CN105732250A - Preparing method for high-purity grifola frondosa polyphenol component - Google Patents
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Abstract
The invention relates to a preparing method for a high-purity grifola frondosa polyphenol component, and belongs to the field of functional factors of functional food and medicine.The preparing method includes the steps that a grifola frondosa polyphenol crude component is prepared through an ultrasonic-assisted extraction technology, biomacromolecules such as polysaccharide and protein are removed with an ultrafiltration membrane retention technology, and finally various high-purity grifola frondosa polyphenol compound monomers are prepared by adopting the macroporous adsorbent resin NKA-II type column chromatography-preparative high performance liquid chromatography series separation and purification technology.The preparing method is fast and efficient, and seventeen grifola frondosa polyphenol compound monomers can be prepared at the same time and high in purity.The prepared grifola frondosa polyphenol compound monomers has a high DPPH free radical and hydroxyl free radical scavenging rate and high reducing power, activity of alpha-glucosidase can be restrained by most grifola frondosa polyphenol compound monomers, and the grifola frondosa polyphenol compound monomers have the physiological effects of effectively protecting human body cell tissue and heart and cerebral vessel circulating system, resisting cancer, delaying ageing and assisting in reducing blood sugar.
Description
Technical field
The present invention relates to the preparation method of a kind of high-purity Grifola frondosa weight polyphenol fraction, belong to functional food functional factor and
Drug world.
Background technology
It is true that Grifola frondosa (Grifola frondosa) belongs to Basidiomycotina Hymenomycetes Aphyllophorales Polyporaceae tree Pseudomonas
Bacterium, primary growth to temperate zone, is distributed mainly on the ground such as Fujian, Hebei, Jilin, Guangxi and Sichuan in subtropical zone in China.As
The food of a kind of preciousness, medicine dual-purpose mushroom fungus, the various nutrients of Grifola frondosa and physiological function become first of various edible fungi of living apart, and have
" edible fungi prince " and the good reputation of " North China Radix Ginseng ".Correlational study shows, Grifola frondosa has enhancing immunity, suppression tumor, resists
Virus, antioxidation is anti-aging, stabilizing blood pressure, reducing blood sugar and blood lipid, improve lipid metabolism and alleviate hepatitis symptom, memory reinforcing etc.
Multiple medicinal health care function.Numerous studies work shows, polyphenols has antioxidation, antibacterial, antimutagenic, antiviral, anti-
The several functions such as tumor, reducing blood sugar and blood fat activity.The most less for the research report of Grifola frondosa polyphenol.Chen XiangDong et al.
Use low pole macroporous adsorbent resin separation from maitake mushroom mycelia crude extract prepare Polyphenols component, it is thus achieved that component be
Brown powder shape product, empirical tests has antioxidation, an antibacterial and α-amylase suppression isoreactivity, but its Grifola frondosa polyphenol prepared
Being mixture, the component of Grifola frondosa polyphenols is further purified and is identified, Grifola frondosa weight polyphenol fraction needs into one
Step separating-purifying (Chen XiangDong, Liu Xiaowen, Wu Wutong. the extraction of Grifola frondosa polyphenol and activity research. food and biotechnology
Report, 2005,24 (4): 26-30.).
The present invention proposes with Grifola Frondosa sporophore as raw material, prepares Grifola frondosa polyphenol group by Ultrasonic Wave-Assisted Extraction technology
Point, then profit retains technology with ultrafilter membrane and removes the biomacromolecule such as polysaccharide therein and albumen, finally uses macroporous absorption tree
The separation purification technique of fat NKA-II column chromatography-preparative high performance liquid chromatography, prepares multiple highly purified Grifola frondosa many
Phenolic compound monomer.Extracting polyphenols the most than ever from maitake mushroom mycelia, this invention uses with ash tree beggar real
Body is raw material, and raw material sources are easier to acquisition and polyphenol content is compared mycelium and come high, and the extraction yield of polyphenol is the highest；With
Existing research report is compared, and uses the Grifola frondosa weight polyphenol fraction obtained by ultrafiltration membrance filter to eliminate in Effects of Extracts of Grifola frondosa on Active
The macromolecular substances such as polysaccharide, albumen, through column chromatography-preparative liquid chromatography series connection thick weight polyphenol fraction of purification techniques, made
The weight polyphenol fraction purity higher (purity is all higher than 95%) obtained, polyphenol antioxidation activity in vitro also significance after purification improves, its
Middle reducing power all than etc. the ascorbic acid of mass concentration (1 mg/mL) strong.
