CN110101728A - Polysaccharide from Portulaca oleracea and general flavone combined extraction method based on micelle medium processing - Google Patents
Polysaccharide from Portulaca oleracea and general flavone combined extraction method based on micelle medium processing Download PDFInfo
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Abstract
The present invention provides a kind of high holosaccharide of efficient purslane and general flavone combined extraction method, it is related to extracting solution and takes off albumen, depigmentation method, it can efficiently and rapidly combined extracting Polysaccharide from Portulaca oleracea and general flavone from purslane in large quantity, and using the albumen and colour component in specific micelle medium extraction single -step operation joint removing Polyose extraction step, the drawbacks of de- albumen, depigmentation need to be carried out independently when avoiding Polysaccharide from Portulaca oleracea purification in the prior art, extraction process is shortened, high-volume is suitble to extract.
Description
Technical field
The invention belongs to technical field of natural product extraction, and in particular to a kind of high holosaccharide of combined extracting purslane and total
The preparation method of flavone extract.
Background technique
Purslane (Portulaca oleracea L.) belongs to portulacaceous plant, and annual meat draft is widely distributed,
Yield is big, is common Chinese herbal medicine and wild dish, one of the wild plant of integration of drinking and medicinal herbs delimited for the Ministry of Public Health.As in tradition
Medicine, purslane have effects that clearing heat and detoxicating, cool blood detumescence, and modern medicine study also has antiatherosclerosis, antibacterial
Anti-inflammatory, hypoglycemic and other effects.
The main active substances being rich in purslane are polysaccharide and flavonoids, additionally containing other groups such as a large amount of protein
Point.Polysaccharide from Portulaca oleracea can promote proliferation of probiotics in enteron aisle, also have effects that reduce blood pressure, blood lipid, therefore effectively extract high-purity
The Polysaccharide from Portulaca oleracea and flavones of degree are of great significance to development and utilization purslane bioactive substance.
Structure is complicated and huge for polysaccharide molecule, and relative molecular mass is up to tens of thousands of hundreds of thousands, with glycosidic bond between structural units
It is connected.There is also the researchs of extensive Polysaccharide from Portulaca oleracea for the prior art.
CN107050041A, which discloses Polysaccharide from Portulaca oleracea, can improve learning and memory in rats ability and sky caused by lead exposure
Between resolution capability damage, and to dendritic spines density caused by lead exposure reduction be significantly improved effect, show Polysaccharide from Portulaca oleracea
There is significant preventive and therapeutic effect to Spatial memory impairment caused by lead.Patent CN103520108A discloses one kind and mentions
It is poor to solve Polysaccharide from Portulaca oleracea stability by preparation for the preparation and its application of the purslane polysaccharide liposome of high immunity,
It is influenced vulnerable to illumination, temperature, oxygen, pH etc., oxidizable decomposition and dissolubility and the low problem of bioavilability.
Enzymatic Extraction Polysaccharide from Portulaca oleracea is generallyd use in the prior art.Other extracting methods further include: overcritical using CO2
Extraction；It is extracted using impulse electric field low temperature；Microwave radiation exaraction etc..108542926 A of CN is related to a kind of purslane extract
Preparation method and pharmaceutical composition and its application, comprising the following steps: weigh purslane powder, Extraction solvent is added, in 30 in degreasing
DEG C~50 DEG C at extract, filter, collect extracting solution, be concentrated under reduced pressure, it is dry to get；Degreasing includes: to be placed in purslane powder
Then refluxing extraction in petroleum ether is volatilized petroleum ether completely in ventilation；Extraction solvent is alcohol solution, alcohol solution it is pure and strong
Degree is 0~75%；Extraction includes: ultrasonic extraction, refluxing extraction.But, polysaccharide complicated by the extract components that this method obtains
Purity is good low, and active material accumulation rate is poor.
CN107746435 A is related to a kind of Polysaccharide from Portulaca oleracea extract and its preparation method and application, specifically discloses one
The preparation method of kind Polysaccharide from Portulaca oleracea extract comprising following steps: 1) smashing it through filter for purslane, water is added, and add
Heat is filtered under diminished pressure after extraction completely to 40-60 DEG C, obtains filtrate I and filter residue I, and filtrate I is baked to thick, addition ethyl alcohol
It is suspended and is dispersed with ultrasonic wave afterwards, added the 0.5-1.5 times of pure water measured of ethanol consumption, be filtered under diminished pressure collection filter cake, dried
After obtain polyoses extract A；2) filter residue I being added in pure water, ultrasonic wave suspends, and enzyme is added and is digested to complete,
Be baked to it is thick, be added ethyl alcohol after with ultrasonic wave be suspended disperse, add the 0.5-1.5 times of pure water measured of ethanol consumption,
It is filtered under diminished pressure collection filter cake, polyoses extract B is obtained after drying；3) polyoses extract A and polyoses extract B mix
To Polysaccharide from Portulaca oleracea extract.
108148148 A of CN is related to a kind of Polysaccharide from Portulaca oleracea Extraction Processes of response phase method optimization, using Box-
Behnken response surface experimental design is analyzed according to Design-Expert 8.0.6 software, extracts purslane to pectase
Polysaccharide process in fresh juicing optimizes, and establishes the regression model that enzymatic isolation method extracts polysaccharide, obtained Enzymatic Extraction dent
Amaranth polysaccharide.
