CN112143769A - A method for preparing radix Puerariae polypeptide extract from radix Puerariae residue and radix Puerariae polypeptide extract prepared thereby - Google Patents
A method for preparing radix Puerariae polypeptide extract from radix Puerariae residue and radix Puerariae polypeptide extract prepared thereby Download PDFInfo
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Abstract
The invention discloses a method for preparing a radix puerariae polypeptide extract by using radix puerariae dregs and the radix puerariae polypeptide extract prepared by the method, wherein the method comprises the following steps: (1) extracting with ethanol solution; (2) extracting with a buffer salt solution; (3) acid precipitation; (4) first dialysis; (5) carrying out enzymolysis; (6) carrying out second dialysis; (7) desalting with ion exchange resin. The method takes the dregs of the pueraria after isoflavone extraction as raw materials, and the pueraria polypeptide extract is prepared by extraction, acid precipitation, primary dialysis, enzymolysis, secondary dialysis and desalination, wherein the protein yield is more than or equal to 2.5 percent, the protein content is more than or equal to 87.3 percent, and the content of polypeptide with the polypeptide molecular weight less than or equal to 2kDa is more than or equal to 95.2 percent; the process has the advantages of low cost, high protein purity, low polypeptide molecular weight, comprehensive utilization of dregs and the like; the kudzu root polypeptide extract prepared by the invention has good antioxidant activity.
Description
Technical Field
The invention relates to the technical field of plant extracts, in particular to a method for preparing a radix puerariae polypeptide extract by using radix puerariae dregs and the radix puerariae polypeptide extract prepared by the method.
Background
Pueraria lobata (Willd.) Ohwi is dry root of Pueraria lobata Ohwi of Leguminosae, and is called as Pueraria lobata Ohwi. Has effects in expelling pathogenic factors from muscles, relieving fever, promoting salivation, quenching thirst, promoting eruption, invigorating yang, relieving diarrhea, dredging meridian passage, and relieving alcoholism. Can be used for treating fever, headache, stiffness and pain of neck and back, thirst, diabetes, measles without adequate eruption, dysentery due to heat evil, diarrhea, vertigo, headache, apoplexy, hemiplegia, thoracic obstruction, cardiodynia, and alcoholic injury. It is recorded in many ancient Chinese important medical records such as Shennong Ben Cao Jing, Shang Han Bing Lun and Ben Cao gang mu. Kudzu root is increasingly paid attention as a plant with homology of medicine and food. Modern researches show that the kudzuvine root contains rich nutrient components such as isoflavone substances, polyphenol, starch, protein, various trace elements and the like, and has high nutritional value and medicinal value. Wherein, the main active substance in the kudzuvine root is isoflavone. Clinical research shows that the kudzu vine root has good curative effect on treating cardiovascular and cerebrovascular diseases and the like.
Various substances such as protein, starch and the like are also remained in the residue left after the extraction of isoflavone components from the kudzuvine root. Protein is an important functional component, and is a research hotspot at home and abroad. Compared with protein, the polypeptide has the characteristics of low molecular weight, contribution to absorption by a human body, multiple special biological activities and the like.
In the prior art, CN1765923A adds water to homogenate kudzuvine root, carries out rotary separation to remove starch, carries out alkali adjustment heat treatment and acid adjustment precipitation to prepare kudzuvine root protein isolate, and the protein content in the prepared protein finished product is more than or equal to 69 percent; the pueraria protein prepared by water extraction, alkali, acid and other treatments has the defects of low protein purity, large molecular weight and the like, the application range of the product is limited, and isoflavone components in the pueraria medicinal materials are not extracted and utilized, so that the resource waste is caused.
