CN109400742A - A kind of dendrobium devonianum refined polysaccharide and its preparation method and application - Google Patents
A kind of dendrobium devonianum refined polysaccharide and its preparation method and application Download PDFInfo
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- CN109400742A CN109400742A CN201811330972.1A CN201811330972A CN109400742A CN 109400742 A CN109400742 A CN 109400742A CN 201811330972 A CN201811330972 A CN 201811330972A CN 109400742 A CN109400742 A CN 109400742A
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- China
- Prior art keywords
- dendrobium devonianum
- refined polysaccharide
- dendrobium
- devonianum
- preparation
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Links
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- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 39
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 33
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Classifications
-
- 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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55583—Polysaccharides
Abstract
The invention discloses a kind of dendrobium devonianum refined polysaccharides and its preparation method and application, the preparation method of dendrobium devonianum refined polysaccharide includes: that dendrobium devonianum is first extracted with water, ethyl alcohol alcohol precipitation is added after extracting solution concentration, precipitating takes off albumen after being dissolved with water, dry Thick many candies, Thick many candies obtain half refined polysaccharide through ion exchange column purification, and half refined polysaccharide obtains dendrobium devonianum refined polysaccharide after purification through gel column again.The dendrobium devonianum refined polysaccharide has enhancing immune, alleviate the effect of physical fatigue, safely and effectively, in the drug and functional food that can be used for preparing the gentle physical fatigue of strengthen immunity, it is alternatively arranged as the application of immunologic adjuvant and the relevant anti-tumor drug of TLR4 that TLR4 agonist is added in vaccine in preparation.
Description
Technical field
The present invention relates to dendrobium devonianum technical fields, and in particular to a kind of dendrobium devonianum refined polysaccharide and preparation method thereof and
Using.
Background technique
TLR4 is the key that cross-film pattern recognition receptors in Toll-like receptor (TLR) family, is mainly expressed in immunocyte,
Also normal epithelium cell and cancer cell are expressed in.TLR4 is by pathogen associated molecular pattern (PAMPs) to including viral, thin
Various invasion foreign pathogens including bacterium, protozoan and fungi are reacted.In addition, it is related by dangerous (or damage)
Molecular pattern (DAMPs) identify endogenous material.These endogenous materials are usually from inflammation, oxidation, damage or necrosis
It is discharged in cell, including beta-alexin, beta-amyloid protein, peroxidase, High mobility group box-1 (HMGB1), heat shock
Albumen (HSPs), hyaluronic acid, heparin sulfate, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 etc..These abilities keep TLR4 anti-as congenital immunity and adaptive immunity
The key regulator answered, and participate in many immune and other diseases.Therefore, TLR4 has become important drug targets.TLR4
Antagonist is being developed for treating diseases associated with inflammation, including asthma, artery sclerosis, diabetes B, autoimmunity and neuritis
Property disease, and TLR4 agonist is (such as true for treating cancer and communicable disease directly or as the immunologic adjuvant in vaccine
Bacterium and helminth).
Dendrobium devonianum is the drying stem of orchid dendrobium devonianum (Dendrobium devonianum Paxton.), again
Name purple dendrobium, purple Pi Lan, fragrant rod grass, Radix Et Rhizoma Rhei, record in " Yunnan Province's Chinese medicine standard ", have reinforcing stomach reg fluid, enriching yin is clear
Heat and other effects.Dendrobium devonianum is recorded in " Yunnan Province's food safety provincial standard " simultaneously, is currently the only by administrative responsibile institution
The dendrobium species for having issued food security standard have increasingly been taken seriously to the research and development of dendrobium devonianum.
Application No. is the Chinese patent applications of 201510040088.4 (Authorization Notice No. CN104628798B) to disclose
The method for preparing anthocyanin and polysaccharide simultaneously from purple dendrobium raw material.It the described method comprises the following steps: 1) preparing Aqueous extracts:
Fresh purple dendrobium stem item is extracted in water, and is separated by solid-liquid separation, gained liquid is Aqueous extracts；2) Aqueous extracts are concentrated: will
Aqueous extracts evaporation obtains concentration Aqueous extracts；3) it extracts polysaccharide: ethyl alcohol being added in concentration Aqueous extracts, solid-liquid point is carried out after standing
From dry obtained solid is polysaccharide；4) anthocyanin is extracted: after step 3) is separated by solid-liquid separation gained liquid removal ethyl alcohol, then will
Solution is drying to obtain anthocyanin.The method can be easily by anthocyani pigment in purple dendrobium and functional active more
Sugar extracts respectively simultaneously, and obtained anthocyani pigment and purity of polysaccharide are higher；Energy consumption is lower in extraction process；This method
Both timeliness can be improved, resource can also be made full use of, with good application prospect.
