A dihydrohuperzine alkaloid complex plant vesicle anti-inflammatory soothing composition and a preparation method and application thereof

Abstract

The application provides an anti-inflammatory soothing composition of dihydrohuperzine alkaloids, a preparation method and application thereof, and the composition comprises olea europaea plant vesicles loaded with dihydrohuperzine alkaloids, chamomile plant vesicles loaded with dihydrohuperzine alkaloids and hamamelis virginiana plant vesicles loaded with dihydrohuperzine alkaloids. The dihydrohuperzine alkaloids are loaded in three composite vesicles, which can break through the stratum corneum barrier, directly reach the epidermal inflammation site, and greatly improve the transdermal absorption efficiency. The dihydrohuperzine alkaloids and the triple plant vesicles are synergistic, multi-target anti-inflammatory soothing is realized, and the anti-inflammatory soothing effect is improved compared with free dihydrohuperzine alkaloids. In the composition, the dihydrohuperzine alkaloids have no irritation and no precipitation in the full concentration range of 0.01% to 5%, are safe and effective, the storage stability is greatly improved, and the long-term storage requirement at room temperature is met.

Description

Technical Field

[0001] This application belongs to the technical field of plant vesicle active components, and particularly relates to a dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition, its preparation method and application. Background Technology

[0002] Environmental pollution, excessive cleansing, and the widespread use of cosmetic procedures have led to an increase in the number of people with sensitive skin. Redness, stinging, damaged skin barrier, and neurogenic itching have become core skin problems. The market demand for gentle, highly active, and universally compatible soothing cosmetic ingredients has surged. Dihydrooat alkaloids (hydroxyphenylpropionamide benzoic acid), as a gentle active ingredient derived from oats, can simultaneously inhibit inflammatory factors and block the nerve stimulation pathways in the skin. With a low onset of action threshold, it is a mainstream active ingredient for sensitive skin repair.

[0003] There are three main ways to apply dihydroacid alkaloids: (1) Direct addition of free dihydroacid alkaloids: The process is simple, but the substance is amphiphilic and cannot penetrate the lipid barrier of the stratum corneum. More than 90% of the activity only stays on the skin surface, and the anti-inflammatory utilization rate is less than 20%. When the addition amount is ≥5%, the formula is prone to separation and precipitation, and at the same time, it stimulates the fragile epidermis, which limits the use of high-efficacy products. (2) Artificially synthesized phospholipid liposome encapsulation: It slightly improves the transdermal ability, but the biocompatibility of synthetic phospholipids is poor, and the capsules are prone to breakage when stored at low temperature. The carrier has no additional skin care effect, the added value of the raw materials is low, and the production cost is high. (3) Delivery of single plant vesicles: Olive vesicles or chamomile vesicles are used to encapsulate the product. A single vesicle only repairs a single target point. The particle size dispersion coefficient is high and the activity encapsulation rate is low. The stability of the system is greatly reduced under high and low concentration formulas, and it is impossible to achieve multiple repairs such as redness reduction, itching relief and barrier repair at the same time.

[0004] Therefore, the application of dihydrooat alkaloids still has problems such as extremely low transdermal efficiency, poor bioavailability, no soothing effect at low dosage, large raw material loss; poor compatibility between high and low concentrations, easy precipitation and skin irritation at high dosages, and inability to meet the needs of both domestic and international formulations. Summary of the Invention

[0005] This application provides a dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition, its preparation method, and its application, to solve the problems existing in related technologies. The technical solution is as follows: In a first aspect, embodiments of this application provide a dihydroacid compound plant vesicle anti-inflammatory and soothing composition, the composition comprising olive plant vesicles loaded with dihydroacid, chamomile plant vesicles loaded with dihydroacid, and witch hazel plant vesicles loaded with dihydroacid.

[0006] In one embodiment, the mass ratio of olive plant vesicle-loaded dihydroacid alkaloids, chamomile plant vesicle-loaded dihydroacid alkaloids, and North American witch hazel plant vesicle-loaded dihydroacid alkaloids is (3-7):(2-4):(1-4).

[0007] Secondly, embodiments of this application provide a method for preparing a dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition, comprising the following steps: The plant vesicle dispersions of olive, chamomile, and witch hazel were ultrasonically activated. Dihydroacid alkaloids were added and subjected to ultrasonic encapsulation and aging; a compound anti-inflammatory and soothing composition of dihydroacid alkaloids and plant vesicles was obtained.

[0008] In one embodiment, the conditions for ultrasonic activation are: ultrasonic treatment at 20-50°C for 1-3 hours at 35-45kHz and 250-350W.

[0009] In one embodiment, the mass ratio of added dihydroaerol alkaloids to fresh vesicles in the plant vesicle dispersion is 1 g: (7-9) g.

[0010] In one embodiment, the ultrasonic encapsulation conditions are as follows: the ultrasonic treatment is carried out at a pressure of <0.01MPa and a temperature of 4-15℃, at a frequency of 35-45kHz and a power of 250-350W for 1-3 hours.

[0011] In one implementation, the aging process involves placing the product at 4°C for 12-36 hours.

[0012] In one implementation, aging also includes centrifugation and concentration processes: Centrifuge at 10000-14000×g for 10-50 min; concentrate the supernatant using a PES ultrafiltration fiber membrane with a molecular weight cutoff of 100kDa until the vesicle particle concentration is ≥2.0×10⁻⁶. 11 particles / mL.

[0013] In one embodiment, the preparation method of olive plant vesicle dispersion, chamomile plant vesicle dispersion, and witch hazel plant vesicle dispersion is as follows: Olive pulp, chamomile inflorescence, and witch hazel branches and leaves were added to water and crushed at low temperature; the crushed liquid was centrifuged in two stages at different speeds, and the supernatant was collected to obtain a plant vesicle dispersion.

[0014] In one embodiment, the mass-to-volume ratio of olive pulp, chamomile inflorescence, witch hazel branches and leaves to water is 1 g: (6-10) mL; The conditions for low-temperature crushing are: crushing at 4-10℃ using a high-speed blender at 8000-12000 rpm for 4-8 minutes; The two-stage differential centrifugation conditions are as follows: First stage centrifugation: 4℃, 2500-3500×g, centrifugation for 10-30 min; Second stage centrifugation: 4℃, 10000-14000×g, centrifugation for 30-50 min.

