Method for testing permeability of olive leaf extract

Abstract

The invention discloses a method for testing the permeability of an olive leaf extract, which comprises the following steps: constructing an initial permeability model, inputting physical and chemical parameters of oleuropein, and predicting the theoretical permeability; carrying out a transdermal experiment on oleuropein by using the pretreated in-vitro animal skin, and measuring the experiment permeability; on the basis of the difference between the experimental permeability and the theoretical permeability, a diffusion coefficient parameter of the initial permeability model is inversely updated through a Bayesian calibration framework, and a second permeability model is generated; adjusting the skin parameters of the second permeation model, constructing a plurality of third permeation models, and calculating the permeability of oleuropein under different skins. Performing initial permeation model prediction and in-vitro animal skin transdermal experiment on the oleuropein respectively to obtain theoretical permeability and experimental permeability respectively, optimizing diffusion coefficient parameters of the initial permeation model according to the experimental permeability, and constructing a plurality of third permeation models corresponding to different skin types; and scientific prediction of the oleuropein permeability under different skin types is realized.

Description

Technical Field

[0001] The invention relates to the technical field of permeation testing, in particular to a method for testing the permeability of an olive leaf extract. Background Art

[0002] Olive leaves are the leaves of the olive tree, which belongs to the genus Oleaceae, and is an evergreen small tree native to the Mediterranean region and now widely planted in subtropical regions around the world. Olive leaves are rich in a variety of active ingredients, including oleuropein, hydroxytyrosol, flavonoids, phenolic compounds, etc. Because of the presence of these active ingredients, olive leaf extract is widely used in cosmetics, with soothing, anti-allergic, anti-inflammatory, whitening, anti-freckle, anti-oxidation and other effects, suitable for all skin types.

[0003] Existing mathematical models (such as QSAR models) are mainly designed for small molecule compounds with a molecular weight of less than 500 Da, assuming that the compounds can freely pass through the pores of the stratum corneum. However, the molecular weight of oleuropein, the active ingredient in olive leaf extract, is as high as 540 Da and contains multiple polar groups, which leads to systematic errors (predicted values ​​are 20-30% higher) in traditional models due to the failure to consider steric hindrance and polar retardation.

[0004] Therefore, it is necessary to provide a method for testing the permeability of olive leaf extract to solve the above-mentioned technical problems. Summary of the invention

[0005] The invention overcomes the shortcomings of the prior art and provides a method for testing the permeability of olive leaf extract.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for testing the permeability of olive leaf extract, comprising the following steps: S1. Construct an initial permeation model, input the physical and chemical parameters of oleuropein, and predict the theoretical permeability; S2. Using the pretreated ex vivo animal skin, conduct a transdermal experiment of oleuropein to determine the experimental permeability; S3, based on the difference between the experimental permeability and the theoretical permeability, the diffusion coefficient parameters of the initial permeability model are updated through the Bayesian calibration framework to generate a second permeability model; S4. Adjust the skin quality parameters of the second permeation model, construct multiple third permeation models, input the physicochemical parameters of oleuropein into each of the third permeation models, and calculate the permeability of oleuropein under different skin qualities.

[0007] In a preferred embodiment of the present invention, the construction of the initial permeation model includes: S11. Define the basic permeability coefficient based on quantitative structure-activity relationship and skin-PAMPA model : , Where D is the diffusion coefficient, K partition is the partition coefficient of stratum corneum / donor solution, and h is the thickness of stratum corneum; , MW is molecular weight (unit Da), log P is the octanol-water partition coefficient, HB is the total number of hydrogen bond donors / acceptors (such as hydroxyl groups, amino groups, etc.), V is the molecular volume, and a1, b1, c1, a2, b2, and c2 are the multivariate linear regression fitting coefficients (the initial values ​​are: a1 = −0.006, b1 = −0.0015, c1 = −3.8, a2 = 0.25, b2 = −,0.12, and c2 = −1.2).