Application No. is the Chinese invention patent application of 201610121973.X (application publication number CN106632708A) public affairs
A kind of method for having opened separating-purifying dendrobium devonianum homogeneous polysaccharide, is first separated with substep alcohol precipitating method, then through gel column
Purifying, can obtain the homogeneous polysaccharide of pure dendrobium devonianum.In addition, the present invention also identifies tooth valve stone using multispectral parsing
The skeleton structure of dry measure used in former times homogeneous polysaccharide, can primary structure precise Identification to the polysaccharide, specify the main polysaccharide in dendrobium devonianum
For acetyl group glucomannans.
Application No. is the Chinese invention patent application of 201710214362.4 (application publication number CN107033253A) public affairs
A kind of purple dendrobium polysaccharide and preparation method thereof with Immune enhancement and hypoglycemic activity has been opened, mentioned, alcohol precipitation, answered by water
Molten, dry or water mentions, ultrafiltration, drying and other steps prepare purple dendrobium polysaccharide, and feature: Wagner's reagent does not develop the color, molecule
Amount is 3 × 105- 5 × 105Da, monosaccharide composition are mainly mannose, acetylated mannose (Acetyl-Man) and contain a small amount of grape
Sugared (Glc) and mannose and glucose ratio of components are greater than 20:1, and glycosidic bond is mainly β-Isosorbide-5-Nitrae-Manp and β-Isosorbide-5-Nitrae-Glcp.The purple
Skin dendrobium polysaccharide has Immune enhancement and hypoglycemic effect, can be used for treating the exploitation of immunocompromised and hyperglycemia Related product.
Summary of the invention
The object of the present invention is to provide a kind of dendrobium devonianum refined polysaccharide and its preparation method and application, the dendrobium devonianums
Refined polysaccharide has the function of that enhancing is immune, and there are also the immune assistants that the polysaccharide is added in vaccine as TLR4 agonist in preparation
The application of agent and the relevant anti-tumor drug of TLR4.
To achieve this purpose, the present invention adopts the following technical scheme: dendrobium devonianum is first extracted with water, added after extracting solution concentration
Ethyl alcohol makes alcohol content reach about 80%, and alcohol precipitation, precipitating takes off albumen after being dissolved with water, dry that Thick many candies, Thick many candies are handed over through ion
It changes column purification and obtains half refined polysaccharide, half refined polysaccharide obtains dendrobium devonianum refined polysaccharide after purification through gel column again.
A kind of preparation method of dendrobium devonianum refined polysaccharide, comprising the following steps:
1) it by dendrobium devonianum raw material pre-treatment, is allowed in powdery, graininess, sheet or strip, the tooth after obtaining pre-treatment
Valve dendrobium nobile；
2) dendrobium devonianum after pre-treatment is placed in container, adds water, extracted, filter out extracting solution；
3) extracting solution is concentrated under reduced pressure into concentrate；
4) ethyl alcohol is added in concentrate, medical fluid alcohol content is made to reach 50%~90%, and alcohol precipitation must precipitate；
5) precipitating is dissolved with water, forms precipitating lysate, is extracted with chloroform-n-butanol mixed liquor, take top layer's liquid
Body is concentrated under reduced pressure, and freeze-drying obtains dendrobium devonianum Thick many candies；
6) dendrobium devonianum Thick many candies are dissolved, loading ion exchange column with water, with ultrapure water elution, collects eluent, benzene
Phenol sulfuric acid method measures absorbance, and each gradient is washed till sugar-free and is detected, and merges identical fraction according to absorption peak, is concentrated under reduced pressure, thoroughly
Analysis, freeze-drying, obtains half refined polysaccharide of dendrobium devonianum；
7) half refined polysaccharide of dendrobium devonianum is dissolved, loading gel column with water, with ultrapure water elution, collects eluent, benzene
Phenol sulfuric acid method measures absorbance, and each gradient is washed till sugar-free and is detected, and merges identical fraction according to absorption peak, is concentrated under reduced pressure, thoroughly
Analysis, freeze-drying, obtains dendrobium devonianum refined polysaccharide.
