CN108948221A - A kind of Cultivation of Dictyophora polysaccharide and its preparation and application with neuroprotective efficacy - Google Patents
A kind of Cultivation of Dictyophora polysaccharide and its preparation and application with neuroprotective efficacy Download PDFInfo
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Abstract
The invention belongs to the field of Chinese medicines, and in particular to a kind of Cultivation of Dictyophora polysaccharide and its preparation and application with neuroprotective efficacy.The preparation method of Cultivation of Dictyophora polysaccharide provided by the invention with neuroprotective efficacy include Cultivation of Dictyophora dries pulverizing, degreasing, water are mentioned, removing protein, ethanol precipitation, then carry out ion exchange, dialysis and gel chromatography process by cation seperation column and anion column respectively and isolate and purify.Preparation process of the present invention is simple, stablizes, is efficient, being suitble to large-scale production; resulting Cultivation of Dictyophora polysaccharide can effectively inhibit neurotoxicity; play good neuroprotection; it can be applied to the exploitation of the ancillary drug and the health food with neuroprotection of nervous system disease, to prevent the nervous system disease and slowing down aging to provide new strategy.
Description
Technical field
The invention belongs to the field of Chinese medicines, and in particular to a kind of Cultivation of Dictyophora polysaccharide and its preparation with neuroprotective efficacy
And application.
Background technique
The nervous system disease is a kind of disease for mainly leading to neurological deficit as pathological characters using neuronal structure destruction
Disease can cause dysfunctions, the lethalities of disabling such as patient's study, memory, speech, emotion and movement higher.With population in the world
The disease incidence of the aggravation of aging degree, the nervous system disease is gradually increasing, it has also become threatens people's physical and mental health and life matter
The outstanding problem of amount.Although increasingly increasing the exploitation of nervous system disease drug investment, at present to such disease before
The occurrence and development process of phase and corresponding prevention still lack concern, therefore there is active development the health care of neuroprotective efficacy to eat
Product are significant to prevention and treatment the nervous system disease.
Dictyophora phalloidea is the traditional rare edible mushroom in China, is known as the good reputations such as " flower of mountain delicacy ", " queen in bacterium ".Studies have shown that
The main chemical compositions of dictyophora phalloidea include the small molecules such as the macromoleculars such as polysaccharide, protein and ketone, terpene, pharmacological activity with
Polysaccharide component is closely related.Existing research shows, dictyophora fungus polysaccharide has immunological regulation, antitumor, anti-aging and anti-oxidant etc. more
Kind biological activity.
Chinese patent CN104262500B discloses " a kind of to have immunocompetent novel red support dictyophora fungus polysaccharide and its preparation
Methods and applications ", the method be using Dictyophora rubrovalvata as raw material, by flooding, Sevage method removing protein, ethanol precipitation,
Then dictyophora fungus polysaccharide is obtained using ion-exchange chromatography, gel filtration chromatography and ultra-filtration and separation after purification.Invention preparation
Dictyophora rubrovalvata polysaccharide can significantly improve NO in mouse macrophage RAW264.7, TNF-α, IL-6 expression, have and good exempt from
Epidemic disease facilitation can be used as the adjuvant therapy medicaments of the immunocompromised diseases such as tumour or improve immunity as having
The functional health-care food or food of effect.But the Dictyophora rubrovalvata polyoses producing method without reference to raw material broken wall, degreasing and
Dialysis treatment, the recovery rate for obtaining polysaccharide are lower and can be containing the oil-soluble impurities such as pigment.
Chinese patent CN103044566B discloses " preparation method that a kind of dictyophora phalloidea water proposes antioxidant polysaccharide in residue ",
This method is the 5%NaOH/0.05%NaBH that preparation is added after grinding dictyophora phalloidea drying4Water mentions in mixed ammonium/alkali solutions, then carries out
The removal, decoloration and the removal of small molecular weight impurity of water-insoluble α-(1 → 3)-d- glucan, finally carry out in polysaccharide extraction liquid
Purifying, dialysis and the freeze-drying of polysaccharide, obtain the dictyophora fungus polysaccharide with antioxidant activity.But the polyoses producing method is not adopted
With raw material broken wall, removing protein, ion exchange and sieve chromatography, the yield and purity of obtained polysaccharide are lower, and this method
It needs to adjust pH, so that whole operation is complex, be difficult to control.
Chinese patent CN104277134B discloses a kind of " system of dictyophora fungus polysaccharide-chelates of zinc with anti-tumor activity
Preparation Method and its application ", this method are that after the dictyophora phalloidea fructification that will be dried crushes, dictyophora fungus polysaccharide is carried out after boiling waterbath polysaccharide
Separation, purifying；Then inorganic zinc source is added in dictyophora fungus polysaccharide solution and carries out chelatropic reaction, through alcohol precipitation, be freeze-dried to obtain bamboo
Sweet-smelling grass polysaccharide-chelates of zinc.The chelate has apparent inhibiting effect to HepG2 cell, and influences less on normal liver cell, tool
There are the potentiality applied to oncotherapy.But the dictyophora fungus polysaccharide preparation method does not use raw material broken wall, degreasing, cation exchange
And dialysis treatment, the yield and purity of obtained polysaccharide are limited.