[0008] S12, introduction of molecular volume blocking factor , the corrected diffusion coefficient D is D vol : , , in, is the pore radius, r is the oleuropein molecular radius; S13, defining polar retardation coefficient based on the number of polar groups , modified distribution coefficient K partition .

[0009] In a preferred embodiment of the present invention, in S13, the polarity retardation coefficient The calculation formula is: , where HBD is the number of hydrogen bond donors and HBA is the number of hydrogen bond acceptors; The correction formula of the distribution coefficient is: , Where e is the fitting coefficient, .

[0010] In a preferred embodiment of the present invention, the theoretical permeability of oleuropein is calculated using the constructed initial permeation model, comprising the following steps: S14. Substitute the physical and chemical parameters of oleuropein into the initial permeability model to calculate the initial permeability coefficient .

[0011] S15. Based on the steady-state diffusion model of Fick's first law and the initial permeability coefficient, the cumulative permeability Q is calculated: , where C0 is the initial concentration of oleuropein, t is the penetration time, and A is the skin contact area.

[0012] S16. Divide the cumulative permeation amount Q by the permeation time t to obtain the permeability per hour.

[0013] In a preferred embodiment of the present invention, the physicochemical parameters of oleuropein include: molecular weight, LogP, polar surface area, total number of hydrogen bond donors / acceptors and molecular volume.

[0014] In a preferred embodiment of the present invention, in S2, the ex vivo animal skin is pig back skin, and the pretreatment steps include: S21. After removing the hair, use sodium sulfide solution to remove the hair, and soak and disinfect with benzalkonium chloride; S22, trim subcutaneous fat, leaving skin thickness of 1.5±0.2mm; S23. Use a treatment solution containing collagenase type IV, saponin, and glycerin to treat the skin in stages with controlled temperature.

[0015] In a preferred embodiment of the present invention, in S23, the temperature control stage includes: Enzyme activation stage: incubate at 35-40℃ with shaking for 10-20min; Lipid removal stage: 41-45°C for 5-15 min; Glycerol infiltration stage: incubate at 3-5°C for 10-30 minutes.

[0016] In a preferred embodiment of the present invention, the treatment solution is prepared by dissolving 0.6-1.2 U / mL collagenase type IV, 2.0-3.0 mg / mL saponin, 80-120 mg / mL glycerol, 1.5-2.5 mg / mL BSA and 0.8-1.2 mg / mL trehalose in HBSS solution. The specific preparation process includes: 1) Take 800 mL of pre-cooled HBSS (containing 0.02% gentamicin, i.e. 0.2 mg / mL), pH 7.4.

[0017] 2) Gradient addition of corresponding ingredients: Glycerol: Add 64-96 mL of glycerol and stir to mix; BSA: weigh 1.5-2.5g BSA and add slowly, avoiding vortexing and foaming; Trehalose: Dissolve 0.8-1.2g trehalose and stir magnetically until transparent; Saponin: add 2.0-3.0g saponin and dissolve in 40℃ water bath for 10 minutes; Collagenase: Weigh the enzyme powder according to the measured specific activity, dissolve it, filter and sterilize it, then add it.

[0018] 3) Add HBSS to 1 L and filter through a 0.22 μm filter membrane.

[0019] In a preferred embodiment of the present invention, the transdermal experiment uses a Franz diffusion cell, which specifically includes: The receiving solution is PBS, and the receiving solution is kept at a constant temperature of 31-32°C and a stirring speed of 100-300rpm; The donor pool oleuropein concentration was 10-100 μg / mL; The sampling time points were 0, 1, 2, 4, 6, 8, 10, 12, and 24 h.

[0020] In a preferred embodiment of the present invention, the calculation of the experimental permeability includes: S27, according to the concentration of oleuropein in the receiving solution C n , calculate the cumulative permeability Q n : , Where A is the effective penetration area; S28. Fitting Q by linear regression n The slope of the -t curve gives the permeability.

[0021] In a preferred embodiment of the present invention, the Bayesian calibration in S3 specifically includes: S31. Define the lognormal prior distribution of the diffusion coefficient D: ; S32. Constructing likelihood function , where the experimental data follows a heteroskedastic normal distribution; S33. Sampling the posterior distribution via Markov Chain Monte Carlo , take the posterior mean to update the diffusion coefficient.

