CN108567836A - A method of combined extraction separation flavones and polysaccharide from hawthorn skin slag - Google Patents
A method of combined extraction separation flavones and polysaccharide from hawthorn skin slag Download PDFInfo
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- CN108567836A CN108567836A CN201810783900.6A CN201810783900A CN108567836A CN 108567836 A CN108567836 A CN 108567836A CN 201810783900 A CN201810783900 A CN 201810783900A CN 108567836 A CN108567836 A CN 108567836A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/46—Eucommiaceae (Eucommia family), e.g. hardy rubber tree
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- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/17—Preparation or pretreatment of starting material involving drying, e.g. sun-drying or wilting
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Abstract
The invention discloses a kind of methods of combined extraction separation flavones and polysaccharide in skin slag from hawthorn, this method is using the hawthorn skin slag after hawthorn wine fermentation as raw material, using the flavones and polysaccharide in ultrasound and enzyme process ancillary technique combined extracting hawthorn skin slag, and according to the different characteristics of flavones and polysaccharide in hawthorn skin slag, using ADSORPTION IN A FIXED BED technology, to in hawthorn skin slag extracting solution flavones and polysaccharide synchronize separation, both Hawthorn Polysaccharides have been isolated and purified, hawthorn flavone extract is obtained again, realize the comprehensive utilization of hawthorn, also solve the wasting of resources and environmental pollution caused by hawthorn skin slag simultaneously.The method extracted amount of the present invention is high, and purity is high, and extraction efficiency is high, extraction process does not need special equipment, of low cost, and approach is provided for combined extraction separation flavones and polysaccharide, also the utilization for hawthorn skin slag provides new approach, has good social and economic benefit.
Description
Technical field
The present invention relates to a kind of methods of combined extraction separation flavones and polysaccharide in skin slag from hawthorn, belong to extraction process skill
Art field.
Background technology
Hawthorn (Crataegus pinnatifida Bunge) also known as hawthorn, large-fruited Chinese hawthorn, rose family hawthorn, on mountain
The ground such as east, Shaanxi, Shanxi, Henan, Jiangsu, Zhejiang, Liaoning, Jilin, Heilungkiang, the Inner Mongol, Hebei are distributed.Haw berry can
Eat it raw or make preserved fruit fruitcake, can be used as medicine after drying, be the distinctive dual-purpose of drug and fruit seeds of China, have reducing blood lipid, blood pressure, cardiac stimulant,
The effects that arrhythmia, while being also the good medicine of spleen benefiting and stimulating the appetite, relieving dyspepsia, phlegm reduction of blood circulation promoting, full, hernia, blood to chest diaphragm spleen
The diseases such as silt, amenorrhoea have fine curative effect.Studies have shown that chemical containing organic acid, triterpenes, flavonoids and polysaccharide etc. in hawthorn
Ingredient.Wherein, flavones and polysaccharide have the function of preferable lowering blood pressure and blood fat.In addition to this, flavone of hawthorn fruit and polysaccharide also have
Other important functions such as prevent diabetes and its complication, treat osteopathy and osteoporosis etc..Therefore, for flavone of hawthorn fruit
Research with polysaccharide is of great significance.
In recent years, with the raising of people's living standard and health care consciousness, Howthorn Wine, fruit vinegar with healthcare function,
Fruit juice is quickly grown in hawthorn processing industry, while also producing a large amount of hawthorn skin slag.Hawthorn skin slag after hawthorn wine fermentation
In still contain abundant active material, developed and used in default of effective, cause the huge wasting of resources and environmental pollution.Cause
This, excavates effective functional component extraction and separation method, can not only improve the comprehensive utilization of hawthorn, but also can reduce environment dirt
Dye.
Currently, it is less for the research of functional mass in hawthorn skin slag, and concentrate on to simple function substance in hawthorn
The research of extraction process, such as the extraction of flavones, three note acid function substances.It is seldom to the research of Hawthorn Polysaccharides extraction process, mesh
It is preceding that there has been no the researchs of functional materials combined extraction separation technology in hawthorn.
