A method for extracting isorhamnetin from seabuckthorn puree

Abstract

The present invention relates to a method for extracting isorhamnetin from seabuckthorn puree, so as to solve the problem that the traditional process only extracts the free isorhamnetin in the seabuckthorn puree, but cannot extract the isorhamnetin in the glycoside compound, resulting in the waste of isorhamnetin resources and the technical problem of reducing the yield of isorhamnetin. The method includes: 1) defatting the seabuckthorn puree; 2) spraying a weakly acidic buffer solution and a surfactant into the defatted raw material, and then adding naringinase and pectinase; 3) adding an ester solvent to the residue after double enzymolysis for extraction, concentrating the extract, adsorption filtering, and collecting the filtrate; 4) adding an electrolyte salting-out agent to the filtrate, extracting with alkaline water, collecting the upper liquid, washing with water to neutrality, concentrating the upper liquid and filtering; 5) recrystallization. The present invention can increase the yield by 30-40%, realize the purification of isorhamnetin, and the isorhamnetin content reaches more than 98%.

Description

Technical Field

[0001] The invention relates to a method for extracting chemical substances from natural substances, and in particular to a method for extracting isorhamnetin from seabuckthorn puree. Background Art

[0002] Seabuckthorn is a shrub or small tree of the genus Hippophae in the family Elaeagnaceae, also known as sour willow and sour thorn. Seabuckthorn fruit is a common medicine used by ancient Tibetan and Mongolian medicine to treat diseases. It has the pharmacological effects of expectoration, lung benefit, stomach nourishment, spleen strengthening, blood circulation and removing bad habits. Seabuckthorn flavonoids are important active ingredients in seabuckthorn, and are used as physical and chemical identification and quality control indicators in preparations and seabuckthorn medicines. Clinically proven seabuckthorn flavonoids are pure natural medicines for the treatment of ischemic heart disease, angina pectoris, myocardial ischemia, chronic heart failure, hyperlipidemia, prevention of atherosclerosis, myocardial infarction, cerebral thrombosis and other diseases, and have certain research significance.

[0003] Flavonoids exist in all parts of seabuckthorn, including 0.3% in juice, 0.8% in seabuckthorn leaves, and 1% in seabuckthorn oil. Seabuckthorn puree is a byproduct of the production process of seabuckthorn juice, and the content of flavonoids can reach 2%, with isorhamnetin as the main active ingredient. Extracting isorhamnetin from seabuckthorn puree will open up new ways for the development and utilization of seabuckthorn resources.

[0004] Chinese patent CN101125843A mentions a method for extracting and refining isorhamnetin from sea buckthorn puree: the puree is defatted by supercritical extraction or organic solvent, then impurities are removed by 0-30% alcohol aqueous solution, then extracted by alkali alcohol, and the filtrate is adjusted to neutral to obtain crude isorhamnetin, which is then recrystallized to obtain an isorhamnetin product with a content of 80-95%.

[0005] Chinese patent CN102533431A relates to a method for continuously extracting and separating seabuckthorn oil and isorhamnetin from seabuckthorn pulp: seabuckthorn fruit is extracted with ethanol, seabuckthorn fruit oil is extracted with an organic solvent, and the insoluble matter is dissolved in methanol or ethanol to obtain crude isorhamnetin, which is then recrystallized using an ethanol-ethyl acetate mixed solvent to obtain isorhamnetin crystals with a purity of 95% and a yield of about 0.4-0.5%.

[0006] Chinese patent CN106146449A mentions a method for preparing isorhamnetin from seabuckthorn pomace and its application: seabuckthorn pomace is extracted with ethanol, concentrated, and deoiled with dichloromethane to obtain crude isorhamnetin, which is then recrystallized with acetone or petroleum ether to obtain isorhamnetin with a content of 70-75% and an effective component recovery rate of 60%.

[0007] Chinese patent CN103435587A mentions a method for preparing isorhamnetin: seabuckthorn pomace is hydrolyzed by biological enzymes, separated by macroporous resins, concentrated and crystallized, and repeatedly recrystallized from ethanol and ethyl acetate to obtain isorhamnetin with a content of 98% and a yield of 0.4-0.5%.

