Water-in-oil-in-water three-phase coated microbead as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a water-in-oil-in-water three-phase coated microbead as well as a preparation method and application thereof. The water-in-oil-in-water three-phase coated microbead is of a spherical structure and sequentially comprises an outer water phase, an inner oil phase and an inner water phase from outside to inside, the outer water phase comprises sodium alginate, agarose, cyclodextrin, glycerin and other components, the inner oil phase comprises caprylic / capric triglyceride, grape seed oil, olive squalane, tocopherol and other components, and the inner water phase comprises sodium alginate, agarose, cyclodextrin, glycerin and other components. And the inner water phase comprises ampelopsis grossedentata extract, astragalus membranacus root extract and other components. According to the invention, an easily oxidized and unstable active matter is fully and firmly protected by adopting a droplet microfluidic coating technology, so that the active matter is free from external stimulation, active ingredients with different effects play multiple roles, and the composition has good anti-inflammatory, soothing and oil-controlling effects and can be applied to the field of cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a water-in-oil-in-water three-phase encapsulated microsphere, its preparation method, and its application. Background Technology

[0002] Hydrogel microcapsules are miniature carriers formed by encapsulating active substances in a hydrophilic three-dimensional network structure, typically with diameters in the micrometer range. Their core characteristics include high biocompatibility, controlled release capability, and responsiveness to environmental factors such as temperature and pH, leading to their widespread applications in drug delivery, cell encapsulation, and sustained-release cosmetics. However, existing technologies still have some limitations for applications involving complex water-in-oil-in-water three-phase encapsulation systems.

[0003] Currently, most hydrogel microcapsules are composed of single polymers such as sodium alginate, chitosan, agar, and gelatin, which react with metal ions (Ca). 2+ Mg 2+ It is obtained through cross-linking. To encapsulate easily oxidized and deteriorated functional active substances, fat-soluble active substances, and plant essential oils, an emulsifier needs to be added and homogenized under high pressure to form a water-oil mixed emulsion before encapsulation. This encapsulation technology has the following drawbacks and shortcomings: ① Existing hydrogel microcapsule technology only utilizes the basic properties of the material itself, without combining it with specific application needs to fully explore its more functions and effects. ② Encapsulating functional active substances, fat-soluble active substances, and plant essential oils requires the use of emulsifiers for high-temperature and high-pressure emulsification and homogenization, which can easily affect some temperature-sensitive active substances, thus greatly weakening the efficacy of encapsulated active substances. ③ Traditional single emulsifiers cannot completely guarantee the full and stable mixing of the water-oil mixed emulsion, resulting in incomplete encapsulation, low encapsulation rate, and other stability problems, as well as low effective utilization of active substances or essential oils, directly affecting the release efficiency and efficacy of active substances. ④ Microcapsule networks composed of single polymers lack the ability to encapsulate multiple components, failing to achieve multi-dimensional skincare effects.

[0004] Microfluidics is a technology that manipulates or controls liquids at the nanoliter or picoliter level within micrometer-scale channels. This technology focuses on constructing microfluidic channel systems to achieve various complex microfluidic manipulation functions. Based on their basic structure, microfluidic chips can be categorized into coaxial focusing, flow focusing, T-type, and Y-type. Current applications in the cosmetics industry include the preparation of multiple emulsion systems, in vitro efficacy testing, in vitro skin models, and the development of active ingredient carriers. Its main application in cosmetics lies in improving product development efficiency and safety testing. Its core advantage lies in the precise manipulation of tiny fluids, enabling efficient preparation and testing. Summary of the Invention

[0005] In order to overcome the shortcomings and drawbacks of existing technologies such as low stability and low retention rate of active ingredients of vine tea extract (vine tea acidic polysaccharide APG-2a) and Astragalus membranaceus root extract, the primary objective of this invention is to provide a water-in-oil-in-water (W / O / W) three-phase encapsulated microbeads, which have good anti-inflammatory, soothing and oil-controlling effects.

[0006] Another objective of this invention is to provide a method for preparing the above-mentioned water-in-oil-in-water three-phase encapsulated microspheres, which can stably encapsulate easily oxidized and discolored active substances in multiple ways.

[0007] Another object of the present invention is to provide the application of the above-mentioned water-in-oil-in-water three-phase encapsulated microbeads in the field of cosmetics.

[0008] The objective of this invention is achieved through the following technical solution: A water-in-oil-in-water (W / O / W) three-phase encapsulated microsphere has a spherical structure, consisting of an outer aqueous phase, an inner oil phase, and an inner aqueous phase from the outside to the inside. The external aqueous phase comprises the following components by mass percentage: sodium alginate 0.3-0.4%, agarose 0.2-0.25%, cyclodextrin 0.5-1.5%, glycerol 5-10%, hexanediol 0.1-0.5%, p-hydroxyacetophenone 0.1-0.5%, and water 40-45%; The inner oil phase comprises the following components by mass percentage: 5-10% caprylic / capric triglycerides, 1-5% grape seed oil, 1.5-5.5% olive squalane, 0.1-0.5% tocopherol (vitamin E), 1-3% natural wax, and 1-10% castor oil / IPDI copolymer; The internal aqueous phase comprises the following components by mass percentage: 0.1-1.5% vine tea extract (vine tea acidic polysaccharide APG-2a), 1-5% Astragalus membranaceus root extract, 0.01-0.05% hyaluronic acid, 2-5% butylene glycol, 0.05-0.1% xanthan gum, and 5-6% water; The total mass percentage of all components in the external aqueous phase, internal oil phase, and internal aqueous phase is 100%.

[0009] The mass ratio of the external aqueous phase, the internal oil phase, and the internal aqueous phase is (3.5-3.6):(1.9-2.0):1.

