CN107686473A - A kind of gynura bicolor polysaccharide polyphenol combined preparation process - Google Patents

A kind of gynura bicolor polysaccharide polyphenol combined preparation process Download PDF

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CN107686473A
CN107686473A CN201710880992.5A CN201710880992A CN107686473A CN 107686473 A CN107686473 A CN 107686473A CN 201710880992 A CN201710880992 A CN 201710880992A CN 107686473 A CN107686473 A CN 107686473A
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gynura bicolor
ethanol
refrigerators
precipitation
polysaccharide
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CN107686473B (en
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付才力
陈雨浓
刘前
王梅
郭雅靖
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Fuzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
    • C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass

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  • Medicines Containing Plant Substances (AREA)
  • Preparation Of Fruits And Vegetables (AREA)
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Abstract

The present invention provides a kind of gynura bicolor polysaccharide polyphenol combined preparation process, and by ethanol water extraction and adsorbing separation extraction method, the preparation of polysaccharide polyphenol is joined together, reduce operating procedure, the utilization rate of resource is improved, adds efficiency, simultaneously effective improves the purity of the polysaccharide polyphenol of extraction.

Description

A kind of gynura bicolor polysaccharide polyphenol combined preparation process
Technical field
The invention belongs to biological technical field, and in particular to a kind of gynura bicolor polysaccharide polyphenol combined preparation process.
Background technology
Gynura bicolor（Gynura bicolor DC）Alias blood skin dish, kwan-yin dish, red wind dish, Gynura bicolor etc., are composite family three Half cultigen cooked in seven grass category with young stem and leaf, perennial root evergreen herbage.Gynura bicolor originates in China, and four The ground such as river, Zhejiang, Guangdong, Guangxi, Hainan, Fujian, Taiwan, or wild or cultivating and growing, widely planted with Sichuan, the band of Chongqing one especially Training is edible.
Gynura bicolor is a kind of edible and medicinal health-care vegetable, and Tender bases of leaf can be cold and dressed with sauce or stir-fried and eaten, at the same rich in crude protein, can Feeding habits fiber, fat, amino acid, alkaloid, Flavonoid substances, volatile oil and mineral matter.The mineral nutrition of gynura bicolor is more rich The micronutrient levels such as richness, particularly iron, copper, zinc are higher, and also containing glycosides displayed composition, this composition is nontoxic, have extension anti- Bad hematic acid and the effect for reducing blood vessel purple scar.Except also be used to produce in China for directly edible or making clean vegetables, gynura bicolor Medicinal liquor and health protection tea, and be used as in Japan preparing natural food pigment.Gynura bicolor nutrition is comprehensive and has healthcare function concurrently, collects " fresh, green, wild " and " nutrition ", " medicinal ", " delicious " are important green health care food resources.
Gynura bicolor contains notoginseng polysaccharide, and it has a variety of bioactive functions closely related with the maintenance of biological function. The compound that notoginseng polysaccharide is made up of many identical or different monose with α or β-glycosidic bond, possess the level Four knot of complexity Structure, there is the effect such as immunological regulation, antiviral, anti-aging, antitumor, radioresistance, in addition, polysaccharide also has anti-freezing The other biological activities such as blood, anti-inflammatory, antibacterial, anticonvulsion, calm, Zhichuan and hypotensive.In recent years, people to the structure of polysaccharide and The research of activity deepens continuously, and further illustrates the relation of polysaccharide mechanism of action and structure, its multifarious physiologically active is more Add and be taken seriously.
The extracting method of plant polyose mainly has solvent extraction method, sour formulation, alkaline extraction, enzymatic isolation method, ultrafiltration, super at present Sound wave reinforcement, microwave method etc..Extraction method of polysaccharides used in the present invention is ethanol water extraction, relative to other several methods, Its reaction condition is gentle, and extraction rate is fast, and the purity of polysaccharide of extraction is high.
Polyphenol substance in gynura bicolor includes Flavonoid substances and anthocyan etc..Plant polyphenol is also known as tannin or tannin, There is the compound of middle growing stage including tannin and with tannin, there is polynary phenolic hydroxyl group and carboxyl structure, main skeleton type is C6- ClType and C6-C3Type.The unique texture of plant polyphenol makes it have multiple efficacies, and anthocyan material is one kind of polyphenol, is tool There is the bioflavonoid of special molecular structure, can effectively remove free radical, there is inoxidizability, antibacterial, anticancer, anti-inflammatory, anti- Viral isoreactivity, tissue can be protected from the oxidative damage of free radical, and can effectively improve microcirculation in human body.
