Targeted mitochondrial composition for delaying cell senescence as well as preparation method and application thereof

Abstract

The invention discloses a targeted mitochondrial composition as well as a preparation method and application thereof. The preparation method comprises the following steps: S1, preparing a phospholipid-fat-soluble component composite membrane; s2, a water phase solution preparation step; step S3, a phospholipid nanoparticle colostrum preparation step; step S4, a chitosan solution preparation step; and step S5, a step of self-assembly of the phospholipid-chitosan composite nanoparticles. According to the mitochondrial targeting composition as well as the preparation method and the application thereof, systematic anti-aging intervention is realized from a cellular level to a human body level by virtue of a synergistic effect of beta-nicotinamide mononucleotide (beta-NMN), bakuchiol and saccharomycetes / rice fermentation product filtrate and in combination with a phospholipid-chitosan composite nano targeting carrier technology.

Description

Technical Field

[0001] This invention belongs to the field of mitochondrial component technology, and relates to a mitochondrial composition, particularly to a targeted mitochondrial composition for delaying cell aging, its preparation method, and its application. Background Technology

[0002] Mitochondria, as the "energy factory" and "metabolic regulatory center" of eukaryotic cells, directly affect cellular vitality and lifespan due to their functional integrity. As organelles with their own independent genomes, mitochondria generate approximately 90% of cellular ATP through oxidative phosphorylation, providing the energy basis for cell proliferation, substance synthesis (such as collagen secretion), and damage repair. Simultaneously, they are both a major source of reactive oxygen species (ROS) and maintain the oxidation-antioxidant balance through antioxidant enzymes such as superoxide dismutase 2 (SOD2). Furthermore, mitochondria achieve the clearance of damaged mitochondria (mitochondrial autophagy) and functional optimization through a dynamic balance between fusion (mediated by MFN1 / 2 and OPA1 proteins) and division (mediated by Drp1 protein). The synergistic effect of these functions is a prerequisite for cells to maintain a "youthful state." Abnormal mitochondrial function will trigger metabolic disorders and activate the aging process.

[0003] A key factor in cellular senescence is the progressive decline in mitochondrial function. When mitochondrial oxidative phosphorylation efficiency decreases, ATP production declines, resulting in insufficient energy for cellular synthesis and repair, forcing the cell into a "low-function senescent state." Simultaneously, excessive accumulation of mitochondrial ROS attacks unprotected mitochondrial DNA (mtDNA), leading to mutations and further reducing oxidative phosphorylation efficiency, creating a vicious cycle of "ROS accumulation → mtDNA damage → more ROS production." In senescent cells, overactivation of the Drp1 protein leads to mitochondrial fragmentation, preventing damage from autophagy. Downregulation of MFN1 / 2 protein expression inhibits mitochondrial fusion, disrupts cristae (the main site of oxidative phosphorylation), and exacerbates ATP production impairment. Furthermore, mitochondria exert pressure through "reverse signals" (such as Ca2+). 2 The ability of mitochondrial biogenesis to regulate nuclear gene expression (including mitochondrial metabolites) also weakens with age, leading to a decline in both the quantity and quality of mitochondria. This cellular senescence is not limited to individual cells but also spreads to neighboring tissues through SASP (IL-6, MMP-1, etc.)-mediated "senescence signal diffusion," accelerating the overall aging process.

[0004] As the largest organ in the human body, skin aging is a macroscopic manifestation of "internal mitochondrial functional decline." The proliferation and differentiation of epidermal keratinocytes (KCs) are highly dependent on mitochondrial ATP. When mitochondria are abnormal, KC proliferation decreases, and the expression of barrier-related proteins such as filaggrin is reduced, leading to thinning of the stratum corneum, increased transepidermal water loss (TEWL), and dry, sensitive skin. The synthesis of collagen and elastic fibers by dermal fibroblasts (FBs) requires a large amount of ATP. When mitochondrial function is abnormal, ATP levels in FBs decrease, reducing collagen synthesis. Simultaneously, ROS accumulation activates matrix metalloproteinases (MMPs), accelerating collagen degradation, ultimately leading to dermal thinning, elastic fiber breakage, skin laxity, and wrinkles. Furthermore, cell proliferation and migration required for skin damage repair (such as wound healing) also depend on mitochondrial energy. Mitochondrial functional decline prolongs the repair cycle, making damage more prone to transforming into chronic inflammation, further accelerating aging.

[0005] Nicotinamide adenine dinucleotide (NAD+) is a core regulator of mitochondrial function. It not only directly affects ATP production as a coenzyme for oxidative phosphorylation, but is also a substrate of SIRT1 (silent information regulator 1)—SIRT1 promotes mitochondrial regeneration by activating PGC-1α through deacetylation. Maintaining a high NAD+ / NADH ratio can inhibit electron leakage from electron transport chain complex I and reduce ROS production. However, NAD+ levels decline significantly with age: after age 30, the activity of its synthases decreases while the activity of its depleting enzymes increases, leading to a decrease of approximately 10%-15% in NAD+ every 10 years. This depletion exacerbates mitochondrial functional decline, becoming a major contributor to aging.

[0006] Furthermore, skin aging requires multi-dimensional intervention from "enhanced energy metabolism, inhibition of oxidative stress, morphological repair, and barrier protection." Most anti-aging technologies focus on superficial interventions such as epidermal barrier repair, anti-oxidation, or collagen replenishment, neglecting the fundamental role of mitochondria as "energy factories" and "metabolic regulatory centers" in cellular aging, and failing to include them as core targets. Even when some technologies involve mitochondrial-related components, the lack of targeted carriers makes it difficult for these components to penetrate the cell membrane and accumulate in the inner mitochondrial membrane, resulting in extremely low effective concentrations and limited functional efficiency.

[0007] Most existing anti-aging technologies only focus on superficial interventions such as epidermal barrier repair, anti-oxidation, or collagen replenishment, failing to consider mitochondria as the core driving target of cellular aging. As the "energy factory" and "metabolic regulation center," the functional decline of mitochondria (such as reduced ATP production, ROS accumulation, and morphological abnormalities) is the root cause of cellular aging. However, current technologies do not design systematic intervention programs targeting key aspects such as mitochondrial energy metabolism, oxidative stress, and homeostasis, thus failing to halt the aging process at its source.

[0008] Even if some technologies involve mitochondrial-related active ingredients, due to the lack of targeted carrier technology, the active ingredients are easily metabolized or retained in other parts of the cell before reaching the mitochondria. The actual effective concentration acting on the mitochondria is extremely low, resulting in limited functional efficiency and failing to fully realize the anti-aging potential.

[0009] In view of this, there is an urgent need to design a new active ingredient in order to overcome at least some of the aforementioned defects of existing active ingredients. Summary of the Invention

[0010] This invention provides a targeted mitochondrial composition for delaying cell aging, its preparation method, and its application, enabling systematic anti-aging intervention from the cellular level to the human body level.

[0011] To solve the above-mentioned technical problems, according to one aspect of the present invention, the following technical solution is adopted:

[0012] A method for preparing a mitochondrial-targeting composition, the method comprising:

[0013] Step S1: Preparation of phospholipid-lipid-soluble component composite membrane;

[0014] Add psoralen to the first organic solvent and stir until completely dissolved to form a first solution; add phospholipids and sterols to the first solution to form a homogeneous second solution; evaporate the second solution until the first organic solvent is completely evaporated, forming a uniform lipid film on the inner wall of the first container, namely a phospholipid-lipid-soluble component composite film.

[0015] Step S2, preparation of aqueous solution;

[0016] β-nicotinamide mononucleotide (NMN) and yeast / rice fermentation product filtrate were dissolved in deionized water to obtain an aqueous solution containing the active ingredients.

[0017] Step S3: Preparation of phospholipid nanoparticle proemulsion;

[0018] Add the aqueous solution obtained in step S2 to the first container after step S1 to form a multilayer phospholipid coarse dispersion; subject the multilayer phospholipid coarse dispersion to ultrasonic treatment to obtain a clear and translucent phospholipid nanoparticle proemulsion, thereby achieving the initial loading of lipid-soluble components embedded in the lipid film and water-soluble components encapsulated in the aqueous core.

[0019] Step S4: Chitosan solution preparation steps;

[0020] Chitosan was added to an acetic acid solution and magnetically stirred until completely dissolved to obtain a chitosan solution; sodium tripolyphosphate (TPP) was dissolved in deionized water and stirred until clear to obtain a TPP solution.

[0021] Step S5: Self-assembly of phospholipid-chitosan composite nanoparticles;

[0022] The phospholipid nanoparticle proemulsion from step S3 is dropped into a chitosan solution. Magnetic stirring causes the positively charged chitosan to electrostatically adsorb onto the negatively charged phospholipid membrane surface, forming a phospholipid nanoparticle-chitosan complex. TPP solution is then dropped into the above mixture and stirred to form a stable chitosan network structure, yielding a mitochondrial-targeting composition.

[0023] In one embodiment of the present invention, in step S1, the first organic solvent includes ethanol, methanol, or a mixture thereof; the phospholipid includes at least one of soybean lecithin, hydrogenated soybean lecithin, and phosphatidylcholine; the sterol includes at least one of phytosterol, soybean sterol, and ergosterol; psoralen is added to the organic solvent and stirred in a water bath until completely dissolved to form a first solution; the phospholipid and sterol are added to the first solution and ultrasonically dispersed to form a homogeneous solution; the solution is transferred to a rotary evaporator and rotary evaporated until the organic solvent is completely evaporated, forming a uniform lipid film on the wall of the lipid film bottle;

[0024] In step S2, β-nicotinamide mononucleotide (NMN) and yeast / rice fermentation product filtrate are dissolved in deionized water, ultrasonically dissolved, and the pH is adjusted to a suitable range to obtain an aqueous solution containing active ingredients; the mass ratio of β-nicotinamide mononucleotide, psoralen, and yeast / rice fermentation product filtrate is (0.01-1.5):(0.01-1.5):(0.1-5);

[0025] In step S3, the aqueous solution from step S2 is added to the lipid film bottle after step S1, and the solution is hydrated by water bath oscillation to form a multilayer coarse dispersion of phospholipids. The coarse dispersion is then transferred to a probe sonicator and sonicated under ice bath conditions to obtain a clear and translucent phospholipid nanoparticle proemulsion, thus achieving the initial loading of lipid-soluble components embedded in the lipid film and water-soluble components encapsulated in the aqueous core.

[0026] In step S4, chitosan is added to an acetic acid solution of a set concentration and stirred magnetically until completely dissolved to obtain a chitosan solution; sodium tripolyphosphate (TPP) is dissolved in deionized water and stirred until clear to obtain a TPP solution.

