Magnolia bark polysaccharide, and preparation method and application thereof

Abstract

The application belongs to the field of chemical industry, and particularly relates to a magnolia officinalis polysaccharide, a preparation method and application thereof, and comprises the following steps: S1: ultrasonic-assisted enzyme extraction of magnolia officinalis polysaccharide: mixing magnolia officinalis powder with cellulase, dissolving in water, and then performing ultrasonic extraction to obtain a magnolia officinalis polysaccharide extract liquid; S2: impurity removal of the magnolia officinalis polysaccharide extract liquid: centrifuging the magnolia officinalis polysaccharide extract liquid obtained in step S1, taking a filtrate, and sequentially performing TCA method protein removal, dialysis, ethanol precipitation and freeze-drying to obtain the magnolia officinalis polysaccharide. Compared with the prior art, the application solves the problems of complex magnolia officinalis polysaccharide preparation, high cost, limited application of the magnolia officinalis polysaccharide and the like in the prior art, realizes the preparation of the magnolia officinalis polysaccharide with simple preparation method, reasonable process, high extraction efficiency and good purification effect, and proposes the application of the magnolia officinalis polysaccharide in hyaluronidase activity inhibition and ultraviolet resistance, thereby expanding the application of the magnolia officinalis polysaccharide in the field of chemical industry.

Description

Technical Field

[0001] This invention belongs to the field of chemical engineering, specifically relating to a polysaccharide of Magnolia biondii, its preparation method, and its application. Background Technology

[0002] Magnolia biondii, a plant belonging to the genus Magnolia in the family Magnoliaceae, has the effect of dispelling wind-cold and clearing nasal passages. Magnolia biondii flowers are particularly effective for nasal congestion, runny nose, and headache caused by wind-cold. Polysaccharides are commonly found in the cells of plants, animals, and microorganisms. They are high-molecular-weight compounds formed by ordinary monosaccharides linked by glycosidic bonds, and play a role in living organisms similar to that of proteins and nucleic acids.

[0003] Modern clinical and pharmacological studies have shown that Magnolia biondii possesses good anti-inflammatory, antihistamine, antihypertensive, and antibacterial effects. Magnolia biondii polysaccharides are mainly found in the cell walls of Magnolia biondii. Currently, the extraction methods for Magnolia biondii polysaccharides are complex, and the purity obtained is low. For example, in the study on the separation, purification, antibacterial, and antioxidant activities of Magnolia biondii polysaccharides, Yang Wen (Shaanxi Normal University) used hot water extraction, resulting in low purity. CN111777691A discloses a method for extracting Magnolia biondii polysaccharides, which involves continuous extraction under subcritical conditions, centrifugation to obtain crude polysaccharides, followed by purification to obtain the final Magnolia biondii polysaccharide. This method has high requirements for production conditions and is costly. Therefore, there is a need to propose a simple process and a high-quality method for preparing Magnolia biondii polysaccharides.

[0004] Furthermore, existing technologies only study the antioxidant and antibacterial activities of Magnolia biondii polysaccharides, which limits the application areas of Magnolia biondii. Summary of the Invention

[0005] The purpose of this invention is to provide a Magnolia biondii polysaccharide, its preparation method, and its applications to solve at least one of the aforementioned problems. This addresses the issues of complex preparation, high cost, and limited application of Magnolia biondii polysaccharide in existing technologies. The invention achieves a simple preparation method, reasonable process, high extraction efficiency, and good purification effect for Magnolia biondii polysaccharide. Furthermore, Magnolia biondii is a traditional Chinese medicinal herb, and its current applications are mainly concentrated in clinical and pharmacological research. This invention also proposes the application of Magnolia biondii polysaccharide in the inhibition of hyaluronidase activity and in anti-ultraviolet radiation, expanding the application of Magnolia biondii polysaccharide in the chemical industry, especially in the cosmetics field.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The first aspect of this invention discloses a method for preparing Magnolia biondii polysaccharide, comprising the following steps:

[0008] S1: Ultrasonic-assisted enzyme extraction of Magnolia biondii polysaccharide: Magnolia biondii powder is mixed with cellulase and dissolved in water, followed by ultrasonic extraction to obtain Magnolia biondii polysaccharide extract solution;

[0009] S2: Purification of Magnolia biondii polysaccharide extract: Centrifuge the Magnolia biondii polysaccharide extract obtained in step S1, and take the filtrate to remove protein by TCA method, dialysis, ethanol precipitation and freeze drying in sequence to obtain Magnolia biondii polysaccharide.

[0010] The prepared Magnolia biondii polysaccharide showed good results in inhibiting hyaluronidase activity and resisting ultraviolet radiation.

