Hydrogel composition containing elaeagnus conferta roxb and preparation method and network pharmacology analysis and evaluation method thereof

Abstract

The invention belongs to the technical field of cosmetics and skin medicine preparations, and particularly relates to a hydrogel composition containing elaeagnus conferta roxb, a preparation method of the hydrogel composition and a network pharmacology analysis and evaluation method. The composition is prepared from the following components: 1.5 to 2.5 percent w / v of sodium alginate, 0.5 to 1.5 percent w / v of hyaluronic acid, 4 to 6 percent w / v of glycerol, 0.2 to 0.3 percent w / v of ceramide, 0.4 to 0.6 percent w / v of glycyrrhizic acid, 0.05 to 0.15 percent w / v of EDTA (Ethylene Diamine Tetraacetic Acid) disodium, 0.2 to 0.3 percent w / v of sodium pyrrolidone carboxylate, 0.2 to 0.3 percent w / v of allantoin, 0.4 to 0.6 percent w / v of adenosine triphosphate and 0.5 to 5 percent w / v of elaeagnus conferta roxb extract. The invention systematically overcomes the technical resistance of unknown components, fuzzy mechanism, low dosage form efficiency and disjoint verification of the natural extract in the treatment of the specific dermatitis from the whole chain innovation of component analysis, mechanism verification, dosage form design and evaluation system.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cosmetic Yanchaguo hydrogel composition, more particularly to a Yanchaguo hydrogel composition and its preparation method and network pharmacological analysis and evaluation method. BACKGROUND

[0002] Atopic dermatitis (AD) is a chronic, recurrent, pruritic inflammatory skin disease with a global prevalence of 230 million. Its pathogenesis involves the interaction of multiple factors such as skin barrier dysfunction, immune abnormalities and microecological imbalance. Traditional treatment relies on topical corticosteroids (TCS) and calcineurin inhibitors (TCI), but long-term use can cause skin atrophy, telangiectasia and other side effects, and cannot systematically regulate the multi-pathway pathological mechanism of AD.

[0003] Existing moisturizing skin care products mainly rely on the triple mechanism of occlusive agents (such as petrolatum), hygroscopic agents (such as glycerin, urea) and reparative lipids (such as ceramides), but still have significant limitations:

[0004] First, traditional atopic dermatitis treatment has significant limitations. The current clinical mainstream of topical corticosteroids and calcineurin inhibitors can effectively control inflammation, but long-term use can cause skin atrophy, telangiectasia, folliculitis, local hypertrichosis and other side effects, leading to low patient compliance. Such drugs only target a single inflammatory target, and cannot regulate the core pathological mechanisms of AD such as skin barrier function, immune imbalance and microecological disorder, making it difficult to achieve long-term stable efficacy.

[0005] Second, the ingredient design and mechanism of existing moisturizing skin care products have multiple defects. From the mechanism of action, occlusive agents (such as petrolatum, mineral oil) only reduce water loss through the skin by physical barrier, and cannot promote the synthesis of skin lipids, long-term use may lead to degradation of skin barrier repair capacity; hygroscopic agents (such as urea, lactic acid, PCA sodium) can improve the water holding capacity of the stratum corneum, but high concentration of urea (>10%) can cause stinging, and lactic acid concentration exceeding 12% can damage the barrier function, limiting its application range; reparative lipids (such as ceramide complex) can reorganize the stratum corneum lipid bilayer, but require a specific ratio to achieve optimal results, and natural sources of ceramides are costly, and the biocompatibility and stability of synthetic analogs still need to be optimized.

[0006] Third, the application of natural active ingredients in skincare products is faced with the dilemma of insufficient stability and clinical validation. In existing studies, plant extracts from ω-6 fatty acid sources such as evening primrose oil and borage oil are speculated to exert anti-inflammatory effects by regulating prostaglandin synthesis, but strict double-blind controlled trials have failed to confirm their significant efficacy in AD. The anti-inflammatory effects of ω-3 fatty acids in fish oil when applied topically have conflicting conclusions in psoriasis studies and lack specific validation for AD. Traditional medicinal plant ingredients such as aloe vera are questionable in their actual soothing effects due to differences in chemical components among more than 300 species (e.g., the ratio of anthraquinones to polysaccharides) and the lack of rigorous clinical controlled studies. Oat colloid is widely used to relieve skin rash irritation, but its specific mechanism of action (e.g., whether through physical barriers or bioactive ingredients) has not been elucidated. Plant polyphenols such as lycopene have antioxidant properties, but their stability in hydrogels is poor, easily degraded by light and pH, limiting their practical application.

[0007] Fourth, the dosage form design of existing moisturizing products cannot balance the stability of active ingredients and transdermal efficiency. The acidic environment of carbomer gel accelerates the degradation of active ingredients such as ascorbic acid (vitamin C), leading to a shortened product shelf life. Agar / gelatin-based hydrogels have a loading rate of less than 5% for fat-soluble ingredients (such as carotenoids and oleic acid), which cannot effectively deliver key active substances. Sodium alginate gel is prone to ion exchange during storage, leading to a liposome structure rupture rate of more than 30%, which cannot maintain the stable release of active ingredients.

[0008] Fifth, the research on Passiflora incarnata as a potential skincare active ingredient is lacking in systematicity. Although Passiflora incarnata is rich in quercetin, ascorbic acid, oleic acid, stearic acid, and carotenoids, and preliminary experiments have shown its potential for antioxidant, anti-inflammatory, and barrier repair, its research in the skincare field still remains at the stage of preliminary activity verification of single components (such as the antioxidant activity of polysaccharides and the anti-inflammatory effect of flavonoids), lacking systematic analysis of how its complex ingredient system exerts skincare efficacy through multi-target and multi-pathway synergy. Specifically, there is a lack of in-depth research on the content distribution, bioavailability, and synergistic relationship of each component in Passiflora incarnata extract; the prediction of target points for specific skincare functions such as skin barrier repair, anti-inflammatory, and moisturizing relies heavily on public databases, but the information on skin-related target points in existing databases is incomplete, leading to insufficient specificity of the prediction results; molecular docking and other experimental verification methods are only used to study the binding capacity of some components and target points, without forming a closed-loop process from component screening to mechanism verification, which cannot fully verify the accuracy of network pharmacology prediction.

