CN104059162B - A kind of ganoderan and preparation method thereof - Google Patents
A kind of ganoderan and preparation method thereof Download PDFInfo
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- CN104059162B CN104059162B CN201410293682.XA CN201410293682A CN104059162B CN 104059162 B CN104059162 B CN 104059162B CN 201410293682 A CN201410293682 A CN 201410293682A CN 104059162 B CN104059162 B CN 104059162B
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Abstract
The invention discloses a kind of ganoderan and preparation method thereof, the preparation method of this ganoderan comprises: Ganoderma sporophore carries out water extraction, obtains extracting residue; Extract residue and carry out micronizing, obtain ultrafine powder; Ultrafine powder is through water extract-alcohol precipitation, and the precipitation collected is ganoderan.The ganoderan beta-glucan content that the present invention prepares reaches more than 50%, has the activity of obvious activating macrophage.
Description
Technical field
The present invention relates to medicinal fungi field of deep, relate in particular to a kind of ganoderan and preparation method thereof.
Background technology
Glossy ganoderma (Ganodermalucidum) is the foremost medicinal fungi of China, because it has the effects such as antitumor, raising is immune, delay senility, is used widely as functional food and medicine.Ganoderan is one of main active ingredient of glossy ganoderma, and pharmacologically active is various, especially raising body's immunity, antitumor etc. in Be very effective.Along with the application of Ganoderma extract at healthcare products and field of medicaments constantly expands, the extracted amount of Ganoderma sporophore increases severely, and causes remaining a large amount of extraction residues, passes into disuse and produce pollution by environment, and cause the very large wasting of resources.
Beta-glucan is one of main component on fungal cell wall, and it is main that its structural backbone connects with β-(1,3) glycosidic link, is considered to main active polysaccharide.Large quantity research prove such polysaccharide can immune cell activated, regulate cytokine secretion, participate in host specificity immunity and non-specific immunity, thus improve body's immunity.The mixed polysaccharide being separated acquisition in recent years in glossy ganoderma is more, less to the research of its beta-glucan.And the extraction of β-1,3-dextran to carry mainly with alkali be main, subsequent treatment process is complicated.
Summary of the invention
The present invention provide firstly a kind of preparation method of ganoderan, and the method comprises the steps:
Ganoderma sporophore carries out water extraction, obtains extracting residue;
Extract residue and carry out micronizing, obtain ultrafine powder;
Ultrafine powder is through water extract-alcohol precipitation, and the precipitation collected is ganoderan.
Specifically, the preparation method of a kind of ganoderan of the present invention, comprises the steps:
Extract residue: Ganoderma sporophore is ground into particle, adds the water of 10-15 times of weight, soak at room temperature 30-60 minute, be heated to boiling, keep 60-120 minute, filter;
Superfine grinding: after extracting residue drying, pulverizes 30-40 minute with micronizing vibrating grinder, crosses 200 mesh sieves, collects ultrafine powder;
Water extract-alcohol precipitation: by ultrafine powder, adds the distilled water of 15-20 times of weight, soak at room temperature 30-60 minute, is heated to boiling, keeps 60-120 minute, centrifugal; By extracting solution, being evaporated to solid-liquid ratio is 1:1-1:2 (mass/volume, unit: kg/liter), add dehydrated alcohol, reach 40%-60% to ethanol weight percent content, room temperature leaves standstill 3-5 hour, centrifugal segregation alcohol precipitation supernatant, collecting precipitation, is required ganoderan after lyophilize.
Present invention also offers a kind of ganoderan, it utilizes the preparation method of above-mentioned ganoderan to prepare.
The ganoderan that the present invention obtains, molecular weight is 180,000-240 ten thousand dalton, and beta-glucan content reaches more than 50%, has the activity of obvious activating macrophage.
The preparation method of ganoderan of the present invention is a kind of method of easy, efficient, pollution-free, applicable large-scale production that polysaccharide content is high; stay-in-grade ganoderan can be obtained by above-mentioned preparation method; yield reaches 0.9%; polysaccharide content is more than 80%, and wherein beta-glucan content reaches more than 50%.
Figure of description
The determination of activity result of Fig. 1 ganoderan stimulated in vitro scavenger cell release NO
Fig. 2 HPLC analyzes the molecular weight distribution collection of illustrative plates of ganoderan
Embodiment:
Red ganoderma sporophore: the Shanghai agriculture glossy ganoderma 1 work song entity that the cultivation of Longquan, Zhejiang Province base obtains
Micronizing vibrating grinder: Sanqing Stainless Steel Apparatus Co., Ltd., Shandong SQW series
Embodiment 1:
1, glossy ganoderma extracts the acquisition of residue: with red ganoderma sporophore for raw material, be ground into coarse grain, take 200g, add 3L water, soak at room temperature 30 minutes, is heated to boiling, keeps 120 minutes, filters.Filter residue repeats above-mentioned steps, then extracts 1 time, after filtration, residue obtainedly dries in 60 DEG C of blast driers.
