An adenosine-undecylenoyl phenylalanine synergistic whitening composition and method of making the same

Abstract

The application discloses an adenosine-undecylenoyl phenylalanine synergistic whitening composition and a preparation method thereof. The whitening active ingredient of the whitening composition is composed of adenosine, glycyrrhizic acid dipotassium, undecylenoyl phenylalanine, nicotinamide and a VC derivative. The whitening active ingredient accounts for 2.35-10.2% of the total mass of the whitening composition in terms of mass fraction. The content ratio of the adenosine and the undecylenoyl phenylalanine is 0.05-1:0.2-2. The adenosine and the undecylenoyl phenylalanine are combined as whitening components and added into a whitening product for the first time. The adenosine can inhibit the activity of melanocytes and directly inhibit the production of melanin from the source. Meanwhile, the whitening mechanism of the undecylenoyl phenylalanine and the nicotinamide is supplemented, synergistic effect is achieved, the generation and deposition of melanin are blocked in a whole link, and the whitening effect is obvious. The product has good stability, high safety, good economic value and market prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an adenosine-undecenoyl phenylalanine synergistic whitening composition and its preparation method. Background Technology

[0002] With the improvement of living standards, people's pursuit of beauty has become increasingly higher, especially women's pursuit of skin whitening, which has reached its peak. Consequently, whitening cosmetics have become incredibly popular. However, many whitening cosmetics currently on the market have the problem of insignificant whitening effects or even no whitening effect at all. The main reasons for this are as follows:

[0003] Firstly, existing whitening ingredients, when at excessively high concentrations, can easily irritate the skin. For example, hydroquinone, vitamin C, and arbutin, when at high concentrations, can cause the stratum corneum to renew too quickly or excessively inhibit melanin production, damaging normal skin cells. Furthermore, some whitening ingredients may become unstable under air or light, potentially causing irritation. Therefore, most existing whitening products contain low levels of whitening ingredients, or only trace amounts (generally not exceeding 0.1%), resulting in products that do not achieve the desired whitening effect.

[0004] Secondly, existing whitening products have a single whitening mechanism and cannot completely block melanin production. Firstly, some existing whitening products contain only one whitening ingredient, while melanin production and deposition is a complex process; single-ingredient whitening products have limited whitening efficacy. Secondly, even though some whitening products on the market contain compound ingredients, their whitening mechanism is mostly to reduce skin pigmentation by inhibiting melanin production or to promote the renewal of the stratum corneum to achieve an immediate whitening effect. This not only fails to inhibit melanin production at its source but may also cause significant damage to the skin. For example, some acid-based whitening products, after use, make the stratum corneum thinner, making the skin more susceptible to external stimuli, reducing its defenses, and ultimately leading to a series of skin problems—a net loss.

[0005] Adenosine is a nucleoside composed of adenine and ribose molecules, playing important biological roles in living organisms. Studies have shown that adenosine plays a crucial role in intracellular and intercellular signal transduction. It can regulate various physiological processes, such as apoptosis, cell cycle regulation, and immune responses, through interaction with adenosine receptors, and even modulate neuronal activity, inhibiting the excitability of the central nervous system. In the cosmetics and skincare industry, adenosine has been reported to be added to products, claiming anti-wrinkle, antioxidant, and skin-soothing effects. However, its use as a skin-whitening ingredient has not been reported.

[0006] Undecenoyl phenylalanine, also known as amide, is a melanocyte-stimulating hormone (α-MSH) receptor antagonist and is used by cosmetic manufacturers as an important whitening agent. However, undecenoyl phenylalanine cannot inhibit the activity of melanocytes, making it difficult to address the problem of melanin regeneration at its source. Therefore, the whitening effect of its products is not ideal.

[0007] In addition, existing whitening products also have the problem of poor stability. This is because the products contain some whitening ingredients that are unstable or easily oxidized. If these ingredients are not fully dissolved or protected, the product will turn black after being left for a long time, or crystallize or precipitate, which will greatly affect the shelf life of the product and is very unfavorable for storage. Summary of the Invention

[0008] To address the shortcomings of existing whitening products, such as low whitening ingredient content, insufficient whitening effect, and limited whitening mechanism that fails to fully block melanin production, this invention provides an adenosine-undecenoyl phenylalanine synergistic whitening composition and its preparation method. This invention, for the first time, combines adenosine as a whitening ingredient with undecenoyl phenylalanine, overcoming the limitations of undecenoyl phenylalanine's whitening mechanism and directly inhibiting melanin production at its source, thus synergistically enhancing the product's whitening effect. The specific technical solution is as follows:

[0009] First, the present invention provides an adenosine-undecenoyl phenylalanine synergistic whitening composition, wherein the whitening active ingredients of the whitening composition include adenosine and undecenoyl phenylalanine; and, by mass fraction: the whitening active ingredients account for 2.35 to 10.2% of the total mass of the whitening composition; and the content ratio of adenosine to undecenoyl phenylalanine is 0.05 to 1: 0.2 to 2.

[0010] The aforementioned adenosine-undecenoyl phenylalanine synergistic whitening composition also includes nicotinamide, VC derivatives and dipotassium glycyrrhizate as its whitening active ingredients; and by mass fraction, the whitening composition also contains 3-22% oil, 2-10% polyol, 1-4% emulsifier, and the balance being purified water.