[0015] In one embodiment, the compounded product is filtered sequentially through a 1.0 μm PTFE membrane and then through a 0.22 μm PES sterilization membrane to obtain a dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition.

[0016] Thirdly, the embodiments of this application provide the application of the above-mentioned dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition in the preparation of anti-inflammatory and soothing products.

[0017] Fourthly, embodiments of this application provide an anti-inflammatory and soothing product comprising the above-mentioned dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition.

[0018] The advantages or beneficial effects of the above technical solutions include at least the following: This application discloses a dihydroacid alkaloid complex plant vesicle anti-inflammatory and soothing composition, comprising olive plant vesicles loaded with dihydroacid alkaloids, chamomile plant vesicles loaded with dihydroacid alkaloids, and witch hazel plant vesicles loaded with dihydroacid alkaloids. The combination of olive plant vesicles, chamomile plant vesicles, and witch hazel plant vesicles loaded with dihydroacid alkaloids solves the problems of easy precipitation and high skin irritation associated with high-concentration dihydroacid alkaloids, making them unsuitable for high-efficacy formulations. It also overcomes the issues of amphiphilic imbalance, weak stratum corneum penetration, and low bioavailability associated with free dihydroacid alkaloids. Furthermore, the combination of these three vesicles provides good carrier stability, as confirmed by accelerated cold and heat cycling tests and long-term storage observations. The synergistic effect of the three plant vesicles achieves multi-target anti-inflammatory and soothing effects.

[0019] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0020] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0021] Figure 1The polydispersity coefficient and zeta potential histogram of the dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition in Examples 3-10 are shown. Figure 2 Bar chart showing the encapsulation efficiency of dihydrooat alkaloids in the dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing compositions of Examples 3-10; Figure 3 The bar chart shows the inhibition rate of NO release from RAW264.7 cells by the dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition of Examples 3-10. Figure 4 Figure 1 shows the results of in vitro skin irritation and human patch safety evaluation of different samples; where A represents the recombinant epidermal cell survival rate of the blank group, free DHA group, and the anti-inflammatory and soothing composition of dihydrooat alkaloids and plant vesicles from Example 3 at different concentrations; B represents the IL-1α content released in the epidermal model induced by the three groups of samples at different concentrations; C represents the skin irritation score of the human patch test at different concentrations; and D represents the in vitro erythrocyte hemolysis rate of the three groups of samples at different concentrations. Figure 5 Comparison of in vitro transdermal permeability of artificial biomimetic skin for blank group, free DHA group, and dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition group of Example 3; where A is the cumulative transdermal amount of active ingredient at different permeation times at 1% active ingredient concentration; B is the total transdermal amount of the three groups of samples at different addition concentrations at the 24h permeation endpoint. Figure 6 The figures show the in vitro anti-inflammatory and soothing efficacy evaluation of the control group, free DHA group, and the anti-inflammatory and soothing composition of dihydrooat alkaloids compound plant vesicles in Example 3 at the cellular, enzyme, and three-dimensional skin model levels. A represents the inhibition rate of NO release from RAW264.7 macrophages by different concentrations of the sample; B represents the inhibition rate of COX-2, a key inflammatory enzyme, by different concentrations of the sample; C represents the TEWL value of the UVB-damaged three-dimensional skin model after treatment with different concentrations of the sample; and D represents the ROS scavenging ability of different concentrations of the sample within the skin model. Figure 7 The graph shows the results of a 28-day human clinical trial on the anti-inflammatory and soothing efficacy of the blank group, the free DHA group, and the dihydrooat alkaloid compound plant vesicle anti-inflammatory and soothing composition from Example 3 in volunteers with sensitive skin; Figure A shows the skin redness index (a) of the three groups of samples with different concentrations after 28 days of continuous use. B represents the subjective stinging VAS scores of the subjects in the three groups at a 1% added concentration. Detailed Implementation

[0022] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0023] The use of free dihydrooat alkaloids differs significantly between domestic and international applications. Domestically, they are typically used in mild, low-concentration skincare products (0.01%), while internationally they are used in high-concentration emergency repair products (10%). However, free dihydrooat alkaloids have extremely low transdermal absorption efficiency and poor bioavailability. Low concentrations offer no soothing effect and result in significant raw material loss; high concentrations, on the other hand, are prone to precipitation and skin irritation. Therefore, conventional use cannot simultaneously meet the formulation requirements of both domestic and international applications. This application provides a dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition, its preparation method, and its application.

[0024] This application provides a dihydroacid alkaloid complex plant vesicle anti-inflammatory and soothing composition, comprising dihydroacid alkaloids loaded on olive plant vesicles, dihydroacid alkaloids loaded on chamomile plant vesicles, and dihydroacid alkaloids loaded on witch hazel plant vesicles. By loading dihydroacid alkaloids onto olive plant vesicles, chamomile plant vesicles, and witch hazel plant vesicles, the compatibility and skin penetration of dihydroacid alkaloids at high concentrations are improved, and bioavailability is enhanced. When used in combination with the three types of vesicles, olive plant vesicles repair the skin barrier, chamomile plant vesicles soothe nerve pain, and witch hazel plant vesicles reduce redness and swelling, while the anti-inflammatory activity of dihydroacid alkaloids is enhanced, achieving multi-target anti-inflammatory and soothing effects.

[0025] In one embodiment, the mass ratio of dihydroacid alkaloids loaded on olive plant vesicles, dihydroacid alkaloids loaded on chamomile plant vesicles, and dihydroacid alkaloids loaded on witch hazel plant vesicles is (3-7):(2-4):(1-4). The three types of vesicles in this application are compounded in a certain proportion. For example, the mass ratios of dihydroacid alkaloids loaded on olive plant vesicles, dihydroacid alkaloids loaded on chamomile plant vesicles, and dihydroacid alkaloids loaded on witch hazel plant vesicles are 7:2:1, 6:3:1, 6:2:2, 5:4:1, 5:3:2, 5:2:3, 4:4:2, 4:3:3, and 3:3:4. The combined use of these three types of vesicles achieves synergistic effects of the three functions and utilizes the complementary lipid membranes of the three vesicles to achieve high encapsulation of dihydroacid alkaloids and composition stability.

