CN114890974A - Method for extracting osthole by response surface design method - Google Patents
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- HPUXDMUGCAWDFW-UHFFFAOYSA-N Osthole Natural products COc1ccc2CCC(=O)Oc2c1C=CC(=O)C HPUXDMUGCAWDFW-UHFFFAOYSA-N 0.000 title claims abstract description 138
- MBRLOUHOWLUMFF-UHFFFAOYSA-N osthole Chemical compound C1=CC(=O)OC2=C(CC=C(C)C)C(OC)=CC=C21 MBRLOUHOWLUMFF-UHFFFAOYSA-N 0.000 title claims abstract description 138
- 230000004044 response Effects 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 43
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 306
- 238000000605 extraction Methods 0.000 claims abstract description 172
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- 238000002474 experimental method Methods 0.000 claims abstract description 28
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- 238000004364 calculation method Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 59
- 238000005303 weighing Methods 0.000 claims description 36
- 239000013558 reference substance Substances 0.000 claims description 29
- 239000002904 solvent Substances 0.000 claims description 25
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 14
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- 239000000463 material Substances 0.000 claims description 5
- 238000002137 ultrasound extraction Methods 0.000 claims description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
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- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 3
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- OGJPXUAPXNRGGI-UHFFFAOYSA-N norfloxacin Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCNCC1 OGJPXUAPXNRGGI-UHFFFAOYSA-N 0.000 description 1
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- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/74—Benzo[b]pyrans, hydrogenated in the carbocyclic ring
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Extraction Or Liquid Replacement (AREA)
Abstract
The invention discloses a method for extracting osthole by using a response surface Design method, which relates to the technical field of extraction and separation of active ingredients of traditional Chinese medicines, and the extraction process comprises the steps of adding methanol in a certain proportion into cnidium fruit powder for reflux extraction, adopting a Box-Behnken response surface Design method of Design-Expert software on the basis of a single-factor experiment, selecting four factors of methanol concentration, extraction time, material-liquid ratio and temperature as response variables, taking an optimal point of the single-factor experiment as a center, taking a horizontal value around the optimal point as the level of a response surface, respectively coding the four factors of-1, 0 and 1, taking the extraction rate of osthole as a response value, carrying out polynomial fitting regression on each factor to obtain a regression equation, and carrying out regression calculation on the equation to obtain the optimal extraction process condition of osthole.
Description
Technical Field
The invention relates to the technical field of extraction and separation of active ingredients of traditional Chinese medicines, in particular to a method for extracting osthole by a response surface design method.
Background
The osthole is derived from dried fruit of cnidium monnieri (L.) DC of Umbelliferae, and has effects of eliminating dampness, dispelling pathogenic wind, killing parasites, relieving itching, warming kidney, and tonifying yang. Modern pharmacological research shows that osthole has good effects in resisting arrhythmia, osteoporosis, AIDS, fungi, tumor, blood pressure, and mutagenesis. The main medicinal active ingredients of the cnidium fruit are coumarin and volatile oil compounds, wherein osthole is the main active ingredient with the highest content, and the content level of osthole in different producing areas is 24.17-189.32 mg.L -1 Within the range.
The prior research methods for extracting and separating osthole from fructus cnidii include ultrasonic-assisted extraction, enzyme-assisted extraction, microwave-assisted extraction, supercritical carbon dioxide extraction, two-aqueous-phase extraction, column separation chromatography and the like, and the extraction methods can extract osthole to a certain extent, but the methods have the defects of high requirements on extraction devices, limited industrial amplification, difficult achievement of laboratory conditions and the like. The optimization of the extraction process of the osthole mainly starts from two aspects of improving the extraction rate and improving the economy of the extraction method. With the development of modern pharmacological experimental techniques and the diversification of research methods, the pharmacological action of cnidium monnieri is gradually explored, the clinical medication advantages shown by cnidium monnieri are gradually shown, and the demand of cnidium monnieri is increasing, so that the development of the technology for improving the extraction and separation of cnidium monnieri is urgent.
Disclosure of Invention
Aiming at the defects in the prior art that the extraction efficiency of the osthole is low, the economic cost is high, part of extraction methods are limited in industrial scale-up production, the laboratory conditions are difficult to achieve and the like, the invention aims to provide a method with simple conditions and high extraction rate, and the osthole is economically and efficiently extracted from the traditional Chinese medicine fructus cnidii.
