Lotus plumule extract, method for preparing the same, and use thereof in cosmetics

Abstract

The application discloses a lotus plumule extract, a preparation method thereof and application of the lotus plumule extract in cosmetics, and comprises the following steps: preparing a natural eutectic solvent; mixing the natural eutectic solvent with water to obtain an extraction agent; mixing the extraction agent with lotus plumule powder, ultrasonic extraction, centrifugal filtration, and obtaining the lotus plumule extract. The preparation method of the lotus plumule extract has the advantages of high extraction efficiency, green environmental protection and good stability of raw materials compared with traditional extraction processes, is favorable for promoting the development of a 'green daily chemical' industry, and can be widely applied to extraction of other plant raw materials. Meanwhile, the extract can be applied to cosmetics, has skin care effects such as whitening, repair, anti-photoaging and the like, and provides scientific basis and technical support for development and high value-added utilization of lotus natural skin care products.

Description

Technical Field

[0001] This invention relates to the field of cosmetic formulations, specifically to a lotus germ extract, its preparation method, and its application in cosmetics. Background Technology

[0002] With increasing demands for safety and environmental protection in daily necessities, the development of extraction processes for many plant and animal extracts has been limited. Therefore, using green and natural solvents for plant extraction has broad application prospects. Lotus seed heart, also known as lotus embryo, is the green young leaf and radicle of the lotus seed, rich in flavonoids, including oxyglycoside and C-glycoside flavonoids, which possess various skin-care benefits. Due to the diverse types and molecular structures of plant flavonoids, their physicochemical properties also vary; even flavonoids from different parts of the same species are not entirely the same, resulting in a lack of universality between green extraction processes. Existing lotus embryo extraction processes often use organic solvents such as methanol, petroleum ether, and ethyl acetate as extraction agents, affecting the green and environmentally friendly characteristics of the product. While solvents such as pure water and ethanol are more environmentally friendly, they suffer from low extraction efficiency. Therefore, developing a natural and green solvent for the efficient extraction of lotus embryo, especially its flavonoid compounds, is crucial.

[0003] Chinese patent CN105343190A discloses a method for preparing flavonoid extract from lotus seed hearts. This patent uses lotus seed hearts as the main raw material, firstly using ethanol solvent extraction, followed by vacuum concentration and extraction with petroleum ether to obtain a crude extract. Then, solvents such as ethyl acetate and n-butanol are prepared in a certain proportion, and high-speed countercurrent chromatography is used to enrich the flavonoid components. This method has advantages such as large preparation volume, simple pretreatment, no adsorption loss, short single separation time, and good separation effect. However, the reagents involved in this process, such as n-butanol, petroleum ether, and ethyl acetate, are still organic reagents, which does not conform to the concept of green and environmentally friendly extraction. Chinese invention patent CN104744367B discloses a flash extraction method for lotus seed heart alkaloids, using a flash extractor, with extraction time of 2-8 minutes, less than 4% of the time required for traditional heating and reflux extraction. This method is convenient for sample post-processing, saves energy, has low production costs, and a short cycle. However, it also uses organic extraction solvents, which does not meet the requirements of green and environmentally friendly extraction. Summary of the Invention

[0004] In order to develop a natural and green solvent for the extraction of lotus embryos, especially for obtaining high levels of flavonoids, the first aspect of the present invention provides a method for preparing lotus embryo extract, comprising the following steps: Preparation of natural eutectic solvents; The extractant is obtained by mixing a natural eutectic solvent with water; The extractant was mixed with lotus germ powder, extracted by ultrasound, and then centrifuged and filtered to obtain lotus germ extract.

[0005] In one embodiment, the pH of the natural eutectic solvent is 2.27-9.01, the viscosity of the natural eutectic solvent at 25°C is 3.61-18.14 mPa·s, the conductivity of the natural eutectic solvent is 14.88-4519 S / m, and the polarity of the natural eutectic solvent is 47.65-49.58 kcal / mol.

[0006] In one embodiment, the natural eutectic solvent comprises a hydrogen bond acceptor and a hydrogen bond donor, wherein the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-4).

[0007] In one embodiment, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1, 1:2, 1:3, or 1:4.

[0008] In one embodiment, the hydrogen bond acceptor includes at least one of betaine or proline.

[0009] In one embodiment, the hydrogen bond donor includes at least one of polyols, natural organic acids, natural sugars, and amines; In one embodiment, the hydrogen bond donor includes at least one of glycerol, ethylene glycol, malic acid, citric acid, glucose, fructose, and urea.

[0010] In one embodiment, the natural eutectic solvent comprises a combination of betaine and glycerol; In one embodiment, the natural eutectic solvent includes a combination of betaine and ethylene glycol; In one embodiment, the natural eutectic solvent comprises a combination of proline and glycerol.

[0011] In a preferred embodiment, the mass ratio of the natural eutectic solvent to water in the extractant is (6-9):(1-4).

[0012] In a preferred embodiment, the mass ratio of the natural eutectic solvent to water in the extractant is (6.5-7.5):(2.5-3.5).

[0013] In a preferred embodiment, the mass ratio of the natural eutectic solvent to water in the extractant is 7:3.

[0014] In a preferred embodiment, the mass ratio of the natural eutectic solvent to water in the extractant is 9:1.

[0015] In a preferred embodiment, the mass ratio of the natural eutectic solvent to water in the extractant is 8:2.

