CN107759647B - Method for simultaneously extracting hyperoside and luteolin from rosa davurica pall - Google Patents
Method for simultaneously extracting hyperoside and luteolin from rosa davurica pall Download PDFInfo
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- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
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- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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Abstract
The invention provides a method for simultaneously extracting hyperin and luteolin from rosa davurica, which comprises the following steps of carrying out degreasing treatment on rosa davurica; then extracting with ethanol water solution under the assistance of complex enzyme and ionic liquid. The invention creatively uses a proper degreasing pretreatment method and the like before the extraction step; the invention creatively uses a method of complex enzyme auxiliary extraction in the extraction step; the invention creatively uses an ionic liquid auxiliary extraction method in the extraction step, and obtains the extraction extract with the highest hyperin and luteolin content, wherein the hyperin extraction rate is more than 33 mug/g, and the luteolin extraction rate is more than 16 mug/g.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a method for simultaneously extracting hyperin and luteolin from rosa davurica pall.
Background
The rose hip is dry mature fruit of Rosa davurica pall of Rosa davurica of Rosaceae, collected in autumn and dried in the sun, mainly produced in northeast, north China, inner Mongolia and other places, and contains chemical components such as general flavone, vitamin, pectin, sugar, saponin and the like. Pharmacological experiments show that the aqueous extract and the alcohol extract of the rose hip can reduce the blood pressure and the cerebrovascular resistance of anesthetized rats, slow the heart rate of anesthetized cats, increase the coronary flow and reduce the coronary resistance; can also reduce oxygen consumption of mice, prolong the survival time of anoxic mice, and resist the effect of isoproterenol on increasing myocardial oxygen consumption; the fructus Rosae Davuricae water extract can be injected intravenously for inhibiting thrombosis in rat. A large number of domestic researches in recent years show that the health-care tea has the effects of resisting aging, enhancing immunity, resisting fatigue, protecting liver and the like.
Disclosure of Invention
In view of the above, the present invention provides a method for simultaneously extracting hyperoside and luteolin from rosa davurica pall, comprising the following steps:
1) degreasing the rose hip;
2) mixing the degreased rose hip with an extraction solvent, adding a complex enzyme, adjusting the pH value of the system and the temperature of a water bath, and performing enzymolysis;
3) adding appropriate amount of ionic liquid into the enzymatic hydrolysate of fructus Rosae Davuricae, adjusting system water bath temperature, and extracting;
4) placing the extracted steep of the rose hip in a volumetric flask for constant volume, and simultaneously measuring the contents of hyperin and luteolin in the extracted steep by adopting a high performance liquid chromatography.
Preferably, in the method for simultaneously extracting hyperoside and luteolin from rosa davurica pall of the present invention, the degreasing treatment of the rosa davurica pall in the step 1) is to naturally air-dry the rosa davurica pall to make the water content less than 7% by weight, and then, the mass ratio of the material to the liquid is 1:3 (mass: volume) of petroleum ether (boiling range is 60-90 ℃) and carrying out reflux degreasing and low-temperature drying to ensure that the water content is lower than 4 weight percent, thus obtaining the pretreated rose hip; more preferably, the reflux degreasing time of the petroleum ether is 2 times, and the degreasing time is 1.5h and 1.0h respectively; more preferably, the temperature of the low-temperature drying is less than 60 ℃.
Preferably, in the method for simultaneously extracting hyperoside and luteolin from rosa davurica pall, the complex enzyme in the step 2) is formed by mixing cellulase and pectinase according to the mass ratio of 2: 1; the extraction solvent in the step 2) is 70% ethanol water solution; the pH value of the system in the step 2) is 5-6, and the water bath temperature is 45-50 ℃. The ionic liquid in the step 3) is 1-butyl-3-methylimidazole nitrate; the temperature of the water bath in the step 3) is 75-80 ℃.
More preferably, the enzymolysis method in step 2) is to mix 70% ethanol water solution with the pretreated rose hip according to a material-to-liquid ratio (mass: volume ratio) of 1:40, adding complex enzyme which is 0.05 percent of the mass of the rose hip, adjusting the pH value of the system to 5-6 by using 20 percent acetic acid aqueous solution, and performing enzymolysis for 3 hours at the water bath temperature of 45-50 ℃.