[0027] In step S5, the phospholipid nanoparticle proemulsion from step S3 is dropped into the chitosan solution. By magnetic stirring, the positively charged chitosan and the negatively charged phospholipid membrane surface are electrostatically adsorbed to form a "phospholipid nanoparticle-chitosan" complex. The TPP solution is dropped into the above mixture and stirred to form a stable chitosan network structure, thus obtaining the mitochondrial targeted composition.

[0028] In one embodiment of the present invention, in step S1, the feeding ratio of psoralen to the first organic solvent is (0.5-1.0) parts by mass : (30-50) parts by volume; the feeding ratio of phospholipids to the first organic solvent is (3.0-5.0) parts by mass : (30-50) parts by volume; the feeding ratio of sterols to the first organic solvent is (0.5-1.0) parts by mass : (30-50) parts by volume; the ultrasonic dispersion conditions are: power 150-200W, time 5-15 minutes; the parameters of the rotary evaporator are: temperature 35-45℃, vacuum degree 0.03-0.07MPa, rotary evaporation time 20-40 minutes.

[0029] In one embodiment of the present invention, in step S2, the feeding ratio of β-nicotinamide mononucleotide (NMN) to deionized water is (0.5-1.0) parts by mass: (50-100) parts by volume, and the feeding ratio of yeast / rice fermentation product filtrate to deionized water is (2-5) parts by mass: (50-100) parts by volume; the aqueous phase ultrasonic dissolution conditions are: power 100-200W, time 5-15 minutes; the pH value is adjusted to 5.0-6.0 with 0.05-0.2M acetic acid solution.

[0030] In step S3, the water bath oscillation hydration conditions are: temperature 35-45℃, rotation speed 150-200rpm, and time 25-30 minutes; the ultrasonic treatment conditions are: ice bath temperature ≤10℃, power 250-350W, pulse mode of working for 2-4 seconds / intermittent for 2-4 seconds, and total time 8-20 minutes.

[0031] In one embodiment of the present invention, in step S4, the chitosan solution is prepared under the following conditions: the amount of chitosan is 0.5-1 parts by mass, the amount of acetic acid solution is 80-120 parts by volume, the concentration is 0.05-0.2M, the pH value is 5.0-5.5, and it is dissolved for 1-1.5 hours under magnetic stirring at a speed of 450-500 rpm.

[0032] The TPP solution preparation conditions are as follows: the amount of TPP is 0.1-0.2 parts by mass, the amount of deionized water is 40-60 parts by volume, the pH is adjusted to 5.0-5.5, and the solution is stirred for 10-15 minutes.

[0033] In step S5, the phospholipid nanoparticle proemulsion is added to the chitosan solution at a rate of 0.5-1 volume parts / minute, the magnetic stirring speed is 300-350 rpm, and the stirring time continues for 30-45 minutes after addition.

[0034] TPP crosslinking and curing conditions: The TPP solution is added to the phospholipid-chitosan complex at a rate of 0.3-0.5 parts per minute, the stirring speed is increased to 450-500 rpm, and stirring is continued for 1-1.5 hours after addition.

[0035] According to another aspect of the present invention, the following technical solution is adopted: a mitochondrial-targeting composition prepared by the above preparation method, wherein the mitochondrial-targeting composition comprises β-nicotinamide mononucleotide (NMN), bakuchiol, and yeast / rice fermentation product filtrate, wherein the β-nicotinamide mononucleotide, bakuchiol, and yeast / rice fermentation product filtrate form NMN-bakuchiol-yeast / rice fermentation product filtrate co-loaded phospholipid-chitosan composite nanoparticles, i.e., the mitochondrial-targeting composition.

[0036] In one embodiment of the present invention, the mass ratio of β-nicotinamide mononucleotide, psoralen, and yeast / rice fermentation product filtrate is (0.01-1.5):(0.01-1.5):(0.1-5).

[0037] In one embodiment of the present invention, the mass ratio of β-nicotinamide mononucleotide, psoralen and yeast / rice fermentation product filtrate is (0.5-1):(0.5-1):(2-3).

[0038] According to another aspect of the present invention, the following technical solution is adopted: the application of the above-mentioned mitochondrial-targeting composition in the preparation of skin care products.

[0039] In one embodiment of the present invention, the skin care product is an essence, cream, lotion, freeze-dried powder, toner, or mask.

[0040] The beneficial effects of this invention are as follows: The mitochondrial-targeting composition, its preparation method, and its application proposed in this invention achieve systematic anti-aging intervention from the cellular level to the human body level through the synergistic effect of β-nicotinamide mononucleotide (β-NMN), psoralen, and yeast / rice fermentation product filtrate, combined with phospholipid-chitosan composite nano-targeting carrier technology.

[0041] (1) Multi-target synergistic repair of mitochondrial core functions.

[0042] Mitochondria, as the cell's "energy factory" and "metabolic regulation center," experience functional decline, which is the root cause of cellular aging. This patent comprehensively improves mitochondrial function through the synergistic effect of three components:

[0043] β-NMN as NAD + Its direct precursor can enhance intracellular NAD+. +It enhances oxidative phosphorylation efficiency, promotes ATP production, activates the SIRT1 / PGC-1α signaling axis, promotes mitochondrial biosynthesis, and inhibits reactive oxygen species (ROS) production at the source, thus blocking the vicious cycle of "ROS accumulation → mitochondrial damage → more ROS production".

[0044] Bakuchiol enhances DNA repair and inhibits inflammation by upregulating the SIRT1 and AMPK pathways, creating a low-inflammatory environment for cells. On the other hand, it initiates autophagy to clear damaged mitochondria, optimizes energy metabolism efficiency, and maintains mitochondrial homeostasis.

[0045] Yeast / rice fermentation product filtrate not only directly repairs mitochondrial morphology and improves electron transport chain function, but also promotes keratinocyte proliferation and barrier protein synthesis, achieving a dual effect of "mitochondrial function repair + barrier protection".

[0046] The three work together to cover the entire chain of "energy supply-oxidation regulation-morphology optimization", fundamentally reversing mitochondrial functional decline and delaying the cellular aging process.

[0047] (2) Targeted delivery technology breaks through the bottleneck of component delivery.

[0048] To address the problem that traditional anti-aging ingredients struggle to penetrate cell membranes and accumulate in the inner mitochondrial membrane, the patented technology utilizes phospholipid-chitosan composite nanocarrier technology.

[0049] The phospholipid membrane encapsulates lipid-soluble components (psoralen), while the aqueous core encapsulates water-soluble components (β-NMN, yeast / rice fermentation product filtrate). Combined with chitosan surface modification, a stable nanoparticle structure is formed, which significantly enhances the components' ability to penetrate cell membranes and their targeted enrichment on the inner mitochondrial membrane.

[0050] This carrier technology effectively solves the shortcomings of traditional ingredients that are easily metabolized or retained in other parts of the cell, and greatly increases the effective concentration of active ingredients in mitochondria, giving full play to their anti-aging potential.

[0051] (3) Enhancement of both skin barrier strengthening and anti-aging effects.

[0052] Based on the repair of mitochondrial function, combined with the direct effect of yeast / rice fermentation product filtrate on the skin barrier, this patent achieves dual anti-aging through "cell-level repair + barrier strengthening":

[0053] Promotes epidermal renewal: Yeast / rice fermentation product filtrate can significantly promote the proliferation of keratinocytes, providing a basis for the dynamic renewal of the skin barrier and improving problems such as thinning of the stratum corneum and dryness caused by decreased cell proliferation capacity.

[0054] Strengthening the barrier structure: By upregulating the synthesis of key barrier proteins such as ceramides and filaggrin, it strengthens the "brick wall structure" of the stratum corneum, enhances the integrity of the skin barrier and its ability to resist external stimuli, reduces transepidermal water loss (TEWL), and relieves skin sensitivity. Attached Figure Description

[0055] Figure 1 This is a flowchart of a method for preparing a mitochondrial-targeting composition according to an embodiment of the present invention.

[0056] Figure 2 This is a schematic diagram of the FLG fluorescence intensity in the negative control group.

[0057] Figure 3 This is a schematic diagram of the fluorescence intensity of FLG in Experiment Example 1.

[0058] Figure 4 This is a schematic diagram of the fluorescence intensity of FLG in Experiment Example 2.

[0059] Figure 5 This is a schematic diagram of the fluorescence intensity of FLG in Experiment Example 3.

[0060] Figure 6 This is a schematic diagram of the wrinkles around the eyes before using the face cream in Example 1.

[0061] Figure 7 This is a schematic diagram showing wrinkles around the eyes after two weeks of using the face cream from Example 1.

[0062] Figure 8 This is a schematic diagram showing wrinkles around the eyes when using the face cream in Example 1. Detailed Implementation

[0063] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0064] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims of the present invention.

[0065] The description in this section pertains to only a few typical embodiments, and the present invention is not limited to the scope of the embodiments described. Substitution of identical or similar prior art methods with some technical features in the embodiments is also within the scope of the description and protection of this invention.

[0066] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, particularly the definitions disclosed in these documents concerning synthetic techniques, product and processing design, polymers, comonomers, initiators, or catalysts in the art. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0067] The numerical ranges in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. A numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided there is an interval of at least two units between any lower and any higher value. For example, if a component, physical, or other property (such as molecular weight, melt index, etc.) is described as 100 to 1000, this means that all individual values, such as 100, 101, 102, etc., are explicitly listed, as well as all subranges, such as 100 to 166, 155 to 170, 198 to 200, etc. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly stated in this application. It should also be noted that the terms "first," "second," etc., used herein are not intended to specify a particular order, but are merely used to distinguish substances with different structures.

[0068] When referring to chemical compounds, unless explicitly stated otherwise, the singular includes all isomers and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively). Additionally, unless explicitly stated otherwise, nouns described with "an," "a," or "the" also include their plural forms.

[0069] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0070] The steps described in the various embodiments in the specification are for illustrative purposes only, and the implementation of this application is not limited by the order of the steps.

[0071] This invention discloses a method for preparing a mitochondrial-targeting composition. Figure 1 This is a flowchart of a method for preparing a mitochondrial-targeting composition according to an embodiment of the present invention; please refer to [link / reference]. Figure 1 The preparation method includes:

[0072]

Step S1

[0073] Add psoralen to the first organic solvent and stir until completely dissolved to form a first solution; add phospholipids and sterols to the first solution to form a homogeneous second solution; evaporate the second solution until the first organic solvent is completely evaporated, forming a uniform lipid film on the inner wall of the first container, namely a phospholipid-lipid-soluble component composite film.

