Anti-aging composition as well as preparation method and application thereof

Abstract

The invention discloses an anti-aging composition as well as a preparation method and application thereof, and belongs to the technical field of functional foods. Comprising the following raw materials: 2 to 4% of sophora flower powder extract, 0.5 to 1.0% of pyrroloquinoline quinone disodium salt, 10 to 16% of marine collagen peptide, 0.5 to 3.0% of microalgae mustard oil nanoparticles, 1 to 3% of fructo-oligosaccharide, 0.1 to 0.3% of a composite thickening agent, 2 to 5% of a natural sweetening agent, 0.2 to 0.6% of a natural acidulant, 0.05 to 0.2% of essence, 1.0 to 3.0% of lecithin, 0.2 to 1.0% of cholesterol, 0.5 to 1.0% of glycerin, and the balance of water. The sophora flower powder extract, the pyrroloquinoline quinine disodium salt and the like are adopted to prepare the nano-liposome, and the nano-liposome is combined with specific raw materials to prepare the composition, so that synergistic anti-aging of multiple active ingredients can be realized, the effective ingredients can be ensured not to be damaged in the stomach, the bioavailability is high, efficient targeted delivery can be realized, and the application prospect is wide. The dual-channel molecules synergistically and effectively resist inflammation and activate mitochondrial functions, so that an excellent systematic anti-aging effect is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to an anti-aging composition and its preparation method and application, belonging to the technical field of functional food. BACKGROUND

[0002] Nowadays, in the field of functional food, anti-aging products as an important category have always occupied a large share of the market. There are many ways to resist aging, mainly divided into two kinds: external and internal. The external way mainly emphasizes protection and repair, such as sun protection, medical beauty intervention, environmental protection, etc.; while the internal anti-aging focuses on regulation and supplementation, such as: supplementing NAD+ precursors, delaying aging by enhancing cell energy metabolism and DNA repair capacity; or taking antioxidant foods (blueberries, tomatoes, carrots), clearing free radicals and reducing the damage of glycation reaction to collagen; controlling sugar intake, reducing the accumulation of advanced glycation end products (AGEs); also keeping adequate water intake, reducing serum sodium concentration to reduce the risk of chronic diseases.

[0003] Among the above-mentioned endogenous anti-aging strategies, oral functional food is popular in the market due to its convenience, high compliance. The current mainstream anti-aging products on the market are mainly based on collagen and single antioxidant ingredients, which have the following main problems: 1. Low bioavailability: Many natural flavonoids are easily enzymatically degraded, combined with food matrix or oxidatively degraded in the gastrointestinal tract, and the oral absorption rate is usually less than 15%; 2. Poor stability of ingredients: PQQ and other mitochondrial coenzymes are partially degraded in strong acidic gastric juice, and high doses may also cause gastrointestinal discomfort, making it difficult to achieve the effective concentration required by cell mitochondria; 3. Lack of precise targeted delivery: Current products are mainly solid powders or simple nanodispersions, which cannot effectively cross the intestinal mucosal barrier and cannot target active ingredients to cell mitochondria, limiting the anti-aging effect.

[0004] Therefore, there is an urgent need for a new anti-aging composition that can improve the oral absorption rate of multiple active ingredients, has good stability, and has targeted delivery capability, to meet the market demand for efficient, safe, and easy-to-use functional foods. SUMMARY

[0005] In order to solve the above problems, an anti-aging composition and its preparation method and application are provided, which uses sophora flower pollen extract, pyrroloquinoline quinone disodium salt, etc. to prepare nanoliposomes and combines with specific raw materials to prepare the composition, which can realize the synergistic anti-aging of multiple active ingredients, ensure that the effective ingredients will not be destroyed in the stomach, has high bioavailability, and can also achieve efficient targeted delivery. The synergistic effect of double-path molecules effectively resists inflammation and activates mitochondrial function, ultimately completes the complementation and synergy of endogenous anti-inflammatory, mitochondrial energy supplement, intestinal health and skin repair, thereby achieving excellent system anti-aging effect.

[0006] According to one aspect of the present application, there is provided an anti-aging composition comprising, by weight percentage, the following raw materials: Sophora japonica pollen extract 2-4%, pyrroloquinoline quinone disodium salt 0.5-1.0%, marine collagen peptide 10-16%, microalgae mustard oil nanoparticle 0.5-3.0%, fructooligosaccharide 1-3%, composite thickening agent 0.1-0.3%, natural sweetener 2-5%, natural sour agent 0.2-0.6%, essence 0.05-0.2%, lecithin 1.0-3.0%, cholesterol 0.2-1.0%, and glycerol 0.5-1.0%, with the balance being water.

[0007] Optionally, the Sophora japonica pollen extract, pyrroloquinoline quinone disodium salt, lecithin, and cholesterol are nanoized to form a nano-liposome.

[0008] Optionally, the nano-liposome has a particle size of 80-120 nm, an average encapsulation rate of no less than 88%, and an average drug loading of no less than 5%.

[0009] Specifically, the Sophora japonica pollen extract (mainly quercetin glycoside) has an encapsulation rate of no less than 87%, the pyrroloquinoline quinone disodium salt (PQQ·2Na) has an encapsulation rate of no less than 89%, the drug loading of PQQ·2Na is ≥5.0%, the drug loading of Sophora japonica pollen extract is ≥7.0%, the nano-liposome has a PDI of ≤0.25, and the Zeta potential is -15 mV to -35 mV.

[0010] The present application effectively isolates enzymatic hydrolysis, oxidation, and acidic degradation by using the nano-liposome bilayer structure to form a physical barrier in the gastrointestinal environment, thereby providing protection for PQQ·2Na and quercetin glycoside in Sophora japonica pollen extract, significantly improving the activity retention rate after oral administration; at the same time, the particle size can avoid being captured by mucous membrane mucus and can enter the intestinal epithelial cells through endocytosis or fusion, thereby delivering the active molecules more directly and at a higher concentration to systemic circulation.

