A composition of three-leaf ginseng sterol and flavone, and a preparation method and application thereof

Abstract

The present application relates to plant extracts and its cosmetic field, specifically discloses a composition of smilax sieboldi miq sterol and flavone and a preparation method and application thereof. The sterol component and the flavone component of smilax sieboldi miq are compounded to form the composition, the mass percentage content of total sterol in the sterol component is greater than or equal to 15%, the mass percentage content of total flavone in the flavone component is greater than or equal to 60%, the sterol component and the flavone component cooperate, can inhibit the release of inflammatory factors and the synthesis of melanin through multi-target synergistic effect, thereby playing a role at two key nodes of inflammatory source and pigment synthesis, blocking the formation of inflammatory pigmentation, thereby the skin symptoms of inflammatory pigmentation can be significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of plant extracts and their cosmetics, specifically relating to a composition of trifolinol and flavonoids, its preparation method and application. Background Technology

[0002] Post-inflammatory hyperpigmentation (PIH) is a skin condition characterized by increased pigmentation following inflammation or damage caused by various endogenous and exogenous factors, such as acne, cosmetic procedures, chemical peels, or barrier damage. It manifests as localized reddish-brown to grayish-black patches, and epidemiological studies show a particularly high prevalence among Asian individuals with Fitzpatrick skin types III–VI. While inflammatory hyperpigmentation tends to resolve spontaneously, it often takes months to years, and in some cases, it can develop into a permanent pigmentation problem. These cosmetic imperfections not only affect a patient's quality of life but can also easily lead to psychological problems such as anxiety and depression.

[0003] The occurrence of inflammatory hyperpigmentation involves not only increased melanin synthesis, but more importantly, the abnormal activation of melanocytes mediated by inflammatory factors. Under the induction of the inflammatory microenvironment, keratinocytes release large amounts of regulatory mediators such as endothelin-1 (ET-1) and prostaglandin E2 (PGE2), which significantly upregulate the expression levels of tyrosinase (TYR) and its related proteins (TYRP1, TYRP2) by activating the microphthalmia-associated transcription factor (MITF) signaling axis, ultimately leading to excessive melanin production and deposition. Currently, most commercially available products focus on directly inhibiting tyrosinase activity (such as arbutin and kojic acid) or accelerating keratin metabolism (such as glycolic acid and salicylic acid). While effective for some pigmentation problems, their intervention effect on inflammatory hyperpigmentation, an inflammation-driven type of hyperpigmentation, is limited. More importantly, many chemically synthesized ingredients have controversies regarding skin irritation, photosensitivity, or long-term safety, making it difficult to meet consumers' needs for gentle, effective, and source-regulating treatments.

[0004] *Tetrastigma hemsleyanum*, a plant belonging to the genus *Tetrastigma* in the Vitaceae family, is rich in flavonoids, especially flavonoid C-glycosides and phytosterols, in its stems and leaves. Previous studies have reported that *Tetrastigma hemsleyanum* extract possesses antioxidant activity; however, current techniques are limited to the extraction and activity studies of single components. There are few reports on the use of *Tetrastigma hemsleyanum* extract to improve inflammatory pigmentation, and there are no reports on the combination of *Tetrastigma hemsleyanum* sterols and flavonoid components for the regulation of inflammatory pigmentation. Furthermore, there are no studies on the synergistic mechanism of these two components or related human efficacy verification. Summary of the Invention

[0005] In view of the technical problems involved in the prior art, the present invention will provide a composition of trifolinol and flavonoids, a preparation method thereof, and its application.

[0006] To achieve the above objectives, the following technical solutions are specifically included: In a first aspect, the present invention provides a composition of trifolin sterol and flavonoids, comprising a trifolin sterol component and a trifolin flavonoid component, wherein the total mass percentage of sterol compounds in the trifolin sterol component is greater than or equal to 15%, and the total mass percentage of flavonoid compounds in the trifolin flavonoid component is greater than or equal to 60%.

[0007] This invention combines the sterol and flavonoid components of *Tripterygium wilfordii* to form a composition. The sterol component contains a total sterol content of ≥15% by mass, and the flavonoid component contains a total flavonoid content of ≥60% by mass. These components are not traditional, simple crude extracts of *Tripterygium wilfordii*. The sterol and flavonoid components work synergistically to inhibit the release of inflammatory factors and melanin synthesis through multiple targets, thereby acting simultaneously at two key nodes: the source of inflammation and pigment synthesis. This blocks the formation of inflammatory pigmentation and significantly improves the symptoms of inflammatory pigmentation.

[0008] Preferably, the mass ratio of the trifolin sterol component to the trifolin flavonoid component is (0.125-8):1, more preferably (0.2-6):1, even more preferably (0.5-5):1, and still more preferably (0.8-3):1. The compound composition of trifolin sterol and trifolin flavonoid is used to improve pigmentation problems caused by inflammatory stimuli such as acne, post-cosmetic procedures, or skin barrier damage. When the two are used together in the above-mentioned mass ratio range, they exhibit a strong synergistic effect in improving inflammatory pigmentation, resulting in a greater degree of improvement in skin inflammatory pigmentation.

[0009] Preferably, in the composition of trifolin sterol and flavonoids, the total mass percentage of sterol compounds is 2%-23%, more preferably 2.6%-21.5%, or even more preferably 4%-16.1%.

[0010] Preferably, in the composition of trifolinol and flavonoids, the total mass percentage of flavonoids is 7%-70%, more preferably 8.2%-66.1%, or even more preferably 24.7%-62%.

[0011] Preferably, the total mass percentage of sterols in the trifolin sterol component is greater than or equal to 20%, and the total mass percentage of sterols in the trifolin sterol component is 20.5%-30%, more preferably 23%-26%.

[0012] Preferably, the total mass percentage of flavonoids in the *Trifolium repens* flavonoid component is greater than or equal to 70%, more preferably 71%-79%, and even more preferably 73%-77%.

[0013] Preferably, the trifolinol component includes at least one of β-sitosterol and stigmasterol; more preferably, the trifolinol component includes β-sitosterol and stigmasterol, and the total mass percentage of β-sitosterol and stigmasterol is 5%-13% based on the dry weight of the trifolinol component, and more preferably 8%-10%.

