Application of hippophae rhamnoides extract in melanin resistance

Abstract

The invention belongs to the technical field of natural product extraction and medicine, and particularly relates to application of a sea-buckthorn extract in resisting melanin. The invention provides an application of a sea-buckthorn extract in preparation of a product for inhibiting melanogenesis. The sea-buckthorn extract is a sea-buckthorn water-phase extract. The sea-buckthorn water-phase extract has a remarkable inhibition effect on tyrosinase, and meanwhile shows good DPPH and ABTS free radical scavenging capacity, so that the sea-buckthorn water-phase extract has the dual effects of resisting oxidation and inhibiting melanin generation key enzyme activity. The sea-buckthorn aqueous phase extract can be used for remarkably reducing the expression of melanin synthesis key genes (Tyr, Trp1, Mitf, Msh and Mc1r) and partial genes (Erk2 and Rsk) of an MAPK pathway. The sea-buckthorn water-phase extract provided by the invention is derived from natural plants, is high in safety and clear in effect, and provides new active ingredients and experimental basis for developing natural medicines, functional cosmetics or health care products with the melanin generation resisting effect.

Description

Application of sea buckthorn extract in anti-melanin Technical Field

[0001] This invention belongs to the field of natural product extraction and pharmaceutical technology, specifically relating to the application of sea buckthorn extract in anti-melanin. Background Technology

[0002] Melanin is an important biological pigment in the skin of animals and humans, but its excessive accumulation can cause various pigmentary disorders, such as melasma and café-au-lait spots, and in more severe cases, it can lead to melanoma, seriously affecting the patient's physical health and quality of life. Melanin production is a complex process regulated by multiple factors. Among them, tyrosinase (TYR), tyrosinase-associated protein-1 (TRP-1), and tyrosinase-associated protein-2 (TRP-2) play important roles in this process. Studies have shown that the activity of tyrosinase is mainly regulated by microphthalmia-associated transcription factor (MITF). Ultraviolet radiation can activate the expression of MITF, which in turn activates the expression of TYP, ultimately promoting melanin production. Research has found that excessive melanin accumulation is often accompanied by increased oxidative stress, while enhanced antioxidant capacity can effectively inhibit the activity of tyrosinase, thereby reducing melanin production. The antioxidant defense system in the skin can not only scavenge free radicals and reduce oxidative damage, but also simultaneously achieve the dual effects of anti-melanin production and enhanced cellular antioxidant capacity by regulating the MITF signaling pathway. This synergistic effect provides a scientific basis for developing active ingredients with both anti-melanin production and antioxidant effects.

[0003] In recent years, with the increasing demand for skin health and whitening effects, the search for safe and effective melanin inhibitors from natural plants has become a research hotspot in the cosmetics and pharmaceutical fields. Sea buckthorn (Hippophaerhamnoides L.), a plant used both as food and medicine, is rich in various bioactive components such as vitamin C, vitamin E, flavonoids, and saponins, and has been proven to have significant antioxidant, anti-inflammatory, and immunomodulatory effects. However, systematic research on the inhibitory effect of sea buckthorn extract on melanin production is still relatively limited, especially regarding the specific inhibitory effect of its different polar sites on tyrosinase activity, its in vivo anti-melanin efficacy, and its mechanism of action. Therefore, developing a natural active ingredient based on sea buckthorn extract that possesses both antioxidant and melanin-inhibiting functions has significant theoretical value and application potential for developing novel, safe, and effective whitening and skincare products. Summary of the Invention

[0004] The purpose of this invention is to provide a safe, efficient, and naturally derived active ingredient that has both melanin-inhibiting and antioxidant effects, its preparation method, and its application.

[0005] Existing technologies generally believe that the whitening effect of sea buckthorn is mainly attributed to its rich content of known antioxidant components such as vitamin C and flavonoids, and related studies have mostly focused on the total extract. However, the applicant, through systematic solvent extraction to obtain different polar fractions of sea buckthorn and conducting parallel activity screening, found that although vitamin E and total flavonoids were most abundant in the ethyl acetate and petroleum ether phases, the fraction with the strongest inhibition of tyrosinase activity was precisely the aqueous extract, and its half-maximal inhibitory concentration (IC50) was the lowest. 50 The aqueous extract showed significantly better performance than other parts. Further research revealed that this aqueous extract not only exhibited excellent tyrosinase inhibitory activity and free radical scavenging ability, but also significantly reduced melanin content and inhibited the expression of related genes in a zebrafish in vivo model, demonstrating clear in vivo whitening activity and good safety. This result is quite different from conventional understanding, indicating that the anti-melanin effect of sea buckthorn is not solely dominated by a single high-content component, but rather stems from a synergistic system composed of multiple water-soluble active components such as vitamin C and saponins in the aqueous phase.

