Aacer saccharin condensed tannin, its preparation method and application, whitening skin care product and pharmaceutical composition

Abstract

The present application provides a Acer truncatum Bunge condensed tannin and a preparation method and application thereof, a whitening skin care product and a pharmaceutical composition, and relates to the technical field of biological medicine. 1 and R 2 are independently hydrogen or hydroxyl, and n is 0-14. The present application explores the melanin production inhibition mechanism of the Acer truncatum Bunge condensed tannin from two aspects. Firstly, the inhibition of the Acer truncatum Bunge condensed tannin on the activity of tyrosine monophenolase and diphenolase and the mechanism of the inhibition are explored. Then, the melanoma cell B16-F10 is used as a cell model to explore the influence and mechanism of the condensed tannin on the melanin production. The results show that the tyrosinase inhibition activity of the Acer truncatum Bunge condensed tannin is strong. Moreover, the Acer truncatum Bunge condensed tannin provided by the present application also has excellent antioxidant property, and has a good application prospect in the aspects of whitening skin care products and pharmaceuticals.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a condensed tannin from Acer truncatum, its preparation method and application, whitening skin care products, and pharmaceutical compositions. Background Technology

[0002] Condensed tannins, also known as proanthocyanidins, are a class of oligomers and polymers found in plants, composed of varying numbers of monomers such as catechins and epicatechins. Compounds polymerized from 2 to 4 monomers are called oligomeric condensed tannins (OPCs), while polymeric condensed tannins (PPCs) can reach a degree of polymerization of ten or higher. Current reports demonstrate that condensed tannins play crucial roles in human immune regulation, skin whitening and melanin inhibition, antioxidation, antibacterial activity, antiviral activity, cardiovascular disease control, and gut microbiota regulation, making them important raw materials for the development of pharmaceuticals, health products, and skin whitening products. The activity of condensed tannins is related to the degree of polymerization, the number and position of hydroxyl groups, the linkage mode, and the spatial configuration. Generally, the reactivity increases with increasing degree of polymerization, while metabolic absorption decreases with increasing degree of polymerization. Therefore, condensed tannins from different plant sources possess different structural and activity characteristics. Currently, the large demand for condensed tannins in China relies primarily on the production of condensed tannins from logging and bark extraction or the direct import of expensive black thorn (Acacia mearnsii) condensed tannins, thus creating a predicament for the sustainable development of related industries. Therefore, exploring healthier, greener, renewable, and sustainable natural plant condensed tannin resources has become a critical issue that urgently needs to be addressed.

[0003] Acer truncatum is a deciduous tree belonging to the Sapindaceae family and the Acer genus. It is an important multifunctional economic tree species in my country. Currently, research on Acer truncatum mainly focuses on the composition, extraction and function of seed oil, determination and isolation of active components in leaves, tissue culture and rapid propagation, etc., while research on the pericarp and seed coat of Acer truncatum is relatively limited. Yang Lianli et al. (Yang Lianli Li. Identification and content determination of tannins in Acer truncatum seed coat [J]. Journal of Xianyang Normal University, 2004, (04): 31-3. Yang Lianli, Guo Naini. Ultrasonic extraction and characterization of tannins in Acer truncatum seed coat [J]. Leather Chemical Industry, 2006, (03): 20-2+42.) identified the tannins in the seed coat of Acer truncatum as high-quality condensed tannins, and found that the tannins were abundant. Wu Weizhong et al. (Wu Weizhong, Li Jun, Xu Jie. Research overview of chemical components of Acer truncatum[J]. Chinese Pharmaceutical Affairs, 2008, (07): 603-9.) found that tannins extracted from Acer truncatum seed coat have sedative, hypnotic, analgesic, anticoagulant and antidiarrheal effects. Fans (FAN H, SUN L, YANGL, et al. Assessment of the bioactive phenolic composition of Acer truncatum seed coat as abyproduct of seed oil[J]. Industrial Crops & Products, 2018, 118: 11-9.) found that the inner seed coat of Acer truncatum is rich in high-quality bioactive phenolic resources, especially the content of condensed tannins is quite high, and it shows excellent antioxidant capacity. Yan et al. (LIANGY, KONGF, MA X, et al. Inhibitory Effect of Acertruncatum Bunge Seed Coat Extract on Fatty Acid Synthase, Differentiation and Lipid Accumulation in 3T3-L1 Adipocytes[J].Molecules,2022,27(4).) found that the inner seed coat extract of Acertruncatum Bunge has inhibitory activity on fatty acid synthase.

[0004] The aforementioned studies mostly used a single solvent to extract phenolic components from the inner seed coat of Acer truncatum. The crude extracts obtained contained condensed tannins as well as phenolic acids, flavonoids, and other components, making it difficult to verify whether condensed tannins were the main contributing substance in subsequent activity tests. Furthermore, there are currently no reports on the isolation, purification, and subsequent structural identification of condensed tannins from Acer truncatum, limiting in-depth research on these tannins. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a *Acer truncatum* condensed tannin, its preparation method and application, whitening skin care products, and pharmaceutical compositions. The *Acer truncatum* condensed tannin provided by this invention has high purity and strong antioxidant and tyrosinase inhibitory activities.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a condensed tannin from Acer truncatum, having the structure shown in Formula I:

[0008]

[0009] In Equation I, R 1 and R 2 It can be either hydrogen or hydroxyl, and the value of n ranges from 0 to 14.

[0010] This invention provides a method for preparing the condensed tannins of Acer truncatum described in the above technical solution, comprising the following steps:

[0011] The inner seed coat powder of Acer truncatum, the extractant and the antioxidant are mixed and extracted to obtain an extract; the extractant is a first organic solvent and water, the first organic solvent includes one or more of acetone, ethanol and n-butanol, and the volume fraction of the first organic solvent in the extractant is 50-100%;

[0012] The extract is concentrated, and the resulting aqueous phase is subjected to a second organic solvent extraction and a third organic solvent extraction in sequence to obtain a crude aqueous extract; the second organic solvent includes petroleum ether and / or cyclohexane; the third organic solvent includes one or more of ethyl acetate, diethyl ether, dichloromethane and trichloromethane;

[0013] The crude extract was dried in aqueous phase and then reconstituted, and purified by Sephadex LH-20 column chromatography to obtain Acer truncatum condensed tannins. The elution method of the Sephadex LH-20 column purification was gradient elution, and the eluents used in the gradient elution were methanol aqueous solution and acetone aqueous solution in sequence. The volume fraction of methanol in the methanol aqueous solution was 20-90%, and the volume fraction of acetone in the acetone aqueous solution was 50-90%.

[0014] Preferably, the solid-liquid ratio of the inner seed coat powder and extractant of Acer truncatum is 1g: 5-50mL.

[0015] Preferably, the antioxidant includes one or more of ascorbic acid, vitamin E, and 2,6-di-tert-butyl-p-cresol;

[0016] The antioxidant is 0.01 to 0.5% of the weight of the inner seed coat powder of Acer truncatum.

[0017] Preferably, the extraction is performed 1 to 3 times, and the extraction time for each extraction is 30 to 120 minutes.

[0018] Preferably, the inner seed coat powder of Acer truncatum is obtained by drying, pulverizing and sieving the inner seed coat raw material of Acer truncatum in sequence;

[0019] The inner seed coat raw material of Acer truncatum includes the inner seed coat of Acer truncatum and / or the inner seed coat by-product after the oil extraction of Acer truncatum.

[0020] The drying temperature is 50–100°C, and the time is 1–8 hours;

[0021] The sieve used for sieving has a mesh size of 40.

