Preparation method of a solanum lycopersicum anthocyanin extract, product and application thereof

Abstract

This invention discloses a method for preparing anthocyanin extract from tree tomato, along with its products and applications, belonging to the field of natural plant extraction and application. The method uses tree tomato peel as raw material, employing an acidic ethanol aqueous solution containing 0.5% (w / v) trifluoroacetic acid as the extraction solvent, at a material-to-liquid ratio of 1:30 (g / mL), at a temperature of 50℃, and for 60 minutes to obtain a high-content anthocyanin extract. This tree tomato anthocyanin extract possesses excellent anti-inflammatory, antioxidant, anti-glycation, elastase-inhibiting, and whitening activities, and exhibits low cytotoxicity, no phototoxicity, and no skin sensitization. The efficient and stable anthocyanin extraction process provided by this invention has high extraction efficiency and good enrichment effect of active ingredients, meeting the requirements for subsequent industrial production. The obtained extract has whitening, anti-aging, and anti-inflammatory effects, providing a high-quality raw material and formulation solution for developing multifunctional and highly safe natural plant-based skincare products.

Description

Technical Field

[0001] This invention relates to the field of natural plant extraction and application, and in particular to a method for preparing anthocyanin extract from tree tomato, its products, and applications. Technical Background

[0002] Tree tomato (Cyphomandra betacea) fruit is rich in anthocyanin active ingredients. Anthocyanins are known to have antioxidant and anti-inflammatory potential, showing promising applications in the field of functional cosmetics. However, related extraction technologies and application research still have significant limitations, restricting the high-value development of this resource.

[0003] Firstly, at the extraction technology level, existing studies mostly directly use conventional organic solvent (such as alcohol) extraction methods, lacking systematic process optimization tailored to the characteristics of tree tomato raw materials. In particular, insufficient research on the synergistic effects of key parameters such as temperature, time, and solid-liquid ratio makes it difficult to establish a stable, efficient, and reproducible standardized extraction process. The result is often significant fluctuations in extract yield and the content of major active ingredients, poor batch-to-batch consistency, and difficulty in achieving stable and efficient large-scale preparation.

[0004] Secondly, in terms of activity evaluation and application research, existing technologies mostly focus only on the basic antioxidant activity of extracts, while lacking sufficient and systematic research on their deeper effects, which are crucial for skin health. This makes it impossible to accurately position their comprehensive value as a multi-functional cosmetic ingredient. Furthermore, there is a gap in the transformation chain from basic research to product application in existing technologies, hindering commercial application.

[0005] Therefore, this application aims to create an extraction method for tree tomato anthocyanins with clearly defined, stable, and scalable process parameters. Based on this method, it systematically and comprehensively evaluates the skin-related activities of tree tomato in various aspects, such as anti-oxidation, anti-inflammation, whitening, and anti-glycation. This will complete an integrated application study from standardized preparation to efficacy verification, which is of urgent need and great significance for fully exploring the commercial value of tree tomato resources and promoting their practical application in the field of high-value-added cosmetics. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing anthocyanin extract from tree tomato, which, through improvements in extraction solvent and extraction conditions, efficiently extracts anthocyanin active ingredients, providing strong support for subsequent research and development and industrial applications.

[0007] To address the aforementioned technical problems, this invention provides a method for preparing anthocyanin extract from tree tomato, the method comprising the following steps:

[0008] S1: Take the peel of the tree tomato fruit, dry it, and crush it to obtain peel powder;

[0009] S2: Prepare an acidic ethanol aqueous solution containing 0.5% (w / v) trifluoroacetic acid as the extraction solvent;

[0010] S3: Mix the dried fruit peel powder with the extraction solvent at a material-to-liquid ratio of 1:25-1:35 (g / mL), extract at 45-55℃ in the dark for 45-75 minutes, perform solid-liquid separation, and collect the extract; the residue should be extracted at least once.

[0011] S4: Combine all extracts and concentrate under reduced pressure to remove ethanol to obtain a concentrated solution;

[0012] S5: Freeze-dry the concentrated liquid to obtain the tree tomato anthocyanin extract.

[0013] Further improvements were made: in step S2, the ethanol-water solution was 50% (v / v) ethanol-water solution; in step S3, the material-to-liquid ratio was 1:30 (g / mL), the extraction temperature was 50℃, the extraction time was 60 minutes per extraction, and extraction was performed twice.

[0014] As a further improvement of the present invention, the present invention also provides a tree tomato anthocyanin extract. This extract is prepared by the above-described method for preparing tree tomato anthocyanin extract, and the tree tomato anthocyanin extract can be used to prepare whitening, anti-aging, or anti-inflammatory cosmetics or pharmaceuticals. Its anti-aging effects can be achieved through multiple mechanisms, including anti-oxidation, anti-glycation, and inhibition of elastase.

[0015] Further improvements include ensuring that the total anthocyanin content in the extract, calculated as cyanidin-3-glucoside, is not less than 300 mg / g.

[0016] As another improvement of the present invention, the present invention also provides a cosmetic composition comprising the above-mentioned tree tomato anthocyanin extract, and cosmetically acceptable excipients.

[0017] As a further improvement, the tree tomato anthocyanin extract is present in the cosmetic composition at a mass percentage of 0.1%-5%.

[0018] Further improvements include formulating the cosmetic composition into a serum, lotion, cream, or mask. In a cosmetic serum formulation containing the extract, the serum significantly enhances the product's in vitro antioxidant and whitening effects, and its actual skin-improving effect has been verified through human trials.

[0019] As another improvement of the present invention, the present invention also provides the application of the above-mentioned tree tomato anthocyanin extract in the preparation of anti-inflammatory cosmetics or pharmaceuticals. Specifically, the extract can significantly inhibit the release of inflammatory factors NO, IL-6 and TNF-α from LPS-induced Raw264.7 cells in a dose-dependent manner within a concentration range of 1-100 μg / mL.

