CN108395459B - Method for extracting phlorizin, astragalin and afzerin from apple flowers by using ionic liquid - Google Patents
Method for extracting phlorizin, astragalin and afzerin from apple flowers by using ionic liquid Download PDFInfo
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Abstract
The invention relates to a method for extracting phlorizin, astragalin and afzerin from apple flowers by using ionic liquid, which comprises the steps of mixing dried apple flower powder with an extracting agent, carrying out ultrasonic extraction at room temperature, centrifuging after the ultrasonic extraction is finished, and taking supernate to obtain the compound apple flower extract; the extractant is formed by mixing ionic liquid and methanol. The total content of phlorizin, astragalin and afzerin in the verification test was 145.4 mg/g. Compared with the traditional extraction method, the total extraction rate of phlorizin, astragalin and afzerin in the apple flowers can reach 25.4 percent after the ionic liquid is added. The result shows that the optimal extraction process is screened by adopting an ionic liquid combined response curved surface method, the methanol extracting solution of the apple flower has a certain activating effect on tyrosinase, and the methanol ionic liquid extracting solution has a certain inhibiting effect on tyrosinase. Provides scientific basis for the development and utilization of the energy of the apple flowers.
Description
Technical Field
The invention belongs to the technical field of medicines or/and health-care products, and particularly relates to a method for extracting phlorizin, astragalin and afzelin from apple flowers by using an ionic liquid combined response curved surface method.
Background
The apple flower is apple of Malus of RosaceaeMalus pumilaDried flowers from Mill. The chemical components of the apple flower mainly comprise dihydrochalcones such as phlorizin and phloretin,and other flavonoid components such as quercetin, kaempferide, rutin, etc. The apple scented tea has the effects of enriching blood, relieving neuralgia and liver spots, improving eyesight, treating black spots, pimples and acnes, beautifying skin, helping digestion, protecting liver, activating blood and the like. Pharmacological studies have shown that: the flavonoids have antioxidant, antiinflammatory, analgesic, immunity regulating, antiaging, blood lipid reducing, and antitumor effects. In addition, the literature reports that some flavonoid compounds can inhibit the synthesis of melanin and can be used for treating pigmentation-related diseases such as chloasma; some flavonoids can stimulate melanin synthesis, and can be used for treating depigmentation diseases such as vitiligo.
The Ionic Liquid (IL) is a novel green organic solvent, also called room temperature molten salt, and consists of specific organic cations with relatively large volume and asymmetric structure and inorganic anions with relatively small volume, and has the characteristics of good thermal stability, chemical stability, wide viscosity range, good adjustability and solubility and the like. Not only reduces the pollution to the environment, but also can destroy the structure of cellulose, thereby promoting the dissolution of effective components and further improving the extraction rate. In recent years, documents report that the ionic liquid is applied to extraction of active compounds of traditional Chinese medicines, and no research on extraction of flavonoid components in apple flowers by the ionic liquid is reported.
Therefore, the invention adopts ionic liquid-ultrasonic wave auxiliary extraction-high performance liquid chromatography, designs a center combination experiment of Box-Benhnken 5 factor 3 level by applying Design-Expert8.06 statistical analysis software, simultaneously measures the content of 3 components of phlorizin, astragalin and afzerin in apple flowers by using an HPLC method, screens out an optimal extraction process, and performs tyrosinase activity research on the components.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for optimally extracting phlorizin, astragalin and afzerin from apple flowers by using ionic liquid, and the extraction method is simple to operate and high in extraction efficiency.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for optimally extracting phlorizin, astragalin and afzerin from apple flower by using ionic liquid comprises the steps of mixing dried apple flower powder with an extracting agent, carrying out ultrasonic extraction at room temperature (25-30 ℃), centrifuging after the ultrasonic extraction is finished, and taking supernate to obtain the finished product.
Specifically, in the above extraction method, the extractant is formed by mixing an ionic liquid and methanol, and the ionic liquid is [ BMIM ] BF4、［BMIM］Br、［BMIM］PF6Or [ HMIM ] PF6(ii) a The concentration of the ionic liquid in the extractant is 0.1 to 1.0 mol/L (of these, 0.4 to 0.8 mol/L is preferred). Unless otherwise specified, methanol is referred to herein as a volume concentration.