[0011] Further, the effective substances in Sophora japonica pollen extract and PQQ·2Na form a double-pathway molecular synergy. First, the NF-κB inflammatory pathway is inhibited. The high concentration of quercetin glycoside and polyphenolic compounds in Sophora japonica pollen extract can bind to cell membrane receptors or intracellular signaling molecules, inhibit IKKβ kinase activity, reduce IκBα phosphorylation and degradation, block the translocation of NF-κB to the nucleus, and thereby reduce the transcription and expression of TNF-α, IL-6, and other pro-inflammatory factors, and slow down the oxidative damage and apoptosis caused by chronic low-grade inflammation.

[0012] Secondly, PGC-1α mitochondrial biosynthesis activation, PQQ·2Na can be used as a coenzyme-like molecule, by activating the AMPK and SIRT1 pathway, promote PGC-1α deacetylation and activation, enhance the transcription of downstream genes such as TFAM, start mitochondrial autophagy and new, enhance mitochondrial membrane potential and respiratory chain activity, enhance ATP synthesis, improve cell energy metabolism, reduce ROS production.

[0013] Alternatively, the Sophora japonica pollen extract is obtained by supercritical CO2 extraction, the extraction conditions are 25-30 MPa, 40-45 ℃, 2-3 h, and the quercetin glycoside content is ≥85%.

[0014] Specifically, the extraction method of Sophora japonica pollen extract is as follows: I. Raw material pretreatment 1. Raw material source: mature Sophora japonica pollen is selected, the moisture content is ≤8%, and the ash content is ≤1.5%.

[0015] 2. Crushing and grading: the pollen is crushed to pass through an 80 mesh (180 µm) screen by air cooling to increase the surface area and improve the extraction efficiency.

[0016] 3. Drying: drying in a 50 ℃ oven for 4 h to make the residual moisture content ≤2%, preventing water condensation in supercritical CO2 and affecting fluid penetration.

[0017] II. Supercritical CO2 extraction 1. Equipment Supercritical CO2 extraction device (with heating, pressurizing and separation module), system volume 500 mL, pressure resistance 35 MPa.

[0018] 2. Loading Load the pretreated Sophora japonica pollen into the extraction cylinder, the loose bulk density is 0.3 g / mL, and there is 20% space to prevent blockage.

[0019] 3. Parameter setting Pressure: initial pressure rise to 25 MPa, the second stage can be adjusted to 30 MPa (fractionated extraction); Temperature: 40 ℃ constant temperature; flow rate: CO2 flow rate 15 kg / h, ethanol 0.5 kg / h; Residence / dynamic time: (1) Static pre-soaking: static for 10 min after pressure rise to 25 MPa, so that CO2 is fully saturated; (2) Dynamic extraction: continuous extraction for 90 min at 25 MPa and 40 ℃; (3) Pressure rise extraction: the pressure is raised to 30 MPa, and dynamic extraction is continued for 60 min.

[0020] (4) Recovery and separation: The eluent was recovered in a 5 MPa, 20°C pressure reduction module.

[0021] 4. Solvent recovery and drying The separated CO2 / ethanol mixture containing active ingredients was rotary evaporated to recover ethanol; the remaining material was spray dried (inlet temperature 180°C, outlet temperature 80°C) to obtain a light yellow powder, which was the Sophora japonica L. extract.

[0022] 5. Content determination (HPLC): Chromatographic conditions: C18 column, flow rate 1.0 mL / min, detection wavelength 360 nm; quercetin glycoside content ≥ 85% (w / w).

[0023] Alternatively, the marine collagen peptide has an average molecular weight of 3-5 kDa.

[0024] Alternatively, the microalgae mustard oil nanoparticle has a particle size of 80-120 nm, and an EPA content of ≥20%.

[0025] Specifically, the preparation method of the microalgae mustard oil nanoparticle is as follows: (I) Raw material composition and characteristics 1. The microalgae oil is a microalgae oil extracted by fermentation of Schizochytrium sp. (split alga), which is rich in EPA (eicosapentaenoic acid) and DHA; after refining, the EPA content is ≥20%, which is the main active fat-soluble component.

[0026] 2. Emulsification wall material Main emulsifier: food-grade lecithin (≥95% purity); co-emulsifier: Tween-80 (polysorbate 80); carrier wall material (for spray drying): malt dextrin (DE 10-12) and gum arabic are used in combination.

[0027] (II) Nanoparticle preparation steps 1. Oil phase preparation Heat the microalgae mustard oil to a flowable state in a 40°C water bath, take 3g; add lecithin (2.5%) and Tween-80 (0.5%) according to the total amount of emulsifier accounting for 10% of the mass of the oil phase, and stir to dissolve uniformly.

[0028] 2. Water phase preparation Dissolve malt dextrin (6g) and gum arabic (1g) in distilled water (90mL) to form a 10% total solid solution; stir until completely dissolved, and control the water phase temperature at 45°C.

[0029] 3. Emulsion formation The oil phase is slowly added into the water phase, pre-emulsified for 3 minutes using a high-speed homogenizer (10000 rpm) to form a coarse emulsion; and then ultrasonically treated in an ice bath for 10 minutes using an ultrasonic disruptor (200 W, 20 kHz) to obtain a uniform nano-particle emulsion.

[0030] 4. Particle size control The particle size of the emulsion obtained after ultrasonic treatment is controlled at 80-120 nm, PDI≤0.25, and the Zeta potential is -20±3 mV.

