Quinoa flavone extracting solution as well as preparation method and application thereof

Abstract

The invention provides a quinoa flavone extracting solution as well as a preparation method and application thereof, and belongs to the technical field of natural plant extraction. The preparation method of the quinoa flavone extracting solution provided by the invention comprises the following steps: mixing quinoa, compound enzyme and water to obtain a mixed solution; performing ultrasonic treatment on the mixed solution to obtain a quinoa flavone extracting solution; the compound enzyme comprises cellulase and pectinase. According to the method, the flavone in the chenopodium quinoa willd is extracted by adopting ultrasonic-compound enzyme synergistically, so that the method has the characteristics of high yield, short extraction time, low energy consumption, large raw material treatment capacity, low possibility of damaging active substances, environmental friendliness, reduction of environmental pollution and the like, the comprehensive utilization of the chenopodium quinoa willd can be enhanced, and the additional value of the chenopodium quinoa willd can be improved.

Description

Technical Field

[0001] This invention belongs to the field of natural plant extraction technology, specifically relating to a quinoa flavonoid extract, its preparation method, and its application. Background Technology

[0002] Flavonoids are a broad category of compounds consisting of two benzene rings with phenolic hydroxyl groups linked by a central three-carbon atom. They are widely found in various plants and berries in nature, and their molecular structures vary. Examples include rutin, hesperidin, quercetin, green tea polyphenols, anthocyanins, and anthocyanic acids. Flavonoids can improve blood circulation and lower cholesterol. Flavonoids in *Swissonia indica* (a type of fruit) also contain a PAF anticoagulant factor, which significantly reduces the incidence and symptoms of cardiovascular and cerebrovascular diseases. Flavonoids can also inhibit the exudation of inflammatory enzymes, promote wound healing, and relieve pain, making them useful in cosmetics for various allergies and sensitivities. Furthermore, flavonoids are powerful antioxidants, effectively scavenging free radicals in the body and inhibiting tyrosinase activity, thus preventing cell degeneration and aging, and are used in anti-aging and whitening cosmetics.

[0003] Quinoa, also known as South American quinoa or quinoa grain, is a herbaceous crop belonging to the Chenopodiaceae family. Quinoa is rich in fiber, a variety of polyunsaturated fatty acids, and generally contains higher levels of various vitamins and minerals than common grains. The main flavonoids in quinoa are quercetin and kaempferol. Quinoa flavonoid extracts exhibit good antioxidant activity, with a higher DPPH scavenging capacity than vitamin C. Darker seeds also have higher flavonoid content and antioxidant activity. In recent years, quinoa has yielded many research results in the fields of nutrition and pharmacology, but research on extraction methods and processes for quinoa flavonoids is still relatively limited. Traditional methods for extracting plant flavonoids include hot water extraction, organic solvent extraction, organic solvent reflux extraction, Soxhlet extraction, and alkaline extraction with acid precipitation. These methods have drawbacks such as long extraction times, low extraction efficiency, and large solvent consumption. Furthermore, most methods use organic solvents for flavonoid extraction, which may pose solvent residue and toxicity risks. Therefore, it is essential to develop new processes for extracting flavonoid antioxidants from quinoa to enhance the comprehensive utilization of quinoa and increase its added value. Summary of the Invention

[0004] The purpose of this invention is to provide a quinoa flavonoid extract, its preparation method, and its applications. The preparation method of the quinoa flavonoid extract provided by this invention is simple to operate, has high extraction efficiency, and can enhance the comprehensive utilization of quinoa and increase its added value.

[0005] This invention provides a method for preparing quinoa flavonoid extract, comprising the following steps: mixing quinoa, a compound enzyme, and water to obtain a mixture; subjecting the mixture to ultrasonic treatment to obtain quinoa flavonoid extract; wherein the compound enzyme includes cellulase and pectinase.

[0006] As a preferred embodiment, the amount of the compound enzyme added is 0.4% to 0.6% of the quinoa mass; the mass-to-volume ratio of quinoa to water is 1 g: 80 to 120 mL.

[0007] As a preferred embodiment, the mass ratio of cellulase to pectinase is 0.6~1.4:1; the enzyme activity of cellulase is 50~200 U / mg, and the enzyme activity of pectinase is 30~100 U / mg.