More preferably, the method for ionic liquid assisted extraction in step 3) is as follows: adding a proper amount of ionic liquid 1-butyl-3-methylimidazole nitrate into the enzymatic hydrolysate of the rose hip to enable the molar concentration of the ionic liquid to be 0.1-0.6 mol/L, then raising the water bath temperature to 75-80 ℃ for extraction for 5 hours, filtering, concentrating the filtrate under reduced pressure, and putting the filtrate in a proper volumetric flask for constant volume to obtain the extraction immersion liquid of the rose hip.
Preferably, in the method for simultaneously extracting hyperoside and luteolin from rosa davurica pall of the present invention, the content of hyperoside and luteolin in the extract liquid is simultaneously determined by the high performance liquid chromatography in the step 4), and the mobile phase is as follows: acetonitrile as mobile phase A and 0.25% phosphoric acid water solution as mobile phase B, and gradient elution (0 min-17% A;22 min-20% A;27 min-24% A;28 min-28% A;36 min-32% A;41 min-35% A).
The invention provides the rosa davurica fruit extraction immersion liquid containing the hyperin and the luteolin, which is obtained by the method, wherein the extraction rate of the hyperin in the rosa davurica fruit is more than 33 mug/g, and the extraction rate of the luteolin is more than 16 mug/g.
Compared with the prior art, the invention has the following advantages: the invention creatively uses a proper degreasing pretreatment method and the like before the extraction step; the invention creatively uses a method of complex enzyme auxiliary extraction in the extraction step; the invention creatively uses an ionic liquid auxiliary extraction method in the extraction step, and obtains the extraction extract with the highest hyperin and luteolin content, wherein the hyperin extraction rate is more than 33 mug/g, and the luteolin extraction rate is more than 16 mug/g.
Detailed Description
The embodiment of the invention provides a method for simultaneously extracting hyperin and luteolin from rosa davurica pall, which comprises the following steps:
1) degreasing the rose hip;
2) then mixing the degreased rose hip with an extraction solvent, adding a complex enzyme, adjusting the pH value of the system and the temperature of a water bath, and carrying out enzymolysis;
3) adding appropriate amount of ionic liquid into the enzymatic hydrolysate of fructus Rosae Davuricae, adjusting system water bath temperature, and extracting;
4) placing the extracted steep of the rose hip in a volumetric flask for constant volume, and simultaneously measuring the contents of hyperin and luteolin in the extracted steep by adopting a high performance liquid chromatography.
Preferably, in an embodiment of the present invention, the degreasing treatment of the rosa davurica pall in the step 1) is to naturally air-dry the rosa davurica pall to make the water content less than 7% by weight, and then, the degreasing treatment is performed in a material-to-liquid ratio of 1:3 (mass: volume) of petroleum ether (boiling range is 60-90 ℃) and the obtained product is subjected to reflux degreasing and low-temperature drying to ensure that the water content is lower than 4 weight percent, thus obtaining the pretreated rose hip.
More preferably, the reflux degreasing time of the petroleum ether is 2 times, and the degreasing time is 1.5h and 1.0h respectively.
More preferably, the temperature of the low-temperature drying is less than 60 ℃.
Preferably, in the method for simultaneously extracting hyperoside and luteolin from rosa davurica pall, the complex enzyme in the step 2) is formed by mixing cellulase and pectinase according to the mass ratio of 2: 1; the extraction solvent in the step 2) is 70% ethanol water solution; the pH value of the system in the step 2) is 5-6, and the water bath temperature is 45-50 ℃.
Preferably, in the method for simultaneously extracting hyperoside and luteolin from rosa davurica pall, the ionic liquid in the step 3) is 1-butyl-3-methylimidazole nitrate; the temperature of the water bath in the step 3) is 75-80 ℃.
More preferably, the enzymolysis method in step 2) is to mix 70% ethanol water solution with the pretreated rose hip according to a material-to-liquid ratio (mass: volume) of the raw materials is 1:40, adding complex enzyme which is 0.05 percent of the mass of the rose hip, adjusting the pH value of the system to be 5-6 by using 20 percent of acetic acid aqueous solution, and performing enzymolysis for 3 hours at the water bath temperature of 45-50 ℃.