[0020] As another improvement of the present invention, the present invention also provides the application of the above-mentioned tree tomato anthocyanin extract in the preparation of anti-aging cosmetics or pharmaceuticals. Specifically, the extract has a strong scavenging ability against DPPH free radicals, and its antioxidant index (AO value) is 0.45, which is superior to glutathione, exhibiting strong antioxidant efficacy; the extract can also effectively inhibit the formation of advanced glycation end products (AGEs), and its IC50 value is [missing information]. 50 The concentration was 19.81 μg / mL, indicating strong anti-glycation activity; the extract also inhibited elastase activity, with an IC50 value of 19.81 μg / mL. 50 With a concentration of 30.58 μg / mL, it can exert anti-aging effects in multiple ways, including anti-oxidation, anti-glycation, and inhibition of elastase.

[0021] As another improvement of the present invention, the present invention also provides the application of the above-mentioned tree tomato anthocyanin extract in the preparation of whitening cosmetics or pharmaceuticals. Specifically, the extract can inhibit the activity of tyrosinase and melanin production in A375 cells in a dose-dependent manner, with an inhibition rate exceeding 50% at a concentration of 100 μg / mL.

[0022] To ensure its safety as a cosmetic ingredient, a systematic toxicological evaluation was also conducted in this embodiment of the invention. For example, cytotoxicity tests showed that it is safe for skin-related cells at effective concentrations; in vitro 3T3 neutral red uptake phototoxicity tests confirmed its lack of phototoxicity (PIF value ≤ 2); and human skin occlusive patch tests further demonstrated its lack of skin sensitization. These systematic research results collectively constitute the scientific foundation for the safe application of this extract.

[0023] With this design, the present invention has at least the following advantages:

[0024] This invention, through screening and research on the extraction process of tree tomato peel, yields a highly efficient and stable anthocyanin extraction process. This process achieves high extraction efficiency and good enrichment of active ingredients, meeting the requirements for subsequent industrial production. The resulting tree tomato anthocyanin extract possesses whitening, anti-aging, and anti-inflammatory effects, making it suitable for application in pharmaceuticals or cosmetics. This provides strong support for the high-value utilization of tree tomato fruit resources and lays a solid foundation for the development of safe, efficient, and multifunctional natural cosmetics. Attached Figure Description

[0025] The above is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0026] Figure 1-3 This is the cytotoxicity evaluation result of the tree tomato anthocyanin extract of the present invention.

[0027] Figure 4 This is the phototoxicity evaluation result of the tree tomato anthocyanin extract of the present invention.

[0028] Figure 5 These are the antioxidant activity test results of the tree tomato anthocyanin extract of this invention.

[0029] Figure 6-8 This is the test result of the tree tomato anthocyanin extract of the present invention inhibiting the release of NO, IL-6 and TNF-α in an LPS-induced Raw264.7 cell model.

[0030] Figure 9 This is the result of the non-enzymatic glycosylation test of the tree tomato anthocyanin extract of the present invention.

[0031] Figure 10 This is the test result of the inhibition rate of the tree tomato anthocyanin extract against elastase activity according to the present invention. Detailed Implementation

[0032] Several typical embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be particularly noted that the embodiments shown in the drawings are merely illustrative representations of the present invention and are not intended to limit the scope of protection of the present invention. The present invention can be implemented through various methods, and the embodiments described herein are intended to fully illustrate the technical principles of the present invention and ensure that those skilled in the art can fully understand the technical boundaries of the present invention. Specific embodiments are described below:

[0033] Example 1: Preparation of anthocyanin extract from tree tomato

[0034] This embodiment aims to describe in detail the preparation process of tree tomato anthocyanin extract, and to screen and determine the optimal extraction process by comparing the extraction efficiency under three different extraction parameters (temperature, time, and material-liquid ratio).

[0035] 1.1 Experimental Materials and Equipment

[0036] Ingredients: Fresh tree tomato fruit, using the peel.

[0037] Main reagents: food-grade ethanol, trifluoroacetic acid (analytical grade), distilled water.

[0038] Main equipment: blower drying oven, Chinese medicine pulverizer, standard sieve (60 mesh), electronic analytical balance (accuracy 0.0001g), constant temperature water bath shaker, rotary evaporator, freeze dryer, vacuum filtration device, pH meter.

[0039] 1.2 Raw material pretreatment

[0040] Wash the peel of fresh tree tomato fruit with deionized water, drain the surface moisture, and dry it in a 50℃ forced-air drying oven until constant weight (moisture content <8%). Grind the dried peel with a traditional Chinese medicine pulverizer, pass it through a 60-mesh standard sieve, and obtain uniform tree tomato peel powder. Store it in a desiccator in a sealed, light-proof container for later use.

[0041] 1.3 Solvent Preparation

[0042] Preparation of extraction solvent: Measure a certain volume of 50% (v / v) ethanol aqueous solution, then add trifluoroacetic acid, stir to dissolve completely, and finally prepare an acidic ethanol extraction solvent containing 0.5% (w / v) trifluoroacetic acid.

[0043] 1.4 Extraction Process

[0044] Extraction was performed using the three sets of parameters described below, with each set of experiments repeated three times.

[0045] Option A: Material-to-liquid ratio 1:30 (g / mL), extraction temperature 40℃, single extraction time 90 min.

[0046] Option B: Material-to-liquid ratio 1:30 (g / mL), extraction temperature 50℃, single extraction time 60 min.

[0047] Option C: Material-to-liquid ratio 1:30 (g / mL), extraction temperature 60℃, single extraction time 60 min.

