Plumulae platycladi extract, preparation method and application thereof

Abstract

The application relates to a lotus plumule extract, a preparation method thereof and application, and relates to the cosmetic field. The lotus plumule extract contains lotus plumule base and rutin and is obtained by extracting the lotus plumule by using a natural eutectic solvent. A cosmetic or skin external agent contains the lotus plumule extract. The lotus plumule extract prepared by the application can be applied to skin external agents and cosmetics and has good antioxidant, anti-inflammatory soothing, repair, improvement of mitochondrial function and anti-aging effects.

Description

Technical Field

[0001] This invention relates to the field of cosmetics, and more particularly to lotus seed heart extract, its preparation method, and its application in cosmetics. Background Technology

[0002] Alkaloids are one of the main active components of lotus seed heart. Currently, approximately 14 alkaloid compounds have been isolated and identified from lotus seed heart, mainly monobenzylisoquinolines, dibenzylisoquinolines, apophenes, and isoquinolines. Flavonoids are another major active component of lotus seed heart. Currently, approximately 30 flavonoid compounds have been isolated and identified from lotus seed heart, mainly flavonoids and their glycosides, and flavonols and their glycosides. There are currently no reports on the use of lotus seed heart extract to protect against UV damage, chemical irritants, and pollutants, or to soothe and repair the skin or improve mitochondrial dysfunction.

[0003] Traditional methods for extracting active ingredients from lotus seed hearts often require long extraction times and high extraction temperatures, which may lead to degradation of the active ingredients and further reduce the quality and efficacy of the extract. CN104744367B discloses a flash extraction method for lotus seed heart alkaloids, with an extraction time less than 4% of that required by traditional heating and reflux extraction. This method can extract lotusine, isolivineine, and methyllotusine. However, this method uses a flash extractor, which is cumbersome, requires specialized equipment, and uses highly toxic hexane and chloroform, which are harmful to human health and the environment. CN105343190B discloses a method for preparing lotus seed heart flavonoid extract, which can separate three chromatographic peaks and simultaneously obtain three flavonoid components. The flavonoid content and yield of the obtained extract are significantly higher than those of existing preparation methods. However, this method requires the use of organic solvents such as ethanol, ethyl acetate, and n-butanol, and the extraction temperature is high (70℃-80℃), which can significantly damage the flavonoid components. There are currently no reports on the use of NaDES to extract and prepare lotus seed heart extract, or on the application of NaDES-prepared lotus seed heart extract in cosmetics for its antioxidant, anti-inflammatory, soothing, repairing, mitochondrial function-improving, and anti-aging effects. Summary of the Invention

[0004] The purpose of this invention is to provide a lotus seed heart extract that contains both alkaloids and bioflavonoids, which can address complex skin problems with multiple targets when applied in cosmetics.

[0005] The present invention also aims to provide a method for preparing lotus seed heart extract, which simultaneously extracts alkaloids and bioflavonoids.

[0006] Another objective of this invention is to provide a cosmetic product for applying the lotus seed heart extract prepared in this invention to cosmetic products.

[0007] A lotus seed heart extract containing lotus seed heart alkaloid and rutin is obtained by extraction from lotus seed heart using a natural eutectic solvent. The natural eutectic solvent contains a hydrogen bond acceptor and a hydrogen bond donor. The hydrogen bond acceptor is at least one of phytosphingosine, glycerophosphoinositol choline salt, and betaine. The hydrogen bond donor is at least one of 2'-fucosylated lactose, lactobionic acid, panthenol, and 1,3-propanediol.

[0008] Preferably, the molar ratio of hydrogen bond acceptor to hydrogen bond donor is 5:1 to 1:6.

[0009] Preferably, the natural eutectic solvent is composed of phytosphingosine and 2'-fucosylated lactose, with a molar ratio of phytosphingosine to 2'-fucosylated lactose of 5:1; or, the natural eutectic solvent is composed of phytosphingosine and lactobionic acid, with a molar ratio of phytosphingosine to lactobionic acid of 2:1; or, the natural eutectic solvent is composed of glycerophosphoinositol choline salt and 2'-fucosylated lactose, with a molar ratio of glycerophosphoinositol choline salt to 2'-fucosylated lactose of 5:1; or, the natural eutectic solvent is composed of glycerophosphoinositol choline salt and lactobionic acid, with a molar ratio of glycerophosphoinositol choline salt to lactobionic acid of 1:1; or, the natural eutectic solvent is composed of betaine and lactobionic acid, with a molar ratio of betaine to lactobionic acid of 1:2.

