Preparation method of solanum photeinocarpum extract and product and application of solanum photeinocarpum extract

Abstract

The invention discloses a preparation method of a solanum photeinocarpum extract as well as a product and application of the solanum photeinocarpum extract, and belongs to the field of plant extraction. The preparation method comprises the following steps: taking solanum photeinocarpum seedlings, crushing, heating with 70% ethanol at 65 DEG C, performing reflux extraction for 3-4 times, combining extracting solutions, and performing vacuum concentration until no alcohol smell exists, thereby obtaining an ethanol extract; the preparation method comprises the following steps: mixing a water phase and an organic phase, dispersing with pure water in a ratio of 1: 1, sequentially extracting with n-hexane, dichloromethane and ethyl acetate, respectively extracting each part for 3-5 times according to a ratio of the organic phase to the water phase of 1: 1-1: 5, and recovering under reduced pressure to obtain an ethyl acetate extract which is used as the solanum photeinocarpum extract. The preparation method adopts a mild process, is safe, controllable and environment-friendly, and is suitable for industrial production. The obtained solanum photeinocarpum extract is low in toxicity and free of sensitization, has remarkable anti-oxidation, anti-inflammatory and anti-aging effects, can be applied to cosmetics such as skin care water aqua and anti-aging face cream, effectively improves the skin moisturizing rate and collagen synthesis amount, and has an anti-light aging effect.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extraction, in particular to a preparation method of Solanum nigrum L. extract, and a product and application thereof. Background Art

[0002] Solanum americanum (Solanum americanum) is a plant of the Solanaceae family, genus Solanum, widely distributed in tropical and subtropical regions. In traditional medicine, the whole herb or fruit has been used to treat inflammation, infection, liver damage, and skin diseases. Modern research suggests that Solanum americanum can lower blood pressure, stabilize blood sugar, prevent cancer, and clear heat and reduce internal heat. However, other studies have shown that Solanum americanum contains toxic substances such as solanine and solanine, which primarily manifest in the nervous and digestive systems, necessitating caution in its use.

[0003] Currently, the extraction methods for Solanum nigrum mainly include traditional water extraction / alcohol extraction, single organic solvent extraction, supercritical CO2 extraction, etc., but they all have defects such as low efficiency, high toxicity, and high cost, and there is a lack of systematic evaluation research on cytotoxicity and sensitization.

[0004] Based on the above research status, the present invention proposes a preparation method of Solanum nigrum L. extract, its product and application, so that it can effectively extract the effective parts of Solanum nigrum L., provide the necessary conditions for exploring the potential activity of its effective ingredients, and achieve the purpose of fully utilizing its antioxidant, anti-inflammatory and anti-aging activities. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method of Solanum nigrum extract, so that the functional active parts thereof can be efficiently extracted while avoiding their toxicity, thereby providing the necessary conditions for exploring their applications in the fields of antioxidant, anti-inflammatory, anti-aging and skin repair, and expanding their utilization potential in the pharmaceutical and cosmetic industries.

[0006] In order to solve the above technical problems, the present invention provides a method for preparing a Solanum nigrum extract, comprising the following steps:

[0007] (1) Take Solanum nigrum seedlings, crush them, add 70% ethanol at a solid-liquid ratio of 1:5 to 1:10, heat and reflux at 65°C for 3 to 4 times, combine the extracts, and concentrate under reduced pressure until there is no alcohol taste, thereby obtaining the Solanum nigrum ethanol extract;

[0008] (2) The ethanol extract obtained in step (1) is dispersed with pure water in a ratio of 1:1, and extracted with n-hexane, dichloromethane, and ethyl acetate in sequence, wherein the volume ratio of the organic phase to the aqueous phase is 1:1 to 1:5, and each organic phase is extracted 3 to 5 times, the ethyl acetate extracts are combined, and the solvent is recovered under reduced pressure to obtain the Solanum nigrum extract.

[0009] Further improvement, step (1) takes out Solanum nigrum seedlings 3-13 days after seedling, crushes them, adds 70% ethanol according to a solid-liquid ratio of 1:10, heats and refluxes at 65°C for extraction 3 times, the first extraction time is 3 hours, the second and third extraction times are 4 hours, the extracts are combined, and concentrated under reduced pressure until there is no alcohol taste, to obtain the Solanum nigrum ethanol extract; in step (2), each organic phase is extracted 3 times, the volume ratio of the organic phase to the aqueous phase in the first extraction is 1:3, and the volume ratio of the organic phase to the aqueous phase in the second and third extractions is 1:2.

[0010] The present invention also provides a Solanum nigrum pauciflorum extract, which is prepared by the above-mentioned preparation method of the Solanum nigrum pauciflorum extract.

[0011] The present invention also provides a functional cosmetic comprising the Solanum nigrum pauciflorum extract and cosmetically acceptable excipients, wherein the Solanum nigrum pauciflorum extract is added in an amount of 0.1 to 10 wt %. The cosmetic has anti-inflammatory, antioxidant, anti-skin aging, and / or long-lasting moisturizing effects.

[0012] Further improvement, the addition amount of the Solanum nigrum extract is 0.5~1 wt%.

[0013] According to a further improvement, the cosmetic is a skin care lotion, a face cream, an emulsion or an essence.

[0014] The present invention also provides a medicine comprising the above-mentioned Solanum nigrum extract and pharmaceutically acceptable excipients, and the medicine has anti-inflammatory, anti-oxidation and anti-skin aging effects.

[0015] The present invention also provides a use of a Solanum nigrum L. extract in the preparation of anti-skin aging drugs or cosmetics, wherein the Solanum nigrum L. extract has non-enzymatic glycation inhibitory activity, elastase inhibitory activity, and MGO-induced BJ cell glycation inhibitory activity.

