CN107686473B - A kind of gynura bicolor polysaccharide polyphenol combined preparation process - Google Patents
A kind of gynura bicolor polysaccharide polyphenol combined preparation process Download PDFInfo
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- CN107686473B CN107686473B CN201710880992.5A CN201710880992A CN107686473B CN 107686473 B CN107686473 B CN 107686473B CN 201710880992 A CN201710880992 A CN 201710880992A CN 107686473 B CN107686473 B CN 107686473B
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- 241000123865 Gynura bicolor Species 0.000 title claims abstract description 71
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 40
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 40
- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 33
- -1 polysaccharide polyphenol Chemical class 0.000 title claims abstract description 10
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims abstract description 38
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 117
- 239000007788 liquid Substances 0.000 claims description 39
- 239000000243 solution Substances 0.000 claims description 35
- 239000006228 supernatant Substances 0.000 claims description 33
- 150000004676 glycans Chemical class 0.000 claims description 30
- 230000001376 precipitating effect Effects 0.000 claims description 30
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 28
- 235000009508 confectionery Nutrition 0.000 claims description 22
- 150000008442 polyphenolic compounds Chemical class 0.000 claims description 22
- 238000010828 elution Methods 0.000 claims description 20
- 238000001914 filtration Methods 0.000 claims description 20
- 239000000126 substance Substances 0.000 claims description 20
- 238000001556 precipitation Methods 0.000 claims description 19
- 238000002390 rotary evaporation Methods 0.000 claims description 19
- 102000004190 Enzymes Human genes 0.000 claims description 18
- 108090000790 Enzymes Proteins 0.000 claims description 18
- 229930014669 anthocyanidin Natural products 0.000 claims description 17
- 150000001452 anthocyanidin derivatives Chemical class 0.000 claims description 17
- 235000008758 anthocyanidins Nutrition 0.000 claims description 17
- 238000002156 mixing Methods 0.000 claims description 16
- 229920005654 Sephadex Polymers 0.000 claims description 15
- 239000012507 Sephadex™ Substances 0.000 claims description 15
- 238000005119 centrifugation Methods 0.000 claims description 12
- 239000000706 filtrate Substances 0.000 claims description 11
- 238000000746 purification Methods 0.000 claims description 11
- 239000003480 eluent Substances 0.000 claims description 10
- 238000013019 agitation Methods 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 238000004140 cleaning Methods 0.000 claims description 8
- 239000012141 concentrate Substances 0.000 claims description 8
- 239000000287 crude extract Substances 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 150000002576 ketones Chemical class 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 21
- 238000000926 separation method Methods 0.000 abstract description 5
- 238000011017 operating method Methods 0.000 abstract description 2
- 238000003809 water extraction Methods 0.000 abstract description 2
- 235000019441 ethanol Nutrition 0.000 description 56
- 238000004519 manufacturing process Methods 0.000 description 28
- 238000011084 recovery Methods 0.000 description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 22
- 238000000034 method Methods 0.000 description 20
- 239000000284 extract Substances 0.000 description 10
- 239000003755 preservative agent Substances 0.000 description 7
- 230000002335 preservative effect Effects 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 5
- 239000004365 Protease Substances 0.000 description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 5
- 235000019419 proteases Nutrition 0.000 description 5
- 241000220215 Moringa Species 0.000 description 4
- 235000011347 Moringa oleifera Nutrition 0.000 description 4
- 235000018553 tannin Nutrition 0.000 description 4
- 229920001864 tannin Polymers 0.000 description 4
- 239000001648 tannin Substances 0.000 description 4
- 241001571466 Potentilla discolor Species 0.000 description 3
- 241000220324 Pyrus Species 0.000 description 3
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 235000021017 pears Nutrition 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 235000003143 Panax notoginseng Nutrition 0.000 description 2
- 241000180649 Panax notoginseng Species 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 229930003935 flavonoid Natural products 0.000 description 2
