Paulownia flower polysaccharide as well as preparation method and application thereof
The preparation of polysaccharide components from paulownia flowers using an ethanol gradient precipitation process solves the problem of the lack of natural tyrosinase inhibitors, provides cosmetic raw materials with significant tyrosinase inhibition and whitening effects, and realizes the high-value utilization of paulownia flower resources.
Patent Information
- Application Number
- CN202511634265.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-07
- Publication Date
- 2026-01-02
AI Technical Summary
Existing chemically synthesized tyrosinase inhibitors pose safety risks, limiting their widespread application in the field of skin-whitening cosmetics. Meanwhile, the tyrosinase inhibitory effects of natural tyrosinase inhibitors such as Amorpha fruticosa polysaccharide and Bletilla striata polysaccharide have not been reported, and there are no reports on the preparation and application of Paulownia tomentosa polysaccharide.
Polysaccharide components were prepared from paulownia flowers using an ethanol gradient precipitation process. Through steps such as sieving, defatting, hydrothermal extraction, fractionation precipitation, and dialysis, paulownia flower polysaccharides with significant tyrosinase inhibitory effects were obtained and compounded with other natural ingredients to form a product that inhibits tyrosinase.
The prepared paulownia flower polysaccharide significantly inhibits tyrosinase activity. It is a natural ingredient with a significant whitening effect and is non-irritating, providing a safe raw material for whitening cosmetics.
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Figure CN121248809A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of Paulownia flowers polysaccharide, in particular to a Paulownia flowers polysaccharide and a preparation method and application thereof. BACKGROUND
[0002] Tyrosinase is a copper-containing metalloproteinase, which widely exists in animals, plants and microorganisms, and plays a key role in the process of melanin biosynthesis and enzymatic browning. In the process of melanin synthesis, it first catalyzes the oxidation of L-tyrosine to L-dopa, showing the monophenolase activity of tyrosinase, and then further oxidizes L-dopa to dopaquinone, showing the diphenolase activity of tyrosinase, and dopaquinone is finally synthesized into melanin through a series of complex biochemical reactions. In the skin tissue, abnormal increase of tyrosinase activity will cause excessive deposition of melanin, leading to pigmentary disorders such as chloasma, freckle, age spot and the like, which seriously affects the skin appearance and health status. In the field of cosmetics, tyrosinase inhibitors as the core whitening ingredients, their market demand is continuously growing with the pursuit of natural whitening products by consumers. At present, there are various types of tyrosinase inhibitors on the market, such as kojic acid, arbutin, L-ascorbic acid, hydroquinone and ellagic acid, etc. However, the tyrosinase inhibitors synthesized by chemical synthesis often have safety problems, which may cause potential harm to human health and environment, limiting their wide application in the field of whitening cosmetics. Therefore, it has become a research hotspot to find safe and effective tyrosinase inhibitors from natural products.
[0003] Plant polysaccharides are a class of natural products with wide sources and high safety, which have various biological activities. It has been reported that Amorpha fruticosa L. flower polysaccharide and Bletilla striata polysaccharide have significant tyrosinase inhibitory effect. Paulownia flowers, as a common plant resource, contain various bioactive components, and its anti-inflammatory and antioxidant effects have been studied, however, whether it has tyrosinase inhibitory effect has not been reported. Ethanol fractionation and alcohol precipitation method is a commonly used method for separation and purification of polysaccharides, which principle is to use ethanol to destroy the hydration layer of polysaccharide molecules, so that its solubility is reduced and precipitates, and by controlling the ethanol concentration, the fractionation of polysaccharides with different molecular weights can be realized. However, there is no related report on the preparation of Paulownia flowers polysaccharide by ethanol fractionation and alcohol precipitation method, and there is no related report on the use of Paulownia flowers polysaccharide as a tyrosinase inhibitor. The present application provides a Paulownia flowers polysaccharide, a preparation method and application thereof in tyrosinase inhibition, which provides a new idea and way for the research and development of tyrosinase inhibitors, and realizes the high-value utilization of Paulownia flowers resources. SUMMARY
[0004] The present application aims to provide a Paulownia flowers polysaccharide and a preparation method and application thereof, which obtains polysaccharide components with significant tyrosinase inhibitory effect by ethanol gradient precipitation process.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing paulownia flower polysaccharide, comprising the following steps: (1) After crushing the paulownia flowers, sieve them and then degrease them to obtain a degreased sample; (2) The defatted sample was extracted using a hydrothermal method and concentrated to obtain a concentrated aqueous extract; (3) The water extract concentrate is fractionated and precipitated, the protein is removed, and then dialyzed and dried to obtain the paulownia flower polysaccharide.
