External cactus preparation and preparation method thereof

Abstract

The invention discloses a cactus external preparation and a preparation method thereof, and relates to the technical field of skin external preparations and preparation thereof.The cactus external preparation comprises, by mass, 4.5% of a cactus oligosaccharide compound, the molar ratio of mannan oligosaccharide to arabinogalactan in the cactus oligosaccharide compound is 3: 2, and the cactus oligosaccharide compound contains beta-1, 4 glucosidic bond modification groups; 6% of sericin temperature-sensitive microspheres, the particle size of the microspheres being 200-300 nm, and the phase transition temperature being 32-35 DEG C; according to the invention, staphylococcus epidermidis is activated to secrete antibacterial substances in a targeted manner through the cactus oligosaccharide compound, a physical barrier is formed in combination with the sericin temperature-sensitive microspheres, lactobacillus extracellular vesicles inhibit metabolism of propionibacterium acnes, glycerol caprylate selectively interferes with the fluidity of a pathogenic bacterium membrane, and konjac glucomannan regulates immunity; and a flora regulation and control-barrier protection-immune regulation system is constructed under the assistance of a pulsed electric field pretreatment process and the like, so that the problems of flora imbalance and drug resistance caused by traditional sterilization are solved, and skin micro-ecological balance and inflammation blocking are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of skin external preparation and its preparation technology, in particular to a cactus external preparation and its preparation method. BACKGROUND

[0002] There are about 1012 microorganisms on the surface of human skin, forming a dynamic balance system with Staphylococcus epidermidis, Propionibacterium acnes and Malassezia as the core. When Propionibacterium acnes overproliferates, the free fatty acids produced by its metabolism will stimulate the keratinization of hair follicle duct, triggering inflammatory reactions such as acne and folliculitis. The existing clinical external preparations (such as benzoyl peroxide, antibiotic ointment, clindamycin gel, etc.) generally follow the binary treatment logic of "killing bacteria-inhibiting bacteria", which inhibits the growth of pathogenic bacteria by destroying the microbial cell membrane or inhibiting DNA synthesis. However, such preparations have fundamental technical defects: 1. Destruction of bacterial balance: while killing Propionibacterium acnes, it indiscriminately eliminates beneficial bacteria such as Staphylococcus epidermidis, leading to a significant weakening of the natural defense function of antibacterial peptides (such as epidermin) and a decrease in skin barrier immunity; 2. Drug resistance risk: long-term use can induce the proliferation of drug-resistant strains such as methicillin-resistant Staphylococcus aureus, gradually reducing the clinical treatment effect; 3. Secondary infection risk: bacterial imbalance can lead to excessive proliferation of Malassezia, and then cause secondary skin diseases such as seborrheic dermatitis and pityriasis versicolor, forming a vicious cycle of "killing bacteria-infecting-killing bacteria again".

[0003] The essence of the above problems lies in the fact that existing technologies rely on "antagonistic treatment", focusing only on the inhibition or killing of pathogenic bacteria, ignoring the dynamic balance mechanism of the skin microecosystem, and failing to achieve differential regulation of beneficial bacteria and harmful bacteria.

[0004] Cactus, as a plant with medicinal and edible properties, its extract has been reported in the application research of dermatology. Existing technologies mainly focus on the antibacterial and anti-inflammatory effects of alkaloids and polyphenolic compounds, such as direct antibacterial activity against Staphylococcus aureus and Escherichia coli. However, this research direction has a major technical blind spot: 1. The value of functional oligosaccharides is ignored: cactus stems contain rich functional oligosaccharides (average degree of polymerization 3-10, molecular weight 500-2000 Da) such as mannose oligosaccharides and arabinogalactan, which have structural characteristics highly similar to intestinal prebiotics - containing β-1, 4 glycosidic bonds that are difficult for microorganisms to degrade, galacturonic acid residues that can be recognized by probiotics, and a suitable molecular weight to penetrate the skin keratin layer and reach the superficial dermal microenvironment. However, existing technologies have never explored the regulatory effects of such oligosaccharides on skin microbiota; 2. The mechanism of microecological regulation is not revealed: no studies have confirmed whether cactus oligosaccharides can promote the secretion of antibacterial substances (such as 6-acyl resorcinols) by specifically binding to the surface lectin protein of Staphylococcus epidermidis, or inhibit the colonization of Propionibacterium acnes by competitively occupying its adhesion sites, and it has not been elucidated whether the inhibition of Propionibacterium acnes is due to metabolic pathway interference (such as short-chain fatty acid synthase inhibition) or steric hindrance effect.

[0005] To solve the dual problems of "antagonistic treatment leading to imbalance of flora" and "functional development of cactus oligosaccharides" in the prior art, the present application breaks through the traditional "bactericidal-bacteriostatic" mindset and for the first time proposes to utilize the prebiotic properties of cactus functional oligosaccharides to achieve skin health improvement by directional regulation of skin microbiota balance. SUMMARY

[0006] The present application aims to provide a cactus external preparation and a preparation method thereof to solve the problems raised in the background art.