[0010] A method for preparing lotus seed heart extract includes the following steps:

[0011] Step 1: Mix the hydrogen bond acceptor and the hydrogen bond donor, and stir continuously at 80℃-150℃ for 90-180 min to obtain a natural eutectic solvent. The hydrogen bond acceptor is at least one of phytosphingosine, glycerophosphoinositol choline salt, and betaine. The hydrogen bond donor is at least one of 2'-fucosylated lactose, lactobionic acid, panthenol, and 1,3-propanediol.

[0012] Step 2: Add water to the natural eutectic solvent to form an extraction solvent with a concentration of 40%-80%. Add lotus seed heart powder to the extraction solvent. The solid-liquid ratio of lotus seed heart powder to extraction solvent is 1g:5mL-1g:50mL. Perform ultrasonic extraction at room temperature for 30 minutes with an ultrasonic power of 200W-800W.

[0013] Step 3: Centrifuge, collect the supernatant and filter to obtain lotus seed heart extract.

[0014] As a preferred embodiment, in step 1, phytosphingosine and 2'-fucosylated lactose are mixed in a molar ratio of 5:1, or glycerophosphoinositol choline salt and 2'-fucosylated lactose are mixed in a molar ratio of 5:1, and stirred at 120°C for 120 min to obtain a natural eutectic solvent; in step 2, 100g of lotus seed heart powder is taken and added to the extraction solvent at a material-to-liquid ratio of 1g:30mL, wherein the concentration of the natural eutectic solvent in the extraction solvent is 60%, and the remainder is water.

[0015] As another preferred embodiment, in step 1, phytosphingosine and lactobionic acid are mixed at a molar ratio of 2:1, or glycerophosphoinositol choline salt and lactobionic acid are mixed at a molar ratio of 1:1, or betaine and lactobionic acid are mixed at a molar ratio of 1:2, and stirred at 120°C for 100 min to obtain a natural eutectic solvent; in step 2, 100g of lotus seed heart powder is taken and added to the extraction solvent at a material-to-liquid ratio of 1g:30mL, wherein the concentration of the natural eutectic solvent in the extraction solvent is 60%, and the remainder is water.

[0016] A cosmetic product containing a lotus seed heart extract prepared by a method for preparing a lotus seed heart extract according to the present invention.

[0017] Preferably, the lotus seed extract accounts for 0.01-10% of the weight of the cosmetic product.

[0018] A topical skin agent containing a lotus seed heart extract prepared by a method for preparing lotus seed heart extract.

[0019] Beneficial Effects: The preparation method of this invention is natural, green, and safe. The lotus seed heart extract prepared by this invention simultaneously yields alkaloids and bioflavonoids, which can target the dual stimulation of photodamage and neurogenic inflammation induced by both UV and chemical substances. It can act on the root cause of complex skin problems at multiple points, simultaneously resolving skin discomfort caused by both chemical and UV irritation. The lotus seed heart extract prepared by this invention can be used in topical skin agents and cosmetics, exhibiting good antioxidant, soothing, repairing, mitochondrial function improvement, and anti-wrinkle effects. Attached Figure Description

[0020] Figure 1 The HPLC chromatogram of the extract obtained in Example 1;

[0021] Figure 2 Typical graph of average fluorescence intensity of TRPV1;

[0022] Figure 3 Typical graph of average fluorescence intensity of CGRP;

[0023] Figure 4 Typical graph of average fluorescence intensity of calcium ions;

[0024] Figure 5 This is a typical image from a scratch test;

[0025] Figure 6 A typical graph showing the red / green average fluorescence intensity ratio of mitochondrial membrane potential;

[0026] Figure 7 Typical graph of average fluorescence intensity of mitochondrial ROS;

[0027] Figure 8 The appearance of emulsion 1 after 3 months under light, constant temperature of 5℃, constant temperature of 25℃, and constant temperature of 50℃;

[0028] Figure 9 The image shows the appearance of emulsion 2 after being exposed to light, constant temperature at 5℃, constant temperature at 25℃, and constant temperature at 50℃ for 3 months. Detailed Implementation

[0029] To make the technical means, creative features, objectives and effects of the invention easier to understand, the invention will be further explained below with reference to specific illustrations.

[0030] A lotus seed heart extract contains both flavonoids (including rutin) and alkaloids (including lotusine). This lotus seed heart extract is obtained from lotus seed hearts using a natural eutectic solvent (NaDES). The natural eutectic solvent contains hydrogen bond acceptors and hydrogen bond donors. The hydrogen bond acceptor is at least one selected from phytosphingosine, glycerophosphoinositol choline salt, and betaine; the hydrogen bond donor is at least one selected from 2'-fucosylated lactose, lactobionic acid, panthenol, and 1,3-propanediol. Preferably, the molar ratio of hydrogen bond acceptor to hydrogen bond donor is 5:1 to 1:6.