Invention content
In view of the deficiencies of the prior art, the object of the present invention is to provide one kind from hawthorn skin slag combined extraction separation flavones
With the method for polysaccharide, separation flavone of hawthorn fruit and polysaccharide can simultaneously, be effectively extracted.
To achieve the goals above, the technical solution adopted in the present invention is：
A method of combined extraction separation flavones and polysaccharide from hawthorn skin slag include the following steps：
(1) hawthorn skin slag pre-processes：
After the hawthorn skin slag filtering after hawthorn wine fermentation, then remove seed is dried in the shade naturally, the hawthorn skin after drying in the shade naturally
Slag, which is put into pulverizer, breaks into powder；
(2) ultrasound enzyme combined extracting：The hawthorn skin slag that step (1) is obtained, according to solid-liquid ratio 1:40 are added 20% second
Alcohol mixes mixing；Then 0.2~0.3% (m/m) cellulase and 0.2~0.25% (v/v) Tween 80 is added, is put into 60~70
30~40min of ultrasonic extraction in DEG C water-bath repeats extraction 2 times, is centrifuged after extraction, obtain supernatant and precipitation, supernatant is
For flavone of hawthorn fruit and polysaccharide extraction liquid；
(3) it is concentrated by evaporation：Flavone of hawthorn fruit and polysaccharide extraction liquid that step (2) obtains are placed on Rotary Evaporators, 50 DEG C
It is poured into culture dish after evaporation and concentration, is put into -80 DEG C of ultra low temperature freezers after freeze overnight, is put into freeze drier and is lyophilized,
Obtain flavone of hawthorn fruit polysaccharide crude extract；
(4) flavone of hawthorn fruit, the separation of polysaccharide, purifying：
(a1) Hawthorn Polysaccharides detach：The flavone of hawthorn fruit polysaccharide crude extract that will be obtained in step (3) is dissolved with 20% ethyl alcohol
Afterwards, upper AB-8 macroporous resin adsorptions are collected eluent, are concentrated under reduced pressure into the 1/4 of hawthorn effluent volume with distillation water elution,
Obtain Hawthorn Polysaccharides concentrate；
(a2) Hawthorn Polysaccharides purify：The Hawthorn Polysaccharides concentrate that will be obtained in step (a1) is added 5~6 times while stirring
The ethyl alcohol of volume centrifuges after standing 12h in 4 DEG C of refrigerators, collects sediment, after then redissolving sediment with distilled water, be added 5
The savage reagents of~6 times of volumes, shaking table concussion, centrifugation are repeated 3 times, merge supernatant, be concentrated under reduced pressure into supernatant volume
5~6 times of volume ethanol dissolvings are added after 1/4, are centrifuged after standing 12h in 4 DEG C of refrigerators, it is Hawthorn Polysaccharides to collect sediment；
(b1) flavone of hawthorn fruit detaches：Step (a1) is distilled to the macroreticular resin after water elution, with 50% ethanol elution, is collected
Eluent is concentrated under reduced pressure, obtains flavone of hawthorn fruit crude extract；
(b2) flavone of hawthorn fruit purifies：The flavone of hawthorn fruit crude extract that step (b2) obtains is dissolved in ethyl alcohol, with 3~5 times
Ethyl alcohol phase is stayed after volume n-hexane extraction, and hawthorn flavone extract is obtained after reduced pressure.
Powder crosses 60 mesh sieve in step (1).
The enzyme activity of step (2) cellulase is 1000U/mg.
The solid-liquid ratio of flavone of hawthorn fruit polysaccharide crude extract and 20% ethyl alcohol is 1 in step (a1)：2.
Distilled water is 3-5BV, elution flow rate 2ml/min in step (a1).
Preferably, distilled water is 3BV in step (a1).
50% ethyl alcohol is 3-5BV, elution flow rate 2ml/min in step (b1).
Preferably, 50% ethyl alcohol is 3BV in step (b1).
Advantageous effect of the present invention：
1, the present invention is using the hawthorn skin slag after hawthorn wine fermentation as raw material, since Hawthorn Fruit Wine has already passed through in the production process
Pectinase treatment, therefore the pectin in hawthorn skin slag is digested, and does not have to fruit then during flavone of hawthorn fruit and Polyose extraction
Glue enzymatic treatment, is effectively simplified the extraction process of flavone of hawthorn fruit and polysaccharide, also solves the problems, such as the recycling of hawthorn skin slag.
