrobium devonianum refined polysaccharide to the zebra fish of the macrophage reduction disease model is determined according to the death number and the toxicity reaction condition of the zebra fish.

3 results of the experiment

The dendrobium devonianum refined polysaccharide does not cause the death of the zebra fish under the concentration of 125, 250, 500, 1000, 1500 and 2000 mug/mL, has no obvious toxic reaction, is similar to a normal control group and a model control group, can determine that the MTC of the dendrobium devonianum refined polysaccharide on the macrophage reduction model zebra fish is 2000 mug/mL, and shows that the dendrobium devonianum refined polysaccharide has high safety in the dosage range. See table 1 for details.

TABLE 1 experiment result of concentration-mortality after treatment of Dendrobii officmalis caulis refined polysaccharide (n is 30)

Test of drug efficacy

Animal experiments prove that the dendrobium devonianum refined polysaccharide has the function of enhancing immunity.

1.1 sample: the dendrobium devonianum refined polysaccharide prepared in the example 1; the dendrobium devonianum homogeneous polysaccharide prepared in the comparative example 1 and the dendrobium devonianum polysaccharide prepared in the comparative example 2.

1.2 Experimental animals: the melanin allele mutant Albino strain zebrafish was bred in a natural pair mating breeding manner. 150 tails in total, the age is 2 days (2dpf) after fertilization, the fish is raised in water for fish culture at the temperature of 28 ℃ (water quality: 200mg of instant sea salt is added into per 1L of reverse osmosis water, the conductivity is 480-510 mu S/cm, the pH is 6.9-7.2, the hardness is 53.7-71.6 mg/L CaCO₃) The license number for experimental animals is as follows: SYXK (Zhe) 2012-0171. The feeding management meets the requirements of international AAALAC certification.

1.3 Main reagents: vinorelbine, colorless solution, lot number 140501, purchased from Jiangsu Haofen pharmaceutical Co., Ltd, stored in a dark place at 4 ℃ in the dark; the solution is diluted with physiological saline to a mother solution with a concentration of 0.05mg/mL for use. Methylcellulose (Sigma, China); neutral red (Sigma, China).

2 method of experiment

150 black pigment allele mutant Albinostrain zebra fishes 2 days (2dpf) after fertilization are randomly selected to be placed in a six-hole plate, 30 zebra fishes are treated in each hole (experimental group), and a zebra fish macrophage reduction model is established by intravenous injection of 0.25 ng/tail dose of vinorelbine. The model zebra fish is respectively dissolved in water and is provided with refined polysaccharide 222 and 667 mu g/mL of dendrobium devonianum, the comparative example 1 group is respectively dissolved in water and is provided with homogeneous polysaccharide 222 and 667 mu g/mL of dendrobium devonianum, the comparative example 2 group is respectively dissolved in water and is provided with polysaccharide 222 and 667 mu g/mL of dendrobium devonianum, meanwhile, a normal control group (zebra fish treated by water for fish culture) and a model control group are set, and the capacity of each hole (experimental group) is 3 mL. After the zebra fish is treated for 2 days, 2 mug/mL neutral red solution is added to dye the zebra fish in vivo, the number (N) of macrophages at the head of the zebra fish is counted after dyeing is finished, and the promotion effect of the dendrobium devonianum refined polysaccharide on the formation of the macrophages is evaluated in a statistical sense. The calculation formula of the formation promoting effect of the dendrobium devonianum refined polysaccharide on the macrophages of the zebra fish is as follows:

statistical analysis using analysis of variance and Dunnett's T-test indicated significant differences with p < 0.05.

3 results of the experiment

Comparing the number (39.9) of the macrophages at the head of the zebra fish in the model control group with the number (61.9) of the macrophages in the normal control group to ensure that the p is less than 0.001, so that the successful construction of the zebra fish macrophage reduction model is prompted; the numbers of the macrophages at the head of the zebra fish in concentration groups of 222 and 667 mu g/mL of the dendrobium devonianum refined polysaccharide are 49.6, 54.5 and 46.6 respectively, compared with a model control group, the p of the concentration group of 222 mu g/mL is less than 0.01, the p of the concentration group of 667 mu g/mL is less than 0.001, the promotion effects on the formation of the macrophages of the zebra fish are 24.3% and 36.6% respectively, the result shows that the dendrobium devonianum refined polysaccharide has obvious promotion effect on the formation of the macrophages, and the result is detailed in table 2.

TABLE 2 evaluation of macrophage formation-promoting action of Dendrobii nobile Lindl refined polysaccharide (n 10)

P <0.01, p <0.001, compared to model control group

As can be seen from table 2, compared with examples 1 and 2, the dendrobium devonianum refined polysaccharide of the present invention shows that the dendrobium devonianum refined polysaccharide has an obvious promotion effect on macrophage formation and an obvious immunity enhancement effect.

Meanwhile, the refined polysaccharide of dendrobium devonianum prepared in the embodiment 1 of the invention is compounded with ginsenoside Rg1, arabinose and galactose, the first group is that the refined polysaccharide of dendrobium devonianum and ginsenoside Rg1 are compounded according to the mass ratio of 1:1 to form effective components, the second group is that the refined polysaccharide of dendrobium devonianum, the ginsenosides Rg1 and arabinose are compounded according to the mass ratio of 1: 1: 0.4: 0.2 to form effective components, then preparing the effective components with the concentrations of 222 and 667 mu g/mL, and repeating the test, the results are shown in Table 3.