Further, the apple flower powder and the extracting agent can be mixed according to the solid-liquid ratio of 1 g: 20-100 mL (preferably 1 g: 60-1 g: 100 mL) of the apple flower powder is added, and the appropriate mesh range of the apple flower powder is 10-90 meshes (preferably 50-70 meshes).
In order to obtain better extraction effect, the ultrasonic extraction time is preferably 10-60 min (wherein, preferably 40-60 min), and the centrifugal rotation speed is preferably 2000-.
The method for extracting phlorizin, astragalin and afzerin from apple flowers by using the ionic liquid has the following steps:
1) firstly, selecting a proper solvent to dissolve the ionic liquid and determining the optimal type of an extracting agent; and respectively comparing methanol, acetonitrile, ethyl acetate, 70% ethanol and water to dissolve the ionic liquid and extract apple flowers, and screening out the methanol to obtain the best extraction rate of the target analyte. Then respectively compare [ BMIM ] BF4Methanol, [ BMIM ] Br/methanol, [ BMIM ] PF6Methanol, [ HMIM ] PF6The extraction rate of target analytes by four extractants of methanol is finally determined to be the best extractant of [ BMIM ] Br/methanol;
2) on the basis of the step 1), designing factor levels of extraction conditions, taking different control parameters in the apple flower extraction process as factors, and determining the factor levels of a response surface method by a single-factor analysis method; after screening the control parameters of a plurality of extraction processes, selecting several control parameters with the largest influence as factors: taking the solid-liquid ratio (times), the concentration (mol/L) of the extracting agent, the ultrasonic time (min), the centrifugal rotating speed (r/min) and the number (meshes) of the crushing mesh as five factors to carry out the test;
3) adopting Design-Expert8.06 to Design a response surface test, and determining the maximum extraction rates of phlorizin, astragalin and afzerin and the corresponding factor levels thereof through variance analysis;
4) extracting phlorizin, astragalin and afzerin in apple flowers under the optimal condition according to the level of a control parameter factor corresponding to the maximum extraction rate of the phlorizin, astragalin and afzerin, and detecting the extraction rate (comparing the peak area of a test solution detected by a high performance liquid chromatograph at 270 nm with the standard curves of the phlorizin, astragalin and the afzerin, and calculating the total extraction amount of the phlorizin, astragalin and the afzerin). Comparing the actual extraction rate with the traditional solvent detection value to determine the advancement of the ionic liquid extraction;
5) and (3) carrying out tyrosinase activity determination on the ionic liquid extract, the methanol extract and the ionic liquid of the apple flower under the same system.
The invention provides a process for optimizing and extracting phlorizin, astragalin and afzerin by utilizing an ionic liquid combined response curved surface experimental method, designs a center combination experiment with a Box-Benhnken 5 factor 3 level by applying Design-Expert8.06 statistical analysis software, simultaneously measures the content of 3 components of phlorizin, astragalin and afzerin in apple flowers by utilizing an HPLC method, screens out an optimal extraction process, and performs tyrosinase activity research on the optimal extraction process. The invention provides a new thought and approach for Chinese medicine researchers, the optimal extraction conditions established by the experiment are scientific and feasible, and the method can be used for optimizing and selecting an extraction technology suitable for industrial expanded production, improving the old process, improving the efficiency and providing an experimental basis for the comprehensive utilization of apple flowers. In addition, the extraction method is simple to operate, high in extraction efficiency and suitable for large-scale industrial production.
Drawings
FIG. 1 is an HPLC chart of an apple flower test sample (a) and a mixed reference sample (b), wherein 1 is phlorizin 2, astragalin 3 and afzerin;
FIG. 2 shows the effect of different extraction solvents on the extraction rate of 3 flavonoids including phlorizin, astragalin and afzerin in apple flowers;
FIG. 3 shows the effect of the kind of extractant on the extraction rate of 3 flavonoid compounds including phlorizin, astragalin and afzerin in apple flowers;
FIG. 4 shows the influence of the number of the crushed apple flowers on the extraction rate of 3 flavonoid compounds including phlorizin, astragalin and afzerin in apple flowers;
FIG. 5 shows the effect of extractant concentration on the extraction rate of phlorizin, astragalin and afzerin 3 flavone compounds in apple flowers;
FIG. 6 is the effect of ultrasonic time on the extraction rate of phlorizin, astragalin and afzerin 3 flavone compounds in apple flowers;
FIG. 7 shows the effect of solid-liquid ratio on the extraction rate of phlorizin, astragalin and afzerin 3 flavone compounds in apple flowers;
FIG. 8 shows the effect of centrifugal speed on the extraction rate of phlorizin, astragalin and afzerin 3 flavone compounds in apple flowers;
FIG. 9 influence of interaction on extraction rate of phlorizin, astragalin and afzerin 3 flavone compounds in apple flowers.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.