2, the present invention uses cellulase and Tween 80 synergy, and cellulase is one group of complex enzyme, can hydrolyze fibre
Dimension, makes plant cell wall destroy, fully discharges cell inclusion, be conducive to the extraction of flavones and polysaccharide in hawthorn skin slag；Tween
80 can be effectively promoted Cellulase Molecules distribution, the probability of ineffective adsorption be reduced, to improve enzymolysis efficiency.
3, the present invention uses the flavones and polysaccharide in ultrasound and enzyme process ancillary technique combined extracting hawthorn skin slag, and according to mountain
The different characteristics of flavones and polysaccharide in short, bristly hair or beard skin slag, using ADSORPTION IN A FIXED BED technology, to flavones in hawthorn skin slag extracting solution and more
Sugar synchronizes separation, has not only isolated and purified Hawthorn Polysaccharides, but also obtain hawthorn flavone extract, realizes the comprehensive profit of hawthorn
With, while also solving the wasting of resources and environmental pollution caused by hawthorn skin slag.
4, method extracted amount of the invention is high, and purity is high, and extraction efficiency is high, and extraction process does not need special equipment, at
This is cheap, and approach is provided for combined extraction separation flavones and polysaccharide, and the also utilization for hawthorn skin slag provides new approach, tool
There is good social and economic benefit.
Description of the drawings
Fig. 1 is the enzyme concentration the selection result of extracting flavonoids in hawthorn skin slag.
Fig. 2 is the concentration of alcohol the selection result of extracting flavonoids in hawthorn skin slag.
Fig. 3 is the extraction time the selection result of extracting flavonoids in hawthorn skin slag.
Fig. 4 is the Extracting temperature the selection result of extracting flavonoids in hawthorn skin slag.
Fig. 5 is the solid-liquid ratio the selection result of extracting flavonoids in hawthorn skin slag.
Fig. 6 is the enzyme concentration the selection result of Polyose extraction in hawthorn skin slag.
Fig. 7 is the concentration of alcohol the selection result of Polyose extraction in hawthorn skin slag.
Fig. 8 is the extraction time the selection result of Polyose extraction in hawthorn skin slag.
Fig. 9 is the Extracting temperature the selection result of Polyose extraction in hawthorn skin slag.
Figure 10 is the solid-liquid ratio the selection result of Polyose extraction in hawthorn skin slag.
Figure 11 is influence of the Tween 80 to extracting flavonoids rate in hawthorn skin slag.
Figure 12 is influence of the Tween 80 to polysaccharide extract rate in hawthorn skin slag.
Figure 13 is the selection result of polysaccharide distilled water elution volume.
Figure 14 is the selection result of flavones ethanol elution volume.
Figure 15 is the selection result of flavones ethanol elution flow velocity.
Specific implementation mode
The specific implementation mode of the present invention is described in further detail with reference to embodiments.