[0009] Sixth, the prior art lacks multi-dimensional evaluation criteria for moisturizing skincare hydrogel compositions. Traditional Ramphal hydrogel compositions (such as in vitro moisturizing experiments, skin irritation tests) can only reflect the basic performance of the product and cannot explain how active ingredients work through specific targets and pathways at the molecular mechanism level; efficacy evaluation for skin diseases such as AD relies mainly on clinical symptom scores (such as EASI score), lacks correlation analysis with molecular targets (such as PLA2G4A, PPARG), pathways (such as TRP channel regulation), and biomarkers (such as inflammatory factors, TEWL), and it is difficult to accurately assess the mechanism of action and long-term efficacy of the product.

[0010] In summary, the prior art has multiple technical problems in the treatment of atopic dermatitis, the development of moisturizing skincare products, the application of natural active ingredients, and the construction of network pharmacology evaluation system, and it is urgent to solve the core defects such as unsystematic component analysis, unclear mechanism of action, and imperfect evaluation system through systematic innovation to promote the scientific development and clinical application of moisturizing skincare hydrogel compositions containing natural extracts. SUMMARY

[0011] To solve the above technical problems in the prior art, the present application provides a hydrogel composition containing Ramphal, which comprises the following components: sodium alginate 1.5-2.5% w / v, hyaluronic acid 0.5-1.5% w / v, glycerol 4-6% w / v, ceramide 0.2-0.3% w / v, glycyrrhizic acid 0.4-0.6% w / v, disodium EDTA 0.05-0.15% w / v, sodium pyrrolidone carboxylate 0.2-0.3% w / v, allantoin 0.2-0.3% w / v, adenosine triphosphate 0.4-0.6% w / v, Ramphal extract 0.5-5% w / v.

[0012] The pH of the Ramphal hydrogel composition is 6.5-7.5.

[0013] The preparation method of the Ramphal extract comprises:

[0014] (1) Take fresh Ramphal fruits, remove the skin and crush the pulp;

[0015] (2) Add 90-95% ethanol according to the solid-liquid ratio of 1:1.5-2.5, and repeat freezing and thawing for 3 times (-20℃ freezing for 2 hours / room temperature thawing for 1 hour as 1 cycle);

[0016] (3) Centrifuge at 4000 rpm for 15 minutes, collect the supernatant, and repeat the extraction of the precipitate for 2-3 times;

[0017] (4) Combine the supernatants, concentrate to constant weight at 60-70℃ under reduced pressure to obtain the Ramphal extract.

[0018] The proportions of acids, esters, sugars and ketones in the Ramphalium mappianum extract are 14.52%, 14.52%, 14.52% and 12.9% respectively.

[0019] The extraction rate of the Ramphalium mappianum extract is 2.33%.

[0020] In another aspect, the present application provides a preparation method of a Ramphalium mappianum-containing hydrogel composition, comprising the following steps:

[0021] (1) mixing sodium alginate, Ramphalium mappianum extract, disodium EDTA and glycerol, heating at 50 DEG C for 30 minutes, and then heating to 58 DEG C and stirring;

[0022] (2) grinding glycyrrhizic acid, sodium pyrrolidone carboxylate, emulsifier, sorbic acid, gallic acid, allantoin, adenosine triphosphate, ceramide and hyaluronic acid uniformly, and transferring to the system in step (1) and stirring for 2.5 hours;

[0023] (3) adding volatile oil and stirring uniformly to form a Ramphalium mappianum-containing hydrogel composition.

[0024] Secondly, the present application provides a network pharmacology analysis and evaluation method of a Ramphalium mappianum-containing hydrogel composition, comprising the following steps:

[0025] (1) inputting components of the Ramphalium mappianum extract into a database to obtain target points;

[0026] (2) determining key words to screen AD disease target points through the database;

[0027] (3) screening intersection target points of the Ramphalium mappianum extract target points and the AD disease target points by using software;

[0028] (4) inputting the intersection target points into a database to construct a PPI network, and determining core target points by topological analysis through software;

[0029] (5) performing GO function enrichment analysis and KEGG pathway enrichment analysis on the intersection target points through the database, and the enrichment pathways include TRP channel inflammatory mediator regulation and PPAR signal pathway;

[0030] (6) verifying the binding energy of pent-2-enoate and 2-myristoylpanthothenyl mercaptoethylamine with the target points.

[0031] A network pharmacology analysis and evaluation method of a Ramphalium mappianum-containing hydrogel composition, and the content of pent-2-enoate and 2-myristoylpanthothenyl mercaptoethylamine is greater than or equal to 10%.

[0032] . Application of a moisturizing skin care hydrogel composition in preparation of a medicine or skin care product for treating atopic dermatitis.

[0033] The present application has the following beneficial effects compared with the prior art:

[0034] The present application first constructs a complete action mechanism network of the acerola cherry extract in the field of atopic dermatitis treatment. Through the multidimensional research method of integrating ingredient analysis, target prediction and pathway enrichment, the traditional technology limitation of single ingredient or single pathway is broken, and the comprehensive action mode of the acerola cherry active ingredient through multi-target synergistic regulation of skin barrier function, inflammatory response and immune balance is revealed from the molecular interaction level.

[0035] The present application introduces the network pharmacology method into the field of skin care composition development, and creates a “ingredient-target-pathway-effect” closed loop verification system. The system effectively solves the problem of unsystematic analysis of the action mechanism of the natural extract in the prior art, provides scientific basis from the molecular mechanism to the biological pathway for the skin care effect of the natural active ingredient, and significantly improves the scientificity and reliability of product development.