[0010] The method for preparing water-in-oil-in-water three-phase encapsulated microspheres includes the following steps: (1) Preparation of external aqueous phase: Take the components of external aqueous phase, agarose and p-hydroxyacetophenone, and add them to the water of external aqueous phase preheated to 85-90℃. Keep warm at 85-90℃ and stir to dissolve to obtain mixture 1; take the components of external aqueous phase, sodium alginate, cyclodextrin, glycerol and hexanediol, and add them to the remaining water of external aqueous phase preheated to 50-60℃. Stir to dissolve to obtain mixture 2; pour mixture 1 into mixture 2 and mix well to obtain external aqueous phase; (2) Preparation of inner oil phase: The components of the inner oil phase, castor oil / IPDI copolymer, caprylic / capric triglyceride and natural wax, are thoroughly stirred and dissolved. Then, the components of the inner oil phase, olive squalane, tocopherol (vitamin E) and grape seed oil, are added and stirred evenly to obtain the inner oil phase. (3) Preparation of the inner aqueous phase: Hyaluronic acid, a component of the inner aqueous phase, was added to butanediol to disperse it. Then xanthan gum and water were added, and the mixture was heated and stirred at 80-85℃ for 15-20 minutes to fully dissolve the hyaluronic acid and xanthan gum. The mixture was then cooled to 45-50℃ and vine tea extract (vine tea acidic polysaccharide APG-2a) and Astragalus membranaceus root extract were added and stirred evenly to obtain the inner aqueous phase. (4) The outer aqueous phase obtained in step (1), the inner oil phase obtained in step (2) and the inner aqueous phase obtained in step (3) are injected into the corresponding microchannels of the microfluidic device, so that the inner aqueous phase and the inner oil phase are mixed to form water-in-oil (W / O) sandwich droplets, and the W / O sandwich droplets are mixed with the outer aqueous phase to form W / O / W droplets. (5) The W / O / W droplets flowed out of the tube and dripped into a 0.15-0.25% (w / w) solution of calcium pyrrolidone carboxylate (PCA-Ca) for further solidification to obtain water-in-oil-in-water (W / O / W) three-phase encapsulated microspheres.

[0011] The amount of water used in the external water phase in step (1) is 10-15%.

[0012] The microfluidic device mentioned in step (4) is a three-phase flow droplet microfluidic device, and its structure is preferably coaxial focusing type, flow focusing type, T-type or Y-type, etc.

[0013] The three-phase flow droplet microfluidic device is preferably composed of a T-shaped channel, wherein the channel includes a main channel and a side channel that are interconnected, wherein the main channel and the side channel are set at 90 degrees; the side channel includes a first side channel and a second side channel, wherein the first side channel and the second side channel are coaxially arranged, and the first side channel is located inside the second side channel; the diameter ratio of the first side channel, the second side channel and the main channel is 1:2:4.

[0014] The W / O / W droplets described in step (4) are preferably prepared by the following method: The inner aqueous phase obtained in step (2) is injected into the first side channel of the three-phase flow droplet microfluidic device, the inner oil phase is injected into the second side channel, and the outer aqueous phase is injected into the main channel; the inner aqueous phase and the inner oil phase form W / O sandwich droplets in the side channel, and the W / O sandwich droplets and the outer aqueous phase are mixed at the junction of the main channel and the side channel to form W / O / W droplets.

[0015] The preferred flow velocity ratio of the internal water phase, internal oil phase, and external water phase in the pipeline is 1:5:10.

[0016] The mass percentage of the pyrrolidone calcium carboxylate solution mentioned in step (5) is 0.15-0.25%.

[0017] The application of the aforementioned water-in-oil-in-water (W / O / W) three-phase encapsulated microbeads in the cosmetics field.

[0018] An essence containing the aforementioned water-in-oil-in-water three-phase encapsulated microbeads.

[0019] The principle of this invention: Vine tea, also known as berry tea, fairy tea, dragon beard tea, longevity vine, and vine matriarch tea, is shaped like a dragon. During processing, its cells break down, allowing the flavonoid active ingredients within the cells to permeate to the surface, forming a layer of "white frost," giving it a unique appearance. Both the tender leaves and mature fruits are edible. The Ming Dynasty medical text *Jiuhuang Bencao* records its use as a substitute food during famines. Modern research shows that its extracts can prevent thrombosis, lower blood sugar, and enhance immunity, and have an adjunctive therapeutic effect on sore throat and hepatitis. Because it is rich in various bioactive components (such as flavonoids, polyphenols, polysaccharides, and proteins), it can effectively inhibit the release of inflammatory factors, reduce skin redness and sensitivity, and also has good oil-controlling properties.

[0020] Astragalus membranaceus is a plant belonging to the genus Astragalus in the legume family. It is the same source of authentic Astragalus listed in the Chinese Pharmacopoeia as Astragalus mongholicus. The Shennong's Classic of Materia Medica first recorded: "Astragalus, sweet in taste, slightly warm. It is used to treat carbuncles, chronic sores, drain pus and relieve pain, treat epilepsy, hemorrhoids, fistulas, and to replenish deficiencies and treat various childhood illnesses." Because it is rich in flavonoids (such as gentianin and verbascoside), saponins (astragaloside A), polysaccharides, and various amino acids and trace elements (iron, zinc, selenium, etc.), it also possesses antioxidant, anti-inflammatory, and immunomodulatory effects. By regulating immunity and improving microcirculation, it can effectively relieve skin sensitivity and inflammation.

[0021] To address skin inflammation caused by various factors, this invention utilizes a combination of *Astragalus membranaceus* root extract (*Acidic Polysaccharide APG-2a*) and *Astragalus membranaceus* root extract to achieve effective oil control, anti-inflammatory, and soothing effects. However, both *Acidic Polysaccharide APG-2a* and *Astragalus membranaceus* root extract are prone to oxidation and discoloration, and are sensitive to metal ions and pH. To address this issue and ensure maximum preservation and utilization of the material's efficacy, this invention employs the following technical means in raw material and process selection: (1) Selection of inner oil phase: This invention selects a combination of natural plant essential oils, grapeseed oil and olive squalane. Grapeseed oil is rich in linoleic acid, which has good effects in soothing and anti-inflammatory, strengthening the skin barrier, and regulating oil. The structure of olive squalane is very similar to that of squalene in human sebum, so it has good repair and soothing effects while enhancing the skin's moisturizing barrier. At the same time, castor oil / IPDI copolymer and natural wax are added to form a stable "isolation film" to more fully encapsulate the active substances in the inner aqueous phase, avoiding the oxidation and discoloration problems caused by external environmental stimuli to vine tea extract (vine tea acidic polysaccharide APG-2a) and Astragalus membranaceus root extract.