[0011] Preferably, in step S1, the ratio of Magnolia biondii powder to water is 1g:10-50mL; the particle size of the Magnolia biondii powder is not greater than 80 mesh; the specific activity of the cellulase is 10,000 U / g, and the amount used is 3wt% of the Magnolia biondii powder.

[0012] Preferably, in step S1, the ratio of Magnolia biondii powder to water is 1g:40mL.

[0013] Preferably, in step S1, the cellulase is dissolved in water to enzymatically hydrolyze the magnolia flower powder; the hydrolysis temperature is 30-70°C and the time is 1 hour.

[0014] Preferably, in step S1, the ultrasonic power of the ultrasonic extraction is 100-500W, the temperature is 50℃, and the time is 20-60min.

[0015] Preferably, in step S1, the ultrasonic power of the ultrasonic extraction is 300W and the time is 40min.

[0016] Preferably, in step S2, the centrifugation speed is 5000 r / min and the time is 15 min;

[0017] In the TCA method for protein removal, the volume fraction of trichloroacetic acid is 18%, the volume ratio of the magnolia polysaccharide extract to trichloroacetic acid is 1:1, the temperature is 4℃, and the time is 4h.

[0018] The dialysis was performed at a molecular weight cutoff of 3500D, a temperature of 4°C, and a time of 4 hours.

[0019] In the ethanol precipitation process, the volume ratio of Magnolia biondii polysaccharide extract to ethanol was 1:5, the temperature was 4℃, and the time was 12h.

[0020] The freeze-drying temperature is -50℃ and the time is 48 hours.

[0021] The second aspect of this invention discloses a magnolia polysaccharide, which is prepared by any of the methods described above.

[0022] The third aspect of this invention discloses the application of the above-mentioned Magnolia biondii polysaccharide in inhibiting hyaluronidase activity, wherein the concentration of Magnolia biondii polysaccharide used is 0.05-0.5 mg / mL.

[0023] The fourth aspect of this invention discloses the application of the above-mentioned Magnolia biondii polysaccharide in anti-ultraviolet radiation, wherein the concentration of Magnolia biondii polysaccharide used is 0.05-1 mg / mL, and it absorbs ultraviolet radiation with a wavelength of 280-400 nm.

[0024] The working principle of this invention is as follows:

[0025] The principle of ultrasound-assisted enzyme extraction of Magnolia biondii polysaccharides is as follows: enzymes can decompose plant tissues, thereby destroying cell structure. Then, the cavitation effect and stirring action of ultrasound can be used to accelerate the release and diffusion of polysaccharides in cells, thereby increasing the yield of polysaccharides.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The Magnolia biondii polysaccharide provided by this invention has a high yield of 15.8% and exhibits excellent hyaluronidase activity inhibition and UV protection, as well as antioxidant properties, making it a promising candidate for application in the cosmetics field. Furthermore, the preparation method of the Magnolia biondii polysaccharide is simple, the process is reasonable, the extraction efficiency is high, and the purification effect is good, providing scientific support for the further development and utilization of Magnolia biondii polysaccharide. Attached Figure Description

[0028] Figure 1 The graph shows the effect of the ratio of Magnolia biondii powder to water on the yield of Magnolia biondii polysaccharides.

[0029] Figure 2 Figure 1 shows the effect of enzymatic hydrolysis temperature on the yield of Magnolia biondii polysaccharide.

[0030] Figure 3 The graph shows the effect of ultrasonic power on the yield of Magnolia biondii polysaccharides.

[0031] Figure 4 The graph shows the effect of ultrasound time on the yield of Magnolia biondii polysaccharides.

[0032] Figure 5 The response surface plot shows the interaction between ultrasonic power and enzymatic hydrolysis temperature.

[0033] Figure 6 The response surface plot shows the interaction between the ratio of Magnolia biondii powder to water and the enzymatic hydrolysis temperature.

[0034] Figure 7 The response surface plot shows the interaction between the ratio of Magnolia biondii powder to water and the ultrasonic power.

[0035] Figure 8 DPPH free radical scavenging capacity diagram;

[0036] Figure 9 A graph showing the free radical scavenging capacity of ABTS;

[0037] Figure 10This is a graph showing the hydroxyl radical scavenging capacity.

[0038] Figure 11 This is a graph showing the inhibition rate of hyaluronidase activity.

[0039] Figure 12 This is a diagram showing the UV protection capability. Detailed Implementation

[0040] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0042] This invention provides a polysaccharide from Magnolia biondii, its preparation method, and its application.

[0043] The following embodiments of the present invention use Magnolia biondii flower buds.

[0044] In this invention, the preferred method for obtaining the magnolia flower powder includes: sequentially freeze-drying and pulverizing the magnolia flower raw material and sieving it to obtain the magnolia flower powder. In this invention, the freeze-drying temperature is preferably -50°C, and the freeze-drying time is preferably 48 hours. This invention does not specifically limit the pulverizing parameters, as long as magnolia flower powder with a particle size ≤ 80 mesh can be obtained.