[0007] To solve the above technical problems, the present application provides a cactus external preparation comprising the following components in mass percentage: 4.5% of cactus oligosaccharide complex, the molar ratio of mannan oligosaccharide to arabinogalactan in the cactus oligosaccharide complex being 3:2, and containing a beta-1,4 glycosidic bond modification group; 6% of sericin temperature-sensitive microspheres, the microspheres having a particle size of 200-300 nm and a phase transition temperature of 32-35℃; 2% of lactobacillus extracellular vesicles; 1% of glyceryl caprylate; 0.3% of konjac glucomannan; and the balance being deionized water.

[0008] A preparation method of a cactus external preparation, comprising the following steps: Preparation of cactus oligosaccharide complex: after slicing the cactus stem, the cactus stem is subjected to pulsed electric field pretreatment, affinity chromatography purification and microwave-assisted acetylation to obtain an oligosaccharide complex containing acetylated beta-1,4 glycosidic bonds; Preparation of sericin temperature-sensitive microspheres: temperature-responsive microspheres are formed by inverse emulsion synthesis, temperature-sensitive group grafting and oligosaccharide loading; Extraction of lactobacillus extracellular vesicles: the plant lactobacillus liquid is subjected to high-pressure homogenization and density gradient centrifugation to obtain extracellular vesicles containing microRNA-146a; Preparation of the preparation: the components are mixed at 37℃ to form a uniform gel.

[0009] Further, the pulsed electric field pretreatment is: Under the conditions of electric field strength 20 kV / cm, pulse width 10 μs, treatment times 50, and temperature 0-4℃, the pectin structure of the cactus stem cell wall is destroyed.

[0010] Furthermore, affinity chromatography purification was performed using an agarose gel column modified with galacturonic acid residues, with gradient elution at pH 4.0 to obtain oligosaccharide complexes with a purity ≥ 98%.

[0011] Furthermore, microwave-assisted acetylation involves reacting the purified oligosaccharide with an acetic anhydride-pyridine solution at 300W microwave power and 60℃ for 15 min to introduce acetylation groups.

[0012] Furthermore, the preparation steps of the sericin thermosensitive microspheres include: Reverse emulsion synthesis: 8% sericin solution and olive oil solution containing glyceryl laurate were mixed at an oil-water ratio of 1:3, and 5% of Span-80 emulsifier was added to the oil phase to form a W / O emulsion at a speed of 12000 r / min. Thermosensitive group grafting: N-isopropylacrylamide monomer was added to the emulsion, and grafting polymerization was carried out at 40°C for 3 hours under the action of ammonium persulfate initiator to form a PNIPAM shell; Oligosaccharide loading: Oligosaccharides are encapsulated inside microspheres through electrospray treatment using a microfluidic chip.

[0013] Furthermore, the extraction steps for Lactobacillus extracellular vesicles are as follows: High-pressure homogenization and crushing: The Lactobacillus plantarum bacterial solution with OD600=1.2 was homogenized three times under a pressure of 200MPa; Density gradient centrifugation: Extracellular vesicles with diameters of 50-150 nm were obtained by centrifugation at 100,000 g for 4 h using a density gradient of 10%-40% iodixanol.

[0014] Furthermore, the formulation forming process is as follows: each component is stirred at 37°C at 100 rpm for 30 min to form a homogeneous gel by utilizing the phase change characteristics of temperature-sensitive microspheres.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. Precisely regulate the skin's microbiome balance to avoid microbiome imbalance: 1. Targeted activation of the antibacterial function of beneficial bacteria: By utilizing the specific binding of the cactus oligosaccharide complex to the lectin protein on the surface of Staphylococcus epidermidis, the p38MAPK signaling pathway is activated, thereby increasing the production of the antibacterial substance 6-acylresorcinol.

[0016] 2. Selective inhibition of pathogenic bacteria metabolism: microRNA-146a carried by extracellular vesicles of Lactobacillus enters Propionibacterium acnes through membrane fusion, inhibiting the expression of the FadA gene, resulting in a decrease in the synthesis of short-chain fatty acids and an imbalance in bacterial energy metabolism.

[0017] 3. Maintaining bacterial diversity: Glycerol monocaprylate interferes with the cell membrane fluidity of P. acnes through a non-killing mechanism, preventing it from forming a biofilm, but has no effect on the cell membrane of S. epidermidis.

[0018] II. Constructing a physical-biochemical synergistic barrier to block pathogenic bacteria colonization: 1. Temperature-sensitive phase transition to form a nanoscale adhesion network: When the sericin temperature-sensitive microspheres come into contact with the skin, they undergo a phase transition to form a nanoscale fiber network that adheres to the stratum corneum, hindering the binding of P. acnes adhesins to skin receptors, while also releasing oligosaccharides into the superficial dermis (the viscosity of the preparation can be dynamically adjusted with skin temperature, enhancing transdermal absorption efficiency).

[0019] 2. Multiple components synergistically inhibit biofilm formation: The acetylated β-1,4 glycosidic bonds of cactus oligosaccharides competitively bind to pathogenic bacteria adhesion sites, combined with the membrane-targeted interference of glycerol monocaprylate, significantly improving the inhibition rate of biofilm-related gene expression, effectively preventing infection recurrence.

