CN114699438A - Dandelion total flavonoids and extraction method and application thereof - Google Patents
Dandelion total flavonoids and extraction method and application thereof Download PDFInfo
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- CN114699438A CN114699438A CN202210456329.3A CN202210456329A CN114699438A CN 114699438 A CN114699438 A CN 114699438A CN 202210456329 A CN202210456329 A CN 202210456329A CN 114699438 A CN114699438 A CN 114699438A
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- dandelion
- total flavonoids
- ethanol
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- extraction
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- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
- A61K36/288—Taraxacum (dandelion)
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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Abstract
The invention relates to dandelion total flavonoids as well as an extraction method and application thereof, belonging to the technical field of extraction of plant active ingredients. The extraction method comprises the following steps: (1) mixing the dandelion powder with ethanol, soaking, extracting, centrifuging, and taking filtrate to obtain a crude dandelion total flavone extract; (2) purifying the crude extract of the total flavonoids of the dandelion, and drying to obtain the total flavonoids of the dandelion; the mass volume ratio of the dandelion powder to the ethanol is 1 g: 20-40 mL; the concentration of the ethanol is 40-60 vt%. The dandelion total flavonoids with oxidation resistance, reducing capability, anti-inflammatory and whitening capability can be extracted according to the method of the invention.
Description
Technical Field
The invention relates to the technical field of extraction of plant active ingredients, in particular to dandelion total flavonoids and an extraction method and application thereof.
Background
Taraxacum mongolicum hand-Mazz (Latin) Compositae, perennial herbaceous plants of Taraxacum, distributed in most areas of the country, blossom in spring and autumn every year, and have long flowering phase. The taraxacum flower has high contents of flavonoids, yellow pigment, phenolic acid compounds, etc. Ancient book of traditional Chinese medicine, Bencao gang mu, records that dandelion has dry nature and mild taste, and has the efficacy of clearing heat and removing toxicity.
The herba Taraxaci contains abundant bioactive components, and the bioactive components are various, and mainly comprise flavonoids, sterols, triterpenes, phenolic acids, pigments, sesquiterpene lactones, coumarins, etc. The natural flavonoid compounds occupy an important position in human dietary structure, and the intake amount of the natural flavonoid compounds is closely related to the occurrence of diseases such as cardiovascular and cerebrovascular diseases and the like. The compounds have strong biological activities of oxidation resistance, tumor resistance, bacteria resistance, inflammation diminishing, gallbladder benefiting, liver protection and the like.
The prior method for extracting the dandelion total flavonoids cannot obtain the dandelion total flavonoids with high content and high purity.
Disclosure of Invention
The invention aims to provide dandelion total flavonoids as well as an extraction method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for extracting dandelion total flavonoids, which comprises the following steps:
(1) mixing the dandelion powder with ethanol, soaking, extracting, centrifuging, and taking filtrate to obtain a crude dandelion total flavone extract;
(2) purifying the crude extract of the total flavonoids of the dandelion, and drying to obtain the total flavonoids of the dandelion;
the mass volume ratio of the dandelion powder to the ethanol is 1 g: 20-40 mL;
the concentration of the ethanol is 40-60 vt%.
Preferably, the particle size of the dandelion powder is less than or equal to 300 μm.
Preferably, the soaking temperature is 20-25 ℃;
the soaking time is 50-70 min.
Preferably, the extraction is flash extractor extraction;
the extraction time is 60-100 s;
the extracted voltage is 95-105V.
Preferably, the rotating speed of the centrifugation is 9000-11000 r/min;
the centrifugation time is 8-12 min.
Preferably, the purification is macroporous resin adsorption purification;
the macroporous resin is D101, AB-8, X-5 or NKA-9.
The invention also provides the dandelion total flavonoids extracted by the extraction method, and the purification rate of the dandelion total flavonoids is more than or equal to 52.94%.
The invention also provides application of the dandelion total flavonoids in preparation of anti-oxidation medicines, health-care foods or cosmetics.
The invention also provides application of the dandelion total flavonoids in preparation of anti-inflammatory drugs, health-care foods or cosmetics.