2, the micronizing of glossy ganoderma residue: dried glossy ganoderma is extracted residue and adopt micronizing vibrating grinder to pulverize 30min, cross 200 mesh sieves, collect ultrafine powder.
3, the extraction and isolation of ganoderan: take above-mentioned glossy ganoderma residue ultrafine powder 150g, add 2L distilled water, normal temperature 60 minutes, is heated to boiling, keeps micro-and boil 60 minutes, centrifugal.Precipitation repeats extraction 1 time, after centrifugal, collect united extraction liquid, being evaporated to solid-liquid ratio is 1:1 (mass/volume, unit: kg/liter), slowly add dehydrated alcohol, limit edged stirs, and reaches 50% to ethanol content, and room temperature leaves standstill 4 hours, centrifugal segregation alcohol precipitation supernatant, collecting precipitation.
4, dry: precipitation is dissolved in water, and volatilizes ethanol and is placed on frozen 2h in-20 DEG C of refrigerators, and then be put into-80 DEG C of refrigerator and cooled and freeze 3h, finally put into freeze drier freeze-drying, namely obtain required ganoderan.
The detection of polysaccharide content:
Measure with phend-sulphuric acid, precision takes this product 10mg and puts in 100ml volumetric flask, and add water about 80ml, is cooled to room temperature, constant volume in 100ml volumetric flask after heating hydrotropy.Accurate pipette samples 1.0m1, puts in 15ml test tube, adds phenol, sulphate reagent respectively, and fully measuring absorbance at 490nm place after reaction, take dextran as the sugared content of standard substance calculation sample.
Beta-glucan assay: beta-glucan content in polysaccharide sample is measured with reference to the method in the yeast of Megazyme company and mushroom beta-glucan detection kit.
The yield preparing gained ganoderan is 0.91%, and polysaccharide content is 82.52%, and beta-glucan content is 53.21%.Embodiment 2:
1, glossy ganoderma extracts the acquisition of residue: with red ganoderma sporophore for raw material, be ground into coarse grain, take 500g, add 6L water, soak at room temperature 40 minutes, is heated to boiling, keeps micro-and boil 120 minutes, filters.Filter residue repeats above-mentioned steps, then extracts 2 times, after filtration, residue obtainedly dries in 60 DEG C of blast driers.
2, the micronizing of glossy ganoderma residue: dried glossy ganoderma is extracted residue and adopt micronizing vibrating grinder to pulverize 40min, cross 200 mesh sieves, collect ultrafine powder.
3, the extraction and isolation of ganoderan: take above-mentioned glossy ganoderma residue ultrafine powder 400g, add 6L distilled water, normal temperature 60 minutes, is heated to boiling, keeps micro-and boil 120 minutes, centrifugal.Precipitation repeats extraction 2 times, after centrifugal, collect united extraction liquid, being evaporated to solid-liquid ratio is 1:1.5 (mass/volume, unit: kg/liter), slowly add dehydrated alcohol, limit edged stirs, and reaches 40% to ethanol content, and room temperature leaves standstill 4 hours, centrifugal segregation alcohol precipitation supernatant, collecting precipitation.
4, dry: precipitation is dissolved in water, and volatilizes ethanol and is placed on frozen 2h in-20 DEG C of refrigerators, and then be put into-80 DEG C of refrigerator and cooled and freeze 3h, finally put into freeze drier freeze-drying, namely obtain required ganoderan.
The yield preparing gained ganoderan is 0.94%, and polysaccharide content is 85.31%, and beta-glucan content is 54.51%.Embodiment 3:
The determination of activity of ganoderan stimulated in vitro scavenger cell release NO:
1, preparation of samples: accurately take ganoderan that embodiment 1 and embodiment 2 prepare in sterilized eppendorf pipe, be mixed with the sample liquid of concentration 5mg/mL with aseptic PBS.With the centrifugal 30min of 15000r/min after abundant dissolving, supernatant is transferred in new sterile eppendorf tubes under aseptic condition, becomes 2mg/mL, 0.5mg/mL, 0.1mg/mL stand-by diluted sample.
2, cell cultures: the RAW264.7 scavenger cell strain in vegetative period of taking the logarithm, with DMEM perfect medium 37 DEG C, containing 5%CO
2secondary Culture under condition, with 0.05% pancreatin or 5%EDTA solution digestion, collecting cell after the centrifugal 3min of suspension 1000rpm/min, counts for subsequent use.