More to plant polyphenol extraction research at present, main extracting method has solvent extraction, ultrasonic wave leaching method, microwave Extraction, adsorbing separation extraction method, separation of metal ions method, biological enzymolysis extraction method etc..More phenol extraction sides used in the present invention Method is adsorbing separation extraction method, it is advantageous that suction-operated of the resin to plant polyphenol be strong and reusable edible, operating condition Gently, recovery rate is higher, and separation product quality is preferable, nontoxic etc..
CN201511008885.0 is related to extraction method of polysaccharides in a kind of moringa seeds, and the powdered moringa seeds after crushing are put Enter in water, soak at a certain temperature certain time, extract, obtain moringa seeds polysaccharide extraction liquid, described moringa seeds Polyose extraction The yield of polysaccharide reaches more than 14% in liquid, though the recovery rate of the method polysaccharide is high, purity is relatively low.CN201611054253.2 A kind of potentilla discolor extraction method of polysaccharides is provided, polysaccharide is extracted from potentilla discolor using biological enzymolysis technology, along with ultrasonic wave Processing, ultrafiltration, alcohol precipitation, quaternary salt deposit, salt is washed, alcohol precipitation refines or macroporous resin column refines, and obtains the higher potentilla discolor of purity Polysaccharide, though the purity of polysaccharide that the method obtains is high, operating process is cumbersome, and cost is higher.CN201210229217.0 provides a kind of The extracting method of pears polyphenol, pears young fruit is removed the peel and is enucleated, is cut into less than 1cm3Fritter, with liquid nitrogen grinding into powder, use 30- 90% ethanol solution carries out extraction 2-6h to the operatic circle, centrifugation, and gained supernatant is pears polyphenol extract solution, and the method is extracted more Though phenol activity is high, while cost is also high, is not suitable for industrialized production, and wasting of resources phenomenon is serious.CN201310300937.6 is Four-o'clock seed will be peeled off, interior flesh is discarded, leaves crust, be dried to constant weight, 80-100 mesh sieves are crossed after crushing, by solid-liquid ratio 1:5- 25 add the ethanol solution that concentration is 30-95%, and the heating extraction 30-150min in 60 DEG C of thermostat water bath, leaching liquor is through taking out Filter, concentration, dry polyphenol extract, the method production cost is low, but the polyphenol purity extracted is not high, and recovery rate is also relatively low.
The content of the invention
The invention provides method prepared by a kind of gynura bicolor polysaccharide polyphenol joint.The method combines the preparation of polysaccharide polyphenol Get up, reduce operating procedure, improve the utilization rate of resource, add efficiency, simultaneously effective improve the polysaccharide of extraction The purity of polyphenol.
To achieve the above object, the present invention uses following technical scheme：
The preparation method comprises the following steps：
（1）After extracing bad leaf and old stalk and the blade in worm hole by fresh gynura bicolor, minced after cleaning, loaded on hermetic bag, 4 Saved backup in DEG C refrigerator.
（2）The gynura bicolor fresh leaf after mincing accurately is weighed, by solid-to-liquid ratio 1:4g/ml adds 50% ethanol water（Second Alcohol：Water（V/V）=1:1）, 3h is extracted in shaking table under conditions of 45 DEG C, after coarse filtration, collects filtrate and filter residue.By filter residue again With 1:2g/ml solid-to-liquid ratio adds 50% ethanol water（Ethanol：Water（V/V）=1:1）Extraction one is repeated under the same conditions It is secondary, filtered after coarse filtration, and extract solution twice is merged.After mixing, with vacuum rotary evaporator concentrate directly To 1/6 that volume is original volume, after concentration crude extract is installed with brown reagent bottle, it is standby to be placed in fridge freshness retaining layer preservation.
（3）Concentrate is taken, adds 95% ethanol solution of 3 times of volumes, after mixing, 150r/min magnetic agitation 30min, Preservative film covers beaker mouth, and normal temperature is stood overnight.Liquid 6000r/min centrifugation 10min are mixed, collect precipitation and supernatant respectively Liquid, precipitation are Thick many candies materials, and supernatant need to further handle to obtain polyphenol substance.
（4）The Thick many candies solution 100mL that compound concentration is 2-3mg/mL, the neutrality that addition enzyme amount is 1000U/g will be precipitated Protease, pH=7 are adjusted, under the conditions of 50 DEG C of enzymes, digest 2h.By the solution after taking off albumen processing, 6000r/min centrifugations 10min, precipitation is collected, is Gynura bicolor polysaccharide.
