CN112979489A - Method for preparing amino acid from notoginseng flower - Google Patents
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Abstract
The invention relates to the technical field of extraction of plant active ingredients, in particular to a method for preparing amino acid from notoginseng flower, which comprises the following steps: (1) drying Notoginseng, pulverizing, adding enzyme and water for enzymolysis, adding ethanol for extraction, filtering to obtain extractive solution, concentrating, and recovering ethanol to obtain extract; (2) mixing the extract with the extractive solution, pouring into a separating funnel, standing for layering, collecting the lower layer liquid, and concentrating to obtain fluid extract; (3) cooling the fluid extract, crystallizing, filtering to obtain supernatant, and diluting with water to obtain diluent; (4) passing the diluent obtained in the step (3) through cation and anion resin for exchange adsorption, then sequentially eluting by using water and an ammonia water solution, and merging and collecting the ammonia water eluent; (5) concentrating the ammonia water eluate under reduced pressure, and freeze drying. The method has the advantages of high yield and purity, short extraction time and low cost.
Description
Technical Field
The invention relates to the technical field of extraction of plant active ingredients, in particular to a method for preparing amino acid from notoginseng flower.
Background
Notoginseng flower is flower of Panax notoginseng (Burk.) F.H.Chen ex C.Chow.) belonging to Araliaceae. The notoginseng flower has good hemostatic effect and tranquilizing and brain nourishing effects, promotes the division and growth of various blood cells, increases the number of the blood cells, can obviously improve the quantity of the red blood cells of the blood loss anemia, and has better treatment effect on the blood loss anemia. Sanchinin amino acid in Notoginseng has hemostatic effect, and gamma-aminobutyric acid has tranquilizing effect. The notoginseng essence has the highest content in notoginseng flower, and the total amino acid content in notoginseng flower is also the highest and higher than that in notoginseng root and stem.
Notoginseng flower is the high content part of notoginseng saponin in the whole plant of notoginseng, so that dozens of saponin components including ginsenoside Rb1, Rb2, Rb3, Rc, Rd and the like are found, wherein the ginsenoside Rb3 and Rc have the highest content and are the first important component of notoginseng flower. In addition, the chemical components in the notoginseng flower also comprise amino acids, flavonoids, polysaccharides, volatile oils, inorganic substances and the like, and the notoginseng flower has wide pharmacological effects. The amino acids are the second important components of Notoginseng, and Notoginseng radix contains more than ten kinds of amino acids, such as arginine, alanine, tyrosine, phenylalanine, proline, valine, cystine, isoleucine, leucine, threonine, lysine, tryptophan, glutamic acid, aspartic acid, etc. Notoginseng root with total amino acid content higher than 10% and amino acid content higher than 7%, and 7 kinds of essential amino acids in Notoginseng.
The amino acid is the second most important active ingredient in the panax notoginseng, such as dencichine, gamma aminobutyric acid and the like, and the content of the amino acid in the panax notoginseng flower in the panax notoginseng whole plant is the highest. At present, few reports are reported on the preparation process for preparing amino acid from notoginseng flower, the traditional small-test analysis preparation method is usually water extraction, organic solvent extraction is carried out to remove components with polarity smaller than that of amino acid, such as saponin, and then the amino acid is prepared by electrophoresis, lead salt precipitation, isoelectric point method and anion or cation exchange method. Ion exchange chromatography reported in literature, wherein imported packing is expensive and is mainly used for preparing amino acid monomers, and common anion or cation exchange resins are also used for preparing total amino acids, such as 201X4 and 001X7, however, when the resins are applied to amino acid preparation, the exchange speed is slow, the loading is fast, the exchange is easy to be incomplete, and the exchange unit amount is very small; according to the characteristics of amino acid, the exchange capacity difference of acidic, basic and neutral amino acid on anions and cations is large, and the exchange speed difference is also large, so that the common anion-cation resin is not ideal for preparing the sanchi flower amino acid.