Immunological regulation and anti-tumor aspect are had focused largely on for the research of dictyophora fungus polysaccharide bioactivity at present, to it in mind
Through the research and application concern deficiency in systemic disease field, the application that there has been no dictyophora fungus polysaccharides in terms of neuroprotection.Meanwhile
At present for dictyophora fungus polysaccharide extraction mostly use greatly crushing, water mention with these easy steps of alcohol precipitation, lack to the fine of raw material
Processing, effectively removing comprehensively and the homogeneity of polysaccharide component is purified to impurity, cause polysaccharide yield and purity by
It influences.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of preparation method of dictyophora fungus polysaccharide, dictyophora fungus polysaccharide is realized
It high efficiency extraction and isolates and purifies, and verify Cultivation of Dictyophora polysaccharide there is defencive function to nervous system.One aspect of the present invention provides
Cultivation of Dictyophora polysaccharide the preparation method comprises the following steps: using Cultivation of Dictyophora as raw material, broken wall crushes after Cultivation of Dictyophora is dried, through degreasing,
Water mentions, removing protein and ethanol precipitation, then carries out ion exchange, dialysis and gel chromatography by cation and anion column respectively
Process isolates and purifies acquisition.Specific steps include:
(1) broken wall crushes after drying Cultivation of Dictyophora at 40-60 DEG C, obtains the Cultivation of Dictyophora powder that partial size is 1-10 μm；
(2) by Cultivation of Dictyophora powder obtained by step (1) with organic solvent degreasing 2-5 times, solid-liquid ratio g/mL is 1:5-1:40,
Skimming temp is 50-90 DEG C, degreasing time 2-6h, obtains degreasing Cultivation of Dictyophora powder；
It (3) is 1:10- by step (2) resulting degreasing Cultivation of Dictyophora powder distillation flooding 2-6h, solid-liquid ratio g/mL
1:40, extraction temperature are 60-100 DEG C, and extracting times are 1-3 times, obtain Aqueous extracts；
(4) de- albumen is carried out to Aqueous extracts obtained by step (3) using Sevage method to handle, Aqueous extracts and Sevage reagent
Volume ratio is 4:1-1:4, is centrifuged 4-8min in 6000-8000r/min after concussion 25-35min, and number of processes is 5-9 times, is obtained de-
Albumen Aqueous extracts；
(5) it utilizes and takes off albumen Aqueous extracts obtained by methanol precipitation step (4), the volume ratio of ethyl alcohol and de- albumen Aqueous extracts is 4:
It is centrifuged 5-10min in 6000-12000r/min after 1-9:1,0-4 DEG C of standing 6-24h, obtains Cultivation of Dictyophora Thick many candies；
(6) cation exchange column is crossed after Cultivation of Dictyophora Thick many candies obtained by step (5) being dissolved in distilled water, using distilled water
Elution is concentrated after collecting eluent, then crosses anion-exchange column, is successively eluted using distilled water and salt ion solution,
It is concentrated after collecting salt ion eluent, the salt ion eluent after must being concentrated；
(7) the salt ion eluent after concentration obtained by step (6) is subjected to flowing water dialysis treatment, the retention molecule of bag filter
Amount is 1.0-3.5kD, is concentrated after collecting trapped fluid, the dialysis trapped fluid after must being concentrated；
(8) solvent resistant column is added in the dialysis trapped fluid after concentration obtained by step (7) and is in charge of company to distill water elution
It is continuous to collect eluent, the polyoses content in eluent is then detected, the eluent that main absorption peak represents is collected, is finally concentrated
And drying, obtain Cultivation of Dictyophora polysaccharide.
Further, Cultivation of Dictyophora described in the step (1) refers to the edible position cap of Cultivation of Dictyophora, bacterium
Skirt, stem, volva or fructification.
Further, organic solvent described in the step (2) is ethyl alcohol, methanol, acetone, ether, petroleum ether or it is mixed
Close liquid.
Further, in the step (6), the material of anion-exchange column and cation exchange column in the step (6)
For cellulose, sephadex, Ago-Gel or resin；The salt ion solution is acetate, phosphate, barbiturates
Salt, citrate, sodium chloride, potassium chloride, ammonium chloride or glycine.
Further, in the step (8), the material of solvent resistant column is sephadex, Ago-Gel, polypropylene
One in acrylamide gel, cross-link dextran and bisacrylamide gel copolymer, Sepharose and glucan covalent bond gel
Kind.
The longuette bamboo obtained using the preparation method of the Cultivation of Dictyophora polysaccharide disclosed by the invention with neuroprotective efficacy
The recovery rate of sweet-smelling grass polysaccharide is high, with high purity, stability is good.
On the other hand, the invention also discloses a kind of Cultivation of Dictyophora polysaccharide with neuroprotective efficacy；
Further, experimental study proves that many kinds of substance can be effectively relieved to human neuroblastoma in Cultivation of Dictyophora polysaccharide
The toxic effect of cell (SH-SY5Y) reduces neurological disease incidence；
Further, Cultivation of Dictyophora polysaccharide can inhibit beta-amyloid protein and 6- hydroxyl dopamine in Caenorhabditis elegans mould
The intracorporal neurotoxicity of type, plays neuroprotection；
Further, Cultivation of Dictyophora polysaccharide can alleviate the intracorporal oxidative stress damage of caenorhabditis elegan disease model and mitochondria
Dysfunction can be used for preventing and treating with protein abnormal aggregation, mitochondria dysfunction, excitatory toxicity, oxidative damage and nerve
First apoptosis etc. is the nervous system disease of major pathologic features, to play the role of neuroprotection and functional rehabilitation.
Another object of the present invention is to provide the Cultivation of Dictyophora polysaccharide with neuroprotection in preparation nervous system
Application in adjuvant object.
A further object of the present invention is to provide the Cultivation of Dictyophora polysaccharide with neuroprotective efficacy to have nerve in preparation
Purposes in the health food of protection effect.
Compared with prior art, the present invention has the advantage that
(1) preparation method of the Cultivation of Dictyophora polysaccharide provided by the invention with neuroprotective efficacy, the method technique
Advantages of simple, stability and high efficiency are suitble to large-scale production.
(2) preparation method of the Cultivation of Dictyophora polysaccharide provided by the invention with neuroprotective efficacy, will in the method
It is broken that wall cell disruption powder is carried out after Cultivation of Dictyophora is dry, makes that raw material availability is higher, the extraction of Cultivation of Dictyophora polysaccharide is more thorough.
(3) preparation method of the Cultivation of Dictyophora polysaccharide provided by the invention with neuroprotective efficacy obtains in the method
Dictyophora phalloidea Thick many candies successively pass through cation seperation column, anion column, dialysis retention and sieve chromatography, make polysaccharide charge property and
Molecular weight more stable uniform, gained Cultivation of Dictyophora purity of polysaccharide are high.