In step 1), dendrobium devonianum is broken into powder, 20~50 meshes is crossed, obtains powdery dendrobium devonianum；Or by dendrobium devonianum
Cut into slices, every with a thickness of 2~5mm, obtain sheet dendrobium devonianum, be cut into above-mentioned thickness, be conducive to effective ingredient
Extraction.
In step 2), the weight ratio of dendrobium devonianum and water after the pre-treatment is 1:10~100, further preferably
1:20~40.
The condition of the extraction are as follows: be heated to 95 DEG C~105 DEG C, heat preservation is extracted 2~4 hours.Pass through above-mentioned condition
Extraction can extract effective ingredient.
The number of the extraction is 2~4 times, is specifically included, and: Xian Jiashui extracts, extracting solution is filtered out, then by filter residue
Water equally extracts again plus equally, and so on, is divided into 2~4 times and obtains extracting solution, last combined extract.
In step 3), the condition of the reduced pressure are as follows: it is concentrated under reduced pressure at 50 DEG C~70 DEG C, further preferably,
It is concentrated under reduced pressure at 60 DEG C~70 DEG C.
In step 4), the medical fluid alcohol content refers to that concentrate is plus the volume of ethyl alcohol in the volume ratio of ethyl alcohol.
In step 5), the volume ratio of chloroform and n-butanol is 3~5:1 in chloroform-n-butanol mixed liquor, further preferably
For 4:1.
The chloroform-n-butanol (volume ratio 4:1) mixed liquor dosage and the volume ratio of precipitating lysate are 1:4.Institute
The condition for the reduced pressure stated are as follows: be concentrated under reduced pressure at 50 DEG C~60 DEG C.
In step 6), step 7), the condition of the reduced pressure are as follows: be concentrated under reduced pressure at 50 DEG C~60 DEG C.Described
Dialysis condition are as follows: for 24 hours with the distilled water dialysis (dialysis retaining molecular weight 7000) of flowing.
For the dendrobium devonianum refined polysaccharide being prepared through high effective liquid chromatography for measuring molecular weight, molecular weight is 8 × 104-5
×106；
The dendrobium devonianum refined polysaccharide being prepared becomes mannose and grape through gas chromatography analysis, monosaccharide group
The molar ratio of sugar, glucose and mannose is 1:10-1:20.
The dendrobium devonianum polysaccharide being prepared is through nuclear magnetic resonance spectroscopy, structure:
Wherein, n is duplicate structural unit number.
The dendrobium devonianum polysaccharide being prepared can be used for preparing the drug and function of the gentle physical fatigue of enhancing immunization
It can property food.Dendrobium devonianum refined polysaccharide in the drug and functional food of the gentle physical fatigue of enhancing immunization
Weight percentage be 0.1%~99.5%.
The drug and functional food of the gentle physical fatigue of enhancing immunization, effective component is mass ratio 1:
1 dendrobium devonianum refined polysaccharide and ginsenoside Rg1；Alternatively, effective component be dendrobium devonianum refined polysaccharide, ginsenoside Rg1,
Arabinose and galactolipin, mass ratio 1:1:0.4:0.2.
Specific result of study of the invention is described below: firstly, it is solidifying to create a kind of ion exchange column combination by research
The method of the purifying dendrobium devonianum polysaccharide of rubber column gel column, is separated to a kind of new dendrobium devonianum refined polysaccharide from dendrobium devonianum, point
Son amount is 9.5 × 104Da, molar ratio 15.85:1, research finds that the dendrobium devonianum refined polysaccharide is TLR4 agonist, in body
In outer and zebra fish body can direct stimulating expression of macrophage activation, it is exciting to show that the dendrobium devonianum refined polysaccharide can be used as TLR4
Agent is used to prepare the immunologic adjuvant being added in vaccine and the relevant anti-tumor drug of TLR4, and the tune as immune function
It saves agent and is used for health food.
Compared with prior art, the present invention has the advantage that
In the present invention, it is currently the only to have that dendrobium devonianum, which is with reinforcing stomach reg fluid, the rare Chinese medicine of nourishing Yin and clearing heat effect,
The dendrobium species of dual-purpose of drug and food qualification.Present invention firstly discloses the methods by using ion exchange column attached gel column purification
Dendrobium devonianum polysaccharide is prepared, because the dendrobium devonianum polysaccharide the Nomenclature Composition and Structure of Complexes being prepared is clear, and there is apparent enhancing to exempt from
Epidemic disease effect has wide application prospect suitable for developing the functional food and drug with enhancing immunization.