The industrial purification difficulty of the notoginseng flower amino acid is mainly in the separation of notoginseng polysaccharide, the notoginseng polysaccharide and notoginseng polysaccharide are both large-polarity water-soluble components, the conventional chemical reagent precipitation method is difficult to industrially implement, an ion exchange method is also used for amino acid purification, the current industrial commonly used ion exchange is mainly large-particle cation resin, the exchange equivalent is small, the particles are large, the exchange speed is slow, the resin consumption is high, and the pH value is adjusted when the ion exchange resin is used.
Chinese patent application CN1865232A discloses a method for extracting dencichine from radix Notoginseng, which belongs to the technical field of medicaments, and comprises the steps of adopting an alcohol solution extraction method, then carrying out water extraction treatment, adding ethanol into an aqueous solution for precipitation, filtering out impurities, extracting the aqueous solution by using n-butyl alcohol, combining aqueous phases, directly removing sugar, phytochrome and the like in the aqueous solution by using an ion exchange resin (such as D001 type ion exchange resin) column process polymerized by taking styrene and diethylbenzene as monomers, precipitating acetone in eluent, and finally carrying out recrystallization to obtain the dencichine with higher purity. The method is safe, low in cost, suitable for industrial production and capable of extracting the dencichine with higher purity. In the prior art, n-butanol is used for extraction, and the n-butanol has special smell and is regurgitated by people; steam may cause drowsiness and dizziness; stimulating the respiratory system and the skin. Moreover, the method is mainly used for extracting the dencichine, and the yield of the total amino acid is low.
Chinese patent application CN107162926A discloses a dencichine extraction method, which comprises extracting dencichine from industrial waste residues of radix Notoginseng, performing subsequent purification, crystallization and other subsequent steps under the condition that the obtained product contains dencichine as verified by thin layer detection during the extraction process, combining the eluents which have only one spot and have the same Rf value as the control of dencichine, and crystallizing. The invention also provides a method for extracting pseudo-ginseng amino acid, which takes industrial pseudo-ginseng waste residue as a raw material, and has the same process steps as the pseudo-ginseng extract extraction method, except that during crystallization, the combined eluent and a pseudo-ginseng extract reference product have the same spots and other spots. The invention further provides application of the obtained dencichine, which can be used for preparing products with hemostatic function, such as toothpaste, band-aid and the like. However, the patent application is also mainly used for extracting the dencichine, and does not disclose how to extract other amino acids, and the purity of the obtained dencichine is low.
Therefore, it is necessary to develop a method for extracting amino acids from notoginseng flowers, which can solve the above-mentioned problems.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for extracting amino acid from notoginseng flower, which has high yield and purity, short extraction time and low cost.
The invention is realized by the following technical scheme:
the notoginseng or notoginseng flower is rich in vegetable protein, the vegetable polypeptide is macromolecular substance, in order to improve the amino acid yield, the protein polypeptide is firstly hydrolyzed into amino acid, so as to improve the amino acid yield, if not hydrolyzed, the macromolecular protein and polypeptide can come down along with the amino acid when the ammonia water is eluted even if the macromolecular protein and polypeptide are absorbed by ion exchange, thereby influencing the amino acid purity; the ion resin adopted by the invention is powder resin, the granularity is very small, the exchange speed is high, the exchange single amount is large, the elution loss solvent is less, and the sample loading speed and the exchange speed are greatly improved by adopting the exchange adsorption of anions and cations.
A method for preparing amino acids from flos Notoginseng comprises the following steps:
(1) drying Notoginseng, pulverizing, adding enzyme and water for enzymolysis, adding ethanol for extraction, filtering to obtain extractive solution, concentrating, and recovering ethanol to obtain extract;
(2) mixing the extract with the extractive solution, pouring into a separating funnel, standing for layering, collecting the lower layer liquid, and concentrating to obtain fluid extract;
(3) cooling the fluid extract, crystallizing, filtering to obtain supernatant, and diluting with water to obtain diluent;
(4) performing exchange adsorption on the diluent obtained in the step (3) through cation resin and anion resin, loading the lower layer of the anion resin and the upper layer of the cation resin during column packing, loading the sample after pure water is balanced, stopping loading the sample after exchange is saturated, sequentially eluting with water and an ammonia water solution, and combining and collecting the ammonia water eluent;
(5) concentrating the ammonia water eluate under reduced pressure to obtain soft extract, and freeze drying.