[0016] The present invention also provides a use of a Solanum nigrum extract in the preparation of moisturizing or long-lasting moisturizing medicines or cosmetics. The Solanum nigrum extract can achieve rapid moisturizing for 2 hours and long-lasting moisturizing for 24 hours.

[0017] The present invention also provides a use of a Solanum nigrum L. extract in the preparation of anti-inflammatory drugs or cosmetics, wherein the Solanum nigrum L. extract can inhibit the expression levels of NO, IL-6 and TNF-α.

[0018] The present invention also provides a use of a Solanum nigrum L. extract in the preparation of antioxidant drugs or cosmetics, wherein the Solanum nigrum L. extract can effectively scavenge DPPH free radicals.

[0019] After adopting such a design, the present invention has at least the following advantages:

[0020] The preparation method of the Solanum nigrum L. extract of the present invention adopts a mild extraction process, avoids the use of toxic and harmful solvents, ensures the safety and environmental friendliness of the product, and has strong controllability of the process flow, which is suitable for industrial production. The preparation method can retain its natural active ingredients to the greatest extent through precise processing and extraction optimization of Solanum nigrum L., effectively improving the biological activity and application value of the extract. The obtained Solanum nigrum L. extract has significant antioxidant, anti-inflammatory, anti-glycation, anti-aging and moisturizing effects, is non-sensitizing, and has no obvious cytotoxicity. It can be used to prepare functional cosmetics with anti-inflammatory, antioxidant, and anti-aging effects, can long-lasting moisturizing, promote skin barrier repair and improve microcirculation, and has huge market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0022] FIG1 shows the antioxidant activity test results of the ethanol extract, n-hexane extract, dichloromethane extract and ethyl acetate extract of Solanum nigrum L. of the present invention.

[0023] Figures 2-4 show the test results of the Solanum nigrum ethanol extract, n-hexane extract, dichloromethane extract, and ethyl acetate extract of the present invention in inhibiting the release of NO, IL-6, and TNF-α in the LPS-induced Raw264.7 cell model.

[0024] Figure 5-7 This is the cytotoxicity evaluation result of the Solanum nigrum extract SA-EA of the present invention.

[0025] Figure 8-9 The results show the effect of the Solanum nigrum extract SA-EA on the sensitization activity of THP-1 cells.

[0026] 10-12 are test results showing that the Solanum nigrum extract SA-EA of the present invention inhibits the release of TNF-α, IL-1β and IL-6 in a CuSO 4 -induced zebrafish inflammation model.

[0027] FIG13 is a test result of the anti-non-enzymatic glycation of the Solanum nigrum extract SA-EA of the present invention.

[0028] Figures 14-15 show the evaluation results of the anti-glycation activity of the Solanum nigrum extract SA-EA of the present invention in the MGO-induced BJ cell glycation model.

[0029] FIG16 shows the test results of the inhibition rate of Solanum nigrum L. extract SA-EA on elastase activity of the present invention.

[0030] Figure 17 These are the test results of the antioxidant effect of the Solanum nigrum extract SA-EA in skin care lotions of the present invention.

[0031] FIG18 shows the test results of the moisturizing effect of the Solanum nigrum extract SA-EA in the skin care lotion of the present invention.

[0032] FIG19 shows the test results of the anti-aging effect of the Solanum nigrum extract SA-EA in skin care creams of the present invention. DETAILED DESCRIPTION

[0033] Exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0034] Example 1: Preparation of Solanum nigrum L. Extract

[0035] 1.1 Extraction

[0036] Take 10.0 kg of Solanum nigrum seedlings 3-13 days after emergence, crush them, add 70% ethanol according to a solid-liquid ratio of 1:10, heat and reflux at 65°C for 3 times, the first extraction time is 3 hours, the second and third extraction times are 4 hours, combine the multiple extracts, and concentrate under reduced pressure until there is no alcohol taste, to obtain 3.1 kg of Solanum nigrum ethanol extract.

[0037] 1.2 Extraction

[0038] The above-mentioned Solanum nigrum ethanol extract was dispersed in pure water at a ratio of 1:1, and then extracted with n-hexane, dichloromethane and ethyl acetate in sequence. Each organic phase was extracted 3 times. The ratio of the organic phase to the aqueous phase in the first extraction was 1:3, and the ratio of the organic phase to the aqueous phase in the second and third extractions was 1:2. The extracts were collected separately, and the solvent was recovered under reduced pressure to obtain 105g of n-hexane extract, 153g of dichloromethane extract and 97g of ethyl acetate extract.

[0039] Example 2: Preparation of Solanum nigrum L. Extract

[0040] 2.1 Extraction

[0041] Take 10.0 kg of Solanum nigrum seedlings 3-13 days after emergence, crush them, add 80% ethanol according to a solid-liquid ratio of 1:5, heat and reflux at 55°C for 4 times, each extraction time is 3 hours, combine multiple extracts, and concentrate under reduced pressure until there is no alcohol taste, to obtain 2.5 kg of Solanum nigrum ethanol extract.

[0042] 2.2 Extraction

[0043] The above-mentioned Solanum nigrum ethanol extract was dispersed in pure water at a ratio of 1:1, and then extracted with n-hexane, dichloromethane and ethyl acetate in sequence. Each organic phase was extracted 4 times. The ratio of organic phase to aqueous phase in the first extraction was 1:1, and the ratio of organic phase to aqueous phase in the next three extractions was 1:3. The extracts were collected separately, and the solvent was recovered under reduced pressure to obtain 138g of n-hexane extract, 117g of dichloromethane extract and 89g of ethyl acetate extract.