[0031] The present application can form a stable small-particle structure of EPA by compounding microalgae mustard oil nano-particles with other raw materials, so that the EPA enters the circulatory system through enteral epithelial endocytosis or chylomicron transport mechanism, thereby significantly improving the absorption rate; at the same time, the EPA can inhibit the synthesis of prostaglandin E2 (PGE2) and reduce the activation of the NF-κB pathway, thereby synergizing with the active ingredients of sophora flower extract to resist inflammation; at the same time, the EPA can improve cardiovascular health, promote skin lipid metabolism, and delay the aging process of the skin.

[0032] Optionally, the composite thickening agent comprises xanthan gum and hydroxypropyl methyl cellulose at a weight ratio of 1:(2-4); the natural sweetener comprises erythritol and monk fruit extract at a weight ratio of (3-4):1; The natural sour agent comprises malic acid and citric acid at a weight ratio of 1:(1-2); and the essence is a natural food-grade essence. The essence can be adjusted according to market tastes, such as green tea mint, citrus, etc.

[0033] Specifically, the high viscosity of xanthan gum and the micellar structure of HPMC together form a three-dimensional network structure, which remains stable under high-temperature sterilization and storage conditions, preventing the degradation or precipitation of the thickening agent. The natural sweetness of monk fruit has a sweetening effect, which can mask the weak fishy smell of marine collagen and microalgae oil. Erythritol is derived from nature and has little gastrointestinal irritation, and is safe for long-term use. The addition of the sour agent optimizes the taste and creates a weak acidic environment, which can optimize the interaction between the nano-liposome and the intestinal mucosa and improve the absorption of active ingredients.

[0034] According to another aspect of the present application, a preparation method of the above-mentioned anti-aging composition is also provided, comprising the following steps: (1) mixing lecithin, cholesterol and glycerol in chloroform, and rotary evaporating to form a uniform lipid dry film; (2) weighing the sophora flower extract and pyrroloquinoline quinone disodium salt separately, and dissolving them in a phosphate buffer to prepare an active ingredient-containing water phase; (3) adding the water phase obtained in step (2) into the lipid dry film obtained in step (1), and standing for hydration at room temperature, and then ultrasonically refining the hydrated suspension; (4) homogenizing the suspension after ultrasonic treatment to obtain a nanoliposome suspension containing sophora japonica pollen extract and pyrroloquinoline quinone disodium salt; (5) adding marine collagen peptide, microalgae mustard oil nanoparticle, fructooligosaccharide, complex thickening agent, natural sweetener, natural acidulant, essence and water into the nanoliposome suspension in sequence, stirring uniformly, and sterilizing by autoclaving to obtain the anti-aging composition.

[0035] Optionally, the rotary evaporation condition in step (1) is 40-45°C in temperature and 120-150 rpm in rotation speed; The standing hydration time in step (3) is 1-2 h, and the ultrasonic parameters are 20-25 kHz and 200-220 W, and the ultrasonic treatment time is 5-8 min; The high-pressure homogenization condition in step (4) is 80-100 MPa in pressure and 3 times in circulation, and the temperature in step (5) is 110-125°C, and the time is 10-15 min.

[0036] According to another aspect of the present application, the anti-aging composition or the anti-aging composition prepared by the above preparation method is also provided for use as a dietary supplement.

[0037] The beneficial effects of the present application include but are not limited to: 1. The anti-aging composition of the present application effectively encapsulates sophora japonica pollen extract and PQQ·2Na in the intermembrane or intramembrane water phase by using nanoliposomes with a bilayer composed of lecithin and cholesterol, so that the active ingredients can be protected from degradation and oxidation in the harsh environment of gastric and intestinal tract, thus maintaining the original activity; and the nanoliposomes can realize efficient delivery across the mucosal barrier by means of “nanocytosis” or fusion with the intestinal epithelial cell membrane, so that the active substances are released into the intestinal cells more quickly. The nanoliposome carrier exhibits sustained-release characteristics, so that the active ingredients are continuously released in the intestinal tract, prolonging the intestinal residence time and achieving good effects.

[0038] 2. The anti-aging composition of the present application can exert the synergistic anti-aging effect of sophora japonica pollen extract and PQQ by compounding them in nanoliposomes. Sophora japonica flavonoids (mainly quercetin glycosides) can inhibit the NF-κB pathway, reduce the expression of pro-inflammatory cytokines (TNF-α, IL-6), alleviate chronic inflammatory state, protect telomere structure and delay cell aging; PQQ, as a mitochondrial bioenergy regulator, can activate the AMPK-SIRT1-PGC-1α pathway, promote mitochondrial biogenesis, and improve cell metabolism and antioxidant capacity, thus delaying the functional decline of the body from the energy level; not only acting on the inflammation pathway and energy metabolism pathway, but also participating in the regulation of cell cycle, apoptosis, DNA repair and redox homeostasis, forming an anti-aging integrated mechanism from the molecular-cell-tissue-system level.

[0039] 3. The anti-aging composition of the present application can promote collagen regeneration, improve skin elasticity and water content by adding marine collagen peptides; microalgae mustard oil nanoparticles provide omega-3 fatty acids (EPA≥20%), which can inhibit the synthesis of PGE2 and leukotriene inflammatory mediators, synergize with sophora flavones to resist inflammation, and at the same time improve the skin lipid barrier and enhance the stability of the cell membrane. Oligofructose regulates intestinal flora, promotes the absorption of active ingredients, and indirectly improves the absorption and transport efficiency of PQQ and flavonoids.