[0074] In one embodiment, the first organic solvent includes ethanol, methanol, or a mixture thereof; the phospholipid includes at least one of soybean lecithin, hydrogenated soybean lecithin, and phosphatidylcholine; the sterol includes at least one of phytosterol, soybean sterol, and ergosterol; psoralen is added to the organic solvent and stirred in a water bath until completely dissolved to form a first solution; the phospholipid and sterol are added to the first solution and ultrasonically dispersed to form a homogeneous solution; the solution is transferred to a rotary evaporator and rotary evaporated until the organic solvent is completely evaporated, forming a uniform lipid film on the wall of the lipid film bottle;

[0075] In one embodiment of the present invention, the feeding ratio of psoralen to the first organic solvent is (0.5-1.0) parts by mass : (30-50) parts by volume; the feeding ratio of phospholipids to the first organic solvent is (3.0-5.0) parts by mass : (30-50) parts by volume; the feeding ratio of sterols to the first organic solvent is (0.5-1.0) parts by mass : (30-50) parts by volume; the ultrasonic dispersion conditions are: power 150-200W, time 5-15 minutes; the parameters of the rotary evaporator are: temperature 35-45℃, vacuum degree 0.03-0.07MPa, rotary evaporation time 20-40 minutes.

[0076] In one embodiment, the mass-to-volume ratio of psoralen to ethanol is (0.5-1.0)g:(30-50)ml; the mass-to-volume ratio of soybean lecithin to ethanol is (3.0-5.0)g:(30-50)ml; the mass-to-volume ratio of phytosterol to ethanol is (0.5-1.0)g:(30-50)ml; the ultrasonic dispersion conditions are: power 150-200W, time 5-15 minutes; the parameters of the rotary evaporator are: temperature 35-45℃, vacuum degree 0.05MPa, rotary evaporation time 20-40 minutes.

[0077]

Step S2

[0078] β-nicotinamide mononucleotide (NMN) and yeast / rice fermentation product filtrate were dissolved in deionized water to obtain an aqueous solution containing the active ingredients.

[0079] In one embodiment, β-nicotinamide mononucleotide (NMN) and yeast / rice fermentation product filtrate were dissolved in deionized water, dissolved by ultrasonication, and the pH was adjusted to a suitable range to obtain an aqueous solution containing the active ingredients.

[0080] In one embodiment of the present invention, in step S2, the feeding ratio of β-nicotinamide mononucleotide (NMN) to deionized water is (0.5-1.0) parts by mass: (50-100) parts by volume, and the feeding ratio of yeast / rice fermentation product filtrate to deionized water is (2-5) parts by mass: (50-100) parts by volume; the aqueous phase ultrasonic dissolution conditions are: power 100-200W, time 5-15 minutes; the pH value is adjusted to 5.0-6.0 with 0.05-0.2M acetic acid solution;

[0081] In one embodiment, in step S2, the mass-to-volume ratio of NMN to deionized water is (0.5-1.0):(50-100) g / mL, and the mass-to-volume ratio of yeast / rice fermentation product filtrate to deionized water is (2-5):(50-100) g / mL; the aqueous phase ultrasonic dissolution conditions are: power 100-200W, time 5-15 minutes; the pH value is adjusted to 5.0-6.0 with 0.05-0.2M acetic acid solution.

[0082] In one embodiment of the present invention, the mass ratio of β-nicotinamide mononucleotide, bakuchiol, and yeast / rice fermentation product filtrate is (0.01-1.5):(0.01-1.5):(0.1-5). In another embodiment, the mass ratio of β-nicotinamide mononucleotide, bakuchiol, and yeast / rice fermentation product filtrate is (0.5-1):(0.5-1):(2-3).

[0083]

Step S3

[0084] Add the aqueous solution obtained in step S2 to the first container after step S1 to form a multilayer phospholipid coarse dispersion; subject the multilayer phospholipid coarse dispersion to ultrasonic treatment to obtain a clear and translucent phospholipid nanoparticle proemulsion, thereby achieving the initial loading of lipid-soluble components embedded in the lipid film and water-soluble components encapsulated in the aqueous core.

[0085] In one embodiment, the aqueous solution from step S2 is added to the lipid film bottle after step S1, and the solution is hydrated by shaking in a water bath to form a milky white multilayer phospholipid coarse dispersion. The coarse dispersion is transferred to a probe sonicator and ultrasonically treated under ice bath conditions to obtain a clear and translucent phospholipid nanoparticle proemulsion, thereby achieving the initial loading of lipid-soluble components embedded in the lipid film and water-soluble components encapsulated in the aqueous core.

[0086] In one embodiment of the present invention, in step S3, the water bath oscillation hydration conditions are: temperature 35-45℃, rotation speed 150-200rpm, and time 25-30 minutes; the ultrasonic treatment conditions are: ice bath temperature ≤10℃, power 250-350W, pulse mode of working for 2-4 seconds / intermittent for 2-4 seconds (e.g., working for 3 seconds / intermittent for 3 seconds), and total time 8-20 minutes.

[0087]

Step S4

[0088] Chitosan was added to an acetic acid solution and magnetically stirred until completely dissolved to obtain a chitosan solution; sodium tripolyphosphate (TPP) was dissolved in deionized water and stirred until clear to obtain a TPP solution.

[0089] In one embodiment, chitosan is added to an acetic acid solution of a set concentration and stirred magnetically until completely dissolved to obtain a chitosan solution; sodium tripolyphosphate (TPP) is dissolved in deionized water and stirred until clear to obtain a TPP solution.

[0090] In one embodiment of the present invention, in step S4, the chitosan solution preparation conditions are as follows: the amount of chitosan is 0.5-1 parts by mass, the amount of acetic acid solution is 80-120 parts by volume, the concentration is 0.05-0.2M, the pH value is 5.0-5.5, and it is dissolved at a magnetic stirring speed of 450-500 rpm for 1-1.5 hours; the TPP solution preparation conditions are as follows: the amount of TPP is 0.1-0.2 parts by mass, the amount of deionized water is 40-60 parts by volume, the pH is adjusted to 5.0-5.5, and it is stirred for 10-15 minutes.

[0091] In one embodiment, in step S4, the chitosan dissolution conditions are as follows: chitosan dosage 0.5-1g, acetic acid solution 80-120mL 0.05-0.2M (pH 5.0-5.5), magnetic stirring speed 450-500rpm, time 1-1.5 hours; the TPP solution preparation conditions are as follows: TPP dosage 0.1-0.2g, deionized water volume 40-60mL (pH 5.0-5.5), stirring for 10-15 minutes.

[0092]

Step S5

[0093] The phospholipid nanoparticle proemulsion from step S3 is dropped into a chitosan solution. Magnetic stirring causes the positively charged chitosan to electrostatically adsorb onto the negatively charged phospholipid membrane surface, forming a phospholipid nanoparticle-chitosan complex. TPP solution is then dropped into the above mixture and stirred to form a stable chitosan network structure, yielding a mitochondrial-targeting composition.

[0094] In one embodiment, the phospholipid nanoparticle proemulsion from step S3 is dropped into a chitosan solution, and the positively charged chitosan is electrostatically adsorbed onto the negatively charged phospholipid membrane surface by magnetic stirring to form a "phospholipid nanoparticle-chitosan" complex; TPP solution is dropped into the above mixture and stirred to form a stable chitosan network structure, thus obtaining a mitochondrial-targeting composition.

[0095] In one embodiment of the present invention, in step S5, the phospholipid nanoparticle proemulsion is added to the chitosan solution at a rate of 0.5-1 volume parts / minute, the magnetic stirring speed is 300-350 rpm, and the stirring time is continued for 30-45 minutes after addition; the TPP crosslinking and curing conditions are as follows: the TPP solution is added to the phospholipid-chitosan complex at a rate of 0.3-0.5 volume parts / minute, the stirring speed is increased to 450-500 rpm, and the stirring is continued for 1-1.5 hours after addition.

[0096] In one embodiment, in step S5, the chitosan-phospholipid electrostatic adsorption conditions are: a dropping rate of 0.5-1 mL / min, a magnetic stirring speed of 300-350 rpm, and a stirring time of 30-45 minutes after dropping; the TPP crosslinking curing conditions are: a dropping rate of 0.3-0.5 mL / min, a stirring speed of 450-500 rpm, and a stirring time of 1-1.5 hours after dropping.

[0097] This invention further discloses a mitochondrial-targeting composition prepared by the above-described method. The mitochondrial-targeting composition comprises β-nicotinamide mononucleotide (NMN), bakuchiol, and yeast / rice fermentation product filtrate. The β-nicotinamide mononucleotide, bakuchiol, and yeast / rice fermentation product filtrate form NMN-bakuchiol-yeast / rice fermentation product filtrate co-loaded phospholipid-chitosan composite nanoparticles.

[0098] This invention provides the above-mentioned mitochondrial-targeting composition and its application.

[0099] This invention provides a composition targeting mitochondria, comprising β-nicotinamide mononucleotide, bakuchiol, and yeast / rice fermentation product filtrate, wherein the β-nicotinamide mononucleotide, bakuchiol, and yeast / rice fermentation product filtrate are prepared into NMN-bakuchiol-yeast / rice fermentation product filtrate co-loaded phospholipid-chitosan composite nanoparticles.

[0100] In one embodiment of the present invention, the mass ratio of β-nicotinamide mononucleotide, bakuchiol, and yeast / rice fermentation product filtrate is (0.01-1.5):(0.01-1.5):(0.1-5). In another embodiment, the mass ratio of β-nicotinamide mononucleotide, bakuchiol, and yeast / rice fermentation product filtrate is (0.5-1):(0.5-1):(2-3).

[0101] This invention further provides the application of a mitochondrial-targeting composition in the preparation of skincare products. These skincare products can be any form of skincare or cosmetic, including toners, serums, lotions, creams, freeze-dried powders, etc. The required base ingredients vary depending on the type of skincare product. The skincare products possess anti-aging effects.

[0102] The present invention also provides a face cream targeting mitochondria, comprising a base excipient and an active ingredient, wherein the active ingredient is NMN-psoralen-yeast / rice fermentation product filtrate co-loaded phospholipid-chitosan composite nanoparticles.

[0103] This invention further discloses a face cream targeting mitochondria, comprising phases A, B, C, D, and E, each comprising the following components in weight percentage:

[0104] Phase A comprises: 5-10% glycerol; 3-10% butylene glycol; 0.5-2% trehalose; 0.5-2% xylitol; 0.01-1% sodium hyaluronate; 0.5-5% nicotinamide; 0.1-5% panthenol; 0.1-2% sodium acrylate / sodium acryloyl dimethyl taurate copolymer; 0.1-2% PEG-100 stearate; 0.01-1% disodium EDTA; and water as the balance.

[0105] Phase B includes: isopropyl isostearate 0.1-5%; caprylic / capric triglyceride 0.1-5%; Butyrospermum Parkii fruit oil 0.1-5%; polydimethylsiloxane 0.1-4%; stearyl alcohol 0.1-2%; cetyl alcohol 0.1-2%; behenyl alcohol 0.1-2%; sorbitan oleate 0.1-2%;

[0106] Phase C includes: 0.1-2% arginine;

[0107] Phase D includes: 3-8% of the active ingredient composition;

[0108] Phase E includes: (daily-use) fragrance 0.1-2%; phenoxyethanol 0.01-1%.