Embodiment 1
A method of combined extraction separation flavones and polysaccharide from hawthorn skin slag include the following steps：
(1) hawthorn skin slag pre-processes：
After the hawthorn skin slag filtered through gauze after hawthorn wine fermentation, then remove seed is dried in the shade naturally, after drying in the shade naturally
Hawthorn skin slag is put into pulverizer break into powder after cross 60 mesh sieve；
(2) ultrasound enzyme combined extracting：The hawthorn skin slag that step (1) is obtained, according to solid-liquid ratio 1:40 (g/ml) are added
20% (v/v) ethyl alcohol mixes mixing；Then mixture 0.25% (m/m) cellulase (1000U/mg) and 0.2% (v/v) is added
Tween 80 is put into ultrasonic extraction 40min in 60 DEG C of water-baths, repeats extraction 2 times, is centrifuged after extraction, obtain supernatant and precipitation,
Supernatant is flavone of hawthorn fruit and polysaccharide extraction liquid；
(3) it is concentrated by evaporation：Flavone of hawthorn fruit and polysaccharide extraction liquid that step (2) obtains are placed on Rotary Evaporators, 50 DEG C
It is poured into culture dish after evaporation and concentration, is put into -80 DEG C of ultra low temperature freezers after freeze overnight, is put into freeze drier and is lyophilized,
Obtain flavone of hawthorn fruit polysaccharide crude extract；
(4) flavone of hawthorn fruit, the separation of polysaccharide, purifying：
(a1) Hawthorn Polysaccharides detach：The flavone of hawthorn fruit polysaccharide crude extract that will be obtained in step (3), with 20% (v/v) ethyl alcohol
According to solid-liquid ratio 1：After 2 (g/ml) dissolvings, upper AB-8 macroporous resin adsorptions distill water elution with 3BV (resin volume), and flow velocity is
2ml/min collects eluent, is concentrated under reduced pressure into the 1/4 of hawthorn effluent volume, obtains Hawthorn Polysaccharides concentrate；
(a2) Hawthorn Polysaccharides purify：5 times of volumes are added in the Hawthorn Polysaccharides concentrate that will be obtained in step (a1) while stirring
Ethyl alcohol, centrifuged after standing 12h in 4 DEG C of refrigerators, collect sediment, after then redissolving sediment with distilled water, 5 times of bodies are added
Long-pending savage reagents, to remove the protein in polysaccharide, after shaking table shakes 30min, 4000r/min centrifuges 20min, repeats 3
It is secondary, merge supernatant, 5 times of volume ethanol dissolvings are added after being concentrated under reduced pressure into the 1/4 of supernatant volume, 12h is stood in 4 DEG C of refrigerators
After centrifuge, collect sediment be Hawthorn Polysaccharides；
(b1) flavone of hawthorn fruit detaches：Step (a1) is distilled to the macroreticular resin after water elution, with 3BV (resin volume) 50%
(v/v) ethanol elution, flow velocity 2ml/min collect eluent, are concentrated under reduced pressure, obtain flavone of hawthorn fruit crude extract；
(b2) flavone of hawthorn fruit purifies：The flavone of hawthorn fruit crude extract that step (b2) obtains is dissolved in ethyl alcohol, with 3 times of volumes
Ethyl alcohol phase is stayed after n-hexane extraction, and to remove the liposoluble substance in flavone of hawthorn fruit crude extract, hawthorn Huang is obtained after reduced pressure
Ketone extract.
Embodiment 2
A method of combined extraction separation flavones and polysaccharide from hawthorn skin slag include the following steps：
(1) hawthorn skin slag pre-processes：
After the hawthorn skin slag filtered through gauze after hawthorn wine fermentation, then remove seed is dried in the shade naturally, after drying in the shade naturally
Hawthorn skin slag is put into pulverizer break into powder after cross 60 mesh sieve；
(2) ultrasound enzyme combined extracting：The hawthorn skin slag that step (1) is obtained, according to solid-liquid ratio 1:40 are added 20% (v/
V) ethyl alcohol mixes mixing；Then 0.2% (m/m) cellulase (1000U/mg) and 0.25% (v/v) Tween 80 is added, is put into 60
Ultrasonic extraction 40min in DEG C water-bath repeats extraction 2 times, is centrifuged after extraction, obtain supernatant and precipitation, supernatant is mountain
Short, bristly hair or beard flavones and polysaccharide extraction liquid；
(3) it is concentrated by evaporation：Flavone of hawthorn fruit and polysaccharide extraction liquid that step (2) obtains are placed on Rotary Evaporators, 50 DEG C
It is poured into culture dish after evaporation and concentration, is put into -80 DEG C of ultra low temperature freezers after freeze overnight, is put into freeze drier and is lyophilized,
Obtain flavone of hawthorn fruit polysaccharide crude extract；
(4) flavone of hawthorn fruit, the separation of polysaccharide, purifying：
(a1) Hawthorn Polysaccharides detach：The flavone of hawthorn fruit polysaccharide crude extract that will be obtained in step (3), with 20% (v/v) ethyl alcohol