[0022] This invention provides the application of the Acer truncatum condensed tannins described in the above-described technical solutions or the Acer truncatum condensed tannins prepared by the above-described technical solutions in the preparation of skin care products, health products or pharmaceuticals.

[0023] Preferably, the skin care products include whitening skin care products and / or anti-aging skin care products;

[0024] The drugs include one or more of the following: human immune-regulating drugs, antioxidant drugs, antitumor drugs, antibacterial drugs, antiviral drugs, drugs for treating cardiovascular diseases, and drugs for regulating intestinal flora.

[0025] This invention provides a whitening skin care product, comprising Acer truncatum condensed tannin and excipients; the Acer truncatum condensed tannin is the Acer truncatum condensed tannin described in the above technical solution or the Acer truncatum condensed tannin prepared by the preparation method described in the above technical solution.

[0026] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, characterized in that the active ingredient comprises the Acer truncatum condensed tannins described in the above-described technical solution or the Acer truncatum condensed tannins prepared by the preparation method described in the above-described technical solution.

[0027] The present invention provides a condensed tannin from Acer truncatum, having the structure shown in Formula I; in Formula I, R 1 and R 2Independently, it can be hydrogen or hydroxyl, with n ranging from 0 to 14. Oxidative stress is one of the main causes of cell damage. The use of antioxidants can effectively regulate cell damage and oxidative stress-mediated metabolic disorders. Oxidation is closely related to the aging process. This invention uses a combination of three methods—DPPH, ABTS, and FRAP—to evaluate the antioxidant capacity and skin care potential of Acer truncatum condensed tannins from different perspectives. The results show that Acer truncatum condensed tannins have excellent antioxidant capacity, which can alleviate stress by reducing the production of free radicals, protect the body from molecular and cellular damage, and enhance the body's immunity. The antioxidant capacity of Acer truncatum condensed tannins has good application prospects in cardioprotection, vascular protection, and diseases such as cancer and aging.

[0028] Tyrosinase (TYR), also known as polyphenol oxidase, is a multi-subunit, multifunctional copper-containing oxidase and a key enzyme in melanin synthesis metabolism. Overexpression or abnormally increased activity of TYR can lead to dysregulation of pigmentation regulation in the human body, resulting in conditions such as age spots, freckles, and melasma. Abnormalities in the body's endocrine function and imbalances in oxidation and antioxidation can promote a series of physiological and biochemical reactions involving tyrosine and tyrosinase, ultimately leading to increased pigmentation, decreased resident bacteria and increased transient bacteria in the affected skin area, ultimately resulting in dysfunction of the skin's immune and metabolic systems. The Acer truncatum condensed tannin provided in this invention is a natural, green, and effective tyrosinase inhibitor that can reduce melanin production, effectively alleviate or avoid the above-mentioned negative effects, and achieve skin whitening and skin care effects. As shown in the test results of the examples, this invention explores the mechanism of melanin production inhibition by condensed tannins of Acer truncatum from two aspects. First, it explores the inhibition of tyrosine monophenolase and bisphenolase activities by condensed tannins of Acer truncatum and the mechanism of this inhibition. Then, using melanoma cells B16-F10 as a cell model, it explores the effect of condensed tannins on melanin production and the mechanism of action. The results show that condensed tannins of Acer truncatum have strong tyrosinase inhibitory activity.

[0029] This invention provides a method for preparing condensed tannins from Acer truncatum as described in the above-mentioned technical solution. Acer truncatum seed coat, as a waste product after seed oil extraction, is renewable annually, has a large resource volume, and will not cause the ecological damage caused by the current widespread use of logging for bark extraction of plant condensed tannin raw materials. This invention uses Acer truncatum seed coat as a raw material to extract, purify, structurally identify, and study the bioactivity of Acer truncatum condensed tannins. The raw material is widely available and low-cost, realizing the resource-based reuse of Acer truncatum seed coat waste, which is green, environmentally friendly, and suitable for industrial production. Attached Figure Description

[0030] Figure 1 The process flow diagram for preparing condensed tannins from Acer truncatum;

[0031] Figure 2MALDI-TOF-MS image of condensed tannins in Acer truncatum;

[0032] Figure 3 The HPLC chromatogram of the product obtained by degradation of condensed tannins in Acer truncatum via benzyl mercaptan degradation method.

[0033] Figure 4 The results show the antioxidant activity of condensed tannins from Acer truncatum. A represents the DPPH-Trolox standard curve, B represents the ABTS-Trolox standard curve, C represents the iron reducing power-Trolox standard curve, D represents the DPPH free radical scavenging results, and E represents the ABTS scavenging results. + Free radical detection results, F represents the iron reducing power detection results;

[0034] Figure 5 The inhibitory effects of condensed tannins from *Acer truncatum* on monophenol tyrosinase and bisphenol tyrosinase are shown in Figure 1. A represents the effect of condensed tannins on the steady-state activity of monophenol tyrosinase; B represents the effect of condensed tannins on the relative activity of bisphenol tyrosinase; C represents the inhibitory mechanism of condensed tannins on bisphenol tyrosinase; D is the double reciprocal plot of the effect of condensed tannins from *Acer truncatum* on bisphenol tyrosinase obtained using the Lineweaver-Burk double reciprocal plot method; and E represents the inhibition constant K for bisphenol tyrosinase. IS F is the inhibition constant K for the enzyme-substrate complex. I ;

[0035] Figure 6 The UV-Vis spectra of L-Try hydroxylation and L-DOPA oxidation catalyzed under the conditions of absence (A) and presence (B) of condensed tannins in Acer truncatum are shown. Wherein, A is the UV-Vis spectrum of L-Try hydroxylation without condensed tannins, B is the UV-Vis spectrum of L-Try hydroxylation in the presence of condensed tannins, C is the UV-Vis spectrum of L-DOPA oxidation without condensed tannins, and D is the UV-Vis spectrum of L-DOPA oxidation in the presence of condensed tannins.

[0036] Figure 7 Cu for condensed tannins of Acer truncatum 2+ Chelation capacity and fluorescence quenching results of its interaction with tyrosinase, where A is the full-wavelength scan of the reaction between condensed tannin and CuCl2, B is the fluorescence spectrum of the reaction between condensed tannin and tyrosinase, and C is the Stern-Volmer plot of fluorescence quenching of tyrosinase by Acer truncatum condensed tannin.

[0037] Figure 8The diagram shows the inhibitory effects of Acer truncatum condensed tannins on tyrosinase activity and melanin production in mouse B16-F10 cells. In the diagram, A represents the inhibitory effect on the proliferation of mouse B16-F10 cells, B represents the inhibitory effect on melanin production in mouse B16-F10 cells, and C represents the inhibitory effect on tyrosinase activity in mouse B16-F10 cells.

[0038] Figure 9 The results show the molecular docking of condensed tannins from Acer truncatum on the inhibition of tyrosinase. In this diagram, A is gallatechin, B is epigallocatechin, C is apocytosine, D is epiapocytosine, E is catechin, and F is epicatechin. Detailed Implementation

[0039] This invention provides a condensed tannin from Acer truncatum, having the structure shown in Formula I:

[0040]

[0041] In Equation I, R 1 and R 2 It can be either hydrogen or hydroxyl, and the value of n ranges from 0 to 14.

[0042] In this invention, in formula I, when R 1 When it is hydrogen, R 2 For hydrogen; or, when R 1 When it is a hydroxyl group, R 2 For hydroxyl groups; or, when R 1 When it is a hydroxyl group, R 2 It is hydrogen.

