CN105732250B - A kind of preparation method of high-purity grifola frondosus weight polyphenol fraction - Google Patents

A kind of preparation method of high-purity grifola frondosus weight polyphenol fraction Download PDF

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CN105732250B
CN105732250B CN201610082919.9A CN201610082919A CN105732250B CN 105732250 B CN105732250 B CN 105732250B CN 201610082919 A CN201610082919 A CN 201610082919A CN 105732250 B CN105732250 B CN 105732250B
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grifola frondosus
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吕旭聪
刘斌
贾瑞博
李燕
周文斌
陈竟豪
林占熺
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FUJIAN JUNZHITANG BIOTECHNOLOGY Co.,Ltd.
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Abstract

The present invention relates to a kind of preparation method of high-purity grifola frondosus weight polyphenol fraction, belong to the function factor and drug field of functional food.The thick component of ash tree flower polyphenol is prepared by Ultrasonic Wave-Assisted Extraction technology, then profit removes the large biological molecules such as polysaccharide and albumen therein with milipore filter retention technology, the separation purification technique of the column chromatography preparative high performance liquid chromatographies of macroporous absorbent resin NKA II is finally used, prepares the grifola frondosus polyphenol compound monomer of a variety of high-purities.Preparation method of the present invention rapidly and efficiently, can prepare 17 kinds of grifola frondosus polyphenol compound monomers simultaneously, and the purity of product is high.Prepared grifola frondosus polyphenol compound monomer is respectively provided with stronger DPPH free radicals and Scavenging action to hydroxyl free radical and stronger reducing power; most of polyphenol compound monomers can suppress α glucosidase activities, have and effectively protect the physiological actions such as human cell tissue, cardiovascular circulation system and anticancer, anti-aging and auxiliary hyperglycemic.

Description

A kind of preparation method of high-purity grifola frondosus weight polyphenol fraction
Technical field
The present invention relates to a kind of preparation method of high-purity grifola frondosus weight polyphenol fraction, belong to functional food function factor and Drug field.
Background technology
Grifola frondosus（Grifola frondosa）It is true to belong to Basidiomycotina Hymenomycetes Aphyllophorales Polyporaceae tree Pseudomonas Bacterium, subtropical zone to temperate zone is mainly grown in, the ground such as Fujian, Hebei, Jilin, Guangxi and Sichuan are distributed mainly in China.As A kind of precious food, medicine dual-purpose gill fungus bacterium, the various nutrients of grifola frondosus and physiological function are known as into first of the various edible mushrooms that live apart " edible mushroom prince " and the good reputation of " North China ginseng ".Correlative study shows that grifola frondosus has strengthen immunity, suppresses tumour, be anti- Viral, anti-oxidant anti-aging, stabilizing blood pressure, reducing blood sugar and blood lipid, improve fat metabolism and alleviate hepatitis symptom, enhancing memory etc. A variety of medicinal health care functions.Numerous studies work shows that polyphenols has anti-oxidant, antibacterial, anti-mutagenesis, antiviral, anti- The multiple functions such as tumour, reducing blood sugar and blood fat activity.Research report both at home and abroad for ash tree flower polyphenol is less.Chen XiangDong et al. Using the macroporous absorbent resin of low pole, separation prepares Polyphenols component from maitake mushroom mycelia crude extract, and the component of acquisition is Brown powder shape product, empirical tests have anti-oxidant, antibacterial and alpha-amylase suppresses isoreactivity, but its obtained ash tree flower polyphenol It is mixture, is not further purified and identifies that grifola frondosus weight polyphenol fraction needs into one to the component of grifola frondosus polyphenols Walk separating-purifying（Chen XiangDong, Liu Xiaowen, the extraction of Wu Wutong ash tree flower polyphenols and activity research food and biotechnics Report, 2005,24 (4): 26-30.）.