[0044] Example 3: Preparation of Solanum nigrum L. Extract

[0045] 3.1 Extraction

[0046] Take 10.0 kg of Solanum nigrum seedlings 3-13 days after emergence, crush them, add 60% ethanol according to a solid-liquid ratio of 1:10, heat and reflux at 75°C for 3 times, the first extraction time is 3 hours, the second and third extraction times are 4 hours, combine the multiple extracts, and concentrate under reduced pressure until there is no alcohol taste, to obtain 2.7 kg of Solanum nigrum ethanol extract.

[0047] 3.2 Extraction

[0048] The above-mentioned Solanum nigrum ethanol extract was dispersed in pure water at a ratio of 1:1, and then extracted with n-hexane, dichloromethane and ethyl acetate in sequence. Each organic phase was extracted 3 times. The ratio of the organic phase to the aqueous phase in the first extraction was 1:1, and the ratio of the organic phase to the aqueous phase in the second and third extractions was 1:3. The extracts were collected separately, and the solvent was recovered under reduced pressure to obtain 112g of n-hexane extract, 134g of dichloromethane extract and 92g of ethyl acetate extract.

[0049] Example 4: Evaluation of the antioxidant activity of Solanum nigrum extracts

[0050] 4.1 Reagents and Materials

[0051] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH), anhydrous ethanol, and a 96-well plate.

[0052] 4.2 Instruments

[0053] Microplate reader, 1 / 100,000 microbalance.

[0054] 4.3 Samples to be tested

[0055] DPPH working solution: 50 μg / mL, prepared in anhydrous ethanol.

[0056] Positive control group: GSH, concentrations of 300, 100, 33.3, 11.1, 3.7, 1.23, and 0.41 μg / mL.

[0057] Test substance working solution:

[0058] The ethanol extract, n-hexane extract, dichloromethane extract, and ethyl acetate extract of Solanum nigrum obtained in Example 1 were prepared with distilled water to prepare test concentrations of 1.23, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg / mL, respectively.

[0059] 4.4 Experimental methods

[0060] Weigh 1.5 mg of DPPH powder into a 50 mL centrifuge tube, wrap in tin foil and protect from light. Add 30 mL of anhydrous ethanol and mix thoroughly. Ultrasonicate to completely dissolve the powder to obtain a 50 μg / mL DPPH working solution. Prepare GSH working solutions and test substance working solutions of corresponding concentrations using a gradient dilution method. Add 50 μL of test substance working solution, 50 μL of GSH working solution, or 50 μL of anhydrous ethanol to each well. Then add 150 μL of DPPH working solution or 150 μL of anhydrous ethanol to each well. After addition, mix using a 96-well microplate mixer and react at room temperature in the dark for 30 minutes. Read the absorbance at a wavelength of 517 nm using a microplate reader.

[0061] The DPPH clearance rate P of the test substance and GSH was calculated using the following formula (1):

[0062] (1)

[0063] Where As represents the absorbance of the mixture of the test substance (or GSH) and DPPH, Ac represents the absorbance of the mixture of the test substance (or GSH) and EtOH, Ab represents the absorbance of the mixture of EtOH and DPPH, and A0 represents the absorbance of the mixture of EtOH and EtOH.

[0064] After calculating the clearance rate, GraphPad Prism software was used to draw a graph, with the final concentration of the sample in the reaction system as the horizontal axis and the clearance rate as the vertical axis. A nonlinear fitting equation was established between the concentration of the test solution and the clearance rate, and the half-maximum clearance amount EC50 was calculated. The antioxidant capacity AO value of the polypeptide or small molecule active substance sample was calculated according to the following formula (2):

[0065] (2)

[0066] EC50(S) represents the concentration (μg / mL) corresponding to the half-clearance rate of the test substance, and EC50(R) represents the concentration (μg / mL) corresponding to the half-clearance rate of GSH.

[0067] 4.4 Experimental Principles and Results

[0068] When a free radical scavenger (reducing agent) is present, DPPH reacts with its single electron pairing, causing the characteristic purple color of the DPPH ethanol solution to gradually disappear, turning it colorless or light yellow, thereby weakening the light absorption of DPPH. Studies have shown that the degree of discoloration of the DPPH ethanol solution is linearly related to the number of electrons it accepts. Therefore, the free radical scavenging ability of the test sample, that is, the antioxidant activity, can be evaluated by measuring the light absorption of the reaction solution of the sample and DPPH at 517nm.

[0069] The test results are shown in Table 1 and Figure 1 shown.

[0070] Table 1 Comparison of antioxidant activity test results of various extraction parts of Solanum nigrum

[0071]

[0072] From Table 1 and Figure 1 The results show that the ethyl acetate extract of Solanum nigrum has a strong ability to scavenge DPPH, and according to the AO value, it can be seen that the AO value of the ethyl acetate extract of Solanum nigrum is small, close to the positive control GSH, indicating that the ethyl acetate extract of Solanum nigrum has strong antioxidant activity.

[0073] The ethanol, hexane, and dichloromethane extracts of Solanum nigrum also have a certain ability to scavenge DPPH. However, the results clearly show that the antioxidant activity of the ethanol extract of Solanum nigrum is lower than that of the ethyl acetate extract of Solanum nigrum, and the DPPH scavenging effects of the hexane and dichloromethane extracts are even worse. This indicates that the ethyl acetate extract of Solanum nigrum has higher research value.

[0074] Example 5: Evaluation of the anti-inflammatory activity of Solanum nigrum extracts

[0075] 5.1 Reagents and Materials

[0076] DMEM high-glucose medium, fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, lipopolysaccharide (LPS), dexamethasone (DEX), Mouse TNF-α ELISA detection kit, Mouse IL-6 ELISA detection kit, NO detection kit.