[0016] In a preferred embodiment, the mass ratio of the natural eutectic solvent to water in the extractant is 7.5:2.5.

[0017] In a preferred embodiment, the mass ratio of the natural eutectic solvent to water in the extractant is 6:4.

[0018] In a preferred embodiment, the mass ratio of the natural eutectic solvent to water in the extractant is 6.5:3.5.

[0019] In one embodiment, the ratio of lotus germ powder to extractant is 1:(20-60)g / mL.

[0020] In one embodiment, the ratio of lotus germ powder to extractant is 1:(20-40)g / mL.

[0021] In one embodiment, the ratio of lotus germ powder to extractant is 1:27.5 g / mL.

[0022] In one embodiment, the ratio of lotus germ powder to extractant is 1:20 g / mL.

[0023] In one embodiment, the ratio of lotus germ powder to extractant is 1:30 g / mL.

[0024] In one embodiment, the ratio of lotus germ powder to extractant is 1:35 g / mL.

[0025] In one embodiment, the ratio of lotus germ powder to extractant is 1:40 g / mL.

[0026] In one embodiment, the ratio of lotus germ powder to extractant is 1:50 g / mL.

[0027] In one embodiment, the temperature of the ultrasonic extraction is 40-80℃.

[0028] In one embodiment, the temperature of the ultrasonic extraction is one of 40°C, 60°C, 70°C, and 80°C.

[0029] In one embodiment, the ultrasonic extraction time is 20-100 min, and the ultrasonic power of the ultrasonic extraction is 180-420 W.

[0030] In one embodiment, the ultrasonic extraction time is 40-80 min, and the ultrasonic power of the ultrasonic extraction is 240-360 W.

[0031] In one embodiment, the ultrasonic extraction time is one of 20 min, 40 min, 60 min, 80 min, or 100 min.

[0032] In one embodiment, the ultrasonic power of the ultrasonic extraction is one of 180 W, 240 W, 300 W, 360 W, and 420 W.

[0033] A second aspect of the present invention provides a lotus germ extract, which is prepared by the method described above.

[0034] A third aspect of the present invention provides an application of a lotus germ extract, which is used in cosmetics.

[0035] Compared with the prior art, the present invention has the following beneficial effects: (1) The method for preparing lotus embryo extract according to the present invention uses a green natural eutectic solvent to extract lotus embryo extract by ultrasonic extraction, which has the advantages of high efficiency, green environmental protection and good raw material stability compared with traditional extraction process.

[0036] (2) The method for preparing lotus embryo extract described in this invention uses a combination of proline and glycerol as a green and natural eutectic solvent, which is conducive to promoting the development of the "green daily chemical" industry, has higher consumer acceptance, and can be widely used in the extraction of other plant raw materials.

[0037] (3) The lotus germ extract prepared by the method of the present invention has a high content of flavonoid compounds, and has repair, whitening and moisturizing effects. It has good safety for cells at an addition amount of 1.2wt% and can significantly inhibit melanin secretion.

[0038] (4) The lotus embryo extract prepared by the method of the present invention has good raw material stability. After the extract is compounded (1wt% extract + 5wt% 1,2-hexanediol + 94wt% water), it does not show significant changes when stored for a certain period of time under light-proof, room temperature, and high temperature (40℃, 45℃, 50℃).

[0039] (5) The method for preparing lotus embryo extract described in this invention has a simple extraction process, controllable cost, and the extractant is safe and environmentally friendly. Attached Figure Description

[0040] Figure 1 The images show physical pictures of the natural eutectic solvents used in Example 1 and Comparative Examples 1-13. From left to right, the images show Comparative Examples 1-7, Example 1, and Comparative Examples 8-13.

[0041] Figure 2The images show the extracts of lotus embryos extracted using natural eutectic solvents, as shown in Example 1 and Comparative Examples 1-13. From left to right, the images represent Comparative Examples 1-7, Example 1, and Comparative Examples 8-13.

[0042] Figure 3 The graph shows a comparison of the total flavonoid content of lotus germ extracts prepared in Example 1 and Comparative Examples 1-13.

[0043] Figure 4 FT-IR spectra of proline and glycerol as solvents, proline and glycerol (1:3 molar ratio, 30wt% aqueous solution).

[0044] Figure 5 The graph shows the total flavonoid content of the lotus germ extracts prepared in Examples 1-5.

[0045] Figure 6 The graph shows the total flavonoid content of lotus germ extracts prepared in Examples 1, 6-9.

[0046] Figure 7 The graph shows the total flavonoid content of lotus germ extracts prepared in Examples 1, 10-13.

[0047] Figure 8 The graph shows the total flavonoid content of lotus germ extracts prepared in Examples 1, 14-17.

[0048] Figure 9 The graph shows the total flavonoid content of lotus germ extracts prepared in Examples 1, 18-21.

[0049] Figure 10 The images show scanning electron microscope (SEM) images of lotus embryo extracts prepared in Example 19, Comparative Example 14, and Comparative Example 15. From left to right, the images show Comparative Example 14, Comparative Example 15, and Example 19. From top to bottom, the images show magnifications of 60x and 200x, respectively.

[0050] Figure 11 The chart shows a comparison of the total flavonoid content of lotus germ extracts prepared in Example 19, Comparative Example 14, and Comparative Example 15.