- 150000002215 flavonoids Chemical class 0.000 description 2
- 235000017173 flavonoids Nutrition 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 150000003839 salts Chemical group 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- GDSOZVZXVXTJMI-SNAWJCMRSA-N (e)-1-methylbut-1-ene-1,2,4-tricarboxylic acid Chemical compound OC(=O)C(/C)=C(C(O)=O)\CCC(O)=O GDSOZVZXVXTJMI-SNAWJCMRSA-N 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 244000022198 Mirabilis jalapa Species 0.000 description 1
- 235000009053 Mirabilis jalapa Nutrition 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000002082 anti-convulsion Effects 0.000 description 1
- 230000003471 anti-radiation Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 229940121357 antivirals Drugs 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000004089 microcirculation Effects 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 238000000874 microwave-assisted extraction Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- OVARTBFNCCXQKS-UHFFFAOYSA-N propan-2-one;hydrate Chemical compound O.CC(C)=O OVARTBFNCCXQKS-UHFFFAOYSA-N 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- IKGXIBQEEMLURG-NVPNHPEKSA-N rutin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-NVPNHPEKSA-N 0.000 description 1
- 235000015067 sauces Nutrition 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Sustainable Development (AREA)
- Medicines Containing Plant Substances (AREA)
- Cosmetics (AREA)
- Preparation Of Fruits And Vegetables (AREA)
Abstract
The present invention provides a kind of gynura bicolor polysaccharide polyphenol combined preparation process, and by ethyl alcohol water extraction and adsorbing separation extraction method, the preparation of polysaccharide polyphenol is joined together, reduce operating procedure, the utilization rate for improving resource, increases efficiency, simultaneously effective improves the purity of the polysaccharide polyphenol of extraction.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of gynura bicolor polysaccharide polyphenol combined preparation process.
Background technique
Gynura bicolor (Gynura bicolor DC) alias blood skin dish, kwan-yin dish, red wind dish, Gynura bicolor etc., are composite family three
Half cultivar cooked in seven grass categories with young stem and leaf, perennial root evergreen herbage.Gynura bicolor is native to China, and four
The ground such as river, Zhejiang, Guangdong, Guangxi, Hainan, Fujian, Taiwan or wild or cultivating and growing are widely planted with Sichuan, one band of Chongqing especially
Training is edible.
Gynura bicolor is a kind of edible and medicinal health-care vegetable, and Tender base of leaf can be cold and dressed with sauce or be stir-fried and eaten, at the same rich in crude protein, can
Feeding habits fiber, fat, amino acid, alkaloid, Flavonoid substances, volatile oil and minerals.The mineral nutrition of gynura bicolor is more rich
Richness, especially iron, copper, the micronutrient levels such as zinc are higher, also contain glycosides displayed ingredient, and this ingredient is nontoxic, have extension anti-
Bad hematic acid and the effect for reducing blood vessel purple scar.In addition to also be used to produce in China for directly edible or production clean vegetables, gynura bicolor
Liquid medicine and health protection tea, and be used as preparing natural food pigment in Japan.Gynura bicolor nutrition is comprehensive and has both healthcare function, collects
" fresh, green, wild " and " nutrition ", " medicinal ", " delicious ", are important green health care food resource.
Gynura bicolor contains notoginseng polysaccharide, with a variety of bioactive functions closely related with the maintenance of biological function.
Notoginseng polysaccharide is to possess complicated level Four knot as many identical or different monosaccharide with compound composed by α or β-glycosidic bond
Structure, have the effects that immunological regulation, it is antiviral, delay senescence, it is antitumor, anti-radiation, in addition to this, polysaccharide also has anticoagulant
The other biological activities such as blood, anti-inflammatory, antibacterial, anticonvulsion, calm, Zhichuan and blood pressure lowering.In recent years, people to the structure of polysaccharide and
Active research deepens continuously, and further illustrates the relationship of polysaccharide mechanism of action and structure, multifarious physiological activity is more
Add and is taken seriously.
