CN104306392A - Application of handkea utriformis polysaccharide in preparing antioxidant - Google Patents
Application of handkea utriformis polysaccharide in preparing antioxidant Download PDFInfo
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Abstract
The invention discloses application of a handkea utriformis polysaccharide in preparing an antioxidant. The inventor collects 14 common wild macro fungi in Lesser Khingan Mountains in Northeast China. The antioxidant activities of 14 macro fungal polysaccharides are tested by use of three methods and results indicate that the handkea utriformis polysaccharide has the highest antioxidant activity; the handkea utriformis polysaccharide has the free radical scavenging activity of 535.8 micro mol/g TEAC, the reduction activity of 5.94mmol Fe/g and the metal chelating ability of 274.8 micro mol Fe<2+>/g. The application of the handkea utriformis polysaccharide in preparing the antioxidant supplies the gaps of macro fungal polysaccharides; especially, the handkea utriformis polysaccharide has anti-tyrosinase activity in addition to high antioxidant activity. The application of the handkea utriformis polysaccharide in preparing the antioxidant lays a foundation for reasonable utilization of wild macro fungi in Northeast China.
Description
Technical field
The present invention relates to the novelty teabag of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide, the Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide that particularly chaps is preparing the application in antioxidant, also relates to the extracting method of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide.
Background technology
Macro fungi (Macrofungi or Macromycetes), refer to that those can form the fungus of macroscopic large-scale sporophore, their majorities belong to the Basidiomycota (Basidiomycota) of mycota, and remaining belongs to Ascomycota (Ascomycota).The macro fungi that sexual spore produces the small club shaped structure in outside is under the jurisdiction of Basidiomycota; Sexual spore produces and is under the jurisdiction of Ascomycota the macro fungi of small cystic structures.
Macro fungi is considered to important natural resources, macro fungi can be divided into edible fungi and medicinal fungus according to purposes.Macro fungi is rich in the nutrient substance such as polysaccharide, aminoacid, vitamin, is considered to the health food of low fat, high nutrition.Some macro fungis have been found to have the effect of antitumor, antioxidation, antibacterial, anti-diabetic and blood fat reducing, and the polysaccharide of these active and macro fungi generations, unsaturated fatty acid, protease isoreactivity composition are relevant.Macro fungi, except eating and be medicinal, is also used in other a lot of aspect.Such as, the active parent nucleus of methoxy acrylic bactericide, separates at first in macro fungi.
Macro fungi can be divided into according to its nutritional mode, parastic macro fungi, saprophytic form macro fungi.Main from living body biological, obtain the macro fungi of nutrition, be called as parastic macro fungi; From non-living body biology, what obtain nutrition is saprophytic fungus.In bacterial parasite, a part of fungus can form symbiosis mycorrhiza with host plant, is called as exotrophic mycorrhiza type macro fungi.Comparatively speaking, then the comparatively easy artificial culture of saprophytic bacteria is bacterial parasite, the more difficult artificial culture of VA Mycorrhizal Fungi.
Our country is vast in territory, be considered to one of the abundantest country of bio-diversity, had more than 3800 kinds by the macro fungi recorded, wherein agarics (e.g., Armillaria mellea) about has 1600 kinds, pore fungi (as, Coriolous Dersicolor (Fr.) Quel fungus) about 1300 kinds, false truffle (e.g., Lasiosphaera nipponica(Kawam.)Y.Kobayasi) about 300 kinds, colloid class (e.g., Auricularia fungus) about 100 kinds.Since ancient times, Chinese just have the tradition utilizing macro fungi.Edible fungi aspect: we ancestors just start the mushroom that searches for food before 6000 to 7000 years, before the history of the mushroom that searches for food of literature record can trace back to more than 2000 year, nearly 900 kinds of the macro fungi that China is known, a lot of macro fungi is wherein had to be cultivated by domestication, China is also the country that whole world edible fungi output is the highest, and the edible fungi exceeding total amount 50% originates from China.Due to having a long way to go of dietary habit between east and west, think poisonous macro fungi in many countries, but just can be eaten after processing in China.Since ancient times, just there is the custom utilizing macro fungi to be used as medicine in China, and such as Ganoderma, these famous Chinese medicines of Cordyceps all belong to macro fungi.
The method that the object of the invention is to be combined with Morphological Identification technology by Molecular Identification technology identifies the wild large-form fungi that 14 kinds of northeast of collecting is common, investigates the multiple biological activity of these 14 kinds of macro fungis.By the research to 14 kinds of wild large-form fungi, for these funguses developed further provide theoretical foundation, make that some are idle, reproducible resource obtains rational exploitation and utilization.
Summary of the invention
Inventor, in the Xiaoxinanlin Mountains, Northeast China, collects 14 kinds of common wild large-form fungi.First these 14 kinds of macro fungis are identified, and their polyoses content, antioxidation, antityrosinase isoreactivity are studied.By morphology and molecular biology two kinds of method qualification macro fungis, find that they belong to 6 sections respectively: wherein porous section 4 kinds, Tricholomataceae 3 kinds, Russulaceae 3 kinds, handle mushroom section 3 kinds, Lycoperdaceae a kind and rivet mushroom section a kind.The crude polysaccharides of these 14 kinds of macro fungis is extracted, then precipitation, defat and dialysis has been carried out to carry out purification to crude polysaccharide extract, obtained the polysaccharide after water miscible purification.