The present invention is proposed using Grifola Frondosa sporophore as raw material, and ash tree flower polyphenol group is prepared by Ultrasonic Wave-Assisted Extraction technology Point, then profit removes the large biological molecules such as polysaccharide and albumen therein with milipore filter retention technology, finally using macroporous absorption tree The separation purification technique of II column chromatographies of fat NKA--preparative high performance liquid chromatography, the grifola frondosus for preparing a variety of high-purities are more Phenolic compound monomer.Compared to polyphenols is extracted from maitake mushroom mycelia in the past, the invention uses real with ash tree beggar Body is raw material, and raw material sources are easier to obtain and polyphenol content comes high compared to mycelium, and the extraction yield of polyphenol is also higher；With Existing research report is compared, and is eliminated using the grifola frondosus weight polyphenol fraction obtained by ultrafiltration membrance filter in Effects of Extracts of Grifola frondosa on Active The macromolecular substances such as polysaccharide, albumen, it is made through column chromatography-preparative liquid chromatography series connection thick weight polyphenol fraction of purification techniques The weight polyphenol fraction purity obtained is higher（Purity is all higher than 95%）, also conspicuousness improves polyphenol antioxidation activity in vitro after purification, its Middle reducing power than etc. mass concentration（1 mg/mL）Ascorbic acid it is strong.
The content of the invention
It is an object of the invention to provide it is a kind of rapidly and efficiently, the grifola frondosus polyphenol compound that purity is high, preparation amount is larger The preparation technology of monomer.
A kind of preparation method of high-purity grifola frondosus weight polyphenol fraction, its preparation process are as follows：
（1）Pretreatment：Grifola Frondosa sporophore dried product is ground into powder, it is standby to cross 40~80 mesh sieves；
（2）Extraction：A certain amount of grifola frondosus powder is taken, is extracted 2-4 times using ultrasonic assistant extractive technique, merges extraction Liquid, residue is removed through plate-frame filtering；
（3）Membrane filtration：Filtered by film filter, retain egg bletilla polysaccharide, the concentrating filter liquor of acquisition, drying Ash tree flower polyphenol crude extract is obtained afterwards；
（4）Isolate and purify：Ash tree flower polyphenol crude extract is redissolved in deionized water, it is efficient using column chromatography-preparative Liquid chromatography tandem separating and purifying technology is isolated and purified.
The condition of ultrasonic wave added of the present invention extraction is：Using water as Extraction solvent, feed liquid weight ratio is 1：10-1： 50,30-70 DEG C of ultrasonic temperature, ultrasonic time 30-90 min, ultrasonic power 100-400w.
Filter membrane of the present invention is the milipore filter that molecular cut off is 5 kD, and drying means is true used by filtrate Vacuum freecing-dry or centrifugal spray drying.
The filler of column chromatography of the present invention is the type macroporous absorbent resins of NKA- II, the BV/h of loading speed 3, during absorption Between 2 h, eluting solvent is 20%-60% ethanol solutions, the BV/h of elution speed 3；Through adsorbing, eluting collection eluent, it is concentrated under reduced pressure To crude product is done to obtain, through 0.45 μm of membrane filtration after being redissolved with 50% methanol.
Liquid phase chromatogram condition of the present invention is：The anti-phase C18 chromatographic columns of preparative, chromatographic column specification：The mm of internal diameter 20, 5 μm, Detection wavelength 280nm, the mL of sample size 1, flow velocity 5-10 mL/min of 250 mm, C18 silica filler particle diameter of length, column temperature 30 DEG C, using A:Methanol-B:0.1% formic acid carries out linear gradient elution, and elution program is
（1）0~30 min：A: 5%+B: 95%→A: 60%+B: 40%；
（2）30~35 min：A: 60%+B: 40%→A: 5%+B: 95%；
（3）35~45 min：A: 5%+B: 95%.