Preferably, the enzyme in step (1) is a mixture of neutral protease and at least one of papain and soybean peptide hydrolase.
The enzyme of the invention is used for hydrolyzing proteins, polypeptides and polysaccharides in the notoginseng flower and combining the proteins.
More preferably, the mass ratio of the at least one of papain and soybean peptide hydrolase to the neutral protease is 1.5-2: 0.5-1.
More preferably, the enzyme is a mixture of papain and neutral protease, and the mass ratio of the papain to the neutral protease is 1.5-2: 0.5-1.
More preferably, the enzyme is a mixture of soybean peptide hydrolase and neutral protease, and the mass ratio of the soybean peptide hydrolase to the neutral protease is 1.5-2: 0.5-1.
Preferably, the amount of the enzyme is 0.1-0.2% of the mass of the dried notoginseng flower.
Preferably, the volume-to-mass ratio of the volume of the water to the mass of the dried notoginseng flower is 6-8 mL/g.
Preferably, the extraction process in the step (1) is specifically as follows: drying Notoginseng flower, pulverizing, adding 0.1-0.2% enzyme and 6-8 times of water for enzymolysis, adding 95% ethanol until the alcohol content of the whole system is 50-65%, extracting for 2-3 times, filtering to obtain extractive solution, concentrating, and recovering ethanol to obtain extract.
More preferably, the step (1) is adding 95% ethanol until the ethanol content is 50-65%, extracting at 45-55 deg.C for 2-3h, filtering to obtain first filter residue and first filtrate, adding 5-7 times of 50-65% ethanol into the first filter residue, extracting at 45-55 deg.C for 1-2h, filtering to obtain second filtrate, mixing the first filtrate and the second filtrate to obtain extractive solution, vacuum concentrating at 40-60 deg.C, and recovering ethanol to 1 times of crude drug mass (1 time of Notoginseng flower mass) to obtain extract.
More preferably, step (1) comprises the steps of:
(1) drying and crushing notoginseng flower, adding 0.1-0.2% enzyme and 6-8 times of water for enzymolysis for 30-50min, adding 95% ethanol until the alcohol content of the whole system is 50-65%, extracting at 45-55 ℃ for 2-3h, filtering to obtain first filter residue and first filtrate, adding 5-7 times of 50-65% ethanol into the first filter residue, extracting at 45-55 ℃ for 1-2h, filtering to obtain second filtrate, combining the first filtrate and the second filtrate to obtain extract, and vacuum concentrating at 40-60 ℃ to recover ethanol to 1 time of crude drug mass (namely 1 time of notoginseng flower mass) to obtain extract.
The "crude drug amount" in the present invention is not particularly specified, and refers to the mass of dried notoginseng flower.
Preferably, the extract in step (2) is a mixed solution of polyethylene glycol 6000, anhydrous disodium hydrogen phosphate and water.
More preferably, the volume ratio of the extract to the extraction liquid in the step (2) is 0.15-0.20: 1.
More preferably, the mass of the polyethylene glycol 6000 is 9-11% of the mass of the extraction liquid.
More preferably, the mass of the anhydrous disodium hydrogen phosphate is 13-15% of the mass of the extraction liquid.
Preferably, the extract and the extract liquid are mixed in the step (2), heated and stirred at 50-60 ℃ for dissolution, then poured into a separating funnel, kept stand for layering, and the lower layer liquid is taken out and concentrated until the Baume degree is 1.1-1.2, so that the clear paste is obtained.
More preferably, the step (2) comprises the steps of:
mixing the extract with an extract liquor, wherein the volume ratio of the extract to the extract liquor is 0.15-0.20:1, the extract liquor is a mixed solution of polyethylene glycol 6000, anhydrous disodium hydrogen phosphate and water, the mass of the polyethylene glycol 6000 is 9-11% of the mass of the extract liquor, the mass of the anhydrous disodium hydrogen phosphate is 13-15% of the mass of the extract liquor, heating and stirring at 50-60 ℃ to dissolve, then pouring into a separating funnel, standing, cooling to room temperature for layering, taking the lower layer liquid, and concentrating until the Baume degree is 1.1-1.2 to obtain the clear paste.