[0006] Therefore, the present invention provides the use of sea buckthorn extract in the preparation of products for inhibiting melanin production; the sea buckthorn extract is an aqueous extract of sea buckthorn.

[0007] In some embodiments, the preparation method of the sea buckthorn aqueous extract includes: extracting sea buckthorn fruit with ethanol, then extracting with petroleum ether, ethyl acetate, and n-butanol, retaining the aqueous phase, concentrating and drying to obtain the extract.

[0008] In some embodiments, the contents of the following components in the aqueous sea buckthorn extract are as follows, based on the dry weight of the extract, wherein a measurement error of ±5% is allowed for each content:

[0009] Vitamin C: 16.6 mg / 100g;

[0010] Vitamin E: 0.465 mg / 100g;

[0011] Total flavonoids: 0.7296 mg / g;

[0012] Total saponins: 31.61-51.28 mg / g.

[0013] In some implementations, the product is used to prevent and / or treat hyperpigmentation disorders.

[0014] In some implementations, the hyperpigmentation disorder is selected from freckles, melasma, liver spots, age spots, solar lentigines, melanosis, Poitz-Yage syndrome, melasma of pregnancy, hyperpigmentation following drug use, and post-inflammatory hyperpigmentation.

[0015] In some implementations, the product is a skin whitening product.

[0016] In some implementations, the product is a product that downregulates the expression of at least one of the genes Tyr, Trp1, Mitf, Msh, Mc1r, Erk2, and Rsk.

[0017] In some implementations, the product is an antioxidant.

[0018] The present invention also provides compositions comprising the aqueous extract of sea buckthorn described herein.

[0019] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0020] In this invention, the carrier includes, but is not limited to, one or more of the following: water, alcohols (such as ethanol and propylene glycol), oils (such as squalane and jojoba oil), esters, silicone oils, surfactants, emulsifiers, thickeners (such as carbomer and xanthan gum), film-forming agents, and solvents.

[0021] In this invention, the excipients include, but are not limited to, one or more of the following: preservatives (such as phenoxyethanol, parabens), chelating agents (such as disodium EDTA), pH adjusters (such as citric acid, triethanolamine), fragrances, colorants, ultraviolet absorbers, moisturizers (such as glycerin, sodium hyaluronate), skin penetration enhancers, and antioxidant stabilizers.

[0022] In some embodiments, the composition may be formulated as a dosage form suitable for topical application, including but not limited to creams, emulsions, gels, serums, masks, solutions, sprays, or patches.

[0023] The beneficial effects of this invention are as follows: This invention provides an aqueous extract derived from natural sea buckthorn. This extract not only has a significant inhibitory effect on tyrosinase, effectively reducing melanin synthesis, but also possesses excellent antioxidant capacity, scavenging free radicals and alleviating oxidative stress. In vitro and in vivo experiments have jointly verified its clear whitening efficacy and good biosafety, and have preliminarily revealed that it works by regulating melanin synthesis-related genes and signaling pathways. This provides high-quality natural raw materials and reliable evidence for the development of whitening and antioxidant cosmetics or drugs that are highly effective, safe, and have a clear mechanism. Attached Figure Description

[0024] Figure 1: Inhibition rate of kojic acid tyrosinase in different polar fractions of sea buckthorn (A. Crude sea buckthorn extract; B. Sea buckthorn petroleum ether phase; C. Sea buckthorn ethyl acetate phase; D. Sea buckthorn n-butanol phase; E. Sea buckthorn aqueous phase; F. Kojic acid).

[0025] Figure 2: DPPH free radical scavenging rate (n=3);

[0026] Figure 3: ABTS radical scavenging rate (n=3);

[0027] Figure 4: Effects of sea buckthorn aqueous extract on zebrafish embryos and melanin content on zebrafish body surface (A. Embryo survival rate; B. Embryo hatching rate; C. Embryo malformation rate; D. Melanin content on zebrafish body surface; n=20).

[0028] Figure 5: Effects of different concentrations of sea buckthorn aqueous extract on melanin production in zebrafish (A. Tyrosinase activity; B. Melanin content (1. Normal control group; 2. Kojic acid positive control group (0.3 mg / mL); 3. Low concentration of sea buckthorn aqueous extract (30 μg / mL); 4. Medium concentration of sea buckthorn aqueous extract (60 μg / mL); 5. High concentration of sea buckthorn aqueous extract (120 μg / mL)).