(4) Cultivation of Dictyophora polysaccharide prepared by the present invention is integrally moved in mammalian neural cell level and Caenorhabditis elegans
It is equal to the neurotoxicity of protein abnormal aggregation, mitochondria dysfunction, excitatory toxicity and oxidative damage induction in object level
It is inhibited, it can be applied to the adjuvant therapy medicaments of the nervous system disease and the health food with neuroprotective efficacy
In, to provide new strategy for the nervous system disease prevention and treatment and successful aging, and deeply opening for natural polysaccharide product can be promoted
Hair and application.
Detailed description of the invention
Fig. 1 is the infrared spectrogram of Cultivation of Dictyophora polysaccharide；
Fig. 2 is that the monosaccharide of Cultivation of Dictyophora polysaccharide forms gas chromatogram.
Specific embodiment
The preparation method of Cultivation of Dictyophora polysaccharide provided by the invention with neuroprotective efficacy, specifically: by longuette bamboo
Broken wall crushes after sweet-smelling grass drying, mentions through degreasing, water, removing protein and ethanol precipitation, then successively passes through cation exchange column and anion
Exchange column, flowing water dialysis and gel chromatography process isolate and purify the Cultivation of Dictyophora polysaccharide for obtaining and having neuroprotective efficacy.
A part of specific embodiment is set forth below the present invention is further detailed, specific item is not specified in embodiment
Part person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer, being can
With the conventional products obtained by commercially available purchase.The embodiment is the preferable embodiment of the present invention, but implementation of the invention
Mode is simultaneously not restricted to the described embodiments, it is other it is any without departing from the spirit and principles of the present invention made by change
Become, modification, substitution, combination, simplify, should be equivalent substitute mode, be included in protection scope of the present invention.
Embodiment 1, a kind of preparation method of the Cultivation of Dictyophora polysaccharide with neuroprotective efficacy
The preparation method of the Cultivation of Dictyophora polysaccharide with neuroprotective efficacy, includes the following steps:
(1) 200g Cultivation of Dictyophora fructification is dry at 40 DEG C, broken wall crushing is carried out to dictyophora phalloidea using micronizer,
Obtain the Cultivation of Dictyophora powder that partial size is 5 μm；
(2) ethyl alcohol is added according to solid-liquid ratio 1:10 (g/mL) in Cultivation of Dictyophora powder, the heating stirring reflux 2h at 75 DEG C,
It is filtered to remove solvent, is repeated the defatting step 3 times, degreasing dictyophora phalloidea powder is obtained；
(3) by degreasing dictyophora phalloidea powder according to solid-liquid ratio 1:15 (g/mL) plus distilled water, heating stirring extracts 3h at 95 DEG C,
Aqueous extracts are collected by filtration, repeat the aqueous extraction step 2 times, Aqueous extracts are concentrated under reduced pressure；
(4) Aqueous extracts will be concentrated, Sevage reagent (chloroform: the volume ratio of n-butanol is 4:1) is added according to volume ratio 1:1,
Centrifugation (6000r/min, 5min) takes supernatant after concussion 30min, repeats the removing protein step 7 time, obtains de- albumen Aqueous extracts；
(5) 4 times of volume ethanols are added in Aqueous extracts according to volume ratio 4:1 for de- albumen Aqueous extracts, after mixing at 4 DEG C
18h is staticly settled, centrifugation (6000r/min, 10min) collects precipitating, obtains Cultivation of Dictyophora Thick many candies after vacuum freeze drying；
(6) Cultivation of Dictyophora Thick many candies are dissolved in after distilled water and cross 732 type cation exchange resins, using 4 times of column volumes
Water elution (flow velocity 2BV/h), is collected after being concentrated under reduced pressure after eluent after DEAE Sepharose FF anion-exchange column, according to
It is secondary that (flow velocity 2.0mL/min) is eluted using water and 1M sodium chloride solution, collect the eluent of 1M sodium chloride solution；
(7) it uses molecular cut off to carry out flowing water dialysis for the bag filter of 3.5kD after eluent is concentrated under reduced pressure, collects retention
It is concentrated under reduced pressure after liquid；
(8) Sephadex G-200 solvent resistant column is added in dialysis trapped fluid and is in charge of continuous collection to distill water elution
Eluent (flow velocity 1.0mL/min) then detects the polyoses content in every pipe eluent using Phenol sulfuric acid procedure, collects main inhale
The eluent that peak represents is received, vacuum freeze drying is finally carried out and obtains Cultivation of Dictyophora polysaccharide.
Embodiment 2, a kind of preparation method of the Cultivation of Dictyophora polysaccharide with neuroprotective efficacy
The preparation method of the Cultivation of Dictyophora polysaccharide with neuroprotective efficacy, includes the following steps:
(1) 200g Cultivation of Dictyophora fructification is dry at 50 DEG C, broken wall crushing is carried out to dictyophora phalloidea using micronizer,
Obtain the Cultivation of Dictyophora powder that partial size is 1；
(2) ethyl alcohol is added according to solid-liquid ratio 1:20g/mL respectively in Cultivation of Dictyophora powder, successively the heating stirring at 80 DEG C
Flow back 3h, is filtered to remove solvent, repeats the defatting step 2 times, obtains degreasing dictyophora phalloidea powder；
(3) by degreasing dictyophora phalloidea powder according to solid-liquid ratio 1:20 (g/mL) plus water, heating stirring extracts 3h, filtering at 85 DEG C
Aqueous extracts are collected, are repeated the aqueous extraction step 3 times, Aqueous extracts are concentrated under reduced pressure；
(4) Aqueous extracts will be concentrated, Sevage reagent (chloroform: the volume ratio of n-butanol is 4:1) is added according to volume ratio 1:1,
Centrifugation (6000r/min, 8min) takes supernatant after concussion 25min, repeats the removing protein step 5 time, obtains de- albumen Aqueous extracts；
(5) 6 times of volume ethanols are added in Aqueous extracts according to volume ratio 6:1 for de- albumen Aqueous extracts, after mixing at 4 DEG C
12h is staticly settled, centrifugation (8000r/min, 5min) collects precipitating, obtains Cultivation of Dictyophora Thick many candies after vacuum freeze drying；
(6) JK008 type cation exchange resin is crossed after Cultivation of Dictyophora Thick many candies being dissolved in distilled water, using 4 times of column volumes
Water elution (flow velocity 2BV/h), collect eluent after be concentrated under reduced pressure, after Q Sepharose XL anion-exchange column, successively
Using water and 1M sodium chloride solution elution (flow velocity 2.0mL/min), the eluent of 1M sodium chloride solution is collected；
(7) it uses molecular cut off to carry out flowing water dialysis for the bag filter of 2.0kD after eluent is concentrated under reduced pressure, collects retention
It is concentrated under reduced pressure after liquid；
(8) dialysis trapped fluid is added and 6 FF solvent resistant column of Sepharose is added in dialysis trapped fluid, with distilled water
Elution, is in charge of continuous collection eluent (flow velocity 1.0mL/min), is then detected using differential refractometer more in every pipe eluent
Sugared content, collects the eluent that major absorbance peak represents, and finally carries out vacuum freeze drying and obtains Cultivation of Dictyophora polysaccharide.