Summary of the invention
It is an object of the invention to provide a kind of rapidly and efficiently, purity is high, preparation amount is bigger Grifola frondosa polyphenol compound
The preparation technology of monomer.
A kind of preparation method of high-purity Grifola frondosa weight polyphenol fraction, its preparation process is as follows:
(1) pretreatment: Grifola Frondosa sporophore dry products is ground into powder, crosses 40～80 mesh sieves standby；
(2) extraction: take a certain amount of Grifola frondosa powder, utilizes ultrasonic assistant extractive technique to extract 2-4 time, united extraction liquid,
Residue is removed through plate-and-frame filtration；
(3) membrane filtration: filtered by film filter, retains egg bletilla polysaccharide, it is thus achieved that concentrating filter liquor, obtain after drying
Obtain Grifola frondosa polyphenol crude extract；
(4) isolated and purified: Grifola frondosa polyphenol crude extract is redissolved in deionized water, use the efficient liquid phase of column chromatography-preparative
Chromatographic tandem separating and purifying technology carries out isolated and purified.
The condition of ultrasonic wave added of the present invention extraction is: with 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.
Filter membrane of the present invention be molecular cut off be the ultrafilter membrane of 5 kD, the drying means that filtrate is used is true
Vacuum freecing-dry or centrifugal spray drying.
The filler of column chromatography of the present invention is NKA-II type macroporous adsorbent resin, and loading speed 3 BV/h, during absorption
Between 2 h, eluting solvent is 20%-60% ethanol solution, elution speed 3 BV/h；Eluent, concentrating under reduced pressure is collected through absorption, eluting
To doing to obtain crude product, through 0.45 μm membrane filtration after redissolving with 50% methanol.
Liquid phase chromatogram condition of the present invention is: preparative anti-phase C18 chromatographic column, chromatographic column specification: internal diameter 20 mm,
Length 250 mm, C18 silica filler particle diameter 5 μm, detects wavelength 280nm, sample size 1 mL, flow velocity 5-10 mL/min, column temperature
30 DEG C, use 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 frondosa weight polyphenol fraction of the present invention is through preparative high performance liquid chromatography purification, according to ultraviolet
Detector spectrogram is collected Grifola frondosa weight polyphenol fraction respectively and is carried out crystallization operation acquisition, 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-Galla Turcica (Galla Helepensis) acyl) glucoside (gallic acid-4-O-(6`-O-galloyl)
Glucoside), caftaric acid (caffeoyl tartaric acid), caffeic acid (caffeic acid), epicatechin
Epicatechol gallate ((-)-epicatechin 3-O-gallate), 5,6,7,3', 4'-pentamethoxyl flavone (sinenstein),
To coumaroyl guinic acid (p-coumaroyl-quinic acid), m-digallic acid (m-digallic acid), dehydrogenation two
Ferulic acid (dehydrodiferulic acid), Quercetin-3-O-β-D-galactopyranoside (quercetin-3-O-β-D-
Galactopyranoside), 3,4,5-trimethoxybenzoic acid (methyl gallate), 5-nonadecyl resorcinol (5-
Nonadecylresorcin), ferulic acid (ferulic acid), p-coumaroyl malic acid (p-coumaroyl malic
Acid) and p-coumaroyl tartaric acid (p-coumaroyl tartaric acid), purity is all higher than 95%.
With DPPH radical scavenging activity, Hydroxyl radical-scavenging ability and reducing power as foundation, it was demonstrated that the ash tree of purification
Flower polyphenol component has stronger antioxidation activity in vitro, can prepare and have the food of antioxidant activity, food ingredient or guarantor
Strong product.
Prove that through experiment in vitro the Grifola frondosa weight polyphenol fraction of purification has stronger suppression to the activity of α-glucosidase
Effect, wherein Quercetin, gallic acid-3-O-(6`-O-Galla Turcica (Galla Helepensis) acyl) glucoside and to Quercetin-3-O-β-D-pyrans half
The IC of lactoside50It is respectively 3.94 μ g/mL, 3.24 μ g/mL and 7.23 μ g/mL, remote to the inhibition of alpha-glucosidase
It is much better than acarbose (IC50=1.04 mg/mL).
It is an advantage of the current invention that: the present invention uses macroporous adsorbent resin column chromatography to tie mutually with preparative high performance liquid chromatography
The technology closed, builds a kind of high-purity Grifola frondosa polyphenol compound monomer preparation method rapidly and efficiently, has separating effect
The advantages such as good, the time is short, and preparation purity is high.
