A method for analyzing anti-aging and skin whitening mechanisms of bergamot extract based on network pharmacology and experimental verification

Abstract

This invention discloses a method for analyzing the anti-aging and skin whitening mechanisms of bergamot extract based on network pharmacology and experimental verification. Belonging to the field of computer-aided drug screening technology, this method uses network pharmacology to predict the core targets of the active ingredients in bergamot and screens the signaling pathways regulated by the bergamot extract. Through DPPH free radical scavenging experiments, mushroom tyrosinase inhibition experiments, B16F10 cell melanin production inhibition experiments, HSF cell type I collagen secretion promotion experiments, and a UVB-induced mouse skin pigmentation model, the method systematically verifies that bergamot extract has multiple effects, including significantly inhibiting melanin synthesis, scavenging free radicals, promoting collagen secretion, and improving skin pigmentation. This invention is the first to combine computational prediction with experimental verification, fully elucidating the whitening and anti-aging potential of bergamot extract and its underlying molecular mechanisms, providing a reliable method and basis for developing novel natural cosmetic raw materials.

Description

Technical Field

[0001] This invention belongs to the field of computer-aided drug screening technology, specifically a method for analyzing the anti-aging and skin whitening mechanisms of bergamot extract based on network pharmacology and experimental verification. Background Technology

[0002] Under normal physiological conditions, hyperpigmentation primarily functions as a barrier against ultraviolet (UVR) radiation penetrating deep into the skin and disrupting circulation, protecting the skin from UV damage. However, excessive melanin production can hinder normal hyperpigmentation, leading to functional disorders such as melasma, solar lentigines, and post-inflammatory hyperpigmentation. Traditional medications for treating hyperpigmentation, such as hydroquinone and mercuric chloride, often cause adverse side effects like skin irritation and contact dermatitis, making them unsuitable for clinical use and treatment of skin diseases. In recent years, traditional Chinese medicine, due to its high efficacy and multi-target effects, has shown potential in treating various skin diseases, making it an important direction for new drug research.

[0003] Buddha's Hand (Citrus medica L. var. sarcodactylis Swingle) is the dried fruit of the citrus plant (Citrus medica L. var. sarcodactylis Swingle). China is the world's largest producer of citrus fruits, with numerous varieties, among which Buddha's Hand is one of the major citrus varieties. The medicinal value of Buddha's Hand was first recorded under the name "Gouyuan" in Chen Zangqi's *Bencao Shiyi* (Supplement to the Compendium of Materia Medica) during the Tang Dynasty, where it was described as having the effects of soothing the liver and relieving depression, and promoting qi circulation. Currently, there are abundant research reports on the chemical composition and pharmacological effects of Buddha's Hand, mainly focusing on the extraction and separation of essential oils, component analysis, and functional activities. Buddha's Hand possesses pharmacological effects such as antioxidant, neuroprotective, anticancer, and anti-inflammatory properties. Traditional Chinese medicine prescriptions containing Buddha's Hand have shown clinical efficacy in treating melasma caused by liver qi stagnation in women. Investigating the effect of Buddha's Hand extract on melanin synthesis is crucial for improving the utilization rate of Buddha's Hand and expanding its application and development value in food, medicine, and cosmetics, and provides reliable experimental evidence for the intervention and treatment of melanin-related diseases. Summary of the Invention

[0004] To address the aforementioned issues, the present invention aims to provide a method for analyzing the anti-aging and skin-whitening mechanisms of bergamot extract based on network pharmacology and experimental verification.

[0005] To achieve the above objectives, the present invention employs the following technical solution: A method for analyzing the anti-aging and skin whitening mechanisms of bergamot extract based on network pharmacology and experimental validation is proposed. This method uses network pharmacology to predict the core targets that the active ingredients of bergamot may act on, screens the signaling pathways regulated by bergamot extract, and then verifies the anti-aging and skin whitening mechanisms of bergamot extract through in vitro activity validation, cellular level validation, and animal model validation.