In order to achieve the above purposes, the technical scheme adopted by the invention is as follows: a method for extracting osthole by using a response surface Design method comprises the steps of adding methanol in a certain proportion into cnidium fruit powder for reflux extraction, selecting four factors including methanol concentration, extraction time, material-liquid ratio and temperature as response variables by using a Box-Behnken response surface Design method of Design-Expert software on the basis of a single-factor experiment, taking an optimal point of the single-factor experiment as a center, respectively taking a horizontal value around the optimal point as the level of a response surface, respectively coding the four factors to be-1, 0 and 1, taking osthole extraction rate as a response value, and performing polynomial fitting regression on the factors to obtain a regression equation:
Y＝6.88-0.1808A+2.8B-0.0625C+3.21D-0.1525AB-0.085AC+0.25AD+0.3775BC+0.845BD+0.025CD+0.0918A2-0.5195B2-0.3483C2-1.81D2；
in the formula, A is the concentration of methanol, B is the extraction time, C is the ratio of material to liquid, D is the temperature, and Y is the extraction rate of osthole;
and (4) performing regression calculation on the equation to obtain the optimal extraction process condition of the osthole.
On the basis of the technical scheme, the optimal extraction process conditions of the osthole are as follows: the extraction time was 1 hour, the liquid-to-feed ratio was 11, the temperature was 58.8 ℃ and the methanol concentration was 78%.
On the basis of the technical scheme, the osthole extraction rate obtained under the optimal extraction process conditions is 14.49% -14.76%, and the antioxidant activity of the osthole is superior to that of ascorbic acid within the concentration range of 40-100 mug/mL.
On the basis of the technical scheme, the chromatographic conditions of the osthole are that a chromatographic column is a C18 analytical column, a mobile phase A is chromatographic grade methanol, a mobile phase B is a 0.1% formic acid solution, the flow rate is 1.0mL/min, the detection wavelength is 314nm, the column temperature is 30 ℃, and the sample injection amount is 20 uL.
On the basis of the technical scheme, Design-Expert software is used for designing the response surface, and system software is used for simulating and generating the optimal extraction process parameters.
On the basis of the technical scheme, the method for the single-factor experiment comprises the following steps:
(1) pretreating the fructus cnidii: crushing dried fruits of cnidium monnieri, sieving the crushed fruits by a No. 3 sieve, and drying the crushed fruits for 4 to 6 hours at the temperature of between 50 and 90 ℃ to obtain dried powder of the cnidium monnieri fruit;
(2) preparation of control solutions: accurately weighing 1.0mg of osthole reference substance, dissolving in 1mL of methanol solution, and preparing into 1.0mg/mL osthole reference substance solution;
(3) preparation of a test solution: extracting osthole by methanol reflux extraction; taking 5.0g of fructus cnidii powder, adding a methanol solution according to a material-to-liquid ratio of 4: 1-12: 1, placing the mixture in a water bath kettle, setting the extraction temperature to be 50-90 ℃, keeping the magnetic stirring speed constant, extracting for 0.5-2 hours, complementing the weight, and filtering to obtain a osthole test solution;
(4) measurement method and wavelength selection: respectively sucking 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL of reference substance solution, placing in a 10mL volumetric flask, adding methanol for dilution to scale, and shaking up; finally, a methanol reagent is used as a blank, an ultraviolet spectrophotometer is adopted to determine an absorption spectrogram at 210-600nm, and the maximum absorption wavelength is 314 nm; finally, 314nm is selected as the measuring wavelength;
(5) and (3) verification of an extraction mode: respectively taking 3 parts of fructus cnidii powder, precisely weighing each part, adding methanol solution with the same material-to-liquid ratio, and respectively performing ultrasonic extraction and methanol solvent reflux for the same time to obtain osthole extracting solutions in different extraction modes; the result shows that the extraction rate of reflux extraction is highest;
(6) selection of an extraction solvent: respectively taking 3 parts of fructus cnidii powder, precisely weighing each part, adding methanol solution and ethanol solution with the same material-to-liquid ratio, controlling other variables to be completely consistent, and respectively performing reflux extraction at 60 ℃ for 1 hour to obtain osthole extracting solutions extracted by different extraction solvents; the result shows that the extraction rate is highest when methanol is used as a solvent;
(7) experiment of extraction times: respectively taking 3 parts of fructus Cnidii powder, precisely weighing each part, adding methanol solution with the same material-to-liquid ratio, controlling other variables to be consistent, respectively extracting under reflux at 60 deg.C for 1 hr, respectively extracting for 1 time, 2 times and 3 times; obtaining osthole extracting solutions under different extraction times; the result shows that the osthole extraction rate is highest when the extraction frequency is 1, so that the subsequent experiment optimization is carried out by selecting the extraction frequency to be 1.