（5）By step（3）Supernatant half after alcohol precipitation centrifugation is rotated at 45 DEG C, after being concentrated to 50ml filterings, By filtrate through the column chromatography continuous purifications of Sephadex LH 20, the ethanol elution first through 1000ml 50%, afterwards with 500ml 70% the third The ketone aqueous solution elutes, and acetone eluant is put into -20 DEG C of refrigerators and preserves 2h through 45 DEG C of rotary evaporations, then is preserved in -80 DEG C of refrigerators It is freeze-dried after 4h, obtains gynura bicolor OPC.
（6）Remaining supernatant after alcohol precipitation is centrifuged also is rotated at 45 DEG C, after being concentrated to 50ml filterings, by filtrate Through SepPakC18 column chromatography continuous purifications, first eluted through the ethanol waters of 1000ml 10%, it is water-soluble with the ethanol of 500ml 80% afterwards Liquid elutes, and 80% ethanol water eluent is put into -20 DEG C of refrigerators and preserves 2h, then preserve 4h in -80 DEG C of refrigerators through 45 DEG C of rotary evaporations After be freeze-dried, obtain gynura bicolor anthocyanidin.
The advantage of the invention is that：The technique and the process integration of polyphenol that gynura bicolor polysaccharide will be prepared, the system of having simplified Standby technique, improves operating efficiency；The wasting of resources is avoided, improves the utilization rate of resource；It can ensure to be carried well simultaneously The purity of the polysaccharide polyphenol taken, improve the rate of recovery.
Embodiment
Embodiment 1
After extracing bad leaf and old stalk and the blade in worm hole by the fresh gynura bicolor in ZhangZhou place of production, mince, be loaded on after cleaning Hermetic bag, save backup in 4 DEG C of refrigerators.
The gynura bicolor fresh leaf after 200g is minced accurately is weighed, by solid-to-liquid ratio 1:It is water-soluble that 4g/ml adds the ethanol of 800mL 50% Liquid（Ethanol：Water（V/V）=1:1）, 3h is extracted in shaking table under conditions of 45 DEG C, after coarse filtration, collects filtrate and filter residue.Will Filter residue is again with 1:The 2g/ml solid-to-liquid ratio addition ethanol waters of 400mL 50%（Ethanol：Water（V/V）=1:1）In identical condition It is lower to repeat extraction once, filtered after coarse filtration, and extract solution twice is merged.After mixing, rotary evaporation in vacuo is used Device carry out concentration until volume be original volume 1/6, will concentration crude extract installed with brown reagent bottle after, be placed in fridge freshness retaining Layer preservation is standby.
150mL concentrates are taken, add the 450mL95% of 3 times of volumes ethanol solution, after mixing, 150r/min magnetic agitations 30min, preservative film capping beaker mouth, normal temperature are stood overnight.Liquid 6000r/min centrifugation 10min are mixed, collect precipitation respectively And supernatant, precipitation are Thick many candies materials, supernatant need to further handle to obtain polyphenol substance.
Obtained Thick many candies precipitation is configured to 2mg/mL Thick many candies solution 100mL, addition enzyme amount is in 1000U/g Property protease, adjust pH=7, under 50 DEG C of enzyme optimum conditions, digest 2h.Solution after processing is placed in centrifuge tube, 6000r/ Min centrifuges 10min, collects precipitation.As a result show, the polysaccharide recovery of the gynura bicolor in ZhangZhou place of production is 80.21%, and purity is 75.56%。
Supernatant after alcohol precipitation is centrifuged respectively takes half after rotating, concentrating, respectively by the Hes of Sephadex LH 20 SepPakC18 column chromatography continuous purifications.The posts of Sephadex LH 20 are after 1000ml 50% ethanol elution with 500ml 70% the third The ketone aqueous solution elutes, and by acetone eluant through 45 DEG C of rotary evaporations, and is put into -20 DEG C of refrigerators and preserves 2h, is preserved in -80 DEG C of refrigerators It is freeze-dried after 4h, obtains gynura bicolor OPC.As a result show, the OPC rate of recovery of the gynura bicolor in ZhangZhou place of production For 70.25%, purity 80.30%.
Meanwhile take the remaining supernatant after alcohol precipitation centrifugation to cross SepPakC18 posts, eluted through the ethanol waters of 1000ml 10% Eluted afterwards with the ethanol waters of 500ml 80%, by 80% ethanol water eluent through 45 DEG C of rotary evaporations, be put into -20 DEG C of refrigerators and preserve 2h, it is freeze-dried after -80 DEG C of refrigerators preserve 4h, obtains gynura bicolor anthocyanidin, as a result show, the gynura bicolor in ZhangZhou place of production The anthocyanidin rate of recovery be 75.77%, purity 75.46%.