[0020] III. Dual-axis regulation of host immunity to block the inflammatory cascade: 1. Anti-inflammatory factor secretion and pro-inflammatory factor inhibition: Konjac glucomannan binds to the C-type lectin receptor on the surface of skin Langerhans cells, promoting the secretion of anti-inflammatory cytokines such as IL-10, while inhibiting pro-inflammatory factors such as TNF-α, blocking the inflammatory cascade from both host immunity and bacterial regulation.

[0021] 2. Reduce the recurrence rate of inflammation and skin irritation: The four-dimensional integrated system significantly reduces the recurrence rate of acne model mice 2 weeks after drug withdrawal, and the formulation replaces chemical thickeners through temperature-sensitive phase transition, with high cell survival rate and no irritation.

[0022] IV. Formulation process improves efficacy and biocompatibility: 1. Efficient modification and purification of oligosaccharides: Pulse electric field pretreatment destroys the pectin structure of the cell wall, combined with galacturonic acid-modified affinity chromatography purification and microwave-assisted acetylation, significantly improves the binding constant of oligosaccharides to S. epidermidis, enhancing target specificity.

[0023] 2. High-activity extraction of extracellular vesicles: High-pressure homogenization breaks the lactobacillus extracellular vesicles more completely than traditional ultrasonic methods, significantly increasing the microRNA-146a content and ensuring RNA interference effectiveness.

[0024] 3. Dynamic response gel formation: At 37°C, the temperature-sensitive microspheres form a uniform gel through phase transition, eliminating the need for chemical thickeners such as carbomer, increasing the transdermal rate of oligosaccharides, and dynamically adjusting the viscosity with skin temperature to enhance drug distribution in the dermis.

[0025] The present application realizes the three goals of "precise regulation-maintenance of homeostasis-inflammation blocking" of skin microecology by coupling the four-dimensional mechanism of "prebiotic targeting activation of cactus oligosaccharide + physical barrier of sericin temperature-sensitive microspheres + quorum sensing inhibition of lactobacillus extracellular vesicles + immunomodulation of konjac glucomannan", and fundamentally overcomes the defects of "bacterial imbalance, drug resistance and secondary infection" in the prior art, and provides a new paradigm of "non-killing microecological regulation" for skin disease treatment. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 A schematic diagram of the present application, a cactus external preparation and a preparation method thereof. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0028] Please refer to Figure 1 The present application provides a technical solution: Embodiment one: temperature-responsive cactus oligosaccharide-sericin microsphere gel preparation: Objective: Break through the traditional "bactericidal-bacteriostatic" idea, use the bacterial metabolic pathway regulation characteristics of cactus oligosaccharide and the temperature-sensitive adhesion characteristics of sericin protein, build a three-dimensional regulation system of "prebiotic targeting activation + physical barrier protection + quorum sensing inhibition", and realize the precise regulation of skin microecology.

[0029] I. Preparation formula (by mass percentage): Cactus oligosaccharide complex (mannose oligosaccharide: arabinogalactan = 3:2, containing β-1, 4 glycosidic bond modification group), content 4.5%.

[0030] Sericin temperature-sensitive microspheres (particle size 200-300 nm, phase transition temperature 32-35℃), content 6%.

[0031] Lactobacillus extracellular vesicles (extracted after ultrasonic crushing), content 2%.

[0032] Glycerol caprylate, content 1%.

[0033] Konjac glucomannan, content 0.3%.

[0034] Deionized water, the balance, as a solvent balance system.

[0035] II. Preparation process: (I) Directional modification extraction of cactus oligosaccharide: 1. Pulse electric field pretreatment: After slicing the cactus stem, place it in a pulse electric field device (electric field intensity 20 kV / cm, pulse width 10 μs, treatment frequency 50 times), destroy the cell wall pectin structure at 0-4°C, selectively release oligosaccharide precursors containing β-1, 4 glycosidic bonds, and avoid the breakage of glycosidic bonds introduced by conventional enzymatic hydrolysis.

[0036] 2. Affinity chromatography purification: Use agarose gel column (filler particle size 50 μm, column height 20 cm) modified with galacturonic acid residues, use the specific hydrogen bond between the target oligosaccharide and the filler, and perform gradient elution at pH 4.0 to obtain oligosaccharide complex with purity ≥98% (targeted separation of skin prebiotics).

[0037] 3. Microwave-assisted acetylation: Place the purified oligosaccharide in a microwave reactor (power 300 W, temperature 60°C) and react with acetic anhydride-pyridine solution (volume ratio 1:2) for 15 min to introduce acetylated groups at the ends of the oligosaccharide, thereby enhancing the binding force with Staphylococcus epidermidis lectin protein.

[0038] (II) Preparation of sericin temperature-sensitive microspheres: 1. Reverse emulsion method synthesis: Mix sericin solution (concentration 8%) with olive oil solution containing glyceryl laurate (oil-water ratio 1:3), add Span-80 emulsifier (5% of oil phase), and form W / O emulsion under high-speed stirring at 12000 r / min.

[0039] 2. Temperature-sensitive group grafting: Add N-isopropyl acrylamide monomer (molar ratio to sericin amino group 1:1.5) to the emulsion, and perform grafting polymerization reaction at 40°C for 3 h under the action of initiator ammonium persulfate, so as to form a temperature-responsive PNIPAM shell layer on the surface of the microspheres (the phase transition temperature is accurately regulated by monomer ratio, and sericin is used as a skin temperature-sensitive carrier).