High-purity grifola frondosus weight polyphenol fraction of the present invention is purified by preparative high performance liquid chromatography, according to ultraviolet Detector spectrogram collects grifola frondosus weight polyphenol fraction and carries out crystallization operation acquisition respectively, and 17 kinds of high-purity weight polyphenol fractions reflect through mass spectrum It is set to：Ellagic acid（ellagic acid）, Quercetin（quercetin）, 4-HBA（4-hydroxybenzoic acid）, gallic acid -3-O- (6`-O- nutgall acyls) glucoside（gallic acid-4-O-(6`-O-galloyl) glucoside）, caftaric acid（caffeoyl tartaric acid）, caffeic acid（caffeic acid）, epicatechin Gallate（(-)-epicatechin 3-O-gallate）, 5,6,7,3', 4'- pentamethoxyl flavones（sinenstein）、 To coumaroyl guinic acid（p-coumaroyl-quinic acid）, m-digallic acid（m-digallic acid）, dehydrogenation two Forulic acid（dehydrodiferulic acid）, Quercetin -3-O- β-D- galactopyranosides（quercetin-3-O-β-D- galactopyranoside）, 3,4,5- trimethoxybenzoic acids（methyl gallate）, 5- nonadecyl resorcinols（5- nonadecylresorcin）, forulic acid（ferulic acid）, to coumaric acyl malic acid（p-coumaroyl malic acid）And to coumaric acyl tartaric acid（p-coumaroyl tartaric acid）, purity is all higher than 95%.
Using DPPH radical scavenging activities, Hydroxyl radical-scavenging ability and reducing power as foundation, it was demonstrated that the ash tree of purifying Flower polyphenol component has stronger antioxidation activity in vitro, can prepare the food with antioxidation activity, food ingredient or guarantor Strong product.
The grifola frondosus weight polyphenol fraction for proving purifying through experiment in vitro has stronger suppression to the activity of α-glucuroide Effect, wherein Quercetin, gallic acid -3-O- (6`-O- nutgall acyls) glucosides and to Quercetin -3-O- β-D- pyrans half The IC of lactoside50Respectively 3.94 μ g/mL, 3.24 μ g/mL and 7.23 μ g/mL, it is remote to the inhibition of alpha-glucosidase It is much better than acarbose（IC50=1.04 mg/mL）.
The advantage of the invention is that：The present invention is mutually tied using macroporous adsorbent resin column chromatography with preparative high performance liquid chromatography The technology of conjunction, a kind of preparation method of high-purity grifola frondosus polyphenol compound monomer rapidly and efficiently is built, there is separating effect Good, the time is short, prepares the advantages such as purity height.
Embodiment
Embodiment one
The preparation of grifola frondosus fine powder：Taking Grifola Frondosa sporophore, it is standby that pulverizer crushed 80 mesh sieves in 50 DEG C of drying.
More phenol extractions：A certain amount of grifola frondosus fine powder is taken, using water as Extraction solvent, solid-liquid ratio 1：30, ultrasonic temperature Under conditions of 70 DEG C, ultrasonic time 60min, ultrasonic power 300w, extracted 2 times using ultrasonic assistant extractive technique, merging carries Liquid is taken, filtered, concentration, dries preparation grifola frondosus Polyphenols crude extract.
Membrane filtration：Grifola frondosus Polyphenols crude extract will be prepared to redissolve, filled by the milipore filter that molecular cut off is 5 kD Put and filtered, retain egg bletilla polysaccharide.Drying means is vacuum freeze drying used by filtrate, and routinely drying condition enters Row drying.
Macroporous adsorbent resin column chromatography：Take the macroporous absorbent resin wet method dress posts of NKA- II pre-processed, after balance on Sample, macroreticular resin loading mass concentration 5 mg/mL, the BV/h of loading speed 3, the h of adsorption time 2, with distilled water with 2 mL/min Flow velocity is rinsed to neutrality, then with 40% ethanol solution（pH 6.0）Eluted with the BV/h of elution speed 3, collect eluent, subtract Pressure concentration, lyophilized, the preparation thick component of grifola frondosus polyphenol compound, through 0.45 μm of membrane filtration after being redissolved with 50% methanol.