[0109] This invention also provides a method for preparing a face cream, comprising the following steps:

[0110] a. Aqueous phase: Place water in a container equipped with a stirring device and stir. After fully wetting and mixing the other components of phase A, add them to the water and stir thoroughly until completely dissolved. Heat to 75-80℃.

[0111] b. Oil phase; Mix the components of phase B and heat to 75-80℃, stirring until completely dissolved;

[0112] c. Emulsification: Add the oil phase obtained in step b to the aqueous phase obtained in step a, stir at 250-350 rpm for 3-5 min, and then homogenize at 2500-3500 rpm for 3-10 min;

[0113] d. Add each component to phase C, stir at 250-350 rpm for 3-5 min, then homogenize at 2500-3500 rpm for 3-10 min, and stir while cooling to 40-50℃;

[0114] e. Add the components in phase D, stir at 250-350 rpm for 3-5 minutes, and then homogenize at 2500-3500 rpm for 3-10 minutes;

[0115] f. After cooling to 35-38℃, add phase E, stir at 250-350 rpm for 3-10 minutes, then homogenize at 2500-3500 rpm for 3-10 minutes to obtain the desired face cream.

[0116] β-Nicotinamide mononucleotide (NMN), as a direct precursor of NAD+ (nicotinamide adenine dinucleotide), can rapidly increase intracellular NAD+ levels, improve oxidative phosphorylation efficiency, and activate SIRT1. On the one hand, NAD+, as a key coenzyme for mitochondrial oxidative phosphorylation, can significantly enhance complex I activity, improve oxidative phosphorylation efficiency, and promote ATP production, providing sustained energy support for biological processes such as collagen secretion and barrier repair. On the other hand, by activating the SIRT1 / PGC-1α signaling axis, it upregulates the expression of nuclear respiratory factors (NRFs) and mitochondrial transcription factor A (TFAM), promoting mitochondrial biosynthesis (including increased quantity and morphological repair) while maintaining high NAD+ levels. + The NMN / NADH ratio inhibits superoxide anion generation at the source of the electron transport chain and synergistically enhances the activity of endogenous antioxidant enzymes such as SOD and glutathione peroxidase, thereby intercepting oxidative damage at its source. The ATP supply increased by NMN can drive dermal fibroblasts to synthesize collagen networks, while inhibiting the activity of matrix metalloproteinases (MMP-1 / MMP-3), reducing the degradation of collagen and elastic fibers.

[0117] Psoralen, also known as psoralen, is one of the main active components of Psoralea corylifolia seeds and belongs to the monoterpenoid class of substances. Psoralen can upregulate SIRT1 (silencing regulatory protein 1) and AMPK (adenosine monophosphate-activated protein kinase), synergistically enhancing cellular energy metabolism and delaying the aging process. Psoralen can significantly upregulate the biological activity of SIRT1 by specifically regulating gene expression. As a deacetylase, SIRT1 can activate multiple key anti-aging pathways: on the one hand, it reduces cell cycle arrest caused by accumulated DNA damage by promoting the recruitment and function of DNA damage repair-related proteins; on the other hand, SIRT1 can inhibit the nuclear translocation of pro-inflammatory transcription factors such as NF-κB, reducing the secretion of inflammatory factors and creating a low-inflammatory repair environment for cells; simultaneously, the synergistic effect of SIRT1 and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1α) can promote mitochondrial biosynthesis, optimize mitochondrial electron transport chain function, and improve cellular energy supply efficiency. The activation of AMPK by psoralen further enhances the ability to regulate energy metabolism. As a core molecule for cellular energy sensing, AMPK can promote the clearance of aging-related substances such as damaged mitochondria and misfolded proteins by initiating autophagy, thus maintaining homeostasis. In addition, AMPK can also optimize the efficiency of cellular glucose uptake and fatty acid oxidation by regulating the activity of key enzymes in glucose and lipid metabolism, thereby reducing oxidative stress caused by metabolic disorders.

[0118] Yeast / rice fermentation product filtrate is a skincare active ingredient prepared through microbial fermentation technology. It is rich in over 90 active ingredients, including α-ketoglutarate, amino acids, vitamins, and minerals, combining naturalness and bioactivity. Yeast / rice fermentation product filtrate exhibits a multi-dimensional mechanism of action involving cell proliferation, barrier strengthening, and energy revitalization. Firstly, yeast / rice ferment filtrate can significantly promote the proliferation of epidermal keratinocytes, providing a foundation for the dynamic renewal of the skin barrier. Secondly, it enhances the production of intercellular lipids by upregulating the expression of serine palmitoyltransferase, a key enzyme in ceramide synthesis, while promoting the synthesis of desmosome proteins (figrin FLG, inner laminarin IVL) and tight junction proteins (Claudin-1, Claudin-4, Occludin), strengthening the "brick wall structure" of the stratum corneum and improving the integrity of the skin barrier and its resistance to external stimuli. Thirdly, yeast / rice ferment filtrate can repair mitochondrial damage, improve mitochondrial morphology, optimize electron transport chain function, and enhance cellular ATP production efficiency, providing continuous energy support for biological processes such as collagen secretion and barrier protein synthesis. In addition, this ingredient can also promote the production of endogenous hyaluronic acid in the skin, reduce transepidermal water loss (TEWL), and achieve deep moisturizing and elasticity maintenance.

[0119] The following detailed description is provided through specific embodiments. Unless otherwise specified, the raw materials and testing methods involved in this invention are commonly used in the field.

[0120] Table 1. Proportions of each component in the face cream formula.

[0121]

[0122]

[0123] Examples 1-3, Comparative Examples 1-7, and Blank Example 1

[0124] The only difference between Example 2 and Example 1 is the addition of each active ingredient, and the only difference between Example 3 and Example 1 is the reduction of each active ingredient.

[0125] In Examples 1-3, the preparation method of NMN-psoralen-yeast / rice fermentation product filtrate co-loaded phospholipid-chitosan composite nanoparticles includes:

[0126] Step S1: Preparation of phospholipid-lipid-soluble component composite membrane

[0127] Add psoralen to ethanol and stir in a water bath until completely dissolved; add soybean lecithin and phytosterol to the above solution and disperse by ultrasonication to form a homogeneous ethanol-lipid solution; transfer the solution to a rotary evaporator and rotary evaporate until the ethanol is completely evaporated, forming a uniform lipid film on the bottle wall;

[0128] NMN and yeast / rice fermentation product filtrate were dissolved in deionized water, ultrasonically dissolved, and the pH was adjusted to a suitable range to obtain an aqueous solution containing active ingredients.

[0129] Step S2: Preparation of phospholipid nanoparticle promulgation

[0130] Add the aqueous solution from step 1 to the lipid film bottle from step 1, and hydrate it by shaking in a water bath to form a multilayer coarse dispersion of phospholipids. Transfer the coarse dispersion to a probe sonicator and sonicate it under ice bath conditions to obtain a clear and translucent phospholipid nanoparticle proemulsion, thus achieving the initial loading of lipid-soluble components embedded in the lipid film and water-soluble components encapsulated in the aqueous core.

[0131] Step S3: Preparation of chitosan solution

[0132] Chitosan was added to a certain concentration of acetic acid solution and magnetically stirred until completely dissolved to obtain a chitosan solution; sodium tripolyphosphate (TPP) was dissolved in deionized water and stirred until clear to obtain a TPP solution.

[0133] Step S4: Self-assembly of phospholipid-chitosan composite nanoparticles

[0134] The phospholipid nanoparticle proemulsion from step 2 was dropped into the chitosan solution. Magnetic stirring was used to cause the positively charged chitosan to electrostatically adsorb onto the negatively charged phospholipid membrane surface, forming a "phospholipid nanoparticle-chitosan" complex. TPP solution was then dropped into the above mixture and stirred to form a stable chitosan network structure.

[0135] In one embodiment of the present invention, in step S1, the mass-to-volume ratio of psoralen to ethanol is 1.0 g: 50 ml; the mass-to-volume ratio of soybean lecithin to ethanol is 3.0 g: 50 ml; the mass-to-volume ratio of phytosterol to ethanol is 0.5 g: 50 ml; the ultrasonic dispersion conditions are: power 200 W, time 5 minutes; the rotary evaporator parameters are: temperature 40℃, vacuum degree 0.05 MPa, rotary evaporation time 30 minutes; the mass-to-volume ratio of NMN to deionized water is 1.0 g: 50 mL; the mass-to-volume ratio of yeast / rice fermentation product filtrate to deionized water is 3 g: 50 mL; the aqueous phase ultrasonic dissolution conditions are: power 150 W, time 5 minutes; and the pH is adjusted to 5.0 using 0.1 M acetic acid solution.

[0136] In step S2, the water bath oscillation hydration conditions are: temperature 40℃, rotation speed 150rpm, time 30 minutes; the ultrasonic treatment conditions are: ice bath (temperature ≤10℃), power 300W, pulse mode (3 seconds working / 3 seconds intermittent), total time 15 minutes.

[0137] In step S3, the chitosan dissolution conditions are as follows: chitosan dosage is 1g, acetic acid solution is 100mL 0.1M (pH 5.0), magnetic stirring speed is 500rpm, and time is 1.5 hours; TPP solution preparation conditions are as follows: TPP dosage is 0.2g, deionized water volume is 50mL (pH 5.0), and stirring is performed for 10 minutes.

[0138] In step S4, the conditions for chitosan-phospholipid electrostatic adsorption are: a dropping rate of 0.5 mL / min, a magnetic stirring speed of 350 rpm, and a stirring time of 45 minutes after dropping; the conditions for TPP crosslinking and curing are: a dropping rate of 0.5 mL / min, a stirring speed of 500 rpm, and a stirring time of 1.5 hours after dropping.

[0139] The difference between Comparative Examples 1-3 and Example 1 is that only the phospholipid-chitosan composite nanoparticles lack the key composition component.

[0140] The only difference between Comparative Example 1 and Example 1 is that the phospholipid-chitosan composite nanoparticles lack the key component yeast / rice fermentation product filtrate, and are instead NMN-psoralen co-loaded phospholipid-chitosan composite nanoparticles.

[0141] A method for preparing NMN-psoralen co-loaded phospholipid-chitosan composite nanoparticles includes the following steps:

[0142] Step S1: Preparation of phospholipid-lipid-soluble component composite membrane

[0143] Add psoralen to ethanol and stir in a water bath until completely dissolved; add soybean lecithin and phytosterol to the above solution and disperse by ultrasonication to form a homogeneous ethanol-lipid solution; transfer the solution to a rotary evaporator and evaporate by rotary evaporation until the ethanol is completely evaporated, forming a uniform lipid film on the bottle wall; dissolve NMN in deionized water, dissolve by ultrasonication, and adjust the pH to a suitable range to obtain an aqueous solution containing the active ingredient.