On the basis of the technical scheme, the method also comprises the following linear relation investigation: precisely weighing 5mg of osthole reference substance, and dissolving with 5mL of methanol solvent to obtain 1mg/mL reference substance stock solution; respectively sucking 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL of reference substance solution, placing in a 10mL volumetric flask, adding methanol to dilute to scale, and shaking up to obtain a series of reference substance solutions; taking the concentration of a reference substance as a horizontal coordinate and taking a peak area as a vertical coordinate; the regression equation Y of the standard curve is 64.979X-342.08(R is 0.9996), and the linear range is 2.2 ug/mL-250 ug/mL.
On the basis of the technical scheme, the method further comprises the following steps:
investigation of methanol concentration: accurately weighing 2.0g of each of 3 parts of osthole powder, wherein the fixed temperature is 70 ℃, and the material-liquid ratio is 1: 8. the extraction time is 1 h; adding 50%, 60%, 70%, 80% and 90% methanol respectively, and examining the influence of the methanol concentration on the extraction rate of osthole;
examination of extraction time: accurately weighing 2.0g of each of 5 parts of osthole powder, wherein the fixed temperature is 70 ℃, and the material-liquid ratio is 1: 8. the methanol concentration is 70%; setting the time of methanol solvent reflux extraction as 0.5h, 1h, 1.5h and 2h respectively, and investigating the influence of the extraction time on the osthole extraction rate;
and (3) investigating liquid-material ratio: accurately weighing 2.0g each of 5 parts of osthole powder, fixing temperature at 70 deg.C, methanol concentration at 70%, and extraction time at 1 hr; setting the material-liquid ratio as 1: 4. 1: 6. 1: 8. 1: 10. 1: 12, investigating the influence of different feed liquid ratios on the extraction rate of the osthole;
temperature investigation: accurately weighing 2.0g of each of 5 parts of osthole powder, fixing the concentration of methanol to be 70%, and the material-liquid ratio to be 1: 8. the extraction time is 1 h; the temperature was set at 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, and 90 deg.C, and the effect of different temperatures on the extraction rate of osthole was examined.
The invention has the beneficial effects that:
the extraction method is simple and easy to implement, mild in condition, clean and pollution-free in experimental process and high in extraction rate, and can be used for extracting osthole in large industrial dose. Meanwhile, an antioxidant experiment shows that the osthole has better antioxidant capacity, and theoretical support is provided for development and utilization of the osthole.
Drawings
FIG. 1 is a flow chart of an extraction and assay experiment of the present invention;
FIG. 2 is a chromatogram peak superposition diagram of osthole reference substance and sample;
FIG. 3 is a chromatogram of an osthole reference substance of the present invention under HPLC-MS conditions;
FIG. 4 is a mass spectrum corresponding to the osthole reference substance of the present invention;
FIG. 5 is an HPLC-MS chromatogram under the optimal extraction conditions of fructus Cnidii of the present invention;
FIG. 6 is a mass spectrum of the present invention under the optimum condition for extracting osthole;
FIG. 7 is a graph of the response surface of the interaction of various factors of the osthole extraction rate according to the present invention;
FIG. 8 is a graph showing the results of oxidation resistance tests of osthole and ascorbic acid.
Detailed Description
The technical scheme and the beneficial effects of the invention are clearer and clearer by further describing the specific embodiments of the invention with the accompanying drawings of the specification. The embodiments described below are exemplary and are intended to be illustrative of the invention, but are not to be construed as limiting the invention.
The embodiment of the invention provides a method for extracting osthole by using a response surface Design method, which comprises the steps of adding methanol in a certain proportion into cnidium fruit powder for reflux extraction, adopting a Box-Behnken response surface Design method of Design-Expert software on the basis of a single-factor experiment, selecting four factors of methanol concentration, extraction time, material-liquid ratio and temperature as response variables, taking an optimal point of the single-factor experiment as a center, respectively taking a horizontal value around the optimal point as the level of a response surface, respectively coding the four factors to be-1, 0 and 1, taking the extraction rate of the osthole as a response value, and carrying out polynomial fitting regression on each factor to obtain a regression equation:
Y＝6.88-0.1808A+2.8B-0.0625C+3.21D-0.1525AB-0.085AC+0.25AD+0.3775BC+0.845BD+0.025CD+0.0918A 2 -0.5195B 2 -0.3483C 2 -1.81D 2 ；
wherein A is methanol concentration, B is extraction time, C is material-liquid ratio, D is temperature, and Y is osthole extraction rate;
and (4) performing regression calculation on the equation to obtain the optimal extraction process condition of the osthole.
