Solanum nigrum nano microcapsule inclusion as well as preparation method and application thereof

Abstract

The invention discloses a solanum nigrum nano microcapsule inclusion as well as a preparation method and application thereof. The preparation method comprises the following steps: extracting solanum nigrum serving as a raw material and ethanol serving as an extraction solvent, synchronously realizing protein flocculent precipitation, polysaccharide complexation removal and free radical scavenging oxidation resistance by utilizing the synergistic effect of a chitosan-lignin compound, mixing with an HP-beta-CD carrier, and carrying out ultrahigh-pressure micro-jet homogeneous crosslinking and freeze drying to obtain the chitosan-lignin-HP-beta-CD / HP-beta-CD / HP-beta-CD / HP-beta-CD / HP-beta-CD composite material. The solanum nigrum nano microcapsule inclusion is obtained. The microcapsule is high in encapsulation efficiency and transdermal absorption rate, and human body tests show that the microcapsule can significantly reduce acne score, regulate grease secretion and improve skin elasticity, and can be used for developing efficient and safe natural acne-removing products.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials and nanotechnology, and particularly relates to a Solanum nigrum nano-microcapsule inclusion body and a preparation method and application thereof. Background Art

[0002] Acne is a common chronic inflammatory skin disease. Existing acne treatment products often rely on chemically synthesized ingredients (such as salicylic acid and benzoyl peroxide), which can be irritating and disrupt the skin barrier. Solanum nigrum L. is rich in flavonoids (such as quercetin and australonidine), which exhibit anti-inflammatory, antibacterial, and antioxidant activities. However, its active ingredients are susceptible to degradation due to factors such as light, temperature, and pH, and traditional dosage forms (such as creams and gels) have low transdermal absorption efficiency.

[0003] Currently, traditional methods are often used to extract Solanum nigrum, resulting in low purity and efficiency. For example, CN200810033619.7 (Chinese patent) discloses a method for preparing Solanum nigrum ethanol extracts, but does not involve nano-microencapsulation technology. Chen Jianxuan (Optimization of Ultrasound-Assisted Extraction of Total Flavonoids from Solanum nigrum Fruits by Response Surface Methodology [J]. Journal of Yangzhou University: Agriculture and Life Sciences, 2015(2):7. DOI:CNKI:SUN:JSNX.0.2015-02-022.) reported on ultrasound-assisted extraction of total flavonoids from Solanum nigrum fruit, but did not involve purification or nano-microencapsulation technology. Currently, nano-microcapsule preparation mostly relies on chitosan or PLGA, resulting in low transdermal absorption and clinical efficacy.

[0004] Existing microencapsulation technologies (such as spray drying and solvent evaporation) suffer from the following drawbacks: 1) uneven particle size: microcapsules prepared by traditional methods are typically >500 nm in size, making them difficult to penetrate the stratum corneum; 2) low encapsulation efficiency: solvent evaporation achieves an encapsulation efficiency of only 50%-70%; and 3) high energy consumption: spray drying requires high temperatures, which damages heat-sensitive components. Ultrahigh-pressure microfluidization (UHPM) achieves nanoscale homogenization through high-pressure fluid shear forces, offering advantages such as environmental friendliness, high efficiency, and minimal damage. However, its systematic application for nanoencapsulation of active ingredients from Solanum nigrum has yet to be seen. Summary of the Invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing Solanum nigrum nano-microcapsule inclusions.

[0006] Another object of the present invention is to provide Solanum nigrum nano-microcapsule inclusions prepared by the method.

[0007] Another object of the present invention is to provide an application of the Solanum nigrum nano-microcapsule inclusion body.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A method for preparing Solanum nigrum nano-microcapsule inclusions comprises the following steps:

[0010] (1) Extraction and purification of active ingredients from Solanum nigrum

[0011] The Solanum nigrum is dried, crushed and sieved, and then an ethanol aqueous solution with a concentration of 50 to 90% by mass is added, and ultrasonic-assisted extraction is performed at 25 to 80° C., centrifuged, and the supernatant is collected;

[0012] (2) Chitosan-lignin one-step pretreatment

[0013] ① Dissolving chitosan in acetic acid solution to obtain a chitosan solution; dissolving lignin in water to obtain a lignin mixed solution; then stirring the chitosan solution and the lignin mixed solution to mix evenly, and spray drying to obtain a chitosan-lignin composite powder; wherein the mass ratio of chitosan to lignin is 1-2:1-2;

[0014] ② Add the chitosan-lignin complex powder to the supernatant extracted in step (1), stir and mix, adjust the pH value to 3.0-7.0, collect the supernatant by centrifugation, discard the precipitate (containing protein, polysaccharide and complex impurities), and obtain the pretreated extract;

[0015] (3) Preparation of nanocapsules

[0016] The pretreated extract obtained in step ② and hydroxypropyl β-cyclodextrin (HP-β-CD) are stirred and mixed in a core-wall mass ratio of 1:1 to 3, and then homogenized for 1 to 10 times under a pressure of 10,000 to 30,000 Psi. The homogenized emulsion is collected and finally freeze-dried to obtain the Solanum nigrum nanocapsule inclusion body.

[0017] The sieving in step (1) is through a 10-100 mesh sieve; preferably through a 20 mesh sieve.

[0018] The concentration of the ethanol aqueous solution in step (1) is preferably 70% by mass.

[0019] The material-liquid ratio of Solanum nigrum to ethanol aqueous solution in step (1) is 1 g: (10-60) mL; preferably 1 g: 20 mL.

[0020] The power of the ultrasonic extraction in step (1) is 200-400W, preferably 300W.

[0021] The temperature of the ultrasonic extraction in step (1) is preferably 50°C.

[0022] The ultrasonic extraction time in step (1) is 0 to 120 minutes (excluding 0); preferably 20 to 30 minutes; more preferably 30 minutes.

[0023] The number of extractions in step (1) is 1 to 6 times (multiple extractions and combined extracts); preferably, the extractions are 2 to 3 times.

[0024] The centrifugal conditions in step (1) are: 4000-10000 rpm, centrifugation for 10-30 min; preferably: 5000 rpm, centrifugation for 15 min.