More preferably, the method for ionic liquid assisted extraction in step 3) is as follows: adding a proper amount of ionic liquid 1-butyl-3-methylimidazole nitrate into the enzymatic hydrolysate of the rose hip to enable the molar concentration of the ionic liquid to be 0.1-0.5 mol/L, then raising the water bath temperature to 75-80 ℃ for extraction for 5 hours, filtering, concentrating the filtrate under reduced pressure, and putting the filtrate in a proper volumetric flask for constant volume to obtain the extraction immersion liquid of the rose hip.
Preferably, in the method for simultaneously extracting hyperoside and luteolin from rosa davurica pall of the present invention, the content of hyperoside and luteolin in the extract liquid is simultaneously determined by the high performance liquid chromatography in the step 4), and the mobile phase is as follows: acetonitrile as mobile phase A and 0.25% phosphoric acid water solution as mobile phase B, and gradient elution (0 min-17% A;22 min-20% A;27 min-24% A;28 min-28% A;36 min-32% A;41 min-35% A).
Preferably, in an embodiment of the present invention, in the step 4), the content of hyperoside and luteolin in the extract is determined simultaneously by high performance liquid chromatography, and the chromatographic conditions are as follows: the chromatographic column is big Neelitet ODS 2C18(4.6 mm × 250mm,5 microns), acetonitrile serving as a mobile phase A, 0.25% phosphoric acid aqueous solution serving as a mobile phase B, and gradient elution (0 min-17% A;22 min-20% A;27 min-24% A;28 min-28% A;36 min-32% A;41 min-35% A), wherein the volume flow rate is 1.0mL/min, the column temperature is 35 ℃, the detection wavelength is 360nm, the sample injection amount is 20 mu L, and under the chromatographic conditions, the separation degrees of chromatographic peaks and adjacent peaks of hyperin and luteolin are both greater than 1.5.
Preferably, in an embodiment of the invention, the content of hyperoside and luteolin in the extract is determined simultaneously by high performance liquid chromatography in the step 4), the peak area and the mass concentration of a chromatographic peak of hyperoside in the range of 0.19-15.20 μ g/mL (R = 0.9981) are in a good linear relationship, and the peak area and the mass concentration of a chromatographic peak of luteolin in the range of 0.19-15.20 μ g/mL (R = 0.9977) are in a good linear relationship; in instrument precision test, the RSD of hyperin reference substance is 2.40%, the RSD of rose hip extract is 2.07%, the RSD of luteolin reference substance is 2.35%, and the RSD of rose hip extract is 2.99%; the stability test result shows that the rose hip extract has good stability within 5 hours and meets the measurement requirement; the result of the repeatability test shows that the hyperoside RSD in the rose hip extract is 1.47%, the luteolin RSD in the rose hip extract is 2.93%, and the result shows that the repeatability is good; the sample application recovery rate test result shows that the average recovery rate of hyperin is 101.07%, the RSD is 1.77%, the average recovery rate of luteolin is 97.49%, the RSD is 1.76%, the recovery rate is good, the method accuracy is high, and the feasibility is achieved.