The lower layer liquid after extraction, standing and layering is disodium hydrogen phosphate water solution containing amino acid, and the upper layer liquid is polyethylene glycol solution containing saponin, flavone, etc.
Preferably, the clear paste in step (3) is cooled at 1-3 ℃, crystallized, filtered and crystallized to obtain supernatant, and diluted by adding 8-10 times of water to obtain diluent.
More preferably, the step (3) comprises the steps of:
cooling the fluid extract at 1-3 deg.C for 24 hr to precipitate disodium hydrogen phosphate crystal, filtering and crystallizing to obtain supernatant, and diluting the supernatant with 8-10 volume times of water to obtain diluent.
Preferably, in step (4), the anionic resin is ZGAPX powder resin, and the cationic resin is ZGCPX-H powder resin.
Preferably, the mass ratio of the anionic resin to the cationic resin is 1: 1.5-2.5.
Preferably, the mass ratio of the total mass of the anion and cation resin to the dried notoginseng flower in the step (1) is 1: 0.5-0.8.
Preferably, the flow rate of the sample is 2 column volumes per hour.
Preferably, the flow rate of elution is 3-4 column volumes per hour.
Preferably, the mass concentration of the ammonia water solution in the step (4) is 15-20%.
More preferably, the step (4) comprises the steps of:
subjecting the diluent obtained in the step (3) to ZGCPX-H cation powder resin and ZGAPX anion powder resin for exchange adsorption, wherein the mass ratio of the total mass of the anion resin and the cation resin to the dried flos Notoginseng obtained in the step (1) is 1:0.5-0.8, the mass ratio of the anion resin to the cation resin is 1:1.5-2.5, loading the anion resin into the lower layer during column loading, loading the cation resin into the upper layer, balancing pure water, loading the diluent at the flow rate of 2 column volumes per hour, detecting ninhydrin during loading, stopping loading after the exchange saturation, standing for 2H, eluting with water and 15-20% ammonia water solution at the flow rate of 3-4 column volumes per hour, washing with water until the diluent is colorless and odorless, eluting with the ammonia water solution, and performing amino acid detection reaction with ninhydrin during ammonia water solution elution, stopping eluting after no amino acid exists, and combining and collecting ammonia water eluents.
Preferably, the aqueous ammonia eluent in step (5) is concentrated under reduced pressure to a water content of less than 50%.
More preferably, the step (5) comprises the steps of:
concentrating the ammonia water eluate at below 60 deg.C under reduced pressure until the water content is below 50% to obtain soft extract, and freeze drying.
The invention has the beneficial effects that:
in order to improve the yield of amino acid, peptide bonds of protein, polypeptide and polysaccharide combined amino acid are hydrolyzed into amino acid so as to improve the yield of amino acid, and even if the macromolecular protein and the polypeptide are absorbed by ion exchange, the macromolecular protein and the polypeptide can be eluted with the amino acid together when the macromolecular protein and the polypeptide are eluted by ammonia water to influence the purity of the amino acid; the ion resin adopted by the invention is powder resin, the granularity is very small, the exchange speed is high, the exchange single amount is large, the elution loss solvent is less, and the sample loading speed and the exchange speed are greatly improved by adopting the exchange adsorption of anions and cations.
Notoginseng radix amino acid is soluble in medium ethanol, and high molecular polysaccharide, tannin, pectin, etc. are insoluble in medium ethanol, so most polysaccharide can not be extracted; according to the invention, by optimizing the extraction process, the saponin, the flavone and other components in the extracting solution are extracted to the upper polyethylene glycol layer, the amino acid is extracted to the lower disodium hydrogen phosphate aqueous solution layer, and the characteristic of low saturation degree of disodium hydrogen phosphate during low-temperature refrigeration is utilized, so that most disodium hydrogen phosphate can be refrigerated and separated out, the desalting effect is achieved, and a small amount of residual disodium hydrogen phosphate can directly flow out along an ion exchange column during ion exchange sampling and washing, and cannot be brought into a final product.