[0008] Seabuckthorn fruit has free isorhamnetin and its glycosides as the dominant active ingredients. In addition to rich free compounds, it also includes glycoside compounds bound by glucose, rhamnose, rutin, etc. However, the common disadvantage of the above existing technologies is that they can only extract free isorhamnetin in seabuckthorn puree, but cannot extract isorhamnetin in glycoside compounds, resulting in a waste of isorhamnetin resources and a reduction in the yield of isorhamnetin. Summary of the invention

[0009] The purpose of the present invention is to provide a method for extracting isorhamnetin from seabuckthorn puree, so as to solve the technical problem that the traditional process can only extract free isorhamnetin in seabuckthorn puree, but cannot extract isorhamnetin in glycoside compounds, resulting in waste of isorhamnetin resources and reduced yield of isorhamnetin.

[0010] In order to achieve the above object, the present invention adopts the following technical scheme:

[0011] A method for extracting isorhamnetin from seabuckthorn puree is characterized in that it comprises the following steps:

[0012] 1) defatting the seabuckthorn puree raw material to obtain a defatted raw material;

[0013] 2) Double enzymatic hydrolysis

[0014] 2.1) Spraying a weakly acidic buffer solution with a pH value of 5.5-6.8 and a mass of 0.8-1.5 times that of the defatted raw material and a surfactant into the defatted raw material; the mass of the surfactant is 0.05-0.20% of the weakly acidic buffer solution;

[0015] 2.2) adding 0.3-1.0% of the weight of the defatted raw material naringinase and pectinase, stirring evenly, and performing enzymolysis at 40-55° C. for 3-6 hours; the mass ratio of the naringinase to the pectinase is 2.6-4.2:1;

[0016] 3) Adsorption and impurity removal

[0017] 3.1) adding an ester solvent to the residue after enzymatic hydrolysis in step 2) for at least 2 extractions, each time for no less than 1 hour, at a temperature of 40-60° C.; the mass of the ester solvent is 3-5 times the mass of the residue after enzymatic hydrolysis;

[0018] 3.2) After the extract is concentrated to 1 / 2-1 / 3 of the original volume, it is adsorbed and filtered to collect the filtrate;

[0019] 4) Extraction and crystallization

[0020] 4.1) adding an electrolyte salting-out agent to the filtrate obtained in step 3.2) at a mass ratio of the electrolyte salting-out agent to the filtrate volume of 0.3-0.5 g:100 ml, then extracting with alkaline water 2-3 times, collecting the upper layer liquid, washing with water until neutral, and then separating the liquids, concentrating the upper layer liquid to a specific gravity of 1.08-1.15;

[0021] 4.2) Keep the concentrated solution warm at 30-40°C for more than 2 hours for crystallization, and filter to obtain crude isorhamnetin crystals;

[0022] 5) Recrystallization

[0023] 5.1) adding an alcohol solvent having a concentration of 60-80% and a mass 10-15 times that of the crude isorhamnetin crystals to the obtained crude isorhamnetin crystals, heating to dissolve, filtering impurities, allowing the crystals to stand and filtering; the alcohol solvent is adjusted to a pH of 9-10 using alkaline water having a mass concentration of 5-10%;

[0024] 5.2) The filtered crystals are washed with water until they become neutral and dried to obtain isorhamnetin crystals.

[0025] Furthermore, in step 2.1), the weakly acidic buffer is a citric acid buffer; and the surfactant is a sodium dodecyl sulfonate, sodium dodecyl sulfate or sodium dodecylbenzene sulfonate surfactant.

[0026] Furthermore, in step 4.1), the electrolyte salting-out agent is sodium chloride, sodium sulfate or magnesium sulfate; and the alkaline water is saturated lime water or a sodium bicarbonate solution with a mass concentration of 1-3%.

[0027] Furthermore, in step 2.1), citric acid buffer and sodium dodecylbenzene sulfonate with a pH of 6.0 are sprayed; the mass of the citric acid buffer and sodium dodecylbenzene sulfonate is equal to the mass of the defatted raw material, and the mass of the sodium dodecylbenzene sulfonate is 0.10% of the citric acid buffer;

[0028] In step 2.2), naringinase and pectinase in an amount of 0.5% of the defatted raw material are added, stirred evenly, and enzymolyzed at 45° C. for 5 h; the mass ratio of naringinase to pectinase is 3.0:1;

[0029] In step 4.1), sodium chloride is added to the filtrate obtained in step 3.2), the mass ratio of sodium chloride to the volume of the filtrate is 0.4 g:100 ml, and then saturated lime water solution is used for extraction three times, and the upper layer liquid is concentrated to a specific gravity of 1.09;

[0030] In step 4.2), the insulation temperature is 40°C.