The invention provides dandelion total flavonoids and an extraction method and application thereof. The method has the following advantages:
the invention utilizes the flash extractor to extract the total flavonoids of the dandelion, and the crude extract of the total flavonoids of the dandelion with the total flavonoids content of not less than 21.15mg/g can be obtained according to the extraction method of the invention. After the crude extract of the total flavonoids of the dandelions obtained by the invention is purified by macroporous resin D101, AB-8, X-5 or NKA-9, the total flavonoids of the dandelions with the purification rate of more than or equal to 52.94 percent can be obtained. The dandelion total flavonoids extracted by the method have strong capacity of removing DPPH free radicals and ABTS free radicals, have strong inhibition rate activity on hyaluronidase and tyrosinase, and can be prepared into medicines, health-care foods and cosmetics.
Drawings
FIG. 1 is a graph showing the influence of the total flavonoids of dandelion in ethanol concentration;
FIG. 2 is a diagram showing the influence of the mass-volume ratio of dandelion powder to ethanol on the total flavone content of dandelion;
FIG. 3 is a graph showing the effect of extraction time on the total flavonoid content of dandelion;
FIG. 4 is a three-dimensional graph of response surface of ethanol concentration, mass-to-volume ratio of dandelion powder and ethanol, and extraction time to total flavonoids content in dandelion;
FIG. 5 is a graph showing the effect of total flavonoids of dandelion extract and VC on scavenging DPPH free radicals;
FIG. 6 is a graph showing the effect of total flavonoids of dandelion extract and VC on scavenging ABTS free radicals;
FIG. 7 is a graph showing the scavenging effect of total flavonoids of dandelion and VC on superoxide anion radicals;
FIG. 8 is a graph showing the effect of reducing power of the dandelion total flavone extract and VC;
FIG. 9 is a graph showing the effect of dandelion total flavonoids extract and dipotassium glycyrrhizinate on hyaluronidase inhibition;
FIG. 10 is a graph showing the effect of dandelion total flavonoids extract and kojic acid on tyrosinase inhibition.
Detailed Description
The invention provides a method for extracting dandelion total flavonoids, which comprises the following steps:
(1) mixing the dandelion powder with ethanol, soaking, extracting, centrifuging, and taking filtrate to obtain a crude dandelion total flavone extract;
(2) purifying the crude extract of the total flavonoids of the dandelion, and drying to obtain the total flavonoids of the dandelion;
in the invention, the mass volume ratio of the dandelion powder to the ethanol is 1 g: 20-40 mL, preferably 1 g: 30 mL; the concentration of the ethanol is 40-60 vt%, and preferably 50 vt%.
In the invention, the particle size of the dandelion powder is less than or equal to 300 μm. In the present invention, the extraction is flash extractor extraction; the extraction time is 60-100 s; the extracted voltage is 95-105V, and preferably 100V. In the invention, the soaking temperature is 20-25 ℃, and preferably 22.5 ℃; the soaking time is 50-70 min, preferably 60 min. In the present invention, the extraction time is preferably 80 s. In the invention, the rotating speed of the centrifugation is 9000-11000 r/min, preferably 10000 r/min; the centrifugation time is 8-12 min, preferably 10 min.
In the invention, the extraction method also comprises the optimization of the concentration of ethanol, the mass-volume ratio of the dandelion powder to the ethanol and the extraction time; the optimization method adopts a response surface method for optimization; the response surface method Design method comprises the steps of determining the optimal levels of ethanol concentration, the mass-volume ratio of dandelion powder to ethanol and extraction time according to a single-factor experiment result, centering on a single-factor experiment optimal point, taking 1 horizontal value around the optimal point as the level of a response surface, carrying out three-factor three-level experiment Design by using Design-Expert software according to an X-Behnken Design principle, checking the correlation between a response value and a predicted value, taking three factors of ethanol concentration A, the mass-volume ratio B of dandelion to ethanol and extraction time C as independent variables, taking total flavone content R as a response value, and establishing a multivariate quadratic equation as follows: r ═ 58.40725+1.88462A +1.19170B +0.44686C +9.00000 × 10-4AB+2.11250×10-3AC+1.75000×10-4BC-0.020907A2-0.020533B2-3.56437×10-3C2. Wherein R is the total flavone extract mg/g of herba Taraxaci, and A, B, C are respectivelyEthanol concentration vt%, feed-liquid ratio (g/mL), extraction time s.