[0043] In this invention, the value range of n is preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; the average degree of polymerization of the Acer truncatum condensed tannin is preferably 12.09.

[0044] In this invention, the structural unit of the Acer truncatum condensed tannin includes an extension unit (the unit within square brackets) and a terminal group (the unit outside square brackets) connected to the extension unit. The terminal unit preferably includes one or more of gallatechin, catechin, and epicatechin. The extension unit preferably includes one or more of epicatechin, catechin, aphthyl, epiafthyl, gallatechin, and epigallocatechin.

[0045] This invention provides a method for preparing the condensed tannins of Acer truncatum described in the above technical solution, comprising the following steps:

[0046] The inner seed coat powder of Acer truncatum, the extractant and the antioxidant are mixed and extracted to obtain an extract; the extractant is a first organic solvent and water, the first organic solvent includes one or more of acetone, ethanol and n-butanol, and the volume fraction of the first organic solvent in the extractant is 50-100%;

[0047] The extract is concentrated, and the resulting aqueous phase is subjected to a second organic solvent extraction and a third organic solvent extraction in sequence to obtain a crude aqueous extract; the second organic solvent includes petroleum ether and / or cyclohexane; the third organic solvent includes one or more of ethyl acetate, diethyl ether, dichloromethane and trichloromethane;

[0048] The crude extract was dried in aqueous phase and then reconstituted, and purified by Sephadex LH-20 column chromatography to obtain Acer truncatum condensed tannins. The elution method of the Sephadex LH-20 column purification was gradient elution, and the eluents used in the gradient elution were methanol aqueous solution and acetone aqueous solution in sequence. The volume fraction of methanol in the methanol aqueous solution was 20-90%, and the volume fraction of acetone in the acetone aqueous solution was 50-90%.

[0049] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0050] This invention involves mixing inner seed coat powder of Acer truncatum, an extractant, and an antioxidant, and then extracting the extract to obtain an extract. The extractant is a first organic solvent and water. The first organic solvent includes one or more of acetone, ethanol, and n-butanol, and the volume fraction of the first organic solvent in the extractant is 50-100%.

[0051] In this invention, the inner seed coat powder of *Acer truncatum* is preferably obtained by sequentially drying, pulverizing, and sieving the inner seed coat raw material of *Acer truncatum*, with the undersized portion being the inner seed coat powder. In this invention, the inner seed coat raw material of *Acer truncatum* includes the inner seed coat of *Acer truncatum* and / or the inner seed coat byproduct after *Acer truncatum* seed oil extraction. In this invention, the drying temperature is preferably 50–100℃, more preferably 60–70℃; the drying time is preferably 1–8 hours, more preferably 3–4 hours. In this invention, the sieve size for sieving is preferably 40 mesh (i.e., the particle size of the inner seed coat powder of *Acer truncatum* is ≥40 mesh). In this invention, the inner seed coat of *Acer truncatum* is preferably obtained by peeling off the inner seed coat of *Acer truncatum* seeds after preliminary drying. In this invention, the preliminary drying temperature is preferably 50–100°C, more preferably 60–70°C; the preliminary drying time is preferably 0.5–4 hours, more preferably 1–2 hours. Under the above conditions, the seeds of *Acer truncatum* are preliminarily dried, and the outer seed coat can be easily removed by sieving, facilitating the subsequent separation of the outer and inner seed coats. In this invention, the separation of the inner seed coat is preferably performed by machine or manually to separate the inner seed coat from the kernel.

[0052] As a waste product after seed oil extraction, the seed coat of Acer truncatum is renewable annually and has a large resource volume. It will not cause the ecological damage caused by the current widespread use of logging for bark extraction of plant condensed tannin raw materials. This invention uses Acer truncatum seed coat as raw material to extract, purify, identify the structure of Acer truncatum condensed tannins and study their bioactivity. The raw material source is wide and the cost is low, realizing the resource-based reuse of Acer truncatum seed coat waste, which is green and environmentally friendly.

[0053] In this invention, the extractant is a first organic solvent and water. The first organic solvent includes one or more of acetone, ethanol, and n-butanol, preferably acetone. The volume fraction of the first organic solvent in the extractant is 50-100%, preferably 60-90%, and more preferably 70-80%. In this invention, the solid-liquid ratio of the inner seed coat powder of *Acer truncatum* to the extractant is preferably 1g:5-50mL, more preferably 1g:10-30mL, and even more preferably 1g:10-20mL.

[0054] In this invention, the antioxidant preferably includes one or more of ascorbic acid, vitamin E, and 2,6-di-tert-butyl-p-cresol, more preferably ascorbic acid. In this invention, the antioxidant is preferably 0.01–0.5% of the weight of the inner seed coat powder of *Acer truncatum*, more preferably 0.1–0.3%, and even more preferably 0.1–0.2%.

[0055] In this invention, the extraction is preferably performed 1 to 3 times, more preferably 2 to 3 times; the extraction time for a single extraction is preferably 30 to 120 minutes, more preferably 40 to 100 minutes, and even more preferably 50 to 60 minutes; the extraction temperature is preferably room temperature. In this invention, the extraction is preferably ultrasonic extraction, and the extraction is preferably performed under sealed conditions to prevent oxidation of condensed tannins.

[0056] After the extraction is completed, the present invention preferably further includes: performing solid-liquid separation on the obtained extraction system, with the resulting liquid component being the extract. The present invention does not have any particular limitation on the solid-liquid separation; any solid-liquid separation method well known to those skilled in the art can be used, such as filtration, vacuum filtration, or centrifugation.

[0057] After obtaining the extract, the present invention concentrates the extract and then sequentially performs a second organic solvent extraction and a third organic solvent extraction to obtain a crude extract aqueous phase. The present invention does not specifically limit the concentration method, as long as the organic phase is removed, such as by rotary evaporation concentration. The concentration temperature is preferably 40–60°C, more preferably 40–50°C. In the present invention, the second organic solvent includes petroleum ether and / or cyclohexane, preferably petroleum ether. In the present invention, the third organic solvent includes one or more of ethyl acetate, diethyl ether, dichloromethane, and trichloromethane, more preferably ethyl acetate. In the present invention, the number of second and third organic solvent extractions is preferably 1–5 times, more preferably 2–4 times, and even more preferably 2–3 times. The present invention can remove chlorophyll, lipophilic phenolic compounds, and small molecule phenolic substances from the aqueous phase through the second and third organic solvent extractions.

[0058] After obtaining the crude extract aqueous phase, the present invention dries and reconstitutes the crude extract aqueous phase, then purifies it using a Sephadex LH-20 column to obtain Acer truncatum condensed tannins. In this invention, the drying preferably includes freeze-drying, and the freeze-drying conditions are not particularly limited; drying to constant weight is sufficient. In this invention, the solvent used for reconstitution is preferably an aqueous methanol solution, wherein the volume fraction of methanol in the aqueous methanol solution is preferably 20-90%, more preferably 30-80%, further preferably 40-70%, and most preferably 50-60%. In this invention, the elution method for purification using the Sephadex LH-20 column is gradient elution, wherein the eluents used in the gradient elution are, sequentially, an aqueous methanol solution and an aqueous acetone solution; the volume fraction of methanol in the aqueous methanol solution is 20-90%, preferably 30-80%, more preferably 40-70%, and further preferably 50-60%; the volume fraction of acetone in the aqueous acetone solution is 50-90%, preferably 55-85%, more preferably 60-80%, and further preferably 70%.