[0077] 5.2 Instruments

[0078] CO2 incubator, biological safety cabinet, inverted microscope, and microplate reader.

[0079] 5.3 Cell lines

[0080] Mouse mononuclear macrophage leukemia cells (Raw264.7) were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences.

[0081] 5.4 Samples to be tested

[0082] LPS working solution: 25 ng / mL, prepared in DMEM medium containing 1% FBS

[0083] Dexamethasone working solution (DEX): 10 μg / mL, prepared using LPS working solution;

[0084] Test substance working solution:

[0085] The ethanol extract, n-hexane extract, dichloromethane extract, and ethyl acetate extract of Solanum nigrum obtained in Example 1 were prepared using LPS working solution to prepare test concentrations of 1, 3, 10, 30, and 100 μg / mL, respectively.

[0086] 5.5 Experimental methods

[0087] Take Raw264.7 cells in good exponential growth phase, use cell scraper to remove cells, and adjust the cell density to 1.5×10 5 / mL cell suspension. Take the above cell suspension and inoculate it into a 48-well plate, 500 μL / well, and incubate it in a 37°C, 5% CO2 incubator for 16-24 hours. When the cell fusion rate reaches 30-50%, remove the supernatant and add 400 μL of the above test sample to each well except the control group. Incubate in a 37°C, 5% CO2 incubator for 24 hours. After incubation, collect the supernatant and centrifuge to remove cell debris. According to the steps in the kit instructions, the concentrations of NO, IL-6 and TNF-α in the supernatant of each group were detected.

[0088] 5.6 Experimental Principles and Results

[0089] Lipopolysaccharide (LPS) can activate the synthesis and release of various cytokines and inflammatory mediators through the cell signaling system. The accumulation of inflammatory factors can cause vasodilation, redness, swelling, stinging, and itching, leading to skin sensitivity. In this study, an LPS-induced inflammation model was established in Raw264.7 cells to evaluate the anti-inflammatory activity of various extracts from Solanum nigrum.

[0090] The test results are shown in Table 2 and Figure 2-4 shown.

[0091] Table 2 Anti-inflammatory activity test results of various extracts from Solanum nigrum

[0092]

[0093] from Figure 2-4 It can be seen that compared with the control group, the expression levels of NO, TNF-α and IL-6 in the Raw264.7 cells in the LPS group were significantly increased after LPS induction, indicating that the Raw264.7 cell inflammation model was successfully established.

[0094] As can be seen from Table 2, at the same test concentration (10 μg / mL), the ethyl acetate extract had a significantly better inhibitory effect on inflammatory factors NO, IL-6, and TNF-α than other extracts. Figure 2-4 As can be seen, the ethyl acetate extract of Solanum nigrum at a concentration of 3-100 μg / mL can significantly inhibit the LPS-induced release of NO, IL-6, and TNF-α in Raw264.7 cells in a dose-dependent manner, indicating that the ethyl acetate extract of Solanum nigrum has excellent anti-inflammatory activity.

[0095] In summary, the ethyl acetate extract of Solanum nigrum L. exhibits stronger biological activity and has higher research value. The following experiments use the ethyl acetate extract of Solanum nigrum L. as the research object of the Solanum nigrum L. extract of this application, referred to as SA-EA.

[0096] Example 6: Cytotoxicity test

[0097] 6.1 Reagents and Materials

[0098] High glucose medium (DMEM), 1640 medium, fetal bovine serum (FBS), DPBS, dimethyl sulfoxide (DMSO), CCK-8.

[0099] 6.2 Instruments

[0100] CO2 incubator, biological safety cabinet, inverted microscope, and microplate reader.

[0101] 6.3 Cell lines

[0102] Mouse monocytic macrophage leukemia cells (Raw264.7), human acute monocytic leukemia cells (THP-1), and human immortalized epidermal cells (HaCaT).

[0103] 6.4 Samples to be tested

[0104] Sample group: Solanum nigrum extract SA-EA obtained in Example 1, tested at concentrations of 300 μg / mL, 100 μg / mL, 30 μg / mL, 10 μg / mL, 3 μg / mL, 1 μg / mL, 0.3 μg / mL, and 0.1 μg / mL.

[0105] Solvent control group: DMEM complete medium or 1640 complete medium;

[0106] Positive control group: DMEM complete medium containing 10% DMSO, or 1640 complete medium containing 10% DMSO;

[0107] Zero adjustment group: DMEM complete medium (without cells) or 1640 complete medium (without cells).

[0108] 6.5 Experimental methods

[0109] Raw264.7 cells, THP-1 cells or HaCaT cells were cultured at a density of 2.5×10 5 100 μL of cell suspension (100 μL / well) was inoculated into each well of a 96-well plate for culture. When the cell fusion rate reached about 40%, the supernatant was removed and samples of different concentrations were added in sequence for treatment with bioactive substances. After 24 h of treatment, CCK-8 reagent was added at a ratio of 1:10. After 40 min, the absorbance of each well at 450 nm was measured using a microplate reader. The cell survival rate was calculated according to the following formula (3):

[0110] (3)

[0111] 6.6 Results

[0112] The CCK-8 assay is a method for detecting cell survival and growth. The measured OD value is proportional to the cell activity. The results of the test sample's toxicity to Raw264.7 cells, THP-1 cells, and HaCaT cells are as follows: Figure 5-7 The results showed that the Solanum nigrum extract SA-EA in the above cells had a cell viability greater than 90% at a concentration of 300 μg / mL and below, and showed no obvious cytotoxicity, indicating that the Solanum nigrum extract SA-EA has a high safety for immune-related cells and skin cells.