[0026] This application provides a method for preparing a dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition, comprising the following steps: The plant vesicle dispersions of olive, chamomile, and witch hazel were ultrasonically activated. Dihydroacid alkaloids were added, ultrasonically encapsulated, aged, and compounded to obtain a dihydroacid alkaloid complex plant vesicle anti-inflammatory and soothing composition.

[0027] This application involves ultrasonic activation of vesicles to open channels in the vesicle membrane. After adding dihydroacid alkaloids, the dihydroacid alkaloids are introduced into the vesicles through the channels under ultrasonic action, utilizing the concentration gradient of dihydroacid alkaloids. During the subsequent aging process, the vesicle membrane channels close, thereby achieving the encapsulation of dihydroacid alkaloids.

[0028] As one implementation method, the conditions for ultrasonic activation are: 35-45kHz, 250-350W, and ultrasonic treatment at 20-50℃ for 1-3 hours.

[0029] As one implementation method, the mass ratio of dihydroaeroxanone to fresh vesicles in the plant vesicle dispersion is 1g:(7-9)g; preferably, it is 1g:8g.

[0030] As one implementation method, ultrasonic encapsulation is performed at a pressure <0.01MPa and a temperature of 4-15℃, at 35-45kHz and 250-350W, for 1-3 hours. The ultrasound is controlled within certain conditions to avoid irreversible damage to the vesicles while simultaneously generating reversible membrane channels.

[0031] One implementation method involves placing the vesicle at 4°C for 12-36 hours. During aging, the membrane channels on the vesicle membrane are restored, ensuring the stability of the subsequent system.

[0032] As one implementation method, aging also includes centrifugation and concentration processes: Centrifuge at 10000-14000×g for 10-50 min; concentrate the supernatant using a PES ultrafiltration fiber membrane with a molecular weight cutoff of 100kDa until the vesicle particle concentration is ≥2.0×10⁻⁶. 11 particles / mL. High-speed centrifugation can remove aggregated particles of free active matter and broken vesicle fragments. Ultrafiltration concentrates the vesicles to a specific concentration for convenient subsequent use, especially for quantitative applications.

[0033] As one embodiment, the preparation method of olive plant vesicle dispersion, chamomile plant vesicle dispersion, and North American witch hazel plant vesicle dispersion is as follows: Olive pulp, chamomile inflorescence, and witch hazel branches and leaves were added to water and crushed at low temperature; the crushed liquid was centrifuged in two stages at different speeds, and the supernatant was collected to obtain a plant vesicle dispersion.

[0034] In this application, olive pulp, chamomile inflorescence, and witch hazel branches and leaves are crushed in water to release the vesicles, and then the vesicles are separated by differential centrifugation to obtain a dispersion of the corresponding plant vesicles.

[0035] As one implementation method, the mass-to-volume ratio of olive pulp, chamomile inflorescence, witch hazel branches and leaves to water is 1 g: (6-10) mL.

[0036] One implementation method involves cryogenic crushing at 4-10°C using a high-speed blender. The blending speed is 8000-12000 rpm for 4-8 minutes. Cryogenic crushing ensures that the vesicles are not damaged by temperature increases during the crushing process.

[0037] As one implementation method, the two-stage differential centrifugation conditions are as follows: First-stage centrifugation: 4℃, 2500-3500×g, centrifugation for 10-30 min; Second-stage centrifugation: 4℃, 10000-14000×g, centrifugation for 30-50 min. The first-stage centrifugation can remove coarse fibers and plant tissue residues; the second-stage centrifugation removes large-particle impurities.

[0038] In this embodiment, the qualification criterion for the vesicle dispersion is: vesicle particle concentration detected by a nanoparticle size analyzer ≥ 1.2 × 10⁻⁶. 11 Particles / mL, PDI ≤ 0.25, Zeta potential ≤ -18mV, must meet these standards before proceeding to the next step.

[0039] In one implementation method, the compounded product is filtered sequentially through a 1.0 μm PTFE membrane and then through a 0.22 μm PES sterile membrane to obtain a dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition. Pre-filtration with a 1.0 μm PTFE membrane removes aggregated particles; final filtration with a 0.22 μm PES sterile membrane removes residual unencapsulated free active substances and microbial impurities.

[0040] As one implementation method, the filtrate is transported to the storage tank through a sterile pipeline. After sampling and testing to ensure that all indicators are qualified, it is aseptically filled, packaged, labeled, and stored in the warehouse.

[0041] This application provides the use of the above-mentioned dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition in the preparation of anti-inflammatory and soothing products.

[0042] This application provides an anti-inflammatory and soothing product comprising the above-mentioned dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition.

[0043] As one implementation method, a complex plant vesicle anti-inflammatory and soothing composition with a dihydroacid alkaloid content of 0.01%-5.0% is added to the anti-inflammatory and soothing product. This application establishes comprehensive dose-response data for a complex plant vesicle anti-inflammatory and soothing composition with a dihydroacid alkaloid content of 0.01%-5.0%, adaptable to cosmetic formulations with different additive standards both domestically and internationally. Within this additive range, it can achieve anti-inflammatory and soothing effects.

[0044] The following specific examples will provide further details.