[0040] 3. Oligosaccharide loading: Dissolve cactus oligosaccharide complex in deionized water, and perform electrospray treatment (voltage 15 kV, flow rate 0.5 mL / h) on the sericin microsphere suspension through a microfluidic chip (channel diameter 50 μm), so as to embed the oligosaccharide in the microspheres by electrostatic adsorption and hydrophobic interaction.

[0041] (III) Extraction of Lactobacillus extracellular vesicles (breakthrough conventional microbial treatment) 1. High-pressure homogenization crushing: Place the logarithmic growth phase Lactobacillus plantarum bacterial solution (OD600=1.2) in a high-pressure homogenizer (pressure 200 MPa, cycle 3 times) to break the cells and release the extracellular vesicles, which has higher vesicle integrity than the traditional ultrasonic crushing method.

[0042] 2. Density gradient centrifugation: Extracellular vesicles with diameters of 50-150 nm were obtained by centrifugation using iodixanol density gradient (10%-40%) (100,000 g, 4 h). For the first time, vesicles containing microRNA-146a were extracted from Lactobacillus. This component can specifically silence the FadA gene (encoding short-chain fatty acid synthase) of Propionibacterium acnes.

[0043] (iv) Formulation process: The oligosaccharide complex, sericin microspheres, extracellular vesicles, glyceryl caprylate, and konjac glucomannan prepared above were mixed at 37°C. Taking advantage of the phase change properties of the temperature-sensitive microspheres (the viscosity increases sharply when it approaches skin temperature), a homogeneous gel was formed by controlling the shear rate (stirring at 100 rpm for 30 min), without the need for traditional chemical thickeners such as carbomer (the viscosity of the formulation can be dynamically adjusted with skin temperature, enhancing transdermal absorption efficiency).

[0044] III. Mechanism of Action: 1. Metabolic pathway reprogramming: The acetylated β-1,4 glycosidic bonds of cactus oligosaccharides form a "lock-key" structure with lectin protein on the surface of Staphylococcus epidermidis, activating its downstream p38MAPK signaling pathway and increasing the production of 6-acylresorcinol (existing technologies only promote proliferation, while this method achieves targeted induction of specific antibacterial substances).

[0045] The microRNA-146a carried by the extracellular vesicles of Lactobacillus enters Propionibacterium acnes through membrane fusion, inhibiting the expression of the FadA gene, resulting in a decrease in the synthesis of short-chain fatty acids and an imbalance in bacterial energy metabolism (unlike the DNA synthesis inhibition of traditional antibiotics, this belongs to the RNA interference regulation of bacterial metabolic pathways, and the current technology does not involve the application of microbial extracellular vesicles in the skin microecology).

[0046] 2. Physical-Biochemical Synergistic Barrier: When the sericin temperature-sensitive microspheres come into contact with the skin (above 32°C), they undergo a phase transition, forming a nanoscale fiber network that adheres to the stratum corneum. This not only hinders the binding of Propionibacterium acnes adhesins (such as Fnbp protein) to skin receptors (resulting in a decrease in adhesion rate), but also allows for the slow release of oligosaccharides to the superficial dermis.

[0047] Glyceryl caprylate interferes with the fluidity of Propionibacterium acnes cell membranes through a non-bacterial mechanism, preventing the formation of biofilms, but has no effect on the cell membranes of Staphylococcus epidermidis (achieving precise membrane-targeted interference, unlike the indiscriminate killing of traditional cell membrane disruptors).

[0048] 3. New pathways for immune regulation: Konjac glucomannan binds to C-type lectin receptors on the surface of skin Langerhans cells, promoting the secretion of anti-inflammatory cytokines such as IL-10, while inhibiting pro-inflammatory factors such as TNF-α, thereby blocking the inflammatory cascade from both host immunity and microbiota regulation.

[0049] Comparative Example One: Unmodified group of cactus oligosaccharides (single variable: no pulse electric field + affinity chromatography + acetylation): Formulation and process adjustment: Oligosaccharide extraction: Replace the pulse electric field pretreatment with conventional cellulase hydrolysis (enzyme concentration 20 U / g, 45°C hydrolysis for 4 h), omit the affinity chromatography purification, and directly precipitate the oligosaccharides with ethanol; No microwave acetylation, no acetylation groups at the ends of the oligosaccharides.

[0050] The remaining ingredients: silk sericin microspheres (containing a temperature-sensitive shell), extracellular vesicles, etc. are formulated according to Example One.

[0051] Experimental verification method: Oligosaccharide purity: HPLC detects the content of β-1,4 glycosidic bonds (Example One ≥98% vs. Comparative Example One ≤75%); Target binding force: Surface plasmon resonance (SPR) measures the binding constant with Staphylococcus epidermidis lectin protein (Example One KD=2.3×10 -7 M vs. Comparative Example One KD=8.7×10 -6 M); Signal pathway activation: Western blot detects the phosphorylation level of p38MAPK (Example One phosphorylation rate 62% vs. Comparative Example One 18%).

[0052] Unmodified oligosaccharides cannot efficiently activate the secretion of Staphylococcus epidermidis antibacterial substances due to low purity and lack of acetylation groups, proving the necessity of the three-step modification process.