[0144] Specific parameters in step S1: Mass-volume ratio of psoralen to ethanol: 1.0g:50ml; Mass-volume ratio of soybean lecithin to ethanol: 3.0g:50ml; Mass-volume ratio of phytosterols to ethanol: 0.5g:50ml; Ultrasonic dispersion conditions: Power 200W, Time 5 minutes; Rotary evaporator parameters: Temperature 40℃, Vacuum 0.05MPa, Rotary evaporation time 30 minutes; Mass-volume ratio of NMN to deionized water: 1.0g:50mL; Aqueous phase ultrasonic dissolution conditions: Power 150W, Time 5 minutes; pH adjustment: Adjusted to 5.0 with 0.1M acetic acid solution.

[0145] Step S2: Preparation of phospholipid nanoparticle promulgation

[0146] Add the aqueous solution from step 1 to the lipid film bottle from step 1, and hydrate it by shaking in a water bath to form a multilayer coarse dispersion of phospholipids. Transfer the coarse dispersion to a probe sonicator and sonicate it under ice bath conditions to obtain a clear and translucent phospholipid nanoparticle proemulsion, thus achieving the initial loading of lipid-soluble components embedded in the lipid film and water-soluble components encapsulated in the aqueous core.

[0147] Specific parameters in step S2:

[0148] Water bath oscillation hydration conditions: temperature 40℃, rotation speed 150rpm, time 30 minutes;

[0149] Ultrasonic treatment conditions: ice bath (temperature ≤10℃), power 300W, pulse mode (3 seconds working / 3 seconds intermittent), total time 15 minutes.

[0150] Step S3: Preparation of chitosan solution

[0151] Chitosan was added to a certain concentration of acetic acid solution and magnetically stirred until completely dissolved to obtain a chitosan solution; sodium tripolyphosphate (TPP) was dissolved in deionized water and stirred until clear to obtain a TPP solution.

[0152] Specific parameters in step S3:

[0153] Chitosan dissolution conditions: 1 g chitosan, 100 mL 0.1 M acetic acid solution (pH 5.0), magnetic stirring speed 500 rpm, time 1.5 hours;

[0154] TPP solution preparation conditions: 0.2g TPP, 50mL deionized water (pH 5.0), stir for 10 minutes.

[0155] S4, self-assembly of phospholipid-chitosan composite nanoparticles

[0156] The phospholipid nanoparticle proemulsion from step 2 was dropped into the chitosan solution. Magnetic stirring was used to cause the positively charged chitosan to electrostatically adsorb onto the negatively charged phospholipid membrane surface, forming a "phospholipid nanoparticle-chitosan" complex. TPP solution was then dropped into the above mixture and stirred to form a stable chitosan network structure.

[0157] Furthermore, the specific parameters in S4 are as follows:

[0158] Chitosan-phospholipid electrostatic adsorption conditions: dropping rate 0.5 mL / min, magnetic stirring speed 350 rpm, stirring time after dropping for 45 minutes;

[0159] TPP crosslinking and curing conditions: dropping rate 0.5 mL / min, stirring speed increased to 500 rpm, and stirring continued for 1.5 hours after dropping.

[0160] The only difference between Comparative Example 2 and Example 1 is that the phospholipid-chitosan composite nanoparticles lack the key component NMN, and are instead phospholipid-chitosan composite nanoparticles co-loaded with psoralen-yeast / rice fermentation product filtrate.

[0161] The preparation method of bakuchiol-yeast / rice fermentation product filtrate co-loaded phospholipid-chitosan composite nanoparticles includes:

[0162] Step S1: Preparation of phospholipid-lipid-soluble component composite membrane

[0163] Add psoralen to ethanol and stir in a water bath until completely dissolved; add soybean lecithin and phytosterol to the above solution and disperse by ultrasonication to form a homogeneous ethanol-lipid solution; transfer the solution to a rotary evaporator and evaporate by rotary evaporation until the ethanol is completely evaporated, forming a uniform lipid film on the bottle wall; dissolve the yeast / rice fermentation product filtrate in deionized water, dissolve by ultrasonication, and adjust the pH to a suitable range to obtain an aqueous solution containing active ingredients.

[0164] Specific parameters in step S1: Mass-volume ratio of psoralen to ethanol: 1.0g:50ml; Mass-volume ratio of soybean lecithin to ethanol: 3.0g:50ml; Mass-volume ratio of phytosterols to ethanol: 0.5g:50ml; Ultrasonic dispersion conditions: power 200W, time 5 minutes; Rotary evaporator parameters: temperature 40℃, vacuum 0.05MPa, rotary evaporation time 30 minutes; Mass-volume ratio of yeast / rice fermentation product filtrate to deionized water: 3g:50mL; Aqueous phase ultrasonic dissolution conditions: power 150W, time 5 minutes; pH adjustment: adjusted to 5.0 with 0.1M acetic acid solution.

[0165] Step S2: Preparation of phospholipid nanoparticle proemulsion;

[0166] Add the aqueous solution from step 1 to the lipid film bottle from step 1, and hydrate it by shaking in a water bath to form a multilayer coarse dispersion of phospholipids. Transfer the coarse dispersion to a probe sonicator and sonicate it under ice bath conditions to obtain a clear and translucent phospholipid nanoparticle proemulsion, thus achieving the initial loading of lipid-soluble components (psoralen) embedded in the lipid film and water-soluble components (yeast / rice fermentation product filtrate) encapsulated in the aqueous core.

[0167] Specific parameters in step S2: Water bath oscillation hydration conditions: temperature 40℃, rotation speed 150rpm, time 30 minutes; Ultrasonic treatment conditions: ice bath (temperature ≤10℃), power 300W, pulse mode (working for 3 seconds / intermittent for 3 seconds), total time 15 minutes.

[0168] Step S3: Preparation of chitosan solution;

[0169] Chitosan was added to a certain concentration of acetic acid solution and magnetically stirred until completely dissolved to obtain a chitosan solution; sodium tripolyphosphate (TPP) was dissolved in deionized water and stirred until clear to obtain a TPP solution.

[0170] Specific parameters in step S3: Chitosan dissolution conditions: 1g chitosan, 100mL 0.1M acetic acid solution (pH 5.0), magnetic stirring speed 500rpm, time 1.5 hours;

[0171] TPP solution preparation conditions: 0.2g TPP, 50mL deionized water (pH 5.0), stir for 10 minutes.

[0172] Step S4: Self-assembly of phospholipid-chitosan composite nanoparticles;

[0173] The phospholipid nanoparticle proemulsion from step 2 was dropped into the chitosan solution. Magnetic stirring was used to cause the positively charged chitosan to electrostatically adsorb onto the negatively charged phospholipid membrane surface, forming a "phospholipid nanoparticle-chitosan" complex. TPP solution was then dropped into the above mixture and stirred to form a stable chitosan network structure.

[0174] Specific parameters in step S4: Chitosan-phospholipid electrostatic adsorption conditions: dropping rate 0.5 mL / min, magnetic stirring speed 350 rpm, stirring time after dropping for 45 minutes; TPP crosslinking curing conditions: dropping rate 0.5 mL / min, stirring speed increased to 500 rpm, stirring time after dropping for 1.5 hours.

[0175] The only difference between Comparative Example 3 and Example 1 is that the phospholipid-chitosan composite nanoparticles lack the key component psoralen, and are NMN-yeast / rice fermentation product filtrate co-loaded with phospholipid-chitosan composite nanoparticles.

[0176] The only difference between Comparative Examples 4-6 and Example 1 is the absence of two key composition components. The only difference between Comparative Example 7 and Example 1 is the replacement of the key component NMN-psoralen-yeast / rice fermentation product filtrate co-loaded phospholipid-chitosan composite nanoparticles with free active ingredients. The only difference between Blank Example 1 and Example 1 is the absence of all key composition components.

[0177] The carrier preparation method is consistent with the preparation method of NMN-psoralen-yeast / rice fermentation product filtrate co-loaded phospholipid-chitosan composite nanoparticles. Only the active ingredient dissolution step in the lipid phase or aqueous phase needs to be adjusted in S1 (the dissolution operation and corresponding parameters of the removed component are deleted). The subsequent steps and parameters in S2-S4 remain completely unchanged and will not be described again here.

[0178] The specific proportions are shown in Table 2, and the preparation method is the same as in Example 1.

[0179] Table 2. Raw material composition ratios of Examples 1-3, Comparative Examples 1-7, and Blank Example 1.

[0180]

[0181]

[0182] Characterization data and effect data of the products in the examples and comparative examples

[0183] Efficacy evaluation test

[0184] Table 3. Distribution ratio of raw materials for cell experiments

[0185]

[0186] I. Verification of Mitochondrial Function Repair

[0187] Epidermal keratinocytes (HaCaT) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator until the logarithmic growth phase.

[0188] Samples from Experimental Examples 1-3, Experimental Comparative Examples 1-7, Experimental Blank Example 1, and the Negative Control Group (Blank) were diluted to the experimental concentration using serum-free DMEM medium; the Negative Control Group was treated with only serum-free DMEM medium.

[0189] 1. ATP content determination

[0190] Digest logarithmic phase cells at 5 × 10⁻⁶ 4 / well density: seeded into 24-well plates (1 mL culture medium per well), and cultured for 24 hours for adhesion;

[0191] Remove the original culture medium, add 1 mL of diluted sample or serum-free culture medium (negative control group) to each well, and incubate at 37°C for 48 hours;

[0192] Remove the culture medium, add 100 μL of cell lysis buffer to each well, and vortex for 10 minutes to lyse the cells;

[0193] Transfer 20 μL of lysis buffer to a 96-well plate, add 100 μL of ATP detection reagent, and incubate in the dark for 10 minutes.

[0194] The luminescence value (RLU) was detected by an ELISA reader, with the negative control group as 100%, and the relative ATP content of the experimental group was calculated.

[0195] 2. ROS level detection

[0196] After cell seeding (same as ATP experiment), culture for 24 hours until cell adhesion, add sample and incubate for 24 hours;

[0197] Remove the culture medium, add 10 μM DCFH-DA probe (diluted with serum-free culture medium) to each well, and incubate at 37°C in the dark for 30 minutes;

[0198] Discard the probe and wash twice with PBS.

[0199] Cells were digested with trypsin, collected by centrifugation (1000 rpm × 5 min), and resuspended in 300 μL PBS;

[0200] The intensity of green fluorescence (excitation light 488 nm, emission light 525 nm) was detected by flow cytometry. The negative control group was taken as 100%, and the ROS inhibition rate of the experimental group was calculated.