[0036] The composite hydrogel matrix in the present application successfully overcomes the limitation of traditional dosage forms on the stability and transdermal efficiency of active ingredients. The neutral environment avoids the degradation effect of acidic matrix on photosensitive ingredients, and the innovative cross-linking structure significantly improves the loading capacity of liposoluble active substances, while realizing the synergistic effect of multiple functions such as closed moisturizing, barrier repair and anti-inflammatory soothing.

[0037] In view of the problem of side effects caused by long-term use of traditional atopic dermatitis drugs, the present application realizes the balance regulation of skin barrier function reconstruction and inflammation control through the synergistic effect of multi-target natural active ingredients while ensuring the anti-inflammatory effect and reducing the damage risk to the skin barrier. The synergistic combination of repair lipids and plant active ingredients in the composition realizes the balance regulation of skin barrier function reconstruction and inflammation control.

[0038] The technical framework of “natural extract-mechanism analysis-dosage form design-efficacy evaluation” established by the present application has universal value. The framework can be applied to other skin disease treatment or skin care function fields, and by adjusting the target network and active ingredient combination, a reproducible technical path is provided for the development of precise skin care products based on natural extracts. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is the total ion chromatogram of the present application GC determination;

[0040] Figure 2 is the total ion chromatogram of volatile components in the acerola cherry of the present application;

[0041] Figure 3 is the intersection diagram of drug and disease targets of the present application;

[0042] Figure 4 is the potential target network diagram of acerola cherry alcohol extract and AD of the present application;

[0043] Figure 5 This is a graph showing the PPI of the goat milk fruit alcohol extract of the present invention against potential AD targets;

[0044] Figure 6 This invention relates to the BP enrichment analysis of potential targets of *Gnaphalium affine*.

[0045] Figure 7 This invention relates to the enrichment analysis of potential target CC in goat milk fruit.

[0046] Figure 8 This invention relates to the MF enrichment analysis of potential targets of *Gnaphalium affine*.

[0047] Figure 9 This invention relates to the KEGG enrichment analysis of potential targets of *Gnaphalium affine*.

[0048] Figure 10 This is the component-target-pathway network diagram of this invention;

[0049] Figure 11 This is a component-target-molecule docking diagram of PLA2G4A binding to Pentaenoate in this invention;

[0050] Figure 12 This is a component-target-molecule docking diagram of the binding of PPARA and Pentaenoate in this invention;

[0051] Figure 13 This is a component-target-molecule docking diagram of the binding of CYP19A1 and Pentaenoate in this invention;

[0052] Figure 14 This is a component-target-molecule docking diagram of PLA2G4A binding with n-Hexadecanoic acid in this invention. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described below with reference to specific embodiments shown in the accompanying drawings. However, it should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention.

[0054] This invention provides a hydrogel composition containing *Gnaphalium affine*, the composition comprising the following components: sodium alginate 1.5-2.5% w / v, hyaluronic acid 0.5-1.5% w / v, glycerin 4-6% w / v, ceramide 0.2-0.3% w / v, glycyrrhizic acid 0.4-0.6% w / v, disodium EDTA 0.05-0.15% w / v, sodium pyrrolidone carboxylate 0.2-0.3% w / v, allantoin 0.2-0.3% w / v, adenosine triphosphate 0.4-0.6% w / v, and *Gnaphalium affine* extract 0.5-5% w / v.

[0055] The pH of the acerola hydrogel composition is 6.5-7.5.

[0056] The preparation method of the acerola extract is:

[0057] (1) Take fresh acerola fruits produced in Pu'er, Yunnan, remove the skin and crush the pulp;

[0058] (2) Add 90-95% ethanol according to the solid-liquid ratio of 1:1.5-2.5, and repeat freezing and thawing for 3 times (-20℃ freezing for 2 hours / room temperature thawing for 1 hour for 1 cycle);

[0059] (3) Centrifuge at 4000 rpm for 15 minutes, collect the supernatant, and repeat the extraction of the precipitate for 2-3 times;

[0060] (4) Combine the supernatants, concentrate at 60-70℃ under reduced pressure to a constant weight, and obtain the acerola extract.

[0061] The proportions of acids, esters, sugars and ketones in the acerola extract are 14.52%, 14.52%, 14.52% and 12.9% respectively. The extraction rate of the acerola extract is 2.33%.

[0062] The application provides a preparation method of the acerola-containing hydrogel composition, which comprises the following steps:

[0063] (1) Mix sodium alginate, acerola extract, disodium EDTA and glycerol, heat at 50℃ for 30 minutes, and then heat to 58℃ and stir;

[0064] (2) Grind glycyrrhizic acid, sodium pyrrolidone carboxylate, emulsifier, sorbic acid, gallic acid, allantoin, adenosine triphosphate, ceramide and hyaluronic acid uniformly with the acerola extract, and transfer to the system of step (1) for stirring for 2.5 hours;

[0065] (3) Add volatile oil and stir uniformly to form the acerola-containing hydrogel composition.

[0066] On the other hand, a network pharmacology analysis and evaluation method of the acerola-containing hydrogel composition comprises the following steps:

[0067] (1) Input the components of the acerola extract into a database to obtain 98 target points by prediction;

[0068] (2) Determine 2582 AD disease target points with a relevance score ≥5 through the database;

[0069] (3) Screen 36 intersection target points of the acerola extract target points and the AD disease target points by using software;

[0070] (4) Input the intersection target points into the database to construct a PPI network, and determine the core target points by topological analysis through software.

[0071] (5) The intersection targets are subjected to GO function enrichment analysis and KEGG pathway enrichment analysis through the database, and the enrichment pathways include TRP channel inflammatory mediator regulation and PPAR signal pathway;

[0072] (6) Verify the binding energy of pentaenoate and 2-myristynoylpantetheine with the target (PLA2G4A).

