CN108653395B - Coupling extraction method for active ingredients of bark of artificial forest - Google Patents
Coupling extraction method for active ingredients of bark of artificial forest Download PDFInfo
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Abstract
The invention belongs to the technical field of plant extraction, and discloses a coupling extraction method for active ingredients of bark of an artificial forest, which comprises the steps of mixing crushed materials of the bark of the artificial forest and a cellulose complex enzyme aqueous solution according to a material-to-liquid ratio of 1: 8-16, carrying out water bath treatment at 40-70 ℃ for 30-60 min, and filtering to obtain filter residue and filtrate; mixing the obtained filter residue with 40-70% ethanol according to the material-liquid ratio of 1: 6-14, performing ultrasonic extraction at 40-60 ℃ for 40-80 min, and filtering to obtain filtrate; the filtrates obtained in the first two times are combined. The enzyme treatment and the ultrasonic-assisted coupled extraction have high yield of the extracted active ingredients, which is more than 20% higher than that of the traditional organic solvent extraction method, and the method is a low-temperature, green and efficient extraction method.
Description
Technical Field
The invention belongs to the technical field of plant extraction, and particularly relates to an efficient extraction method for active ingredient enzyme treatment and ultrasonic-assisted coupling of artificial forest bark.
Background
China is one of the largest countries in the world where artificial forests are planted. The artificial forest is a forest which is built and cultivated by adopting methods and technical measures such as artificial sowing, planting or cuttage and is built by artificial afforestation or artificial re-afforestation. The artificial forest can be introduced into new species or enhanced management of native tree species, and is classified into artificial masson pine forest, artificial fir forest, artificial poplar forest, artificial eucalyptus forest and the like according to tree species. The fast-growing tree species of the artificial forest has the advantages of fast growth, rapid development, more earning foreign exchange and ecological benefit.
The non-wood resources such as bark, branches and leaves of the artificial forest in China are rich, but the utilization rate of the non-wood resources is low, and the non-wood resources are not well utilized. For example, eucalyptus bark is a waste material in the pulp industry, which accounts for 5.97% of the total biomass of eucalyptus, and most of the waste materials are directly thrown away, thereby not only causing resource waste, but also bringing about environmental pollution. The eucalyptus bark contains rich high-added-value bioactive components, such as phenolic compounds, tannins, flavonoids, monoterpene compounds, triterpenic acid and the like, has the bioactive effects of resisting tumors, resisting bacteria, reducing blood sugar, resisting oxidation and the like, and has wide application prospect. The poplar has become the main afforestation tree species of the artificial forest in China, and is mainly used for ecological protection forest, three-north protection forest, agriculture and forestry protection forest, industrial forest and the like. A large amount of bark waste is generated in the processing process of poplar wood, and is not fully utilized at present. Black wattle (Acacia mearensii) is a tree species of Acacia of Mimosaceae, a fast-growing, evergreen arbor, native to Australia, and introduced in Guangdong, Guangxi, Fujian, Yunnan, etc. of China after 1950. The bark of black wattle contains chemical substances such as oligomeric proanthocyanidin, flavone, anthraquinone, triterpenoid saponin, polysaccharide, gum and protein besides polyphenol compounds. The proanthocyanidin in the bark of black wattle has various physiological activities, such as antidiabetic, antiinflammatory, antioxidant, anticancer, cardiovascular and cerebrovascular disease preventing and treating bioactivity, etc.
At present, the active ingredients of the bark of the artificial forest are mainly extracted by a traditional solvent extraction method, an ultrasonic-assisted extraction method and a supercritical fluid extraction method. The supercritical fluid extraction method has high equipment cost and low production capacity. The traditional solvent extraction method has the defects of high solvent consumption, high extraction temperature, long extraction time, low extraction rate, high loss of effective components, high production cost and the like, and has poor feasibility in industrial production.
Disclosure of Invention
Aiming at the problems of low utilization rate of non-woody resource bark of the artificial forest and low extraction rate of active substances in China, the invention provides a method for coupling and extracting the active ingredients of the bark of the artificial forest, which obviously improves the extraction efficiency of active substances such as flavone, proanthocyanidin and the like.