[0029] Figure 6: Results of sea buckthorn aqueous extract on the expression of melanin-related genes in zebrafish (1. Normal control group; 2. Kojic acid positive control group (0.3 mg / mL); 3. Low concentration of sea buckthorn aqueous extract (30 μg / mL); 4. Medium concentration of sea buckthorn aqueous extract (60 μg / mL); 5. High concentration of sea buckthorn aqueous extract (120 μg / mL)). Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All features disclosed in this specification, or steps in all disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0032] Example 1

[0033] 1. Experimental Section

[0034] 1.1 Experimental Materials, Reagents and Instruments

[0035] Sea buckthorn fruit was purchased from Qinghai Kangpu Biotechnology Co., Ltd.; tyrosinase was purchased from Sigma-Aldrich; L-DOPA and kojic acid (KA) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; ascorbic acid was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; reagents for total RNA extraction, reverse transcription, primers, etc., BCA protein concentration assay kit (Shanghai Beyotime Biotechnology Co., Ltd.), total antioxidant capacity test kit (Nanjing Jiancheng Bioengineering Institute); wild-type AB strain zebrafish (Hubei Chuangxin Biotechnology Co., Ltd.); E3 culture medium (NaCl 0.29 g / L, KCl 0.013 g / L, CaCl2・2H2O 0.048 g / L, MgCl2・6H2O 0.082 g / L, pH 7.2, prepared with deionized water).

[0036] Zebrafish farming system (Beijing Aisheng Technology Development Co., Ltd.), small-scale traditional Chinese medicine grinder (Beijing Zhongxing Weiye Reagent Instrument Co., Ltd.), rotary evaporator (Shanghai Ailang Instrument Co., Ltd.), vacuum freeze dryer (Beijing Sihuan Scientific Instrument Factory Co., Ltd.), multifunctional microplate reader (Epoch2, Berten Instruments, Inc., USA), high-speed refrigerated centrifuge (5430R, Eppendorf GmbH, Germany), ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.), ultraviolet spectrophotometer (Shanghai Yidian Scientific Instrument Co., Ltd.).

[0037] 1.2 Experimental Methods

[0038] 1.2.1 Preparation of different polarity parts of sea buckthorn

[0039] Select sea buckthorn fruits and grind them into a fine powder. Weigh the sea buckthorn powder and add 70% ethanol at a material-to-liquid ratio of 1:8 (g / mL). Heat and soak for 96 h. Filter while hot. Place the filtrate in a rotary evaporator and concentrate under reduced pressure to obtain a crude extract of sea buckthorn fruits. Further extract the obtained extract using petroleum ether, ethyl acetate, and water-saturated n-butanol (1:1, v / v). Shake and mix for 30 s, let stand at room temperature for 6 h, and extract three times with each solvent. Combine the extracts with the same solvent, retain the upper aqueous phase extract, evaporate and concentrate to obtain extracts of different polar fractions.

[0040] 1.2.2 In vitro tyrosinase inhibition experiment of different polarity sites of sea buckthorn

[0041] Tyrosinase activity was determined by ultraviolet spectrophotometry. Using ultrapure water as the solvent, extracts from different polar fractions of sea buckthorn and kojic acid were prepared into a series of sample solutions with mass concentrations of 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg / mL, respectively. A 100 U / mL tyrosinase solution and a 1 mg / mL levodopa substrate solution were prepared using disodium hydrogen phosphate-citrate buffer. Each experiment included three replicates and one baseline (without tyrosinase solution, replaced by buffer solution), and a control group of enzyme reaction wells without samples was also included. 100 μL of sample solution was added to each tube sequentially. 50 μL of tyrosinase solution was added to the assay wells (the baseline wells were filled with buffer solution), and the mixture was incubated at 37°C in the dark for 10 min. Then, 200 μL of levodopa solution was added, and the mixture was incubated at 37°C in the dark for 30 min. The absorbance was then measured at 475 nm. The absorbance was calculated according to formula (1). .

[0042]

[0043] In formula (1): T is the absorbance value of the sample, T0 is the absorbance value of the sample background, C is the absorbance value of the control group and C0 is the absorbance value of the control group background.

[0044] 1.2.3 In vitro antioxidant activity experiment of sea buckthorn aqueous extract

[0045] (1) Determination of DPPH free radical scavenging rate

[0046] Dissolve 1 mg of DPPH (1,1-diphenyl-2-picrylhydrazyl radical) solid in 24 mL of methanol, sonicate for 5 min, shake thoroughly to mix, store in the dark, and use within 5 h. Take 1 mL of the above DPPH solution, dilute with 0.5 mL of methanol, and adjust the absorbance to 0.6-1.0.

[10] Ascorbic acid and sea buckthorn aqueous extract solutions in methanol were prepared at concentrations of 0, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 mg / mL. 100 μL of each sample and 200 μL of DPPH solution were added to each well of a 96-well plate, and the plates were incubated at 37 °C with shaking for 30 min. The absorbance of the samples was measured at 517 nm. The formula for calculating the DPPH free radical scavenging rate is shown below:

[0047]

[0048] In formula (2): D is the DPPH free radical scavenging rate; A1 is the absorbance of the sample; A2 is the absorbance of the blank sample; A3 is the absorbance of the control group.