The extracting method of plant polyose mainly has solvent extraction method, sour formulation, alkaline extraction, enzymatic isolation method, ultrafiltration, surpasses at present
Sound wave reinforcement, microwave method etc..Extraction method of polysaccharides used in the present invention is ethyl alcohol water extraction, relative to other several methods,
Its reaction condition is mild, 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 substance 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 can protect tissue 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..Polyphenol extraction side used in the present invention
Method is adsorbing separation extraction method, it is advantageous that resin is strong to the suction-operated of plant polyphenol and recyclable, operating condition
Mildly, 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 smashed powdered moringa seeds are put
Enter in water, impregnate certain time at a certain temperature, extracts, obtain moringa seeds polysaccharide extraction liquid, the moringa seeds Polyose extraction
The yield of polysaccharide reaches 14% or more in liquid, though the recovery rate of the method polysaccharide is high, purity is lower.CN201611054253.2
A kind of potentilla discolor extraction method of polysaccharides is provided, extracts polysaccharide from potentilla discolor using biological enzymolysis technology, adds ultrasonic wave
Processing, ultrafiltration, alcohol precipitation, quaternary salt deposit, salt is washed, alcohol precipitation refines or macroporous resin column purification, obtains the higher potentilla discolor of purity
Polysaccharide, though the purity of polysaccharide that the method obtains is high, operating process is cumbersome, higher cost.CN201210229217.0 provides one kind
Pears young fruit peeled and cored is cut into less than 1cm by the extracting method of pears polyphenol3Fritter use 30- with liquid nitrogen grinding at powder
90% ethanol solution carries out extraction 2-6h to the operatic circle, centrifugation, and gained supernatant is pears polyphenol extracting solution, which extracts 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, drying to constant weight, 80-100 mesh is smashed it through, by solid-liquid ratio 1:5-
25 are added 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.
Summary of the invention
The present invention provides a kind of methods of gynura bicolor polysaccharide polyphenol joint preparation.The method combines the preparation of polysaccharide polyphenol
Get up, reduce operating procedure, improve the utilization rate of resource, increase efficiency, simultaneously effective improves the polysaccharide of extraction
The purity of polyphenol.
To achieve the above object, the invention adopts the following technical scheme:
The preparation method comprises the following steps:
(1) it after extracing bad leaf and old stalk and the blade in worm hole for fresh gynura bicolor, is minced after cleaning, loaded on sealing
Bag, it is saved backup in 4 DEG C of refrigerators.
(2) the gynura bicolor fresh leaf after mincing is accurately weighed, 50% ethanol water (second is added by solid-to-liquid ratio 1:4g/ml
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.Again by filter residue
It repeats to extract one at identical conditions with the solid-to-liquid ratio addition 50% ethanol water (ethyl alcohol: water (V/V)=1:1) of 1:2g/ml
It is secondary, it is filtered after coarse filtration, and extracting solution twice is merged.After mixing, be concentrated with vacuum rotary evaporator straight
It is spare to be placed in fridge freshness retaining layer preservation after installing concentration crude extract with brown reagent bottle to 1/6 that volume is original volume.
(3) concentrate is taken, 95% ethanol solution of 3 times of volumes is added, after mixing, 150r/min magnetic agitation 30min,
Preservative film covers beaker mouth, and room temperature is stood overnight.It is mixed liquid 6000r/min centrifugation 10min, collects precipitating and supernatant respectively
Liquid, precipitating are Thick many candies substances, and supernatant need to be further processed 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 adjusts pH=7, under the conditions of 50 DEG C of enzymes, digests 2h.It will be through treated solution, the 6000r/min centrifugation of de- albumen
10min collects precipitating, is Gynura bicolor polysaccharide.
(5) the supernatant half after the centrifugation of step (3) alcohol precipitation is rotated at 45 DEG C, after being concentrated to 50ml filtering,
Filtrate is chromatographed into continuous purification through 20 column of Sephadex LH, ethanol elution first through 1000ml 50%, afterwards with 500ml 70% the third
The elution of ketone aqueous solution, acetone eluant are put into -20 DEG C of refrigerators and save 2h, then save in -80 DEG C of refrigerators through 45 DEG C of rotary evaporations
It is freeze-dried after 4h, obtains gynura bicolor procyanidine.
(6) the remaining supernatant after being centrifuged alcohol precipitation also rotates at 45 DEG C, after being concentrated to 50ml filtering, by filtrate
Continuous purification is chromatographed through SepPakC18 column, is first eluted through 10% ethanol water of 1000ml, it is water-soluble with 80% ethyl alcohol of 500ml afterwards
Liquid elution, 80% ethanol water eluent is put into -20 DEG C of refrigerators and saves 2h through 45 DEG C of rotary evaporations, then saves 4h in -80 DEG C of refrigerators
After be freeze-dried, obtain gynura bicolor anthocyanidin.
The present invention has the advantages that by the process integration of the technique and polyphenol that prepare gynura bicolor polysaccharide, the system of having simplified
Standby technique, improves work efficiency；The wasting of resources is avoided, the utilization rate of resource is improved；It can guarantee to be mentioned well simultaneously
The purity of the polysaccharide polyphenol taken improves the rate of recovery.