Specifically, the optimal extraction process conditions of the osthole are as follows: the extraction time is 1 hour, the liquid-material ratio is 14:1, the temperature is 64 ℃, and the methanol concentration is 75.8%.
Specifically, the osthole extraction rate obtained under the optimal extraction process conditions is 14.49% -14.76%, and the antioxidant activity of the osthole is superior to that of ascorbic acid within the concentration range of 40-100 mug/mL.
Specifically, the chromatographic conditions of the osthole are that a chromatographic column is a C18 analytical column, a mobile phase A is chromatographic grade methanol, a mobile phase B is a 0.1% formic acid solution, the flow rate is 1.0mL/min, the detection wavelength is 314nm, the column temperature is 30 ℃, and the sample injection amount is 20 uL.
Specifically, Design-Expert software is used for designing a response surface, and system software is used for simulating and generating the optimal extraction process parameters.
Specifically, the method of the single-factor experiment comprises the following steps:
(1) pretreating the fructus cnidii: pulverizing dried fruit of cnidium fruit, sieving with No. 3 sieve, and drying at a certain temperature for a certain time to obtain dried powder of cnidium fruit;
(2) preparation of control solutions: accurately weighing 1.0mg of osthole reference substance, dissolving in 1mL of methanol solution, and preparing into 1.0mg/mL osthole reference substance solution;
(3) preparation of a test solution: extracting osthole by methanol reflux extraction; taking 5.0g of fructus Cnidii powder, adding methanol solution at a certain ratio, placing in a water bath, extracting at a certain temperature, magnetic stirring speed and extraction time, filtering, and supplementing the weight to obtain osthole test solution;
(4) measurement method and wavelength selection: respectively sucking 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL of reference substance solution, placing in a 10mL volumetric flask, adding methanol for dilution to scale, and shaking up; finally, a methanol reagent is used as a blank, an ultraviolet spectrophotometer is adopted to determine an absorption spectrogram at 210-600nm, and the maximum absorption wavelength is 314 nm; finally, 314nm is selected as the measuring wavelength;
(5) and (3) verification of an extraction mode: respectively taking 3 parts of fructus cnidii powder, precisely weighing each part, adding methanol solution with the same material-to-liquid ratio, and respectively performing ultrasonic extraction and methanol solvent reflux for the same time to obtain osthole extracting solutions in different extraction modes; the result shows that the extraction rate of reflux extraction is highest;
(6) selection of an extraction solvent: respectively taking 3 parts of fructus cnidii powder, precisely weighing each part, adding methanol solution and ethanol solution with the same material-to-liquid ratio, controlling other variables to be completely consistent, and respectively performing reflux extraction at 60 ℃ for 1 hour to obtain osthole extracting solutions extracted by different extraction solvents; the result shows that the extraction rate is highest when methanol is used as a solvent;
(7) experiment of extraction times: respectively taking 3 parts of fructus Cnidii powder, precisely weighing each part, adding methanol solution with the same material-to-liquid ratio, controlling other variables to be consistent, respectively extracting under reflux at 60 deg.C for 1 hr, respectively extracting for 1 time, 2 times and 3 times; obtaining osthole extracting solutions under different extraction times; the result shows that the osthole extraction rate is highest when the extraction frequency is 1, so that the subsequent experiment optimization is carried out by selecting the extraction frequency to be 1.
Specifically, the method also comprises the following linear relation investigation: precisely weighing 5mg of osthole reference substance, and dissolving with 5mL of methanol solvent to obtain 1mg/mL reference substance stock solution; respectively sucking 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL of reference substance solution, placing in a 10mL volumetric flask, adding methanol to dilute to scale, and shaking up to obtain a series of reference substance solutions; taking the concentration of a reference substance as a horizontal coordinate and taking a peak area as a vertical coordinate; the regression equation Y of the standard curve is 64.979X-342.08(R is 0.9996), and the linear range is 2.2 ug/mL-250 ug/mL.
