CN110101728B - Combined extraction method of purslane polysaccharide and total flavonoids based on micelle medium treatment - Google Patents
Combined extraction method of purslane polysaccharide and total flavonoids based on micelle medium treatment Download PDFInfo
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Abstract
The invention provides a high-efficiency combined extraction method of high-purity purslane polysaccharide and total flavonoids, and relates to a method for deproteinizing and depigmenting an extract, which can efficiently and quickly combine and extract purslane polysaccharide and total flavonoids from purslane in large batch, and adopts a specific micelle medium to extract protein and pigment components in a single-step operation combined removal polysaccharide extraction step, so that the defect that the deproteinization and the pigment removal are required to be carried out independently in the purification of purslane polysaccharide in the prior art is avoided, the extraction process is shortened, and the method is suitable for large-batch extraction.
Description
Technical Field
The invention belongs to the technical field of natural product extraction, and particularly relates to a preparation method for extracting purslane high-purity polysaccharide and total flavonoid extract in a combined manner.
Background
Purslane (Portulaca oleracea L.) belongs to Portulacaceae, has annual meat quality and wide distribution and high yield, is common Chinese herbal medicine and wild vegetable, and is one of medicinal and edible wild plants defined by Ministry of health. As a traditional Chinese medicine, the purslane has the effects of clearing away heat and toxic materials, cooling blood and relieving swelling, and also has the effects of resisting atherosclerosis, resisting bacteria and diminishing inflammation, reducing blood sugar and the like in modern medical research.
The purslane is rich in polysaccharide and flavonoid as main active substances, and also contains a large amount of other components such as protein and the like. The purslane polysaccharide can promote the proliferation of probiotics in intestinal tracts and has the effects of reducing blood pressure and blood fat, so that the effective extraction of high-purity purslane polysaccharide and flavone has important significance for developing and utilizing purslane bioactive substances.
The molecular structure of the polysaccharide is complex and huge, the relative molecular mass is as high as tens of thousands and hundreds of thousands, and the structural units are connected by glycosidic bonds. Extensive studies of purslane polysaccharides also exist in the prior art.
CN107050041A discloses that purslane polysaccharide can improve the learning and memory ability and spatial resolution ability damage of rats caused by lead exposure, and has a significant improvement effect on the density reduction of dendritic spines caused by lead exposure, which indicates that purslane polysaccharide has a significant prevention and treatment effect on the spatial learning and memory ability damage caused by lead. Patent CN103520108A discloses preparation and application of purslane polysaccharide liposome for improving immunity, and solves the problems of poor stability, susceptibility to illumination, temperature, oxygen, pH and the like, susceptibility to oxidative decomposition, and low solubility and bioavailability of purslane polysaccharide through a preparation.
In the prior art, the purslane polysaccharide is generally extracted by adopting an enzyme method. Other extraction methods further include: supercritical CO2 extraction; low-temperature extraction by adopting a pulse electric field; microwave-assisted extraction, and the like. CN 108542926A relates to a preparation method of purslane extractive, a pharmaceutical composition and application thereof, comprising the following steps: weighing purslane powder, degreasing, adding an extraction solvent, extracting at 30-50 ℃, filtering, collecting an extracting solution, concentrating under reduced pressure, and drying to obtain the purslane powder; degreasing comprises the following steps: placing herba Portulacae powder in petroleum ether, extracting under reflux, and volatilizing petroleum ether completely at ventilation position; the extraction solvent is an alcohol-water solution, and the alcohol concentration of the alcohol-water solution is 0-75%; the extraction comprises the following steps: ultrasonic extraction and reflux extraction. However, the extracts obtained by this method are complex in composition, good in purity of polysaccharides and poor in enrichment of active substances.
CN 107746435A relates to a purslane polysaccharide extract and a preparation method and application thereof, in particular to a preparation method of the purslane polysaccharide extract, which comprises the following steps: 1) crushing purslane, filtering, adding water, heating to 40-60 ℃, leaching completely, filtering under reduced pressure to obtain filtrate I and filter residue I, baking the filtrate I to be viscous, adding ethanol, suspending and dispersing by ultrasonic waves, adding purified water with the amount of 0.5-1.5 times that of the ethanol, filtering under reduced pressure to collect a filter cake, and drying to obtain a polysaccharide extract A; 2) adding the filter residue I into purified water, suspending by ultrasonic waves, adding enzyme for enzymolysis completely, baking to be viscous, adding ethanol, suspending and dispersing by ultrasonic waves, adding purified water with the amount of 0.5-1.5 times of that of the ethanol, filtering under reduced pressure, collecting a filter cake, and drying to obtain a polysaccharide extract B; 3) mixing the polysaccharide extract A and the polysaccharide extract B to obtain the purslane polysaccharide extract.
CN 108148148A relates to a purslane polysaccharide enzymatic extraction process optimized by a response surface method, which adopts Box-Behnken response surface experimental Design and carries out analysis according to Design-Expert 8.0.6 software to optimize the process of extracting polysaccharide from fresh purslane juice by pectinase, establish a regression model of extracting polysaccharide by an enzymatic hydrolysis method and obtain purslane polysaccharide extracted by the enzymatic method.