[0006] Specifically, the following steps are included: 1) Prediction of active ingredients and targets: Screening for active ingredients in bergamot extract using network pharmacology and predicting their potential targets; 2) Disease target acquisition and intersection analysis: Search for disease targets related to "hyperpigmentation" from the GeneCards, OMIM, and PharmGKB databases, and find the intersection between the active ingredient targets and the disease targets; 3) Core target screening and pathway analysis: The active ingredient-disease target protein interaction network was constructed using the STRING database. The core targets were screened using Cytoscape software. The core targets were annotated with GO function and enriched using the DAVID database. The signaling pathways regulated by the bergamot extract were screened. 4) In vitro activity verification: Antioxidant capacity was evaluated by DPPH free radical scavenging experiment; whitening potential was initially screened by mushroom tyrosinase inhibition experiment; 5) Cellular level verification: The inhibitory effect of bergamot extract on melanin production was verified using mouse melanoma cells B16F10; the effect of bergamot extract on HSF cell proliferation and type I collagen secretion was verified using human skin fibroblasts (HSF). 6) Animal model validation: A UVB-induced mouse skin pigmentation model was established to validate the anti-aging and skin whitening mechanism of bergamot extract.

[0007] The bergamot extract mentioned in step 1) is either an alcoholic extract or an aqueous extract of bergamot.

[0008] In step 1), the active ingredients in bergamot extract were screened using the TCMSP database with oral bioavailability ≥30% and drug-likeness ≥0.14.

[0009] The active ingredient mentioned in step 1) is one or more of the following: geraniol, 5,2',6'-trihydroxy-7,8-dimethoxyflavone, sitosterol, oleic acid, enzyme inhibitor complex, 5,2',5'-trihydroxy-6,7,8-dimethoxyflavone and squalene.

[0010] The platforms for predicting potential targets mentioned in step 1) are TCMSP and Swiss Target Prediction.

[0011] The core targets mentioned in step 3) include AR, SRC, MM7, P9, ESR1, AKT1, MAPK3, VEGFA, and IL6.

[0012] The signal paths described in step 3) include the PI3K-Akt signal path and the MAPK signal path.

[0013] The safe medicinal concentration of the bergamot alcohol extract is no higher than 20 mg / mL, and the safe medicinal concentration of the bergamot water extract is no higher than 40 mg / mL.

[0014] The present invention has the following advantages over the prior art: This invention systematically analyzes the anti-aging and skin whitening mechanisms of bergamot extract using network pharmacology methods. It reveals for the first time that bergamot alcohol extract has dual effects of skin whitening and anti-aging, and establishes a complete and verifiable efficacy evaluation method system, thereby overcoming the shortcomings of existing technologies that have a single understanding of the efficacy of bergamot extract and a fragmented evaluation system. Compared to existing technologies, this invention not only verifies a clear whitening effect by inhibiting tyrosinase activity, reducing melanin synthesis, and downregulating the expression of key genes such as MITF, but also confirms its anti-aging potential by promoting the secretion of type I collagen by HSF cells, achieving integrated development of multiple skin benefits from a single natural ingredient. Furthermore, this invention provides a progressive and standardized screening process, from initial screening using in vitro antioxidant and enzyme inhibition, to cell model verification of B16F10 cell melanin production inhibition and HSF cell collagen secretion promotion, and finally to in vivo confirmation of a UVB-induced mouse skin pigmentation model. This method is rigorous, reliable, and can be widely applied to the efficacy evaluation of natural products. Finally, this invention demonstrates through in vivo experiments that FAE can significantly improve UVB-induced skin pigmentation, reduce plasma TYR and IL-18 levels, and increase SOD activity and skin collagen content, providing solid experimental evidence for its development into a safe and effective cosmetic or topical drug ingredient. Attached Figure Description