[0025] The concentration of the acetic acid solution in step (2) ① is 0.8% to 2.0% by mass, preferably 1% by mass.

[0026] The concentration of the chitosan solution in step (2) ① is 0.8% to 2.0% by mass, preferably 1% by mass.

[0027] The concentration of the lignin mixed solution in step (2) ① is 0.5% to 3.0% by mass, preferably 1% by mass.

[0028] The mass ratio of chitosan to lignin described in step (2)① is preferably 1:1.

[0029] The lignin described in step (2)① is alkali lignin (also known as alkaline lignin, kraft lignin).

[0030] The stirring conditions described in step (2) ① are: 300-600 rpm, stirring for 1-3 hours; preferably: 500 rpm, stirring for 2 hours.

[0031] The spray drying conditions described in step (2) ① are: inlet temperature 140-180°C, outlet temperature 70-90°C, feed rate 3-8 mL / min, and atomizing pressure 0.3-0.8 MPa; preferably: inlet temperature 150°C, outlet temperature 80°C, feed rate 5 mL / min, and atomizing pressure 0.4-0.6 MPa.

[0032] The amount of chitosan-lignin complex described in step (2) ② is calculated based on the final concentration of the chitosan-lignin complex in the system being 0.5-5% by mass; preferably, the amount of chitosan-lignin complex is calculated based on the final concentration of the chitosan-lignin complex in the system being 1.5% by mass.

[0033] The stirring conditions in step (2) ② are: stirring at 25° C. and 200-400 rpm for 1-3 hours; preferably: stirring at 25° C. and 300 rpm for 1 hour.

[0034] The pH value in step (2) ② is adjusted using HCl solution and NaOH solution; preferably, 1 mol / L HCl solution and NaOH solution are used for adjustment.

[0035] The pH value adjustment described in step (2) ② is preferably to adjust the pH value to 5.0.

[0036] The centrifugal conditions described in step (2) ② are: 4° C., 4000-10000 rpm, 10-30 min; preferably: 4° C., 8000 rpm, 20 min.

[0037] The mass ratio of the pretreated extract in step (3) to the core wall of hydroxypropyl β-cyclodextrin (HP-β-CD) is preferably 1:2.

[0038] The homogenization in step (3) is performed using an ultra-high pressure homogenizer (UHPM homogenizer).

[0039] The homogenization pressure in step (3) is 20,000 to 30,000 Psi, more preferably 30,000 Psi.

[0040] The number of homogenization steps in step (3) is preferably 1 to 5 times, more preferably 3 times.

[0041] The freezing in step (3) can be performed by filling the emulsion into freeze-drying bottles with a liquid layer thickness of ≤1 cm, and then freezing.

[0042] The freezing conditions described in step (3) are: freezing at -60 to -90°C for 2 to 6 hours (freezing until completely solidified); preferably: freezing at -80°C for 3 hours (freezing until completely solidified).

[0043] The drying described in step (3) is vacuum drying.

[0044] The vacuum drying conditions are: first drying at -20°C and ≤0.1mbar for 8 to 16 hours, then drying at 25°C and ≤0.01mbar for 4 to 8 hours (total drying time 12 to 24 hours); preferably: first drying at -20°C and ≤0.1mbar for 12 hours, then drying at 25°C and ≤0.01mbar for 6 hours (total drying time 18 hours).

[0045] The average particle size of the Solanum nigrum nano-microcapsule inclusions in step (3) is 10 to 1000 nm, preferably 10 to 150 nm (PDI ≤ 0.2, encapsulation efficiency ≥ 90%), and more preferably 30 nm.

[0046] A Solanum nigrum nano-microcapsule inclusion body is prepared by any of the methods described above.

[0047] The application of the Solanum nigrum nano-microcapsule inclusions in the preparation of acne-removing, anti-oxidation, anti-aging and / or antibacterial products.

[0048] The products include medicines (drugs), daily chemical products (cosmetics, skin care products), etc.

[0049] The bacteria include Propionibacterium acnes and the like.

[0050] The product may further include auxiliary materials (carriers) and the like.

[0051] An oil-in-water emulsion (O / W type) containing the above-mentioned Solanum nigrum nano-microcapsule inclusions contains the following components, calculated by mass percentage: 1% of the above-mentioned Solanum nigrum nano-microcapsule inclusions, 10% caprylic / capric triglyceride, 5% glycerol, 3% Croda A165, 0.30% carbomer 980, 0.30% triethanolamine, 0.50% p-hydroxyacetophenone, and water makes up to 100%.

[0052] The water is preferably deionized water.

[0053] The method for preparing the oil-in-water emulsion containing Solanum nigrum nano-microcapsule inclusions comprises the following steps:

[0054] (i) heating caprylic / capric triglyceride and Croda A165 to 80±5° C. to dissolve and prepare an oil phase;

[0055] (ii) dispersing glycerol and carbomer 980 in water and heating to 80 ± 5 °C to prepare an aqueous phase;

[0056] (iii) slowly adding the oil phase to the water phase, homogenizing and emulsifying, cooling to 40±5°C, adding the above-mentioned Solanum nigrum nanocapsules, stirring and mixing evenly, then adding triethanolamine to adjust the pH to 5.5-6.0, and adding p-hydroxyacetophenone and stirring evenly to obtain the product.

[0057] The homogenization conditions in step (iii) are preferably: homogenization at 10,000 rpm for 5 minutes.

[0058] The stirring speed in step (iii) is preferably 500 rpm.

[0059] The oil-in-water emulsion containing the Solanum nigrum nano-microcapsule inclusions is used in the preparation of acne-removing, anti-oxidation, anti-aging and / or antibacterial products.

[0060] The present invention has the following advantages and effects compared to the prior art:

[0061] 1. This invention utilizes an optimized extraction process (70% ethanol ultrasonic extraction), a chitosan-lignin pretreatment step, and subsequent mixing with an HP-β-CD carrier. Ultrahigh-pressure microfluidization homogenization and crosslinking are followed by freeze-drying to produce Solanum nigrum nanocapsules. The microcapsules achieve an encapsulation efficiency exceeding 90%, and a transdermal absorption rate 110% higher than that of the free extract. Human trials have shown that they can significantly reduce acne scores (p < 0.01), regulate oil secretion, and improve skin elasticity. This invention provides a highly efficient and safe technical solution for the development of natural acne-removing products.