[0031] A method for preparing lotus seed heart extract includes the following steps:

[0032] Step 1: Mix hydrogen bond acceptor and hydrogen bond donor in a molar ratio of 5:1-1:6 (preferably 5:1) and stir continuously for 90-180 min at 80℃-150℃ (preferably 120℃) to obtain a natural eutectic solvent. The hydrogen bond acceptor is at least one of phytosphingosine, glycerophosphoinositol choline salt, and betaine. The hydrogen bond donor is at least one of 2'-fucosylated lactose, lactobionic acid, panthenol, and 1,3-propanediol.

[0033] Step 2: Add water to the natural eutectic solvent to form an extraction solvent with a concentration of 40%-80%. Add lotus seed heart powder to the extraction solvent. The solid-liquid ratio of lotus seed heart powder to the extraction solvent is 1g:5mL-1g:50mL (preferably 1:30g / mL). Perform ultrasonic extraction at room temperature for 30 minutes with an ultrasonic power of 200W-800W (preferably 400W).

[0034] Step 3: Centrifuge, collect the supernatant and filter to obtain lotus seed heart extract.

[0035] Example 1

[0036] A method for preparing lotus seed heart extract, comprising the following steps:

[0037] 1) Mix phytosphingosine and 2'-fucosylated lactose in a molar ratio of 5:1 and stir at 120°C for 120 min to obtain NaDES;

[0038] 2) Take 100g of lotus seed heart powder and add extraction solvent (NaDES concentration is 60%, the rest is water) at a material-to-liquid ratio of 1:30g / mL. Extract at room temperature using ultrasound for 30min.

[0039] 3) Centrifuge the extract, filter the supernatant to obtain lotus seed heart extract.

[0040] Example 2

[0041] Referring to the method for preparing lotus seed heart extract described in Example 1, the NaDES system was replaced with phytosphingosine and lactobionic acid (molar ratio 2:1), the time in step 1 was changed to 100 min, and the rest remained unchanged.

[0042] Example 3

[0043] Referring to the method for preparing lotus seed heart extract described in Example 1, the NaDES system was replaced with phytosphingosine and panthenol (molar ratio 1:2), the temperature in step 1 was changed to 100°C, and the rest remained unchanged.

[0044] Example 4

[0045] Referring to the method for preparing lotus seed heart extract described in Example 1, the NaDES system was replaced with phytosphingosine and 1,3-propanediol (molar ratio 1:6), and the temperature and time in step 1 were replaced with 80℃ and 90min, while the rest remained unchanged.

[0046] Example 5

[0047] Referring to the method for preparing lotus seed heart extract described in Example 1, the NaDES system was replaced with glycerophosphate inositol choline salt and 2'-fucosylated lactose (molar ratio 5:1), while the rest remained unchanged.

[0048] Example 6

[0049] Referring to the method for preparing lotus seed heart extract described in Example 1, the NaDES system was replaced with glycerophosphate inositol choline salt and lactobionic acid (molar ratio 1:1), the time in step 1 was changed to 100 min, and the rest remained unchanged.

[0050] Example 7

[0051] Referring to the method for preparing lotus seed heart extract described in Example 1, the NaDES system was replaced with glycerophosphate inositol choline salt and panthenol (molar ratio 1:2), the temperature in step 1 was changed to 100°C, and the rest remained unchanged.

[0052] Example 8

[0053] Referring to the method for preparing lotus seed heart extract described in Example 1, the NaDES system was replaced with glycerophosphate inositol choline salt and 1,3-propanediol (molar ratio 1:4), and the temperature and time in step 1 were replaced with 80℃ and 90min, while the rest remained unchanged.

[0054] Example 9

[0055] Referring to the method for preparing lotus seed heart extract described in Example 1, the NaDES system was replaced with betaine and 2'-fucosylated lactose (molar ratio 3:1), while the rest remained unchanged.

[0056] Example 10

[0057] Referring to the method for preparing lotus seed heart extract described in Example 1, the NaDES system was replaced with betaine and lactobionic acid (molar ratio 1:2), the time in step 1 was changed to 100 min, and the rest remained unchanged.

[0058] Example 11

[0059] Referring to the method for preparing lotus seed heart extract described in Example 1, the NaDES system was replaced with betaine and panthenol (molar ratio 1:2), the temperature in step 1 was changed to 100°C, and the rest remained unchanged.