[0008] As a preferred embodiment, the power of the ultrasonic treatment is 200~500 W; the temperature of the ultrasonic treatment is 40~60℃; and the time of the ultrasonic treatment is 30~150 min.

[0009] As a preferred embodiment, the pH of the mixture is 4.5 to 6.5.

[0010] As a preferred embodiment, the ultrasonic treatment further includes enzyme inactivation and separation; the enzyme inactivation temperature is 80~100℃; and the enzyme inactivation time is 10~20 min.

[0011] As a preferred embodiment, the separation includes: centrifuging and filtration of the enzyme-inactivated extract; the filter membrane used for filtration has a pore size of 0.22~0.8 μm.

[0012] The present invention also provides a quinoa flavonoid extract, which is obtained by the preparation method described above, and the yield of the quinoa flavonoid extract is 8.38~9.30 mg / g.

[0013] The present invention also provides the application of quinoa flavonoid extract obtained by the preparation method described above in cosmetics.

[0014] As a preferred embodiment, the cosmetics include at least one of the following: antioxidant, anti-aging, and whitening cosmetics.

[0015] Beneficial Effects: This invention provides a method for preparing quinoa flavonoid extract, comprising the following steps: mixing quinoa, a compound enzyme, and water to obtain a mixture; subjecting the mixture to ultrasonic treatment to obtain quinoa flavonoid extract; the compound enzyme includes cellulase and pectinase. The method for preparing quinoa flavonoid extract provided by this invention utilizes the high specificity of enzymes reacting with cell walls to disrupt cell walls and release flavonoids. The cavitation effect of ultrasound achieves localized high temperature and high pressure in the aqueous solution, and the mechanical disturbance effect of ultrasound further accelerates the diffusion of quinoa flavonoid extract from quinoa into the solvent, shortening the extraction time. This invention employs a synergistic extraction method of ultrasound and compound enzymes to extract flavonoids from quinoa, resulting in high yield, short extraction time, low energy consumption, large raw material processing capacity, preservation of active substances, and environmental friendliness, reducing environmental pollution. It enhances the comprehensive utilization of quinoa and increases its added value. The results of the examples show that the yield of flavonoids in quinoa in this invention is 8.38~9.30 mg / g; the flavonoid extract obtained from quinoa has a better DPPH free radical scavenging rate than VC and has the ability to inhibit tyrosinase activity. It can be used as a functional additive in food and cosmetics, and has the effects of scavenging free radicals, anti-aging and whitening. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The standard curve for rutin; Figure 2 A standard curve showing the DPPH free radical scavenging rate of VC standard; Figure 3 The scavenging rate of DPPH free radicals by the extract obtained in Example 4 is shown. Figure 4 The figure shows the inhibition of tyrosinase activity by the extract obtained in Example 4. Detailed Implementation

[0018] This invention provides a method for preparing quinoa flavonoid extract, comprising the following steps: mixing quinoa, a compound enzyme, and water to obtain a mixture; subjecting the mixture to ultrasonic treatment to obtain quinoa flavonoid extract; wherein the compound enzyme includes cellulase and pectinase. This invention utilizes the synergistic effect of ultrasound and a compound enzyme to extract flavonoids from quinoa, significantly improving the yield of flavonoids from quinoa. The extraction method of this invention features short extraction time, low energy consumption, large raw material processing capacity, minimal damage to active substances, and is environmentally friendly, reducing environmental pollution, thus possessing significant industrial application value.

[0019] Unless otherwise specified, all raw materials involved in this invention can be obtained through conventional commercial means.

[0020] This invention involves mixing quinoa, a compound enzyme, and water to obtain a mixture. In one embodiment, the quinoa includes quinoa seeds. In another embodiment, the quinoa includes quinoa flour; the particle size of the quinoa flour is 60-200 mesh. In specific embodiments of this invention, the particle size of the quinoa flour can be any value within the range of 60-200 mesh, for example, 60, 70, 80, 90, 100, 120, 130, 150, 160, 180, or 200 mesh. This invention has found that 60-mesh powder has a smaller particle size and a correspondingly larger specific surface area, which accelerates the diffusion of active ingredients such as flavonoids from the plant cells into the solvent, thus shortening the extraction time.