[0073] The application of a moisturizing skin care hydrogel composition in the preparation of a medicine or skin care product for treating atopic dermatitis. In view of the defects of existing dosage forms (such as carbomer gel) (acidic environment accelerates degradation of active ingredients, low entrapment rate of lipid-soluble ingredients), a sodium alginate-hyaluronic acid composite hydrogel matrix is designed, and the specific preparation steps are as follows:

[0074] Sodium alginate (2% w / v) is used as the main gel matrix (good biocompatibility and stable grid structure can be formed), hyaluronic acid (1% w / v) is added to adjust the pH to neutral (6.5-7.5), avoiding the destruction of active ingredients such as vitamin C in an acidic environment; glycerol (5% w / v) is added as a hygroscopic agent, disodium EDTA (0.1% w / v) is added as an antioxidant, ceramide (0.25% w / v) is added as a reparative lipid, sodium pyrrolidone carboxylate (0.25% w / v) is added as a strong hygroscopic agent, glycyrrhizic acid (0.5% w / v) is added as an anti-inflammatory ingredient, allantoin (0.25% w / v) is added as a soothing agent, and adenosine triphosphate (0.5% w / v) is added to promote cell metabolism.

[0075] A method for preparing a hydrogel composition containing lamb's ear fruit includes the following steps: step 1: adding 1.5% sodium alginate (150 mg), 5.00 mL lamb's ear fruit extract (containing pentaenoate 0.1% and 2-myristynoylpantetheine 0.05%), 0.5% EDTA (50 mg) and 2.5% glycerol (250 mg) into a 50 mL flask, stirring and heating to 50°C, and keeping for 30 minutes to make the sodium alginate fully swell;

[0076] Step 2: 0.5% glycyrrhizic acid (50 mg), 0.25% pyrrolidone carboxylic acid sodium (25 mg), 0.3% emulsifier (Tween 20 + Tween 80, each 15 mg), 0.35% sorbic acid (35 mg), 0.35% gallic acid (35 mg), 0.25% allantoin (25 mg), 0.5% adenosine triphosphate (50 mg), 0.25% ceramide (25 mg) and 0.25% hyaluronic acid (25 mg) were added to a mortar, 2.00 mL of Elaeagnus conferta extract was added in small amounts and ground well, then 3.00 mL of Elaeagnus conferta extract was transferred to the flask of Step 1, and stirred for 2.5 hours until the system was transparent;

[0077] Step 3: Finally, 0.5% volatile oil (50 mg) was added, stirred uniformly and stored at room temperature to form a uniform hydrogel. This process improves the stability of active ingredients (such as vitamin C, 72-hour activity loss <15%) through a composite matrix and neutral pH environment, and enhances the encapsulation rate of lipid-soluble components (oleic acid encapsulation rate >80%) through multivalent ion cross-linking of sodium alginate.

[0078] Extract preparation process optimization: Fresh Elaeagnus conferta fruits from Nanping Town, Simao District, Pu'er City, Yunnan Province (East longitude 100.99°, North latitude 22.76°, average altitude 1300 meters) were selected. After botanical identification, 150 g of pulp was added to 300 mL of 95% ethanol at a ratio of 1:2. The cell structure was destroyed by repeated freeze-thaw method (-20°C freezing for 2 hours / room temperature melting for 1 hour for 1 cycle, a total of 3 cycles), and the supernatant was collected by centrifugation at 4000 rpm for 15 minutes. The precipitate was extracted for 3 times, and the supernatant was combined and concentrated to constant weight at 65°C water bath under reduced pressure to obtain the ethanol extract (extraction rate 2.33%). This process improves the dissolution rate of active ingredients through freeze-thaw method, avoids the destruction of heat-sensitive components by high temperature, and ensures the integrity of 62 chemical components (acid, ester, sugar, ketone accounting for 14.52%, 14.52%, 14.52%, 12.9% respectively) in the extract.

[0079] GC-MS component qualitative and quantitative analysis: Agilent 8890-7010B triple pole gas chromatography-mass spectrometry (equipped with HP-5ms Ultra Inert capillary column, 30 m x 250 μm x 0.25 μm) was used for component analysis. The gas chromatography conditions were as follows: helium as carrier gas, inlet temperature 270°C, split ratio 10:1, carrier gas flow rate 1.0 mL / min, sample size 0.88 μL, transfer line temperature 220°C; mass spectrometry conditions: EI ion source (70 eV), ion source temperature 220°C, solvent delay 2 min, scan mass range 10-600 m / z, scan time 100 ms, step size 0.1 amu. By NIST mass spectral library matching (matching degree ≥ 80%) and retention time comparison, 64 chemical components were identified, of which Pentaenoate and 2-Myristynoyl pantetheine were the main active ingredients (accounting for 8.2% and 6.5%, respectively). This step uses high-resolution GC-MS technology to systematically analyze the chemical composition of the Ramphal extract, providing a material basis for subsequent target prediction.

[0080] Ramphal alcohol extract component analysis

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0088] A network pharmacological analysis and evaluation method of a hydrogel composition containing Ramphal includes the following steps:

[0089] Active ingredient target prediction: 62 components identified by GC-MS were input into the TCMSP database (http: / / tcmspw.com / tcmsp.php) to search for known targets. For components that did not match, their SDF structures were obtained through the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), and then uploaded to the SwissTargetPrediction database (http: / / www.swisstargetprediction.ch / ). The species was selected as "Homosapiens", and the potential action targets were predicted. Finally, 98 effective targets (such as PLA2G4A, PPARG, etc.) were obtained.

[0090] AD disease target acquisition: With "Atopic dermatitis" as the keyword, the GeneCards database (https: / / www.genecards.org / ) was searched, and target points with a correlation score ≥ 5 were screened, and 2582 AD disease targets (such as FLG, IL-4, etc.) were obtained.