[0059] After purification using the Sephadex LH-20 column, the present invention preferably further includes: collecting the acetone-water eluent and freeze-drying it to obtain Acer truncatum condensate tannins. The present invention does not have specific limitations on the freeze-drying conditions; drying to constant weight is sufficient.

[0060] This invention provides the application of the *Acer truncatum* condensed tannins described in the above-described technical solutions, or the *Acer truncatum* condensed tannins prepared by the above-described preparation methods, in the preparation of skincare products, health products, or pharmaceuticals. In this invention, the skincare products preferably include whitening skincare products and / or anti-aging skincare products. In this invention, the pharmaceuticals preferably include one or more of the following: human immune-regulating drugs, antioxidant drugs, antitumor drugs, antibacterial drugs, antiviral drugs, drugs for treating cardiovascular diseases, and intestinal flora regulating drugs.

[0061] This invention provides a whitening skincare product, comprising Acer truncatum condensed tannin and excipients; the Acer truncatum condensed tannin is the Acer truncatum condensed tannin described in the above technical solution or the Acer truncatum condensed tannin prepared by the preparation method described in the above technical solution. This invention does not have any special limitations on the excipients; cosmetic excipients well known to those skilled in the art can be used.

[0062] This invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, characterized in that the active ingredient comprises *Acer truncatum* condensed tannin as described in the above-described technical solution or *Acer truncatum* condensed tannin prepared by the preparation method described in the above-described technical solution. This invention does not specifically limit the pharmaceutically acceptable excipients; any pharmaceutically acceptable excipient well known to those skilled in the art can be used. This invention does not specifically limit the dosage form and administration method of the pharmaceutical composition; any dosage form and administration method well known to those skilled in the art can be used.

[0063] To further illustrate the present invention, the following detailed description of the condensed tannins of Acer truncatum, their preparation methods, and applications is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1

[0065] (1) According to Figure 1 The process shown is for preparing Acer truncatum condensed tannins.

[0066] Obtaining the inner seed coat of Acer truncatum: Acer truncatum seeds were initially dried in a 60℃ oven for 2 hours. The inner seed coat was then separated from the kernel, and the obtained inner seed coat was dried in a 60℃ oven for 4 hours. After pulverizing, it was passed through a 40-mesh sieve. The portion passing through the sieve was the inner seed coat powder of Acer truncatum, which was stored in a -20℃ refrigerator for later use.

[0067] Extraction of condensed tannins from the inner seed coat of *Acer truncatum*: Inner seed coat powder, 70 v / v acetone aqueous solution, and ascorbic acid were mixed and sealed with plastic wrap. The sample was extracted using an ultrasonic extractor for 60 min, filtered, and the residue was extracted twice more. The three filtrates were combined to obtain the extract. The solid-liquid ratio of *Acer truncatum* inner seed coat powder to 70 v / v acetone aqueous solution was 1 g: 10 mL, and the mass of ascorbic acid was 0.1% of the mass of *Acer truncatum* inner seed coat powder.

[0068] Isolation and purification of condensed tannins from the inner seed coat of *Acer truncatum*: The organic phase of the extract was removed by rotary evaporation at 40℃. The remaining aqueous phase was extracted three times each with petroleum ether and ethyl acetate to obtain a crude aqueous extract. The crude aqueous extract was freeze-dried to constant weight and dissolved in 50 v / v% methanol aqueous solution. Purification was performed using a Sephadex LH-20 column, eluting sequentially with 50 v / v% methanol aqueous solution and 70 v / v% acetone aqueous solution. The 70% acetone aqueous fraction was collected and freeze-dried to constant weight to obtain the condensed tannins from *Acer truncatum*.

[0069] (2) Structural analysis of condensed tannins from Acer truncatum (sample)

[0070] Dissolve 1 mg of Acer truncatum condensed tannin in 1 mL of 70 v / v acetone aqueous solution to obtain the sample solution.

[0071] Determination of total phenolic content in condensed tannins of Acer truncatum: 40 μL of 25 v / v % Folin-Ciocalteu solution was added to a 96-well plate, followed by 20 μL of diluted sample solution (dilution factors of 2, 4, 8, and 16) and standard solution, and mixed thoroughly. The mixture was incubated at room temperature for 5 min, then 140 μL of Na₂CO₃ solution (700 mM) was added, and the plate was shaken at 500 rpm for 30 s to ensure thorough mixing. The plate was then incubated in a 40℃ oven for 30 min in the dark. Detection was performed using a microplate reader at 765 nm wavelength. Each sample was repeated three times. A standard curve was established with catechin concentration as the x-axis and absorbance (OD) as the y-axis. The catechin content in the sample solution was read from the standard curve. The total phenolic content was expressed as catechin equivalent (CE), in mg CE / g.

[0072] Determination of condensed tannin content in Acer truncatum: 20 μL of diluted sample solution (dilution factors of 2, 4, 8, and 16) and standard solution were added to a 96-well plate. Then, 120 μL of 4 w / v % vanillin methanol solution was added, mixed thoroughly, and shaken at 200 rpm for 1 min. Next, 60 μL of concentrated hydrochloric acid (original solution) was added, mixed thoroughly, and shaken at 200 rpm for 5 min. The mixture was incubated at room temperature in the dark for 15 min, and the results were detected using a microplate reader at 500 nm. Each sample was repeated three times. A standard curve was established with the concentration of catechin standards as the x-axis (X) and the absorbance value (OD) as the y-axis (Y). The catechin content in the sample solution was read from the standard curve. The condensed tannin content was expressed as catechin equivalent (CE), expressed as mg CE / g.

[0073] Table 1. Total phenolic and condensed tannin content in the inner seed coat of Acer truncatum.

[0074] Condensed tannins 7.9 499.797±12.145 727.139±69.278

[0075] Table 1 shows that the total phenol content of the condensed tannin is about 50 wt%, and the overall tannin content is about 72.7%, which is over 70%, indicating high purity.

[0076] The laser was used with a wavelength of 337 nm and a pulse width of 3 ns. In reflection mode, the accelerating voltage was 20.0 kV and the reflecting voltage was 23.0 kV. The scanning range in positive ion mode was 500–4000 Da. The prepared Acer truncatum condensed tannin was dissolved in acetone to prepare a 10 mg / mL sample solution. The matrix was 2,5-dihydroxybenzoic acid (DHB), which was dissolved in acetone to prepare a 10 mg / mL matrix solution. The cation reagent was cesium chloride (CsCl) to prepare a 1.52 mg / mL cesium chloride solution. After deionizing the sample and matrix with a strongly acidic cation exchange resin, the sample solution (10 mg / mL), cesium chloride solution (1.52 mg / mL), and matrix solution (10 mg / mL) were mixed at a volume ratio of 1:1:3. 1 μL of the mixture was loaded onto the sample, and the solvent was evaporated at room temperature before analysis. Figure 2 The image shows the MALDI-TOF-MS diagram of condensed tannins in Acer truncatum. The results indicate that condensed tannins with a degree of polymerization of 2–16 (i.e., n = 0–14) are mainly distributed in the inner seed coat of Acer truncatum.

[0077] Benzylthiol thiolysis condensed tannins: The prepared Acer truncatum condensed tannins were degraded by thiolysis, and the thiolysis products were analyzed using Re-HPLC-ESI-MS. Acer truncatum condensed tannin solution (5 mg / mL, solvent: 50 v / v% methanol aqueous solution) was mixed with 3.3 v / v% hydrochloric acid methanol solution and 5 v / v% benzylthiol methanol solution. The mixture was heated at 40 °C for 30 min, then cooled to room temperature. The solution was filtered through a 0.45 μm membrane filter. 500 μL of the treated sample solution (thiolysis-treated condensed tannin solution) was loaded for Re-HPLC-ESI-MS analysis.