Specific embodiment
Embodiment 1
After extracing bad leaf and old stalk and the blade in worm hole for the fresh gynura bicolor in ZhangZhou place of production, minced after cleaning,
Loaded on hermetic bag, saved backup in 4 DEG C of refrigerators.
The gynura bicolor fresh leaf after 200g is minced is accurately weighed, it is water-soluble that 50% ethyl alcohol of 800mL is added by solid-to-liquid ratio 1:4g/ml
Liquid (ethyl alcohol: water (V/V)=1:1), extracts 3h under conditions of 45 DEG C in shaking table, after coarse filtration, collects filtrate and filter residue.It will
Filter residue is again with the solid-to-liquid ratio addition 50% ethanol water of 400mL (ethyl alcohol: water (V/V)=1:1) of 1:2g/ml in identical condition
It is lower to repeat to extract once, it is filtered after coarse filtration, and extracting 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 spare.
150mL concentrate is taken, the ethanol solution of the 450mL95% of 3 times of volumes is added, after mixing, 150r/min magnetic agitation
30min, preservative film cover beaker mouth, and room temperature is stood overnight.It is mixed liquid 6000r/min centrifugation 10min, collects precipitating respectively
And supernatant, precipitating are Thick many candies substances, supernatant need to be further processed to obtain polyphenol substance.
Obtained Thick many candies precipitating is configured to the Thick many candies solution 100mL of 2mg/mL, addition enzyme amount is in 1000U/g
Property protease, adjust pH=7, under 50 DEG C of enzyme optimum conditions, digest 2h.By treated, solution is placed in centrifuge tube, 6000r/
Min is centrifuged 10min, collects precipitating.The results show that the polysaccharide recovery of the gynura bicolor in ZhangZhou place of production is 80.21%, purity is
75.56%。
After supernatant after alcohol precipitation is centrifuged takes half to be rotated, be concentrated, pass through 20 He of Sephadex LH respectively
SepPakC18 column chromatographs continuous purification.20 column of Sephadex LH is after the ethanol elution of 1000ml 50% with 500ml 70% the third
The elution of ketone aqueous solution by acetone eluant through 45 DEG C of rotary evaporations, and is put into -20 DEG C of refrigerators and saves 2h, saves in -80 DEG C of refrigerators
It is freeze-dried after 4h, obtains gynura bicolor procyanidine.The result shows that the procyanidine rate of recovery of the gynura bicolor in ZhangZhou place of production
It is 70.25%, purity 80.30%.
Meanwhile the remaining supernatant after taking alcohol precipitation to be centrifuged crosses SepPakC18 column, elutes through 10% ethanol water of 1000ml
It is eluted afterwards with 80% ethanol water of 500ml, by 80% ethanol water eluent through 45 DEG C of rotary evaporations, is put into -20 DEG C of refrigerators and saves
2h is freeze-dried after -80 DEG C of refrigerators save 4h, gynura bicolor anthocyanidin is obtained, the results show that 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 for the fresh gynura bicolor in the Guangxi place of production, minced after cleaning,
Loaded on hermetic bag, saved backup in 4 DEG C of refrigerators.
The gynura bicolor fresh leaf after 200g is minced is accurately weighed, it is water-soluble that 800mL50% ethyl alcohol is added by solid-to-liquid ratio 1:4g/ml
Liquid (ethyl alcohol: water (V/V)=1:1), extracts 3h under conditions of 45 DEG C in shaking table, after coarse filtration, collects filtrate and filter residue.It will
Filter residue adds 400mL50% ethanol water (ethyl alcohol: water (V/V)=1:1) at identical conditions with the solid-to-liquid ratio of 1:2g/ml again
It repeats to extract once, be filtered after coarse filtration, and extracting 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 spare.
150mL concentrate is taken, the ethanol solution of the 450mL95% of 3 times of volumes is added, after mixing, 150r/min magnetic agitation
30min, preservative film cover beaker mouth, and room temperature is stood overnight.It is mixed liquid 6000r/min centrifugation 10min, collects precipitating respectively
And supernatant, precipitating are Thick many candies substances, supernatant need to be further processed to obtain polyphenol substance.