In the invention, the method for measuring the content of the total flavonoids in the crude total flavonoid extract adopts an aluminum trichloride color development method for detection. The detection method comprises the following steps: transferring the crude extract of total flavonoids into a measuring cylinder, and accurately measuring the volume of the crude extract of total flavonoids to obtain a stock solution for measuring the content of the total flavonoids in the dandelion. Transferring 2mL of stock solution by a pipette, adding into a test tube, adding 2mL of 1% aluminum trichloride, developing for 10min, adjusting to zero by taking 75 vt% ethanol solution as blank reference, and measuring the absorbance of the component to be measured by using an enzyme-labeling instrument at the wavelength of 425 nm. Obtaining a regression equation according to the absorbance (Y) and the concentration (X) by taking the reed as a standard substance: y is 8.4731X-0.0031, R2The concentration was calculated as 0.9997, and the total flavone content was total flavone mass (mg)/dandelion powder mass (g).
In the invention, the purification is macroporous resin adsorption purification; the macroporous resin is D101, AB-8, X-5 or NKA-9.
The invention also provides the dandelion total flavonoids extracted by the extraction method, and the purification rate of the dandelion total flavonoids is more than or equal to 52.94%.
The invention also provides application of the dandelion total flavonoids in preparation of anti-oxidation medicines, health-care foods or cosmetics.
The invention also provides application of the dandelion total flavonoids in preparation of anti-inflammatory drugs, health-care foods or cosmetics.
The embodiments of the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
In the embodiment of the invention, the method for measuring the content of the total flavonoids in the crude total flavonoid extract adopts an aluminum trichloride color development method for detection. The detection method comprises the following steps: transferring the crude extract of total flavonoids into a measuring cylinder, and accurately measuring the volume of the crude extract of total flavonoids to obtain a stock solution for measuring the content of the total flavonoids in the dandelion. Transferring 2mL of stock solution by a pipette, adding 2mL of 1% aluminum trichloride, developing for 10min, adjusting to zero by using 75 vt% ethanol solution as blank reference blank, and measuring the light absorption of the component to be measured at a wavelength of 425nm by using an enzyme-labeling instrumentAnd (4) degree. Obtaining a regression equation according to the absorbance (Y) and the concentration (X) by taking the reed as a standard substance: y is 8.4731X-0.0031, R2The concentration was calculated as 0.9997, and the total flavone content was total flavone mass (mg)/dandelion powder mass (g).
Example 1 response surface experiment optimization extraction parameters
(1) Single factor experiment
Accurately weighing 5g of herba Taraxaci powder with particle size of 300 μm, adding 100mL of ethanol, soaking at 25 deg.C for 60min, and extracting with a flash extractor at 100V for 60s, wherein the ethanol concentration is 40 vt%, 50 vt%, 60 vt%, 70 vt%, and 80 vt%, respectively. And (3) investigating the influence of the ethanol concentration on the content of the total flavonoids in the crude extract of the dandelion. The results are shown in FIG. 1, and the total flavonoids in the crude extract of dandelion reached the maximum at an ethanol concentration of 50 vt%.
Accurately weighing 5g of dandelion powder with the particle size of 300 mu m, adding 60 vt% ethanol, soaking for 60min at 25 ℃, extracting for 60s by using a flash extractor with the voltage of 100V, and respectively weighing 1g of dandelion powder and ethanol according to the mass-to-volume ratio of dandelion powder to ethanol: 10mL, 1 g: 20mL, 1 g: 30mL, 1 g: 40mL, 1 g: ethanol is added in an amount of 50mL, and the influence of the mass volume ratio of the dandelion powder and the ethanol on the total flavone content in the crude dandelion extract is examined. The results are shown in figure 2, wherein the mass-volume ratio of the dandelion powder to the ethanol is 1 g: the total flavone in the crude extract of dandelion reaches the maximum when the total flavone is 30 mL.