Instruments and reagents.
Astragalin (batch 17042804) was purchased from Goodpastel Biotechnology Inc., and its HPLC purity was greater than 98%; the purity of the afzerin and the phlorizin is more than 98 percent by HPLC detection. 1-hexyl-3-methylimidazolium hexafluorophosphate [ HMIM ] PF6 (Sammerfell scientific Co., U.S.), brominated 1-butyl-3-methylimidazolium [ BMIM ] Br (Merck-Liquid, Germany), 1-butyl-3-methylimidazolium tetrafluoroborate [ BMIM ] BF4 (Merck-Liquid, Germany), 1-butyl-3-methylimidazolium hexafluorophosphate [ BMIM ] PF6 (Merck-Liquid, Germany). Acetonitrile, methanol (HPLC grade), phosphoric acid (Fuyu fine chemical Co., Ltd., Tianjin), and water as purified water (Hangzhou Waha Baili food Co., Ltd.).
Shimadzu LC-20AT series high performance liquid chromatograph (Shimadzu, Japan): an LC-20AT liquid chromatogram infusion pump, a CTO-10AS column incubator, an SPD-20A ultraviolet detector and an LC-Solution chromatogram data processing system; KQ-500DB type ultrasonic cleaner (ultrasonic instruments ltd, kunshan, jiangsu); a hundred thousand class electronic balance model AB135-S (mettler-toledo instruments ltd, switzerland).
Experimental methods and results.
2.1 establishment of the method for measuring the content of phlorizin, astragalin and afzerin.
2.1.1 preparation of control solutions
Taking appropriate amount of phlorizin, astragalin and afzerin, precisely weighing, adding methanol for dissolving, and making into mixed reference substance solutions with concentrations of 0.204, 0.196 and 0.199 mg/mL respectively.
2.1.2 preparation of test solutions
Precisely weighing 10 mg of apple flower sample powder (70 meshes), adding an extracting agent (the extracting agent is formed by mixing ionic liquid [ BMIM ] Br and methanol, the concentration of the ionic liquid [ BMIM ] Br in the extracting agent is 0.52 mol/L.) 1mL, performing ultrasonic extraction at room temperature for 60min, centrifuging at 7000 r/min for 3 min, sucking supernatant, filtering through a 0.22 mu m microporous filter membrane, and taking filtrate to obtain a sample solution (the crushing mesh number, the extracting agent, the solid-liquid ratio, the ultrasonic time and the centrifugal rotating speed are all the optimal conditions for screening).
2.1.3 chromatographic conditions and System suitability experiments.
TABLE 1 chromatographic conditions for apple flower assay
According to the above chromatographic conditions, the reference solution and the test solution are injected into the sample at a volume of 10 μ L, and the chromatogram is recorded, as shown in FIG. 1. Under the chromatographic condition, chromatographic peaks of phlorizin, astragalin and afzelin in the sample are consistent with the peak time of a reference substance, and the separation degree of the three compounds is good.
2.1.4 Linear relationship investigation.
Precisely absorbing mixed reference substance solutions with different concentrations, performing sample injection measurement under the chromatographic condition of 2.1.3, and recording peak areas of the components. By peak area (Y) As ordinate, sample introduction qualityXμ g) is the abscissa, and the regression equation is plotted: phlorizin:Y=643736X+158998，r= 0.9987; astragalin:Y=518051X+145776，r= 0.9979; african bean glycosideY=835408X-359973，r= 0.9942. The result shows that the sampling amounts of phlorizin, astragalin and afzerin are in good linear relation with the peak area response values when the sampling amounts are 0.4080-14.28 mug, 0.3920-13.72 mug and 0.3980-13.93 mug respectively.
2.1.5 repeat experiments.