[0062] 2. The present invention utilizes the synergistic effect of a chitosan-lignin complex (1:1, w / w) to simultaneously achieve protein flocculation and precipitation, polysaccharide complexation removal, and free radical scavenging and antioxidant effects through the complexation of chitosan (positively charged) and lignin (containing phenolic hydroxyl groups), breaking through the limitation of traditional processes where a single material only targets specific impurities. Chitosan (cationic) and lignin (anionic) form a stable complex with neutralized charge at a 1:1 ratio, avoiding self-aggregation. The flocculation effect of chitosan complements the antioxidant and complexing capabilities of lignin, covering a wider range of impurity types. Experimental results show that at this ratio, the flavonoid-lignin complexation constant reaches its peak, and the active ingredient protection is optimal. When the ratio deviates from 1:1, the excess charge leads to a decrease in the solubility or dispersibility of the complex (for example, at 2:1, there is excess chitosan, the solution viscosity increases, and it can be used in high-protein impurity systems to prioritize protein removal; if the active ingredient is easily oxidized (such as polyphenols), a ratio of 1:2 can be preferred, but a dispersant (such as 0.1% Tween 80) must be added to inhibit aggregation).

[0063] 3. The present invention completes the complexation and decontamination in one step by adjusting the pH to 5.0. Under the condition of pH 5.0, the amino groups of chitosan are protonated (NH3 + ) forms hydrogen bonds and electrostatic complex networks with the phenolic hydroxyl groups (-OH) of lignin, selectively binds to flavonoids, and significantly improves the complex stability (binding constant K = 1.2×10 4 M -1 , 3 times higher than that of a single component). The present invention completes complexation and purification in a single step at pH 5.0, breaking through the bottleneck of traditional multi-step purification.

[0064] 4. The present invention realizes the multifunctional integration of impurity removal, anti-oxidation and activity protection through chemical complementation, the protein / polysaccharide removal rate is increased by 10%, the flavonoid oxidation loss rate is reduced to below 5%, the encapsulation rate exceeds 90%, and the alkaloid residue is reduced to 5ppm, eliminating the addition of antioxidants and multi-stage purification steps.

[0065] 5. The present invention is based on ultra-high pressure microfluidization technology (UHPM). By optimizing the active ingredient extraction process and nanotechnology, it significantly improves the encapsulation rate, stability, transdermal absorption efficiency and bioavailability of Solanum nigrum flavonoid compounds, solving the problems of poor stability of active ingredients and low transdermal absorption efficiency in traditional technologies. It is suitable for the development of efficient and safe natural acne-removing products. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 1 is the particle size distribution diagram of the nanocapsules prepared in Example 1. DETAILED DESCRIPTION

[0067] The present invention will be described in further detail below in conjunction with the examples, but embodiments of the present invention are not limited thereto. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually based on conventional experimental conditions or the experimental conditions recommended by the manufacturer. Unless otherwise stated, the reagents and raw materials used in the present invention can be obtained commercially.

[0068] Example 1

[0069] This example provides a method for preparing Solanum nigrum nanocapsules based on UHPM, addressing the issues of poor stability of active ingredients and low transdermal absorption efficiency in traditional technologies, and verifying their effectiveness in anti-acne cosmetics. The specific steps are as follows:

[0070] 1. Extraction and purification of active ingredients from Solanum nigrum

[0071] (1) High-efficiency ultrasonic extraction of Solanum nigrum flavonoids

[0072] ① Raw material processing: Dry and crush the Solanum nigrum (from Foshan, Guangdong) and pass through a 20-mesh sieve;

[0073] ② Extraction process: Take 20.0 g of Solanum nigrum powder, add 400 mL of 70% ethanol aqueous solution by mass (solid-liquid ratio 1:20), ultrasonic-assisted extraction for 20 to 30 minutes (20 minutes in this experiment), ultrasonic power 200 to 400 W (300 W in this experiment), temperature 50°C, then centrifuge (5,000 rpm, 15 min, 25°C), and take the supernatant; add 400 mL of 70% ethanol aqueous solution to the residue, repeat ultrasonic extraction once, and combine the two supernatants (or: repeat ultrasonic extraction twice and combine the three supernatants);

[0074] (2) Chitosan-lignin one-step pretreatment

[0075] ① Chitosan-lignin composite: Chitosan (100-300 kDa, Sigma-Aldrich C3646-100G) was dissolved in 1% (w / v) acetic acid solution to obtain a 1% (w / v) chitosan solution; lignin (alkaline lignin, Sigma-Aldrich 471003-100G) was dissolved in deionized water to obtain a 1% (w / v) lignin mixed solution; the two solutions were then mixed in a chitosan:lignin mass ratio of 1:1, magnetically stirred (500 rpm, 2 hours), and spray dried (inlet temperature: 150°C; outlet temperature: 80°C; feed rate: 5 mL / min; atomization pressure: 0.4-0.6 MPa) to obtain chitosan-lignin composite powder.

[0076] ② Add chitosan-lignin complex to the supernatant extracted in step (1) (stirring speed 300 rpm, temperature 25°C, time 1 hour), then adjust the pH to 5.0 with 1M HCl / NaOH, to a final concentration of 1.5% (w / v) chitosan-lignin complex. Centrifuge again (centrifugation conditions: 8,000 rpm, 20 minutes, 4°C), collect the supernatant, and discard the precipitate (containing protein, polysaccharide, and complex impurities) to obtain the pretreated extract.

[0077] 2. Preparation of nanocapsules

[0078] (3) HP-β-CD carrier mixture

[0079] Carrier material: hydroxypropyl β-cyclodextrin (HP-β-CD, purchased from Shanxi Lisheng Biotechnology Co., Ltd.), core wall (extract: HP-β-CD) mass ratio 1:2;

[0080] Homogenization process: The extract pretreated in step (2) was mixed with HP-β-CD, stirred magnetically (500 rpm, 30°C) for 1 hour until completely dissolved, and filtered using a 0.45 μm nylon filter membrane to remove undissolved particles.