[0022] (2) Selection of external aqueous phase: This invention selects a combination of cyclodextrin, sodium alginate, and agarose. Cyclodextrin is a rigid cyclic oligosaccharide formed by a small number (6-8) of glucose monomers linked by glycosidic bonds. It has a unique cyclic conical structure and a hydrophobic inner cavity and a hydrophilic outer surface. This structure allows it to encapsulate molecules of various sizes and polarities (guest molecules). When the cyclodextrin inclusion complex is applied to the skin, the active ingredients encapsulated in the cavity need to be gradually released, which can stabilize easily decomposed active ingredients. Sodium alginate is a natural high-molecular-weight polysaccharide, mainly extracted from brown algae (such as kelp and giant kelp). It has good biocompatibility and biodegradability, and good gelling and film-forming properties. Agarose is a natural polysaccharide extracted from red algae (such as Gracilaria). Its main components are galactose and its derivatives. It has good moisturizing, gelling, and film-forming properties. Therefore, when used in combination, cyclodextrin can stabilize and regulate the active ingredients in the inner oil phase, while sodium alginate and agarose form a tight and stable network structure to encapsulate the inner oil phase. Finally, the mixture is further solidified in a PCA-Ca (calcium pyrrolidone carboxylate) aqueous solution to obtain the final water-in-oil-in-water three-phase encapsulated microspheres. The combination of sodium alginate, agarose, and cyclodextrin not only increases the encapsulation stability but also further improves bioavailability.

[0023] (3) This invention employs droplet microfluidics to form microspheres through W / O / W three-phase flow encapsulation. Figure 1 and Figure 2 Compared with existing production processes, it can achieve more precise and stable encapsulation and utilization of active ingredients.

[0024] The present invention has the following advantages and effects compared with the prior art: (1) The present invention uses droplet microfluidic encapsulation technology to fully and firmly protect the easily oxidized and unstable active ingredients, so as to protect them from external stimuli (such as heat, pH, light and oxidation), and the active ingredients with different functions can play multiple roles. When applied to cosmetics, it not only plays an effective role, but also solves the problem of some easily oxidized and unstable active ingredients causing discoloration and taste changes in the formula.

[0025] (2) Sodium alginate, agarose, cyclodextrin and other polymers are used in synergy to more firmly seal and encapsulate while further improving the utilization of active ingredients, avoiding waste and damage to the skin barrier.

[0026] (3) The encapsulation material selected in this invention is of natural origin, conforms to the current green and environmentally friendly skin care, and has good biocompatibility.

[0027] (4) The present invention uses droplet microfluidic technology for encapsulation, which is efficient and simple, accurately protects the efficacy of active ingredients and is easy to industrialize. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the W / O / W microsphere structure, where 1: internal aqueous phase, 2: internal oil phase, and 3: external aqueous phase.

[0029] Figure 2 This is a schematic diagram of a three-phase flow droplet microfluidic device and the preparation of W / O / W microbeads, where 4: main channel, 5: first side channel, and 6: second side channel.

[0030] Figure 3 This is a graph showing the results of the RAW264.7 macrophage nitric oxide release inhibition assay.

[0031] Figure 4 This is a graph showing the relative lipid content within human sebaceous gland cells. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0033] like Figure 2 As shown in the embodiment, the three-phase flow droplet microfluidic device is composed of a T-shaped channel, wherein the channel includes a main channel 4 and a side channel that are interconnected, wherein the main channel 4 and the side channel are set at 90 degrees; the side channel includes a first side channel 5 and a second side channel 6, wherein the first side channel 5 and the second side channel 6 are coaxially arranged, and the first side channel 5 is located inside the second side channel 6; the diameter ratio of the first side channel 5, the second side channel 6 and the main channel 4 is 1:2:4.

[0034] The extraction process of vine tea extract (vine tea acidic polysaccharide APG-2a) in the examples is as follows: (1) After washing the fresh leaves of vine tea, they are transferred to a belt dryer and dried at 60°C with forced air until the moisture content is ≤8%. They are then coarsely crushed to 20 mesh (0.8 mm). Then, they are further processed by an air jet mill to a particle size ≤50 μm (passing through a 200 mesh sieve) to destroy the cell wall structure and increase the dissolution surface area. (2) Mix the crushed vine tea and n-hexane in step (1) at a ratio of 1:8 and defatt it at 50°C for 1 hour; filter, discard the filtrate, and evaporate the solvent from the residue for later use; add deionized water and sonicate (40KHZ for 35 minutes), centrifuge, and obtain the supernatant. (3) After concentrating the supernatant under reduced pressure, add 95% ethanol (volume fraction), let stand at 4°C for 12 hours, and collect the precipitate by centrifugation, which is crude polysaccharide. (4) The crude polysaccharide was redissolved in water and the protein was removed by the Sevag method. Specifically, chloroform and n-butanol were mixed at a volume ratio of 4:1 and added to the crude polysaccharide solution. The mixture was shaken for 30 min and centrifuged to remove the protein layer. 1 wt.% of activated carbon was added to the system and stirred at 70°C for 40 min. The mixture was then filtered to obtain a pale yellow polysaccharide solution. (5) Further concentration under reduced pressure, followed by fine purification using ion exchange resin. Specifically, DEAE-agarose gel CL-6B column was used with sodium chloride solution (0-2 mol / L) as the eluent for gradient elution. The elution peak solution (containing the target substance) in tubes 97-136 was collected and finally freeze-dried to obtain the finished product of vine tea acid polysaccharide APG-2a.