[0113] Example 7: Evaluation of THP-1 Cell Sensitivity of Solanum nigrum Extract

[0114] 7.1 Reagents and Materials

[0115] 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin (P / S), β-mercaptoethanol (β-ME), DPBS, bovine serum albumin (BSA), dinitrochlorobenzene (DNCB), lactic acid (LA), 7-AAD, FITC anti-human CD86 Antibody, PE anti-human CD54 Antibody, 5-FITC Mouse IgG1 κ Isotype Control Antibody, PE Mouse IgG1 κ Isotype Control Antibody.

[0116] 7.2 Instruments

[0117] CO2 incubator, biological safety cabinet, inverted microscope, flow cytometer.

[0118] 7.3 Cell lines

[0119] Human acute monocytic leukemia cells (THP-1) were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0120] 7.4 Samples to be tested

[0121] Blank control group: 1640 complete medium;

[0122] Solvent control group: 0.2% DMSO, prepared using 1640 complete medium;

[0123] Positive control group: 3.125 μg / mL DNCB, prepared in 1640 complete medium;

[0124] Negative control group: 1 mg / mL lactic acid, prepared in 1640 complete medium;

[0125] Administration group: The Solanum nigrum extract SA-EA obtained in Example 1 was prepared at concentrations of 100, 50, 25, and 12.5 μg / mL using 1640 complete culture medium.

[0126] 7.5 Experimental methods

[0127] Take THP-1 cells in good exponential growth phase, centrifuge to remove supernatant and resuspend in culture medium to adjust the cell density to 1.5×10 6 / mL, take the cell suspension and inoculate it in a 48-well plate, 200 μL / well. According to the experimental group, prepare the corresponding test solution with a 2-fold concentration. Add 200 μL / well of the prepared test sample to the corresponding well and incubate in a 37°C, 5% CO2 incubator for 24 hours. After incubation, collect the cell suspension in each well and divide it into two parts, one for CD86 and CD54 staining, and the other for isotype control staining. After staining, fix the cells with paraformaldehyde, and use flow cytometry to detect the mean fluorescence intensity (MFI) of FITC, PE, and 7-AAD dyes in each group of cells. Then use the following formula (4) to calculate the relative fluorescence intensity (RFI) of each group of test substances.

[0128] (4)

[0129] 7.6 Experimental Principles and Results

[0130] When allergens come into contact with the skin, dendritic cells differentiate and mature during their migration to lymphoid organs, and upregulate the expression of a series of surface molecules. By staining the cell surface molecules CD86 and CD54 with fluorescent antibody dyes and measuring them with flow cytometry, it can be determined whether the test substance is allergenic.

[0131] The experimental results are shown in Figure 8-9 The positive control group produced positive expression in the cells (RFICD86 > 150, RFICD54 > 200) and the cell viability was > 50%; the negative control group produced negative expression in the cells (RFICD86 < 150, RFICD54 < 200) and the cell viability was > 50%; the solvent control group produced negative expression in the cells (RFICD86 < 150, RFICD54 < 200) and the cell viability was > 90%, indicating that the cells passed the stability test.

[0132] It can also be seen that at all tested concentrations of Solanum nigrum extract SA-EA, the CD86 and CD54 expressions of THP-1 cells were negative, and the cell viability was > 90%, indicating that Solanum nigrum extract SA-EA had no allergenicity.

[0133] Example 8: Evaluation of the anti-inflammatory activity of Solanum nigrum extract in zebrafish

[0134] 8.1 Reagents and Materials

[0135] 0.1% DMSO solution (for sample dissolution), E3 culture medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4), PBS buffer, copper sulfate (CuSO4), dexamethasone (DEX), TNF-α ELISA kit, IL-6 ELISA kit, IL-1β ELISA kit.

[0136] 8.2 Instruments

[0137] Biochemical incubator, centrifuge, and microplate reader.

[0138] 8.3 Experimental Animals

[0139] The zebrafish used in this test were wild-type AB strain zebrafish (Danio rerio) (3 days old) provided by Yunnan Yunke Specialty Plant Detection Co., Ltd.

[0140] 8.4 Samples to be tested

[0141] Model group: 0.05 mM CuSO4;

[0142] Positive control group: 0.05 mM CuSO4+Dex 10 μM;

[0143] Low, medium and high dose groups of Solanum nigrum extract SA-EA: 0.05 mM CuSO4+SA-EA 5 μg / mL (low); 0.05 mM CuSO4+ SA-EA 10 μg / mL (medium); 0.05 mM CuSO4+ SA-EA 20 μg / mL (high).

[0144] 8.5 Experimental methods

[0145] 3 dpf zebrafish embryos were randomly divided into 30 groups and treated according to the aforementioned groups. The embryos were then incubated for 24 hours. After treatment, the embryos were washed three times with PBS and homogenized. The supernatants were collected after centrifugation and the concentrations of TNF-α, IL-6, and IL-1β in the supernatants of each group were determined according to the ELISA kit instructions.

[0146] 8.6 Experimental Principles and Results

[0147] Cu²⁺ can stimulate the zebrafish immune system, promote the migration of neutrophils to the tail, simulate acute inflammatory response, and promote the secretion of inflammatory factors (TNF-α, IL-6, IL-1β) in the body. Figure 10-12 As shown in the results, compared with the control group, the levels of TNF-α, IL-6, and IL-1β in the zebrafish model group after CuSO4 induction were significantly increased, indicating that the in vivo inflammation model of zebrafish was successfully established. SA-EA, a Solanum nigrum extract, significantly inhibited the release of TNF-α, IL-6, and IL-1β in a dose-dependent manner at concentrations of 5-20 μg / mL. This suggests that SA-EA, a Solanum nigrum extract, has excellent anti-inflammatory activity in vivo, possibly by inhibiting inflammatory signaling pathways and reducing the secretion of inflammatory factors.