Although exist in the prior art it is a variety of extract Polysaccharide from Portulaca oleracea preparation methods, or purity it is not high or
It is complicated for operation, it is unfavorable for industrially being mass produced.Therefore, it is necessary to develop, a kind of extraction process is succinct, and extraction conditions are mild
Polysaccharide from Portulaca oleracea industrial extraction method.
Purslane leaf portion is rich in flavone compound.Flavone compound has multiple biological activities, such as antibacterial, disappears
It is scorching, disease-resistant become, decompression, it is clearing heat and detoxicating, etc., additionally it is anti-oxidant, in terms of it is also effective, be a kind of natural organic
Antioxidant.Such as 107595903 A of CN provides a kind of the extraction method of anti-inflammation components and answering for anti-inflammation components from purslane
With this method comprises the step of: purslane herb drying and crushing is obtained purslane powder；By purslane powder acidic ethanol
Ultrasonic wave extraction obtains ethanol extract；Ethanol extract is concentrated under reduced pressure into no alcohol taste, adds water to be configured to solution and crosses macropore tree
Rouge absorption, washes with water macroreticular resin after crude extract solution is excessively complete, then use pH value for 5~6 and acetonitrile: the volume ratio of n-butanol
Mixed liquor for 1:6-8 is eluant, eluent, affords eluent；Concentrated dry purslane anti-inflammation components again.
Although the prior art has Polysaccharide from Portulaca oleracea and flavones the report of extraction, two as purslane are main greatly
Active component is wanted, does not there is from purslane herb in large quantity while extracting high-purity Polysaccharide from Portulaca oleracea and flavones also so far
Report, and the extraction of one-component results in the waste of rest activity substance.Therefore, it is necessary on the basis of existing technology, mention
For a kind of efficiently succinct, combined technique of separation and purification Polysaccharide from Portulaca oleracea and general flavone for being easy to realize industrial production,
It solves to extract the waste of component present in polysaccharide and Flavonoid substances, polysaccharide and the separation of flavonoids difficulty from purslane and extract pure
Spend low problem.
Summary of the invention
In order to overcome prior art defect, the present invention provides a kind of high holosaccharide of efficient purslane and general flavone is combined and mentioned
Take method, can efficiently and rapidly combined extracting Polysaccharide from Portulaca oleracea and general flavone from purslane in large quantity, and use glue
Shu Jiezhi extracts the albumen and colour component in single -step operation joint removing Polyose extraction step, avoids dent in the prior art
The drawbacks of de- albumen, depigmentation need to be carried out independently when the high-purity purification of amaranth polysaccharide, shortens extraction process, and high-volume is suitble to extract.
Present invention process is to carry out albumen, pigment using the micelle medium extract liquor of special ratios in liquid of extracting polysaccharide
Removing processing, wherein micelle medium extract liquor is dissolved in heptane, 50% butanol in the mixed solvent by cetyl trimethylammonium bromide
It obtains, wherein the mass fraction of cetyl trimethylammonium bromide in the solution is 3-10%.
Technical solution of the present invention is as follows.
A kind of Polysaccharide from Portulaca oleracea and general flavone combined extraction method, comprising the following steps:
(1): ungrease treatment, condensed skimmed treatment fluid, drying defatted purslane are carried out to purslane；
(2): grease removal being carried out to ungrease treatment liquid, macroreticular resin gradient elution extracts Portulaca Total Flavone；
(3): extracting Thick many candies from degreasing purslane；
(4): preparing micelle medium extract liquor, salinization, the double de- processing of extraction are carried out to the Thick many candies solution after enzymatic hydrolysis；
(5): extract liquor carries out dialysis removal of impurities, removes small molecular weight impurity；
(6): being concentrated by evaporation, decrease temperature crystalline, to obtain high-purity Polysaccharide from Portulaca oleracea.
In addition, the invention also includes optional following steps:
(7): to the processing of gained Polysaccharide from Portulaca oleracea further alcohol precipitation, further increasing purity of polysaccharide.
Wherein, in step (1), the purslane can be dry purslane stem and leaf part, be also possible to containing clean root
The herb in portion.The ungrease treatment is handled using ethyl alcohol or petroleum ether.
Wherein, in step (2), the extract liquor after grease removal carries out gradient elution with large pore resin absorption column, collects general flavone
Component, and collect non-flavonoid component.
Wherein, in step (3), the Polyose extraction uses high temperature processing step, and extracting solution includes merging under above-mentioned elution
The step of non-flavonoid component come.
It wherein, include that enzymolysis processing is carried out to the protein impurities in Thick many candies using protease in the step in step (4)
The step of.The enzymatic hydrolysis is carried out according to conventional method in that art, and it is papain, neutral proteinase, alkalinity that protease, which is selected from,
At least one of protease.
Wherein, in step (5), the dialysis is to remove the small molecular weight impurities such as mono-and di-saccharides, sodium chloride.
Specifically, technical solution of the present invention is as follows.
A kind of Polysaccharide from Portulaca oleracea and general flavone combined extraction method, specifically include following steps.
Step (1): ungrease treatment is carried out to purslane:
S1: purslane herb smashed 40-60 mesh through constant temperature drying, obtained purslane powder；
S2: 85-99% ethyl alcohol leaching 3-12h is added in purslane powder in Soxhlet extractor, carries out Soxhlet at 50-60 DEG C and mentions
Take 1-5h.