In the prior art, CN111087479A extracts oxidized starch and kudzu root polypeptide from kudzu root crude starch, and comprises the steps of suspension, sieving, sedimentation, solid-liquid separation, concentration, drying and the like, wherein the content of the prepared kudzu root oxidized starch is more than 90%, and the content of the kudzu root polypeptide is more than 10%; the prepared pueraria polypeptide has low protein content, and the molecular weight of the polypeptide in a finished product is uneven without hydrolysis, separation and purification treatment of the protein.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the technical defects of the background technology and provide a method for preparing a radix puerariae polypeptide extract by using radix puerariae dregs and the radix puerariae polypeptide extract prepared by the method. The method takes the dregs of the pueraria after isoflavone extraction as raw materials, and the pueraria polypeptide extract is prepared by extraction, acid precipitation, primary dialysis, enzymolysis, secondary dialysis and desalination, wherein the protein yield is more than or equal to 2.5 percent, the protein content is more than or equal to 87.3 percent, and the content of polypeptide with the polypeptide molecular weight less than or equal to 2kDa is more than or equal to 95.2 percent; the process has the advantages of low cost, high protein purity, low polypeptide molecular weight, comprehensive utilization of dregs and the like; the kudzu root polypeptide extract prepared by the invention has good antioxidant activity.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for preparing radix Puerariae polypeptide extract from radix Puerariae residue comprises the following steps:
(1) ethanol solution extraction: crushing and sieving the cleaned and dried kudzu root medicinal material, adding 65-80% ethanol aqueous solution which is 10-15 times (v/m) of the medicinal material, refluxing and extracting for 3-5 h, filtering and drying;
(2) extraction with a buffer salt solution: stirring and extracting the kudzu vine root dregs subjected to alcohol extraction for 3-5 hours at 20-40 ℃ by using 10-15 times (v/m) of phosphoric acid buffer solution containing 0.05-0.1% of NaCl by mass fraction and having a concentration of 0.1-0.5 mol/L, pH of 6.5-7.5, and centrifuging after extraction is finished, and reserving a centrifugal liquid;
(3) acid precipitation: regulating the pH of the centrifugate to 3.5-5.0 by using a hydrochloric acid solution, putting the centrifugate into a refrigerator with the temperature of 3-5 ℃, standing for 12-24 hours, centrifuging, and keeping a precipitate;
(4) first dialysis: dissolving the acid precipitation precipitate with appropriate amount of water, and properly adjusting pH to make it fully dissolved; putting the solution into proper purified water by using a dialysis membrane with the molecular weight cutoff of 3500Da for dialysis for 10-20 h, and fully dialyzing to keep the membrane internal solution;
(5) enzymolysis: regulating the pH value of the membrane internal solution to 3.5-5.0 by using hydrochloric acid solution, adding acid protease with the amount of 0.1-0.5% of the medicinal material, carrying out enzymolysis for 2-4 h at 40-60 ℃, and stabilizing the pH value of the enzymolysis solution by adding a proper amount of acid and alkali in the enzymolysis process; after enzymolysis is finished, adjusting the pH value of the solution after enzymolysis to 6.5-7.5 by using NaOH solution, adding neutral protease or papain with the amount of 0.1-0.5% of the amount of medicinal materials, carrying out enzymolysis for 2-4 h at 40-60 ℃, and stabilizing the pH value of the enzymolysis solution by adding a proper amount of acid and alkali in the enzymolysis process; after enzymolysis is finished, boiling the solution to inactivate enzyme;
(6) and (3) second dialysis: putting the enzymatic hydrolysate into proper purified water for dialysis for 10-20 h by using a dialysis membrane with the molecular weight cutoff of 2000Da, and reserving the liquid outside the membrane;
(7) desalting with ion exchange resin: desalting the second dialysate by mixed bed ion exchange resin, wherein the resin is mixed resin prepared by Amberlite MB20 type mixed ion exchange resin or 732 cation exchange resin and D301-G type anion exchange resin according to a ratio of 2: 3(v/v), the column passing speed is 8-15 times column volume/h, the resin dosage is 1/30-1/10 of the weight of the radix puerariae decoction dregs, and the effluent liquid from the column is concentrated, dried and crushed in vacuum to obtain the radix puerariae polypeptide extract.
Preferably, in the step (1), the mesh number during sieving is 30-60 meshes.
Preferably, in the step (1), the drying method includes drying or naturally airing the ethanol remaining in the filter residue in a forced air drying oven at 50-60 ℃.
Preferably, in the step (3), the mixture is placed into a refrigerator at 4 ℃ and is kept still for 12 hours.
Preferably, in the step (5), the time for boiling to inactivate the enzyme is 10-15 min.
A radix Puerariae polypeptide extract is prepared by the above method for preparing radix Puerariae polypeptide extract from radix Puerariae residue.
In the technical scheme, the percentage is mass percentage.