[0060] Example 12

[0061] Referring to the method for preparing lotus seed heart extract described in Example 1, the NaDES system was replaced with betaine and 1,3-propanediol (molar ratio 1:3), and the temperature and time in step 1 were replaced with 80℃ and 90min, respectively, while the rest remained unchanged.

[0062] Comparative Example 1

[0063] Referring to the method for preparing lotus seed heart extract described in Example 1, the extraction solvent was replaced with a 70% aqueous ethanol solution, while the rest remained unchanged.

[0064] Comparative Example 2

[0065] A lotus seed heart extract was prepared according to the existing technology CN105343190B (a method for preparing lotus seed heart flavonoid extract). This method uses a high extraction temperature (70-80℃), and the resulting extract is prone to containing a large number of impurities, such as chlorophyll, resin, tannins, proteins, and mucilage. This invention can efficiently extract rutin and lotus seed heart alkaloids at room temperature, avoiding the degradation of active ingredients caused by high temperatures.

[0066] Comparative Example 3

[0067] A lotus seed heart extract was prepared according to the existing technology CN1117735C (Preparation method for extracting and purifying isolidinine and lotusine from lotus seed heart). The extraction solvent used in this method is methanol, which is highly toxic to humans. The NaDES used in this invention is composed of natural cosmetic active ingredients, is non-toxic, and biodegradable, avoiding the residual risks and environmental pollution problems of traditional organic solvents (such as methanol and high-concentration ethanol). NaDES does not need to be removed from the extraction system and can be directly used in cosmetics.

[0068] Extraction rate: According to the Pharmacopoeia of the People's Republic of China: 2020 edition, the extraction rate was determined by high performance liquid chromatography (General Rule 0512). The lotus seed heart extract was diluted with the mobile phase and filtered through a membrane. The extract was then detected by high performance liquid chromatography. Rutin and lotus seed alkaloid standards were used as references. The contents of rutin and lotus seed alkaloid were obtained based on the peak area and the predetermined standard curve. The extraction rates of rutin and lotus seed alkaloid were then calculated.

[0069] Rutin extraction rate (mg / g)

[0070] Lotus seed extract rate (mg / g)

[0071] In the formula, Y1 and Y2 are the mass concentrations (mg / mL) of rutin and lotusine in the lotus seed heart extract, D is the volume of the extract (mL), and M is the mass (g) of the lotus seed heart.

[0072] Table 1 is a comparison table of the extraction rate test results of the effective components in each embodiment.

[0073]

[0074] As shown in Table 1, the extraction rates of rutin and lotusine in Example 1 were increased by 74.6% and 197.4% respectively compared with Comparative Example 1; the extraction rates of rutin and lotusine in Example 2 were increased by 45.2% and 222.2% respectively compared with Comparative Example 1. In all examples, the extraction rates of rutin and lotusine were significantly higher than those of 70% ethanol extraction, achieving the goal of fully extracting lotus seed heart raw materials.

[0075] Cytotoxicity test: Step 1. Seed cells in 96-well plates and incubate at 37°C in 5% CO2 for 24 h. Grouping is shown in Table 2 below.

[0076] Table 2 is a summary table of the cytotoxicity test groupings.

[0077]

[0078] Step 2. Sample feeding: Add fresh culture medium containing the corresponding concentration of sample to the sample group. Replace the normal control group and blank control group with fresh culture medium and continue to incubate at 37℃ and 5% CO2 for 24h.

[0079] Step 3. After incubation, add MTT solution to each well and continue culturing for 4 hours.

[0080] Step 4. Remove the culture medium, add DMSO solution, shake to mix, and measure the absorbance value at 490 nm.

[0081]

[0082] OD490: Absorbance value at 490 nm.

[0083] Table 3 shows the cytotoxicity test results for each example:

[0084]

[0085] All examples showed no cytotoxicity at a concentration of 0.2%, indicating that they are safe and non-irritating as cosmetic ingredients.

[0086] HPLC chromatogram testing: The determination was performed according to the high-performance liquid chromatography method (General Rule 0512), with a detection wavelength of 282 nm. The test results of the extract obtained in Example 1 are as follows... Figure 1 As shown.

[0087] Efficacy test:

[0088] Experiment 1: Relative expression levels of IL-1α and IL-6 genes

[0089] Step 1. Seed cells in 6-well plates and incubate at 37°C with 5% CO2 for 24 hours.

[0090] Table 4 is a summary table of experimental groups for the relative expression levels of IL-1α and IL-6 genes.

[0091]

[0092] Step 2. After incubation, remove the culture medium, wash with D-Hanks 1-2 times, and then expose the sample group, positive control group, and model control group to UVB irradiation at 80 mJ / cm². 2 .