Step (2): grease removal is carried out to ungrease treatment liquid, extracts Portulaca Total Flavone:
S1: the vacuum distillation of ungrease treatment liquid obtains general flavone primary extract concentrate；
S2: concentrate 65-75% ethyl alcohol is diluted, petroleum ether extraction, grease removal；
S3: the extract liquor after degreasing carries out gradient elution with large pore resin absorption column, successively uses deionized water, 50% second
Alcohol, 75% ethyl alcohol are eluted, and ultraviolet detection scans absorption peak in elution process, until eluent is colourless and occurs without absorption peak
When, stop elution, collects eluent containing flavones respectively and be free of flavones eluent；
S4: received eluent containing flavones is collected, and Portulaca Total Flavone class compound crystal is precipitated in evaporation.
Wherein, the ultraviolet detection method of eluate sample are as follows: take eluent in colorimetric cylinder, successively plus 5% sodium nitrite
Solution, 10% aluminum nitrate solution, 4% sodium hydroxide solution, shake up placement, are diluted with 75% ethyl alcohol.In ultraviolet specrophotometer
On in scanning absorption peak in 460~560nm wave-length coverage.
Step (3): Thick many candies are extracted from degreasing purslane:
S1: the purslane powder of ungrease treatment is mixed with deionized water, the high-temperature process 1-2h at 115-121 DEG C,
Then ultrasonic extraction 2-3 times, filtering, combined extract；
S2: initial concentration: extracting solution is merged with the eluent without flavones that above-mentioned resin elution step is collected,
It is concentrated by evaporation in evaporator to 50% volume hereinafter, obtaining initial concentration liquid；
S3: by initial concentration liquid high speed centrifugation, collecting supernatant, concentrated by rotary evaporation, and 95% ethyl alcohol of filtrate will be concentrated at 4 DEG C
Or dehydrated alcohol carries out alcohol precipitation, obtains Thick many candies precipitating, is successively eluted with dehydrated alcohol, acetone, drying is slightly more to get purslane
Sugar.
Step (4): preparing micelle medium extract liquor, carries out salinization, the double de- processing of extraction to the Thick many candies solution after enzymatic hydrolysis:
S1: micelle medium extract liquor is prepared:
Take the mixing that appropriate cetyl trimethylammonium bromide is dissolved in heptane, the butanol solution of 50% volume fraction forms molten
It in agent, mixes well, obtains micelle medium extract liquor, wherein the mass fraction of cetyl trimethylammonium bromide in the solution is
3-10%；
S2: enzymatic hydrolysis: being dissolved in distilled water for purslane Thick many candies, is configured to Thick many candies solution, and proteolytic enzyme enzymatic hydrolysis is added
2-4h is reacted, enzyme deactivation is cooled to room temperature；NaCl to mass fraction 3-5% is added to enzymolysis liquid, mixes, carries out salinization；
S3: micelle medium extract liquor being added into enzymolysis liquid, acutely concussion extraction 10-15min, then high speed centrifugation point
Layer, takes lower layer's solution, obtains the polysaccharide extraction liquid of the impurity such as de- albumen, pigment.
Wherein, micelle medium extract liquor additional amount is the 20-25% of enzymolysis liquid volume.
Step (5): extract liquor carries out dialysis removal of impurities, removes small molecular weight impurity:
Polysaccharide solution through double de- extraction processings is evaporated concentration, then in the bag filter of 1-5KD molecular cut off
Middle distilled water dialysis desalination and small molecular weight impurity, obtain the polysaccharide solution of dialysis treatment.
Step (6): being concentrated by evaporation to saturation crystallization, is cooled to 0-5 DEG C of crystallization, dehydrated alcohol washing, to obtain high-purity
Polysaccharide from Portulaca oleracea.
The invention also includes optional following steps:
Step (7): alcohol precipitation: being configured to solution with deionized water for gained polysaccharide, with 5-10 times of volume dehydrated alcohol alcohol precipitation
16-24h is filtered, and dehydrated alcohol washing is dried in vacuo to get high-purity Polysaccharide from Portulaca oleracea.
Specifically, step (1)-(2) operating process is as follows.
(S1) the clean purslane herb after removing dirt drying smashed 40-60 mesh, obtained in 55-60 DEG C of constant temperature drying
Purslane powder.
(S2) smashed purslane powder bag is weighed in filter paper, and 85- is added in gauze package in Soxhlet extractor
The alcohol dipping 3-12h of 99% volume carries out Soxhlet extraction 1-5h at 50-60 DEG C；Wherein, the feed liquid quality of purslane and solvent
Volume ratio is 1g:5-15ml；It is preferred that 1g:5-10ml.
Wherein, the preferably ethyl alcohol of 90% or more volume fraction is as Extraction solvent.
(S3) after Soxhlet extraction, filter packet is taken out, low temperature drying is dry, the purslane medicinal material powder after obtaining degreasing；
Extracting solution is filtered, filtrate decompression is distilled, alcohol solvent is recycled, obtains concentrate, i.e. general flavone primary extract solution.
(S4) general flavone first extract 65-75% ethyl alcohol is diluted, with petroleum ether extraction 3 times, each 10-15min, is carried out
Grease removal processing, while removing the fat-soluble pigments such as chlorophyll, carrotene.
(S5) extract liquor after extracting degreasing carries out gradient elution with large pore resin absorption column, successively uses deionized water, 50%
Ethyl alcohol, 75% ethyl alcohol are eluted, and eluent UV spectrophotometer measuring scanning Flavonoid substances is taken to inhale in elution process
Peak is received, when eluent is colourless and occurs without absorption peak, stops elution, eluent containing flavones is collected respectively and is washed without flavones
De- liquid；The eluent containing flavones of collection is converged, heating evaporation solvent is until be precipitated purslane flavonoid crystal.