Although various preparation methods for extracting purslane polysaccharide exist in the prior art, the purity is not high, or the operation is complex, so that the method is not beneficial to industrial large-scale production. Therefore, there is a need to develop an industrial extraction method of purslane polysaccharide, which has a simple extraction process and mild extraction conditions.
The leaf part of herba Portulacae is rich in flavonoids. The flavonoid compound has various biological activities, such as antibiosis, antiphlogosis, anti-pathological changes, blood pressure reduction, heat clearing and detoxifying, and the like, and also has effective effects in the aspects of antioxidation, anticancer and the like, and is a natural organic antioxidant. For example, CN 107595903 a provides a method for extracting anti-inflammatory components from purslane and the application of the anti-inflammatory components, the method comprises the following steps: drying and pulverizing whole purslane herb to obtain purslane powder; ultrasonically extracting purslane powder with acidic ethanol to obtain an ethanol extract; concentrating the ethanol extract under reduced pressure until no alcohol smell exists, adding water to prepare a solution, adsorbing the solution by macroporous resin, washing the macroporous resin by water after the crude extract solution passes through, and then adopting a solvent with the pH value of 5-6 and the volume ratio of acetonitrile to n-butanol of 1: 6-8, eluting to obtain eluent; concentrating and drying to obtain the anti-inflammatory component of purslane.
Although the prior art reports about extraction of both purslane polysaccharide and flavone, as two major active components of purslane, there has been no report of simultaneous extraction of high-purity purslane polysaccharide and flavone from whole purslane herb in large batch, and extraction of a single component results in waste of the remaining active substances. Therefore, on the basis of the prior art, a combined process method for separating and purifying purslane polysaccharide and total flavonoids, which is efficient and simple and is easy to realize industrial production, is needed to be provided, and the problems of component waste, difficult separation of polysaccharide and flavonoids and low extraction purity existing in the extraction of polysaccharide and flavonoids from purslane are solved.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides an efficient combined extraction method of high-purity purslane polysaccharide and total flavonoids, which can efficiently and quickly extract purslane polysaccharide and total flavonoids from purslane in a combined manner in large batch, and adopts micellar medium extraction and single-step operation to remove protein and pigment components in the polysaccharide extraction step in a combined manner, so that the defect that protein and pigment removal need to be carried out independently during high-purity purification of purslane polysaccharide in the prior art is overcome, the extraction process is shortened, and the method is suitable for large-batch extraction.
The process comprises the step of removing protein and pigment from a micelle medium extraction liquid in a polysaccharide crude extract according to a specific proportion, wherein the micelle medium extraction liquid is obtained by dissolving cetyl trimethyl ammonium bromide in a mixed solvent of heptane and 50% butanol, and the mass fraction of the cetyl trimethyl ammonium bromide in the solution is 3-10%.
The technical scheme of the invention is as follows.
A method for extracting purslane polysaccharide and total flavonoids in a combined way comprises the following steps:
(1): defatting herba Portulacae, concentrating defatting solution, and drying defatted herba Portulacae;
(2): degreasing the degreasing treatment liquid, performing gradient elution by using macroporous resin, and extracting total flavonoids of the purslane;
(3): extracting crude polysaccharide from defatted purslane;
(4): preparing micelle medium extraction liquid, and performing salinization and extraction double-removal treatment on the crude polysaccharide solution after enzymolysis;
(5): dialyzing the extract to remove impurities and removing small molecular impurities;
(6): evaporating, concentrating, cooling and crystallizing to obtain high-purity purslane polysaccharide.
In addition, the present invention also includes the optional steps of:
(7): the obtained purslane polysaccharide is further subjected to alcohol precipitation treatment, so that the purity of the polysaccharide is further improved.
In the step (1), the purslane can be dry purslane stem and leaf parts, or whole plants containing clean roots. The degreasing treatment adopts ethanol or petroleum ether for treatment.
Wherein, in the step (2), the extraction liquid after degreasing is subjected to gradient elution by a macroporous adsorption resin column, total flavone components are collected, and non-flavone components are collected.
Wherein, in the step (3), the polysaccharide extraction adopts a high-temperature treatment step, and the extracting solution comprises a step of combining the eluted non-flavone components.
Wherein, in the step (4), the step comprises the step of carrying out enzymolysis treatment on protein impurities in the crude polysaccharide by using protease. The enzymolysis is carried out according to the conventional method in the field, and the protease is at least one selected from papain, neutral protease and alkaline protease.
Wherein, in the step (5), the dialysis is carried out to remove micromolecular impurities such as monosaccharide, disaccharide, sodium chloride and the like.
Specifically, the technical scheme of the invention is as follows.
A combined extraction method of purslane polysaccharide and total flavonoids specifically comprises the following steps.