The technical scheme of the invention is as follows:
a coupling extraction method of active ingredients of bark of artificial forest comprises the following steps:
(1) mixing the crushed materials of the bark of the artificial forest and a cellulose compound enzyme aqueous solution according to the material-liquid ratio of 1: 8-16, carrying out enzymolysis for 30-60 min under the condition of water bath at the temperature of 40-70 ℃, and filtering to obtain enzymolysis filter residues and enzymolysis filtrate;
(2) mixing the enzymolysis filter residue obtained in the step (1) with 40-70% by volume of ethanol according to the material-liquid ratio of 1: 6-14, performing ultrasonic extraction at 40-60 ℃ for 40-80 min, and filtering to obtain a filtrate;
(3) and (3) combining the filtrates in the step (1) and the step (2) to obtain a total filtrate containing the bark active ingredients.
Preferably, the cellulose complex enzyme comprises cellulase and hemicellulase.
Preferably, the enzyme activity of the cellulose complex enzyme is 30000-40000U/g.
Preferably, the mass of the cellulose complex enzyme accounts for 0.1-0.4% of the dry weight of the bark powder.
Preferably, the mass concentration of the cellulose complex enzyme aqueous solution is 0.005-0.03%.
Preferably, the frequency of the ultrasound in the step (2) is 30-50 KHz.
Preferably, the bark is eucalyptus bark, poplar bark or acacia bark.
Preferably, the active ingredient is one or more of flavone, proanthocyanidin and polyphenol.
Preferably, the total filtrate is subjected to reduced pressure concentration and liquid-liquid extraction, and then is separated by macroporous adsorption resin to obtain the active ingredient.
Compared with the prior art, the invention has the following advantages:
the invention provides a coupling extraction method of active ingredients of bark of an artificial forest, which comprises the following steps: mixing the crushed materials of the bark of the artificial forest and a cellulose compound enzyme aqueous solution according to the material-liquid ratio of 1: 8-16, carrying out enzymolysis for 30-60 min under the condition of water bath at the temperature of 40-70 ℃, and filtering to obtain filter residues and filtrate; mixing the obtained filter residue with 40-70% by volume of ethanol according to the material-liquid ratio of 1: 6-14, performing ultrasonic extraction at 40-60 ℃ for 40-80 min, and filtering to obtain filtrate; combining the two filtrates to obtain a total filtrate containing bark active components. The extraction method of the invention has high yield of extracted active ingredients, which is more than 20% higher than the extraction yield of the traditional organic solvent and more than 3% higher than the extraction yield of the ultrasonic-assisted extraction method. The invention is a low-temperature, green and efficient extraction method. The method overcomes the defects that the extraction rate of the active ingredients is limited by the intracellular and extracellular equilibrium concentration, the extraction time is long, more solvents are consumed, the extraction efficiency is low, the active ingredients are easy to lose, and the like, has the characteristics of strong universality of raw materials, and has wide application prospect in the processing production of the active substances of the barks of the non-wood resources of the artificial forest.
Detailed Description
The invention provides a coupling extraction method of active ingredients of bark of an artificial forest, which comprises the following steps:
(1) mixing the crushed materials of the bark of the artificial forest and a cellulose compound enzyme aqueous solution according to the material-liquid ratio of 1: 8-16, carrying out enzymolysis for 30-60 min under the condition of water bath at the temperature of 40-70 ℃, and filtering to obtain enzymolysis filter residues and enzymolysis filtrate;
(2) mixing the enzymolysis filter residue obtained in the step (1) with 40-70% by volume of ethanol according to the material-liquid ratio of 1: 6-14, performing ultrasonic extraction at 40-60 ℃ for 40-80 min, and filtering to obtain a filtrate;
(3) and (3) combining the filtrates in the step (1) and the step (2) to obtain a total filtrate containing the bark active ingredients.
The invention has no special limitation on the types of the barks of the artificial forests, and the barks of the tree species planted in the artificial forests in the field can be extracted by the extraction method of the invention to extract the active ingredients. In the embodiment of the invention, 3 kinds of bark active ingredients of artificial woods of eucalyptus, poplar and acacia negra are preferably subjected to enzyme treatment and alcohol ultrasonic-assisted coupled extraction. The extraction method of the invention has no special limitation on the types of the extracted active substances, and the extracted active substances can be one or more of flavone, proanthocyanidin and polyphenol according to the different types of the active ingredients of the bark in different tree species. In the specific embodiment of the invention, the extraction rate of the extraction method is compared with that of the conventional extraction method by taking total flavonoids, proanthocyanidins and plant polyphenols as the marks of the active ingredients of the barks of eucalyptus, poplar and acacia mearnsii respectively. It should be noted that the extraction method of the present invention can also be used for extracting active ingredients from bark of forest trees grown under natural conditions.