Detailed description of the invention
Fig. 1 is the gas chromatogram of dendrobium devonianum refined polysaccharide prepared by the embodiment of the present invention 1；
Fig. 2 is dendrobium devonianum refined polysaccharide prepared by the embodiment of the present invention 11H-NMR spectrum (500MHz)；
Fig. 3 is dendrobium devonianum refined polysaccharide prepared by the embodiment of the present invention 113C-NMR spectrogram (125MHz)；
Fig. 4 is that dendrobium devonianum refined polysaccharide prepared by the embodiment of the present invention 1 is thin as the external evoked macrophage of TLR-4 agonist
The experimental result picture of born of the same parents' activation；
Wherein a is direct action diagram of the dendrobium devonianum refined polysaccharide to TLR4 in Fig. 4, and abscissa is refined polysaccharide concentration,
Ordinate is TLR4 with respect to stimulus intensity；B is influence diagram of the dendrobium devonianum refined polysaccharide to TNF secretion-α in Fig. 4, and abscissa is
Refined polysaccharide concentration, ordinate are the content of TNF-α.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments to the present invention make into
One step explanation, but these embodiments must not be used to explain limiting the scope of the invention.
Embodiment 1
The present embodiment dendrobium devonianum refined polysaccharide, is prepared according to following concrete modes:
1) dendrobium devonianum raw material is crushed, crosses 40 mesh screens, obtains powdery dendrobium devonianum, it is spare；
2) dendrobium devonianum of powdery is placed in container, adds the water of 40 times of dendrobium devonianum weight amounts, 100 DEG C~105 DEG C guarantors
Temperature is extracted 3 hours, is filtered to get filtrate, and filter residue for another example extract once by this repetition, is filtered to get filtrate, is merged extracting solution twice；
3) extracting solution is concentrated under reduced pressure at a temperature of 60 DEG C~70 DEG C into concentrate；
4) dehydrated alcohol that 4 times of amounts are added in concentrate makes medical fluid alcohol content reach 80%, and alcohol precipitation must precipitate；
5) precipitating is dissolved with water, forms precipitating lysate, is extracted with chloroform-n-butanol (volume ratio 4:1) mixed liquor,
Chloroform-n-butanol (volume ratio 4:1) mixed liquor dosage and the volume ratio of precipitating lysate are 1:4, and top layer's liquid adds equally
The chloroform of volume-n-butanol mixed liquor repeats aforesaid operations 6 times, top layer's liquid is finally taken to subtract at a temperature of 50 DEG C~60 DEG C
Pressure concentration, freeze-drying obtain dendrobium devonianum Thick many candies；
6) dendrobium devonianum Thick many candies are dissolved with water, loading DEAE-Sepharose Fast Flow column, with ultrapure washing
De-, automatic collector is collected, and wriggling flow rate pump is 1mL/min, and 8min collects a pipe.Phenol sulfuric acid procedure measures absorbance, each
Gradient is washed till sugar-free and is detected, and merges identical fraction according to absorption peak, is concentrated under reduced pressure at a temperature of 50 DEG C~60 DEG C, then with flowing
Distilled water dialysis (dialysis retaining molecular weight 7000) for 24 hours, freeze-drying obtains half refined polysaccharide of dendrobium devonianum；
7) half refined polysaccharide of dendrobium devonianum is dissolved with water, loading Sephadex G-200 column, with ultrapure water elution, automatically
Collector is collected, and wriggling flow rate pump is 1mL/min, and 11min collects a pipe.Phenol sulfuric acid procedure measures absorbance, and each gradient is washed
It is detected to sugar-free, identical fraction is merged according to absorption peak, is concentrated under reduced pressure at a temperature of 50 DEG C~60 DEG C, then with the distillation of flowing
For 24 hours, freeze-drying obtains dendrobium devonianum refined polysaccharide for water dialysis (dialysis retaining molecular weight 7000)；
8) for dendrobium devonianum refined polysaccharide through high effective liquid chromatography for measuring molecular weight, molecular weight is 9.5 × 104。
9) the dendrobium devonianum refined polysaccharide being prepared is through gas chromatography analysis, and gas chromatogram is as shown in Figure 1, its list