[0014] Preferably, the flavonoid component of *Trifolium repens* comprises flavonoid C glycosides. Specifically, the flavonoid component of *Trifolium repens* comprises at least one of purslane, isopurslane, vitexin, and isovitexin. More preferably, the flavonoid component of *Trifolium repens* comprises purslane, isopurslane, vitexin, and isovitexin, and the total mass percentage of flavonoid C glycosides of purslane, isopurslane, vitexin, and isovitexin, based on the weight of the flavonoid component of *Trifolium repens*, is 30%-35%.

[0015] Secondly, the present invention provides a method for preparing the composition of trifolinol and flavonoids, comprising the following steps: S1. The stems and leaves of *Trifolium repens* are washed, dried, crushed and sieved to obtain *Trifolium repens* powder. S2. The *Trifolium repens* powder is mixed with a mixed solution of ethanol and water, and then subjected to reflux extraction, filtration, and concentration to obtain a first type of extract. S3. Disperse the first type of extract in water, then add petroleum ether for extraction, and collect the petroleum ether phase and the aqueous alcohol phase; S4. The petroleum ether phase is concentrated to obtain a second type of extract. The second type of extract is mixed with an alkaline solution and subjected to a saponification reaction to obtain a saponified product. The saponified product is then subjected to dilution, extraction, collection of the extract, washing, and concentration to obtain a third type of extract. The third type of extract is dissolved in ethyl acetate and then filtered, crystallized, and dried to obtain the trifolin sterol component. S5. The aqueous-alcohol phase is dried to remove residual petroleum ether, ethanol is added and mixed evenly, and then allowed to stand, solid-liquid separation is performed, the supernatant is collected and concentrated to obtain the fourth type of extract; the fourth type of extract, a mixed solvent of ethyl acetate and n-butanol and sodium chloride are mixed, extracted, the organic phase is collected and concentrated to obtain the fifth type of extract; the fifth type of extract is dispersed in water, eluted, the eluent is collected and concentrated and freeze-dried to obtain the trifolin flavonoid component; S6. Mix the trifolin sterol component and the trifolin flavonoid component evenly to obtain a composition of trifolin sterol and flavonoid.

[0016] In the method of this invention, the aerial parts of *Trifolium repens* are first extracted with an aqueous ethanol solution and concentrated to obtain a crude extract. The aqueous dispersion of the crude extract is then extracted with petroleum ether to obtain a petroleum ether phase (source of sterol components) and a hydro-alcohol phase (source of flavonoid components). The petroleum ether phase is subjected to saponification, extraction, and crystallization to obtain the sterol components. The hydro-alcohol phase is subjected to alcohol precipitation for impurity removal and extraction to obtain the flavonoid components. The sterol components and flavonoid components are mixed to obtain the composition of this invention. The method of this invention is green, simple, scalable, and has a high extraction rate. It can obtain sterol components with a total sterol content ≥20% and flavonoid components with a total flavonoid content ≥70%, achieving the goal of controllable preparation of the composition of this invention.

[0017] Preferably, in step S1, the mesh size of the sieve is 20-100 mesh.

[0018] Preferably, in step S2, based on 1000g of the *Trifolium repens* powder, the volume of the mixed solution of ethanol and water is 5-50L.

[0019] Preferably, in step S2, the volume ratio of ethanol to water in the mixed solution of ethanol and water is 1:(0.5-1.2).

[0020] Preferably, in step S2, the reflux extraction temperature is 60-80℃, the reflux extraction is repeated multiple times, the number of reflux extractions is 1-5 times, the time of each reflux extraction is 1-3 hours, and the extracts from multiple reflux extractions are combined and filtered; the mesh size of the filter is 100-300 mesh.

[0021] Preferably, in steps S2-S5, each of the concentrations is independently selected from vacuum concentration at 40-60°C.

[0022] Preferably, in step S3, the volume of water used in the extraction is 1-3 times that of the first type of extract.

[0023] Preferably, in step S3, the volume of petroleum ether used in the extraction is 0.8-2 times the total volume of the first type of extract and water.

[0024] Preferably, in step S3, the extraction is repeated multiple times, the number of extractions is 2-3 times, and the extraction time for each extraction is 10-30 minutes. After extraction, the petroleum ether phase and the aqueous alcohol phase are collected by standing and separating. Then, the petroleum ether phase and the aqueous alcohol phase extracted multiple times are combined.

[0025] Preferably, in step S4, the alkaline solution comprises an ethanol solution of potassium hydroxide or sodium hydroxide, and the mass percentage of alkaline substances in the alkaline solution is 5%-20%.

[0026] Preferably, in step S4, the volume of the alkaline solution is 8-15 times the volume of the second type of extract.

[0027] Preferably, in step S4, the temperature of the saponification reaction is 50-70°C, and the time of the saponification reaction is 0.5-3 hours.

[0028] Preferably, in step S4, the solvent used in the dilution process is water, and the volume of water is 0.8-1.2 times the volume of the saponified product.

[0029] Preferably, in step S4, the solvent used in the extraction process is petroleum ether, the extraction is repeated multiple times, the number of extractions is 2-3 times, the extraction time for each extraction is 10-30 minutes, and after the extraction, the extracts from the multiple extractions are combined.

[0030] Preferably, in step S4, the washing liquid is water, and the washing is performed until the solution is neutral.

[0031] Preferably, in step S4, the dissolution temperature is 40-60℃, and the filtration temperature is 30-60℃.

[0032] Preferably, in step S4, the volume of the ethyl acetate is 5-15 times the volume of the third type of extract.

[0033] Preferably, in step S4, the crystallization temperature is 1-6℃, the crystallization is multiple crystallization, the number of crystallizations is 2-5 times, the time for each crystallization is 12-24 hours, and after crystallization, the crystals from multiple crystallizations are combined; the drying is vacuum drying, and the temperature of vacuum drying is 40-60℃.

[0034] Preferably, in step S5, the drying is vacuum rotary evaporation, the temperature of the vacuum rotary evaporation is 40-60℃, and the time of the vacuum rotary evaporation is 5-20 minutes.

[0035] Preferably, in step S5, the final concentration of ethanol in the system after uniform mixing is 50% to 80% (v / v).