Next, the present invention tests the antioxidant activity of 14 kinds of macro fungi polysaccharide, for scavenging free radicals ABTS by three kinds of methods
+after the purification of active aspect macro fungi, the activity of polysaccharide PPS is 16.9 to 535.8 μm of ol/g TEAC, in iron ion reducing power, after purification, the activity of polysaccharide PPS is 0.23 to 5.94mmol Fe/g, and in ferrous ion chelating ability, after purification, the activity of polysaccharide PPS is 0.70 to 484.61 μm of ol Fe
2+the antioxidant activity that/g finds that there is 6 kinds of macro fungi polysaccharide is comparatively strong, and these 6 kinds of macro fungis can eat.These 6 kinds of funguses are be full of cracks Lasiosphaera Seu Calvatia, mastoid process handle mushroom, Armillaria ostoyae, parasol mushroom, Lepista mucla (Bull.:Fr.) Cooke and yellow Armillariella respectively.The strongest is be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi (Handkea utriformis), and its Free-radical scavenging activity is 535.8 μm of ol/g TEAC, and reducing power activity is 5.94mmol Fe/g, and metal chelating makes a concerted effort to be 274.8 μm of ol Fe
2+/ g is the front two that is positioned at 14 kinds of macro fungis.
Therefore, on the basis of this research, the present invention proposes be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide and preparing the application in antioxidant.
In the present invention, preferably, described be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide extracts by the following method and obtains:
(1) by the sporophore of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi, dry under 30-50 DEG C of condition, until quality no longer changes, with pulverizer the sporophore grinds of drying;
(2) with acetone by the lixiviate three times under 20-30 DEG C of condition of the be full of cracks Lasiosphaera Seu Calvatia mycopowder of drying, each 2-4 hour, centrifugally removes supernatant, precipitation boiling water extraction 3 hours, centrifugal, after removing precipitation, collects supernatant;
(3) step (2) is collected the supernatant obtained to mix with the volume ratio of ethanol according to 1:4-6, at 0-10 DEG C, make polysaccharide precipitation, centrifugalize, obtain precipitation;
(4) resolution of precipitate is in water, then uses lyophilization, obtains the crude extract of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide.
In the present invention, preferred, described method also comprises and will obtain the further deproteinization purification of crude extract, the dialysis of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide and again utilize alcohol settling, and concrete operations are:
(1) chloroform and n-butyl alcohol are hybridly prepared into Sevag reagent, then the crude extract of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide are mixed with Sevag reagent, stir, centrifugal; Solution divides three layers, and upper strata is polysaccharide solution, and lower floor is organic reagent, and intermediate layer is the albumin layer removed, and collects the polysaccharide solution on upper strata;
(2) by collect polysaccharide solution according to step (1) method reprocessing, until remove protein;
(3) dialyse with deionized water, the molecular cut off of bag filter is 3500 dalton, and remove micromolecule, the polysaccharide solution of the purification of acquisition, adds the ethanol of 5 times of volumes, at 4 DEG C, makes polysaccharide precipitation, centrifugalize, obtains precipitation; The precipitation obtained, then use water dissolution, lyophilizing, be the polysaccharide after water miscible purification.
Wherein, preferably, described Sevag reagent is formed according to volume ratio 5:1 mixed preparing chloroform and n-butyl alcohol.
Wherein, preferably, being first dissolved in a small amount of water by the crude extract of described be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide, is then that 5:1 mixes with Sevag reagent according to volume ratio.
Wherein, preferably, in step (3), also comprise the precipitation that will obtain, then use water dissolution, and then add the ethanol of 5 times of volumes, at 4 DEG C, make polysaccharide precipitation, centrifugalize, obtain precipitation, more fully to remove alcohol soluble substance, finally will be precipitated and dissolved in water, lyophilizing, is the polysaccharide after water miscible purification again.
The present invention is also tested for 14 kinds of macro fungi polysaccharide concentrations when 1mg/ml to the suppression ratio of tryrosinase, wherein be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi and the suppression ratio of Gomphidius rutilus to tryrosinase have exceeded 50%, be respectively 61% and 85%, by testing the suppression ratio of polysaccharide to tryrosinase of these two kinds of other concentration of macro fungi, obtain their IC
50value can reach 0.78mg/ml and 0.46mg/ml, and especially the activity of the polysaccharide of Gomphidius rutilus is even close to arbutin IC
50value (0.43mg/ml).Further experiment find, Gomphidius rutilus should belong to compound tyrosinase inhibitor.
Further, the invention allows for a kind of extracting method of macro fungi polysaccharide, it is characterized in that utilizing acetone, Sevag reagent, the method for ethanol and dialysis remove and comprise nonpolar, that low pole material, albumen, alcohol soluble substance and molecular weight are less than 3500 daltonian micromolecule non-polysaccharose substance, specifically comprise the following steps:
(1) by the sporophore of macro fungi, dry under 30-50 DEG C of condition, until quality no longer changes, with pulverizer the sporophore grinds of drying;
(2) with acetone by the lixiviate three times under 20-30 DEG C of condition of the macro fungi mycopowder of drying, each 2-4 hour, centrifugally removes supernatant, then uses boiling water extraction 3 hours, centrifugal, after removing precipitation, collects supernatant;
(3) step (2) is collected the supernatant obtained to mix with the volume ratio of ethanol according to 1:4-6, at 0-10 DEG C, make polysaccharide precipitation, centrifugalize, obtain precipitation;
(4) resolution of precipitate is in water, then uses lyophilization, obtains the crude extract of macro fungi polysaccharide.