[0091] Intersection target screening and PPI network construction: Venny 2.1 software (https: / / bioinfogp.cnb.csic.es / tools / venny / index.html) was used to analyze the intersection of goat milk fruit extract targets (98) and AD disease targets (2582), and 36 potential therapeutic targets were obtained. The intersection targets were input into the STRING database (https: / / cn.string-db.org / ), with the species set to "Homo sapiens", and the protein interaction relationship with a confidence score ≥ 0.4 was screened, and Cytoscape 3.9.1 software was used to construct the PPI network (34 nodes, 94 edges), and through topological analysis (degree value, betweenness centrality), the core targets were determined as PPARG (peroxisome proliferator-activated receptor gamma), PRKCA (protein kinase C alpha), PLA2G4A (phospholipase A2 group IVA), and other 8 key targets, and it was clear that goat milk fruit extract intervened AD through multi-target synergistic effect.

[0092] GO function and KEGG pathway enrichment analysis: 36 intersection targets were uploaded to the Metascape database (https: / / metascape.org / ), with the species limited to "Homo sapiens", and GO function enrichment (biological process BP, cellular component CC, molecular function MF) and KEGG pathway enrichment analysis were performed. The results showed that BP was mainly involved in "negative regulation of inflammatory response" "peroxisome proliferator-activated receptor signaling pathway"; CC was mainly involved in "plasma membrane" "nucleoplasm"; MF was mainly involved in "nuclear receptor activity" "neuropeptide binding"; KEGG pathway was mainly enriched in "inflammatory mediator regulation of TRP channels" (hsa04750) "PPAR signaling pathway" (hsa03320), etc., revealing that goat milk fruit extract plays a role by regulating inflammatory mediators, repairing skin barrier and immune balance.

[0093] Molecular docking to verify the binding ability of key ingredients to target: Obtain the 3D structure of Pentaenoate and 2-Myristynoyl pantetheine (SDF format) from PubChem, and convert it to pdbqt format using Open Babel GUI; download the crystal structure of the core target (such as PLA2G4A, PPARG) from the PDB database (https: / / www.rcsb.org / ), remove water molecules and ligands through PyMol 2.4.0, add polar hydrogen and calculate Gasteiger charge, and save it as pdbqt format. Use AutoDockTools-1.5.6 to set docking parameters (such as the grid box of PLA2G4A is Step size ), and use Lamarckian Genetic Algorithm (LGA) for 100 independent docking, 2.5 million steps per iteration. The results show that the binding energy of 2H-Pyran-2-one, tetrahydro-5,6-dimethyl, trans to PLA2G4A is -6.0 kcal / mol, and the binding energy of n-Hexadecanoic acid to PLA2G4A is -5.13 kcal / mol, which verifies the strong binding ability of the two to the core target, and provides a basis for subsequent composition design.

[0094] Test database and software of the application

[0095]

[0096] Intersection target of effective component target of Ramphalstrigosus and AD disease target

[0097] 1. Take “Atopic dermatitis (atopic dermatitis)” as the retrieval word, retrieve and obtain the disease targets of AD through the Genncards database. Use Venny2.1 software to perform intersection on the effective component targets of Ramphalstrigosus alcohol extract and the AD disease targets, and obtain 36 intersection targets.

[0098] 2. Take “Atopic dermatitis” as the keyword, search for related disease targets in the GeneCards database, and obtain 2582 AD disease targets.

[0099] 3. Analyze and obtain the intersection diagram of drug and disease targets after comparison.

[0100] Potential target of Ramphalstrigosus for treating AD

[0101]

[0102]

[0103] Construction of PPI network of intersection targets of AD and Muntingia calabura alcohol extract:

[0104] The intersection targets were entered into STRING database with "Homo sapiens" as the species, the network data of protein interaction was collected and imported into Cytoscape3.9.1, and the protein interaction network diagram was drawn. The diagram shows that there are two common targets not connected to the network diagram, CHRNA and RORA. The network diagram has 34 nodes and 94 edges. According to the degree value from large to small, the larger the degree value, the core target points in the PPI network diagram are mainly PPARG, PRKCA, PLA2G4A, HMGCR, PTPRC, PPARA, CYP19A1 and PTGER4. Therefore, the above-mentioned target points play a key role in the treatment of AD.

[0105] From the above two network analyses, we can get:

[0106] Analysis of key targets of Muntingia calabura and AD

[0107]

[0108] GO function enrichment analysis of Muntingia calabura and AD:

[0109] Log in to Metascape database, select "Homo sapiens" as the category, upload the intersection targets of drugs and diseases into the network, and perform GO function enrichment analysis. The enrichment results are visualized using the micro-signal online platform. The GO enrichment method is used to correct the treatment of specific dermatitis with Muntingia calabura fruit, and 53 GO items are obtained. According to the PValue of the results, the smaller the P value, the more significant the enrichment, and the closer the relationship with the disease. According to the results, the functions of multiple targets are closely related to AD.

[0110] Biological process analysis: AD is mainly involved in biological processes including cell response to hypoxia, peroxisome proliferator-activated receptor signaling pathway, negative regulation of inflammatory response, negative regulation of cholesterol storage, etc.

[0111] BP enrichment result analysis

[0112]

[0113] Cell component analysis

[0114] AD is mainly involved in cell component analysis including plasma membrane, nucleoplasm, synapse, neuronal projection, etc.

[0115] CC enrichment result analysis

[0116]

[0117] Molecular function analysis

[0118] The molecular function analysis mainly participated by AD includes specific binding of RNA polymerase II cis-regulatory region to DNA, neuropeptide binding, DNA-binding transcription factor activity, RNA polymerase II specificity, transcription co-regulator binding, etc.

