Cardiospermum halicacabum extract and application of cardiospermum halicacabum extract in preparation of cosmetics or medicines for relieving light aging damage

Abstract

The invention discloses a cardiospermum halicacabum extract, which is prepared by the following steps: (1) taking cardiospermum halicacabum powder, adding 70-90% ethanol aqueous solution, heating and refluxing for 40-50 minutes under the condition of 65-71 DEG C, and filtering to obtain filtrate and decoction dregs; (2) flushing the medicine residues with a 70-90% ethanol aqueous solution to obtain a flushing solution, combining the flushing solution with the filtrate obtained in the step (1) to obtain a combined filtrate, adding the 70-90% ethanol aqueous solution into the combined filtrate to complement the weight lost due to ethanol volatilization, shaking uniformly, and filtering to obtain a supernatant; and (3) enabling the supernate obtained in the step (2) to pass through a microfiltration membrane with the size of 0.45 mu m. The preparation method of the cardiospermum halicacabum extract is simple, and the content of myricetin, luteolin and apigenin in the cardiospermum halicacabum extract is high. The invention further discloses application of the cardiospermum halicacabum extract in preparation of cosmetics or medicines for relieving light aging damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and particularly relates to Cardiospermum halicacabum extract and its application in preparing cosmetics or drugs for reducing photoaging damage. Background Art

[0002] Ultraviolet (UV) exposure, especially UVB, is the main cause of skin photoaging. Ultraviolet rays are mainly divided into UVA (320 - 400 nm), UVB (280 - 320 nm), and UVC (200 - 280 nm) according to wavelength. The sunburn ability of UVB is more than 1000 times higher than that of UVA, and it can cause sunburn. Reactive oxygen species (ROS) induced by ultraviolet rays, such as singlet oxygen, superoxide anion, and hydrogen peroxide, can damage proteins, DNA, and lipids, promote the expression of matrix metalloproteinases (MMPs), destroy collagen and elastic fibers, and accelerate skin aging. Therefore, it is necessary to externally supplement antioxidants to scavenge ROS, inhibit lipid peroxidation reactions, and inhibit the generation of free radicals by combining with transition metals through the Fenton reaction.

[0003] In the prior art, externally supplemented non - enzymatic antioxidants are mainly synthesized by photosynthetic plants. Therefore, it is crucial to seek natural plant extracts that can protect the skin from ultraviolet damage and reduce photoaging damage. Summary of the Invention

[0004] The purpose of the present invention is to provide Cardiospermum halicacabum extract and its application in preparing cosmetics or drugs for reducing photoaging damage. By developing Cardiospermum halicacabum extract and using the prepared Cardiospermum halicacabum extract for reducing photoaging damage, the purpose of using the natural plant component Cardiospermum halicacabum extract to prepare cosmetics or drugs for reducing photoaging damage is achieved.

[0005] Cardiospermum halicacabum L. is a perennial plant of the Sapindaceae family, widely distributed in tropical and subtropical regions of Asia and Africa. In the prior art, Cardiospermum halicacabum extract has the effects of inhibiting the activity of collagen - degrading enzymes, promoting wound healing, antibacterial, and anti - inflammatory, and is used in traditional Indian medicine to treat rheumatism, low back pain, neurological diseases, orchitis, and epididymitis.

[0006] According to the first aspect of the present invention, there is provided Cardiospermum halicacabum extract, which is prepared by the following steps: (1) Take Cardiospermum halicacabum powder, add an ethanol aqueous solution of 70 - 90%, heat - reflux at 65 - 71 °C for 40 - 50 min, filter to obtain a filtrate and medicinal residues; (2) Rinse the medicinal residues with 70 - 90% ethanol aqueous solution to obtain a rinsing solution. Combine the rinsing solution with the filtrate obtained in step (1) to get a combined filtrate. Then add 70 - 90% ethanol aqueous solution to the combined filtrate to make up for the weight loss due to ethanol volatilization. Shake well and filter to obtain a supernatant. (3) Filter the supernatant obtained in step (2) through a 0.45 μm microporous membrane to obtain the product.

[0007] The preparation method of the Cardiospermum halicacabum L. extract of the present invention is simple. Using Cardiospermum halicacabum L. as the raw material, through heating and refluxing with 70 - 90% ethanol aqueous solution as the extraction solution, filtering to obtain a filtrate and medicinal residues. By combining the filtrate and the rinsing solution obtained from washing the medicinal residues to get a combined filtrate, and then using a microporous membrane to filter the supernatant obtained from filtering the combined filtrate to prepare the Cardiospermum halicacabum L. extract. The Cardiospermum halicacabum L. extract prepared by the preparation method of the present invention contains high contents of myricitrin, luteolin and apigenin, and the contents of myricitrin, luteolin, apigenin and the total extraction value are much higher than those of the Cardiospermum halicacabum L. extracts prepared with other ethanol concentrations, showing good application potential in reducing photoaging damage.