Specifically, still include:
investigation of methanol concentration: accurately weighing 2.0g of each of 3 parts of osthole powder, wherein the fixed temperature is 70 ℃, and the material-liquid ratio is 1: 8. the extraction time is 1 h; adding 50%, 60%, 70%, 80% and 90% methanol respectively, and examining the influence of the methanol concentration on the extraction rate of osthole;
examination of extraction time: accurately weighing 2.0g of each of 5 parts of osthole powder, wherein the fixed temperature is 70 ℃, and the material-liquid ratio is 1: 8. the methanol concentration is 70%; setting the time of methanol solvent reflux extraction as 0.5h, 1h, 1.5h and 2h respectively, and investigating the influence of the extraction time on the osthole extraction rate;
and (3) investigating liquid-material ratio: accurately weighing 2.0g each of 5 parts of osthole powder, fixing temperature at 70 deg.C, methanol concentration at 70%, and extraction time at 1 hr; setting the material-liquid ratio as 1: 4. 1: 6. 1: 8. 1: 10. 1: 12, investigating the influence of different feed liquid ratios on the extraction rate of the osthole;
temperature investigation: accurately weighing 5 parts of osthole powder, 2.0g of each powder, fixing the concentration of methanol to be 70%, and mixing the materials in a liquid ratio of 1: 8. the extraction time is 1 h; the temperature was set at 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, and 90 deg.C, and the effect of different temperatures on the extraction rate of osthole was examined.
The present invention will be further illustrated by specific examples, with reference to fig. 1 to 7.
1. Preparation of instruments and reagents:
1.1 Instrument: DF-101S heat collection type constant temperature heating magnetic stirrer (Wuhan Keel instrument equipment Co., Ltd.), SHZ-D circulating water type vacuum pump (Zhongyi Yuhua instrument Co., Ltd.), TU-1901 double-beam ultraviolet visible light photometer (Beijing Puyan general instrument Co., Ltd.), RE-52AA rotary evaporator (Shanghai Yarong instrument Co., Ltd.), triple quadrupole liquid chromatography-mass spectrometer (American Saimei Feishai), C18 chromatographic column (4.6mm, 4.6 x 200mm) and C18 chromatographic column (1.7um, 2.1 x 100 mm).
1.2 reagent: chromatographically pure methanol (norfloxacin); fructus Cnidii (Shiweiben local product, identified by Onagawa Wen teacher of Chinese materia Medica of Hubei pharmaceutical institute, and belonging to cnidium of Umbelliferae); potassium persulfate (mclin); ascorbic acid standards (mclin); DPPH free radical scavenging capacity kit (michelin); hydroxy radical scavenging ability kit (Mecline), osthole reference sample (Vickqi Biotechnology Co., Ltd., Sichuan province), formic acid (chemical reagent Co., Ltd., national drug group)
2. The method and the result are as follows:
(1) pretreating the fructus cnidii: pulverizing fructus Cnidii, sieving with No. 3 sieve, and drying at a certain temperature for a certain time to obtain fructus Cnidii dried powder.
(2) Preparation of control solutions: accurately weighing 1.0mg of osthole reference substance, dissolving in 1mL of methanol solution, and preparing into osthole reference substance solution with concentration of 1.0 mg/mL.
(3) Preparation of a test solution: extracting osthole with methanol under reflux. Taking 5.0g of fructus Cnidii powder, adding methanol solution at a certain ratio, placing in a water bath, extracting at a certain temperature, magnetic stirring speed and extraction time, filtering, and supplementing the weight to obtain osthole test solution.
(4) Measurement method and wavelength selection: 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL of the control solutions are respectively sucked and placed in a 10mL volumetric flask, diluted to the scale by adding methanol and shaken up. Finally, taking a methanol reagent as a blank, and irradiating an ultraviolet spectrophotometer to determine an absorption spectrogram at the wavelength of 210-600 nm; finally 314nm was chosen as the measurement wavelength.
(5) And (3) linear relation investigation: precisely weighing osthole control 5mg, and dissolving with 5mL methanol solvent to obtain 1mg/mL control stock solution. 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL of the control solutions are respectively sucked and placed in a 10mL volumetric flask, diluted to the scale by adding methanol and shaken up. A series of control solutions were obtained. The control concentration (ug/ml) was plotted on the abscissa and the peak area was plotted on the ordinate. The standard curve yields the regression equation Y-64.979X-342.08 (R-0.9996). The linear range is 2.2ug/mL to 250 ug/mL.
(6) Single factor investigation:
verification of an extraction mode: respectively taking 3 parts of fructus cnidii powder, precisely weighing each part, adding methanol solution with the same material-to-liquid ratio, and respectively performing ultrasonic extraction and methanol solvent reflux for the same time to obtain osthole extracting solutions in different extraction modes; the result shows that the extraction rate of reflux extraction is highest, so methanol reflux extraction is selected as the extraction mode of osthole.