[0045] The PES ultrafiltration fiber membrane used in this application is a Cobetter Biophsep™ Lab+mPES hollow fiber ultrafiltration module, model HFELP00030530P; the membrane material is modified polyethersulfone (mPES); the molecular weight cutoff is 100 kDa; the membrane type is a hollow fiber ultrafiltration membrane; the effective filtration area is 800 cm²; and the inner diameter of the membrane fibers is 0.5 mm. The PTFE filter membrane is Tianjin Jinteng JTFA0210, made of hydrophobic polytetrafluoroethylene, with a nominal pore size of 1.0μm; The PES sterilization filter membrane is made of Maiborei MSP013022S, a hydrophilic polyethersulfone material with a nominal pore size of 0.22μm and is sterile grade; the ultrafiltration centrifuge tube is Amicon Ultra-0.5mL 100K ultrafiltration centrifuge tube (UFC510096, made of regenerated cellulose membrane material, with a nominal molecular weight cutoff of 100kDa). Example 1 Commercially available olive pulp, chamomile inflorescences, and witch hazel branches and leaves were added to a crusher at a ratio of 1g:8mL (raw material weight: purified water) and crushed at 4℃. After crushing, the mixture was subjected to two-stage differential centrifugation. The first stage centrifugation conditions were: 4℃, 3000×g, centrifugation for 20 min, and the supernatant was collected. The second stage centrifugation conditions were: 4℃, 12000×g, centrifugation for 40 min, and the supernatant was collected. This yielded olive plant vesicle dispersions, chamomile plant vesicle dispersions, and witch hazel plant vesicle dispersions. The concentration of vesicle particles detected by the nanoparticle size analyzer is ≥1.2×10⁻⁶. 11 Particles / mL, PDI ≤ 0.25, Zeta potential ≤ -18mV, vesicle dispersion is qualified; Take a certain volume of vesicle dispersion, centrifuge at 100,000g for 70 minutes at 4℃, and the precipitate is fresh vesicles. Weigh the fresh vesicles to obtain their mass. When using, convert the volume of dispersion to be used according to the corresponding relationship.

[0046] Example 2 Vesicle activation: Olive plant vesicle dispersion, chamomile plant vesicle dispersion and North American witch hazel plant vesicle dispersion were respectively transferred into an ultrasonic reactor and ultrasonically treated at room temperature for 2 hours at 40 kHz and 300 W. Single-component encapsulation preparation: The fresh vesicle content in the plant vesicle dispersion was determined according to the method in Example 1; the corresponding volume of the vesicle dispersion was measured according to the ratio of dihydroacid alkaloid to fresh plant vesicle weight = 1g: 8g; dihydroacid alkaloid powder with a purity ≥98% was mixed with the three plant vesicle dispersions respectively, and the mixed system was transferred into a closed ultrasonic encapsulation device. The pressure was controlled to be <0.01MPa and the temperature to be <15℃, and the active substance was encapsulated by ultrasound; after the encapsulation was completed, the system was aged at 4℃ for 24h. High-speed centrifugation purification: After aging, the system was centrifuged at 12000×g for 30min; Membrane concentration: PES ultrafiltration fiber membrane with a molecular weight cutoff of 100 kDa was used for concentration at room temperature until the vesicle particle concentration was ≥2.0 × 10⁻⁶. 11 Particles / mL were used to obtain concentrated olive plant vesicle-loaded dihydroacid alkaloids (OLV@DHA), chamomile plant vesicle-loaded dihydroacid alkaloids (CHV@DHA), and North American witch hazel plant vesicle-loaded dihydroacid alkaloids (HAMV@DHA).

[0047] Example 3 Olive plant vesicle loaded with dihydroacid, chamomile plant vesicle loaded with dihydroacid, and witch hazel plant vesicle loaded with dihydroacid at a mass ratio of 5:3:2 were mixed and stirred at low speed of 150 r / min for 30 min until homogeneous. The mixture was pre-filtered using a 1.0μm PTFE membrane and then terminally filtered through a 0.22μm PES sterilization membrane to obtain a dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition.

[0048] Example 4 The vesicles of olive plant loaded with dihydroacid, vesicles of chamomile plant loaded with dihydroacid, and vesicles of witch hazel plant loaded with dihydroacid were prepared in a mass ratio of 7:2:1.

[0049] Example 5 The vesicles of olive plant loaded with dihydroacid alkaloids, the vesicles of chamomile plant loaded with dihydroacid alkaloids, and the vesicles of witch hazel plant loaded with dihydroacid alkaloids were prepared in a mass ratio of 6:3:1.

[0050] Example 6 The vesicles of olive plant loaded with dihydroacid, vesicles of chamomile plant loaded with dihydroacid, and vesicles of witch hazel plant loaded with dihydroacid were prepared in a mass ratio of 5:4:1.

[0051] Example 7 The vesicles of olive plant loaded with dihydroacid alkaloids, the vesicles of chamomile plant loaded with dihydroacid alkaloids, and the vesicles of witch hazel plant loaded with dihydroacid alkaloids were prepared in a mass ratio of 5:2:3.

[0052] Example 8 The vesicles of olive plant loaded with dihydroacid alkaloids, the vesicles of chamomile plant loaded with dihydroacid alkaloids, and the vesicles of witch hazel plant loaded with dihydroacid alkaloids were prepared in a mass ratio of 4:4:2.

[0053] Example 9 The vesicles of olive plant loaded with dihydroacid alkaloids, the vesicles of chamomile plant loaded with dihydroacid alkaloids, and the vesicles of witch hazel plant loaded with dihydroacid alkaloids were prepared in a mass ratio of 4:3:3.

[0054] Example 10 The vesicles of olive plant loaded with dihydroacid alkaloids, the vesicles of chamomile plant loaded with dihydroacid alkaloids, and the vesicles of witch hazel plant loaded with dihydroacid alkaloids were prepared in a mass ratio of 3:3:4.

[0055] 1. The polydispersity index (PDI) and zeta potential of the dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing compositions of Examples 3-10 were measured using a nanoparticle size potentiometer. The nanoparticle size potentiometer was a Malvern Panalytical (UK) Zetasizer Nano ZS90; the light source was a He-Ne laser, 633 nm; the detection temperature range was 4-90℃, and a constant temperature of 25℃ could be set (25℃ was used in this application); the results are as follows. Figure 1 As shown.

[0056] Figure 1 In the PDI results, it was found that under different vesicle loading ratios of dihydrooat alkaloids, the PDI of Examples 3-10 was all <0.2, and the vesicle system was generally stable. Among them, the PDI value of Example 3 with a ratio of 5:3:2 was the lowest, indicating that the vesicle particle size was the most uniform and the system had the best storage stability. The large differences in the proportions of Example 4 (7:2:1) and Example 10 (3:3:4) caused a slight increase in particle size dispersion.