[0045] The polysaccharides obtained by the extraction methods in the following embodiments of the present invention are total polysaccharides from Magnolia biondii.

[0046] The polysaccharide extraction rate determination method in the following embodiments of the present invention is as follows:

[0047] Transfer 400 μL of the sample to be tested (Magnolia biondii polysaccharide extract) into a 5 mL test tube, add 200 μL of 6 wt% phenol solution to the test tube, mix thoroughly, and then quickly add 1 mL of concentrated sulfuric acid. Let stand for 20 min, then heat in a boiling water bath for 15 min, remove and let stand for 1 h to cool to room temperature. Subsequently, measure the absorbance at a wavelength of 490 nm using an ELISA reader. According to the glucose standard curve, the yield of Magnolia biondii polysaccharide in the extract was obtained.

[0048] Plotting the glucose standard curve:

[0049] Using the absorbance of glucose concentration at 490 nm as the dependent variable and glucose concentration as the independent variable, a linear regression equation was obtained, and the linear relationship was good.

[0050] In this invention, the preparation method of the Magnolia biondii polysaccharide includes the following steps:

[0051] Magnolia biondii powder, cellulase, and water were mixed and subjected to ultrasonic extraction. The extraction process of Magnolia biondii polysaccharide was analyzed and optimized using response surface methodology to obtain a Magnolia biondii polysaccharide extract solution. The solution was centrifuged, filtered, impurity removed, and freeze-dried to obtain the Magnolia biondii polysaccharide.

[0052] The ratio of Magnolia biondii powder to water is 1g:(10-50)mL; the enzymatic hydrolysis temperature is 10-50℃.

[0053] The ultrasonic extraction power was 100–500 W; the ultrasonic extraction time was 20–60 min; the centrifugation speed was 5000 r / min and the time was 15 min; the TCA protein removal parameters were: trichloroacetic acid volume fraction of 18%, volume ratio of magnolia polysaccharide extract to trichloroacetic acid solution of 1:1, temperature of 4℃, and time of 4 h; the dialysis parameters were: molecular weight cutoff of dialysis bag of 3500D, temperature of 4℃, and time of 4 h; the ethanol precipitation parameters were: volume ratio of magnolia polysaccharide extract to ethanol solution of 1:5, temperature of 4℃, and time of 12 h; the freeze-drying parameters were: temperature of -50℃ and time of 48 h.

[0054] In this invention, the preferred ratio of the amount of Magnolia biondii powder to water is 1g:(10-50)mL, more preferably 1g:(30-50)mL, even more preferably 1g:(40-50)mL, and most preferably 40mL.

[0055] In this invention, the enzymatic hydrolysis temperature is preferably 30-70°C, more preferably 40-60°C, more preferably 50-60°C, and most preferably 50°C.

[0056] In this invention, the power of the ultrasonic extraction is preferably 100-500W, more preferably 200-400W, more preferably 300-400W, and most preferably 300W.

[0057] In this invention, the ultrasonic extraction time is preferably 20 to 60 minutes, and more preferably 40 minutes.

[0058] In this invention, the preferred parameters for ultrasonic extraction include: the ratio of Magnolia biondii powder to water is 1g:40mL; the enzymatic hydrolysis temperature is 50℃; the ultrasonic extraction power is 300W; and the ultrasonic extraction time is 40min.

[0059] Other extraction conditions during the extraction process were fixed as follows: cellulase addition of 3wt%, enzymatic hydrolysis time of 1h, and ultrasonic temperature of 50℃. After extraction, the extraction liquid was centrifuged by ultrasonic extraction, the upper liquid was filtered, and the filtrate was subjected to TCA protein removal, dialysis, ethanol precipitation, and freeze drying to obtain the Magnolia biondii polysaccharide.

[0060] In this invention, the centrifugation speed is preferably 5000 r / min, and the centrifugation time is preferably 15 min.

[0061] In this invention, the vacuum filter paper is a rapid qualitative filter paper.

[0062] In this invention, the preferred parameters for protein removal by the TCA method are: trichloroacetic acid volume fraction of 18%, volume ratio of magnolia polysaccharide extract to trichloroacetic acid solution of 1:1, temperature of 4°C, and time of 4 hours.

[0063] In this invention, the molecular weight cutoff of the dialysis bag used for dialysis is preferably 3500D, the temperature is preferably 4℃, and the time is preferably 4h.

[0064] In this invention, the preferred parameters for ethanol precipitation are: a volume ratio of Magnolia biondii polysaccharide extract to ethanol solution of 1:5, a temperature of 4°C, and a time of 12 hours.

[0065] In this invention, the freeze-drying parameters are preferably -50°C and 48h.