The basic principle of the invention is as follows:
the protein is one of the main components of the kudzuvine root, accounts for about 7 to 12 percent of the dry weight of the kudzuvine root, and the protein is extracted from the kudzuvine root dregs after the extraction of ethanol water solution by using buffer salt solution with certain concentration; adjusting pH value to precipitate protein in the extractive solution within isoelectric point; dialyzing the acid precipitation to remove small molecular impurities and salt, and hydrolyzing proteins in the precipitation by using proteolytic enzyme to hydrolyze large molecular weight proteins into small molecular weight polypeptides; dialyzing the solution after dissolving the polypeptide through a dialysis membrane, transferring the small molecular polypeptide out of the dialysis membrane, and leaving impurities with large molecular weight and a part of protein which is not completely enzymolyzed in the membrane; and (3) treating the membrane external liquid by desalting resin, removing salt in the membrane external liquid, concentrating the sample, and drying in vacuum to obtain the pueraria polypeptide extract.
Compared with the prior art, the invention has the beneficial effects that:
(1) the method takes the dregs of the pueraria after isoflavone extraction as raw materials, and the pueraria polypeptide extract is prepared by extraction, acid precipitation, primary dialysis, enzymolysis, secondary dialysis and desalination, wherein the protein yield is more than or equal to 2.5 percent, the protein content is more than or equal to 87.3 percent, and the content of polypeptide with the polypeptide molecular weight less than or equal to 2kDa is more than or equal to 95.2 percent;
(2) the process has the advantages of low cost, high protein purity, low polypeptide molecular weight, comprehensive utilization of dregs and the like;
(3) the kudzu root polypeptide extract prepared by the invention has good antioxidant activity;
(4) the invention has simple and convenient process and is easy for industrialized production;
(5) the invention can comprehensively utilize the dregs and the waste materials generated in the production process of the kudzuvine root, and can develop new products and increase the enterprise benefit.
Drawings
FIG. 1 shows DPPH radical scavenging effect of the polypeptides extract of Pueraria lobata prepared in example 2 and comparative example 3 of the present invention;
FIG. 2 shows the scavenging effect of pueraria polypeptide extracts on hydroxyl radical (. OH) prepared in example 2 and comparative example 3 of the present invention;
FIG. 3 shows the reducing power of the polypeptides extract of Pueraria lobata obtained in example 2 and comparative example 3 of the present invention;
FIG. 4 shows the reducing power of Vc as a positive control.
Detailed Description
For a better understanding of the present invention, reference is made to the following detailed description and accompanying drawings. It is to be understood that these examples are for further illustration of the invention and are not intended to limit the scope of the invention. In addition, it should be understood that the invention is not limited to the above-described embodiments, but is capable of various modifications and changes within the scope of the invention.
Example 1
Pulverizing cleaned and dried radix Puerariae, sieving to 60 mesh, adding 10 times (v/m) of 80% ethanol water solution, reflux extracting for 3 hr, filtering, and drying the residual ethanol in filter residue in 60 deg.C blast drying oven. Extracting the dried radix Puerariae residue with 15 times (v/m) of 0.05% NaCl-containing 6.5 mol/L, pH phosphoric acid buffer solution at 25 deg.C under stirring for 5 hr. After extraction, centrifugation is carried out, and the centrifuged clear liquid is reserved. Adjusting pH of the centrifugate to 3.5 with hydrochloric acid solution, standing in a refrigerator at 4 deg.C for 12 hr, centrifuging, and retaining precipitate. Dissolving the acid precipitation with appropriate amount of water, adjusting pH to dissolve completely, dialyzing the solution with dialysis membrane with cut-off molecular weight of 3500Da in appropriate amount of purified water for 20 hr, and retaining membrane fluid. Regulating pH of the membrane internal solution to 3.5 with hydrochloric acid solution, adding acid protease with 0.1% of medicinal material amount, performing enzymolysis at 55 deg.C for 4h, and adding appropriate amount of acid and alkali during enzymolysis to stabilize pH value of the enzymolysis solution. After enzymolysis is finished, adjusting the pH value of the solution after enzymolysis to 7.0 by using NaOH solution, adding neutral protease with the amount of 0.1 percent of the amount of the medicinal material, performing enzymolysis for 4 hours at 50 ℃, and stabilizing the pH value of the enzymolysis solution by adding a proper amount of acid and alkali in the enzymolysis process. After the enzymolysis is finished, the solution is boiled for 10min to inactivate the enzyme. And (3) putting the enzymatic hydrolysate into proper purified water for dialysis for 20 hours by using a dialysis membrane with the molecular weight cutoff of 2000Da, and reserving the liquid outside the membrane. Desalting the second dialysate with mixed ion exchange resin of Amberlite MB20 type, wherein the column passing speed is 10 times of column volume/h, and the resin dosage is 1/10 of radix Puerariae residue weight. Concentrating the effluent of the lower column under reduced pressure, vacuum drying, and pulverizing to obtain radix Puerariae polypeptide extract.