Wherein, the ultraviolet detection method of eluate sample are as follows: take eluent 1mL in 25mL colorimetric cylinder, add 5% nitrous acid
Sodium solution 0.1mL shakes up and places 5min；Add 10% aluminum nitrate solution 0.1mL, shakes up and place 5min；Add 4% sodium hydroxide solution
0.5mL shakes up and places 5min, is then diluted to 15mL with 75% ethyl alcohol, shakes up and places 10min.On ultraviolet specrophotometer
In scanning absorption peak in 460~560nm wave-length coverage.
In the present invention, based on raw material, general flavone yield is up to 15mg/g or more, in terms of general flavone theoretical value, extracts
Rate about 80%.
In the present invention, high performance liquid chromatography measures 80% or more the purity of flavone compound in crystal, can directly use
In antibacterial, anti-inflammatory purposes.
Specifically, step (3)-(4) operating process is as follows.
(S1) by the purslane powder of above-mentioned steps ungrease treatment and deionized water by the ratio of solid-liquid ratio 1g:15-30ml
Example mixing, autoclave high temperature at 115-121 DEG C handle 1-2h, after high-temperature process at 90-100 DEG C ultrasonic extraction
2-3 times, each 30-60min, filtering, which merges, collects extracting solution.
(S2) extracting solution that above-mentioned steps obtain is mixed with what above-mentioned resin elution step was collected without flavones eluent
Merge, concentration is evaporated in thin film evaporator and removes excessive moisture, the vacuum degree of thin film evaporator is 0-0.05MP, outlet
Temperature is 55 DEG C -65 DEG C.
(S3) initial concentration liquid is centrifuged 20-30min with 8000-10000rpm, collects supernatant, concentrated by rotary evaporation to raw medicine
Concentration filtrate with the dehydrated alcohol of 5-10 times of volume is carried out alcohol precipitation 2 times at 4 DEG C, each 12-16h by 0.5-2 times of material amount；It closes
And Thick many candies precipitate, and are successively eluted with dehydrated alcohol, acetone, it is dry to get purslane Thick many candies.
(S4) it prepares micelle medium extract liquor: appropriate cetyl trimethylammonium bromide being taken to be dissolved in heptane, 50% butanol group
At in the mixed solvent, mix well, obtain micelle medium extract liquor, wherein cetyl trimethylammonium bromide is in the solution
Mass fraction is 3-10%；
Wherein, 50% butanol: the volume ratio of heptane is 1:3-5, preferably 1:3-4.
Wherein, preferably the mass fraction of cetyl trimethylammonium bromide in the solution is 3-5%.
(S5) purslane Thick many candies are dissolved in distilled water, are configured to Thick many candies solution, proteolytic enzyme is added in enzyme and fits item
Enzyme digestion reaction 2-4h is vibrated under part, then boiling water enzyme deactivation is cooled to room temperature；Wherein, Thick many candies liquid quality fraction 10-50mg/
Ml, preferably 10-30mg/ml；
Wherein it is preferred to press (3-5) × 10 when enzymatic hydrolysis4Papain is added into Thick many candies solution in the ratio of U/g polysaccharide
PH 7.0, hydrolase digests 2-3h at 55-60 DEG C.
(S6) NaCl to mass fraction 3-5% is added to enzymolysis liquid and carries out salinization processing, mix, then add into enzymolysis liquid
Enter the micelle medium extract liquor of 20-25% volume, acutely concussion extraction 10-15min, then high speed centrifugation is layered, organic layer is abandoned,
Lower layer's solution is taken, the polysaccharide extraction liquid of the impurity such as deproteination and pigment is obtained.
Specifically, step (5)-(7) operating process is as follows.
(S1) gained is evaporated concentration through the polysaccharide solution of double de- extraction processings, in the bag filter of 1-5KD interception
Middle distilled water 2-3 desalination of dialysis and small molecular weight impurity, obtain the polysaccharide solution of dialysis treatment；
(S2) polysaccharide solution of dialysis treatment is concentrated by evaporation in crystallization kettle, cooling is carried out after a large amount of crystallizations and continues to tie
Crystalline substance cools down 0-3 DEG C of crystallization temperature, and dehydrated alcohol washs after crystal is precipitated, and drying obtains Polysaccharide from Portulaca oleracea after purification.
Through calculating, polysaccharide extract rate is up to 8% or more, 95% or more purity.
Preferably, the present invention still further comprises alcohol precipitation processing step: by gained polysaccharide deionized water dissolving, using 5-10
Times volume dehydrated alcohol alcohol precipitation 16-24h is filtered, and dehydrated alcohol washing is dried in vacuo to get high-purity Polysaccharide from Portulaca oleracea, purity
97% or more.
In the present invention, albumen, pigment, polyoses content be can be used under conventional method in that art measurement.For example, benzene can be used
Phenol-sulfuric acid method surveys sugared content, and the specific method is as follows: with glucose as a standard product, accurately weighs the dry glucose to constant weight, adds
Water, which is settled in volumetric flask, obtains reference substance solution.Glucose control product solution is drawn in volumetric flask, water supplement obtains difference
The glucose dilution of concentration.It takes dilution that 5% phenol solution, the concentrated sulfuric acid is added respectively, stands, be cooled to room temperature after shaking up
Absorbance is surveyed at 485-490nm afterwards, draws standard curve.Polysaccharide from Portulaca oleracea solution is prepared, measures light absorption value according to the above method,
Total starches content is calculated according to standard curve.