It is prepared by high-purity polyphenol compound monomer：The type macroporous adsorbent resin column chromatography isolates of NKA- II further across Preparative high performance liquid chromatography is purified, and grifola frondosus weight polyphenol fraction, concentrated drying, system are collected respectively according to UV-detector spectrogram Standby high-purity grifola frondosus polyphenol compound monomer.Preparative liquid chromatography condition is defined as：The anti-phase C18 chromatographic columns of preparative, Chromatographic column specification：The mm of internal diameter 20,5 μm, Detection wavelength 280nm, the mL of sample size 1 of 250 mm, C18 silica filler particle diameter of length, Flow velocity 5-10 mL/min, 30 DEG C of column temperature, using A:Methanol-B:0.1% formic acid carries out linear gradient elution, and elution program is：
（1）0~30 min：A: 5%+B: 95%→A: 60%+B: 40%；
（2）30~35 min：A: 60%+B: 40%→A: 5%+B: 95%；
（3）35~45 min：A: 5%+B: 95%.
The HPLC-MS analysis of high-purity grifola frondosus polyphenol compound monomer：Liquid phase chromatogram condition： The anti-phase C18 column chromatographies post specification of preparative：5 μm mm of internal diameter 4.6, length 250 of mm, C18 silica filler particle diameter, detection Wavelength 280nm, sample size 20 μ L, the mL/min of flow velocity 0.8,30 DEG C of column temperature, using A:Methanol-B:0.1% formic acid enters line Property gradient elution, elution program are：
（1）0~30 min：A: 5%+B: 95%→A: 60%+B: 40%；
（2）30~35 min：A: 60%+B: 40%→A: 5%+B: 95%；
（3）35~45 min：A: 5%+B: 95%.
Mass Spectrometry Conditions are：Spraying（IS）Voltage is 3.0 kV, hole voltage 20V, 130 DEG C of the temperature of ESI ion guns, is dissociated 350 DEG C of temperature, mass spectral analysis is carried out in negative ion mode with reference to spectrum, mass spectral analysis scope 50 arrives 600m/z.
By corresponding mass spectrogram, the retention time and Information in Mass Spectra of Main Basiss chromatographic peak including molecular weight information etc., Pass through European Phenol-Explorer polyphenol database website http://phenol-explorer.eu/ and documentation ＆ info are carried out The identification of phenolic compound, the results showed that the weight polyphenol fraction that macroporous absorbent resin is adsorbed in -40% ethanol eluate includes：Tan is spent Acid（ellagic acid）, Quercetin（quercetin）, 4-HBA（4-hydroxybenzoic acid）, do not eat Sub- acid -3-O- (6`-O- nutgall acyls) glucoside（gallic acid-4-O-(6`-O-galloyl)glucoside）, coffee Coffee acyl tartaric acid（caffeoyl tartaric acid）, caffeic acid（caffeic acid）, L-Epicatechin gallate （(-)-epicatechin 3-O-gallate）, 5,6,7,3', 4'- pentamethoxyl flavones（sinenstein）, to coumaric acyl Kui Buddhist nun's acid（p-coumaroyl-quinic acid）, m-digallic acid（m-digallic acid）, the forulic acid of dehydrogenation two （dehydrodiferulic acid）, Quercetin -3-O- β-D- galactopyranosides（quercetin-3-O-β-D- galactopyranoside）, 3,4,5- trimethoxybenzoic acids（methyl gallate）, 5- nonadecyl resorcinols（5- nonadecylresorcin）, forulic acid（ferulic acid）, to coumaric acyl malic acid（p-coumaroyl malic acid）And to coumaric acyl tartaric acid（p-coumaroyl tartaric acid）.
The macroporous absorbent resin of table 1 adsorbs the Mass Spectrometric Identification result of weight polyphenol fraction in -40% ethanol eluate
The antioxidation in vitro experiment of the ash tree flower polyphenol of test example 1
Using DPPH radical scavenging activities, Hydroxyl radical-scavenging ability and reducing power as foundation, it was demonstrated that the ash tree of purifying Flower polyphenol component has stronger antioxidation activity in vitro, can prepare the food with antioxidation activity, food ingredient or guarantor Strong product.