[0031] The naringinase can simultaneously and effectively degrade residues such as glucose, rhamnose and rutin on the isorhamnetin mother core structure, and then compounded with pectinase, the yield of isorhamnetin is increased through double enzyme conversion; sodium chloride is added because sodium chloride is easily available, cheap and has the best effect; a 40°C heat preservation crystallization process is adopted to make isorhamnetin crystallize and precipitate, while small polar impurities are not easy to precipitate, thereby achieving the purification of isorhamnetin.

[0032] Further, in step 3.1), the ester solvent is ethyl acetate or methyl acetate;

[0033] In step 3.2), the adsorption filtration is specifically as follows: adding alumina and chitosan to the concentrated solution, stirring at 50-60°C for 1 hour, filtering, and collecting the filtrate; the amounts of alumina and chitosan are 1-3% and 0.5-1.5% of the mass of the residue after enzymatic hydrolysis in step 2), respectively.

[0034] Furthermore, in step 5.1), the alcohol solvent is methanol, ethanol or isopropanol, and the alkaline water is sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution or potassium carbonate solution.

[0035] Furthermore, in step 3.1), ethyl acetate 4 times the mass of the residue after enzymatic hydrolysis was added for extraction, the extraction times were 3 times, each time for 1.5 hours, and the extraction temperature was 50°C;

[0036] In step 3.2), 2% of the weight of the residue after enzymatic hydrolysis in step 2) and 1.0% of chitosan were added, and stirred at 55° C. for 1 h;

[0037] In step 5.1), an ethanol solvent having a concentration of 60% and a mass 13 times that of crude isorhamnetin crystals is added; the pH value of the ethanol solvent is adjusted using a potassium hydroxide aqueous solution having a mass concentration of 5%.

[0038] Alumina is used to adsorb phenolic impurities such as coumarin and a small amount of quercetin in seabuckthorn puree, and chitosan is used to remove terpenes or steroid compounds in seabuckthorn puree. It has good impurity adsorption performance and simplifies the subsequent purification process.

[0039] Furthermore, in step 2), after step 2.2), there is a step 2.3), in which the enzyme is inactivated at 78-82°C for 8-12 minutes.

[0040] Furthermore, in step 1), the degreasing is performed by supercritical extraction; the supercritical extraction pressure is 20-35 MPa, the extraction temperature is 40-50° C., and the extraction time is 3-5 h.

[0041] Furthermore, in step 1), the supercritical extraction pressure is 30 MPa, the extraction temperature is 45° C., and the extraction time is 4 h;

[0042] In step 2.3), the enzyme was inactivated at 80°C for 10 min.

[0043] Beneficial effects of the present invention:

[0044] 1. The weakly acidic buffer in the present invention is used to protect the double enzyme from inactivation, and the surfactant is used to improve the enzymatic hydrolysis of the double enzyme; naringinase can simultaneously and effectively degrade the residues of glucose, rhamnose and rutin on the isorhamnetin parent nucleus structure, realize the hydrolysis of glycoside compounds, and then compound pectinase, through the double enzyme conversion, the isorhamnetin with original biological activity can be efficiently extracted, thereby increasing the yield of isorhamnetin by about 20%.

[0045] 2. In the present invention, an ester solvent is added to the residue after enzymatic hydrolysis to significantly remove most of the water-soluble impurities; by adding an electrolyte salting-out agent, the solubility of isorhamnetin in the alkaline water phase is reduced by utilizing the "salting-out effect", thereby improving the extraction rate and significantly improving the isorhamnetin content; by utilizing the heat preservation crystallization process, isorhamnetin is crystallized and precipitated, while small polar impurities are not easily precipitated and remain in the mother liquor, thereby achieving the purification of isorhamnetin.

[0046] 3. The present invention utilizes the special adsorption performance and dosage of alumina to adsorb phenolic impurities such as coumarin and a small amount of quercetin in seabuckthorn puree; utilizes the effects of hydroxyl groups, amino groups and other groups in chitosan to effectively remove terpenes or steroid compounds in seabuckthorn puree, is easy to operate, has good impurity adsorption performance, and simplifies subsequent purification processes.