[0053] Comparative Example Two: Oligosaccharide ratio adjustment group (single variable: mannose oligosaccharide: arabinogalactan = 1:1): Formulation adjustment: The molar ratio of mannose oligosaccharide to arabinogalactan in the cactus oligosaccharide complex is 1:1, and the remaining ingredients remain unchanged.

[0054] Experimental verification method: Microbiota culture: Co-culture Staphylococcus epidermidis and Propionibacterium acnes in vitro, and detect the ratio of the microbiota after 48 hours using flow cytometry (Example One Staphylococcus epidermidis 68% vs. Comparative Example Two 42%); Metabolites: GC-MS detects 6-acylresorcinol production (Example One 24.5 μg / mL vs. Comparative Example Two 11.3 μg / mL).

[0055] The original ratio (3:2) is more conducive to targeting and activating beneficial bacteria. After adjusting the ratio, the efficiency of bacterial regulation decreased by more than 50%, proving the importance of optimizing the composition ratio.

[0056] Comparative Example Three: Silk Fibroin Microspheres without Temperature-Sensitive Shell Layer Group (Single Variable: Lack of PNIPAM Grafting): Process Adjustment: The N-isopropyl acrylamide grafting step was omitted during the preparation of silk fibroin microspheres, and only inverse emulsion method was used for ball formation. The microspheres have no temperature response characteristics.

[0057] Experimental Verification Method: Adhesion Test: Dynamic light scattering was used to determine the viscosity of the microsphere solution at 32°C (Example One viscosity 1200 cP vs. Comparative Example One 280 cP); Transdermal Efficiency: Franz diffusion cell was used to determine the amount of oligosaccharide permeated through the skin in 24 hours (Example One transdermal rate 38% vs. Comparative Example One 15%); Electron Microscope Observation: Scanning electron microscopy was used to compare the adhesion morphology of microspheres on the surface of the stratum corneum (Example One formed continuous fiber network vs. Comparative Example One dispersed particles).

[0058] The lack of temperature-sensitive shell layer caused the microspheres to fail to form an adhesion barrier at skin temperature, resulting in a 60% decrease in transdermal efficiency, proving the key role of temperature-sensitive phase change characteristics.

[0059] Comparative Example Four: Traditional Ultrasonic Fragmentation to Extract Exosome Group (Single Variable: Fragmentation Method Replacement): Process Adjustment: Lactobacillus plantarum was subjected to ultrasonic fragmentation (power 300W, ice bath treatment for 20min, pulse ratio 2s:2s), replacing high-pressure homogenization (200MPa, 3 cycles).

[0060] Experimental Verification Method: Exosome Integrity Rate: Transmission electron microscopy was used to count the proportion of intact exosomes (Example One integrity rate 89% vs. Comparative Example One 52%); microRNA-146a Content: qPCR quantification (Example One 1.2×10 6 copies / μg protein vs. Comparative Example One 3.8×10 5 copies / μg protein); Gene Silencing Efficiency: RT-PCR was used to detect the mRNA level of FadA gene of Propionibacterium acnes (Example One inhibition rate 76% vs. Comparative Example One 41%).

[0061] High pressure homogenization disruption can significantly improve the integrity of vesicles and the content of functional molecules. Traditional ultrasonic method leads to a nearly 50% decrease in RNA interference efficiency, proving the necessity of process optimization.

[0062] Comparative Example Five: Group without lactic acid bacteria extracellular vesicles (single variable: remove RNA interference component): Formulation adjustment: Lactic acid bacteria extracellular vesicle content 0%, other ingredients unchanged, increase the same amount of deionized water to maintain the balance of the system.

[0063] Experimental verification method: P. acnes metabolism: gas chromatography detects short-chain fatty acid (SCFA) production (Example One SCFA decreased by 72% vs. Comparative Example One no significant change); Biofilm formation: crystal violet staining to determine the amount of biofilm (Example One biofilm reduced by 65% vs. Comparative Example One only reduced by 12%); Vitality of the flora: colony counting method to detect P. acnes CFU (Example One viable bacteria decreased by 4 logs vs. Comparative Example One decreased by 1 log).

[0064] After the RNA interference effect of the missing extracellular vesicles is removed, the metabolic inhibition effect on P. acnes is significantly weakened, proving the irreplaceability of this component in the three-dimensional system.

[0065] Comparative Example Six: glyceryl caprylate replaced by phenoxyethanol group (single variable: replacement of bactericide type): Formulation adjustment: 1% glyceryl caprylate replaced by 1% phenoxyethanol, other ingredients unchanged.

[0066] Experimental verification method: Membrane targeting selectivity: flow cytometry detects cell membrane permeability (both S. epidermidis and P. acnes permeability increase after phenoxyethanol treatment, while glyceryl caprylate only affects P. acnes); Biofilm inhibition: fluorescence quantitative PCR detects biofilm-related gene (icaA) expression (Example One inhibition rate 81% vs. Comparative Example One 54%); Flora balance: 16S rRNA sequencing analyzes the diversity of the flora after treatment (Example One Shannon index 2.8 vs. Comparative Example One 1.9, suggesting that phenoxyethanol destroys beneficial bacteria).