[0049] (2) ABTS + Free radical scavenging assay

[0050] Antioxidant activity was determined using the ABTS free radical scavenging method. Under the action of a specific oxidant, ABTS was oxidized to produce green ABTS. + Free radical cations. When antioxidants are present in the system, ABTS can be inhibited. + The formation of ABTS radicals was observed. Trolox standards were prepared into solutions with concentrations of 1.5, 1.2, 0.9, 0.6, 0.3, and 0.1 mM according to the kit instructions. Test samples were prepared into solutions with concentrations of 1.0, 0.8, 0.6, 0.4, 0.2, and 0 mg / mL. The ABTS radical scavenging rate was detected using the ABTS kit method, and absorbance was measured at 405 nm. The formula for calculating the ABTS radical scavenging rate is shown below:

[0051]

[0052] 1.2.4 Zebrafish Experiment

[0053] (1) Determination of the maximum tolerated concentration of sea buckthorn aqueous extract

[0054] Adult zebrafish were cultured in a water circulation system maintained at a water temperature of 27 ± 1 °C, a pH of 7.2 ± 0.2, and a cycle of 14 h light / 10 h dark. The adult fish were placed in spawning boxes the night before spawning. After natural mating and spawning the following day, embryos were collected. Eight hours later, normal zebrafish embryos were cultured in 6-well plates, 20 embryos per well. The experimental groups received 0.015, 0.03, 0.06, 0.12, 0.24, and 0.48 mg / mL of sea buckthorn aqueous extract, while the positive control group received 0.1, 0.3, and 0.6 mg / mL of KA. An E3 culture medium without the test substance was established as a blank control group (Con group). Zebrafish mortality was recorded daily. After 72 hours, the cumulative mortality rate, hatching rate, and malformation rate for each group were calculated. All experiments were conducted in accordance with standard ethical guidelines for laboratory animals and under the supervision of the Animal Ethics Committee of the Northwest Plateau Institute of Biology (July 2024).

[0055] (2) Determination of tyrosinase activity and melanin content in zebrafish

[0056] Twenty normal zebrafish embryos aged 8 h were placed in each well of a 6-well plate. Embryos cultured with different concentrations of sea buckthorn aqueous extract served as experimental groups (30 μg / mL, 60 μg / mL, and 120 μg / mL), embryos cultured with 0.3 mg / mL KA served as the positive control group, and embryos without any treatment served as the blank control group. At 72 h, images were taken using a stereomicroscope, and image analysis was performed using ImageJ software. The images were converted to 8-bit grayscale images, and after background removal (parameter set to 130 pixels), a threshold parameter was set, and the total area of ​​melanin spots in the zebrafish was calculated. The differences in the ratio of melanin area to total area among different concentration treatment groups were compared. At least six spots were calculated for each group, and the average value was taken.

[0057] At the end of the experiment, zebrafish juveniles were collected, frozen in liquid nitrogen, weighed, and 100 μL of PBS buffer was added. After homogenization, the mixture was centrifuged at 12,000 rpm for 15 min at 4 ℃ to separate the supernatant and precipitate. 350 μL of 1 M NaOH solution (containing 10% DMSO) was added to the precipitate, and the mixture was incubated in a water bath at 80 ℃ for 1 h. The absorbance was measured at 405 nm to calculate the melanin content. 20 μL of the supernatant and 180 μL of levodopa (4 mM) were added to a 96-well plate and incubated at 37 ℃ in the dark for 30 min. The absorbance was measured at 475 nm to calculate the tyrosinase activity inhibition rate.

[0058]

[0059] In equation (4): A1 is the absorbance value of the sample at a wavelength of 405 nm and A2 is the absorbance value of the blank control.

[0060]

[0061] In equation (5): A1 is the absorbance value of the sample at a wavelength of 475 nm and A2 is the absorbance value of the blank control.

[0062] (3) Real-time quantitative PCR analysis of the expression of melanin-related genes in zebrafish

[0063] Total RNA was extracted using RNAiso Plus reagent, and purified by chloroform extraction, isopropanol precipitation, and washing with 75% ethanol. The purified RNA was then dissolved in ddH2O for later use. RNA concentration and purity (A) were determined using a Nanodrop 1000 spectrophotometer. 260 / A 280The ratio was adjusted to ensure RNA integrity and uniform concentration. Using the extracted RNA as a template, the reaction system was prepared according to the reverse transcription kit instructions, and the reverse transcription reaction was performed on a gene amplification instrument (37℃ 15 min → 85℃ 5 s). After synthesizing cDNA, it was stored at -20℃. Using cDNA as a template, the qPCR reaction system was prepared, and 10 μL of the reaction system was added to each well of a 96-well plate for programmed amplification (primers were synthesized by Shanghai Bioengineering Co., Ltd., and the sequences are shown in Table 1).