The present invention has the following beneficial effects with respect to the prior art:
1) present invention develops the synchronous technique for extracting Polysaccharide from Portulaca oleracea and general flavone, avoids and extracts list in the prior art
Waste of one component for other components；It especially for the polysaccharide adulterated during to extracting flavonoids, is recycled, is had
Effect improves polysaccharide extract rate；
2) present invention has selected specific solvent burden ratio and CTAB content, can effectively go by micelle medium extraction process
The impurity such as isolating protein and pigment, avoid the destruction of polysaccharide structures, and effectively reduce the loss of polysaccharide, and gained polysaccharide is pure
Degree is high.
3) the synchronous technique for extracting general flavone and polysaccharide of the present invention, it is safe and efficient, energy saving, substantially reduce polysaccharide purification
Time adapts to the requirement being industrially mass produced without complicated purifier apparatus.
Specific embodiment
Below by specific embodiment, the present invention is described in detail, but these embodiments are not to of the invention
Protection scope constitutes any type of restriction.
Embodiment 1
(1) the clean purslane herb 1kg after removing dirt drying smashed 60 meshes, and obtained horse in 55 DEG C of constant temperature drying 3h
Bitterroot powder, is wrapped in filter paper, external application gauze package, 6h is impregnated with the ethyl alcohol 10L of 90% volume, in Soxhlet extractor
Soxhlet extraction 3h is carried out at 55 DEG C.
(2) after Soxhlet extraction, filter packet is taken out, powder is dried into drying, the purslane medicinal material powder after obtaining degreasing；
Filtrate decompression is distilled to recover alcohol solvent by micro-filtrate membrane filtration extracting solution, obtains the concentrate solution of general flavone primary extract about
510ml；Concentrate is diluted to 1L with 75% ethyl alcohol, with the concussion of 400ml petroleum ether extraction 3 times, each 15min, carries out grease removal
Processing, while removing the fat-soluble pigments such as chlorophyll, carrotene, the extract liquor after obtaining degreasing.
(3) extract liquor after extracting degreasing carries out gradient elution with D140 type large pore resin absorption column, successively with deionized water,
50% ethyl alcohol, 75% ethyl alcohol are eluted, and eluent UV spectrophotometer measuring is taken to scan flavonoids object in elution process
Matter absorption peak is simultaneously collected eluent, stops elution when eluent is colourless and occurs without absorption peak, collects obtain respectively
Eluent containing flavones and be free of flavones eluent；The eluent containing flavones of collection is converged, it is straight that solvent is evaporated in thin film evaporator
To precipitation purslane flavonoid crystal 16.1g, yield 16.1mg/g.
Wherein, the ultraviolet detection method of eluate sample are as follows: take eluent 1mL in 25mL colorimetric cylinder, add 5% nitrous acid
Sodium solution 0.1mL shakes up and places 5min；Add 10% aluminum nitrate solution 0.1mL, shakes up and place 5min；Add 4% sodium hydroxide solution
0.5mL shakes up and places 5min, is then diluted to 15mL with 75% ethyl alcohol, shakes up and places 10min.On ultraviolet specrophotometer
Scan absorption peak.
(4) the purslane powder of above-mentioned steps ungrease treatment and deionized water are mixed in the ratio of solid-liquid ratio 1g:20ml
It closes, handles 2h, ultrasonic extraction 3 times at 90 DEG C after high-temperature process, each 45min, mistake in 121 DEG C of autoclave high temperatures
Filter merges and collects extracting solution.
(5) extracting solution for obtaining above-mentioned steps merges with what above-mentioned steps (3) were collected without flavones eluent,
It is evaporated concentration in thin film evaporator, removes excessive moisture, the vacuum degree of thin film evaporator is 0.03MP, outlet temperature 55
DEG C, obtain initial concentration liquid about 3L.
(6) by initial concentration liquid with 8000rpm high speed centrifugation 30min, supernatant is collected, concentrated by rotary evaporation to 1L will at 4 DEG C
Filtrate is concentrated and carries out alcohol precipitation 2 times with the dehydrated alcohol of 8 times of volumes, each 16h；Obtained Thick many candies are precipitated and are merged, are successively used
Dehydrated alcohol, acetone elution, it is dry, obtain purslane Thick many candies；Meanwhile cetyl trimethylammonium bromide being taken to be dissolved in 50% fourth
In the double solvents of alcohol, heptane by the mixing of 1:3 volume ratio, mixes well, obtain cetyl trimethylammonium bromide mass fraction
It is spare for 5% micelle medium extract liquor.
(7) above-mentioned steps are obtained into purslane Thick many candies and is dissolved in the Thick many candies solution that distilled water is configured to 30mg/ml, by 5
×104Proteolytic enzyme papain is added in the ratio of U/g polysaccharide thereto, and enzyme digestion reaction 4h, boiling water are vibrated in the case where 7.0,55 DEG C of pH
Enzyme deactivation is cooled to room temperature.
(8) it is added into enzymolysis liquid and analyzes pure grade NaCl to the progress salinization processing of mass fraction 4%, mixed, be then added
The micelle medium extract liquor of 20% volume, acutely concussion extraction 10min carries out double de- extraction processings, and then high speed centrifugation is layered,
Organic layer is abandoned, the polysaccharide extraction liquid of the impurity such as deproteination and pigment is obtained.