Wherein, preferably, also comprise further for the crude extract obtaining macro fungi polysaccharide deproteinization purification, dialysis and again utilize alcohol settling, concrete operations are:
(1) chloroform and n-butyl alcohol are hybridly prepared into Sevag reagent, then the crude extract of macro fungi polysaccharide are mixed with Sevag reagent, stir, centrifugal; Solution divides three layers, and upper strata is polysaccharide solution, and lower floor is organic reagent, and intermediate layer is the albumin layer removed, and collects the polysaccharide solution on upper strata;
(2) by collect polysaccharide solution according to step (1) method reprocessing, until remove protein;
(3) dialyse with deionized water, the molecular cut off of bag filter is 3500 dalton, and remove micromolecule, the polysaccharide solution of the purification of acquisition, adds the ethanol of 5 times of volumes, at 4 DEG C, makes polysaccharide precipitation, centrifugalize, obtains precipitation.The precipitation obtained, then use water dissolution, lyophilizing, be the polysaccharide after water miscible purification.
Wherein, preferably, described Sevag reagent is formed according to volume ratio 5:1 mixed preparing chloroform and n-butyl alcohol; Preferred, the crude extract of described be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide is first dissolved in a small amount of water, is then that 5:1 mixes with Sevag reagent according to volume ratio.
Wherein, preferably, in step (3), also comprise the precipitation that will obtain, then use water dissolution, and then add the ethanol of 5 times of volumes, at 4 DEG C, make polysaccharide precipitation, centrifugalize, obtain precipitation, more fully to remove alcohol soluble substance, finally will be precipitated and dissolved in water, lyophilizing, is the polysaccharide after water miscible purification again.
The blank of some macro fungi polysaccharide researches has been filled up in proposition of the present invention, polysaccharide as the Lasiosphaera Seu Calvatia that chaps, mastoid process handle mushroom and yellow Armillariella seldom saw report in the past, find that their polysaccharide has antioxidant activity by this research, the Lasiosphaera Seu Calvatia that especially chaps has antityrosinase activity concurrently except antioxidant activity is comparatively strong.Although also find the polysaccharide once studied mistake of some macro fungis in addition, found its new activity by this research, as Gomphidius rutilus has good antityrosinase activity.Proposition of the present invention is that the Appropriate application of Northeast China wild large-form fungi provides the foundation.
Accompanying drawing explanation
Fig. 1 is the Technology Roadmap that in embodiment 1 prepared by polysaccharide;
Fig. 2 is 3 kinds of Antioxidative Activity Determination results of macro fungi polysaccharide;
Fig. 3 is the antityrosinase determination of activity result of macro fungi polysaccharide (1mg/ml).
Detailed description of the invention
Below in conjunction with embodiment to above-mentioned being described in more detail with other technical characteristic and advantage of the present invention.Should be understood that; the embodiment of the following stated; only that the preferred embodiment of the present invention is described; not scope of the present invention is limited; under not departing from the present invention and designing the prerequisite of spirit; the various distortion that those of ordinary skill in the art make technical scheme of the present invention and improvement, all should fall in protection domain that claims of the present invention determines.
Experiment material, reagent and instrument involved in the embodiment of the present invention
1, test material
The JIUYUE of 2011, have collected the common wild large fungi in 14 kinds of northeast in the Xiaoxinanlin Mountains, Northeast China.
2, reagent and instrument
Reagent: the analytical pure such as acetone, dehydrated alcohol, chloroform, phenol, from Beijing chemical reagents corporation.The E.C. 3.4.21.64 of Molecular Identification, the reagent such as Taq enzyme, dNTP is from Beijing Tian Gen biochemical technology company limited.Tryrosinases etc. are from Sigma chemical reagent company limited.Glass rod, triangular flask, beaker, condensing tube, the glass drying oven such as glue head dropper, pipet, volumetric flask, bottle,suction, blue lid bottle, cillin bottle, glass desicator, round-bottomed flask, from Shu Niu glass apparatus company limited.Rotary Evaporators RE-52AA is from Shanghai Yarong Biochemical Instrument Plant.Shu Mei KQ-600DE numerical control supersonic cleaning apparatus, from Kunshan Ultrasonic Instruments Co., Ltd..85-2 constant temperature blender with magnetic force is from Haimen kylin medical apparatus factory.98-1-C numeral control-temperature electric heating cover and 101-1AB air dry oven are from Tianjin Stettlen Instrument Ltd., freezer dryer, from Bo Yikang experimental apparatus company limited, the multiplex vacuum pump of SHB-III circulating water type is from Nanjing Wen Er instrument and equipment company limited.Refrigerated centrifuger, from Hunan Xiang Yi centrifuge Instrument Ltd..The pipettor of various range, from Shanghai big dragon armarium company limited.Electrophoresis equipment is from Liuyi Instruments Plant, Beijing.PCR instrument PCR System9700, from Canadian Applied Biosystems company.
The Molecular Identification of embodiment 1 macro fungi
Method:
The Molecular Identification of macro fungi, utilize round pcr, select ITS (Internal Transcribed Spacer, the ITS internal transcribed spacer) sequence of universal primer amplification macro fungi, then to carry out checking order and comparison has come, concrete steps are as follows:
1. the macro fungi of collecting is dry at 35 DEG C in air dry oven.