Sugar, which is organized, becomes mannose and glucose, and the molar ratio of glucose and mannose is 1:15.85；
10) dendrobium devonianum refined polysaccharide further confirms that the monosaccharide components of polysaccharide are mainly sweet dew through nuclear magnetic resonance spectroscopy
Sugar, the absorption peak containing acetyl group in polysaccharide.1H-NMR spectrum (500MHz) is shown in Fig. 2,13C-NMR spectrogram (125MHz) is shown in Fig. 3,
The structural unit of polysaccharide is as follows:
Embodiment 2
The present embodiment dendrobium devonianum refined polysaccharide, is prepared according to following concrete modes:
1) by dendrobium devonianum feed stock chip, the sheet dendrobium devonianum with a thickness of 2~5mm is obtained；
2) dendrobium devonianum of sheet is placed in container, adds the water of 30 times of dendrobium devonianum weight amounts, 100 DEG C~105 DEG C guarantors
Temperature is extracted 3 hours, is filtered to get filtrate, and filter residue for another example extract once by this repetition, is filtered to get filtrate, is merged extracting solution twice；
3) extracting solution is concentrated under reduced pressure at a temperature of 60 DEG C~70 DEG C into concentrate；
4) dehydrated alcohol that 4 times of amounts are added in concentrate makes medical fluid alcohol content reach 80%, and alcohol precipitation must precipitate；
5) precipitating is dissolved with water, is extracted with chloroform-n-butanol (volume ratio 4:1) mixed liquor, chloroform-n-butanol (body
Product is than 4:1) mixed liquor dosage and the volume ratio of precipitating lysate be 1:4, top layer's liquid adds the positive fourth of the chloroform-of same volume
Alcohol mixed liquor repeats aforesaid operations 6 times, top layer's liquid is finally taken to be concentrated under reduced pressure at a temperature of 50 DEG C~60 DEG C, is freeze-dried,
Obtain dendrobium devonianum Thick many candies；
6) dendrobium devonianum Thick many candies are dissolved with water, loading DEAE-Sepharose Fast Flow column, with ultrapure washing
De-, automatic collector is collected, and wriggling flow rate pump is 1mL/min, and 8min collects a pipe.Phenol sulfuric acid procedure measures absorbance, each
Gradient is washed till sugar-free and is detected, and merges identical fraction according to absorption peak, is concentrated under reduced pressure at a temperature of 50 DEG C~60 DEG C, then with flowing
Distilled water dialysis (dialysis retaining molecular weight 7000) for 24 hours, freeze-drying obtains dendrobium devonianum refined polysaccharide.
Comparative example 1
It is prepared according to following concrete modes:
According to application No. is the Chinese invention patent Shens of 201610121973.X (application publication number CN106632708A)
Please specification embodiment 1 prepare dendrobium devonianum homogeneous polysaccharide.
Comparative example 2
It is prepared according to following concrete modes:
By application No. is the Chinese invention patent applications of 201710214362.4 (application publication number CN107033253A)
Specification embodiment 1 prepares purple dendrobium polysaccharide.
Safety testing
Animal experiment proves that dendrobium devonianum refined polysaccharide has very high safety.
1 experimental material
1.1 samples: dendrobium devonianum refined polysaccharide prepared by embodiment 1.
1.2 experimental animals: melanin allelic variants Albino strain zebra fish, with natural paired cross breeding side
Formula carries out.Totally 240 tail, age are after fertilization 2 days (2dpf), raising (water quality: every 1L reverse osmosis water in 28 DEG C of fish culture water
Middle addition 200mg Instant Ocean, conductivity are 480~510 μ S/cm；PH is 6.9~7.2；Hardness is 53.7~71.6mg/L
CaCO3), experimental animal uses credit number are as follows: SYXK (Zhejiang) 2012-0171.Feeding management meets international AAALAC certification
It is required that.
1.3 main agents: vinorelbine, colourless solution, 140501,4 DEG C of lot number are protected from light closed preservation；Face used time life
Reason salt water is diluted to the mother liquor that concentration is 0.05mg/mL.
1.4 key instruments: disecting microscope (SZX7, OLYMPUS, Japan)；Be connected with microscope camera (VertA1,
China)；6 orifice plates (Fisher Scientific, China)；Precision electronic balance (CP214, OHAUS, America)；It is micro-
Injection instrument (IM-300, Narishige)；It draws needle instrument (PC-10, Narishige, Japan).