[0035] The extraction process of Astragalus membranaceus root extract in the examples is as follows: (1) The roots of Astragalus membranaceus were screened and cleaned, then transferred to a belt dryer and dried with hot air circulation at 60°C until the moisture content was ≤8%. They were then coarsely pulverized to 20 mesh (0.8 mm). Then, they were processed by an air jet mill to a particle size ≤50 μm (passing through a 200 mesh sieve) to destroy the cell wall structure and increase the dissolution surface area. (2) Mix the crushed Astragalus membranaceus root from step (1) with hexane at a ratio of 1:8 and defatt it at 50°C for 1 hour; filter, discard the filtrate, and evaporate the solvent from the residue for later use; add an enzymatic hydrolysate containing 0.05-0.07 wt.% cellulase (50 U / mg) and 0.03-0.05 wt.% papain (800 U / mg), sonicate (16 kHz for 40 min), and centrifuge to obtain the supernatant; (3) The supernatant was filtered through a microfiltration ceramic membrane (to remove tannins and plant fibers), and then concentrated through a spiral wound ultrafiltration membrane; then 0.8 wt.% activated carbon was added, stirred at 70°C for 40 min, filtered through a plate and frame filter until colorless, dehydrated and concentrated through a nanofiltration membrane, and finally freeze-dried under vacuum to obtain the finished product of Astragalus membranaceus root extract.

[0036] In the following examples, the cyclodextrin was hydroxypropyl β-cyclodextrin, the natural wax was carnauba wax, purchased from Guangzhou Limeda Import & Export Trading Co., Ltd., the silicone elastomer (DOWSIL™ 9040) was purchased from Dow Chemical (Shanghai) Co., Ltd., and other reagents were common reagents, all of which are commercially available.

[0037] Example 1 (1) Preparation of external aqueous phase: According to Table 1, weigh the components of the internal aqueous phase, agarose and p-hydroxyacetophenone, and add them to hot water preheated to 85-90℃ (the amount used is 15% of the total amount of water used in the external aqueous phase). Keep the temperature at 85-90℃ and stir to dissolve to obtain mixture 1, which is ready for use. According to Table 1, weigh the components of the internal aqueous phase, sodium alginate, cyclodextrin, glycerol and hexanediol, and add them to the remaining hot water preheated to 50-60℃ (the amount used is 27.8%). Stir to dissolve to obtain mixture 2. Pour mixture 1 (the dissolved agarose solution) into mixture 2 and mix well to obtain the external aqueous phase.

[0038] (2) Preparation of inner oil phase: The components of the inner oil phase, castor oil / IPDI copolymer, caprylic / capric triglyceride and natural wax, were thoroughly stirred and dissolved according to Table 1. Then, olive squalane, tocopherol (vitamin E) and grape seed oil were added and stirred evenly to obtain the inner oil phase.

[0039] (3) Preparation of the inner aqueous phase: Weigh the hyaluronic acid powder of the inner aqueous phase according to Table 1 and add it to butanediol to disperse it. Then add xanthan gum and deionized water, heat and stir at 80-85℃ for 20 minutes to fully dissolve hyaluronic acid and xanthan gum. Then cool down to 45-50℃ and add vine tea extract (vine tea acidic polysaccharide APG-2a) and Astragalus membranaceus root extract and stir evenly to obtain the inner aqueous phase.

[0040] (4) Inject the external aqueous phase obtained in step (1), the internal oil phase obtained in step (2), and the internal aqueous phase obtained in step (3) into the corresponding microchannels of the three-phase flow droplet microfluidic device respectively. Figure 2 The inner aqueous phase is injected into the first side channel 5 of the three-phase flow droplet microfluidic device, the inner oil phase is injected into the second side channel 6, and the outer aqueous phase is injected into the main channel 4. The inner aqueous phase and the inner oil phase form W / O sandwich droplets in the side channels. The W / O sandwich droplets and the outer aqueous phase mix at the junction of the main channel and the side channels to form W / O / W droplets. The flow velocity ratio of the inner aqueous phase, the inner oil phase and the outer aqueous phase in the channels is 1:5:10.

[0041] (5) W / O / W droplets flowed out of the tube and dripped into the PCA-Ca (pyrrolidone calcium carboxylate) aqueous solution for further solidification, resulting in water-in-oil-in-water (W / O / W) three-phase encapsulated microbeads. Example 2

[0042] The specific preparation method is the same as in Example 1, and the composition and content of the raw materials are shown in Table 1. Example 3

[0043] The specific preparation method is the same as in Example 1, and the composition and content of the raw materials are shown in Table 1.

[0044] Table 1. Raw material composition and content in Examples 1-3

[0045] Comparative Example 1: Removed vine tea extract The specific preparation method is the same as in Example 1, and the composition and content of the raw materials are shown in Table 2.

[0046] Comparative Example 2: Astragalus root extract without membrane capsules The specific preparation method is the same as in Example 1, and the composition and content of the raw materials are shown in Table 2.

[0047] Table 2. Raw material composition and content of Comparative Example 1 and Comparative Example 2

[0048] Comparative Example 3: Carrageenan replaces sodium alginate The specific preparation method is the same as in Example 1, and the composition and content of the raw materials are shown in Table 3.

[0049] Table 3. Raw material composition and content of Comparative Example 3

[0050] Comparative Example 4: Gellan gum replaced agarose The specific preparation method is the same as in Example 1, and the composition and content of raw materials are shown in Table 4.

[0051] Table 4. Raw material composition and content of Comparative Example 4

[0052] Comparative Example 5: Silicone elastomer replacing castor oil / IPDI copolymer The specific preparation method is the same as in Example 1, and the composition and content of raw materials are shown in Table 5.

[0053] Table 5. Raw material composition and content of Comparative Example 5

[0054] Comparative Example 6: Replacing Natural Wax with Synthetic Wax The specific preparation method is the same as in Example 1, and the composition and content of raw materials are shown in Table 6.