[0040] 4. The preparation method of the anti-aging composition of the present application is mature, and the components of the formula can be produced in sterile conditions under food GMP standard conditions, which has good industrial transformation prospects. BRIEF DESCRIPTION OF DRAWINGS

[0041] The drawings described herein are used to provide further understanding of the present application, and form a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application, and do not constitute improper limitations on the present application. In the drawings: Figure 1 It is a skin horny layer water content experimental result line graph (test group + control group overall subject population); Figure 2 It is a skin horny layer water content experimental result line graph (all subjects over the age of 30); Figure 3 It is a skin horny layer water content experimental result line graph (different populations over the age of 30 and under the age of 30 in the test group); Figure 4 It is a skin elasticity experimental result line graph (all subjects under the age of 30 compared); Figure 5 It is a skin elasticity experimental result line graph (all subjects over the age of 30 compared); Figure 6 It is a crow's feet wrinkle depth experimental result line graph (all subjects under the age of 30 compared); Figure 7 It is a crow's feet wrinkle depth experimental result line graph (all subjects over the age of 30 compared); Figure 8 It is a comparison chart of the crow's feet wrinkles of the subject with volunteer number 2024120302042 (age> 30); Figure 9 It is a comparison chart of the skin fineness of the subject with volunteer number 2024120302038 (age> 30). DETAILED DESCRIPTION

[0042] The application will be described in greater detail below with reference to the following Examples, but the application is not limited to these Examples.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or materials used in the present application can be purchased through conventional routes, and unless otherwise specified, the reagents or materials used in the present application are used according to the conventional manner in the art or according to the product instructions. In addition, any method and material similar or equivalent to those described can be applied to the method of the present application. The preferred implementation methods and materials described in the present patent are only for demonstration.

[0044] The sources and models of the raw materials in the present application are as follows: Sophora flower pollen source: dried flower buds of Sophora japonica L. of Leguminosae; pyrroloquinoline quinone disodium salt model: PQQ-2Na-99 (food grade), Shanghai Baixin Biological Technology Co., Ltd.; marine collagen peptide: deep-sea cod skin enzymolysis, Qingdao Haida Biological Group Co., Ltd.; fructooligosaccharide FOS-95, Bowling Bio-technology Co., Ltd.; xanthan gum, hydroxypropyl methylcellulose model: XanHPMC®-130 (food grade), CP Kelco (USA); erythritol Erythritol-99, Bowling Bio-technology Co., Ltd.; monk fruit extract, Hunan Huacheng Biological Resources Co., Ltd.; malic acid, Ningxia Jiazhu Chemical Co., Ltd.; citric acid, Anhui Fengyuan Biological Technology Co., Ltd. Room temperature in the present application is 25°C.

[0045] Method parameters for HPLC determination of Sophora flower pollen extract: chromatographic column: C18 reversed-phase column (250 mm x 4.6 mm, 5 µm packing), column temperature maintained at 30°C. Mobile phase: methanol: 0.1% acetic acid water = 60:40 (by volume), eluted with isocratic. Flow rate 1.0 mL / min, detection wavelength 360 nm (maximum absorption wavelength of quercetin glycoside); injection volume: 10 µL for sample and standard solution. Sample preparation: take 0.01 g of freeze-dried extract powder in 10 mL of methanol and ultrasonic for 15 min, dilute to 10 mL and filter through a 0.45 µm filter membrane.

[0046] Method parameters for HPLC determination of pyrroloquinoline quinone disodium salt: 1. Instrument and chromatographic column Instrument model: Agilent 1260 Infinity II; Chromatographic column: Zorbax Eclipse Plus C18 (4.6 mm x 150 mm, 5 µm).

[0047] 2. Mobile phase and gradient Mobile phase A: 0.1% (v / v) formic acid in water; Mobile phase B: high purity acetonitrile; Gradient program: 0-5 min: A→95% (isocratic elution); 5-15 min: linear gradient from 95% A→80% A; 15-20 min: 80% A hold; 20-22 min: return to 95% A; 22-25 min: re-equilibrate to 95% A.

[0048] 3. Chromatographic conditions Flow rate: 1.0 mL / min; column temperature: 30±1°C; detection wavelength: 330 nm (PQQ maximum absorption peak); injection volume: 10 µL.

[0049] 4. Standard curve and quantification Standard preparation: PQQ·2Na standard (≥98%) was weighed and accurately prepared into 5, 10, 20, 50, and 100 µg / mL solutions, respectively; injection determination: each concentration was injected in triplicate, and the peak area was recorded; curve fitting: the standard curve was plotted with concentration as the abscissa and peak area as the ordinate, the linear range was 5-100 µg / mL, the goodness of fit R 2 ≥ 0.999; sample determination: the sample to be determined was determined under the same conditions, and the content (mg / g) was calculated.

[0050] Example 1 Anti-aging composition 1# Anti-aging composition 1# is prepared from the following raw materials: by weight percentage, it is prepared from the following raw materials: Sophora japonica pollen extract 3%, pyrroloquinoline quinone disodium salt 0.8%, marine collagen peptide 12%, microalgae mustard oil nanoparticle 2.0%, fructooligosaccharide 2%, composite thickening agent 0.2%, natural sweetener 3%, natural sour agent 0.4%, essence 0.1%, lecithin 2.0%, cholesterol 0.6%, and glycerol 0.7%, with the balance being water.