Embodiment 2
After extracing bad leaf and old stalk and the blade in worm hole by the fresh gynura bicolor in the Guangxi place of production, mince, be loaded on after cleaning Hermetic bag, save backup in 4 DEG C of refrigerators.
The gynura bicolor fresh leaf after 200g is minced accurately is weighed, by solid-to-liquid ratio 1:It is water-soluble that 4g/ml adds 800mL50% ethanol Liquid（Ethanol：Water（V/V）=1:1）, 3h is extracted in shaking table under conditions of 45 DEG C, after coarse filtration, collects filtrate and filter residue.Will Filter residue is again with 1:2g/ml solid-to-liquid ratio addition 400mL50% ethanol waters（Ethanol：Water（V/V）=1:1）Under the same conditions Repeat extraction once, filtered after coarse filtration, and extract solution twice is merged.After mixing, vacuum rotary evaporator is used Carry out concentration until volume be original volume 1/6, will concentration crude extract installed with brown reagent bottle after, be placed in fridge freshness retaining layer Preservation is standby.
150mL concentrates are taken, add the 450mL95% of 3 times of volumes ethanol solution, after mixing, 150r/min magnetic agitations 30min, preservative film capping beaker mouth, normal temperature are stood overnight.Liquid 6000r/min centrifugation 10min are mixed, collect precipitation respectively And supernatant, precipitation are Thick many candies materials, supernatant need to further handle to obtain polyphenol substance.
Obtained Thick many candies precipitation is configured to 3mg/mL Thick many candies solution 100mL, addition enzyme amount is in 1000U/g Property protease, adjust pH=7, under 50 DEG C of enzyme optimum conditions, digest 2h.Solution after processing is placed in centrifuge tube, 6000r/ Min centrifuges 10min, collects precipitation.As a result show, the polysaccharide recovery of the gynura bicolor in the Guangxi place of production is 75.57%, and purity is 70.99%。
Supernatant after alcohol precipitation is centrifuged respectively takes half after rotating, concentrating, respectively by the Hes of Sephadex LH 20 SepPakC18 column chromatography continuous purifications.The posts of Sephadex LH 20 use the water of 750ml 70% after 1000ml 50% ethanol elution Acetone elutes, and by acetone eluant through 45 DEG C of rotary evaporations, and is put into -20 DEG C of refrigerators and preserves 2h, after -80 DEG C of refrigerators preserve 4h It is freeze-dried, obtains gynura bicolor OPC, the results showed that, the OPC rate of recovery of the gynura bicolor in the Guangxi place of production is 60.11%, purity 70.32%.
Meanwhile take the remaining supernatant after alcohol precipitation centrifugation to cross SepPakC18 posts, eluted through the ethanol waters of 750ml 10% Eluted afterwards with the ethanol waters of 500ml 80%, by 80% ethanol water eluent through 45 DEG C of rotary evaporations, be put into -20 DEG C of refrigerators and preserve 2h, it is freeze-dried after -80 DEG C of refrigerators preserve 4h, obtains gynura bicolor anthocyanidin, as a result show, the gynura bicolor in the Guangxi place of production The anthocyanidin rate of recovery be 70.89%, purity 75.33%.
Embodiment 3
After extracing bad leaf and old stalk and the blade in worm hole by the fresh gynura bicolor in the Zhejiang place of production, mince, be loaded on after cleaning Hermetic bag, save backup in 4 DEG C of refrigerators.
The gynura bicolor fresh leaf after 200g is minced accurately is weighed, by solid-to-liquid ratio 1:It is water-soluble that 4g/ml adds 800mL50% ethanol Liquid（Ethanol：Water（V/V）=1:1）, 3h is extracted in shaking table under conditions of 45 DEG C, after coarse filtration, collects filtrate and filter residue.Will Filter residue is again with 1:2g/ml solid-to-liquid ratio addition 400mL50% ethanol waters（Ethanol：Water（V/V）=1:1）Under the same conditions Repeat extraction once, filtered after coarse filtration, and extract solution twice is merged.After mixing, vacuum rotary evaporator is used Carry out concentration until volume be original volume 1/6, will concentration crude extract installed with brown reagent bottle after, be placed in fridge freshness retaining layer Preservation is standby.