The detection calculation shows that the protein yield of the kudzu root polypeptide extract is 2.9%, the protein content is 87.5%, and the ratio of the polypeptide molecular weight less than or equal to 2kDa is 95.2%.
Example 2
Pulverizing cleaned and dried radix Puerariae, sieving to 60 mesh, adding 15 times (v/m) 70% ethanol water solution, reflux extracting for 5 hr, filtering, and naturally drying the residual ethanol in the residue. Extracting radix Puerariae residue with 15 times (v/m) of 0.1% NaCl-containing 7.0 mol/L, pH phosphoric acid buffer solution at 25 deg.C under stirring for 5 hr, centrifuging, and collecting the centrifugate. Adjusting pH of the centrifugate to 4.0 with hydrochloric acid solution, standing in a refrigerator at 4 deg.C for 12 hr, centrifuging, and retaining precipitate. Dissolving the acid precipitate with appropriate amount of water, and adjusting pH to dissolve completely. The solution is put into proper purified water by a dialysis membrane with the molecular weight cutoff of 3500Da for dialysis for 12 hours and is fully dialyzed, and the membrane internal solution is reserved. Regulating pH of the membrane internal solution to 4.0 with hydrochloric acid solution, adding acid protease with 0.3% of medicinal material amount, performing enzymolysis at 55 deg.C for 3h, and stabilizing pH value of the enzymolysis solution by adding appropriate amount of acid and alkali during enzymolysis. After enzymolysis is finished, adjusting the pH value of the solution after enzymolysis to 7.0 by using NaOH solution, adding neutral protease with the amount of 0.3 percent of the amount of the medicinal material, performing enzymolysis for 3 hours at 55 ℃, and stabilizing the pH value of the enzymolysis solution by adding a proper amount of acid and alkali in the enzymolysis process. After the enzymolysis is finished, the solution is boiled for 10min to inactivate the enzyme. And (3) putting the enzymatic hydrolysate into proper purified water for dialysis for 12h by using a dialysis membrane with the molecular weight cutoff of 2000Da, and reserving the liquid outside the membrane. Desalting the second dialysate with mixed bed ion exchange resin (732 cation exchange resin and D301-G anion exchange resin at a ratio of 2: 3(v/v), wherein the column passing speed is 12 times column volume/h, and the resin amount is 1/20 of radix Puerariae residue weight. Concentrating the effluent liquid, vacuum drying, and pulverizing to obtain radix Puerariae polypeptide extract.
The detection calculation shows that the protein yield of the kudzu root polypeptide extract is 2.5%, the protein content is 87.3%, and the ratio of the molecular weight of the polypeptide less than or equal to 2kDa is 96.8%.
Example 3
Pulverizing cleaned and dried radix Puerariae, sieving to 40 mesh, adding 15 times (v/m) 70% ethanol water solution, reflux extracting for 5 hr, filtering, and naturally drying the residual ethanol in the residue. Extracting radix Puerariae residue with 15 times (v/m) of 0.05% NaCl-containing 7.5 mol/L, pH phosphoric acid buffer solution at 30 deg.C under stirring for 5 hr, centrifuging, and collecting the centrifugate. Adjusting pH of the centrifugate to 3.5 with hydrochloric acid solution, standing in a refrigerator at 4 deg.C for 12 hr, centrifuging, and retaining precipitate. Dissolving the acid precipitate with appropriate amount of water, and adjusting pH to dissolve completely. The solution is put into proper purified water by a dialysis membrane with the molecular weight cutoff of 3500Da for dialysis for 20 hours and is fully dialyzed, and the membrane internal solution is reserved. Regulating pH of the membrane internal solution to 3.5 with hydrochloric acid solution, adding acid protease with 0.3% of medicinal material amount, performing enzymolysis at 55 deg.C for 3h, and stabilizing pH value of the enzymolysis solution by adding appropriate amount of acid and alkali during enzymolysis. After enzymolysis is finished, adjusting the pH value of the solution after enzymolysis to 7.0 by using NaOH solution, adding papain with the amount of 0.5 percent of the medicinal material, carrying out enzymolysis for 3h at 50 ℃, and stabilizing the pH value of the enzymolysis solution by adding a proper amount of acid and alkali in the enzymolysis process. After the enzymolysis is finished, the solution is boiled for 15min to inactivate the enzyme. And (3) putting the enzymatic hydrolysate into proper purified water for dialysis for 18h by using a dialysis membrane with the molecular weight cutoff of 2000Da, and reserving the liquid outside the membrane. Desalting the second dialysate with mixed ion exchange resin of Amberlite MB20 type, wherein the column passing speed is 10 times of column volume/h, and the resin dosage is 1/10 of radix Puerariae residue weight. Concentrating the effluent liquid, vacuum drying, and pulverizing to obtain radix Puerariae polypeptide extract.