[0047] 4. The present invention adopts alkali alcohol recrystallization process to make the isorhamnetin content reach more than 98%, and the yield is increased by 30-40% compared with the traditional one. The operation process is simple, energy consumption is low, resources are saved, production cost is low, and it is more suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The present invention is a flow chart of a method for extracting isorhamnetin from seabuckthorn puree;

[0049] Figure 2 HPLC chart of the defatted raw material in Example 1 of the present invention;

[0050] Figure 3 HPLC diagram of the residue after enzymatic hydrolysis in Example 1 of the present invention;

[0051] Figure 4 HPLC chart of the product obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0052] The present invention is described in detail below through specific embodiments.

[0053] Example 1

[0054] The present invention discloses a method for extracting isorhamnetin from seabuckthorn puree, comprising the following steps:

[0055] 1) Defatted sea buckthorn puree

[0056] 10 kg of seabuckthorn puree raw material was taken and defatted by supercritical extraction to obtain defatted raw material; the supercritical extraction pressure was 20-35 MPa, the extraction temperature was 40-50°C, and the extraction time was 3-5 h. In this embodiment, the supercritical extraction pressure was 30 MPa, the extraction temperature was 45°C, and the extraction time was 4 h. The defatted raw material was analyzed by HPLC, and the results were as follows: Figure 2 shown.

[0057] 2) Double enzymatic hydrolysis

[0058] 2.1) spraying a weak acidic buffer solution with a pH value of 5.5-6.8 and a mass of 0.8-1.5 times that of the defatted raw material and a surfactant into the defatted raw material in step 1), wherein the mass of the surfactant is 0.05-0.20% of the weak acidic buffer solution. In this embodiment, a citric acid buffer solution with a pH value of 6.0 and a mass equal to that of the defatted raw material and sodium dodecylbenzene sulfonate are sprayed, and the mass of the sodium dodecylbenzene sulfonate is 0.10% of the citric acid buffer solution.

[0059] 2.2) 0.3-1.0% of naringinase and pectinase by weight of the defatted raw material, the mass ratio of naringinase to pectinase is 2.6-4.2:1, the mixture is stirred evenly, and enzymolysis is performed at 40-55° C. for 3-6 h. In this embodiment, 0.5% of naringinase and pectinase by weight of the defatted raw material, the mass ratio of naringinase to pectinase is 3.0:1, the mixture is stirred evenly, and enzymolysis is performed at 45° C. for 5 h.

[0060] 2.3) The enzyme was inactivated at 80°C for 10 min. The residue after enzymatic hydrolysis was analyzed by HPLC. The results were as follows: Figure 3 As shown, from Figure 2 and Figure 3 According to the test results, the isorhamnetin content increased from 0.657% to 0.802% through enzymatic hydrolysis, an increase of more than 20%.

[0061] 3) Adsorption and impurity removal

[0062] 3.1) Add an ester solvent to the residue after enzymatic hydrolysis in step 2) for extraction, wherein the mass of the ester solvent is 3-5 times the mass of the residue after enzymatic hydrolysis, and the extraction temperature is 40-60°C. In this embodiment, ethyl acetate is added for extraction, and the mass of ethyl acetate is 4 times the mass of the residue after enzymatic hydrolysis, and the extraction temperature is 50°C; extract 3 times, each time for 1.5h.

[0063] 3.2) After the extract is concentrated to 1 / 2-1 / 3 of the original volume, it is adsorbed and filtered to collect the filtrate;

[0064] Adsorption filtration is specifically as follows: adding alumina and chitosan to the concentrated solution, wherein the amounts of alumina and chitosan are 1-3% and 0.5-1.5% of the mass of the residue after enzymatic hydrolysis in step 2), respectively; stirring at 50-60° C. for 1 hour; and filtering. In this embodiment, 2% alumina and 1.0% chitosan of the mass of the residue after enzymatic hydrolysis in step 2) are added, and stirring is performed at 55° C. for 1 hour.