Step (1): carrying out degreasing treatment on purslane:
s1: drying the whole purslane at constant temperature, and crushing and sieving by a 40-60-mesh sieve to obtain purslane powder;
s2: adding 85-99% ethanol into herba Portulacae powder, soaking for 3-12 hr, and performing Soxhlet extraction at 50-60 deg.C for 1-5 hr.
Step (2): degreasing the degreasing treatment liquid, and extracting purslane total flavonoids:
s1: distilling the degreasing treatment solution under reduced pressure to obtain a concentrated solution of a primary extract of the total flavonoids;
s2: diluting the concentrated solution with 65-75% ethanol, extracting with petroleum ether, and removing fat;
s3: gradient eluting the degreased extract with macroporous adsorbent resin column, sequentially eluting with deionized water, 50% ethanol and 75% ethanol, scanning absorption peak by ultraviolet detection during elution, stopping elution until the eluate is colorless and no absorption peak appears, and respectively collecting eluate containing flavone and eluate without flavone;
s4: collecting the received eluent containing flavone, and evaporating to separate out purslane total flavonoid compound crystals.
Wherein, the ultraviolet detection method of the eluent sample comprises the following steps: and (3) putting the eluent into a colorimetric tube, sequentially adding a 5% sodium nitrite solution, a 10% aluminum nitrate solution and a 4% sodium hydroxide solution, shaking uniformly, standing, and diluting with 75% ethanol. Scanning an absorption peak in a wavelength range of 460-560 nm on an ultraviolet spectrophotometer.
And (3): extraction of crude polysaccharide from defatted purslane:
s1: mixing the degreased purslane powder with deionized water, treating at the high temperature of 115-121 ℃ for 1-2h, then carrying out ultrasonic extraction for 2-3 times, filtering, and combining the extracting solutions;
s2: primary concentration: mixing the extract with the eluate containing no flavone, and concentrating by evaporation in an evaporator to below 50% volume to obtain primary concentrated solution;
s3: centrifuging the primary concentrated solution at high speed, collecting supernatant, concentrating by rotary evaporation, precipitating the concentrated filtrate with 95% ethanol or anhydrous ethanol at 4 deg.C to obtain crude polysaccharide precipitate, sequentially leaching with anhydrous ethanol and acetone, and drying to obtain herba Portulacae crude polysaccharide.
And (4): preparing micelle medium extraction liquid, and performing salinization and extraction double-removal treatment on the crude polysaccharide solution after enzymolysis:
s1: preparing micelle medium extraction liquid:
dissolving a proper amount of cetyl trimethyl ammonium bromide in a mixed solvent consisting of heptane and 50% volume fraction butanol solution, and fully and uniformly mixing to obtain micelle medium extract, wherein the mass fraction of the cetyl trimethyl ammonium bromide in the solution is 3-10%;
s2: enzymolysis: dissolving crude purslane polysaccharide in distilled water to prepare a crude polysaccharide solution, adding proteolytic enzyme for enzymolysis reaction for 2-4h, inactivating enzyme, and cooling to room temperature; adding NaCl into the enzymolysis liquid until the mass fraction is 3-5%, uniformly mixing, and salinizing;
s3: adding micelle medium extract into the enzymolysis solution, extracting for 10-15min under intense shaking, centrifuging at high speed for layering, and taking the lower layer solution to obtain polysaccharide extract without protein, pigment and other impurities.
Wherein the addition amount of the micelle medium extraction liquid is 20-25% of the volume of the enzymolysis liquid.
And (5): dialyzing the extract to remove impurities and removing small molecular impurities:
evaporating and concentrating the polysaccharide solution subjected to double extraction, and dialyzing with distilled water in a dialysis bag with molecular weight cut-off of 1-5KD to remove salt and small molecular impurities to obtain the polysaccharide solution subjected to dialysis.
And (6): evaporating and concentrating to saturation crystallization, cooling to 0-5 ℃ for crystallization, and washing with absolute ethyl alcohol to obtain the high-purity purslane polysaccharide.
The present invention also includes the optional steps of:
and (7): alcohol precipitation: preparing the obtained polysaccharide into a solution by using deionized water, precipitating with 5-10 times of volume of absolute ethyl alcohol for 16-24h, filtering, washing with absolute ethyl alcohol, and drying in vacuum to obtain the high-purity purslane polysaccharide.
Specifically, the operational flow of steps (1) to (2) is as follows.
(S1) taking the clean purslane whole plant after dust removal and drying, drying at the constant temperature of 55-60 ℃, and crushing through a 40-60-mesh sieve to obtain purslane powder.
(S2) weighing the crushed purslane powder, wrapping the purslane powder in filter paper, wrapping the purslane powder with gauze, adding 85-99% by volume of ethanol into a Soxhlet extractor, soaking for 3-12h, and carrying out Soxhlet extraction at 50-60 ℃ for 1-5 h; wherein the mass volume ratio of the purslane to the solvent is 1g:5-15 ml; preferably 1g:5-10 ml.
Among them, ethanol with a volume fraction of 90% or more is preferable as the extraction solvent.