[0081] (4) Ultra-high pressure microfluidization homogeneous cross-linking

[0082] The mixed solution obtained in step (3) was injected into the liquid storage tank of the UHPM homogenizer and the homogenization process was started (pressure 30,000 PSi, homogenization 3 times). Circulating cooling was used to ensure that the temperature was ≤ 10°C to avoid thermal degradation. The homogenized emulsion was collected and immediately proceeded to the next step. Nanocapsules with a particle size of approximately 30 nm were produced.

[0083] (5) Freeze-drying

[0084] The nanocapsules (emulsion) prepared in step (3) were divided into freeze-drying bottles (liquid layer thickness ≤ 1 cm) and freeze-dried; wherein, the pre-freezing temperature was -80°C for 3 hours (until completely solidified); vacuum drying was performed at -20°C, ≤ 0.1 mbar, for 12 hours; and at 25°C, ≤ 0.01 mbar, for 6 hours (total drying time 18 hours);

[0085] 3. Physical and chemical properties and application effects

[0086] (1) Characterization of physical and chemical properties

[0087] Particle size and PDI: Dynamic light scattering (DLS) was used to measure the particle size distribution of the nanocapsules after freeze-drying in step (4). Figure 1 ).

[0088] Stability: The particle size change of the freeze-dried nanocapsules was measured after an accelerated test (40°C / 75% RH, 90 days). The results showed that the particle size change was <5% and the encapsulation efficiency was >90%.

[0089] The results are shown in Table 1. SEM observations showed that the microcapsules had regular morphology and were uniformly dispersed, which was consistent with the DLS data (CV < 10%), indicating that the technology had high reproducibility.

[0090] Table 1: SEM-displayed morphology of nanocapsules (scale: 100 nm)

[0091] Observation indicators Qualitative description quantitative data Micromorphology Spherical, smooth surface, no aggregation Sphericity index ≥0.92 Dispersibility Evenly dispersed, no visible agglomerates Coefficient of dispersion (CV) <10% Dimensional consistency The particle size distribution is consistent with the DLS results (30±1nm) The average particle size measured by SEM is 31±2nm

[0092] (2) Efficacy verification

[0093] The nanocapsules freeze-dried in step (5) and the extract in step (2) were subjected to efficacy verification experiments, specifically as follows:

[0094] 1) In vitro transdermal test:

[0095] Commercially available fresh suckling pig skin was fixed in a Franz diffusion cell with the stratum corneum facing upward (donor cell). 5 mL of preheated PBS (37°C) was injected into the receiving cell and magnetically stirred (600 rpm). The cells were equilibrated for 30 minutes to ensure skin hydration (stable electrical resistance). The experiment was divided into two groups, and the drug administration was as follows: experimental group: the reconstituted nanocapsule solution (the dried nanocapsules were reconstituted with PBS buffer at a concentration of 20 μL / cm 2 ) was evenly applied to the skin surface; the control group was treated with free extract (equal volume and active ingredient concentration). Samples were collected at 0, 2, 4, 8, 12, and 24 hours. Three areas of the same skin sample were tested, and the mean ± SD was calculated.

[0096] The Franz diffusion cell method showed that the transdermal absorption rate of the nano-microcapsules was 68.2%, which was 110% higher than that of the unencapsulated extract (32.5%) (Table 2), verifying the synergistic effect of nano-technology.

[0097] Table 2: Comparison of in vitro transdermal absorption curves (nanocapsules vs. free extracts)

[0098] Time (h) Cumulative release rate of nanocapsules (%) Cumulative release rate of free extract (%) Release rate increase (%) 0 0.0±0.0 0.0±0.0 - 2 12.5±1.2 5.8±0.8 +116％ 4 25.3±2.1 11.4±1.1 +122％ 8 42.7±2.5 18.9±1.6 +126％ 12 55.1±3.0 24.3±2.0 +127％ 24 68.2±3.5 32.5±2.8 +110％

[0099] 2) Human trials:

[0100] First, prepare an oil-in-water emulsion (O / W type) containing 1% nanocapsules. The formula is shown in Table 3. The specific preparation process is as follows: Heat caprylic / capric triglyceride and Croda A165 to 80°C to dissolve to form the oil phase. Disperse glycerol and carbomer 980 in deionized water and heat to 80°C to form the water phase. Slowly add the oil phase to the water phase and emulsify it using a homogenizer (10,000 rpm, 5 minutes). After cooling to 40°C, add the freeze-dried nanocapsule powder and stir (500 rpm) until uniform. Adjust the pH to 5.5-6.0 (consistent with the physiological pH of the skin) with triethanolamine, add parahydroxyacetophenone, stir evenly, and discharge.

[0101] Table 3 Composition of oil-in-water emulsion (O / W type) containing 1% nanocapsules

[0102] Element Ratio (w / w%) supplier Freeze-dried nanocapsules 1％ The freeze-dried product of step (5) Caprylic / capric triglyceride 10％ BASF (Myritol 318) glycerin 5％ Sigma-Aldrich (G7893) Hoda A165 3％ Guangzhou Jiasheng Chemical Co., Ltd. Carbomer 980 0.30％ Lubrizol (Carbopol 980) triethanolamine 0.30％ Wanhua Chemical Group Co., Ltd. p-Hydroxyacetophenone 0.50％ Guangzhou Baiyu Biotechnology Co., Ltd. Deionized water Top up to 100% -

[0103] Twenty patients with mild to moderate acne (volunteers, aged 18-30 years) used an oil-in-water emulsion (O / W type) containing 1% nanocapsules, applied daily after cleansing in the morning and evening (dosage standard: approximately 0.25g (half FTU) each time, once in the morning and evening) for 16 weeks. The 20 subjects were treated and observed once a month. The skin oil tester ( ) to measure oil secretion, using a skin moisture tester ( Acne was assessed using the IGA (5-point scale): mild: grade 2 (small number of non-inflammatory lesions); moderate: grade 3 (many non-inflammatory and inflammatory lesions).