Embodiment 3, a kind of preparation method of the Cultivation of Dictyophora polysaccharide with neuroprotective efficacy
The preparation method of the Cultivation of Dictyophora polysaccharide with neuroprotective efficacy, includes the following steps:
(1) 200g Cultivation of Dictyophora fructification is dry at 50 DEG C, broken wall crushing is carried out to dictyophora phalloidea using micronizer,
Obtain the Cultivation of Dictyophora powder that partial size is 10 μm；
(2) petroleum ether is added according to solid-liquid ratio 1:15g/mL respectively in Cultivation of Dictyophora powder, successively heats and stirs at 50 DEG C
Reflux 6h is mixed, solvent is filtered to remove, is repeated the defatting step 5 times, degreasing dictyophora phalloidea powder is obtained；
(3) by degreasing dictyophora phalloidea powder according to solid-liquid ratio 1:30 (g/mL) plus water, heating stirring extracts 2h, mistake at 100 DEG C
Aqueous extracts are collected in filter, are repeated the aqueous extraction step 2 times.Aqueous extracts are concentrated under reduced pressure；
(4) Aqueous extracts will be concentrated, Sevage reagent (chloroform: the volume ratio of n-butanol is 4:1) is added according to volume ratio 4:1,
Centrifugation (8000r/min, 4min) takes supernatant after concussion 30min, repeats the removing protein step 6 time, obtains de- albumen Aqueous extracts；
(5) 9 times of volume ethanols are added in Aqueous extracts according to volume ratio 9:1 for de- albumen Aqueous extracts, after mixing at 4 DEG C
8h is staticly settled, centrifugation (12000r/min, 5min) collects precipitating, obtains Cultivation of Dictyophora Thick many candies after vacuum freeze drying；
(6) Cultivation of Dictyophora Thick many candies are dissolved in after distilled water and cross D113 type cation exchange resin, using 4 times of column volumes
Water elution (flow velocity 2BV/h) is concentrated under reduced pressure after collecting eluent, after DEAE-52 cellulose anion exchange column, successively uses
Water and 1M sodium chloride solution elution (flow velocity 2.0mL/min), collect the eluent of 1M sodium chloride solution；
(7) it uses molecular cut off to carry out flowing water dialysis for the bag filter of 3.0kD after eluent is concentrated under reduced pressure, collects retention
It is concentrated under reduced pressure after liquid；
(8) dialysis trapped fluid is added and Sepharose 6B solvent resistant column is added in dialysis trapped fluid, to distill washing
It is de-, it is in charge of continuous collection eluent (flow velocity 1.0mL/min), the polysaccharide in every pipe eluent is then detected using differential refractometer
Content, collects the eluent that major absorbance peak represents, and finally carries out vacuum freeze drying and obtains Cultivation of Dictyophora polysaccharide.
Embodiment 4, a kind of preparation method of the Cultivation of Dictyophora polysaccharide with neuroprotective efficacy
The preparation method of the Cultivation of Dictyophora polysaccharide with neuroprotective efficacy, includes the following steps:
(1) 200g Cultivation of Dictyophora fructification is dry at 60 DEG C, broken wall crushing is carried out to dictyophora phalloidea using micronizer,
Obtain the Cultivation of Dictyophora powder that partial size is 5 μm；
(2) ethyl alcohol is added according to solid-liquid ratio 1:40 (g/mL) in Cultivation of Dictyophora powder, the heating stirring reflux 2h at 90 DEG C,
It is filtered to remove solvent, is repeated the defatting step 3 times, degreasing dictyophora phalloidea powder is obtained；
(3) by degreasing dictyophora phalloidea powder according to solid-liquid ratio 1:40 (g/mL) plus distilled water, heating stirring is extracted at 100 DEG C
Aqueous extracts are collected by filtration in 3h, repeat the aqueous extraction step 1 time.Aqueous extracts are concentrated under reduced pressure；
(4) Aqueous extracts will be concentrated, Sevage reagent (chloroform: the volume ratio of n-butanol is 4:1) is added according to volume ratio 1:4,
Centrifugation (6000r/min, 5min) takes supernatant after concussion 35min, repeats the removing protein step 9 time, obtains de- albumen Aqueous extracts；
(5) 9 times of volume ethanols are added in Aqueous extracts according to volume ratio 9:1 for de- albumen Aqueous extracts, after mixing at 4 DEG C
6h is staticly settled, centrifugation (12000r/min, 5min) collects precipitating, obtains Cultivation of Dictyophora Thick many candies after vacuum freeze drying；
(6) Cultivation of Dictyophora Thick many candies are dissolved in after distilled water and cross 732 type cation exchange resins, using 4 times of column volumes
Water elution (flow velocity 2BV/h), is collected after being concentrated under reduced pressure after eluent after DEAE Sepharose FF anion-exchange column, according to
It is secondary that (flow velocity 2.0mL/min) is eluted using water and 1M sodium chloride solution, collect the eluent of 1M sodium chloride solution；
(7) it uses molecular cut off to carry out flowing water dialysis for the bag filter of 3.5kD after eluent is concentrated under reduced pressure, collects retention
It is concentrated under reduced pressure after liquid；
(8) Sephadex G-200 solvent resistant column is added in dialysis trapped fluid and is in charge of continuous collection to distill water elution
Eluent (flow velocity 1.0mL/min) then detects the polyoses content in every pipe eluent using Phenol sulfuric acid procedure, collects main inhale
The eluent that peak represents is received, vacuum freeze drying is finally carried out and obtains Cultivation of Dictyophora polysaccharide.