Accurately weighing 5g of dandelion powder with the particle size of 300 mu m, wherein the mass volume ratio of the dandelion powder to the ethanol is 1 g: adding 60 vt% ethanol into 20mL, soaking at 25 deg.C for 60min, and extracting with flash extractor at 100V for 20, 40, 60, 80, and 100s respectively. And (3) investigating the influence of the extraction time on the content of the total flavonoids in the crude extract of the dandelion. The result is shown in FIG. 3, and the total flavone content in the crude extract of dandelion reaches the maximum when the extraction time is 80 s.
(2) Response surface method for optimizing and extracting parameters
The optimal ethanol concentration is determined according to the single-factor experiment result and is 50 vt%, and the mass-volume ratio of the dandelion powder to the ethanol is 1 g: centering on 30mL and extraction time 80s, respectively taking 1 horizontal value around the optimal point as the level of the response surface, respectively coding-1, 0 and 1, and setting the specific factor level as shown in Table 1Shown in the figure. Carrying out three-factor three-level experimental Design by using Design-Expert software according to an X-Behnken Design principle, wherein three factors of ethanol concentration A, a mass-volume ratio B of dandelion to ethanol and extraction time C are independent variables, total flavone content R is used as a response value, the experimental scheme Design and response value results are shown in a table 2, the results are shown in a figure 4, and the obtained multivariate quadratic equation is established as follows: r ═ 58.40725+1.88462A +1.19170B +0.44686C +9.00000 × 10-4AB+2.11250×10-3AC+1.75000×10-4BC-0.020907A2-0.020533B2-3.56437×10-3C2. In the formula, R is the extraction amount mg/g of total flavonoids of dandelion, and A, B, C is the concentration (vt%) of ethanol, the mass-volume ratio g/mL of dandelion powder and ethanol, and the extraction time s respectively. The results of the regression equation analysis of variance are shown in table 3.
TABLE 1 design of the level of response surface experiment factors
TABLE 2 response surface design and response values
TABLE 3 variance regression results of regression equation
The said p <0.05, p <0.01, p < 0.001.
Tables 2-3 and FIG. 4 show that the influence of three factors on the total flavone content is: c extraction time>The mass-volume ratio of the dandelion powder to the ethanol>A concentration of ethanol. High correlation coefficient (R)20.985) indicates that,the experimental response values have good correlation with the predicted response values. P<0.001 shows that each dependent variable and each variable have obvious correlation, and the established model has significance. Mistyped item P>0.05, the model is well fitted and has no significant difference, and the model and a regression equation can be used for analyzing the content of the total flavonoids in the dandelion. The optimal parameters obtained by the model design are as follows: the ethanol concentration is 49.68 vt%, and the mass volume ratio of the dandelion powder to the ethanol is 1 g: 30.44mL, extraction time 78.15 s. The theoretical content of the total flavonoids in the crude extract of the total flavonoids in the dandelion obtained by the optimal parameters is 24.00 mg/g.
And (3) extracting the total flavonoids in the dandelion according to the obtained optimal parameter setting extraction method, and finally obtaining the actual content of the total flavonoids in the crude dandelion extract, wherein the actual content is 23.89 +/-0.6391 mg/mL. The content of the total flavonoids extracted from the theoretical value and the actual value is basically consistent, which indicates that the designed model is suitable for extracting the total flavonoids of the dandelion.
Example 2
After the dandelion total flavonoids are extracted according to the optimal extraction parameters obtained in the example 1, four different types of macroporous adsorption resins D101, AB-8, X-5 and NKA-9 are selected to adsorb the dandelion total flavonoids extract, and the macroporous adsorption resin with the best adsorption and analysis effects is selected by taking the adsorption amount and the analysis rate of the total flavonoids as indexes to carry out subsequent experiments.
Respectively weighing 1g of pretreated macroporous resin: AB-8, D101, X-5 and NKA-9 are put into a 50mL conical flask, 10mL dandelion total flavone crude extract is added, the mixture is put on a shaker at 25 ℃ and 120r/min for adsorption for 12h, and the supernatant is taken to determine the total flavone content. And (3) carrying out suction filtration on the adsorbed resin, washing residual liquid on the surface of the resin with distilled water, pouring the resin into a 100mL conical flask, adding 20mL of 70% ethanol solution, placing the conical flask on a shaker at 25 ℃ and 120r/min for resolving for 12 hours, and taking supernatant to determine the content of the total flavonoids. Calculating the adsorption rate, the resolution ratio and the purification rate of the dandelion total flavonoids by different macroporous resins according to the following formulas. The results are shown in Table 4.