[0011] The aforementioned adenosine-undecenoyl phenylalanine synergistic whitening composition comprises, wherein the oil is one or more of octyldodecyl alcohol, polydimethylsiloxane, caprylic / capric triglyceride, tocopheryl acetate, and bisabolol; the polyol is one or more of glycerol, glyceryl polyether-26, 1,3-butanediol, 1,3-propanediol, 1,2-hexanediol, and 1,2-pentanediol; and the emulsifier is one or more of Tween-20 with low oleic acid content, polyglycerol-10 stearate, glyceryl caprylate, cetearyl glucoside & cetearyl alcohol, arachidonic acid & behenol & arachidonic acid glucoside.

[0012] Preferably, in the aforementioned adenosine-undecenoyl phenylalanine synergistic whitening composition, the emulsifier includes octyldodecyl alcohol, and the content ratio of undecenoyl phenylalanine to octyldodecyl alcohol is 0.2-2:1-10.

[0013] Preferably, in the aforementioned adenosine-undecenoyl phenylalanine synergistic whitening composition, the oil includes Tween-20 with low oleic acid content, and the content ratio of undecenoyl phenylalanine to emulsifier is 1:1 to 2.

[0014] More preferably, the aforementioned adenosine-undecenoyl phenylalanine synergistic whitening composition comprises, by mass fraction, the following components:

[0015] Whitening active ingredients: Adenosine 0.05-1%, Undecenoyl Phenylalanine 0.2-2%, Niacinamide 2-5%, Vitamin C derivative 0.05-2%, Dipotassium glycyrrhizate 0.05-0.2%;

[0016] Oils and fats: 1-10% octyl dodecanol, 0.5-3% polydimethylsiloxane, 0.5-3% caprylic / capric triglyceride, 0.5-3% tocopheryl acetate, 0.5-3% bisabolol;

[0017] Polyols: glycerol 2-5%, glycerol polyether 2-5%, 1,3-butanediol 2-5%, 1,3-propylene glycol 2-5%, 1,2-hexanediol 0.2-3%, 1,2-pentanediol 0.5-2%;

[0018] Emulsifiers: Tween-20 with low oleic acid content 0.5-2%, polyglycerol-10 stearate 0.5-2%, glyceryl caprylate 0.5-2%, cetearyl glucoside & cetearyl alcohol 0.5-3%, arachidonic acid & behenol & arachidonic acid glucoside 0.5-3%.

[0019] More preferably, the aforementioned adenosine-undecenoyl phenylalanine synergistic whitening composition comprises, by weight fraction, the following substances:

[0020] Whitening active ingredients: Adenosine 0.2%, Dipotassium glycyrrhizate 0.2%, Undecenoyl phenylalanine 1%, Niacinamide 3%, Vitamin C derivative 1%;

[0021] Oils and fats: 5% octyl dodecanol, 1% polydimethylsiloxane, 2% caprylic / capric triglyceride, 0.5% tocopheryl acetate, 0.5% bisabolol;

[0022] Polyols: glycerol 2%, glycerol polyether-26 5%, 1,3-butanediol 2%, 1,3-propanediol 2%, 1,2-hexanediol 0.8%, 1,2-pentanediol 0.5%;

[0023] Emulsifiers: Tween-20 with low oleic acid content 1%, polyglycerol-10 stearate 0.5%, glyceryl caprylate 0.5%, cetearyl glucoside & cetearyl alcohol 2%, arachidonic acid & behenol & arachidonic acid glucoside 2%.

[0024] Secondly, the present invention also provides a method for preparing the aforementioned adenosine-undecenoyl phenylalanine synergistic whitening composition, comprising the following steps:

[0025] 1) Disperse undecenoyl phenylalanine, a whitening active ingredient, in oil to obtain solution I;

[0026] 2) Mix all components of the emulsifier evenly at 60-80℃ to obtain solution II;

[0027] 3) Mix solution I and solution II thoroughly to obtain solution III, which serves as the oil phase, for later use;

[0028] 4) Mix the polyol and the remaining whitening active ingredients evenly at 60-80℃ to obtain solution IV as the aqueous phase, for later use;

[0029] 5) At 60-80℃, add oil phase solution III to aqueous phase solution IV, stir and mix evenly, and then homogenize to ensure that the oil phase is completely coated by the aqueous phase, thereby obtaining the adenosine-undecenoyl phenylalanine synergistic whitening composition.

[0030] As a preferred technical solution, in step 1), the preparation process of solution I is as follows: firstly, at 40-60°C, undecenoyl phenylalanine is dispersed in octyldodecyl alcohol, and heated while stirring until undecenoyl phenylalanine is completely dissolved and the solution is colorless and transparent, then other oil components are added and mixed evenly.

[0031] As a preferred technical solution, in step 5), the stirring speed for uniform mixing is controlled at 50-300 r / min, and the stirring time is 5-20 min; the homogenization speed is controlled at 2000-6000 r / min, and the homogenization time is 2-10 min.

[0032] The beneficial effects of this invention are:

[0033] 1) This invention is the first to add adenosine as a whitening ingredient to a whitening product, and combines it with undecenoyl phenylalanine to compensate for the whitening mechanism defects of undecenoyl phenylalanine, synergistically improving the whitening effect of the product. Adenosine can inhibit the activity of melanocytes, directly inhibiting melanin production at its source; simultaneously, undecenoyl phenylalanine acts on the biochemical reactions at each stage of melanin formation induced by α-MSH, intercepting melanin production signals, controlling the binding of α-MSH (melanocyte-stimulating hormone) to melanin-producing factors, and effectively inhibiting tyrosinase activity, thereby achieving a complete blockade of melanin production and deposition, resulting in a better whitening effect for the product.