[0057] The Zeta potential results showed that the potentials of all examples were negative, indicating that the vesicle system had electrostatic repulsion stability. Example 3, with a ratio of 5:3:2, had the largest absolute negative potential value, indicating the best electrostatic stability and making it less prone to aggregation and stratification. The absolute potential values ​​of the other ratios were relatively low, but the Zeta potentials were all less than -18mV, indicating that the vesicle system was relatively stable.

[0058] Therefore, the dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing compositions of Examples 3-10 have a particle size PDI≤0.25 and a Zeta potential <-18mV, and the vesicle dispersions are qualified.

[0059] 2. Separation of free active substances using ultrafiltration centrifugation: Ultrafiltration centrifuge tubes with a molecular weight cutoff of 100 kDa were used. The sample was added to the ultrafiltration centrifuge tube and centrifuged at 8000 × g for 15 min. Free dihydroacid alkaloids permeated through the membrane and entered the filtrate, while the dihydroacid alkaloids encapsulated in the vesicles were retained in the upper concentrate. The concentration of free dihydroacid alkaloids in the filtrate was determined by HPLC (Agilent 1260 Infinity II high-performance liquid chromatograph (equipped with a DAD detector, Waters XBridge C18 column, detection wavelength 280 nm, external standard method)). An equal volume of the original sample was taken and subjected to methanol vortex lysis with ultrasonication (Shanghai Keda SK3300H ultrasonic cleaner, vortexing for 5 min, followed by ultrasonication at 300W in an ice-water bath for 30 min) to fully release the dihydroacid alkaloids encapsulated within the vesicles. The total dihydroacid alkaloid concentration in the sample was determined by HPLC. Encapsulation efficiency (%) = (Total DHA concentration) (Free DHA concentration) ÷ Total DHA concentration × 100%. Results are as follows: Figure 2 As shown.

[0060] Figure 2 The encapsulation results showed that the encapsulation rate of dihydroacid alkaloids in Example 3, with a ratio of 5:3:2, reached the peak value (89.7%) of Examples 3-10. In Examples 4 (7:2:1) and 10 (3:3:4), the single vesicles were excessive, and the encapsulation rate was slightly lower than that of the balanced ratio group. This proves that the reasonable and balanced combination of the three plant vesicles is beneficial to improving the encapsulation capacity of dihydroacid alkaloids.

[0061] 3. Construction of an LPS-induced RAW264.7 cell inflammation model; RAW264.7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in a Thermo 3111 CO2 incubator at 37℃ and 5% CO2 saturated humidity; the inflammation model was established using Sigma L2880 lipopolysaccharide LPS. After incubation with the dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition from Examples 3-10, the NO content in the supernatant was detected using a Beyotime S0021 NO detection kit (Griess colorimetric method) and a Thermo Multiskan FC microplate reader at a wavelength of 540nm. The NO inhibition rate was calculated; the results are as follows. Figure 3 As shown.

[0062] Figure 3The NO inhibition rate results showed that the NO inhibition rate of Example 3 with a ratio of 5:3:2 was significantly higher than that of Examples 4-10, and the in vitro anti-inflammatory activity was the strongest. The anti-inflammatory activity was weakened in the compound system with excessive single vesicles or two types of vesicles, which confirmed that the three plant vesicles had a significant synergistic effect in anti-inflammatory compounding within a certain ratio.

[0063] 4. Skin safety evaluation experiment (1) Detection of recombinant human epidermal cell viability and IL-1α: The commercial EpiDerm three-dimensional human epidermal model (MatTek, EPI-200) was used, and three test systems were set up: ① Blank group (deionized water + propylene glycol only, propylene glycol concentration was 0.01%, 0.1%, 1%, 5%); ② Free DHA group: free dihydroacid alkaloid concentration was 0.01%, 0.1%, 1%, 5% respectively, solvent was deionized water; ③ Complex vesicle group: OLV@D was concentrated in the ultrafiltration process of Example 2. The dihydroacid alkaloids in HA, CHV@DHA, and HAMV@DHA were 5% by mass. After being compounded according to the proportions in Example 3, they were diluted with water to dihydroacid alkaloid concentrations of 0.01%, 0.1%, and 1%, respectively, resulting in compound vesicle groups with dihydroacid alkaloid concentrations of 0.01%, 0.1%, 1%, and 5%. Human epidermal cells were incubated at these three different concentrations for 24 hours. Cell viability was detected by the MTT assay (Beyotime, C0009MTT cell proliferation assay kit, using a Thermo Multiskan FC microplate reader at 570 nm wavelength), and the release of the stimulating factor IL-1α was detected by an ELISA kit (R&D Systems, DLA00, using a Thermo Multiskan FC microplate reader at 450 nm wavelength). The results are as follows: Figure 4 As shown.

[0064] Cell survival rate: The cell survival rate of the blank group remained stable at over 97% at all concentrations, with no cytotoxicity; the cell survival rate of the free DHA group decreased significantly with increasing concentration, with a survival rate of only 72% at a 5% concentration, indicating significant cell stimulation; at the same concentration, the cell survival rate of the complex vesicle group was much higher than that of the free DHA group, with a survival rate of nearly 90% at a high concentration of 5%, indicating that vesicle encapsulation significantly reduced the cytotoxicity of the active ingredient.

[0065] IL-1α inflammatory factor: The release of IL-1α in the free DHA group increased significantly with concentration, and the release of IL-1α at a concentration of 5% was more than 4 times that of the control group; the release of IL-1α in the compound vesicle group increased only slightly across the entire concentration gradient, proving that plant vesicles can reduce the inflammatory stimulation of epidermal cells by active substances.