The detection calculation shows that the protein yield of the kudzu root polypeptide extract is 3.2%, the protein content is 88.4%, and the ratio of the molecular weight of the polypeptide to the molecular weight of the polypeptide is less than or equal to 2kDa is 96.6%.
Comparative example 1
Pulverizing cleaned and dried radix Puerariae, sieving to 40 mesh, adding 15 times (v/m) 70% ethanol water solution, reflux extracting for 5 hr, filtering, and naturally drying the residual ethanol in the residue. Extracting radix Puerariae residue with 15 times (v/m) of water at 50 deg.C under stirring for 5 hr, centrifuging, and collecting the centrifugate. Adjusting pH of the centrifugate to 3.5 with hydrochloric acid solution, standing in a refrigerator at 4 deg.C for 12 hr, centrifuging, and retaining precipitate. Dissolving the acid precipitate with appropriate amount of water, and adjusting pH to dissolve completely. The solution is put into proper purified water by a dialysis membrane with the molecular weight cutoff of 3500Da for dialysis for 20 hours and is fully dialyzed, and the membrane internal solution is reserved. Regulating pH of the membrane internal solution to 3.5 with hydrochloric acid solution, adding acid protease with 0.3% of medicinal material amount, performing enzymolysis at 55 deg.C for 3h, and stabilizing pH value of the enzymolysis solution by adding appropriate amount of acid and alkali during enzymolysis. After enzymolysis is finished, adjusting the pH value of the solution after enzymolysis to 7.0 by using NaOH solution, adding papain with the amount of 0.5 percent of the medicinal material, carrying out enzymolysis for 3h at 50 ℃, and stabilizing the pH value of the enzymolysis solution by adding a proper amount of acid and alkali in the enzymolysis process. After the enzymolysis is finished, the solution is boiled for 15min to inactivate the enzyme. And (3) putting the enzymatic hydrolysate into proper purified water for dialysis for 18h by using a dialysis membrane with the molecular weight cutoff of 5000Da, and reserving the liquid outside the membrane. Desalting the second dialysate with mixed ion exchange resin of Amberlite MB20 type, wherein the column passing speed is 10 times of column volume/h, and the resin dosage is 1/10 of radix Puerariae residue weight. Concentrating the effluent liquid, vacuum drying, and pulverizing to obtain radix Puerariae polypeptide extract.
The detection calculation shows that the protein yield of the kudzu root polypeptide extract is 1.7%, the protein content is 48.4%, and the ratio of the molecular weight of the polypeptide to the molecular weight of the polypeptide is less than or equal to 2kDa is 74.1%.
Comparative example 2
Pulverizing cleaned and dried radix Puerariae, sieving to 60 mesh, adding 10 times (v/m) of 80% ethanol water solution, reflux extracting for 3 hr, filtering, and drying the residual ethanol in filter residue in 60 deg.C blast drying oven. Extracting the dried radix Puerariae residue with 15 times (v/m) of 0.1mol/L, pH 6.5 phosphoric acid buffer solution containing 0.1% NaCl at 25 deg.C under stirring for 5 hr. After extraction, centrifugation is carried out, and the centrifuged clear liquid is reserved. Adjusting pH of the centrifugate to 3.5 with hydrochloric acid solution, standing in a refrigerator at 4 deg.C for 12 hr, centrifuging, and retaining precipitate. Dissolving the acid precipitation with appropriate amount of water, adjusting pH to dissolve completely, dialyzing the solution with dialysis membrane with cut-off molecular weight of 3500Da in appropriate amount of purified water for 20 hr, and retaining membrane fluid. Regulating pH of the membrane internal solution to 3.5 with hydrochloric acid solution, adding acid protease with 0.1% of medicinal material amount, performing enzymolysis at 55 deg.C for 4h, and adding appropriate amount of acid and alkali during enzymolysis to stabilize pH value of the enzymolysis solution. After enzymolysis is finished, adjusting the pH value of the solution after enzymolysis to 7.0 by using NaOH solution, adding neutral protease with the amount of 0.1 percent of the amount of the medicinal material, performing enzymolysis for 4 hours at 50 ℃, and stabilizing the pH value of the enzymolysis solution by adding a proper amount of acid and alkali in the enzymolysis process. After enzymolysis, boiling the solution for 10min to inactivate enzyme, concentrating under reduced pressure, vacuum drying, and pulverizing to obtain radix Puerariae polypeptide extract.