[0051] Wherein, the Sophora japonica extract, pyrroloquinoline quinone disodium salt and lecithin, cholesterol are nanoized to form nano-liposomes. Dynamic light scattering (DLS) determination, the particle size of the nano-liposomes is 80-120 nm, PDI≤0.25; HPLC determination of Sophora japonica extract (mainly quercetin glycosides) encapsulation efficiency 87%, pyrroloquinoline quinone disodium salt (PQQ·2Na) encapsulation efficiency 89%, drug loading PQQ·2Na 5.0%, Sophora japonica extract 7.0%. Sophora japonica extract is obtained by supercritical CO2 extraction by the above method, the content of quercetin glycosides is ≥85%; marine collagen peptide average molecular weight 3-5 kDa; microalgae mustard oil nanoparticle particle size 80-120 nm, EPA content ≥20%, PDI≤0.25; complex thickening agent includes xanthan gum and hydroxypropyl methyl cellulose, weight ratio 1:3; natural sweetener includes erythritol and monk fruit extract, weight ratio 3:1; natural acidifier includes malic acid and citric acid, weight ratio 1:1; essence is natural food grade essence.

[0052] The preparation method comprises the following steps: (1) lecithin, cholesterol and glycerol are mixed and dissolved in chloroform, and rotary evaporation is performed to form a uniform lipid dry film; (2) Sophora japonica extract and pyrroloquinoline quinone disodium salt are weighed and dissolved in phosphate buffer to prepare an aqueous phase containing active ingredients; (3) The aqueous phase obtained in step (2) is added to the lipid dry film obtained in step (1), and after standing at room temperature, the hydrated suspension is ultrasonically refined; (4) The suspension after ultrasonic treatment is subjected to high-pressure homogenization to obtain a nano-liposome suspension loaded with Sophora japonica extract and pyrroloquinoline quinone disodium salt; (5) marine collagen peptide, microalgae mustard oil nanoparticle, fructooligosaccharide, complex thickening agent, natural sweetener, natural acidifier, essence and water are sequentially added to the nano-liposome suspension, stirred uniformly, and then high-pressure sterilized to obtain an anti-aging composition 1#.

[0053] In step (1), the rotary evaporation conditions are temperature 42℃, rotation speed 130 rpm; in step (3), the standing hydration time is 1.5 h, the ultrasonic parameters are 22 kHz, 210 W, and the ultrasonic treatment time is 6 min; in step (4), the high-pressure homogenization conditions are pressure 90 MPa and cycle 3 times; in step (5), the temperature is 120℃ and the time is 12 min.

[0054] Example 2 Anti-aging composition 2# Anti-aging composition 2# is prepared by the following raw materials: by weight percentage, including the following raw materials: Sophora japonica pollen extract 2%, pyrroloquinoline quinone disodium salt 0.5%, marine collagen peptide 16%, microalgae mustard oil nanoparticle 3.0%, fructooligosaccharide 1%, composite thickening agent 0.3%, natural sweetener 2%, natural acidifier 0.6%, essence 0.05%, lecithin 1.0%, cholesterol 1.0% and glycerol 0.5%, the balance is water.

[0055] Among them, Sophora japonica pollen extract, pyrroloquinoline quinone disodium salt and lecithin, cholesterol are nano-processed to form nanoliposomes. Dynamic light scattering (DLS) measurement, the particle size of nanoliposomes is 80-120 nm, PDI≤0.25; HPLC measurement Sophora japonica pollen extract (mainly quercetin glycoside) encapsulation rate 88%, pyrroloquinoline quinone disodium salt (PQQ·2Na) encapsulation rate 90%, drug loading PQQ·2Na 5.5%, Sophora japonica pollen extract 8%. Sophora japonica pollen extract is obtained by supercritical CO2 extraction by the above method, the content of quercetin glycoside is ≥85%; marine collagen peptide average molecular weight 3-5 kDa; microalgae mustard oil nanoparticle particle size 80-120 nm, EPA content ≥20%, PDI≤0.25; composite thickening agent includes xanthan gum and hydroxypropyl methyl cellulose, weight ratio 1:2; natural sweetener includes erythritol and monk fruit extract, weight ratio 4:1; natural acidifier includes malic acid and citric acid, weight ratio 1:2; essence is natural food grade essence.

[0056] The preparation method comprises the following steps: (1) Mix lecithin, cholesterol and glycerol in chloroform, and rotary evaporation to form a uniform lipid dry film; (2) Weigh Sophora japonica pollen extract and pyrroloquinoline quinone disodium salt separately, and dissolve them in phosphate buffer to prepare an aqueous phase containing active ingredients; (3) Add the aqueous phase obtained in step (2) to the lipid dry film obtained in step (1), and stand at room temperature after hydration, and then ultrasonic thinning of the hydrated suspension; (4) Homogenize the suspension after ultrasonic treatment to obtain a nanoliposome suspension loaded with Sophora japonica pollen extract and pyrroloquinoline quinone disodium salt; (5) Add marine collagen peptide, microalgae mustard oil nanoparticle, fructooligosaccharide, composite thickening agent, natural sweetener, natural acidifier, essence and water to the nanoliposome suspension in turn, stir uniformly, and then autoclave to obtain anti-aging composition 2#.

[0057] The rotation evaporation condition in step (1) is 40℃ and 120 rpm; the standing hydration time in step (3) is 1 h, and the ultrasonic parameters are 20 kHz and 200 W, and the ultrasonic treatment time is 5 min; the high-pressure homogenization condition in step (4) is 80 MPa and 3 cycles; and the temperature in step (5) is 110℃ and the time is 15 min.

[0058] Example 3 Anti-aging composition 3 The anti-aging composition 3 is prepared from the following raw materials by weight percentage: Sophora japonica pollen extract 4%, pyrroloquinoline quinone disodium salt 1.0%, marine collagen peptide 10%, microalgae mustard oil nanoparticle 3.0%, fructooligosaccharide 3%, composite thickening agent 0.3%, natural sweetener 2%, natural acidifier 0.2%, essence 0.05%, lecithin 3.0%, cholesterol 1.0%, and glycerol 1.0%, and the balance is water.