（1）Remove the measure of DPPH free radical abilities（DPPH methods）
2.0mL various concentrations testing sample solutions are taken, add the mL of 0.2 mmol/L DPPH ethanol solutions 2.0, mixing The min of avoid light place 30 after uniformly, determines its light absorption value at the nm of wavelength 517.Sample is replaced as sky using the % ethanol of 2.0 mL 95 White control；Using 2.0 mL samples and the % alcohol mixeding liquids of 2.0 mL 95 as sample controls, to eliminate the shadow of sample itself color Ring.The calculation formula of DPPH clearance rates is：
The IC50 of 17 kinds of grifola frondosus polyphenolic substances is respectively less than 0.20 mg/mL after measured.This result can be seen that grifola frondosus Polyphenol has certain scavenging action to DPPH free radicals.
（2）The measure of hydroxyl radical free radical ability
The mL of various concentrations testing sample solution 1 is taken, adds 9 mmol/L FeSO41 mL, 9 mmol/L salicylic acids-second The mL of alcoholic solution 1, finally add 8.8 mmol/L H2O2 1 mL starts reaction, 30 min is reacted under 37 DEG C of water-baths, in 510 nm The light absorption value of lower each strength solution of measure.Due to H2O2With Fe2+Mixing can produce OH, and salicylic acid can catch OH and produce coloured Material, because sample itself has light absorption value, then with 9 mmol/L FeSO41 mL, 9 mmol/L salicylic acids-ethanol solution 1 ML each adds the control absorption value of the mL of sample solution 1 and the mL of distilled water 1 of various concentrations as sample（That is distilled water generation For 8.8 mmol/L H2O2）.OH clearance rates are calculated as follows：
The IC50 of 17 kinds of grifola frondosus polyphenolic substances is less than 0.12 mg/mL after measured, and polyphenol is to H2O2/Fe2+ System passes through OH caused by Fenton reactions has scavenging action.
（3）The measure of reducing power
0.2 mol/L pH 6.6 phosphate buffer 2.5mL and the testing sample of various concentrations are separately added into test tube Solution 1mL, adds the potassium ferricyanide solutions of 2.5 mL 1%, well mixed after reacting 20 min in 50 DEG C of water-baths.It is fast after taking-up Quickly cooling is but and the addition % trichloroacetic acid terminating reactions of 2. 5mL 10,3500 r/min centrifuge 10 min.The mL of supernatant 2.5 is taken, 2.5 mL distilled water and the % liquor ferri trichloridis of 0.5 mL 0.1 are added, 10 min are stood after mixing, are determined at 700 nm each The light absorption value of solution.Light absorption value is bigger, and explanation reducing power is stronger.The calculation formula of reducing power is：Reducing power=A samples-A is empty In vain.
After measured the reducing power of 17 kinds of grifola frondosus polyphenolic substances than etc. mass concentration（1 mg/mL）Ascorbic acid By force.
The external alpha-glucosaccharase enzyme level experiment of the ash tree flower polyphenol of test example 2
Alpha-glucosidase activity determines：In 10 mM that 1 mL is prepared with 50 mM phosphate buffers (pH 7.4) In pNPG substrate systems, add 5 μ L alpha-glucosaccharase enzyme solutions, it is rapid to mix, by Shimadzu Corporation UV-2600 is ultraviolet can See that extinction value changes per minute carry out Indicator Reaction speed ν values at spectrophotometer detection 400nm, so as to characterize the big of enzyme activity It is small.Measurement temperature is 37 DEG C.
By the high-purity grifola frondosus weight polyphenol fraction of preparation, the concentration soluble in water that is configured to is 50 mg/mL solution respectively, with 50 MM pH 6.5 PBS buffer solutions are diluted to concentration as 0.001 mg/mL, 0.005 mg/mL, 0.01 mg/mL, 0.05 respectively mg/mL、0.1 mg/mL、0.2 mg/mL、0.4 mg/mL、0.6 mg/mL、0.8 mg/mL、1.0 mg/mL、2.0 mg/mL、 4.0 mg/mL, 6.0 mg/mL, 8.0 mg/mL and 10.0 mg/mL.Take the μ L of solution 10 of each concentration and isometric α-grape Glucosides enzyme liquid mixes, and enzyme activity system is made, enzyme activity is determined after 30 DEG C of insulation 2h.Enzyme activity determination method is same as above.α during measure- Final concentration of 0.2 U/mL of glucuroide, substrate pNPG concentration are 10 mM.Each enzyme activity system is calculated according to measurement result Enzyme activity.Enzyme activity × 100% of enzyme activity/alpha-glucosidase of enzyme activity=each enzyme activity system.