[0201] 3. Detection of mitochondrial membrane potential (ΔΨm)

[0202] Cells were seeded and sampled (same as in the ATP experiment), and incubated for 48 hours.

[0203] Remove the culture medium, add 10 μg / mL JC-1 probe (diluted with serum-free culture medium) to each well, and incubate at 37°C in the dark for 20 minutes;

[0204] Discard the probe and wash twice with PBS.

[0205] Cells were digested with trypsin, collected by centrifugation, and resuspended in 300 μL PBS;

[0206] Flow cytometry was used to detect red fluorescence (590 nm) and green fluorescence (525 nm), and the red / green fluorescence ratio was calculated (the higher the ratio, the more stable the membrane potential).

[0207] 4. Detection of mitochondrial biosynthesis and homeostatic proteins

[0208] Cells were seeded in 6-well plates (density 1×10⁶). 5 ( / well), add sample and incubate for 48 hours;

[0209] Remove the culture medium, wash twice with PBS, add 100 μL of RIPA lysis buffer (containing protease inhibitor), and lyse on ice for 30 minutes.

[0210] Centrifuge at 4℃ (12000 rpm × 15 minutes), collect the supernatant, and determine the protein concentration using the BCA method;

[0211] Protein samples (30 μg / well) were subjected to SDS-PAGE electrophoresis (10% separating gel) and then transferred to a PVDF membrane;

[0212] Block with 5% skim milk powder for 1 hour, add primary antibody (SIRT1, PGC-1α, TFAM, MFN1, Drp1, diluted 1:1000), and incubate overnight at 4°C;

[0213] Wash the membrane 3 times with TBST (5 minutes each time), add HRP-labeled secondary antibody (1:5000 dilution), and incubate at room temperature for 1 hour;

[0214] ECL chemiluminescent solution was used for development, and the gray values ​​of the bands were analyzed using ImageJ software (with GAPDH as an internal reference to calculate the relative expression level).

[0215] Table 4. Experimental values ​​for mitochondrial function repair verification

[0216]

[0217] As shown in Table 4, β-NMN, psoralen, and yeast / rice fermentation product filtrate, when co-loaded on a nanocarrier, significantly reversed mitochondrial dysfunction through multi-target synergy.

[0218] 1. ATP content

[0219] The relative ATP content of Experimental Example 1 (5% full-component nanoparticle group) was 152±3.2%, significantly higher than that of Experimental Comparative Example 1 (118±2.5% of yeast / rice fermentation product filtrate group), Experimental Comparative Example 2 (105±2.0% of NMN group), and Experimental Comparative Example 3 (112±2.2% of psoralen group), which lacked a single component. It also far exceeded that of Experimental Comparative Examples 4 to 6 (92-98%), which lacked two components, and was only slightly lower than that of Experimental Example 2 (8% high-concentration group) (178±4.1%), demonstrating concentration dependence. The relative ATP content of Experimental Example 3 (2% low-concentration group) was 131±2.8%. Although the effect was weakened due to the reduced total amount of components, it was still significantly better than that of Experimental Comparative Examples 1 to 6, illustrating the necessity of the synergistic effect of all components for ATP generation. In Experimental Comparative Example 7 (free component group), due to the lack of targeted delivery by the nanocarrier, the component was difficult to accumulate in the mitochondria, and its ATP content was 121±3.0%, only 79% of that in Experimental Example 1. This verifies the key role of the nanocarrier in increasing the effective concentration through targeted delivery. Experimental Blank Example 1 (empty carrier group), lacking effective components, had an ATP content of 98±2.1%, which was close to the 100±2.0% of the negative control group, showing no significant difference. This verifies that the carrier itself has no additional efficacy, eliminating the interference of the carrier on the experimental results.

[0220] 2. ROS inhibition rate

[0221] The ROS inhibition rate of Experiment 1 was 42±2.1%, and that of Experiment 2 was 55±3.0%, both significantly higher than those of Comparative Example 1 (yeast / rice fermentation product filtrate group lacking a single component, 23±1.5%), Comparative Example 2 (NMN group lacking a single component, 18±1.2%), and Comparative Example 3 (bakuchiol group lacking a single component, 22±1.4%). The inhibition rates of Comparative Examples 4 to 6 (lacking two components) were 5%-8%, close to the 8±0.9% of the blank sample, indicating that the lack of components led to almost complete loss of oxidative stress regulation. The inhibition rate of Comparative Example 7 (free component group) was 30±2.0%, only 71% of that of Experiment 1, further confirming the synergistic effect of the nanocarrier on ROS source interception. The ROS inhibition rate of Blank Example 1 (empty carrier group) was 8±0.9%, with no significant difference from the 0% of the negative control group, again verifying that the carrier itself has no additional efficacy.

[0222] 3. JC-1 Red / Green Ratio

[0223] The JC-1 red / green ratio in Experiment 1 was 2.7±0.12, and in Experiment 2 it was 3.2±0.15, both significantly higher than the control groups. Specifically, the ratios for control groups lacking a single component were 1.6±0.08 in control group 1 (yeast / rice fermentation product filtrate), 1.3±0.06 in control group 2 (NMN), and 1.5±0.07 in control group 3 (psoralen), only 60%-63% of those in Experiment 1. The ratios for control groups lacking two components (groups 4-6) were 1.0-1.1, not significantly different from the negative control group's 1.0±0.04, indicating that component deficiency leads to severe disruption of mitochondrial membrane potential. The ratio for control group 7 (free component group) was 2.0±0.10, lower than that of Experiment 1 (a 26% decrease), further demonstrating the crucial role of nanocarriers in maintaining membrane potential.

[0224] 4. Expression of SIRT1 and PGC-1α proteins

[0225] In Experiment 1, the relative expression level of SIRT1 was 2.3±0.15 and the relative expression level of PGC-1α was 2.1±0.13, which were significantly higher than those of Experiment 1 (1.5±0.10 for lacking a single component), Experiment 2 (1.2±0.08 for lacking NMN), and Experiment 3 (1.4±0.09 for lacking bakuchiol) which lacked a single component. The relative expression level of SIRT1 in Experiment 4 to 6, which lacked two components, was 1.0-1.1, which was close to that of the experimental blank (1.1±0.08), indicating that the lack of components led to the failure of SIRT1 / PGC-1α axis activation. The relative expression levels of SIRT1 in experimental control group 7 (free component group) were 1.7±0.12 and PGC-1α were 1.6±0.11, which were only 74%-76% of those in experimental case 1. This further verifies that the nanocarrier enhances the regulatory ability of mitochondrial biosynthesis by increasing the enrichment of components.

[0226] II. Verification of Skin Barrier Function Strengthening

[0227] 1. Keratinocyte proliferation assay (CCK-8 assay)

[0228] Resuscitate HaCaT cells and culture them in DMEM medium containing 10% FBS to the logarithmic growth phase;

[0229] Digest cells, at 5 × 10 3 The culture medium was seeded at a density of 100 μL per well in a 96-well plate and incubated for 24 hours to allow the culture to adhere to the plate.

[0230] Remove the original culture medium, add 100 μL of diluted sample or serum-free culture medium (negative control group) to each well, and set up 3 replicates;

[0231] After 48 hours of incubation, add 10 μL of CCK-8 reagent to each well and incubate at 37°C in the dark for 2 hours.

[0232] The absorbance at OD450nm was measured using an ELISA reader, with the negative control group as 100%, and the cell proliferation rate was calculated.

[0233] 2. Barrier protein expression detection (immunofluorescence assay)

[0234] Cells were seeded in confocal culture dishes (density 2 × 10⁶). 4 / dish), add sample and incubate for 48 hours;

[0235] Remove the culture medium, fix with 4% paraformaldehyde for 15 minutes, and wash twice with PBS;

[0236] Permeabilize with 0.1% Triton X-100 for 5 minutes, then wash twice with PBS;

[0237] Block with 5% BSA for 30 minutes, add primary antibody (FLG, Claudin-1, 1:200 dilution), and incubate overnight at 4°C;

[0238] Wash 3 times with PBS (5 minutes each time), add Alexa Fluor 488 labeled secondary antibody (1:500 dilution), and incubate at room temperature in the dark for 1 hour;

[0239] Nuclear staining with DAPI (1 μg / mL) for 5 minutes, followed by washing twice with PBS;

[0240] The fluorescence intensity was observed by laser confocal microscopy (excitation light 488nm / 405nm), and the relative expression level was calculated using ImageJ software (with the negative control group as 100%).

[0241] Table 5. Numerical values ​​from the experiment verifying skin barrier function enhancement.

[0242]

[0243] From the data in Table 5 and Figure 1-4 It can be seen that β-NMN, psoralen, and yeast / rice fermentation product filtrate, when co-loaded with nanocarriers, showed significant advantages in indicators such as keratinocyte proliferation and barrier protein expression, verifying the synergistic effect of β-NMN, psoralen, and yeast / rice fermentation product filtrate and the synergistic value of nanocarrier targeted delivery for skin barrier strengthening.

[0244] 1. HaCaT cell proliferation rate

[0245] The HaCaT cell proliferation rate of Experiment 1 (5% full-component nanoparticle group) was 132±2.8%, significantly higher than that of Experiment 1 (110±2.0% without yeast / rice fermentation product filtrate group), Experiment 2 (98±1.8% without NMN group), and Experiment 3 (105±2.0% without psoralen group), which lacked a single component. The proliferation rates of Experiment 4 to 6 (85-90%) lacking two components were close to that of the control group (95±1.8%), indicating that component deficiency significantly reduced cell proliferation. The proliferation rate of Experiment 2 (8% high concentration group) was 145±3.5%, showing better results due to increased component concentration. The proliferation rate of Experiment 3 (2% low concentration group) was 123±2.2%, although the effect was weakened due to the reduced total component amount, it was still better than most of the control groups, demonstrating the necessity of concentration dependence and synergistic effects of full-component synthesis. The proliferation rate of experimental control example 7 (free component group) was 120±2.5%, lower than that of experimental example 1 (9% decrease), verifying that the nanocarrier enhanced the cell proliferation promotion effect by increasing the enrichment of components. The proliferation rate of experimental blank example 1 (empty carrier group) was 95±1.8%, which was not significantly different from the 100±2.0% of the negative control group, excluding the interference of the carrier itself on cell proliferation.