2 experimental methods
240 tail after fertilization, 2 days (2dpf) melanin allelic variants Albino strain zebra fish are randomly selected in six
In orifice plate, every hole (experimental group) handles 30 tail zebra fish, and intravenous injection gives vinorelbine 0.25ng/ tail dosage and establishes zebra
Fish macrophage reduces disease model.Model zebra fish respectively it is water-soluble give dendrobium devonianum refined polysaccharide 125,250,500,1000,
1500 and 2000 μ g/mL concentration, while Normal group (fish culture water handles zebra fish) and model control group, every hole are set
(experimental group) capacity is 3mL.During the experiment, the death condition of zebra fish is observed and recorded daily and removes dead zebra
Fish.After dendrobium devonianum refined polysaccharide is handled 3 days, the zebra fish The dead quantity and toxicity profile of each experimental group are counted, according to zebra
The The dead quantity and toxic reaction situation of fish determine that dendrobium devonianum refined polysaccharide reduces disease model zebra fish most to macrophage
Big tolerable concentration (MTC).
3 experimental results
Dendrobium devonianum refined polysaccharide does not cause zebra fish under 125,250,500,1000,1500 and 2000 μ g/mL concentration
Death, and have no that overt toxicity reacts, it is similar to Normal group and model control group, it may be determined that dendrobium devonianum refined polysaccharide pair
The MTC that macrophage reduces disease model zebra fish is 2000 μ g/mL, shows that dendrobium devonianum refined polysaccharide has in the dosage range
There is very high security.See Table 1 for details.
" concentration-death rate " experimental result (n=30) after the processing of 1. dendrobium devonianum refined polysaccharide of table
Pharmacodynamic test
Animal experiment proves that dendrobium devonianum refined polysaccharide has the function of that enhancing is immune.
1.1 samples: dendrobium devonianum refined polysaccharide prepared by embodiment 1；The dendrobium devonianum homogeneous polysaccharide of the preparation of comparative example 1,
Purple dendrobium polysaccharide prepared by comparative example 2.
1.2 experimental animals: melanin allelic variants Albino strain zebra fish, with natural paired cross breeding side
Formula carries out.Totally 150 tail, age are after fertilization 2 days (2dpf), raising (water quality: every 1L reverse osmosis water in 28 DEG C of fish culture water
Middle addition 200mg Instant Ocean, conductivity are 480~510 μ S/cm；PH is 6.9~7.2；Hardness is 53.7~71.6mg/L
CaCO3), experimental animal uses credit number are as follows: SYXK (Zhejiang) 2012-0171.Feeding management meets international AAALAC certification
It is required that.
1.3 main agents: vinorelbine, colourless solution, lot number 140501, purchased from the gloomy limited public affairs of medicine company share of Jiangsu person of outstanding talent
Department, 4 DEG C are protected from light closed preservation；Face the mother liquor that the used time is 0.05mg/mL at concentration with normal saline dilution.Methylcellulose
(Sigma, China)；Dimethyl diaminophenazine chloride (Sigma, China).
1.4 key instruments: disecting microscope (SZX7, OLYMPUS, Japan)；Be connected with microscope camera (VertA1,
China)；6 orifice plates (Fisher Scientific, China)；Precision electronic balance (CP214, OHAUS, America)；It is micro-
Injection instrument (IM-300, Narishige)；It draws needle instrument (PC-10, Narishige, Japan).
2 experimental methods
150 tail after fertilization, 2 days (2dpf) melanin allelic variants Albino strain zebra fish are randomly selected in six
In orifice plate, every hole (experimental group) handles 30 tail zebra fish, and intravenous injection gives vinorelbine 0.25ng/ tail dosage and establishes zebra
Fish macrophage reduces disease model.Model zebra fish is water-soluble respectively to give dendrobium devonianum refined polysaccharide 222,667 μ g/mL concentration,
1 group of comparative example is water-soluble respectively to give dendrobium devonianum homogeneous polysaccharide 222,667 μ g/mL concentration, and 2 groups of comparative example water-soluble respectively to give purple
Skin dendrobium polysaccharide 222,667 μ g/mL concentration, while Normal group (fish culture water handles zebra fish) and model comparison are set
Group, every hole (experimental group) capacity are 3mL.After zebra fish is handled 2 days, 2 μ g/mL neutral red solutions is added, living body is carried out to zebra fish
Dyeing counts zebra fish head macrophage quantity (N) after dyeing, evaluates dendrobium devonianum refined polysaccharide with statistical significance
The facilitation that macrophage is formed.Dendrobium devonianum refined polysaccharide forms facilitation calculation formula to zebra fish macrophage
It is as follows:
With variance analysis and Dunnett ' s T- inspection carry out statistical analysis, p < 0.05 shows with significant difference.