The grifola frondosus weight polyphenol fraction for proving purifying through experiment in vitro has stronger suppression to the activity of α-glucuroide Effect, wherein Quercetin, gallic acid -3-O- (6`-O- nutgall acyls) glucosides and to Quercetin -3-O- β-D- pyrans half The IC of lactoside50Respectively 3.94 μ g/mL, 3.24 μ g/mL and 7.23 μ g/mL, it is remote to the inhibition of alpha-glucosidase It is much better than acarbose（IC50=1.04 mg/mL）.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, it should all belong to the covering scope of the present invention.

Claims (2)

  1. A kind of 1. preparation method of high-purity grifola frondosus weight polyphenol fraction, it is characterised in that：Comprise the following steps：
    （1）Pretreatment：Grifola Frondosa sporophore dried product is ground into powder, it is standby to cross 40~80 mesh sieves；
    （2）Extraction：Take grifola frondosus powder to add in distilled water, extracted 2-4 times using ultrasonic assistant extractive technique, merge extraction Liquid, residue is removed through plate-frame filtering；
    （3）Membrane filtration：Filtered by film filter, retain egg bletilla polysaccharide, the concentrating filter liquor of acquisition, obtained after drying Obtain ash tree flower polyphenol crude extract；
    （4）Isolate and purify：Ash tree flower polyphenol crude extract is redissolved in deionized water, using column chromatography-preparative efficient liquid phase Chromatographic tandem separating and purifying technology is isolated and purified；
    The condition of described ultrasonic wave added extraction is：Using water as Extraction solvent, feed liquid weight ratio is 1：10-1：50, ultrasonic temperature 30-70 DEG C, ultrasonic time 30-90 min, ultrasonic power 100-400w；
    Described filter membrane is the milipore filter that molecular cut off is 5 kD, and drying means is vacuum freeze drying used by filtrate Or centrifugal spray drying；
    The filler of described column chromatography is：The type macroporous absorbent resins of NKA- II, the BV/h of loading speed 3, the h of adsorption time 2, elution Solvent is 20%-60% ethanol solutions, the BV/h of elution speed 3；Through adsorbing, eluting collection eluent, it is concentrated under reduced pressure into slightly dry Product, through 0.45 μm of membrane filtration after being redissolved with 50% methanol；
    The anti-phase C18 chromatographic columns of preparative, chromatographic column specification：5 μm mm of internal diameter 20, length 250 of mm, C18 silica filler particle diameter, Detection wavelength 280nm, the mL of sample size 1, flow velocity 5-10 mL/min, 30 DEG C of column temperature, using A:Methanol-B:0.1% formic acid is carried out Linear gradient elution, elution program are：
    （1）0~30 min：A: 5%+B: 95%→A: 60%+B: 40%；
    （2）30~35 min：A: 60%+B: 40%→A: 5%+B: 95%；
    （3）35~45 min：A: 5%+B: 95%.
  2. A kind of 2. preparation method of high-purity grifola frondosus weight polyphenol fraction according to claim 1, it is characterised in that：NKA- II type macroporous adsorbent resin column chromatography isolate purifies further across preparative high performance liquid chromatography, is composed according to UV-detector Figure collects grifola frondosus weight polyphenol fraction and carries out crystallization operation respectively, obtains 17 kinds of high-purity weight polyphenol fractions：Ellagic acid, Quercetin, 4- Hydroxybenzoic acid, gallic acid -3-O- (6`-O- nutgall acyls) glucoside, caftaric acid, caffeic acid, epicatechin Gallate, 5,6,7,3', 4'- pentamethoxyls flavones, to coumaroyl guinic acid, m-digallic acid, the forulic acid of dehydrogenation two, It is Quercetin -3-O- β-D- galactopyranosides, 3,4,5- trimethoxybenzoic acids, 5- nonadecyls resorcinol, forulic acid, right Coumaric acyl malic acid and 95% is all higher than to coumaric acyl tartaric acid, purity.
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