[0036] Preferably, in step S5, the settling time is 12-24 hours.

[0037] Preferably, in step S5, the solid-liquid separation method is centrifugation, the centrifugation speed is 3000-5000 rpm, and the centrifugation time is 1-10 min.

[0038] Preferably, in step S5, when collecting and concentrating the supernatant, the supernatant is concentrated to 0.3-0.8 times its original volume.

[0039] Preferably, in step S5, the volume of the mixed solvent of ethyl acetate and n-butanol is 1-3 times the volume of the fourth type of extract.

[0040] Preferably, in step S5, the volume ratio of ethyl acetate to n-butanol in the mixed solvent of ethyl acetate and n-butanol is (2-10):1.

[0041] Preferably, in step S5, the mass percentage of sodium chloride is 1%-5% based on the total mass of the fourth type of extract, the mixed solvent of ethyl acetate and n-butanol, and sodium chloride.

[0042] Preferably, in step S5, the extraction is repeated multiple times, the number of extractions is 2-3 times, the extraction time for each extraction is 10-30 minutes, after the extraction, the organic phases after multiple extractions are combined, each extraction is carried out under shaking conditions, and after each extraction, the organic phase is collected by standing to achieve layering.

[0043] Preferably, in step S5, in the water dispersion of the fifth type of extract, the mass-to-volume ratio of the fifth type of extract to water is 1:(3-8), with units of g / mL.

[0044] Preferably, in step S5, the stationary phase in the elution is a resin column, the mass of which is 5-10 times the mass of the fifth type of extract, and the mobile phase is an aqueous solution of ethanol. The elution includes a first elution and a second elution. In the first elution, the mobile phase is an aqueous solution of ethanol with a volume fraction of 20%-30%, and in the second elution, the mobile phase is an aqueous solution of ethanol with a volume fraction of 60%-80%.

[0045] Preferably, in step S5 and step S6, the mass ratio of the trifolin sterol component and the trifolin flavonoid component is (0.125-8):1, and more preferably (1-2):1.

[0046] Thirdly, the present invention provides the use of the aforementioned trifolinol and flavonoid composition in at least one of the following ACs for non-disease treatment purposes, or in the preparation of products having at least one of the following ADs, characterized in that, A. Inhibits or reduces melanin production in the skin; B. Inhibit the secretion of endothelin-1 (ET-1) and / or prostaglandin E2 (PGE2) by skin keratinocytes. C. Inhibits the expression of MITF and its downstream target genes TYR, TYRP1 and TYRP2 in skin melanocytes; D. Improves post-inflammatory hyperpigmentation of the skin.

[0047] In the process of inflammatory hyperpigmentation, the MITF, TYR, TYRP1, and TYRP2 genes work synergistically to achieve multi-level regulation of melanin synthesis: MITF, as a core regulatory factor, can be activated by inflammatory factors (such as ET-1), thereby initiating the expression of key enzyme genes such as TYR, TYRP1, and TYRP2; among them, TYR encodes tyrosinase, which is the rate-limiting enzyme in melanin synthesis, catalyzing the conversion of tyrosine to dopa and dopaquinone; TYRP1 and TYRP2 participate in the polymerization and stabilization of melanin, jointly affecting the intensity and persistence of hyperpigmentation. The composition of this invention can simultaneously inhibit the ET-1 / PGE2 inflammatory pathway and the MITF / TYR melanin synthesis pathway, achieving a synergistic intervention targeting both inflammation and pigmentation; and in human skin efficacy tests, it has been demonstrated that products containing the composition of this invention can significantly increase skin ITA° value and reduce MI value, effectively improving symptoms of inflammatory hyperpigmentation such as post-acne scars and post-cosmetic procedures.

[0048] Preferably, the post-inflammatory hyperpigmentation is caused by inflammation following acne, cosmetic procedures, or dermatitis / eczema.

[0049] Fourthly, the present invention provides a topical skin product comprising the aforementioned composition of trifolinol and flavonoids.

[0050] Preferably, based on the total mass of the topical skin product, the mass percentage of the composition of trifolinol and flavonoids is 0.01%-5%, more preferably 0.05%-5%, more preferably 0.1%-5%, more preferably 0.15%-3%, and more preferably 0.15%-1%.

[0051] Preferably, the dosage form of the topical skin product is selected from at least one of toner, serum, cream, mask, spray, dressing, and gel.

[0052] In some embodiments, when the dosage form of the topical skin product is a serum, the serum comprises the following components in weight percentages: chelating agent 0.01%-0.5%, moisturizer 1%-20%, rheology modifier 0-2%, pH buffer 0-1%, preservative 0.01%-1%, the composition of trifolinol and flavonoids 0.05%-6%, solubilizer 0-2%, and the balance being water.

[0053] Topical skin products can be formulated in various dosage forms and formulated with different commonly used adjuvants, such as the exemplary components of the serum formulations described above, but are not limited to these components. More specifically, the chelating agent includes, but is not limited to, EDTA-2Na; the moisturizer includes, but is not limited to, at least one of glycerin, 1,2-pentanediol, and 1,3-propanediol; the rheology modifier includes, but is not limited to, at least one of carbomer, xanthan gum, and ammonium acryloyldimethyl taurate / VP copolymer; the pH adjuster includes, but is not limited to, at least one of citric acid and sodium citrate; the preservative includes, but is not limited to, at least one of phenoxyethanol and p-hydroxyacetophenone; and the solubilizer includes, but is not limited to, at least one of PEG-40 hydrogenated castor oil and PEG-60 hydrogenated castor oil.

[0054] For example, the preparation method of the essence includes the following steps: mixing water, chelating agent, moisturizer, rheology modifier, and pH adjuster at 80-85°C to obtain phase A; cooling phase A to 40-45°C, adding preservative, and mixing evenly to obtain a mixture AB; mixing the solubilizer and the composition of trifolinol and flavonoids at 50-65°C to obtain phase C; and mixing the mixture AB and phase C at 40-60°C to obtain the essence.