(9) gained is subjected to thin film evaporation through the polysaccharide solution about 4L of double de- extraction processings and is concentrated into 1L or so, then existed
It is dialysed 3 desalinations and small molecular weight impurity in the bag filter that interception molecular weight is 3000 with distilled water, each dialysis time 12h,
Obtain the polysaccharide solution of dialysis treatment；
(10) polysaccharide solution of above-mentioned dialysis treatment is first concentrated by evaporation in crystallization kettle, a large amount of crystalline substances is precipitated wait start
Stopping heating when body, carry out decrease temperature crystalline, cools down 3 DEG C of crystallization temperature, crystal is washed after being precipitated with dehydrated alcohol, it dries,
Obtain Polysaccharide from Portulaca oleracea 90.2g after purification, polysaccharide extract rate 9.02%, purity 95.2%.
The fungistatic effect of Portulaca Total Flavone class extract manufactured in the present embodiment carries out as follows.
Test method: the flavonoids activity extract in the present embodiment being dissolved with dimethyl sulfoxide, micro-filtrate membrane filtration, preparation
At the initial concentration of 1mg/mL.Sterilizing test tubes 10 are taken, successively half times of dilution (i.e. 0.5mg/mL, 0.25mg/mL, with such
Push away), wherein a pipe is as blank control.So that culture solution and bacteria suspension total amount of liquid that every pipe is added are 1mL.Test tube is put into
Test tube observing bacterial growth situation is taken out in 37 DEG C of constant incubator cultures afterwards for 24 hours.If test tube liquid muddiness indicates no bacteriostasis；If
Test tube is limpid, indicates that bacterial growth is suppressed, can inhibit the medical fluid of the greatest dilution of bacterial growth, as the sample
Minimal inhibitory concentration.
The experimental results showed that extractive of general flavone obtained by the present embodiment is 0.25mg/ to Escherichia coli minimum inhibitory concentration
Ml is 0.12mg/ml to staphylococcus aureus minimum inhibitory concentration, has excellent fungistatic effect.
Embodiment 2
(1) the clean purslane herb 5kg after removing dirt drying, smashed 60 meshes, was wrapped in filter paper, external application yarn
Cloth package impregnates 6h with the ethyl alcohol 50L of 95% volume, carries out Soxhlet extraction 4h, raw material point three at 60 DEG C in Soxhlet extractor
It criticizes and extracts respectively；
(2) after Soxhlet extraction, filter packet is taken out, powder is dried drying by the purslane medicinal material powder after obtaining degreasing,
It is spare；Filtrate decompression is distilled to recover alcohol solvent by micro-filtrate membrane filtration extracting solution, obtains general flavone primary extract concentrate solution about
2L；Concentrate is diluted to 3.5L with 75% ethyl alcohol, carries out grease removal processing for 3 times with the concussion extraction of 2L petroleum ether, each 15min,
Extract liquor after obtaining degreasing.
(3) by the extract liquor concentrated by rotary evaporation after degreasing, gradient elution is carried out with large pore resin absorption column, successively uses deionization
Water, 50% ethyl alcohol, 75% ethyl alcohol are eluted, and eluent UV spectrophotometer measuring is taken to scan flavonoids in elution process
Material absorbing peak is simultaneously collected eluent, stops elution when eluent is colourless and occurs without absorption peak, collects respectively
To eluent containing flavones and be free of flavones eluent；The eluent containing flavonoids of collection is converged, is evaporated in thin film evaporator molten
Agent is until be precipitated purslane flavonoid crystal 84.3g, yield 16.8mg/g.
Wherein, the ultraviolet detection method of eluate sample is the same as described by embodiment 1.
(4) the purslane powder of above-mentioned ungrease treatment and deionized water are mixed in the ratio of solid-liquid ratio 1g:20ml,
118 DEG C of autoclave high temperature handles 2h, ultrasonic extraction 3 times at 90 DEG C after high-temperature process, and each 30min is filtered, and closes
And collect extracting solution.
(5) obtained extracting solution is merged with what above-mentioned steps (3) were collected without flavones eluent, in thin film evaporation
It is evaporated concentration in device, removes excessive moisture, the vacuum degree of thin film evaporator is 0.02MP, and outlet temperature is 60 DEG C, is obtained
Initial concentration liquid about 10L.
(6) initial concentration liquid is centrifuged in batches with 8000rpm high speed centrifugation 30min, collects supernatant, concentrated by rotary evaporation is extremely
Concentration filtrate with the dehydrated alcohol of 8 times of volumes is carried out alcohol precipitation in two batches at 4 DEG C by 3L, and alcohol precipitation 2 times, each 20h；By what is obtained
Thick many candies precipitating merges, and is successively eluted with dehydrated alcohol, acetone, dry, obtains purslane Thick many candies；Meanwhile taking cetyl
Trimethylammonium bromide is dissolved in the double solvents that 50% butanol, heptane are mixed by 1:4 volume ratio, is mixed well, is obtained hexadecane
The micelle medium extract liquor that base trimethylammonium bromide mass fraction is 4%, it is spare.
(7) above-mentioned steps are obtained into purslane Thick many candies and is dissolved in the Thick many candies solution that distilled water is configured to 50mg/ml, by 3
×104Proteolytic enzyme papain is added in the ratio of U/g polysaccharide thereto, and enzyme digestion reaction 4h, boiling water are vibrated in the case where 7.0,55 DEG C of pH
Enzyme deactivation is cooled to room temperature.