According to solid-liquid ratio 1：After 2 dissolvings, upper AB-8 macroporous resin adsorptions distill water elution, flow velocity 2ml/ with 4BV (resin volume)
Min collects eluent, is concentrated under reduced pressure into the 1/4 of hawthorn effluent volume, obtains Hawthorn Polysaccharides concentrate；
(a2) Hawthorn Polysaccharides purify：5 times of volumes are added in the Hawthorn Polysaccharides concentrate that will be obtained in step (a1) while stirring
Ethyl alcohol, centrifuged after standing 12h in 4 DEG C of refrigerators, collect sediment, after then redissolving sediment with distilled water, 5 times of bodies are added
Long-pending savage reagents, to remove the protein in polysaccharide, after shaking table shakes 30min, 4000r/min centrifuges 20min, repeats 3
It is secondary, merge supernatant, 5 times of volume ethanol dissolvings are added after being concentrated under reduced pressure into the 1/4 of supernatant volume, 12h is stood in 4 DEG C of refrigerators
After centrifuge, collect sediment be Hawthorn Polysaccharides；
(b1) flavone of hawthorn fruit detaches：Step (a1) is distilled to the macroreticular resin after water elution, with 4BV (resin volume) 50%
Ethanol elution, flow velocity 2ml/min collect eluent, are concentrated under reduced pressure, obtain flavone of hawthorn fruit crude extract；
(b2) flavone of hawthorn fruit purifies：The flavone of hawthorn fruit crude extract that step (b2) obtains is dissolved in ethyl alcohol, with 4 times of volumes
Ethyl alcohol phase is stayed after n-hexane extraction, and to remove the liposoluble substance in flavone of hawthorn fruit crude extract, hawthorn Huang is obtained after reduced pressure
Ketone extract.
Embodiment 3
A method of combined extraction separation flavones and polysaccharide from hawthorn skin slag include the following steps：
(1) hawthorn skin slag pre-processes：
After the hawthorn skin slag filtered through gauze after hawthorn wine fermentation, then remove seed is dried in the shade naturally, after drying in the shade naturally
Hawthorn skin slag is put into pulverizer break into powder after cross 60 mesh sieve；
(2) ultrasound enzyme combined extracting：The hawthorn skin slag that step (1) is obtained, according to solid-liquid ratio 1:40 are added 20% (v/
V) ethyl alcohol mixes mixing；Then 0.3% (m/m) cellulase (1000U/mg) and 0.2% (v/v) Tween 80 is added, is put into 70
Ultrasonic extraction 40min in DEG C water-bath repeats extraction 2 times, is centrifuged after extraction, obtain supernatant and precipitation, supernatant is mountain
Short, bristly hair or beard flavones and polysaccharide extraction liquid；
(3) it is concentrated by evaporation：Flavone of hawthorn fruit and polysaccharide extraction liquid that step (2) obtains are placed on Rotary Evaporators, 50 DEG C
It is poured into culture dish after evaporation and concentration, is put into -80 DEG C of ultra low temperature freezers after freeze overnight, is put into freeze drier and is lyophilized,
Obtain flavone of hawthorn fruit polysaccharide crude extract；
(4) flavone of hawthorn fruit, the separation of polysaccharide, purifying：
(a1) Hawthorn Polysaccharides detach：The flavone of hawthorn fruit polysaccharide crude extract that will be obtained in step (3), with 20% (v/v) ethyl alcohol
According to solid-liquid ratio 1：After 2 dissolvings, upper AB-8 macroporous resin adsorptions distill water elution, flow velocity 2ml/ with 5BV (resin volume)
Min collects eluent, is concentrated under reduced pressure into the 1/4 of hawthorn effluent volume, obtains Hawthorn Polysaccharides concentrate；
(a2) Hawthorn Polysaccharides purify：6 times of volumes are added in the Hawthorn Polysaccharides concentrate that will be obtained in step (a1) while stirring
Ethyl alcohol, centrifuged after standing 12h in 4 DEG C of refrigerators, collect sediment, after then redissolving sediment with distilled water, 6 times of bodies are added
Long-pending savage reagents, to remove the protein in polysaccharide, after shaking table shakes 30min, 4000r/min centrifuges 20min, repeats 3
It is secondary, merge supernatant, 6 times of volume ethanol dissolvings are added after being concentrated under reduced pressure into the 1/4 of supernatant volume, 12h is stood in 4 DEG C of refrigerators
After centrifuge, collect sediment be Hawthorn Polysaccharides；
(b1) flavone of hawthorn fruit detaches：Step (a1) is distilled to the macroreticular resin after water elution, with 5BV (resin volume) 50%
Ethanol elution, flow velocity 2ml/min collect eluent, are concentrated under reduced pressure, obtain flavone of hawthorn fruit crude extract；
(b2) flavone of hawthorn fruit purifies：The flavone of hawthorn fruit crude extract that step (b2) obtains is dissolved in ethyl alcohol, with 5 times of volumes
Ethyl alcohol phase is stayed after n-hexane extraction, and to remove the liposoluble substance in flavone of hawthorn fruit crude extract, hawthorn Huang is obtained after reduced pressure
Ketone extract.