[0006] Preferably, a 50-70 mesh sieve is used when sieving in step (1).
[0007] As a preferred embodiment, the degreasing treatment method in step (1) is as follows: the crushed paulownia flowers and petroleum ether are mixed at a ratio of 1g: 8~10mL, refluxed at 65℃ for 1.5~2.5 h, filtered and the filtrate is discarded, the residue is dried and mixed with 90~100% ethanol at a ratio of 1g: 8~10mL, refluxed at 75~85℃ for 1.5~2.5 h, filtered and the filtrate is discarded, and the residue is dried at 50~60℃ to obtain the degreased sample.
[0008] Preferably, the specific method for hydrothermal extraction in step (2) is as follows: ① Mix the defatted sample and water at a ratio of 1g:8~10mL, extract in a water bath at 85~95℃ for 1.5~2.5 h, and filter to obtain filtrate 1 and residue 1; ② Centrifuge filtrate 1 at 3500~4500 r / min for 8~12 min to obtain filtrate 2 and residue 2; ③ Extract residue 1 and residue 2 again following steps ① to ② to obtain filtrate 3 and residue 3; ④ Combine filtrate 2 and filtrate 3 to obtain paulownia flower aqueous extract.
[0009] As a preferred embodiment, the concentration method in step (2) is as follows: the aqueous extract of paulownia flowers is concentrated to 15-25% of its original volume at 60-70°C to obtain the aqueous concentrate; the concentration is performed using a rotary evaporator.
[0010] Preferably, the method for graded precipitation in step (3) is as follows: A. Add ethanol to the water extract concentrate to make the ethanol concentration in the solution 35~45%, let it stand at 2~6℃ for 10~14h, and separate the supernatant 1 and 35~45% precipitate. B. Add ethanol to supernatant 1 to make the concentration of ethanol in the solution 55~65%, let it stand at 2~6℃ for 10~14h, and separate supernatant 2 and 55~65% precipitate. C. Add ethanol to supernatant 2 to make the concentration of ethanol in the solution 75~85%, let it stand at 2~6℃ for 10~14h, and separate supernatant 3 and 75~85% precipitate. The precipitate in step (3) is deproteinized using the Savage method; the dialysis bag with a molecular weight cutoff of 3000~4000 Da is used during the dialysis in step (3); and the drying in step (3) is performed by freeze drying.
[0011] The present invention also provides paulownia flower polysaccharide prepared by the aforementioned preparation method.
[0012] The present invention also provides the application of the aforementioned paulownia flower polysaccharide in the preparation of products that inhibit tyrosinase activity.
[0013] The present invention also provides a product for inhibiting tyrosinase activity, comprising the following raw materials in parts by weight: 1-1.6 parts of paulownia flower polysaccharide, 2-3 parts of aloe vera extract, 4.5-5.5 parts of snow lotus extract, 2.2-2.6 parts of saponin extract, 1.6-2.0 parts of scutellaria baicalensis extract, 0.2-0.5 parts of myricetin, 0.8-1.2 parts of quercetin, 0.3-0.5 parts of proanthocyanidins, and 0.5-0.7 parts of luteolin.
[0014] Preferably, the product also contains excipients acceptable in the cosmetics field.