[0008] In some embodiments, in step (1), the mass ratio of the Cardiospermum halicacabum L. powder to the volume of 70 - 90% ethanol aqueous solution is 1:(25 - 35) g / mL.

[0009] In some embodiments, in step (1), the mass ratio of the Cardiospermum halicacabum L. powder to the volume of 70 - 90% ethanol aqueous solution is 1:30 g / mL.

[0010] In some embodiments, the mass ratio of the Cardiospermum halicacabum L. powder to the volume of 70 - 90% ethanol aqueous solution used for rinsing the medicinal residues is 1:(3 - 5) g / mL.

[0011] It should be noted that in step (2), the purpose of adding 70 - 90% ethanol aqueous solution to the combined filtrate is to make up for the weight loss due to ethanol volatilization, and the addition amount of 70 - 90% ethanol aqueous solution is determined according to the volatilization amount of ethanol during the preparation of the Cardiospermum halicacabum L. extract.

[0012] In some embodiments, in step (3), after filtering the supernatant through a 0.45 μm microporous membrane, lyophilization is also carried out. Specifically, after filtering the supernatant through a 0.45 μm microporous membrane, lyophilization is carried out under the condition of - 45~ - 35°C.

[0013] According to the second aspect of the present invention, there is provided the application of the above - mentioned Cardiospermum halicacabum L. extract in the preparation of cosmetics or drugs for reducing photoaging damage. The Cardiospermum halicacabum L. extract has antioxidant activity and can repair photoaged cells. Therefore, it can be used as an active ingredient in cosmetics or drugs for reducing photoaging damage to prepare cosmetics or drugs for reducing photoaging damage.

[0014] In some embodiments, the cosmetics or drugs for reducing photoaging damage are cosmetics or drugs having one or more of the following effects: (a) antioxidant activity; (b) improving the cell proliferation dysfunction of photoaged cells; (c) reducing the oxidative damage of photoaged cells.

[0015] In some embodiments, the cosmetics or drugs for reducing photoaging damage are cosmetics or drugs having one or more of the following effects: (a) scavenging free radicals; (b) reducing ability; (c) increasing the cell viability of photoaged cells; (d) preventing photoaged cells from arresting at the S phase; (e) reducing the ROS level in photoaged cells.

[0016] It should be noted that the reducing ability of the cosmetics or drugs for reducing photoaging damage means that the cosmetics or drugs for reducing photoaging damage have the ability to reduce oxidized substances (such as high-valent metal ions, free radicals, etc.) to low-valent states by providing electrons or hydrogen atoms, which is one of the important indicators for evaluating antioxidant activity.

[0017] In some embodiments, the photoaged cells are photoaged cells induced by UVB. Specifically, the photoaged cells are photoaged human skin fibroblasts (HSF cells) induced by UVB.

[0018] In some embodiments, the cosmetics for reducing photoaging damage are at least one of lotion, cream, essence, freeze-dried powder, paste, aqueous solution, facial mask, and spray.

[0019] The beneficial effects of the present invention include: (1) The preparation method of the Cardiospermum halicacabum extract of the present invention is simple. The Cardiospermum halicacabum extract prepared by this preparation method contains high contents of myricitrin, luteolin, and apigenin, and the contents of myricitrin, luteolin, and apigenin and the total extraction value are much higher than those of the Cardiospermum halicacabum extracts prepared with other ethanol concentrations, showing good application potential in reducing photoaging damage.