[0034] 2) The whitening composition of the present invention controls the ratio of adenosine to undecenoyl phenylalanine at 0.05-1:0.2-2, which not only makes the product have a better whitening effect, but also allows it to react with undecenoyl phenylalanine to achieve esterification, reducing the cytotoxicity and irritation of undecenoyl phenylalanine, so that the product achieves the purpose of whitening without irritation, gentle skin care, and can be used with confidence by people with sensitive skin.

[0035] 3) The whitening composition of the present invention uses octyldodecyl alcohol to dissolve undecenoyl phenylalanine and adenosine, which not only increases the solubility of undecenoyl phenylalanine and adenosine, but also octyldodecyl alcohol is fat-soluble and similar to the skin structure, which has a good transdermal penetration promoting effect, improves the transdermal penetration rate of whitening ingredients, further enhances the whitening effect, and promotes the stability of the product.

[0036] 4) The whitening composition of the present invention encapsulates the whitening ingredients through the combination of oils and emulsifiers, ensuring that the composition is mild, stable and does not precipitate, thus further improving the stability of the whitening product; at the same time, it ensures the antioxidant properties of adenosine, improves the antioxidant capacity of the whitening composition, protects the whitening composition from oxidation, and ensures the stability of the entire whitening composition.

[0037] Overall, the product of this invention has good temperature resistance, good light stability, good performance, and high safety, and has good economic value and market prospects. Attached Figure Description

[0038] Figure 1 The results of the cytotoxicity test of the adenosine-containing whitening composition of the present invention;

[0039] Figure 2 The results of the chicken embryo chorioallantoic membrane irritation test of the adenosine-containing whitening composition of the present invention are shown. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments.

[0041] Example 1

[0042] This embodiment addresses the problems of existing whitening products on the market, such as insufficient whitening ingredient concentration, high concentrations of whitening ingredients causing significant skin irritation, single whitening ingredients, a limited whitening mechanism that fails to block melanin production across the entire process, significant skin damage from immediate whitening products, and poor product stability. It proposes an adenosine-undecenoyl phenylalanine synergistic whitening composition and its preparation method. For the first time, adenosine is added as a whitening ingredient and combined with undecenoyl phenylalanine to directly inhibit melanin production at its source, improving product efficacy while enhancing the overall stability of the whitening composition.

[0043] The adenosine-containing whitening composition described in this embodiment comprises adenosine and undecenoyl phenylalanine as its whitening active ingredients. The whitening active ingredients account for 2.35–10.2% of the total mass of the whitening composition by mass fraction, and the ratio of adenosine to undecenoyl phenylalanine is 0.05–1:0.2–2. Furthermore, the whitening active ingredients of this composition also include niacinamide, vitamin C derivatives, and dipotassium glycyrrhizate. The composition also contains 3–22% oil, 2–10% polyol, 1–4% emulsifier, and the remainder is purified water by mass fraction. Adenosine inhibits the activity of melanocytes, directly suppressing melanin production at its source. Simultaneously, the whitening mechanisms of undecenoyl phenylalanine and niacinamide synergistically enhance the whitening effect, achieving a complete blockade of melanin formation and deposition, resulting in a significant whitening effect. In addition, since undecenoyl phenylalanine has strong cytotoxicity and irritation, the adenosine-containing whitening composition described in this embodiment adds adenosine, and the content ratio of adenosine to undecenoyl phenylalanine is 0.05-1:0.2-2. Through the esterification of the two, the cytotoxicity and irritation of undecenoyl phenylalanine can be reduced, so that the product achieves the purpose of whitening without irritation, gentle skin care, and can be used with confidence by people with sensitive skin.

[0044] In the adenosine-containing whitening composition described in this embodiment, the oil is one or more of octyldodecyl alcohol, polydimethylsiloxane, caprylic / capric triglyceride, tocopheryl acetate, and bisabolol; the polyol is one or more of glycerol, glyceryl polyether-26, 1,3-butanediol, 1,3-propanediol, 1,2-hexanediol, and 1,2-pentanediol; the emulsifier is one or more of Tween-20 with low oleic acid content, polyglycerol-10 stearate, glyceryl caprylate, cetearyl glucoside & cetearyl alcohol, arachidonic acid & behenol & arachidonic acid glucoside. The content ratio of undecenoyl phenylalanine to octyldodecyl alcohol is 0.2-2:1-10, and the content ratio of undecenoyl phenylalanine to emulsifier is 1:1-2. Octyldodecyl alcohol, combined with undecenoyl phenylalanine, not only increases the solubility of both adenosine and undecenoyl phenylalanine, but also, due to its lipid solubility and similarity to skin structure, octyldodecyl alcohol exhibits good transdermal penetration enhancement, improving the transdermal absorption rate of whitening ingredients and further enhancing their whitening effect. Furthermore, the combination of oils and emulsifiers encapsulates the whitening ingredients, ensuring a gentle and stable composition without precipitation, thus improving the stability of the whitening product. Simultaneously, adenosine's antioxidant properties enhance the antioxidant capacity of the whitening composition, protecting it from oxidation and improving the overall stability of the whitening composition.