Preferably, in an embodiment of the present invention, in the step 4), the content of hyperoside and luteolin in the extract is determined simultaneously by high performance liquid chromatography, and the results of three batches of process stability verification tests show that: the method has the advantages that the extraction rate of hyperin in the rosa davurica pall is more than 33 mug/g, and the extraction rate of luteolin is more than 16 mug/g.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: extraction of hyperin and luteolin from fructus Rosae Davuricae
1. Pre-treating the rose hip: naturally airing the rosa davurica pall to enable the water content to be lower than 7% by weight, then carrying out reflux degreasing on the rosa davurica pall by using petroleum ether (the boiling range is 60-90 ℃) with the material-liquid ratio of 1:3 (mass: volume), and drying the rosa davurica pall at low temperature to enable the water content to be lower than 4% by weight to obtain the pretreated rosa davurica pall. Naturally drying the rose hip until the water content is lower than 7%, then carrying out reflux degreasing for 2 times (1.5 h and 1.0h each time) by using petroleum ether (60-90 ℃) with the material-liquid ratio of 1:3 (mass: volume), mainly removing lipid, pigment and the like in the rose hip, drying the degreased rose hip in a drying oven at the temperature lower than 60 ℃ until the water content is lower than 4%, and obtaining the pretreated rose hip;
2. enzymolysis: mixing 70% ethanol water solution with the pretreated rose hip according to the material-liquid ratio (mass: volume) of 1:40, adding a cellulase and pectinase complex enzyme (mass ratio of 2:1) which is 0.05% of the mass of the rose hip, adjusting the pH value of the system to 5-6 by using 20% acetic acid water solution, and performing enzymolysis for 3 hours at the water bath temperature of 45-50 ℃;
3. auxiliary extraction by ionic liquid: adding a proper amount of ionic liquid 1-butyl-3-methylimidazole nitrate into the enzymatic hydrolysate of the rose hip to enable the molar concentration of the ionic liquid to be 0.4mol/L, then raising the water bath temperature to 75-80 ℃ for extraction for 5h, filtering, concentrating the filtrate under reduced pressure, and putting the filtrate in a proper volumetric flask for constant volume to obtain the extraction immersion liquid of the rose hip.
Content determination: absorbing a proper amount of rose hip extract obtained by extraction, filtering the rose hip extract by a 0.45 mu m organic filter membrane to be used as a test solution, then injecting 20 mu L of sample according to chromatographic conditions, measuring peak areas of chromatographic peaks of hyperin and luteolin, and calculating the mass concentration of the hyperin and luteolin in the extract by adopting an external standard method, thereby calculating the extraction rate of the hyperin and luteolin, wherein the extraction rate of the hyperin and luteolin is 34.62 mu g/g and 17.01 mu g/g respectively.
Weighing a proper amount of dried rose hip powder according to the influence of an extraction solvent on the extraction effect, putting the rose hip powder into a round-bottomed flask, adding an ethanol aqueous solution with the volume fractions of 0%, 10%, 30%, 50%, 70% and 90% of ethanol according to the feed-liquid ratio of 1:20 (g: mL), adding a complex enzyme (cellulase: pectinase =2: 1; mass ratio) with the mass fraction of 0.05% of the mass of rose hip, adjusting the pH value of the system to 5-6 by using a 20% acetic acid aqueous solution, carrying out enzymolysis for 3h at the water bath temperature of 45-50 ℃, adding a proper amount of ionic liquid 1-butyl-3-methylimidazole nitrate so that the molar concentration of the ionic liquid is 0.1mol/L, raising the water bath temperature to 75-80 ℃, carrying out extraction for 5h, filtering, carrying out reduced-pressure concentration on the filtrate, putting the filtrate into a proper volumetric flask for constant volume sampling, carrying out sample injection according to the chromatographic condition of 20 muL, measuring peak areas of hyperin and luteolin chromatographic peaks, and calculating the mass concentration of the hyperin and luteolin in the extracted immersion liquid by adopting an external standard method, thereby calculating the extraction rate of the hyperin and the luteolin, wherein when the extraction solvent is 70% ethanol water solution by volume fraction of ethanol, the extraction rate of the hyperin and the luteolin is the highest, and the extraction rate is respectively 25.47 microgram/g and 9.51 microgram/g.
Weighing a proper amount of dried rose hip powder under the influence of enzyme types on the extraction effect, putting the rose hip powder into a round-bottomed flask, adding 70% ethanol aqueous solution according to the material-liquid ratio of 1:20 (g: mL), respectively adding cellulase, pectinase and a complex enzyme of the cellulase and the pectinase, wherein the mass ratio of the cellulase to the pectinase is 1:1, 2:1, 3: 1, 1:2 and 1:3, the pH value of the system is adjusted to 5-6 by using 20% acetic acid aqueous solution, carrying out enzymolysis for 3h at the water bath temperature of 45-50 ℃, then adding a proper amount of ionic liquid 1-butyl-3-methylimidazole nitrate to ensure that the molar concentration of the ionic liquid is 0.1mol/L, then raising the water bath temperature to 75-80 ℃ for extraction for 5h, filtering, carrying out reduced-pressure concentration on the filtrate, putting the filtrate into a proper volumetric flask for sample injection, then carrying out sample injection according to the chromatographic condition of 20 mu L, measuring peak areas of hyperin and luteolin chromatographic peaks, and calculating the mass concentration of the hyperin and luteolin in the extracted immersion liquid by adopting an external standard method, thereby calculating the extraction rate of the hyperin and the luteolin, and as a result, when the mass ratio of the cellulase to the pectinase is 2:1, the extraction rate of the hyperin and the luteolin is the highest, and the extraction rate is respectively 25.55 microgram/g and 9.48 microgram/g.
