CN112516224B - Method for extracting high-purity coumarin from girald daphne bark - Google Patents
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Abstract
The invention belongs to the field of natural product chemistry, and particularly relates to a method for extracting high-purity coumarin from girald daphne bark, which comprises the following steps: (1) supercritical fluid extraction; (2) dissolving the ionic liquid; (3) separating the extract; (4) refining the extract. The invention obtains a primary extract by supercritical fluid extraction, dissolves the extract in ionic liquid, filters, extracts the filtrate by mixed solvent to obtain the girald daphne herb essence, and further refines by a macroporous adsorption resin chromatography method. The invention solves the defects of low purity and difficult impurity removal of the previous daphne giraldii nitsche extraction product, can obtain a high-purity target product, and is particularly suitable for the preparation and scientific research of high-purity medicaments.
Description
Technical Field
The invention belongs to the field of natural product chemistry, and particularly relates to a method for extracting high-purity coumarin from girald daphne bark.
Background
The cortex Daphne Giraldii Nitsche is stem bark and root bark of Daphne giraldii Nitsche, Daphne Shaannsi and Daphne giraldii Nitsche of Thymelaeaceae. Huangrui Xiang is distributed in Shaanxi, Gansu, Qinghai, Sichuan and other places. Shangan Rue Xiang is distributed in Shaanxi, Gansu, Sichuan, Yunnan, Tibet, etc. The daphne odora is distributed in Shaanxi, Gansu, Sichuan and Yunnan provinces. Has the effects of dispelling pathogenic wind, dredging collaterals, removing blood stasis and relieving pain, and can be used for treating rheumatalgia, numbness of limbs, headache, stomach pain, lumbago and traumatic injury.
Daphne giraldii nitsche has complex components and mainly contains chemical components such as coumarins, diterpenes, lignins, flavonoids, anthraquinones, sterols and the like, wherein the most main effective components are daphnetin, daphnetin B, daphnetin-8-O-glucoside, 7-OH coumarins and other coumarins, in the existing preparation of daphne giraldii nitsche, the components such as daphnetin and the like are also used as index components for controlling the quality of medicines, meanwhile, the daphne giraldii nitsche medicinal material belongs to toxic medicinal materials, and the toxic diterpene components are detected by the extraction process of the daphne giraldii nitsche, so that harmful impurities are reduced from the source, the daphne giraldii nitsche mainly comprises daphne genuinin and genidin, and the structural formula is as follows.
Chinese patent CN102008599B discloses a method for preparing girald daphne bark extract, which comprises reflux-extracting with ethanol solution, and refining with macroporous adsorbent resin, wherein the obtained girald daphne bark extract has low purity and can not effectively remove impurities.
Chinese patent application CN108721439A discloses another preparation method of daphne giraldii nitsche extract, which comprises the steps of reflux extraction by using ethanol solution, extraction by sequentially using petroleum ether, ethyl acetate and water saturated n-butanol, and refining by macroporous adsorption resin, thus effectively removing partial impurities, such as toxic diterpenoid components, but failing to obtain good removal effect.
In conclusion, the existing quality control method only measures the content of daphnetin, daphnetin-8-O-glucoside and 7-OH coumarin in medicinal materials, extracts and preparations, and fails to control the quality of toxic components of the medicinal materials, so that the preparations have certain gastrointestinal irritation or skin irritation anaphylactic reaction, therefore, a method capable of comprehensively controlling the active components of the medicinal materials, effectively ensuring the curative effect of the medicinal materials and comprehensively controlling the toxic components of the medicinal materials can effectively control the toxic reaction.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method which can overcome the defects of low purity and difficult impurity removal of daphne giraldii nitsche extraction products and obtain high-purity target products, and the method is particularly suitable for the preparation and scientific research of high-purity medicaments.