6 parts of apple flower sample is accurately weighed, the sample is prepared according to the 2.1.2 conditions, 10 mu L of sample is injected according to the chromatographic conditions under 2.1.3 items, and the result shows that: RSD of phlorizin, astragalin and afzerin are respectively 1.16%, 1.8% and 2.1%, which shows that the test method has good repeatability.
2.1.6 precision experiments.
Precisely sucking 10 mu L of mixed reference substance solution, and continuously injecting samples for 6 times according to chromatographic conditions under 2.1.3, wherein the result shows that: RSDs of phlorizin, astragalin and afzerin are 0.61%, 0.73% and 0.51% respectively, which shows that the precision of the instrument is good and the quantity of the substance can be accurately reflected.
2.1.7 stability experiments.
Precisely weighing 1 part of apple flower, preparing a sample according to the conditions of 2.1.2, injecting 10 mu L of the sample for 0, 4, 8, 12, 16, 20 and 24 hours according to the chromatographic conditions of 2.1.3, and displaying the result: the RSD of phlorizin, astragalin and afzerin is 2.58%, 0.97% and 1.42% respectively, which indicates that phlorizin, astragalin and afzerin in the test solution are basically stable within 24 h.
2.1.8 sample recovery test.
Weighing 6 parts of an apple flower sample with known contents of phlorizin, astragalin and asfomin, preparing a sample solution according to the conditions of 2.1.2, measuring the contents of phlorizin, astragalin and asfomin in the sample, adding standard substances which are equal to 80% of the mass of the phlorizin, astragalin and asfomin in the sample, respectively, injecting 10 mu L of the sample according to the chromatographic conditions of 2.1.3, wherein the average sample injection recovery rates of the phlorizin, astragalin and the asfomin are 97.70%, 101.32% and 106.0%, and the RSD values are 0.90%, 1.23% and 2.08%, respectively, and the accuracy of the test result of the reaction meets the requirement.
2.2 Single factor test.
2.2.1 selection of extractant.
The choice of the type of extractant has a large impact on the extraction yield of the target analyte. In this experiment, [ BMIM ] was selected first]BF4，[BMIM]Br，[BMIM]PF6，[HMIM]PF6 4 types of ionic liquids are used as extracting agents, and the 4 types of ionic liquids are imidazoles, are stable in air and solution and can be competitively combined with lignocellulose, so that the cellulose is efficiently dissolved, components in plant cells are dissolved out more completely, and the extraction rate is increased. However, since ionic liquids are mostly viscous liquids, and [ BMIM ]]Br is in a solid crystal state, so that the selection of a proper solvent for dissolving the ionic liquid is particularly important. Experiments comparing 5 solvents of 70% ethanol, methanol, acetonitrile, ethyl acetate and water, samples were prepared under the conditions of 2.1.2 above, and analyzed by injection HPLC under the 2.1.3 chromatographic conditions above, to compare the contents of phlorizin, astragalin and afzelin in the 3 target analytes. The result shows that acetonitrile can only extract two flavonoid components in apple flower, and as shown in figure 2, methanol has the best extraction rate on target analytes, so methanol is selected as an extraction solvent.
FIG. 3 compares BMIM]BF4Methanol, [ BMIM ]]Br/methanol, [ BMIM]PF6Methanol, [ HMIM ]]PF6The results of the extraction of target analytes with 4 extraction solvents in methanol show that [ BMIM ]]The extraction rate of the target analyte by Br/methanol is highest, which is probably related to the self-composition structure of the ionic liquid. Therefore [ BMIM ] was selected in this experiment]Br/Methanol is used as an extractant.
2.2.2 selection of the number of crushing meshes.
According to the above test method, the influence of the number of pulverization mesh of 10, 40, 50, 60, 70 and 90 mesh on the extraction amount of the target analyte was examined under the same test conditions. As shown in fig. 4, the extraction amount tends to increase with the increase of the number of pulverization meshes, and the extraction rate of the target analyte reaches the maximum when the number of pulverization meshes is 60. This is probably because the ionic liquid has viscosity, and the chemical components in the sample are more easily extracted as the particle size of the powder is gradually reduced, but when the particle size of the powder is too small, the ionic liquid is easily agglomerated, and the release of the chemical components is hindered.
2.2.3 selection of extractant concentration.