The invention optimizes the composition of the extract liquor and obviously improves the yield and the purity of the amino acid.
The invention optimizes the composition of enzyme and the specific process of alcohol extraction, and obviously improves the yield of amino acid.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The ZGCPX-H cation powder resin and the ZGAPX anion powder resin are purchased from Zhejiang Kongguang industry GmbH.
Example 1
A method for preparing amino acids from flos Notoginseng comprises the following steps:
(1) drying and crushing notoginseng flower, adding 0.1% enzyme (the enzyme is a mixture of papain and neutral protease, the mass ratio of the papain to the neutral protease is 1.5:1) and 6 times of water for enzymolysis for 30min, then adding 95% ethanol until the alcohol content of the whole system is 50%, then extracting at 45 ℃ for 2h, filtering to obtain a first filter residue and a first filtrate, adding 5 times of 50% ethanol into the first filter residue, extracting at 45 ℃ for 1h, filtering to obtain a second filtrate, combining the first filtrate and the second filtrate to obtain an extracting solution, and performing vacuum concentration at 40 ℃ to recover ethanol to 1 time of crude drug mass to obtain an extract;
(2) mixing the extract with an extract liquor, wherein the volume ratio of the extract to the extract liquor is 0.15:1, the extract liquor is a mixed solution of polyethylene glycol 6000, anhydrous disodium hydrogen phosphate and water, the mass of the polyethylene glycol 6000 is 9% of the mass of the extract liquor, the mass of the anhydrous disodium hydrogen phosphate is 13% of the mass of the extract liquor, heating, stirring and dissolving at 50 ℃, then pouring into a separating funnel, standing, cooling to room temperature for layering, taking down the liquid of the lower layer, and concentrating until the Baume degree is 1.1 to obtain clear paste;
(3) cooling the fluid extract at 1 deg.C for 24 hr to precipitate disodium hydrogen phosphate crystal, filtering and crystallizing to obtain supernatant, and diluting the supernatant with 8 volume times of water to obtain diluent;
(4) performing exchange adsorption on the diluent obtained in the step (3) through ZGCPX-H cation powder resin and ZGAPX anion powder resin, wherein the mass ratio of the total mass of the anion powder resin and the cation powder resin to the dried flos notoginseng obtained in the step (1) is 1:0.5, the mass ratio of the anion powder resin to the cation powder resin is 1:1.5, the anion resin is used for filling the lower layer when a column is filled, the cation resin is used for filling the upper layer, the diluent is used for sampling at the flow rate of 2 column volumes per hour after pure water is balanced, ninhydrin detection is performed when the sample is filled, the sample is stopped when the exchange is saturated, water and 15% ammonia water solution are sequentially used for elution at the flow rate of 3 column volumes per hour after standing for 2 hours, the ammonia water is used for washing until the water is colorless and odorless, the ammonia water solution is used for elution, ninhydrin detection reaction is performed when the ammonia water solution is used for elution, and the elution is stopped when no amino acid is present, combining and collecting ammonia water eluent;
(5) concentrating the ammonia water eluate at 60 deg.C under reduced pressure until the water content is less than 50% to obtain soft extract, and freeze drying.
