CN115812889A - Application of acanthopanax senticosus extract in preparation of tyrosinase inhibitor product - Google Patents
Application of acanthopanax senticosus extract in preparation of tyrosinase inhibitor product Download PDFInfo
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- CN115812889A CN115812889A CN202211417072.7A CN202211417072A CN115812889A CN 115812889 A CN115812889 A CN 115812889A CN 202211417072 A CN202211417072 A CN 202211417072A CN 115812889 A CN115812889 A CN 115812889A
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- 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
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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Abstract
The invention relates to an application of an acanthopanax senticosus extract in preparation of tyrosinase inhibitor products, belonging to the fields of cosmetics and foods. The application of the acanthopanax senticosus extract in preparing tyrosinase inhibitor products is characterized in that the acanthopanax senticosus extract is obtained by the following method: mixing acanthopanax fruit powder with an ethanol water solution with a certain concentration, and carrying out ultrasonic treatment for a certain time to obtain an extracting solution; filtering, distilling and drying the obtained extract to obtain the acanthopanax ethanol extract. The in vitro enzyme activity test result proves that: the acanthopanax ethanol extract has certain inhibition activity on tyrosinase under different concentrations when L-dopa and L-tyrosine are used as substrates, the inhibition effect of the acanthopanax ethanol extract on tyrosine is very obvious when the acanthopanax ethanol extract and L-tyrosine are used as main active components, and the acanthopanax ethanol extract and the L-dopa and L-tyrosine have obvious reversible competitive inhibition effect on tyrosinase. Therefore, the acanthopanax ethanol extract is an excellent tyrosinase natural inhibitor and has the potential of being further developed into beauty cosmetics or food preservatives.
Description
Technical Field
The invention relates to an application of an acanthopanax senticosus extract in preparation of tyrosinase inhibitor products, belonging to the fields of cosmetics and foods.
Background
Tyrosinase is the major rate-limiting enzyme in the process of melanin production, which is formed by tyrosine through a series of chemical reactions. In melanocyte, tyrosine is hydroxylated by tyrosinase to generate dopa and dopaquinone, which is converted into indoloquinone by oxidation and decarboxylation, and finally indoloquinone is polymerized into melanin. Inhibition of tyrosinase has been established as one of the main strategies for regulating melanin production, and this inhibitor is used to treat hyperpigmentation and other adverse effects from melanin production. There are a variety of drugs for inhibiting melanin production, which can treat excessive production of melanin or human skin pigmentation, such as natural plant-derived arbutin, ascorbic acid, etc.
At present, many tyrosinase inhibitors are limited in application due to safety, so that the search for natural effective tyrosinase inhibitors has become a research and development trend in the food and cosmetic industries. At present, no research and development aiming at inhibiting tyrosinase activity by acanthopanax senticosus extract is available. The invention provides a preparation method of an acanthopanax ethanol extract and a new application of the acanthopanax ethanol extract as a tyrosinase inhibitor. Through in vitro tyrosinase inhibition activity research, the acanthopanax ethanol extract has obvious tyrosinase inhibition activity, the acanthopanax glycoside E and syringin which are main active components have obvious inhibition effects on tyrosine, and the acanthopanax glycoside E and the syringin which have obvious reversible competitive inhibition effects on tyrosinase can be used as a natural tyrosinase inhibitor. Can be developed into functional foods and cosmetics with whitening function and food antistaling agent for preventing food browning, and provides reliable basis for developing health products, foods and cosmetics.
Disclosure of Invention
The invention aims to provide a new application of an acanthopanax ethanol extract as a tyrosinase inhibitor. A tyrosinase activity inhibition experiment using L-dopa and L-tyrosine as substrates is established, an acanthopanax ethanol extract is used as a natural inhibitor, and the inhibition mechanism of two main active ingredients on tyrosinase is discussed.
The application of the acanthopanax senticosus extract in preparing tyrosinase inhibitor products is that the acanthopanax senticosus extract is obtained by the following method: mixing acanthopanax fruit powder with an ethanol water solution with a certain concentration, and carrying out ultrasonic treatment for a certain time to obtain an extracting solution; filtering, distilling and drying the obtained extract to obtain the acanthopanax ethanol extract.
Preferably, the volume fraction of ethanol in the ethanol aqueous solution is 30-80%; the mass-volume ratio of the acanthopanax fruit powder to the ethanol water solution is 1g.
Preferably, the ultrasonic treatment time is 180min to 250min.