[0022] In a preferred embodiment of the present invention, in S4, the skin quality parameters include: The thickness of the stratum corneum, h, can be adjusted in the range of 10-30 μm; Lipid content L, adjustment range is 4-17%; Among them, the partition coefficient is further adjusted based on lipid content: , in, , L 默认 =10%, β is the lipid sensitivity coefficient.

[0023] In a preferred embodiment of the present invention, the skin types include: dry skin, normal skin, oily skin and sensitive skin.

[0024] In a preferred embodiment of the present invention, the parameter combinations for different skin types include: Dry skin: stratum corneum thickness 18-22 μm, lipid content 4-6%; Normal skin: stratum corneum thickness 14-16 μm, lipid content 9-11%; Oily skin: stratum corneum thickness 10-12 μm, lipid content 14-17%; Sensitive skin: stratum corneum thickness 16-19μm, lipid content 7-9%.

[0025] In a preferred embodiment of the present invention, the method is applicable to the development of cosmetics or medicines, and the requirements of oleuropein permeability of different skin types can be adapted by adjusting the concentration of penetration enhancer or the type of carrier in the formula.

[0026] The present invention solves the defects existing in the background technology and has the following beneficial effects: (1) The present invention provides a method for testing the permeability of olive leaf extract, wherein an initial permeability model prediction and an in vitro animal skin permeation experiment are performed on oleuropein to obtain a theoretical permeability and an experimental permeability, respectively. The diffusion coefficient parameters of the initial permeability model are optimized according to the experimental permeability, and a plurality of third permeability models corresponding to different skin types are constructed, thereby achieving a scientific prediction of the permeability of oleuropein under different skin types. This method not only solves the problem that the existing data model is not suitable for the permeability test of oleuropein with a large molecular weight and multiple polar groups, but also can quickly and comprehensively evaluate the permeability of oleuropein under different skin types.

[0027] (2) The present invention uses a Bayesian calibration framework to reversely map the experimentally obtained permeation data to the diffusion coefficient in the initial permeation model. This process closely combines the initial model that originally relied solely on data prediction with the actual transdermal experimental verification data, thereby generating a second permeation model that integrates theory and practice. This significantly improves the prediction accuracy of the permeation model and solves the problem that the existing mathematical model is not applicable to oleuropein permeability testing.

[0028] (3) The present invention aims to adjust the barrier properties of ex vivo pig skin to a level close to that of human skin by subjecting it to a series of pretreatment steps. Specifically, collagenase treatment is used during the treatment process to accurately calibrate the stratum corneum porosity of ex vivo pig skin; at the same time, saponin is used to effectively remove excess lipids. On this basis, the transepidermal water loss value of ex vivo pig skin is further reduced by the application of glycerol penetration and cryoprotection technology, making it close to the actual level of human skin. This series of treatment measures ensures that the deviation between the permeability measurement results of oleuropein and the actual value of the human body is less than 10%, thereby significantly improving the accuracy of the transdermal experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work. Figure 1 is a flow chart of a permeability testing method according to a preferred embodiment of the present invention; Figure 2 1 is a diagram of a method for pretreating in vitro animal skin according to a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0032] In addition, the terms "first", "second", etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, a feature defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0033] like Figure 1 As shown, the present invention provides a method for testing the permeability of olive leaf extract, comprising the following steps: S1. Construct an initial permeation model, input the physicochemical parameters of oleuropein, and predict the theoretical permeability of oleuropein; S2. Using the pretreated ex vivo animal skin, a transdermal experiment of oleuropein was performed to determine the experimental permeability of oleuropein; S3, based on the difference between the experimental permeability and the theoretical permeability, the diffusion coefficient parameters of the initial permeability model are updated through the Bayesian calibration framework to generate a second permeability model; S4. Adjust the skin quality parameters in the second permeation model, construct multiple third permeation models, input the physicochemical parameters of oleuropein into each of the third permeation models, and calculate the permeability of oleuropein under different skin qualities.