Screening experiment
1, ultrasound and the condition of enzyme process ancillary technique
(1) influence of the various extracting conditions (being not added with Tween 80) to extracting flavonoids in hawthorn skin slag
According to experiment of single factor, volume fraction of ethanol, solid-liquid ratio, enzyme concentration, Extracting temperature, extraction time are sieved
Choosing, the optimised process that can obtain extracting flavonoids in hawthorn skin slag are (Fig. 1-5)：Volume fraction of ethanol is 20%, solid-liquid ratio 1/40
(g/ml), plus cellulose enzyme amount is 0.4%, 60 DEG C of ultrasonic extraction 50min, repeats to extract 2 times, flavones carries under the extraction conditions
It is 5.21% to take rate.
(2) influence of the various extracting conditions (being not added with Tween 80) to Polyose extraction in hawthorn skin slag
According to experiment of single factor, volume fraction of ethanol, solid-liquid ratio, enzyme concentration, Extracting temperature, extraction time are sieved
Choosing, the optimised process that can obtain Polyose extraction in hawthorn skin slag are (Fig. 6-10)：Volume fraction of ethanol is 20%, solid-liquid ratio 1/40
(g/ml), plus cellulose enzyme amount is 0.4%, 60 DEG C of ultrasonic extraction 50min, repeats to extract 2 times, polysaccharide carries under the extraction conditions
It is 9.18% to take rate.
(3) influence of the Tween 80 to flavones and Polyose extraction in hawthorn skin slag
Volume fraction of ethanol is 20%, solid-liquid ratio is 1/40 (g/ml), under 60 DEG C of ultrasound conditions, different enzyme concentrations are detected
After 0.2% Tween 80 is added in extracting solution, Tween 80 is to flavones in hawthorn skin slag and polysaccharide extract rate under different ultrasonic times
It influences (Figure 11-12)：When cellulase additive amount is 0.25%, and Tween 80 additive amount is 0.2%, (other extraction conditions are not
Become), 60 DEG C of ultrasonic extraction 40min, extracting flavonoids rate is 5.19%, and the recovery rate of polysaccharide is 9.22%.This explanation, is added tween
The adsorption efficiency after 80s for improving enzyme, can effectively reduce enzyme dosage and extraction time.
2, the screening of resin
According to the different characteristics of flavones and polysaccharide in hawthorn skin slag, the suction-operated of 7 kinds of resins is screened, macropore tree
Fat does not have adsorptivity, Hawthorn Polysaccharides that can be trapped in the gap of macroreticular resin Hawthorn Polysaccharides, when with distillation water elution
Hawthorn Polysaccharides can be eluted, and flavone of hawthorn fruit is attracted on macroreticular resin, to achieve the effect that detach Hawthorn Polysaccharides, knot
Fruit is shown in Table 1.Table 1 the result shows that, macroreticular resin AB-8 is higher than other trees to the adsorbance, adsorption rate and resolution factor of flavone of hawthorn fruit
Fat, therefore select AB-8 macroreticular resins separation flavone of hawthorn fruit and polysaccharide.