[0093] Step 3. After modeling, the normal control group was replaced with fresh culture medium, the model control group was replaced with fresh culture medium containing CAP (15 μmol / L), the positive control group was added with fresh culture medium containing CAP (15 μmol / L) and dexamethasone (0.01%), and the sample group was added with fresh culture medium containing CAP (15 μmol / L) and the sample. Three biological replicates were performed and incubated at 37°C and 5% CO2 for 24 h.

[0094] Step 4. Extract total RNA from each experimental group, synthesize cDNA, and use q-PCR to detect the gene expression of β-actin and the target gene.

[0095] Step 5. Using β-actin as an internal reference for gene expression, calculate the relative RNA expression level of the target gene. , , , In the formula: Q: relative expression levels of IL-1α and IL-6 genes.

[0096] Table 5 shows the comparison of the relative expression levels of the IL-1α gene.

[0097]

[0098] Table 6 shows the comparison of the relative expression levels of the IL-6 gene.

[0099]

[0100] The above results indicate that lotus seed heart extract has a soothing effect.

[0101] Experiment 2: TRPV1 immunofluorescence and CGRP immunofluorescence

[0102] Step 1. Seed cells in 12-well plates and incubate at 37°C with 5% CO2 for 24 hours.

[0103] Table 7 is a summary table of immunofluorescence experiment groups.

[0104]

[0105] Step 2. After incubation, remove the culture medium, wash with D-Hanks 1-2 times, and then expose the sample group, positive control group, and model control group to UVB irradiation at 80 mJ / cm². 2 .

[0106] Step 3. After modeling, the normal control group was replaced with fresh culture medium, the model control group was replaced with fresh culture medium containing CAP (15 μmol / L), the positive control group was added with fresh culture medium containing CAP (15 μmol / L) and 4-tert-butylcyclohexanol (15.6 μg / mL), and the sample group was added with fresh culture medium containing CAP (15 μmol / L) and the sample. Three biological replicates were performed, and the samples were incubated at 37°C and 5% CO2 for 24 h.

[0107] Step 4. After incubation, discard the solution and proceed with washing, fixation, permeabilization, and antibody incubation. Take images under a fluorescence microscope and use ImageJ software for quantitative analysis of the immunofluorescence results.

[0108]

[0109] In the formula: S: average fluorescence intensity of TRPV1 and average fluorescence intensity of CGRP.

[0110] Figure 2 This is a typical graph showing the average fluorescence intensity of TRPV1.

[0111] Table 8 shows the comparison of TRPV1 fluorescence detection results.

[0112]

[0113] Figure 3 This is a typical graph showing the average fluorescence intensity of CGRP.

[0114] Table 9 shows the comparison of CGRP fluorescence detection results.

[0115]

[0116] The above results indicate that lotus seed heart extract has a soothing effect.

[0117] Experiment 3: Calcium Ion Flow-Fluorescence Developing Method

[0118] Step 1. Seed cells in 12-well plates and incubate at 37°C with 5% CO2 for 24 hours.

[0119] Table 10 is a summary table of experimental groups.

[0120]

[0121] Step 2. After incubation, remove the culture medium, replace the normal control group with fresh culture medium, replace the model control group with fresh culture medium containing CAP (15 μmol / L), add fresh culture medium containing CAP (15 μmol / L) and 4-tert-butylcyclohexanol (15.6 μg / mL) to the positive control group, and add fresh culture medium containing CAP (15 μmol / L) and the sample to the sample group. Perform 3 biological replicates and continue incubation at 37℃ and 5% CO2 for 24 h.

[0122] Step 3. After incubation, discard the solution and proceed with washing, fixation, permeabilization, and antibody incubation. Take images under a fluorescence microscope and use ImageJ software for quantitative analysis of the immunofluorescence results.

[0123]

[0124] In the formula: S: average fluorescence intensity of calcium ions.

[0125] Figure 4 This is a typical graph showing the average fluorescence intensity of calcium ions.

[0126] Table 11 shows a comparison of the average fluorescence intensity detection results of calcium ions.

[0127]

[0128] Compared with the model control group, the relative expression levels of IL-1α and IL-6 genes in the sample group were significantly downregulated, with improvement rates of 22% and 30%, respectively. The average fluorescence intensity of TRPV1, CGRP, and calcium ions were significantly reduced compared with the model control group, with improvement rates of 18%, 21%, and 36%, respectively, revealing that lotus seed heart extract has a soothing effect.

[0129] Experiment 4: Healing Area

[0130] Step 1. Seed cells in 12-well plates and incubate at 37°C with 5% CO2 for 24±2 hours.