[0104] The results showed that the acne score decreased from the baseline of 3.2±0.5 to 0.8±0.3 (p<0.01), and the acne score decreased by 60%-100% (Table 4); the oil secretion decreased from 6.5±1.2 to 4.1±0.9 (p<0.05), and the oil secretion decreased by 33.3%-37.5%; the skin moisture content increased from 35%±5% to 48%±6% (p<0.01), and the skin moisture content increased by 12.5%-50%, and the elasticity increased by 40%-100%, indicating that the product is effective.

[0105] Table 4: Changes in acne scores in human trials (n=20)

[0106] Time (week) Acne score (mean ± SD) Oil secretion (mean ± SD) Skin moisture content (mean ± SD) 0 3.2±0.5 6.5±1.2 35％±5％ 4 2.1±0.4 5.2±0.8 42％±6％ 8 1.5±0.3 4.7±0.7 46％±5％ 16 0.8±0.3 4.1±0.9 48％±6％

[0107] Note: Acne is evaluated using the IGA scoring system (5-point system).

[0108] 3) Comparison of Acne-Removing Efficacy (Nanocapsules vs. Free Extracts)

[0109] Referring to the human test method in step 2) above, 60 patients with mild to moderate acne (volunteers, aged 18-30 years) were randomly divided into three groups. They were treated with the oil-in-water emulsion (O / W type) containing 1% nanocapsules prepared in step 2), the Solanum nigrum extract prepared in step (2), and a commercially available 2% salicylic acid anti-acne cream, respectively. The patients were applied to their faces every morning and evening after cleansing for 16 weeks. The oil secretion, skin moisture content, and erythema area were then measured (using the VISIA-CR system). The specific groupings are as follows:

[0110] Experimental group: using oil-in-water emulsion (O / W type) containing 1% nanocapsules (n=20);

[0111] Control group 1: using a free Solanum nigrum extract emulsion (referring to the preparation method of an oil-in-water emulsion (O / W type) containing 1% nanocapsules (formula see Table 3), except that the freeze-dried product in step (5) was replaced with the extract obtained after the chitosan-lignin one-step pretreatment in step (2), and the remaining preparation steps were the same) (n=20);

[0112] Control group 2: used commercially available 2% salicylic acid anti-acne cream (n=20).

[0113] Experimental period: 16 weeks (baseline + follow-up every 4 weeks).

[0114] The results are shown in Table 5: The results show that the nano-microcapsule group prepared by the present invention is significantly better than the control group in acne score and oil control (p < 0.01).

[0115] Table 5: Comparison of acne removal efficacy

[0116]

[0117] Note: Data are mean ± SD, *p < 0.05 (vs. emulsion in the free Solanum nigrum extract group), **p < 0.01 (vs. emulsion in the free Solanum nigrum extract group).

[0118] Example 2

[0119] 1. Optimization of extraction and purification process and control experiments

[0120] 1. Optimization of ethanol concentration

[0121] The method of Example 1 was followed, except that the concentration of the 70% ethanol aqueous solution in step (1) was replaced with 50% and 90%, respectively. The remaining steps were the same as in Example 1. The flavonoid yield and purity of the extract after the chitosan-lignin pretreatment in step (2) were determined by spectrophotometry (using rutin as the standard). Three replicates were performed.

[0122] Flavonoid yield (Yield, %) = C·V / W × 100%;

[0123] Where, C: flavonoid concentration of the extract (mg / mL), V: total volume of the extract (mL), W: mass of the raw material (mg);

[0124] Flavonoid purity (Purity,%) = C·V / M×100%;

[0125] Where, C: flavonoid concentration of the extract (mg / mL), V: total volume of the extract (mL), M: dry weight of the extract (mg).

[0126] The results are shown in Table 6. The results showed that the flavonoid yield and purity extracted by 70% ethanol were significantly higher than those of other concentrations (p<0.05), and 70% ethanol was selected after optimization.

[0127] Table 6

[0128] Ethanol concentration (%) Flavonoid yield (mg / g) purity(%) Significance (vs. 70%) 50％ 8.2±0.3 78.5±2.1 p<0.05 70％ 12.5±0.5 95.2±1.8 - 90％ 10.1±0.4 82.3±2.3 p<0.05

[0129] 2. Optimization of extraction temperature and time

[0130] According to the method of Example 1, the extraction temperature in step (1) was set to 25°C, 50°C, and 80°C, and the extraction time was set to 15, 30, and 60 min (see Table 7 for details). The remaining steps were the same as in Example 1. The flavonoid yield of the extract after the chitosan-lignin one-step pretreatment in step (2) was determined using the same spectrophotometric method as described above.

[0131] The results are shown in Table 7. The results showed that the optimal extraction condition was 50°C for 30 min, with the highest yield and no significant difference compared with the extended extraction time (60 min) (p>0.05).

[0132] Table 7

[0133] Temperature (℃) Time (min) Flavonoid yield (mg / g) Significance (vs.50℃×30min) 25℃ 30 6.8±0.2 p<0.01 50℃ 30 12.5±0.5 - 80℃ 30 9.3±0.4 p<0.05 50℃ 15 8.1±0.3 p<0.01 50℃ 60 11.2±0.4 p>0.05

[0134] 2. Optimization and control experiments of chitosan-lignin one-step pretreatment process

[0135] 1. Use different materials to remove impurities

[0136] According to the method of Example 1, the chitosan-lignin complex in step (2) was replaced by chitosan and lignin, respectively, and the remaining steps were the same as in Example 1. The protein removal rate, polysaccharide removal rate and DPPH clearance rate of the extract after pretreatment in step (2) were measured respectively. The experiment was repeated three times.

[0137] Protein removal rate was determined using the Bicinchoninic Acid Assay (BCA) kit (ThermoFisher, 23225); protein removal rate (%) = (1-before C treatment / after C treatment) × 100%;

[0138] The polysaccharide removal rate was determined using the phenol-sulfuric acid method; polysaccharide removal rate (%) = (1-before C treatment / after C treatment) × 100%;

[0139] DPPH radical scavenging rate was determined by photometry, referring to Brand-Williams et al. (1995). DPPH radical reagent (Sigma, D9132) was prepared into a 0.1 mM ethanol solution and measured by photometry; DPPH radical scavenging rate (%) = (1-A sample / A blank) × 100%.