[0246] 2. FLG fluorescence intensity

[0247] The FLG (figrin) fluorescence intensity in Experiment 1 was 150±3.5%, and in Experiment 2 (8% high concentration group) it was 165±4.2%. Both were significantly higher than those in Comparative Example 1 (yeast / rice fermentation product filtrate group) lacking a single component (120±2.5%), Comparative Example 2 (NMN group) lacking NMN (108±2.1%), and Comparative Example 3 (bakuchiol group) lacking psoralen (115±2.3%). Comparative Examples 4 to 6 (98-102%) lacking two components were close to the 105±2.1% of the blank sample (empty carrier group), indicating that component deficiency led to a near-complete loss of barrier protein synthesis ability. The FLG fluorescence intensity in Comparative Example 7 (free component group) was 135±3.0%, only 90% of that in Experiment 1, further confirming the synergistic effect of the nanocarrier on barrier protein synthesis. The FLG intensity of blank example 1 (empty vector group) was 105±2.1%, which was not significantly different from the negative control group's 100±1.9%, verifying that the vector itself had no additional efficacy.

[0248] 3. Claudin-1 fluorescence intensity

[0249] The Claudin-1 fluorescence intensity in Experiment 1 was 142±3.1%, and in Experiment 2 (8% high concentration group) it was 158±3.8%, both significantly higher than the control group. Among them, the control group lacking a single component, Experiment 1 (lacked yeast / rice fermentation product filtrate group), Experiment 2 (lacked NMN group), and Experiment 3 (lacked psoralen group) were 110±2.1%, only 81%-73% of Experiment 1. The control groups lacking two components, Experiment 4 to 6 (90-95%), showed no significant difference from the negative control group (100±2.0%), indicating that component deficiency severely damaged the tight junction structure. The Claudin-1 fluorescence intensity in Experiment 7 (free component group) was 128±2.8%, lower than Experiment 1 (a 10% decrease), further demonstrating the crucial role of nanocarriers in maintaining barrier function.

[0250] The synergistic effect of β-NMN, bakuchiol, and yeast / rice fermentation product filtrate promotes keratinocyte proliferation and enhances the expression of filaggrin and tight junction proteins, thereby strengthening the entire skin barrier chain. Combined with the targeted delivery of nanocarriers, the bioavailability of each component is significantly improved, thus showing advantages far exceeding those of single-component deficiency, multi-component deficiency, and free components in key indicators such as cell proliferation and barrier protein synthesis. This provides experimental support for improving skin barrier function and delaying aging at the cellular level.

[0251] III. Human Efficacy Testing

[0252] Characterization data and effect data of the products in the examples and comparative examples

[0253] (1) Test samples: face creams of Examples 1-3, Comparative Examples 1-7 and Blank Example 1 as described in Table 2.

[0254] (2) Subjects: 110 healthy female subjects aged 30-60 years were recruited (10 subjects per group). Inclusion criteria included: forehead wrinkles grade 3-6, nasolabial folds grade 1-3, crow's feet wrinkles grade 2-4; mean F4 score of cheeks >6 or mean R2 score ≤0.65; positive lactic acid stinging test (total score ≥3 points). Each group used the same formula product. Written informed consent was obtained. Before enrollment, subjects were asked a series of questions regarding their medical history and health status according to the inclusion and exclusion criteria. Simultaneously, a conformity assessment and skin color test were performed on the test sites, and the results were recorded.

[0255] Environmental conditions: The visual assessment and instrument testing were conducted in an environment with a temperature of 21±1℃ and a relative humidity of 50±10%RH. The visual assessment was conducted under constant lighting conditions (fluorescent tubes or LED lights with a color temperature of 5500~6500K). Subjects were required to adapt to these environmental conditions for at least 30 minutes before assessment and testing could be performed.

[0256] (3) Test method: Apply the sample twice a day, morning and evening. Apply an appropriate amount of the test sample to the face. Apply sunscreen to the entire face every morning.

[0257] Results were tested at the following three time periods: before sample use (D0), 2 weeks after sample use (W1), and 4 weeks after sample use (W4).

[0258] (4) The test results can be calculated using the following formula:

[0259] Rate of change = (Analysis value after product use - Analysis value before product use) ÷ Analysis value before product use × 100%.

[0260] 1) Stratum corneum moisture content: Measured using a Corneometer CM825 (the higher the value, the better the moisturizing effect).

[0261] 2) Transepidermal water loss (TEWL): Measured using Tewameter™ Hex (the lower the value, the stronger the barrier function).

[0262] 3) Skin elasticity (R2 value): Measured using a Cutometer MPA580 (the higher the R2 value, the better the elasticity).

[0263] 4) Skin firmness (F4 value): Measured using a Cutometer MPA580 (the lower the F4 value, the better the firmness).

[0264] 5) Wrinkle parameters: The number of crow's feet was analyzed using the EvaFACE optical imaging system (the lower the value, the more significant the improvement in wrinkles).

[0265] Table 6. Change Rate of Human Efficacy Test Indicators

[0266]

[0267] From the data in Table 6 and Figure 5-7 It can be seen that, in terms of stratum corneum moisture content, Example 1 showed an increase of 12.8±1.5% and 28.3±2.1% in moisture content at W1 (1 week of use) and W4 (4 weeks of use), respectively; Example 2 showed a better increase due to the increased ingredient concentration, with an increase of 15.5±1.8% in W1 and 36.1±2.5% in W4; Example 3 showed a slightly lower increase due to the reduced total amount of ingredients, with an increase of 9.2±1.2% in W1 and 23.8±1.8% in W4. The comparative examples lacking single / multiple ingredients or free components showed an increase in moisture content of only 30%-60% of the examples. For example, Comparative Example 1 showed an increase of 16.7±1.5% at W4; the blank example, as an empty carrier group, only showed an increase of 8.5±0.3%, verifying the key role of full ingredient synergy and nanocarriers in moisturizing effect.

[0268] Regarding transepidermal water loss (TEWL), the TEWL of Example 1 decreased by 8.1±1.0% and 19.9±1.6% at W1 and W4, respectively; Example 2, due to its higher concentration, showed better results, with a decrease of 8.9±1.2% at W1 and 24.0±2.0% at W4; Example 3, due to its lower concentration, showed slightly weaker results, with a decrease of 7.2±0.7% at W1 and 15.6±1.2% at W4. The comparative groups lacking components or containing free components showed only 30%-50% reduction in TEWL compared to the examples. For instance, Comparative Example 1 showed a decrease of 10.8±1.1% at W4; the blank example, as an empty carrier group, only showed a decrease of 5.2±0.2%, indicating that the absence of components or carriers significantly weakens the barrier repair capacity.

[0269] Regarding skin elasticity (R² value), the R² value of Example 1 increased by 5.6±0.6% and 12.7±1.0% at W1 and W4, respectively. Example 3 showed a more significant improvement in elasticity due to its high concentration, with an increase of 6.3±0.7% at W1 and 15.8±1.3% at W4. Example 3, with its slightly weaker effect due to its low concentration, showed an increase of 5.2±0.4% at W1 and 9.9±0.8% at W4. The comparative examples lacking components or containing free components showed only 50%-70% of the improvement in R² value compared to the examples. For instance, Comparative Example 1 showed an increase of 7.9±0.7% at W4; the blank example, as an empty carrier group, only showed an increase of 4.3±0.2%, verifying the core role of component synergy and nanocarriers in maintaining elasticity.

[0270] Regarding the volume of nasolabial folds, in Example 1, the volume of nasolabial folds decreased by 1.9±0.2% and 5.4±0.4% at W1 and W4, respectively; in Example 2, due to the higher concentration, the improvement was even better, with a decrease of 2.2±0.3% at W1 and 6.0±0.5% at W4; in Example 3, due to the lower concentration, the effect was slightly weaker, with a decrease of 1.6±0.1% at W1 and 5.0±0.3% at W4. The comparative examples lacking components or containing free components showed a volume reduction of only 40%-60% of that in the examples. For example, in Comparative Example 1, the volume decreased by 4.3±0.3% at W4; the blank example, as an empty carrier group, showed no improvement at all, with an increase of 0.7±0.05% at W4, indicating that the lack of components led to a significant decrease in the anti-wrinkle effect.

[0271] Regarding skin gloss, Example 1 showed an increase of 10.2±1.2% and 27.5±2.0% in gloss at W1 and W4, respectively; Example 2, due to its high concentration, showed a better gloss improvement, with an increase of 13.1±1.4% in W1 and 31.2±2.3% in W4; Example 3, due to its low concentration, showed a slightly weaker effect, with an increase of 7.8±0.9% in W1 and 23.6±1.5% in W4. The comparative examples lacking components or containing free components showed only 30%-50% of the gloss improvement of the examples. For example, Comparative Example 1 showed an improvement of 17.8±1.3% at W4; the blank example, as an empty carrier group, only showed an improvement of 7.7±0.3%, further confirming the necessity of component synergy and nanocarriers for gloss optimization.

[0272] Regarding the length of crow's feet wrinkles, in Example 1, the length of crow's feet wrinkles was shortened by 5.8±0.3% and 8.2±0.5% at W1 and W4, respectively; in Example 2, due to the higher concentration, the improvement was even better, with W1 shortening by 6.5±0.4% and W4 shortening by 9.0±0.6%; in Example 3, due to the lower concentration, the effect was slightly weaker, with W1 shortening by 5.2±0.2% and W4 shortening by 7.5±0.4%. The comparative groups lacking components or containing free components showed a length reduction of only 50%-70% of that in the examples. For example, in Comparative Example 1, the length was shortened by 5.8±0.3% at W4; the blank example, as an empty carrier group, only shortened by 2.5±0.1%, indicating that the absence of components or carrier significantly weakened the anti-wrinkle effect.

[0273] In summary, the synergistic effect of β-NMN, bakuchiol, and yeast / rice fermentation product filtrate, combined with the targeted delivery of phospholipid-chitosan composite nanocarriers, significantly improved skin hydration, barrier function, elasticity, firmness, and anti-wrinkle indicators in human efficacy tests. The effect far exceeded that of single-component deficiency, multi-component deficiency, and free component groups, achieving cellular-level anti-aging, epidermal rejuvenation, and barrier homeostasis reconstruction, verifying the anti-aging effectiveness of this technology from the cellular level to the human body.

[0274] In summary, the mitochondrial-targeting composition, its preparation method, and its application proposed in this invention achieve systematic anti-aging intervention from the cellular level to the human body level through the synergistic effect of β-nicotinamide mononucleotide (β-NMN), psoralen, and yeast / rice fermentation product filtrate, combined with phospholipid-chitosan composite nano-targeting carrier technology.

[0275] (1) Multi-target synergistic repair of mitochondrial core functions.

[0276] Mitochondria, as the cell's "energy factory" and "metabolic regulation center," experience functional decline, which is the root cause of cellular aging. This patent comprehensively improves mitochondrial function through the synergistic effect of three components:

[0277] β-NMN as NAD + Its direct precursor can enhance intracellular NAD+. + It enhances oxidative phosphorylation efficiency, promotes ATP production, activates the SIRT1 / PGC-1α signaling axis, promotes mitochondrial biosynthesis, and inhibits reactive oxygen species (ROS) production at the source, thus blocking the vicious cycle of "ROS accumulation → mitochondrial damage → more ROS production".