[0015] This invention provides a paulownia flower polysaccharide, its preparation method, and its application. The preparation method of the paulownia flower polysaccharide includes the following steps: (1) pulverizing paulownia flowers and sieving them, followed by defatting to obtain a defatted sample; (2) extracting the defatted sample using a hydrothermal method, concentrating it to obtain a concentrated aqueous extract; (3) subjecting the concentrated aqueous extract to ethanol fractionation precipitation, removing proteins, dialyzing, and drying to obtain the paulownia flower polysaccharide. This invention prepares fractionated polysaccharides from paulownia flowers. In vitro experiments show that they have tyrosinase inhibitory effects, thus showing promise for developing whitening cosmetics. The fractionated polysaccharides obtained by separation are compounded to form a composition that can significantly inhibit tyrosinase. The composition is natural, widely available, has significant whitening effects, and is non-irritating. Attached Figure Description
[0016] Figure 1 FT-IR image of paulownia flower polysaccharide; Figure 2 Chromatograms of 15 monosaccharide standards; Figure 3 Chromatogram of the monosaccharide composition of 40% Paulownia flower polysaccharide; Figure 4 Chromatogram of the monosaccharide composition of 60% Paulownia flower polysaccharide; Figure 5Chromatogram of the monosaccharide composition of 80% Paulownia flower polysaccharide; Figure 6 Analysis of the tyrosinase inhibitory effect of paulownia flower polysaccharides; Figure 7 To analyze the reversibility of tyrosinase inhibition by paulownia flower polysaccharide; Figure 8 Lineweaver-Burk plot showing the inhibitory effect of paulownia flower polysaccharide on tyrosinase activity. Detailed Implementation
[0017] This invention provides a method for preparing paulownia flower polysaccharide, comprising the following steps: (1) After crushing the paulownia flowers, sieve them and then degrease them to obtain a degreased sample; (2) The defatted sample was extracted using a hydrothermal method and concentrated to obtain a concentrated aqueous extract; (3) The water extract concentrate is fractionated and precipitated, the protein is removed, and then dialyzed and dried to obtain the paulownia flower polysaccharide.
[0018] In this invention, a 50-70 mesh sieve is preferably used when sieving in step (1), and a 60 mesh sieve is more preferably used.
[0019] In this invention, the degreasing treatment method in step (1) is preferably as follows: pulverized paulownia flowers and petroleum ether are mixed at a ratio of 1g:8~10mL, refluxed at 65℃ for 1.5~2.5 h, filtered and the filtrate is discarded, the residue is dried and mixed with 90~100% ethanol at a ratio of 1g:8~10mL, refluxed at 75~85℃ for 1.5~2.5 h, filtered and the filtrate is discarded, and the residue is dried at 50~60℃ to obtain a degreased sample. More preferably, the method is as follows: pulverized paulownia flowers and petroleum ether are mixed at a ratio of 1g:10mL, refluxed at 65℃ for 2 h, filtered and the filtrate is discarded, the residue is dried and mixed with 95% ethanol at a ratio of 1g:10mL, refluxed at 80℃ for 2 h, filtered and the filtrate is discarded, and the residue is dried at 55℃ to obtain a degreased sample.
[0020] In this invention, the preferred method for hydrothermal extraction in step (2) is as follows: ① Mix the defatted sample and water at a ratio of 1g:8~10mL, extract in a water bath at 85~95℃ for 1.5~2.5 h, and filter to obtain filtrate 1 and residue 1; ② Centrifuge filtrate 1 at 3500~4500 r / min for 8~12 min to obtain filtrate 2 and residue 2; ③ Extract residue 1 and residue 2 again following steps ① to ② to obtain filtrate 3 and residue 3; ④ Combine filtrate 2 and filtrate 3 to obtain paulownia flower aqueous extract; Further preferred options are: ① Mix the defatted sample and water at a ratio of 1g:10mL, extract in a water bath at 90℃ for 2 hours, and filter to obtain filtrate 1 and residue 1; ② Centrifuge filtrate 1 at 4000 r / min for 10 min to obtain filtrate 2 and residue 2; ③ Extract residue 1 and residue 2 again following steps ① to ② to obtain filtrate 3 and residue 3; ④ Combine filtrate 2 and filtrate 3 to obtain paulownia flower aqueous extract.
[0021] In this invention, the preferred method for concentration in step (2) is to concentrate the aqueous extract of paulownia flowers to 15-25% of the original volume at 60-70°C to obtain an aqueous concentrate. More preferably, the method is to concentrate the aqueous extract of paulownia flowers to 20% of the original volume at 65°C to obtain an aqueous concentrate. A rotary evaporator is used for concentration.