[0078] Re-HPLC-ESI-MS analysis was performed using an Agilent system with 0.5 v / v% trifluoroacetic acid (TFA)-ultrapure water and CH3CN as the mobile phase. The flow rate was set to 1 mL / min, and the gradient elution program was as follows: from 0 to 45 min, the volume fraction of CH3CN linearly increased from 12% to 80%; from 45 to 50 min, the volume fraction of CH3CN linearly decreased from 80% to 12%. The column temperature was 25℃, the negative ion mode was used, the detection wavelength was 280 nm, and the UV spectral range was 200–600 nm.

[0079] Calculation of average degree of polymerization (mDP): mDP = peak area of ​​extended unit / peak area of ​​terminal unit + 1.

[0080] Figure 3 The HPLC chromatogram of the product obtained by degradation of condensed tannins from Acer truncatum using benzyl mercaptan is shown. The HPLC chromatogram mainly shows 10 single peaks (peak 10 being benzyl mercaptan). The distribution of constituent units, including terminal and extended units, after hydrolysis of Acer truncatum is shown in Table 2. The terminal units are gallatechin, catechin, and epicatechin, while the extended units are mainly epicatechin, accompanied by catechin, afoetin, epiafoetin, gallatechin, and epigallocatechin. The average degree of polymerization is 12.09. Therefore, the condensed tannins and their composition structure are shown in Formula I and Table 3.

[0081] Table 2. Analysis results of products obtained from the degradation of condensed tannins in Acer truncatum using the benzyl thiol degradation method.

[0082]

[0083] Table 3. Composition and Structure of Condensed Tannins from Acer truncatum (Formula I)

[0084] -H -H (Table) Afotoxin Progeranium -OH -OH (Table) Gallatechin Original delphinidin -OH -H (Table) Catechins Proanthocyanidins

[0085] Test Example 1

[0086] 1. Antioxidant activity experiment of condensed tannins from Acer truncatum

[0087] (1) Determination of DPPH free radical scavenging ability

[0088] Using ultrapure water as a blank control, 10 μL of appropriately diluted sample solution and Trolox solution were added to a 96-well plate, mixed with 40 μL of freshly prepared 1 mM (mmol / L) DPPH methanol solution, followed by the addition of 190 μL of methanol. The mixture was shaken at 200 rpm for 1 min to ensure homogeneity, and incubated at room temperature in the dark for 30 min. The absorbance was measured at 517 nm. The DPPH radical scavenging capacity of the sample was expressed as Trolox equivalent (TE).

[0089] (2) ABTS + Free radical scavenging capacity determination

[0090] Equal volumes of 7 mM ABTS solution and 2.4 mM K₂S₂O₈ solution were mixed and incubated at room temperature for 12–16 h. The absorbance of the mixture was measured at 734 nm using a 722 UV-Vis spectrophotometer (Shanghai Youke Instrument Co., Ltd., China). The absorbance was adjusted to 0.7 ± 0.02 with methanol, which became the ABTS working solution. 5 μL of methanol, appropriately diluted sample solution, and Trolox solution were added to a 96-well plate, mixed thoroughly with 200 μL of freshly prepared ABTS working solution, and incubated at room temperature for 5 min in the dark. Finally, the absorbance at 734 nm was measured. Sample ABTS + Free radical scavenging capacity is expressed as Trolox equivalent (TE).

[0091] (3) Ferric reducing ability of plasma (FRAP)

[0092] Accurately measure 40 μL of the methanol solution and blank solution (distilled water) of the sample to be tested, then add 100 μL of phosphate buffer (0.2 M, pH = 6.6) and 100 μL of 1% (w / v) potassium ferricyanide solution. Mix well and incubate at 50°C for 20 min. Then add 50 μL of 10% (w / v) trichloroacetic acid solution and incubate at room temperature for 10 min. Take 100 μL of the above solution, add 100 μL of distilled water and 20 μL of 0.1% (w / v) ferric chloride solution, mix well, and measure the absorbance at 700 nm using a microplate reader. The test results are expressed as Trolox equivalent (TE).

[0093] Figure 4 The results show the antioxidant activity of condensed tannins from Acer truncatum. A represents the DPPH-Trolox standard curve, B represents the ABTS-Trolox standard curve, C represents the iron reducing power-Trolox standard curve, D represents the DPPH free radical scavenging results, and E represents the ABTS scavenging results. + The results of free radical detection are shown, and F represents the result of iron reducing power detection. The results indicate that the condensed tannins of Acer truncatum have strong antioxidant capacity, and their antioxidant equivalents measured in the DPPH, ABTS, and FRAP systems are 305.7349, 242.8354, and 968.3861 μmol trolox / g, respectively.

[0094] 2. Inhibitory effect of condensed tannins from Acer truncatum on monophenol tyrosinase and bisphenol tyrosinase

[0095] (1) Inhibitory effect of condensed tannins from Acer truncatum on monophenol tyrosinase

[0096] Using 6 mM L-tyrosine as a substrate, 6.7 μL of sample solution at different concentrations (final concentrations of 0, 20, 40, 80, 100, and 120 μg / mL) and 3.3 μL of monophenol tyrosinase solution (final concentration of 80 U / mL) were added sequentially to a 96-well plate in a 200 μL reaction system. The plate was incubated at 37°C for 10 min, and finally, 20 μL of L-tyrosine solution (final concentration of 6 mM, pH = 6.8) was added. The absorbance was measured at 475 nm every 30 s for a total of 15 min. A blank control was prepared using 50 mM PBS (pH = 6.8) instead of tyrosinase, and a negative control was prepared using 50 mM PBS (pH = 6.8) instead of the sample. Three samples were prepared for each group. The inhibitory effect on monophenol tyrosinase was determined by IC50. 50 Value representation.

[0097] (2) Inhibitory effect of Acer truncatum condensed tannins on bisphenol tyrosinase

[0098] Using 0.5 mM L-DOPA as a substrate, 6.7 μL of sample solutions at different concentrations (final concentrations of 0, 20, 40, 80, 100, and 120 μg / mL) and 3.3 μL of tyrosinase solution (final concentration of 80 U / mL) were added sequentially to a 96-well plate in a 200 μL reaction system. The plate was incubated at 37 °C for 10 min, and finally 20 μL of L-DOPA solution (final concentration of 0.5 mM, pH = 6.8) was added. The absorbance was measured at 475 nm. In the experiment investigating the inhibitory effect of Acer truncatum condensed tannins on bisphenol tyrosinase, the initial slope of the kinetic curve was used as an indicator of bisphenol tyrosinase activity, i.e., ΔOD / ΔT, where ΔT = 30 s (the time interval from the addition of L-DOPA to the completion of the first data reading after 10 min of bisphenol tyrosinase incubation was 30 s). Each experiment was repeated three times.

[0099] Figure 5 The inhibitory effects of condensed tannins from *Acer truncatum* on monophenol tyrosinase and bisphenol tyrosinase are shown in Figure 1. A represents the effect of condensed tannins on the steady-state activity of monophenol tyrosinase; B represents the effect of condensed tannins on the relative activity of bisphenol tyrosinase; C represents the inhibitory mechanism of condensed tannins on bisphenol tyrosinase; D is the double reciprocal plot of the effect of condensed tannins from *Acer truncatum* on bisphenol tyrosinase obtained using the Lineweaver-Burk double reciprocal plot method; and E represents the inhibition constant K for bisphenol tyrosinase. IS F is the inhibition constant K for the enzyme-substrate complex. I .