The detection calculation shows that the protein yield of the kudzu root polypeptide extract is 1.3%, the protein content is 42.9%, and the ratio of the molecular weight of the polypeptide to the molecular weight of the polypeptide is less than or equal to 2kDa is 55.7%.
Comparative example 3
Pulverizing cleaned and dried radix Puerariae, sieving to 60 mesh, adding 15 times (v/m) 70% ethanol water solution, reflux extracting for 5 hr, filtering, and naturally drying the residual ethanol in the residue. Extracting radix Puerariae residue with 15 times (v/m) of 0.05% NaCl-containing phosphate buffer solution with concentration of 0.1mol/L, pH of 6.5 at 25 deg.C under stirring for 5 hr, centrifuging after extraction, and retaining centrifugate. Adjusting pH of the centrifugate to 4.0 with hydrochloric acid solution, standing in a refrigerator at 4 deg.C for 12 hr, centrifuging, and retaining precipitate. Dissolving the acid precipitate with appropriate amount of water, and adjusting pH to dissolve completely. The solution is put into proper purified water by a dialysis membrane with the molecular weight cutoff of 3500Da for dialysis for 12 hours and is fully dialyzed, and the membrane internal solution is reserved. Regulating the pH value of the membrane internal solution to 9.0 by using NaOH solution, adding alkaline protease with the amount of 0.1 percent of the medicinal material, carrying out enzymolysis for 3h at 55 ℃, and stabilizing the pH value of the enzymolysis solution by adding a proper amount of acid and alkali in the enzymolysis process. After the enzymolysis is finished, the solution is boiled for 10min to inactivate the enzyme. And (3) putting the enzymatic hydrolysate into proper purified water for dialysis for 12h by using a dialysis membrane with the molecular weight cutoff of 5000Da, and reserving the liquid outside the membrane. Desalting the second dialysate with mixed bed ion exchange resin (732 cation exchange resin and D301-G anion exchange resin at a ratio of 2: 3(v/v), wherein the column passing speed is 12 times column volume/h, and the resin amount is 1/20 of radix Puerariae residue weight. Concentrating the effluent liquid, vacuum drying, and pulverizing to obtain radix Puerariae polypeptide extract.
The detection calculation shows that the protein yield of the kudzu root polypeptide extract is 2.3%, the protein content is 58.3%, and the ratio of the molecular weight of the polypeptide to the molecular weight of the polypeptide is not more than 2kDa is 66.7%.
Effects of the embodiment
1. Determination of protein content, protein yield and ratio of polypeptide molecular weight less than or equal to 2kDa in radix Puerariae polypeptide extract prepared in examples 1-3 and comparative examples 1-3
(1) Related detection method for pueraria polypeptide extract
The protein content detection method comprises the following steps: the determination method is based on GB 5009.5-2016;
the molecular weight of the polypeptide is less than or equal to 2 kDa: the determination method is based on GB 31645-2018.
(2) Formula for calculation
Protein content ═ (weight of protein in extract/weight of extract) × 100%;
the protein yield is (weight of extract x protein content in extract/amount of drug material) x 100%.
The protein content, the protein yield and the ratio of the polypeptide molecular weight less than or equal to 2kDa in the pueraria polypeptide extract prepared in the examples 1-3 and the comparative examples 1-3 are shown in the following table.
2. Experiment of in vitro antioxidant activity of kudzu root polypeptide extract
2.1 Experimental materials for radix Puerariae polypeptide extract
Kudzu root polypeptide extract 1: example 2 preparation of kudzu root polypeptide extract sample; kudzu root polypeptide extract 2: the pueraria polypeptide extract sample prepared in comparative example 3.