[0022] In this invention, the fractional precipitation method described in step (3) is preferably: A. Add ethanol to the water extract concentrate to make the ethanol concentration in the solution 35~45%, let it stand at 2~6℃ for 10~14h, and separate the supernatant 1 and 35~45% precipitate. B. Add ethanol to supernatant 1 to make the concentration of ethanol in the solution 55~65%, let it stand at 2~6℃ for 10~14h, and separate supernatant 2 and 55~65% precipitate. C. Add ethanol to supernatant 2 to make the concentration of ethanol in the solution 75~85%, let it stand at 2~6℃ for 10~14h, and separate supernatant 3 and 75~85% precipitate. Further preferred options are: A. Ethanol was added to the concentrated aqueous extract to make the ethanol concentration in the solution 40%, and the solution was allowed to stand at 4°C for 12 hours. The supernatant 1 and 40% precipitate were then separated. B. Add ethanol to supernatant 1 to make the concentration of ethanol in the solution 60%, let it stand at 4℃ for 12h, and separate supernatant 2 and 60% precipitate. C. Add ethanol to the supernatant 2 to make the concentration of ethanol in the solution 80%, let it stand at 4℃ for 12h, and separate the supernatant 3 and 80% precipitate.
[0023] In step (3), the dialysis bag used during dialysis is preferably 3000~4000 Da, and more preferably 3500 Da; in step (3), the drying is preferably done by freeze drying.
[0024] The present invention also provides paulownia flower polysaccharide prepared by the aforementioned preparation method.
[0025] The present invention also provides the application of the aforementioned paulownia flower polysaccharide in the preparation of products that inhibit tyrosinase activity.
[0026] The present invention also provides a product for inhibiting tyrosinase activity, comprising the following raw materials in parts by weight: 1-1.6 parts of paulownia flower polysaccharide, 2-3 parts of aloe vera extract, 4.5-5.5 parts of snow lotus extract, 2.2-2.6 parts of saponin extract, 1.6-2.0 parts of scutellaria baicalensis extract, 0.2-0.5 parts of myricetin, 0.8-1.2 parts of quercetin, 0.3-0.5 parts of proanthocyanidins, and 0.5-0.7 parts of luteolin.
[0027] In this invention, the product preferably comprises the following raw materials in parts by weight: 1.3 parts of paulownia flower polysaccharide, 2.5 parts of aloe vera extract, 5 parts of snow lotus extract, 2.4 parts of saponin extract, 1.8 parts of scutellaria baicalensis extract, 0.3-0.4 parts of myricetin, 1 part of quercetin, 0.4 parts of proanthocyanidins, and 0.6 parts of luteolin.
[0028] In this invention, the product also contains excipients acceptable in the cosmetics field.
[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0030] Example 1: Preparation of Paulownia flower ethanol fractionated polysaccharide
[0031] The dried paulownia flowers were pulverized and passed through a 60-mesh sieve. The dried paulownia flower powder was mixed with petroleum ether at a ratio of 1:10 (w:v), and refluxed at 65°C for 2 hours to remove impurities and alcohol-soluble pigments. The mixture was then filtered, the filtrate was discarded, and the residue was collected and dried. The degreased paulownia flower powder was then mixed with 95% ethanol at a ratio of 1:10 (w:v), and refluxed at 80°C for 2 hours to remove small molecule impurities and alcohol-soluble pigments. The mixture was then filtered, the filtrate was discarded, and the residue was dried in an oven at 55°C and sealed for later use.
[0032] Take the defatted sample and add the specified amount of pure water at a material-to-liquid ratio of 1:10 (g / mL). Extract in a water bath at 90 ℃ for 2 h. After extraction, filter out the supernatant to obtain the filter residue and the preliminarily separated supernatant. Centrifuge the supernatant at 4000 r / min for 10 min to completely separate the filter residue and filtrate. Repeat the extraction on the filter residue once more. Combine the extracts obtained from the two extractions and concentrate them to 1 / 5 of the original volume using a rotary evaporator at 65 ℃ to obtain the concentrated water extract for later use.