Obtained Thick many candies precipitating is configured to the Thick many candies solution 100mL of 3mg/mL, addition enzyme amount is in 1000U/g
Property protease, adjust pH=7, under 50 DEG C of enzyme optimum conditions, digest 2h.By treated, solution is placed in centrifuge tube, 6000r/
Min is centrifuged 10min, collects precipitating.The results show that the polysaccharide recovery of the gynura bicolor in the Guangxi place of production is 75.57%, purity is
70.99%。
After supernatant after alcohol precipitation is centrifuged takes half to be rotated, be concentrated, pass through 20 He of Sephadex LH respectively
SepPakC18 column chromatographs continuous purification.20 column of Sephadex LH uses 70% water of 750ml after the ethanol elution of 1000ml 50%
Acetone elution by acetone eluant through 45 DEG C of rotary evaporations, and is put into -20 DEG C of refrigerators and saves 2h, after -80 DEG C of refrigerators save 4h
It is freeze-dried, obtains gynura bicolor procyanidine, the results showed that, the procyanidine rate of recovery of the gynura bicolor in the Guangxi place of production is
60.11%, purity 70.32%.
Meanwhile the remaining supernatant after taking alcohol precipitation to be centrifuged crosses SepPakC18 column, elutes through 10% ethanol water of 750ml
It is eluted afterwards with 80% ethanol water of 500ml, by 80% ethanol water eluent through 45 DEG C of rotary evaporations, is put into -20 DEG C of refrigerators and saves
2h is freeze-dried after -80 DEG C of refrigerators save 4h, gynura bicolor anthocyanidin is obtained, the results show that 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 for the fresh gynura bicolor in the Zhejiang place of production, minced after cleaning,
Loaded on hermetic bag, saved backup in 4 DEG C of refrigerators.
The gynura bicolor fresh leaf after 200g is minced is accurately weighed, it is water-soluble that 800mL50% ethyl alcohol is added by solid-to-liquid ratio 1:4g/ml
Liquid (ethyl alcohol: water (V/V)=1:1), extracts 3h under conditions of 45 DEG C in shaking table, after coarse filtration, collects filtrate and filter residue.It will
Filter residue adds 400mL50% ethanol water (ethyl alcohol: water (V/V)=1:1) at identical conditions with the solid-to-liquid ratio of 1:2g/ml again
It repeats to extract once, be filtered after coarse filtration, and extracting 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 spare.
150mL concentrate is taken, the ethanol solution of the 450mL95% of 3 times of volumes is added, after mixing, 150r/min magnetic agitation
30min, preservative film cover beaker mouth, and room temperature is stood overnight.It is mixed liquid 6000r/min centrifugation 10min, collects precipitating respectively
And supernatant, precipitating are Thick many candies substances, supernatant need to be further processed to obtain polyphenol substance.
Obtained Thick many candies precipitating is configured to the Thick many candies solution 100mL of 2.5mg/mL, addition enzyme amount is 1000U/g's
Neutral proteinase adjusts pH=7, under 50 DEG C of enzyme optimum conditions, digests 2h.By treated, solution is placed in centrifuge tube,
6000r/min is centrifuged 10min, collects precipitating.The results show that the polysaccharide recovery of the gynura bicolor in the Zhejiang place of production is 70.33%, it is pure
Degree is 65.49%.
After supernatant after alcohol precipitation is centrifuged takes half to be rotated, be concentrated, pass through 20 He of Sephadex LH respectively
SepPakC18 column chromatographs continuous purification.20 column of Sephadex LH uses 70% water of 800ml after the ethanol elution of 1000ml 50%
Acetone elution by acetone eluant through 45 DEG C of rotary evaporations, and is put into -20 DEG C of refrigerators and saves 2h, after -80 DEG C of refrigerators save 4h
It is freeze-dried, obtains gynura bicolor procyanidine, the results showed that, the procyanidine rate of recovery of the gynura bicolor in the Zhejiang place of production is
60.15%, purity 65.37%.
Meanwhile the remaining supernatant after taking alcohol precipitation to be centrifuged crosses SepPakC18 column, elutes through 10% ethanol water of 800ml
It is eluted afterwards with 80% ethanol water of 500ml, by 80% ethanol water eluent through 45 DEG C of rotary evaporations, is put into -20 DEG C of refrigerators and saves
2h is freeze-dried after -80 DEG C of refrigerators save 4h, gynura bicolor anthocyanidin is obtained, the results show that 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 for the fresh gynura bicolor in Hainan place of production, minced after cleaning,
Loaded on hermetic bag, saved backup in 4 DEG C of refrigerators.