[0021] In one embodiment, the composite enzyme includes cellulase and pectinase. In another embodiment, the mass ratio of cellulase to pectinase is 0.6–1.4:1. In specific embodiments of the present invention, the mass ratio of cellulase to pectinase can be any value within the range of 0.6–1.4:1, such as 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, or 1.4:1. Adjusting the ratio of cellulase to pectinase within this range can effectively address fluctuations in cell wall composition from different raw materials, eliminating the need for overly complex formulation screening for specific raw materials and demonstrating excellent process adaptability. In one embodiment, the cellulase activity is 50-200 U / mg. In a specific embodiment of the present invention, the cellulase activity can be any value within the range of 50-200 U / mg, such as 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 U / mg. In another embodiment, the pectinase activity is 30-100 U / mg. In a specific embodiment of the present invention, the pectinase activity can be any value within the range of 30-100 U / mg, such as 30, 40, 50, 60, 70, 80, 90, or 100 U / mg. The present invention utilizes the high specificity of enzymes in reacting with cell walls to disrupt cell walls and release flavonoids, thereby increasing the yield of flavonoids in quinoa. Cellulase hydrolyzes the cellulose in quinoa, completely exposing the protoplasts (containing flavonoids) encased within the plant cell walls. Pectinase, on the other hand, hydrolyzes the pectin in the intercellular layer, disrupting cell adhesion and causing cells to separate, thereby releasing flavonoids from the tissue mass. This invention further improves the yield of flavonoids from quinoa by controlling the relative amounts of the complex enzymes.

[0022] In one embodiment, the amount of the compound enzyme added is 0.4% to 0.6% of the quinoa mass. In a specific embodiment of the present invention, the amount of the compound enzyme added can be any value within the range of 0.4% to 0.6% of the quinoa mass, such as 0.4%, 0.45%, 0.5%, 0.55%, or 0.6%. In one embodiment, the mass-to-volume ratio of quinoa to water is 1 g: 80 to 120 mL. In a specific embodiment of the present invention, the mass-to-volume ratio of quinoa to water can be any ratio within the range of 1 g: 80 to 120 mL, such as 1 g: 80 mL, 1 g: 90 mL, 1 g: 100 mL, 1 g: 110 mL, or 1 g: 120 mL. The cellulose and pectin content in the quinoa seed coat and cell wall is relatively stable. The enzyme addition amount of 0.4% to 0.6% is just enough to provide sufficient enzyme activity units to saturate the enzymatically biodegradable sites in the substrate. Within this enzyme dosage range, the enzymatic hydrolysis reaction is mild and controllable, achieving selective cell wall disruption rather than excessive hydrolysis. Both cellulase and pectinase are hydrolases, and their catalytic reactions require the participation of water molecules. This feed-to-liquid ratio range ensures that the system has sufficient water activity (Aw), allowing the enzyme molecules to maintain their native conformation and the hydrated state of their catalytically active centers, thus avoiding loss of enzyme activity due to insufficient water.

[0023] After mixing quinoa, a compound enzyme, and water to obtain a mixture, the method further includes: adjusting the pH of the mixture; the adjustment includes using a sodium carbonate / citric acid solution; as one embodiment, the pH of the mixture is 4.5~6.5; in a specific embodiment of the present invention, the pH of the mixture can be any value from 4.5 to 6.5, for example 4.5, 4.6, 4.8, 5.0, 5.2, 5.4, 5.5, 5.6, 5.8, 6.0, 6.2, 6.3, 6.4, or 6.5. For cellulase, the effective pH range of most commercial cellulases is 4.0~6.5, and the optimal pH range is between 4.5~5.5, while the optimal pH of pectinase is usually around 5.0. Under the condition of pH 5.5, not only is the enzyme activity high, but its spatial conformation is also more stable, which is conducive to continuous catalytic reaction.