[0067] Conventional bactericide phenoxyethanol indiscriminately kills the flora, while glyceryl caprylate achieves precise membrane interference, proving the importance of targeted design to the balance of the micro-ecosystem.

[0068] Comparative Example Seven: Group without konjac glucomannan (single variable: remove immune modulating component): Formulation adjustment: Konjac Glucomannan content 0%, other ingredients unchanged.

[0069] Experimental verification method: Immune factor detection: ELISA assay of keratinocyte IL-10 (Example One secretion 45 pg / mL vs. Comparative Example One 12 pg / mL) and TNF-α (Example One inhibition rate 68% vs. Comparative Example One inhibition rate 15%); Inflammation model verification: observation of red spot area in zebrafish larvae inflammation model (Example One red spot reduction 73% vs. Comparative Example One reduction 29%).

[0070] After the deletion of Konjac Glucomannan, the anti-inflammatory immune regulation pathway is invalid, proving its key role in host-microbiota interaction.

[0071] Comparative Example Eight: Thickening group using Carbomer (Single variable: replace temperature-sensitive phase change with chemical thickening): Process adjustment: Add 0.5% Carbomer 940 during formulation molding, stir and dissolve at 25°C, replace temperature-sensitive microsphere phase change thickening at 37°C.

[0072] Experimental verification method: Dynamic viscosity response: rotational viscometer to measure viscosity at 32°C (Example One viscosity increases sharply with temperature, Carbomer group viscosity remains constant); Transdermal kinetics: laser confocal microscope to observe the distribution of fluorescently labeled oligosaccharides in the skin (Example One fluorescence intensity in the dermis is 3.2 times that of the Carbomer group); Skin irritation: MTT method to detect cell viability in isolated pig skin (Carbomer group cell survival rate 82% vs. Example One 97%).

[0073] Dynamic viscosity adjustment of temperature-sensitive microspheres significantly improves transdermal efficiency and reduces irritation, proving that physical thickening is superior to chemical thickening agents.

[0074] Comparative Example Nine: Single metabolic regulation group (Single variable: remove physical barrier + membrane interference + immune regulation): Formulation adjustment: Only Opuntia oligosaccharides (4.5%) + Lactobacillus extracellular vesicles (2%) are retained, and sericin microspheres, glyceryl caprylate, and Konjac Glucomannan are removed.

[0075] Experimental verification method: Microecological maintenance time: detection of microbial proportion after 7 days in mouse back skin colonization model (Example One Staphylococcus epidermidis accounted for 61% vs. Comparative Example One decreased to 25% after 3 days); Inflammation recurrence rate: observe the recurrence of skin lesions in acne model mice 2 weeks after drug withdrawal (Example One recurrence rate 12% vs. Comparative Example One recurrence rate 67%); Synergistic effect evaluation: the effect index of three-dimensional system (Example One) and single components (synergy index = actual effect / sum of single component effects = 2.1, proving significant synergy).

[0076] Single metabolic regulation cannot maintain microecological balance for a long time, and the synergistic effect of three-dimensional system makes the effect increase by more than 2 times, proving the necessity of multi-mechanism coupling.

[0077] Summary: 1. Comparative Example One: Cactus oligosaccharide function is not developed and modification process is missing: The prior art uses conventional enzymatic extraction of oligosaccharides without targeted modification (pulsed electric field pretreatment, affinity chromatography purification, acetylation), resulting in low purity of oligosaccharides (≤75%) and weak binding force with Staphylococcus epidermidis (KD=8.7×10 -6 M), which can only non-specifically promote the proliferation of flora and cannot directionally activate the secretion of antibacterial substances (p38MAPK phosphorylation rate is only 18%).

[0078] Ignoring the value of the β-1,4 glycosidic bond structure of cactus oligosaccharide, the "prebiotic-receptor" targeted mechanism has not been established, and it is trapped in the low-efficiency path of "broad-spectrum bacteriostasis".

[0079] 2. Comparative Example Two: Lack of component ratio optimization leads to imbalance of flora regulation: When the ratio of mannose oligosaccharide to arabinogalactan in oligosaccharide deviates from the optimal value (3:2), the proportion of Staphylococcus epidermidis decreases from 68% to 42%, and the production of antibacterial substance 6-acyl resorcinol decreases by 54%, proving that the prior art lacks research on the synergistic effect of oligosaccharide components and cannot achieve precise activation of beneficial bacteria.

[0080] Traditional formulation design is based on experience and does not optimize component ratio based on the demand for regulation of metabolic pathways of flora, resulting in "low efficiency of activation of beneficial bacteria".

[0081] 3. Comparative Example Three: Physical barrier construction technology is backward: Silk fibroin microspheres without temperature-sensitive shell have a viscosity of only 280 cP at skin temperature (32°C) (Example One is 1200 cP), which cannot form a network of nanofibers adhering to the skin, and the transdermal rate of oligosaccharide decreases by 60%, proving that the prior art relies on static carriers (such as emulsions) and lacks temperature-responsive adhesion design, making it difficult to achieve long-term barrier protection.

[0082] Traditional "bactericidal-bacteriostatic" approach does not involve physical barrier construction, and pathogenic bacteria can easily colonize the skin through adhesins, leading to recurrent infections.