[0100] Depend on Figure 5 As shown in A and B, the condensed tannins of Acer truncatum exhibit strong inhibitory effects on both monophenol tyrosinase and bisphenol tyrosinase. Specifically, its IC50 inhibitory effect on monophenol tyrosinase is...50 The value was 30.375 μg / mL, while the IC50 for bisphenol tyrosinase was... 50 The value was 97.859 μg / mL.

[0101] 3. The inhibitory mechanism and type of inhibition of bisphenol tyrosinase by condensed tannins in Acer truncatum

[0102] (1) The inhibitory mechanism of condensed tannins in Acer truncatum on bisphenol tyrosinase

[0103] In the inhibition mechanism of bisphenol tyrosinase by condensed tannins from Acer truncatum, using 0.5 mM L-DOPA as a substrate, in a 200 μL reaction system, the concentration of bisphenol tyrosinase was varied. 6.7 μL of sample solution at different concentrations (final concentrations of 0, 20, 40, 80, and 100 μg / mL) and 3.3 μL of tyrosinase solution (final concentrations of 0, 20, 40, 80, and 100 U / mL) were added sequentially to a 96-well plate. The plate was incubated at 37℃ for 10 min, and finally 20 μL of substrate L-DOPA (final concentration) was added. The absorbance of the condensed tannin at 475 nm (concentration 0.5 mM, pH = 6.8) was measured to determine the relationship between the enzyme-catalyzed reaction rate and the enzyme concentration. Based on this relationship, the inhibitory mechanism of condensed tannin on bisphenol tyrosinase was determined. Y: enzyme-catalyzed reaction rate; X: enzyme concentration. If the sample solutions of different concentrations form a set of straight lines passing through the origin, the inhibitory effect of condensed tannin on bisphenol tyrosinase by Acer truncatum is reversible. If the sample solutions of different concentrations form a set of parallel straight lines, the inhibitory effect of condensed tannin on bisphenol tyrosinase by Acer truncatum is irreversible.

[0104] Depend on Figure 5 As shown in C, the inhibitory effect of Acer truncatum condensed tannins on bisphenol tyrosinase is reversible. This indicates that the inhibitory effect of Acer truncatum condensed tannins on bisphenol tyrosinase is not achieved by reducing the effective amount of enzyme, but by reducing the catalytic efficiency of the enzyme.

[0105] (2) Inhibition type and inhibition constant of Acer truncatum condensed tannins on bisphenol A

[0106] In a 200 μL reaction system, the concentration of substrate L-DOPA was varied (final concentrations were 0.5, 0.25, 0.167, 0.125, and 0.0833 mM), while the enzyme concentration remained constant (final concentration of 80 U / mL). Sample solutions (final concentrations of 0, 20, 40, 80, and 100 μg / mL) and enzyme solutions were added sequentially to 96-well plates. The plates were incubated at 37 °C for 10 min, and finally, substrate solutions of different concentrations were added. The absorbance was measured at 475 nm to determine the effect of different substrate concentrations on the activity of bisphenol tyrosinase. The Lineweaver-Burk double reciprocal plot method was used, where Y represents the reciprocal of the reaction rate and X represents the reciprocal of the substrate concentration, to determine the type of inhibition. If the lines representing different sample concentrations intersect the Y-axis, it indicates competitive inhibition; if they intersect the X-axis, it indicates typical non-competitive inhibition; if they intersect in the second or third quadrant, it indicates mixed inhibition; intersecting in the second quadrant indicates mixed competitive and non-competitive inhibition, and intersecting in the third quadrant indicates mixed anti-competitive and non-competitive inhibition; if the lines are parallel, it indicates anti-competitive inhibition. By plotting the slope and intercept of the lines against the effector concentration twice, the inhibitory constant (K) of the effector on tyrosinase can be calculated. I ) and the inhibition constant of enzyme substrate complex (K) IS ).

[0107] In the kinetic study of the inhibitory effect of condensed tannins from Acer truncatum on bisphenol tyrosinase, Figure 5 Figure D shows the double reciprocal plot of the condensed tannins from *Acer truncatum* against bisphenol tyrosinase obtained using the Lineweaver-Burk double reciprocal plot method. The straight lines obtained from 1 / V versus 1 / S intersect in the second quadrant, indicating that the inhibition type of condensed tannins from *Acer truncatum* against bisphenol tyrosinase is competitive-non-competitive inhibition, i.e., a mixed inhibitory effect. This means that the condensed tannins extracted from the inner seed coat of *Acer truncatum* can bind to both bisphenol tyrosinase and its substrate complex. When the condensed tannin structure contains only one type, such as type b condensed tannins, it tends to be competitive inhibition. Condensed tannins of the same type inhibit tyrosinase in the same way because their structures are very similar. When condensed tannins appear in multiple structures simultaneously (such as type a and type b coexisting), the inhibition of bisphenol tyrosinase by condensed tannins is mainly a mixed inhibition. Figure 5 Plotting the intercept of the straight line in D against the concentration of the sample inhibitor yields the inhibition constant KIS of the sample inhibitor on bisphenol tyrosinase, as shown below. Figure 5 As shown in E; Figure 5 Plotting the slope of the straight line in D against the concentration of the sample inhibitor yields the inhibition constant K of the sample inhibitor on the enzyme-substrate complex. I ,like Figure 5As shown in Figure F. Table 4 lists a series of data on the inhibitory effect of Acer truncatum condensed tannins on mono- and bisphenol tyrosinase, from which the inhibition constant K of the sample inhibitor on bisphenol tyrosinase can be obtained. I The concentration was 32.5 μg / mL, and the inhibition constant of the sample inhibitor on the enzyme and substrate complex was K. IS The concentration was 120 μg / mL, which clearly shows that K I Less than K IS This indicates that the binding affinity of Acer truncatum condensed tannins to bisphenol tyrosinase is greater than their binding affinity to the enzyme and substrate complex. A summary of the above inhibition types and inhibition constants is shown in Table 4.

[0108] Table 4. Inhibition types and inhibition constants of mono- and bisphenol tyrosinase by condensed tannins from Acer truncatum.

[0109]

[0110] 4. Effects of condensed tannins from Acer truncatum on the oxidation spectra of L-Try and L-DOPA catalyzed by tyrosinase

[0111] This study investigated the UV-Vis spectra of L-Try hydroxylation and L-DOPA oxidation catalyzed by condensed tannins from Acer truncatum under both presence and absence conditions to demonstrate the effect of condensed tannins on L-Try hydroxylation and L-DOPA oxidation. The specific method was as follows: The reaction system consisted of 6 mM L-Try, 1.25 mM L-DOPA, 10 U / mL tyrosinase, and 50 mM PBS (pH = 6.8) phosphate buffer solution. The spectral changes of the tyrosinase-catalyzed products after the addition of condensed tannins were measured using a microplate reader within the wavelength range of 320–800 nm.

[0112] Figure 6 The UV-Vis spectra of L-Try hydroxylation and L-DOPA oxidation catalyzed under the conditions of absence (A) and presence (B) of condensed tannins in Acer truncatum are shown. Wherein, A is the UV-Vis spectrum of L-Try hydroxylation without condensed tannins, B is the UV-Vis spectrum of L-Try hydroxylation in the presence of condensed tannins, C is the UV-Vis spectrum of L-DOPA oxidation without condensed tannins, and D is the UV-Vis spectrum of L-DOPA oxidation in the presence of condensed tannins.