[0064]

[0065] 1.2.5 Statistics and Analysis

[0066] Statistical analysis and graphing were performed. One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for pairwise comparisons. A p-value < 0.05 was considered statistically significant. Quantitative analysis was performed on the graphs.

[0067] 2 Results Analysis

[0068] 2.1 Preparation of extracts from different polar fractions of sea buckthorn

[0069] 2.5 kg of dried sea buckthorn fruit was weighed, pulverized, and extracted with 70% ethanol, yielding 1.2 kg of residue, with a recovery rate of approximately 52%. After evaporation and concentration, the residue was dissolved in pure water to a final volume of 2 L. The solution was then extracted sequentially with petroleum ether, ethyl acetate, and n-butanol, ultimately yielding four polar fractions: 13.3 g of petroleum ether phase, 20.8 g of ethyl acetate phase, 286.7 g of n-butanol phase extract, and 948.7 g of aqueous phase extract.

[0070] 2.2 Inhibitory effect of different polarity sites of sea buckthorn on in vitro tyrosinase activity

[0071] The results are shown in Figure 1. When the concentration of the aqueous phase of sea buckthorn was 0.2, 0.8, and 3.2 µg / mL, the tyrosinase activity decreased by 33.83%, 50.27%, and 70.11%, respectively, indicating that the aqueous phase of sea buckthorn had a significant inhibitory effect on tyrosinase. The n-butanol and petroleum ether phases of sea buckthorn had weaker inhibitory effects on tyrosinase activity, decreasing by 32.02% and 52.94%, respectively, at 3.2 µg / mL. The ethyl acetate phase showed no inhibitory effect. Further measurements showed that the IC50 values ​​of the aqueous phase, petroleum ether phase, and the positive control drug kojic acid inhibited tyrosinase activity. 50 The values ​​were 0.7768, 2.1690 and 0.1058 µg / mL, respectively. The n-butanol phase did not achieve the half-maximal effect, while the ethyl acetate phase showed no inhibitory effect.

[0072] The above results indicate that the aqueous extract of sea buckthorn has a strong inhibitory effect on tyrosinase activity. Therefore, subsequent experiments will investigate the anti-melanin production effect of the aqueous extract of sea buckthorn.

[0073] 2.3 In vitro antioxidant activity of sea buckthorn aqueous extract

[0074] (1) Determination of DPPH free radical scavenging rate

[0075] The results are shown in Figure 2. Both the aqueous extract of sea buckthorn and the positive control ascorbic acid showed significant DPPH radical scavenging ability, and the aqueous extract of sea buckthorn exhibited strong antioxidant capacity in the concentration range of 1.0 mg / mL to 3 mg / mL. The DPPH radical half-maximal scavenging concentrations (IC50) of the aqueous extract of sea buckthorn and ascorbic acid are shown in Figure 2. 50 The concentrations were 0.6215 mg / mL and 0.0436 mg / mL, respectively, indicating that the aqueous extract of sea buckthorn has significant antioxidant capacity.

[0076] (2) ABTS free radical scavenging rate determination

[0077] The results are shown in Figure 3. The aqueous extract of sea buckthorn has the ability to scavenge ABTS free radicals, and its IC50 value is [missing information]. 50 The concentration was 3.3342 mg / mL, while the IC50 of ascorbic acid was... 50 The concentration was 0.0692 mg / mL, and the clearance rate reached over 90% at 0.15 mg / mL. In conclusion, the aqueous extract of sea buckthorn possesses certain antioxidant capabilities.

[0078] 2.4 Effect of Seabuckthorn Aqueous Extract on Anti-melanin Activity in Zebrafish Embryos

[0079] (1) Effects of sea buckthorn aqueous extract on zebrafish embryos

[0080] As shown in Figure 4A, the survival rate of zebrafish embryos decreased significantly with increasing concentration of the aqueous extract of sea buckthorn. The blank control group showed no embryo mortality, with a survival rate of 100%. The average survival rates of zebrafish embryos in the 30, 60, 120, 240, and 480 μg / mL aqueous extract groups were 96.25%, 92.5%, 86.25%, 47.5%, and 7.5%, respectively. When the drug concentration was ≥120 μg / mL, the mortality rate of zebrafish embryos increased significantly (P<0.01). Therefore, subsequent experiments selected concentrations ≤120 μg / mL for efficacy evaluation.