In the formula: q is the adsorption amount mg/g; a is the adsorption rate of flavone; p is the analytic amount mg/g; d is the resolution; n is the purification rate%; c0The initial flavone concentration is mg/mL; crThe concentration of the flavone in the adsorption solution is mg/mL; v1The volume is mL of the adsorption solution; m is the dry weight g of the resin; cdThe concentration of the flavone in the resolution solution is mg/mL; v2The volume of the solution is mL.
TABLE 4 comparison of adsorption and desorption effects of different types of macroporous adsorbent resins
Table 4 shows that different types of macroporous resin have different adsorption rates on the total flavonoids of dandelion but have little difference. The type of macroporous resin with the best adsorption effect is AB-8.
Example 3
According to the optimal parameters of the embodiment 1, the ethanol concentration is 49.68 vt%, and the mass volume ratio of the dandelion powder to the ethanol is 1 g: 30.44mL for 78.15s, and extracting the dandelion total flavonoids to obtain the dandelion total flavonoids crude extract. Adsorbing the crude extract of the total flavonoids of the dandelion by using AB-8 type macroporous resin, and then freezing and drying to obtain the total flavonoids of the dandelion.
(1) Determination of DPPH (dipeptidyl peptidase) removing capability of dandelion total flavonoids
Preparing the dandelion total flavonoids into sample solutions of 0mg/mL, 0.02mg/mL, 0.04mg/mL, 0.06mg/mL, 0.08mg/mL and 0.10mg/mL respectively by using absolute ethyl alcohol, taking 2mL of each concentration sample solution respectively, adding 2.0mL of DPPH solution (prepared by using absolute ethyl alcohol) with the concentration of 0.1mmol/L, and carrying out dark reaction for 30min at the temperature of 25 ℃. Measuring absorbance value at 517nm, marking as A, adjusting to zero by using 2.0mL of anhydrous ethanol and 2.0mL of distilled water as blank group, using 2.0mL of sample solution and 2.0mL of anhydrous ethanol as blank control group, measuring absorbance value at 517nm as Ai, using the blank control group to remove absorbance existing in total flavone sample, using 2.0mL of LDPPH solution and 2.0mL of anhydrous ethanol as model control group, measuring absorbance value at 517nm as A0. The clearance calculation formula is:
EDPPH＝[1-(Ai-A))/A0]×100％
in the formula: eDPPHClearance of DPPH radicals,%;
a is the light absorption value of 2mL of sample solution added with 2mL of DPPH working solution;
Aiadding 2mL of absolute ethyl alcohol into 2mL of sample solution to obtain a light absorption value;
A0absorbance of 2mL absolute ethanol was added to 2mL DPPH solution.
Triplicate measurements were averaged to compare the effect of VC at 0. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL, 12. mu.g/mL, 16. mu.g/mL, and 20. mu.g/mL on DPPH clearance. The results are shown in FIG. 5.
FIG. 5 shows that the dandelion total flavonoids scavenge the IC of DPPH free radicals50IC with value of 36.56 mug/mL and DPPH free radical scavenging effect of VC50The value is 7.38 mu g/mL, which indicates that the dandelion total flavone obtained by the application has stronger ability of eliminating DPPH free radicals.
(2) Determination of ABTS eliminating capacity of dandelion total flavone
The determination method of ABTS free radical clearance rate comprises the following steps: mixing the ABTS solution with the concentration of 7mmol/L and the potassium persulfate solution with the concentration of 2.45mmol/L according to the volume ratio of 1:1 to prepare a cation working solution of the ABTS free radical, and keeping out of the sun for 12-14 h to excite the ABTS free radical. ABTS free radicals are diluted by PBS until the light absorption value is 0.7 +/-0.02 to prepare ABTS working solution.