[0066] (2) Human patch test: 60 healthy volunteers were divided into two groups: a 0.01%, 0.1%, 1%, and 5% concentration composite vesicle group and a 0.01%, 0.1%, 1%, and 5% concentration free DHA group. Patches were applied to the back for 24 hours. After patch removal, the volunteers were continuously observed. Skin irritation scores were calculated at 72 hours using the national standard 0-3 scoring system (0 = no irritation, 1 = mild irritation, 2 = moderate irritation, 3 = severe irritation). The results are as follows: Figure 4 As shown.

[0067] Human patch irritation score: The irritation scores of the blank group and the compound vesicle group were both 0 points, and there were no erythema, edema, or papules on the human skin; 1% of the free DHA group showed mild irritation and 5% showed mild to moderate irritation; it is confirmed that high concentrations of free DHA have the risk of skin sensitization and formulation precipitation. The vesicle delivery system of this invention completely eliminates the skin irritation of high concentrations of active ingredients.

[0068] (3) Red blood cell hemolysis test: In vitro red blood cell suspensions were co-incubated with 0.01%, 0.1%, 1%, and 5% free DHA groups, 0.01%, 0.1%, 1%, and 5% complex vesicle groups, and blank groups, respectively, and the hemolysis rate was detected. 2% (v / v) New Zealand rabbit red blood cell saline suspension was mixed with equal volumes of 0.01%, 0.1%, 1%, and 5% free DHA groups, 0.01%, 0.1%, 1%, and 5% complex vesicle groups, and blank saline groups, respectively; 1% Triton X-100 was set as a positive hemolysis control group. After incubating each group of samples in a 37°C water bath for 1 hour, centrifuge them at 3000 rpm for 10 minutes at room temperature using an Eppendorf 5810R benchtop refrigerated centrifuge. Transfer the supernatant to a Corning 3599 96-well clear plate and measure the absorbance of the supernatant at 540 nm using a Thermo Multiskan FC microplate reader. Calculate the hemolysis rate using the formula: Hemolysis rate (%) = (Absorbance of sample group) (Absorbance of blank control group) / (Absorbance of positive control group) (Absorbance of blank control group) × 100%; results are as follows: Figure 4 As shown, a lower hemolysis rate indicates less irritation to the skin and mucous membranes.

[0069] Red blood cell hemolysis rate: The hemolysis rate of the free DHA group increased sharply with concentration, and the hemolysis rate of 5% concentration was close to 9%; the hemolysis rate of the complex vesicle group was less than 5% at all concentrations, and the mucosal irritation was significantly reduced, making it suitable for formulas of delicate skin products such as serums, masks, and eye area products.

[0070] Therefore, the three plant vesicles in the dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition of this application can significantly reduce the irritation of dihydrooat alkaloids to cells, skin, and mucous membranes, and are safe for human use in the full addition range of 0.01%-5%, with no potential risk of sensitization.

[0071] 5. Franz in vitro transdermal permeation experiment The Tianjin Zhengtong TK-12D vertical Franz diffusion cell and PermeGear Strat-M were used. ® An animal-free, artificial biomimetic skin membrane separates the supply and receiving pools; the diffusion pool has an effective permeation area of ​​3.14 cm². 2 The receiving cell has a volume of 7 mL, the constant temperature water bath is 37℃, and the magnetic stirring speed is 300 rpm. Subject sample setup: ① Blank group; ② Free DHA group; ③ Complex vesicle group. (Same as "4. Skin safety evaluation experiment").

[0072] Time gradient permeation experiment: Diffusion experiments were conducted using a blank group (1% propylene glycol), a free DHA group (1% dihydroazoline), and a complex vesicle group (1% dihydroazoline). The cumulative transdermal absorption rate (μg / cm³) of the blank group, free DHA group, and complex vesicle group was measured at 2h, 4h, 8h, 12h, and 24h. 2 ); the results are as follows Figure 5 As shown.

[0073] Concentration gradient permeation experiment: Total transdermal volume was measured after 24 hours at concentrations of 0.01%, 0.1%, 1%, and 5%; the receiving solution was pH 7.4 PBS buffer, and the mixture was stirred at 37℃. The content of active ingredients penetrating the membrane was quantitatively determined by HPLC. Each group had n=6. Results are as follows: Figure 5 As shown.

[0074] Based on the time gradient penetration results: the blank group showed almost no penetration of active ingredients; the free DHA group exhibited an amphiphilic imbalance, making it difficult to penetrate the stratum corneum, with a cumulative transdermal absorption of only 40 μg / cm³ over 24 hours. 2 The transdermal transdermal absorption rate of the encapsulated group was significantly higher than that of the free group at all time points, and the cumulative transdermal transdermal absorption rate over 24 hours was approximately 2.3 times that of the free group; this demonstrates that plant vesicles can carry insoluble DHA to penetrate the cuticle barrier and continuously improve transdermal efficiency.

[0075] Concentration gradient permeation results: The transdermal penetration of all three groups of samples increased in a dose-dependent manner with increasing concentration; at the same concentration, the transdermal penetration of the composite vesicle group was always significantly higher than that of the free DHA group; the free DHA group at a low concentration of 0.01% had almost no effective permeation, while the composite vesicle group could still achieve transmembrane delivery of active ingredients, solving the industry pain point that low-addition formulations cannot achieve the desired effect of active ingredients.

[0076] Therefore, the combined plant vesicles of olive, chamomile, and witch hazel can significantly enhance the skin penetration ability of dihydrooat alkaloids, achieving efficient transdermal absorption at both high and low concentrations and amplifying the utilization rate of anti-inflammatory activity.

[0077] 6. Validation of in vitro anti-inflammatory and soothing mechanism Samples: Three test systems were set up: ① blank group; ② free DHA group; ③ complex vesicle group. (Same as "4. Skin safety evaluation experiment").

[0078] (1) NO inhibition experiment of RAW264.7 cells: RAW264.7 cells in logarithmic growth phase were used with 1×10 5 Cells were seeded per well in 96-well plates. After adhesion, 1 μg / mL LPS was added to construct an LPS-induced macrophage inflammation model. Gradual concentrations of test samples were added simultaneously, with 1 μg / mL dexamethasone as a positive control. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h. Cell supernatant was collected, and the NO content of each concentration was measured using a Griess method NO detection kit. Absorbance was read at 540 nm using a Thermo Multiskan FC microplate reader to calculate the NO inhibition rate. 1% panthenol was used as a positive control. Results are as follows: Figure 6 As shown.