Detailed description of the invention
Embodiment one
The preparation of Grifola frondosa fine powder: taking Grifola Frondosa sporophore in 50 DEG C of drying, it is standby that 80 mesh sieves pulverized by pulverizer.
Many phenol extractions: take a certain amount of Grifola frondosa fine powder, with water as Extraction solvent, solid-liquid ratio is 1:30, ultrasonic temperature
70 DEG C, ultrasonic time 60min, under conditions of ultrasonic power 300w, utilize ultrasonic assistant extractive technique to extract 2 times, merging carries
Take liquid, through filtering, concentrating, be dried preparation Grifola frondosa Polyphenols crude extract.
Membrane filtration: preparation Grifola frondosa Polyphenols crude extract is redissolved, is filled by the ultrafilter membrane that molecular cut off is 5 kD
Put and filter, retain egg bletilla polysaccharide.The drying means that filtrate is used is vacuum lyophilization, and drying condition enters routinely
Row is dried.
Macroporous adsorbent resin column chromatography: take pretreatment good NKA-II macroporous adsorbent resin wet method dress post, after balance on
Sample, macroporous resin loading mass concentration 5 mg/mL, loading speed 3 BV/h, adsorption time 2 h, with distilled water with 2 mL/min
Flow velocity rinses to neutral, then with 40% ethanol solution (pH 6.0) with elution speed 3 BV/h eluting, collection eluent, subtract
Pressure concentrates, lyophilizing, prepares the thick component of Grifola frondosa polyphenol compound, through 0.45 μm membrane filtration after redissolving with 50% methanol.
Prepared by high-purity polyphenol compound monomer: NKA-II type macroporous adsorbent resin column chromatography separator further across
Preparative high performance liquid chromatography purification, collects Grifola frondosa weight polyphenol fraction respectively according to UV-detector spectrogram, concentrated dry, system
Standby high-purity Grifola frondosa polyphenol compound monomer.Preparative liquid chromatography condition is defined as: preparative anti-phase C18 chromatographic column,
Chromatographic column specification: internal diameter 20 mm, length 250 mm, C18 silica filler particle diameter 5 μm, detects wavelength 280nm, sample size 1 mL,
Flow velocity 5-10 mL/min, column temperature 30 DEG C, use 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 frondosa polyphenol compound monomer: liquid phase chromatogram condition:
Preparative anti-phase C18 column chromatography post specification: internal diameter 4.6 mm, length 250 mm, C18 silica filler particle diameter 5 μm, detection
Wavelength 280nm, sample size 20 μ L, flow velocity 0.8 mL/min, column temperature 30 DEG C, use A: methanol-B:0.1% formic acid carries out line
Property gradient elution, 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%.
Mass Spectrometry Conditions is: spraying (IS) voltage is 3.0 kV, and the ionogenic temperature of hole voltage 20V, ESI 130 DEG C is dissociated
Temperature 350 DEG C, carries out mass spectral analysis, mass spectral analysis scope 50 to 600m/z in conjunction with spectrum at negative ion mode.
By corresponding mass spectrum, retention time and the Information in Mass Spectra of Main Basis chromatographic peak include molecular weight information etc.,
Carried out by Europe Phenol-Explorer polyphenol database website http://phenol-explorer.eu/ and documentation & info
The qualification of phenolic compound, result shows that the weight polyphenol fraction in absorption with macroporous adsorbent resin-40% ethanol elution includes: tan flower
Acid (ellagic acid), Quercetin (quercetin), 4-HBA (4-hydroxybenzoic acid), no food
Sub-acid-3-O-(6`-O-Galla Turcica (Galla Helepensis) acyl) 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 flavone (sinenstein), to coumaric acyl Kui
Buddhist nun acid (p-coumaroyl-quinic acid), m-digallic acid (m-digallic acid), dehydrogenation two ferulic acid
(dehydrodiferulic acid), Quercetin-3-O-β-D-galactopyranoside (quercetin-3-O-β-D-
Galactopyranoside), 3,4,5-trimethoxybenzoic acid (methyl gallate), 5-nonadecyl resorcinol (5-
Nonadecylresorcin), ferulic acid (ferulic acid), p-coumaroyl malic acid (p-coumaroyl malic
And p-coumaroyl tartaric acid (p-coumaroyl tartaric acid) acid).