The selection result of table 1, resin
| Resin | Adsorbance (mg/g) | Adsorption rate (%) | Resolution factor (%) |
| LS300B | 25.25 | 51.64 | 87.27 |
| LS800 | 24.58 | 50.27 | 88.61 |
| LS803 | 25.62 | 52.39 | 87.27 |
| LX28 | 18.52 | 37.87 | 82.02 |
| LX68M | 19.44 | 39.76 | 78.01 |
| LX32 | 19.33 | 39.52 | 79.61 |
| AB-8 | 28.22 | 57.72 | 90.81 |
3, the screening of polysaccharide distilled water elution volume
When the distilled water flowed through is 3BV, the yield of polysaccharide reaches 98.7%, continues growing distilled water effluent volume,
Polysaccharide yield is almost unchanged, therefore distills best, the result is shown in Figure 13 when water elution dosage is 3BV.Hawthorn is detached to obtain under this condition
Purity of polysaccharide is 81.89%, yield 98.7%.
4, the screening of flavones eluate concentration
(table 2) is screened by the ethanol eluate to various concentration, wherein 50% ethanol eluate flavones resolution factor is up to
92.36%, continuing growing concentration of alcohol AB-8 influences less flavone of hawthorn fruit resolution factor, therefore selects 50% ethyl alcohol as elution
Liquid.Wherein flow velocity is 2ml/min, elution volume 3BV.Detach under this condition flavone of hawthorn fruit purity is 64.53%, yield is
91.58%.
Table 2, ethanol elution agent concentration screening result
| AB-8 | Resolution factor (%) |
| Water | 3.19 |
| 20% ethyl alcohol | 90.98 |
| 50% ethyl alcohol | 92.36 |
| 70% ethyl alcohol | 92.75 |
| 100% ethyl alcohol | 92.10 |
5, the screening of flavones ethanol eluate volume
When 50% ethyl alcohol flowed through is 3BV, the yield of flavones reaches 91.58%, continues growing ethanol eluate volume,
Flavones yield is almost unchanged, thus 50% ethanol elution dosage be 3BV when best, the result is shown in Figure 14.
6, the screening of flavones ethanol elution flow velocity
When the elution flow rate of 50% ethyl alcohol is 2ml/min, the resolution factor of flavones reaches 92.36%, improves elution flow rate,
Resolution factor decline, therefore 50% ethanol elution flow velocity be 2ml/min when best, the result is shown in Figure 15.
The foregoing is merely the embodiments that the present invention is best, and for those skilled in the art, the present invention can have
Various modifications and variations.All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on, should all
It is included within protection scope of the present invention.
Claims (8)
1. a kind of method of combined extraction separation flavones and polysaccharide in skin slag from hawthorn, which is characterized in that include the following steps：
(1) hawthorn skin slag pre-processes：
After the hawthorn skin slag filtering after hawthorn wine fermentation, then remove seed is dried in the shade naturally, the hawthorn skin slag after drying in the shade naturally is put
Enter and breaks into powder in pulverizer；
(2) ultrasound enzyme combined extracting：The hawthorn skin slag that step (1) is obtained, according to solid-liquid ratio 1:40, which are added 20% ethyl alcohol, mixes
Close mixing；Then 0.2~0.3% (m/m) cellulase and 0.2~0.25% (v/v) Tween 80 is added, is put into 60~70 DEG C of water
30~40min of ultrasonic extraction in bath repeats extraction 2 times, is centrifuged after extraction, obtain supernatant and precipitation, supernatant is mountain
Short, bristly hair or beard flavones and polysaccharide extraction liquid；
(3) it is concentrated by evaporation：Flavone of hawthorn fruit and polysaccharide extraction liquid that step (2) obtains are placed on Rotary Evaporators, are concentrated by evaporation
After pour into culture dish, be put into -80 DEG C of ultra low temperature freezers after freeze overnight, be put into freeze drier and be lyophilized, obtain hawthorn
Flavones polysaccharide crude extract；
(4) flavone of hawthorn fruit, the separation of polysaccharide, purifying：
(a1) Hawthorn Polysaccharides detach：The flavone of hawthorn fruit polysaccharide crude extract that will be obtained in step (3), after being dissolved with 20% ethyl alcohol, on