[0113] Depend on Figure 6 As shown in section A, when there is no condensation tannin in Acer truncatum, a series of characteristic peaks appear at a wavelength of 475 nm. From... Figure 6 As shown in Figure B, after adding Acer truncatum condensed tannins, a similar characteristic peak appeared at 475 nm, but the absorption intensity was significantly weakened, indicating that the addition of Acer truncatum condensed tannins significantly reduced the L-Try hydroxylation products. Figure 6As shown in C and D, the oxidation spectrum of L-DOPA changes with and without the addition of condensed tannins are similar to those of L-Try hydroxylation, further demonstrating that the condensed tannins of Acer truncatum have a significant inhibitory effect on the catalytic activity of tyrosinase.

[0114] 5. Cu of condensed tannins in Acer truncatum 2+ Analysis of chelating capacity and its interaction with tyrosinase using fluorescence quenching method

[0115] (1)Cu 2+ chelating ability

[0116] Methods: The copper ion chelating ability of Acer truncatum condensed tannins was determined using a fluorescence spectrophotometer (F-4600, Tokyo, Japan). The reaction system consisted of 0.35 mL of Acer truncatum condensed tannin (100 μg / mL) solution (solvent pH = 6.8, 50 mM PBS) mixed with 0.35 mL of copper chloride solutions with concentrations of 5, 10, 15, and 20 mM, and 0.93 mL of PBS (50 mM, pH = 6.8) in phosphate buffer solution. The fluorescence intensity of the mixture was measured in the range of 290–500 nm, with an excitation wavelength of 280 nm.

[0117] Figure 7 Cu for condensed tannins of Acer truncatum 2+ Chelating capacity and fluorescence quenching results of its interaction with tyrosinase, where A is the full-wavelength scan of the reaction between condensed tannin and CuCl2, B is the fluorescence spectrum of the reaction between condensed tannin and tyrosinase, and C is the Stern-Volmer plot of fluorescence quenching of tyrosinase by Acer truncatum condensed tannin.

[0118] Results: Tyrosinase is a polyphenol oxidase containing copper atoms. The inhibitory effect of Acer truncatum condensed tannins on tyrosinase is closely related to the ability of the condensed tannins to chelate copper atoms, such as... Figure 7 As shown in Figure A, the fluorescence intensity of the condensed tannins in Acer truncatum gradually decreased with the addition of different concentrations of copper ions, indicating that the condensed tannins in Acer truncatum interact with copper ions. Therefore, the inhibitory effect of the condensed tannins in Acer truncatum on tyrosinase is likely due to the chelation of copper atoms in tyrosinase by the condensed tannins.

[0119] (2) Fluorescence quenching analysis of interaction with tyrosinase

[0120] Methods: The fluorescence intensity of tyrosinase under the action of Acer truncatum condensed tannins was measured using a fluorophotometer. The reaction system consisted of 70 μL of tyrosinase solution (346 U / mL) and 10 μL of Acer truncatum condensed tannin solutions (pH 6.8, 50 mM PBS) at concentrations of 6.25, 12.5, 25, 50, and 100 μg / mL, respectively. The mixture was incubated at 37 °C for 1 min, and the fluorescence intensity was measured in the wavelength range of 290–500 nm for excitation and emission. The slit width for both excitation and emission was 10 nm.

[0121] Results: Various amino acid residues in tyrosinase possess intrinsic fluorescence, emitting fluorescence in the ultraviolet region. The binding of condensed tannins from *Acer truncatum* to tyrosinase causes changes in protein structure, leading to fluorescence quenching. Figure 7 As shown in Figure B, with the continuous increase of the concentration of condensed tannins in Acer truncatum, the peak shape of the fluorescence spectrum remained unchanged, but the intensity decreased with increasing concentration. This indicates that the condensed tannins in Acer truncatum formed a complex with tyrosinase, leading to a change in the conformation of tyrosinase. Figure 7 As shown in Figure C, the good linear relationship between F0 / F and [Q] indicates that the fluorescence quenching of Acer truncatum condensation tannin tyrosinase is a single type of quenching, either static or dynamic.

[0122] 6. Inhibitory effect of Acer truncatum condensed tannins on tyrosinase activity and melanin production in B16 mouse melanoma cells.

[0123] (1) Effects of Acer truncatum condensed tannins on the proliferation of B16 mouse melanoma cells (B16-F10)

[0124] Methods: B16-F10 mouse melanoma cells were cultured and, when the cell density reached 80-90% and the cells were in good growth condition, they were inoculated at a concentration of 1.0 × 10⁶ cells / mL. 5 The cells were seeded at a concentration of 100 μL / mL in 96-well plates. After culturing for 12 h, condensed tannins of different concentrations were added, with 6 replicates for each concentration. The cells were cultured for another 12 h, then 20 μL of LMTT (5 mg / mL, prepared with PBS) was added, and the cells were cultured for another 4 h. The culture medium was removed, and 150 μL of DMSO solution was added to dissolve the blue-purple crystals. After repeated pipetting and mixing, the cells were detected at 570 nm using a microplate reader. 0.1 v / v% DMSO was used as a blank control, and the cell viability was set at 100%.

[0125] Results: The effect of condensed tannins from Acer truncatum on mouse melanoma cells was detected by the MTT assay. Figure 8As shown in Figure A, after 48 hours of treatment, the proliferation rate of cells treated with 6.25–50 μg / mL Acer truncatum condensed tannins was over 80%, indicating that within this concentration range, Acer truncatum condensed tannins had a relatively small effect on the proliferation of melanocyte B16-F10 cells. Subsequent melanin-related synthesis experiments were conducted within the concentration range that had the least impact on melanocytes.

[0126] (2) Effects of Acer truncatum condensed tannins on melanin production in mouse B16-F10 cells

[0127] Methods: Cells in the logarithmic growth phase were seeded into 6-well plates and cultured overnight. After the culture medium was removed, DMEM solution with sample concentrations of 0, 6.25, 12.5, 25, and 50 μg / mL was added, respectively. After culturing at 37°C for 48 h, the cells were washed with PBS, scraped off with a cell scraper, and collected into EP tubes by centrifugation. The supernatant was discarded, and NaOH solution was added to the tubes. The tubes were heated in a 100°C water bath for 1 h, vortexed for 1 min every 10 min. After cooling to room temperature, the tubes were centrifuged at 12000 rpm for 20 min and transferred to 96-well plates. The absorbance at 475 nm was measured.

[0128] Results: Microscopic observation of melanin production in B16-F10 cells treated with condensed tannins from Acer truncatum for 48 hours was performed. Figure 8 As shown in Figure B, in the untreated control group, obvious melanin clusters were observed in the cells, and melanin accumulation was also visible intracellularly. Cells treated with the sample, especially those with a condensed tannin concentration of 50 μg / mL, showed significant inhibition of melanin production compared to the control group. Figure 8 As shown in Figure C, compared with the control group without medication, the amount of melanin production gradually decreased with the increase of the concentration of Acer truncatum condensed tannin. The melanin inhibition rate of 50 μg / mL Acer truncatum condensed tannin reached 55%, indicating that Acer truncatum condensed tannin has an inhibitory effect on melanin in mouse B16-F10 cells.