[0081] (2) Effect of sea buckthorn aqueous extract on melanin area on zebrafish body surface

[0082] As shown in Figure 4B, compared with the control group, the melanin content of zebrafish treated with kojic acid and 30, 60, and 120 μg / mL of sea buckthorn aqueous extract decreased by 12.11%, 10.77%, 13.67%, and 11.74%, respectively. The results indicate that sea buckthorn aqueous extract can significantly inhibit melanin production in zebrafish, and its anti-melanin effect is similar to that of the positive control group.

[0083] (3) Effect of sea buckthorn aqueous extract on anti-melanin effect in zebrafish

[0084] The results of the inhibition rate of zebrafish tyrosinase activity in different treatment groups are shown in Figure 5A. Compared with the blank group, the aqueous extract of sea buckthorn significantly inhibited the tyrosinase activity in zebrafish (P < 0.05) in a dose-dependent manner. At a concentration of 30 μg / mL, the relative inhibition rate of tyrosinase activity reached 10%, which was significantly different from the blank control group (P < 0.05). The melanin content in zebrafish is shown in Figure 5B. The aqueous extract of sea buckthorn had a significant dose-dependent inhibitory effect on melanin synthesis (P < 0.05). Compared with the blank control group, the melanin inhibition rates of the 30, 60, and 120 μg / mL aqueous extract treatment groups were 90%, 84.22%, and 65.32% of those of the blank group, respectively.

[0085] 2.5 Effects of sea buckthorn aqueous extract on the expression of melanin-related genes in zebrafish

[0086] As shown in Figure 6 (AE), compared with the blank control group, the expression levels of Tyr, Trp1, Msh, Mitf, and Mc1r genes in the test group treated with sea buckthorn aqueous extract were significantly reduced (P < 0.01), and this reduction showed a certain dose-dependent effect. As shown in Figure 6 (FG), the sea buckthorn aqueous extract also significantly regulated the MAPK signaling pathway-related genes Jnk1, Erk2, and Rsk (P < 0.01), significantly upregulating the transcription level of Jnk1 while downregulating the transcription levels of Erk2 and Rsk.

[0087] Example 2: Determination of the contents of vitamin C, vitamin E, total flavonoids, and total saponins in sea buckthorn extract.

[0088] 1. Experimental Methods

[0089] (1) Determination of Vitamin C Content in Sea Buckthorn Extract

[0090] The content of ascorbic acid in food was determined according to the national food safety standard (method 1: high performance liquid chromatography).

[0091] Sample preparation: Add an equal mass of 20 g / L metaphosphoric acid to the solid sample and homogenize.

[0092] Weigh an appropriate amount of sample, dilute to 50 mL with 20 g / L metaphosphoric acid, extract by sonication for 5 min, centrifuge at 4000 r / min for 5 min, and filter the supernatant through a 0.45 μm filter membrane for analysis (to measure free ascorbic acid). Take 20 mL of the centrifuged supernatant, add 10 mL of 40 g / L L-cysteine ​​solution, adjust the pH to 7.0-7.2, shake to reduce, then adjust the pH to 2.5-2.8 and dilute to volume. Filter through a membrane for analysis.

[0093] Chromatographic conditions settings, column: reversed-phase C24 18 Chromatography. Mobile phase: methanol-water (95:5 v / v), isocratic elution. Mobile phase was potassium dihydrogen phosphate-hexadecyltrimethylammonium bromide buffer (pH 2.5-2.8):methanol = 98:2, flow rate 0.7 mL / min, detection wavelength: 245 nm (characteristic absorption wavelength of vitamin C), column temperature, injection volume 10 μL. Prepare a mixed standard series of 0-50 μg / mL ascorbic acid, inject and plot a standard curve.

[0094] Calculation formula

[0095]

[0096] In formula (6): X: ascorbic acid content mg / 100g; c: concentration μg / mL obtained from the standard curve; V: volume of fixed volume mL; f: dilution factor; m: sample amount g or mL; the total amount minus the free content is the dehydroascorbic acid content.

[0097] (2) Determination of Vitamin E Content in Sea Buckthorn Extract

[0098] According to national food safety standards, the content of vitamins A, D, and E in food is determined using the first method: reversed-phase high-performance liquid chromatography.

[0099] Weigh a certain amount of sample into a saponification bottle, add ascorbic acid ethanol solution (to prevent vitamin E oxidation), then add potassium hydroxide solution, shake well, and place in a water bath for reflux saponification to convert vitamin E in the sample from the ester bonded state to the free state.