Example 2
A method for preparing amino acids from flos Notoginseng comprises the following steps:
(1) drying and crushing notoginseng flower, adding 0.2% enzyme (the enzyme is a mixture of soybean peptide hydrolase and neutral protease, the mass ratio of the soybean peptide hydrolase to the neutral protease is 2:0.5) and 8 times of water for enzymolysis for 50min, adding 95% ethanol until the alcohol content of the whole system is 65%, then extracting at 55 ℃ for 3h, filtering to obtain first filter residue and first filtrate, adding 7 times of 65% ethanol into the first filter residue, extracting at 55 ℃ for 2h, filtering to obtain second filtrate, combining the first filtrate and the second filtrate to obtain an extracting solution, and performing vacuum concentration at 60 ℃ to recover ethanol to 1 time of crude drug mass to obtain an extract;
(2) mixing the extract with an extract liquor, wherein the volume ratio of the extract to the extract liquor is 0.20:1, the extract liquor is a mixed solution of polyethylene glycol 6000, anhydrous disodium hydrogen phosphate and water, the mass of the polyethylene glycol 6000 is 11% of the mass of the extract liquor, the mass of the anhydrous disodium hydrogen phosphate is 15% of the mass of the extract liquor, heating, stirring and dissolving at 60 ℃, then pouring into a separating funnel, standing, cooling to room temperature for layering, taking down the liquid of the lower layer, and concentrating until the Baume degree is 1.2 to obtain clear paste;
(3) cooling the fluid extract at 3 deg.C for 24 hr to precipitate disodium hydrogen phosphate crystal, filtering and crystallizing to obtain supernatant, and diluting the supernatant with 10 volume times of water to obtain diluent;
(4) performing exchange adsorption on the diluent obtained in the step (3) through ZGCPX-H cation powder resin and ZGAPX anion powder resin, wherein the mass ratio of the total mass of the anion powder resin and the cation powder resin to the dried flos notoginseng obtained in the step (1) is 1:0.8, the mass ratio of the anion powder resin to the cation powder resin is 1:2.5, loading the anion resin into the lower layer during column loading, loading the cation resin into the upper layer, balancing pure water, loading the diluent at the flow rate of 2 column volumes per hour, detecting ninhydrin during loading, stopping loading after the exchange saturation, standing for 2 hours, sequentially eluting with water and 20% ammonia water solution at the flow rate of 4 column volumes per hour, washing with water until the water is colorless and odorless, then eluting with the ammonia water solution, performing amino acid detection reaction by using ninhydrin during ammonia water solution elution, stopping elution after no amino acid exists, combining and collecting ammonia water eluent;
(5) concentrating the ammonia water eluate at 50 deg.C under reduced pressure until the water content is less than 50% to obtain soft extract, and freeze drying.
Example 3
A method for preparing amino acids from flos Notoginseng comprises the following steps:
(1) drying and crushing notoginseng flower, adding 0.15% enzyme (the enzyme is a mixture of papain and neutral protease, the mass ratio of the papain to the neutral protease is 1.5:0.7) and 7 times of water for enzymolysis for 40min, adding 95% ethanol until the alcohol content of the whole system is 60%, then extracting at 50 ℃ for 2.5h, filtering to obtain a first filter residue and a first filtrate, adding 6 times of 60% ethanol into the first filter residue, extracting at 50 ℃ for 1.5h, filtering to obtain a second filtrate, combining the first filtrate and the second filtrate to obtain an extracting solution, and performing vacuum concentration at 50 ℃ to recover ethanol to 1 time of crude drug mass to obtain an extract;
(2) mixing the extract with an extract liquor, wherein the volume ratio of the extract to the extract liquor is 0.18:1, the extract liquor is a mixed solution of polyethylene glycol 6000, anhydrous disodium hydrogen phosphate and water, the mass of the polyethylene glycol 6000 is 10% of the mass of the extract liquor, the mass of the anhydrous disodium hydrogen phosphate is 14% of the mass of the extract liquor, heating, stirring and dissolving at 55 ℃, then pouring into a separating funnel, standing, cooling to room temperature for layering, taking down the liquid of the lower layer, and concentrating until the Baume degree is 1.1 to obtain clear paste;
(3) cooling the fluid extract at 2 deg.C for 24 hr to precipitate disodium hydrogen phosphate crystal, filtering and crystallizing to obtain supernatant, and diluting the supernatant with 9 volume times of water to obtain diluent;
(4) performing exchange adsorption on the diluent obtained in the step (3) through ZGCPX-H cation powder resin and ZGAPX anion powder resin, wherein the mass ratio of the total mass of the anion powder resin and the cation powder resin to the dried flos notoginseng obtained in the step (1) is 1:0.6, the mass ratio of the anion powder resin to the cation powder resin is 1:2, the anion resin is used for filling a lower layer when a column is filled, the cation resin is used for filling an upper layer, the diluent is subjected to pure water balance and then is subjected to sample loading at the flow rate of 2 column volume amounts/hour, ninhydrin detection is performed when the sample loading is performed, the sample loading is stopped after the exchange saturation, water and 18% ammonia water solution are sequentially adopted for elution after the pure water is washed to be colorless and odorless, the ammonia water solution is adopted for elution, ninhydrin is used for amino acid detection reaction when the ammonia water solution is eluted, and the elution is stopped after no amino acid is generated, combining and collecting ammonia water eluent;
(5) concentrating the ammonia water eluate at 40 deg.C under reduced pressure until the water content is less than 50% to obtain soft extract, and freeze drying.