The preparation method of the crushed material of the bark of the artificial forest is not particularly limited and can be prepared by adopting the conventional method of the technicians in the field. Preferably, the bark of the artificial forest is mechanically peeled, the bark is air-dried and is crushed by a crusher to obtain crushed material of the bark of the artificial forest. The particle size of the crushed pieces of the bark of the artificial forest is not specially limited, and in principle, the smaller the particle size is, the more beneficial the dissolution of active substances in the bark is. In the invention, the particle size of the crushed materials of the bark of the artificial forest is preferably 1-10 mm, and more preferably 3-5 mm.
In the invention, the crushed materials of the bark of the artificial forest are mixed with the cellulose compound enzyme aqueous solution to obtain a mixed material. The feed-liquid ratio of the crushed materials of the bark of the artificial forest and the cellulose compound enzyme aqueous solution is preferably 1: 8-16, and more preferably 1: 10-13. The mass concentration of the cellulose complex enzyme aqueous solution is preferably 0.005-0.025%, and more preferably 0.01-0.015%. The enzyme activity of the cellulose complex enzyme is preferably 30000-40000U/g, and more preferably 32000-35000U/g. The cellulose compound enzyme is preferably compounded by cellulase and hemicellulase. The cellulose complex enzyme of the invention carries out enzymolysis on cellulose and hemicellulose components in the cell wall structure of plant cells, thereby reducing mass transfer resistance, fully releasing active substances in the plant cells, and better protecting the activity of the active substances, thereby improving the extraction efficiency and extraction rate of the active substances. In the invention, the mass of the cellulose compound enzyme accounts for 0.1-0.4% of the dry weight of the bark raw material, and more preferably 0.15-0.3%. The pH value of the mixed material is preferably 5-6.
The invention carries out water bath enzymolysis treatment on the mixture of the crushed materials of the bark of the artificial forest and the cellulose compound enzyme aqueous solution. The temperature of the mixed solution of the crushed materials of the bark of the artificial forest and the cellulose compound enzyme aqueous solution for water bath enzymolysis is preferably 50-65 ℃, and the time of the enzymolysis is preferably 40-50 min. Filtering the mixed solution after enzymolysis, and filtering to obtain enzymolysis filter residue and enzymolysis filtrate. The filtration method is not particularly limited in the present invention, and a conventional filtration method in the art may be employed.
In the invention, the enzymolysis filter residue is subjected to ultrasonic extraction in an ethanol solution, the diffusion and mixing of a target component in a solvent are improved by utilizing secondary effects such as ultrasonic cavitation, mechanical vibration and the like, the extraction efficiency is improved, the extraction time is shortened, and meanwhile, the material structure and the biological activity of important active substances such as flavonoids, proanthocyanidins, plant polyphenols and the like are not influenced. In the invention, the mass volume ratio of the filter residue to the ethanol solution is 1: 6-14, preferably 1: 7-13, and more preferably 1: 8-12. The volume concentration of the ethanol is preferably 50-60%. The temperature of the ultrasonic treatment is preferably 45-55 ℃, and the time of the ultrasonic treatment is preferably 50-70 min, and more preferably 60 min. The ultrasonic frequency of the ultrasonic treatment is preferably 30-50 KHz, and more preferably 35-45 KHz. And filtering the material subjected to ultrasonic treatment to obtain filtrate and filter residue.
Combining the two filtrates to obtain a total filtrate containing bark active components.
The active ingredients in the total filtrate can be further separated and extracted by a person skilled in the art according to a conventional method in the field, the step of the invention is not particularly limited, and the active ingredients are preferably obtained by concentrating the total filtrate under reduced pressure, performing liquid-liquid extraction, and separating by macroporous adsorption resin.