2.2DPPH free radical scavenging assay
Accurately sucking prepared sample solutions of 0, 0.2, 0.4, 0.6, 0.8 and 1.0mg/mL of kudzu root polypeptide extract 1, 2 of kudzu root polypeptide extract and 2mL of positive control Vc into dry and clean glass test tubes with plugs respectively, adding 2mL of DPPH ethanol solution with the mass fraction of 0.004% into each test tube respectively under the condition of keeping out of the sun, uniformly mixing, reacting for 30min in a dark environment, measuring the absorbance value at the wavelength of 517nm after the reaction is finished, repeating the test for 3 times, and taking the average value of the result. The clearance calculation formula is as follows:
in the formula: a. the0The absorbance value of 2mL of pure water and 2mL of DPPH ethanol solution is added; a. the1Adding 2mL of DPPH ethanol solution absorbance value into 2mL of solution to be detected; a. the2The absorbance value of the solution to be detected is 2mL and the absolute ethyl alcohol solution is 2 mL.
2.3 measurement of hydroxyl radical (. OH) scavenging action
1mL of 9mmol/L FeSO4Sequentially adding the solution and 2mL of 9mmol/L salicylic acid-ethanol solution into each dry and clean glass test tube with a plug, mixing, adding 2mL of prepared radix Puerariae polypeptide extract 1, radix Puerariae polypeptide extract 2 and positive control Vc solution with mass concentrations of 0, 0.5, 1.0, 1.5, 2.0 and 2.5mg/mL respectively, and finally adding 2mL of 8.8mmol/L H2O2The solution was started and allowed to react at room temperature for 1h, and its absorbance was measured at a wavelength of 510 nm. The test was repeated 3 times and the results averaged. The clearance calculation formula is as follows:
in the formula: a. the1For adding polysaccharide solution and H with different mass concentrations2O2Absorbance of the solution; a. the2For adding an equal volume of H2O instead of H2O2Absorbance of the solution；A0For adding an equal volume of H2O-substituted polysaccharide solution and H2O2Absorbance of the solution.
2.4 measurement of reducing ability
Respectively sucking 1mL of prepared sample solution of radix Puerariae polypeptide extract 1 and 2 and 1mL of positive control Vc solution (the concentrations are respectively 0, 20, 40, 60, 80 and 100 mu g/mL) with mass concentrations of 0, 0.1, 0.2, 0.3, 0.4 and 0.5mg/mL, adding into each dry and clean glass test tube with plug, 2.5mL of phosphate buffer (pH 6.6, 0.2mol/L) and 2.5mL of 1% potassium ferricyanide solution were added to each tube, mixed well, and then subjected to water bath at 50 ℃ for 20min, then respectively adding 2.5mL of trichloroacetic acid solution with the mass fraction of 10%, centrifuging for 10min at the rotating speed of 3000r/min by a centrifuge, respectively and precisely absorbing 2.5mL of supernatant by a pipette gun, respectively adding 2.5mL of pure water and 2.5mL of prepared 0.1% FeCl3 solution, uniformly mixing, standing at room temperature for 30min, and measuring the absorbance value of the solution at the wavelength of 700 nm. The test was repeated 3 times and the results averaged.
2.5 results of antioxidant experiments
2.5.1DPPH radical scavenging action
The rate of DPPH radical scavenging is related to the ability of the antioxidant to provide hydrogen.
As can be seen from figure 1, the pueraria polypeptide extract 1 and the pueraria polypeptide extract 2 both have a certain DPPH free radical scavenging effect, the pueraria polypeptide extract 1 has a scavenging capacity obviously higher than that of the pueraria polypeptide extract 2, and when the concentration is 1.0mg/ml, the scavenging rates of the pueraria polypeptide extract 1 and the pueraria polypeptide extract 2 on DPPH free radicals are respectively 98.6% and 87.9%.
2.5.2 hydroxyl radical (. OH) scavenging action
As can be seen from FIG. 2, the pueraria polypeptide extract 1 and the pueraria polypeptide extract 2 have a scavenging effect on hydroxyl radicals (. OH), the scavenging capacity of the pueraria polypeptide extract 1 is obviously higher than that of the pueraria polypeptide extract 2, and when the concentration is 2.5mg/ml, the scavenging rates of the pueraria polypeptide extract 1 and the pueraria polypeptide extract 2 on DPPH radicals are 96.3% and 79.7% respectively.
2.5.3 reducing ability
As can be seen from fig. 3, the pueraria polypeptide extract 1 and the pueraria polypeptide extract 2 have a certain degree of reducing power, and the reducing power of the pueraria polypeptide extract 1 is higher than that of the pueraria polypeptide extract 2, and when the concentration is 0.5mg/ml, the absorbance values of the pueraria polypeptide extract 1 and the pueraria polypeptide extract 2 are 0.54 and 0.32 respectively, but the reducing power is lower than that of the positive control Vc.