Embodiment 5, the yield of Cultivation of Dictyophora polysaccharide, purity and structural analysis
(1) infrared spectroscopy detects
By Cultivation of Dictyophora polysaccharide made from embodiment 1, takes 2.5mg that KBr powder dry in right amount is added, ground in mortar
Tabletting in tablet press machine is added after uniformly, thickness about 1mm is made, the transparent tabletting that diameter is about 1cm or so.Use Fourier transform infrared
Spectrometer is to tabletting in 400-4000cm-1Section is scanned, and acquires the infrared absorption pattern of sample, with Nexus system software
Map is analyzed and processed.
For the infrared spectrogram of Cultivation of Dictyophora polysaccharide as shown in Figure 1, abscissa is wavelength in figure, ordinate is light transmittance.?
3428cm-1The wider and strong peak at place represents the stretching vibration of hydroxyl.In 2922cm-1And 1411cm-1The bands of a spectrum at place generation respectively
The stretching vibration and deformation vibration of table C-H.1635cm-1And 1066cm-1The peak at place is respectively the stretching vibration of C=O and C-O, table
It is bright that there are carboxyls.800cm-1α type glycosidic bond absorption peak between the band and saccharide residue at place is consistent.
(2) monosaccharide composition analysis
Cultivation of Dictyophora polysaccharide made from embodiment 1 is hydrolyzed with 2M sulfuric acid, reacts 6h at 100 DEG C of tube sealing, then use carbon
Sour barium neutralizes.It is freeze-dried after supernatant is collected by centrifugation, sample is handled with sugared nitrile acetic ester derivative method, finally uses gas
Phase chromatographic determination monosaccharide component.
For the monosaccharide composition gas chromatogram of Cultivation of Dictyophora polysaccharide as shown in Fig. 2, abscissa is the time in figure, ordinate is electricity
Pressure.The monosaccharide composition of Cultivation of Dictyophora polysaccharide is mainly mannose (75.6%), further includes a small amount of galactolipin (5.7%), rock algae
Sugared (3.2%), glucose (7.9%), xylose (2.2%), rhamnose (4.1%) and glucuronic acid (1.3%).
Comparative example 1, a kind of preparation method of the Cultivation of Dictyophora polysaccharide with neuroprotective efficacy
Preparation method is similar to Example 1.
The difference from embodiment 1 is that step (1) the midi skirt dictyophora phalloidea powder does not carry out broken wall treatment.Comparative example 2,
A kind of preparation method of the Cultivation of Dictyophora polysaccharide with neuroprotective efficacy
Preparation method is similar to Example 1.
The difference from embodiment 1 is that step (6) the midi skirt dictyophora phalloidea Thick many candies do not pass through cation exchange column, and
Step (7) and (8) processing are not carried out.
Test example one, the detection of Cultivation of Dictyophora polysaccharide yield
1, test material: Cultivation of Dictyophora polysaccharide prepared by embodiment 1 and comparative example 1.
2, test method: polysaccharide yield %=Cultivation of Dictyophora polysaccharide quality/Cultivation of Dictyophora material quality × 100%.
3, test result: test result is as shown in table 1.
1 Cultivation of Dictyophora polysaccharide yield comparison result of table
| Project | Embodiment 1 | Comparative example 1 |
| Dictyophora fungus polysaccharide yield | 3.5% | 2.7% |
Table 1 the results show that Cultivation of Dictyophora polysaccharide made from embodiment 1 yield be 3.5%, comparative example 1 obtain longuette
The yield of dictyophora fungus polysaccharide is 2.7%, illustrates that wall breaking technology can significantly improve the recovery rate of polysaccharide.
Test example two, the analysis of Cultivation of Dictyophora purity of polysaccharide
1, test material: Cultivation of Dictyophora polysaccharide prepared by embodiment 1 and test example 2.
2, test method: weighing dextrose standard sample 50mg first and be dissolved in 100mL water, respectively draw 0,0.02mL,
0.04mL, 0.08mL, 0.1mL, 0.12mL, 0.16mL, 0.2mL solution add water to mend to 0.2mL, are then added in 24 orifice plates
5% phenol of 0.5mL and the 1.0mL concentrated sulfuric acid are placed at room temperature for 30min after shaken well, measure light absorption value in 490nm, calculate standard
Curve.Then appropriate Cultivation of Dictyophora polysaccharide dissolution is weighed, 0.2mL solution is drawn, 5% phenol of 0.5mL and the dense sulphur of 1.0mL is added
Acid is placed at room temperature for 30min after shaken well, measures light absorption value in 490nm, standard curve is utilized to calculate polyoses content.
3, test result: test result is as shown in table 2.
2 Cultivation of Dictyophora purity of polysaccharide of table analyzes result
| Project | Embodiment 1 | Comparative example 2 |
| Dictyophora fungus polysaccharide content | 90.6% | 78.2% |
Table 2 is the results show that Cultivation of Dictyophora polysaccharide made from embodiment 1, polyoses content 90.6%.Comparative example 2 obtains
Cultivation of Dictyophora polysaccharide, polyoses content 78.2% illustrates while using anion-exchange column and cation exchange column, dialysis
Retention and sieve chromatography processing can significantly improve purity of polysaccharide compared with the simple processing by anion-exchange column.