[0055] Compared with the prior art, the present invention has the following beneficial effects: The present invention combines the sterol component of *Trifolium repens* with the flavonoid component of *Trifolium repens* to form a composition, wherein the total sterol content in the sterol component is greater than or equal to 15% by mass, and the total flavonoid content in the flavonoid component is greater than or equal to 60% by mass. The sterol component and the flavonoid component work synergistically to inhibit the release of inflammatory factors and melanin synthesis through multi-target synergistic inhibition, thereby playing a role at two key nodes of inflammation source and pigment synthesis, blocking the formation of inflammatory pigmentation, and thus significantly improving the skin symptoms of inflammatory pigmentation. Attached Figure Description

[0056] Figure 1 The total ion chromatogram of the trifolin sterol fraction prepared in Example 1 is shown in the GC-MS chromatogram.

[0057] Figure 2 The image shows the HPLC chromatogram of the flavonoid component of *Trifolium repens* prepared in Example 1.

[0058] Figure 3 Comparison of inflammatory pigmentation and skin tone improvement in a typical subject before (left) and after (right) 28 days of using the serum containing the composition of Example 1 of this invention. Detailed Implementation

[0059] To better illustrate the purpose, technical solution, and advantages of this invention, specific embodiments will be used to further explain the invention below. Unless otherwise specified, the test methods used in the embodiments and / or comparative examples are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0060] Unless otherwise specified, the sterol component and flavonoid component of the present invention are obtained by the preparation methods of the following embodiments of the present invention.

[0061] The following involves some raw material information: Commercially available phytosterols: soybean source, β-sitosterol ≥50wt%, purchased from Haisif. B16 cells were purchased from Shangen Biotech, SNL-115; HaCaT cells were purchased from Shangen Biotech, SNL-163; B16 cell culture medium (SNLM-115) and HaCaT cell culture medium (SNLM-163) were both purchased from Shangen Biotech; MTT solution was purchased from Solarbio, M8180; DMEM medium was purchased from Shangen Biotech, SNM-002D; fetal bovine serum was purchased from Shangen Biotech; LPS was purchased from Solarbio, L8880; human endothelin-1 (ET-1) ELISA kit was purchased from Lianke Biotech, EK1314; PGE2 ELISA kit was purchased from Lianke Biotech, EK8103; trypsin was purchased from Solarbio, T1300; reverse transcription kit was purchased from TaKaRa, DRR036A; SYBR Green qPCR... Mix was purchased from Aikerui, AG11701; upstream and downstream primers MITF, TYR, TYRP1, TYRP2 and primers for the internal reference gene β-actin were all synthesized by Sangon Biotech; phosphate-buffered saline (PBS): pH 7.4. Unless otherwise specified, all raw materials used in the embodiments and comparative examples of this invention are commercially available, and the same raw materials were used in all parallel experiments. "v / v" in this document indicates a volume ratio.

[0062] Example 1 S1. Take the stems and leaves of *Trifolium repens*, wash them, dry them naturally in the sun, pulverize them, and pass them through a 40-mesh sieve to obtain *Trifolium repens* powder. S2. Weigh 1000g of the *Trifolium repens* powder obtained in step S1, add 20L of 50% ethanol aqueous solution (v / v), and reflux extract twice in a constant temperature water bath at 70℃ for 2 hours each time. The reflux extraction conditions are the same each time. Combine the extracts, filter through a 200-mesh filter, and concentrate under reduced pressure at 55℃ until there is no alcohol taste to obtain the first type of extract. S3. The first type of extract is suspended in 2 times the volume of deionized water to obtain a mixed system. An equal volume of petroleum ether is added to the mixed system and the mixture is shaken and extracted for 20 minutes. The mixture is allowed to stand and separate into layers. The upper petroleum ether phase is collected and the lower aqueous alcohol phase is reserved for later use. The extraction is repeated twice with petroleum ether under the same conditions each time. The petroleum ether phase or aqueous alcohol phase from the repeated extractions is combined.

[0063] S4. The petroleum ether phase was concentrated under reduced pressure at 40°C to a paste-like consistency, yielding a second type of paste. Ten times the volume of a 12wt% potassium hydroxide ethanol solution was added, and the mixture was saponified at 65°C for 1.5 hours. After cooling to room temperature, a mixed system was obtained. An equal volume of deionized water was added to the mixed system for dilution. An equal volume of petroleum ether was then added to the mixed system for extraction with shaking for 20 minutes. The mixture was allowed to stand and separate into layers, and the upper petroleum ether phase was collected. The extraction was repeated once more with petroleum ether under the same conditions each time. The petroleum ether phases from the repeated extractions were combined, washed with water until neutral, and concentrated under reduced pressure at 45°C to dryness, yielding a third type of paste. The obtained product was dissolved in 10 times the volume of ethyl acetate at 55°C upon heating. The mixture was filtered while hot, and the filtrate was allowed to stand at 4°C for crystallization for 18 hours. The crystals were collected by filtration. This crystallization process was repeated twice, with the same conditions each time. The crystals were combined and dried under reduced pressure at 45°C to obtain 10.94 g of the trifolin sterol fraction.

[0064] S5. Remove residual petroleum ether by rotary evaporation under reduced pressure at 45°C for 10 minutes, add anhydrous ethanol, and adjust the ethanol concentration in the mixture to 75% (v / v). Stir well and let stand at room temperature for 18 hours. Centrifuge at 4000 rpm for 10 minutes to remove the precipitate and collect the supernatant.

[0065] S6. Concentrate the supernatant under reduced pressure to half of its original volume to obtain the fourth type of extract (aqueous phase); add ethyl acetate-n-butanol mixed solvent (volume ratio 4:1), the volume of the mixed solvent being 1.5 times the volume of the fourth type of extract, and add 3wt% sodium chloride to the fourth type of extract. Shake thoroughly for 15 minutes, allow to stand and separate into layers, collect the upper organic phase, repeat the extraction 3 times, and combine the organic phases.

[0066] S7. The organic phase was concentrated under reduced pressure at 45℃ to a paste-like consistency, yielding the fifth type of extract. Deionized water was added at a mass-to-volume ratio of 1:5 (g / mL) to disperse and dissolve the extract. The solution was then passed through a D101 macroporous adsorption resin column (resin volume 8 times the mass of the extract) as the stationary phase. Impurities were removed by elution with a 25% ethanol-water solution (v / v) of 4 times the resin column volume (BV). Flavonoids were then enriched by elution with a 70% ethanol-water solution (v / v) of 4 times the resin column volume (BV), and the eluent was collected. The collected solution was concentrated under reduced pressure at 45℃ and freeze-dried to finally obtain 29.83 g of the *Trifolium repens* flavonoid fraction.