[0020] (2) The Cardiospermum halicacabum extract of the present invention can be used as an active ingredient in cosmetics or drugs for reducing photoaging damage to prepare cosmetics or drugs for reducing photoaging damage. The cosmetics or drugs for reducing photoaging damage have antioxidant activity and can repair photoaged cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 are HPLC diagrams of different solutions. Among them, Diagram A is the HPLC diagram of the test solution in high-performance liquid chromatography detection, Diagram B is the HPLC diagram of the reference solution in high-performance liquid chromatography detection, and Diagram C is the HPLC diagram of the blank solvent in high-performance liquid chromatography detection. In Diagram A and Diagram B, 1 is the chromatographic peak of myricitrin, 2 is the chromatographic peak of luteolin, and 3 is the chromatographic peak of apigenin; Figure 2 HPLC chromatograms for the system durability test. Among them, Figure A is the HPLC chromatogram of the Inertsil ODS-SP-C18 column for the system durability test, Figure B is the HPLC chromatogram of the Diamonsil-C18 column for the system durability test, and Figure C is the HPLC chromatogram of the InertSustain AQ-C18 column for the system durability test. In Figure C, 1 is the chromatographic peak of myricitrin, 2 is the chromatographic peak of luteolin, and 3 is the chromatographic peak of apigenin; Figure 3 Total extracted ion current chromatograms. Among them, Figure A is the total extracted ion current chromatogram of the Cardiospermum halicacabum extract obtained in Example 1 of the present invention in the positive ion mode, and Figure B is the total extracted ion current chromatogram of the Cardiospermum halicacabum extract obtained in Example 1 of the present invention in the negative ion mode; Figure 4 The DPPH radical scavenging rate of the Cardiospermum halicacabum extract in the antioxidant activity test of the Cardiospermum halicacabum extract; Figure 5 The ABTS radical scavenging rate of the Cardiospermum halicacabum extract in the antioxidant activity test of the Cardiospermum halicacabum extract; Figure 6 The ferric reducing antioxidant power of the Cardiospermum halicacabum extract in the antioxidant activity test of the Cardiospermum halicacabum extract; Figure 7 OD detection results of each group of HSF cells induced by UVB-induced photoaging in the test of the effect of the Cardiospermum halicacabum extract on the survival rate of HSF cells induced by UVB-induced photoaging; Figure 8 Cell cycle histograms of HSF cells detected by PI single staining combined with flow cytometry in the test of the effect of the Cardiospermum halicacabum extract on the cell cycle of HSF cells induced by UVB-induced photoaging. Among them, Figure A is the cell cycle histogram of the normal group, Figure B is the cell cycle histogram of the model group, and Figure C is the cell cycle histogram of the drug administration group; Figure 9 In the test of the effect of the Cardiospermum halicacabum extract on the cell cycle of HSF cells induced by UVB-induced photoaging, from Figure 8 The proportions of G0 / G1 phase cells and S phase cells in each group of HSF cells obtained from the results; Figure 10 FITC-A diagrams of each group in the test of the effect of the Cardiospermum halicacabum extract on the ROS level of HSF cells induced by UVB-induced photoaging. Among them, Figure A is the FITC-A diagram of the blank group, Figure B is the FITC-A diagram of the model group, Figure C is the FITC-A diagram of the VE group, Figure D is the FITC-A diagram of the 25 μg / mL CHE group, Figure E is the FITC-A diagram of the 50 μg / mL CHE group, and Figure F is the FITC-A diagram of the 100 μg / mL CHE group; Figure 11ROS levels in HSF cells of each group in the experiment on the effect of Cardiospermum halicacabum extract on ROS levels in UVB-induced photoaged HSF cells; Figure 12 Detection results of fluorescence signals of HSF cells in each group in the experiment on the effect of Cardiospermum halicacabum extract on ROS levels in UVB-induced photoaged HSF cells. Detailed implementation manners

[0022] The present invention will be further described in detail below with reference to the accompanying drawings, but the implementation manners of the present invention are not limited thereto. The raw materials and reagents involved in the following examples can be obtained from commercial channels. The process steps not disclosed in the examples are prior art.

[0023] In the present invention, Cardiospermum halicacabum is purchased from Guilin, Guangxi, and is identified by Chief Pharmacist Yuan Xiaohong of Guangdong Provincial Hospital of Traditional Chinese Medicine as the dried whole herb of the plant Cardiospermum halicacabum L. of the Sapindaceae family.

[0024] Example 1 The Cardiospermum halicacabum extract provided in this example is prepared through the following steps: Take 1 g of Cardiospermum halicacabum powder, accurately weigh it, place it in a 100 mL round-bottom flask, add 30 mL of 70% ethanol aqueous solution, heat under reflux at 68°C for 45 min, filter to obtain a filtrate and residues. The filtrate is placed in a volumetric flask, and the residues are rinsed with 3 mL of 70% ethanol aqueous solution. The obtained rinsing solution is incorporated into the above volumetric flask, and the weight loss due to ethanol volatilization is made up with 70% ethanol aqueous solution. Shake well, filter, take the supernatant and pass it through a 0.45 μm microporous filter membrane to obtain the Cardiospermum halicacabum extract.

[0025] Example 2 The Cardiospermum halicacabum extract provided in this example is prepared through the following steps: After passing the Cardiospermum halicacabum powder through a 20-mesh sieve, weigh 1 g accurately, place it in a 100 mL round-bottom flask, add 30 mL of 70% ethanol aqueous solution, heat under reflux at 68°C for 45 min, filter to obtain a filtrate and residues. The filtrate is placed in a volumetric flask, and the residues are rinsed with 3 mL of 70% ethanol aqueous solution. The obtained rinsing solution is incorporated into the above volumetric flask, and the weight loss due to ethanol volatilization is made up with 70% ethanol aqueous solution. Shake well, filter, take the supernatant and pass it through a 0.45 μm microporous filter membrane, and lyophilize to obtain the Cardiospermum halicacabum extract.

[0026] The obtained Cardiospermum halicacabum extract is lyophilized and reserved. It should be noted that the purpose of lyophilization is to retain the physical, chemical, and biological activities of the Cardiospermum halicacabum extract, and the lyophilization temperature is -40°C.