[0065] 4) Extraction and crystallization

[0066] 4.1) adding an electrolyte salting-out agent (dissolved in advance with a small amount of water) to the filtrate obtained in step 3.2), wherein the mass ratio of the electrolyte salting-out agent to the volume of the filtrate is 0.3-0.5 g:100 ml, and then extracting with alkaline water for 2-3 times. In this embodiment, sodium chloride is added, and the mass ratio of sodium chloride to the volume of the filtrate is 0.4 g:100 ml. Extracting with saturated lime water solution for 3 times; collecting the upper liquid, washing with water until neutral, and then separating the liquids, concentrating the upper liquid to a specific gravity of 1.08-1.15, and concentrating the upper liquid to a specific gravity of 1.09 in this embodiment.

[0067] 4.2) Keep the concentrated solution warm at 30-40°C for more than 2 hours for crystallization. In this embodiment, the warming temperature is 40°C. Filter to obtain crude isorhamnetin crystals.

[0068] 5) Recrystallization

[0069] 5.1) adding an alcohol solvent having a concentration of 60-80% and a mass of 10-15 times that of the crude isorhamnetin crystals to the crude isorhamnetin crystals obtained in step 4.2), the alcohol solvent being adjusted to a pH of 9-10 using alkaline water having a mass concentration of 5-10%, in this embodiment, an ethanol solvent having a concentration of 60% and a mass of 13 times that of the crude isorhamnetin crystals is added, and the pH value of the ethanol solvent is adjusted using a potassium hydroxide aqueous solution having a mass concentration of 5%; heating to dissolve, filtering impurities, allowing the crystals to stand and filtering.

[0070] 5.2) The filtered crystals are washed with water until they become neutral and dried to obtain isorhamnetin crystals.

[0071] 64.9 g of isorhamnetin crystals were obtained. The isorhamnetin content in the isorhamnetin crystals was 99.06%, and the yield was 0.649%. The obtained isorhamnetin crystals were subjected to HPLC analysis. The results were as follows: Figure 4 shown.

[0072] Example 2

[0073] The method steps of this embodiment are the same as those of Embodiment 1, except that:

[0074] In step 1), the supercritical extraction pressure is 35 MPa, the extraction temperature is 40° C., and the extraction time is 3 h.

[0075] In step 2.1), citric acid buffer solution with a pH value of 5.5 and a mass of 1.5 times that of the defatted raw material and sodium dodecyl sulfate are sprayed, and the mass of the sodium dodecyl sulfate is 0.20% of the citric acid buffer solution.

[0076] In step 2.2), naringinase and pectinase in an amount equal to the mass of the defatted raw material are added, the mass ratio of naringinase to pectinase is 2.6:1, and enzymolysis is carried out at 55° C. for 3 h.

[0077] In step 2.3), the enzyme was inactivated at 78°C for 12 min.

[0078] In step 3.1), the mass of ethyl acetate is 5 times the mass of the residue after enzymatic hydrolysis, and the extraction temperature is 60°C.

[0079] In step 3.2), 3% of the weight of the residue after enzymatic hydrolysis in step 2) and 1.5% of chitosan were added and stirred at 50°C.

[0080] In step 4.1), the mass ratio of sodium chloride to the volume of the filtrate is 0.5 g:100 ml, and a sodium bicarbonate solution with a mass concentration of 3% is used for extraction, and the upper layer liquid is concentrated to a specific gravity of 1.12.

[0081] In step 5.1), an ethanol solvent having a concentration of 80% and a mass of 15 times that of crude isorhamnetin crystals is added, and the pH value of the ethanol solvent is adjusted using a sodium carbonate aqueous solution having a mass concentration of 5%.

[0082] 61.5 g of isorhamnetin crystals were obtained, the isorhamnetin content in the isorhamnetin crystals was 98.75%, and the yield was 0.615%.

[0083] Example 3

[0084] The method steps of this embodiment are the same as those of Embodiment 1, except that:

[0085] In step 1), the supercritical extraction pressure is 20 MPa, the extraction temperature is 50° C., and the extraction time is 5 h.

[0086] In step 2.1), citric acid buffer solution with a pH value of 5.5 and a mass of 0.8 times that of the defatted raw material and sodium dodecyl sulfate are sprayed, and the mass of sodium dodecyl sulfate is 0.05% of the citric acid buffer solution.

[0087] In step 2.2), 0.3% of the weight of the defatted raw material is added with naringinase and pectinase, the weight ratio of naringinase to pectinase is 4.2:1, and the enzymolysis is carried out at 40° C. for 6 h.

[0088] In step 2.3), the enzyme was inactivated at 82°C for 8 min.