Preferably, the distillation step is: and (3) carrying out rotary evaporation on the filtered extracting solution at the temperature of 40-55 ℃, and removing ethanol in the extracting solution to obtain the acanthopanax ethanol crude extract.
Preferably, the drying step is: vacuum drying or freeze drying the obtained radix Acanthopanacis Senticosi crude extract at low temperature to obtain radix Acanthopanacis Senticosi ethanol extract.
In the application of the invention, the tyrosinase activity is obviously inhibited by the acanthopanax senticosus ethanol extract in a concentration-dependent manner when the L-dopa and the L-tyrosine are taken as substrates, and the concentration (IC) of the acanthopanax senticosus ethanol extract is reduced by half when the tyrosinase activity is reduced by half 50 ) 1.77 plus or minus 0.96mg/ml and 1.86 plus or minus 0.75mg/ml respectively; when L-dopa and L-tyrosine are used as substrates, the inhibition rates of the acanthopanax ethanol extract on tyrosinase are 34.51 +/-2.05% and 40.86 +/-0.03%, 55.31 +/-0.6% and 40.38 +/-2.84%, 84.33 +/-2.71% and 88.03 +/-0.78%, 90.47 +/-0.87% and 94.87 +/-0.30% respectively under different concentrations (1 mg/ml, 2.5mg/ml, 5mg/ml and 7.5 mg/ml).
In the application of the invention, the main active components of the acanthopanax senticosus extract, namely eleutheroside E and syringin, are inhibition rate IC of tyrosinase 50 The values are respectively 11.13 +/-0.50 mu M and 15.41 +/-0.22 mu M, and the inhibition rates are respectively 51.53 +/-0.01 percent and 55.03 +/-3.38 percent at the concentration of 20 mu M; and tyrosinase kinetic experiment results show that eleutheroside E and syringin are reversible competitive inhibition reactions to tyrosinase.
Specifically, the invention provides an application of acanthopanax senticosus extract in preparing food preservative products.
Specifically, the invention provides application of the acanthopanax senticosus extract in preparation of whitening and brightening daily chemical washing products, wherein the daily chemical washing products are facial cleanser, face cream, hand cream, lotion and facial mask.
Specifically, the invention provides application of the acanthopanax senticosus extract in preparing whitening and brightening functional food, wherein the dosage form of the functional food is solid powder, oral beverage, tablet candy, food preservative film additive and the like.
The invention has the beneficial effects that: the invention provides a preparation method of an acanthopanax ethanol extract and a new application of the acanthopanax ethanol extract as a tyrosinase inhibitor. Through in vitro tyrosinase inhibition research, the acanthopanax ethanol extract has obvious tyrosinase inhibition activity, can effectively inhibit the generation of melanin and is used as a natural tyrosinase inhibitor. The product can be developed into functional food and cosmetics with whitening function and food preservative for preventing food browning, and provides reliable basis for developing health products, foods and cosmetics of Acanthopanax senticosus. The acanthopanax ethanol extract provided by the invention can be used as a natural plant tyrosinase inhibitor, and has the advantages of simple extraction process, low production cost and easiness in industrial production.
Drawings
FIG. 1 shows the effect of different concentrations of ethanol extract of Acanthopanax senticosus on tyrosinase activity when L-dopa is used as a substrate;
FIG. 2 shows the effect of different concentrations of ethanol extract of Acanthopanax senticosus harms on tyrosinase activity when L-tyrosine is used as substrate;
FIG. 3 shows the inhibitory effect of eleutheroside E on tyrosinase at different concentrations with L-dopa as a substrate (A); a relation graph (B) of the reaction speed of eleutheroside E and the tyrosinase activity; a Lineweaver-Burk penultimate curve (C) of eleutheroside E at different substrate L-dopa concentrations;
FIG. 4 shows the inhibitory effect of syringin on tyrosinase (A) with L-dopa as substrate at different concentrations; a relational graph (B) of the reaction speed of the syringin and the tyrosinase activity; a Lineweaver-Burk double reciprocal curve (C) of syringin at different substrate L-dopa concentrations;
note: the letters in the graph represent significant differences between the comparative groups within the 95% confidence interval, P <0.05, P <0.01, P <0.001, and P <0.0001, and statistical calculations were performed using the analysis of variance ANOVA method.
Detailed Description
The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.
The test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
One of the specific implementation modes is as follows:
the invention provides an acanthopanax ethanol extract, which is prepared by the following method:
step 1: taking Acanthopanax (Acanthopanax senticosus) fruit powder, mixing the sample from the original planting base of Heilongjiang soup with ethanol water solution with certain concentration, and performing ultrasonic treatment for a certain time to obtain an extracting solution.