[0048] Detailed operating procedures (taking Option B as an example):

[0049] Weighing and mixing: Accurately weigh 10.00 g (accurate to 0.01 g) of dried tree tomato peel powder and place it in a 500 mL stoppered conical flask. Add 300 mL of pre-prepared acidic ethanol extraction solvent containing 0.5% (w / v) trifluoroacetic acid at a material-to-liquid ratio of 1:30.

[0050] Extraction: Seal the conical flask and place it in a constant temperature water bath shaker set to 50℃. Set the rotation speed to 120 rpm and start timing. Extract for 60 minutes in the dark.

[0051] Solid-liquid separation: Immediately after extraction, vacuum filtration is performed using a Buchner funnel lined with filter paper to separate the residue from the extract. The filtrate is collected.

[0052] Secondary extraction: Transfer all the filter residue back to the original conical flask, add 300 mL of extraction solvent again, and repeat the above extraction (50℃, 60 min) and filtration steps.

[0053] Filtrate Combination: Combine the filtrates obtained from the two extractions to obtain the total extract.

[0054] Concentration: Transfer the combined total extract to a rotary evaporator and concentrate under reduced pressure at a water bath temperature of 50°C until all ethanol is recovered, yielding a concentrated solution (approximately 50-80 mL remaining).

[0055] Drying: The concentrate was transferred to a freeze-drying tray and pre-frozen at -50°C for 4 hours, then freeze-dried (cold trap temperature -80°C, vacuum degree <10 Pa) to constant weight to obtain a deep purple-red to purple-black tree tomato anthocyanin crude extract powder 2.

[0056] Weighing and Storage: Accurately weigh the crude extract powder and calculate the yield. Store the sample at -20℃ in a sealed container, protected from light, until analysis.

[0057] Following the specific operating steps described above, and operating according to the extraction parameters of schemes A and C, we obtained crude anthocyanin extract powder 1 and crude anthocyanin extract powder 3 from tree tomatoes.

[0058] 1.5 Evaluation Indicators and Calculation Methods

[0059] Preliminary HPLC analysis revealed that the anthocyanins in the extract powder were mainly cyanidin-3-glucoside.

[0060] The formula for calculating the extract yield is:

[0061]

[0062] Total anthocyanin content: determined by pH differential method.

[0063] Accurately weigh an appropriate amount of extract powder, dissolve and dilute it separately with potassium chloride-hydrochloric acid buffer at pH 1.0 and acetate-sodium acetate buffer at pH 4.5.

[0064] The absorbance (A) of the two solutions was measured at wavelengths of 520 nm and 700 nm, respectively.

[0065] The formula for calculating total anthocyanins is:

[0066]

[0067] in: ; MW: molecular weight of cyanidin-3-glucoside (449.2 g / mol); DF: dilution factor; V: final volume (L); ε: molar extinction coefficient of cyanidin-3-glucoside (26,900 L / mol·cm); l: optical path length of cuvette (1 cm); m: sample mass (g).

[0068] Results are expressed as milligrams (mg / g) of cyanidin-3-glucoside equivalents per gram of extract.

[0069] 1.6 Results and Analysis

[0070] The results of the three extraction processes are compared in the table below (data is the average of three parallel experiments ± standard deviation):

[0071] Table 1. Effects of different extraction process parameters on the yield and content of anthocyanins in *Tomato*.

[0072] plan extract Extract yield (%) Total anthocyanin content (mg / g) A Tree tomato anthocyanin crude extract powder 1 8.2 ± 0.3 241 ± 8 B Tree tomato anthocyanin crude extract powder 2 11.8 ± 0.4 308 ± 10 C Tree tomato anthocyanin crude extract powder 3 9.5 ± 0.3 265 ± 9

[0073] 1.7 Conclusion

[0074] Experimental results show that, under the same extraction cycle, Scheme B significantly outperforms Schemes A and C in both extract yield and total anthocyanin content. This indicates that the extraction process achieves an optimal balance between dissolution efficiency and prevention of thermal degradation, maximizing the extraction and retention of anthocyanin active ingredients in tree tomato peel. Therefore, Scheme B is determined to be the optimized extraction process of this invention.

[0075] Example 2: Stability verification of the optimal extraction process

[0076] This embodiment aims to conduct three independent repeated experiments on the optimal process (Scheme B) selected in Example 1 to verify the stability of the process.

[0077] 2.1 Experimental Design

[0078] Following the process flow and parameters of Scheme B in Example 1, three independent preparation experiments (numbered B-1, B-2, and B-3) were conducted. Each experiment used a new batch of raw materials, and minor adjustments were made within the allowed parameter ranges to simulate normal fluctuations in actual production:

[0079] Material-to-liquid ratio: 1:30 (g / mL)

[0080] Extraction temperature: Three experiments were conducted at 48℃, 50℃, and 52℃ respectively;

[0081] Extraction time: Three experiments were conducted at 55 min, 60 min, and 65 min respectively;

[0082] Other conditions: Extraction was performed twice, protected from light, and subsequent concentration and freeze-drying steps were exactly the same.

[0083] 2.2 Experimental Results

[0084] The results of three independent replicate experiments are as follows:

[0085] Table 2. Extraction results of three repeated experiments for the optimal process (Scheme B)

[0086]

[0087] 2.3 Conclusion

[0088] The extract yield (11.5%-11.8%) and total anthocyanin content (302 mg / g-310 mg / g) of the three repeated experiments remained highly consistent, with RSDs of less than 2% and 1.5%, respectively, indicating that the optimal extraction process (Scheme B) of the present invention has excellent stability and reproducibility.

[0089] The total anthocyanin content measured in three repeated experiments ranged from a minimum of 302 mg / g to a maximum of 310 mg / g, both exceeding 300 mg / g. Subsequent examples used the tree tomato anthocyanin extract obtained through this extraction process, designated CB-M008.