[0140] The results are shown in Table 8.

[0141] Table 8: Impurity removal effects of chitosan-lignin composites and single materials

[0142]

[0143] 2. Using different ratios of chitosan and lignin

[0144] The performance tests of complexes with different ratios reveal the effects of ratios on impurity removal, antioxidant properties and encapsulation efficiency. That is, according to the method of Example 1, the chitosan and lignin mass ratios in the chitosan-lignin complex in step (2) are replaced with 2:1 and 1:2, respectively. The remaining steps are the same as in Example 1. The protein removal rate, polysaccharide removal rate, alkaloid residue, antioxidant capacity (DPPH clearance rate, flavonoid oxidation loss rate) and nano-microcapsule encapsulation efficiency, polydispersity index (PDI) and stability (placed at 40°C for 90 days) of the extract after pretreatment in step (2) were measured respectively. The experiment was repeated three times. Among them,

[0145] The protein removal rate, polysaccharide removal rate and DPPH clearance rate are the same as those in step 1 above;

[0146] Alkaloid residues were determined using HPLC-UV (referenced to a solanine standard, Agilent Eclipse XDB-C18 (4.6 × 150 mm, 5 μm), phase A: 0.1% (v / v) phosphoric acid aqueous solution, phase B: acetonitrile). Residual amount (mg / g) = C·V / W; where C is the HPLC concentration (mg / mL), V is the volume of the extract (mL), and W is the weight of the raw material (g).

[0147] Flavonoid oxidation loss rate, the flavonoid content before and after treatment was determined; loss rate = (1-before C treatment / after C treatment) × 100%;

[0148] Encapsulation efficiency (EE%), free drug concentration (Cfree) and total drug concentration (Ctotal) were measured; EE% = (1-Cfree / Ctotal) × 100%;

[0149] Polydispersity index (PDI) determination, dynamic light scattering (DLS, Malvern Zetasizer Nano ZS);

[0150] Stability (90 days at 40°C): Particle size change, initial (D0) and endpoint (Dt) particle sizes were measured by DLS.

[0151] The results are shown in Tables 9 to 11.

[0152] Table 9: Decontamination efficiency of complexes with different ratios

[0153] Chitosan:lignin (w / w) Protein removal rate (%) Polysaccharide removal rate (%) Alkaloid residue (ppm) 2:1 88±2 60±3 10±1 1:1 82±3 75±2 5±0.3 1:2 70±2 85±3 3±0.5

[0154] Table 10: Comparison of antioxidant capacity

[0155] Chitosan:lignin (w / w) DPPH clearance rate (%) Flavonoid oxidation loss rate (%) 2:1 65±2 15±1 1:1 85±2 5±0.5 1:2 92±1 3±0.3

[0156] Table 11: Effect on nanocapsule encapsulation efficiency

[0157] Chitosan:lignin (w / w) Encapsulation efficiency (%) PDI Stability (40℃ / 90 days) 2:1 88±1 0.22±0.02 Particle size increase +12% 1:1 92±1 0.15±0.01 +6.7％ 1:2 85±2 0.25±0.03 +18％

[0158] It can be seen from Table 9 that: when the chitosan ratio is high (2:1), the protein removal rate is the highest (88%), but the polysaccharide removal rate is low (60%); when the lignin ratio is high (1:2), the polysaccharide and alkaloid removal rates are optimal (85%, 3ppm), but the protein removal rate decreases (70%); when the mass ratio of chitosan to lignin is 1:1, the comprehensive performance is the best, and the protein, polysaccharide and alkaloid removal rates are balanced (82%, 75%, 5ppm).

[0159] It can be seen from Table 10 that the higher the lignin ratio, the stronger the antioxidant capacity (DPPH clearance rate is 92% at 1:2), but the flavonoid oxidation loss rate does not decrease linearly; when the mass ratio of chitosan and lignin is 1:1, the best balance is achieved between antioxidant capacity (85%) and activity protection (loss rate is 5%).

[0160] It can be seen from Table 11 that when the mass ratio of chitosan to lignin is 1:1, the encapsulation efficiency is the highest (92%), the particle size distribution is the narrowest (PDI = 0.15), and the stability is the best; while a high ratio of lignin (1:2) will cause microcapsule aggregation due to excessive hydrophobicity, reducing the encapsulation efficiency.

[0161] 3. Use different pH values

[0162] According to the method of Example 1, the pH value in step (2) was adjusted to 3.0, 5.0, and 7.0 respectively, and the remaining steps were the same as in Example 1. The complex stability (binding constant K, M -1 ) and flavonoid retention rate (%). The experiment was repeated three times.

[0163] Complex stability (binding constant K) was determined using UV-visible spectrophotometry. Flavonoid solutions of varying concentrations (0-100 μM) were prepared in various pH buffers (formic acid-sodium formate buffer (pH 3.0), acetic acid-sodium acetate buffer (pH 5.0), and phosphate buffer saline (PBS) (pH 7.0). Equal volumes of chitosan-lignin complex solution (mass ratio of chitosan to lignin 1:1, complex solution concentration 1 mg / mL) were added, and the mixtures were incubated at 25°C for 30 minutes. The absorbance (A) was measured, and a curve plotting the absorbance versus flavonoid concentration was used to generate the binding constant K.

[0164] Flavonoid retention rate (%) = before C treatment / after C treatment × 100%.

[0165] The results are shown in Table 12.

[0166] Table 12: Effect of pH on flavonoid-lignin complexation

[0167] pH Complex stability (binding constant K,M-1) Flavonoid retention rate (%) 3.0 <![CDATA[3.5×10 3 ]]> 78±2 5.0 <![CDATA[1.2×10 4 ]]> 95±1 7.0 <![CDATA[6.8×10 3 ]]> 85±2

[0168] 4. Use different carrier types

[0169] Following the method of Example 1, the carrier material HP-β-CD in step (3) was replaced with "HP-β-CD + chitosan (HP-β-CD:chitosan = 9:1; w / w)" or "HP-β-CD + chitosan-lignin complex prepared in step (2) ① of Example 1 (HP-β-CD:complex = 3:7; w / w)". The remaining steps were the same as in Example 1. The encapsulation efficiency, PDI, and alkaloid residue of the nanocapsules after freeze-drying in step (5) were measured. The experiment was repeated three times.