[0089] In step 3.1), the mass of ethyl acetate is 3 times the mass of the residue after enzymatic hydrolysis, and the extraction temperature is 40°C.

[0090] In step 3.2), 1% of alumina and 0.5% of chitosan are added based on the weight of the residue after enzymatic hydrolysis in step 2), and stirred at 50°C.

[0091] In step 4.1), the ratio of sodium chloride mass to filtrate volume is 0.3 g:100 ml.

[0092] In step 4.2), the insulation temperature is 30°C.

[0093] In step 5.1), a methanol solvent having a concentration of 80% and a mass 10 times that of the crude isorhamnetin crystals is added, and the pH value of the methanol solvent is adjusted using a sodium hydroxide aqueous solution having a mass concentration of 5%.

[0094] 63.3 g of isorhamnetin crystals were obtained, the isorhamnetin content in the isorhamnetin crystals was 98.46%, and the yield was 0.633%.

[0095] Example 4

[0096] The method steps of this embodiment are the same as those of Embodiment 1, except that:

[0097] In step 1), the supercritical extraction pressure is 22 MPa and the extraction time is 3.5 h.

[0098] In step 2.1), citric acid buffer solution with a pH value of 6.6 and a mass of 1.4 times that of the defatted raw material and sodium dodecyl sulfate are sprayed, and the mass of the sodium dodecyl sulfate is 0.15% of the citric acid buffer solution.

[0099] In step 2.2), 0.4% of the mass of naringinase and pectinase based on the mass of the defatted raw material was added, the mass ratio of naringinase to pectinase was 3.5:1, and enzymolysis was carried out at 50° C. for 4 h.

[0100] In step 3.1), the mass of ethyl acetate is 5 times the mass of the residue after enzymatic hydrolysis, and the extraction temperature is 60°C.

[0101] In step 3.2), 1% of alumina and 1.5% of chitosan by weight of the residue after enzymatic hydrolysis in step 2) are added and stirred at 60°C.

[0102] In step 4.1), the mass ratio of sodium chloride to the volume of the filtrate is 0.5 g:100 ml, and a sodium bicarbonate solution with a mass concentration of 2.5% is used for extraction, and the upper layer liquid is concentrated to a specific gravity of 1.11.

[0103] In step 5.1), a methanol solvent having a concentration of 70% and a mass 15 times that of the crude isorhamnetin crystals is added.

[0104] 60.8 g of isorhamnetin crystals were obtained, the isorhamnetin content in the isorhamnetin crystals was 98.79%, and the yield was 0.608%.

[0105] Example 5

[0106] The method steps of this embodiment are the same as those of Embodiment 1, except that:

[0107] In step 1), the extraction temperature is 40° C. and the extraction time is 5 h.

[0108] In step 2.1), citric acid buffer solution with a pH value of 6.5 and a mass of 0.9 times that of the defatted raw material and sodium dodecylbenzene sulfonate are sprayed, and the mass of the sodium dodecylbenzene sulfonate is 0.08% of the citric acid buffer solution.

[0109] In step 2.2), 0.8% of the mass of the defatted raw material naringinase and pectinase were added, the mass ratio of naringinase to pectinase was 4:1, and enzymolysis was carried out at 50° C. for 3 h.

[0110] In step 3.1), the mass of ethyl acetate is 5 times the mass of the residue after enzymatic hydrolysis.

[0111] In step 3.2), 3% of the weight of the residue after enzymatic hydrolysis in step 2) and 1.0% of chitosan were added and stirred at 60°C.

[0112] In step 4.1), the ratio of sodium chloride mass to filtrate volume is 0.36 g:100 ml.

[0113] In step 5.1), an ethanol solvent having a concentration of 70% and a mass 12 times that of the crude isorhamnetin crystals is added, and the pH value of the ethanol solvent is adjusted using a sodium carbonate aqueous solution having a mass concentration of 5%.

[0114] 62.7 g of isorhamnetin crystals were obtained, the isorhamnetin content in the isorhamnetin crystals was 98.38%, and the yield was 0.627%.

[0115] Comparative Example 1

[0116] The method steps of this embodiment are the same as those of Embodiment 1, except that:

[0117] In step 1), the extraction temperature is 40° C. and the extraction time is 5 h.

[0118] Step 2) double enzymatic hydrolysis process was not implemented.