The gynura bicolor fresh leaf after 200g is minced is accurately weighed, it is water-soluble that 800mL50% ethyl alcohol is added by solid-to-liquid ratio 1:4g/ml
Liquid (ethyl alcohol: water (V/V)=1:1), extracts 3h under conditions of 45 DEG C in shaking table, after coarse filtration, collects filtrate and filter residue.It will
Filter residue adds 400mL50% ethanol water (ethyl alcohol: water (V/V)=1:1) at identical conditions with the solid-to-liquid ratio of 1:2g/ml again
It repeats to extract once, be filtered after coarse filtration, and extracting 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 spare.
150mL concentrate is taken, the ethanol solution of the 450mL95% of 3 times of volumes is added, after mixing, 150r/min magnetic agitation
30min, preservative film cover beaker mouth, and room temperature is stood overnight.It is mixed liquid 6000r/min centrifugation 10min, collects precipitating respectively
And supernatant, precipitating are Thick many candies substances, supernatant need to be further processed to obtain polyphenol substance.
Obtained Thick many candies precipitating is configured to the Thick many candies solution 150mL of 2mg/mL, addition enzyme amount is in 1000U/g
Property protease, adjust pH=7, under 50 DEG C of enzyme optimum conditions, digest 2h.By treated, solution is placed in centrifuge tube, 6000r/
Min is centrifuged 10min, collects precipitating.The results show that the polysaccharide recovery of the gynura bicolor in Hainan place of production is 75.09%, purity is
80.55%。
Supernatant after alcohol precipitation is centrifuged takes half, after being rotated, being concentrated, passes through 20 He of Sephadex LH respectively
SepPakC18 column chromatographs continuous purification.20 column of Sephadex LH uses 70% water of 500ml after the ethanol elution of 1000ml 50%
Acetone elution by acetone eluant through 45 DEG C of rotary evaporations, and is put into -20 DEG C of refrigerators and saves 2h, after -80 DEG C of refrigerators save 4h
It is freeze-dried, obtains gynura bicolor procyanidine, the results showed that, the procyanidine rate of recovery of the gynura bicolor in Hainan place of production is
70.46%, purity 75.91%.
Meanwhile the remaining supernatant after taking alcohol precipitation to be centrifuged crosses SepPakC18 column, elutes through 10% ethanol water of 1000ml
It is eluted afterwards with 80% ethanol water of 500ml, by 80% ethanol water eluent through 45 DEG C of rotary evaporations, is put into -20 DEG C of refrigerators and saves
2h is freeze-dried after -80 DEG C of refrigerators save 4h, gynura bicolor anthocyanidin is obtained, the results show that 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 for the fresh gynura bicolor in the Taiwan place of production, minced after cleaning,
Loaded on hermetic bag, saved backup in 4 DEG C of refrigerators.
The gynura bicolor fresh leaf after 200g is minced is accurately weighed, it is water-soluble that 800mL50% ethyl alcohol is added by solid-to-liquid ratio 1:4g/ml
Liquid (ethyl alcohol: water (V/V)=1:1), extracts 3h under conditions of 45 DEG C in shaking table, after coarse filtration, collects filtrate and filter residue.It will
Filter residue adds 400mL50% ethanol water (ethyl alcohol: water (V/V)=1:1) at identical conditions with the solid-to-liquid ratio of 1:2g/ml again
It repeats to extract once, be filtered after coarse filtration, and extracting 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 spare.
150mL concentrate is taken, the ethanol solution of the 450mL95% of 3 times of volumes is added, after mixing, 150r/min magnetic agitation
30min, preservative film cover beaker mouth, and room temperature is stood overnight.It is mixed liquid 6000r/min centrifugation 10min, collects precipitating respectively
And supernatant, precipitating are Thick many candies substances, supernatant need to be further processed to obtain polyphenol substance.