[0090] Example 3: Cytotoxicity Test

[0091] 3.1 Reagents and Materials

[0092] High glucose medium (DMEM), fetal bovine serum (FBS), DPBS, dimethyl sulfoxide (DMSO), and CCK-8.

[0093] 3.2 Instruments

[0094] CO2 incubator, biosafety cabinet, inverted microscope, ELISA reader.

[0095] 3.3 Cell lines

[0096] Mouse monocytic leukemia cells (Raw264.7), human acute monocytic leukemia cells (THP-1), and human immortalized epidermal cells (HaCaT).

[0097] 3.4 Samples to be tested

[0098] Sample group: Tree tomato anthocyanin extract CB-M008, tested at concentrations of 300 μg / mL, 100 μg / mL, 30 μg / mL, 10 μg / mL, 3 μg / mL, and 1 μg / mL.

[0099] Solvent control group: DMEM complete culture medium;

[0100] Positive control group: DMEM complete medium containing 10% DMSO;

[0101] Zeroing group: DMEM complete culture medium (cell-free).

[0102] 3.5 Experimental Methods

[0103] Raw264.7 cells, THP-1 cells, or HaCaT cells were cultured at a density of 2.5 × 10⁻⁶ cells / year. 5 100 μL of cell suspension per well was seeded into each well of a 96-well plate and cultured until the cell confluence reached approximately 40%. The supernatant was discarded, and different concentrations of the sample were added sequentially for treatment with bioactive substances. After 24 h of treatment, CCK-8 reagent was added at a 1:10 ratio. After 40 min, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula:

[0104]

[0105] 3.6 Results

[0106] The CCK-8 assay is a method for detecting cell viability and growth; the measured OD value is directly proportional to cell activity. For example... Figure 1-3 As shown, the results of the test sample on the cytotoxicity of Raw264.7 cells, THP-1 cells and HaCaT cells showed that the cell viability of the extract was greater than 80% in the above cells at concentrations of 100 μg / mL and below, and no obvious cytotoxicity was observed, indicating that the extract has high safety for immune-related cells and skin cells.

[0107] Example 4: Phototoxicity test

[0108] 4.1 Reagents and Materials

[0109] High glucose medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, neutral red, glacial acetic acid, anhydrous ethanol, chlorpromazine hydrochloride (CPZ), and HEPES.

[0110] 4.2 Instruments

[0111] CO2 incubator, biosafety cabinet, inverted microscope, ELISA reader.

[0112] 4.3 Cell lines

[0113] Mouse embryonic fibroblasts (BALB / C 3T3).

[0114] 4.4 Samples to be tested

[0115] Sample group: Tree tomato anthocyanin extract CB-M008, tested at concentrations of 1 μg / mL, 3 μg / mL, 10 μg / mL, 30 μg / mL, 100 μg / mL, 300 μg / mL, and 1000 μg / mL;

[0116] Blank control group: DMEM medium containing 10% FBS;

[0117] Baseline group: DMEM medium containing 10% FBS (cell-free).

[0118] 4.5 Experimental Methods

[0119] Cells were arranged at a density of 2×10⁻⁶. 5 100 μL of cell suspension per well was seeded into each 96-well cell culture plate. When the cell confluence reached approximately 100%, the supernatant was removed. The test samples were then added sequentially for 1 h of bioactive treatment. Cell culture plates containing samples requiring light treatment were then irradiated with UVA; those without light were placed in an incubator for further culture. After UVA irradiation, the culture medium was removed and the plates were washed. DMEM medium was added, and the 96-well plates were placed in a CO2 incubator (37℃, 5% CO2) for 18–22 h of further culture.

[0120] After culture, add neutral red staining solution and incubate for another 3 hours in a cell culture incubator. After incubation, completely remove the neutral red working solution and wash the cells. Then add neutral red desorption solution (distilled water: ethanol: acetic acid = 49:50:1) to completely lyse the cells, and measure the absorbance at 540 nm using a microplate reader. Calculate the cell viability of the positive control or sample cells using the following formula:

[0121]

[0122] Then, the half-maximal inhibitory concentration (IC50) of cells under UVA irradiation (+Irr) and no irradiation (-Irr) conditions was calculated separately. 50 Calculate the photostimulation factor (PIF) using the following formula:

[0123]

[0124] 4.6 Results

[0125] Some drugs, upon exposure to light, transition from a stable state to an excited state, thereby causing damage to the body. This experiment, based on the "Cosmetic Safety Technical Specifications" (2015 edition), uses the in vitro 3T3 neutral red uptake phototoxicity test method for cosmetic chemical raw materials to determine the changes in the ability of BALB / C 3T3 fibroblasts to absorb neutral red or their cytotoxicity after combined exposure to the test substance and ultraviolet radiation, in order to assess whether the substance is phototoxic.

[0126] The photostimulation factor (PIF) value of a typical phototoxic drug CPZ is usually ≥5, indicating phototoxicity. Experimental results are as follows... Figure 4 As shown, the photostimulation factor (PIF) value of the tomato anthocyanin extract CB-M008 was calculated to be approximately 1.07≤2, indicating that the extract has no phototoxicity.

[0127] Example 5: Evaluation of antioxidant activity

[0128] 5.1 Reagents and Materials

[0129] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH), anhydrous ethanol, 96-well plate.

[0130] 5.2 Instruments

[0131] ELISA reader, 0.0001 g / L microbalance.

[0132] 5.3 Samples to be tested

[0133] DPPH working solution: 50 μg / mL, prepared with anhydrous ethanol;

[0134] Tree tomato anthocyanin extract CB-M008: tested concentrations of 1.25, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg / mL, prepared with distilled water;

[0135] Positive control group: GSH, with concentrations of 300, 100, 33.3, 11.1, 3.7, 1.25, and 0.41 μg / mL.