[0045] The whitening composition described in this embodiment, by mass fraction, comprises the following substances: Whitening active ingredients: adenosine 0.05-1%, dipotassium glycyrrhizate 0.05-0.2%, undecanoyl phenylalanine 0.2-2%, nicotinamide 2-5%, VC derivative 0.05-2%; Oils: octyl dodecanol 1-10%, polydimethylsiloxane 0.5-3%, caprylic / capric triglyceride 0.5-3%, tocopheryl acetate 0.5-3%, bisabolol 0.5-3%; Polyols: glycerin 2-5%, glyceryl polyether-26 2-5%, 1,3-butanediol 2-5%, 1,3-propylene glycol 2-5%, 1,2-hexanediol 0.2-3%, 1,2-pentanediol 0.5-2%; Emulsifiers: Tween-20 with low oleic acid content 0.5-2%, polyglycerol-10 Stearate 0.5-2%, glyceryl caprylate 0.5-2%, cetearyl glucoside & cetearyl alcohol 0.5-3%, arachidonic acid & behenol & arachidonic acid glucoside 0.5-3%. Preferably, it is composed of the following substances: whitening active ingredients: adenosine 0.2%, dipotassium glycyrrhizate 0.2%, undecanoyl phenylalanine 1%, nicotinamide 3%, VC derivative 1%; oils: octyl dodecanol 5%, polydimethylsiloxane 1%, caprylic / capric triglyceride 2%, tocopheryl acetate 0.5%, bisabolol 0.5%; polyols: glycerin 2%, glyceryl polyether-26 5%, 1,3-butanediol 2%, 1,3-propanediol 2%, 1,2-hexanediol 0.8%, 1,2-pentanediol 0.5%; emulsifiers: low oleic acid content Tween-20 1%, polyglycerol-10 stearate 0.5%, glyceryl caprylate 0.5%, cetearyl glucoside & cetearyl alcohol 2%, arachidonic acid & behenol & arachidonic acid glucoside 2%.

[0046] The whitening composition described in this embodiment is prepared by the following steps:

[0047] 1) Disperse undecenoyl phenylalanine, a whitening active ingredient, in oil to obtain solution I; the specific preparation process is as follows: first, disperse undecenoyl phenylalanine in octyldodecyl alcohol at 40-60℃, and heat while stirring until undecenoyl phenylalanine is completely dissolved and the solution is colorless and transparent, then add other oil components and mix evenly.

[0048] 2) Mix all components of the emulsifier evenly at 60-80℃ to obtain solution II;

[0049] 3) Mix solution I and solution II thoroughly to obtain solution III, which is the oil phase, and set aside for later use;

[0050] 4) Mix the polyol and the remaining whitening active ingredients at 60-80℃ to obtain solution IV, which is the aqueous phase, for later use;

[0051] 5) At 60–80°C, add the oil phase to the aqueous phase, stir and mix thoroughly, then homogenize to ensure the oil phase is completely coated by the aqueous phase, thus obtaining the whitening composition. The stirring speed for uniform mixing is controlled at 50–300 r / min, and the stirring time is 5–20 min; the homogenization speed is controlled at 2000–6000 r / min, and the homogenization time is 2–10 min.

[0052] Example 2

[0053] This embodiment describes the preparation of a whitening product according to the preparation method described in Example 1, as follows:

[0054] First, weigh out the following substances separately:

[0055] Whitening active ingredients: Adenosine 0.2g, Undecylenoyl Phenylalanine 1g, Dipotassium Glycyrrhizate 0.2g, Niacinamide 3g, Vitamin C derivative 1g;

[0056] Oils and fats: 5g octyl dodecanol, 1g polydimethylsiloxane, 2g caprylic / capric triglyceride, 0.5g tocopheryl acetate, 0.5g bisabolol;

[0057] Polyols: glycerol 2g, glycerol polyether-26 5g, 1,3-butanediol 2g, 1,3-propanediol 2g, 1,2-hexanediol 0.8g, 1,2-pentanediol 0.5g;

[0058] Emulsifiers: 1g of low oleic acid content Tween-20, 0.5g of polyglycerol-10 stearate, 0.5g of glyceryl caprylate, 2g of cetearyl glucoside & cetearyl alcohol, and 2g of arachidonic acid & behenol & arachidonic acid glucoside.

[0059] The preparation process is as follows: First, at 45°C, undecenoyl phenylalanine, a whitening active ingredient, is dispersed in octyldodecanol while stirring and heating until the undecenoyl phenylalanine is completely dissolved and the solution is colorless and transparent. Then, other oil components are added and mixed evenly to obtain solution I. Simultaneously, the emulsifier components are mixed evenly at 70°C to obtain solution II. Then, solution I and solution II are mixed evenly to obtain solution III, which is the oil phase, for later use. Simultaneously, the polyol components and the remaining whitening active ingredients are mixed evenly at 70°C to obtain solution IV, which is the aqueous phase, for later use. Finally, at 70°C, the oil phase is added to the aqueous phase, and the stirring speed is controlled at 200 r / min for 15 min. Then, homogenization is performed at a speed controlled at 5000 r / min for 10 min to ensure that the oil phase is completely coated by the aqueous phase, thus obtaining the whitening composition.

[0060] Example 3

[0061] This example investigates the effect of adenosine on the cytotoxic effects of different concentrations of undecenoyl phenylalanine. In this example, the formulations of each product are identical to those in Example 2, except for the ratio of adenosine to undecenoyl phenylalanine, which differs from Example 1 (combination 7).