[0015] Figure 1 Schematic diagram of the DPPH radical scavenging activity of FAE and FWE; Figure 2 (a) shows the effect of FAE on the activity of mushroom tyrosinase in vitro; (b) shows the effect of FWE on the activity of mushroom tyrosinase in vitro. Figure 3 (a) shows the effect of different concentrations of FAE on the viability of B16F10 cells; (b) shows the effect of different concentrations of FEW on the viability of B16F10 cells. Figure 4(a) shows the effect of different concentrations of FAE on tyrosinase in B16F10 cells; (b) shows the effect of different concentrations of FWE on tyrosinase in B16F10 cells; (c) shows the distribution of melanin in B16F16 cells observed under a microscope after culturing with FAE or FWE for 24 h; (d) shows the effect of different concentrations of FAE on melanin content in B16F16 cells after culturing for 24 h; (e) shows the effect of different concentrations of FWE on melanin content in B16F16 cells after culturing for 24 h. Figure 5 Mapping of mRNA expression levels of TYR, TYRP-1, TYRP-2, and MITF using Real-time PCR analysis; Figure 6 The effect of different concentrations of FAE and FWE on HSF cell viability is shown in the figure. Figure 7 The effect of different concentrations of FAE and FWE on changes in COL1α1 in HSF cells is shown in the figure. Figure 8 The diagram shows the effect of FAE on UV-induced melanin over-deposition; (a) is the Masson-Fontana staining diagram; (b) is the relative melanin expression diagram. Figure 9 The graph shows the effect of FAE on the levels of TYR, IL-18, and SOD in mouse plasma. Figure 10 The figure shows the effect of FAE on type I collagen content in mouse skin tissue. Detailed Implementation

[0016] To better understand the technical solution of the present invention, the following detailed embodiments further illustrate the above-mentioned content of the present invention. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention.

[0017] In the following examples, dried Buddha's Hand fruit was purchased from Anguo Tongli Traditional Chinese Medicine Co., Ltd. Reagents and cells: DPPH (Sangon Biotech); mushroom tyrosinase, L-DOPA (Solepro); Mouse melanoma cells B16F10 (Chinese Academy of Sciences Cell Bank); DMEM high glucose medium, fetal bovine serum (FBS) (Gibco); CCK-8 kit (Beyotime); TRIzol reagent (Invitrogen); Reverse transcription and qPCR kit (Nearshore Protein); Human skin fibroblast HSF; Human COL1α1 ELISA kit (Sangon Biotech); SPF-grade male C57BL / 6 mice, 6-8 weeks old; Arbutin (standard); Mouse TYR, IL-18, SOD, type I collagen ELISA kit (Huamei Biotechnology); Masson-Fontana melanin staining kit (Huamei Biotechnology).

[0018] Preparation of bergamot alcohol extract (FAE): The bergamot was ground and pulverized and passed through a 60-mesh sieve. 10g of bergamot powder was weighed and 200mL of 70% ethanol was added at a feeding ratio of 1:20 (g / mL). The mixture was thoroughly mixed and sonicated for 1h. The mixture was centrifuged at 8000r / min for 15min. The supernatant was collected and the extract was filtered using a Buchner funnel. The extract was concentrated under reduced pressure at 50℃ to twice the weight of the crude drug, with a crude drug concentration of 0.5g / ml. The extract was stored at 4℃.

[0019] Preparation of Buddha's Hand Water Extract (FWE): Weigh 10g of Buddha's Hand powder after sieving, add 200mL of pure water at a ratio of 1:20, sonicate for 1h, centrifuge at 8000r / min for 15min, collect the supernatant and filter the extract using a Buchner funnel, concentrate under reduced pressure at 65℃ to twice the weight of the crude drug using a reflux condenser, the crude drug concentration is 0.5g / ml, and store at 4℃.

[0020] Example 1: Potential efficacy prediction analysis based on network pharmacology 1) Active Ingredient and Target Prediction: Using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), potential active ingredients of Buddha's Hand were screened based on oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.14. A total of 7 ingredients were identified, including diosmetin, sitosterol alpha1, and 5,2',6'-trihydroxy-7,8-dimethoxyflavone. The targets of these ingredients were predicted using TCMSP and the SwissTargetPrediction platform, resulting in 259 potential targets after deduplication.