The technical scheme of the invention is as follows:
a method for extracting high-purity coumarin from daphne giraldii nitsche is characterized by comprising the following steps:
s1: supercritical fluid extraction; pulverizing cortex Daphne Giraldii Nitsche, sieving with 50 mesh sieve, and passing through supercritical fluid CO2Extracting to obtain a primary extract of girald daphne bark;
s2: dissolving the ionic liquid: placing the primary extract of girald daphne bark obtained in the step S1 in ionic liquid, simultaneously performing microwave treatment, wherein the mass of the ionic liquid is 3-5 times of that of the primary extract of girald daphne bark, and filtering to obtain filtrate which is a treatment product of girald daphne bark;
s3: separating an extract: adding the obtained cortex Daphne Giraldii Nitsche processed product in step S2 into mixed solvent, extracting for 3 times, and separating organic phase to obtain cortex Daphne Giraldii Nitsche coumarin;
s4: refining the extract: and (4) performing chromatography on the girald daphne herb essence obtained in the step (S3) by adopting macroporous adsorption resin, collecting the washed eluent, and performing reduced pressure concentration and drying to finally obtain the high-purity girald daphne herb essence.
Further, the conditions of the supercritical fluid extraction in the step S1 are as follows: controlling the temperature of the extraction kettle to be 45-50 ℃, the pressure of the extraction kettle to be 25-30MPa, the extraction time to be 1-2h, and setting the flow of carbon dioxide to be 20-25 kg/h.
Further, the ionic liquid in the step S2 is 1-butyl-3-methylimidazole chlorine salt water solution with the concentration of 0.8-1.5 mol/L.
Further, the microwave treatment conditions in the step S2 are that the treatment temperature is 40-45 ℃, the treatment time is 10-25min, and the microwave power is 200-400W.
Further, the mixed solvent in the step S3 is prepared from ethyl acetate and ethanol according to a mass ratio of 1: 2-5.
Further, the amount of the mixed solvent of the step S3 is 3 to 10 times of the mass of the processed product of the daphne giraldii nitsche obtained in the step S2.
Further, in the step S4, the macroporous adsorbent resin chromatography step includes washing the macroporous adsorbent resin with water, and then washing the macroporous adsorbent resin with a resolving liquid.
Further, the resolving liquid is a methanol water solution with the volume fraction of 60-75%.
The macroporous adsorption resin used in the method for extracting high-purity coumarin from girald daphne is D-101 type, is purchased from the environmental protection science and technology limited of Xian Wei Hua, and the raw materials such as ethyl acetate, ethanol and the like are all known commercial common raw materials and can be purchased from the market.
Compared with the prior art, the invention has the following advantages:
(1) the invention obtains the preliminary extract by the supercritical fluid extraction mode, dissolves the extract in the ionic liquid, filters, extracts the filtrate by the mixed solvent, and finally refines the extract by the macroporous absorption resin.
(2) The invention creatively combines and applies supercritical fluid extraction, ionic liquid extraction and mixed solvent extraction, and finally, the product is refined through macroporous adsorption resin, so that the high-purity daphnetin extract can be obtained, the content of four coumarins such as daphnetin, daphnetin-8-O-beta-D-glucoside, 7-OH coumarin and the like can reach 88.1 percent through experiment verification, the detection of toxic diterpenoid components can know that the content of toxic components is extremely low, and the content of daphnetin and genistein is only 0.06 percent through experiment verification.
(3) The extraction efficiency is greatly improved by using the ionic liquid, the content is ensured, and simultaneously, higher purity is ensured.
(4) The invention solves the defects of low purity and difficult impurity removal of the prior daphne giraldii nitsche extract, can obtain a high-purity target product, is particularly suitable for the preparation and scientific research of high-purity medicaments, and has wide application prospect.
Detailed Description
The present invention will be further explained by way of specific embodiments in the form of examples. The scope of the above-described subject matter of the present invention is not limited to the following examples.