[0027] Example 3 The Cardiospermum halicacabum extract provided in this example is prepared through the following steps: Take 1 g of Cardiospermum halicacabum powder, accurately weigh it, place it in a 100 mL round-bottom flask, add 30 mL of 90% ethanol aqueous solution, heat under reflux at 68 °C for 45 min, filter to obtain a filtrate and residues. Transfer the filtrate into a volumetric flask, wash the residues with 3 mL of 90% ethanol aqueous solution, combine the obtained washing solution into the above volumetric flask, make up the weight lost due to ethanol volatilization with 90% ethanol aqueous solution, shake well, filter, take the supernatant and pass it through a 0.45 μm microporous filter membrane to obtain the Cardiospermum halicacabum extract.

[0028] Comparative Example 1 The Cardiospermum halicacabum extract provided in this comparative example is prepared through the following steps: Take 1 g of Cardiospermum halicacabum powder, accurately weigh it, place it in a 100 mL round-bottom flask, add 30 mL of water, heat under reflux at 68 °C for 45 min, filter to obtain a filtrate and residues. Transfer the filtrate into a volumetric flask, wash the residues with 3 mL of water, combine the obtained washing solution into the above volumetric flask, make up the weight lost due to ethanol volatilization with water, shake well, filter, take the supernatant and pass it through a 0.45 μm microporous filter membrane to obtain the Cardiospermum halicacabum extract.

[0029] Comparative Example 2 The Cardiospermum halicacabum extract provided in this comparative example is prepared through the following steps: Take 1 g of Cardiospermum halicacabum powder, accurately weigh it, place it in a 100 mL round-bottom flask, add 30 mL of 30% ethanol aqueous solution, heat under reflux at 68 °C for 45 min, filter to obtain a filtrate and residues. Transfer the filtrate into a volumetric flask, wash the residues with 3 mL of 30% ethanol aqueous solution, combine the obtained washing solution into the above volumetric flask, make up the weight lost due to ethanol volatilization with 30% ethanol aqueous solution, shake well, filter, take the supernatant and pass it through a 0.45 μm microporous filter membrane to obtain the Cardiospermum halicacabum extract.

[0030] Comparative Example 3 The Cardiospermum halicacabum extract provided in this comparative example is prepared through the following steps: Take 1 g of Cardiospermum halicacabum powder, accurately weigh it, place it in a 100 mL round-bottom flask, add 30 mL of 50% ethanol aqueous solution, heat under reflux at 68 °C for 45 min, filter to obtain a filtrate and residues. Transfer the filtrate into a volumetric flask, wash the residues with 3 mL of 50% ethanol aqueous solution, combine the obtained washing solution into the above volumetric flask, make up the weight lost due to ethanol volatilization with 50% ethanol aqueous solution, shake well, filter, take the supernatant and pass it through a 0.45 μm microporous filter membrane to obtain the Cardiospermum halicacabum extract.

[0031] I. High Performance Liquid Chromatography (HPLC) Detection 1. Instruments: Agilent 1200 high performance liquid chromatograph (Agilent, USA); XS-205 electronic balance (Mettler, Switzerland).

[0032] 2. Reagents Myricitrin (Lot No.: DO1031EA14), luteolin (Lot No.: JB242594), and apigenin (Lot No.: M29GB150104) were all purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; methanol (Lot No.: D807297) was purchased from Merck, and phosphoric acid (Lot No.: P816339) was purchased from Macklin. Methanol was of chromatographic grade, and water was self-made ultrapure water (meeting the standard: GB / T 33087-2016 "Specifications and Test Methods for High Purity Water for Instrumental Analysis"), and other reagents were all of analytical grade.

[0033] 3. Solution Preparation (1) Test solution: The Cardiospermum halicacabum extract of Example 1 was used as the test solution.

[0034] (2) Reference solution: Appropriate amounts of myricitrin, luteolin, and apigenin were taken as references, accurately weighed, placed in a 10 mL volumetric flask, dissolved and made up to the mark with methanol to prepare reference solutions containing myricitrin (4500 μg / mL), luteolin (1600 μg / mL), and apigenin (4800 μg / mL) per milliliter respectively, and stored in the dark in a refrigerator at 4°C for later use.

[0035] (3) Blank solvent: Methanol.

[0036] 4. Chromatographic Conditions Chromatographic column: InertSustain AQ-C18 column (4.6 mm × 250 mm, 5 μm), linear gradient elution, and the elution program is shown in Table 1; mobile phase A is methanol, mobile phase B is phosphoric acid-water (volume ratio of the two is 1000:1), the flow rate is 1 mL / min; the detection wavelength is 350 nm; the column temperature is 30°C; the injection volume is 10 μL.

[0037] Table 1 Elution Program

[0038] 5. HPLC Detection Results Under the above chromatographic conditions, the test solution, reference solution, and blank solvent were injected respectively, and the HPLC detection results are shown in Figure 1 . Figure 1Among them, Figure A is the HPLC chromatogram of the test solution; Figure B is the HPLC chromatogram of the reference solution; Figure C is the HPLC chromatogram of the blank solvent. It can be observed that myricitrin, luteolin and apigenin can be detected in the test solution and the reference solution. The peak emergence times of the test solution and the reference solution are consistent, and the peak shapes are good. The blank solvent does not interfere with the determination of the analytes (myricitrin, luteolin and apigenin) in the test solution and the reference solution. The above results indicate that the Cardiospermum halicacabum L. extract in Example 1 contains myricitrin, luteolin and apigenin.