[0079] NO inhibitory activity: No inhibitory effect in the blank group; the inhibition rate of the free DHA group and the complex vesicle group increased significantly with increasing concentration; at the same concentration, the NO inhibition rate of the complex vesicle group was much higher than that of the free DHA group and the positive control panthenol; the inhibition rate of the 5% concentration complex vesicle group can reach 70%, which strongly inhibits the release of inflammatory mediators.

[0080] (2) COX-2 inflammatory enzyme inhibition experiment: A recombinant human COX-2 enzyme inhibitor screening kit was used. Test samples of varying concentrations were added to the enzyme reaction system. After incubation at 37℃ for 10 min, arachidonic acid substrate was added to initiate the enzymatic reaction. After colorimetric termination, the absorbance was detected at 590 nm using a Thermo Multiskan FC microplate reader, and the COX-2 enzyme inhibition rate was calculated. The results are as follows: Figure 6 As shown.

[0081] COX-2 enzyme inhibition: COX-2 is the core target of skin redness and swelling. The free DHA group only had a moderate inhibitory effect; the complex vesicle group showed an enzyme inhibition capacity of more than 40% at each concentration, and the inhibition rate exceeded 60% at high concentrations, which confirmed the soothing and anti-redness and swelling advantages from an enzymatic perspective.

[0082] (3) Three-dimensional full-thickness skin TEWL barrier repair experiment: The MatTek EFT-400 three-dimensional full-thickness skin model was used, and a 310nm UVB ultraviolet lamp was used at 300mJ / cm 2A skin photodamage model was constructed by dose irradiation. Gradual concentrations of test samples were applied to the skin surface and incubated at 37°C for 24 hours. Transdermal water loss (TEWL) values ​​were measured using a Tewameter TM300 (Courage+Khazawa, Germany) transdermal water loss meter. The results are as follows: Figure 6 As shown; the lower the value, the stronger the skin barrier repair ability.

[0083] Skin barrier repair TEWL: After UVB modeling, the TEWL of the blank group remained at around 26, indicating severe barrier damage; the free DHA group showed a slight reduction in moisture loss; the TEWL of the complex vesicle group continued to decrease with concentration, reaching only 7.8 at a 5% concentration, indicating significant repair of the stratum corneum barrier integrity, with both repair and soothing effects.

[0084] (4) DCFH-DH reactive oxygen species scavenging experiment: The ability of the skin model to scavenge reactive oxygen species was detected by using a recombinant skin model. An oxidative stress model was constructed by UVB irradiation. After adding the test sample and incubating together, the DCFH-DA fluorescent probe (DCFH-DA reactive oxygen species detection kit, Beyotime) was loaded. After incubation at 37℃ for 20 min, the sample was washed with PBS buffer and the fluorescence intensity was detected by a fluorescence microplate reader (S0033S fluorescence microplate reader, BioTek Synergy H1) at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. The ROS scavenging rate was calculated. The higher the scavenging rate, the stronger the soothing and antioxidant effect.

[0085] ROS scavenging ability: The free DHA group had only a moderate antioxidant effect; the complex vesicle group had a significantly improved reactive oxygen species scavenging ability, which could alleviate the sensitivity to oxidative stress caused by ultraviolet rays and external stimuli.

[0086] Therefore from Figure 6 The results showed that, with the same amount of added compound vesicles, the anti-inflammatory, antioxidant, and barrier repair effects were significantly better than those of free dihydroaerol alkaloids, and the efficacy was positively correlated with the concentration.

[0087] 7. 28-day human clinical trial for sensitive skin 240 volunteers with sensitive skin that is prone to redness and stinging due to damaged facial barrier were recruited and randomly divided into three groups: ① blank group; ② free DHA group; ③ complex vesicle group (same as "4. Skin safety evaluation experiment"); 20 people in each group; the test sample was applied to the face every morning and evening for 28 consecutive days; Objective instrument testing: Facial redness index (a) was measured using a German Courage+Khazawa CR-400 colorimeter (CIELAB color space, 8mm measuring aperture). (The lower the value, the milder the redness); Subjective evaluation: Visual analogue score (VAS) was used (0 = no stinging, 10 = severe stinging). Three groups of samples with a core 1% concentration were selected to compare the improvement in subjective stinging effect. Results are as follows: Figure 7 As shown.

[0088] from Figure 7 Skin redness index a As you can see, in the blank group, the redness index remained stable at around 14 at all concentrations, with no improvement in redness on sensitive skin; in the free DHA group, redness gradually decreased with increasing concentration, but the improvement was limited; in the compound vesicle group, the redness index decreased significantly with increasing concentration, and at a high concentration of 5%, the redness index decreased even further. With just 2.3, facial redness almost completely subsided; a very low additive concentration of 0.01% is sufficient to achieve noticeable redness reduction, making it suitable for low-additive, affordable skincare products.

[0089] Based on the subjective stinging VAS score: the stinging scores of subjects in the blank group and the free DHA group were all higher than 4 points, and the daily burning and stinging sensations were obvious; the score of the complex vesicle group was only about 0.7, and the subjects' facial stinging, burning and tightness sensations basically disappeared, and the subjective relief was significantly better than that of the free DHA group and the blank group.

[0090] Therefore, the dihydrooat alkaloid compound plant vesicle anti-inflammatory and soothing composition can significantly reduce skin redness and relieve burning and stinging. It is effective at a low concentration of 0.01%, and the repair effect is optimal at a high concentration of 5%. Both high and low concentrations have excellent soothing and repairing effects and are suitable for use in all types of cosmetics at home and abroad.