[0136] 5.4 Experimental Methods

[0137] Weigh 1.5 mg of DPPH powder into a 50 mL centrifuge tube, wrap it in aluminum foil to protect it from light, add 30 mL of anhydrous ethanol, vortex to mix, and sonicate to completely dissolve the powder to obtain a 50 μg / mL DPPH working solution. Prepare corresponding concentrations of GSH working solution and test substance working solution using a serial dilution method. Add 50 μL of test substance working solution, or 50 μL of GSH working solution, or 50 μL of anhydrous ethanol to each well, and then add 150 μL of DPPH working solution or 150 μL of anhydrous ethanol to each well. After adding the samples, mix them using a 96-well microplate mixer, react at room temperature in the dark for 30 min, and read the absorbance at a wavelength of 517 nm using a microplate reader.

[0138] The DPPH clearance rate P of the test substance and GSH was calculated using the following formula:

[0139]

[0140] Where As represents the absorbance of the test substance (or GSH) and DPPH mixture, Ac represents the absorbance of the test substance (or GSH) and EtOH mixture, Ab represents the absorbance of the EtOH and DPPH mixture, and A0 represents the absorbance of the EtOH and EtOH mixture.

[0141] After calculating the clearance rate, GraphPad Prism software was used to plot the data. The final concentration of the sample in the reaction system was plotted on the x-axis, and the clearance rate on the y-axis. A nonlinear fitting equation was established between the concentration of the test solution and the clearance rate, and the half-maximal clearance (EC) was calculated. 50 The antioxidant capacity (AO) value of peptide or small molecule active substance samples is calculated using the following formula:

[0142]

[0143] EC 50 (S) represents the concentration (μg / mL) corresponding to the half-maximal clearance of the test substance, and EC50(R) represents the concentration (μg / mL) corresponding to the half-maximal clearance of GSH.

[0144] 5.5 Results

[0145] When free radical scavengers (reducing agents) are 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 pale yellow, thus weakening the light absorption of DPPH. Studies have shown that the degree of fading of the DPPH ethanol solution is linearly related to the number of electrons it accepts. Therefore, the ability of the test sample to scavenge free radicals, i.e., the magnitude of its antioxidant activity, can be evaluated by measuring the light absorption at 517 nm of the sample's reaction solution with DPPH.

[0146] The test results are as follows Figure 5 As shown, the tree tomato anthocyanin extract CB-M008 exhibited significant DPPH free radical scavenging ability. Its scavenging activity was superior to the positive control glutathione (GSH), specifically demonstrated by a lower EC50 value (11.52 μg / mL) and a higher antioxidant index (AO = 0.45). This indicates that the extract possesses strong antioxidant activity.

[0147] Example 6: Evaluation of anti-inflammatory activity

[0148] 6.1 Reagents and Materials

[0149] DMEM high glucose medium, fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, lipopolysaccharide (LPS), dexamethasone (DEX), Mouse TNF-α ELISA kit, Mouse IL-6 ELISA kit, NO detection kit.

[0150] 6.2 Instruments

[0151] CO2 incubator, biosafety cabinet, inverted microscope, ELISA reader.

[0152] 6.3 Cell lines

[0153] Mouse mononuclear macrophage leukemia cells (Raw264.7) were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0154] 6.4 Sample to be tested

[0155] LPS working solution: 25 ng / mL, prepared using DMEM medium containing 1% FBS;

[0156] Dexamethasone working solution (Dex): 10 μg / mL, prepared using LPS working solution;

[0157] Tree tomato anthocyanin extract CB-M008: tested at concentrations of 1, 3, 10, 30, and 100 μg / mL, prepared using LPS working solution.

[0158] 6.5 Experimental Methods

[0159] Raw 264.7 cells in good exponential growth phase were collected, detached using a cell scraper, centrifuged, and then adjusted to a cell density of 1.5 × 10⁻⁶. 5 Cell suspension was prepared at a concentration of 500 μL / well in 48-well plates and incubated at 37°C with 5% CO2 for 16-24 h. Once cell confluence reached 30-50%, the supernatant was discarded. Except for the control group, 400 μL of the test sample was added to each well, and the plates were incubated at 37°C with 5% CO2 for 24 h. After incubation, the supernatant was collected, centrifuged to remove cell debris, and the concentrations of NO, IL-6, and TNF-α in the supernatant were measured according to the kit instructions.

[0160] 6.6 Experimental Principles and Results

[0161] Lipopolysaccharide (LPS) can activate the synthesis and release of various cytokines and inflammatory mediators through cell signaling systems. The accumulation of inflammatory factors can cause symptoms such as vasodilation, redness, swelling, stinging, and itching, leading to a state of skin irritation and sensitivity. This experiment used LPS-induced Raw 264.7 cells to establish an inflammation model and evaluated the anti-inflammatory activity of the extract. The experimental results are as follows: Figure 6-8As shown, compared with the control group, the expression levels of NO, TNF-α, and IL-6 in Raw 264.7 cells were significantly increased after LPS induction, indicating that the Raw 264.7 cell inflammation model was successfully established. Extract concentrations of 1-100 μg / mL significantly inhibited LPS-induced release of NO, IL-6, and TNF-α from Raw 264.7 cells. This indicates that the tree tomato anthocyanin extract of this invention possesses good anti-inflammatory activity.

[0162] Example 7: Evaluation of non-enzymatic glycation inhibition activity

[0163] 7.1 Reagents and Materials

[0164] Bovine serum albumin (BSA), D-anhydrous glucose, DPBS, penicillin / streptomycin (P / S).

[0165] 7.2 Instruments

[0166] ELISA reader, 0.0001 g / L microbalance.