[0039] 6. Methodological investigation 6.1 Investigation of linear relationship Respectively take 0.50, 1.00, 2.00, 4.00, 6.00, 8.00 mL of the reference solution, place them in 10 mL volumetric flasks, add methanol to dilute to the mark, and inject for detection respectively. The regression equations and linear ranges of each analyte are obtained. The results are shown in Table 2.

[0040] Table 2 Regression equations and linear ranges

[0041] It can be seen from Table 2 that the r values of each reference substance are not lower than 0.9993, indicating that there is a good linear relationship between each analyte and the peak area within their respective mass concentration ranges.

[0042] 6.2 Precision test Take the same test solution and inject it continuously for 6 times. Detect and record the peak areas of each chromatographic peak according to the above chromatographic conditions. The RSD values of the peak areas of myricitrin, apigenin and luteolin are calculated to be 0.45%, 0.82% and 0.90% respectively. The results indicate that the instrument precision is good. 6.3 Stability test Prepare 6 parallel test solutions and inject them at 0, 2, 4, 6, 8, 12, 24 h after preparation respectively. Detect and record the peak areas of the chromatographic peaks according to the above chromatographic conditions. The RSD values of the peak areas of myricitrin, apigenin and luteolin are calculated to be 1.35%, 2.53% and 2.03% respectively. The results indicate that the test solution has good stability.

[0043] 6.4 Repeatability test Prepare 6 parallel test solutions and inject them for detection according to the above chromatographic conditions. Calculate and take the average value. The contents of myricitrin, apigenin and luteolin are 99.43, 29.12, 83.66 μg / mg respectively, and the RSD values are 0.51%, 0.62% and 0.18% respectively. The results indicate that the repeatability of this test is good. 6.5 Spiked recovery test Six samples of Cardiospermum halicacabum with known content, 0.5 g each, were placed in a 30 mL volumetric flask, 1.0 mL of the reference solution was added, and injection detection was carried out according to the above chromatographic conditions. The recovery rate was calculated, and the results are shown in Table 3. The test results show that the detection method is accurate and reliable.

[0044] Table 3 Results of recovery test of added samples

[0045] 6.6 System durability test To investigate the influence of the chromatographic column on the separation of the test solution, according to the above chromatographic conditions, Inertsil ODS-SP-C18 column (4.6 mm×150 mm, 5 μm), Diamonsil-C18 column (4.6 mm×250 mm, 5 μm) and InertSustain AQ-C18 column (4.6 mm×250 mm, 5 μm) were used for injection respectively, and the HPLC detection results are shown in Figure 2 . Figure 2 In it, Figure A is the HPLC chart of Inertsil ODS-SP-C18 column; Figure B is the HPLC chart of Diamonsil-C18 column; Figure C is the HPLC chart of InertSustain AQ-C18 column. It can be seen from Figure 2 that using InertSustain AQ-C18 column can not only ensure the separation of each analyte, and the peak shapes and resolution of each analyte are good, but also shorten the analysis time. Therefore, InertSustain AQ-C18 column is selected as the chromatographic column for HPLC detection of the test solution.

[0046] 7. Influence of different extraction solutions on the contents of various components of Cardiospermum halicacabum The extracts of Cardiospermum halicacabum in Example 1, Example 3, and Comparative Examples 1-3 were injected and detected according to the above chromatographic conditions, and 3 repeated tests were carried out, and the data were recorded as the mean ± standard deviation. The extraction results of each analyte are shown in Table 4.

[0047] Table 4 Extraction results of each analyte (n = 3, )

[0048] As can be seen from Table 4, there are certain differences in the extraction values of myricitrin, luteolin, apigenin, and the total extraction value in the obtained Cardiospermum halicacabum extract with the change of ethanol concentration in the extraction solution. Among them, the extraction value of myricitrin in the Cardiospermum halicacabum extract obtained with 70% aqueous ethanol solution in Example 1 is the highest, which is 3297.17±26.91 μg / mg. The extraction values of luteolin, apigenin, and the total extraction value in the Cardiospermum halicacabum extract obtained with 90% aqueous ethanol solution in Example 3 are the highest. The extraction value of luteolin is 1373.57±18.69 μg / mg, the extraction value of apigenin is 5065.74±27.54 μg / mg, and the total extraction value is 7663.14±31.67 μg / mg. The extraction values of myricitrin, luteolin, apigenin, and the total extraction value of Comparative Examples 1-3 are relatively low, indicating that the contents of myricitrin, luteolin, and apigenin in the Cardiospermum halicacabum extract obtained by extraction with 70% and 90% aqueous ethanol solutions are high, showing good application potential in reducing photoaging damage.