Table 3(n ═ 3)

As can be seen from Table 3, the compound effective component has more obvious promotion effect on macrophage formation, more obvious immunity enhancing effect and obvious effect.

Test of drug efficacy

Experiments prove that the dendrobium devonianum refined polysaccharide prepared in example 1 is a TLR4 agonist and can directly induce macrophage activation.

1 method of experiment

1.1 cell lines and cell cultures

RAW264.7 cells (purchased from Shanghai bioscience research institute of Chinese academy of sciences) were cultured in DMEM medium supplemented with 10% heat-inactivated FBS, 100U/mL penicillin and 100. mu.g/mL streptomycin. HEK-BLUE^TMhTLR4 cells (professor Thomas C.Mitchell, university of Louis Verlag, USA) supplemented with 10% heat-inactivated FBS, 100U/mL penicillin, 100. mu.g/mL streptomycinAnd 0.4% HEK-Blue^TMThe culture in DMEM medium with antibiotic mixture was selected. Cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator.

1.2 measurement of cytokine secretion from RAW264.7 cells

RAW264.7 cells (5X 10)⁵One) was inoculated in a 24-well flat-bottom plate for 24h, and then DvP-1 (Dendrobii nobile Lindl refined polysaccharide), LPS, PMB (10. mu.g/mL) were added to the plate to a final volume of 2 mL. The culture supernatant was collected and the content of the cytokine TNF-. alpha.was determined by ELISA reagents.

1.3TLR4 activation assay

HEK-BLUE^TMHTLR4 cells (1X 10)⁴Individual cells) were seeded in 96-well plates. After 2h incubation DvP-1, LPS (positive control) was added to a final volume of 200. mu.l, incubation was continued for 24h, the supernatant was collected and quantified with QUANTI-blue^TMThe reaction was carried out, and the absorbance at 630nm was measured.

2 results of the experiment

The dendrobium devonianum refined polysaccharide is a TLR4 agonist and can directly induce macrophage activation.

We used HEK293 cell line HEK-BLUE stably expressing TLR4^TMThe direct effect of dendrobium devonianum refined polysaccharide (DvP-1) on TLR4 was studied by hTLR4 cells, and the result (figure 4a) shows that similar to positive control group LPS, DvP-1 can significantly stimulate TLR4 activation in a concentration-dependent manner at the concentration of 3.125-50 mug/mL (P)<0.001). Further using a macrophage RAW264.7 research, it is found that DvP-1 also significantly stimulates TNF-alpha secretion from RAW264.7 cells in a concentration-dependent manner. And the effect was not affected by perennial mycin b (pmb). These results indicate that DvP-1 is a TLR4 agonist and directly stimulates macrophage activation.

Claims

1. A preparation method of dendrobium devonianum refined polysaccharide is characterized by comprising the following steps:

the alcohol content of the liquid medicine refers to the volume of the concentrated solution and the ethanol in the volume ratio of the ethanol;

5) dissolving the precipitate with water to form a precipitate dissolved solution, and mixing the precipitate dissolved solution with a solvent in a volume ratio of 4: 1, extracting by using a chloroform-n-butanol mixed solution, wherein the volume ratio is 4: 1, the volume ratio of the chloroform-n-butanol mixed solution to the precipitation dissolving solution is 1:4, the uppermost layer liquid is added with the chloroform-n-butanol mixed solution with the same volume, the operation is repeated for 6 times, and finally, the uppermost layer liquid is taken to be decompressed and concentrated at the temperature of 50-60 ℃, and freeze drying is carried out to obtain the dendrobium devonianum crude polysaccharide;

6) dissolving the dendrobium devonianum crude polysaccharide with water, loading a DEAE-Sepharose Fast Flow column, eluting with ultrapure water, collecting with an automatic collector, measuring the absorbance by a phenol-sulfuric acid method, washing each gradient until sugar is detected, combining the same fractions according to an absorption peak, concentrating under reduced pressure at the temperature of 50-60 ℃, dialyzing with flowing distilled water for 24 hours, dialyzing with a dialysis membrane with the molecular weight cutoff of 7000, and freeze-drying to obtain the dendrobium devonianum semi-refined polysaccharide;

7) dissolving semi-refined polysaccharide of Dendrobium devonianum with water, loading on a Sephadex G-200 column, eluting with ultrapure water, collecting with an automatic collector, controlling the flow rate of a peristaltic pump to be 1mL/min, collecting a tube for 11min, measuring the absorbance by a phenol-sulfuric acid method, washing each gradient until sugar is detected, combining same fractions according to absorption peaks, concentrating under reduced pressure at the temperature of 50-60 ℃, dialyzing with flowing distilled water for 24h, intercepting the molecular weight of a dialysis membrane to be 7000, and freeze-drying to obtain the refined polysaccharide of Dendrobium devonianum;

8) the molecular weight of the refined polysaccharide of Dendrobium devonianum is 9.5 × 10⁴The monosaccharide consists of mannose and glucose, and the molar ratio of the glucose to the mannose is 1: 15.85.

2. the application of the dendrobium devonianum refined polysaccharide prepared by the preparation method of claim 1 in preparing functional foods and medicines for enhancing immunity and relieving physical fatigue is characterized in that the functional foods and the medicines for enhancing immunity and relieving physical fatigue comprise the dendrobium devonianum refined polysaccharide and ginsenoside Rg1 in a mass ratio of 1: 1; or the effective components are dendrobium devonianum refined polysaccharide, ginsenoside Rg1, arabinose and galactose, and the mass ratio is 1: 1: 0.4: 0.2.