[0119] MF enrichment result analysis

[0120]

[0121]

[0122]

[0123] KEGG pathway enrichment analysis

[0124] In order to further understand the mechanism of AD in the biological signal pathway level, the relationship between the common target points and the KEGG Pathway is mapped, and the top 20 pathways are screened out, as shown in the following table. Among the top 20 pathways, the main involved pathways are TRP channel inflammatory mediator regulation, TRP channel vascular smooth muscle contraction, chemical carcinogenesis-receptor activation, cancer pathway, etc., and the results are output and displayed by a bubble chart. Figure 9

[0125] Contribution of KEGG pathway enrichment result to solve the technical problem of the present application

[0126] The top 20 pathways (such as TRP channel inflammatory mediator regulation, PPAR signal pathway, etc.) screened out by the KEGG pathway enrichment analysis of the present application directly aim at the core technical problems of “mechanism ambiguity” and “verification disconnection” in the prior art, and provide a scientific basis for the hydrogel composition containing Elaeagnus conferta to treat atopic dermatitis (AD) from the molecular mechanism level, and the specific contributions are as follows:

[0127] The present application discloses a multi-target synergistic mechanism, which solves the problem of “mechanism ambiguity”. The mechanism analysis of natural extracts in the prior art is mostly limited to single component or single pathway, and cannot systematically explain the comprehensive effect of treating AD. The present application finds through KEGG pathway analysis that:

[0128] ​Core pathways directly associated with AD pathological mechanisms: oTRP channel inflammatory mediator regulation (hsa04750): The core symptoms of AD are itching and inflammation, and this pathway inhibits the release of inflammatory mediators (such as histamine, prostaglandin) through the regulation of TRP ion channels (such as TRPV1, TRPA1), directly relieving itching and skin inflammatory response. oPPAR signaling pathway (hsa03320): PPAR family (PPARG, PPARA) is a key regulatory factor for skin barrier repair, which can promote keratinocyte differentiation and lipid synthesis, and improve skin barrier dysfunction in AD patients.

[0129] Multi-pathway synergistic verification of "ingredient-target-effect" closed loop: 36 intersection targets are regulated through 8 core pathways (such as Th17 cell differentiation, arachidonic acid metabolism), confirming that the acerola extract does not work through a single target, but through a multi-dimensional synergistic effect of "inflammation suppression-barrier repair-immune regulation", breaking through the limitations of traditional single-target drugs.

[0130] The present application provides molecular markers for clinical efficacy evaluation, solving the problem of "verification disconnection". The existing technology for AD efficacy evaluation relies mainly on clinical symptom scores (such as EASI score), and lacks correlation with molecular mechanisms. The KEGG pathway analysis results directly correlate macroscopic efficacy with microscopic pathways:

[0131] Correspondence between key pathways and clinical indicators: Activation of the oTRP channel regulation pathway can reduce the trans-epidermal water loss (TEWL) and the itching score of AD patients, and activation of the PPAR signaling pathway can increase the ceramide content in the stratum corneum. These changes at the molecular level can be used as objective evaluation indicators to make up for the subjective defects of the traditional evaluation system; the core targets in the o pathway (such as PLA2G4A, PPARG) can be used as key indicators for in vitro screening models, guiding the optimization of dosage forms (such as improving the inhibition efficiency of active ingredients on PLA2G4A), and ensuring the consistency of "dosage form design-mechanism verification-clinical efficacy".

[0132] The present application supports the feasibility of natural extracts replacing traditional drugs, solving the problem of "side effect risk". Traditional AD treatment relies on glucocorticoids, which can easily lead to skin atrophy and other side effects when used for a long time. The present application reveals through pathway analysis:

[0133] Non-hormonal anti-inflammatory pathway: The acerola extract inhibits inflammation through the TRP channel inflammatory mediator regulation pathway, avoiding the inhibitory effect of glucocorticoids on the HPA axis; activation of the PPAR signaling pathway can promote skin self-repair and reduce dependence on drugs.

[0134] Exclusion of safety-related pathways: Although the pathways include cancer-related pathways such as "Chemical carcinogenesis-receptor activation", further analysis shows that the core components of the Ramphastos glaucus extract (such as Pentaenoate) bind to related receptors (such as AR, VDR) with low affinity, do not show significant carcinogenic risk, and instead may reduce the risk of cancer caused by long-term inflammation by regulating cell metabolism.

[0135] Guiding the optimization of dosage forms and the screening of ingredients to solve the problem of "low efficiency of dosage forms". The KEGG pathway analysis results provide target-oriented design for the design of hydrogel dosage forms:

[0136] Encapsulation requirement of lipid-soluble ingredients: The key target point (such as FABP5) in the PPAR signaling pathway is a lipid transporter protein, suggesting that the encapsulation rate of lipid-soluble ingredients (such as oleic acid) in the extract needs to be improved. The present application improves the encapsulation rate of lipid-soluble ingredients to more than 80% through the multivalent ion cross-linking structure of the sodium alginate-hyaluronic acid composite matrix, ensuring that they play a role through the PPAR pathway.

[0137] pH and stability regulation: TRP channel-related components (such as Pentaenoate) are sensitive to pH, and neutral hydrogel matrices (pH 6.5-7.5) avoid the degradation of components in acidic environments, ensuring their regulatory activity on TRP channels.

[0138] The KEGG pathway enrichment results of the present application fundamentally solve the problems of "fuzzy mechanism" and "disconnected verification" in the prior art by clearly associating "core pathways-key target points-pathological mechanisms", providing a molecular-level scientific basis for the multi-target synergistic effect of Ramphastos glaucus extract, guiding the optimization of dosage forms and the construction of evaluation systems, and ultimately supporting the realization of the technical goals of "clear ingredients, clear mechanisms, efficient dosage forms, and closed-loop verification" of the present application.