[0067] S8. Mix the trifolin sterol component and the trifolin flavonoid component at a mass ratio of 0.5:1, grind them in a mortar until they are evenly mixed, and obtain the composition.

[0068] Example 2 The difference between this embodiment and embodiment 1 is that the mass ratio of the trifolin sterol component and the trifolin flavonoid component is different in step S9. In this embodiment, the mass ratio of the trifolin sterol component and the trifolin flavonoid component is 1:1, and the rest are the same.

[0069] Example 3 The difference between this embodiment and embodiment 1 is that the mass ratio of the trifolin sterol component and the trifolin flavonoid component is different in step S9. In this embodiment, the mass ratio of the trifolin sterol component and the trifolin flavonoid component is 2:1, and the rest are the same.

[0070] Example 4 The difference between this embodiment and Example 1 is that the mass ratio of the trifolin sterol component and the trifolin flavonoid component is different in step S9. In this embodiment, the mass ratio of the trifolin sterol component and the trifolin flavonoid component is 0.2:1, and the rest are the same.

[0071] Example 5 The difference between this embodiment and embodiment 1 is that the mass ratio of the trifolin sterol component and the trifolin flavonoid component is different in step S9. In this embodiment, the mass ratio of the trifolin sterol component and the trifolin flavonoid component is 0.125:1, and the rest are the same.

[0072] Example 6 The difference between this embodiment and embodiment 1 is that the mass ratio of the trifolin sterol component and the trifolin flavonoid component is different in step S9. In this embodiment, the mass ratio of the trifolin sterol component and the trifolin flavonoid component is 8:1, and the rest are the same.

[0073] Comparative Example 1 The difference between this comparative example and Example 1 is that this comparative example did not perform steps S5-S9, and only obtained the trifolin sterol component.

[0074] Comparative Example 2 The difference between this comparative example and Example 1 is that this comparative example did not perform step S4, and only obtained the trifoliate flavonoid component.

[0075] Comparative Example 3 The difference between this comparative example and Example 1 is that the composition of this comparative example is obtained by mixing the trifolin sterol component from step S4 of Example 1 with commercially available soy isoflavones (total flavonoids ≥80wt%, Sigma) at a mass ratio of 0.5:1 at room temperature.

[0076] Comparative Example 4 The difference between this comparative example and Example 1 is that the composition of this comparative example is obtained by mixing commercially available phytosterols and the trifoliate flavonoid component in step S8 of Example 1 at a mass ratio of 0.5:1 at room temperature.

[0077] Comparative Example 5 The difference between this comparative example and Example 1 is that steps S3-S9 were not performed in this comparative example, and the first type of extract in step S2 was freeze-dried to obtain a crude extract of *Tripterygium wilfordii*. This crude extract contains various components of *Tripterygium wilfordii*, such as sterols, flavonoids, and polysaccharides, and is a total extract of *Tripterygium wilfordii* without separation and purification.

[0078] Performance testing: (1) Using β-sitosterol as a reference standard, the trifolinol component of Example 1 was qualitatively and quantitatively analyzed by gas chromatography-mass spectrometry (GC-MS) using the external standard method. The results are as follows: Figure 1 As shown. The results indicated that the main detectable sterol components were β-sitosterol and stigmasterol, with a combined content of 9.71 wt%. The remaining components were mainly fat-soluble components coexisting with sterols, primarily including fatty acids and triterpenoids. Using β-sitosterol as a control, the total sterol content of the trifolin sterol component in Example 1 was determined to be 24.12 wt% (all on dry weight) using the vanillin-perchloric acid colorimetric method.

[0079] (2) Using rutin as a reference standard, the total content of flavonoids in the *Trifolium repens* flavonoid fraction prepared in Example 1 was tested by ultraviolet-visible spectrophotometry (UV-Vis). The test results showed that the total content of flavonoids was 74.35 wt%. Simultaneously, the composition of flavonoids in the flavonoid fraction was analyzed by high-performance liquid chromatography (HPLC), and the test results are as follows: Figure 2 As shown in the figure. The results indicate that the flavonoid components contain arbutin, isoarbutin, vitexin, and isovitexin, with a total content of 33.47 wt% for the four flavonoid C glycosides; the remaining components are mainly phenolic acids (such as chlorogenic acid) and polysaccharides that coexist with the flavonoid components.

[0080] (3) Cytotoxicity test: Using the products from the examples and comparative examples as test substances, the cytotoxicity against B16 mouse melanoma cells and HaCaT human keratinocytes was evaluated by the MTT assay to determine the safe concentration range.

[0081] Take B16 cells or HaCaT cells in the logarithmic growth phase, at a density of 5 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours until complete cell adhesion. The original culture medium was discarded, and 100 μL of fresh culture medium (the corresponding specialized medium for the cell type) containing different concentrations of the test substance were added to each well, with concentration gradients of 50 µg / mL, 100 µg / mL, and 200 µg / mL for the sample groups. An equal volume of fresh culture medium without the test substance was added to the control group. Simultaneously, blank wells containing only culture medium (without cell seeding) were set up as the control group. After treatment, the cells were incubated for another 48 hours.

[0082] After incubation, add 20 µL of 5 mg / mL MTT solution to each well, vortex to mix, and continue incubation for 4 hours. Carefully discard the supernatant from the wells, add 150 µL of dimethyl sulfoxide to each well, and shake on a shaker at low speed for 10 minutes to fully dissolve the formazan crystals produced in the reaction. Finally, use a microplate reader to measure the absorbance values ​​of each well in the sample group, control group, and blank group at a wavelength of 490 nm. Calculate the relative cell viability based on the absorbance values ​​using the following formula.

[0083] .

[0084] The cytotoxicity results of the components in Examples 1-6 and Comparative Examples 1-5 are shown in Table 1. The results show that the cell viability of each test substance is >90% at ≤200 μg / mL, indicating no significant cytotoxicity.