[0024] After obtaining the mixture of quinoa powder and compound enzyme, the present invention performs ultrasonic treatment. As one embodiment, the power of the ultrasonic treatment is 200-500 W; in a specific embodiment of the present invention, the power of the ultrasonic treatment can be any value within the range of 200-500 W, for example, 200, 300, 400, or 500 W. As one embodiment, the temperature of the ultrasonic treatment is 40-60°C; in a specific embodiment of the present invention, the temperature of the ultrasonic treatment can be any value within the range of 40-60°C, for example, 40, 42, 45, 47, 50, 52, 55, 58, or 60°C. As one embodiment, the time of the ultrasonic treatment is 30-150 min; in a specific embodiment of the present invention, the time of the ultrasonic treatment can be any value within the range of 30-150 min, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 min. As one embodiment, the frequency of the ultrasound is 40 kHz. This invention utilizes the cavitation effect of ultrasound to achieve localized high temperature and high pressure in an aqueous solution. Combined with the mechanical disturbance effect of ultrasound, this accelerates the diffusion of quinoa flavonoid extract from quinoa flour into the solvent, shortening the extraction time and synergistically improving flavonoid yield with a compound enzyme. By controlling the conditions of ultrasonic treatment, this invention further improves the yield and extraction efficiency of flavonoids from quinoa.

[0025] After ultrasonic treatment, the present invention preferably performs enzyme inactivation on the ultrasonically treated extract to obtain an enzyme-inactivated extract. As one embodiment, the enzyme inactivation method includes water bath heating. As one embodiment, the enzyme inactivation temperature is 80-100℃. In a specific embodiment of the present invention, the enzyme inactivation temperature can be any value within the range of 80-100℃, such as 80, 85, 90, 95, or 100℃. As one embodiment, the enzyme inactivation time is 10-20 min. In a specific embodiment of the present invention, the enzyme inactivation time can be any value within the range of 10-20 min, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 min. During enzyme inactivation, excessively low temperature or excessively short inactivation time will lead to insufficient enzyme inactivation, resulting in over-enzymatic hydrolysis. The present invention, through appropriate enzyme inactivation temperature and time, can quickly and effectively terminate the enzyme reaction and avoid over-enzymatic hydrolysis.

[0026] The present invention obtains an enzyme-inactivated extract and then separates the enzyme-inactivated extract. As one embodiment, the separation includes centrifugation and filtration of the enzyme-inactivated extract. As one embodiment, the centrifugation conditions are: centrifugation speed 6000-10000 rpm, centrifugation temperature 0-4℃, and centrifugation time 5-10 min. In a specific embodiment of the present invention, the centrifugation speed can be any value within the range of 6000-10000 rpm, for example, 6000, 7000, 8000, 9000, or 10000 rpm; the centrifugation temperature can be any value within the range of 0-4℃, for example, 0, 1, 2, 3, or 4℃; and the centrifugation time can be any value within the range of 5-10 min, for example, 5, 6, 7, 8, 9, or 10 min. In a specific embodiment of the present invention, the centrifugation conditions are: centrifugation speed 10000 rpm, centrifugation temperature 4℃, and centrifugation time 10 min. The centrifugation method of the present invention can remove impurities and effectively retain flavonoid active ingredients.

[0027] In this invention, the supernatant is preferably filtered after centrifugation. As one embodiment, the pore size of the filter membrane used for filtration is 0.22~0.8 μm. In specific embodiments of this invention, the pore size of the filter membrane can be any value within the range of 0.22~0.8 μm, such as 0.22, 0.30, 0.45, 0.6, or 0.8 μm. If the pore size of the filter membrane is too small, the flavonoid extract may not be able to pass through the membrane, leading to the loss of the extract. If the pore size is too large, it may result in insufficient impurity removal, affecting the determination of flavonoid content. This invention limits the pore size of the filter membrane to facilitate the full permeation of flavonoid components and the removal of impurities, thereby improving the accuracy and stability of subsequent flavonoid content determination.

[0028] This invention provides a quinoa flavonoid extract, which is obtained by the preparation method described above, and the yield of the quinoa flavonoid extract is 8.38~9.30 mg / g.

[0029] This invention also provides the application of quinoa flavonoid extract obtained by the preparation method of the above-mentioned technical solution in cosmetics. In this invention, the cosmetics include at least one of the following: antioxidant, anti-aging, and whitening cosmetics; the cosmetics can be one or more of the following: creams, lotions, toners, serums, gels, masks, and facial cleansers.