(S3) after the Soxhlet extraction is finished, taking out the filter bag, and drying at low temperature to obtain degreased purslane medicinal material powder; filtering the extractive solution, distilling the filtrate under reduced pressure, and recovering ethanol solvent to obtain concentrated solution, i.e. primary extract solution of total flavonoids.
(S4) diluting the primary extract with 65-75% ethanol, extracting with petroleum ether for 3 times (10-15 min each time), and removing fat-soluble pigment such as chlorophyll and carotene.
(S5) gradient eluting the degreased extract with macroporous adsorption resin column, sequentially eluting with deionized water, 50% ethanol and 75% ethanol, detecting and scanning flavonoid absorption peak of the eluent with an ultraviolet spectrophotometer during the elution process, stopping the elution until the eluent is colorless and no absorption peak appears, and respectively collecting the eluent containing flavone and the eluent not containing flavone; converging the collected flavone-containing eluents, and heating to evaporate the solvent until the purslane flavonoid compound crystal is separated out.
Wherein, the ultraviolet detection method of the eluent sample comprises the following steps: taking 1mL of eluent to a 25mL colorimetric tube, adding 0.1mL of 5% sodium nitrite solution, shaking uniformly and standing for 5 min; adding 0.1mL of 10% aluminum nitrate solution, shaking uniformly and standing for 5 min; adding 0.5mL of 4% sodium hydroxide solution, shaking, standing for 5min, diluting with 75% ethanol to 15mL, shaking, and standing for 10 min. Scanning an absorption peak in a wavelength range of 460-560 nm on an ultraviolet spectrophotometer.
In the invention, based on the raw materials, the yield of the total flavone can reach more than 15mg/g, and the extraction rate is about 80 percent based on the theoretical value of the total flavone.
In the invention, the purity of the flavonoid compound in the crystal is over 80 percent measured by high performance liquid chromatography, and the flavonoid compound can be directly used for bacteriostasis and anti-inflammation.
Specifically, the operation flow of steps (3) to (4) is as follows.
(S1) mixing the purslane powder degreased in the step with deionized water according to the ratio of 1g to 15-30ml, carrying out high-temperature treatment in a high-pressure reaction kettle at the temperature of 115-121 ℃ for 1-2h, carrying out ultrasonic extraction at the temperature of 90-100 ℃ for 2-3 times after the high-temperature treatment, each time for 30-60min, filtering, combining and collecting the extracting solution.
(S2) mixing the extract obtained in the step with the eluent which is collected in the resin elution step and does not contain flavone, evaporating and concentrating in a thin film evaporator to remove excessive water, wherein the vacuum degree of the thin film evaporator is 0-0.05MP, and the outlet temperature is 55-65 ℃.
(S3) centrifuging the primary concentrated solution at 8000-; mixing the crude polysaccharide precipitates, sequentially leaching with absolute ethyl alcohol and acetone, and drying to obtain the purslane crude polysaccharide.
(S4) preparing a micelle medium extraction liquid: dissolving a proper amount of cetyl trimethyl ammonium bromide in a mixed solvent consisting of heptane and 50% butanol, and fully and uniformly mixing to obtain a micelle medium extract, wherein the mass fraction of the cetyl trimethyl ammonium bromide in the solution is 3-10%;
wherein, 50% butanol: the volume ratio of heptane is 1:3-5, preferably 1: 3-4.
Among them, the mass fraction of cetyltrimethylammonium bromide in the solution is preferably 3 to 5%.
(S5) dissolving the purslane crude polysaccharide in distilled water to prepare a crude polysaccharide solution, adding proteolytic enzyme, carrying out oscillation enzymolysis reaction for 2-4h under an appropriate enzyme condition, then inactivating the enzyme with boiling water, and cooling to room temperature; wherein, the mass fraction of the crude polysaccharide solution is 10-50mg/ml, preferably 10-30 mg/ml;
wherein, preferably, the enzymolysis is carried out according to (3-5) multiplied by 104Adding papain into the crude polysaccharide solution at a ratio of U/g polysaccharide, and performing enzymolysis at 55-60 deg.C and pH of 7.0 for 2-3 h.
(S6) adding NaCl into the enzymolysis liquid until the mass fraction is 3-5%, performing salinization treatment, mixing uniformly, then adding micelle medium extraction liquid with the volume of 20-25% into the enzymolysis liquid, violently shaking and extracting for 10-15min, then centrifuging at a high speed for layering, removing an organic layer, taking the lower layer solution, and obtaining the polysaccharide extraction liquid without protein, pigment and other impurities.
Specifically, the operational flow of steps (5) to (7) is as follows.
(S1) evaporating and concentrating the obtained polysaccharide solution subjected to double extraction, and dialyzing with distilled water in a dialysis bag with cut-off of 1-5KD for 2-3 times to remove salt and small molecular impurities to obtain polysaccharide solution subjected to dialysis;
(S2) evaporating and concentrating the polysaccharide solution after dialysis treatment in a crystallization kettle, cooling for continuous crystallization after a large amount of crystallization, cooling for crystallization at the temperature of 0-3 ℃, washing with absolute ethyl alcohol after crystal precipitation is finished, and drying to obtain the purified purslane polysaccharide.