[0051] Figure 12 Comparative liquid chromatography fingerprints of lotus germ extracts prepared in Example 19, Comparative Example 14, and Comparative Example 15.

[0052] Figure 13 The image shows the qualitative analysis of the lotus germ extract prepared in Example 19 by liquid chromatography.

[0053] Figure 14The natural eutectic solvents of Comparative Examples 2-13 were adjusted to have a molar ratio of hydrogen bond acceptor to hydrogen bond donor of 1:3. Physical images of the natural eutectic solvents of Comparative Examples 1 and Example 1 are also shown. From left to right, the images are Comparative Examples 1-7, Example 1, and Comparative Examples 8-13.

[0054] Figure 15 This is a graph showing the results of the cell whitening activity test.

[0055] Figure 16 This is a graph showing the results of a cell scratch repair test.

[0056] Figure 17 This is a graph showing the results of cell safety testing.

[0057] Figure 18 The graph shows the results of the stability test.

[0058] Figure 19 This is a graph showing the results of a UVA safe irradiation dose test.

[0059] Figure 20 The figure shows the test results of HFF cell survival rate at different concentrations of total flavonoids from lotus embryo.

[0060] Figure 21 The image shows the test results of the photoprotection of lotus embryo flavonoids on UVA-induced fibroblasts.

[0061] Figure 22 Microscopic image of cell morphology after lotus embryo flavonoid treatment.

[0062] Figure 23 The image shows the results of SOD enzyme content testing in cells treated with lotus embryo flavonoids.

[0063] Figure 24 The figure shows the results of MDA content testing in cells treated with lotus embryo flavonoids.

[0064] Figure 25 The image shows the results of IL-6 content testing in cells treated with lotus embryo flavonoids.

[0065] Figure 26 The figure shows the results of TNF-α content testing in cells treated with lotus embryo flavonoids.

[0066] Figure 27 The image shows the results of the MMP-1 content test in cells treated with lotus embryo flavonoids.

[0067] Figure 28 The image shows the results of the MMP-3 content test in cells treated with lotus embryo flavonoids. Detailed Implementation

[0068] Example 1 A method for preparing lotus embryo extract includes the following steps: Preparation of natural eutectic solvents; The extractant is obtained by mixing a natural eutectic solvent with water; Accurately weigh lotus embryo powder, add extractant, vortex mix the two, extract using ultrasonic-assisted extraction, centrifuge at 8000 rpm for 20 min, retain the supernatant, and filter the supernatant using a 0.45 μm aqueous polyethersulfone needle filter to obtain lotus embryo extract.

[0069] The natural eutectic solvent comprises proline as a hydrogen bond acceptor and glycerol as a hydrogen bond donor, in a molar ratio of 1:3.

[0070] The method for preparing the natural eutectic solvent is as follows: proline and glycerol are mixed in a molar ratio and heated and stirred at 80°C until clear and transparent to obtain the natural eutectic solvent.

[0071] The natural eutectic solvent, after preparation, is vacuum dried for more than 12 hours to remove excess moisture and ensure no crystal precipitation, before being used for subsequent extraction experiments.

[0072] The mass ratio of the natural eutectic solvent to water in the extractant is 7:3.

[0073] The ratio of lotus germ powder to extractant is 1:30 g / mL.

[0074] The ultrasonic extraction temperature is 40℃, the ultrasonic extraction time is 40 min, and the ultrasonic power of the ultrasonic extraction is 240 W.

[0075] Example 2 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the ultrasonic extraction temperature is 50℃.

[0076] Example 3 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the ultrasonic extraction temperature is 60℃.

[0077] Example 4 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the ultrasonic extraction temperature is 70℃.

[0078] Example 5 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the ultrasonic extraction temperature is 80℃.

[0079] Example 6 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 3, except that the ultrasonic extraction time is 20 min.

[0080] Example 7 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 3, except that the ultrasonic extraction time is 60 min.

[0081] Example 8 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 3, except that the ultrasonic extraction time is 80 min.

[0082] Example 9 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 3, except that the ultrasonic extraction time is 100 min.

[0083] Example 10 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 7, except that the ratio of lotus embryo powder to extractant is 1:20 g / mL.

[0084] Example 11 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 7, except that the ratio of lotus embryo powder to extractant is 1:40 g / mL.

[0085] Example 12 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 7, except that the ratio of lotus embryo powder to extractant is 1:50g / mL.

[0086] Example 13 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 7, except that the ratio of lotus embryo powder to extractant is 1:60g / mL.

[0087] Example 14 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 7, except that the mass ratio of natural eutectic solvent to water in the extractant is 9:1.

[0088] Example 15 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 7, except that the mass ratio of natural eutectic solvent to water in the extractant is 8:2.

[0089] Example 16 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 7, except that the mass ratio of natural eutectic solvent to water in the extractant is 7.5:2.5.

[0090] Example 17 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 7, except that the mass ratio of natural eutectic solvent to water in the extractant is 6:4.

[0091] Example 18 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 7, except that the ultrasonic power of the ultrasonic extraction is 180 W.

[0092] Example 19 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 7, except that the ultrasonic power of the ultrasonic extraction is 300 W.

[0093] Example 20 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 7, except that the ultrasonic power of the ultrasonic extraction is 360 W.

[0094] Example 21 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 7, except that the ultrasonic power of the ultrasonic extraction is 420 W.