[0148] Example 9: Evaluation of the non-enzymatic saccharification inhibitory activity of Solanum nigrum extract

[0149] 9.1 Reagents and Materials

[0150] Bovine serum albumin (BSA), D-anhydrous glucose, DPBS, penicillin / streptomycin (P / S).

[0151] 9.2 Instruments

[0152] Microplate reader, 1 / 100,000 microbalance.

[0153] 9.3 Samples to be tested

[0154] BSA working solution: 40 μg / mL, prepared in DPBS containing 1% P / S;

[0155] Glucose working solution: 480 μg / mL, prepared in DPBS containing 1% P / S;

[0156] Test samples: Solanum nigrum extract SA-EA obtained in Example 1, with concentrations of 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg / mL, prepared in DPBS containing 1% P / S.

[0157] 9.4 Experimental Methods

[0158] According to Table 3 below, add the prepared SA-EA solutions of varying concentrations, BSA working solution, glucose working solution, and DPBS to a 96-well plate. Seal the plates with sealing film and incubate in a 37°C incubator in the dark for 7 days. After incubation, measure fluorescence intensity using a microplate reader with an excitation wavelength of 370 nm and an emission wavelength of 440 nm.

[0159] Table 3 Composition ratios of the SA-EA non-enzymatic glycation inhibitory activity of Solanum nigrum extract

[0160]

[0161] The AGEs inhibition rate (P) and half-maximal inhibitory concentration (IC50) of the test sample were calculated according to the following formula (5).

[0162] (5)

[0163] Among them, T, T0, C, and C0 represent the fluorescence intensities of the sample group, sample background group, glycation reaction group, and BSA group under the conditions of excitation wave 370 nm and emission wave 440 nm, respectively.

[0164] 9.5 Experimental Principles and Results

[0165] Non-enzymatic glycation (NEG) refers to the non-enzymatic condensation reaction between carbonyl groups on reducing sugars (such as glucose and fructose) and free amino groups on macromolecules such as proteins, lipids, or nucleic acids. The stable covalent adducts formed by this reaction are called advanced glycation end products (AGEs). This study evaluated the anti-glycation ability of each test substance by measuring its ability to inhibit the production of AGEs.

[0166] The experimental results are as follows Figure 13As shown in the results, SA-EA, an extract from Solanum nigrum L., has significant non-enzymatic glycation inhibitory activity, with a half maximal inhibitory concentration (IC50) of 95.89 μg / mL.

[0167] Example 10: Evaluation of the anti-glycation activity of Solanum nigrum extract

[0168] 10.1 Reagents and Materials

[0169] DMEM high glucose medium, fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, methylglyoxal (MGO), aminoguanidine hydrochloride (AG), and fluorescent probe (H2DCFDA).

[0170] 10.2 Instruments

[0171] CO2 incubator, biological safety cabinet, and fluorescence inverted microscope.

[0172] 10.3 Cell lines

[0173] Human skin fibroblasts (BJ) were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0174] 10.4 Samples to be tested

[0175] Methylglyoxal (MGO) working solution: 800 μM, prepared in DMEM complete medium;

[0176] Aminoguanidine (AG) working solution: 30 μg / mL, prepared using methylglyoxal working solution;

[0177] Test sample: Solanum nigrum extract SA-EA obtained in Example 1, with test concentrations of 0.5, 1, and 2 μg / mL, prepared using methylglyoxal working solution.

[0178] 10.5 Experimental Methods

[0179] Take BJ cells in good exponential growth phase, use 0.25% Trypsin-EDTA to detach the cells from the culture dish, and adjust the cell concentration to 3×10 4Prepare a cell suspension at a concentration of 1 mL / well. Inoculate the cell suspension into a 12-well plate at 1 mL / well and incubate in a 37°C, 5% CO2 incubator for 16-24 hours. Once the cell confluence reaches 60%, remove the supernatant and, except for the control group, add 1 mL of the test sample to each well. Incubate in a 37°C, 5% CO2 incubator for 24 hours. After incubation, remove the supernatant and wash with DPBS. Add 500 μL of the prepared ROS staining solution (10 μM H2DCFDA) to each well and incubate in a 37°C, 5% CO2 incubator for 30 minutes in the dark. After incubation, photograph the cells under a fluorescence microscope and analyze the mean fluorescence intensity using Image J.

[0180] 10.6 Experimental Principles and Results

[0181] Advanced glycation endproducts (AGEs), pathological products of non-enzymatic glycation reactions, directly deposit in skin tissue, interact with the receptor for advanced glycation endproducts (RAGE), induce oxidative damage and pigmentation, and mediate irreversible cross-linking of collagen and elastin, leading to structural damage, loss of elasticity, and wrinkle formation, thus exerting multiple negative effects on skin health. In this study, human primary skin fibroblasts (BJ) were glycated using methylglyoxal. After incubation with the test samples for 24 hours, the production of reactive oxygen species (ROS) in BJ cells was measured to evaluate the anti-glycation activity of the test substances.

[0182] The experimental results are as follows Figure 14-15 As shown in the results, compared with the control group, the fluorescence intensity of ROS in BJ cells was significantly enhanced after MGO induction in the MGO group, indicating that the BJ cell glycation model was successfully established. SA-EA, a Solanum nigrum extract, significantly reduced the fluorescence intensity of ROS at concentrations of 0.5, 1, and 2 μg / mL, indicating that SA-EA, a Solanum nigrum extract, has the effect of inhibiting MGO-induced glycation in BJ cells.

[0183] Example 11: Evaluation of Elastase Inhibitory Activity of Solanum nigrum Extract

[0184] 11.1 Reagents and Materials

[0185] Elastase, N-succinyl-Ala-Ala-Ala-pNA (NSAAAP), 1 M Tris-HCl buffer (pH = 8.0), and DPBS.