[0119] In step 3.1), the mass of ethyl acetate is 5 times the mass of the defatted raw material.

[0120] In step 3.2), 3% of alumina and 1.0% of chitosan by weight of the defatted raw material were added and stirred at 60°C.

[0121] In step 4.1), the ratio of sodium chloride mass to filtrate volume is 0.36 g:100 ml, and the upper liquid is concentrated to a specific gravity of 1.08.

[0122] In step 5.1), an ethanol solvent having a concentration of 70% and a mass 12 times that of the crude isorhamnetin crystals is added, and the pH value of the ethanol solvent is adjusted using a sodium carbonate aqueous solution having a mass concentration of 5%.

[0123] 53.0 g of isorhamnetin crystals were obtained, the isorhamnetin content in the isorhamnetin crystals was 98.12%, and the yield was 0.53%.

[0124] Comparative Example 2

[0125] The method steps of this embodiment are the same as those of Embodiment 1, except that:

[0126] In step 1), the extraction temperature is 40° C. and the extraction time is 5 h.

[0127] In step 2.1), citric acid buffer solution with a pH value of 6.5 and a mass of 0.9 times that of the defatted raw material and sodium dodecylbenzene sulfonate are sprayed, and the mass of the sodium dodecylbenzene sulfonate is 0.08% of the citric acid buffer solution.

[0128] In step 2.2), 0.8% of the mass of the defatted raw material naringinase and pectinase were added, the mass ratio of naringinase to pectinase was 4.0:1, and enzymolysis was carried out at 50° C. for 3 h.

[0129] In step 3.1), the mass of ethyl acetate is 5 times the mass of the residue after enzymatic hydrolysis.

[0130] In step 3.2), 3% of the weight of the residue after enzymatic hydrolysis in step 2) and 1.0% of chitosan were added and stirred at 60°C.

[0131] In step 4.1), no sodium chloride is added to the filtrate obtained in step 3.2), and the upper liquid is concentrated to a specific gravity of 1.10.

[0132] In step 5.1), an ethanol solvent having a concentration of 70% and a mass 12 times that of the crude isorhamnetin crystals is added, and the pH value of the ethanol solvent is adjusted using a sodium carbonate aqueous solution having a mass concentration of 5%.

[0133] 58.5 g of isorhamnetin crystals were obtained, the isorhamnetin content in the isorhamnetin crystals was 98.27%, and the yield was 0.585%.

[0134] The concentration methods in the above embodiments all adopt single-effect evaporation equipment for concentration, and the analysis and detection methods of Examples 2-5 and Comparative Examples 1-2 are the same as those of Example 1.

[0135] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for extracting isorhamnetin from seabuckthorn puree, It is characterized in that The following steps are involved: 1) defatting the seabuckthorn puree raw material to obtain a defatted raw material; 2) Double enzymatic hydrolysis 2.1) Spraying a weakly acidic buffer solution with a pH value of 5.5-6.8 and a mass of 0.8-1.5 times that of the defatted raw material and a surfactant into the defatted raw material; the mass of the surfactant is 0.05-0.20% of the total amount of the weakly acidic buffer solution; 2.2) adding 0.3-1.0% of the weight of the defatted raw material naringinase and pectinase, stirring evenly, and performing enzymolysis at 40-55° C. for 3-6 hours; the mass ratio of the naringinase to the pectinase is 2.6-4.2:1; 3) Adsorption and impurity removal 3.1) adding an ester solvent to the residue after enzymatic hydrolysis in step 2.2) for at least 2 extractions, each time for no less than 1 hour, at a temperature of 40-60° C.; the mass of the ester solvent is 3-5 times the mass of the residue after enzymatic hydrolysis; 3.2) After the extract is concentrated to 1 / 2-1 / 3 of the original volume, it is adsorbed and filtered to collect the filtrate; 4) Extraction and crystallization 4.1) adding an electrolyte salting-out agent to the filtrate obtained in step 3.2) at a mass ratio of the electrolyte salting-out agent to the filtrate volume of 0.3-0.5 g:100 ml, then extracting with alkaline water 2-3 times, collecting the upper layer liquid, washing with water until neutral, and then separating the liquids, concentrating the upper layer liquid to a specific gravity of 1.08-1.15; 4.2) Keep the concentrated solution warm at 30-40°C for more than 2 hours for crystallization, and filter to obtain crude isorhamnetin crystals; 5) Recrystallization 5.1) adding an alcohol solvent with a concentration of 60-80% and a mass 10-15 times that of the crude isorhamnetin crystals to the crude isorhamnetin crystals, heating to dissolve, filtering impurities, leaving the crystals to stand and filtering; the alcohol solvent is adjusted to a pH of 9-10 with alkaline water with a mass concentration of 5-10%; 5.2) The filtered crystals are washed with water until they become neutral and dried to obtain isorhamnetin crystals.