[0095] Comparative Example 1 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the natural eutectic solvent includes betaine as hydrogen bond acceptor and glycerol as hydrogen bond donor, with a molar ratio of 1:3.

[0096] Comparative Example 2 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the natural eutectic solvent includes betaine as hydrogen bond acceptor and ethylene glycol as hydrogen bond donor, with a molar ratio of 1:4.

[0097] Comparative Example 3 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the natural eutectic solvent includes betaine as hydrogen bond acceptor and malic acid as hydrogen bond donor, with a molar ratio of 1:1.

[0098] Comparative Example 4 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the natural eutectic solvent includes betaine as hydrogen bond acceptor and citric acid as hydrogen bond donor, with a molar ratio of 1:1.

[0099] Comparative Example 5 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the natural eutectic solvent includes betaine as hydrogen bond acceptor and glucose as hydrogen bond donor, with a molar ratio of 1:1.

[0100] Comparative Example 6 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the natural eutectic solvent includes betaine as hydrogen bond acceptor and fructose as hydrogen bond donor, with a molar ratio of 1:1.

[0101] Comparative Example 7 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the natural eutectic solvent includes betaine as hydrogen bond acceptor and urea as hydrogen bond donor, with a molar ratio of 1:1.

[0102] Comparative Example 8 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the natural eutectic solvent includes proline as a hydrogen bond acceptor and ethylene glycol as a hydrogen bond donor, with a molar ratio of 1:4.

[0103] Comparative Example 9 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the natural eutectic solvent includes proline as hydrogen bond acceptor and malic acid as hydrogen bond donor, with a molar ratio of 1:1.

[0104] Comparative Example 10 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the natural eutectic solvent includes proline as hydrogen bond acceptor and citric acid as hydrogen bond donor, with a molar ratio of 1:1.

[0105] Comparative Example 11 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the natural eutectic solvent includes proline as a hydrogen bond acceptor and glucose as a hydrogen bond donor, with a molar ratio of 1:1.

[0106] Comparative Example 12 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the natural eutectic solvent includes proline as hydrogen bond acceptor and fructose as hydrogen bond donor, with a molar ratio of 1:1.

[0107] Comparative Example 13 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 1, except that the natural eutectic solvent includes proline as hydrogen bond acceptor and urea as hydrogen bond donor, with a molar ratio of 1:1.

[0108] Comparative Example 14 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 19, except that the natural eutectic solvent is replaced with pure water.

[0109] Comparative Example 15 A method for preparing lotus embryo extract, the specific implementation method is the same as in Example 19, except that the natural eutectic solvent is replaced with a 70wt% ethanol aqueous solution.

[0110] Performance testing 1. Test the physicochemical properties of the natural eutectic solvents of Example 1 and Comparative Examples 1-13, including pH, viscosity, conductivity, and polarity.

[0111] pH value: The pH value of the solvent is measured using a pH meter. The pH meter probe is completely immersed in the solution to be tested, and the value is counted after it stabilizes.

[0112] Viscosity determination: Measured using a rheometer with a CP50-1 rotor (1° cone plate, 50 mm diameter), a test gap of 0.098 mm, and a shear rate of 100 s⁻¹. -1 The test duration was 30 seconds, the operating temperature was set to 298.15 K, and each group was repeated three times to record the values.

[0113] Conductivity measurement: The conductivity of the solvent was measured using a conductivity meter. The electrode was cleaned with pure water and wiped until no moisture remained. The electrode was completely immersed in the solvent to be tested, and the electrode was stirred slightly to ensure full contact between the electrode and the solvent. The working temperature was set to 298.15 K. Each group was repeated three times. The value was recorded after the reading stabilized.

[0114] Polarity determination: The polarity of the solvent was determined using the Nile Red (NR) staining method. 500 μL of NR was pipetted into a clean centrifuge tube and vacuum dried until the methanol was completely evaporated. 1 mL of the NaDES sample was added, and the mixture was sonicated in a water bath for 20 min to ensure thorough mixing of NaDES and NR. 200 μL of each sample was transferred to a 96-well plate, and a multi-mode microplate reader was used to perform a full-wavelength spectral scan. The maximum absorption wavelength was recorded, with a wavelength scan interval of 3 nm. The operating temperature was set to 298.15 K. Each group was tested in triplicate. The values ​​were recorded and calculated using the following formula: E NR (kcal / mol) = h× c×NA / λ max =28591 / λ max in, h E is Planck's constant. NRFor Moore's transition, c is the speed of light, λ max (nm) is the maximum absorption wavelength, and NA is Avogadro's constant.

[0115] The test results are shown in Table 1. The correlation analysis between the physicochemical properties of the natural eutectic solvent and the extraction efficiency of total flavonoids from lotus embryo is shown in Table 2.

[0116] Table 1

[0117] Table 2

[0118] Because natural eutectic solvents generally have high viscosity, which can lead to inaccurate index measurements, 30% (w / w) of purified water was added before testing to dilute the solvent and facilitate the measurement. Table 2 shows that, overall, the extraction efficiency of total flavonoids from lotus germ is negatively correlated with pH, ​​viscosity, and conductivity, but positively correlated with polarity. The pH of the solvent for extracting flavonoids is generally best kept below 7. In the experiment, except for the proline + urea combination, the pH of all other solvent combinations was below 7, and their corresponding flavonoid extraction efficiencies were relatively high. Lower solvent viscosity leads to higher flavonoid extraction efficiency, mainly because lower solvent viscosity increases the solute diffusion coefficient, reduces mass transfer resistance, and facilitates substance extraction.