Obtained Thick many candies precipitating is configured to the Thick many candies solution 150mL of 3mg/mL, addition enzyme amount is in 1000U/g
Property protease, adjust pH=7, under 50 DEG C of enzyme optimum conditions, digest 2h.By treated, solution is placed in centrifuge tube, 6000r/
Min is centrifuged 10min, collects precipitating.The results show that the polysaccharide recovery of the gynura bicolor in the Taiwan place of production is 75.06%, purity is
70.47%。
After supernatant after alcohol precipitation is centrifuged takes half to be rotated, be concentrated, pass through 20 He of Sephadex LH respectively
SepPakC18 column chromatographs continuous purification.20 column of Sephadex LH uses 70% water of 750ml after the ethanol elution of 1000ml 50%
Acetone elution by acetone eluant through 45 DEG C of rotary evaporations, and is put into -20 DEG C of refrigerators and saves 2h, after -80 DEG C of refrigerators save 4h
It is freeze-dried, obtains gynura bicolor procyanidine, the results showed that, the procyanidine rate of recovery of the gynura bicolor in the Taiwan place of production is
55.15%, purity 60.84%.
Meanwhile the remaining supernatant after taking alcohol precipitation to be centrifuged crosses SepPakC18 column, elutes through 10% ethanol water of 750ml
It is eluted afterwards with 80% ethanol water of 500ml, by 80% ethanol water eluent through 45 DEG C of rotary evaporations, is put into -20 DEG C of refrigerators and saves
2h is freeze-dried after -80 DEG C of refrigerators save 4h, gynura bicolor anthocyanidin is obtained, the results show that 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 for the fresh gynura bicolor in the Guangdong place of production, minced after cleaning,
Loaded on hermetic bag, saved backup in 4 DEG C of refrigerators.
The gynura bicolor fresh leaf after 200g is minced is accurately weighed, it is water-soluble that 800mL50% ethyl alcohol is added by solid-to-liquid ratio 1:4g/ml
Liquid (ethyl alcohol: water (V/V)=1:1), extracts 3h under conditions of 45 DEG C in shaking table, after coarse filtration, collects filtrate and filter residue.It will
Filter residue adds 400mL50% ethanol water (ethyl alcohol: water (V/V)=1:1) at identical conditions with the solid-to-liquid ratio of 1:2g/ml again
It repeats to extract once, be filtered after coarse filtration, and extracting 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 spare.
150mL concentrate is taken, the ethanol solution of the 450mL95% of 3 times of volumes is added, after mixing, 150r/min magnetic agitation
30min, preservative film cover beaker mouth, and room temperature is stood overnight.It is mixed liquid 6000r/min centrifugation 10min, collects precipitating respectively
And supernatant, precipitating are Thick many candies substances, supernatant need to be further processed to obtain polyphenol substance.
The gynura bicolor in the Guangdong place of production is mentioned through water, after alcohol precipitation, and obtained Thick many candies precipitating is configured to the thick more of 2.5mg/mL
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.
By treated, solution is placed in centrifuge tube, and 6000r/min is centrifuged 10min, collects precipitating.The results show that the purple in the Guangdong place of production
The polysaccharide recovery for carrying on the back dish is 70.80%, purity 65.79%.
After supernatant after alcohol precipitation is centrifuged takes half to be rotated, be concentrated, pass through 20 He of Sephadex LH respectively
SepPakC18 column chromatographs continuous purification.20 column of Sephadex LH uses 70% water of 800ml after the ethanol elution of 1000ml 50%
Acetone elution by acetone eluant through 45 DEG C of rotary evaporations, and is put into -20 DEG C of refrigerators and saves 2h, after -80 DEG C of refrigerators save 4h
It is freeze-dried, obtains gynura bicolor procyanidine, the results showed that, the procyanidine rate of recovery of the gynura bicolor in the Guangdong place of production is
60.21%, purity 60.75%.