(8) it is added into enzymolysis liquid and analyzes pure grade NaCl to the progress salinization processing of mass fraction 3%, mixed, be then added
The micelle medium extract liquor of 25% volume, acutely concussion extraction 15min carries out double de- extraction processings, and then high speed centrifugation is layered,
Organic layer is abandoned, the polysaccharide extraction liquid of the impurity such as deproteination and pigment is obtained.
(9) gained is subjected in thin film evaporator thin film evaporation through the polysaccharide solution of double de- extraction processings and is concentrated into the left side 3L
Then the right side is dialysed in the bag filter that interception molecular weight is 5000 with distilled water 3 desalinations of dialysis and small molecular weight impurity every time
Time 16h obtains the polysaccharide solution of dialysis treatment.
(10) polysaccharide solution of above-mentioned dialysis treatment is first concentrated by evaporation in crystallization kettle, a large amount of crystalline substances is precipitated wait start
Stopping heating when body, carry out decrease temperature crystalline, cools down 1 DEG C of crystallization temperature, crystal is washed after being precipitated with dehydrated alcohol, it dries,
Obtain Polysaccharide from Portulaca oleracea 466.1g after purification, polysaccharide extract rate 9.32%, purity 95.5%.
(11) alcohol precipitation is handled: after at room temperature dissolving polysaccharide obtained by step (10) just with deionized water, with 10 times of volumes
Dehydrated alcohol alcohol precipitation for 24 hours, filters, dehydrated alcohol washing, vacuum drying, and the high-purity Polysaccharide from Portulaca oleracea being further purified is more
Sugared content 97.6%.
Comparative example 1
The micelle medium extract liquor of double de- extracting operations in 1 step of embodiment (8) is replaced with into Sevage reagent (chlorine
It is imitative: n-butanol=4.5:1), and handled without salinization；Remaining step and basic operation condition are constant, obtain comparative example
1。
The comparative example obtains Polysaccharide from Portulaca oleracea extract 87.3g, purity of polysaccharide 78.2%, containing compared with polyprotein and color
Plain impurity.
Comparative example 2
On the basis of comparative example 1, by Sevage reagent (chloroform: n-butanol=4.5:1) extracting operation repeat into
Twice, remaining step and basic operation condition are constant, obtain comparative example 2 for row.
The comparative example obtains Polysaccharide from Portulaca oleracea extract 85.6g, purity of polysaccharide 86.7%, still containing compared with polyprotein and
Pigment impurity.
Comparative example 3
On the basis of comparative example 1, what nonjoinder step (3) was collected in step (5) is free of flavones eluent group
Point, remaining step and operating condition are constant, obtain comparative example 3.
The comparative example obtains Polysaccharide from Portulaca oleracea extract 71.2g, polysaccharide extract rate 7.12%.
Above embodiments not limit technical solution of the present invention, and those skilled in the art can be to aforementioned each reality
It applies technical solution documented by example to modify, these modify or replace the range without departing from technical solution of the present invention.
Claims (9)
1. a kind of Polysaccharide from Portulaca oleracea and general flavone combined extraction method comprising extracted in Polyose extraction operation using micelle medium
The step of taking processing, wherein micelle medium extraction solution by cetyl trimethylammonium bromide is dissolved in heptane, volume fraction is
The in the mixed solvent of 50% butanol aqueous solution composition obtains；It is characterized in that, the described method comprises the following steps:
Step (1): ungrease treatment is carried out to purslane；
Step (2): carrying out grease removal to ungrease treatment liquid, and it is always yellow to carry out gradient elution extraction purslane with large pore resin absorption column
Ketone collects eluent containing flavones respectively and is free of flavones eluent；
Step (3): Thick many candies are extracted from degreasing purslane, process is as follows:
S1: the purslane powder of ungrease treatment is mixed with deionized water, the high-temperature process 1-2h at 115-121 DEG C, then
Ultrasonic extraction 2-3 times, filtering, combined extract；
S2: initial concentration: extracting solution is merged with the eluent without flavones that above-mentioned resin elution step is collected, is being steamed
It sends out in device and is concentrated by evaporation to 50% volume hereinafter, obtaining initial concentration liquid；
S3: by initial concentration liquid high speed centrifugation, collecting supernatant, concentrated by rotary evaporation, at 4 DEG C will concentration filtrate with 95% ethyl alcohol or nothing
Water-ethanol carries out alcohol precipitation, obtains Thick many candies precipitating, and successively with dehydrated alcohol, acetone elution precipitating, it is slightly more to be dried to obtain purslane
Sugar；
Step (4): preparing micelle medium extract liquor, carries out salinization, the double de- processing of extraction, process to the Thick many candies solution after enzymatic hydrolysis
It is as follows:
S1: micelle medium extract liquor is prepared:
It takes appropriate cetyl trimethylammonium bromide to be dissolved in heptane, 50% butanol in the mixed solvent, mixes well, obtain micella Jie
Matter extract liquor, wherein the mass fraction of cetyl trimethylammonium bromide in the solution is 3-10%；
Preferably, cetyl trimethylammonium bromide mass fraction is 3-5%；
S2: being dissolved in distilled water for purslane Thick many candies and be configured to Thick many candies solution, and proteolytic enzyme is added and carries out enzymatic hydrolysis 2-4h, goes out
Enzyme is cooled to room temperature；NaCl to mass fraction 3-5% is added to enzymolysis liquid, mixes, carries out salinization；
S3: micelle medium extract liquor being added into enzymolysis liquid, acutely concussion extraction 10-15min, and then high speed centrifugation is layered, and is obtained
To the polysaccharide extraction liquid of the impurity such as de- albumen, pigment；
Step (5): extract liquor carries out dialysis removal of impurities, removes small molecular weight impurity:
The above-mentioned polysaccharide solution by double de- extraction processings is evaporated concentration, is then the saturating of 1-5 KD in molecular cut off
It analyses in bag with distilled water dialysis desalination and small molecular weight impurity, obtains the polysaccharide solution of dialysis treatment；
Step (6): it is concentrated by evaporation crystallization, 0-5 DEG C is cooled to and continues to crystallize, crystal is washed with dehydrated alcohol, to obtain pure
Change Polysaccharide from Portulaca oleracea.