[0030] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 Quinoa seeds were washed, dried, and then freeze-dried under vacuum until they were ground into quinoa powder with a particle size of 60 mesh. Quinoa powder was added to deionized water at a mass-to-volume ratio of 1 g:100 mL, along with 4 mg of a complex enzyme consisting of cellulase and pectinase. The cellulase activity was 200 U / mg, and the pectinase activity was 100 U / mg, with a mass ratio of 1:1. Sodium carbonate / citric acid solution was added to adjust the pH to 4.5. After stirring thoroughly, the mixture was subjected to ultrasonic treatment at a frequency of 40 kHz, a power of 400 W, a temperature of 50℃, and a time of 30 min to obtain an ultrasonically treated solution. The ultrasonically treated solution was then subjected to enzyme inactivation at 90℃ for 15 min to obtain an enzyme-inactivated extract. The enzyme-inactivated extract was centrifuged at 10000 r / min for 10 min at 4℃. The supernatant was filtered through a 0.45 mL alumina filter. The quinoa flavonoid extract was obtained by filtration through a µm filter membrane.

[0032] The flavonoid content in quinoa was determined spectrophotometrically. Quinoa powder:deionized water 1 g:100 mL was used, and 1% (v / v) HCl-ethanol solution was added. Extraction was carried out by reflux for 2 h at a constant temperature of 70℃. The crude quinoa flavonoid extract was first centrifuged using a high-speed centrifuge until the liquid was clear and transparent. The residue was collected, and an equal volume of the above extraction solvent was added for repeated reflux extraction. The extracts were collected and combined, and the solvent was distilled off under reduced pressure at 60℃. When no liquid residue remained in the rotary evaporator flask, the solids on the flask wall were dissolved with a small amount of 95% ethanol. The flask was then stored at 4℃ for later use.

[0033] Preparation of rutin standard solution: Accurately weigh 5 mg of rutin standard, dissolve it in 95% ethanol, and dilute to a 25 mL volumetric flask to obtain a 0.2 mg / mL standard solution. Determination of maximum absorption wavelength: Use 95% ethanol solution as a blank control. Select a scanning range of 190–900 nm to first obtain an absorption curve for the blank solution. After calibrating the blank of the UV spectrophotometer, scan the sample, still within the 190–900 nm range. This yields an absorption curve for the rutin standard solution. Select and record the maximum absorption wavelength on this curve. In this experiment, the maximum absorption peak of the rutin standard solution was determined to be 365 nm.

[0034] Preparation of the rutin standard curve: Accurately pipette 0.0, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of rutin standard solution into six 25 mL volumetric flasks. Add 1 mL of NaNO₂ solution to each flask, shake well, and let stand for 6 min. Then add 1 mL of Al(NO₃)₃ solution to each flask, shake well, and let stand for 6 min. Next, add 10 mL of NaOH solution to each flask. Finally, dilute to the mark with distilled water, shake well, and let stand for 15 min. Measure the absorbance at 365 nm using a UV spectrophotometer. Plot the standard curve with absorbance on the ordinate and concentration on the abscissa to obtain the standard regression equation. See [link to standard curve]. Figure 1 .

[0035] Using 95% ethanol as a blank control, the absorbance was measured at 365 nm, and the mass concentration of flavonoids in quinoa was obtained by using the rutin standard curve.

[0036] The yield of flavonoids in quinoa was calculated using the following formula: Flavonoid yield: W = C∙V∙N / 1000M; In the formula, W: flavonoid yield (mg / g); C: flavonoid concentration (mg / L); V: extract volume (mL); N: dilution factor; M: sample weight (g), referring to the mass of quinoa seeds when preparing quinoa flavonoid extract; The results showed that the yield of flavonoids in quinoa in this embodiment was 8.62 mg / g.

[0037] Example 2 The preparation method was the same as in Example 1, except that the pH was adjusted to 5.5 using sodium carbonate / citric acid solution, and the yield of flavonoids in quinoa was 9.01 mg / g.

[0038] Example 3 The preparation method was the same as in Example 2, except that the ultrasonic treatment time was 120 min, and the yield of flavonoids in quinoa was 9.24 mg / g.

[0039] Example 4 The preparation method was the same as in Example 3, except that the amount of compound enzyme added was 6 mg, and the yield of flavonoids in quinoa was 93.3 mg / g.

[0040] Comparative Example 1 The preparation method was the same as in Example 4, except that the complex enzyme was omitted, and the yield of flavonoids in the quinoa was 7.52 mg / g.