The method detects the content of active ingredients in the combined filtrate and calculates the extraction rate. The results show that: the method for extracting the active ingredients (total flavonoids, proanthocyanidins and plant polyphenols) from the bark of the artificial forest such as eucalyptus, poplar, black wattle and the like has high extraction yield which is more than 20% higher than that of the conventional organic solvent extraction method and more than 3% higher than that of the conventional ultrasonic-assisted extraction method, and is a low-temperature, green and high-efficiency method for extracting the active ingredients from the bark of the artificial forest.
The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.
In the following examples of the present invention, the cellulose complex enzyme was cellulase and hemicellulase, and the enzyme activity was 32000U/g, which was purchased from Kangdien Bio Inc.
Example 1
Extraction of general flavone from eucalyptus bark
Respectively taking 3g (accurate to 0.001) of eucalyptus bark powder raw materials, putting the raw materials into a 100mL conical flask, adding 0.2mg/mL cellulose complex enzyme solution according to the material-to-liquid ratio of 1:14, carrying out water bath treatment at 50 ℃ for 45min, filtering, transferring the filtrate into a 100mL volumetric flask, adding 60% ethanol solution with the same volume as the cellulose complex enzyme solution into filter residues, carrying out ultrasonic extraction at 50 ℃ for 45min (ultrasonic frequency of 40KHz), filtering, combining the two filtrates, and fixing the volume to 100 mL.
Comparative examples 1 to 1
Extraction of general flavone from eucalyptus bark
Respectively taking 3 parts of 3g (accurate to 0.001) eucalyptus bark powder raw materials, putting the raw materials into a 100mL conical flask, adding water according to the material-liquid ratio of 1:14, soaking for 45min at 50 ℃, filtering, transferring the filtrate into a 100mL volumetric flask, adding 60% ethanol solution with the material-liquid ratio of 1:14 into filter residues, carrying out water bath extraction for 45min at 50 ℃, filtering, combining the two filtrates, and fixing the volume to 100 mL.
Comparative examples 1 to 2
Extraction of general flavone from eucalyptus bark
Respectively taking 3g (accurate to 0.001) of eucalyptus bark powder raw materials, putting the raw materials into a 100mL conical flask, adding water according to the material-liquid ratio of 1:14, performing ultrasonic treatment for 45min (ultrasonic frequency of 40KHz) at 50 ℃, filtering, transferring the filtrate into a 100mL volumetric flask, adding 60% ethanol solution with the material-liquid ratio of 1:14 into filter residues, performing ultrasonic extraction for 45min (ultrasonic frequency of 40KHz) at 50 ℃, filtering, combining the two filtrates, and fixing the volume to 100 mL.
Rutin is used as a standard substance, the content of flavone in the filtrate obtained in example 1, comparative example 1-1 and comparative example 1-2 is measured by a sodium nitrite-aluminum nitrate method, and the extraction yield of the flavone is calculated. The specific determination method is as follows:
the invention adopts NaNO2-Al(NO3)3The method for determining the content of the total flavonoids in the eucalyptus bark extract by a colorimetric method comprises the following steps:
weighing 20mg rutin standard substance dried at 105 deg.C to constant mass, placing in 50mL volumetric flask, dissolving with 60% ethanol solution and fixing volume to obtain a solution with mass concentration of 0.4g.L-1Rutin standard solution. Accurately transferring 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4mL of rutin standard solution, placing in a 10mL volumetric flask, respectively adding 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, 0.8 and 0.6mL of 60% ethanol solution, adding 5% NaNO20.5mL of the solution was shaken up, left for 6min, and 10% Al (NO) was added3)30.5mL of the solution is shaken up and placed for 6min, 4mL of 4% NaOH solution is added, the volume is fixed to the scale by 60% ethanol solution, and the solution is shaken up and placed for 15 min. The absorbance of the corresponding reagent was measured at a wavelength of 510nm, using the reagent as a blank. Drawing a standard curve by taking the absorbance (A) as a vertical coordinate and the rutin mass concentration (X) as a horizontal coordinate to obtain a regression equation of the total flavone standard curve, wherein the regression equation is that A is 2.7014X-0.0518, and a correlation coefficient R20.9998, and the linear range is 0-0.06 g.L-1。
And (3) calculating yield: diluting the extractive solution by a certain amount, collecting 1mL of diluted solution, and adding NaNO2-Al(NO3)3Measuring absorbance by colorimetry, calculating the yield of the total flavone according to a standard curve, and calculating the yield of the total flavone according to a formula.