[0100] After cooling, the saponified solution was transferred to a separatory funnel, and anhydrous diethyl ether was added for fractional extraction. The ether extracts were combined. The extract was washed repeatedly with distilled water until neutral. After dehydration with anhydrous sodium sulfate, the solution was concentrated under reduced pressure to remove the diethyl ether, yielding a vitamin E concentrate. The concentrate was then brought to a final volume with a methanol-water mixture and filtered through a 0.45 μm organic filter membrane for instrumental analysis.

[0101] Chromatographic conditions settings, column: reversed-phase C24 18Chromatographic column (e.g., 250 mm × 4.6 mm, 5 μm). Mobile phase: methanol-water (95:5 v / v), isocratic elution. Flow rate: 1.0 mL / min. Column temperature: 30 ℃. Detection wavelength: 292 nm (characteristic absorption wavelength of vitamin E). Injection volume: 10-20 μL.

[0102] Prepare a series of concentration gradients of mixed standard solutions of α-, β-, γ-, and δ-tocopherols. Inject and detect the samples under the chromatographic conditions described above, and record the peak areas of each tocopherol. Plot a standard curve with the standard solution concentration on the x-axis and the peak area on the y-axis, and calculate the regression equation.

[0103] The test sample solution was injected under the same chromatographic conditions, and the peak area of ​​each tocopherol was recorded. The concentration of each tocopherol in the sample was calculated using the regression equation of the standard curve. The total vitamin E content is the sum of the contents of the four tocopherols, calculated using the following formula:

[0104]

[0105] In formula (7): X: the content of vitamin E in the sample, in mg / 100g; c: the concentration of vitamin E in the sample solution obtained from the standard curve, in mg / mL; V: the volume of the sample after dilution, in mL; f: the dilution factor; m: the sample weight, in g.

[0106] (3) Determination of total flavonoid content in sea buckthorn extract

[0107] A modified aluminum nitrate-sodium nitrite colorimetric method was used, with rutin as the standard, to prepare a 0.50 mg / mL solution. Accurately pipette 0, 0.4, 0.8, 1.20, 1.60, and 2.0 mL of the stock solution into 25 mL volumetric flasks, and add water to a final volume of 10.0 mL. Add 1.0 mL of 5% sodium nitrite solution sequentially, shake well, and let stand for 6 min; add 1.0 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min; finally, add 4.0 mL of 20% sodium hydroxide solution, add water to the mark, shake well, and let stand for 15 min. Using a blank as a reference, measure the absorbance of each tube at a wavelength of 510 nm. Plot a standard curve with rutin mass (mg) on ​​the x-axis and absorbance on the y-axis.

[0108] Accurately weigh an appropriate amount of sea buckthorn seed meal extract, dissolve it in a small amount of ethanol and dilute to volume to prepare a sample solution of appropriate concentration. Accurately pipette 2.0 mL of the sample solution into a 25 mL volumetric flask, and perform the same procedure as described above to determine the absorbance. Calculate the total flavonoid content (m1) in the sample solution according to the regression equation of the standard curve. All determinations were performed in triplicate.

[0109]

[0110] In formula (8): m1: total flavonoid content in the sample solution, mg; m: mass of the sample, g.

[0111] (4) Determination of total saponin content in sea buckthorn extract

[0112] Preparation of standard and sample solutions: Accurately weigh 2.0 mg of oleanolic acid standard, dissolve it in a small amount of ethanol, and dilute to a final volume in a 10 mL volumetric flask to prepare a 0.20 mg / mL oleanolic acid standard solution. Prepare sample solutions of specific concentrations based on the mass of each dried sample, and record their concentrations.

[0113] Construction of the standard curve: Accurately pipette 0.25, 0.50, 1.00, 1.50, and 2.00 mL of oleanolic acid standard solution into dry, stoppered test tubes. Evaporate to dryness in an 85°C water bath. After cooling (oven drying), add 0.4 mL of 5% vanillin-glacial acetic acid solution (0.5 g vanillin plus 10 mL glacial acetic acid, sonicated in the dark) and 1.6 mL of perchloric acid sequentially. Incubate in a 70°C water bath for 10 min, cool in water for 10 min, add 10 mL of glacial acetic acid, shake well, and let stand for 15 min. Using a 1 cm cuvette, adjust the zero point with a reagent blank and measure the absorbance at a wavelength of 560 nm. Plot the standard curve with absorbance A as the x-axis and the mass of oleanolic acid standard as the y-axis.

[0114] Sample determination method: Pipette 0.20 mL of sample solution into dry stoppered test tubes, follow the steps in the standard curve above, adjust the zero point with reagent blank solution, measure the absorbance of the sample at a wavelength of 560 nm, and calculate the mass m2 of total saponins in the sample solution according to the standard curve.