[0049] II. Mass Spectrometry Detection Based on the information comparison of UPLC-Q-TOF-MS technology, spectral library, and reference substances (chlorogenic acid, quercetin, rutin, kaempferol, luteolin, luteoloside, calycosin, quercitrin, isoquercitrin, myricitrin, apigenin), the components of the Cardiospermum halicacabum extract obtained in Example 1 were qualitatively identified, and the results are as Figure 3 shown in Table 5 and Table 6.

[0050] Table 5 Compound-related Information of Cardiospermum halicacabum Extract in Positive Ion Mode

[0051] Table 6 Compound-related Information of Cardiospermum halicacabum Extract in Negative Ion Mode

[0052] Next, the Cardiospermum halicacabum extract prepared in Example 2 was used for multiple tests to study the antioxidant activity of the Cardiospermum halicacabum extract and the effect of the Cardiospermum halicacabum extract on UVB-induced photoaged HSF cells.

[0053] III. Antioxidant Activity of Cardiospermum halicacabum Extract 1. Test Method (1) DPPH Method: Using Cardiospermum halicacabum L. extract as the test sample and VC as the positive control test sample, dilute the Cardiospermum halicacabum L. extract and VC with 75% ethanol aqueous solution into Cardiospermum halicacabum L. extract solutions and VC solutions with different mass concentrations (0.25, 0.5, 1.0, 2.0, 2.5 mg / mL). Weigh 5 mg of DPPH precisely into a 250 mL volumetric flask, and make up the volume with 75% ethanol aqueous solution to obtain the DPPH solution, which is kept in the dark for later use. Precisely pipette 2 mL of Cardiospermum halicacabum L. extract solution or VC solution with different mass concentrations, add 2 mL of DPPH solution, shake well, and react in the dark at room temperature for 30 min. Measure the absorbance of the test sample at 517 nm. Using 75% ethanol aqueous solution plus DPPH solution as the blank and the test sample solution plus 75% ethanol aqueous solution as the control, repeat the experiment 3 times for each sample, take the average value, and calculate the DPPH scavenging rate according to the following formula: DPPH scavenging rate (%) = [1 - (Ai - Aj) / A0] × 100% Formula (I), In Formula (I), A0 is the absorbance of the blank group; Ai is the absorbance of the test sample; Aj is the absorbance of the control group.

[0054] (2) ABTS method: First, dilute the Cardiospermum halicacabum L. extract with 75% ethanol aqueous solution into test sample solutions with different concentrations (0.25, 0.5, 1.0, 2.0, 2.5 mg / mL), using VC as the positive control test sample. Secondly, prepare the ABTS + solution according to the instructions provided in the kit. Finally, dilute VC with 75% ethanol aqueous solution to different concentrations (0.25, 0.5, 1.0, 2.0, 2.5 mg / mL) to prepare the positive control test sample solution. In the experiment, mix 80 μL of the test sample solution or positive control test sample solution with 120 μL of the ABTS + solution, and pipette it onto a 96-well plate. Using 75% ethanol aqueous solution plus ABTS + solution as the blank and the test sample solution plus 75% ethanol aqueous solution as the control, measure the absorbance of each well at 734 nm within 10 minutes. Repeat the experiment 3 times for each test sample, take the average value, and calculate the ABTS scavenging rate according to the following formula: ABTS scavenging rate (%) = [1 - (Ai - Aj) / A0] x 100% Formula (II), In Formula (II), A0 is the absorbance of the blank group; Ai is the absorbance of the test sample; Aj is the absorbance of the control group.

[0055] (3) FRAP method: Under acidic conditions, antioxidants can reduce Ferric-tripyridyltriazine (Fe 3+-TPTZ) produces blue Fe 2+ -TPTZ, which has a maximum light absorption at 593 nm. Using vitamin C (VC) as a positive control, different mass concentrations (0.25, 0.5, 1.0, 2.0, 2.5 mg / mL) of VC and Cardiospermum halicacabum extract were added to Fe 3+ -TPTZ. After adding Fe 3+ -TPTZ to each solution and incubating at room temperature for 5 minutes, the absorbance value was measured with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 593 nm to obtain the iron-reducing antioxidant capacity of the solution.

[0056] 2. Test results The antioxidant activities of Cardiospermum halicacabum extract were verified by DPPH, ABTS, and FRAP methods respectively, and the results are as follows Figures 4 - 6 shown. Figure 4 is the DPPH radical scavenging rate of Cardiospermum halicacabum extract, Figure 5 is the ABTS radical scavenging rate of Cardiospermum halicacabum extract, Figure 6 is the iron-reducing antioxidant capacity of Cardiospermum halicacabum extract. As can be seen from the figure, Cardiospermum halicacabum extract shows an obvious dose-dependent relationship in DPPH radical scavenging ability, ABTS radical scavenging ability, and iron-reducing antioxidant capacity (FRAP value). Each index increases with the increase in the concentration of Cardiospermum halicacabum extract. When the concentration is 2.5 mg / mL, the DPPH scavenging rate, ABTS scavenging rate, and iron-reducing antioxidant capacity are the highest, indicating that Cardiospermum halicacabum extract exhibits good in vitro antioxidant activity, especially in iron-reducing antioxidant capacity, and also has a certain degree of total reducing ability.