[0041] Comparative Example 2 The preparation method was the same as in Example 4, except that the ultrasonic treatment was omitted, and the yield of flavonoids in the quinoa was 7.88 mg / g.

[0042] Comparative Example 3 The preparation method was the same as in Example 4, except that the ultrasonic treatment was omitted and only an equal amount of cellulase was added to replace the complex enzyme. The yield of flavonoids in quinoa was 7.32 mg / g.

[0043] Comparative Example 4 The preparation method was the same as in Example 4, except that the ultrasonic treatment was omitted and only an equal amount of pectinase was added to replace the complex enzyme. The yield of flavonoids in quinoa was 6.91 mg / g.

[0044] Comparative Example 5 The preparation method was the same as in Example 4, except that an equal amount of cellulase was added to replace the complex enzyme, resulting in a flavonoid yield of 8.02 mg / g in quinoa.

[0045] Comparative Example 6 The preparation method was the same as in Example 4, except that an equal amount of pectinase was added to replace the complex enzyme, resulting in a flavonoid yield of 7.71 mg / g in quinoa.

[0046] As shown by the results of the above examples and comparative examples, the flavonoid yields in quinoa from Examples 1-4 were significantly higher than those from Comparative Examples 1-6. Ultrasonic treatment helps to break down plant cell walls; omitting ultrasonic treatment hinders flavonoid dissolution. The compound enzyme has a synergistic effect compared to a single enzyme, resulting in a higher flavonoid yield. Therefore, the ultrasonic-compound enzyme synergistic extraction method of the present invention can effectively improve the flavonoid yield in quinoa.

[0047] Test Example 1 uses the quinoa flavonoid extract prepared in Example 4 as a representative for performance evaluation.

[0048] 1. Evaluation of antioxidant properties The antioxidant capacity of the quinoa flavonoid extract in Example 4 was determined according to the DPPH free radical scavenging method, and the absorbance was measured at a wavelength of 519 nm using a UV spectrophotometer. A DPPH free radical scavenging rate scavenging curve was plotted using vitamin C as a standard to create a standard curve for vitamin C, and the antioxidant activity of the quinoa extract was compared and studied. Figure 2 A standard curve showing the DPPH free radical scavenging rate of VC standard.

[0049] The specific experimental steps for DPPH free radical scavenging rate are as follows: Prepare a solution with a concentration of 2×10 using anhydrous ethanol. -4Take 2 mL of the prepared DPPH solution (D0110, Shanghai Yika Biotechnology Co., Ltd.) at mol / L and put it into a test tube. Add 2 mL of deionized water and shake well to make a total of 4 mL. Measure the absorbance at 517 nm as A0. Take 2 mL of the prepared DPPH solution and put it into a test tube. Add 2 mL of the test sample reaction solution diluted 100 times. After standing in the dark at 37℃ for 30 min, measure the absorbance at 517 nm as A1.

[0050] The formula for calculating the clearance rate is: Clearance rate (%) = (A0 - A1) ÷ A0 × 100%; Note: A higher clearance rate indicates a stronger antioxidant capacity.

[0051] according to Figure 1 The half-inhibitory concentration (IC50) of VC for scavenging free radicals was calculated using a nonlinear curve fitting method. 50 The value was 0.1 mg / mL; the scavenging rate of the extract obtained in Example 4 above against DPPH free radicals was determined, and the results are shown in [the table below]. Figure 3 .Depend on Figure 3 It can be seen that the scavenging rate of free radicals increases with the increase of quinoa flavonoid concentration, and the calculated half-inhibitory concentration (IC50) of quinoa flavonoids for scavenging free radicals is higher. 50 The value was 0.006 mg / mL. Compared to the half-inhibitory concentration (IC50) of vitamin C for free radical scavenging, quinoa flavonoids showed an IC50 value of 0.006 mg / mL. 50 ICs smaller than VC 50 The lower the half-inhibitory concentration, the stronger the scavenging ability. Therefore, quinoa flavonoids have a stronger ability to scavenge free radicals than vitamin C.