[0115]

[0116] In equation (9): X i Total soap content in the sample, expressed in mg / 100g or mg / 100mL; C i : After background correction, the mass of oleanolic acid in the test solution is calculated from the standard curve, in mg; V: the dilution volume of the test sample, in mL; V0: the volume of the sample solution used for color development, in mL; m: the sample amount, in g or mL.

[0117] 2. Results Analysis

[0118] The contents of four active ingredients in sea buckthorn extracts of different polarities were systematically determined, and the distribution of each ingredient in different polar fractions showed significant differences:

[0119] (1) Vitamin C (Table 2): It was mainly concentrated in the aqueous phase, with a content of 16.6 mg / 100g, which was significantly higher than that in the n-butanol phase (8.11 mg / 100g), ethyl acetate phase (2.51 mg / 100g) and the undetected petroleum ether phase, indicating that vitamin C, as a water-soluble component, was mainly retained in the aqueous phase.

[0120] (2) Vitamin E (Table 3): It exhibits typical fat-soluble distribution characteristics, with the highest content in the petroleum ether phase (336 mg / 100g), followed by the ethyl acetate phase (51.0 mg / 100g), while the content in the aqueous phase is extremely low (0.465 mg / 100g), indicating that vitamin E is mainly found in non-polar and weakly polar sites.

[0121] (3) Total flavonoids (Table 4): The highest content was found in the ethyl acetate phase (52.27 mg / g), followed by the n-butanol phase (23.31 mg / g), and the lowest content was found in the aqueous phase (0.7296 mg / g). This distribution pattern indicates that flavonoids are more soluble in moderately to strongly polar organic solvents.

[0122] (4) Total saponins (Table 5): They were detected in different polar fractions, with higher contents in petroleum ether and ethyl acetate phases (test values ​​of 184.28 mg / g and 135.77 mg / g, respectively). A considerable amount of saponins (31.61-51.28 mg / g) was also found in the aqueous phase, indicating that saponin components have good solubility in different polar solvents.

[0123] It is worth noting that analysis of the content of vitamin C, vitamin E, total flavonoids, and total saponins in each polar fraction revealed that, based solely on the distribution of a single high-content active ingredient, the ethyl acetate and petroleum ether phases, rich in flavonoids and vitamin E, should theoretically possess strong antioxidant and potential whitening activities. While the aqueous phase is rich in vitamin C, its flavonoid and vitamin E contents are significantly lower, and according to conventional understanding, its overall anti-melanin activity may not be optimal. However, the tyrosinase inhibition experiment in Example 1 and the in vivo zebrafish experiment consistently showed that the fraction with the strongest anti-melanin activity was not the fraction predicted above, but rather the aqueous extract. This result clearly contradicts expectations based on the content of a single component, indicating that the anti-melanin efficacy of sea buckthorn cannot be simply attributed to the independent action of any single high-content star ingredient, but is more likely due to the comprehensive biological effect produced by the synergistic system composed of vitamin C, saponins, and other water-soluble components in the aqueous extract.

[0124]

[0125] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of sea buckthorn extract in the preparation of products for inhibiting melanin production; wherein the sea buckthorn extract is an aqueous extract of sea buckthorn.

2. The application according to claim 1, characterized in that, The preparation method of the sea buckthorn aqueous extract includes: extracting sea buckthorn fruit with ethanol, then extracting with petroleum ether, ethyl acetate and n-butanol, retaining the aqueous phase and concentrating and drying to obtain the extract.

3. The application according to claim 2, characterized in that, Based on the dry weight of the aqueous extract of sea buckthorn, the contents of the following components in the aqueous extract of sea buckthorn are as follows, wherein the measurement error of each content is allowed to be ±5%: Vitamin C: 16.6 mg / 100g; Vitamin E: 0.465 mg / 100g; Total flavonoids: 0.7296 mg / g; Total saponins: 31.61-51.28 mg / g.

4. The application according to claim 1, characterized in that, The product is used for the prevention and / or treatment of hyperpigmentation disorders.

5. The application according to claim 4, characterized in that, The hyperpigmentation disorders mentioned are selected from freckles, melasma, liver spots, age spots, solar lentigines, melanosis, Poitz-Yage syndrome, melasma of pregnancy, hyperpigmentation after drug use, and post-inflammatory hyperpigmentation.

6. The application according to claim 1, characterized in that, The product in question is a skin whitening product.

7. The application according to claim 1, characterized in that, The product is a product that downregulates the expression of at least one of the following genes: Tyr, Trp1, Mitf, Msh, Mc1r, Erk2, and Rsk.

8. The application according to claim 1, characterized in that, The product in question is an antioxidant.

9. A composition, characterized in that, The composition comprises the sea buckthorn aqueous extract of claim 1.

10. The composition according to claim 9, characterized in that, It also includes pharmaceutically acceptable carriers or excipients.

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