[0055] Table 6. Raw material composition and content of Comparative Example 6

[0056] Comparative Example 7: The production process for all raw materials used in Example 3 was replaced with direct emulsification homogenization and encapsulation. According to Example 3, an external aqueous phase, an internal oil phase, and an internal aqueous phase were prepared respectively. The internal aqueous phase was added to the internal oil phase, and after stirring, emulsifying, and homogenizing using conventional methods, it was added to the external aqueous phase and stirred, emulsified, and homogenized further. Effect Example

[0057] The water-in-oil-in-water triple-phase encapsulated microbeads prepared in Examples 1-3 and Comparative Examples 1-7 were added to a prepared essence for efficacy verification. The essence contained the following components by mass percentage: 83.6% pure water, 0.5% p-hydroxyacetophenone, 5% glycerin, 0.2% carbomer, 0.2% arginine, 10% water-in-oil-in-water triple-phase encapsulated microbeads, and 0.5% hexanediol. The essence was prepared as follows: carbomer, p-hydroxyacetophenone, glycerin, and pure water were added to a container and heated to 85°C with stirring to disperse. Homogenization was performed at a low speed of 3000-5000 r / min. The temperature was lowered to 60-65°C to defoam, and arginine was added for neutralization. The temperature was lowered to 45-50°C, and water-in-oil-in-water triple-phase encapsulated microbeads and hexanediol were added to obtain the microbead essences of Examples 1-3 and Comparative Examples 1-7, respectively.

[0058] I. Stability testing and active ingredient detection According to the "Technical Guidelines for Stability Testing and Evaluation of Cosmetics," the microbead essences of Examples 1-3 and Comparative Examples 1-7 were subjected to six different conditions for a three-month testing period: room temperature (25°C, protected from light), high temperature (45°C, protected from light), low temperature (-10°C, protected from light), low temperature (5°C, protected from light), light exposure (30°C), and alternating hot and cold cycles (45°C for 24 hours, -10°C for 24 hours, protected from light). The appearance, color, and odor were examined to evaluate the stability of the samples. Simultaneously, at the end of the fourth and eleventh weeks of observation, high-performance liquid chromatography (HPLC) was used to detect changes in the content of water-in-oil-in-water encapsulated microbead active ingredients in the essences to evaluate the stability of the water-in-oil-in-water microbead active ingredient content. The specific method is as follows: 1. Detection Target and Principle The active ingredient content of *Astragalus membranaceus* extract (acidic polysaccharide APG-2a) and *Astragalus membranaceus* extract in the aqueous phase of water-in-oil-in-water microbeads was determined by HPLC. The microbeads have a three-layer structure of "outer aqueous phase-inner oil phase-inner aqueous phase". It is necessary to first destroy the outer aqueous phase (sodium alginate, agarose, etc.) and the inner oil phase (natural wax, squalane, etc.) to release the active ingredient in the inner aqueous phase. Then, the target component is separated by HPLC, and the content is calculated by standard sample comparison method (qualitative analysis by retention time and quantitative analysis by peak area).

[0059] 2. Reagents and Instruments 2.1 Reagents Standards: Vine Tea Extract (TCI, Tokyo Kasei; purity ≥98%), Astragalus membranaceus Extract (TCI, Tokyo Kasei; purity ≥98%).

[0060] Extraction solvents: 50% methanol (chromatographic grade methanol + ultrapure water, v / v), 0.1 mol / L sodium citrate solution (to destroy the external aqueous phase), and n-hexane (analytical grade, to remove the internal oil phase).

[0061] Mobile phase: *Tea vine extract* detection: methanol-0.1% phosphoric acid aqueous solution (40:60, v / v); *Astragalus membranaceus* extract detection: acetonitrile-water (32:68, v / v).

[0062] Other: ultrapure water (18.2 MΩ·cm), 0.22 μm organic phase filter membrane, solid phase extraction column (C18, 500 mg / 6 mL, for purification).

[0063] 2.2 Instruments High-performance liquid chromatograph (with UV detector), C18 column (250mm×4.6mm, 5μm), electronic balance (accuracy 0.0001g), ultrasonic extractor, high-speed centrifuge (12000r / min), vortex mixer, solid-phase extraction device and nitrogen blower, etc.

[0064] 3. Experimental Methods 3.1 Sample pretreatment (10 microbead essences under 6 stability conditions): Accurately weigh 1.0 g of the microbead essence sample and place it in a 50 mL centrifuge tube. Add 5 mL of 0.1 mol / L sodium citrate solution (to disrupt the outer aqueous phase), vortex to mix, and let stand at room temperature for 10 min. Observe the dissolution of the outer layer of microbeads (in a loose state). Add 5 mL of n-hexane (to dissolve the inner oil phase components such as oil and squalane), vortex for 2 min, and centrifuge at 4℃ and 8000 r / min for 10 min. Discard the upper n-hexane phase (inner oil phase impurities) and retain the lower aqueous phase (containing the active ingredients of the inner aqueous phase and the residues of the outer aqueous phase). Add 10 mL of 50% methanol to the retained lower aqueous phase (to extract the inner aqueous phase extracts of *Tea japonica* and *Astragalus membranaceus*), and sonicate for 30 min (power 300 W, temperature 30℃, to avoid high temperature damage to the active ingredients). Centrifuge at 4℃ and 12000 r / min for 15 min, and take the supernatant and place it in a 25 mL volumetric flask. The residue was extracted once with 5 mL of 50% methanol. After centrifugation, the supernatants were combined and diluted to 25 mL with 50% methanol. The mixture was shaken well to obtain the crude extract.

[0065] 3.2 Sample purification (10 microbead essences under 6 stability conditions): Activate the solid-phase extraction column (C18), load 5 mL of crude extract onto the column, and after the solution has completely passed through the column, rinse with 5 mL of ultrapure water (to remove polar impurities) and discard the eluent; elute with 8 mL of 70% (v / v) methanol (collect the target active substance), concentrate the eluent to near dryness using a nitrogen blower, and make up to 2 mL with 50% methanol. Filter through a 0.22 μm organic phase membrane and proceed with analysis.

[0066] 3.3 Preparation of standard working solution: Vine tea extract: Accurately weigh 10.0 mg of vine tea extract standard, dissolve it in 50% methanol and dilute to 10 mL to obtain a concentration of 1.0 mg / mL. Store at 4℃ protected from light. Take 0.1, 0.2, 0.5, 1.0 and 2.0 mL of each solution and dilute to 10 mL with 50% methanol to obtain a series of standard solutions with concentrations of 10, 20, 50, 100 and 200 μg / mL.

[0067] Astragalus membranaceus extract: Accurately weigh 10.0 mg of Astragalus membranaceus extract standard, dissolve it in methanol and dilute to 10 mL to obtain a concentration of 1.0 mg / mL. Store at 4℃ protected from light. Take 0.05, 0.1, 0.2, 0.5 and 1.0 mL of each solution and dilute to 10 mL with methanol to obtain a series of standard solutions with concentrations of 5, 10, 20, 50 and 100 μg / mL.