Selecting an extraction solvent: respectively taking 3 parts of fructus cnidii powder, precisely weighing each part, adding methanol solution and ethanol solution with the same material-to-liquid ratio, controlling other variables to be completely consistent, and respectively performing reflux extraction at 60 ℃ for 1 hour to obtain osthole extracting solutions extracted by different extraction solvents; the results show that methanol is the solvent of highest extraction yield, so methanol solvent was chosen.
3. Experiment of extraction times: respectively taking 3 parts of fructus Cnidii powder, precisely weighing each part, adding methanol solution with the same material-to-liquid ratio, controlling other variables to be consistent, respectively extracting under reflux at 60 deg.C for 1 hr, respectively extracting for 1 time, 2 times, and 3 times. Obtaining osthole extracting solutions under different extraction times; the result shows that the osthole extraction rate is highest when the extraction frequency is 1, so that the subsequent experiment optimization is carried out by selecting the extraction frequency to be 1.
Investigation of methanol concentration: accurately weighing 2.0g of each of 3 parts of osthole powder, wherein the fixed temperature is 70 ℃, and the material-liquid ratio is 1: 8. the extraction time is 1 h. Methanol was added at concentrations of 50%, 60%, 70%, 80%, and 90%, respectively, and the effect of methanol concentration on the osthole extraction rate was examined.
Investigation of extraction time: accurately weighing 2.0g of each of 5 parts of osthole powder, wherein the fixed temperature is 70 ℃, and the material-liquid ratio is 1: 8. the methanol concentration was 70%. The time for methanol solvent reflux extraction is set to be 0.5h, 1h, 1.5h and 2h respectively, and the influence of the extraction time on the osthole extraction rate is examined.
Investigating liquid-material ratio: accurately weighing 5 parts of osthole powder 2.0g each, fixing temperature at 70 deg.C, methanol concentration at 70%, and extraction time at 1 hr. Setting the material-liquid ratio as 1: 4. 1: 6. 1: 8. 1: 10. 1: 12, investigating the influence of different feed liquid ratios on the extraction rate of the osthole.
Sixthly, temperature investigation: accurately weighing 2.0g of each of 5 parts of osthole powder, fixing the concentration of methanol to be 70%, and the material-liquid ratio to be 1: 8. the extraction time is 1 h. The temperature was set at 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, and 90 deg.C, and the effect of different temperatures on the extraction rate of osthole was examined.
(1) Box-Behnken response surface investigation and optimization
Selecting factor level and designing experiment: the factors and levels of the experiment are determined by examining the single factor, namely, four factors of methanol concentration (A), extraction time (B), material-liquid ratio (C) and temperature (D) are selected as response variables, an optimal point of the single factor experiment is taken as a center, a level value is taken from the upper part and the lower part of the optimal point as the level of a response surface, the levels are respectively coded as-1, 0 and 1, and the response surface analysis of 29 test points with four factors and three levels is designed by taking the extraction rate of osthole as an examination index. The center and level of the experimental design are shown in Table 1, and the design combination and osthole extraction rate are shown in Table 2.
TABLE 1Box-Behnken response surface method experiment factors and levels
Table 2 response surface experimental design groupings and results
Establishing a regression model and performing correlation analysis: and performing polynomial fitting regression on the experimental result by using Design Expert software to obtain a quadratic polynomial regression equation of independent variable extraction time (A), liquid-material ratio (B), temperature (C), methanol concentration (D) and dependent variable osthole extraction rate (Y) as follows:
Y＝6.88-0.1808A+2.8B-0.0625C+3.21D-0.1525AB-0.085AC+0.25AD+0.3775BC+0.845BD+0.025CD+0.0918A 2 -0.5195B 2 -0.3483C 2 -1.81D 2 .
the analysis of variance is shown in Table 3.
TABLE 3 results of analysis of variance of response surface
Note: p<0.01 is extremely significant, P<0.05 is significant: r 2 ＝0.9628，R 2 Adj ＝0.9256，CV＝14.14％。
As can be seen from the data in table 3, the P value of the model is less than 0.0001, the simulated regression equation is significant, the mismatching term is 0.3863 and is greater than 0.05, and the mismatching term is not significant, which indicates that the fitting degree of the regression equation is good and has statistical significance. The decision coefficient R2 is 0.9628, indicating a closer proximity between the measured and predicted values. The correction coefficient R2Adj is 0.9256, which shows that the four extraction factors have 92.56% influence on the extraction effect of osthole, and the experiment has higher accuracy. The coefficient of variation CV was 14.14%, which was less than 15%, indicating good stability of the experiment.