[0170] The results are shown in Table 13.

[0171] Table 13: Comparison of alkaloid residues (nanocapsules)

[0172]

[0173] 3. Optimization of Nanocapsule Preparation Process and Control Experiment

[0174] 1. Preparation of nanocapsules under different pressures

[0175] According to the method of Example 1, the homogenization pressure in step (4) was adjusted to 10,000 Psi and 20,000 Psi, respectively, and the remaining steps were the same as in Example 1. Dynamic light scattering (DLS) was used to determine the particle size distribution of the nanocapsules after freeze-drying in step (5). The experiment was repeated three times. The results are shown in Table 15. The results showed that pressure was negatively correlated with particle size, with the minimum particle size (30 nm) reached at 30,000 PSi, PDI ≤ 0.16, and the particle size decreased by 20-50 nm for every 10,000 Psi increase in pressure (Table 14). Conclusion: At 30,000 PSi, the particle size was the smallest (30 nm), the PDI was the lowest (0.16), and the encapsulation efficiency was the highest (92.5%), which was significantly better than other pressure conditions.

[0176] Table 14: Effect of ultrahigh pressure microfluidization pressure on particle size and encapsulation efficiency

[0177]

[0178]

[0179] 2. Effect of different homogenization times on particle size uniformity

[0180] Following the method of Example 1, the number of homogenization cycles in step (4) was adjusted to 1 and 5 times, respectively. The remaining steps were the same as in Example 1. Dynamic light scattering (DLS) was used to measure the particle size distribution of the nanocapsules after freeze-drying in step (5). The experiment was repeated three times. The results are shown in Table 15. The results showed that the optimal particle size uniformity (PDI = 0.16) was achieved with 3 homogenization cycles, and increasing the number to 5 cycles did not significantly improve the particle size uniformity (p > 0.05).

[0181] Table 15: Effect of ultra-high pressure microfluidization times on particle size

[0182] Homogenization times Average particle size (nm) PDI Significance (vs. 3 times) 1 time 45±2 0.22±0.02 p<0.01 3 times 30±1 0.16±0.01 - 5 times 28±1 0.17±0.01 p>0.05

[0183] 3. Effect of different core-wall mass ratios on encapsulation efficiency

[0184] Following the method of Example 1, the core-to-wall (extract: HP-β-CD) mass ratio in step (3) was adjusted to 1:1 and 1:3, respectively. The remaining steps were the same as in Example 1. The encapsulation efficiency and drug loading of the nanocapsules after freeze-drying in step (5) were measured. The experiment was repeated three times. The results are shown in Table 16. The results showed that the encapsulation efficiency and drug loading reached their peak values ​​when the core-to-wall ratio was 1:2, which was significantly better than the other ratios (p < 0.05).

[0185] Table 16: Effects of different core-to-wall mass ratios on encapsulation efficiency and drug loading

[0186] Core-to-wall ratio Encapsulation efficiency (%) Drug loading (%) Significance (vs. 1:2) 1:1 82.4±1.5 8.3±0.3 p<0.01 1:2 92.5±1.2 9.8±0.4 - 1:3 88.1±1.8 7.6±0.2 p<0.05

[0187] 4. Comparative analysis of hydroxypropyl β-cyclodextrin (HP-β-CD) and other core wall materials

[0188] According to the method of Example 1, the carrier material hydroxypropyl β-cyclodextrin (HP-β-CD) in step (3) was replaced with chitosan (CS) (100-300 kDa, Sigma-Aldrich C3646-100G) and polylactic-co-glycolic acid (PLGA) (molecular weight: 10,000-20,000, Shanghai MacLean Biochemical Technology Co., Ltd. P879466), respectively. The remaining steps were the same as in Example 1, and the encapsulation efficiency and retention rate were tested. The experiment was repeated three times. The results are shown in Table 17.

[0189] Results showed that HP-β-CD exhibited a mass loss of only 5.1% at 100°C, significantly lower than chitosan (15.2%) and PLGA (28.4%). The encapsulation retention rate remained at 88.4% at 100°C, significantly higher than that of other materials. HP-β-CD exhibited structural stability under ultrahigh-pressure microfluidization at 30,000 PSi, protecting thermally unstable components, whereas chitosan is susceptible to shear degradation.

[0190] Table 17: Comparative analysis of different core wall materials

[0191]

[0192] Note: Data are mean ± SD (n = 3), * indicates p < 0.01 compared with the control group (ANOVA test).

[0193] 5. Other Potential Applications

[0194] Antibacterial efficacy: The Solanum nigrum nanocapsules prepared according to Example 1 achieved a 92.4% inhibition rate against Propionibacterium acnes at a concentration of 1 mg / mL (inhibition rate assay (96-well plate microtiter plate method), sample:bacteria solution = 1:1). The balance between therapeutic efficacy and safety was over 1.5 times that of the free extract. The results are shown in Table 18. Among them:

[0195] Experimental materials: Propionibacterium acnes ATCC 6919, 0.05% chlorhexidine gluconate (C832370, Shanghai MacLean Biochemical Technology Co., Ltd., diluted with PBS), free extract (the extract of the chitosan-lignin one-step pretreatment in step (2) of Example 1), and the freeze-dried nanocapsules in step (5) of Example 1 (diluted with PBS).