The neuroprotective efficacy research of test example three, Cultivation of Dictyophora polysaccharide
In order to verify Cultivation of Dictyophora polysaccharide provided by the invention with neuroprotective efficacy, this test example utilizes above-mentioned implementation
The laboratory sample of example has carried out human neuroblastoma cells and Caenorhabditis elegans experiment.
1, the cytotoxicity experiment of human neuroblastoma cells
(1) test material: Cultivation of Dictyophora polysaccharide prepared by embodiment 1；Aβ42；3-nitropropionic acid；Pidolidone；Hundred grass
It is withered.
(2) experimental method:
Blank group, control group and dictyophora fungus polysaccharide group are arranged first: blank group is normal person's neuroblastoma cell SH-
SY5Y, control group and experimental group are respectively to utilize 50 μM of A β 42, hundred grass of 7.5mM3- nitropropionic acid, 10mML- glutamic acid and 0.8mM
The SH-SY5Y cell of modeling after withered processing.
Further, cell culture medium culture will be added in blank group and control group, be separately added into implementation in each experimental group
Cultivation of Dictyophora polysaccharide made from example 1, Cultivation of Dictyophora polysaccharide are dissolved with cell culture medium, and ultimate density is respectively 1,2,4mg/
mL。
Further, after above-mentioned each experimental group being incubated for 36h at 37 DEG C, MTT solution is added and continues to be incubated for 4h, is incubated for knot
It is inhaled after beam and abandons supernatant, addition DMSO shaking measures light absorption value at 570nm, and versus cell vigor is medicine group light absorption value and sky
The ratio of white group of light absorption value.
(3) experimental result
Test result is as shown in Table 3-6, and data are indicated with mean ± SEM, using one-wayANOVA and post-hoc
Test counts group difference.
3 Cultivation of Dictyophora polysaccharide of table alleviates A β 42 to the toxic effect of SH-SY5Y cell
Table 3 is the results show that compared to the blank group, and through 50 μM of A β 42, treated that SH-SY5Y cell viability is aobvious in control group
Work is reduced to 43.7%.2mg/mL and 4mg/mL Cultivation of Dictyophora polysaccharide treated experimental group is added, SH-SY5Y cell viability is aobvious
It writes and improves, versus cell energy value is respectively increased from 43.7% to 57.8% and 65.7%.
4 Cultivation of Dictyophora polysaccharide of table alleviates 3-nitropropionic acid to the toxic effect of SH-SY5Y cell
Table 4 the results show that compared to the blank group, in control group after the processing of 7.5mM 3-nitropropionic acid SH-SY5Y cell
Vigor significantly reduces.Compared with the control group, cell viability can be significantly improved by being handled using 4mg/mL Cultivation of Dictyophora polysaccharide, relatively
Cell viability value is improved from 48.4% to 60.8%.
5 Cultivation of Dictyophora polysaccharide of table alleviates Pidolidone to the toxic effect of SH-SY5Y cell
Table 5 is the results show that compared to the blank group, and through 10mM Pidolidone, treated that SH-SY5Y cell viability significantly drops
It is low.Compared with the control group, the SH-SY5Y cell viability of 1mg/mL and 2mg/mL Cultivation of Dictyophora polysaccharide is added from control group
35.8% is respectively increased to 50.3% and 50.5%.
6 Cultivation of Dictyophora polysaccharide of table alleviates paraquat to the toxic effect of SH-SY5Y cell
Table 6 is the results show that compared to the blank group, the SH-SY5Y cell viability handled through 0.8mM paraquat significantly reduces.
From the result of experimental group can be seen that Cultivation of Dictyophora polysaccharide concentration be 1,2,4mg/mL under conditions of be able to suppress paraquat
Cytotoxicity, to improve cell activity, versus cell energy value is respectively increased from 42.8% to 55.7%, 58.1% and
60.5%.
The experiment of 2.L1 phase CL4176 caenorhabditis elegan
(1) test material: Cultivation of Dictyophora polysaccharide prepared by embodiment 1.
(2) experimental method:
Blank group, control group and dictyophora fungus polysaccharide group are arranged first: blank group is untreated Wild-type C nematode, control
Group and dictyophora fungus polysaccharide group are L1 phase CL4176 caenorhabditis elegan.
Further, nematode culture medium culture is added in blank group and control group, is separately added into implementation in each experimental group
Cultivation of Dictyophora polysaccharide made from example 1, Cultivation of Dictyophora polysaccharide are dissolved with culture medium, and ultimate density is respectively 1,2,4mg/mL.
Further, 23 DEG C are warming up to after caenorhabditis elegan being cultivated 36h at 15 DEG C, it is elegant from every 10h since the 40h
Habronemic paralysis situation simultaneously counts paralysis rate.
Further, 23 DEG C are warming up to after caenorhabditis elegan being cultivated 36h at 15 DEG C, caenorhabditis elegan is collected after 60h, is added
PBS buffer solution (contains 1% polysorbas20), is then transferred in glass homogenizer and is homogenized on ice, obtains caenorhabditis elegan homogenate, receives
Collection homogenate adds DCFH-DA probe solution (indicator of active oxygen radical level), and incubation is protected from light at 37 DEG C, is then used
Fluorescence microplate reader detects fluorescent value, and excitation wavelength 485nm, launch wavelength 530nm utilize more each reality of relative intensity of fluorescence
Test a group oxidation resistance.
Further, it takes caenorhabditis elegan homogenate to be added in 96 orifice plate of black, adds mitochondrial membrane potential detection examination
Agent JC-1 dyeing liquor measures red fluorescence (excitation wavelength 525nm, launch wavelength with fluorescence microplate reader after vibrating 30s respectively
590nm) with green fluorescence (excitation wavelength 490nm, launch wavelength 530nm), finally calculates red green fluorescence ratio and represent line grain
Body level of membrane potential.
(3) experimental result
As shown in table 7-9, data are indicated experimental result with mean ± SEM, using one-wayANOVA and post-hoc
Test counts group difference.