Step 2: filtering the extractive solution, and distilling the solvent under reduced pressure to obtain radix Acanthopanacis Senticosi ethanol crude extract.
And step 3: taking the acanthopanax ethanol crude extract, and drying to obtain the acanthopanax ethanol extract.
Preferably, in the preparation method of the acanthopanax ethanol extract provided by the invention, the volume fraction of ethanol in the ethanol water solution is 30-80%; the mass-volume ratio of the acanthopanax fruit powder to the ethanol water solution is 1: 10-1, and the ultrasonic time is 180-250 min.
Preferably, the volume fraction of ethanol in the ethanol water solution is 70%, and the mass-volume ratio of the acanthopanax fruit powder to the ethanol water solution is 1:25, the ultrasonic time is 180min.
Preferably, in the method for preparing the ethanol extract of acanthopanax senticosus provided by the invention, the step 2 is specifically to filter the obtained extracting solution, perform rotary evaporation at 40-55 ℃, remove the ethanol in the extracting solution obtained in the step 1 and obtain the crude ethanol extract of acanthopanax senticosus.
Preferably, in the method for preparing an ethanol extract of acanthopanax senticosus provided by the invention, the drying step in the step 3 is specifically to obtain the ethanol extract of acanthopanax senticosus after vacuum drying or low-temperature freeze drying of the obtained crude extract of acanthopanax senticosus.
Preferably, the drying method of the acanthopanax senticosus ethanol extract is vacuum drying.
The invention also aims to provide the acanthopanax senticosus extract prepared by the method and application of the acanthopanax senticosus extract in preparing tyrosinase inhibitor products.
Example 1
A method for preparing radix Acanthopanacis Senticosi ethanol extract comprises adding radix Acanthopanacis Senticosi fruit powder into ethanol water solution, ultrasonically mixing, filtering, rotary steaming, and lyophilizing to obtain radix Acanthopanacis Senticosi ethanol extract, which comprises the following steps:
weighing 20g of acanthopanax fruit powder, adding 500ml of 70% ethanol solution, and performing ultrasonic treatment for 180min at 45 ℃ to obtain an acanthopanax ethanol crude extract. Filtering the crude extract, and rotary evaporating at 40 deg.C until ethanol solution is completely removed. Vacuum drying and lyophilizing the rotary-steamed extract to obtain radix Acanthopanacis Senticosi ethanol extract, collecting, and storing at-80 deg.C.
1. Tyrosinase inhibitory activity assay:
tyrosinase is the main rate-limiting enzyme in the process of generating melanin, the catalytic action of tyrosinase mainly occurs in two reactions of converting tyrosine into dopa and converting dopa into dopaquinone in the process of generating melanin by tyrosinase reaction, wherein the dopaquinone is a colored substance and has a maximum absorption peak at the wavelength of 475 nm. Therefore, the inhibition effect of the sample on tyrosinase is calculated by measuring the amount of dopaquinone by using L-dopa or L-tyrosine as a substrate.
Mixing 111 μ L of 2.5mM L-dopa or L-tyrosine solution with 200 μ L of 0.1M phosphate buffer solution, incubating at 25 deg.C, adding 11 μ L of radix Acanthopanacis Senticosi extract solution (concentration of 1mg/mL, 2.5mg/mL, 5mg/mL, 7.5 mg/mL) and 11 μ L of tyrosinase aqueous solution (1000 units/mL), mixing 333 μ L of total reaction system, incubating for 540s, and detecting absorbance at 475 nm. Kojic acid served as a positive control for this experiment.
Tyrosinase activity = (A sample/A control). Times.100% (absorbance as average)
The experimental data provided by the invention are that the figures 1 and 2 respectively show the inhibition rate of the acanthopanax ethanol extract on tyrosinase when L-dopa and L-tyrosine are used as substrates, and the finding shows that the acanthopanax ethanol extract has good tyrosinase inhibition effect on both L-dopa and L-tyrosine, and shows good correlation with the concentration of the acanthopanax ethanol extract, namely, the inhibition rate also shows a corresponding increasing trend along with the increase of the concentration of the acanthopanax ethanol extract.