[0059] The Sophora japonica pollen extract, pyrroloquinoline quinone disodium salt, lecithin, and cholesterol are nanoized to form nanoliposomes. Dynamic light scattering (DLS) determination shows that the particle size of the nanoliposomes is 80-120 nm, and the PDI is ≤0.25. HPLC determination shows that the Sophora japonica pollen extract (mainly quercetin glycoside) has an encapsulation rate of 90%, and the pyrroloquinoline quinone disodium salt (PQQ·2Na) has an encapsulation rate of 91%. The drug loading of PQQ·2Na is 6.0%, and the drug loading of Sophora japonica pollen extract is 8.0%. The Sophora japonica pollen extract is obtained by supercritical CO2 extraction by the above method, and the quercetin glycoside content is ≥85%. The marine collagen peptide has an average molecular weight of 3-5 kDa. The microalgae mustard oil nanoparticle has a particle size of 80-120 nm, an EPA content of ≥20%, and a PDI of ≤0.25. The composite thickening agent includes xanthan gum and hydroxypropyl methyl cellulose at a weight ratio of 1:4. The natural sweetener includes erythritol and monk fruit extract at a weight ratio of 3:1. The natural acidifier includes malic acid and citric acid at a weight ratio of 1:1. The essence is a natural food-grade essence.

[0060] The preparation method includes the following steps: (1) Mix the lecithin, cholesterol, and glycerol in chloroform, and rotationally evaporate to form a uniform lipid dry film; (2) Weigh the Sophora japonica pollen extract and pyrroloquinoline quinone disodium salt separately, and dissolve them in a phosphate buffer to prepare an aqueous phase containing active components; (3) Add the aqueous phase obtained in step (2) to the lipid dry film obtained in step (1), stand for hydration at room temperature, and then finely disperse the hydrated suspension by ultrasonic treatment; (4) Homogenize the suspension after ultrasonic treatment to obtain a nanoliposome suspension loaded with Sophora japonica pollen extract and pyrroloquinoline quinone disodium salt; (5) adding marine collagen peptide, microalgae mustard oil nanoparticles, fructooligosaccharide, complex thickening agent, natural sweetener, natural acidulant, essence and water into the nanoliposome suspension in sequence, stirring uniformly, and then high-pressure sterilization to obtain anti-aging composition 3#.

[0061] In the step (1), the rotary evaporation condition is 45℃ and the rotation speed is 150 rpm; in the step (3), the standing hydration time is 2 h, and the ultrasonic parameters are 20 kHz and 200 W, and the ultrasonic treatment time is 5 min; in the step (4), the high-pressure homogenization condition is 100 MPa and the cycle is 3 times; in the step (5), the temperature is 125℃ and the time is 10 min.

[0062] Comparative anti-aging composition 1 The difference between Comparative Example 1 and Example 1 is that the sophora flower pollen extract is not added in Comparative Example 1.

[0063] Comparative anti-aging composition 2 The difference between Comparative Example 2 and Example 1 is that lecithin, cholesterol and glycerol are not added in Comparative Example 2, and the nanoliposome is not prepared.

[0064] Comparative anti-aging composition 3 The difference between Comparative Example 3 and Example 1 is that the microalgae mustard oil nanoparticles are not added in Comparative Example 3.

[0065] Experimental example I. Subject experiment A total of 80 subjects were recruited in this experiment and randomly divided into 2 groups. All subjects signed the informed consent form before the experiment. All subjects were required to clean the facial skin and wipe it clean with dry facial tissues before the test. The test site was exposed to the test environment for at least 20 min before testing. The skin keratin layer water content and skin elasticity of the subjects were detected, and the facial images of the subjects were collected as initial values. The inter-group control method was used, in which 1 group of subjects drank anti-aging composition 1# (hereinafter referred to as “test group”), and the other group of subjects drank comparative anti-aging composition 2# (hereinafter referred to as “control group”). One bottle was drunk after each meal or before going to bed every day, and the continuous drinking lasted for 4 weeks. All skin indicators were detected again at 1 week, 2 weeks and 4 weeks after starting to drink.

[0066] (1) Effect of anti-aging composition on skin keratin layer water content The experimental results of the overall subject population of the two groups are shown in Figure 1 , the experimental results of the population of all subjects aged 30 years old or above are shown in Figure 2 , and the comparison chart of different populations of subjects aged 30 years old or above and below 30 years old in the test group is shown in Figure 3 .

[0067] From the above figure, it can be seen that, compared with the control group, the test group population under the age of 30 or 30 and above has a trend of increasing the water content of the stratum corneum within 4 weeks of continuous taking, among which the population over the age of 30 has a more significant effect on increasing the water content of the stratum corneum (increasing by 9.63%~14.05%).

[0068] (2) Effect of Anti-aging Composition on Skin Elasticity Figure 4 For all subjects under the age of 30, the experimental results are compared, Figure 5 For all subjects over the age of 30, the experimental results are compared.

[0069] The initial value of skin elasticity before taking the sample is set to 100%. As can be seen from the figure, compared with the control group, the test group population under the age of 30 or 30 and above has a trend of increasing the skin elasticity within 4 weeks of continuous taking, and the population under the age of 30 has a more significant effect on increasing the skin elasticity (increasing by 5.03%~9.51%).

[0070] (3) Effect of Anti-aging Composition on the Depth of Crow's Feet Figure 6 For all subjects under the age of 30, the experimental results are compared, Figure 7 For all subjects over the age of 30, the experimental results are compared.

[0071] The initial value of the average depth of crow's feet before taking the sample is set to 100%. As can be seen from the figure, compared with the control group, the population under the age of 30 began to reduce the depth of crow's feet after 2 weeks of continuous taking of the anti-aging oral liquid, and the population of 30 and above had a relatively low level of crow's feet depth within 4 weeks of continuous taking. Within the test group, different age groups have different effects on reducing the depth of wrinkles. In the early stage of taking (within 1 week), the younger the age (<30 years old), the faster the effect, and with the continuous taking of the anti-aging oral liquid, the older the age (≥35 years old), the more obvious the trend of improving the depth of crow's feet, but there is no significant difference between groups.