[0119] 2. Examples 1 and 13: Physical images of the natural eutectic solvents are shown below. Figure 1 The figures, from left to right, are Comparative Examples 1-7, Example 1, and Comparative Examples 8-13.

[0120] 3. Examples 1 and 1-13: Extracts from lotus embryos obtained using natural eutectic solvents (see attached images). Figure 2 The figures, from left to right, are Comparative Examples 1-7, Example 1, and Comparative Examples 8-13.

[0121] 4. The total flavonoid content of lotus embryo extracts from Examples 1 and Comparative Examples 1-13 was determined by high performance liquid chromatography. The test results are shown below. Figure 3 .

[0122] The method for determining flavonoid content by high performance liquid chromatography is as follows: The chromatographic operation was performed on an Agilent 1260 high-performance liquid chromatograph using a COSMOSIL Cholester C18 column (4.6 × 250 mm, 5 μm). The mobile phases were acetonitrile (phase A) and 1% formic acid in water (phase B). The elution gradient program was: 0 min, 5 vol% A; 65 min, 30 vol% A; 75 min, 95 vol% A; 80 min, 5 vol% A; 85 min, 5 vol% A. The flow rate was 0.8 mL / min, the column temperature was 32 °C, the injection volume was 10 μL, and the detection wavelengths were 280 nm and 350 nm.

[0123] The content of flavonoid compounds was determined using a semi-quantitative method based on standards. 1 mg of schaftoside standard was accurately weighed and added to 1 mL of methanol to prepare a 1 mg / mL schaftoside stock solution. This stock solution was then diluted proportionally to prepare a series of standard solutions with different concentrations: 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, and 0.015625 mg / mL. The solutions were then analyzed using liquid chromatography, and a standard curve for schaftoside was plotted.

[0124] 5. The FT-IR spectra of the solvents proline, glycerol, proline and glycerol (1:3 molar ratio), and proline and glycerol (1:3 molar ratio, 30wt% aqueous solution) were tested. The test results are shown in [reference needed]. Figure 4 .

[0125] 6. The total flavonoid content of Examples 1-21 was determined by high performance liquid chromatography. The test results for Examples 1-5 are shown below. Figure 5 The test results for Examples 1 and 6-9 are shown below. Figure 6 The test results for Examples 1 and 10-13 are shown below. Figure 7 The test results for Examples 1 and 14-17 are shown below. Figure 8 The test results for Examples 1 and 18-21 are shown below. Figure 9 .

[0126] 7. Scanning electron micrographs of lotus embryo extracts from Examples 19, 14, and 15 are shown below. Figure 10 From left to right, they are Comparative Example 14, Comparative Example 15, and Example 19.

[0127] 8. The total flavonoid content of lotus embryo extracts from Examples 19, 14, and 15 was determined by high performance liquid chromatography (HPLC). The test results are shown in [Figure number missing]. Figure 11 .

[0128] 9. A comparison of the liquid chromatography fingerprint results of lotus germ extracts from Examples 19, 14, and 15 is shown in the figure. Figure 12 .

[0129] 10. The qualitative analysis of the lotus embryo extract in Example 19 by ultra-high performance liquid chromatography-mass spectrometry is shown in [the figure]. Figure 13 The qualitative analysis data are shown in Table 3.

[0130] The qualitative method using ultra-high performance liquid chromatography-mass spectrometry is as follows: 1) Chromatographic conditions: The column was a COSMOSIL Cholester C18 (4.6 × 250 mm, 5 μm). The mobile phases were acetonitrile (phase A) and 1% formic acid in water (phase B). The elution gradient program was: 0 min, 5 vol% A; 65 min, 30 vol% A; 75 min, 95 vol% A; 80 min, 5 vol% A; 85 min, 5 vol% A. The flow rate was 0.8 mL / min, the column temperature was set at 32℃, the injection volume was 10 μL, and the detection wavelengths were 280 nm and 350 nm.

[0131] 2) Mass spectrometry conditions: Chromatographic parameters were the same as above, with a flow rate of 0.4 mL / min and an injection volume of 1 μL. Full scan mode (PI and NI) was used. Mass spectrometry detection parameters were: scan range 100-1000 m / z; capillary voltage: 3 kV for PI mode and 2.5 kV for NI mode; cone voltage: 10 V for PI mode and 70 V for NI mode; collision energy: 15 eV for PI mode and 30 eV for NI mode; solvent gas removal temperature: 350℃; ion source temperature: 150℃.

[0132] Table 3

[0133] 11. The molar ratio of the natural eutectic solvent in Comparative Examples 2-13 was adjusted to 1:3 for hydrogen bond acceptors and hydrogen bond donors. Physical images of the natural eutectic solvents in each group, as well as Comparative Example 1 and Example 1, are shown below. Figure 14 The figures, from left to right, represent Comparative Examples 1-7, Example 1, and Comparative Examples 8-13. Except for betaine-glycerol (Comparative Example 1), all other groups showed varying degrees of crystal precipitation or were unable to synthesize a solvent, making them unsuitable for use as extraction agents.