Example 1 method for extracting high-purity coumarin from daphne giraldii nitsche
A method for extracting high-purity coumarin from daphne giraldii nitsche comprises the following steps:
s1: supercritical fluid extraction: pulverizing cortex Daphne Giraldii Nitsche, sieving with 50 mesh sieve, and passing through supercritical fluid CO2Extracting to obtain a primary extract of girald daphne bark, wherein the supercritical fluid extraction conditions are as follows: controlling the temperature of the extraction kettle to be 45 ℃, the pressure of the extraction kettle to be 25MPa, the extraction time to be 1h, and setting the flow of carbon dioxide to be 20 kg/h;
s2: dissolving the ionic liquid: placing the primary extract of the girald daphne bark obtained in the step S1 in ionic liquid, wherein the ionic liquid is 0.8mol/L of 1-butyl-3-methylimidazolium chloride aqueous solution, the mass of the ionic liquid is 3 times of that of the primary extract of the girald daphne bark, simultaneously performing microwave treatment at the treatment temperature of 40 ℃, the treatment time of 10min and the microwave power of 200W, and filtering to obtain filtrate which is a treatment product of the girald daphne bark;
s3: separating an extract: adding the treated product of girald daphne obtained in the step S2 to a mixed solvent prepared from ethyl acetate and ethanol in a mass ratio of 1: 2, extracting for 3 times, and separating an organic phase to obtain the daphne giraldii essence, wherein the amount of the mixed solvent is 3 times of the mass of a treatment product of the daphne giraldii nitsche;
s4: refining the extract: and (4) performing chromatography on the girald daphne bark essence obtained in the step (S3) by adopting macroporous adsorption resin, washing the macroporous adsorption resin by using water, washing the macroporous adsorption resin by using a methanol aqueous solution with the volume fraction of 60% as an analytic solution, collecting the washed eluent, and performing reduced pressure concentration and drying to finally obtain the high-purity girald daphne bark essence.
Example 2A method for extracting high purity coumarin from Daphne giraldii Nitsche
A method for extracting high-purity coumarin from cortex Daphne Giraldii Nitsche comprises the following steps:
s1: supercritical fluid extraction: pulverizing cortex Daphne Giraldii Nitsche, sieving with 50 mesh sieve, and passing through supercritical fluid CO2Extracting to obtain a primary extract of girald daphne bark, wherein the supercritical fluid extraction conditions are as follows: controlling the temperature of the extraction kettle to be 50 ℃, the pressure of the extraction kettle to be 30MPa, the extraction time to be 2 hours, and setting the flow of carbon dioxide to be 25 kg/h;
s2: dissolving the ionic liquid: placing the primary extract of the girald daphne bark obtained in the step S1 in ionic liquid, wherein the ionic liquid is 1.5 mol/L1-butyl-3-methylimidazolium chloride aqueous solution, the mass of the ionic liquid is 3 times of that of the primary extract of the girald daphne bark, simultaneously performing microwave treatment, the treatment temperature is 45 ℃, the treatment time is 25min, the microwave power is 400W, and filtering to obtain filtrate which is a treatment product of the girald daphne bark;
s3: separating an extract: adding the treated product of girald daphne obtained in the step S2 to a mixed solvent prepared from ethyl acetate and ethanol in a mass ratio of 1: 5, extracting for 3 times, and separating an organic phase to obtain the daphne giraldii nitsche coumarin, wherein the amount of the mixed solvent is 10 times of the mass of the treatment product of the daphne giraldii nitsche;
s4: refining the extract: and (4) performing chromatography on the girald daphne bark essence obtained in the step (S3) by adopting macroporous adsorption resin, washing the macroporous adsorption resin by using water, then washing the macroporous adsorption resin by using a methanol aqueous solution with the volume fraction of 75% as an analytic solution, collecting the washed eluent, concentrating under reduced pressure, and drying to finally obtain the high-purity girald daphne bark essence.