As shown in FIG. 5, when the concentration of the extractant is 0.1 to 1.0 mol/L, the extraction rate of the target analyte tends to increase with the increase of the concentration of the extractant, and tends to decrease with the further increase of the concentration of the extractant. This is probably because the solvent diffusion decreases with the increase of the ionic liquid concentration, and it is difficult to enter the medicinal material, and the ingredients in the medicinal material cannot be sufficiently extracted, resulting in a decrease in the extraction rate. Therefore, 0.6 mol/L was selected as the optimum extractant concentration.
2.2.4 selection of ultrasound time.
10, 20, 30, 40, 50 and 60min are respectively selected as ultrasonic time, the concentration of the extracting agent is 0.6 mol/L, and the influence of different ultrasonic time on the extraction rate of the target analyte is compared according to the test conditions, and the result is shown in FIG. 6. The extraction rate of the target analyte basically shows a gradually increasing trend along with the prolonging of the ultrasonic time; when the ultrasonic time is 50 min, the extraction rate of the target analyte reaches the maximum, and then the extraction rate of the target analyte shows a descending trend along with the increase of the time. The above reasons may be due to the fact that the structure of the ionic liquid and the target analyte is destroyed by the ultrasound for a long time, and the specific reasons need further research.
2.2.5 selection of solid to liquid ratio.
The extraction rates of the ionic liquid for the target analyte were examined at solid-to-liquid ratios of 1:20, 1:40, 1:50, 1:60, 1:80, and 1: 100. As a result, as shown in FIG. 7, the extraction rate reached a maximum value when the solid-to-liquid ratio reached 1:80, and the extraction rate decreased conversely when the ratio was increased further. This may be due to the ionic liquid itself and the physical properties.
2.2.6 selection of centrifuge rotation speed.
Under the optimum conditions optimized above, 2000, 4000, 6000, 7000, 8000 and 9000 r.min were selected-1Examining the effect of centrifugation speed on the extraction rate of target analytes, as shown in FIG. 8, the extraction rate was 8000 r.min-1The time reaches the maximum, so the test is selected to be 8000 r.min-1The center point "0" level as the level of the response surface factor.
And 2.3 optimizing the experimental design by using a response surface method.
According to the design principle of a Box-Benhnken central combined test, the total content of phlorizin, astragalin and afzerin in apple flowers is taken as a response value (Y), 5 factors including the crushing mesh number (A), the concentration (B) of an extracting agent, the ultrasonic time (C), the solid-liquid ratio (D) and the centrifugal rotating speed (E) are selected, and a response surface analysis method of 5 factors and 3 levels is adopted. The test factors and level design are shown in table 2.
TABLE 2 design table of response surface factor level
In order to examine the influence of various influencing factors and interaction thereof on the extraction process, the Design Expert8.06 software is used for carrying out multiple regression fitting on the data in the table 2 to obtain a quadratic polynomial regression model equation Y =125.10+4.05A-0.95B-1.97C +23.88D + E-2.93AB-1.38AC +12.54AD +0.88AE-3.29BC-3.52BD +1.65BE +2.43CD-6.81CE +0.47DE +1.42A2-0.89B2-2.68C2+24.76 D2-3.28E2. Analysis of variance of the fitted quadratic polynomial model is shown in Table 3, model P<0.0001 indicates high significance, and can be used for predicting response values; the model mismatching item is not obvious, which shows that the fitting degree of the regression equation in the whole regression space is good, and the multivariate correlation systemNumber R2=0.9572， R2adj=0.9230， R2pre =0.8472, indicating that the model fits well to the experimental reality. In the response surface method, the model coefficient effect marks the representation of a response value, and when a coefficient has a positive effect, the response mode is increased along with the increase of a variable; when a coefficient has a negative effect, the response mode decreases as the variable increases. The higher the absolute value of the coefficient, the more important the weight of the response variable. The solid-liquid ratio in this test is the most significant factor, so the solid-liquid ratio has the greatest influence on the whole test.
TABLE 3 regression equation significance test
Significant pole(s) ((P<0.001) is highly significant (P<0.01: (significantly:)P<0. 05) 。
2.3.1 response surface plot analysis.