[0134] 12. Whitening Activity Test: The lotus embryo extract prepared in Example 3 was prepared into a 1.2 wt% solution as Experiment Group 1, and the lotus embryo extract prepared in Comparative Example 15 (70 wt% ethanol aqueous solution extraction) was prepared into a 1.2 wt% solution as Experiment Group 2. The α-MSH (α-melanin-stimulating hormone) model served as a negative control, 1 wt% arbutin aqueous solution served as a positive control, and water served as a blank control. Cellular whitening activity was tested, and the relative melanin secretion was measured. The method for the cellular whitening activity test referred to the experimental method in "T / SHRH 027-2019 In Vitro Test of B16 Cell Melanin Synthesis Inhibition Experiment," and the specific test protocol is as follows.

[0135]

[0136] Test results are available Figure 15 .

[0137] The results showed that the 1.2wt% lotus embryo extract prepared in this application significantly inhibited melanin secretion in B16 mouse melanoma cells, with a statistically significant difference compared to the negative control group (P<0.05). Parallel experiments using 1wt% arbutin as a reference also showed a significant difference compared to the negative control group. In conclusion, both 1.2wt% lotus embryo extract and 1wt% arbutin possess whitening effects and can be used as whitening active ingredients in cosmetics and other fields.

[0138] 13. Scratch Repair Experiment: The lotus embryo extract prepared in Example 3 was prepared into 0.625wt% and 1.2wt% aqueous solutions as Experiment Group 1, and the lotus embryo extract prepared in Comparative Example 15 (70wt% ethanol aqueous solution extraction) was prepared into 0.625wt% and 1.2wt% aqueous solutions as Experiment Group 2. A scratch repair experiment was conducted to test cell migration rate. The testing method for cell scratch repair efficacy referred to the experimental method in "T / ZHCA 020-2022*Human Fibroblast Migration Ability Experiment", and the specific testing protocol is as follows.

[0139]

[0140] Test results are available Figure 16 .

[0141] The results showed that the 1.2wt% lotus embryo extract prepared in this application significantly increased the cell migration rate by 88.6% compared with the blank control group and by 113.47% compared with the 1.2wt% lotus embryo extract group of comparative example 12, indicating that it has a certain repair effect on cells.

[0142] 14. Safety Testing: The lotus embryo extract prepared in Example 3 was prepared into aqueous solutions of 0.04 wt%, 0.08 wt%, 0.16 wt%, 0.31 wt%, 0.63 wt%, 1.25 wt%, 2.5 wt%, and 5 wt% to test cell safety. The cell safety testing method referred to the experimental method in GB / T 16886.5-2017 Biological Evaluation of Medical Devices. The test results are shown in […]. Figure 17 .

[0143] The results showed that the safe addition range of the lotus germ extract prepared in this application for cells is 0-1.25 wt%.

[0144] 15. Stability Test: A solution composed of 1 wt% lotus embryo extract, 5 wt% 1,2-hexanediol, and 94 wt% water prepared in Example 19 was stored for one month under the following conditions: -18°C, light exposure, protection from light, room temperature (25°C), and high temperature (40°C, 45°C, 50°C). The appearance was observed. The test results are shown in […]. Figure 18 .

[0145] like Figure 18 As shown: Top: Initial state; Bottom: State after one month of storage; From left to right: -18℃, light exposure, light avoidance, room temperature (25℃), high temperature (40℃, 45℃, 50℃) conditions.

[0146] The results showed that the 1wt% lotus embryo extract solution prepared in this application was slightly turbid at -18℃, and showed slight fading under light but not obvious. It did not show significant changes under light-protected, room temperature, and high temperature (40℃, 45℃, 50℃) conditions, and had good stability.

[0147] 16. The lotus embryo extract prepared in Example 19 was purified at a loading concentration of 0.53 mg / mL, with a loading volume to resin volume ratio of 3 mg:1 mL and a loading flow rate of 1 BV / h. After loading, the sample was allowed to stand for 12 h for complete adsorption. After adsorption, the column was eluted with 5 column volumes of distilled water, followed by 8 column volumes of 50 wt% ethanol-water elution for flavonoids, both at a flow rate of 1 BV / h. The eluent was collected, and most of the solvent was removed using a vacuum rotary evaporator. Finally, the eluent was freeze-dried to obtain lotus embryo flavonoids.

[0148] A standard UVA irradiation method was employed to simulate UVA photodamage to skin cells in the natural environment. First, HFF cells were cultured to the logarithmic growth phase to ensure healthy cell growth. Then, 100 µL of cell suspension was seeded into 96-well plates and cultured for 24 h. The supernatant was discarded, and 100 µL of PBS was added. The plates were then irradiated under a UV lamp with different doses of UVA. After UV irradiation, cells were cultured for another 4 h, and changes in cell viability were measured using the CCK-8 assay. The UVA irradiation dose at which 50% viability was achieved was selected as the modeling condition.

[0149] Results: Using 2-10 mJ / cm 2 HFF cell survival was negatively correlated with UVA dose; that is, as the irradiation dose increased, the HFF cell survival rate decreased. In this study, the UVA irradiation dose was 6 mJ / cm². 2 The cell viability was 58%. At this point, the cell viability was higher than 50%, and there was significant cell damage, which was used to establish a HFF photoaging model of fibroblasts. The test results are shown below. Figure 19 .

[0150] 16.1 Test the fibroblast proliferation activity of total flavonoids from lotus embryo.