[0131] Table 12 is a summary table of experimental groups for healing area.

[0132]

[0133] Step 2. Scratching: Wash with D-Hanks 1-2 times. Add fresh culture medium to the normal control wells, add fresh culture medium containing the corresponding concentration of the sample to the sample group, and add fresh culture medium containing EGF to the positive control group. Incubate at 37℃ and 5% CO2 for 24±2h. Take pictures at 0h and 24h after drug administration and record the scratch area.

[0134] Step 3. ImageJ analyzes the scratch area of ​​each group and calculates the healing area (Y).

[0135] Y: Healing area.

[0136] Figure 5 This is a classic diagram of a scratch test.

[0137] Table 13 shows a comparison of the results of healing area detection.

[0138]

[0139] Compared with the normal control group, the healing area of ​​the sample group was significantly increased, with an improvement rate of 49%; the relative expression level of S100A7 gene was significantly downregulated compared with the model control group, with an improvement rate of 22%, revealing that lotus seed heart extract has a repairing effect.

[0140] Experiment 5: Relative Expression Level of S100A7 Gene

[0141] Step 1. Seed cells in 6-well plates and incubate at 37°C with 5% CO2 for 24 hours.

[0142] Table 14 is a summary table of experimental groups for the relative expression level of the S100A7 gene.

[0143]

[0144] Step 2. After incubation, remove the culture medium, wash with D-Hanks 1-2 times, and then expose the sample group, positive control group, and model control group to UVB irradiation at 80 mJ / cm². 2 .

[0145] Step 3. After modeling, the normal control group was replaced with fresh culture medium, the model control group was replaced with fresh culture medium containing CAP (15 μmol / L), the positive control group was added with fresh culture medium containing CAP (15 μmol / L) and ectoine (0.05%), and the sample group was added with fresh culture medium containing CAP (15 μmol / L) and the sample. Three biological replicates were performed and incubated at 37°C and 5% CO2 for 24 h.

[0146] Step 4. Extract total RNA from each experimental group, synthesize cDNA, and use q-PCR to detect the gene expression of β-actin and the target gene.

[0147] Step 5. Using β-actin as an internal reference for gene expression, calculate the relative RNA expression level of the target gene. , , , In the formula: Q: relative expression level of S100A7 gene.

[0148] Table 15 shows the comparison of the relative expression levels of the S100A7 gene.

[0149]

[0150] The above results indicate that lotus seed heart extract has restorative effects.

[0151] Experiment 6: Mitochondrial membrane potential, red and green fluorescence intensity, and mitochondrial ROS fluorescence intensity

[0152] Step 1. Seed cells in 12-well plates and incubate at 37°C with 5% CO2 for 24 hours.

[0153] Table 16 is a summary table of fluorescence intensity experiment groupings:

[0154]

[0155] Step 2. After incubation, remove the culture medium, wash with D-Hanks 1-2 times, and then expose the sample group, positive control group, and model control group to UVB irradiation at 80 mJ / cm². 2 .

[0156] Step 3. After modeling, the normal control group was replaced with fresh culture medium, the model control group was replaced with fresh culture medium containing CAP (15 μmol / L), the positive control group was added with fresh culture medium containing CAP (15 μmol / L) and EGCG (0.002%), and the sample group was added with fresh culture medium containing CAP (15 μmol / L) and the sample. Three biological replicates were performed and incubated at 37°C and 5% CO2 for 24 h.

[0157] Step 4. After incubation, stain with JC-1 and observe and photograph under a fluorescence microscope.

[0158] Step 5. ImageJ analyzes the fluorescence intensity (S) of each group and calculates the red / green average fluorescence intensity ratio of the mitochondrial membrane potential.

[0159]

[0160]

[0161] In the formula: P: the ratio of red to green average fluorescence intensity of mitochondrial membrane potential.

[0162] Step 6. After incubation, discard the solution, stain with the Mitoso™ Red mitochondrial superoxide detection kit, take pictures with a fluorescence microscope, and analyze the fluorescence intensity using ImageJ software.

[0163] In the formula: P: average fluorescence intensity of mitochondrial ROS.

[0164] Figure 6 This is a typical graph showing the red / green average fluorescence intensity ratio of mitochondrial membrane potential.

[0165] Table 17 compares the results of red-green fluorescence intensity detection of mitochondrial membrane potential.

[0166]

[0167] Figure 7 This is a typical graph showing the average fluorescence intensity of mitochondrial ROS.

[0168] Table 18 shows a comparison of mitochondrial ROS fluorescence intensity detection results.