3 experimental results
Model control group zebra fish head macrophage quantity (39.9) compared with Normal group (61.9) p <
0.001, prompt zebra fish macrophage to reduce the success of disease model construction；Dendrobium devonianum refined polysaccharide 222,667 μ g/mL concentration groups
Zebra fish head macrophage quantity is respectively 49.6,54.5 and 46.6, compared with model control group, 222 μ g/mL concentration groups
P < 0.01,667 μ g/mL concentration group p < 0.001, to zebra fish macrophage formed facilitation be respectively 24.3%,
36.6%, display dendrobium devonianum refined polysaccharide, which forms macrophage, has apparent facilitation, and as a result see Table 2 for details.
2. dendrobium devonianum refined polysaccharide of table forms facilitation evaluation experimental result (n=10) to macrophage
Compared with model control group, p < 0.001 * * p < 0.01, * * *
As can be seen from Table 2, dendrobium devonianum refined polysaccharide of the present invention shows dendrobium devonianum essence compared to embodiment 1 and embodiment 2
Polysaccharide processed, which forms macrophage, has apparent facilitation, has apparent enhancing immunization.
Meanwhile the dendrobium devonianum refined polysaccharide for preparing the embodiment of the present invention 1 and ginsenoside Rg1, arabinose, gala
Sugar compounding, first group is to compound dendrobium devonianum refined polysaccharide with ginsenoside Rg1 1:1 in mass ratio, forms effective component, the
Two groups are by dendrobium devonianum refined polysaccharide and ginsenoside Rg1, arabinose, and galactolipin 1:1:0.4:0.2 in mass ratio is compounded,
Effective component is formed, is made into effective component concentration 222,667 μ g/mL concentration later, carries out repeating test, the results are shown in Table 3.
Table 3 (n=3)
As shown in Table 3, using the effective component after compounding, being formed to macrophage has more obvious facilitation,
With more obvious enhancing immunization, significant effect.
Pharmacodynamic test
The dendrobium devonianum refined polysaccharide for being verified by experiments the preparation of embodiment 1 is TLR4 agonist, can directly induce macrophage thin
Born of the same parents' activation.
1 experimental method
1.1 cell lines and cell culture
RAW264.7 cell (be purchased from Chinese Academy of Sciences Shanghai bioscience research institute), 10% heat inactivation FBS of addition,
It is cultivated in the DMEM culture medium of 100U/mL penicillin and 100 μ g/mL streptomysins.HEK-BLUETM- hTLR4 cell (U.S. Louis
Si Weier university Thomas professor C.Mitchell present), in addition 10% heat inactivation FBS, 100U/mL penicillin, 100 μ g/
ML streptomysin and 0.4%HEK-BlueTMIt selects to cultivate in the DMEM culture medium of antibiotic cocktail.Cell is in 37 DEG C, 5%CO2
It is cultivated in incubator.
The measurement of 1.2RAW264.7 cell secretion of cytokines
By RAW264.7 cell (5 × 105It is a) it is inoculated in 24 hole flat bottom plate cultures for 24 hours, DvP-1 (tooth valve is then added
Dendrobium nobile refined polysaccharide), LPS, PMB (10 μ g/ml), final volume 2mL.Medium supernatant is collected, is measured with ELISA reagent
The content of cytokine TNF-α.
1.3TLR4 activation determination
By HEK-BLUETM- HTLR4 cell (1 × 104A cell) it is inoculated in 96 hole plates.After cultivating 2h, DvP- is added
1, LPS (positive control), final volume are 200 μ l, continue culture for 24 hours, supernatant are collected, with quantitative QUANTI-blueTMInstead
It answers, absorbance is measured at 630nm.
2 experimental results
Dendrobium devonianum refined polysaccharide is TLR4 agonist, and can directly inducing macrophage activation.