Weighing a proper amount of dried rose hip powder under the influence of enzymolysis time on extraction effect, putting the rose hip powder into a round-bottomed flask, adding 70% ethanol aqueous solution according to a feed-liquid ratio of 1:20 (g: mL), adding complex enzyme (cellulase: pectinase =2: 1; mass ratio) which is 0.05% of the mass of the rose hip, adjusting the pH value of the system to be 5-6 by using 20% acetic acid aqueous solution, carrying out enzymolysis for 1, 2, 3, 4 and 5 hours under the condition that the water bath temperature is 45-50 ℃, then adding a proper amount of ionic liquid 1-butyl-3-methylimidazole nitrate to ensure that the molar concentration of the ionic liquid is 0.1mol/L, then raising the water bath temperature to 75-80 ℃ for extraction for 5 hours, filtering, carrying out reduced pressure concentration on filtrate, placing the filtrate into a proper volumetric flask for constant volume, carrying out sample injection for 20 mu L according to chromatographic conditions, determining peak areas of hyperoside and luteolin, and calculating the mass concentration of the hyperin and the luteolin in the extracted immersion liquid by adopting an external standard method, thereby calculating the extraction rate of the hyperin and the luteolin, and as a result, when the enzymolysis time is 3h, the extraction rate of the hyperin and the luteolin is the highest and is respectively 26.01 mug/g and 9.55 mug/g, the enzymolysis time is continuously prolonged, and the extraction rate of the hyperin and the luteolin is slightly increased.
Weighing a proper amount of dried rose hip powder according to the influence of the molar concentration of the ionic liquid on the extraction effect, putting the rose hip powder into a round-bottomed flask, adding 70% ethanol aqueous solution according to the feed-liquid ratio of 1:20 (g: mL), adding complex enzyme (cellulase: pectinase =2: 1; mass ratio) which is 0.05% of the mass of the rose hip, adjusting the pH value of the system to 5-6 by using 20% acetic acid aqueous solution, carrying out enzymolysis for 3h at the water bath temperature of 45-50 ℃, then adding a proper amount of ionic liquid 1-butyl-3-methylimidazole nitrate to ensure that the molar concentration of the ionic liquid is 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6mol/L respectively, then raising the water bath temperature to 75-80 ℃ for extraction and volume fixing for 5h, filtering, carrying out reduced pressure concentration on the filtrate, putting the filtrate into a proper volumetric flask, then carrying out sample injection according to the chromatographic condition of 20 mu L, and measuring the peak areas of the chromatographic peaks of the hyperin and luteolin, the mass concentrations of the hyperin and the luteolin in the extracted immersion liquid are calculated by adopting an external standard method, so that the extraction rates of the hyperin and the luteolin are calculated, and as a result, when the molar concentration of the ionic liquid is 0.4mol/L, the extraction rates of the hyperin and the luteolin are the highest and are respectively 29.06 mug/g and 12.21 mug/g, the molar concentration of the ionic liquid is continuously improved, and the extraction rates of the hyperin and the luteolin are not obviously increased.