[0167] 7.3 Sample to be tested

[0168] BSA working solution: 40 μg / mL, prepared using DPBS containing 1% P / S;

[0169] Glucose working solution: 480 μg / mL, prepared using DPBS containing 1% P / S;

[0170] The sample to be tested was tree tomato anthocyanin extract CB-M008, prepared at concentrations of 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg / mL using DPBS containing 1% P / S.

[0171] 7.4 Experimental Methods

[0172] According to the table below, different concentrations of the prepared extract, BSA working solution, glucose working solution, and DPBS were added to 96-well plates, respectively. After sealing with sealing film, the plates were incubated at 37°C in the dark for 7 days. After incubation, the fluorescence intensity was detected using a microplate reader at an excitation wavelength of 370 nm and an emission wavelength of 440 nm.

[0173] Table 3. Component ratios for the evaluation experiment of non-enzymatic glycation inhibition activity of anthocyanin extracts.

[0174]

[0175] Calculate the AGEs inhibition rate (P) and half-maximal inhibitory concentration (IC50) of the test sample using the following formulas. 50 ).

[0176]

[0177] Wherein, T, T0, C, and C0 represent the fluorescence intensity of the sample group, the sample background group, the saccharification reaction group, and the BSA group under the conditions of excitation wave 370 nm and emission wave 440 nm, respectively.

[0178] 7.5 Experimental Principles and Results

[0179] Non-enzymatic glycation (NEG) refers to the non-enzymatic condensation reaction between the carbonyl group of reducing sugars (such as glucose and fructose) and the free amino group of macromolecules such as proteins, lipids, or nucleic acids. The stable covalent additions generated by this reaction are called advanced glycation end products (AGEs). This experiment assesses the anti-glycation ability of each test substance by measuring its ability to inhibit the formation of AGEs. The experimental results are as follows: Figure 9 As shown, the anthocyanin extract of *Tomato japonica* exhibits significant non-enzymatic glycation inhibitory activity, with a half-maximal inhibitory concentration (IC50) of [missing value]. 50 The concentration was 19.81 μg / mL.

[0180] Example 8: Evaluation of elastase inhibitory activity

[0181] 8.1 Reagents and Materials

[0182] Elastase, N-succinyl-Ala-Ala-Ala-pNA (NSAAAP), 1 M Tris-HCl buffer (pH= 8.0), DPBS.

[0183] 8.2 Instruments

[0184] ELISA reader, 0.0001 g / L microbalance.

[0185] 8.3 Sample to be tested

[0186] NSAAAP working solution: 0.5 mg / mL, prepared using 50 mM Tris-HCl buffer;

[0187] Tree tomato anthocyanin extract CB-M008: test concentrations were 0.41, 1.23, 3.7, 11.1, 33.33, 100, and 300 μg / mL, prepared using 50 mM Tris-HCl buffer;

[0188] elastase working solution: 5 U / mL, prepared using 50 mM Tris-HCl buffer.

[0189] 8.4 Experimental Methods

[0190] According to the table below, prepare the elastase working solution, different concentrations of tomato anthocyanin extract solution, and 50 mM Tris-HCl buffer were added to each well of a 96-well plate. The plates were incubated at 25°C for 10 min, then NSAAAP working solution or 50 mM Tris-HCl buffer was added, and the plates were incubated at 25°C for another 30 min. After incubation, the absorbance was measured at 410 nm using a microplate reader.

[0191] Table 4. Group ratios for evaluating the elastase inhibitory activity of tree tomato anthocyanin extract.

[0192]

[0193] Calculate the elastase activity (P) and half-maximal inhibitory concentration (IC50) of the sample using the following formula. 50 :

[0194]

[0195] Where T, T0, C, and C0 represent the absorbance of the sample group, the background sample group, the reaction group, and the solvent group at 410 nm, respectively.

[0196] 8.5 Experimental Principles and Results

[0197] Inhibiting elastase activity is beneficial for improving skin aging. This experiment used NSAAAP as a substrate. Elastase can be hydrolyzed in 50mM Tris-HCl buffer at pH 8.0 to Nsuccinyl-Ala-Ala-Ala and p-nitroaniline (pNA). The absorbance of pNA can be measured at 410 nm using a spectrophotometer. Test substances with elastase-inhibiting activity can reduce elastin degradation, thereby lowering the absorbance. This experiment evaluated the inhibitory effect of test substances on elastase activity based on changes in absorbance. Experimental results are as follows: Figure 10 As shown, elastase activity (P) decreases with increasing extract concentration, and its half-maximal inhibitory concentration (IC50) decreases. 50 The concentration was 30.58 μg / mL. These results indicate that the tree tomato anthocyanin extract of the present invention possesses anti-aging effects by inhibiting elastase.

[0198] Example 9: Evaluation of Whitening Activity

[0199] 9.1 Reagents and Materials

[0200] DMEM high glucose medium, fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, trypsin, L-DOPA, Triton X-100, sodium hydroxide (NaOH), dimethyl sulfoxide (DMSO), and kojic acid.

[0201] 9.2 Instruments

[0202] CO2 incubator, biosafety cabinet, inverted microscope, ELISA reader, cell counter.

[0203] 9.3 cell lines

[0204] Human malignant melanoma cells (A375) were purchased from the American Type Culture Collection (ATCC).

[0205] 9.4 Sample to be tested

[0206] working solution of tree tomato anthocyanin extract: diluted to the final test concentration of 25, 50, or 100 μg / mL using DMEM medium containing 1% FBS.

[0207] Positive control working solution (kojic acid): Prepared into a working solution of 100 μg / mL using DMEM medium containing 1% FBS.