The pH of the 1M concentration of 2.10mL is the Tris-Hcl of 8.0, the pH adding the 0.5M concentration of 20ml is the EDTA of 8.0, add the NaCl of the 5M concentration of 5ml again, add sterilized water and be settled to 100ml, be configured to DNA extraction buffer 100ml (namely, 100mM Tris-HCl, 100mM EDTA pH8.0,250mM NaCl).
3. get the sporophore of 50mg macro fungi, grind into powder in liquid nitrogen, be loaded in the centrifuge tube of 1.5ml, add 500 μ l Extraction buffers, with turbula shaker mixing, resuspended.
4. add the SDS of 10% of 125 μ l, and then to add 12.5 μ l concentration be the E.C. 3.4.21.64 of 20mg/ml, 37 DEG C of water-bath 1h.
Under 5.12000rpm, centrifugal 3min, gets supernatant with pipettor, abandons precipitation.500 μ l isopropyl alcohols are added, mixing, 5 to 10min under room temperature in supernatant.
Under 6.12000rpm, centrifugal 5min, discards supernatant with pipettor, and 70% ethanol of precipitation 1ml is washed once, and under 12000rpm, centrifugal 3min, discards supernatant with pipettor.Stay precipitation, natural drying, obtain DNA crude extract.
7. in DNA crude extract, add the TE of 500 μ l, after abundant dissolving DNA, carry out phenol and imitate extracting.Once, under 12000rpm, centrifugal 5min gets supernatant to the saturated phenol extracting of equal-volume Tris; Once, 12000rpm is centrifugal, and lower 5min gets supernatant to equal-volume phenol chloroform extraction; Once, 12000rpm is centrifugal, and 3min gets supernatant to equal-volume chloroform, removes impurity.
8. add the NaAc of the 3M concentration of 1/10 volume, the dehydrated alcohol mixing of 2 times of volumes, ice bath 30min to 1h precipitates DNA, and under 12000rpm, centrifugal 5min, removes supernatant with pipettor.Precipitation (perhaps invisible) 70% ethanol 1mL cleaning once (uses rifle head), drying about 10min under room temperature.
The TE dissolving DNA of 9.30-50 μ l, adds the RNA enzyme of 1 to 2 μ l, at 37 DEG C of water-bath 1h.1.5 μ l bromjophenol blues add 5 μ L samples, carry out electrophoresis.
10. STb gene is diluted 50 times, with fungus universal primer ITS1 (5 '-TCCGATGGTGAACCTGCGG-3 ') and ITS4 (5 '-TCCTCCGCTTATTGATATGC-3 ') for amplimer pair, carry out PCR reaction, the ITS sequence of amplification targeted fungal, reaction system is as follows: 10 times of buffer 25 μ l, dNTP (10mM) 1.0 μ l, primer 1 (10 μMs) 2.5 μ l, primer 2 (10 μMs) 2.5 μ l, template DNA (10ng/ μ L) 1.0 μ l, Taq DNA polymer (2.5u/ μ L) 0.5 μ l, sterile deionized water 25 μ l.
The above-mentioned solution of 11. mixing, after centrifugal, puts into PCR instrument.React by following setting: 3min at 94 DEG C, then repeat circulation (circulation: at 94 DEG C, the 1min in bracket; Then at 52 DEG C, 1min; At 72 DEG C, 2min) 30 times, then 8min at 72 DEG C.
The ITS sequence that electrophoresis detection amplifies is carried out, purification of target sequence after 12.PCR completes.
ITS sequence after 13. purification utilizes primer I TS1 and ITS4 to check order, and sequencing result utilizes the BLAST of http://www.ncbi.nlm.nih.gov website (Basic Local Alignment Search Tool) to compare.
14. combining forms are observed, qualification macro fungi, submit to sequencing result in the GenBank of http://www.ncbi.nlm.nih.gov website, obtain DNA sequence accession number (Accession Number).
Result:
The Molecular Identification result of macro fungi is as shown in table 1 below:
The Molecular Identification result table of table 1 macro fungi
Table 1 presents the result of strain identification.Qualification, first by Morphological Characteristics, identifies wild large-form fungi.Then, by the nearly step card qualification result of molecular biology method.Find in by ITS sequence comparison process, the similarity of the fungus ITS sequence wherein in 13 kinds of macro fungis and GenBank has exceeded 96%, wherein has the similarity of 5 kinds of macro fungis to reach 100%.No. ten its similarity of macro fungi is only had to be 93%, lower, but pass through Morphological comparison, substantially Russula fungus can be confirmed as, do not get rid of the novel bacterial that it is a Russula, again further new result out before, temporarily with the Russula persicina that property is the highest similarly, it is named.According to our qualification result, 14 kinds of funguses are divided into 6 sections, wherein Polyporaceae (Polyporaceae) has 4 kinds, Tricholomataceae (Tricholomataceae) have 3 kinds, Russulaceae (Russulaceae) have 3 kinds, handle mushroom section (Lepiotaceae) have 3 kinds, Lycoperdaceae (Lycoperdaceae) have a kind with rivet mushroom section (Gomphidiaceae) have a kind.Wherein the Russula (Russula) of Russulaceae has 3 kinds, (Daedaleopsis) of Polyporaceae, the Lepiota (Macrolepiota) of Tricholomataceae have 2 kinds, other 7 kinds of bacterium are respectively from different genus.