Weighing a proper amount of dried rose hip powder under the influence of feed liquid ratio on extraction effect, putting the rose hip powder into a round-bottomed flask, adding 70% ethanol aqueous solution according to the feed liquid ratio of 1:5, 1:10, 1:20, 1:30, 1:40 and 1:50 (g: mL), adding complex enzyme (cellulase: pectinase =2: 1; mass ratio) which is 0.05% of the mass of rose hip, adjusting the pH value of the system to 5-6 by using 20% acetic acid aqueous solution, performing enzymolysis for 3h at the water bath temperature of 45-50 ℃, adding a proper amount of ionic liquid 1-butyl-3-methylimidazole nitrate so that the molar concentration of the ionic liquid is 0.4mol/L, raising the water bath temperature to 75-80 ℃, extracting for 5h, filtering, concentrating the filtrate under reduced pressure, putting the filtrate into a proper volumetric flask, injecting 20 mu L under chromatographic conditions, measuring the peak areas of the hypericin and the luteolin chromatographic peak, the mass concentration of the hyperin and the luteolin in the extracted immersion liquid is calculated by adopting an external standard method, so that the extraction rate of the hyperin and the luteolin is calculated, and as a result, when the material-liquid ratio is 1:40, the extraction rate of the hyperin and the luteolin is high and is respectively 31.15 microgram/g and 14.08 microgram/g, and the extraction rate of the hyperin and the luteolin is slightly increased and is smaller with the increase of the material-liquid ratio.
Weighing a proper amount of dried rose hip powder under the influence of the ionic liquid auxiliary extraction time on the extraction effect, putting the rose hip powder into a round-bottomed flask, adding 70% ethanol aqueous solution according to the material-to-liquid ratio of 1:40 (g: mL), adding complex enzyme (cellulase: pectinase =2: 1; mass ratio) which is 0.05% of the mass of rose hip, adjusting the pH value of the system to 5-6 by using 20% acetic acid aqueous solution, carrying out enzymolysis for 3h at the water bath temperature of 45-50 ℃, then adding a proper amount of ionic liquid 1-butyl-3-methylimidazole nitrate to enable the molar concentration of the ionic liquid to be 0.4mol/L, then raising the water bath temperature to 75-80 ℃, extracting 1, 2, 3, 4, 5 and 6h respectively, filtering, concentrating the filtrate under reduced pressure, putting the filtrate into a proper volumetric flask for constant volume, then feeding mu L according to the chromatographic condition of 20 samples, measuring the peak areas of hypericin and luteolin chromatographic peaks, the mass concentration of the hyperin and the luteolin in the extracted immersion liquid is calculated by adopting an external standard method, so that the extraction rate of the hyperin and the luteolin is calculated, and as a result, when the ionic liquid auxiliary extraction time is 5 hours, the extraction rate of the hyperin and the luteolin is higher and is respectively 32.64 microgram/g and 15.21 microgram/g, and the extraction rate of the hyperin and the luteolin is slightly increased and is smaller with the extension of the extraction time.
Weighing a proper amount of dried rose hip powder according to the influence of the ionic liquid auxiliary extraction water bath temperature on the extraction effect, putting the rose hip powder into a round bottom flask, adding 70% ethanol water solution according to the material-to-liquid ratio of 1:40 (g: mL), adding complex enzyme (cellulase: pectinase =2: 1; mass ratio) which is 0.05% of the mass of rose hip, adjusting the pH value of the system to 5-6 by using 20% acetic acid water solution, carrying out enzymolysis for 3h at the water bath temperature of 45-50 ℃, then adding a proper amount of ionic liquid 1-butyl-3-methylimidazole nitrate to ensure that the molar concentration of the ionic liquid is 0.4mol/L, then respectively controlling the water bath temperature to be 50-60 ℃, 60-70 ℃, 70-75 ℃, 75-80 ℃, 80-85 ℃, extracting for 5h, filtering, carrying out reduced pressure concentration on the filtrate, putting the filtrate into a proper volumetric flask, then carrying out sample injection according to the chromatographic condition of 20 mu L, measuring peak areas of hyperin and luteolin chromatographic peaks, and calculating the mass concentration of the hyperin and luteolin in the extracted immersion liquid by adopting an external standard method, thereby calculating the extraction rate of the hyperin and the luteolin, and as a result, when the temperature of the ionic liquid auxiliary extraction water bath is 75-80 ℃, the extraction rate of the hyperin and the luteolin is the highest, namely 32.29 mug/g and 15.37 mug/g respectively.
Through multiple experiments, the enzyme species is fixed as complex enzyme (cellulase: pectinase =2: 1; mass ratio), the ionic liquid auxiliary extraction time is fixed for 5 hours, when the ionic liquid auxiliary extraction water bath temperature is fixed to 75-80 ℃, the extraction rate of hyperin and luteolin is high, and in order to further optimize the extraction process conditions, four-factor three-level orthogonal experiments are carried out by selecting ethanol volume fraction A (65%, 70% and 75%), material-liquid ratio B (1: 35, 1:40 and 1: 45), enzymolysis time C (3.0, 3.5 and 4.0 hours) and ionic liquid molar concentration (0.35, 0.4 and 0.45 mol/L).