Preparing the dandelion total flavonoids into sample solutions of 0mg/mL, 0.04mg/mL, 0.08mg/mL, 0.12mg/mL, 0.16mg/mL, 0.20mg/mL and 0.24mg/mL respectively by using absolute ethyl alcohol, taking 0.2mL of each concentration sample solution respectively, adding 2mLABTS working solution respectively, mixing uniformly, keeping out of the sun, reacting at 37 ℃ for 10min, and measuring the light absorption value A at 734 nm; respectively taking 0.5mL of the sample solution with each concentration, respectively adding 2mLPBS, after reaction, measuring the light absorption value A at 734nmiThen, 2mL of LaBTS working solution and 0.5mL of deionized water are respectively used for reaction, and the absorbance value A is measured at 734nm0As a reference. The clearance calculation formula is:
EABTS＝[1-(Ai-A))/A0]×100％
in the formula: eABTSClearance of ABTS free radicals,%;
a is the light absorption value of 0.5mL of sample solution added with 2mL of ABTS working solution;
Aiadding the light absorption value of 2mL PBS for 0.5mL sample solution;
A0absorbance of 0.5mL deionized water was added to 2mL of the ABTS solution.
Triplicate measurements were averaged to compare the effect of VC at 0. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL, 12. mu.g/mL, 16. mu.g/mL, and 20. mu.g/mL on ABTS clearance. The results are shown in FIG. 6.
FIG. 6 shows that the total flavonoids of dandelion eliminate the IC of ABTS free radical50IC with value of 73.36 mu g/mL and ABTS free radical scavenging effect of VC50The value is 10.73 mu g/mL, which indicates that the dandelion total flavone obtained by the application has stronger capability of eliminating ABTS free radicals.
(3) Determination of capacity of total flavonoids of dandelion for eliminating superoxide anion free radicals
Extracting herba Taraxaci with anhydrous ethanolSample solutions of 0mg/mL, 2mg/mL, 4mg/mL, 6mg/mL, 8mg/mL and 10mg/mL were prepared, 1mL of each concentration sample solution was added with 6.50mL of 0.05mol/L Tris-HCl buffer (pH 8.2) and 0.40mL of 25mmol/L pyrogallol solution, the mixture was mixed and reacted in a water bath at 25 ℃ for 5min, and 1.00mL of 8mmol/L HCl solution was added to terminate the reaction. Determination of the Absorbance A at 325nmi. Superoxide anion clearance was calculated as follows:
Esuperoxide anion＝[1-(Ai-A))/A0]×100％
In the formula: eSuperoxide anionIs the clearance rate for superoxide anion radical,%;
a is the light absorption value of sample solution without adding pyrogallol;
Aiadding pyrogallol and the light absorption value of a sample solution;
A0the absorbance value of the solution without adding the pyrogallol is shown.
The results are shown in FIG. 7, where the scavenging effect of VC at 0. mu.g/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1.0mg/mL, 1.2mg/mL and 1.4mg/mL is compared by averaging three replicates.
FIG. 7 shows that total flavonoids of dandelion scavenge superoxide anion radical IC50VC with a value of 3.07mg/mL scavenges the IC of superoxide anion radical50The value is 0.26mg/mL, which indicates that the dandelion total flavone obtained by the application has stronger capacity of eliminating superoxide anion free radicals.
(4) Determination of reduction power of dandelion total flavone
The dandelion total flavonoids were prepared into sample solutions of 0mg/mL, 4mg/mL, 8mg/mL, 12mg/mL and 16mg/mL with absolute ethanol, 1mL of each sample was added with 2.5mL of 0.2mol/LpH ═ 6.6 phosphate buffer, and then 2.5mL of 1% potassium ferricyanide solution was added, and after mixing uniformly, the mixture was reacted in a water bath at 50 ℃ for 20 min. After being taken out, the mixture is rapidly cooled and added with 2.5mL of 10% trichloroacetic acid to stop the reaction, and the mixture is centrifuged at 3500r/min for 10 min. Collecting supernatant 2.5mL, adding distilled water 2.5mL and 0.5mL of 0.1% ferric trichloride solution, mixing, standing for 10min, and measuring absorbance A of each solution at 700nm wavelength700nm. The absorbance value of the solution without sample is used as a blank control. The total reducing power is reflected by an absorbance value, and the higher the absorbance value is, the stronger the reducing power is. The formula for calculating the reducing power is as follows:
A700nm＝Asample (I)-ABlank space；
The average value is taken by testing in parallel for three times,
as shown in FIG. 8, the results were compared with the reducing power of VC at 0mg/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, and 1.0 mg/mL.