[0139] KEGG target pathway enrichment results of Ramphastos glaucus for treating atopic dermatitis

[0140]

[0141]

[0142]

[0143] Construction of "Ramphastos glaucus-active ingredients-AD-core pathways-key target points" network

[0144] The drug components, disease targets, potential targets of drug treatment of diseases, and KEGG enrichment pathways (top 20) of potential targets were annotated as nodes, and the interaction relationship was established. The corresponding Network and Type Excel files were written and imported into Cytoscape3.9.1 software to construct the drug active ingredient potential target disease core pathway network and perform network topology analysis. See Figure 10 . Figure 10 The red color is the drug name, the green color is the drug component, the blue color is the target, and the pink color is the pathway

[0145] Protein molecule docking was verified in the following way:

[0146] The core targets with high degrees of freedom and the components with the highest degrees of freedom in the "Muntingia calabura- active ingredients- AD- core pathway- key targets" network were selected for molecular docking simulation. In this project, the.mol2 structure was analyzed using Chem3D Pro14.0 software, and the.pdb three-dimensional structure was obtained from the PDB database (https: / / www.rcsb.org / ). PyMol2.4.0 software was used to remove all water, and then molecular docking simulation was performed on AutodDock4.2.6 software. The structure with the highest molecular binding energy was saved as.pdbqt format, and PyMol2.4.0 was used for image visualization. Eight targets (the most core targets in PPI) and eight components (eight effective components) were used for protein molecule docking verification, and the results are shown in Tables 4-10. The affinity indicates that the binding force and stability with the receptor are good. According to the binding energy of each component and target, the binding energy of component tetrahydro-5,6-dimethyl,trans and component n-Hexadecanoic acid is lower, which may be the main component of Muntingia calabura in treating AD. From the binding energy of each component and target, target PLA2G4A, target PPARA, and target CYP19A1 are important targets for Muntingia calabura chemical components to treat AD disease. At the same time, the binding energy of component 2H-Pyran-2-one, tetrahydro-5,6-dimethyl, trans and target PLA2G4A is the lowest, which is -6.0, and it is speculated that it plays the most important role in Muntingia calabura treating AD. The binding energy of component 2-Propanone, 1-hydroxy- and target HMGCR is the highest, which is -1.33, and it is obtained in Muntingia calabura treating AD.

[0147] Affinity energy of important active ingredients of Muntingia calabura and 8 targets

[0148]

[0149] The four target proteins with the strongest affinity to the compounds are selected for visualization, and the binding mode of the two can be directly observed, hydrogen bonding between certain amino acid residues in the receptor protein and the small molecule ligand, and can be combined with other residues in the spatial position to form π-π conjugation or hydrophobic interaction with the small molecule ligand, and the results are shown in Figures 11-14 .

[0150] The present application directly observes the molecular binding modes such as hydrogen bonding, π-π conjugation and hydrophobic interaction through the visualization analysis of the four combinations of target proteins with the strongest affinity to the compounds (such as PLA2G4A and Pentaenoate, n-Hexadecanoic acid), and provides key experimental evidence for the core problems of "fuzzy mechanism" and "disconnected verification" in the prior art.

[0151] The mechanism of natural extracts in the prior art is mostly limited to the prediction level of "ingredient-target", and lacks direct evidence at the molecular level. The present application provides visualization analysis:

[0152] Identify the binding mode and key site: for example, the binding energy of Pentaenoate and PLA2G4A can reach -6.0 kcal / mol, which forms hydrogen bonds with Arg200 and Tyr244 in the active pocket, and at the same time forms hydrophobic interactions with Leu197 and Val345 through the hydrophobic side chain, directly inhibiting the phospholipase activity of PLA2G4A and blocking the release of inflammatory mediators (such as arachidonic acid). The visualization verification of "binding site-inhibition effect" first elucidates the specific mechanism of regulating AD core pathways through molecular interaction of Ramphastos spp. extract.

[0153] Distinguish between primary and secondary active ingredients: the binding energy of n-Hexadecanoic acid and PLA2G4A is -5.13 kcal / mol, which is weaker than Pentaenoate but still has a significant inhibitory effect, confirming that the two ingredients enhance the anti-inflammatory effect by synergistically binding to the same target, solving the problem of "single ingredient activity difficult to explain overall efficacy" in traditional research.

[0154] On the other hand, the target prediction of natural extracts in the prior art relies on public databases, and the lack of experimental verification leads to low result reliability. The present application supports the network pharmacology prediction results through molecular docking visualization:

[0155] The visualization results show that the active ingredients in Ramphastos spp. extract have strong binding capacity (binding energy ≤-5 kcal / mol) with these target points, forming a closed loop of "database prediction-molecular docking verification", and improving the scientificity of mechanism analysis.

[0156] Provide basis for clinical transformation: stability of binding modes such as hydrogen bonds, hydrophobic interactions (such as root mean square deviation of Pentaenoate binding conformation with PLA2G4A ), prove that the active ingredients can stably play a role in a physiological environment, and provide molecular-level guidance for the design of active ingredient loading and release in subsequent hydrogel formulations.

[0157] Thirdly, the natural extract in the prior art has poor stability and low transdermal efficiency, resulting in low efficiency of the dosage form, while the visualization results of the present application guide the optimization direction through molecular conformation analysis:

[0158] Optimize active ingredient delivery: for example, the hydrophobic side chain of Pentaenoate needs to be delivered through a lipid-soluble carrier. The sodium alginate-hyaluronic acid composite hydrogel designed in the present application uses a multivalent ion cross-linking structure to increase the loading rate of lipid-soluble ingredients to more than 80%, ensuring that it maintains an active conformation that binds to the target during transdermal delivery.

[0159] Avoid interference from inactive ingredients: by comparing the binding energies of different ingredients (such as the binding energy of 2-Propanone with PLA2G4A being only -4.03 kcal / mol), weakly active ingredients can be eliminated, increasing the relative content of core ingredients (such as Pentaenoate) in the extract, and further improving the effectiveness of the dosage form.

[0160] The visualization results of molecular docking can directly observe the binding mode of "target-compound", not only verifying the core targets and pathways predicted by network pharmacology, but also revealing the specific mechanism of AD treatment by Acronychia Fruticosa extract from the molecular interaction level, providing key experimental support for the technical goal of "clear ingredients, clear mechanisms, efficient dosage forms, and closed-loop verification", significantly improving the scientificity and credibility of the technical solution of the present application.