The polysaccharide extraction rate can reach more than 8% and the purity can reach more than 95% through measurement and calculation.
Preferably, the method further comprises the step of alcohol precipitation treatment: dissolving the obtained polysaccharide with deionized water, precipitating with 5-10 times volume of anhydrous ethanol for 16-24h, vacuum filtering, washing with anhydrous ethanol, and vacuum drying to obtain high purity herba Portulacae polysaccharide with purity of more than 97%.
In the present invention, the contents of protein, pigment and polysaccharide can be determined by the conventional method in the field. For example, the sugar content can be measured by the phenol-sulfuric acid method, which comprises the following steps: taking glucose as a standard substance, accurately weighing the glucose dried to a constant weight, and adding water to a constant volume to obtain a reference substance solution. And (4) sucking the glucose reference substance solution into a volumetric flask, and adding water to obtain glucose diluents with different concentrations. And respectively adding the diluent into 5% phenol solution and concentrated sulfuric acid, shaking uniformly, standing, cooling to room temperature, measuring absorbance at 485-490nm, and drawing a standard curve. Preparing a purslane polysaccharide solution, measuring a light absorption value according to the method, and calculating the total polysaccharide content according to a standard curve.
Compared with the prior art, the invention has the following beneficial effects:
1) the invention develops the process for synchronously extracting the purslane polysaccharide and the total flavone, and avoids the waste of other components caused by extracting a single component in the prior art; particularly, the polysaccharide doped in the flavone extraction process is recovered, so that the polysaccharide extraction rate is effectively improved;
2) by means of the micelle medium extraction process, the specific solvent ratio and CTAB content are selected, so that the impurities such as protein, pigment and the like can be effectively removed, the damage of a polysaccharide structure is avoided, the loss of polysaccharide is effectively reduced, and the purity of the obtained polysaccharide is high.
3) The process for synchronously extracting the total flavone and the polysaccharide has the advantages of safety, high efficiency and energy conservation, greatly shortens the purification time of the polysaccharide, does not need complex purification equipment, and is suitable for the requirement of industrial large-scale production.
Detailed Description
The present invention will be described in detail with reference to specific examples, but the scope of the present invention is not limited to these examples in any way.
Example 1
(1) Taking 1kg of clean purslane whole plant after dust removal and drying, drying at the constant temperature of 55 ℃ for 3h, crushing and sieving with a 60-mesh sieve to obtain purslane powder, wrapping the purslane powder in filter paper, wrapping the purslane powder with gauze, soaking the purslane powder in 10L of ethanol with the volume of 90% for 6h, and performing Soxhlet extraction in a Soxhlet extractor at the temperature of 55 ℃ for 3 h.
(2) After the Soxhlet extraction is finished, taking out the filter bag, and drying the powder to obtain degreased purslane medicinal material powder; filtering the extractive solution with microfiltration membrane, distilling the filtrate under reduced pressure to recover ethanol solvent to obtain concentrated solution of total flavone initial extract about 510 ml; diluting the concentrated solution to 1L with 75% ethanol, extracting with 400ml petroleum ether under shaking for 3 times, each time for 15min, removing fat, and removing fat soluble pigment such as chlorophyll and carotene to obtain defatted extractive solution.
(3) Gradient eluting the degreased extract with D140 type macroporous adsorbent resin column, sequentially eluting with deionized water, 50% ethanol, and 75% ethanol, detecting and scanning flavonoid absorption peak with ultraviolet spectrophotometer during the elution process, collecting eluate until the eluate is colorless and no absorption peak appears, and collecting eluate containing flavone and eluate without flavone respectively; converging the collected eluent containing flavone, and evaporating the solvent in a thin film evaporator until 16.1g of purslane flavonoid compound crystals are separated out, wherein the yield is 16.1 mg/g.
Wherein, the ultraviolet detection method of the eluent sample comprises the following steps: taking 1mL of eluent to a 25mL colorimetric tube, adding 0.1mL of 5% sodium nitrite solution, shaking uniformly and standing for 5 min; adding 0.1mL of 10% aluminum nitrate solution, shaking uniformly and standing for 5 min; adding 0.5mL of 4% sodium hydroxide solution, shaking, standing for 5min, diluting with 75% ethanol to 15mL, shaking, and standing for 10 min. The absorption peak was scanned on a uv spectrophotometer.
(4) Mixing the purslane powder degreased in the above steps with deionized water according to the ratio of material to liquid of 1g to 20ml, carrying out high-temperature treatment in a high-pressure reaction kettle at 121 ℃ for 2h, carrying out ultrasonic extraction at 90 ℃ for 3 times after the high-temperature treatment, carrying out 45min each time, filtering, combining and collecting the extracting solutions.