[0021] 2) Disease target acquisition and intersection analysis: Using "hyperpigmentation" as the keyword, pigmentation-related disease targets were retrieved from the GeneCards, OMIM, and PharmGKB databases. After integration and deduplication, 1422 disease targets were obtained. The intersection of the Buddha's Hand component targets and the disease targets was taken to obtain 52 common targets.

[0022] 3) Core Target Screening and Pathway Analysis: Fifty-two intersecting targets were submitted to the STRING database to construct a protein-protein interaction network, with a confidence level >0.7. The network file was imported into Cytoscape 3.9.1 software, and the "NetworkAnalyzer" plugin was used to calculate topological parameters such as node degree values. Twenty-three genes with degree values ​​greater than the median were selected as core targets, including AR, SRC, MMP9, AKT1, and MAPK3. KEGG pathway enrichment analysis was performed on the core targets using the DAVID database (P<0.05). The results showed that these targets were significantly enriched in pathways closely related to cell proliferation, apoptosis, and melanin synthesis, such as the PI3K-Akt signaling pathway, MAPK signaling pathway, and VEGF signaling pathway.

[0023] 4) Predicted Conclusions: The above analysis predicts that the active ingredients in Buddha's Hand may affect melanocyte function by acting on multiple core targets and regulating signaling pathways such as PI3K-Akt and MAPK. This prediction provides a directional basis for subsequent experimental research targeting melanin synthesis.

[0024] Example 2: In vitro activity verification DPPH free radical scavenging assay: A 0.1 mM DPPH working solution was prepared using anhydrous ethanol. In a 96-well plate, 10 μL of FAE or FWE sample solution serially diluted with DMSO (final concentrations equivalent to crude drug concentrations of 5, 10, 20, 40, and 80 mg / mL) was added to each well, followed by 190 μL of DPPH working solution. The control wells were treated with 10 μL of DMSO instead of the sample solution, and the blank wells were treated with 190 μL of anhydrous ethanol instead of the DPPH working solution. After mixing, the mixture was reacted at room temperature in the dark for 30 min, and the absorbance (OD) was measured at 517 nm. The free radical scavenging rate was calculated using the formula: Scavenging rate (%) = [1 - (OD experiment - OD control) / OD blank] × 100%. Three replicates were used for each concentration. The detection results are shown below. Figure 1 As shown, by Figure 1 It can be seen that both FAE and FWE exhibit concentration-dependent antioxidant activity. The DPPH scavenging rate of FAE is 20.72%~78.99%, while that of FWE is 6.19%~72.87%. At the same concentration, the antioxidant capacity of FAE is stronger than that of FWE (P<0.05).

[0025] Mushroom tyrosinase inhibition assay: 0.5 mg / mL L-DOPA solution and 100 U / mL mushroom tyrosinase solution were prepared using PBS. The assay was performed in a 96-well plate: A (sample background wells): 40 μL sample + 70 μL PBS + 20 μL PBS; B (sample reaction wells): 40 μL sample + 30 μL PBS + 40 μL L-DOPA + 20 μL enzyme solution; C (control background wells): 40 μL PBS + 70 μL PBS + 20 μL PBS; D (control reaction wells): 40 μL PBS + 30 μL PBS + 40 μL L-DOPA + 20 μL enzyme solution. After adding the enzyme solution, the mixture was immediately mixed and incubated at 37°C for 10 min. The OD value was measured at 475 nm. 1 mg / mL kojic acid was used as a positive control. The formula for calculating the tyrosinase inhibition rate is: Inhibition rate (%) = [1 - (OD-B – OD-A) / (OD-D – OD-C)] × 100%. The results are as follows: Figure 2 As shown, by Figure 2 It can be seen that FAE can inhibit mushroom tyrosinase activity in a concentration-dependent manner; while FWE did not show a significant inhibitory effect within the same test range (P>0.05).