150mL concentrates are taken, add the 450mL95% of 3 times of volumes ethanol solution, after mixing, 150r/min magnetic agitations 30min, preservative film capping beaker mouth, normal temperature are stood overnight.Liquid 6000r/min centrifugation 10min are mixed, collect precipitation respectively And supernatant, precipitation are Thick many candies materials, supernatant need to further handle to obtain polyphenol substance.
Obtained Thick many candies precipitation is configured to 2.5mg/mL Thick many candies solution 100mL, addition enzyme amount is 1000U/g's Neutral proteinase, pH=7 are adjusted, under 50 DEG C of enzyme optimum conditions, digest 2h.Solution after processing is placed in centrifuge tube, 6000r/min centrifuges 10min, collects precipitation.As a result show, the polysaccharide recovery of the gynura bicolor in the Zhejiang place of production is 70.33%, pure Spend for 65.49%.
Supernatant after alcohol precipitation is centrifuged respectively takes half after rotating, concentrating, respectively by the Hes of Sephadex LH 20 SepPakC18 column chromatography continuous purifications.The posts of Sephadex LH 20 use the water of 800ml 70% after 1000ml 50% ethanol elution Acetone elutes, and by acetone eluant through 45 DEG C of rotary evaporations, and is put into -20 DEG C of refrigerators and preserves 2h, after -80 DEG C of refrigerators preserve 4h It is freeze-dried, obtains gynura bicolor OPC, the results showed that, the OPC rate of recovery of the gynura bicolor in the Zhejiang place of production is 60.15%, purity 65.37%.
Meanwhile take the remaining supernatant after alcohol precipitation centrifugation to cross SepPakC18 posts, eluted through the ethanol waters of 800ml 10% Eluted afterwards with the ethanol waters of 500ml 80%, by 80% ethanol water eluent through 45 DEG C of rotary evaporations, be put into -20 DEG C of refrigerators and preserve 2h, it is freeze-dried after -80 DEG C of refrigerators preserve 4h, obtains gynura bicolor anthocyanidin, as a result show, the gynura bicolor in the Zhejiang place of production The anthocyanidin rate of recovery be 65.02%, purity 60.99%.
Embodiment 4
After extracing bad leaf and old stalk and the blade in worm hole by the fresh gynura bicolor in Hainan place of production, mince, be loaded on after cleaning Hermetic bag, save backup in 4 DEG C of refrigerators.
The gynura bicolor fresh leaf after 200g is minced accurately is weighed, by solid-to-liquid ratio 1:It is water-soluble that 4g/ml adds 800mL50% ethanol Liquid（Ethanol：Water（V/V）=1:1）, 3h is extracted in shaking table under conditions of 45 DEG C, after coarse filtration, collects filtrate and filter residue.Will Filter residue is again with 1:2g/ml solid-to-liquid ratio addition 400mL50% ethanol waters（Ethanol：Water（V/V）=1:1）Under the same conditions Repeat extraction once, filtered after coarse filtration, and extract solution twice is merged.After mixing, vacuum rotary evaporator is used Carry out concentration until volume be original volume 1/6, will concentration crude extract installed with brown reagent bottle after, be placed in fridge freshness retaining layer Preservation is standby.
150mL concentrates are taken, add the 450mL95% of 3 times of volumes ethanol solution, after mixing, 150r/min magnetic agitations 30min, preservative film capping beaker mouth, normal temperature are stood overnight.Liquid 6000r/min centrifugation 10min are mixed, collect precipitation respectively And supernatant, precipitation are Thick many candies materials, supernatant need to further handle to obtain polyphenol substance.
Obtained Thick many candies precipitation is configured to 2mg/mL Thick many candies solution 150mL, addition enzyme amount is in 1000U/g Property protease, adjust pH=7, under 50 DEG C of enzyme optimum conditions, digest 2h.Solution after processing is placed in centrifuge tube, 6000r/ Min centrifuges 10min, collects precipitation.As a result show, the polysaccharide recovery of the gynura bicolor in Hainan place of production is 75.09%, and purity is 80.55%。
Supernatant after alcohol precipitation is centrifuged respectively takes half, after rotating, concentrating, respectively by the Hes of Sephadex LH 20 SepPakC18 column chromatography continuous purifications.The posts of Sephadex LH 20 use the water of 500ml 70% after 1000ml 50% ethanol elution Acetone elutes, and by acetone eluant through 45 DEG C of rotary evaporations, and is put into -20 DEG C of refrigerators and preserves 2h, after -80 DEG C of refrigerators preserve 4h It is freeze-dried, obtains gynura bicolor OPC, the results showed that, the OPC rate of recovery of the gynura bicolor in Hainan place of production is 70.46%, purity 75.91%.