[0278] Bakuchiol enhances DNA repair and inhibits inflammation by upregulating the SIRT1 and AMPK pathways, creating a low-inflammatory environment for cells. On the other hand, it initiates autophagy to clear damaged mitochondria, optimizes energy metabolism efficiency, and maintains mitochondrial homeostasis.

[0279] Yeast / rice fermentation product filtrate not only directly repairs mitochondrial morphology and improves electron transport chain function, but also promotes keratinocyte proliferation and barrier protein synthesis, achieving a dual effect of "mitochondrial function repair + barrier protection".

[0280] The three work together to cover the entire chain of "energy supply-oxidation regulation-morphology optimization", fundamentally reversing mitochondrial functional decline and delaying the cellular aging process.

[0281] (2) Targeted delivery technology breaks through the bottleneck of component delivery.

[0282] To address the problem that traditional anti-aging ingredients struggle to penetrate cell membranes and accumulate in the inner mitochondrial membrane, this invention employs phospholipid-chitosan composite nanocarrier technology:

[0283] The phospholipid membrane encapsulates lipid-soluble components (psoralen), while the aqueous core encapsulates water-soluble components (β-NMN, yeast / rice fermentation product filtrate). Combined with chitosan surface modification, a stable nanoparticle structure is formed, which significantly enhances the components' ability to penetrate cell membranes and their targeted enrichment on the inner mitochondrial membrane.

[0284] This carrier technology effectively solves the shortcomings of traditional ingredients that are easily metabolized or retained in other parts of the cell, and greatly increases the effective concentration of active ingredients in mitochondria, giving full play to their anti-aging potential.

[0285] (3) Enhancement of both skin barrier strengthening and anti-aging effects.

[0286] Based on the repair of mitochondrial function, combined with the direct effect of yeast / rice fermentation product filtrate on the skin barrier, this patent achieves dual anti-aging through "cell-level repair + barrier strengthening":

[0287] Promotes epidermal renewal: Yeast / rice fermentation product filtrate can significantly promote the proliferation of keratinocytes, providing a basis for the dynamic renewal of the skin barrier and improving problems such as thinning of the stratum corneum and dryness caused by decreased cell proliferation capacity.

[0288] Strengthening the barrier structure: By upregulating the synthesis of key barrier proteins such as ceramides and filaggrin, it strengthens the "brick wall structure" of the stratum corneum, enhances the integrity of the skin barrier and its ability to resist external stimuli, reduces transepidermal water loss (TEWL), and relieves skin sensitivity.

[0289] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0290] The description and application of the present invention herein are illustrative and not intended to limit the scope of the invention to the embodiments described above. Effects or advantages involved in the embodiments may not be apparent due to various factors, and the description of effects or advantages is not intended to limit the embodiments. Variations and modifications of the embodiments disclosed herein are possible, and various substitutions and equivalents of the components in the embodiments are well known to those skilled in the art. It should be apparent to those skilled in the art that the invention can be implemented in other forms, structures, arrangements, proportions, and with other components, materials, and parts without departing from the spirit or essential characteristics of the invention. Other variations and modifications can be made to the embodiments disclosed herein without departing from the scope and spirit of the invention.

Claims

1. A method of preparing a targeted mitochondrial composition, comprising, The preparation method comprises: Step S1, a phospholipid-lipid-soluble component composite membrane preparation step; The psoralen is added into a first organic solvent and stirred until completely dissolved to form a first solution; the phospholipid and the sterol are added into the first solution to form a second solution; and the second solution is evaporated until the first organic solvent is completely volatilized to form a uniform lipid membrane on the inner wall of a first container, i.e. a phospholipid-lipid-soluble component composite membrane; Step S2, a water phase solution preparation step; The β-nicotinamide mononucleotide NMN and the yeast / rice fermentation product filtrate are dissolved in deionized water to obtain a water phase solution containing active ingredients; Step S3, a phospholipid nanoparticle initial milk preparation step; The water phase solution prepared in step S2 is added into the first container after step S1 to form a multi-layer phospholipid coarse dispersion liquid; and the multi-layer phospholipid coarse dispersion liquid is subjected to ultrasonic treatment to obtain a clear and translucent phospholipid nanoparticle initial milk; Step S4, a chitosan solution preparation step; The chitosan is added into an acetic acid solution and magnetically stirred until completely dissolved to obtain a chitosan solution; and the sodium tripolyphosphate TPP is dissolved in deionized water and stirred until clear to obtain a TPP solution; Step S5, a phospholipid-chitosan composite nanoparticle self-assembly step; The phospholipid nanoparticle initial milk of step S3 is dropped into the chitosan solution to cause electrostatic adsorption between the positively charged chitosan and the negatively charged phospholipid membrane surface to form a phospholipid nanoparticle-chitosan composite; and the TPP solution is dropped into the phospholipid-chitosan composite to form a stable chitosan network structure through stirring to obtain a mitochondrion-targeting composition.

2. The preparation method of the mitochondrion-targeting composition according to claim 1, characterized in that: In step S1, the first organic solvent comprises ethanol, methanol or a mixed solvent thereof; the phospholipid comprises at least one of soybean lecithin, hydrogenated soybean lecithin and phosphatidylcholine; the sterol comprises at least one of phytosterol, soybean sterol and ergosterol; the psoralen is added into an organic solvent and stirred in a water bath until completely dissolved to form a first solution; the phospholipid and the sterol are added into the first solution to form a uniform solution through ultrasonic dispersion; and the solution is transferred to a rotary evaporator for rotary evaporation until the organic solvent is completely volatilized to form a uniform lipid membrane on the wall of a lipid membrane bottle; In step S2, the β-nicotinamide mononucleotide NMN and the yeast / rice fermentation product filtrate are dissolved in deionized water and ultrasonically dissolved to adjust the pH to a suitable range to obtain a water phase solution containing active ingredients; In step S3, the water phase solution of step S2 is added into the lipid membrane bottle after step S1, and a multi-layer phospholipid coarse dispersion liquid is formed through water bath oscillation and hydration; the coarse dispersion liquid is transferred to a probe ultrasonic instrument for ultrasonic treatment under ice bath conditions to obtain a clear and translucent phospholipid nanoparticle initial milk; In step S4, the chitosan is added into an acetic acid solution with a set concentration and magnetically stirred until completely dissolved to obtain a chitosan solution; and the sodium tripolyphosphate TPP is dissolved in deionized water and stirred until clear to obtain a TPP solution; In the step S5, the phospholipid nanoparticle initial milk of the step S3 is dripped into the chitosan solution, and the positively charged chitosan is electrostatically adsorbed to the negatively charged phospholipid membrane surface through magnetic stirring to form a "phospholipid nanoparticle-chitosan" complex; the TPP solution is dripped into the above phospholipid-chitosan complex, and stirring is performed to form a stable chitosan network structure, thereby obtaining a mitochondrion-targeting composition.

3. The preparation method of the mitochondrion-targeting composition according to claim 2, characterized in that: In the step S1, the feeding ratio of the first organic solvent to the bakuchiol is (0.5-1.0) mass parts:(30-50) volume parts; the feeding ratio of the first organic solvent to the phospholipid substance is (3.0-5.0) mass parts:(30-50) volume parts; the feeding ratio of the first organic solvent to the sterol substance is (0.5-1.0) mass parts:(30-50) volume parts; the ultrasonic dispersion condition is: power 150-200 W, time 5-15 minutes; and the parameters of the rotary evaporator are: temperature 35-45℃, vacuum degree 0.03-0.07 MPa, and rotary evaporation time 20-40 minutes.

4. The preparation method of the mitochondrion-targeting composition according to claim 2, characterized in that: In the step S2, the feeding ratio of deionized water to β-nicotinamide mononucleotide (NMN) is (0.5-1.0) mass parts:(50-100) volume parts, and the feeding ratio of deionized water to the yeast / rice fermentation product filtrate is (2-5) mass parts:(50-100) volume parts; the water-phase ultrasonic dissolution condition is: power 100-200 W, time 5-15 minutes; and the pH value is adjusted to 5.0-6.0 by using a 0.05-0.2 M acetic acid solution; In the step S3, the water-bath oscillation hydration condition is: temperature 35-45℃, rotation speed 150-200 rpm, and time 25-30 minutes; and the ultrasonic treatment condition is: ice-bath temperature ≤10℃, power 250-350 W, pulse mode: working 2-4 seconds / intermittent 2-4 seconds, and total time 8-20 minutes.

5. The preparation method of the mitochondrion-targeting composition according to claim 2, characterized in that: In the step S4, the chitosan solution preparation condition is: the amount of chitosan is 0.5-1 mass part, the amount of acetic acid solution is 80-120 volume parts, the concentration of the acetic acid solution is 0.05-0.2 M, the pH value is 5.0-5.5, and the dissolution time under magnetic stirring at a rotation speed of 450-500 rpm is 1-1.5 hours; The TPP solution preparation condition is: the amount of TPP is 0.1-0.2 mass part, the amount of deionized water is 40-60 volume parts, the pH value is adjusted to 5.0-5.5, and the stirring time is 10-15 minutes; In the step S5, the dripping speed of the phospholipid nanoparticle initial milk into the chitosan solution is 0.5-1 volume part / minute, the magnetic stirring rotation speed is 300-350 rpm, and the stirring time after dripping is 30-45 minutes. TPP cross-linking and curing conditions: the speed of dropping the TPP solution into the phospholipid-chitosan complex is 0.3-0.5 volume parts per minute, the stirring speed is raised to 450-500 rpm, and the stirring is continued for 1-1.5 hours after the dropping.

6. A targeted mitochondria composition for delaying cell aging, prepared by the method of any one of claims 1 to 5. The targeting mitochondria composition comprises β-nicotinamide mononucleotide NMN, bakuchiol and yeast / rice fermentation product filtrate, which form NMN-bakuchiol-yeast / rice fermentation product filtrate co-loaded phospholipid-chitosan complex nanoparticles, i.e. the targeting mitochondria composition.

7. The targeting mitochondria composition according to claim 6, wherein: The mass ratio of the β-nicotinamide mononucleotide, bakuchiol and yeast / rice fermentation product filtrate is (0.01-1.5): (0.01-1.5)：(0.1-5)。 8. The targeting mitochondria composition according to claim 6, wherein: The mass ratio of the β-nicotinamide mononucleotide, bakuchiol and yeast / rice fermentation product filtrate is (0.5-1): (0.5-1)：(2-3)。 9. Use of the targeting mitochondria composition according to any one of claims 6-8 for preparing a skin care product.

10. The use according to claim 9, wherein: The skin care product is an essence or a cream or a lotion or a lyophilized powder or a skin care water or a mask.

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