[0151] Healthy HFF cells were cultured for 24 h and then treated with different concentrations (0 µg / mL, 62.5 µg / mL, 100 µg / mL, 125 µg / mL, 200 µg / mL, 250 µg / mL, 400 µg / mL, and 500 µg / mL) of lotus embryo flavonoids. The lotus embryo flavonoids were pre-dissolved in pure water and then serially diluted with fetal bovine serum-free DMEM medium. After treatment with 100 µL of lotus embryo flavonoids for 24 h, 10 µL of CCK-8 reagent was added to each well, and the cells were incubated for 2 h. The absorbance was measured at 450 nm and converted to cell viability. IC50 was selected. 80 and 1 / 2 IC 80 The concentration of lotus embryo flavonoids at the specified concentration was used as the concentration for subsequent experiments. The cell viability was calculated using the following formula: Cell viability = [(OD450 of damaged wells - OD450 of blank wells) / (OD450 of control group - OD450 of blank wells)] × 100%.

[0152] Results: When the concentration of lotus embryo flavonoids reached 250 μg / mL, the cell viability was 80%, and no significant cytotoxicity was observed at this concentration, making it suitable for subsequent experiments. The cell viability of HFF cells at different concentrations of lotus embryo flavonoids is shown in the figure below. Figure 20 .

[0153] 16.2 Determination of the protective effect of lotus embryo flavonoids against UVA-induced photodamage to fibroblasts.

[0154] Methods: Healthy HFF cells were cultured for 24 h, and 100 µL of lotus embryo flavonoids were added to each well for 24 h. Afterward, the cells were placed under a UV lamp with the lamp 15 cm away for UVA irradiation at a dose of 6 J / cm². 2 After irradiation, the cells were cultured in an incubator for 24 h, and the cell viability was detected using the CCK-8 assay.

[0155] Results: The control group, which had not undergone UVA irradiation or sample treatment, had a cell viability of 100%, while the model group had a viability of 6 mJ / cm². 2 Survival rate decreased significantly to 51% under UVA stimulation, and after a significance analysis with the control group, it was found that... p A value less than 0.001 indicates statistical significance, signifying successful model establishment. Furthermore, in the sample group treated with lotus germ flavonoids before UVA irradiation, the HFF cell survival rate showed a certain degree of increase, with a highly significant difference compared to the model group, indicating that lotus germ flavonoids have a photoprotective effect on HFF cells. The IC50 value... 80 The corresponding flavonoid concentration compared to 1 / 2 IC 80 The light protection effect is good, so IC was selected subsequently. 80 The corresponding flavonoid concentrations were used as the treatment concentrations for the sample groups. The photoprotective effect of lotus germ flavonoids on UVA-induced fibroblasts was investigated. The test results are shown below. Figure 21 .

[0156] 16.3 Observation of cell morphology after lotus embryo flavonoid treatment.

[0157] Methods: Healthy HFF cells were seeded into 6-well plates with 6 accessory wells. Each well contained 2 mL of DMEM culture medium. The plates were incubated for 24 h. The culture medium was then aspirated, and lotus embryo flavonoids were added for pretreatment for 24 h. The culture medium was then aspirated again, and 800 μL of pre-chilled PBS was added. The plates were then placed under a UV lamp for UVA irradiation for 24 h at a dose of 6 J / cm². 2 After aspirating the culture medium, replace it with an equal volume of PBS. Use an inverted microscope to randomly select fields of view to take pictures of the cells in order to observe their condition.

[0158] Results: Compared with the control group of HFF cells, after UVA irradiation, fibroblasts showed a significant decrease in number, an increase in the number of dead cells, irregular cell morphology, indistinct outlines, and a reduction or even disappearance of the elongated spindle shape, exhibiting morphological changes typical of aging cells. In contrast, compared with the UVA-only irradiation group, cells pretreated with lotus germ flavonoids for 24 hours before UVA irradiation showed an increased number of cells that retained their elongated spindle shape, had fewer dead cells, and were arranged more regularly. Based on the cell number and morphological results, UVA irradiation caused significant damage to HFF cells, while pretreatment with lotus germ flavonoids mitigated the damage caused by UVA to fibroblasts. Using the classic antioxidant L-ascorbic acid (Vc) as a positive control, the morphological state of Vc-treated HFF cells was similar to that of cells treated with lotus germ flavonoids, further demonstrating the photoprotective effect of lotus germ flavonoids. Test results are shown below. Figure 22 .

[0159] 16.4 Oxidative stress test of cells treated with lotus embryo flavonoids.

[0160] Methods: The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected using SOD and MDA assay kits. HFF cells were seeded in 6-well plates, treated with lotus embryo flavonoids, and then irradiated with UVA. The treated HFF cells were placed in a low-temperature environment, and 100 μL of cell lysis buffer was added. Finally, the cells were centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant was used for assay. The specific procedures were performed according to the instructions of the SOD and MDA assay kits from Beijing Bairui Biotechnology Co., Ltd.

[0161] Results: The SOD enzyme activity in the UVA model group was significantly reduced, indicating that UVA causes significant oxidative damage to HFF cells. The SOD enzyme content in the lotus germ flavonoid treatment group was significantly increased compared to the model group, reaching 58 U / mg. This suggests that lotus germ flavonoids can inhibit oxidative stress levels in photoaged cells by increasing SOD enzyme content. Test results are shown below. Figure 23 .