[0072] (4) Comparison of images of some subjects After the experiment, 2 volunteers were randomly selected from the test group, and their crow's feet and skin fineness were analyzed through facial photos within 4 weeks. The volunteer number selected for the comparison of crow's feet is 2024120302042 (age > 30 years old); the volunteer number selected for the comparison of skin fineness is 2024120302038 (age > 30 years old).

[0073] Figure 8Figure 2 shows the comparison of the eye wrinkle condition of the volunteer numbered 2024120302042 before taking the anti-aging composition 1# oral liquid (T0) and after taking it for 4 weeks. Figure 9 Figure 3 shows the comparison of the facial skin fineness of the volunteer numbered 2024120302038 before taking the anti-aging composition 1# oral liquid (T0) and after taking it for 4 weeks.

[0074] It can be seen that after taking the anti-aging oral liquid for 1 week, the eye wrinkles began to change from deep to shallow, and tended to fade, especially after taking it for 2 weeks, the eye wrinkles were gradually filled, and the improvement of the eye wrinkles continued throughout the test period. After taking the anti-aging oral liquid for 4 weeks, the roughness of the skin texture of the volunteer was significantly reduced, the smoothness of the skin surface was effectively improved, and the skin fineness was effectively improved.

[0075] II. In vitro cell level evaluation experiment 1. Experimental method In this experiment, HaCaT (human keratinocytes) was used to evaluate anti-inflammatory and mitochondrial activation related indicators (NF-κB, PGC-1α), and L929 (mouse fibroblasts) was used to evaluate collagen I secretion (dermal repair). Each group has at least 3 independent biological repeats.

[0076] 2. Sample preparation and physicochemical characterization Obtain the finished oral liquid sample (Examples 1-3 and Comparative Examples 1-3), sterilize at 121°C, and cool. Mother liquor preparation: Take an appropriate amount of finished product, dilute with sterile PBS, centrifuge (12000xg, 15min) to remove large particles, and take the supernatant as the mother liquor (sterile operation). Equivalent dilution: calculate the required volume according to the PQQ·2Na content in the formula, dilute the mother liquor to equivalent PQQ=10μM. After preparation and filtration through a 0.22μm filter membrane, store at 4°C, and warm to room temperature before use. Dilution calculation: if the PQQ content in the mother liquor is Amg / mL, the molar mass of PQQ is about 330 g / mol, then the required dilution factor N= (A mg / mL) / (10μM x 0.33 mg / L)=C_m / (0.0033 mg / mL), calculate the volume according to this.

[0077] 3. Cell culture and general conditions Culture medium: DMEM (high sugar) + 10% FBS + 1% penicillin / streptomycin. Culture conditions: 37°C, 5% CO2 humidified incubator. Passage number: cells used in experiments are not more than 20 passages.

[0078] Pre-treatment: Cells were plated overnight to log phase. Treatment time: NF-κΒ (6h, TNF-α induction with simultaneous treatment), PGC-1a (24h treatment), Collagen I (L929, 48h treatment).

[0079] 4. Model setup (inflammation / oxidative stress) Inflammation model (NF-κΒ): Induced with TNF-a 10 ng / mL (treatment samples were added simultaneously), incubated for 6h.

[0080] Oxidative stress model (PGC-1a): Induced with H202 100 μΜ for 1h, followed by 24h treatment with samples.

[0081] Model group setting explanation: In the NF-κΒ reporter assay, the model (TNF-a stimulation without sample) NF-κΒ activity was set as the reference (160%).

[0082] 5. Procedure and parameters for each assay 5.1 Cell viability (CCK-8) Purpose: To evaluate the cytotoxicity of the treatment concentrations and to determine the safe dose.

[0083] Procedure: Cells were seeded in 96-well plates at 5 x 10 3 cells / well and incubated overnight.

[0084] The medium was replaced with treatment (equivalent PQQ: 5, 10, 20 μΜ for each group), 4 wells per group, and incubated for 24h. The medium was removed and 100 μΐ^ of fresh medium + 10 μΐ^ of CCK-8 was added, incubated at 37°C for 1.5h. The OD 450nm was read using a microplate reader and the viability was calculated.

[0085] Criteria: If the 10 μΜ group has a viability of >90%, it is considered safe for functional experiments.

[0086] 5.2 NF-κΒ reporter gene activity Purpose: To quantitatively measure the NF-κΒ pathway activity (anti-inflammatory effect).

[0087] Procedure: HaCaT cells were seeded in 24-well plates (1 x 10 5 cells / well) and cultured until 60-70% confluence. The NF-κΒ-Luc reporter plasmid was transfected with Lipofectamine and co-transfected with Renilla internal control.

[0088] After 24h of transfection, TNF-a (10 ng / mL) was added to induce and samples (equivalent PQQ 10 μΜ) were added simultaneously, incubated for 6h.

[0089] Cells were collected and luciferase (Firefly) and Renilla activities were measured using Dual-Luciferase Assay according to the manufacturer's instructions. Results were normalized to Firefly / Renilla.

[0090] Expression: TNF-α, vector only (100%) represents relative activity. To ensure transfection efficiency and Renilla internal control stability, the same batch was processed in parallel.

[0091] 5.3 PGC-1a mRNA (qRT-PCR) Objective: To detect the transcriptional changes of PGC-1a, a key regulator of mitochondrial biogenesis (reflecting the effect of PQQ).