[0169]

[0170] The results showed that the red / green average fluorescence intensity ratio of mitochondrial membrane potential in the sample group was significantly upregulated compared with the model control group, with an improvement rate of 99%; the average fluorescence intensity of mitochondrial ROS in the sample plant extract group was significantly downregulated compared with the model control group, with an improvement rate of 31%; and the relative expression level of CI standard deviation 2 gene in the sample plant extract group was significantly upregulated compared with the model control group, with an improvement rate of 43%. These results revealed that lotus seed heart extract has the effects of promoting cell energy, anti-oxidation, and improving mitochondrial function.

[0171] Experiment 7: CI Standard Deviation 2 Gene Relative Expression Level

[0172] Step 1. Seed cells in 6-well plates and incubate at 37°C with 5% CO2 for 24 hours.

[0173] Table 19 is a summary table of experimental groups for CI standard deviation and relative gene expression levels.

[0174]

[0175] Step 2. After incubation, remove the culture medium, wash with D-Hanks 1-2 times, and then expose the sample group, positive control group, and model control group to UVB irradiation at 80 mJ / cm². 2 .

[0176] Step 3. After modeling, the normal control group was replaced with fresh culture medium, the model control group was replaced with fresh culture medium containing CAP (15 μmol / L), the positive control group was added with fresh culture medium containing CAP (15 μmol / L) and EGCG (0.002%), and the sample group was added with fresh culture medium containing CAP (15 μmol / L) and the sample. Three biological replicates were performed and incubated at 37°C and 5% CO2 for 24 h.

[0177] Step 4. Extract total RNA from each experimental group, synthesize cDNA, and use q-PCR to detect the gene expression of β-actin and the target gene.

[0178] Step 5. Using β-actin as an internal reference for gene expression, calculate the relative RNA expression level of the target gene.

[0179] , , , In the formula: Q: CI standard deviation 2 gene relative expression level.

[0180] Table 20 shows the comparison of the results of CI standard deviation and relative gene expression levels.

[0181]

[0182] The above results indicate that lotus seed heart extract has the effect of improving mitochondrial function.

[0183] A cosmetic product containing a lotus seed heart extract prepared by the method of the present invention. Preferably, the lotus seed heart extract accounts for 0.01-10% of the weight of the cosmetic product. Preferably, the cosmetic product is a toner, lotion, cream, mask, gel, or serum. A topical skin agent containing a lotus seed heart extract prepared by the method of the present invention. Preferably, the topical skin agent is an ointment, cream, gel, lotion, film-forming agent, or solution.

[0184] Stability test of lotus seed heart extract: The lotus seed heart extracts prepared in Examples 1 and 2 were added to the emulsion at an amount of 0.2%, as shown in the table below.

[0185] Table 21 shows the comparison of emulsion components.

[0186]

[0187] Emulsion 1 and Emulsion 2 were placed under light, constant temperature of 5℃, constant temperature of 25℃, and constant temperature of 50℃ for 3 months, respectively. Figure 7 The images show the appearance of emulsion 1 after 3 months of exposure to light, constant temperature at 5°C, constant temperature at 25°C, and constant temperature at 50°C. Figure 8 The images show the appearance of emulsion 2 after 3 months of exposure to light, constant temperature at 5℃, constant temperature at 25℃, and constant temperature at 50℃. Stability results indicate that emulsions 1 and 2 showed slight discoloration under light exposure, but remained almost unchanged under the other conditions.

[0188] This invention targets the dual stimulation of photodamage and neurogenic inflammation induced by both UV radiation and chemical substances. The lotus seed heart extract effectively alleviates neurogenic inflammation and reduces skin redness and sensitivity by inhibiting the relative expression of TRPV1 and CGRP and the release of inflammatory factors. Simultaneously, it improves mitochondrial membrane potential, reduces ROS levels, protects cellular energy metabolism, and resists photodamage. Furthermore, the extract accelerates cell migration and promotes skin barrier repair. In summary, the lotus seed heart extract exhibits multiple effects, including soothing inflammation, maintaining mitochondrial homeostasis, and barrier repair. This invention also demonstrates its repairing effects through HaCaT cell scratch assays. By detecting the relative expression level of the S100A7 gene and using a HaCaT cell model stimulated by UVB and capsaicin, its inhibitory effects on TRPV1, calcium ion influx, CGRP, IL-1α, and IL-6 are demonstrated, indicating its anti-photodamage, anti-inflammatory, and soothing effects. Furthermore, by improving mitochondrial function and detecting ROS scavenging activity and CI standard deviation 2 gene relative expression levels, its anti-wrinkle and anti-aging effects are confirmed.