[0062] Table 1. Effects of adenosine on the cytotoxic effects of different concentrations of undecenoylphenylalanine

[0063]

[0064] Evaluation method: The MTT assay was used to detect the toxic effects of each formulation on human skin fibroblasts (HSF cells). Specific experiments are as follows:

[0065] Remove the cryopreservation tubes and quickly place them in a 37°C water bath, shaking occasionally to rapidly thaw them for approximately 60–90 seconds. After sterilization, transfer the cell suspension to a centrifuge tube using a pipette, add 10 mL of culture medium, centrifuge at 1000 rpm for 5 minutes, aspirate the supernatant, resuspend the cells in culture medium, and then transfer them to a cell culture flask and incubate in an incubator. Observe cell growth daily and change the medium and passage as needed. Digest and collect cells in the logarithmic growth phase, centrifuge, resuspend the cells in culture medium, and adjust the cell density to 6 × 10⁶ cells / mL. 4 Cells / mL were added to 96-well plates at a rate of 100 μL / well. The edge wells were filled with sterile PBS, and the plates were then incubated. After 24 h of incubation, 100 μL of culture medium containing each combination was added to the corresponding well position in the 96-well plate, and the plates were incubated again. 24 h or 48 h after drug addition, 100 μL of culture medium containing MTT (5 mg / mL, i.e., 0.5% MTT) was added to each well, and the plates were incubated for another 4 h. Afterward, the culture medium was aspirated, and 150 μL of dimethyl sulfoxide was added to each well. The plates were then shaken at low speed for 10 min on a shaker to fully dissolve the crystals. The absorbance of each well was measured at 450 nm using an ELISA reader.

[0066] The results showed that undecenoyl phenylalanine exhibited significant cytotoxic effects on HSF cells at a concentration of 0.5%, while 0.2% adenosine inhibited its cytotoxic effects. Even at a concentration of 2%, no significant cytotoxicity was observed. Specific results are as follows: Figure 1 As shown.

[0067] Example 4

[0068] This embodiment investigates the effect of different proportions of whitening components on the whitening efficacy of an adenosine-containing whitening composition. In this embodiment, the whitening products used for investigation differed only in the content of each component of the whitening active ingredient; the contents of the other components and the preparation methods were the same as in Example 2. The proportions of the whitening active ingredients in each product, expressed as mass fractions, are shown in Table 2.

[0069] Table 2. Different proportions of whitening active ingredients

[0070]

[0071] Methods of evaluation: The effects of each formulation on melanin production and tyrosinase activity in B16-F10 cells were determined using the L-DOPA method and the NaOH lysis method. Specific experiments are as follows:

[0072] Remove the cryopreservation tubes and quickly place them in a 37°C water bath, shaking occasionally to rapidly thaw them, approximately 60-90 seconds. After sterilization, transfer the cell suspension to a centrifuge tube using a pipette, add 10 mL of culture medium, centrifuge at 1000 rpm for 5 minutes, aspirate the supernatant, resuspend the cells in culture medium, and then transfer them to a cell culture flask and incubate in an incubator. Observe cell growth daily, changing the medium and passaged as needed.

[0073] Detection of melanin production by NaOH pyrolysis method:

[0074] B16-F10 cells were seeded at a 25 cm² incubator. 2 In the culture flask, 1×10⁶ cells per well 6 Cells were cultured in 1640 medium. After 24 h of inoculation, 5 mL of each cell was replaced with high-glucose DMEM medium containing different whitening ingredients as shown in Table 2. A 1640 medium group was also included as a negative control. After 48 h of treatment, the cells were digested and counted. The cells were uniformly diluted to a density of 8.5 × 10⁶ cells / mL with 1 mol / L sodium hydroxide solution (containing 0.1% Triton-100). The cells were then incubated at 80 °C for 1 h, centrifuged at 10,000 rpm for 10 min, and 100 μL was transferred to a microplate. Triple replicates were performed, and the absorbance of each group was measured at 405 nm.

[0075] L-DOPA method for determining tyrosinase activity:

[0076] B16-F10 cells were seeded at 75 cm⁻¹ 2 In cell culture flasks, the culture medium was 1640. After culturing for 24 h, when the cell confluence was close to 80% or higher, the cells were digested, counted, and resuspended in PBS buffer at a density of 8 × 10⁶ cells / mL. 6Cells / ml: Take 400 μl of cell suspension and quickly place it in liquid nitrogen for 2 min, then thaw rapidly in a 37℃ water bath. Repeat this process three times. Centrifuge at 12000 rpm for 15 min and collect the supernatant to obtain crude tyrosinase extract. Use 0.1% L-DOPA solution as substrate for assay. Preheat both enzyme and substrate to 37℃. Add 50 μl of substrate L-DOPA, then 50 μl of enzyme extract and 50 μl of each formulation sample to the microplate. Perform triple replicates. Mix all reaction components thoroughly and quickly place the plate in a microplate reader. Read the absorbance at 450 nm, taking readings every 30 s to measure the change in OD value over 30 min.

[0077] The aqueous phase content of the adenosine-containing whitening composition prepared above was investigated, and the results are shown in Table 3.

[0078] Table 3. Effects of different ratios of whitening active ingredients on the inhibition of melanocyte and tyrosinase activity in adenosine-containing whitening compositions.

[0079]

[0080] As shown in Table 3, the combination obtained by using a mixture of adenosine, dipotassium glycyrrhizate, undecenoyl phenylalanine, nicotinamide, and VC derivative as whitening active ingredients has a good inhibitory effect on melanin content and tyrosinase activity. The inhibitory effect shows an asymmetric parabolic trend with the addition ratio. Among them, formulation 4 (adenosine 0.2%, dipotassium glycyrrhizate 0.2%, undecenoyl phenylalanine 1%, nicotinamide 3%, VC derivative 1%) has the highest inhibition rate, with a melanin content inhibition rate of 52.64% and a tyrosinase activity inhibition rate of 36.78%, showing the most significant effect.

[0081] Example 5

[0082] This embodiment is based on the formulation of Example 4, examining the effects of different whitening active ingredients on melanin content and tyrosinase activity. In this embodiment, except for the different proportions of the whitening active ingredients compared to Example 3, the other components and preparation methods are the same as in Example 2. The proportions of the whitening active ingredients in this embodiment are shown in Table 4.