[0066] The present invention also provides a magnolia polysaccharide, wherein the parameters in the preparation method of the magnolia polysaccharide are the same as those in the above technical solution, and will not be repeated here.

[0067] The Magnolia biondii polysaccharide provided by this invention has excellent hyaluronidase activity inhibition and UV protection; at the same time, it also has good antioxidant properties.

[0068] Example 1

[0069] The purchased raw material, Magnolia biondii, is fresh flower buds of Magnolia denudata. After removing a small amount of leaves and impurities, it is freeze-dried at -50℃ for 48 hours. The raw material is then pulverized using a pulverizer and passed through an 80-mesh sieve to obtain Magnolia biondii powder, which is stored at -20℃.

[0070] Take Magnolia biondii powder, add 3wt% 10,000 U / g cellulase, mix and dissolve it evenly with deionized water at a certain material-to-liquid ratio, enzymatically hydrolyze it in a constant temperature water bath for 1 hour, remove it and place it in a boiling water bath at 100℃ for 10 minutes to inactivate the cellulase; then perform extraction in an ultrasonic extraction device at 50℃; after ultrasonication, centrifuge at 5000 r / min for 15 minutes, collect the upper liquid and filter it, and use the obtained filtrate as the material solution for Magnolia biondii polysaccharide extract.

[0071] The content of Magnolia biondii polysaccharide in the extract was determined according to the aforementioned method, and the polysaccharide content was calculated according to the glucose standard curve to obtain the polysaccharide extraction rate.

[0072] Single-factor experiment on the extraction of Magnolia biondii polysaccharides:

[0073] 1.1 Effect of solid-liquid ratio on the extraction rate of Magnolia biondii polysaccharide from solid solution

[0074] The experiment was conducted with 3 wt% cellulase, a hydrolysis temperature of 50℃, a hydrolysis time of 1 h, and ultrasonication at 50℃, 300 W, and for 40 min. 1 g of Magnolia biondii powder and deionized water were mixed thoroughly at mass / volume ratios (solid-liquid ratios) of 1:10, 1:20, 1:30, 1:40, and 1:50 (g:mL). The absorbance values ​​of the five groups of Magnolia biondii polysaccharide extracts were measured, and curves were plotted to study the effect of different solid-liquid ratios on the extraction rate of Magnolia biondii polysaccharides. Each experiment was repeated three times. The results are as follows: Figure 1 As shown.

[0075] from Figure 1 It can be seen that different material-to-liquid ratios result in different extraction rates of Magnolia biondii polysaccharides. The highest yield was achieved at a material-to-liquid ratio of 1:40 (g:mL), with an extraction rate of 14.05%. As the material-to-liquid ratio increases, the concentration gradient from the cell to the solvent increases, leading to a continuous increase in polysaccharide extraction rate. However, once the material-to-liquid ratio reaches 1:40 (g:mL), further increasing the solvent volume actually decreases the extraction rate of Magnolia biondii polysaccharides. This may be due to the precipitation of other substances in the Magnolia biondii powder, such as polyphenols and flavonoids, which hinder the extraction and separation of polysaccharides, thus reducing the extraction rate. Therefore, a material-to-liquid ratio of 1:40 (g:mL) is the optimal extraction ratio for Magnolia biondii polysaccharides.

[0076] 1.2 Effect of enzymatic hydrolysis temperature on the extraction rate of Magnolia biondii polysaccharide from the extract solution

[0077] The experimental material-to-liquid ratio was set at 1:40 g / mL, the amount of cellulase added was 3 wt%, the enzymatic hydrolysis time was 1 h, and the ultrasonic temperature was 50℃, the power was 300 W, and the time was 40 min. The enzymatic hydrolysis temperature was set at 30℃, 40℃, 50℃, 60℃, and 70℃. The absorbance values ​​of the five groups of Magnolia biondii polysaccharide extracts were measured, and curves were plotted to study the effect of different material-to-liquid ratios on the extraction rate of Magnolia biondii polysaccharide. Each experiment was repeated three times. The results are as follows: Figure 2 As shown.

[0078] from Figure 2It can be seen that the highest yield of Magnolia biondii polysaccharide in the extract was 14.48% at an enzymatic hydrolysis temperature of 50℃. As the hydrolysis temperature increased, the yield of Magnolia biondii polysaccharide gradually decreased, possibly because higher temperatures damaged the structure of both the polysaccharide and the cellulase, leading to a lower extraction rate. Therefore, 50℃ is the optimal enzymatic hydrolysis temperature for Magnolia biondii polysaccharide.