[0186] 11.2 Instruments

[0187] Microplate reader, 1 / 100,000 microbalance.

[0188] 11.3 Samples to be tested

[0189] NSAAAP working solution: 0.5 mg / mL, prepared in 50 mM Tris-HCl buffer;

[0190] Test sample: Solanum nigrum extract SA-EA obtained in Example 1, tested at concentrations of 0.41, 1.23, 3.7, 11.1, 33.33, 100, and 300 μg / mL, prepared in 50 mM Tris-HCl buffer;

[0191] Elastase working solution: 5 U / mL, prepared in 50 mM Tris-HCl buffer.

[0192] 11.4 Experimental Methods

[0193] According to Table 4 below, add the prepared elastase working solution, various concentrations of Solanum nigrum extract (SA-EA), and 50 mM Tris-HCl buffer to a 96-well plate and incubate at 25°C for an additional 10 minutes. Then, add NSAAAP working solution or 50 mM Tris-HCl buffer and incubate at 25°C for an additional 30 minutes. After incubation, measure absorbance at 410 nm using a microplate reader.

[0194] Table 4 Composition ratios of SA-EA elastase inhibitory activity of Solanum nigrum extract

[0195]

[0196] The elastase activity (P) and half-maximal inhibitory concentration (IC50) of the test sample were calculated according to the following formula (6):

[0197] (6)

[0198] Wherein, T, T0, C, and C0 represent the absorbance of the sample group, sample background group, reaction group, and solvent group at 410 nm, respectively.

[0199] 11.5 Experimental Principles and Results

[0200] Inhibiting elastase activity can improve skin aging. In this experiment, NSAAAP was used as a substrate. Elastase can hydrolyze it in 50 mM Tris-HCl buffer (pH 8.0) to Nsuccinyl-Ala-Ala-Ala and p-nitroaniline (pNA). The absorbance of pNA can be measured at 410 nm in a spectrophotometer. Test substances that inhibit elastase activity can reduce elastin degradation, thereby lowering absorbance. This experiment evaluated the inhibitory effect of the test substance on elastase activity based on changes in absorbance.

[0201] The experimental results are as follows Figure 16 As shown, the elastase activity (P) decreased with increasing SA-EA concentration, and its half-maximal inhibitory concentration IC50 was 41.07 μg / mL, indicating that the Solanum nigrum L. extract SA-EA of the present invention has the anti-aging effect of inhibiting elastase.

[0202] Example 12: Application of Solanum nigrum L. extract in skin care lotion

[0203] 12.1 Formulation Design

[0204] Skin care lotion: The weight percentage ratio of each raw material is: Solanum nigrum extract SA-EA 0.5%, glycerin 5%, sodium hyaluronate 0.5%, panthenol 2%, β-glucan 1%, disodium EDTA 0.1%, phenoxyethanol 0.5%, and the balance is deionized water.

[0205] Comparative skin care lotion: The weight percentage ratio of each raw material is: glycerin 5%, sodium hyaluronate 0.5%, panthenol 2%, β-glucan 1%, disodium EDTA 0.1%, phenoxyethanol 0.5%, and the balance is deionized water.

[0206] 12.2 Preparation

[0207] Prepare the aqueous phase: Dissolve glycerin, sodium hyaluronate, panthenol, β-glucan, and disodium EDTA in deionized water, heat to 75°C, and stir until completely dissolved.

[0208] Cooling and mixing: After cooling to 40°C, add SA-EA (not added in the comparison skin care lotion) and phenoxyethanol, and stir homogenously (500 rpm × 10 minutes);

[0209] Filtration and filling: Pass through a 0.22 μm microporous filter membrane and fill into a sterile container to obtain a light yellow transparent skin care lotion and a comparative skin care lotion.

[0210] 12.3 Efficacy Testing

[0211] 12.3.1 Antioxidant Capacity Evaluation

[0212] 12.3.1.1 Methods:

[0213] The DPPH radical scavenging activity evaluation experimental method of Example 4 was used to dilute the skin care lotion to a sample concentration of 0.1%, 0.5%, 1%, 2%, and 5%, and the comparative skin care lotion was diluted to a sample concentration of 5%, and the DPPH scavenging rates of different test samples were tested.

[0214] 12.3.1.2 Results:

[0215] The experimental results are as follows Figure 17 As shown in the figure, compared with the control skin care lotion, the skin care lotion containing SA-EA (0.1%-5%) can effectively scavenge DPPH free radicals in a concentration-dependent manner. At a dilution concentration of 5%, the effect is close to that of vitamin C, and it has excellent antioxidant activity.

[0216] 12.3.2 Evaluation of Skin Moisturizing Effect

[0217] 12.3.2.1 Subjects:

[0218] Ten healthy volunteers (aged 25-45 years) who had not used any products (cosmetics or topical medications) for 2-3 days were selected. The test area was kept away from water for 1-3 hours before the test. The inner forearms of both hands were cleaned and dried 1 hour before the test, and six 3cm x 3cm test areas were marked. Before the test, the subjects sat quietly in a standard room with a constant temperature of 20-22°C and a constant humidity of 40-60% for 30 minutes. They were prohibited from drinking water and remained relaxed.

[0219] 12.3.2.2 Test method:

[0220] A 5% diluted test sample solution and a control skin care lotion were quickly and evenly applied once to three test areas at a concentration of (2.0±0.1) mg / cm². A skin moisture content meter (Corneometer CM 825) was used to measure the test area's skin moisture content at 0h, 2h, 8h, and 24h. The average value was taken to determine the subject's skin moisture content.