[0026] Example 3 Cellular Level Validation: Effect of Citrus medica extract on melanin production in B16F10 cells 1) Cytotoxicity assay (CCK-8 assay): B16F10 cells were seeded at a density of 5 × 10³ cells per well in 96-well plates. After 24 h of culture, the medium was replaced with 100 μL of FAE (5, 10, 20, 40 mg / mL) or FWE (10, 20, 40, 80 mg / mL) per well, and cultured for another 24 h. 10 μL of CCK-8 solution was added to each well, and after incubation for 2 h, the OD value was measured at 450 nm. Each group had 5 replicates, and the cells were incubated in a cell culture incubator for 24 h. The following groups were established: Blank wells: Contain only culture medium, no cells; Control group: Cells + complete culture medium without bergamot extract; Drug administration group: cells + culture medium containing different concentrations of FAE or FWE.

[0027] The cell viability calculation formula is: Cell viability (%) = (OD-treated group - OD blank wells) / (OD control group - OD blank wells) × 100%. Results are as follows: Figure 3 As shown, by Figure 3It was found that the cell viability of FAE (5 mg / ml, 10 mg / ml, 20 mg / ml) was 60-100%, and that of FEW (10 mg / ml, 20 mg / ml, 40 mg / ml) was 74-100%. When the concentration of FAE was not higher than 20 mg / ml or the concentration of FWE was not higher than 40 mg / ml, the cell viability was close to or exceeded 80%. Subsequent cell experiments selected concentrations with cell viability higher than 80%.

[0028] 2) Intracellular tyrosinase activity assay: B16F10 cells were seeded in 6-well plates (3×10⁻⁶ cells / well). 5 / well), after overnight culture, each well was treated with medium containing safe concentrations of FAE (5, 10, 20 mg / mL) or FWE (10, 20, 40 mg / mL) for 24 h. A blank control group and a kojic acid (2 mg / mL) positive control group were included. After treatment, the wells were washed with PBS, and 500 μL of PBS lysis buffer containing 1% Triton X-100 was added to each well. Lysis was performed at -80°C. The supernatant was collected by centrifugation and mixed with an equal volume of 2 mg / mL L-DOPA solution. The mixture was incubated at 37°C for 1 h, and the OD value was measured at 475 nm. Protein concentration was corrected using the BCA method. The detection results are as follows: Figure 4 As shown in (a, b), by Figure 4 (a, b) shows that, compared with the control group, different concentrations of FAE and FWE can significantly inhibit intracellular tyrosinase activity (P<0.05). The concentration of FAE is only half that of FWE, but their ability to inhibit tyrosinase activity is comparable.

[0029] 3) Intracellular melanin content determination: Cell processing was the same as in step 2). Cells were digested and collected, washed with PBS, and 600 μL of 1M NaOH solution containing 10% DMSO was added to the cell pellet. The mixture was heated in an 80°C water bath for 1 hour until the melanin was completely dissolved. After cooling, 180 μL was taken and the OD value was measured at 405 nm. The relative content was calculated with the melanin content of the control group as 100%. The detection results are as follows: Figure 4 As shown in (d, e), by Figure 4 As shown in (d, e), FAE and FWE can reduce melanin content in a concentration-dependent manner. The melanin content of the 20 mg / mL FAE treatment group decreased to (58.5 ± 4.2)% of that of the control group (P<0.01).

[0030] 4) Microscopic observation of melanin production in cells: Cells were evenly seeded on six-well plates and cultured for 24 h. Then, bergamot extract was added to the wells and incubated for another 24 h. Cells were observed under a microscope and photographed. Results are as follows: Figure 4 As shown in (c), by Figure 4 (c) It can be seen that FAE and FWE treatment can reduce melanin production in B16F10 cells.