Influence of the 7 Cultivation of Dictyophora polysaccharide of table to CL4176 caenorhabditis elegan paralysis rate
Table 7 is the results show that compared with the control group, being added the processing of 2mg/mL and 4mg/mL Cultivation of Dictyophora polysaccharide, luring in heating
After leading the expression of A β 42 50h and 60h, the paralysis rate significant decrease of CL4176 caenorhabditis elegan (50h: is reduced to from 33.8% respectively
23.1% and 19.8%；60h: it is reduced to 63.1% and 60.3%) respectively from 78.9%, shows that Cultivation of Dictyophora polysaccharide is able to suppress
The neurotoxicity that A β abnormal aggregation mediates.
Influence of the 8 Cultivation of Dictyophora polysaccharide of table to CL4176 caenorhabditis elegan activity in vivo oxygen free radicals
Table 8 shows A β in CL4176 the results show that the relative fluorescence of blank group and control group is respectively 41.1 and 54.5
Great expression results in oxidative stress in nematode body.Using 2mg/mL and 4mg/mL Cultivation of Dictyophora polysaccharide treated experimental group,
Relative fluorescence drops to 41.7 and 42.7 respectively, shows that Cultivation of Dictyophora polysaccharide can significantly reduce in CL4176 caenorhabditis elegan body
Active oxygen radical it is horizontal, there is good internal antioxidant activity.
Influence of the 9 Cultivation of Dictyophora polysaccharide of table to CL4176 caenorhabditis elegan mitochondrial membrane potential
Table 9 shows that A beta-aggregation can the results show that the red green fluorescence ratio of blank group and control group is respectively 1.39 and 1.04
Cause mitochondrial membrane potential abnormal, to damage mitochondrial function.After being handled using 2mg/mL and 4mg/mL Cultivation of Dictyophora polysaccharide
Experimental group, red green fluorescence ratio is respectively increased to 1.33 and 1.29, illustrates that the polysaccharide can improve CL4176 caenorhabditis elegan body
Inner mitochondria film potential level has the function of restoring mitochondrial function.
The experiment of 3.L3 phase BZ555 caenorhabditis elegan
(1) test material: Cultivation of Dictyophora polysaccharide prepared by embodiment 1；6- hydroxyl dopamine.
(2) experimental method:
Blank group, control group and dictyophora fungus polysaccharide group are arranged first: blank group is untreated Wild-type C nematode, control
Group and dictyophora fungus polysaccharide group are that the L3 phase BZ555 caenorhabditis elegan of 1h is handled using 50mM 6- hydroxyl dopamine.
Further, nematode culture medium culture is added in blank group and control group, is separately added into reality in dictyophora fungus polysaccharide group
Cultivation of Dictyophora polysaccharide made from example 1 is applied, Cultivation of Dictyophora polysaccharide is dissolved with culture medium, and ultimate density is respectively 1,2,4mg/mL.
Further, each experimental group is collected into caenorhabditis elegan after 20 DEG C of culture 72h, addition sodium azide solution makes beautiful
It is transferred on agarose pad after nematode paralysis, in fluorescence microscopy microscopic observation caenorhabditis elegan head GFP fluorescence.Using Image
6.0 software of Pro Plus calculates relative intensity of fluorescence, and gained fluorescence intensity level represents cell activity.
Further, each experimental group is collected into after 20 DEG C of culture 72h caenorhabditis elegan, PBS buffer solution is added and (is spat containing 1%
20) temperature, is then transferred in glass homogenizer and is homogenized on ice, collect homogenate and add DCFH-DA probe solution, at 37 DEG C
It is protected from light incubation, then detects fluorescent value (excitation wavelength 485nm, launch wavelength 530nm) with fluorescence microplate reader, relative intensity of fluorescence
Represent caenorhabditis elegan activity in vivo oxygen free radicals.
Further, it takes caenorhabditis elegan homogenate to be added in 96 orifice plate of black, adds JC-1 dyeing liquor, vibrate 30s
Measure red fluorescence (excitation wavelength 525nm, launch wavelength 590nm) and green fluorescence (excitation wave respectively with fluorescence microplate reader afterwards
Long 490nm, launch wavelength 530nm), the ratio of last red fluorescence and green fluorescence is mitochondrial membrane potential level.
(3) experimental result
As shown in table 10-12, data are indicated experimental result with mean ± SEM, using one-wayANOVA and post-hoc
Test counts group difference.
10 Cultivation of Dictyophora polysaccharide of table inhibits 6- hydroxyl dopamine in the intracorporal neurotoxicity of BZ555 nematode
Table 10 is the results show that compared to the blank group, the control group BZ555 caenorhabditis elegan of 50mM 6- hydroxyl dopamine processing
Head fluorescent value significantly reduces, and illustrates that 6- hydroxyl dopamine causes damage to dopamine neuron.2mg/mL and 4mg/ is added
After the processing of mL Cultivation of Dictyophora polysaccharide, the head fluorescent value through 6- hydroxyl dopamine modeling processing BZ555 is obviously increased (from 35.2%
It is respectively increased to 55.7% and 61.1%), shows that Cultivation of Dictyophora polysaccharide can effectively antagonize the neurotoxicity of 6- hydroxyl dopamine.
BZ555 caenorhabditis elegan activity in vivo oxygen radical water of the 11 Cultivation of Dictyophora polysaccharide of table to 6- hydroxyl dopamine modeling
Flat influence
Table 11 is the results show that compared to the blank group, and treated that control group living radical level is aobvious for 6- hydroxyl dopamine
It writes and increases and (improve from 38.9 to 60.5), illustrate that 6- hydroxyl dopamine can cause oxidative stress.Using 2mg/mL and 4mg/mL
Cultivation of Dictyophora polysaccharide treated experimental group, DCF relative fluorescence drop to 50.7 and 47.4 respectively, illustrate Cultivation of Dictyophora polysaccharide
The active oxygen radical level that the induction of 6- hydroxyl dopamine can be resisted increases, and further demonstrates that Cultivation of Dictyophora polysaccharide tool of the present invention
There is good internal antioxidant activity.