The response surface plot is shown in fig. 9, which shows the effect and interaction of the independent variables versus the response values. In fig. 9, a and c show that the response value increases linearly with the increase of the crushing mesh number at a certain ultrasonic time; when the crushing mesh number is not changed, the influence on the extraction amount is not obvious along with the increase of the concentration of the ionic liquid or the prolonging of the ultrasonic time. b. d, e and g, as another factor is determined, the larger the solid-liquid ratio is, the higher the extraction rate of the target analyte is, which may be that when the solid-liquid ratio is larger, the contact area and the contact chance of the apple flower and the solvent are relatively larger, so that the effective component can be contacted with the solvent more and dissolved better, and at the same time, the amount of the effective component dissolved in the solvent is increased by the increase of the amount of the solvent, so that the target analyte can be dissolved in the solvent more. The graph f shows that the interaction between the centrifugal speed and the ultrasonic time has little influence on the extraction rate of the target analyte.
And 2.3.2, optimizing extraction process parameters and verifying a model.
According to response surface analysis, the optimal extraction conditions of the apple flowers are obtained: 70 meshes of crushing mesh, 0.52 mol/L of extracting agent, 59.52 min of ultrasonic treatment, 1:99.90 of solid-to-liquid ratio and 7052.37 r/min of centrifugal rotation speed, and due to the limitation of practical operation, the optimal conditions are changed into: the crushing mesh number is 70 meshes, the concentration of an extracting agent is 0.52 mol/L, the ultrasonic time is 60min, the solid-liquid ratio is 1:100, the centrifugal rotation speed is 7000 r/min, the total content of phlorizin, astragalin and afzelin in the apple flower is measured to be 145.4 mg/g, and the difference is not large compared with the calculated value of a model, so that the extraction parameters obtained by adopting a response surface method are accurate and reliable, and the practical application value is realized.
2.4 tyrosinase activity assay.
mu.L of phosphate buffer (pH 6.8), 5. mu.L of sample solution (or methanol), 25. mu.L of 0.2 U.mL-1The tyrosinase-containing aqueous solution was incubated at 37 ℃ for 10 min, and then 0.5 g.L was added-1L-dopa solution 25 μ L, shaking, reacting at 37 deg.C for 5 min, and measuring absorbance at 492 nm with enzyme labeling instrument. And simultaneously, carrying out parallel tests of a mixed extracting solution of ionic liquid [ BMIM ] Br and methanol, a pure methanol extracting solution and the pure ionic liquid [ BMIM ] Br under the same system, and adding an activator of 8-methoxypsoralen (8-MOP) as a positive control group.
Table 4 the activation rates of the different substances on tyrosinase (X ± s,n =3)
note that 8-MOP is a positive control; comparison with 8-MOP*** P≤0. 001
Table 5 the activation rates of various solvent extracts on tyrosinase (X ± s,n =3)
note that comparison with Ionic liquids*** P≤0. 001
Determining tyrosinase activity under different final concentrations of phlorizin, astragalin and afzelin by in vitro tyrosinase dopa rate oxidation micro-scale methodInfluence of sex. The results are shown in Table 4, and compared with the positive control 8-MOP, the tyrosinase activation rates of astragalin and afzelin are higher than those of the positive control at the same final concentration; the activation rate of phlorizin on tyrosinase at final concentrations of 1 mmol/L and 0.5 mmol/L is remarkably higher than that of a positive control (PLess than or equal to 0.001), whereas at a final concentration of 2 mmol/L, phlorizin does not activate tyrosine as well as the positive control (PLess than or equal to 0.001), on the contrary, it has very strong inhibition effect on tyrosinase. This indicates that the concentration has a different direction of action on the activity of tyrosinase. The influence of different solvent extracts of apple flowers on the activity of tyrosinase is determined, the results are shown in Table 5, and under the same condition, the activation rate of the methanol extract on the tyrosinase is very obviously higher than that of the ionic liquid (II)PLess than or equal to 0.001); the methanol ionic liquid extract has very obvious inhibition effect on tyrosinase (P≦ 0.001), which may be because methanol extracts more substances that activate tyrosinase activity, while ionic liquid extracts more substances that inhibit tyrosinase activity.
Comparative example 1
A method for extracting phloridzin, astragalin and afzerin from apple flower by using ionic liquid comprises precisely weighing apple flower sample powder (50 mesh) 0.5 g, adding 20 mL of methanol, performing ultrasonic extraction at room temperature for 30 min, centrifuging at 3000 r/min for 3 min, and sucking supernatant.