Meanwhile the remaining supernatant after taking alcohol precipitation to be centrifuged crosses SepPakC18 column, elutes through 10% ethanol water of 800ml
It is eluted afterwards with 80% ethanol water of 500ml, by 80% ethanol water eluent through 45 DEG C of rotary evaporations, is put into -20 DEG C of refrigerators and saves
2h is freeze-dried after -80 DEG C of refrigerators save 4h, gynura bicolor anthocyanidin is obtained, the results show that 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, procyanidine and anthocyanidin
Yield and purity, it is known that the recovery rate of polysaccharide and polyphenol of the invention is relatively high.Embodiment 1 and 4 is first group, embodiment 2
It is second group with 5, embodiment 3 and 6 is third group, and three groups compare, polysaccharide, procyanidine 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, procyanidine with
And the extracting method of anthocyanidin has very high efficiency.Wherein, the optimum extraction process of polysaccharide are as follows: 2mg/mL Thick many candies concentration
100mL, the additive amount of neutral proteinase are 1000U/g, and pH=7 digest 2h under 50 DEG C of enzyme optimum conditions.It will that treated be molten
Liquid is placed in centrifuge tube, and 6000r/min is centrifuged 10min；The optimum extraction process of procyanidine are as follows: 20 column of Sephadex LH warp
It is eluted after the ethanol elution of 1000ml 50% with 70% water acetone of 500ml, by acetone eluant through 45 DEG C of rotary evaporations, and is put
Enter -20 DEG C of refrigerators and save 2h, is freeze-dried after -80 DEG C of refrigerators save 4h；The optimum extraction process of anthocyanidin are as follows: ethyl alcohol
Eluent crosses SepPakC18 column, is eluted after the elution of 10% ethanol water of 1000ml with 80% ethanol water of 500ml, will
80% ethanol water eluent is put into -20 DEG C of refrigerators and saves 2h through 45 DEG C of rotary evaporations, is freezed after -80 DEG C of refrigerators save 4h
It is dry.
Claims (1)
1. a kind of gynura bicolor polysaccharide polyphenol combined preparation process, characterized by the following steps:
(1) it after the blade that fresh gynura bicolor is extractd bad leaf, old stalk and worm hole, is minced after cleaning, is loaded on hermetic bag, 4
It is saved backup in DEG C refrigerator；
(2) the gynura bicolor fresh leaf after mincing is accurately weighed, 50% ethanol water is added by solid-to-liquid ratio 1:4 g/mL, at 45 DEG C
Under conditions of 3h is extracted in shaking table, after coarse filtration, collect filtrate and filter residue；Filter residue is added again with the solid-to-liquid ratio of 1:2 g/mL
50% ethanol water repeats to extract once at identical conditions, is filtered after coarse filtration, and extracting solution twice is closed
And；After mixing, with vacuum rotary evaporator carry out concentration until volume be the 1/6 of original volume, will be concentrated crude extract brown
After reagent bottle installs, it is spare to be placed in fridge freshness retaining layer preservation；
(3) concentrate is taken, 95% ethanol solution of 3 times of volumes is added, after mixing, 150r/min magnetic agitation 30min is fresh-keeping
Film covers beaker mouth, and room temperature is stood overnight；It is mixed liquid 6000r/min centrifugation 10min, collects precipitating and supernatant respectively,
Precipitating is Thick many candies substance, and supernatant need to be further processed 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 adjusts pH=7, under the conditions of 50 DEG C of enzymes, digests 2h；It will be centrifuged 10min through de- albumen treated solution, 6000r/min,
Precipitating is collected, is Gynura bicolor polysaccharide；
(5) the supernatant half after the centrifugation of step (3) alcohol precipitation is rotated at 45 DEG C, after being concentrated to 50ml filtering, will be filtered
Liquid chromatographs continuous purification through 20 column of Sephadex LH, first the ethanol solution elution through 1000ml 50%, afterwards with 500ml 70% the third
The elution of ketone aqueous solution, acetone eluant are put into -20 DEG C of refrigerators and save 2h, then save in -80 DEG C of refrigerators through 45 DEG C of rotary evaporations
It is freeze-dried after 4h, obtains gynura bicolor procyanidine；
(6) the remaining supernatant after being centrifuged alcohol precipitation also rotates at 45 DEG C, and after being concentrated to 50ml filtering, filtrate is passed through
SepPakC18 column chromatographs continuous purification, first elutes through 10% ethanol water of 1000ml, uses 80% ethanol water of 500ml afterwards
Elution, 80% ethanol water eluent are put into -20 DEG C of refrigerators and save 2h through 45 DEG C of rotary evaporations, then after -80 DEG C of refrigerators save 4h
It is freeze-dried, obtains gynura bicolor anthocyanidin.
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| 紫背菜提取物降血糖作用的实验研究;余小平;《中华中医药学刊》;20110731;第29卷(第7期);1652-1654 * |
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