According to the magnitude of the P value, the influence of various factors on the extraction rate of the osthole is as follows: b ═ D>A>C, the first time liquid-material ratio (B) and the methanol concentration (D) of the equation have obvious influence on the extraction rate, and the second time A 2 、B 2 、C 2 The influence on the extraction rate is weak.
Analysis of two-factor interaction response surface: a 3D response surface diagram of 6 interactive item effects is shown in fig. 7. Wherein the gradient of the interaction between the liquid-to-feed ratio and the methanol concentration is larger. Indicating that the effect between the liquid-to-feed ratio and the methanol concentration is significant. The gradients of contour maps of 3D response surface maps of other interaction items are small, which indicates that the interaction items have no significant influence on the extraction rate of osthole. The reasonability of the design of the Box-Behnken response surface method is further shown, and the accuracy of the model is high. The response surface of the interaction of the factors is shown in fig. 7.
(9) Verification of the optimal extraction process: the optimized optimal process conditions obtained by Design Expert software are as follows: the extraction time is 1 hour, the liquid-material ratio is 14:1, the temperature is 64 ℃, and the methanol concentration is 75.8%. Three verification tests are carried out under the condition, 14.49%, 14.73% and 14.76% of the osthole is extracted, the average extraction rate is 14.66%, the comparison is close to 11.78% predicted by a model, the relative standard deviation is 1.00% and is less than 2%, the fitting degree of an equation is good, the optimized process reliability is high, and the osthole can be used for extracting osthole in large industrial dose.
(10) Determination of antioxidant capacity of osthole
The results of the osthole on DPPH and ABTS + clearance are shown in FIG. 8. The results show that as the concentration of osthole increases, the clearance for both DPPH and ABTS + increases. Meanwhile, the oxidation resistance of osthole is found to be stronger than that of ascorbic acid at low concentration (40-100 mu g/mL) and weaker than that of ascorbic acid at high concentration. Wherein the clearance of ABTS is always higher in this mass concentration range.
In the description of the specification, reference to the description of "one embodiment", "preferably", "an example", "a specific example" or "some examples", etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention, and schematic representations of the terms in this specification do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The present invention is not limited to the above-described embodiments, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and such modifications and improvements are also considered to be within the scope of the present invention. Those not described in detail in this specification are within the skill of the art.
Claims (8)
1. A method for extracting osthole by a response surface design method is characterized by comprising the following steps: the extraction process comprises the steps of adding methanol in a certain proportion into fructus cnidii powder for reflux extraction, on the basis of a single-factor experiment, adopting a Box-Behnken response surface Design method of Design-Expert software, selecting four factors of methanol concentration, extraction time, feed-liquid ratio and temperature as response variables, taking an optimal point of the single-factor experiment as a center, respectively taking a horizontal value around the optimal point as the level of a response surface, respectively coding the four factors to be-1, 0 and 1, taking the extraction rate of osthole as a response value, and performing polynomial fitting regression on the four factors to obtain a regression equation:
Y＝6.88-0.1808A+2.8B-0.0625C+3.21D-0.1525AB-0.085AC+0.25AD+0.3775BC+0.845BD+0.025CD+0.0918A 2 -0.5195B 2 -0.3483C 2 -1.81D 2 ；
in the formula, A is the concentration of methanol, B is the extraction time, C is the ratio of material to liquid, D is the temperature, and Y is the extraction rate of osthole;
and (4) performing regression calculation on the equation to obtain the optimal extraction process condition of the osthole.
2. The method for extracting osthole by response surface design according to claim 1, wherein the optimum extraction process conditions of osthole are as follows: the extraction time is 1 hour, the liquid-material ratio is 14:1, the temperature is 64 ℃, and the methanol concentration is 75.8%.
3. The method for extracting osthole by response surface design according to claim 2, wherein: the osthole extraction rate obtained under the optimal extraction process conditions is 14.49% -14.76%, and the antioxidant activity of the osthole is superior to that of ascorbic acid within the concentration range of 40-100 mug/mL.
4. The method for extracting osthole by response surface design according to claim 1, wherein the method comprises the following steps: the chromatographic conditions of osthole are that a chromatographic column is a C18 analytical column, a mobile phase A is chromatographic grade methanol, a mobile phase B is a 0.1% formic acid solution, the flow rate is 1.0mL/min, the detection wavelength is 314nm, the column temperature is 30 ℃, and the sample injection amount is 20 uL.
5. The method for extracting osthole by response surface design according to claim 1, wherein the method comprises the following steps: Design-Expert software is used for designing a response surface, and system software is used for simulating and generating the optimal extraction process parameters.