(5) Mixing the extractive solution obtained in the above step with the eluate containing no flavone collected in the above step (3), concentrating by evaporation in a thin film evaporator to remove excessive water, wherein the vacuum degree of the thin film evaporator is 0.03MP, and the outlet temperature is 55 deg.C to obtain about 3L of primary concentrated solution.
(6) Centrifuging the primary concentrated solution at 8000rpm for 30min, collecting supernatant, concentrating to 1L by rotary evaporation, and precipitating the concentrated filtrate with 8 times volume of anhydrous ethanol at 4 deg.C for 2 times, each time for 16 hr; mixing the obtained crude polysaccharide precipitates, sequentially leaching with absolute ethyl alcohol and acetone, and drying to obtain the purslane crude polysaccharide; and meanwhile, dissolving cetyl trimethyl ammonium bromide in a composite solvent formed by mixing 50% of butanol and heptane according to the volume ratio of 1:3, and fully and uniformly mixing to obtain micelle medium extract with the mass fraction of the cetyl trimethyl ammonium bromide being 5% for later use.
(7) Dissolving the crude polysaccharide of herba Portulacae obtained by the above steps in distilled water to obtain crude polysaccharide solution of 30mg/ml, and mixing at 5 × 104Adding papain at U/g polysaccharide ratio, performing enzymolysis at 55 deg.C and pH 7.0 for 4 hr, inactivating enzyme with boiling water, and cooling to room temperature.
(8) Adding analytical pure grade NaCl into the enzymatic hydrolysate until the mass fraction is 4%, performing salinization treatment, mixing uniformly, then adding 20% of micelle medium extraction liquid, performing intense shaking extraction for 10min, performing double-extraction treatment, then performing high-speed centrifugation and layering, and removing an organic layer to obtain a polysaccharide extraction liquid with impurities such as protein, pigment and the like removed.
(9) Performing thin film evaporation concentration on about 4L of the obtained polysaccharide solution subjected to double extraction treatment to about 1L, dialyzing with distilled water in a dialysis bag with a cut-off molecular weight of 3000 for 3 times to remove salt and small molecular impurities, wherein the dialysis time is 12h each time to obtain the polysaccharide solution subjected to dialysis treatment;
(10) and (3) evaporating and concentrating the polysaccharide solution subjected to dialysis treatment in a crystallization kettle, stopping heating when a large amount of crystals begin to precipitate, cooling and crystallizing at the temperature of 3 ℃, washing with absolute ethyl alcohol after the crystals are precipitated, and drying to obtain 90.2g of purified purslane polysaccharide, wherein the polysaccharide extraction rate is 9.02% and the purity is 95.2%.
The bacteriostatic effect of the purslane total flavonoid extract prepared in the present example was performed by the following steps.
The test method comprises the following steps: the flavonoid active extract in this example was dissolved in dimethyl sulfoxide and filtered through a microfiltration membrane to an initial concentration of 1 mg/mL. 10 sterile tubes were then serially diluted half-fold (i.e., 0.5mg/mL,0.25mg/mL, and so on), with one tube serving as a blank. The total amount of the culture solution and the bacterial suspension liquid added to each tube was set to 1 mL. And (3) putting the test tube into a constant-temperature incubator, culturing at 37 ℃ for 24h, and taking out the test tube to observe the growth condition of bacteria. If the test tube liquid is turbid, no bacteriostatic action is shown; if the test tube is clear, the bacteria growth is inhibited, and the maximum dilution liquid medicine capable of inhibiting the bacteria growth is the minimum inhibitory concentration of the sample.
Experimental results show that the total flavone extract obtained in the embodiment has the minimum inhibitory concentration of 0.25mg/ml to escherichia coli and 0.12mg/ml to staphylococcus aureus, and has excellent inhibitory effect.
Example 2
(1) Taking 5kg of clean purslane herb after dust removal and drying, crushing and sieving the purslane herb with a 60-mesh sieve, wrapping the purslane herb in filter paper, externally wrapping the purslane herb with gauze, soaking the purslane herb with 50L of ethanol with the volume of 95% for 6 hours, performing Soxhlet extraction on the purslane herb in a Soxhlet extractor at the temperature of 60 ℃ for 4 hours, and extracting the raw materials in three batches respectively;
(2) after the Soxhlet extraction is finished, taking out the filter bag to obtain degreased purslane medicinal material powder, and drying the powder for later use; filtering the extractive solution with microfiltration membrane, distilling the filtrate under reduced pressure to recover ethanol solvent to obtain 2L of concentrated solution of primary extract of total flavonoids; diluting the concentrated solution to 3.5L with 75% ethanol, extracting with 2L petroleum ether under shaking for 3 times for removing fat, 15min each time, to obtain defatted extractive solution.
(3) Performing rotary evaporation and concentration on the degreased extract, performing gradient elution by using a macroporous adsorption resin column, sequentially eluting by using deionized water, 50% ethanol and 75% ethanol, detecting and scanning flavonoid absorption peaks of the eluent by using an ultraviolet spectrophotometer in the elution process, collecting the eluent until the eluent is colorless and no absorption peak appears, stopping elution, and respectively collecting the eluent containing flavone and the eluent not containing flavone; converging the collected eluent containing flavonoids, and evaporating the solvent in a thin film evaporator until 84.3g of purslane flavonoid compound crystals are separated out, wherein the yield is 16.8 mg/g.