[0085] Table 1. Effects of different test substances on cell viability of B16 cells and HaCaT cells. (4) Experiment on inhibiting the release of inflammatory factors: The effects of the products from the examples and comparative examples on the secretion of ET-1 and PGE2 by lipopolysaccharide (LPS) in HaCaT cells were investigated, including the following procedures: HaCaT cells in the logarithmic growth phase were harvested and processed at a density of 5 × 10⁶ cells per well. 4Cells were seeded at a density of 1000 μg / mL in 24-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours until complete cell adhesion. The experiment included a blank control group, a model group, a positive control group, and a sample group, with 6 replicates per group. After discarding the original culture medium, the cells were washed twice with phosphate-buffered saline (PBS). The blank control group was treated with serum-free DMEM medium, the model group with serum-free DMEM medium containing LPS (final concentration 1 μg / mL), the positive control group with serum-free DMEM medium containing LPS (final concentration 1 μg / mL) and dexamethasone (final concentration 1 μM), and the sample groups with serum-free DMEM medium containing LPS (final concentration 1 μg / mL) and 200 μg / mL of the test substance (the compositions or products from Examples 1-6 and Comparative Examples 1-2). After incubation for another 48 hours, the cell supernatant was collected and centrifuged at 3000 rpm for 10 minutes at 4°C to remove cell debris. The human endothelin-1 (ET-1) ELISA kit and the PGE2 ELISA kit were used, and the concentrations of ET-1 and PGE2 were measured separately according to the kit instructions. The inhibition rate of inflammatory factors was calculated according to the following formula, and the experiment was independently repeated three times.

[0086] .

[0087] The experimental results are shown in Table 2. The results indicate that when the sterol component and the flavonoid component of *Trifolium repens* were combined at a mass ratio of (0.2-2) (Examples 1-4), the inhibition rates of ET-1 and PGE2 were significantly higher than those of either component alone (p<0.01), demonstrating a synergistic effect. The synergistic effect was strongest when the mass ratio of the sterol component to the flavonoid component was 0.5:1. Comparative Examples 1-5 did not show a significant synergistic effect.

[0088] Table 2. Inhibitory effects of different test substances on inflammatory factors ET-1 and PGE2 (Mean±SD, n=3) Note: ** p<0.01, compared with Comparative Example 1 (trifolinol component alone); △ p<0.05, △△ p<0.01, compared with Comparative Example 2 (Trifolium repens flavonoid component alone). The inhibition rates of all test compounds were significantly higher than those of the model group (p<0.01, the inhibition rate of the model group was defined as 0, and the data are not listed in the table). Dexamethasone was used as a positive control and compared with Comparative Examples 1 and 2, respectively.

[0089] (5) Experiment on melanin pathway regulation in HaCaT-B16 co-culture system By simulating the inflammatory pigmentation microenvironment, the synergistic inhibitory effect of the combination of trifolinol and flavonoids on signal transduction between LPS-activated keratinocytes and melanocytes was verified.

[0090] Using 6.5mm Transwell cells (polyester membrane, 0.4μm pore size), the upper layer has 5×10 cells per pore. 4 HaCaT cells were seeded at a density of 10 cells / well, and the lower 6-well plate was filled with 2 × 10 cells / well. 5 B16 cells were seeded at a density of [number] cells per cell, with both layers containing 10% fetal bovine serum (FBS) in DMEM medium (1.5 mL for the upper layer and 2.5 mL for the lower layer). The cells were co-cultured at 37°C in a 5% CO2 incubator for 24 hours until complete cell adhesion. After co-culture, the original medium was discarded, and both layers were replaced with serum-free DMEM medium, and cultured for another 2 hours. The experiment included a blank control group, a model group, a sample group, and a positive control group, with 6 replicates per group (3 independent replicates). In this study, the blank control group had serum-free DMEM medium (containing neither LPS nor the test substance) added to the upper layer; the model group had serum-free DMEM medium containing LPS (final concentration 1 μg / mL) added to the upper layer; the sample group had serum-free DMEM medium containing LPS (final concentration 1 μg / mL) and the test substance (Examples 1, 2, 5, Comparative Example 1 or Comparative Example 2, all with a final concentration of 200 μg / mL) added to the upper layer; the positive control group had serum-free DMEM medium containing LPS (final concentration 1 μg / mL) and α-arbutin (final concentration 100 μg / mL) added to the upper layer; and all sample groups had serum-free DMEM medium added to the lower layer.

[0091] After each sample group was processed, they were co-cultured for 48 hours, with the upper drug-containing medium replaced every 24 hours (the lower medium was not replaced). After co-culture, the upper chamber was discarded, and the lower B16 cells were washed twice with PBS. B16 cells were collected by digestion with 0.25% (w / v) trypsin, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS and counted. After resuspending and washing once more with PBS, the cell pellet was used for RNA extraction.

[0092] Total RNA was extracted from B16 cells according to the Trizol instructions. RNA purity and concentration were measured using a Nanodrop 2000 micro-spectrophotometer. After determining RNA integrity, it was used as a template for reverse transcription. 1 μg of total RNA was used as a template and reverse transcribed using a reverse transcription kit according to the kit's instructions on a PCR instrument to obtain cDNA, which was then stored at -20℃ for later use. The cDNA was used as a template for RT-qPCR to detect the expression of the target gene. The reaction mixture was 20 μL, containing 10 μL of SYBR Green qPCR Mix, 0.8 μL each of forward and reverse primers (MITF, TYR, TYRP1, TYRP2 and primers for the internal control gene β-actin, all at a concentration of 10 μmol / L), 2 μL of cDNA template, and 6.4 μL of enzyme-free water. The reaction program was: 95℃ pre-denaturation for 1 minute, 95℃ denaturation for 5 seconds, and 60℃ annealing and extension for 30 seconds, for a total of 40 cycles. β-actin was used as the internal control gene, and RT-qPCR was performed to detect the expression of the target gene. （-ΔΔCt） The relative expression levels of the MITF, TYR, TYRP1, and TYRP2 genes were calculated using a method. Each sample was tested in triplicate, and the experiment was independently repeated three times.