[0196] Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination:

[0197] Take 10 sterile test tubes (numbered 1-10) and perform serial dilution: add 1.6 mL of fortified Clostridium broth (RCM) to test tube 1, then add 100 μL of Solanum nigrum nanocapsule stock solution (concentration 20 mg / mL), mix well to obtain a starting concentration solution of 1 mg / mL; transfer 1 mL of the mixture from tube 1 to test tube 2 (pre-filled with 1 mL of fresh RCM broth), mix well to reduce the concentration to 0.5 mg / mL; repeat the above steps and transfer to test tube 10 in sequence to form a concentration gradient: 1→0.5→0.25→0.125→0.0625→0.031→0.015→0.0078→0.0039→0.002 mg / mL (10 concentration gradients); add bacteria culture: add 100 μL of standard bacterial suspension (Propionibacterium acnes, 1.5×10 8 CFU / mL), mix thoroughly, and place in an anaerobic jar (37°C, 48 hours). The free extract was used as a control, and a 0.05% (w / v) chlorhexidine solution was used as a positive control. The experiment was repeated three times. MIC (Minimum Inhibitory Concentration): After incubation, observe the turbidity of the test tube. The lowest concentration at which no turbidity is visible is the MIC value. MBC (Minimum Bactericidal Concentration): Take a tube containing a concentration of the MIC value or above, spread it on an RCM plate, and incubate anaerobically for 72 hours. The lowest concentration at which no colonies grow is the MBC value.

[0198] Table 18

[0199] sample Antibacterial rate (%) MIC (μg / mL) MBC (μg / mL) Nanocapsule group 92.4±3.6 32 64 0.05% chlorhexidine 85.1±2.9 8 16 Free extract 63.7±5.2 128 >256

[0200] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing Solanum nigrum nano-microcapsule inclusions, characterized in that: The steps include: (1) Extraction and purification of active ingredients from Solanum nigrum The Solanum nigrum is dried, crushed and sieved, and then an ethanol aqueous solution with a concentration of 50 to 90% by mass is added, and ultrasonic-assisted extraction is performed at 25 to 80° C., centrifuged, and the supernatant is collected; (2) Chitosan-lignin one-step pretreatment ① Dissolving chitosan in acetic acid solution to obtain a chitosan solution; dissolving lignin in water to obtain a lignin mixed solution; then stirring the chitosan solution and the lignin mixed solution to mix evenly, and spray drying to obtain a chitosan-lignin composite powder; wherein the mass ratio of chitosan to lignin is 1-2:1-2; ② Add the chitosan-lignin complex powder to the supernatant extracted in step (1), stir and mix, adjust the pH value to 3.0-7.0, and collect the supernatant by centrifugation to obtain a pretreated extract; (3) Preparation of nanocapsules The pretreated extract obtained in step ② and hydroxypropyl β-cyclodextrin are stirred and mixed in a core-wall mass ratio of 1:1 to 3, and then homogenized for 1 to 10 times under a pressure of 10,000 to 30,000 Psi. The homogenized emulsion is collected and finally freeze-dried to obtain the Solanum nigrum nanocapsule inclusion body.

2. The method according to claim 1, wherein: The amount of chitosan-lignin complex described in step (2) ② is calculated based on its final concentration in the system being 0.5-5% by mass; The homogenization pressure in step (3) is 20,000 to 30,000 psi; The number of homogenization steps in step (3) is 1 to 5 times.

3. The method according to claim 2, wherein: The amount of chitosan-lignin complex described in step (2) ② is calculated based on its final concentration in the system being 1.5% by mass; The mass ratio of chitosan to lignin in step (2) ① is 1:1; The mass ratio of the pretreated extract described in step (3) to the core wall of hydroxypropyl β-cyclodextrin is 1:2; The homogenization pressure in step (3) is 30000 Psi; The homogenization in step (3) was repeated three times.

4. The method according to claim 1, wherein: The solid-liquid ratio of Solanum nigrum to ethanol aqueous solution in step (1) is 1 g: 10-60 mL; The concentration of the ethanol aqueous solution in step (1) is 70% by mass; The concentration of the acetic acid solution in step (2) ① is 0.8% to 2.0% by mass; The concentration of the chitosan solution in step (2) ① is 0.8% to 2.0% by mass; The concentration of the lignin mixed solution described in step (2) ① is 0.5% to 3.0% by mass.

5. The method according to claim 1, wherein: The spray drying conditions described in step (2) ① are: inlet temperature 140-180°C, outlet temperature 70-90°C, feed rate 3-8 mL / min, and atomization pressure 0.3-0.8 MPa; The freezing conditions described in step (3) are: freezing at -60 to -90°C for 2 to 6 hours; The drying in step (3) is vacuum drying; the vacuum drying conditions are: first drying at -20°C and ≤0.1 mbar for 8 to 16 hours, and then drying at 25°C and ≤0.01 mbar for 4 to 8 hours.

6. The method according to claim 1, wherein: The sieving in step (1) is through a 10-100 mesh sieve; The power of the ultrasonic extraction in step (1) is 200-400W; The temperature of the ultrasonic extraction in step (1) is 50°C; The ultrasonic extraction time in step (1) is 0 to 120 minutes, excluding 0; The number of extractions in step (1) is 1 to 6 times; The centrifugal conditions in step (1) are: 4000-10000 rpm, centrifugation for 10-30 min; The stirring conditions described in step (2) ① are: 300-600 rpm, stirring for 1-3 hours; The stirring conditions described in step (2) ② are: 25° C., 200-400 rpm, stirring for 1-3 h; The centrifugal conditions described in step (2) ② are: 4° C., 4000-10000 rpm, 10-30 min.

7. A Solanum nigrum nano-microcapsule inclusion, characterized by: It is prepared by the method according to any one of claims 1 to 6.

8. An oil-in-water emulsion containing the Solanum nigrum nano-microcapsule inclusions according to claim 7, characterized in that: Calculated by mass percentage, the invention contains the following components: 1% Solanum nigrum nano-microcapsule inclusions, 10% caprylic / capric triglyceride, 5% glycerol, 3% Croda A165, 0.30% carbomer 980, 0.30% triethanolamine, 0.50% p-hydroxyacetophenone, and water is added to 100%.

9. Use of at least one of the Solanum nigrum nano-microcapsule inclusions according to claim 7 and the oil-in-water emulsion containing the Solanum nigrum nano-microcapsule inclusions according to claim 8 in the preparation of anti-acne, antioxidant, anti-aging and / or antibacterial products.

10. The use according to claim 9, characterized in that: The products include medicines and daily chemical products.

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