[0208] 9.5 Experimental Methods

[0209] A375 cells were planted at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 cells / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h to allow cell adhesion. Cells were then treated for 72 h with culture medium containing different concentrations of tomato anthocyanin extract (25, 50, 100 μg / mL) or a positive control kojic acid (100 μg / mL). After washing the cells with PBS, 100 μL of PBS containing 1% Triton X-100 was added to each well for repeated freeze-thaw lysis. 80 μL of cell lysate was transferred to a new well, and 20 μL of freshly prepared L-DOPA solution (2.5 mg / mL) was added. The plates were immediately incubated at 37°C. Absorbance was measured at 475 nm using a microplate reader at 0 and 60 minutes of reaction. The increase in absorbance over 60 minutes (ΔOD475) characterized tyrosinase activity. For the determination of intracellular melanin content: another batch of cells treated with the same method for 72 h were digested, collected, washed with PBS and counted. The precipitate of an equal number of cells was resuspended in 200 μL of 1 M NaOH solution (containing 10% DMSO) and heated in an 80°C water bath for 1 h to completely dissolve the melanin. After cooling, the absorbance was measured at a wavelength of 405 nm to reflect the relative melanin content.

[0210] 9.6 Experimental Principles and Results

[0211] The key rate-limiting enzyme in melanin production is tyrosinase. By detecting the activity of this enzyme and the content of the final product, melanin, the skin-whitening effect of a sample can be directly assessed.

[0212] The experimental results (see Table 5 below) showed that, compared with the blank control group, the anthocyanin extract of *Tomato japonica* significantly and dose-dependently inhibited the activity of tyrosinase in A375 cells and reduced melanin synthesis. At a concentration of 100 μg / mL, its inhibition rate against tyrosinase reached 50.2%, and its inhibition rate against melanin production reached 56.3%, comparable to the positive control kojic acid. This indicates that the extract of this invention has clear skin-whitening activity.

[0213] Table 5. Effects of anthocyanin extract from *Tomato* on tyrosinase activity and melanin production in A375 cells.

[0214]

[0215] Example 10: Skin sensitization evaluation (human closed patch test)

[0216] To assess the safety of tree tomato anthocyanin extract for use in cosmetics, a closed patch test on human skin was conducted in accordance with the requirements of the "Cosmetic Safety Technical Specifications".

[0217] 10.1 Test Substances and Grouping:

[0218] Experimental group: an aqueous solution containing 1.2% tree tomato anthocyanin extract (prepared using the extract prepared in Scheme B of Example 1).

[0219] Negative control group: The matrix solution was completely identical to that of the experimental group (containing no active ingredients).

[0220] Positive control group: 0.5% (w / v) sodium dodecyl sulfate aqueous solution, as the standard irritant.

[0221] 10.2 Methods

[0222] Experimental design: A standard Finn Chamber® spot tester was used. 20 μL of the test substance was quantitatively added to the spot tester compartment using a precision pipette.

[0223] Application: The loaded patch array is applied to a healthy, undamaged area of ​​the subject's back and secured with hypoallergenic adhesive tape to ensure a good seal. The application period is 24 hours.

[0224] Observation and evaluation: At 30 minutes (to eliminate the effect of pressure marks), 24 hours and 48 hours after removal of the spot tester, each test site was independently and blinded by a trained dermatologist under standard light conditions.

[0225] Skin reaction score: The score is based on the criteria recommended by the International Contact Dermatitis Study Group (ICDRG), as follows:

[0226] (-) Negative reaction: No changes in the skin.

[0227] (±) Suspicious reaction: only slight erythema.

[0228] (+) Weak positive reaction: erythema, infiltration, and a small number of papules.

[0229] (++) Strong positive reaction: erythema, infiltration, papules, vesicles.

[0230] (+++) Extremely strong positive reaction: obvious erythema and infiltration, with confluent vesicles or bullae appearing.

[0231] Result determination: If the skin reaction score reaches (+) or above during the 48-hour observation after the removal of the spot tester, the subject is considered to have a positive reaction to the test substance.

[0232] The formula for calculating skin reaction rate is:

[0233] .

[0234] 10.3 Results

[0235] The experimental results are shown in the table below. The reaction rates of both the experimental group containing tree tomato anthocyanin extract and the negative control group (matrix) were extremely low, with no statistically significant difference, while the positive control group exhibited the expected irritant reaction. This indicates that under the experimental conditions, the tree tomato anthocyanin extract is not sensitizing to the skin.

[0236] Table 6 Results of human skin occlusive patch test

[0237]

[0238] Example 11: Application of Tree Tomato Anthocyanin Extract in Whitening and Antioxidant Serum

[0239] 11.1 Formulation Design

[0240] Whitening and Antioxidant Essence: Tomato anthocyanin extract 1.2%, niacinamide 3.0%, glycerin 5.0%, panthenol 1.0%, ascorbate glucoside 2.0%, xanthan gum 0.2%, 1,2-hexanediol 1.0%, phenoxyethanol 0.5%, balance deionized water.

[0241] Comparative serum (base control): Except for the absence of tree tomato anthocyanin extract, the other ingredients and their contents are exactly the same as the above-mentioned "whitening and antioxidant serum".

[0242] 11.2 Preparation method

[0243] Aqueous phase preparation: Dissolve glycerol, panthenol, ascorbate glucoside, and nicotinamide in a portion of deionized water, heat to 75-80℃ and stir until completely dissolved.

[0244] Homogenization and mixing: After cooling to 40-45℃, add xanthan gum and homogenize by stirring to fully disperse and swell it. Then add tree tomato anthocyanin extract (omit this step for comparison serum) and stir well.

[0245] Final adjustment and filling: Continue cooling to 30-35℃, add 1,2-hexanediol and phenoxyethanol, and stir well. Finally, add the remaining deionized water to make up to the full volume, homogenize, filter through a 0.22 μm microporous membrane, and fill into light-proof containers to obtain a purple-red transparent essence.