Comparative example 1
The difference from the example 3 is only that the mass ratio of the papain to the neutral protease is 1.5:0.2, and the rest conditions are the same.
Comparative example 2
The difference from the example 3 is only that the mass ratio of the papain to the neutral protease is 1.5:2, and the rest conditions are the same.
Comparative example 3
The difference from example 3 is only that anhydrous disodium hydrogen phosphate in the extract was replaced with ammonium sulfate of equal mass, and the other conditions were the same.
Comparative example 4
The only difference from example 3 is that the cation exchange resin used in step (4) is 001x12 cation exchange resin, no anion exchange resin is used, and the rest conditions are the same, specifically as follows:
a method for preparing amino acids from flos Notoginseng comprises the following steps:
(1) drying and crushing notoginseng flower, adding 0.15% enzyme (the enzyme is a mixture of papain and neutral protease, the mass ratio of the papain to the neutral protease is 1.5:0.7) and 7 times of water for enzymolysis for 40min, adding 95% ethanol until the alcohol content of the whole system is 60%, then extracting at 50 ℃ for 2.5h, filtering to obtain a first filter residue and a first filtrate, adding 6 times of 60% ethanol into the first filter residue, extracting at 50 ℃ for 1.5h, filtering to obtain a second filtrate, combining the first filtrate and the second filtrate to obtain an extracting solution, and performing vacuum concentration at 50 ℃ to recover ethanol to 1 time of crude drug mass to obtain an extract;
(2) mixing the extract with an extract liquor, wherein the volume ratio of the extract to the extract liquor is 0.18:1, the extract liquor is a mixed solution of polyethylene glycol 6000, anhydrous disodium hydrogen phosphate and water, the mass of the polyethylene glycol 6000 is 10% of the mass of the extract liquor, the mass of the anhydrous disodium hydrogen phosphate is 14% of the mass of the extract liquor, heating, stirring and dissolving at 55 ℃, then pouring into a separating funnel, standing, cooling to room temperature for layering, taking down the liquid of the lower layer, and concentrating until the Baume degree is 1.1 to obtain clear paste;
(3) cooling the fluid extract at 2 deg.C for 24 hr to precipitate disodium hydrogen phosphate crystal, filtering and crystallizing to obtain supernatant, and diluting the supernatant with 9 volume times of water to obtain diluent;
(4) subjecting the diluent obtained in the step (3) to exchange adsorption by 001x12 cation exchange resin, wherein the mass ratio of the total mass of the 001x12 cation exchange resin to the dried notoginseng flower in the step (1) is 1:0.6, after pure water is balanced, loading the diluent at the flow rate of 2 column volumes per hour, detecting ninhydrin during loading, stopping loading after the exchange is saturated, standing for 2 hours, eluting with water and 18% ammonia water solution in sequence at the flow rate of 3.5 column volumes per hour, eluting with ammonia water solution after water is washed to be colorless and odorless, performing amino acid identification reaction by ninhydrin during eluting with ammonia water solution, stopping elution after no amino acid exists, combining and collecting ammonia water eluate;
(5) concentrating the ammonia water eluate at 40 deg.C under reduced pressure until the water content is less than 50% to obtain soft extract, and freeze drying.
Test example 1
Yield and purity test of amino acids prepared in examples 1 to 3 and comparative examples 1 to 3: the product was measured using a Hitachi L-8900 amino acid analyzer according to GB/T5009.124-2003 using a mixed amino acid standard solution as a control, and the results are shown in Table 1.
TABLE 1 yield and purity testing of amino acids
From the above table, it can be seen that:
1. the amino acid prepared by the method has high content and the yield is over 90 percent.