Orthogonal test results and process verification test: the orthogonal test result shows that the optimal extraction condition is A2B2C1D2The method comprises the steps of extracting 70% ethanol water solution as a solvent, wherein the material-liquid ratio is 1:40, the enzyme type is complex enzyme (cellulase: pectinase =2: 1; mass ratio), the enzymolysis time is 3 hours, the ionic liquid molar concentration is 0.4mol/L, the ionic liquid auxiliary extraction water bath temperature is 75-80 ℃, and the ionic liquid auxiliary extraction time is 5 hours. The extraction rates of hyperin are 35.48 mug/g, 33.98 mug/g and 36.29 mug/g respectively according to the results of the three process verification tests, and the extraction rates of luteolin are 16.61 mug/g, 17.37 mug/g and 17.02 mug/g respectively, which shows that the process is stable and feasible.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A method for simultaneously extracting hyperoside and luteolin from Rosa davurica pall comprises the following steps:
1) degreasing the rose hip;
2) mixing the degreased rose hip with an extraction solvent, adding a complex enzyme, adjusting the pH value of the system and the temperature of a water bath, and performing enzymolysis; the compound enzyme is prepared from cellulase and pectinase according to the mass ratio of 2:1, mixing; the extraction solvent is 70% ethanol water solution; the pH value of the system is 5-6, and the water bath temperature is 45-50 ℃;
3) adding ionic liquid into the enzymatic hydrolysate of the rose hip, and then adjusting the water bath temperature of the system for extraction; the ionic liquid is 1-butyl-3-methylimidazole nitrate; the temperature of the water bath is 75-80 ℃; the extraction method comprises the following steps: adding ionic liquid 1-butyl-3-methylimidazole nitrate into the enzymatic hydrolysate of the rose hip to enable the molar concentration of the ionic liquid to be 0.1-0.5 mol/L, then raising the temperature of a water bath to 75-80 ℃, extracting for 5 hours, filtering, concentrating the filtrate under reduced pressure, and fixing the volume to a proper volumetric flask to obtain an extraction immersion liquid of the rose hip;
4) the volume of the extracted immersion liquid of the rose hip is determined to a volumetric flask, and the content of hyperin and luteolin in the extracted immersion liquid is simultaneously measured by adopting a high performance liquid chromatography.
2. The method as claimed in claim 1, wherein the degreasing treatment of the rosa davurica pall in the step 1) is natural drying of the rosa davurica pall to make the water content less than 7% by weight, then refluxing and degreasing with petroleum ether with a boiling range of 60 ℃ to 90 ℃ in a mass-to-liquid ratio of 1:3, drying at low temperature to make the water content less than 4% by weight, and obtaining the pretreated rosa davurica pall.
3. The method according to claim 2, wherein the reflux degreasing time of the petroleum ether is 2 times, and the degreasing time is 1.5h and 1.0h respectively; the low temperature drying temperature is lower than 60 ℃.
4. The method as claimed in claim 1, wherein the enzymolysis method in step 2) is to mix 70% ethanol water solution with the pretreated rose hip according to the mass-to-volume ratio of 1:40, add complex enzyme with the mass of 0.05% of the rose hip, adjust the pH value of the system to 5-6 with 20% acetic acid water solution, and perform enzymolysis for 3 hours at the water bath temperature of 45-50 ℃.
5. The method as claimed in claim 1, wherein the content of hyperoside and luteolin in the extractive solution is determined simultaneously by high performance liquid chromatography in step 4), and the mobile phase is as follows: acetonitrile as mobile phase A, 0.25% phosphoric acid water solution as mobile phase B, gradient elution procedure: 0 min-17% A;22 min-20% A;27 min-24% A;28 min-28% A;36 min-32% A;41min- -35% A.
6. The method according to claim 1, wherein the extraction rate of hyperoside in the rose hip is more than 33 μ g/g and the extraction rate of luteolin is more than 16 μ g/g.
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