[0079] 1.3 Effect of ultrasonic power on the extraction rate of Magnolia biondii polysaccharide from the extract solution

[0080] The experimental material-to-liquid ratio was set at 1:40 g / mL, the amount of cellulase added was 3 wt%, the enzymatic hydrolysis temperature was 50℃, the enzymatic hydrolysis time was 1 h, and the ultrasonic temperature was 50℃ for 40 min. The ultrasonic power was set to 100 W, 200 W, 300 W, 400 W, and 500 W, and the absorbance values ​​of the five groups of Magnolia biondii polysaccharide extracts were measured. Curve graphs were plotted to study the effect of different ultrasonic powers on the extraction rate of Magnolia biondii polysaccharide. Each experiment was repeated three times. The results are as follows: Figure 3 As shown.

[0081] Depend on Figure 3 It was found that as the ultrasonic power increased from 100W to 300W, the yield of Magnolia biondii polysaccharide in the extract gradually increased, reaching a maximum of 14.04% at 300W. However, with further increases in ultrasonic power, the yield of Magnolia biondii polysaccharide decreased. This is likely because the cavitation effect and mechanical action generated by ultrasound in the extraction solution effectively broke down the cell walls of the Magnolia biondii powder, causing the polysaccharide components to be in a free state and dissolve into the extraction solvent. Therefore, a certain power can promote polysaccharide extraction; excessively high power causes the destructive effect of ultrasound on Magnolia biondii polysaccharide to outweigh its dissolution effect, thus affecting the polysaccharide extraction rate. Therefore, 300W is the optimal ultrasonic extraction power for Magnolia biondii polysaccharide.

[0082] 1.4 Effect of ultrasonic time on the extraction rate of Magnolia biondii polysaccharide from the extract solution

[0083] The experimental material-to-liquid ratio was set at 1:40 (g / mL), the amount of cellulase added was 3wt%, the enzymatic hydrolysis temperature was 50℃, the enzymatic hydrolysis time was 1 h, and the ultrasonic temperature was 50℃ with a power of 300 W. Different ultrasonic times of 20, 30, 40, 50, and 60 min were set, and the absorbance values ​​of the five groups of Magnolia biondii polysaccharide extracts were measured. Curve graphs were plotted to study the effect of different ultrasonic times on the extraction rate of Magnolia biondii polysaccharide. Each experiment was repeated three times. The results are as follows: Figure 4 As shown.

[0084] from Figure 4It can be seen that the yield of Magnolia biondii polysaccharide in the extract reaches its maximum at 40 min, reaching 15.29%, with increasing ultrasonic time. Afterward, the yield decreases with further increasing ultrasonic time. This is likely because most of the polysaccharides have already dissolved by 40 min; continuing the ultrasonic time might lead to polysaccharide degradation or the dissolution of other impurities. Overall, the ultrasonic time has a minimal impact on the extracted Magnolia biondii polysaccharide content. Therefore, 40 min is determined to be the optimal ultrasonic extraction time for Magnolia biondii polysaccharides.

[0085] Example 2

[0086] Response surface methodology for optimizing the extraction process of Magnolia biondii polysaccharides

[0087] Response surface methodology was performed using the results of the single-factor experiment in Example 1.

[0088] 1. Selection of factor levels in response surface methodology

[0089] Based on the central composite design principle, and considering the results of the single-factor influence experiments, three conditions that have a significant impact on the yield of Magnolia biondii polysaccharide were selected as influencing factors: material-liquid ratio (A), ultrasonic extraction power (B), and enzymatic hydrolysis temperature (C). Based on the single-factor experiments, a three-factor, three-level response surface methodology was used for the experimental design. The analysis factors and level design are shown in Table 1.

[0090] Table 1. Response surface methodology factors and levels for extraction process optimization

[0091]

[0092] 2. Response surface methodology results

[0093] The results of the analysis of variance for Magnolia biondii polysaccharide were obtained using the central composite model and are shown in Table 2.

[0094] Table 2 Results of Optimized Response Surface Experiment

[0095]

[0096] From Table 2, Figures 5-7 It can be seen that the order of influence of the experimental factors on the yield of Magnolia biondii polysaccharide is B>A>C, i.e., ultrasonic power>material-liquid ratio>enzymatic hydrolysis temperature. The formula for calculating the yield (Y) of Magnolia biondii polysaccharide is: Y=+16.26-0.2439A+0.7450B-0.1762C-0.2908AB-0.3570AC+0.5944BC-2.41A 2 -2.60B 2 -2.32C 2 .

[0097] Response surface methodology analysis revealed the following optimal extraction parameters for Magnolia biondii polysaccharides: ultrasonic power 318.11 W, solid-liquid ratio 1:39.24 g / mL, and enzymatic hydrolysis temperature 49.89℃. Under these optimal extraction conditions, the theoretical yield of Magnolia biondii polysaccharides is 15.78%. Considering the need for simplicity and convenience in practical operation, and taking into account various factors that may affect the process, the optimal extraction parameters are: ultrasonic power 300 W, solid-liquid ratio 1:40 g / mL, and enzymatic hydrolysis temperature 50℃.