The method for detecting the eluate sample by ultraviolet light is the same as that described in example 1.
(4) Mixing the degreased herba Portulacae powder with deionized water at a ratio of 1g to 20ml, treating at high temperature in a high-pressure reaction kettle at 118 deg.C for 2 hr, performing ultrasonic extraction at 90 deg.C for 3 times (each for 30 min), filtering, mixing, and collecting extractive solutions.
(5) Mixing the obtained extractive solution with the eluate containing no flavone collected in step (3), concentrating by evaporation in a thin film evaporator to remove excessive water, wherein the vacuum degree of the thin film evaporator is 0.02MP, and the outlet temperature is 60 deg.C to obtain about 10L of primary concentrated solution.
(6) Centrifuging the primary concentrated solution at 8000rpm for 30min at high speed, centrifuging in batches, collecting supernatant, concentrating to 3L by rotary evaporation, precipitating the concentrated filtrate with 8 times volume of anhydrous ethanol at 4 deg.C in two batches, and precipitating with ethanol for 2 times, each time for 20 hr; mixing the obtained crude polysaccharide precipitates, sequentially leaching with absolute ethyl alcohol and acetone, and drying to obtain the purslane crude polysaccharide; and meanwhile, dissolving cetyl trimethyl ammonium bromide in a composite solvent mixed by 50% of butanol and heptane according to the volume ratio of 1:4, and fully and uniformly mixing to obtain micelle medium extract with the mass fraction of the cetyl trimethyl ammonium bromide being 4% for later use.
(7) Dissolving the crude polysaccharide of herba Portulacae obtained by the above steps in distilled water to obtain crude polysaccharide solution of 50mg/ml, and mixing at a ratio of 3 × 104Adding papain at U/g polysaccharide ratio, performing enzymolysis at 55 deg.C and pH 7.0 for 4 hr, inactivating enzyme with boiling water, and cooling to room temperature.
(8) Adding analytical pure grade NaCl into the enzymatic hydrolysate until the mass fraction is 3%, performing salinization treatment, mixing uniformly, then adding 25% of micelle medium extract, performing intense shaking extraction for 15min, performing double-extraction treatment, then performing high-speed centrifugation and layering, and removing an organic layer to obtain a polysaccharide extract without impurities such as protein, pigment and the like.
(9) And (3) performing thin-film evaporation concentration on the obtained polysaccharide solution subjected to double-extraction treatment in a thin-film evaporator to about 3L, and dialyzing with distilled water in a dialysis bag with a cut-off molecular weight of 5000 for 3 times to remove salt and small molecular impurities, wherein the dialysis time is 16h each time, so as to obtain the polysaccharide solution subjected to dialysis treatment.
(10) Evaporating and concentrating the dialyzed polysaccharide solution in a crystallization kettle, stopping heating when a large amount of crystals begin to precipitate, cooling and crystallizing at the temperature of 1 ℃, washing with absolute ethyl alcohol after the crystals are precipitated, and drying to obtain 466.1g of purified purslane polysaccharide, wherein the extraction rate of the polysaccharide is 9.32%, and the purity is 95.5%.
(11) Alcohol precipitation treatment: and (3) dissolving the polysaccharide obtained in the step (10) with deionized water at room temperature, precipitating with 10 times of volume of absolute ethanol for 24h, filtering, washing with absolute ethanol, and drying in vacuum to obtain further purified high-purity purslane polysaccharide with polysaccharide content of 97.6%.
Comparative example 1
The micellar medium extraction solution from the double extraction in step (8) of example 1 was replaced by Sevage reagent (chloroform: n-butanol: 4.5:1) and no salination was performed; the remaining steps and basic operating conditions were unchanged, giving comparative example 1.
This comparative example yielded a purslane polysaccharide extract of 87.3g, with a polysaccharide purity of 78.2%, containing more protein and pigment impurities.
Comparative example 2
On the basis of comparative example 1, the Sevage reagent (chloroform: n-butanol ═ 4.5:1) extraction operation was repeated twice, and the remaining steps and basic operating conditions were unchanged to obtain comparative example 2.
This comparative example yielded 85.6g purslane polysaccharide extract, 86.7% polysaccharide purity, still containing more protein and pigment impurities.
Comparative example 3
On the basis of comparative example 1, the fraction of the eluate collected in step (3) and not containing flavones was not combined in step (5), and the rest of the steps and operating conditions were unchanged, to obtain comparative example 3.
This comparative example yielded 71.2g of purslane polysaccharide extract with 7.12% polysaccharide extraction.
The above embodiments do not limit the technical solutions of the present invention, and a person skilled in the art may modify the technical solutions described in the above embodiments without departing from the scope of the technical solutions of the present invention.