Through detection, the following results are obtained: the total content of phlorizin, astragalin and afzerin in the supernatant was 82.77mg/g (n =3).
Example 1
A method for extracting phloridzin, astragalin and afzelin from apple flower with ionic liquid comprises mixing 0.5 g dried apple flower powder (50 mesh) with 20 mL of extractant (the extractant is ionic liquid [ BMIM ] BF)4Mixing with methanol to obtain ionic liquid BMIM BF4The concentration was 0.7 mol/L. ) Mixing, performing ultrasonic extraction at room temperature for 30 min, centrifuging at 3000 r/min for 3 min, and collecting supernatant.
Through detection, the following results are obtained: the total content of phlorizin, astragalin and afzerin in the supernatant was 88.54 mg/g (n =3).
Example 2
A method for extracting phlorizin, astragalin and afzerin from apple flowers by using ionic liquid comprises the steps of mixing 0.5 g of dried apple flower powder (50 meshes) with 20 mL of extracting agent (the extracting agent is formed by mixing ionic liquid [ BMIM ] Br and methanol, the concentration of the ionic liquid [ BMIM ] Br in the extracting agent is 0.7 mol/L.), carrying out ultrasonic extraction at room temperature for 30 min, centrifuging at 3000 r/min for 3 min after the ultrasonic extraction is finished, and taking supernatant fluid to obtain the finished product.
Through detection, the following results are obtained: the total content of phlorizin, astragalin and afzerin in the supernatant was 99.98 mg/g (n =3).
Example 3
A method for extracting phloridzin, astragalin and afzelin from apple flower with ionic liquid comprises mixing 0.5 g dried apple flower powder (50 mesh) with 20 mL of extractant (the extractant is ionic liquid [ BMIM ] BF)6Mixing with methanol to obtain ionic liquid BMIM BF6The concentration was 0.7 mol/L. ) Mixing, performing ultrasonic extraction at room temperature for 30 min, centrifuging at 3000 r/min for 3 min, and collecting supernatant.
Through detection, the following results are obtained: the total content of phlorizin, astragalin and afzerin in the supernatant was 92.66 mg/g (n =3).
Example 4
A method for extracting phloridzin, astragalin and afzelin from apple flower with ionic liquid comprises mixing 0.5 g dried apple flower powder (50 mesh) with 20 mL of extractant (the extractant is ionic liquid [ HMIM ] BF)6Mixing with methanol to obtain ionic liquid [ HMIM ] BF6The concentration was 0.7 mol/L. ) Mixing, performing ultrasonic extraction at room temperature for 30 min, centrifuging at 3000 r/min for 3 min, and collecting supernatant.
Through detection, the following results are obtained: the total content of phlorizin, astragalin and afzerin in the supernatant was 90.96 mg/g (n =3).
Comparative example 2
A method for extracting phloridzin, astragalin and afzerin from apple flower by using ionic liquid comprises precisely weighing 10 mg of apple flower sample powder (70 mesh), adding 1mL of methanol, performing ultrasonic extraction at room temperature for 60min, centrifuging at 7000 r/min for 3 min, and sucking supernatant to obtain the product.
Through detection, the following results are obtained: the total content of phlorizin, astragalin and afzerin in the supernatant was 115.7mg/g (n =3).
Example 5
A method for extracting phlorizin, astragalin and afzerin from apple flowers by using ionic liquid comprises the steps of mixing 10 mg of dried apple flower powder (70 meshes) with 1mL of extracting agent (the extracting agent is formed by mixing ionic liquid [ BMIM ] Br with methanol, the concentration of the ionic liquid [ BMIM ] Br in the extracting agent is 0.52 mol/L.), carrying out ultrasonic extraction at room temperature for 60min, centrifuging at 7000 r/min for 3 min after the ultrasonic extraction is finished, and taking supernatant fluid to obtain the finished product.
Through detection, the following results are obtained: the total content of phlorizin, astragalin and afzerin in the supernatant was 145.4 mg/g (n =3).