2.6 summary
From the in vitro antioxidant activity test results of the pueraria polypeptide extract, compared with the comparative example 3, the content of protein and the molecular weight of polypeptide in the pueraria polypeptide extract prepared in the example 2 are less than or equal to 2kDa, so that the antioxidant activity of the pueraria polypeptide extract prepared in the example 2 is better.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.
Claims (6)
1. A method for preparing a radix puerariae polypeptide extract by using radix puerariae dregs is characterized by comprising the following steps:
(1) ethanol solution extraction: crushing and sieving the cleaned and dried kudzu root medicinal material, adding 65-80% ethanol water in an amount which is 10-15 times that of the medicinal material, performing reflux extraction for 3-5 hours, filtering, and drying;
(2) extraction with a buffer salt solution: stirring and extracting the kudzu vine root dregs subjected to alcohol extraction for 3-5 h at 20-40 ℃ by using 10-15 times of phosphoric acid buffer solution containing 0.05-0.1% of NaCl by mass and having a concentration of 0.1-0.5 mol/L, pH of 6.5-7.5, centrifuging after extraction is finished, and reserving centrifugate;
(3) acid precipitation: regulating the pH of the centrifugate to 3.5-5.0 by using a hydrochloric acid solution, putting the centrifugate into a refrigerator with the temperature of 3-5 ℃, standing for 12-24 hours, centrifuging, and keeping a precipitate;
(4) first dialysis: dissolving the acid precipitation precipitate with appropriate amount of water, and properly adjusting pH to make it fully dissolved; putting the solution into proper purified water by using a dialysis membrane with the molecular weight cutoff of 3500Da for dialysis for 10-20 h, and fully dialyzing to keep the membrane internal solution;
(5) enzymolysis: regulating the pH value of the membrane internal solution to 3.5-5.0 by using hydrochloric acid solution, adding acid protease with the amount of 0.1-0.5% of the medicinal material, carrying out enzymolysis for 2-4 h at 40-60 ℃, and stabilizing the pH value of the enzymolysis solution by adding a proper amount of acid and alkali in the enzymolysis process; after enzymolysis is finished, adjusting the pH value of the solution after enzymolysis to 6.5-7.5 by using NaOH solution, adding neutral protease or papain with the amount of 0.1-0.5% of the amount of medicinal materials, carrying out enzymolysis for 2-4 h at 40-60 ℃, and stabilizing the pH value of the enzymolysis solution by adding a proper amount of acid and alkali in the enzymolysis process; after enzymolysis is finished, boiling the solution to inactivate enzyme;
(6) and (3) second dialysis: putting the enzymatic hydrolysate into proper purified water for dialysis for 10-20 h by using a dialysis membrane with the molecular weight cutoff of 2000Da, and reserving the liquid outside the membrane;
(7) desalting with ion exchange resin: desalting the second dialysate by mixed bed ion exchange resin, wherein the resin is Amberlite MB20 type mixed ion exchange resin or mixed resin prepared by 732 cation exchange resin and D301-G type anion exchange resin according to a ratio of 2: 3, the column passing speed is 8-15 times of column volume/h, the resin dosage is 1/30-1/10 of the weight of the radix puerariae decoction dregs, and the effluent liquid from the column is concentrated, dried and crushed in vacuum to obtain the radix puerariae polypeptide extract.
2. The method for preparing the pueraria lobata polypeptide extract by using the pueraria lobata dregs as claimed in claim 1, wherein in the step (1), the sieving is performed by 30-60 meshes.
3. The method for preparing the pueraria lobata polypeptide extract by using the pueraria lobata dregs as claimed in claim 1, wherein in the step (1), the drying method comprises drying or naturally drying the ethanol remaining in the filter residue in a forced air drying oven at 50-60 ℃.
4. The method for preparing pueraria lobata polypeptide extract using pueraria lobata dregs as claimed in claim 1, wherein in the step (3), the pueraria lobata is left to stand in a refrigerator at 4 ℃ for 12 hours.
5. The method for preparing pueraria lobata polypeptide extract from pueraria lobata dregs as claimed in claim 1, wherein in the step (5), the enzyme is boiled for 10-15 min.
6. A radix puerariae polypeptide extract is characterized by being prepared by the method for preparing the radix puerariae polypeptide extract by using radix puerariae dregs as claimed in any one of claims 1 to 5.
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