[0129] (3) Effects of Acer truncatum condensed tannins on tyrosinase activity in mouse melanoma cells

[0130] Methods: Logarithmic growth phase cells were seeded in 6-well plates and cultured overnight. After the culture medium was removed, DMEM solution with concentrations of 0, 6.25, 12.5, 25, and 50 μg / mL was added. After culturing at 37°C for 48 h, the cells were washed with PBS, scraped off with a cell scraper, and collected into EP tubes by centrifugation. The supernatant was discarded, and 1% Triton X-100 solution was added to the tubes. The cells were repeatedly frozen and thawed at -80°C to ensure complete lysis. The supernatant was transferred to a clean centrifuge tube, and the protein concentration of the supernatant was determined using a BCA kit. The total protein concentration in the cells was adjusted to be consistent. L-DOPA and enzyme extract were added to a 200 μL reaction system, and the cells were incubated at 37°C for 30 min. The absorbance was measured at 475 nm.

[0131] Results: The effect of *Acer truncatum* condensed tannins on tyrosinase in mouse melanoma cells was characterized by extracting crude enzyme solutions from mouse melanoma cells treated with different concentrations of *Acer truncatum* condensed tannins and measuring the oxidation rate of L-DOPA. Figure 8 As shown in Figure C, the tyrosinase activity gradually decreased with the increase of the concentration of Acer truncatum condensed tannin, and the decrease was concentration-dependent. When the highest concentration of Acer truncatum condensed tannin was 50 μg / mL, the tyrosinase activity decreased to 57%.

[0132] 7. Molecular docking investigation on the inhibitory effect of condensed tannins from Acer truncatum on tyrosinase.

[0133] First, the two-dimensional structures of each monomer unit of C / EC (catechin / epicatechin), GC / EGC (gallotatechin / epigallotatechin), and AF / EAF (aflavin / epiafotaxime) were downloaded from PubChem. These were then converted into three-dimensional structures using Chem3D. Try (PDB code 2Y9W) was downloaded from the Protein DataBank. Molecular docking was performed using catechin / epicatechin, gallotatechin / epigallotatechin, and afotaxime / epiafotaxime as ligands, and tyrosinase as the acceptor. AutoDockTools (Version 1.5.6) and AutoDock Vina (Version 4.2) were used. The results were visualized and interaction analyzed using PyMOL (Version 2.4.1) and Discovery Studio Visualizer (Version 4.5). The combination structure with the lowest docking energy in the output results was selected. Binding activity is negatively correlated with binding energy; the lower the binding energy, the more stable the docking module.

[0134] Molecular docking technology was used to study the molecular mechanism of condensed tannins from Acer truncatum inhibiting tyrosinase, such as... Figure 9The diagram shows the molecular docking results of gallatechin (A), epigallocatechin (B), avocadoin (C), epiavocadoin (D), catechin (E), and epicatechin (F) with tyrosinase (PDB: 2Y9W). The active site pocket of the tyrosinase catalytic center consists of two Cu... 2+ Composed of six histidine residues, including His61, His85, His94, His259, His263, and His296, the molecular docking results show that afrocin and catechin bind to the active sites of His259, His263, and His296 in tyrosinase, respectively. In addition, gallatechin / epigalocatechin, epiafrocin, and epicatechin bind to amino acid residues such as GLU356, LYS379, and ASP357 in tyrosinase, respectively. This provides a reasonable explanation for a more efficient understanding of the inhibitory effect of Acer truncatum condensed tannins on tyrosinase.

[0135] In summary, this invention uses the inner seed coat of *Acer truncatum* as the research object. Condensed tannins are separated, extracted, and purified using a 70 v / v acetone aqueous solution, and the contents of total phenols and condensed tannins are determined. Subsequently, the structure of *Acer truncatum* condensed tannins is further determined using reversed-phase HPLC-ESI-MS, MALDI-TOF-MS, and UV-Vis techniques. Then, the bioactivity of *Acer truncatum* condensed tannins is investigated from two aspects. First, the antioxidant capacity of *Acer truncatum* condensed tannins is evaluated from different perspectives using a combination of DPPH, ABTS, and FRAP methods. Then, the inhibition of tyrosine monophenolase and bisphenolase activities by condensed tannins and the mechanism of this inhibition are determined. Finally, using melanoma cells B16-F10 as a cell model, the effects and mechanisms of action of condensed tannins on melanin production are explored. By establishing and optimizing the extraction and separation method of condensed tannins from Acer truncatum, the raw material source is biomass-rich and renewable, which is more environmentally friendly than existing sources. The obtained condensed tannins from Acer truncatum have high purity, unique structure (degree of polymerization and composition), strong antioxidant activity and tyrosinase inhibitory activity, and have advantages in biological activity.

[0136] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing condensed tannins from Acer truncatum, comprising the following steps: The inner seed coat powder of Acer truncatum, the extractant, and the antioxidant are mixed and extracted to obtain an extract; the extractant is a first organic solvent and water, wherein the first organic solvent is one or more of acetone, ethanol, and n-butanol, and the volume fraction of the first organic solvent in the extractant is 50-100%; The extract is concentrated, and the resulting aqueous phase is subjected to a second organic solvent extraction and a third organic solvent extraction in sequence to obtain a crude aqueous extract; the second organic solvent is petroleum ether and / or cyclohexane; the third organic solvent is one or more of ethyl acetate, diethyl ether, dichloromethane and trichloromethane; The crude extract was dried in aqueous phase and then reconstituted, followed by purification using a Sephadex LH-20 column to obtain Acer truncatum condensed tannins. The Sephadex LH-20 column purification employed gradient elution, with methanol-water solution and acetone-water solution as the eluents, respectively. The methanol-water solution contained 20-90% methanol by volume, and the acetone-water solution contained 50-90% acetone by volume. The condensed tannins of Acer truncatum have the structure shown in Formula I: Equation I; In Equation I, R 1 and R 2 It can be hydrogen or hydroxyl independently, and the value of n ranges from 0 to 14.

2. The preparation method according to claim 1, characterized in that, The solid-liquid ratio of the inner seed coat powder and extractant of Acer truncatum is 1 g: 5~50 mL.

3. The preparation method according to claim 1, characterized in that, The antioxidant is one or more of ascorbic acid, vitamin E and 2,6-di-tert-butyl-p-cresol; The antioxidant is 0.01-0.5% of the weight of the inner seed coat powder of Acer truncatum.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The extraction is performed 1 to 3 times, and the extraction time for each extraction is 30 to 120 minutes.

5. The preparation method according to claim 1, characterized in that, The inner seed coat powder of Acer truncatum is obtained by drying, pulverizing and sieving the inner seed coat raw material of Acer truncatum in sequence. The inner seed coat raw material of Acer truncatum is the inner seed coat of Acer truncatum and / or the inner seed coat by-product after pressing Acer truncatum seed oil. The drying temperature is 50~100℃, and the time is 1~8 h; The sieve used for sieving has a mesh size of 40.

6. The use of the Acer truncatum condensed tannins prepared by the preparation method according to any one of claims 1 to 5 in the preparation of whitening skin care products or anti-aging skin care products.

7. The use of the Acer truncatum condensed tannin prepared by the preparation method according to any one of claims 1 to 5 in the preparation of antioxidant drugs.

8. The use of the Acer truncatum condensed tannin prepared by the preparation method according to any one of claims 1 to 5 in the preparation of tyrosinase inhibitors.

9. The use of the Acer truncatum condensed tannin prepared by the preparation method according to any one of claims 1 to 5 in the preparation of a drug for inhibiting melanoma cells.

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