[0246] 11.3 Efficacy Test

[0247] 11.3.1 Evaluation of in vitro antioxidant capacity

[0248] Methods: The DPPH free radical scavenging method was used. The above-mentioned essence samples were diluted to concentrations of 0.5%, 1%, 2%, and 5% (v / v), with vitamin C (Vc) as a positive control. The absorbance at 517 nm was measured, and the scavenging rate was calculated.

[0249] Results: The whitening and antioxidant essence of this invention exhibited significant DPPH free radical scavenging ability in a dose-dependent manner. At a test concentration of 5%, its scavenging rate reached (84.7 ± 2.9)%, significantly higher than that of the comparative essence at the same concentration (15.3 ± 2.1)% (p < 0.01), demonstrating that the addition of tree tomato anthocyanin extract greatly enhanced the in vitro antioxidant activity of the formula.

[0250] 11.3.2 Evaluation of the effect of in vitro whitening

[0251] Methods: A melanoma cell (A375) model was established. Two serums were diluted to 5% (v / v) with culture medium as test samples. Their inhibition rates on tyrosinase activity and melanin production in A375 cells were measured (method as described in Example 9).

[0252] Results: Compared with the blank control group and the control serum group, the serum treatment of the present invention can significantly inhibit the tyrosinase activity (inhibition rate: (48.5 ± 4.1)%) and melanin synthesis (inhibition rate: (52.1 ± 3.8)%) of A375 cells, and the effect is significantly better than the control serum with no significant inhibitory effect (p < 0.01), which confirms that the formula has a clear in vitro whitening effect.

[0253] 11.3.3 Human Efficacy Evaluation (Skin Brightness and Skin Tone Improvement Test)

[0254] Subjects: 35 healthy female volunteers aged 30-50 years with dull skin and signs of photoaging were recruited.

[0255] Methods: A randomized, double-blind, half-face controlled method was used. Volunteers applied the essence of this invention to one side of their face and a control essence to the other side, twice daily for 8 weeks. At weeks 0, 4, and 8, the number of brown spots on the face was assessed using a skin image analyzer (VISIA-CR), and the individual type angle (ITA°) value was measured using a skin color meter to quantify skin brightness.

[0256] Subjective evaluation: At the end of the trial, a questionnaire was used to investigate the subjects' subjective feelings about the product in terms of moisturizing, brightening and overall satisfaction.

[0257] Results: After 8 weeks of use, compared with the side using the control serum, the skin brightness (ITA° value) on the side using the serum of this invention increased by an average of +13.5%, and the number of facial brown spots decreased by an average of -18.2%, with statistically significant differences (p < 0.01). Subjective questionnaires showed that over 80% of volunteers felt that the skin tone on the side using the serum of this invention was brighter and more even. This indicates that the serum containing tree tomato anthocyanin extract can effectively improve dull skin, brighten skin tone, and has significant whitening and antioxidant effects.

[0258] 11.4 Summary

[0259] The above experimental results show that the essence containing the tree tomato anthocyanin extract of this invention has significantly better in vitro antioxidant and whitening activities, as well as skin tone improvement effects than the matrix formula without the extract, demonstrating the key efficacy contribution of the extract in the formula.

[0260] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, or alterations made by those skilled in the art using the disclosed technical content shall fall within the protection scope of the present invention.

Claims

1. A method for preparing an anthocyanin extract from tree tomato, characterized in that, The preparation method includes the following steps: S1: Take the peel of the tree tomato fruit, dry it, and crush it to obtain peel powder; S2: Prepare an acidic ethanol aqueous solution containing 0.5% (w / v) trifluoroacetic acid as the extraction solvent; S3: Mix the dried fruit peel powder with the extraction solvent at a material-to-liquid ratio of 1:25-1:35 (g / mL), extract at 45-55℃ in the dark for 45-75 minutes, perform solid-liquid separation, and collect the extract; the residue should be extracted at least once. S4: Combine all extracts and concentrate under reduced pressure to remove ethanol to obtain a concentrated solution; S5: Freeze-dry the concentrated liquid to obtain the tree tomato anthocyanin extract.

2. The method for preparing the tree tomato anthocyanin extract according to claim 1, characterized in that, In step S2, the ethanol-water solution is a 50% (v / v) ethanol-water solution; in step S3, the material-to-liquid ratio is 1:30 (g / mL), the extraction temperature is 50℃, the extraction time is 60 minutes per extraction, and extraction is performed twice.

3. A tree tomato anthocyanin extract, characterized in that, The anthocyanin extract is prepared by the method described in claim 1 or 2, and the anthocyanin extract can be used to prepare whitening, anti-aging or anti-inflammatory cosmetics or pharmaceuticals.

4. The tree tomato anthocyanin extract according to claim 3, characterized in that, The total anthocyanin content in the extract, calculated as cyanidin-3-glucoside, shall not be less than 300 mg / g.

5. A cosmetic composition, characterized in that, It contains the tree tomato anthocyanin extract as described in claim 3 or 4, and cosmetic-acceptable excipients.

6. The cosmetic composition according to claim 5, characterized in that, The tree tomato anthocyanin extract is present in the cosmetic composition at a mass percentage of 0.1%-5%.

7. The cosmetic composition according to claim 5, characterized in that, The cosmetic composition is formulated as a serum, lotion, cream, or mask.

8. The use of the tree tomato anthocyanin extract according to claim 3 or 4 in the preparation of anti-inflammatory cosmetics or pharmaceuticals.

9. The use of the tree tomato anthocyanin extract according to claim 3 or 4 in the preparation of anti-aging cosmetics or pharmaceuticals.

10. The use of the tree tomato anthocyanin extract according to claim 3 or 4 in the preparation of whitening cosmetics or pharmaceuticals.

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