Meanwhile take the remaining supernatant after alcohol precipitation centrifugation to cross SepPakC18 posts, eluted through the ethanol waters of 1000ml 10% Eluted afterwards with the ethanol waters of 500ml 80%, by 80% ethanol water eluent through 45 DEG C of rotary evaporations, be put into -20 DEG C of refrigerators and preserve 2h, it is freeze-dried after -80 DEG C of refrigerators preserve 4h, obtains gynura bicolor anthocyanidin, as a result show, the gynura bicolor in Hainan place of production The anthocyanidin rate of recovery be 70.33%, purity 75.67%.
Embodiment 5
After extracing bad leaf and old stalk and the blade in worm hole by the fresh gynura bicolor in the Taiwan place of production, mince, be loaded on after cleaning Hermetic bag, save backup in 4 DEG C of refrigerators.
The gynura bicolor fresh leaf after 200g is minced accurately is weighed, by solid-to-liquid ratio 1:It is water-soluble that 4g/ml adds 800mL50% ethanol Liquid（Ethanol：Water（V/V）=1:1）, 3h is extracted in shaking table under conditions of 45 DEG C, after coarse filtration, collects filtrate and filter residue.Will Filter residue is again with 1:2g/ml solid-to-liquid ratio addition 400mL50% ethanol waters（Ethanol：Water（V/V）=1:1）Under the same conditions Repeat extraction once, filtered after coarse filtration, and extract solution twice is merged.After mixing, vacuum rotary evaporator is used Carry out concentration until volume be original volume 1/6, will concentration crude extract installed with brown reagent bottle after, be placed in fridge freshness retaining layer Preservation is standby.
150mL concentrates are taken, add the 450mL95% of 3 times of volumes ethanol solution, after mixing, 150r/min magnetic agitations 30min, preservative film capping beaker mouth, normal temperature are stood overnight.Liquid 6000r/min centrifugation 10min are mixed, collect precipitation respectively And supernatant, precipitation are Thick many candies materials, supernatant need to further handle to obtain polyphenol substance.
Obtained Thick many candies precipitation is configured to 3mg/mL Thick many candies solution 150mL, addition enzyme amount is in 1000U/g Property protease, adjust pH=7, under 50 DEG C of enzyme optimum conditions, digest 2h.Solution after processing is placed in centrifuge tube, 6000r/ Min centrifuges 10min, collects precipitation.As a result show, the polysaccharide recovery of the gynura bicolor in the Taiwan place of production is 75.06%, and purity is 70.47%。
Supernatant after alcohol precipitation is centrifuged respectively takes half after rotating, concentrating, respectively by the Hes of Sephadex LH 20 SepPakC18 column chromatography continuous purifications.The posts of Sephadex LH 20 use the water of 750ml 70% after 1000ml 50% ethanol elution Acetone elutes, and by acetone eluant through 45 DEG C of rotary evaporations, and is put into -20 DEG C of refrigerators and preserves 2h, after -80 DEG C of refrigerators preserve 4h It is freeze-dried, obtains gynura bicolor OPC, the results showed that, the OPC rate of recovery of the gynura bicolor in the Taiwan place of production is 55.15%, purity 60.84%.
Meanwhile take the remaining supernatant after alcohol precipitation centrifugation to cross SepPakC18 posts, eluted through the ethanol waters of 750ml 10% Eluted afterwards with the ethanol waters of 500ml 80%, by 80% ethanol water eluent through 45 DEG C of rotary evaporations, be put into -20 DEG C of refrigerators and preserve 2h, it is freeze-dried after -80 DEG C of refrigerators preserve 4h, obtains gynura bicolor anthocyanidin, as a result show, the gynura bicolor in the Taiwan place of production The anthocyanidin rate of recovery be 65.66%, purity 60.35%.
Embodiment 6
After extracing bad leaf and old stalk and the blade in worm hole by the fresh gynura bicolor in the Guangdong place of production, mince, be loaded on after cleaning Hermetic bag, save backup in 4 DEG C of refrigerators.
The gynura bicolor fresh leaf after 200g is minced accurately is weighed, by solid-to-liquid ratio 1:It is water-soluble that 4g/ml adds 800mL50% ethanol Liquid（Ethanol：Water（V/V）=1:1）, 3h is extracted in shaking table under conditions of 45 DEG C, after coarse filtration, collects filtrate and filter residue.Will Filter residue is again with 1:2g/ml solid-to-liquid ratio addition 400mL50% ethanol waters（Ethanol：Water（V/V）=1:1）Under the same conditions Repeat extraction once, filtered after coarse filtration, and extract solution twice is merged.After mixing, vacuum rotary evaporator is used Carry out concentration until volume be original volume 1/6, will concentration crude extract installed with brown reagent bottle after, be placed in fridge freshness retaining layer Preservation is standby.