The Mass Spectrometric Identification result of weight polyphenol fraction in table 1 absorption with macroporous adsorbent resin-40% ethanol elution
The antioxidation in vitro test of test example 1 Grifola frondosa polyphenol
With DPPH radical scavenging activity, Hydroxyl radical-scavenging ability and reducing power as foundation, it was demonstrated that the Grifola frondosa of purification is many
Phenol component has stronger antioxidation activity in vitro, can prepare and have the food of antioxidant activity, food ingredient or health product.
(1) mensuration (DPPH method) of DPPH free radical ability is removed
Take 2.0mL variable concentrations testing sample solution, add 0.2 mmol/L DPPH ethanol solution 2.0 mL, mix homogeneously
Rear lucifuge places 30 min, measures its light absorption value at wavelength 517 nm.Replace sample right for blank with 2.0 mL 95 % ethanol
According to；With 2.0 mL samples and 2.0 mL 95 % alcohol mixeding liquids as sample controls, to eliminate the impact of sample intrinsic colour.
The computing formula of DPPH clearance rate is:
The IC50 of 17 kinds of Grifola frondosa polyphenolic substances is respectively less than 0.20 mg/mL after measured.This result can be seen that Grifola frondosa polyphenol
DPPH free radical there is certain scavenging action.
(2) mensuration of hydroxyl radical free radical ability
Take variable concentrations testing sample solution 1 mL, add 9 mmol/L FeSO41 mL, 9 mmol/L salicylic acid-ethanol are molten
Liquid 1 mL, finally adds 8.8 mmol/L H2O2 1 mL starts reaction, reacts 30 min under 37 DEG C of water-baths, surveys under 510 nm
The light absorption value of fixed each strength solution.Due to H2O2With Fe2+Mixing can produce OH, and salicylic acid can catch OH and produce colored substance
Matter, owing to sample itself exists light absorption value, then with 9 mmol/L FeSO41 mL, 9 mmol/L salicylic acid-ethanol solution 1
ML each adds sample solution 1 mL and distilled water 1 mL comparison absorption value (the i.e. distilled water generation as sample of variable concentrations
For 8.8 mmol/L H2O2).It is calculated as follows OH clearance rate:
The IC50 of 17 kinds of Grifola frondosa polyphenolic substances is less than 0.12 mg/mL after measured, and polyphenol is to H2O2/Fe2+ System is passed through
The produced OH of Fenton reaction has scavenging action.
(3) mensuration of reducing power
Phosphate buffer 2.5mL and the testing sample solution of variable concentrations of 0.2 mol/L pH 6.6 it is separately added in test tube
1mL, adds 2.5 mL 1% potassium ferricyanide solutions, reacts 20 min after mix homogeneously in 50 DEG C of water-baths.After taking-up the coldest
But and add 2. 5mL 10 % trichloroacetic acids terminate reaction, 3500 r/min are centrifuged 10 min.Take supernatant 2.5 mL, add
2.5 mL distilled water and 0.5 mL 0.1 % liquor ferri trichloridi, stand 10 min after mixing, measures each solution at 700 nm
Light absorption value.Light absorption value the biggest explanation reducing power is the strongest.The computing formula of reducing power is: reducing power=A sample-A is blank.
After measured the reducing power of 17 kinds of Grifola frondosa polyphenolic substances all than etc. the ascorbic acid of mass concentration (1 mg/mL)
By force.
The external alpha-glucosidase inhibition test of test example 2 Grifola frondosa polyphenol
Alpha-glucosidase activity measures: the 10 mM pNPG prepared with 50 mM phosphate buffers (pH 7.4) at 1 mL
In substrate system, add 5 μ L alpha-glucosaccharase enzymatic solution, mix rapidly, divided by Shimadzu Corporation's UV-2600 UV, visible light
At light photometer detection 400nm, light absorption value per minute change carrys out Indicator Reaction speed ν value, thus characterizes the size of enzyme activity.Survey
Fixed temperature is 37 DEG C.