[0093] The test results are shown in Table 3. Compared with the model group, all tested samples significantly inhibited the expression of the above-mentioned genes (p<0.01). Among them, the inhibitory effect of Comparative Example 2 (Trifoliol flavonoid component alone) was better than that of Comparative Example 1 (Trifoliol sterol component alone). The inhibitory effects of Example 1 (Trifoliol sterol component: Trifoliol flavonoid component = 0.5:1) and Example 2 (Trifoliol sterol component: Trifoliol flavonoid component = 1:1) were the strongest and significantly better than that of Comparative Example 2 (Trifoliol flavonoid component alone) (p<0.01). This indicates that the optimal ratio of Trifoliol sterol and flavonoids (0.5:1, 1:1) can effectively downregulate the expression of melanin synthesis-related genes by synergistically blocking the inflammatory signal transduction between keratinocytes and melanocytes.

[0094] Table 3. Relative expression levels of melanin-related genes in B16 cells after treatment with different test substances (Mean±SD, n=3) Note: Compared with the blank control group, ## P<0.01; compared with the model group, ** P<0.01; compared with Comparative Example 2, △ P<0.05, △△ P<0.01.

[0095] (6) Melanin Synthesis Inhibition Experiment Using B16 cells as a model, this study investigated the inhibitory effect of a combination of trifolin sterol and flavonoids on melanin synthesis in cells.

[0096] Take B16 cells in logarithmic growth phase and use 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 μg / mL in 6-well plates and incubated at 37°C in a 5% (v / v) CO2 incubator for 24 hours until the cells were fully adherent. The original culture medium was discarded. Serum-free DMEM medium was added to the blank control group, serum-free DMEM medium containing the corresponding test substance (200 μg / mL, the product of the example or comparative example) was added to the sample group, and serum-free DMEM medium containing α-arbutin (100 μg / mL) was added to the positive control group. Each group was divided into 3 replicates. The cells were incubated for another 48 hours.

[0097] After culture, cells were collected from 6-well plates by digestion with 0.25% (w / v) trypsin. After centrifugation and discarding the supernatant, cells were resuspended in PBS buffer and washed twice. After centrifugation and discarding the supernatant again, 200 μL of 1 mol / L NaOH solution containing 10% (v / v) dimethyl sulfoxide (DMSO) was added, and cells were lysed in an 80°C water bath for 2 hours. Finally, 100 μL of cell lysate was transferred to 96-well plates, and the absorbance was measured at 405 nm using a microplate reader. The melanin synthesis inhibition rate of the sample group and the positive control group was calculated according to the following formula. All experiments were independently repeated in triplicate.

[0098] .

[0099] The results of the melanin synthesis inhibition experiment are shown in Table 4. The results show that the inhibitory effect of Comparative Example 2 (trifoliol flavonoid component alone) is better than that of Comparative Example 1 (trifoliol sterol component alone). When the trifoliol component and the trifoliol flavonoid component are combined at a mass ratio of (0.2-2):1 (Examples 1-4), the inhibition rate of melanin synthesis is significantly higher than that of either component alone in Comparative Example 2 (p<0.01), showing a synergistic effect. The optimal combination ratio is 0.5:1 (Example 1).

[0100] Table 4. Inhibitory effects of different test substances on melanin synthesis in B16 cells (Mean±SD, n=3) Note: Compared with Comparative Example 2 (Trifolium repens flavonoids used alone), * P<0.05, ** P<0.01.

[0101] (7) Human patch irritation test The skin safety of the compound combination of trifolin sterol and trifolin flavonoids was evaluated by human skin patch test.

[0102] 1. Testing Method Thirty-two subjects aged 18–60 years with no skin diseases, allergies, or weakened immune systems were selected. Before testing, the inner upper arm (where the skin was confirmed to be in good condition) was thoroughly cleaned with water and dried. 20 μL of the example composition dissolved in butylene glycol was evenly applied to the patch applicator filter paper (a control group containing only butylene glycol was also included). The treated patch was then smoothly applied to the subject's test site, and the palm was gently pressed for 10–15 seconds to ensure a tight fit with the skin without air bubbles or loosening. After 24 hours, the patch was carefully removed, and any remaining sample was gently rinsed with water and dried. The skin condition at the test site was observed and recorded at 30 minutes, 24 hours, and 48 hours after patch removal.

[0103] According to the International Contact Dermatitis Study Group (ICDRG) classification: Grade 0: No reaction; Grade I: Mild erythema (suspected reaction); Grade II: Obvious erythema and papules (weak positive reaction); Grade III: Erythema, papules, and vesicles (strong positive reaction); Grade IV: Severe erythema, bullae, and erosions (very strong positive reaction). If a Grade II or higher reaction occurs, it indicates that the sample poses a risk of causing skin irritation or allergic reaction.

[0104] 2. Result Evaluation A total of 32 samples were collected. For any patch area, if irritation symptoms (such as erythema) appeared at any time point (30 minutes, 24 hours, and 48 hours after patch removal), the most severe reaction grade was recorded. The patch test results are shown in Table 5.

[0105] The results showed that the compound compositions of trifolinol and flavonoids provided in Examples 1-4 (compound ratio of 0.2-2:1) all exhibited Grade 0 reactions at concentrations of 0.2wt%, 0.5wt%, and 2.0wt% (solvent is butanediol), and were non-irritating to human skin, demonstrating good safety.

[0106] Table 5 Results of human patch tests for different test substances (8) Human efficacy test 1. Preparation of samples of serum 1 and serum 2 Both serum 1 and serum 2 are prepared according to the following steps: S1. According to the formula in Table 6, add the raw material of phase A into the reactor and stir and mix at 80°C; after cooling to 45°C, add the raw material of phase B and continue stirring until the system is uniform to obtain the AB mixed system. S2. Take another raw material from phase C, heat it at 60°C until it is completely homogeneous, then slowly add it dropwise to the AB mixture, stir evenly, and cool it down to below 30°C to obtain the finished essence.