The preparation of embodiment 2 extract
Method:
1, the preparation of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polyoses extract
(1) sporophore of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi will gathered in forest, dries under 40 DEG C of conditions, until quality no longer changes, with pulverizer the mushroom grinds of drying.
(2) with acetone by the lixiviate three times under 25 DEG C of conditions of the be full of cracks Lasiosphaera Seu Calvatia mycopowder of drying, each 3 hours, centrifugally remove supernatant, precipitation boiling water extraction 3 hours, centrifugal, after removing precipitation, collect supernatant;
(3) mixing collecting the supernatant obtained with the volume ratio of ethanol according to 1:5, at 4 DEG C, making polysaccharide precipitation.Centrifugalize, obtains precipitation.
(4) resolution of precipitate is in water, then uses lyophilization, obtains the crude extract (Crude Polysaccharide, CPS) of polysaccharide.
(5) Sevag reagent is used, deproteinization:
The n-butyl alcohol of 5 times of volume of chloroform and 1 times of volume is mixedly configured into Sevag reagent, then the crude extract obtaining polysaccharide is first dissolved in a small amount of water, then be that 5:1 carries out mixing (the extract sample of 5 times of volumes mixes with the Sevag reagent of 1 times of volume) with Sevag reagent according to volume ratio, stir, centrifugal.Solution divides three layers, and upper strata is polysaccharide solution, and lower floor is organic reagent, and intermediate layer is the albumin layer removed, and collects upper strata polysaccharide solution;
(6) by collect upper strata polysaccharide solution according to step (5) reprocessing, until remove protein (there is no protein adsorption at 260nm place).
(7) carry out dialyse (molecular cut off adopting bag filter is 3500 dalton) with deionized water, remove micromolecule.The polysaccharide solution of the purification obtained, adds the ethanol of 5 times of volumes, and 4 DEG C obtain precipitation, use water dissolution again, and then add the ethanol of 5 times of volumes, at 4 DEG C, make polysaccharide precipitation, centrifugalize, obtains precipitation, more fully to remove alcohol soluble substance, finally will be precipitated and dissolved in again in water, lyophilizing, is the polysaccharide after water miscible purification (Partially purified PS, PPS).
2, the mensuration of the carbohydrate amount of polysaccharide
Carbohydrate amount (the Yemm EW and Willis AJ of polysaccharide in CPS and PPS is measured with anthrone test, The estimation of carbohydrates in plant extracts by anthrone.Biochem J57:508 (1954) .), standard curve is done with glucose, the allocation plan of anthrone solution is dissolved in the water of 22ml by the anthrone of 0.2g, then add the sulphuric acid of 78ml.The anthrone solution getting 0.5ml adds the polysaccharide sample solution of 0.1ml.Mixed liquor heats 10min in boiling water, and then is cooled to indoor temperature.Do not add the blank of sample in contrast with one, measure absorbance at 620nm place.In the preparation and content analysis of polysaccharide, be provided with and repeat three biologys.
3, according to the method described above (flow chart as shown in Figure 1) carried out the mensuration of the extraction of all the other 13 macro fungi polysaccharide and the carbohydrate amount of polysaccharide respectively.
Result:
Table 2 presents 14 kinds of macro fungis, the content of the carbohydrate of the polyoses extract PPS after purification.The carbohydrate of the PPS after purification generally than crude polysaccharides CPS significantly low 10% to 70%, this may be in dialysis, eliminate the low oligosaccharide of molecular weight and monosaccharide causes.That carbohydrate content is the highest is birch pleat pore fungi (L.betulina) approximately 147mg/g, and minimum is that be full of cracks Lasiosphaera Seu Calvatia (H.utriformis) is lower than 4mg/g.According to the result of table 2, after not finding crude polysaccharides CPS or purification, there are clear and definite relation in polysaccharide PPS carbohydrate content and section between belonging to, and namely do not find that the polyoses content of certain section fungus or certain genus fungus is apparently higher than other section or genus.
The carbohydrate content of polysaccharide after table 2 crude polysaccharides and purification
The meansigma methods that in meansigma methods ± SD (n=3) and same hurdle, different letter (a-i) marks represent significant difference (p<0.05) .*CPS represent the crude extract * * PPS of polysaccharide represent water miscible purification after polysaccharide.
The mensuration of embodiment 3 antioxidant activity
Antioxidant activity is measured by free radical scavenging power, reducing power and metal chelating three kinds of methods of making a concerted effort.Polysaccharide PPS after purification, by soluble in water in advance, is configured to the solution that concentration is 4mg/ml.All antioxidation experiments are all provided with and repeat three biologys.
Method:
1, Free-radical scavenging activity
Free radical scavenging experiment is the experiment of a kind of antioxidation, the present invention is by removing ABTS[2, 2 '-azinobis (3-ethylbenzothiazoline-6-sulfonic acid), ABTS] free radical, measure color change experiment and carry out (Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C.Antioxidant activity applying an improved ABTS radical cation decolorization assay.Free Radical Biol Med26:1231-1237 (1999) .Shao P, Chen XX and Sun P.Chemical characterization, antioxidant and antitumor activity of sulfated polysaccharide from Sargassum horneri.Carbohyd Polym 105:260-269 (2014) .).Free radical ABTS
+generation, by utilizing potassium peroxydisulfate (Potassium persulfate, K
2s
2o
8) oxidation ABTS come.In this experiment, final concentration is the ABTS solution of 7.4mM and final concentration is react 16h under the potassium peroxydisulfate room temperature of 2.45mM, obtains blue-green solution.Get the ABTS of 20 μ l
+solution is added in polysaccharide sample, and being diluted with water in 734nm absorption value is 0.70 (± 0.02), and 2h cultivated by mixed liquor, then reads and absorption value under recording 734nm.Quino dimethylacrylate [(S)-(2)-6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid, trolox], i.e. the analog of water miscible vitamin E, as positive control, and the antioxidant activity of polysaccharide sample is converted into the antioxidant activity of the trolox of equivalent, i.e. TEAC μm of ol trolox/g (Trolox Equivalent Antioxidant Capacity, TEAC).