2. As shown in comparative example 4, when the broad-spectrum macroporous ion exchange resin 001X12 was used for exchanging amino acids, the large-particle resin was likely to cause dead adsorption due to its large particles, and the dead adsorption was heavy, so that the flow rate was slow during loading, the loading rate was fast, and the leakage was likely to occur, the amount of solvent used during analysis was large, and the yield after analysis was low.
The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.
Claims (10)
1. A method for preparing amino acid from notoginseng flower, which is characterized by comprising the following steps:
(1) drying Notoginseng, pulverizing, adding enzyme and water for enzymolysis, adding ethanol for extraction, filtering to obtain extractive solution, concentrating, and recovering ethanol to obtain extract;
(2) mixing the extract with the extractive solution, pouring into a separating funnel, standing for layering, collecting the lower layer liquid, and concentrating to obtain fluid extract;
(3) cooling the fluid extract, crystallizing, filtering to obtain supernatant, and diluting with water to obtain diluent;
(4) performing exchange adsorption on the diluent obtained in the step (3) through cation resin and anion resin, loading the lower layer of the anion resin and the upper layer of the cation resin during column packing, loading the sample after pure water is balanced, stopping loading the sample after exchange is saturated, sequentially eluting with water and an ammonia water solution, and combining and collecting the ammonia water eluent;
(5) concentrating the ammonia water eluate under reduced pressure to obtain soft extract, and freeze drying.
2. The method according to claim 1, wherein the enzyme in step (1) is a mixture of neutral protease and at least one of papain and soybean peptide hydrolase; the mass ratio of at least one of the papain and the soybean peptide hydrolase to the neutral protease is 1.5-2: 0.5-1.
3. The method according to claim 1, wherein the extraction process in the step (1) is as follows: drying Notoginseng flower, pulverizing, adding 0.1-0.2% enzyme and 6-8 times of water for enzymolysis, adding 95% ethanol until the alcohol content of the whole system is 50-65%, extracting for 2-3 times, filtering to obtain extractive solution, concentrating, and recovering ethanol to obtain extract.
4. The method as claimed in claim 3, wherein the step (1) comprises adding 95% ethanol until the ethanol content is 50-65%, extracting at 45-55 deg.C for 2-3h, filtering to obtain first residue and first filtrate, adding 5-7 times of 50-65% ethanol into the first residue, extracting at 45-55 deg.C for 1-2h, filtering to obtain second filtrate, mixing the first filtrate and the second filtrate to obtain extractive solution, and vacuum concentrating at 40-60 deg.C to recover ethanol to 1 times of crude drug mass to obtain extract.
5. The method according to claim 1, wherein the extraction liquid in the step (2) is a mixed solution of polyethylene glycol 6000, anhydrous disodium hydrogen phosphate and water; the volume ratio of the extract to the extract is 0.15-0.20: 1; the mass of the polyethylene glycol 6000 is 9-11% of the mass of the extraction liquid; the mass of the anhydrous disodium hydrogen phosphate is 13-15% of the mass of the extraction liquid.
6. The method according to claim 1, wherein the extract is mixed with the extract in step (2), heated and stirred at 50-60 ℃ for dissolution, then poured into a separating funnel, kept stand for layering, and the lower layer liquid is taken out and concentrated to the Baume degree of 1.1-1.2 to obtain the clear paste.
7. The method according to claim 1, wherein in step (3), the fluid extract is cooled at 1-3 ℃, crystallized, filtered and crystallized to obtain supernatant, and diluted with 8-10 times of water to obtain diluted solution.
8. The process of claim 1, wherein in step (4) the anionic resin is ZGAPX powder resin and the cationic resin is ZGCPX-H powder resin; the mass ratio of the anion resin to the cation resin is 1: 1.5-2.5; the flow rate of the sample is 2 column volumes per hour; the elution flow rate is 3-4 column volumes per hour.
9. The method according to claim 1, wherein the mass concentration of the aqueous ammonia solution in the step (4) is 15 to 20%.
10. The process of claim 1, wherein in step (5) the aqueous ammonia eluent is concentrated under reduced pressure to a water content of less than 50%.
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