The results are shown in FIG. 1, with L-dopa as the substrateThe inhibition rate of the acanthopanax ethanol extract is 34.51 +/-2.05% of the lowest value of the measured inhibition rate when the concentration of the acanthopanax ethanol extract is 1mg/ml, 90.47 +/-0.87% of the highest inhibition rate when the acanthopanax ethanol extract is 7.5mg/ml, and IC 50 The value was 1.77. + -. 0.96mg/ml. As shown in FIG. 2, the lowest inhibition rate of the ethanol extract of Acanthopanax senticosus was 40.86. + -. 0.03% at a concentration of 1mg/ml, the highest inhibition rate was 94.87. + -. 0.30% at 7.5mg/ml, and IC was 94.87. + -. 0.30% at L-tyrosine as the substrate 50 The value was 1.86. + -. 0.75mg/ml.
Therefore, the acanthopanax senticosus extract is an excellent tyrosinase natural inhibitor and can be developed into functional food with a whitening function, cosmetic or food preservative for preventing food browning.
2. Tyrosinase kinetics assay:
in order to judge whether the reaction of eleutheroside E and syringin on tyrosinase is reversible, eleutheroside E and syringin (20 μ M, 10 μ M, 5 μ M, 2.5 μ M, 1.25 μ M) with different concentrations are added into a reaction system, when the concentration of substrate L-dopa is unchanged (2.5 mM), the addition amount of tyrosinase (1000U, 500U, 250U, 125U) is changed, and the relationship between the activity of tyrosine catalysis substrate oxidation and the enzyme amount is determined. The v value in the figure indicates the change in absorbance per minute at a wavelength of 475nm and E represents the activity of the enzyme.
A series of intersecting straight lines can be obtained by a Lineweaver-Burk double reciprocal diagram method, and the Mie constant K of tyrosinase oxidized L-DOPA can be obtained by combining a quadratic diagram method m Maximum velocity V max And the suppression constant K i Isokinetic parameters, so that the inhibition type of the inhibitor on the tyrosinase activity can be judged, and the possible inhibition mechanism can be analyzed according to the inhibition type.
Specifically, 111. Mu.L of L-DOPA (0.125 mM, 0.25mM, 0.5mM, 1mM, 2 mM) solutions with different concentrations and 0.1M phosphate buffer (211. Mu.L) are mixed uniformly and incubated at 25 ℃, then 11. Mu.L of aqueous solution of tyrosinase (1000 units/mL) is added, 333. Mu.L of the total reaction system is mixed uniformly, absorbance value of a sample is detected at 475nm after incubation for 540s, and influence of inhibitors with different concentrations on enzymatic reaction rate of L-DOPA with different concentrations is measured. According to a Lineweaver-Burk double reciprocal mapping method, enzyme inhibition kinetic parameters are calculated, and the inhibition type and the inhibition mechanism of the inhibitor on the activity of tyrosinase are obtained.
To evaluate the type of inhibition of tyrosinase by eleutheroside E and syringin, lineweaver-Burk double reciprocal plot method was used. The Lineweaver-Burk equation for competitive inhibition kinetics analysis is given by:
wherein v is the reaction rate, K m Is the Michaelis constant, V, of tyrosinase max To maximize the reaction rate, K i Is the inhibition constant of the inhibitor, [ S ]]As substrate L-dopa concentration, [ I]Is the concentration of the inhibitor.
FIGS. 3A,4A the results are the inhibition rates of different concentrations of eleutheroside E and syringin (1.25. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M) on tyrosinase with L-dopa as substrate, both of which were found to exhibit a certain degree of dose dependence with IC's of both 50 The values were 11.13. + -. 0.50 and 15.41. + -. 0.22. Mu.M, respectively. Wherein the inhibition rate is 34.12 + -0.23% when the concentration of eleutheroside E is 1.25 μ M, and 51.53 + -0.08% when the concentration is 20 μ M. The syringin concentration is increased from 1.25 to 20 mu M, the inhibition rate is increased from 10.36 +/-2.15% to 55.03 +/-4.83%, and the concentration is dependent.
As can be seen from fig. 3B and 4B, the straight line passes through the coordinate axis far point. And along with the increase of the enzyme activity, the slope of the straight line is reduced along with the continuous increase of the concentrations of the eleutheroside E and the syringin, which proves that the inhibition effect of the eleutheroside E and the syringin on the tyrosinase is a reversible process.
As shown in FIGS. 3C and 4C, the concentration of immobilized enzyme in the activity-measuring system, the concentration of substrate L-dopa, and the influence of different concentrations of eleutheroside E and syringin (20. Mu.M, 10. Mu.M, 5. Mu.M, 2.5. Mu.M, 1.25. Mu.M) on the enzyme activity were measured. It can be seen that the intersection of the lines is on the y-axis, with K m V of both changes in value max The values were unchanged, indicating that eleutheroside E and syringin are both typical competitive inhibitions. It K i The values are 0.364 and 0.591, respectively, indicating that the binding affinity of eleutheroside E is superior to that of syringin.