[0068] 3.4 Standard curve plotting and sample determination Inject a series of standard working solutions and record the peak area. Plot a standard curve with the standard concentration (μg / mL) as the x-axis and the peak area as the y-axis, and calculate the regression equation (y=ax+b) and the correlation coefficient (R² must be ≥0.999). Inject the test solution after "sample purification" and perform three parallel determinations, recording the retention time (qualitative) and peak area of ​​the target peak.

[0069] 3.5 Result Calculation Formula for calculating the content (by mass fraction, %) of active ingredients in water-in-oil-in-water three-phase encapsulated microspheres: Content (%) – (C×V×D) / (m×10) 6 ) × 100%

[0070] In the formula, C: concentration of active ingredient in the test solution calculated from the standard curve (μg / mL); V: final volume of sample (2mL); D: total dilution factor; m: sample weight of microbead essence (g); 10 6 Unit conversion factor (μg→g).

[0071] Table 7. Stability of microbead essence samples at room temperature.

[0072] Table 8. Stability of microbead essence samples under high temperature conditions

[0073] Table 9. Stability of microbead essence samples under low temperature conditions

[0074] Table 10. Stability of microbead essence samples under low temperature conditions

[0075] Table 11 Stability of microbead essence samples under light conditions

[0076] Table 12 Stability of microbead essence samples under thermal cycling conditions

[0077] The stability test results (Table 7-12) show that Examples 1-3 and Comparative Examples 1 and 2 exhibit good stability. However, Comparative Examples 3-7, due to the substitution of raw materials and production processes, showed unstable encapsulation, including surface cracks, damage, incompatibility of the oil phase within the microspheres, and oil leakage. High-performance liquid chromatography (HPLC) was used to detect the active ingredients of the water-in-oil-in-water three-phase encapsulated microspheres under six different stability conditions at the end of the fourth and eleventh weeks. The results showed that the active ingredient content in Examples 1-3 was stable, while the active ingredient content in Comparative Examples 1-7 decreased to varying degrees due to differences in encapsulation raw materials and production processes. This indicates that the selection and combination of raw materials in the external aqueous phase have a significant impact on the encapsulation properties of the microspheres.

[0078] II. Soothing Efficacy Test 1. Experimental Objective and Principle This invention uses T / GDCDC 030-2023 "Test of Soothing Efficacy of Cosmetics: Determination of Inhibition of Nitric Oxide (NO) Release from Macrophages in Vitro" to test the NO inhibition effect of the water-in-oil-in-water three-phase encapsulated microbead essence.

[0079] Bacterial lipopolysaccharide (LPS) stimulates macrophages (RAW264.7 cell line), inducing the release of the inflammatory mediator nitric oxide (NO). NO is readily oxidized to NO in vivo or in aqueous solution. 2- Under acidic conditions, NO 2- The product undergoes a diazo reaction with diazonium salt sulfonamide to generate a diazo compound, which further couples with theaylvinyldiamine. The concentration of the product generated in this reaction is linearly related to the NO concentration, with a maximum absorption peak at 550 nm. The optical density (OD) value at 550 nm is measured using a microplate reader to calculate the relative NO content. The NO inhibition rate of the sample group after administration is measured to evaluate the inhibitory effect of the sample on NO release, thereby evaluating whether the sample has a soothing effect.

[0080] 2. Experimental materials, reagents, and equipment 2.1 Experimental Materials and Reagents Mouse mononuclear macrophage leukemia cells RAW264.7 were derived from icell. Fetal bovine serum (FBS, Pronosai), DMEM high-glucose medium (DMEM, Pronosai), thiazolyl blue (MTT, BioFroxx), LPS (Solarbio), dexamethasone (Solarbio), trypsin / EDTA solution (Gibco), phosphate-buffered saline (PBS, Pronosai), and a nitric oxide (NO) assay kit (Bioswamp) were used.

[0081] 2.2 Test Instruments and Equipment Carbon dioxide incubator, biosafety cabinet, inverted phase contrast microscope, ELISA reader, electronic balance, low-speed centrifuge, cell plate shaker, vortex mixer, pipette.

[0082] 3. Test Methods 3.1 Cytotoxicity test methods This experiment used MTT assay to detect cell viability and screen the maximum safe concentration of cells for administration. A blank control (culture medium) and a zeroing well (PBS) were included. Sample concentrations were set as follows: 15%, 6.98%, 3.25%, 1.51%, 0.70%, 0.32%, 0.15%, and 0.07% (v / v), with six replicate wells for each concentration. Specific operating procedures are as follows: (1) Seeding: RAW264.7 cells in the logarithmic growth phase were digested and seeded into 96-well plates, 100 µL (1×10⁻⁶) per well. 5 (cells / mL), ensuring uniform cell adhesion, and incubate the culture plate in a 37°C, 5% CO2 incubator for 24 hours.

[0083] (2) Sample feeding: After the cells have grown for 24 hours, the supernatant was discarded and DMEM high glucose serum medium containing different concentrations of the test substance was added. The culture medium was then placed in a 37°C, 5% carbon dioxide incubator for 24 hours.

[0084] (3) Detection: After culturing cells for 24 h, wash twice with PBS, add 50 µL / well MTT working solution, and continue culturing in a CO2 incubator for 6 h; discard the MTT solution, add 150 µL / well DMSO, shake for 5 min, and use 570 nm as the reference wavelength to read the OD value using a microplate reader. 570nm The relative cell viability was calculated using the following formula: Cell viability (%) = (OD sample / OD blank) × 100%

[0085] The cytotoxicity test results are shown in Table 13 below. As can be seen from the table, the cell survival rate was highest when the test sample concentration was 1.51%, and this test concentration can be used for further experiments.

[0086] Table 13 Results of RAW264.7 macrophage toxicity assay

[0087] 3.2 Macrophage RAW264.7 Nitric Oxide (NO) Release Inhibition Assay (1) Cell seeding: Add 100 µL of 1×10⁶ cells per well of a 96-well plate. 5The cells were suspended in RAW264.7 cell suspension at 37°C and 5% CO2 for 24 hours to ensure uniform cell adhesion.