[0031] 5) RT-qPCR detection of melanin synthesis-related gene expression: Cell treatment was the same as in step 2). Total RNA was extracted using the TRIzol method, and its concentration and purity were determined (A260 / A280 between 1.8 and 2.0). 1 μg of RNA was used for reverse transcription. qPCR was performed using SYBR Green. Primer sequences are as follows: MITF-F: 5'-AGTACAGGAGCTGGAGATG-3', MITF-R: 5'-GTGAGATCCAGAGTTGTCGT-3'; TYR-F: 5'-CTGCCAACGATCCTATCTCCT-3', TYR-R: 5'-GGTTATGTCCAATGGGGTGCATT-3'; TYRP-1-F: 5'-AAGGTTACAGTGCTCCACG-3', TYRP-1-R: 5'-GGTTTGTCCTCCCGTTCCAT-3'; TYRP-2-F: 5'-CAGGAATGCACTGGAAGGGT-3', TYRP-2-R: 5'-ACCAAAACACAGGGTCGTT-3'; GAPDH-F: 5'-GGACCTCATGGCCTACATGG-3', GAPDH-R: 5'-TAGGGCTCTCTCTGCTCAGT-3'.

[0032] Reaction conditions: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, 40 cycles. The relative gene expression level was calculated using the 2^(-ΔΔCt) method. The results are as follows: Figure 5 As shown, by Figure 5 It was found that FAE significantly downregulated the mRNA expression of MITF, TYR, TYRP-1, and TYRP-2 (P<0.05). FWE downregulated the expression of TYR, TYRP-1, and MITF, but had no significant effect on the expression of TYRP-2.

[0033] Example 4 Cell-level validation: Effects of bergamot extract on HSF cell proliferation and type I collagen secretion 1) Cytotoxicity assay: HSF cells were seeded at 5 × 10³ cells per well in 96-well plates and cultured for 24 h. Then, the cells were treated with medium containing FAE (2, 4, 8, 16 mg / mL) or FWE (2, 4, 8 mg / mL) for 24 h, respectively. Cell viability was assessed using the CCK-8 assay. Results are as follows: Figure 6 As shown, by Figure 6 It is known that FAE is non-toxic to cells at concentrations of 2-8 mg / mL, and has a slight proliferative effect at concentrations of 4-8 mg / mL; FWE concentrations have no significant effect on cell viability.

[0034] 2) Detection of COL1α1 secretion (ELISA method): HSF cells were seeded in 6-well plates (3×10⁻⁶ cells / well). 5 After incubating overnight, the culture medium was replaced with serum-free medium containing FAE (2, 4, 8 mg / mL) or FWE (2, 4, 8 mg / mL), and cultured for another 24 hours. Cell supernatant was collected and centrifuged at 3000 rpm for 10 min to remove impurities. The ELISA kit was strictly followed according to the instructions: add standards and samples → add biotinylated antibody → add HRP-labeled streptavidin → add TMB substrate for color development → add stop solution → measure OD value at 450 nm. The concentration of COL1α1 in the sample was calculated based on the standard curve. Results are as follows: Figure 7 As shown, by Figure 7 It was found that FAE significantly promoted the secretion of COL1α1 by HSF cells in a concentration-dependent manner. The secretion amount in the 8 mg / mL FAE group was (182±15)% of that in the control group (P<0.01); there was no significant difference between the FWE treatment group and the control group.

[0035] Example 5: Animal Model Validation: In vivo amelioration of UVB-induced skin pigmentation in mice by bergamot extract. (I) Establishment and Administration of Skin Pigmentation Model: After one week of acclimatization, mice were treated with a shaver and depilatory cream on their backs, with a hair removal area of ​​approximately 2cm × 2cm. They were randomly divided into 5 groups (n=10): normal control group (Con, no irradiation + application of saline), model group (Model, irradiation + application of saline), positive control group (Pos, irradiation + application of 3% arbutin), low-dose FAE group (FAE-L, irradiation + application of 20mg / mL FAE), and high-dose FAE group (FAE-H, irradiation + application of 40mg / mL FAE). Except for the normal control group, all other groups received UVB irradiation, with an initial dose of 50mJ / cm², increasing by 10mJ / cm² every 2 days until reaching 100mJ / cm², then maintaining this dose once daily, 5 days a week, for 4 weeks. Starting from week 5, each group of mice received 100μL of the corresponding medication or saline solution applied to the hair removal area twice daily for 4 consecutive weeks.