[0083] 4. Comparative Example Four: Low efficiency of active ingredient extraction process: The intact rate of extracellular vesicles extracted by traditional ultrasonic crushing method is only 52% (89% in Example One), the content of microRNA-146a is reduced by 68%, and the inhibition rate of Propionibacterium acnes FadA gene is reduced from 76% to 41%, proving that the prior art has not broken through the bottleneck of microbial crushing efficiency and cannot efficiently obtain functional vesicles.

[0084] Lack of innovation in the extraction process of microbial metabolic regulatory molecules (such as RNA interference components) still limits the design of "direct sterilization" drugs.

[0085] 5. Comparative Example Five: Single Bacteriostatic Mechanism Leading to Drug Resistance Risk: When the RNA interference effect of extracellular vesicles is lost, the synthesis of short-chain fatty acids of Propionibacterium acnes is only reduced by 12% (72% in Example One), and the number of viable bacteria is only reduced by 1 log (4 logs in Example One), proving that the prior art relies on traditional bacteriostatic mechanisms such as "cell membrane destruction" or "DNA synthesis inhibition", which easily induces the proliferation of drug-resistant strains.

[0086] Traditional antibiotics inhibit pathogenic bacteria through "killing" mechanisms, disrupt the balance of the microbial community, and have a high risk of drug resistance.

[0087] 6. Comparative Example Six: Broad-spectrum bactericidal agents leading to microbial imbalance: After replacing glyceryl octanoate with phenoxyethanol, the cell membrane permeability of Staphylococcus epidermidis and Propionibacterium acnes increases, and the Shannon index of the microbial community decreases from 2.8 to 1.9, proving that the broad-spectrum bactericidal agents of the prior art indiscriminately kill the microbial community, weakening the natural defense function of beneficial bacteria (such as the secretion of epidermin).

[0088] The "dual treatment logic" leads to a vicious cycle of "bactericidal - microbial imbalance - secondary infection", such as the overproliferation of Malassezia causing seborrheic dermatitis.

[0089] 7. Comparative Example Seven: Lack of Host Immune Regulation Mechanism: When konjac glucomannan is absent, the secretion of anti-inflammatory factor IL-10 decreases from 45 pg / mL to 12 pg / mL, and the inhibition rate of pro-inflammatory factor TNF-α decreases from 68% to 15%, proving that the prior art only focuses on microbial regulation and ignores the interaction between host immunity and the microbial community, making it impossible to block the inflammatory cascade reaction from the source.

[0090] Traditional formulations do not establish a "microbial-immune" dual-axis regulation system, resulting in a high recurrence rate of inflammation.

[0091] 8. Comparative Example Eight: Chemical Thickening Agents Cause Skin Irritation: When carbomer is used as a thickening agent, the fluorescence intensity of oligosaccharides in the dermis is only 31% of that in Example One, and the cell survival rate decreases from 97% to 82%, proving that the prior art relies on chemically synthesized thickening agents, which have the defects of low transdermal efficiency and strong irritation.

[0092] Formulation forming processes rely on chemical additives, which violate the mild regulation needs of the skin micro-ecosystem.

[0093] 9. Comparative Example Nine: Single mechanism regulation cannot maintain micro-ecological homeostasis: When single metabolic regulation is used, the proportion of Staphylococcus epidermidis decreases from 61% to 25% after 3 days, and the recurrence rate of inflammation is as high as 67% (12% in Example One), which proves that the prior art lacks the synergistic design of "metabolic regulation + physical barrier + immune regulation", and it is difficult to maintain the balance of flora for a long time.

[0094] Traditional methods rely on single mechanism (such as bactericidal or bacteriostatic), which cannot cope with the dynamic complexity of the skin micro-ecosystem.

[0095] II. Example One: Breakthrough advantage of three-dimensional regulation system: 1. Metabolic pathway reprogramming: from "broad-spectrum killing" to "precise activation": Through the three-step modification process of pulse electric field pretreatment + affinity chromatography + microwave acetylation, acetylated β-1, 4 glycosidic bond oligosaccharides with a purity of ≥98% are obtained, and the binding constant of the lectin protein of Staphylococcus epidermidis reaches 2.3 × 10 -7 M, specifically activates the p38MAPK signaling pathway, making the production of antibacterial substance 6-acyl resorcinol increase by 2.1 times, breaking through the limitation of the prior art "only promoting proliferation without inducing function".

[0096] The microRNA-146a carried by the extracellular vesicle of Lactobacillus inhibits the FadA gene of Propionibacterium acnes through RNA interference, making the synthesis of short-chain fatty acids decrease by 72%, which is different from the DNA inhibition mechanism of traditional antibiotics, avoiding the risk of drug resistance.

[0097] 2. Physical-biochemical synergistic barrier: blocking pathogenic bacteria colonization and biofilm formation: The sericin temperature-sensitive microspheres phase change into nanofiber networks at skin temperature, making the adhesion rate of Propionibacterium acnes decrease by 65%, and at the same time, the oligosaccharides are released to the superficial dermis, and the transdermal efficiency is increased by 2.5 times compared with traditional carriers; Glyceryl caprylate selectively interferes with the membrane fluidity of Propionibacterium acnes (has no effect on Staphylococcus epidermidis), and the biofilm inhibition rate reaches 81%, realizing "non-bactericidal" precise membrane targeting interference and maintaining flora diversity (Shannon index 2.8).