[0098] Example 3

[0099] Preparation of Magnolia biondii polysaccharide:

[0100] Magnolia biondii polysaccharide was prepared using the extraction conditions obtained from response surface methodology optimization in Example 2. Fresh Magnolia biondii leaves and impurities were removed and freeze-dried at -50°C for 48 hours. The raw material was then pulverized using a pulverizer and passed through an 80-mesh sieve to obtain Magnolia biondii powder, which was stored at -20°C. The Magnolia biondii powder was then mixed with 3 wt% 10,000 U / g cellulase and dissolved in deionized water at a ratio of 1:40 (g:mL). The mixture was then enzymatically hydrolyzed in a constant temperature water bath at 50°C for 1 hour. After hydrolysis, the mixture was placed in a boiling water bath at 100°C for 10 minutes to inactivate the cellulase. Subsequently, water bath extraction was performed using an ultrasonic extraction device. The ultrasonic temperature was 50°C, the ultrasonic power was 300W, and the ultrasonic time was 40 minutes. After ultrasonication, the mixture was centrifuged at 5000 r / min for 15 minutes, and the supernatant was collected and filtered to obtain the Magnolia biondii polysaccharide extract.

[0101] The yield of Magnolia biondii polysaccharide in the extract was determined to be 15.8% using the aforementioned method.

[0102] Example 4

[0103] Purification of Magnolia biondii polysaccharide extract solution:

[0104] The Magnolia biondii polysaccharide extract obtained in Example 3 was concentrated and purified by: taking a certain volume of the Magnolia biondii polysaccharide extract and adding a 18% trichloroacetic acid solution at a volume ratio of 1:1, shaking thoroughly, and allowing the solution to stand at 4°C for 4 hours after mixing; after standing, centrifuging at 5000 r / min for 15 min to remove protein precipitate, repeating the operation twice with a 18% trichloroacetic acid solution; and slowly adding a 20% NaOH solution to the polysaccharide extract. Stir well and adjust the pH to neutral; select a 3500D dialysis bag, check for leaks, fill it with the above treatment solution, and dialyze at 4℃ for 4 hours, then continue dialysis for 2 hours and replace the entire dialysis solution; add about 5 times the volume of anhydrous ethanol to the dialyzed solution, stir well, and let it stand at 4℃ for 12 hours; centrifuge the solution after ethanol precipitation at 5000 r / min for 15 min, take the precipitate, wash it three times, add an appropriate amount of deionized water, and rotary evaporate the solution to about 5 mL. Freeze-dry it at -50℃ for 48 hours to obtain Magnolia biondii polysaccharide.

[0105] Preparation of Magnolia biondii polysaccharide solutions: Magnolia biondii polysaccharide was dissolved in deionized water to obtain Magnolia biondii polysaccharide solutions with concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 and 1 mg / mL.

[0106] Example 5

[0107] Antioxidant activity assay:

[0108] 1. DPPH free radical scavenging rate determination:

[0109] a. Weigh 2 mg of DPPH powder and dissolve it in anhydrous ethanol. After sonicating for 5 min, observe that the DPPH is completely dissolved, and then add anhydrous ethanol to make up to 25 mL. Take 2 mL of this solution and measure its absorbance at 517 nm using an ELISA reader. Adjust the absorbance of the DPPH-ethanol solution to between 1.2 and 1.3, and store it in the dark.

[0110] b. According to Table 3, transfer different concentrations of Magnolia biondii polysaccharide solution, the same concentration of vitamin C solution, and other reagents into test tubes. Vitamin C is used as a positive control. After adding the reagents, mix well and react in the dark for 30 minutes. Measure the absorbance at 517 nm and calculate the DPPH free radical scavenging ability of the Magnolia biondii polysaccharide solution. Each group has three parallel experiments.

[0111] The DPPH free radical scavenging capacity of Magnolia biondii polysaccharide solution is calculated by the following formula:

[0112]

[0113] In the formula: A1 is the absorbance of the DPPH solution containing the sample; A2 is the absorbance of the deionized water containing the sample; A3 is the absorbance of the deionized water containing DPPH.

[0114] Table 3 DPPH free radical scavenging experiment

[0115]

[0116] The results are as follows Figure 8 As shown. From Figure 8 It can be seen that when the concentration of Magnolia biondii polysaccharide solution is 0.05–0.5 mg / mL, the scavenging rate of DPPH free radicals gradually increases with increasing concentration. However, when a certain mass concentration (0.4 mg / mL) is reached, the scavenging rate tends to level off with increasing concentration. The highest DPPH free radical scavenging efficiency, reaching 80.72%, is achieved when the concentration of Magnolia biondii polysaccharide solution is equal to 0.5 mg / mL. Compared with the positive control vitamin C, the DPPH free radical scavenging ability of Magnolia biondii polysaccharide solution is slightly weaker, but it exhibits a certain antioxidant effect.