We express the HEK293 cell line HEK-BLUE of TLR4 with stablizingTM- hTLR4 cell research dendrobium devonianum purification
Direct effect of the polysaccharide (DvP-1) to TLR4, as a result (Fig. 4 a) shows similar to positive controls LPS, DvP-1 3.125~
The TLR4 activation (P < 0.001) that energy concentration dependant manner significantly stimulates between 50 μ g/mL concentration.Further use macrophage
RAW264.7 is the study found that DvP-1 also concentration dependent significantly stimulates RAW264.7 cell TNF secretion-α.And the effect does not have
There is the influence by many years rhzomorph B (PMB)., the result shows that DvP-1 is TLR4 agonist, energy directly stimulating expression of macrophage is living for these
Change.
Claims (10)
1. a kind of preparation method of dendrobium devonianum refined polysaccharide, which comprises the following steps:
1) it by dendrobium devonianum raw material pre-treatment, is allowed in powdery, graininess, sheet or strip, the tooth valve stone after obtaining pre-treatment
Dry measure used in former times；
2) dendrobium devonianum after pre-treatment is placed in container, adds water, extracted, filter out extracting solution；
3) extracting solution is concentrated under reduced pressure into concentrate；
4) ethyl alcohol is added in concentrate, medical fluid alcohol content is made to reach 50~90%, and alcohol precipitation must precipitate；
5) precipitating is dissolved with water, forms precipitating lysate, is extracted with chloroform-n-butanol mixed liquor, top layer's liquid is taken to subtract
Pressure concentration, freeze-drying obtain dendrobium devonianum Thick many candies；
6) dendrobium devonianum Thick many candies are dissolved, loading ion exchange column with water, with ultrapure water elution, collects eluent, phenol sulphur
Acid system measures absorbance, and each gradient is washed till sugar-free and is detected, and merges identical fraction according to absorption peak, is concentrated under reduced pressure, and dialysis is cold
It is lyophilized dry, obtains half refined polysaccharide of dendrobium devonianum；
7) half refined polysaccharide of dendrobium devonianum is dissolved, loading gel column with water, with ultrapure water elution, collects eluent, phenol sulphur
Acid system measures absorbance, and each gradient is washed till sugar-free and is detected, and merges identical fraction according to absorption peak, is concentrated under reduced pressure, and dialysis is cold
It is lyophilized dry, obtains dendrobium devonianum refined polysaccharide.
2. the preparation method of dendrobium devonianum refined polysaccharide according to claim 1, which is characterized in that described in step 2)
Pre-treatment after dendrobium devonianum and water weight ratio be 1:10~100；
The condition of the extraction are as follows: be heated to 95 DEG C~105 DEG C, heat preservation is extracted 2~4 hours；
The number of the extraction is 2~4 times.
3. the preparation method of dendrobium devonianum refined polysaccharide according to claim 1, which is characterized in that described in step 3)
Reduced pressure condition are as follows: be concentrated under reduced pressure at 50 DEG C~70 DEG C.
4. the preparation method of dendrobium devonianum refined polysaccharide according to claim 1, which is characterized in that described in step 5)
Chloroform-n-butanol mixed liquor in the volume ratio of chloroform and n-butanol be 4:1；
The volume ratio of chloroform-n-butanol mixed liquor dosage and precipitating lysate is 1:4；
The condition of the reduced pressure are as follows: be concentrated under reduced pressure at 50 DEG C~60 DEG C.
5. the preparation method of dendrobium devonianum refined polysaccharide according to claim 1, which is characterized in that step 6), step 7)
In, the condition of the reduced pressure are as follows: be concentrated under reduced pressure at 50 DEG C~60 DEG C；
The dialysis condition are as follows: for 24 hours with the distilled water dialysis of flowing, retaining molecular weight 7000 of dialysing.
6. the dendrobium devonianum refined polysaccharide of described in any item preparation method preparations according to claim 1~5.
7. dendrobium devonianum refined polysaccharide according to claim 6, which is characterized in that the dendrobium devonianum refined polysaccharide
Molecular weight is 8 × 104-5×106, monosaccharide group becomes mannose and glucose, and the molar ratio of glucose and mannose is 1:10-
1:20。
8. dendrobium devonianum refined polysaccharide according to claim 6 is in the medicine of the gentle physical fatigue of preparation enhancing immunization
Application in object and functional food.
9. application according to claim 8, which is characterized in that the medicine of the gentle physical fatigue of enhancing immunization
The weight percentage of dendrobium devonianum refined polysaccharide is 0.1%~99.5% in object and functional food.
10. dendrobium devonianum refined polysaccharide according to claim 6 is preparing immunologic adjuvant and TLR4 as TLR4 agonist
Application in relevant anti-tumor drug.
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