2, reducing power is active
The resistance to oxidation FRAP that the measurement of reducing power is reduced by iron ion tests (Ferric Reducing Antioxidant Power, FRAP) (Benzie IFF and Strain JJ, The ferric reducing ability of plasma as a measure of " antioxidant power ": the FRAP assay.Analytical Biochemistry239:70-76 (1996) .Yap YHY, Tan NH, Fung SY, Aziz AA., Tan CS and Ng ST, Nutrient composition, antioxidant properties, and anti-proliferative activity of Lignosus rhinoceros Cooke sclerotium.J Sci Food Agr93:2945-2952 (2013) .), make Fe (TPTZ)
2 3+be reduced into Fe (TPTZ)
2 2+, the latter has navy blue, has absworption peak at 593nm.Concentration, before experiment, is the TPTZ[2 of 10mM, 4,6-Tris (2-pyridyl)-s-triazine by FRAP reagent matching while using] to be added to concentration be remove wiring solution-forming one in the HCl solution of 40mM to solution, solution two is the FeCl of concentration 20mM
36H
2o, the pH of solution three is 3.6, and concentration is the acetate buffer of 300mM, and solution one, solution two and solution three, mix according to the ratio of 1:1:10,37 DEG C of temperature baths, stand-by.Sample thief 30 μ l mixes with the water of 90 μ l, then adds the FRAP reagent reacting 2h of 900 μ l, then measures light absorption value at 595nm.Use FeSO
4criterion curve, active representation unit is a μm ol FeSO
4/ g sample.
3, metal chelating is made a concerted effort active
Metal-chelating resists oxidation experiment strenuously, by (the Decker EA and Welch B that the chelating ability measuring ferrous ion has come, Role of ferritin as a lipid oxidation catalyst in muscle food.J Agri Food Chem38:674 – 677 (1990) .Liu W, Wang H, Yao W, Gao X and Yu L, Effects of Sulfation on the Physicochemical and Functional Properties of a Water-Insoluble Polysaccharide Preparation from Ganoderma lucidum.J Agr Food Chem58:3336-3341 (2010) .), select ethylenediaminetetraacetic acid (Ethylene Diamine Tetraacetic Acid, EDTA) as positive control.The sample getting 200 μ l mixes with the deionized water of 740 μ l and be then that 2mM ferrous chloride reacts 1 minute with the concentration of 20 μ l, be that luxuriant and rich with fragrance hello piperazine (Ferrozine) of 5mM reacts 20min with 40 μ l concentration, absorption value is measured, with μm ol Fe under 562nm
2+/ g represents the sequestering power of sample.
Result:
Table 3 and Fig. 2 present the experimental result of 3 kinds of antioxidant activity of polysaccharide PPS after 14 kinds of macro fungi purification.Fig. 2 is left, the removing ABTS free radical activity represented, and active unit is TEAC (Ttrolox Equivalent Antioxidant Capacity); In Fig. 2, the different result that has been different antioxidant activity result presentation represented.For scavenging free radicals ABTS
+after the purification of active aspect macro fungi, the activity of polysaccharide PPS is 16.9-535.8 μm of ol/g TEAC, in iron ion reducing power, after purification, the activity of polysaccharide PPS is 0.23 to 5.94mmol Fe/g, and in ferrous ion chelating ability, after purification, the activity of polysaccharide PPS is 0.70 to 484.61 μm of ol Fe
2+/ g.On the whole, the polysaccharide anti-oxidative activity from Polyporaceae and Russulaceae fungus is stronger.The polysaccharide PPS after the purification of 4 kinds of macro fungis be full of cracks Lasiosphaera Seu Calvatia, mastoid process handle mushroom, Armillaria ostoyae and parasol mushroom is had all to be positioned at first six digits in any 3 kinds of antioxidation experiment.Lepista mucla (Bull.:Fr.) Cooke has the second high metallic reducing power (4.65mmol Fe/g) in addition, and yellow Armillariella has the strongest metal chelation abilities (484.6 μm of ol Fe
2+/ g), these six kinds of mushrooms above-mentioned, namely fungus be full of cracks Lasiosphaera Seu Calvatia, mastoid process handle mushroom, Armillaria ostoyae, parasol mushroom, Lepista mucla (Bull.:Fr.) Cooke and yellow Armillariella, have stronger antioxidant activity.Chap in 14 kinds of macro fungis Lasiosphaera Seu Calvatia purification after polysaccharide PPS antioxidant activity the strongest.Other extract (non-polyoses extract or non-water extract) of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi was once in the news and had antioxidant activity or other is active, and the polysaccharide after this research to be Late Cambrian chap Lasiosphaera Seu Calvatia purification has stronger antioxidant activity (free radical scavenging power, reducing power and ion chelating power).Except be full of cracks Lasiosphaera Seu Calvatia, other 5 kinds have active compared with the polysaccharide anti-oxidative of the macro fungi mastoid process handle mushroom of strong anti-oxidative activity, Armillaria ostoyae, parasol mushroom, Lepista mucla (Bull.:Fr.) Cooke and yellow Armillariella, are also be found first.In fact, research for the polysaccharide of be full of cracks Lasiosphaera Seu Calvatia, mastoid process handle mushroom and yellow Armillariella is all considerably less, more have no the research of its activity, but the polysaccharide according to our these three kinds of macro fungis of result has good antioxidant activity, they are all again edible fungi simultaneously, have very much the potentiality as antioxidant exploitation.