Calculating the yield of the total flavone according to the standard curve, and calculating the yield of the total flavone according to the formula (1).
In the formula: y-total flavone yield,%; c-the mass concentration of the total flavonoids is mg/L; v-total volume of the extraction solution to be tested, mL; m-mass of raw material, g; w-water content of raw material,%; n-dilution factor.
The results of the total flavone yield are shown in Table 1.
TABLE 1 comparison of enzyme treatment/ultrasonic-assisted coupled extraction of total flavonoids from eucalyptus bark
As can be seen from table 1: example 1 (enzyme water bath treatment + 60% ethanol solution ultrasonic extraction) the highest yield of total flavonoids was 3.87%, which was 24.04% higher than the traditional extraction method of comparative example 1-1, and 3.48% higher than the ultrasonic-assisted extraction of comparative example 1-2. Therefore, the enzyme treatment/ultrasonic wave auxiliary coupling extraction method in the embodiment 1 is the best extraction method of the total flavonoids in the eucalyptus bark.
Example 2
Extraction of general flavone from eucalyptus bark
Respectively taking 3g (accurate to 0.001) of eucalyptus bark powder raw materials, putting the raw materials into a 100mL conical flask, adding 0.1mg/mL cellulose complex enzyme solution (the enzyme amount accounts for 0.2% of the absolute dry raw material) according to the material-to-liquid ratio of 1:14, adjusting the pH value of the mixed solution to be 5, carrying out water bath treatment at 60 ℃ for 55min, filtering, transferring the filtrate into a 100mL volumetric flask, adding 60% ethanol solution with the same volume as the cellulose complex enzyme solution into filter residues, carrying out ultrasonic extraction at 50 ℃ for 60min (ultrasonic frequency of 40KHz), filtering, combining the filtrates of the two times, and keeping the volume to 100 mL. The yield of the total flavone is 3.92 percent, which is 25.64 percent higher than that of the traditional extraction method (example 1-1).
Example 3
Extraction of proanthocyanidin from bark of black wattle
Respectively taking 3 parts of 5g (accurate to 0.001) black wattle bark powder raw material in a conical flask, and mixing the raw material according to a material-liquid ratio of 1: adding 0.1mg/mL cellulose complex enzyme solution, performing water bath treatment at 50 ℃ for 30min, filtering, transferring the filtrate to a 100mL volumetric flask, adding 50% ethanol solution with the same volume as the cellulose complex enzyme solution into the filter residue, performing ultrasonic extraction at 50 ℃ for 40min (ultrasonic frequency 40KHz), filtering, combining the two filtrates, and diluting to 100 mL. And (3) carrying out rotary evaporation on 5mL of extracting solution, drying at low temperature in vacuum, preparing a dried sample into a 1mg/mL solution, and measuring the content of proanthocyanidin by using a vanillin-sulfuric acid method.
Comparative example 3-1
Extraction of proanthocyanidin from bark of black wattle
Respectively taking 3 parts of 5g (accurate to 0.001) black wattle bark powder raw material in a conical flask, and mixing the raw material according to a material-liquid ratio of 1: adding water into the filter residue, soaking at 50 ℃ for 30min, filtering, transferring the filtrate into a 100mL volumetric flask, and adding a material-liquid ratio of 1: 11, extracting with 50% ethanol solution at 50 deg.C for 40min, filtering, mixing the two filtrates, and diluting to 100 mL. And (3) carrying out rotary evaporation on 5mL of extracting solution, drying at low temperature in vacuum, preparing a dried sample into a 1mg/mL solution, and measuring the content of proanthocyanidin by using a vanillin sulfuric acid method.
Comparative examples 3 to 2
Extraction of proanthocyanidin from bark of black wattle
Respectively taking 3 parts of 5g (accurate to 0.001) black wattle bark powder raw material in a conical flask, and mixing the raw material according to a material-liquid ratio of 1: adding water, carrying out ultrasonic treatment for 30min (ultrasonic frequency 40KHz) at 50 ℃, filtering, transferring the filtrate into a 100mL volumetric flask, and adding a material-liquid ratio of 1: 11, ultrasonic extracting at 50 deg.C for 40min (ultrasonic frequency 40KHz), filtering, mixing the filtrates, and diluting to 100 mL. And (3) carrying out rotary evaporation on 5mL of extracting solution, drying at low temperature in vacuum, preparing a dried sample into a 1mg/mL solution, and measuring the content of proanthocyanidin by using a vanillin sulfuric acid method.