[0033] Take the extract obtained in the previous step, add 95% ethanol to make the ethanol concentration in the solution 40%, let it stand at 4℃ for 12 hours, centrifuge to obtain precipitate one and supernatant one. The obtained precipitate one is 40% ethanol graded paulownia flower polysaccharide. Take the supernatant one obtained in the previous step, gradually add 95% ethanol to make the ethanol concentration in the solution 60%, let it stand at 4℃ for 12 hours, centrifuge to obtain precipitate two and supernatant two. The obtained precipitate two is 60% ethanol graded paulownia flower polysaccharide. Take the supernatant two obtained in the previous step, gradually add 95% ethanol to make the ethanol concentration in the solution 80%, let it stand at 4℃ for 12 hours, centrifuge to obtain precipitate three and supernatant three. The obtained precipitate three is 80% ethanol graded paulownia flower polysaccharide (40% ethanol graded paulownia flower polysaccharide, 60% ethanol graded paulownia flower polysaccharide and 80% ethanol graded paulownia flower polysaccharide are collectively referred to as paulownia flower ethanol graded polysaccharide).
[0034] 40%, 60%, and 80% ethanol fractionated paulownia flower polysaccharides were added to distilled water and completely dissolved. Proteins were removed using the Sevage method, and the polysaccharide solution was dialyzed (dialysis bag with a relative molecular weight cutoff of 3500 Da) for 48 hours with distilled water. After dialysis, the dialysate was freeze-dried to obtain 40%, 60%, and 80% purified polysaccharides, which were named PFP-40, PFP-60, and PFP-80, respectively. Figure 1 FT-IR image of paulownia flower polysaccharide Figures 2-5 It is the monosaccharide composition of paulownia flower polysaccharide.
[0035] Depend on Figure 1 As shown, the peak structures of the three paulownia flower polysaccharides obtained after alcohol precipitation fractionation are similar. The absorption peak at 3380 cm⁻¹ is the stretching vibration absorption peak of the -OH group between or within the paulownia flower polysaccharide molecules; the absorption peak at 2930 cm⁻¹ is the stretching vibration absorption peak of the alkyl C-H group; the absorption peak at 1638 cm⁻¹ is the asymmetric stretching vibration absorption peak of the carboxyl group -COOH group, indicating that all three components have uronic acid groups; the absorption peak at 1426 cm⁻¹ is the bending vibration absorption peak of the alkyl C-H group; the absorption peaks at 1000-1200 cm⁻¹ indicate the possible presence of C-O-C ether bonds, C-O-H stretching vibrations, C-C and ring vibrations, indicating that the three paulownia flower polysaccharides obtained after alcohol precipitation fractionation are pyranose sugars.
[0036] Figure 2 The elution order of the monosaccharide standards is shown in Figure 3, compared with the elution time of the standards. Figure 4 and Figure 5The presence of distinct response peaks for fucose, rhamnose, arabinose, glucosamine hydrochloride, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid indicates that the paulownia flower polysaccharides are heteropolysaccharides composed of various monosaccharides. Table 1 shows that the three components contain the same types of monosaccharides, but the proportions of each monosaccharide differ. This indicates that fractional alcohol precipitation did not alter the monosaccharide composition of the obtained polysaccharide groups, but it did change the molar percentages of the monosaccharides. Furthermore, it can be seen that the three paulownia flower polysaccharides mainly contain the galactose component.
[0037] Table 1. Composition of monosaccharides in Paulownia flower polysaccharides
[0038] Example 2: Analysis of the inhibitory effect of paulownia flower polysaccharide on tyrosinase
[0039] Take the PFP-40, PFP-60, and PFP-80 prepared in the previous step, dissolve them in 0.1M PBS buffer to prepare a 6.4 mg / mL solution, and then dilute it 2 / 3 to 3.2, 1.6, 0.8, 0.4, and 0.2 mg / mL, respectively. Prepare a 5 mg / mL L-tyrosine solution, a 0.1 mol / L phosphate buffer, and a 500 μ / mL tyrosinase solution. Referring to the reagent addition amounts in Table 2, add the L-tyrosine solution, sample solution / solvent, and PBS buffer to each well in sequence, mix thoroughly, and incubate at 37 ℃ for 10 min. Then, add 20 μL of tyrosinase solution to each well in sequence, mix well at 37 ℃ for 5 min, and immediately place the well in a microplate reader for measurement at a wavelength of 475 nm. The calculation formula is shown in Equation 1: Formula 1.