2. the method according to claim 1, wherein further including to the processing of gained Polysaccharide from Portulaca oleracea further alcohol precipitation
The step of, process are as follows: by gained polysaccharide deionized water dissolving, with 5-10 times of volume dehydrated alcohol or 95% ethyl alcohol alcohol precipitation 16-
For 24 hours, it filters, dehydrated alcohol washing is dried in vacuo to get high-purity Polysaccharide from Portulaca oleracea.
3. the method according to claim 1, wherein detailed process is as follows for the step (1):
S1: purslane herb smashed 40-60 mesh through constant temperature drying, obtained purslane powder；
S2: 85-99% ethyl alcohol leaching 3-12h is added in purslane powder in Soxhlet extractor, carries out Soxhlet extraction 1- at 50-60 DEG C
5h；
Wherein, the feed liquid mass volume ratio of purslane and solvent is 1g:5-15ml；It is preferred that 1g:5-10ml.
4. the method according to claim 1, wherein detailed process is as follows for the step (2):
S1: the vacuum distillation of ungrease treatment liquid obtains general flavone primary extract concentrate；
S2: concentrate 65-75% ethyl alcohol is diluted, petroleum ether is used to extract grease removal as extractant；
S3: the extract liquor after degreasing carries out gradient elution with large pore resin absorption column, successively with deionized water, 50% ethyl alcohol,
75% ethyl alcohol is eluted, and ultraviolet detection scans absorption peak in elution process, when eluent is colourless and occurs without absorption peak,
Stop elution, collect eluent containing flavones respectively and is free of flavones eluent；
S4: received eluent containing flavones is collected, and Portulaca Total Flavone class compound crystal is precipitated in evaporation.
5. the method according to claim 1, wherein micelle medium extract liquor additional amount is enzymatic hydrolysis in step (4)
The 20-25% of liquid product.
6. the method according to claim 1, wherein step (4) prepares the mixed solvent of micelle medium extract liquor
In 50% butanol: the volume ratio of heptane be 1:3-5, preferably 1:3-4.
7. a kind of extracting method of the high holosaccharide of purslane, which is characterized in that extracted in Polyose extraction operation using micelle medium
Processing is taken, the mixing that wherein micelle medium extract liquor by cetyl trimethylammonium bromide is dissolved in heptane, 50% butanol forms is molten
It is obtained in agent, wherein the mass fraction of cetyl trimethylammonium bromide in the solution is 3-10%；And include the following steps:
(1) Thick many candies are extracted from degreasing purslane；
(2) micelle medium extract liquor is prepared, salinization, the double de- processing of extraction are carried out to the Thick many candies solution after enzymatic hydrolysis；
(3) extract liquor carries out dialysis removal of impurities, removes small molecular weight impurity；
(4) it is concentrated by evaporation, decrease temperature crystalline obtains Polysaccharide from Portulaca oleracea.
8. the method according to the description of claim 7 is characterized in that further including the further extraction purification purslane from degreaser
General flavone.
9. the Polysaccharide from Portulaca oleracea extract that method described in -7 is prepared according to claim 1.
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| CN110592167A (en) * | 2019-08-27 | 2019-12-20 | 杨凌萃健生物工程技术有限公司 | Purslane polypeptide extract and preparation method thereof |
| CN110669151A (en) * | 2019-11-06 | 2020-01-10 | 河南理工大学 | Purslane polysaccharide extract, preparation method and application thereof |
| CN112704650A (en) * | 2020-12-24 | 2021-04-27 | 江苏瑞霆生物科技有限公司 | Preparation method of purslane extract, product and application thereof |
| CN113265874A (en) * | 2021-05-15 | 2021-08-17 | 浙江世纪晨星纤维科技有限公司 | Novel high-antibacterial anti-ultraviolet polyester-viscose yarn |
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| CN110592167A (en) * | 2019-08-27 | 2019-12-20 | 杨凌萃健生物工程技术有限公司 | Purslane polypeptide extract and preparation method thereof |
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| CN113265874A (en) * | 2021-05-15 | 2021-08-17 | 浙江世纪晨星纤维科技有限公司 | Novel high-antibacterial anti-ultraviolet polyester-viscose yarn |
| CN113265874B (en) * | 2021-05-15 | 2023-02-28 | 浙江世纪晨星纤维科技有限公司 | Novel high-antibacterial anti-ultraviolet polyester-viscose yarn |
| CN118649109A (en) * | 2024-08-20 | 2024-09-17 | 广州华淼生物科技研究院有限公司 | Extraction method of anti-drop composition, anti-drop composition and application |
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