Example 3A method for extracting high purity coumarin from Daphne giraldii Nitsche
A method for extracting high-purity coumarin from daphne giraldii nitsche comprises the following steps:
s1: supercritical fluid extraction: pulverizing cortex Daphne Giraldii Nitsche, sieving with 50 mesh sieve, and passing through supercritical fluid CO2Extracting to obtain a primary extract of girald daphne bark, wherein the supercritical fluid extraction conditions are as follows: controlling the temperature of the extraction kettle to be 45 ℃, the pressure of the extraction kettle to be 30MPa, the extraction time to be 2 hours, and setting the flow of carbon dioxide to be 25 kg/h;
s2: dissolving the ionic liquid: placing the primary extract of the girald daphne bark obtained in the step S1 in ionic liquid, wherein the ionic liquid is 1.2 mol/L1-butyl-3-methylimidazolium chloride aqueous solution, the mass of the ionic liquid is 5 times of that of the primary extract of the girald daphne bark, simultaneously performing microwave treatment at the treatment temperature of 40 ℃, the treatment time of 20min and the microwave power of 300W, and filtering to obtain filtrate which is a treatment product of the girald daphne bark;
s3: separating an extract: adding the treated product of girald daphne obtained in the step S2 to a mixed solvent prepared from ethyl acetate and ethanol in a mass ratio of 1: 3, extracting for 3 times, and separating an organic phase to obtain the daphne giraldii nitsche coumarin, wherein the amount of the mixed solvent is 6 times of the mass of the treatment product of the daphne giraldii nitsche;
s4: refining the extract: and (4) performing chromatography on the girald daphne bark essence obtained in the step (S3) by adopting macroporous adsorption resin, washing the macroporous adsorption resin by using water, then washing the macroporous adsorption resin by using a methanol aqueous solution with the volume fraction of 70% as a resolving solution, collecting the washed eluent, and performing reduced pressure concentration and drying to finally obtain the high-purity girald daphne bark essence.
Comparative example 1 method for extracting coumarin from girald daphne bark
A method for extracting coumarin from daphne giraldii nitsche comprises the following steps:
s1: supercritical fluid extraction: pulverizing cortex Daphne Giraldii Nitsche, sieving with 50 mesh sieve, and passing through supercritical fluid CO2Extracting to obtain a primary extract of girald daphne bark, wherein the supercritical fluid extraction conditions are as follows: controlling the temperature of the extraction kettle to be 45 ℃, the pressure of the extraction kettle to be 30MPa, the extraction time to be 2 hours, and setting the flow of carbon dioxide to be 25 kg/h;
s2: separating an extract: adding the primary extract of girald daphne obtained in the step S2 into a mixed solvent, wherein the mixed solvent is prepared from ethyl acetate and ethanol according to a mass ratio of 1: 3, extracting for 3 times, and separating an organic phase to obtain the daphne giraldii essence, wherein the amount of the mixed solvent is 6 times of the mass of a treatment product of the daphne giraldii nitsche;
s3: refining the extract: and (4) performing chromatography on the girald daphne bark essence obtained in the step (S3) by adopting macroporous adsorption resin, washing the macroporous adsorption resin by using water, washing the macroporous adsorption resin by using a methanol aqueous solution with the volume fraction of 70% as an analytic solution, collecting the washed eluent, and performing reduced pressure concentration and drying to finally obtain the high-purity girald daphne bark essence.
Comparative example 1 is similar to example 3 except that comparative example 1 does not have the ionic liquid dissolution and microwave treatment of the primary extract of girald daphne bark of step S2 in example 3.