[0189] The foregoing has shown and described the basic principles, main features, and advantages of the invention. Those skilled in the art should understand that the invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made without departing from the spirit and scope of the invention, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection of the invention is defined by the appended claims and their equivalents.

Claims

1. A lotus seed heart extract, characterized in that, Containing lotusine and rutin, it is extracted from lotus seed hearts using a natural eutectic solvent. The natural eutectic solvent contains hydrogen bond acceptors and hydrogen bond donors. The hydrogen bond acceptor is at least one of phytosphingosine, glycerophosphoinositol choline salt, and betaine. The hydrogen bond donor is at least one of 2'-fucosylated lactose, lactobionic acid, panthenol, and 1,3-propanediol.

2. The lotus seed heart extract according to claim 1, characterized in that, The molar ratio of hydrogen bond acceptor to hydrogen bond donor is 5:1 to 1:

6.

3. The lotus seed heart extract according to claim 2, characterized in that, The natural eutectic solvent is composed of phytosphingosine and 2'-fucosylated lactose, with a molar ratio of phytosphingosine to 2'-fucosylated lactose of 5:

1. Alternatively, the natural eutectic solvent is composed of phytosphingosine and lactobionic acid, with a molar ratio of phytosphingosine to lactobionic acid of 2:

1. Alternatively, the natural eutectic solvent is composed of glycerophosphoinositol choline salt and 2'-fucosylated lactose, with a molar ratio of glycerophosphoinositol choline salt to 2'-fucosylated lactose of 5:1; Alternatively, the natural eutectic solvent is composed of glycerophosphoinositol choline salt and lactobionic acid, with a molar ratio of glycerophosphoinositol choline salt to lactobionic acid of 1:

1. Alternatively, the natural eutectic solvent is composed of betaine and lactobionic acid in a molar ratio of 1:

2.

4. A method for preparing lotus seed heart extract, characterized in that, Includes the following steps: Step 1: Mix the hydrogen bond acceptor and the hydrogen bond donor, and stir continuously at 80℃-150℃ for 90-180 min to obtain a natural eutectic solvent. The hydrogen bond acceptor is at least one of phytosphingosine, glycerophosphoinositol choline salt, and betaine. The hydrogen bond donor is at least one of 2'-fucosylated lactose, lactobionic acid, panthenol, and 1,3-propanediol. Step 2: Add water to the natural eutectic solvent to form an extraction solvent with a concentration of 40%-80%. Add lotus seed heart powder to the extraction solvent. The solid-liquid ratio of lotus seed heart powder to extraction solvent is 1g:5mL-1g:50mL. Perform ultrasonic extraction at room temperature for 30 minutes with an ultrasonic power of 200W-800W. Step 3: Centrifuge, collect the supernatant and filter to obtain lotus seed heart extract.

5. The method for preparing lotus seed heart extract according to claim 4, characterized in that, The molar ratio of hydrogen bond acceptor to hydrogen bond donor is 5:1 to 1:

6.

6. The method for preparing lotus seed heart extract according to claim 5, characterized in that, In step 1, phytosphingosine and 2'-fucosylated lactose are mixed in a molar ratio of 5:1, or glycerophosphoinositol choline salt and 2'-fucosylated lactose are mixed in a molar ratio of 5:1, and stirred at 120°C for 120 min to obtain a natural eutectic solvent. In step 2, take 100g of lotus seed heart powder and add it to the extraction solvent at a material-to-liquid ratio of 1g:30mL. The concentration of the natural eutectic solvent in the extraction solvent is 60%, and the remainder is water.

7. The method for preparing lotus seed heart extract according to claim 5, characterized in that, In step 1, phytosphingosine and lactobionic acid are mixed in a molar ratio of 2:1, or glycerophosphoinositol choline salt and lactobionic acid are mixed in a molar ratio of 1:1, or betaine and lactobionic acid are mixed in a molar ratio of 1:

2. The mixture is stirred at 120°C for 100 min to obtain a natural eutectic solvent. In step 2, take 100g of lotus seed heart powder and add it to the extraction solvent at a material-to-liquid ratio of 1g:30mL. The concentration of the natural eutectic solvent in the extraction solvent is 60%, and the remainder is water.

8. A cosmetic product, characterized in that, The extract contains a lotus seed heart extract prepared by the method described in any one of claims 4-7.

9. A cosmetic product according to claim 8, characterized in that, The lotus seed heart extract accounts for 0.01-10% of the weight of the cosmetic product by weight.

10. A topical skin agent, characterized in that, The extract contains a lotus seed heart extract prepared by the method described in any one of claims 4-7.

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