[0107] Table 6. Essence Raw Material Ratio 2. Testing Methods Sixty participants (Fitzpatrick type III-IV, 18–45 years old) with stable post-acne hyperpigmentation were recruited and randomly assigned to two groups of 30 each. Group 1 applied Serum 1 containing the composition of Example 1 (trifolinol to trifolin flavonoid ratio of 0.5:1) to the entire face (focusing on acne scars) morning and evening. Group 2 used Serum 2 containing the composition of Comparative Example 1 (trifolinol only), for 28 consecutive days. Skin ITA° (Individual Typology Angle) and skin melanin index (MI) were measured on the participants' facial areas before product use (T0) and 28 days after product use (T28). The average values ​​before and after testing were calculated for all participants. The rate of change of ITA° or MI value was calculated using the following formula: the average measurement at T0 represents the average result of all participants before sample application on the test day (day 0), and the average measurement at T28 represents the average result of all participants 28 days after sample application. The skin indices at T0 and T28 were compared using a paired-samples t-test for statistical analysis. * P<0.05, ** P<0.01.

[0108] .

[0109] ITA° value represents skin brightness; the higher the value, the more even and radiant the skin tone. Meanwhile, MI value represents the melanin content in the skin; the lower the value, the less melanin and the whiter the skin tone.

[0110] 3. Experimental Results The results are shown in Tables 7 and 8.

[0111] Table 7. ITA° values ​​and their rate of change (Mean±SD) of subjects measured at 0 days and 28 days. Table 8. MI values ​​and their rate of change (Mean ± SD) of subjects measured at 0 days and 28 days. The results showed that, compared with T0, subjects using serum 1 had a significantly higher ITA° value at T28 (27.28%, p<0.01) and a significantly lower MI value (-19.79%, p<0.01); subjects using serum 2 also showed an increased ITA° value (8.21%, p<0.05) and a decreased MI value (-10.56%, p<0.05). Specifically, the ITA° increase rate (27.28%) in serum 1 group was significantly higher than that in serum 2 group (8.21%), and the MI value reduction rate (-19.79%) was also superior to that in serum 2 group (-10.56%). These results indicate that the combination of trifolin sterol and trifolin flavonoids has a synergistic effect, and its efficacy in improving post-inflammatory hyperpigmentation is significantly better than that of trifolin sterol alone.

[0112] Additionally, a typical subject's facial improvement before and after using the gel sample 28 days prior is provided, such as... Figure 3 As shown, after 28 days of use of the serum containing the composition of Example 1, the subjects experienced a significant reduction in post-inflammatory hyperpigmentation of the face.

[0113] This indicates that after continuous use of the serum containing 0.2% of the composition of Example 1 for 28 days, it can significantly increase the skin's ITA° value and reduce the MI value, demonstrating that the combination of trifolinol and flavonoids has a definite effect in improving inflammatory pigmentation, effectively reducing skin pigmentation and melanin content, and can be used to improve skin condition.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A composition of trifolinol and flavonoids, characterized in that, It includes a trifolin sterol component and a trifolin flavonoid component, wherein the total mass percentage of sterol compounds in the trifolin sterol component is greater than or equal to 15%, and the total mass percentage of flavonoid compounds in the trifolin flavonoid component is greater than or equal to 60%.

2. The composition of trifolinol and flavonoids as described in claim 1, characterized in that, The mass ratio of the trifolin sterol component to the trifolin flavonoid component is (0.125-8):

1.

3. The composition of trifolinol and flavonoids as described in claim 2, characterized in that, The mass ratio of the trifolin sterol component to the trifolin flavonoid component is (0.2-6):

1.

4. The composition of trifolinol and flavonoids as described in claim 1, characterized in that, The trifolin sterol component includes at least one of β-sitosterol and stigmasterol; and / or, the trifolin flavonoid component includes at least one of vitexin, isovitexin, vitexin, and isovitexin.

5. A method for preparing the composition of trifolinol and flavonoids according to any one of claims 1-4, characterized in that, Includes the following steps: S1. The stems and leaves of *Trifolium repens* are washed, dried, crushed and sieved to obtain *Trifolium repens* powder. S2. The *Trifolium repens* powder is mixed with a mixed solution of ethanol and water, and then subjected to reflux extraction, filtration, and concentration to obtain a first type of extract. S3. Disperse the first type of extract in water, then add petroleum ether for extraction, and collect the petroleum ether phase and the aqueous alcohol phase; S4. Concentrate the petroleum ether phase to obtain a second type of extract, mix the second type of extract with an alkaline solution, and carry out a saponification reaction to obtain the saponified product. The saponified product was successively diluted, extracted, the extract was collected, washed and concentrated to obtain a third type of extract; the third type of extract was dissolved in ethyl acetate, and then filtered, crystallized and dried to obtain the trifolin sterol component; S5. The aqueous alcohol phase is dried to remove petroleum ether, ethanol is added and mixed evenly, and then allowed to stand, solid-liquid separation is performed, the supernatant is collected and concentrated to obtain the fourth type of extract; the fourth type of extract, a mixed solvent of ethyl acetate and n-butanol and sodium chloride are mixed, extracted, the organic phase is collected and concentrated to obtain the fifth type of extract. The fifth type of extract was dispersed in water, then eluted, the eluent was collected, concentrated and freeze-dried to obtain the trifoliate flavonoid component; S6. Mix the trifolin sterol component and the trifolin flavonoid component evenly to obtain a composition of trifolin sterol and flavonoid.

6. The use of the composition of trifolinol and flavonoids according to any one of claims 1-4 in at least one of the following ACs for non-disease treatment purposes, or in the preparation of products having at least one of the following ADs, characterized in that, A. Inhibits or reduces melanin production in the skin; B. Inhibit the secretion of endothelin-1 and / or prostaglandin E2 by keratinocytes in the skin; C. Inhibits the expression of MITF and its downstream target genes TYR, TYRP1 and TYRP2 in skin melanocytes; D. Improves post-inflammatory hyperpigmentation of the skin.

7. The application as described in claim 6, characterized in that, The aforementioned post-inflammatory hyperpigmentation is caused by inflammation following acne, cosmetic procedures, or dermatitis / eczema.

8. A topical skin product, characterized in that, Compositions comprising trifolinol and flavonoids as described in any one of claims 1-3.

9. The topical skin product as described in claim 8, characterized in that, Based on the total mass of the topical skin product, the mass percentage of the composition of trifolinol and flavonoids is 0.01%-5%.

10. The topical skin product as described in claim 9, characterized in that, The dosage form of the topical skin product is selected from at least one of the following: toner, serum, cream, mask, spray, dressing, and gel.

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