[0083] Table 4. Proportions of Different Whitening Active Ingredients

[0084]

[0085] The inhibition of melanin content and tyrosinase activity of the adenosine-containing whitening compositions prepared by formulas 7-15 were investigated. The experimental procedures were the same as in Example 2, and the results are shown in Table 5.

[0086] Table 5. Inhibition rates of different whitening active ingredients on melanin content and tyrosinase activity

[0087]

[0088] As shown in Table 5, adenosine has a strong inhibitory effect on melanin content; dipotassium glycyrrhizate, undecenoyl phenylalanine, nicotinamide, and VC derivatives have varying degrees of inhibitory effects on tyrosinase activity; the combination of adenosine and undecenoyl phenylalanine significantly enhances the inhibition rate of melanin content and tyrosinase activity compared to using them alone; the high inhibition rate obtained by the combination of whitening active ingredients from different pathways in this invention is evident.

[0089] Example 6

[0090] This example investigates the solubility of undecenoyl phenylalanine in different oils and the permeation-enhancing effect of oils on undecenoyl phenylalanine. The formulation ratios in this example are consistent with formulation 4 in Example 4, and the preparation method is consistent with Example 2. The different oils are shown in Table 6 below.

[0091] Table 6. Solubility of undecenoyl phenylalanine in different oils

[0092]

[0093] Table 6 shows that octyl dodecanol, dibutyl adipate, and ethylhexyl methoxycinnamate can all dissolve undecenoyl phenylalanine. The undecenoyl phenylalanine was dissolved using these three oils, and its transdermal properties were tested. The experimental steps are as follows:

[0094] 1) Place piglet skin (0.8-1.0 mm thick) in 0.05 M pH 7.4 PBS buffer at room temperature for 30 min to equilibrate.

[0095] 2) Preheat the transdermal diffusion device and set the temperature to 32℃.

[0096] 3) Fill the receiving cell with PBS buffer and place the rotor inside. Place the balanced skin on the support of the Franz diffusion cell with the stratum corneum facing upward and clamp it tightly. Apply 0.05g of sample evenly to the skin. Set up parallel experimental groups at the same time.

[0097] 4) Set the stirring speed to 400 rpm and the transdermal absorption time to 24 h.

[0098] 5) When the transdermal absorption time is over, extract the permeate sample from the receiving cell.

[0099] 6) Determination of undecenoyl phenylalanine content. High-performance liquid chromatography (HPLC) was used. A DAISOPAKSP-100-5-ODS-P column (4.6 mm ID × 250 mm) was selected. Mobile phase A consisted of 11.45 g sodium acetate, 0.5 ml triethylamine, and 0.2 ml 0.1% EDTA aqueous solution, diluted to 1000 ml. After adjusting the pH to 6.4 with acetic acid, 940 ml of the diluted solution was added to 60 ml acetonitrile. Mobile phase B consisted of 600 ml acetonitrile, 400 ml water, and 0.2 ml 0.1% EDTA. Sample derivatization was performed. Take 40 μL of sample solution, add 300 μL of methanol, mix well, add 40 μL of derivatizing agent (PITC:methanol:triethylamine = 1:8:1), mix well, and let stand at room temperature for 30 min. Then add 600 μL of diluent (0.05 M pH 7.4 phosphate buffer), mix well, and extract with 1000 μL of n-hexane. Filter the lower layer through a 0.22 μm filter membrane. Elution method: mobile phase A from 100% to 0%, mobile phase B from 0% to 100% for 0–15 min. Flow rate set to 1.0 mL / min, UV wavelength set to 254 nm, injection volume 10 μL.

[0100] 7) Calculate the transdermal transdermal dose of undecenoyl phenylalanine per unit time based on the contact surface area between the skin and the sample solution, expressed as transdermal efficiency in μg / cm2*h.

[0101] The results of the transdermal test are shown in Table 7.

[0102] Table 7. Results of three oils promoting transdermal absorption of undecenoyl phenylalanine

[0103]

[0104] Because undecenoyl phenylalanine has a molecular weight of 331.45 and possesses a certain degree of transdermal permeability, and because it is oil-soluble, three oils listed in Table 7 were used to dissolve the undecenoyl phenylalanine before conducting transdermal tests. Table 7 shows that octyldodecyl alcohol has a good transdermal permeability-enhancing effect on undecenoyl phenylalanine.

[0105] Example 7

[0106] This example investigates the effect of preparation methods on the stability of adenosine-containing skin-whitening compositions, examining their high and low temperature stability, light stability, centrifugal stability, and pH effects. The conditions were as follows: First, one portion of each prepared adenosine-containing skin-whitening composition was stored at a constant temperature of 8℃ as a control sample. The remaining portions were divided into four parts and then treated as follows: 1) frozen at -15℃ for one month; 2) frozen at -15℃ and then thawed at 45℃, repeated for one month; 3) stored at 45℃ for three months; 4) centrifuged at 2000 rpm for 30 minutes; 5) exposed to natural light for one month. After each treatment, the presence of stratification, discoloration, and precipitation in each adenosine-containing skin-whitening composition was observed, and microbial activity was investigated.

[0107] The formulation in this embodiment is the same as formulation 4 in Example 3. The preparation method is identical to that in Example 3, except for the dissolution method of undecenoyl phenylalanine. The experimental design is as follows: undecenoyl phenylalanine is directly dispersed in oils (method as described in Example 3); undecenoyl phenylalanine is directly dispersed in an emulsifier; undecenoyl phenylalanine is directly dispersed in an aqueous phase.

[0108] Table 8. Dispersed undecenoyl phenylalanine

[0109]

[0110] After preparation, the stability and microbiological properties of the three adenosine-containing whitening compositions A to C were investigated, and the results are shown in Tables 9 and 10.