[0092] Procedure: HaCaT was treated for 24h (sample / control). Total RNA was extracted (Trizol) and cDNA was synthesized using a reverse transcription kit. qPCR (SYBR Green) was performed using the following primers: PGC-1a F / R; GAPDH internal control.

[0093] Cycling conditions: 95°C, 3min; 95°C, 15s, 60°C, 30s, for 40 cycles.

[0094] 5.4 Collagen I (COL I) secretion (ELISA, L929) Objective: To quantify the production of collagen I by fibroblasts (an indicator of dermal repair).

[0095] Procedure: L929 was seeded in 24-well plates (1 x 10^5 cells / well) and treated for 48h after adhesion.

[0096] Supernatant was collected and OD was read according to the ELISA kit (PICP) instructions. ng / mL was calculated.

[0097] Quality control: Standard curve R 2 ≥ 0.99, blank / negative control.

[0098] 6. Experimental data Table 1. Data for each indicator

[0099] (Note: *p <0.05; **p <0.01 compared to the model group.) From the above results, it can be seen that the anti-aging composition prepared by using the raw materials and method defined in the present application exhibits significant and multi-target anti-aging effects at the cellular level. The NF-κB activity of Example 1 is down-regulated from 160% of the model to 95.0%, p<0.01, indicating that the composition has obvious anti-inflammatory effect; the PGC-1a of Example 1 is up-regulated by 2.45 times, p<0.01, indicating that PQQ can effectively activate mitochondrial function in the carrier and formula, and play a complementary energy role; the collagen I of Example 1 is increased from 120 ng / mL of the control group to 220 ng / mL, p<0.01, indicating that it has good effect on skin repair / anti-wrinkle.

[0100] In summary, the present application overcomes the problems of low bioavailability and weak targeting of single component by optimizing the formula and matching the corresponding preparation method, and realizes the synergistic anti-aging of multiple active ingredients.

[0101] The above only describes the embodiments of the present application and is not intended to limit the present application. Various modifications and changes can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the scope of the claims of the present application.

Claims

1. An anti-aging composition, characterized in that, By weight percentage, it is prepared from the following raw materials: 2-4% Sophora japonica pollen extract, 0.5-1.0% disodium pyrroloquinoline quinone, 10-16% marine collagen peptides, 0.5-3.0% microalgae mustard oil nanoparticles, 1-3% fructooligosaccharides, 0.1-0.3% compound thickener, 2-5% natural sweetener, 0.2-0.6% natural acidulant, 0.05-0.2% flavoring, 1.0-3.0% lecithin, 0.2-1.0% cholesterol, and 0.5-1.0% glycerin, with the balance being water.

2. The anti-aging composition according to claim 1, characterized in that, The sophora japonica flower extract, disodium pyrrolidone, lecithin, and cholesterol are nano-sized to form nanoliposomes.

3. The anti-aging composition according to claim 2, characterized in that, The nanoliposomes have a particle size of 80-120 nm, an average encapsulation efficiency of not less than 88%, and an average drug loading of not less than 5%.

4. The anti-aging composition according to claim 1, characterized in that, The Sophora japonica pollen extract was obtained by supercritical CO2 extraction under the following conditions: 25-30 MPa, 40-45℃, 2-3 h, with a quercetin glycoside content ≥85%.

5. The anti-aging composition according to claim 1, characterized in that, The marine collagen peptides have an average molecular weight of 3-5 kDa.

6. The anti-aging composition according to claim 1, characterized in that, The microalgae mustard oil nanoparticles have a particle size of 80-120 nm and an EPA content of ≥20%.

7. The anti-aging composition according to claim 1, characterized in that, The composite thickener comprises xanthan gum and hydroxypropyl methylcellulose in a weight ratio of 1:(2-4). The natural sweetener comprises erythritol and monk fruit extract in a weight ratio of (3-4):1; The natural acidulant includes malic acid and citric acid in a weight ratio of 1:(1-2); the flavoring is a natural food-grade flavoring.

8. A method for preparing an anti-aging composition according to any one of claims 1-7, characterized in that, Includes the following steps: (1) Mix lecithin, cholesterol and glycerol in chloroform and rotary evaporate to form a uniform lipid dry film; (2) Weigh the Sophora japonica pollen extract and the disodium salt of pyrroloquinoline quinone separately, and then dissolve them together in phosphate buffer to prepare an aqueous phase containing the active component; (3) Add the aqueous phase obtained in step (2) to the lipid dry membrane obtained in step (1), let it stand at room temperature to hydrate, and then sonicate the hydrated suspension. (4) The sonicated suspension was homogenized under high pressure to obtain a nanoliposome suspension loaded with Sophora japonica pollen extract and disodium pyrroloquinoline quinone. (5) Marine collagen peptides, microalgae mustard oil nanoparticles, fructooligosaccharides, composite thickeners, natural sweeteners, natural acidulants, flavorings and water are added sequentially to the nanoliposome suspension. After stirring evenly, the mixture is sterilized under high pressure to obtain the anti-aging composition.

9. The preparation method according to claim 8, characterized in that, In step (1), the rotary evaporation conditions are a temperature of 40-45℃ and a rotation speed of 120-150 rpm; In step (3), the hydration time is 1-2 hours, the ultrasonic parameters are 20-25 kHz, 200-220 W, and the ultrasonic treatment is 5-8 minutes. In step (4), the conditions for high-pressure homogenization are a pressure of 80-100 MPa and three cycles; in step (5), the temperature is 110-125℃ and the time is 10-15 min.

10. The application of the anti-aging composition according to any one of claims 1-7 or the anti-aging composition prepared by any one of the preparation methods according to claims 8-9, characterized in that, The anti-aging composition is used in dietary supplements.

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