Claims (4)
1. A method for extracting herba Portulacae polysaccharide and total flavonoids comprises extracting with micelle medium in polysaccharide extraction operation, wherein the micelle medium is obtained by dissolving cetyl trimethyl ammonium bromide in heptane and butanol water solution with volume fraction of 50%; characterized in that the method comprises the following steps:
step (1): carrying out degreasing treatment on the purslane, and specifically comprising the following steps:
s1: drying the whole purslane at constant temperature, and crushing and sieving by a 40-60-mesh sieve to obtain purslane powder;
s2: adding 85-99% ethanol into herba Portulacae powder, soaking for 3-12 hr, and performing Soxhlet extraction at 50-60 deg.C for 1-5 hr; wherein the mass volume ratio of the purslane to the solvent is 1g:5-15 ml;
step (2): degreasing the degreasing treatment solution, performing gradient elution by using a macroporous adsorption resin column to extract total flavonoids from the purslane, and respectively collecting the flavonoid-containing eluate and the flavonoid-free eluate;
the specific process of the step (2) is as follows:
s1: distilling the degreasing treatment solution under reduced pressure to obtain a concentrated solution of a primary extract of the total flavonoids;
s2: diluting the concentrated solution with 65-75% ethanol, and extracting with petroleum ether as extractant to remove fat;
s3: gradient eluting the degreased extract with macroporous adsorbent resin column, sequentially eluting with deionized water, 50% ethanol and 75% ethanol, scanning absorption peak by ultraviolet detection during elution, stopping elution until the eluate is colorless and no absorption peak appears, and respectively collecting eluate containing flavone and eluate without flavone;
s4: collecting the received eluent containing flavone, evaporating to separate out purslane total flavonoid compound crystals;
and (3): the process for extracting crude polysaccharide from defatted purslane is as follows:
s1: mixing the degreased purslane powder with deionized water, treating at the high temperature of 115-121 ℃ for 1-2h, then carrying out ultrasonic extraction for 2-3 times, filtering, and combining the extracting solutions;
s2: primary concentration: mixing the extract with the eluate containing no flavone, and concentrating by evaporation in an evaporator to below 50% volume to obtain primary concentrated solution;
s3: centrifuging the primary concentrated solution at high speed, collecting supernatant, concentrating by rotary evaporation, precipitating the concentrated filtrate with 95% ethanol or anhydrous ethanol at 4 deg.C to obtain crude polysaccharide precipitate, sequentially leaching the precipitate with anhydrous ethanol and acetone, and drying to obtain crude polysaccharide of herba Portulacae;
and (4): preparing micelle medium extraction liquid, and performing salinization and extraction double-removal treatment on the crude polysaccharide solution after enzymolysis, wherein the process comprises the following steps:
s1: preparing micelle medium extraction liquid:
dissolving a proper amount of cetyl trimethyl ammonium bromide in a mixed solvent of heptane and 50% butanol, and fully and uniformly mixing to obtain a micelle medium extract, wherein the mass fraction of the cetyl trimethyl ammonium bromide is 3-5%;
s2: dissolving crude purslane polysaccharide in distilled water to prepare a crude polysaccharide solution, adding proteolytic enzyme for enzymolysis for 2-4h, inactivating enzyme, and cooling to room temperature; adding NaCl into the enzymolysis liquid until the mass fraction is 3-5%, uniformly mixing, and salinizing;
s3: adding micelle medium extract into the enzymolysis solution, extracting for 10-15min by intense shaking, and then centrifuging at high speed for layering to obtain polysaccharide extract without protein and pigment impurities;
and (5): dialyzing the extract to remove impurities and removing small molecular impurities:
evaporating and concentrating the polysaccharide solution subjected to double extraction, and dialyzing with distilled water in a dialysis bag with cut-off molecular weight of 1-5KD to remove salt and small molecular impurities to obtain polysaccharide solution subjected to dialysis;
and (6): evaporating, concentrating, crystallizing, cooling to 0-5 deg.C, crystallizing, and washing with anhydrous ethanol to obtain purified herba Portulacae polysaccharide.
2. The method of claim 1, further comprising the step of subjecting the purslane polysaccharides to a further alcohol precipitation process comprising: dissolving the obtained polysaccharide with deionized water, precipitating with 5-10 times volume of anhydrous ethanol or 95% ethanol for 16-24 hr, vacuum filtering, washing with anhydrous ethanol, and vacuum drying to obtain high purity herba Portulacae polysaccharide.
3. The method as claimed in claim 1, wherein the micellar medium extraction solution is added in the amount of 20-25% by volume of the enzymatic hydrolysate in step (4).
4. The method of claim 1, wherein the ratio of 50% butanol in the mixed solvent of the micellar medium extraction solution prepared in the step (4): the volume ratio of heptane is 1: 3-5.
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| CN109575152A (en) * | 2019-01-28 | 2019-04-05 | 潍坊医学院 | The method of efficient fast eliminating albumen and pigment from acanthopanax giraldii harms polysaccharose extracting solution |
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