[0040] In the formula, A1 is the absorbance of the final mixed solution, A2 is the absorbance of PBS instead of L-tyrosine solution, and A0 is the absorbance of PBS instead of sample.
[0041] Table 2. Procedure for determining tyrosinase inhibitory activity
[0042] Table 3 shows the inhibition rates of tyrosinase on fractionated paulownia flower polysaccharide extracts at different concentrations of 40%, 60%, and 80% ethanol, and the inhibition rate curves are shown in the figure. Figure 6 As shown.
[0043] Table 3. Inhibition rate of tyrosinase by different concentrations of paulownia flower polysaccharide extracts
[0044] Depend on Figure 6It was found that the inhibition rate of paulownia flower polysaccharides on tyrosinase gradually increased with increasing concentration, indicating that the inhibitory effect of paulownia flower polysaccharides on tyrosinase was dose-dependent. The IC50 values of PFP-40, PFP-60, and PFP-80 on tyrosinase were calculated to be 10.38 mg / mL, 7.61 mg / mL, and 3.63 mg / mL, respectively. Paulownia flower polysaccharides fractionated with 80% ethanol showed a more significant inhibitory effect on tyrosinase.
[0045] Example 3: Reversibility analysis of tyrosinase inhibition by paulownia flower polysaccharide
[0046] PFP-80 was prepared into solutions of different concentrations (0, 1.6, 3.2, and 6.4 mg / mL) using PBS. The concentration of the substrate L-tyrosine was fixed at 0.5 mg / mL. Enzyme activity was determined by detecting the changes in absorbance of different tyrosinases (0, 0.125, 0.25, 0.375, and 0.5 kU / mL) at each polysaccharide gradient solution. The relationship between enzyme activity and enzyme concentration at different polysaccharide concentrations was used to determine whether the inhibition was reversible. Absorbance was measured at 475 nm using a microplate reader every 30 seconds for a total of 20 minutes. Figure 7 It can be seen that the initial reaction rate increases with the increase of enzyme activity and decreases with the increase of inhibitor concentration. The slopes of the fitted lines are not equal and intersect at the origin, indicating that the paulownia flower polysaccharide binds to tyrosinase in a reversible manner. That is, the paulownia flower polysaccharide binds to the enzyme through non-covalent bonds, does not react chemically with the enzyme, and will not permanently inactivate the tyrosinase.
[0047] Example 4: Analysis of the Inhibition Types of Tyrosinase by Paulownia Flower Polysaccharides
[0048] Tyrosinase activity was fixed at 0.5 kU / mL. The effects of different gradient polysaccharide solutions (0, 1.6, 3.2, and 6.4 mg / mL) on the enzymatic reaction rate of different concentrations of L-tyrosine (0.125, 0.25, 0.375, and 0.5 mg / mL) were determined, following the same detection procedure as in Example 3. A graph was plotted between the reciprocal of the initial reaction rate (1 / [V0]) and the reciprocal of the substrate concentration (1 / [S]), yielding a set of curves. The ordinate intercept of the curve was 1 / Vm, from which the value of Vm could be calculated. The Michaelis constant Km was then calculated using the Michaelis equation. The inhibition type of tyrosinase was determined by the trends in the values of Km and Vm. Figure 8 It can be seen that by plotting the double reciprocal curve equation using Lineweaver-Burk, a set of straight lines intersecting in the second quadrant were obtained. As the polysaccharide concentration increases, the Vmax value continuously decreases while the Km value continuously increases. Therefore, it is inferred that the inhibition type of tyrosinase by paulownia flower polysaccharide is a mixed inhibition.
[0049] Example 2 demonstrates that the three ethanol-graded Paulownia flower polysaccharides prepared in Example 1 have tyrosinase inhibitory effects, thus showing promise for development into whitening cosmetics. Examples 2, 3, and 4 demonstrate that the 80% ethanol-graded Paulownia flower polysaccharide prepared in Example 1 has a stronger tyrosinase inhibitory ability and interacts with tyrosinase through a mixed inhibition mechanism. These results provide a certain theoretical basis for the development of new whitening cosmetic raw materials.