[0057] IV. Effects of Cardiospermum halicacabum extract on UVB-induced photoaged HSF cells 1. Establishment of a UVB-induced photoaged HSF cell model UVB is a key factor in the process of photoaging. Therefore, in this study, UVB was used as a physical factor to induce a cell photoaging model. The irradiation wavelength was 302 nm, and the irradiation intensity was fixed at 160 J / cm 2 , and six time points of 0, 30, 60, 90, 120, and 150 s were set respectively to determine the modeling time of UVB. The results showed that after UVB irradiation, the survival rate of HSF cells decreased extremely significantly under the modeling conditions of 30 - 150 s. The survival rate of HSF cells decreased with the increase in the UVB irradiation duration. 60 s was selected as the modeling condition for UVB-induced photoaged HSF cells in subsequent experiments.

[0058] 2. Effects of Cardiospermum halicacabum extract on the survival rate of UVB-induced photoaged HSF cells 2.1 Test method After successful model establishment, the UVB-induced photoaged HSF cells were divided into a drug administration group and a blank group. Different concentrations of Cardiospermum halicacabum extract (100, 150, 200, 300, 400, 600 μg / mL) were added to the UVB-induced photoaged HSF cells in the drug administration group, and no Cardiospermum halicacabum extract was added to the blank group. After 12 hours of culture, the OD values of HSF cells in each group were measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) with the MTT method.

[0059] 2.2 Test results The OD test results of the UVB-induced photoaged HSF cells in each group are as Figure 7 shown. From Figure 7 it can be seen that the OD value measured at a concentration of 100 μg / mL of Cardiospermum halicacabum extract (CHE) is greater than the OD values measured at other concentrations. When the concentration of Cardiospermum halicacabum extract is 150 μg / mL or higher, the measured OD value is even lower than that of the blank group without Cardiospermum halicacabum extract, indicating that too high a concentration of Cardiospermum halicacabum extract may have an adverse effect on UVB-induced photoaged HSF cells.

[0060] 3. Effects of Cardiospermum halicacabum extract on the cell cycle of UVB-induced photoaged HSF cells 3.1 Test method It was divided into a normal group (group C), a model group (group M), and a drug administration group (CHE group). The normal group was HSF cells, and the model group and the drug administration group used the successfully modeled UVB-induced photoaged HSF cells. Among them, Cardiospermum halicacabum extract with a concentration of 100 μg / mL was added to the drug administration group. After 24 hours of culture of HSF cells in each group, flow cytometry and PI single staining were used to study the proportions of G0 / G1 phase and S phase cells in HSF cells in each group.

[0061] 3.2 Test results Figure 8 and Figure 9 show the effects of Cardiospermum halicacabum extract on the cell cycle of HSF cells. Figure 8 It is a histogram of the cell cycle of HSF cells detected by PI single staining combined with flow cytometry. Among them, Figure A is the histogram of the cell cycle of the normal group, Figure B is the histogram of the cell cycle of the model group, and Figure C is the histogram of the cell cycle of the drug administration group. Figure 9 It shows the proportions of G0 / G1 phase cells and S phase cells in HSF cells in each group. From Figure 8 and Figure 9It can be seen that, compared with the normal group, the proportion of HSF cells in the G0 / G1 phase in the model group was significantly decreased, while the proportion of cells in the S phase was significantly increased, indicating that UVB irradiation can induce cell cycle arrest of HSF cells in the S phase. Compared with the model group, the proportion of HSF cells in the G0 / G1 phase was significantly increased, and the proportion of cells in the S phase was significantly decreased after administration of 100 μg / mL Cardiospermum halicacabum extract, suggesting that Cardiospermum halicacabum extract has a tendency to prevent UVB-induced photoaged HSF cells from arresting in the S phase, indicating that Cardiospermum halicacabum extract can significantly improve the fibroblast proliferation dysfunction caused by UVB.

[0062] 4. Effects of Cardiospermum halicacabum extract on ROS levels in UVB-induced photoaged HSF cells 4.1 Experimental method It was divided into a blank group (Control group), a model group (Model group), a drug administration group (CHE group) and a VE group. HSF cells were used in the blank group, and UVB-induced photoaged HSF cells with successful modeling were used in the model group, the drug administration group and the VE group. Cardiospermum halicacabum extract with concentrations of 25, 50, and 100 μg / mL was added to the HSF cells in the drug administration group, and 100 μmol vitamin E was added to the HSF cells in the VE group. After culturing for 24 hours, the levels of ROS in HSF cells of each group were detected by flow cytometry.