3, preparation of reagents and Specification Curve of Increasing
Griess reagent: add 6.25mlH in beaker
3pO
3, adding distilled water 250ml, adding the naphthylethyylene-diaminedihydrochloride magnetic stirring apparatus of sulfanilamide and 0.25g of 2.5g respectively to all dissolving, brown reagent bottle 4 DEG C of Refrigerator stores.
Specification Curve of Increasing: the sodium nitrite solution being made into different concns, concentration gradient is 0,5,10,15,20,25,30,35,40 μM totally nine; Get
in 96 orifice bores, add 50 μ LGriess reagent, measure 543nm light absorption value, 3 repetitions of each concentration of typical curve, according to light absorption value drawing standard curve.
4, the mensuration of sample stimulus scavenger cell release NO amount: collect RAW264.7 cell, with colourless RPMI1640 (10% foetal calf serum+1% antibiotic liquid) substratum by cell dilution to 5 × 10
5/ mL, adds 96 orifice plates, and every hole adds 180 μ L, and then adds
testing sample, positive control is LPS (1 μ g/mL), cultivates 48 hours for 37 DEG C.Get
supernatant is in 96 orifice bores, and add 50 μ lGriess reagent, room temperature incubates bath 10 minutes, measures 543nm light absorption value.The burst size of cell NO is calculated according to typical curve.
The results are shown in accompanying drawing 1, result can be found out thus, the ganoderan of embodiment 1 and embodiment 2 gained has obvious promoter action to RAW264.7 scavenger cell strain release NO, and what obviously can strengthen scavenger cell engulfs kill capability, thus the anti-tumor capacity of enhancing body.
Embodiment 4:
The molecular weight analyse of ganoderan
1, test method: high performance liquid chromatography coupling multiple angle laser light scattering instrument analytical method (HPSEC-MALLS)
2, sample pre-treatments: precision takes the polysaccharide 5mg of embodiment 1, adds 1mL phosphate buffered saline buffer, makes it fully dissolve, the centrifugal 20min of sample liquid 12000rpm, gets supernatant liquor, sample introduction analysis.
3, analysis condition: U.S. WatersHPLC high performance liquid chromatograph (Waters2695); Chromatographic column is TSK-GEL series G6000PWXL gel column, and specification is 7.8mm × 300mm (TOSOH, Japan); Column temperature: 35 DEG C; Moving phase: 0.15mol/LNaNO
3and 0.05mol/LNaH
2pO
4, (pH=7,0.02% sodium azide); Flow velocity: 0.5mL/min; Sample size: 100 μ L; Detector: 2414 differential refraction detectors, 8 multi-angle laser scatterometers (Wyatt, HELEOS8), laser detector optical source wavelength selects 623.8nm.Polysaccharide refractive index increment in the solution (dn/dc) calculates according to 0.146mL/g.
4, data processing: use Astra (version6.1.1, WyattTechnology, SantaBarbara, CA) data analysis software gather light scattering data and analyze, calculate molecular weight.
Molecular weight analyse the results are shown in Figure 2, learns that the main molecules amount distribution range of this polysaccharide is 18 ten thousand to 240 ten thousand dalton by calculating.
Claims (2)
1. a preparation method for ganoderan, is characterized in that comprising the steps:
Extract residue: Ganoderma sporophore is ground into coarse grain, adds the water of 10-15 times of weight, soak at room temperature 30-60 minute, be heated to boiling, keep 60-120 minute, filter;
Superfine grinding: after extracting residue drying, pulverizes 30-40 minute with micronizing vibrating grinder, crosses 200 mesh sieves, collects ultrafine powder;
Water extract-alcohol precipitation: by ultrafine powder, adds the distilled water of 15-20 times of weight, soak at room temperature 30-60 minute, is heated to boiling, keeps 60-120 minute, centrifugal; By extracting solution, being evaporated to solid-liquid ratio is 1:1-1:2, mass/volume, unit: kg/liter, adds dehydrated alcohol, reaches 40%-60% to ethanol weight percent content, room temperature leaves standstill 3-5 hour, centrifugal segregation alcohol precipitation supernatant liquor, collecting precipitation, is required ganoderan after lyophilize.
2. a ganoderan, it is prepared by method described in claim 1.
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| CN108267533A (en) * | 2018-04-23 | 2018-07-10 | 福建农大菌草技术开发公司 | A kind of method for building up of ganoderma lucidum molecular weight characteristic collection of illustrative plates and its application |
| CN109481478A (en) * | 2018-11-29 | 2019-03-19 | 杨凌萃健生物工程技术有限公司 | A kind of residual ganoderan extract of low agriculture and preparation method thereof |
| CN113151372B (en) * | 2021-05-18 | 2022-09-16 | 青岛润达生物科技有限公司 | Preparation process of high-purity ganoderma lucidum polysaccharide |
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