[0091] 8. Composition stability test The free dihydrooat alkaloid solution, the single plant vesicle drug delivery system, the binary compound vesicle drug delivery system (olive vesicles@DHA:chamomile vesicles@DHA=5:3, olive vesicles@DHA:witch hazel vesicles@DHA=5:2, chamomile vesicles@DHA:witch hazel vesicles@DHA=3:2), and the ternary compound plant vesicle composition of Example 3 of this invention were sealed and packaged separately. Two types of stability test conditions were set: ① cold and hot cycling: alternating between -10℃ and 45℃, switching the temperature every 12 hours, for a total of 15 cycles; ② long-term constant temperature storage: standing at 25℃ and 60% relative humidity for 90 days. During the experiment, the presence of stratification, precipitation, flocculation, and crystallization in the system was observed visually. The vesicle particle size and PDI changes were detected by dynamic light scattering using Malvern Zetasizer Nano ZS (detection temperature 25℃; scattering angle 173°). The content of dihydrooat alkaloids was determined by external standard method using high performance liquid chromatography (Agilent 1260 Infinity II, C18 column, 280nm UV detection), and the retention rate of active ingredients was calculated.

[0092] Test results: Crystallization occurred after 5 cycles of hot and cold cycling of the free dihydroagar alkaloid aqueous solution, and the retention rate of active ingredients was less than 72% after 30 days of storage at room temperature; the particle size of the single and binary vesicle systems gradually increased during long-term storage; after 15 cycles of hot and cold cycling and 90 days of storage at room temperature, the ternary compound system of this invention showed no stratification, precipitation, or crystallization, and the vesicle particle size and PDI did not change significantly, with a dihydroagar alkaloid retention rate of not less than 91%, proving that the ternary plant vesicle compound can effectively improve the storage stability of the system.

[0093] In summary, the dihydroacid alkaloid complex plant vesicle anti-inflammatory and soothing composition of this application comprises dihydroacid alkaloids loaded onto olive plant vesicles, dihydroacid alkaloids loaded onto chamomile plant vesicles, and dihydroacid alkaloids loaded onto witch hazel plant vesicles. Encapsulating dihydroacid alkaloids in these three complex vesicles allows them to penetrate the stratum corneum barrier, directly reaching the epidermal inflammatory sites, significantly improving transdermal absorption efficiency. The synergistic effect of the vesicles and dihydroacid alkaloids enhances the anti-inflammatory and soothing effect by 40% compared to free dihydroacid alkaloids. Furthermore, the dihydroacid alkaloids in the composition are non-irritating and do not precipitate within the entire concentration range of 0.01%-5%, ensuring safety and effectiveness. Storage stability is greatly improved, meeting the requirements for long-term storage at room temperature for cosmetics.

[0094] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0095] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0096] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition, characterized in that, The composition includes olive plant vesicle-loaded dihydroacid alkaloids, chamomile plant vesicle-loaded dihydroacid alkaloids, and witch hazel plant vesicle-loaded dihydroacid alkaloids.

2. The dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition according to claim 1, characterized in that, The mass ratio of dihydroacid alkaloids loaded on olive plant vesicles, dihydroacid alkaloids loaded on chamomile plant vesicles, and dihydroacid alkaloids loaded on witch hazel plant vesicles was (3-7):(2-4):(1-4).

3. A method for preparing a dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition, characterized in that, Includes the following steps: The plant vesicle dispersions of olive, chamomile, and witch hazel were ultrasonically activated. Dihydroacid alkaloids were added and subjected to ultrasonic encapsulation and aging; a compound anti-inflammatory and soothing composition of dihydroacid alkaloids and plant vesicles was obtained.

4. The method for preparing the dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition according to claim 3, characterized in that, The conditions for ultrasonic activation are: ultrasonic treatment at 20-50℃ for 1-3 hours at 35-45kHz and 250-350W. The mass ratio of added dihydroaeroxan to fresh vesicles in the plant vesicle dispersion was 1g:(7-9)g; Ultrasonic encapsulation was carried out under conditions of pressure <0.01MPa and system temperature of 4-15℃; the conditions for ultrasonic encapsulation were: 35-45kHz, 250-350W, and ultrasonic treatment for 1-3h. The aging process involves placing the container at 4°C for 12-36 hours.

5. The method for preparing the dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition according to claim 3, characterized in that, Aging also includes centrifugation and concentration processes: Centrifuge at 10000-14000×g for 10-50 min; concentrate the supernatant using a PES ultrafiltration fiber membrane with a molecular weight cutoff of 100kDa until the vesicle particle concentration is ≥2.0×10⁻⁶. 11 particles / mL.

6. The method for preparing the dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition according to claim 3, characterized in that, The preparation methods for olive plant vesicle dispersions, chamomile plant vesicle dispersions, and witch hazel plant vesicle dispersions are as follows: Olive pulp, chamomile inflorescence, and witch hazel branches and leaves were added to water and crushed at low temperature; the crushed liquid was centrifuged in two stages at different speeds, and the supernatant was collected to obtain a plant vesicle dispersion.

7. The method for preparing the dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition according to claim 3, characterized in that, The mass-to-volume ratio of olive pulp, chamomile inflorescence, witch hazel branches and leaves to water was 1 g: (6-10) mL. The conditions for low-temperature crushing are: at 4-10℃, crushing is performed using a high-speed blender at 8000-12000 rpm for 4-8 minutes; The two-stage differential centrifugation conditions are as follows: First stage centrifugation: 4℃, 2500-3500×g, centrifugation for 10-30 min; Second stage centrifugation: 4℃, 10000-14000×g, centrifugation for 30-50 min.

8. The method for preparing the dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition according to claim 3, characterized in that, After compounding, the mixture was filtered sequentially through a 1.0μm PTFE membrane and then through a 0.22μm PES sterilization membrane to obtain a dihydrooat alkaloid compound plant vesicle anti-inflammatory and soothing composition.

9. The application of the dihydrooat alkaloid complex plant vesicle anti-inflammatory and soothing composition prepared by the method of any one of claims 1 or 2 or claims 3-8 in the preparation of anti-inflammatory and soothing products.

10. An anti-inflammatory and soothing product, characterized in that, The dihydroacid alkaloid complex plant vesicle anti-inflammatory and soothing composition is prepared by the method comprising the dihydroacid alkaloid complex plant vesicle anti-inflammatory and soothing composition according to claim 1 or 2 or any one of claims 3-8.

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