Influence of the 12 Cultivation of Dictyophora polysaccharide of table to 6- hydroxyl dopamine modeling BZ555 caenorhabditis elegan mitochondrial membrane potential
Table 12 shows 6- hydroxyl the results show that the red green fluorescence ratio of blank group and control group is respectively 1.58 and 1.01
Dopamine can destroy mitochondrial membrane potential.It is red green glimmering using 2mg/mL and 4mg/mL Cultivation of Dictyophora polysaccharide treated experimental group
Light ratio is respectively increased to 1.41 and 1.44, illustrates that Cultivation of Dictyophora polysaccharide of the present invention can improve 6- hydroxyl dopamine stress
Mitochondrial membrane potential is horizontal in BZ555 caenorhabditis elegan body, further confirms that it has the function of restoring mitochondrial function.
In summary test result shows that Cultivation of Dictyophora polysaccharide can inhibit multiple compounds in human nerve cell level
Neurotoxicity, while can inhibit in whole animal level A β abnormal aggregation mediate neurotoxicity, effectively antagonize 6- hydroxyl
The neurotoxicity of base dopamine, and have effects that mitigate oxidative stress damage and restore mitochondrial function.Therefore longuette bamboo
Sweet-smelling grass polysaccharide has good internal and external neuroprotection, has in the development and application of nerve protection medicine and health care product
Vast potential for future development.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (8)
1. a kind of preparation method of the Cultivation of Dictyophora polysaccharide with neuroprotective efficacy, which is characterized in that with Cultivation of Dictyophora be original
Material is prepared by following method: broken wall crushes after Cultivation of Dictyophora is dried, and mentions through degreasing, water, removing protein and ethanol precipitation, then
Successively isolated and purified by cation exchange column and anion-exchange column, flowing water dialysis and gel chromatography process.
2. the preparation method of the Cultivation of Dictyophora polysaccharide according to claim 1 with neuroprotective efficacy, which is characterized in that
The method includes the following steps:
(1) broken wall crushes after drying Cultivation of Dictyophora at 40-60 DEG C, obtains the Cultivation of Dictyophora powder that partial size is 1-10 μm；
(2) by Cultivation of Dictyophora powder obtained by step (1) with organic solvent degreasing 2-5 times, solid-liquid ratio g/mL is 1:5-1:40, degreasing
Temperature is 50-90 DEG C, degreasing time 2-6h, obtains degreasing Cultivation of Dictyophora powder；
It (3) is 1:10-1:40 by step (2) resulting degreasing Cultivation of Dictyophora powder distillation flooding 2-6h, solid-liquid ratio g/mL,
Extraction temperature is 60-100 DEG C, and extracting times are 1-3 times, obtains Aqueous extracts；
(4) it carries out de- albumen to Aqueous extracts obtained by step (3) using Sevage method to handle, the volume of Aqueous extracts and Sevage reagent
4-8min is centrifuged in 6000-8000r/min after 25-35min than shaking for 4:1-1:4, and number of processes is 5-9 times, obtains de- albumen
Aqueous extracts；
(5) it utilizes and takes off albumen Aqueous extracts obtained by methanol precipitation step (4), the volume ratio of ethyl alcohol and de- albumen Aqueous extracts is 4:1-9:
It is centrifuged 5-10min in 6000-12000r/min after 1,0-4 DEG C of standing 6-24h, obtains Cultivation of Dictyophora Thick many candies；
(6) cation exchange column is crossed after Cultivation of Dictyophora Thick many candies obtained by step (5) being dissolved in distilled water, is washed using distillation
It is de-, it is concentrated after collecting eluent, then crosses anion-exchange column, successively eluted using distilled water and salt ion solution, received
It is concentrated after collection salt ion eluent, the salt ion eluent after must being concentrated；
(7) the salt ion eluent after concentration obtained by step (6) is subjected to flowing water dialysis treatment, the molecular cut off of bag filter is
1.0-3.5kD is concentrated, the dialysis trapped fluid after must being concentrated after collecting trapped fluid；
(8) solvent resistant column is added in the dialysis trapped fluid after concentration obtained by step (7) and is in charge of continuous receipts to distill water elution
Collect eluent, then detect the polyoses content in eluent, collects the eluent that main absorption peak represents, be finally concentrated and done
It is dry, obtain Cultivation of Dictyophora polysaccharide.
3. the preparation method of the Cultivation of Dictyophora polysaccharide according to claim 2 with neuroprotective efficacy, which is characterized in that
Cultivation of Dictyophora in the step (1) refers to edible position cap, indusium, stem, volva or the fructification of Cultivation of Dictyophora.
4. the preparation method of the Cultivation of Dictyophora polysaccharide according to claim 2 with neuroprotective efficacy, which is characterized in that
Organic solvent is selected from ethyl alcohol, methanol, acetone, ether, petroleum ether or its mixed liquor in the step (2).
5. the preparation method of the Cultivation of Dictyophora polysaccharide according to claim 2 with neuroprotective efficacy, which is characterized in that
In the step (6) material of anion-exchange column and cation exchange column be cellulose, sephadex, Ago-Gel or
Resin；The salt ion solution be acetate, phosphate, barbiturate, citrate, sodium chloride, potassium chloride, ammonium chloride or
Glycine.
6. the preparation method of the Cultivation of Dictyophora polysaccharide according to claim 2 with neuroprotective efficacy, which is characterized in that
The material of solvent resistant column is that sephadex, Ago-Gel, polyacrylamide gel, crosslinking Portugal are poly- in the step (8)
One of sugar and bisacrylamide gel copolymer, Sepharose and glucan covalent bond gel.
7. the preparation method of the Cultivation of Dictyophora polysaccharide according to claim 1-6 with neuroprotection is made
Cultivation of Dictyophora polysaccharide preparation the nervous system disease ancillary drug in application.
8. the preparation method of the Cultivation of Dictyophora polysaccharide according to claim 1-6 with neuroprotection is made
Cultivation of Dictyophora polysaccharide preparation have neuroprotective efficacy health food in purposes.
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Application publication date: 20181207 |