It can also be concluded that syringin reacts faster than eleutheroside E at the inhibition rate of tyrosinase, but eleutheroside E is slightly better than syringin in terms of binding affinity with the enzyme. This conclusion can be studied later.
Application example 1
A preparation method of a strippable pasty facial mask gel containing an acanthopanax ethanol extract comprises the following steps:
heating sterile deoxygenated water 56.6% and polyvinyl alcohol 15% together to 90 deg.C, slowly stirring (60 rpm), adding glycerol 5% after polyvinyl alcohol is completely swelled, stirring well, and cooling to 50 deg.C; fully mixing 3% of acanthopanax ethanol extract, 0.4% of tween-80 and 20% of sterile deoxygenated water, slowly adding the cooled polyvinyl alcohol-glycerol aqueous solution, and then uniformly stirring; then degassing, and filtering by using a 220-mesh pipeline filter after degassing; sterilizing by gamma ray irradiation, and aseptically canning to obtain the final product.
Claims (10)
1. The application of the acanthopanax senticosus extract in preparing tyrosinase inhibitor products is characterized in that: the acanthopanax extract is obtained by the following method: mixing acanthopanax fruit powder with an ethanol water solution with a certain concentration, and carrying out ultrasonic treatment for a certain time to obtain an extracting solution; filtering, distilling and drying the obtained extract to obtain the acanthopanax ethanol extract.
2. Use according to claim 1, characterized in that: the volume fraction of ethanol in the ethanol aqueous solution is 30-80%; the mass-volume ratio of the acanthopanax fruit powder to the ethanol water solution is 1g.
3. Use according to claim 1, characterized in that: the ultrasonic treatment time is 180-250 min.
4. Use according to claim 1, characterized in that: the distillation steps are as follows: and (3) carrying out rotary evaporation on the filtered extracting solution at the temperature of 40-55 ℃, and removing ethanol in the extracting solution to obtain the acanthopanax ethanol crude extract.
5. Use according to claim 1 or 4, characterized in that: the drying step is as follows: vacuum drying or freeze drying the obtained radix Acanthopanacis Senticosi crude extract at low temperature to obtain radix Acanthopanacis Senticosi ethanol extract.
6. Use according to claim 1, characterized in that: the tyrosinase activity is significantly inhibited by the ethanol extract of Acanthopanax senticosus in a concentration-dependent manner when L-dopa and L-tyrosine are used as substrates, and the concentration (IC) of the ethanol extract of Acanthopanax senticosus is reduced by half 50 ) 1.77 plus or minus 0.96mg/ml and 1.86 plus or minus 0.75mg/ml respectively; the inhibition rates of the acanthopanax ethanol extract on tyrosinase under different concentrations (1 mg/ml, 2.5mg/ml, 5mg/ml and 7.5 mg/ml) by taking L-dopa and L-tyrosine as substrates are 34.51 +/-2.05 percent and 40.86 +/-0.03 percent, 55.31 +/-0.6 percent and 40.38 +/-2.84 percent, 84.33 +/-2.71 percent and 88.03 +/-0.78 percent, 90.47 +/-0.87 percent and 94.87 +/-0.30 percent respectively.
7. Use according to claim 1, characterized in that: the main active ingredients of the acanthopanax senticosus extract, namely eleutheroside E and syringin, are the inhibition rate IC of tyrosinase 50 The values are respectively 11.13 +/-0.50 mu M and 15.41 +/-0.22 mu M, and the inhibition rates are respectively 51.53 +/-0.01 percent and 55.03 +/-3.38 percent at the concentration of 20 mu M; and tyrosinase kinetic experiment results show that eleutheroside E and syringin are reversible competitive inhibition reactions to tyrosinase.
8. Application of radix Acanthopanacis Senticosi extract in preparing food antistaling agent product is provided.
9. The application of the acanthopanax extract in preparing whitening and brightening daily chemical washing products is characterized in that: the daily chemical cleaning and caring product is facial cleanser, facial cream, hand cream, lotion, and facial mask.
10. The application of the acanthopanax senticosus extract in preparing whitening and brightening functional food is characterized in that: the functional food can be solid powder, oral beverage, tablet candy, and food preservative film additive.
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