[0088] (2) Solution preparation: Prepare the test substance and positive control according to Table 14.

[0089] Table 14 Experimental Groups and Concentration Settings

[0090] (3) Drug administration: Discard the culture medium in the 96-well plate and carry out induction and drug administration. Add sample solution to the test well according to Table 15, add negative control solution to the negative control well, add positive control solution to the positive control well, and add complete culture medium to the blank control well, 100µL per well; then place the 96-well plate in a carbon dioxide incubator for 24±2h.

[0091] (4) Collection of cell supernatant: After the incubation culture is completed, collect 100µL of cell culture supernatant from each well into a 1.5mL sterile centrifuge tube and freeze it in an ultra-low temperature freezer at -80℃.

[0092] (5) Griss test: The Griss test should be performed in accordance with the instructions of the nitric oxide test kit.

[0093] (6) Formula for calculating NO inhibition rate: Inhibition rate (%) = (1 – T / C) × 100%

[0094] In the formula: T is the mean OD value of the sample group; C is the mean OD value of the negative control group.

[0095] Results of RAW264.7 macrophage nitric oxide (NO) release inhibition assay: Figure 3 As shown. By Figure 3 It can be seen that the present invention has significant effects in anti-inflammatory and soothing aspects. The relative NO inhibition rate of Examples 1-3 (microbead essence 1-3) is better than that of Comparative Examples 1-7 (microbead essence 4-10), indicating that different encapsulation materials will affect the efficacy of microbeads.

[0096] III. Oil Control Efficacy Test 1. Experimental Objective and Principle SZ95 cells are immortalized human facial sebaceous gland cell lines. Excessive lipid secretion from human sebaceous gland cells is a prerequisite for acne development, and acne patients generally have overactive facial sebaceous glands. Human sebaceous gland cells can serve as a cell model for studying the oil-controlling ability of cosmetics. The efficacy of the test sample in oil control is evaluated by measuring the intracellular lipid content of human sebaceous gland cells after administration of the test substance, comparing it to that of the negative control and the test substance. This experiment followed the laboratory method (HMC-WI-039 determination of intracellular lipid content in human sebaceous gland cells), comparing the intracellular lipid content of the test sample with that of the negative control. If the intracellular lipid content of the test sample was significantly lower than that of the negative control (p < 0.05), the test sample was considered to have a certain oil-controlling effect.

[0097] 2. Experimental materials, reagents, and equipment 2.1 Experimental Materials and Reagents Human sebaceous gland cells: SZ95 cells (DSMZ), DMEM high glucose medium (DMEM, Pronosai), 4% paraformaldehyde (mass percentage concentration), trypsin, Oil Red O staining kit (Zhongqiao Xinzhou), thiazolyl blue (MTT, BioFroxx), phosphate buffer (PBS).

[0098] 2.2 Instruments and Equipment RT-6100 ELISA reader, cell culture plates, pipettes, CO2 incubator, biosafety cabinet, inverted microscope.

[0099] 3. Test Methods 3.1 Cell viability assay This experiment used MTT assay to detect cell viability and screen the maximum safe concentration of cells for administration. A blank control (culture medium) and a zeroing well (PBS) were included. Sample concentrations were set as follows: 15%, 6.98%, 3.25%, 1.51%, 0.70%, 0.32%, 0.15%, and 0.07%, with six replicate wells for each concentration. The specific operating steps are as follows: (1) Inoculation: Human sebaceous gland cells in the logarithmic growth phase were digested and inoculated into 96-well plates. 100 µL (1×10⁻⁶) was added to each well. 5 (cells / mL), ensuring uniform cell adhesion, and incubate the culture plate in a 37°C, 5% CO2 incubator for 24 hours.

[0100] (2) Sample feeding: After the cells have grown for 24 hours, the supernatant is discarded and serum-containing basal culture medium containing different concentrations of the test substance is added. Eight concentrations are set for each test sample. The cells are incubated in a carbon dioxide incubator for 24 hours.

[0101] (3) Detection: After culturing cells for 24 h, wash twice with PBS, add 50 µL / well MTT working solution, and continue culturing in a CO2 incubator for 6 h; discard the MTT solution, add 150 µL / well DMSO, shake for 5 min, and read the OD using a microplate reader. 490nm The relative cell viability was calculated using the following formula: Cell viability (%) = (OD sample / OD blank) × 100%

[0102] The cytotoxicity test results are shown in Table 15 below. Based on the relative cell viability results, the concentration of the test substance with no cytotoxicity was selected as 1.51%, and the following oil-control efficacy test can be conducted based on this concentration.

[0103] Table 15 Results of SZ95 cell viability assay

[0104] 3.2 Determination of intracellular lipid content in human sebaceous gland cells (1) Cell plating: Human sebaceous gland cells were plated in 96-well plates at a density of 100 µL / well and a density of 1 × 10⁻⁶ m² / well. 5 Cells / mL were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0105] (2) Solution preparation: Set up blank control (only staining solution without cells), negative control (untreated normal sebaceous gland cells), positive control (10µg / mL insulin-stimulated normal sebaceous gland cells), and sample group (1.51% microbead essence solution and untreated normal sebaceous gland cells).

[0106] (3) Discard the culture medium in the 96-well plate from step (1) and replace it with basal culture medium containing different concentrations of test samples. The positive control group was given basal culture medium containing 10 µg / mL insulin, and the samples were given basal culture medium containing 1.51% microbead essence. The negative control group was given only basal culture medium, 100 µL per well. After 24 h, fix with 4% paraformaldehyde for 30 min, and finally detect OD using an Oil Red O staining kit. 490nm .

[0107] (4) Formula for calculating the relative lipid content in human sebaceous gland cells: Relative lipid content in human sebaceous gland cells (%) = (Lipid content in sample cells / Lipid content in negative control cells) × 100%

[0108] The results of the relative lipid content in human sebaceous gland cells are as follows: Figure 4 As shown. From Figure 4It can be seen that the relative lipid content in the sebaceous gland cells of the examples and comparative examples is lower than that of the negative control group. Therefore, it can be considered that the microbeads of the present invention have good oil control ability. Moreover, the data of the example group is better than that of the comparative example group, indicating that different materials will also affect the encapsulated active ingredients.