[0036] (II) Sample collection: Blood was collected from the eyeballs 24 hours after the last administration, and the plasma was separated by centrifugation and stored at -80℃. Mice were euthanized, and full-thickness skin tissue was taken from the back. One part was fixed with 4% paraformaldehyde, and the other part was frozen at -80℃.

[0037] (III) Detection indicators: 1) Melanin staining of skin tissue: Fixed tissue was embedded in paraffin and sectioned. The procedure was performed according to the Masson-Fontana staining kit instructions: dewaxing to water → immersion in ammoniacal silver solution at 58°C in the dark for 30 min → washing with distilled water → treatment with 5% sodium thiosulfate for 2 min → rinsing with running water → counterstaining with 0.1% nuclear solid red for 5 min → dehydration, clearing, and mounting. The melanin granules appeared black under an optical microscope. Semi-quantitative analysis of the integrated optical density (IOD) of melanin in the basal layer of the epidermis was performed using Image-Pro Plus software.

[0038] 2) Detection of plasma biochemical indicators and skin collagen content: Using the corresponding mouse ELISA kit, the levels of TYR, IL-18, and SOD activity in plasma were strictly detected according to the instructions. Frozen skin tissue was taken, homogenized with pre-cooled PBS, centrifuged, and the supernatant was collected. The content of type I collagen in the supernatant was detected using the same method with an ELISA kit, and the total protein concentration was determined by the BCA method for correction.

[0039] The results are as follows: ① Effect of bergamot extract on UV-induced hypermelanin deposition: Results are as follows Figure 8 As shown, by Figure 8 (a, b) shows that dark brown pigmented patches appeared on the back skin of mice in the model group, while the pigmentation was significantly reduced in FAE-L, FAE-H and positive control groups. Masson-Fontana staining showed that a large number of melanin granules were deposited in the basal layer of the epidermis and hair follicles in the model group; compared with the model group, the epidermal melanin deposition in the FAE treatment group was significantly reduced (P<0.01).

[0040] ② Effects of bergamot extract on TYR, IL-18, and SOD in mouse plasma: Results are as follows Figure 9 As shown, by Figure 9 It was found that, compared with the normal group, the plasma TYR and IL-18 levels in the model group were significantly increased (P<0.001), and SOD activity was significantly decreased (P<0.05). Compared with the model group, the plasma TYR and IL-18 levels in FAE-L, FAE-H, and the positive control group were significantly decreased (P<0.05 or P<0.01), and SOD activity was significantly increased (P<0.05).

[0041] ③ Effects of bergamot extract on type I collagen in mouse skin tissue: Results are as follows Figure 10 As shown, by Figure 10 It was found that, compared with the normal group, the content of type I collagen in the skin of the model group was significantly decreased (P<0.05). Compared with the model group, the content of type I collagen in the skin of FAE-L, FAE-H and positive control groups was significantly increased (P<0.05).

[0042] In summary, the network pharmacology analysis in Example 1 provided predictions of potential targets and pathway directions for the study. Subsequent systematic experimental verification in Examples 2-5 demonstrated that the bergamot alcohol extract (FAE) prepared in this invention possesses clear free radical scavenging ability and tyrosinase inhibitory activity. It can effectively inhibit melanin production by downregulating the expression of key genes involved in melanin synthesis and promote collagen secretion from skin fibroblasts. In animals, it significantly improves UVB-induced skin pigmentation, regulates related oxidative stress and inflammatory markers, and increases skin collagen content, thus comprehensively exerting skin whitening and anti-aging effects. The bergamot water extract (FWE) also exhibited certain whitening activity. The complete methodological system provided by this invention, from predictive analysis to in vitro and in vivo verification, is scientifically reliable and lays a solid foundation for the application of bergamot extract in the field of whitening and anti-aging.