2. A method for extracting isorhamnetin from seabuckthorn puree according to claim 1, Features: In step 2.1), the weakly acidic buffer is a citric acid buffer; and the surfactant is a sodium dodecyl sulfonate, sodium dodecyl sulfate or sodium dodecylbenzene sulfonate surfactant.

3. A method for extracting isorhamnetin from seabuckthorn puree according to claim 1 or 2, Features: In step 4.1), the electrolyte salting-out agent is sodium chloride, sodium sulfate or magnesium sulfate; the alkaline water is saturated lime water or a sodium bicarbonate solution with a mass concentration of 1-3%.

4. A method for extracting isorhamnetin from seabuckthorn puree according to claim 3, Features: In step 2.1), spraying a citric acid buffer solution and sodium dodecylbenzene sulfonate with a pH of 6.0; the mass of the citric acid buffer solution and the sodium dodecylbenzene sulfonate is equal to the mass of the defatted raw material, and the mass of the sodium dodecylbenzene sulfonate is 0.10% of the citric acid buffer solution; In step 2.2), naringinase and pectinase in an amount of 0.5% of the defatted raw material are added, stirred evenly, and enzymolyzed at 45° C. for 5 h; the mass ratio of naringinase to pectinase is 3.0:1; In step 4.1), sodium chloride is added to the filtrate obtained in step 3.2), the mass ratio of sodium chloride to the volume of the filtrate is 0.4 g:100 ml, and then saturated lime water solution is used for extraction three times, and the upper layer liquid is concentrated to a specific gravity of 1.09; In step 4.2), the insulation temperature is 40°C.

5. A method for extracting isorhamnetin from seabuckthorn puree according to claim 4, Features: In step 3.1), the ester solvent is ethyl acetate or methyl acetate; In step 3.2), the adsorption filtration is specifically as follows: adding alumina and chitosan to the concentrated solution, stirring at 50-60°C for 1 hour, filtering, and collecting the filtrate; the amounts of alumina and chitosan are 1-3% and 0.5-1.5% of the mass of the residue after double enzymatic hydrolysis in step 2), respectively.

6. A method for extracting isorhamnetin from seabuckthorn puree according to claim 5, Features: In step 5.1), the alcohol solvent is methanol, ethanol or isopropanol, and the alkaline water is sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution or potassium carbonate solution.

7. A method for extracting isorhamnetin from seabuckthorn puree according to claim 6, Features: In step 3.1), ethyl acetate 4 times the mass of the residue after enzymatic hydrolysis was added for extraction, the extraction times were 3 times, each time for 1.5 hours, and the extraction temperature was 50°C; In step 3.2), 2% of the weight of the residue after enzymatic hydrolysis in step 2) and 1.0% of chitosan were added, and stirred at 55° C. for 1 h; In step 5.1), an ethanol solvent having a concentration of 60% and a mass 13 times that of crude isorhamnetin crystals is added; the pH value of the ethanol solvent is adjusted using a potassium hydroxide aqueous solution having a mass concentration of 5%.

8. A method for extracting isorhamnetin from seabuckthorn puree according to claim 7, Features: In step 2), after step 2.2), there is further step 2.3), in which the enzyme is inactivated at 78-82°C for 8-12 minutes.

9. A method for extracting isorhamnetin from seabuckthorn puree according to any one of claims 1 to 8, Features: In step 1), the degreasing is carried out by supercritical extraction; the supercritical extraction pressure is 20-35MPa, the extraction temperature is 40-50°C, and the extraction time is 3-5h.

10. A method for extracting isorhamnetin from seabuckthorn puree according to claim 9, Features: In step 1), the supercritical extraction pressure is 30 MPa, the extraction temperature is 45° C., and the extraction time is 4 h; In step 2.3), the enzyme was inactivated at 80°C for 10 min.

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