The antioxidant activity of table 3 macro fungi polysaccharide
The meansigma methods that in mean+SD (n=3) and same hurdle, different letter (a-n) marks represents significant difference (p<0.05).* TEAC refers to total antioxidant capacity; * positive control: it is to Tyrosinase inhibition assays for ferrous metal chelating agen that watermiscible vitamin E is used for ABTS and FRAP, EDTA.
The mensuration of embodiment 4 antityrosinase activity
Method:
The mensuration of the antityrosinase activity of polysaccharide sample, by (the Baurin N that spectrophotometer measurement has come from the change of the restraint of tyrosinase activity of macro fungi, Arnoult E, Scior T, Do QT and Bernard P, Preliminary screening of some tropical plants for anti-tyrosinase activity.J Ethnopharmacol82:155 – 158 (2002) .Chien C, Tsai M, Chen C, Chang S and Tseng C, Effects on tyrosinase activity by the extracts of Ganoderma lucidum and related mushrooms.Mycopathol166:117 – 120 (2008) .Chiari ME, Joray MB, Ruiz G, Palacios SM and Carpinella MC, Tyrosinase inhibitory activity of native plants from central Argentina:Isolation of an active principle from Lithrea molleoides.Food Chem120:10 – 14 (2010) .Yu P and Sun H, Purification of a fucoidan from kelp polysaccharide and its inhibitory kinetics for tyrosinase.Carbohyd Polym99:278-283 (2014) .).With pH be 6.8, concentration is the solution that tryrosinase to be configured to concentration 400U/ml by the phosphate buffer of 200mM.The tyrosinase solution getting 50 μ l mixes with the phosphate buffer of 200 μ l, then adds the polysaccharide sample solution of 250 μ l, makes the final concentration of polysaccharide sample be 1mg/mL or other working concentration.Mixed liquor is reacted 90min at 25 DEG C, and period is with slowly stirring.Then the concentration adding 500 μ l is the TYR of 0.03% (or other working concentration TYR), absorption value is measured immediately under 475nm, calculate the amount that dopachrome is formed, for measuring the absorption value change of tyrosinase activity record 20min.The absorbance value of the 475nm that record different time provides, will not add (suppression ratio is 0%) in contrast of polysaccharide sample, and arbutin, as positive control, calculates antityrosinase active.In experiment, be provided with and repeat three biologys.
Meansigma methods, standard deviation and IC
50value, is calculated by Microsoft Excel2010.One factor analysis of variance (One way analysis of variance, LSD test, P<0.05) calculated by SPSS version13.0 (Statistical Package for Social Sciences).
Result:
It is the good source of antityrosinase compounds that natural product is recognized, on the one hand they can be used for treating the disease that tryrosinase causes, on the other hand can by as whitening agent (Kim YJ and Uyama H, Tyrosinase inhibitors from natural and synthetic sources:structure, inhibition mechanism and perspective for the future.Cell Mol Life Sci62:1707-1723 (2005) .Chang ST, An updated review of tyrosinase Inhibitors.Int J Mol Sci10:2440 – 2475 (2009) .Smit N, Vicanova J and Pavel S, The Hunt for Natural Skin Whitening Agents.Int J Mol Sci10:5326-5349 (2009) .Ismaya WR, Rozeboom HJ, Weijn A, Mes JJ, Fusetti F, Wichers HJ and Dijkstra BW, Crystal Structure of Agaricus bisporus Mushroom Tyrosinase:Identity of the Tetramer Subunits and Interaction with Tropolone.Biochem50:5477-5486 (2011) .).Table 4 and Fig. 3 present the polysaccharide PPS after 14 kinds of wild large-form fungi purification when the 1mg/ml that concentration is, the experimental result of antityrosinase.Can find according to table 4, it is active that be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi and Gomphidius rutilus have the strongest antityrosinase, they can reach 61% and 85% when 1mg/ml respectively to the suppression ratio of tryrosinase, and other 12 kinds of macro fungis when 1mg/ml to the suppression ratio of tryrosinase all below 40%.Further experiment, after recording the purification of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi, polysaccharide PPS is to concentration, i.e. IC in the suppression of tryrosinase
50value can reach 0.78mg/ml, and the activity of Gomphidius rutilus is stronger, i.e. IC
50value can reach 0.46mg/ml.The positive control arbutin of this experiment is 86.5% when 1mg/ml to the activity of tryrosinase.
The antityrosinase activity (1mg/ml) of table 4 macro fungi polysaccharide
The meansigma methods that in mean+SD (n=3) and same hurdle, different letter (a-k) marks represents significant difference (p<0.05).
Claims (10)
1. the Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide that chaps is preparing the application in antioxidant.