[0109] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A water-in-oil-in-water triphasic encapsulated microbead, characterized in that The spherical structure is sequentially provided with an outer water phase, an inner oil phase and an inner water phase from outside to inside; The outer water phase comprises the following components by mass percentage: 0.3-0.4% of sodium alginate, 0.2-0.25% of agarose, 0.5-1.5% of cyclodextrin, 5-10% of glycerol, 0.1-0.5% of hexylene glycol, 0.1-0.5% of p-hydroxyacetophenone and 40-45% of water; The inner oil phase comprises the following components by mass percentage: 5-10% of caprylic / capric triglyceride, 1-5% of grape seed oil, 1.5-5.5% of olive squalane, 0.1-0.5% of tocopherol, 1-3% of natural wax and 1-10% of castor oil / IPDI copolymer; The inner water phase comprises the following components by mass percentage: 0.1-1.5% of ampelopsis grossedentata extract, 1-5% of astragalus membranaceus root extract, 0.01-0.05% of hyaluronic acid, 2-5% of butanediol, 0.05-0.1% of xanthan gum and 5-6% of water; The total mass percentage of all components of the outer water phase, the inner oil phase and the inner water phase is 100%.

2. The water-in-oil-in-water three-phase wrapped microbead according to claim 1, characterized in that: The mass ratio of the outer water phase, the inner oil phase and the inner water phase is (3.5-3.6):(1.9-2.0):

1.

3. Process for the preparation of water-in-oil-in-water triphasic encapsulated microbeads according to claim 1 or 2, characterized in that Comprises the following steps: (1) Preparation of the outer water phase: the components of the outer water phase, agarose and p-hydroxyacetophenone, are added to the partial water of the outer water phase preheated to 85-90 DEG C, and dissolved under stirring at 85-90 DEG C to obtain a mixed solution 1; the components of the outer water phase, sodium alginate, cyclodextrin, glycerol and hexylene glycol, are added to the remaining water of the outer water phase preheated to 50-60 DEG C, and dissolved under stirring to obtain a mixed solution 2; the mixed solution 1 is poured into the mixed solution 2 and stirred uniformly to obtain the outer water phase; (2) Preparation of the inner oil phase: the components of the inner oil phase, castor oil / IPDI copolymer, caprylic / capric triglyceride and natural wax, are fully stirred and dissolved, and then the components of the inner oil phase, olive squalane, tocopherol and grape seed oil, are stirred uniformly to obtain the inner oil phase; (3) Preparation of the inner water phase: the component of the inner water phase, hyaluronic acid, is added to butanediol to disperse, and then xanthan gum and water are added, and the mixture is heated and stirred at 80-85 DEG C for 15-20 min to fully dissolve the hyaluronic acid and the xanthan gum; the temperature is lowered to 45-50 DEG C, and ampelopsis grossedentata extract and astragalus membranaceus root extract are added and stirred uniformly to obtain the inner water phase; (4) The outer water phase prepared in step (1), the inner oil phase prepared in step (2) and the inner water phase prepared in step (3) are respectively injected into the corresponding microchannels of a microfluidic device, so that the inner water phase and the inner oil phase are mixed to form water-in-oil sandwich droplets, the water-in-oil sandwich droplets are mixed with the outer water phase to form W / O / W droplets; (5) The W / O / W droplets are dropped into a calcium pyrrolidone carboxylic acid solution from the pipe opening for further solidification to obtain the water-in-oil-in-water three-phase wrapped microbead.

4. The preparation method of the water-in-oil-in-water three-phase wrapped microbead according to claim 3, characterized in that: In step (4), the microfluidic device is a three-phase flow droplet microfluidic device, and the structure is coaxial focusing type, flow focusing, T-type or Y-type. 5.The method of claim 4, wherein the W / O / W triphasic encapsulated microbeads are prepared by the following steps: (1) preparing an inner aqueous phase; (2) preparing an inner oil phase; (3) preparing an outer aqueous phase; (4) preparing W / O / W droplets by the following method: injecting the inner aqueous phase into a first side channel of a triphasic flow droplet microfluidic device, injecting the inner oil phase into a second side channel, and injecting the outer aqueous phase into a main channel; the inner aqueous phase and the inner oil phase form W / O sandwich droplets in the side channels, and the W / O sandwich droplets mix with the outer aqueous phase at the intersection of the main channel and the side channels to form W / O / W droplets; and (5) preparing the W / O / W triphasic encapsulated microbeads by the following method: injecting the W / O / W droplets into a continuous phase, and then removing the continuous phase. 6.The method of claim 5, wherein the triphasic flow droplet microfluidic device comprises a T-shaped channel, wherein the channel comprises a main channel and side channels that are in communication with each other, the main channel and the side channels are arranged at 90 degrees, the side channels comprise a first side channel and a second side channel, and the first side channel is coaxially arranged inside the second side channel. 7.The method of claim 6, wherein the ratio of the diameters of the first side channel, the second side channel, and the main channel is 1:2:

4. 8.The method of claim 6, wherein the W / O / W droplets in step (4) are prepared by the following method: injecting the inner aqueous phase prepared in step (2) into the first side channel of the triphasic flow droplet microfluidic device, injecting the inner oil phase into the second side channel, and injecting the outer aqueous phase into the main channel; the inner aqueous phase and the inner oil phase form W / O sandwich droplets in the side channels, and the W / O sandwich droplets mix with the outer aqueous phase at the intersection of the main channel and the side channels to form W / O / W droplets. 9.The method of claim 6, wherein the ratio of the flow rates of the inner aqueous phase, the inner oil phase, and the outer aqueous phase in the channels is 1:5:

10. 10.The use of the W / O / W triphasic encapsulated microbeads of claim 1 or 2 in the field of cosmetics. Cosmetics comprising the W / O / W triphasic encapsulated microbeads of claim 1 or 2. ​ ​ ​ 10. A serum, characterized in that ​

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