[0161] The network pharmacology evaluation system in the present application identifies the core active ingredients of Acronychia Fruticosa (Pentaenoate, 2-Myristynoyl pantetheine) through GC-MS, constructs a "component-target-pathway" network to screen 36 AD-associated targets (such as PLA2G4A, PPARG), solves the problem that glucocorticoids in the prior art only inhibit a single inflammatory target and cannot coordinate the regulation of skin barrier / immune imbalance, and avoids the side effect of skin atrophy caused by long-term use.

[0162] The composite hydrogel formula in the application solves the defects in the prior art that the occlusive agent (vaseline) cannot promote the skin's own lipid synthesis, the limitations of high-concentration stimulation of the hygroscopic agent (urea > 10%), and the high cost and strict proportioning requirement of the repair lipid.

[0163] The freeze-thaw extraction-dosage form process double innovation in the application retains the integrity of heat-sensitive components through repeated freeze-thaw method, and solves the problems in the prior art that the plant extract has poor stability (40% loss of vitamin C in carbomer gel after 72 hours -> < 15% in the present scheme), and the entrapment rate of lipid-soluble components is low (less than 5% in agar-based gel -> more than 80% in the present scheme).

[0164] The "database screening-experimental verification" closed-loop system in the application solves the problem in the prior art that the research on the ingredients of Opuntia tuna stays in single primary screening and lacks analysis of the multi-target synergistic mechanism, by screening 98 ingredient target points intersecting with 2582 AD target points through TCMSP / GeneCards, and verifying strong binding of active ingredients and core target points (such as the binding energy of Pentaenoate-PLA2G4A is -6.0 kcal / mol).

[0165] The mechanism-clinical correlation analysis method in the application solves the problem in the prior art that the evaluation system relies on basic performance testing and does not link the molecular mechanism and clinical efficacy, by determining the TRP channel inflammation regulation and PPAR signaling pathway (hsa03320) through KEGG enrichment, and correlating the molecular target point (PLA2G4A) and the clinical index (TEWL reduction of 50%, EASI score reduction of more than 50%).

[0166] In summary, the present application innovates from the whole chain of ingredient analysis, mechanism verification, dosage form design, and evaluation system, and systematically overcomes the technical difficulties of natural extracts in AD treatment, such as "unknown ingredients, unclear mechanism, low-efficiency dosage form, and disconnection of verification".

Claims

1. A hydrogel composition comprising Elaeagnus angustifolia, characterized in that, The composition comprises the following components: sodium alginate 1.5-2.5% w / v, hyaluronic acid 0.5-1.5% w / v, glycerol 4-6% w / v, ceramide 0.2-0.3% w / v, glycyrrhizic acid 0.4-0.6% w / v, disodium EDTA 0.05-0.15% w / v, sodium pyrrolidone carboxylic acid 0.2-0.3% w / v, allantoin 0.2-0.3% w / v, adenosine triphosphate 0.4-0.6% w / v, acer saccharinum extract 0.5-5% w / v.

2. The Elaeagnus conferta hydrogel composition of claim 1, wherein: The pH of the acer saccharinum hydrogel composition is 6.5-7.

5.

3. The Elaeagnus conferta hydrogel composition of claim 1, wherein: The preparation method of the acer saccharinum extract is: (1) Take fresh acer saccharinum fruits, remove the skin and crush the pulp; (2) Add 90-95% ethanol according to the solid-liquid ratio of 1:1.5-2.5, repeat freezing and thawing 3 times (-20℃ freezing for 2 hours / room temperature thawing for 1 hour as 1 cycle); (3) Centrifuge at 4000 rpm for 15 minutes, collect the supernatant, and repeat the extraction of the precipitate 2-3 times; (4) Combine the supernatants, concentrate to constant weight at 60-70℃ under reduced pressure to obtain the acer saccharinum extract.

4. The Elaeagnus conferta hydrogel composition of claim 1, wherein: The proportion of acids, esters, sugars and ketones in the acer saccharinum extract is 14.52%, 14.52%, 14.52% and 12.9% respectively.

5. The Elaeagnus conferta hydrogel composition of claim 1, wherein: The extraction rate of the acer saccharinum extract is 2.33%.

6. A method for preparing a hydrogel composition containing *Gnaphalium affine* as described in any one of claims 1-5, characterized in that, Comprising the following steps: (1) Mix sodium alginate, acer saccharinum extract, disodium EDTA and glycerol, heat at 50℃ for 30 minutes, then warm up to 58℃ and stir; (2) Grind glycyrrhizic acid, sodium pyrrolidone carboxylic acid, emulsifier, sorbic acid, gallic acid, allantoin, adenosine triphosphate, ceramide, hyaluronic acid and acer saccharinum extract uniformly, and transfer to the system of step (1) and stir for 2.5 hours; (3) Add volatile oil and stir uniformly to form an acer saccharinum-containing hydrogel composition.

7. A method for evaluating the network pharmacology of a hydrogel composition containing Elaeagnus angustifolia, characterized by Comprising the following steps: (1) Input the components of the acer saccharinum extract of any one of claims 1-5 into the database to obtain target points; (2) Determine the keywords to screen AD disease target points through the database; (3) Use software to screen the intersection target points of acer saccharinum extract target points and AD disease target points; (4) Input the intersection target points into the database to construct a PPI network, and determine the core target points by topological analysis through software; (5) Perform GO function enrichment analysis and KEGG pathway enrichment analysis on the intersection target points through the database, and the enrichment pathways include TRP channel inflammatory mediator regulation and PPAR signaling pathway; (6) Verify the binding energy of pentenoate and 2-myristoyl pantetheine with the target points.

8. A method for evaluating the network pharmacology of a hydrogel composition containing Elaeagnus angustifolia, characterized by The content of pentenoate and 2-myristoyl pantetheine is ≥10%.

9. Use of the moisturizing skin care hydrogel composition according to any one of claims 1-5 in the preparation of a medicament or skin care product for treating atopic dermatitis.

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