To be configured to concentration be 50 mg/mL solution, with 50 by the most soluble in water for the high-purity Grifola frondosa weight polyphenol fraction of preparation
The PBS buffer of mM pH 6.5 be diluted to respectively concentration be 0.001 mg/mL, 0.005 mg/mL, 0.01 mg/mL, 0.05
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 solution 10 μ L of each concentration and isopyknic α-Fructus Vitis viniferae
Glycosidase liquid mixes, and makes enzyme live body system, measures enzyme activity after 30 DEG C of insulation 2h.Enzyme activity determination method is ibid.α during mensuration-
Final concentration of 0.2 U/mL of glucosidase, substrate pNPG concentration is 10 mM.Each enzyme live body system is calculated according to measurement result
Enzyme activity.Enzyme activity × 100% of the enzyme activity/alpha-glucosidase of enzyme activity=each enzyme live body system.
Prove that through experiment in vitro the Grifola frondosa weight polyphenol fraction of purification has stronger suppression to the activity of α-glucosidase
Effect, wherein Quercetin, gallic acid-3-O-(6`-O-Galla Turcica (Galla Helepensis) acyl) glucoside and to Quercetin-3-O-β-D-pyrans half
The IC of lactoside50It is respectively 3.94 μ g/mL, 3.24 μ g/mL and 7.23 μ g/mL, 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 impartial changes done according to scope of the present invention patent with
Modify, all should belong to the covering scope of the present invention.
Claims (6)
1. the preparation method of a high-purity Grifola frondosa weight polyphenol fraction, it is characterised in that: comprise the following steps:
(1) pretreatment: Grifola Frondosa sporophore dry products is ground into powder, crosses 40～80 mesh sieves standby；
(2) extraction: take Grifola frondosa powder and add in distilled water, utilizes ultrasonic assistant extractive technique to extract 2-4 time, united extraction
Liquid, removes residue through plate-and-frame filtration；
(3) membrane filtration: filtered by film filter, retains egg bletilla polysaccharide, it is thus achieved that concentrating filter liquor, obtain after drying
Obtain Grifola frondosa polyphenol crude extract；
(4) isolated and purified: Grifola frondosa polyphenol crude extract is redissolved in deionized water, use the efficient liquid phase of column chromatography-preparative
Chromatographic tandem separating and purifying technology carries out isolated and purified.
The preparation method of a kind of high-purity Grifola frondosa weight polyphenol fraction the most according to claim 1, it is characterised in that: described
Ultrasonic wave added extraction condition be: with 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.
The preparation method of a kind of high-purity Grifola frondosa weight polyphenol fraction the most according to claim 1, it is characterised in that: described
Filter membrane be molecular cut off be the ultrafilter membrane of 5 kD, the drying means that filtrate is used is vacuum lyophilization or centrifugal spray
Mist is dried.
The preparation method of a kind of high-purity Grifola frondosa weight polyphenol fraction the most according to claim 1, it is characterised in that: described
The filler of column chromatography be: NKA-II type macroporous adsorbent resin, loading speed 3 BV/h, adsorption time 2 h, eluting solvent is
20%-60% ethanol solution, elution speed 3 BV/h；Collect eluent through absorption, eluting, be evaporated to do to obtain crude product, with 50%
Through 0.45 μm membrane filtration after methanol redissolution.
The preparation method of a kind of high-purity Grifola frondosa weight polyphenol fraction the most according to claim 1, it is characterised in that described
Liquid phase chromatogram condition is: preparative anti-phase C18 chromatographic column, chromatographic column specification: internal diameter 20 mm, length 250 mm, C18 silica gel are filled out
Material particle diameter 5 μm, detection wavelength 280nm, sample size 1 mL, flow velocity 5-10 mL/min, column temperature 30 DEG C, employing 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 preparation method of a kind of high-purity Grifola frondosa weight polyphenol fraction the most according to claim 1, it is characterised in that: NKA-
II type macroporous adsorbent resin column chromatography separator, further across preparative high performance liquid chromatography purification, is composed according to UV-detector
Figure is collected Grifola frondosa weight polyphenol fraction respectively and is carried out crystallization operation, obtains 17 kinds of high-purity weight polyphenol fractions: ellagic acid, Quercetin, 4-
Hydroxy benzoic acid, gallic acid-3-O-(6`-O-Galla Turcica (Galla Helepensis) acyl) glucoside, caftaric acid, caffeic acid, epicatechin
Epicatechol gallate, 5,6,7,3', 4'-pentamethoxyl flavone, to coumaroyl guinic acid, m-digallic acid, dehydrogenation two ferulic acid,
Quercetin-3-O-β-D-galactopyranoside, 3,4,5-trimethoxybenzoic acid, 5-nonadecyl resorcinol, ferulic acid, right
Coumaric acyl malic acid and p-coumaroyl tartaric acid, purity is all higher than 95%.
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