[0052] 2. Evaluation of whitening effect The extract obtained in Example 4 was subjected to a tyrosinase activity inhibition experiment to determine the whitening ability of quinoa flavonoids. The specific operation is as follows: Accurately measure 0.5 mL of substrate L-DOPA (Levodopa, Adamas, catalog number: 68867B) into a test tube, then add 0.25 mL of the test sample at different concentrations (0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 μg / mL). Mix thoroughly using a vortex mixer and incubate at 30°C for 5 min. Afterward, add 0.25 mL of tyrosinase solution (Shanghai Titan Technology Co., Ltd., catalog number: 014344194) to the reaction system and immediately incubate at 30°C for another 10 min. Measure the absorbance at 475 nm after the reaction. An equal volume of distilled water was used as a blank control, an equal volume of buffer solution was used as a background control, and kojic acid was used as a positive control. IC50 50The value is defined as the concentration of sample required to reduce tyrosinase activity by 50%. Results are shown below. Figure 4 .from Figure 4 It can be seen that the quinoa flavonoid extract obtained by the ultrasonic-compound enzyme synergistic extraction method of the present invention has a good inhibitory effect on tyrosinase activity, that is, it has a good whitening effect, which further confirms that the preparation method of the quinoa flavonoid extract of the present invention has a good extraction effect.

[0053] The inhibition rate is calculated using the following formula: Inhibition rate (%) = [1 - (A - A0) / (C - C0)] × 100% A: Sample group; A0: Sample background group; C: Blank group; C0: Blank background group.

[0054] In summary, the quinoa flavonoid extract preparation method of the present invention achieves a quinoa flavonoid yield of up to 9.30 mg / g, exhibits a better DPPH free radical scavenging rate than VC, and demonstrates a good inhibition rate against tyrosinase. The quinoa flavonoid extract obtained by the present invention can be added to cosmetics as a functional ingredient, possessing free radical scavenging and whitening effects. Furthermore, the extraction method of the present invention has the advantages of short extraction time, high flavonoid yield, and strong antioxidant properties of flavonoids.

[0055] Application Example 1 The quinoa flavonoid extract prepared in Example 4 was used to prepare the essence, and the formula is shown in Table 1.

[0056] Table 1. Preparation Formula of Quinoa Whitening and Anti-Wrinkle Essence

[0057] Preparation process: 1. Weigh 85 g of deionized water and 0.1 g of carbomer (Lubrizol, product number: 0022) in advance; stir the deionized water at 600 rpm until it does not splash out of the beaker, then slowly pour the weighed carbomer into the deionized water and stir at 600 rpm for about 5 minutes.

[0058] 2. Weigh out all components except triethanolamine, reduce the stirring speed to 200 rpm, add the remaining components to the well-dispersed carbomer solution, and add water to make up the difference.

[0059] 3. After the system is evenly dispersed, add triethanolamine dropwise until the pH of the system is neutral and the system becomes significantly viscous.

[0060] The entire process can be carried out without heating, or with appropriate heating to dissolve some poorly soluble components. It should be noted that triethanolamine needs to be added dropwise after heating and cooling to 45°C; additionally, the transparent gel prepared by this process contains air bubbles, but these bubbles will disappear automatically after standing for a period of time.

[0061] Comparative Example 7 An essence, with the same components and preparation method as in Application Example 1, except that quinoa flavonoid extract is not added.

[0062] Test Example 2: Human Test of Whitening Efficacy Sample group: Quinoa whitening and anti-wrinkle essence (application example 1); Blank group: Blank serum (comparative ratio 6).

[0063] Thirty-six female volunteers aged 25-40 were selected. After cleansing their faces, the volunteers were stabilized in an environment with a temperature of 20-22℃ and a humidity of 40%-60% for 30 minutes. Following the product instructions, volunteers applied the sample group serum to one half of their face and the blank group serum to the other half. Non-invasive follow-up tests were performed on the test areas before use and at weeks 2 and 4. The tests included instrumental testing and image capture.

[0064] Test method: A Mexameter (Courage & Khazaka, Germany) probe for both red and melanin was used. The test area was the pigmentation on the left and right sides of the face. Three parallel tests were performed, and the average value was taken. The unit is au. The less melanin, the brighter the skin.

[0065] The changes in skin melanin content in the test volunteers before and after using the quinoa whitening and anti-wrinkle essence prepared in Application Example 1 of this invention and the blank essence prepared in Comparative Example 1 are shown in Table 2.