FIG. 8 shows that the reduction power of dandelion total flavonoids is 3.00 at a concentration of 6mg/mL and 3.25 at a concentration of 1mg/mL, which indicates that the dandelion total flavonoids obtained by the present application have a strong reduction power.
Example 4
According to the optimal parameters of the embodiment 1, the ethanol concentration is 49.68 vt%, and the mass volume ratio of the dandelion powder to the ethanol is 1 g: 30.44mL for 78.15s, and extracting the dandelion total flavonoids to obtain the crude dandelion total flavonoids extract. Adsorbing the crude extract of the total flavonoids of the dandelion by using AB-8 type macroporous resin, and then freezing and drying to obtain the total flavonoids of the dandelion.
Determination of hyaluronic acid inhibition rate of dandelion total flavonoids
The dandelion total flavonoids were prepared into sample solutions of 0mg/mL, 2mg/mL, 4mg/mL, 6mg/mL, 8mg/mL, 10mg/mL and 12mg/mL using absolute ethanol. Taking 0.5mL of sample solution with different concentrations, adding 0.1mL of 0.25mmol/L calcium chloride solution and 0.5mL of hyaluronidase solution, and carrying out heat preservation culture at 37 ℃ for 40 min; then adding 0.5mL of sodium hyaluronate solution, and carrying out heat preservation culture at 37 ℃ for 40 min; adding 0.1mL of 0.4mol/L sodium hydroxide solution and 0.5mL of acetylacetone solution, heating in a boiling water bath for 15min, and immediately cooling for 5min by using ice water; then adding 1.0mL of Ellisib reagent, standing at normal temperature for 10min for color development, and then rapidly determining the absorbance of the reaction mixed solution at 585nm by using a microplate reader.
Calculating the formula: hyaluronidase inhibition (%) - (A-B) - (C-D) ]/(A-B) × 100%
In the formula: a is the light absorption value of blank group, and the blank group is that acetic acid buffer solution is used for replacing sample liquid;
b is the light absorption value of a blank control group, and the blank control group is that acetic acid buffer solution is used for replacing sample solution and hyaluronidase solution;
c is the light absorption value of the sample group;
d is the light absorption value of the sample control group, and the sample control group is formed by replacing the hyaluronidase solution with acetic acid buffer solution.
The results of the three parallel tests were averaged to compare the inhibitory effects of dipotassium glycyrrhizinate at 0.4mg/mL, 0.8mg/mL, 1.2mg/mL, 1.6mg/mL, 2.0mg/mL, 2.4mg/mL and 2.8mg/mL on hyaluronidase. The results are shown in FIG. 9.
FIG. 9 shows that the dandelion total flavonoids inhibit the hyaluronidase activity IC50IC with value of 3.44mg/mL and with dipotassium glycyrrhizinate for inhibiting hyaluronidase activity50The value is 1.55mg/mL, which shows that the dandelion total flavonoids have stronger capability of inhibiting the activity of the hyaluronidase.
Example 5
According to the optimal parameters of the embodiment 1, the ethanol concentration is 49.68 vt%, and the mass volume ratio of the dandelion powder to the ethanol is 1 g: 30.44mL for 78.15s, and extracting the dandelion total flavonoids to obtain the crude dandelion total flavonoids extract. Adsorbing the crude extract of the total flavonoids of the dandelion by using AB-8 type macroporous resin, and then freezing and drying to obtain the total flavonoids of the dandelion.