[0111] Table 9. Effects of different preparation methods on microorganisms in adenosine-containing skin whitening compositions

[0112]

[0113] Table 10. Effect of different preparation methods on the stability of adenosine-containing skin whitening compositions

[0114]

[0115] As shown in Tables 9 and 10, different preparation methods did not affect the microorganisms of the present invention, but had varying degrees of impact on stability. Specifically, composition A, in which undecenoyl phenylalanine was dispersed in oils, showed no abnormalities under the observed conditions; composition B, in which undecenoyl phenylalanine was dispersed in an emulsifier, showed precipitation after one month at -15°C, and discoloration after one month of cycling at -15°C to 45°C, three months at 45°C, and one month under natural light; composition C, in which undecenoyl phenylalanine was dispersed in an aqueous phase, showed precipitation after one month at -15°C, discoloration and precipitation after one month of cycling at -15°C to 45°C, and discoloration after three months at 45°C and one month under natural light. This demonstrates that undecenoyl phenylalanine dispersed in oils exhibits good stability.

[0116] Example 8

[0117] Experiment 1 in this example investigated the irritant effect of undecenoyl phenylalanine and the inhibitory effect of adenosine on undecenoyl phenylalanine; Experiment 2 compared the irritant effects of ordinary Tween-20 (oleic acid content <11%) and Tween-20 with low oleic acid content (oleic acid content <5%). This example used the chicken embryo chorioallantoic membrane assay for testing.

[0118] The formulation and preparation process of this embodiment refer to formulation 4 in embodiment 3. The design of experiment 1 is shown in Table 11, and the design of experiment 2 is shown in Table 12.

[0119] Table 11. Samples from the chicken embryo chorioallantoic membrane test

[0120]

[0121] Table 12. Chicken embryo chorioallantoic membrane test samples

[0122]

[0123] The procedure for the chicken embryo chorioallantoic membrane test is as follows:

[0124] 1) Take fertilized chicken embryos that have been incubated for 9 days, check the eggs by candling, and mark the location of the air cell on the surface of the eggshell.

[0125] 2) Use dental serrated curved tweezers to peel off the marked part of the eggshell to expose the white egg membrane. Handle with care to avoid damaging the integrity of the egg membrane.

[0126] 3) Use a pipette to add a small amount of 0.9% sodium chloride solution to moisten the egg membrane, then pour off the excess solution.

[0127] 4) Carefully remove the inner lining with tweezers, ensuring the vascular membrane is not damaged. At the same time, observe the integrity of the chicken embryo's blood vessels.

[0128] 5) Take a photo to record the state of the chicken embryo's blood vessels before adding the sample, and add 0.3 ml of sample to evenly cover at least 50% of the CAM surface.

[0129] 6) Observe the CAM reaction immediately after the test substance is applied for 5 minutes. Six chicken embryos are used for each group of samples. Record the observation results and calculate the average score.

[0130] 7) Based on vascular bleeding, vascular dissolution, and clotting time, score according to the following formula:

[0131] Stimulus score (IS) = (301-sec H) × 5 / 300 + (301-sec L) × 7 / 300 + (301-sec C) × 9 / 300

[0132] In the formula, sec H is the average time to observe bleeding, sec L is the average time to observe vascular dissolution, and sec C is the average time to observe coagulation. The time unit is seconds (s).

[0133] 8) The results of the stimuli rating are shown in Table 13 and Figure 2 As shown.

[0134] Table 13. Stimulus Rating Results

[0135]

[0136] As shown in Table 13 and Figure 2 The experimental results shown indicate that Experiment 1 demonstrates the inhibitory effect of adenosine on the irritant effect of undecenoyl phenylalanine; Experiment 2 demonstrates that Tween-20 with low oleic acid content is non-irritating, while ordinary Tween-20 is irritating. Based on this, the Tween-20 with low oleic acid content in this invention is an essential oil phase substance.

[0137] Example 9

[0138] This example verifies the safety of the adenosine-containing whitening composition of the present invention. In this example, the adenosine-containing whitening composition prepared according to formulation 4 of Example 4 is used as the test substance for patch testing, and its preparation method is consistent with that in Example 2. Details are as follows:

[0139] Select an area not exceeding 50mm 2A qualified spot treatment device with a depth of approximately 1 mm was used. The test substance (the present invention includes an adenosine-containing whitening composition) was added to the spot treatment device at a dosage of 0.020 g, with a blank sample added as a negative control. The spot treatment devices containing the test substance and the negative control were applied to the flexor side of the subject's forearm and removed after 24 hours. The subject's skin reaction was observed according to the standards in Table 14 at 30 minutes (after the indentation disappeared), 24 hours, and 48 hours after spot treatment device removal, and the results were recorded as shown in Tables 15 and 16.

[0140] Table 14. Grading Criteria for Skin Reactions in Occlusive Patch Tests

[0141]

[0142] Table 15. Raw data from subjects in the closed patch test

[0143]

[0144] Table 16. Results of the closed patch test

[0145]

[0146] As can be seen from Tables 13 to 15, the adenosine-containing whitening composition of the present invention showed a grade 0 reaction in 33 people and a grade 1-4 reaction in 0 people in the patch test. Therefore, the adenosine-containing whitening composition of the present invention is non-irritating and suitable for use by sensitive people.

[0147] In summary, this invention presents an adenosine-containing whitening composition. Through a scientific combination of different whitening active ingredients and a special preparation method, the product exhibits a stable emulsion phase, excellent temperature resistance, and does not show stratification, discoloration, or precipitation even under low-temperature (-15℃) freezing or high-temperature (45℃) storage. It ensures the patch is non-irritating and hypoallergenic, guaranteeing product safety. Suitable for people with sensitive skin, it possesses good economic value and market prospects.