[0043] While the specific embodiments of the present invention have been described above, they are not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. A method for analyzing the anti-aging and skin-whitening mechanisms of bergamot extract based on network pharmacology and experimental verification, characterized in that: This method involves predicting the core targets of the active ingredients in bergamot through network pharmacology, screening the signaling pathways regulated by bergamot extract, and then verifying the anti-aging and skin whitening mechanisms of bergamot extract through in vitro activity verification, cellular level verification, and animal model verification.

2. The method for analyzing the anti-aging and skin-whitening mechanisms of bergamot extract based on network pharmacology and experimental verification as described in claim 1, characterized in that: Specifically, the following steps are included: 1) Prediction of active ingredients and targets: Screening for active ingredients in bergamot extract using network pharmacology and predicting their potential targets; 2) Disease target acquisition and intersection analysis: Search for disease targets related to "hyperpigmentation" from the GeneCards, OMIM, and PharmGKB databases, and find the intersection between the active ingredient targets and the disease targets; 3) Core target screening and pathway analysis: The active ingredient-disease target protein interaction network was constructed using the STRING database. The core targets were screened using Cytoscape software. The core targets were annotated with GO function and enriched using the DAVID database. The signaling pathways regulated by the bergamot extract were screened. 4) In vitro activity verification: Antioxidant capacity was evaluated by DPPH free radical scavenging experiment; whitening potential was initially screened by mushroom tyrosinase inhibition experiment; 5) Cellular level verification: The inhibitory effect of bergamot extract on melanin production was verified using mouse melanoma cells B16F10; the effect of bergamot extract on HSF cell proliferation and type I collagen secretion was verified using human skin fibroblasts (HSF). 6) Animal model validation: A UVB-induced mouse skin pigmentation model was established to validate the anti-aging and skin whitening mechanism of bergamot extract.

3. The method for analyzing the anti-aging and skin-whitening mechanisms of bergamot extract based on network pharmacology and experimental verification as described in claim 2, characterized in that: The bergamot extract mentioned in step 1) is either an alcoholic extract or an aqueous extract of bergamot.

4. The method for analyzing the anti-aging and skin-whitening mechanisms of bergamot extract based on network pharmacology and experimental verification as described in claim 2, characterized in that: In step 1), the active ingredients in bergamot extract were screened using the TCMSP database with oral bioavailability ≥30% and drug-likeness ≥0.

14.

5. The method for analyzing the anti-aging and skin-whitening mechanisms of bergamot extract based on network pharmacology and experimental verification as described in claim 2, characterized in that: The active ingredient mentioned in step 1) is one or more of the following: geraniol, 5,2',6'-trihydroxy-7,8-dimethoxyflavone, sitosterol, oleic acid, enzyme inhibitor complex, 5,2',5'-trihydroxy-6,7,8-dimethoxyflavone and squalene.

6. The method for analyzing the anti-aging and skin-whitening mechanisms of bergamot extract based on network pharmacology and experimental verification as described in claim 2, characterized in that: The platforms for predicting potential targets mentioned in step 1) are TCMSP and SwissTarget Prediction.

7. The method for analyzing the anti-aging and skin-whitening mechanisms of bergamot extract based on network pharmacology and experimental verification as described in claim 2, characterized in that: The core targets mentioned in step 3) include AR, SRC, MM7, P9, ESR1, AKT1, MAPK3, VEGFA, and IL6.

8. The method for analyzing the anti-aging and skin-whitening mechanisms of bergamot extract based on network pharmacology and experimental verification as described in claim 2, characterized in that: The signal paths described in step 3) include the PI3K-Akt signal path and the MAPK signal path.

9. The method for analyzing the anti-aging and skin-whitening mechanisms of bergamot extract based on network pharmacology and experimental verification as described in claim 3, characterized in that: The safe medicinal concentration of the bergamot alcohol extract is no higher than 20 mg / mL, and the safe medicinal concentration of the bergamot water extract is no higher than 40 mg / mL.

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