[0117] 2. ABTS free radical scavenging rate determination:

[0118] a. Preparation of ABTS free radical working solution: Mix 5 mL of 7 mmol / L ABTS solution with 5 mL of 245 mmol / L potassium persulfate solution until homogeneous. Let stand in the dark for 12 h, then transfer 5 mL to a 50 mL centrifuge tube. Dilute with deionized water and measure the absorbance at 734 nm using an ELISA reader. Adjust the absorbance of the ABTS free radical working solution to 0.7 ± 0.02.

[0119] b. Accurately weigh different concentrations of Magnolia biondii polysaccharide solution, the same concentration of vitamin C solution, and other reagents into test tubes according to Table 4. Vitamin C is used as a positive control. After adding the reagents, mix well and let stand. React at room temperature in the dark for 6 minutes. Measure the absorbance at 734 nm and calculate the ABTS free radical scavenging ability of the Magnolia biondii polysaccharide solution. Each group should have three parallel experiments.

[0120] The scavenging ability of Magnolia biondii polysaccharide solution against ABTS free radicals was calculated using the following formula:

[0121]

[0122] In the formula: A4 is the absorbance of the ABTS solution containing the sample; A5 is the absorbance of the deionized water containing the sample; A6 is the absorbance of the deionized water containing ABTS.

[0123] Table 4 ABTS Free Radical Scavenging Experiment

[0124]

[0125]

[0126] The results are as follows Figure 9 As shown. From Figure 9 It can be seen that when the concentration of Magnolia biondii polysaccharide solution is 0.05–0.5 mg / mL, the scavenging rate of ABTS free radicals gradually increases with increasing concentration, reaching a maximum of 99.23% at a concentration of 0.5 mg / mL. Compared with the positive control vitamin C, the 0.5 mg / mL Magnolia biondii polysaccharide solution exhibits stronger ABTS free radical scavenging ability and demonstrates excellent antioxidant effect.

[0127] 3. Hydroxyl radical scavenging rate determination:

[0128] According to Table 5, accurately weigh different concentrations of Magnolia biondii polysaccharide solution and the same concentration of Vitamin C solution into test tubes. Add 2 mmol / L FeSO4 solution, 6 mmol / L salicylic acid solution and deionized water into the test tubes in sequence, shake well, add 1 mmol / L H2O2 to start the process, and incubate at 37℃ for 30 min. After taking it out, measure the absorbance at 510 nm using an ELISA reader to calculate the hydroxyl radical scavenging ability of Magnolia biondii polysaccharide solution. Vitamin C is a positive control. Three parallel experiments were performed for each group.

[0129] The scavenging ability of Magnolia biondii polysaccharide solution against hydroxyl radicals is calculated using the following formula:

[0130]

[0131] In the formula: A7 is the absorbance of the sample solution; A8 is the absorbance of deionized water instead of H2O2 solution; A9 is the absorbance of the blank control;

[0132] Table 5 Hydroxyl radical scavenging experiment

[0133]

[0134] The results are as follows Figure 10 As shown. From Figure 10 It can be seen that when the concentration of Magnolia biondii polysaccharide solution is 0.05–0.5 mg / mL, the scavenging rate of hydroxyl radicals gradually increases with increasing concentration, reaching a maximum of 45.42% at 0.5 mg / mL. Vitamin C has a strong scavenging ability against hydroxyl radicals; when the concentration of Vitamin C is 0.1 mg / mL, the scavenging effect has reached approximately 60%. Compared with the positive control group (Vc), the scavenging effect of Magnolia biondii polysaccharide solution on hydroxyl radicals is slightly weaker, but it still has a certain antioxidant effect.

[0135] Example 6

[0136] Hyaluronidase activity inhibition rate determination:

[0137] a. Prepare 0.1M glacial acetic acid solution, 0.1M sodium acetate solution, 0.5M sodium carbonate solution, 10M hydrochloric acid solution, 0.1M acetate buffer solution, 4000U / mL hyaluronidase solution, 12.5mM calcium chloride solution, sodium hyaluronate solution, 0.4M sodium hydroxide solution, acetylacetone solution, and p-dimethylaminobenzaldehyde solution separately for later use.

[0138] b. Add reagents according to Table 6. The sample group consists of Magnolia biondii polysaccharide solution and dipotassium glycyrrhizate solution (DPG). Dipotassium glycyrrhizate solution is used as a positive control for hyaluronidase activity inhibition. After the reaction, the absorbance is measured at 585 nm using an ELISA reader. The hyaluronidase activity inhibition capacity of Magnolia biondii polysaccharide solution is calculated. Each group has three parallel experiments.