[0050] Example 5
[0051] 1. Preparation of aloe vera extract: Fresh aloe vera is crushed and then added to 20 times its weight of a 40% ethanol solution. The mixture is then subjected to ultrasonic extraction at 400W for 30 minutes. After filtration, the filtrate is evaporated, and the resulting powder is the aloe vera extract.
[0052] 2. Preparation of snow lotus extract and scutellaria baicalensis extract: The method is the same as that for aloe vera extract.
[0053] 3. The saponin extract was purchased from Shanyang Lianfeng Biotechnology Co., Ltd.
[0054] Example 6 Preparation of whitening products
[0055] (1) Weigh out the raw materials according to the following proportions: 1.3 parts of Paulownia flower polysaccharide (80% ethanol graded Paulownia flower polysaccharide), 2.5 parts of aloe vera extract, 5 parts of snow lotus extract, 2.4 parts of water soap extract, 1.8 parts of Scutellaria baicalensis extract, 0.3 parts of myricetin, 1 part of quercetin, 0.4 parts of proanthocyanidins, and 0.6 parts of luteolin. Mix them to obtain the mixture. (2) Add 80 times the amount of water to the mixture to form a suspension, add carbomer (the ratio of carbomer to water is 0.1g:20mL), and add 10% sodium hydroxide solution to obtain whitening gel.
[0056] Example 7
[0057] 1. Microbial testing
[0058] The whitening gel prepared in Example 6 was tested for total bacterial count, thermotolerant coliforms, and Staphylococcus aureus. The results are shown in Table 4.
[0059] Table 4
[0060] 2. Heavy metal detection
[0061] The whitening gel prepared in Example 6 was tested for mercury, lead, arsenic, and cadmium. The results are shown in Table 5.
[0062] Table 5
[0063] 3. Safety inspection
[0064] (1) Subjects: 23 female volunteers were recruited and screened, aged between 28 and 41.
[0065] Note: Volunteers meeting any of the following criteria will be excluded from this study: ① Individuals with facial skin conditions that may affect their judgment of test results; ② Individuals with highly allergic constitutions; ③Those who have participated in drug clinical trials or other trials within the past 45 days.
[0066] (2) Experimental method: Each volunteer was given the whitening gel prepared in Example 6. The volunteers applied the gel according to their usual cosmetic application habits, applying it to the back of their hands and behind their ears. The area applied by each volunteer was 1 cm². 1cm.
[0067] After application, the application sites of each volunteer were observed. The results showed that no redness or swelling or other discomfort occurred at the application sites of any volunteer. The gel was well absorbed, indicating that the whitening gel prepared in Example 6 of this invention has good safety.
[0068] 4. Whitening test
[0069] (1) Subjects: 23 female volunteers as described in section 3 above.
[0070] (2) Experimental method: Before the start of the experiment, the moisture content, gloss, texture and elasticity of each volunteer's skin were measured and recorded; (3) How to use Application area: Face Usage frequency and cycle: Use for 28 days, once in the morning and once in the evening.
[0071] Instructions for use: After cleansing, take about 2g of the whitening gel prepared in Example 6 and gently massage it onto the face until absorbed. Do not use other skin care products during the trial period.
[0072] (4) Experimental results: Two participants dropped out during the experiment, and 21 volunteers completed the entire experimental cycle. After the experiment, the remaining volunteers’ skin moisture content, gloss, texture and elasticity were tested again to compare the changes.
[0073] The results showed that all 21 volunteers reported significant whitening and freckle-removing effects. The volunteers' average skin moisture content increased by 43.29%, average brightness increased by 26.10%, and average texture decreased by 29.84%.
[0074] As can be seen from the above embodiments, the present invention provides a paulownia flower polysaccharide, its preparation method, and its application. The preparation method of the paulownia flower polysaccharide includes the following steps: (1) pulverizing paulownia flowers and sieving them, followed by defatting to obtain a defatted sample; (2) extracting the defatted sample using a hydrothermal method, concentrating it to obtain a concentrated aqueous extract; (3) classifying and precipitating the concentrated aqueous extract, removing proteins, dialyzing, and drying to obtain the paulownia flower polysaccharide. The present invention prepares paulownia flower polysaccharide from paulownia flowers. In vitro experiments show that it has tyrosinase inhibitory effects, thus showing promise for developing whitening cosmetics. The composition obtained by compounding the isolated paulownia flower polysaccharide can significantly inhibit tyrosinase. The composition is natural, widely available, has significant whitening effects, and is non-irritating.