[0162] The MDA content in the UVA model group was 71 μM / mg higher than that in the control group, reaching a highly significant level, indicating that UVA increased the oxidative stress level of HFF cells. The sample group treated with lotus germ flavonoids had an MDA content of 80 μM / mg, which was also highly significant compared to the model group, indicating that lotus germ flavonoids have a certain inhibitory effect on MDA. Test results are shown below. Figure 24 .

[0163] 16.5 Inflammatory factor test of cells treated with lotus germ flavonoids.

[0164] Methods: TNF-α and IL-6 levels in fibroblasts were detected. Pretreatment was the same as above. The specific procedures were performed according to the instructions of the TNF-α and IL-6 detection kit from Beijing Bairui Biotechnology Co., Ltd.

[0165] Results: In the photodamage-induced HFF cell model, the relative expression level of IL-6 in the model group was significantly higher than that in the control group. p <0.001 indicates that the inflammatory response has been successfully activated under this model. After intervention with lotus germ flavonoids, IL-6 levels significantly decreased to 1.56 ( p The result (<0.001) confirms that it achieves photoprotective effects on HFF cells at the anti-inflammatory level. Test results are shown below. Figure 25 .

[0166] The significant analysis of the relative TNF-α content between the control group and the model group showed that the photodamage model successfully induced an increase in TNF-α levels in HFF cells. The relative content of lotus embryo flavonoids compared to the model group was 1.8, demonstrating that the total flavonoids in lotus embryos possess anti-inflammatory and photoprotective potential. The test results are shown below. Figure 26 .

[0167] 16.6 Detection of matrix metalloproteinases in cells treated with lotus embryo flavonoids.

[0168] Methods: MMP-1 and MMP-3 factors in fibroblasts were detected. Pretreatment was the same as above. Specific procedures were performed according to the instructions of the MMP-1 and MMP-3 detection kit from Beijing Bairui Biotechnology Co., Ltd.

[0169] Results: Compared with the control group, the expression of MMP-1 and MMP-3 in the UVA-induced photoaging model group was significantly increased. p The result (<0.001) confirmed that UVA radiation successfully induced photoaging damage in HFF cells and activated the expression of key enzymes involved in matrix degradation. In the lotus embryo flavonoid treatment group, the expression of both MMP-1 and MMP-3 was effectively inhibited, revealing that lotus embryo flavonoids can exert a photoprotective effect on HFF cells by downregulating UVA-induced overexpression of MMP-1 and MMP-3, thereby effectively mitigating UV-induced extracellular matrix degradation. The test results are shown below. Figure 27 , Figure 28 .

[0170] Note: Figure 21 , Figure 23-28 The t-test was used to analyze each group. ### express p <0.001, correlation with the control group (no UVA irradiation, no sample treatment); *** express p<0.001, correlation with the treatment group (UVA irradiation, no sample treatment).

Claims

1. A method for preparing lotus embryo extract, characterized in that, Includes the following steps: Preparation of natural eutectic solvents; The extractant is obtained by mixing a natural eutectic solvent with water; The extractant was mixed with lotus germ powder, extracted by ultrasound, and then centrifuged and filtered to obtain lotus germ extract; The natural eutectic solvent comprises hydrogen bond acceptors and hydrogen bond donors, wherein the molar ratio of the hydrogen bond acceptors to the hydrogen bond donors is 1:3; The hydrogen bond acceptor is proline; the hydrogen bond donor is glycerol. The mass ratio of the natural eutectic solvent to water in the extractant is (6.5-7.5):(2.5-3.5). The ratio of lotus germ powder to extractant is 1:(20-40)g / mL; The ultrasonic extraction time is 40-80 min; The ultrasonic power extracted by ultrasound is 240-360 W.

2. The method for preparing lotus embryo extract according to claim 1, characterized in that, The pH of the natural eutectic solvent is 2.27-9.01, the viscosity of the natural eutectic solvent at 25°C is 3.61-18.14 mPa·s, the conductivity of the natural eutectic solvent is 14.88-4519 S / m, and the polarity of the natural eutectic solvent is 47.65-49.58 kcal / mol.

3. The method for preparing lotus embryo extract according to claim 1, characterized in that, The mass ratio of the natural eutectic solvent to water in the extractant is at least one of 6.5:3.5, 7:3, or 7.5:2.

5.

4. The method for preparing lotus embryo extract according to claim 1, characterized in that, The ratio of lotus germ powder to extractant is at least one of 1:20 g / mL, 1:30 g / mL, or 1:40 g / mL.

5. The method for preparing lotus embryo extract according to claim 4, characterized in that, The ultrasonic extraction time is at least one of 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min or 80 min.

6. The method for preparing lotus embryo extract according to claim 1, characterized in that, The ultrasonic power of the ultrasonic extraction is 250-350W.

7. The method for preparing lotus embryo extract according to claim 1, characterized in that, The ultrasonic extraction temperature is at least one of 260W, 270W, 280W, 290W, 300W, 310W, 320W, 330W, or 340W.

8. The method for preparing lotus embryo extract according to claim 1, characterized in that, The temperature for ultrasonic extraction is 40-80℃.

9. A lotus germ extract, characterized in that, The lotus embryo extract was prepared using the method described in any one of claims 1-8.

10. An application of the lotus embryo extract according to claim 9, characterized in that, The lotus germ extract is used in cosmetics.

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