150mL concentrates are taken, add the 450mL95% of 3 times of volumes ethanol solution, after mixing, 150r/min magnetic agitations 30min, preservative film capping beaker mouth, normal temperature are stood overnight.Liquid 6000r/min centrifugation 10min are mixed, collect precipitation respectively And supernatant, precipitation are Thick many candies materials, supernatant need to further handle to obtain polyphenol substance.
The gynura bicolor in the Guangdong place of production is configured to the thick more of 2.5mg/mL after water extraction, alcohol precipitation, by obtained Thick many candies precipitation Sugar juice 150mL, the neutral proteinase that addition enzyme amount is 1000U/g, adjusts pH=7, under 50 DEG C of enzyme optimum conditions, digests 2h. Solution after processing is placed in centrifuge tube, 6000r/min centrifugation 10min, collects precipitation.As a result show, the purple in the Guangdong place of production The polysaccharide recovery for carrying on the back dish is 70.80%, purity 65.79%.
Supernatant after alcohol precipitation is centrifuged respectively takes half after rotating, concentrating, respectively by the Hes of Sephadex LH 20 SepPakC18 column chromatography continuous purifications.The posts of Sephadex LH 20 use the water of 800ml 70% after 1000ml 50% ethanol elution Acetone elutes, and by acetone eluant through 45 DEG C of rotary evaporations, and is put into -20 DEG C of refrigerators and preserves 2h, after -80 DEG C of refrigerators preserve 4h It is freeze-dried, obtains gynura bicolor OPC, the results showed that, the OPC rate of recovery of the gynura bicolor in the Guangdong place of production is 60.21%, purity 60.75%.
Meanwhile take the remaining supernatant after alcohol precipitation centrifugation to cross SepPakC18 posts, eluted through the ethanol waters of 800ml 10% Eluted afterwards with the ethanol waters of 500ml 80%, by 80% ethanol water eluent through 45 DEG C of rotary evaporations, be put into -20 DEG C of refrigerators and preserve 2h, it is freeze-dried after -80 DEG C of refrigerators preserve 4h, obtains gynura bicolor anthocyanidin, as a result show, the gynura bicolor in the Guangdong place of production The anthocyanidin rate of recovery be 60.45%, purity 65.89%.
By embodiment 1 ~ 6, compare returning for the polysaccharide extracted from different sources gynura bicolor, OPC and anthocyanidin Yield and purity, it is known that polysaccharide of the invention and the recovery rate of polyphenol are of a relatively high.Embodiment 1 and 4 is first group, embodiment 2 It it is second group with 5, embodiment 3 and 6 is the 3rd group, and three groups compare, polysaccharide, OPC and cyanine in embodiment 1 and 4 Element the rate of recovery and purity significantly improved compared with other two groups, therefore, the present invention designed by gynura bicolor in polysaccharide, OPC with And the extracting method of anthocyanidin has very high efficiency.Wherein, the optimum extraction process of polysaccharide is：2mg/mL Thick many candies concentration 100mL, the addition of neutral proteinase is 1000U/g, pH=7, under 50 DEG C of enzyme optimum conditions, digests 2h.Will be molten after processing Liquid is placed in centrifuge tube, 6000r/min centrifugations 10min；The optimum extraction process of OPC is：The posts of Sephadex LH 20 pass through Eluted with the water acetone of 500ml 70% after 1000ml 50% ethanol elution, by acetone eluant through 45 DEG C of rotary evaporations, and put Enter -20 DEG C of refrigerators and preserve 2h, be freeze-dried after -80 DEG C of refrigerators preserve 4h；The optimum extraction process of anthocyanidin is：Ethanol Eluent crosses SepPakC18 posts, is eluted after the elution of the ethanol waters of 1000ml 10% with the ethanol waters of 500ml 80%, will 80% ethanol water eluent is put into -20 DEG C of refrigerators and preserves 2h, freezed after -80 DEG C of refrigerators preserve 4h through 45 DEG C of rotary evaporations Dry.