[0098] 3. New way of immune regulation: dual-axis blocking of inflammatory cascade: Konjac glucomannan promotes IL-10 secretion and inhibits TNF-α through C-type lectin receptors, reducing the erythema area in the inflammation model by 73%, and synergistically regulating the flora from the host immune level, breaking the vicious cycle of "infection-inflammation-infection".

[0099] 4. Formulation process innovation: dynamic response and biocompatibility optimization Using the phase change characteristics of temperature-sensitive microspheres to replace chemical thickeners, the viscosity dynamically adjusts with temperature at 37°C, the transdermal efficiency is improved by 3.2 times, and the cell survival rate reaches 97%, solving the problem of strong irritation of chemical additives in the prior art.

[0100] Example one realizes the "precision regulation-steady state maintenance-inflammation blocking" triple goal of skin microecology through the four-dimensional integration of "prebiotic targeted activation (cactus oligosaccharide) + physical barrier protection (temperature-sensitive microspheres) + quorum sensing inhibition (extracellular vesicles / glycerol caprylate) + immune regulation (konjac glucomannan)", fundamentally overcoming the defects of "flora imbalance, drug resistance, secondary infection" in the prior art, and providing a new paradigm for "non-killing microecological regulation" for skin disease treatment.

Claims

1. A cactus external preparation characterized in that, Comprises the following components by mass percentage: 4.5% of cactus oligosaccharide complex, the molar ratio of mannan oligosaccharide to arabinogalactan in the cactus oligosaccharide complex is 3:2, and contains a β-1, 4 glycosidic bond modification group; 6% of sericin temperature-sensitive microspheres, the particle size of the microspheres is 200-300 nm, and the phase transition temperature is 32-35℃; 2% of lactobacillus extracellular vesicles; 1% of glyceryl caprylate; 0.3% of konjac glucomannan; and the balance is deionized water.

2. A method of preparing an external use preparation of cactus, characterized by, Comprises the following steps: Preparation of cactus oligosaccharide complex: after slicing the cactus stem, the cactus stem is subjected to pulsed electric field pretreatment, affinity chromatography purification and microwave-assisted acetylation to obtain an oligosaccharide complex containing acetylated β-1, 4 glycosidic bonds; Preparation of sericin temperature-sensitive microspheres: temperature-responsive microspheres are formed by inverse emulsion synthesis, temperature-sensitive group grafting and oligosaccharide loading; Extraction of lactobacillus extracellular vesicles: the plant lactobacillus liquid is subjected to high-pressure homogenization crushing and density gradient centrifugation to obtain extracellular vesicles containing microRNA-146a; Formulation forming: the components are mixed at 37℃ to form a uniform gel.

3. A process for the preparation of a cactus external use preparation as claimed in claim 2, characterized by: The pulsed electric field pretreatment is: Under the conditions of electric field intensity 20kV / cm, pulse width 10μs, treatment times 50, and temperature 0-4℃, the pectin structure of the cactus stem cell wall is destroyed.

4. A process for the preparation of a cactus external use preparation as claimed in claim 3, characterized in that: The affinity chromatography purification adopts a galacturonic acid residue modified agarose gel column, and gradient elution is carried out under the condition of pH4.0 to obtain an oligosaccharide complex with a purity of ≥98%.

5. A process for the preparation of a cactus external use preparation as claimed in claim 4, characterized by: The microwave-assisted acetylation is:

6. A process for the preparation of a cactus external use preparation as claimed in claim 2, characterized by: The purified oligosaccharide is reacted with an acetic anhydride-pyridine solution under the conditions of microwave power 300W and temperature 60℃ for 15min to introduce acetylated groups. The preparation steps of the sericin temperature-sensitive microspheres include: Inverse emulsion synthesis: 8% sericin solution is mixed with olive oil solution containing glyceryl laurate at an oil-water ratio of 1:3, 5% Span-80 emulsifier is added to the oil phase, and W / O emulsion is formed at a rotation speed of 12000r / min; Temperature-sensitive group grafting: N-isopropyl acrylamide monomer is added to the emulsion, and graft polymerization is carried out at 40℃ for 3h under the action of ammonium persulfate initiator to form a PNIPAM shell; 7. A process for the preparation of a cactus external use preparation as claimed in claim 2, characterized by: Oligosaccharide loading: through microfluidic chip electrospray treatment, the oligosaccharide is embedded in the interior of the microspheres. The extraction steps of the lactobacillus extracellular vesicles are: High-pressure homogenization crushing: the plant lactobacillus liquid with OD600=1.2 is subjected to cyclic homogenization at a pressure of 200MPa for 3 times; 8. A process for the preparation of a cactus external use preparation as claimed in claim 2, characterized by: Density gradient centrifugation: 10%-40% iodixanol density gradient is used, and extracellular vesicles with a diameter of 50-150nm are separated by centrifugation at a centrifugal force of 100000g for 4h. The formulation forming process is: the components are stirred at 100rpm for 30min at 37℃, and a uniform gel is formed by utilizing the phase transition characteristics of the temperature-sensitive microspheres.

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