[0139] The inhibition rate of hyaluronidase by Magnolia biondii polysaccharide reflects the strength of its anti-inflammatory activity; the higher the inhibition rate, the stronger the anti-inflammatory activity. The inhibition rate of hyaluronidase activity of Magnolia biondii polysaccharide solution is calculated according to the following formula:

[0140]

[0141] In the formula: T is the absorbance of the sample group, T0 is the absorbance of the sample control group, C is the absorbance of the negative control group, and C0 is the absorbance of the blank control group.

[0142] Table 6 Hyaluronidase Activity Inhibition Experiment

[0143]

[0144]

[0145] The results are as follows Figure 11 As shown. From Figure 11 It can be seen that when the concentration of Magnolia biondii polysaccharide solution is 0.05–0.5 mg / mL, its inhibitory effect on hyaluronidase activity gradually increases with increasing concentration. At a concentration of 0.5 mg / mL, the inhibition rate of hyaluronidase activity by the Magnolia biondii polysaccharide solution reaches 54.03%. This indicates that the Magnolia biondii polysaccharide solution has a good inhibitory effect on hyaluronidase.

[0146] Example 7

[0147] Determination of UV protection ability:

[0148] Based on spectroscopic methods, the ultraviolet transmittance of the sample within a specific wavelength range was measured, and the UV resistance of Magnolia biondii polysaccharide was evaluated using an enzyme-linked immunosorbent assay (ELISA) reader.

[0149] This invention measures the ultraviolet transmittance of different concentrations of Magnolia biondii polysaccharide solutions in the UVB region (280–320 nm) and UVA region (320–400 nm), i.e., the wavelength range of 280–400 nm. The ultraviolet absorbance is calculated using the following formula to evaluate the sun protection performance of Magnolia biondii polysaccharide.

[0150] UV absorptivity = (1 - transmittance) × 100%

[0151] The results are as follows Figure 12 As shown. From Figure 12 It can be seen that when the concentration of the magnolia polysaccharide solution is 0.05–1 mg / mL, its absorption rate in the 280–400 nm wavelength range gradually increases with increasing concentration, while the absorption rate at the same concentration decreases with increasing wavenumber. The anti-ultraviolet effect is best when the concentration of the magnolia polysaccharide solution is equal to 1 mg / mL, with an average absorption rate of 100% in the UVB region and an average absorption rate greater than 86.44% in the UVA region, indicating a certain protective effect in the 280–400 nm wavelength range.

[0152] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. Use of a magnolia polysaccharide in a cosmetic for inhibiting hyaluronidase activity, characterized in that, The concentration of Magnolia biondii polysaccharide used is 0.05~0.5 mg / mL; The preparation method of the aforementioned Magnolia biondii polysaccharide includes the following steps: S1: Ultrasonic-assisted enzyme extraction of Magnolia biondii polysaccharide: Magnolia biondii powder is mixed with cellulase and dissolved in water, followed by ultrasonic extraction to obtain Magnolia biondii polysaccharide extract solution; S2: Purification of Magnolia biondii polysaccharide extract: Centrifuge the Magnolia biondii polysaccharide extract obtained in step S1, and take the filtrate to remove protein by TCA method, dialysis, ethanol precipitation and freeze drying in sequence to obtain Magnolia biondii polysaccharide; The ratio of Magnolia biondii powder to water is 1g:10~50mL; the particle size of Magnolia biondii powder is not greater than 80 mesh; the specific activity of cellulase is 10,000 U / g, and the amount used is 3wt% of Magnolia biondii powder. The cellulase is dissolved in water to enzymatically hydrolyze the Magnolia biondii powder; the enzymatic hydrolysis temperature is 30~70℃, and the time is 1h. The ultrasonic extraction process involves an ultrasonic power of 100-500W, a temperature of 50℃, and a time of 20-60 minutes. Magnolia biondii originates from the magnolia buds of Magnolia denudata.

2. Use according to claim 1, characterized in that, In step S1, the ratio of Magnolia biondii powder to water is 1g:40mL.

3. Use according to claim 1, characterized in that, In step S1, the ultrasonic power for ultrasonic extraction is 300W and the time is 40min.

4. Use according to claim 1, characterized in that, In step S2, the centrifugation speed is 5000 r / min and the time is 15 min; In the TCA method for protein removal, the volume fraction of trichloroacetic acid is 18%, the volume ratio of the magnolia polysaccharide extract to trichloroacetic acid is 1:1, the temperature is 4℃, and the time is 4h. The dialysis was performed at a molecular weight cutoff of 3500D, a temperature of 4°C, and a time of 4 hours. In the ethanol precipitation process, the volume ratio of Magnolia biondii polysaccharide extract to ethanol was 1:5, the temperature was 4℃, and the time was 12h. The freeze-drying temperature is -50℃ and the time is 48 hours.

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