Claims (1)

  1. A kind of 1. gynura bicolor polysaccharide polyphenol combined preparation process, it is characterised in that：Comprise the following steps：
    （1）After the blade that fresh gynura bicolor is extractd to bad leaf, old stalk and worm hole, minced after cleaning, loaded on hermetic bag, 4 Saved backup in DEG C refrigerator；
    （2）The gynura bicolor fresh leaf after mincing accurately is weighed, by solid-to-liquid ratio 1:4 g/mL add 50% ethanol water, at 45 DEG C Under conditions of 3h is extracted in shaking table, after coarse filtration, collect filtrate and filter residue；By filter residue again with 1:2 g/mL solid-to-liquid ratio addition 50% ethanol water repeats extraction once under the same conditions, is filtered after coarse filtration, and extract solution twice is closed And；After mixing, with vacuum rotary evaporator carry out concentration until volume be the 1/6 of original volume, will concentrate crude extract brown After reagent bottle installs, it is standby to be placed in fridge freshness retaining layer preservation；
    （3）Take concentrate, add 95% ethanol solution of 3 times of volumes, after mixing, 150r/min magnetic agitations 30min is fresh-keeping Film covers beaker mouth, and normal temperature is stood overnight；Liquid 6000r/min centrifugation 10min are mixed, collect precipitation and supernatant respectively, Precipitation is Thick many candies material, and supernatant need to further handle to obtain polyphenol substance；
    （4）The Thick many candies solution 100mL that compound concentration is 2-3mg/mL, the neutral protein that addition enzyme amount is 1000U/g will be precipitated Enzyme, pH=7 are adjusted, under the conditions of 50 DEG C of enzymes, digest 2h；By the solution after taking off albumen processing, 6000r/min centrifuges 10min, Precipitation is collected, is Gynura bicolor polysaccharide；
    （5）By step（3）Supernatant half after alcohol precipitation centrifugation is rotated at 45 DEG C, after being concentrated to 50ml filterings, will be filtered Liquid is through the column chromatography continuous purifications of Sephadex LH 20, the first ethanol solution elution through 1000ml 50%, afterwards with 500ml 70% the third The ketone aqueous solution elutes, and acetone eluant is put into -20 DEG C of refrigerators and preserves 2h through 45 DEG C of rotary evaporations, then is preserved in -80 DEG C of refrigerators It is freeze-dried after 4h, obtains gynura bicolor OPC；
    （6）Remaining supernatant after alcohol precipitation is centrifuged also is rotated at 45 DEG C, and after being concentrated to 50ml filterings, filtrate is passed through SepPakC18 column chromatography continuous purifications, first eluted through the ethanol waters of 1000ml 10%, use the ethanol waters of 500ml 80% afterwards Elution, 80% ethanol water eluent are put into -20 DEG C of refrigerators and preserve 2h through 45 DEG C of rotary evaporations, then after -80 DEG C of refrigerators preserve 4h It is freeze-dried, obtains gynura bicolor anthocyanidin.
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CN108586415A (en) * 2018-04-18 2018-09-28 安徽农业大学 The method for preparing high-purity cyanidin from violet cabbage
CN108586414A (en) * 2018-04-18 2018-09-28 安徽农业大学 The method for preparing high-purity delphinidin from purple eggplant
CN111685311A (en) * 2020-07-02 2020-09-22 中国计量大学 Processing method of gynura bicolor and lentinus edodes lotus root starch and product thereof
CN113024679A (en) * 2021-02-25 2021-06-25 广西壮族自治区农业科学院 Method for extracting selenium polysaccharide and polyphenol from selenium-rich moringa seeds
CN114478823A (en) * 2022-03-11 2022-05-13 四川省原子能研究院 Method for preparing pseudo-ginseng polysaccharide with high anti-aging activity by irradiation

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108586415A (en) * 2018-04-18 2018-09-28 安徽农业大学 The method for preparing high-purity cyanidin from violet cabbage
CN108586414A (en) * 2018-04-18 2018-09-28 安徽农业大学 The method for preparing high-purity delphinidin from purple eggplant
CN111685311A (en) * 2020-07-02 2020-09-22 中国计量大学 Processing method of gynura bicolor and lentinus edodes lotus root starch and product thereof
CN113024679A (en) * 2021-02-25 2021-06-25 广西壮族自治区农业科学院 Method for extracting selenium polysaccharide and polyphenol from selenium-rich moringa seeds
CN114478823A (en) * 2022-03-11 2022-05-13 四川省原子能研究院 Method for preparing pseudo-ginseng polysaccharide with high anti-aging activity by irradiation
CN114478823B (en) * 2022-03-11 2023-10-10 四川省原子能研究院 Method for preparing pseudo-ginseng polysaccharide with high anti-aging activity by using irradiation

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