[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing paulownia flower polysaccharide, characterized in that, Includes the following steps: (1) After crushing the paulownia flowers, sieve them and then degrease them to obtain a degreased sample; (2) The defatted sample was extracted using a hydrothermal method and concentrated to obtain a concentrated aqueous extract; (3) The water extract concentrate is fractionated and precipitated, the protein is removed, and then dialyzed and dried to obtain the paulownia flower polysaccharide.
2. The preparation method according to claim 1, characterized in that, When sieving in step (1), a 50-70 mesh sieve is used.
3. The preparation method according to claim 2, characterized in that, The degreasing treatment method described in step (1) is as follows: the crushed paulownia flowers and petroleum ether are mixed at a ratio of 1g: 8~10mL, refluxed at 65℃ for 1.5~2.5h, filtered and the filtrate is discarded, the residue is dried and mixed with 90~100% ethanol at a ratio of 1g: 8~10mL, refluxed at 75~85℃ for 1.5~2.5h, filtered and the filtrate is discarded, and the residue is dried at 50~60℃ to obtain the degreased sample.
4. The preparation method according to claim 3, characterized in that, The specific method of hydrothermal extraction described in step (2) is as follows: ① Mix the defatted sample and water at a ratio of 1g:8~10mL, extract in a water bath at 85~95℃ for 1.5~2.5 h, and filter to obtain filtrate 1 and residue 1; ② Centrifuge filtrate 1 at 3500~4500 r / min for 8~12 min to obtain filtrate 2 and residue 2; ③ Extract residue 1 and residue 2 again following steps ① to ② to obtain filtrate 3 and residue 3; ④ Combine filtrate 2 and filtrate 3 to obtain paulownia flower aqueous extract.
5. The preparation method according to claim 4, characterized in that, The concentration method described in step (2) is as follows: the aqueous extract of paulownia flowers is concentrated to 15-25% of its original volume at 60-70℃ to obtain the aqueous concentrate; the concentration is carried out using a rotary evaporator.
6. The preparation method according to claim 5, characterized in that, The fractional precipitation method described in step (3) is as follows: A. Add ethanol to the water extract concentrate to make the ethanol concentration in the solution 35~45%, let it stand at 2~6℃ for 10~14h, and separate the supernatant 1 and 35~45% precipitate. B. Add ethanol to supernatant 1 to make the concentration of ethanol in the solution 55~65%, let it stand at 2~6℃ for 10~14h, and separate supernatant 2 and 55~65% precipitate. C. Add ethanol to supernatant 2 to make the concentration of ethanol in the solution 75~85%, let it stand at 2~6℃ for 10~14h, and separate supernatant 3 and 75~85% precipitate. The precipitate in step (3) is deproteinized using the Savage method; the dialysis bag with a molecular weight cutoff of 3000~4000 Da is used during the dialysis in step (3); and the drying in step (3) is performed by freeze drying.
7. Paulownia flower polysaccharide prepared by the preparation method according to any one of claims 1 to 6.
8. The use of the paulownia flower polysaccharide according to claim 7 in the preparation of a product that inhibits tyrosinase activity.
9. A product that inhibits tyrosinase activity, characterized in that, The raw materials include the following parts by weight: 1-1.6 parts of the paulownia flower polysaccharide as described in claim 7, 2-3 parts of aloe vera extract, 4.5-5.5 parts of snow lotus extract, 2.2-2.6 parts of saponin extract, 1.6-2.0 parts of scutellaria baicalensis extract, 0.2-0.5 parts of myricetin, 0.8-1.2 parts of quercetin, 0.3-0.5 parts of proanthocyanidins, and 0.5-0.7 parts of luteolin.
10. The product according to claim 9, characterized in that, The product also contains excipients acceptable in the cosmetics industry.