2. apply as claimed in claim 1, it is characterized in that described be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide extracts by the following method and obtain:
(1) by the sporophore of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi, dry under 30-50 DEG C of condition, until quality no longer changes, with pulverizer the sporophore grinds of drying;
(2) with acetone by the lixiviate three times under 20-30 DEG C of condition of the be full of cracks Lasiosphaera Seu Calvatia mycopowder of drying, each 2-4 hour, centrifugally removes supernatant, precipitation boiling water extraction 3 hours, centrifugal, after removing precipitation, collects supernatant;
(3) step (2) is collected the supernatant obtained to mix with the volume ratio of ethanol according to 1:4-6, at 0-10 DEG C, make polysaccharide precipitation, centrifugalize, obtain precipitation;
(4) resolution of precipitate is in water, then uses lyophilization, obtains the crude extract of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide.
3. apply as claimed in claim 2, characterized by further comprising and will obtain the further deproteinization purification of crude extract, the dialysis of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide and again utilize alcohol settling, concrete operations be:
(1) chloroform and n-butyl alcohol are hybridly prepared into Sevag reagent, then the crude extract of be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide are mixed with Sevag reagent, stir, centrifugal; Solution divides three layers, and upper strata is polysaccharide solution, and lower floor is organic reagent, and intermediate layer is the albumin layer removed, and collects the polysaccharide solution on upper strata;
(2) by collect polysaccharide solution according to step (1) method reprocessing, until remove protein;
(3) dialyse with deionized water, the molecular cut off of bag filter is 3500 dalton, and remove micromolecule, the polysaccharide solution of the purification of acquisition, adds the ethanol of 5 times of volumes, at 4 DEG C, makes polysaccharide precipitation, centrifugalize, obtains precipitation; The precipitation obtained, then use water dissolution, lyophilizing, be the polysaccharide after water miscible purification.
4. apply as claimed in claim 3, it is characterized in that described Sevag reagent is formed according to volume ratio 5:1 mixed preparing chloroform and n-butyl alcohol.
5. applying as claimed in claim 3, it is characterized in that the crude extract of described be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide to be first dissolved in a small amount of water, is then that 5:1 mixes with Sevag reagent according to volume ratio.
6. apply as claimed in claim 3, it is characterized in that in step (3), also comprise the precipitation that will obtain, use water dissolution again, and then add the ethanol of 5 times of volumes, at 4 DEG C, make polysaccharide precipitation, centrifugalize, obtain precipitation, more fully to remove alcohol soluble substance, finally will be precipitated and dissolved in again in water, lyophilizing, is the polysaccharide after water miscible purification.
7. the extracting method of a macro fungi polysaccharide, it is characterized in that utilizing acetone, Sevag reagent, the method for ethanol and dialysis remove and comprise nonpolar, that low pole material, albumen, alcohol soluble substance and molecular weight are less than 3500 daltonian micromolecule non-polysaccharose substance, specifically comprise the following steps:
(1) by the sporophore of macro fungi, dry under 30-50 DEG C of condition, until quality no longer changes, with pulverizer the sporophore grinds of drying;
(2) with acetone by the lixiviate three times under 20-30 DEG C of condition of the macro fungi mycopowder of drying, each 2-4 hour, centrifugally removes supernatant, then uses boiling water extraction 3 hours, centrifugal, after removing precipitation, collects supernatant;
(3) step (2) is collected the supernatant obtained to mix with the volume ratio of ethanol according to 1:4-6, at 0-10 DEG C, make polysaccharide precipitation, centrifugalize, obtain precipitation;
(4) resolution of precipitate is in water, then uses lyophilization, obtains the crude extract of macro fungi polysaccharide.
8. method as claimed in claim 7, characterized by further comprising further for the crude extract obtaining macro fungi polysaccharide deproteinization purification, dialysis and again utilize alcohol settling, concrete operations are:
(1) chloroform and n-butyl alcohol are hybridly prepared into Sevag reagent, then the crude extract of macro fungi polysaccharide are mixed with Sevag reagent, stir, centrifugal; Solution divides three layers, and upper strata is polysaccharide solution, and lower floor is organic reagent, and intermediate layer is the albumin layer removed, and collects the polysaccharide solution on upper strata;
(2) by collect polysaccharide solution according to step (1) method reprocessing, until remove protein;
(3) dialyse with deionized water, the molecular cut off of bag filter is 3500 dalton, and remove micromolecule, the polysaccharide solution of the purification of acquisition, adds the ethanol of 5 times of volumes, at 4 DEG C, makes polysaccharide precipitation, centrifugalize, obtains precipitation; The precipitation obtained, then use water dissolution, lyophilizing, be the polysaccharide after water miscible purification.
9. method as claimed in claim 8, is characterized in that described Sevag reagent is formed according to volume ratio 5:1 mixed preparing chloroform and n-butyl alcohol; Preferably, the crude extract of described be full of cracks Lasiosphaera nipponica(Kawam.)Y.Kobayasi polysaccharide is first dissolved in a small amount of water, is then that 5:1 mixes with Sevag reagent according to volume ratio.
10. method as claimed in claim 8, is characterized in that in step (3), also comprises the precipitation that will obtain, use water dissolution again, and then add the ethanol of 5 times of volumes, at 4 DEG C, make polysaccharide precipitation, centrifugalize, obtain precipitation, more fully to remove alcohol soluble substance, finally will be precipitated and dissolved in again in water, lyophilizing, is the polysaccharide after water miscible purification.
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