[0066] Table 2 Results of skin melanin parameter test

[0067] Note: p-value, week n vs. before use, paired t-test; significance, "ns" indicates no statistical difference, p≥0.05; "Indicates a significant difference, 0.01 ≤ p < 0.05;" "0.001≤p<0.01; " ", p<0.001.

[0068] The results in Table 2 show that the quinoa flavonoid extract prepared in this invention can significantly reduce the melanin content in the facial skin of the test volunteers, with a melanin reduction of about 33.59%, demonstrating excellent whitening effects.

[0069] Test Example 3: Human Testing of Anti-wrinkle Efficacy Sample group: Quinoa whitening and anti-wrinkle essence (application example 1); Blank group: Blank serum (comparative ratio 6).

[0070] Thirty-six female volunteers aged 40-50 years were selected. After cleansing their faces, the volunteers were stabilized in an environment with a temperature of 20-22℃ and a humidity of 40%-60% for 30 minutes. Following the product instructions, volunteers applied the sample group serum to one half of their face and the blank group serum to the other half. Non-invasive follow-up tests were performed on the test areas before use and at weeks 2 and 4. The tests included instrumental testing and image capture.

[0071] Test Method: A VC20 plus skin microscope and a SELS (Courage & Khazaka, Germany) active skin surface analysis system were used. The test area was the outer corner of the eye. Three images were taken in the measurement area using the Macro mode of the VC20 plus, and the data were analyzed. The mean of the three data points was used to evaluate the texture. Unit: au The changes in skin wrinkle parameters of the test volunteers before and after using the quinoa whitening and anti-wrinkle essence prepared in Example 1 and the blank essence prepared in Comparative Example 7 are shown in Table 3.

[0072] Table 3 Results of Skin Wrinkle Parameter Test

[0073] Note: p-value, week n vs. before use, paired t-test; significance, "ns" indicates no statistical difference, p≥0.05; "Indicates a significant difference, 0.01 ≤ p < 0.05;" "0.001≤p<0.01; " ", p<0.001.

[0074] The results in Table 3 show that the quinoa flavonoid extract prepared in this invention can significantly reduce the wrinkle area on the face of the test volunteers, with a wrinkle improvement rate of 34.18% after 4 weeks of use, demonstrating excellent anti-wrinkle effects.

[0075] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing quinoa flavonoid extract, characterized in that, The process includes the following steps: mixing quinoa, a compound enzyme, and water to obtain a mixture; subjecting the mixture to ultrasonic treatment to obtain a quinoa flavonoid extract; the compound enzyme includes cellulase and pectinase.

2. The preparation method according to claim 1, characterized in that, The amount of the compound enzyme added is 0.4% to 0.6% of the quinoa mass; the mass-to-volume ratio of quinoa to water is 1 g: 80 to 120 mL.

3. The preparation method according to claim 1 or 2, characterized in that, The mass ratio of cellulase to pectinase is 0.6~1.4:1; the enzyme activity of cellulase is 50~200 U / mg, and the enzyme activity of pectinase is 30~100 U / mg.

4. The preparation method according to claim 1, characterized in that, The power of the ultrasonic treatment is 200~500 W; the temperature of the ultrasonic treatment is 40~60℃; and the time of the ultrasonic treatment is 30~150 min.

5. The preparation method according to claim 1, characterized in that, The pH of the mixture is 4.5 to 6.

5.

6. The preparation method according to claim 1, characterized in that, The ultrasonic treatment further includes enzyme inactivation and separation; the enzyme inactivation temperature is 80~100℃; the enzyme inactivation time is 10~20 min.

7. The preparation method according to claim 6, characterized in that, The separation includes: centrifuging and filtration of the enzyme-inactivated extract; the filter membrane used for filtration has a pore size of 0.22~0.8 μm.

8. A quinoa flavonoid extract, characterized in that, The quinoa flavonoid extract is obtained by the preparation method according to any one of claims 1 to 8, and the yield of the quinoa flavonoid extract is 8.38 to 9.30 mg / g.

9. The application of the quinoa flavonoid extract obtained by the preparation method according to any one of claims 1 to 7 in cosmetics.

10. The application according to claim 9, characterized in that, The types of cosmetics include at least one of the following: antioxidant, anti-aging, and whitening cosmetics.

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