The method for measuring the proanthocyanidin content in the black wattle bark extract by a vanillin sulfuric acid method comprises the following steps:
blank: 0.5ml methanol +2.5ml 3% vanillin +2.5ml 30% sulphuric acid;
experimental groups: 0.5ml of sample/methanol +2.5ml of 3% vanillin/methanol +2.5ml of 30% sulfuric acid/methanol, and after a light-shielding reaction at 30 ℃ for 20min, the light absorption value at 500nm is measured.
Preparing a standard curve by using catechin as a standard substance: y-4.0208 x-0.0071 (R)20.9999) the proanthocyanidin content in the black wattle bark extract was calculated. Wherein: y-absorbance, Abs; x- -mass concentration of substance, mg/mL
The formula for calculating the extraction yield of the proanthocyanidins from the black wattle bark is as follows:
in the formula: g1-bark mass, g;
G2-extract mass, g;
w is the water content of bark,%;
c- -proanthocyanidin content in the extract,%.
The extraction yield of proanthocyanidin from black wattle bark is shown in table 2.
TABLE 2 comparison of proanthocyanidin enzyme treatment/ultrasonic-assisted coupled extraction methods in Acacia Nervitae bark
As can be seen from table 2: example 3 (aqueous enzyme bath treatment + 50% ethanol solution ultrasonic extraction) the highest proanthocyanidin extraction yield was 5.68%, which was 20.17% higher than that of the conventional solvent extraction method of comparative example 3-1 (water extraction + 50% ethanol extraction), and 5.97% higher than that of the ultrasonic assisted extraction of comparative example 3-2 (aqueous solution ultrasonic + 50% ethanol ultrasonic).
Example 4
Extraction of plant polyphenol from poplar bark
Placing 2g (accurate to 0.001) of poplar bark powder raw material into a 50mL conical flask, adding 0.12mg/mL cellulose complex enzyme solution (the material-liquid ratio is 1:14, the enzyme amount accounts for 0.18 percent of the weight of the absolute dry raw material), carrying out water bath treatment for 50min at 55 ℃, filtering, transferring the filtrate into a 100mL volumetric flask, adding 60 percent ethanol solution with the same volume as the cellulose complex enzyme aqueous solution into filter residues, carrying out ultrasonic-assisted extraction for 60min (ultrasonic frequency is 40KHz) at 55 ℃, filtering, combining the filtrates of the two times, fixing the volume to 100mL, taking 0.5mL of extracting solution, carrying out Folin-Ciocalteu method to determine the polyphenol content, and calculating the extraction yield of the plant polyphenol.
Comparative example 4-1
Extraction of plant polyphenol from poplar bark
Taking 2g (accurate to 0.001) of poplar bark powder raw material, adding water according to the material-liquid ratio of 1:14 into a 50mL conical flask, soaking for 50min at 55 ℃, filtering, transferring the filtrate into a 100mL volumetric flask, adding 60% ethanol solution with the material-liquid ratio of 1:14 into the filter residue, extracting for 60min in a water bath at 55 ℃, filtering, combining the filtrates, and fixing the volume to 100 mL. Taking 0.5mL of extracting solution to carry out Folin-Ciocalteu method to determine the polyphenol content, and calculating the extraction yield of the plant polyphenol.
Comparative examples 4 to 2
Extraction of plant polyphenol from poplar bark
Taking 2g (accurate to 0.001) of poplar bark powder raw material, adding water into a 50mL conical flask according to the material-liquid ratio of 1:14, carrying out ultrasonic treatment for 50min (ultrasonic frequency of 40KHz) at 55 ℃, filtering, transferring the filtrate into a 100mL volumetric flask, adding 60% ethanol solution into the obtained filter residue according to the material-liquid ratio of 1:14, carrying out ultrasonic extraction for 60min (ultrasonic frequency of 40KHz) at 55 ℃, filtering, combining the two filtrates, and fixing the volume to 100 mL. Taking 0.5mL of extracting solution to carry out Folin-Ciocalteu method to determine the polyphenol content, and calculating the extraction yield of the plant polyphenol.