AB-8 macroporous resin adsorptions are collected eluent, are concentrated under reduced pressure into the 1/4 of hawthorn effluent volume, obtain mountain with distillation water elution
Short, bristly hair or beard polysaccharide concentrate；
(a2) Hawthorn Polysaccharides purify：5~6 times of volumes are added in the Hawthorn Polysaccharides concentrate that will be obtained in step (a1) while stirring
Ethyl alcohol, centrifuged after standing 12h in 4 DEG C of refrigerators, collect sediment, after then redissolving sediment with distilled water, be added 5~6
The savage reagents of times volume, shaking table concussion, centrifugation are repeated 3 times, merge supernatant, be concentrated under reduced pressure into the 1/4 of supernatant volume
5~6 times of volume ethanol dissolvings are added afterwards, are centrifuged after standing 12h in 4 DEG C of refrigerators, it is Hawthorn Polysaccharides to collect sediment；
(b1) flavone of hawthorn fruit detaches：Step (a1) is distilled to the macroreticular resin after water elution, with 50% ethanol elution, collects elution
Liquid is concentrated under reduced pressure, obtains flavone of hawthorn fruit crude extract；
(b2) flavone of hawthorn fruit purifies：The flavone of hawthorn fruit crude extract that step (b2) obtains is dissolved in ethyl alcohol, with 3~5 times of volumes
Ethyl alcohol phase is stayed after n-hexane extraction, and hawthorn flavone extract is obtained after reduced pressure.
2. the method for combined extraction separation flavones and polysaccharide in the skin slag according to claim 1 from hawthorn, which is characterized in that
Powder crosses 60 mesh sieve in step (1).
3. the method for combined extraction separation flavones and polysaccharide in the skin slag according to claim 1 from hawthorn, which is characterized in that
The enzyme activity of step (2) cellulase is 1000U/mg.
4. the method for combined extraction separation flavones and polysaccharide in the skin slag according to claim 1 from hawthorn, which is characterized in that
The solid-liquid ratio of flavone of hawthorn fruit polysaccharide crude extract and 20% ethyl alcohol is 1 in step (a1)：2.
5. the method for combined extraction separation flavones and polysaccharide in the skin slag according to claim 1 from hawthorn, which is characterized in that
Distilled water is 3-5BV, elution flow rate 2ml/min in step (a1).
6. the method for combined extraction separation flavones and polysaccharide in the skin slag according to claim 5 from hawthorn, which is characterized in that
Preferably, distilled water is 3BV in step (a1).
7. the method for combined extraction separation flavones and polysaccharide in the skin slag according to claim 1 from hawthorn, which is characterized in that
50% ethyl alcohol is 3-5BV, elution flow rate 2ml/min in step (b1).
8. the method for combined extraction separation flavones and polysaccharide in the skin slag according to claim 7 from hawthorn, which is characterized in that
Preferably, 50% ethyl alcohol is 3BV in step (b1).
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| CN110643655A (en) * | 2019-10-23 | 2020-01-03 | 河北科技大学 | Hawthorn flavone chelated sugar and preparation method and application thereof |
| CN112155192A (en) * | 2020-08-26 | 2021-01-01 | 芝麻官食品有限公司 | Preparation method of crystal hawthorn jelly |
| CN115448901A (en) * | 2022-09-27 | 2022-12-09 | 天津冠芳可乐饮料有限公司 | Extraction process of hawthorn flavone and preparation method of hawthorn flavone-containing beverage |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110643655A (en) * | 2019-10-23 | 2020-01-03 | 河北科技大学 | Hawthorn flavone chelated sugar and preparation method and application thereof |
| CN110643655B (en) * | 2019-10-23 | 2022-04-26 | 河北科技大学 | Hawthorn flavone chelated sugar and preparation method and application thereof |
| CN112155192A (en) * | 2020-08-26 | 2021-01-01 | 芝麻官食品有限公司 | Preparation method of crystal hawthorn jelly |
| CN115448901A (en) * | 2022-09-27 | 2022-12-09 | 天津冠芳可乐饮料有限公司 | Extraction process of hawthorn flavone and preparation method of hawthorn flavone-containing beverage |
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