Comparative example 2, method for extracting coumarin from daphne giraldii nitsche
A method for extracting coumarin from cortex Daphne Giraldii Nitsche comprises the following steps:
s1: supercritical fluid extraction: pulverizing cortex Daphne Giraldii Nitsche, sieving with 50 mesh sieve, and passing through supercritical fluid CO2Extracting to obtain a primary extract of girald daphne bark, wherein the supercritical fluid extraction conditions are as follows: controlling the temperature of the extraction kettle to be 45 ℃, the pressure of the extraction kettle to be 30MPa, the extraction time to be 2 hours, and setting the flow of carbon dioxide to be 25 kg/h;
s2: dissolving the ionic liquid: placing the primary extract of girald daphne obtained in the step S1 in ionic liquid, wherein the ionic liquid is 1.2 mol/L1-butyl-3-methylimidazolium chloride aqueous solution, the mass of the ionic liquid is 5 times of that of the primary extract of girald daphne obtained, and filtering to obtain filtrate which is a treatment product of girald daphne;
s3: separating an extract: adding the treated product of girald daphne obtained in the step S2 to a mixed solvent prepared from ethyl acetate and ethanol in a mass ratio of 1: 3, extracting for 3 times, and separating an organic phase to obtain the daphne giraldii essence, wherein the amount of the mixed solvent is 6 times of the mass of a treatment product of the daphne giraldii nitsche;
s4: refining the extract: and (4) performing chromatography on the girald daphne bark essence obtained in the step (S3) by adopting macroporous adsorption resin, washing the macroporous adsorption resin by using water, washing the macroporous adsorption resin by using a methanol aqueous solution with the volume fraction of 70% as an analytic solution, collecting the washed eluent, and performing reduced pressure concentration and drying to finally obtain the high-purity girald daphne bark essence.
Comparative example 2 is similar to example 3 except that comparative example 2 step S2 was not treated with microwaves after exposure to the ionic liquid.
Test example 1 measurement of content of Daphne giraldii Nitsche extract
Test subjects: coumarins obtained in examples 1-3 and comparative examples 1-2.
The test method comprises the following steps:
(1) chromatographic conditions and system applicability test: the instrument comprises: acquity UPLC I-Class (Waters, USA);
a chromatographic column: acquity UPLC BEH C18 (2.1X 100mm,1.7 μm); methanol-0.05% phosphoric acid solution (18: 82) is used as a mobile phase; the detection wavelength was 327 nm. The number of theoretical plates should not be less than 9000 calculated by daphnetin peak.
(2) Preparation of control solutions: accurately weighing appropriate amount of daphnetin, daphnetin-8-O-beta-D-glucoside, daphnetin A and daphnetin B reference, adding methanol to obtain mixed solution containing 40 μ g, 35 μ g and 25 μ g per 1mL, and shaking.
(3) Preparation of a test solution: precisely weighing about 0.2g of the prepared high-content coumarin, placing the high-content coumarin into a round-bottomed bottle with a plug, precisely adding 100mL of 80% ethanol in volume fraction, sealing the plug, weighing, heating and refluxing for 2 hours, cooling, weighing again, complementing the loss weight with 80% ethanol, shaking up, precisely weighing 10mL, evaporating to dryness, adding 50% methanol into residues to dissolve, transferring to a 10mL measuring flask, adding 50% methanol to scale, shaking up, filtering, and taking the subsequent filtrate to obtain the coumarin.
(4) The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
And (3) test results: as shown in table 1.
Table 1: content determination of coumarin in girald daphne bark
As can be seen from table 1, the extraction method of daphne giraldii nitsche coumarin provided by the invention has high extraction efficiency, can obtain high-content coumarins, and particularly, in example 3, the total content of four coumarins can reach 88.1%, which is obviously higher than that in comparative example 1 and comparative example 2; the comparative example 1, which did not undergo ionic liquid and microwave treatment, directly extracted the active ingredients to lose and reduce the content, whereas the comparative example 2, which did not undergo microwave treatment, failed to fully react with ionic liquid in the primary extract of daphne giraldii nitsche, affecting the final extraction effect.
Test example 2 measurement of impurity content of Daphne giraldii Nitsche extract
Test subjects: coumarins from examples 1-3 and comparative example 1.