Determination of tyrosinase inhibition rate by dandelion total flavone
Preparing 10mg/mL, 12mg/mL, 14mg/mL, 16mg/mL, 18mg/mL, 20mg/mL and 22mg/mL sample solutions by using absolute ethyl alcohol, taking 0.5mL of each concentration sample solution, adding 0.5mL of 100U/mL tyrosinase solution, shaking and uniformly mixing, placing in a 37 ℃ thermostat for 10min, adding 1mL of 1mg/mL levodopa solution, uniformly mixing, continuously reacting at 37 ℃ for 10min, and then rapidly determining the absorbance of the reaction mixed solution at 480nm by using a microplate reader. Calculating the formula: tyrosinase inhibition (%) [ (a-B) - (C-D) ]/(a-B) × 100%;
in the formula:
a is the light absorption value of blank group, and the blank group is that acetic acid buffer solution is used for replacing sample liquid;
b is the light absorption value of a blank control group, and the blank control group is that acetic acid buffer solution is used for replacing sample liquid and hyaluronidase solution;
c is the light absorption value of the sample group;
d is the light absorption value of the sample control group, and the sample control group is formed by replacing the hyaluronidase solution with acetic acid buffer solution.
The results of the three parallel tests were averaged, and the inhibition of tyrosine by kojic acid at different concentrations was used as a comparison. The results are shown in FIG. 10.
FIG. 10 shows that the taraxacum total flavonoids inhibit tyrosinase activity IC50IC with a value of 14.86mg/mL for kojic acid inhibiting hyaluronidase Activity50The value is 57.53 mug/mL, which shows that the dandelion total flavone has stronger ability of inhibiting the tyrosinase activity.
From the above embodiments, the invention provides a dandelion total flavone and its extraction method and application. The dandelion total flavonoids extracted by the method have the capability of removing DPPH, ABTS and superoxide anion free radicals, also have the reducing capability and have the capability of inhibiting hyaluronidase and tyrosinase.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A method for extracting total flavonoids from dandelion is characterized by comprising the following steps:
(1) mixing the dandelion powder with ethanol, soaking, extracting, centrifuging, and taking filtrate to obtain a crude dandelion total flavone extract;
(2) purifying the crude extract of the total flavonoids of the dandelion, and drying to obtain the total flavonoids of the dandelion;
the mass volume ratio of the dandelion powder to the ethanol is 1 g: 20-40 mL;
the concentration of the ethanol is 40-60 vt%.
2. The extraction method according to claim 1, wherein the particle size of the dandelion powder is less than or equal to 300 μm.
3. The extraction method according to claim 2, wherein the soaking temperature is 20-25 ℃;
the soaking time is 50-70 min.
4. The extraction method according to claim 3, characterized in that the extraction is a flash extractor extraction;
the extraction time is 60-100 s;
the extracted voltage is 95-105V.
5. The extraction method according to claim 4, wherein the rotation speed of the centrifugation is 9000-11000 r/min;
the centrifugation time is 8-12 min.
6. The extraction method according to any one of claims 1 to 5, wherein the purification is macroporous resin adsorption purification;
the macroporous resin is D101, AB-8, X-5 or NKA-9.
7. The method for extracting dandelion total flavonoids according to any one of claims 1 to 6, wherein the purification rate of dandelion total flavonoids is not less than 52.94%.
8. Use of the dandelion total flavonoids according to claim 7 in the preparation of anti-oxidant drugs, health foods or cosmetics.
9. Use of the dandelion total flavonoids according to claim 7 in the preparation of anti-inflammatory drugs, health foods or cosmetics.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102133254A (en) * | 2011-03-16 | 2011-07-27 | 天津工业大学 | Extraction method of dandelion total flavonoids |
| CN103070899A (en) * | 2012-12-13 | 2013-05-01 | 大兴安岭林格贝有机食品有限责任公司 | Method for separating and purifying total flavone of dandelion |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102133254A (en) * | 2011-03-16 | 2011-07-27 | 天津工业大学 | Extraction method of dandelion total flavonoids |
| CN103070899A (en) * | 2012-12-13 | 2013-05-01 | 大兴安岭林格贝有机食品有限责任公司 | Method for separating and purifying total flavone of dandelion |
Non-Patent Citations (1)
| Title |
|---|
| 赵惠茹等: "大孔吸附树脂分离纯化蒲公英中总黄酮的工艺研究", 《中国药业》 * |
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