[0063] 4.2 Experimental results The flow cytometric detection results of HSF cells in each group are shown in Figures 10 - 12 . From Figure 10 and Figure 11 it can be seen that, compared with the model group, the levels of ROS in HSF cells in the drug administration group and the VE group were both decreased. Among them, when the concentration of Cardiospermum halicacabum extract was 50 and 100 μg / mL, the ROS levels of HSF cells were significantly decreased, P < 0.05, P < 0.01, with statistical significance. From Figure 12 it can be seen that the fluorescence signal of the model group was the most obvious, followed by the CHE group with a Cardiospermum halicacabum extract concentration of 25 μg / mL. In contrast, the fluorescence signals of the CHE group and the VE group with Cardiospermum halicacabum extract concentrations of 50 and 100 μg / mL were not obvious. The above results indicate that Cardiospermum halicacabum extract can reduce the ROS levels of UVB-induced photoaged HSF cells.

[0064] Application Example 1 This application example provides a cream for reducing photoaging damage, and the formula is shown in Table 7.

[0065] Table 7 Formula table of the cream for reducing photoaging damage

[0066] The preparation method of the cream for reducing photoaging damage includes the following steps: (1) Preparation of the prefabricated phase: Weigh the raw materials according to the formula in Table 7, and mix the raw materials separately to obtain Phase A1, Phase A2, Phase A3, Phase B, Phase C, and Phase D; then pre-stir Phase A3 to make it evenly dispersed; pre-heat Phase B to about 78 °C, stir it until it is completely dissolved, and keep it at a constant temperature for standby; heat Phase C to about 58 °C, pre-stir it until it is completely dissolved, and cool it to room temperature for standby.

[0067] (2) Take a clean beaker, put Phase A1, Phase A2, and Phase A3 into the beaker at room temperature to obtain a mixed system. While stirring, heat the mixed system to about 86 °C, keep it warm for 20 minutes, and homogenize it appropriately for 1 - 2 minutes until there are no lumps in the mixed system.

[0068] (3) Add Phase B to the mixed system, stir evenly, and then homogenize it for 2 - 3 minutes until the mixed system becomes a delicate paste.

[0069] (4) Cool the mixed system to about 58 °C, and then add Phase C to the mixed system, stir and mix evenly.

[0070] (5) Cool the mixed system to about 46 °C, and then add Phase D to the mixed system, stir and mix evenly to obtain the anti-photoaging damage cream.

[0071] The above are only some embodiments of the present invention. For those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. Cardiospermum halicacabum extract, characterized in that, It is prepared by the following steps: (1) Take Cardiospermum halicacabum L. powder, add 70 - 90% ethanol aqueous solution, heat under reflux at 65 - 71 °C for 40 - 50 min, filter to obtain a filtrate and medicinal residues; (2) Rinse the medicinal residues with 70 - 90% ethanol aqueous solution to obtain a rinsing solution. Combine the rinsing solution with the filtrate obtained in step (1) to obtain a combined filtrate. Then add 70 - 90% ethanol aqueous solution to the combined filtrate to make up for the weight loss due to ethanol volatilization, shake well, filter to obtain a supernatant; (3) Pass the supernatant obtained in step (2) through a 0.45 μm microporous membrane to obtain the product.

2. The Cardiospermum halicacabum extract according to claim 1, wherein In step (1), the mass ratio of the Cardiospermum halicacabum L. powder to the volume of the 70 - 90% ethanol aqueous solution is 1:(25 - 35) g / mL.

3. The Cardiospermum halicacabum extract according to claim 1 or 2, characterized in that, The mass ratio of the Cardiospermum halicacabum L. powder to the volume of the 70 - 90% ethanol aqueous solution used to rinse the medicinal residues is 1:(3 - 5) g / mL.

4. The Cardiospermum halicacabum extract according to claim 1 or 2, characterized in that, In step (3), after passing the supernatant through a 0.45 μm microporous membrane, it is also freeze - dried, and the freeze - drying temperature is - 45~ - 35 °C.

5. Use of the Cardiospermum halicacabum L. extract according to any one of claims 1 - 4 in the preparation of a cosmetic or drug for reducing photoaging damage.

6. The application according to claim 5, characterized in that, The cosmetic or drug for reducing photoaging damage is a cosmetic or drug having one or more of the following effects: (a) antioxidant activity; (b) improving the cell proliferation dysfunction of photoaged cells; (c) reducing the oxidative damage of photoaged cells.

7. The application according to claim 6, wherein The cosmetic or drug for reducing photoaging damage is a cosmetic or drug having one or more of the following effects: (a) scavenging free radicals; (b) reducing ability; (c) increasing the cell survival rate of photoaged cells; (d) preventing photoaged cells from arresting in the S phase; (e) reducing the intracellular ROS level of photoaged cells.

8. The application according to claim 6 or 7, characterized in that, The photoaged cells are photoaged cells induced by UVB.

9. The application according to claim 5, characterized in that, The cosmetic for reducing photoaging damage is at least one of emulsion, cream, essence, freeze - dried powder, ointment, aqueous solution, facial mask, spray.

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