The test method comprises the following steps:
(1) chromatographic conditions are as follows:
the instrument comprises the following steps: acquity UPLC I-Class (Waters, USA);
a chromatographic column: acquity UPLC BEH C18 (2.1X 100mm,1.7 μm);
mobile phase: 0.1% formic acid aqueous solution A-acetonitrile B, gradient elution;
gradient conditions: 0-10 min: 30-95% B, 10-10.5 min: 95-30% of B,10.5-14 min: 30% of B; column oven: 40 ℃; flow rate: 0.4 mL/min; sample introduction amount: 2 μ L.
(2) Preparation of control solutions: accurately weighing appropriate amount of Daphne genkwa and genistein reference substances, adding methanol to obtain 1 μ g/mL solution, and shaking.
(3) Preparation of a test solution: precisely weighing about 0.2g of the prepared high-content coumarin, placing the high-content coumarin into a round-bottomed bottle with a plug, precisely adding 100mL of 80% ethanol, sealing the plug, weighing, heating and refluxing for 2 hours, cooling, weighing again, supplementing the lost weight with 80% ethanol, shaking up, precisely weighing 10mL, evaporating to dryness, dissolving the residue with 50% methanol, transferring to a 10mL measuring flask, adding 50% methanol to the scale, shaking up, filtering, and taking the subsequent filtrate to obtain the coumarin.
(4) The determination method comprises the following steps: precisely sucking 2 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
And (3) test results: as shown in table 2.
Table 2: determination result of impurity content in girald daphne bark
As can be seen from table 2, examples 1 to 3 can significantly reduce the impurity content compared to comparative example 1, which shows that the impurity content can be significantly reduced and the purity of coumarin in the girald daphne bark extract can be improved by the mixed solvent extraction after the treatment with the ionic liquid, and the method is suitable for the preparation and scientific research of high-purity girald daphne bark drugs, and develops a new idea of an extraction process of the traditional Chinese medicine mainly containing coumarin.
Claims (1)
1. A method for extracting high-purity coumarin from daphne giraldii nitsche is characterized by comprising the following steps:
s1: supercritical fluid extraction: pulverizing cortex Daphne Giraldii Nitsche, sieving with 50 mesh sieve, extracting with supercritical fluid CO2, controlling the temperature of the extraction kettle at 45-50 deg.C, the pressure of the extraction kettle at 25-30MPa, the extraction time at 1-2 hr, and setting the flow rate of carbon dioxide at 20-25kg/h to obtain primary extract of cortex Daphne Giraldii Nitsche;
s2: dissolving the ionic liquid: placing the primary extract of the girald daphne obtained in the step S1 in 0.8-1.5mol/L of ionic liquid of 1-butyl-3-methylimidazolium chloride aqueous solution, simultaneously performing microwave treatment, wherein the conditions of the microwave treatment in the step S2 are that the treatment temperature is 40-45 ℃, the treatment time is 10-25min, the microwave power is 200-400W, the mass of the ionic liquid is 3-5 times of that of the primary extract of the girald daphne, and filtering, wherein the obtained filtrate is a treatment product of the girald daphne;
s3: separating an extract: adding the treated product of girald daphne obtained in step S2 to a mixture of ethyl acetate and ethanol in a mass ratio of 1: 2-5, the amount of the mixed solvent is 3-10 times of the mass of the treatment product of the girald daphne bark obtained in the step S2, the extraction is carried out for 3 times, and the organic phase is separated to obtain the girald daphne bark essence;
s4: refining the extract: and (4) performing chromatography on the girald daphne bark essence obtained in the step (S3) by using a D-101 type macroporous adsorption resin, washing the D-101 type macroporous adsorption resin by using water, washing the D-101 type macroporous adsorption resin by using a methanol water solution with the volume fraction of 60-75% as an analytic solution, collecting the washed eluent, concentrating under reduced pressure, and drying to finally obtain the high-purity girald daphne bark essence.
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