[0148] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and not restrictive in all respects. Furthermore, it should be understood that although this specification describes embodiments, it does not encompass only one technical solution. This descriptive method is merely for clarity, and those skilled in the art should consider the specification as a whole. The technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. The application of adenosine and undecenoyl phenylalanine in the preparation of a skin-whitening composition, characterized in that: The whitening active ingredients of the whitening composition include adenosine and undecenoyl phenylalanine; and, by mass fraction: The whitening active ingredient accounts for 2.35% to 10.2% of the total mass of the whitening composition; The content ratio of adenosine to undecenoyl phenylalanine is 0.05-1:0.2-2.

2. The application according to claim 1, characterized in that: The whitening active ingredients in the whitening composition also include niacinamide, vitamin C derivatives, and dipotassium glycyrrhizate; Furthermore, by mass fraction, the whitening composition also contains Oil content 3-22%, Polyols 2-10%, Emulsifier 1-4%, The remainder is purified water.

3. The application according to claim 2, characterized in that: The oil is one or more of the following: octyldodecyl alcohol, polydimethylsiloxane, caprylic / capric triglyceride, tocopheryl acetate, and bisabolol. The polyol is one or more of glycerol, glycerol polyether-26, 1,3-butanediol, 1,3-propanediol, 1,2-hexanediol, and 1,2-pentanediol; The emulsifier is one or more of the following: Tween-20, polyglycerol-10 stearate, glyceryl caprylate, cetearyl glucoside & cetearyl alcohol, arachidonic acid & behenol & arachidonic acid glucoside, which have low oleic acid content.

4. The application according to claim 3, characterized in that: The emulsifier includes octyldodecyl alcohol, and the content ratio of undecenoyl phenylalanine to octyldodecyl alcohol is 0.2-2:1-10.

5. The application according to claim 4, characterized in that: The oil includes Tween-20 with low oleic acid content, and the ratio of undecenoyl phenylalanine to emulsifier is 1:1 to 2.

6. The application according to claim 5, characterized in that: The whitening composition comprises the following ingredients by mass fraction: Whitening active ingredients: Adenosine 0.05-1%, Undecenoyl Phenylalanine 0.2-2%, Niacinamide 2-5%, Vitamin C derivative 0.05-2%, Dipotassium glycyrrhizate 0.05-0.2%; Oils and fats: 1-10% octyl dodecanol, 0.5-3% polydimethylsiloxane, 0.5-3% caprylic / capric triglyceride, 0.5-3% tocopheryl acetate, 0.5-3% bisabolol; Polyols: glycerol 2-5%, glycerol polyether 2-5%, 1,3-butanediol 2-5%, 1,3-propylene glycol 2-5%, 1,2-hexanediol 0.2-3%, 1,2-pentanediol 0.5-2%; Emulsifiers: Tween-20 with low oleic acid content 0.5-2%, polyglycerol-10 stearate 0.5-2%, glyceryl caprylate 0.5-2%, cetearyl glucoside & cetearyl alcohol 0.5-3%, arachidonic acid & behenol & arachidonic acid glucoside 0.5-3%.

7. The application according to claim 6, characterized in that: The whitening composition comprises, by weight fraction, the following substances: Whitening active ingredients: Adenosine 0.2%, Dipotassium glycyrrhizate 0.2%, Undecenoyl phenylalanine 1%, Niacinamide 3%, Vitamin C derivative 1%; Oils and fats: 5% octyl dodecanol, 1% polydimethylsiloxane, 2% caprylic / capric triglyceride, 0.5% tocopheryl acetate, 0.5% bisabolol; Polyols: glycerol 2%, glycerol polyether-26 5%, 1,3-butanediol 2%, 1,3-propanediol 2%, 1,2-hexanediol 0.8%, 1,2-pentanediol 0.5%; Emulsifiers: Tween-20 with low oleic acid content 1%, polyglycerol-10 stearate 0.5%, glyceryl caprylate 0.5%, cetearyl glucoside & cetearyl alcohol 2%, arachidonic acid & behenol & arachidonic acid glucoside 2%.

8. The application according to claim 7, characterized in that: The preparation method of the whitening composition includes the following steps: 1) Disperse undecenoyl phenylalanine, a whitening active ingredient, in oil to obtain solution I; 2) Mix all components of the emulsifier evenly at 60-80℃ to obtain solution II; 3) Mix solution I and solution II thoroughly to obtain solution III, which serves as the oil phase, for later use; 4) Mix the polyol and the remaining whitening active ingredients evenly at 60-80℃ to obtain solution IV as the aqueous phase, for later use; 5) At 60-80℃, add oil phase solution III to aqueous phase solution IV, stir and mix evenly, and then homogenize to ensure that the oil phase is completely coated by the aqueous phase, thereby obtaining the adenosine-undecenoyl phenylalanine synergistic whitening composition.

9. The application according to claim 8, characterized in that: In step 1), the preparation process of solution I is as follows: first, at 40-60°C, undecenoyl phenylalanine is dispersed in octyldodecyl alcohol, and heated while stirring until undecenoyl phenylalanine is completely dissolved and the solution is colorless and transparent, then other oil components are added and mixed evenly.

10. The application according to claim 8, characterized in that: In step 5), the stirring speed for uniform mixing is controlled at 50-300 r / min, and the stirring time is 5-20 min; the homogenization speed is controlled at 2000-6000 r / min, and the homogenization time is 2-10 min.

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