[0221] 12.3.2.3 Results:

[0222] The experimental results are as follows Figure 18 As shown in the figure, compared with the control skin care lotion, the skin care lotion with added SA-EA has a significantly increased moisture content 2 hours after application, indicating that Solanum nigrum extract SA-EA can quickly replenish skin moisture and still maintain the increased moisture after 24 hours, indicating that the skin care lotion containing SA-EA also has a long-lasting moisturizing effect.

[0223] Example 13: Application of Solanum nigrum L. extract in anti-aging cream

[0224] 13.1 Formulation Design

[0225] Anti-aging cream: The weight percentage ratio of each raw material is: Solanum nigrum extract SA-EA 1%, squalane 8%, shea butter 5%, cetearyl alcohol 3%, tocopheryl acetate 0.5%, xanthan gum 0.3%, 1,2-hexanediol 1%, and the balance is deionized water.

[0226] Comparative cream: The weight percentage ratio of each raw material is: squalane 8%, shea butter 5%, cetearyl alcohol 3%, tocopheryl acetate 0.5%, xanthan gum 0.3%, 1,2-hexanediol 1%, and the balance is deionized water.

[0227] 13.2 Preparation

[0228] Preparation of oil phase: Heat squalane, shea butter, and cetearyl alcohol to 75°C to melt;

[0229] Preparation of aqueous phase: Heat deionized water, xanthan gum, and 1,2-hexanediol to 75°C and stir to dissolve;

[0230] Emulsification: Slowly add the water phase into the oil phase and homogenize and emulsify (8000 rpm × 3 minutes);

[0231] Add active ingredients: SA-EA (not added to the control cream) and tocopheryl acetate when the temperature drops to 45°C;

[0232] Filling: After cooling to room temperature, the mixture was filled to obtain a milky white anti-aging cream paste and a comparison cream paste.

[0233] 13.3 Anti-aging efficacy test

[0234] 13.3.1 Model: UVB-induced HDF cell senescence model;

[0235] 13.3.2 Methods:

[0236] The cell supernatant was collected and centrifuged to remove impurities. The collagen I content of the anti-aging cream and the control cream was detected using an ELISA kit.

[0237] 13.3.3 Results:

[0238] The experimental results are as follows Figure 19 As shown in the data, compared with the control cream (collagen I secretion of 47.5±2.9 ng / mL), the collagen I secretion of the cream containing 1% SA-EA increased to 73.4±3.7 ng / mL after treatment, close to the level of Vc, and significantly increased by 67.6% compared with the model group (collagen I secretion of 43.8±3.4 ng / mL) (P < 0.05), indicating that the cream containing SA-EA has significant anti-photoaging effect.

[0239] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art can make some simple modifications, equivalent changes or modifications based on the technical content disclosed above, which all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a Solanum nigrum extract, characterized in that: The following steps are involved: (1) Take Solanum nigrum seedlings, crush them, add 60-80% ethanol at a solid-liquid ratio of 1:5-1:10, heat and reflux at 55-75°C for 3-4 times, combine the extracts, and concentrate under reduced pressure until there is no alcohol taste, thereby obtaining the Solanum nigrum ethanol extract; (2) The ethanol extract obtained in step (1) is dispersed with pure water in a ratio of 1:1, and extracted with n-hexane, dichloromethane, and ethyl acetate in sequence, wherein the volume ratio of the organic phase to the aqueous phase is 1:1 to 1:5, and each organic phase is extracted 3 to 5 times, the ethyl acetate extracts are combined, and the solvent is recovered under reduced pressure to obtain the Solanum nigrum extract.

2. The method for preparing the Solanum nigrum L. extract according to claim 1, wherein: The invention relates to a method for preparing a Solanum nigrum L. ethanol extract from a plant of 3 to 13 days after seedling emergence, crushing the plant, adding 70% ethanol at a solid-liquid ratio of 1:10, heating and refluxing the plant at 65°C for 3 times, wherein the first extraction time is 3 hours, and the second and third extraction times are 4 hours, combining the extracts, and concentrating under reduced pressure until there is no alcohol taste, thereby obtaining a Solanum nigrum L. ethanol extract. In the method for preparing a Solanum nigrum L. ethanol extract, each organic phase is extracted 3 times, wherein the volume ratio of the organic phase to the aqueous phase in the first extraction is 1:3, and the volume ratio of the organic phase to the aqueous phase in the second and third extractions is 1:

2.

3. A Solanum nigrum extract, characterized in that: The invention is prepared by the preparation method of the Solanum nigrum extract according to claim 1 or 2.

4. A functional cosmetic, characterized in that: The cosmetic comprises the Solanum nigrum extract according to claim 3 and cosmetically acceptable excipients, wherein the Solanum nigrum extract is added in an amount of 0.1 to 10 wt %, and has anti-inflammatory, antioxidant, anti-skin aging and / or long-lasting moisturizing effects.

5. The functional cosmetic according to claim 4, characterized in that The addition amount of the Solanum nigrum extract is 0.5-1 wt %, and the cosmetic is a skin care lotion, a face cream, an emulsion or an essence.

6. A medicine, characterized in that: The medicine comprises the Solanum nigrum extract according to claim 3 and pharmaceutically acceptable excipients, and has anti-inflammatory, antioxidant and anti-skin aging effects.

7. Use of the Solanum nigrum L. extract according to claim 3 in the preparation of anti-skin aging medicines or cosmetics.

8. Use of the Solanum nigrum extract according to claim 3 in the preparation of moisturizing or long-lasting moisturizing medicines or cosmetics.

9. Use of the Solanum nigrum L. extract according to claim 3 in the preparation of anti-inflammatory drugs or cosmetics.

10. Use of the Solanum nigrum extract according to claim 3 in the preparation of antioxidant drugs or cosmetics.

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