Example 6
A method for extracting phloridzin, astragalin and afzelin from apple flower with ionic liquid comprises mixing 10 mg dried apple flower powder (70 mesh) with 1mL of extractant (the extractant is ionic liquid [ BMIM ] BF)4Mixing with methanol to obtain ionic liquid BMIM BF4The concentration was 0.52 mol/L. ) Mixing, performing ultrasonic extraction at room temperature for 60min, centrifuging at 7000 r/min for 3 min after the ultrasonic extraction is finished, and collecting supernatant.
Through detection, the following results are obtained: the total content of phlorizin, astragalin and afzerin in the supernatant was 121.8 mg/g (n =3).
And (4) conclusion: the method adopts ionic liquid-ultrasonic assisted extraction-high performance liquid chromatography for the first time, takes [ BMIM ] Br/methanol solution as an extracting agent, the crushing mesh is 70 meshes, the concentration of the extracting agent is 0.52 mol/L, the ultrasonic time is 60min, the solid-to-liquid ratio is 1:100, and the centrifugal rotation speed is 7000 r/min, so that the extraction rate of phlorizin, astragalin and afungin in the apple flowers is improved by 25.4 percent, and a theoretical basis can be provided for the development and utilization of the apple flowers. However, no regularity of the type of ionic liquid and the different active ingredients has been found herein, and further intensive research is required. The invention provides a new thought and approach for Chinese medicine researchers, the established optimal extraction conditions are scientific and feasible, and the method can be used for optimizing and selecting an extraction technology suitable for industrial expanded production, improving the old process, improving the efficiency and providing experimental basis for comprehensive utilization of apple flowers.
The results of the invention show that: astragalin and afzelin have obvious activation effect on tyrosinase, and the activation rate is continuously increased along with the increase of concentration, so that the astragalin and the afzelin are expected to be developed into a medicament for treating leucoderma; phlorizin has inhibition effect on tyrosinase at the final concentration of 2 mmol/L; the tyrosinase was activated at final concentrations of 1 mmol/L and 0.5 mmol/L, but the activation increased with decreasing concentration, probably because phlorizin at different concentrations affected tyrosinase activity by different mechanisms, and further studies were possible.
Claims (4)
1. A method for extracting phlorizin, astragalin and afzerin from apple flower by using ionic liquid is characterized in that dried apple flower powder is mixed with an extracting agent and then is subjected to ultrasonic extraction at room temperature, and after the ultrasonic extraction is finished, the apple flower powder is centrifuged, and supernatant is taken to obtain the finished product;
the extractant is formed by mixing ionic liquid and methanol, and the ionic liquid is [ BMIM ] BF4、［BMIM］Br、［BMIM］PF6Or [ HMIM ] PF6(ii) a The concentration of the ionic liquid in the extractant is 0.1-1.0 mol/L.
2. The method for extracting phlorizin, astragalin and afzerin from apple flowers by using ionic liquid as claimed in claim 1, wherein the ratio of apple flower powder to extractant is 1 g: adding 20-100 mL of apple flower powder with mesh number of 10-90.
3. The method for extracting phlorizin, astragalin and afzerin from apple flowers by using ionic liquid as claimed in any one of claims 1 to 2, wherein the ultrasonic extraction time is 10-60 min.
4. The method for extracting phlorizin, astragalin and afzerin from apple flowers by using ionic liquid as claimed in claim 3, wherein the centrifugal rotation speed is 2000-9000 r/min.
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| CN103864864A (en) * | 2014-03-27 | 2014-06-18 | 江苏斯威森生物医药工程研究中心有限公司 | Method for efficiently extracting phlorizin from plants |
| CN107029686A (en) * | 2017-04-01 | 2017-08-11 | 大连理工大学 | Hydrophilic Interaction Chromatography stationary phase, preparation method and applications based on amine functionalized imidazole ion liquid |
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| CN103864864A (en) * | 2014-03-27 | 2014-06-18 | 江苏斯威森生物医药工程研究中心有限公司 | Method for efficiently extracting phlorizin from plants |
| CN107029686A (en) * | 2017-04-01 | 2017-08-11 | 大连理工大学 | Hydrophilic Interaction Chromatography stationary phase, preparation method and applications based on amine functionalized imidazole ion liquid |
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| Fruit Seeds of the Rosaceae Family: A Waste, New Life, or a Danger to Human Health?;Matejia Senica et al.;《Journal of Agricultural and Food Chemistry》;20171110;第65卷(第48期);第10621-10629页 * |
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