6. The method for extracting osthole by response surface design method as claimed in claim 1, wherein the method of single factor experiment comprises:
(1) pretreating the fructus cnidii: pulverizing dried fruit of cnidium fruit, sieving with No. 3 sieve, and drying at a certain temperature for a certain time to obtain dried powder of cnidium fruit;
(2) preparation of control solutions: accurately weighing 1.0mg of osthole reference substance, dissolving in 1mL of methanol solution, and preparing into 1.0mg/mL osthole reference substance solution;
(3) preparation of a test solution: extracting osthole by methanol reflux extraction; taking 5.0g of fructus Cnidii powder, adding methanol solution at a certain ratio, placing in a water bath, extracting at a certain temperature, magnetic stirring speed and extraction time, filtering, and supplementing the weight to obtain osthole test solution;
(4) measurement method and wavelength selection: respectively sucking 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL of reference substance solution, placing in a 10mL volumetric flask, adding methanol for dilution to scale, and shaking up; finally, a methanol reagent is used as a blank, an ultraviolet spectrophotometer is adopted to determine an absorption spectrogram at 210-600nm, and the maximum absorption wavelength is 314 nm; finally, 314nm is selected as the measuring wavelength;
(5) and (3) verification of an extraction mode: respectively taking 3 parts of fructus cnidii powder, precisely weighing each part, adding methanol solution with the same material-to-liquid ratio, and respectively performing ultrasonic extraction and methanol solvent reflux for the same time to obtain osthole extracting solutions in different extraction modes; the result shows that the extraction rate of reflux extraction is highest;
(6) selection of an extraction solvent: respectively taking 3 parts of fructus cnidii powder, precisely weighing each part, adding methanol solution and ethanol solution with the same material-to-liquid ratio, controlling other variables to be completely consistent, and respectively performing reflux extraction at 60 ℃ for 1 hour to obtain osthole extracting solutions extracted by different extraction solvents; the result shows that the extraction rate is highest when methanol is used as a solvent;
(7) experiment of extraction times: respectively taking 3 parts of fructus Cnidii powder, precisely weighing each part, adding methanol solution with the same material-to-liquid ratio, controlling other variables to be consistent, respectively extracting under reflux at 60 deg.C for 1 hr, respectively extracting for 1 time, 2 times and 3 times; obtaining osthole extracting solutions under different extraction times; the result shows that the osthole extraction rate is highest when the extraction frequency is 1, so that the subsequent experiment optimization is carried out by selecting the extraction frequency to be 1.
7. The method for extracting osthole by response surface design according to claim 6, further comprising the step of examining the linear relationship: precisely weighing 5mg of osthole reference substance, and dissolving with 5mL of methanol solvent to obtain 1mg/mL reference substance stock solution; respectively sucking 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL of reference substance solution, placing in a 10mL volumetric flask, adding methanol to dilute to scale, and shaking up to obtain a series of reference substance solutions; taking the concentration of a reference substance as a horizontal coordinate and taking a peak area as a vertical coordinate; the regression equation Y of the standard curve is 64.979X-342.08(R is 0.9996), and the linear range is 2.2 ug/mL-250 ug/mL.
8. The method for extracting osthole by response surface design according to claim 6, further comprising:
investigation of methanol concentration: accurately weighing 2.0g of each of 3 parts of osthole powder, wherein the fixed temperature is 70 ℃, and the material-liquid ratio is 1: 8. the extraction time is 1 h; adding 50%, 60%, 70%, 80% and 90% methanol respectively, and examining the influence of methanol concentration on osthole extraction rate;
examination of extraction time: accurately weighing 2.0g of each of 5 parts of osthole powder, wherein the fixed temperature is 70 ℃, and the material-liquid ratio is 1: 8. the methanol concentration is 70%; setting the time of methanol solvent reflux extraction as 0.5h, 1h, 1.5h and 2h respectively, and investigating the influence of the extraction time on the osthole extraction rate;
and (3) investigating liquid-material ratio: accurately weighing 2.0g each of 5 parts of osthole powder, fixing temperature at 70 deg.C, methanol concentration at 70%, and extraction time at 1 hr; setting the material-liquid ratio as 1: 4. 1: 6. 1: 8. 1: 10. 1: 12, investigating the influence of different feed liquid ratios on the extraction rate of the osthole;
temperature investigation: accurately weighing 2.0g of each of 5 parts of osthole powder, fixing the concentration of methanol to be 70%, and the material-liquid ratio to be 1: 8. the extraction time is 1 h; the temperature was set at 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, and 90 deg.C, and the effect of different temperatures on the extraction rate of osthole was examined.
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