The method for determining the plant polyphenol content in the poplar bark extracting solution by the Folin-Ciocalteu method comprises the following steps:
blank: 25ml of a 10% solution of Folin-phenol +2ml of 7.5% Na2CO3Solution +0.5ml distilled water;
experimental groups: 25mL of a 10% Folin-phenol solution +2mL of 7.5% Na2CO3Adding 0.5ml of the extractive solution, heating in 50 deg.C water bath for 5min, naturally cooling, and measuring absorbance at 760 nm; according to the standard curve of gallic acid as standard product, Y is 0.0011x-0.0113(R20.9993) the polyphenol content of the poplar bark plants was calculated.
The formula for calculating the extraction yield of the polyphenol of the poplar bark plant is as follows:
in the formula: g1 is bark mass, G;
g2 is the volume of polyphenol extract, mL;
w is the bark water content,%;
c is the polyphenol content in the extract, mu g/mL.
The result of the polyphenol extraction yield of poplar bark plants is shown in table 3.
TABLE 3 comparison of the plant Polyphenol enzyme treatment/ultrasonic wave assisted coupling extraction method in Populus bark
As can be seen from Table 3, the yield of the plant polyphenol extracted in example 4 (aqueous solution treatment with complex enzyme + ultrasonic extraction with 60% ethanol solution) is 3.21%, which is 26.38% higher than that of the traditional solvent extraction method (aqueous soaking treatment + aqueous solution extraction with 60% ethanol solution) in comparison with the conventional solvent extraction method in comparison with 4-1, and 6.29% higher than that of the ultrasonic-assisted extraction method in comparison with 4-2 (aqueous solution ultrasonic + ultrasonic extraction with 60% ethanol). Therefore, the best method for extracting the polyphenol of the poplar bark plants is to use 60 percent ethanol solution for ultrasonic extraction after the treatment of the complex enzyme aqueous solution in the embodiment 4 of the invention.
The method is used for carrying out enzyme treatment and ultrasonic-assisted coupling extraction on 3 kinds of artificial forest non-woody resource bark active ingredients of eucalyptus, poplar and black wattle, and the result shows that: the extraction method has the highest extraction yield of 3 artificial forest bark active ingredients (total flavonoids, proanthocyanidins and plant polyphenols) of eucalyptus, poplar and black wattle, and the extraction yield is higher than that of the traditional organic solvent by more than 20 percent and is higher than that of ultrasonic-assisted extraction by more than 3 percent. The technology of the invention is a low-temperature, green and efficient extraction method. The method overcomes the defects that the extraction rate of the active ingredients is limited by the intracellular and extracellular equilibrium concentration, the extraction time is long, the consumption of solvents is high, the extraction efficiency is low, the active ingredients are easy to lose, and the like, has the characteristics of strong universality of raw materials, and has wide application prospect in the processing production of the active substances of the barks of the non-wood resources of the artificial forest.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (2)
1. A coupling extraction method of active ingredients of bark of artificial forest comprises the following steps:
(1) mixing the crushed materials of the bark of the artificial forest and a cellulose compound enzyme aqueous solution according to the material-liquid ratio of 1:14, carrying out enzymolysis for 30-60 min under the condition of water bath at the temperature of 40-70 ℃, and filtering to obtain enzymolysis filter residues and enzymolysis filtrate;
(2) mixing the enzymolysis filter residue obtained in the step (1) with 40-70% by volume of ethanol according to the material-liquid ratio of 1: 6-14, performing ultrasonic extraction at 45-55 ℃ for 40-80 min, and filtering to obtain a filtrate;
(3) combining the filtrates of step (1) and step (2) to obtain a total filtrate containing bark active ingredients;
the cellulose compound enzyme comprises cellulase and hemicellulase;
the bark is eucalyptus bark or poplar bark;
the active ingredients are one or two of flavone and polyphenol;
the mass of the cellulose compound enzyme accounts for 0.1-0.4% of the dry weight of the bark powder;
the mass concentration of the cellulose complex enzyme aqueous solution is 0.005-0.03%;
the enzyme activity of the cellulose compound enzyme is 32000U/g;
the ultrasonic frequency in the step (2) is 40 KHz.
2. The method of claim 1, wherein the total filtrate is concentrated under reduced pressure, subjected to liquid-liquid extraction, and then separated by macroporous adsorbent resin to obtain the active ingredient.
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