[0034] It is worth noting that oleuropein in olive leaf extract is a secoiridoid glycoside compound with a large molecular weight and a complex glycoside structure. Specifically, it has a large molecular weight and contains multiple polar groups. Existing mathematical models are usually based on small molecule compounds with a molecular weight of less than 500 Da (such as traditional drugs or cosmetic active ingredients), while oleuropein has a molecular weight of up to 540 Da, and its diffusion rate across the stratum corneum is significantly reduced, resulting in a systematic error in the diffusion coefficient of the existing mathematical model due to the failure to consider the steric hindrance effect of the macromolecules. In other words, it will lead to a high prediction result and cannot be applied to the permeability test of oleuropein.

[0035] The present invention performs initial permeation model prediction and in vitro animal skin transdermal experiment on oleuropein, respectively, obtains theoretical permeability and experimental permeability, optimizes the diffusion coefficient parameter of the initial permeation model according to the experimental permeability, and constructs a plurality of third permeation models corresponding to different skin qualities, thereby achieving scientific prediction of the permeability of oleuropein under different skin qualities. It not only solves the problem that the existing data model is not suitable for the permeability test of oleuropein with a large molecular weight and containing multiple polar groups, but also can quickly and comprehensively evaluate the permeability of oleuropein under different skin qualities. These prediction results provide important references for the application of olive leaf extract in the fields of cosmetics, medicines, etc., help developers to formulate product formulas and usage plans more accurately, meet the needs of people with different skin qualities, and promote in-depth research and wide application of olive leaf extract.

[0036] The above steps are described in detail below.

[0037] Step S1, constructing an initial permeability model, and inputting the physicochemical parameters of oleuropein to predict the theoretical permeability of oleuropein.

[0038] The construction of the initial penetration model includes: S11. Using the quantitative structure-activity relationship (QSAR) model as the core framework and combining the skin permeation prediction algorithm of the skin-PAMPA model, a basic permeation model is constructed: , , in, is the basic permeability coefficient, in cm / h, representing the permeation rate per unit time and unit concentration gradient; D is the diffusion coefficient, K partition is the partition coefficient of stratum corneum / donor solution, and h is the thickness of stratum corneum; MW is molecular weight (unit Da), log P is the octanol-water partition coefficient, HB is the total number of hydrogen bond donors / acceptors (such as hydroxyl groups, amino groups, etc.), V is the molecular volume, and a1, b1, c1, a2, b2, and c2 are the multivariate linear regression fitting coefficients (the initial values ​​are: a1 = −0.006, b1 = −0.0015, c1 = −3.8, a2 = 0.25, b2 = −,0.12, and c2 = −1.2).

[0039] S12. According to the large molecular weight characteristics of oleuropein, the basic permeation model is modified; The molecular size directly affects the diffusion coefficient through steric hindrance. The basic permeation model assumes that the compound freely passes through the stratum corneum pores (the default pore radius is ), but when the oleuropein molecular radius r is close to the pore size, a barrier factor needs to be introduced: , which quantifies the degree to which molecular size inhibits diffusion rate (when , the blocking factor is 0 and the penetration stops); Based on the molecular volume V m（ obtained by molecular dynamics simulation): , unit: nm; For oleuropein (V m =620Å 3 =0.62nm 3 ): ; Introducing the barrier factor into the diffusion coefficient D, we get the diffusion coefficient D after molecular volume correction. vol : ; S13. Based on the number of hydroxyl groups and glycosidic bonds of oleuropein, define the polar retardation coefficient: , where HBD is the number of hydrogen bond donors and HBA is the number of hydrogen bond acceptors; Correction of the distribution coefficient according to the polarity retardation coefficient: , where e is the fitting coefficient (e=-2.1), is the experimental calibration coefficient, which is obtained by fitting the permeation data of oleuropein and glycoside compounds with similar structures (such as paeoniflorin and gardeniaside); S14. Integrate the corrected diffusion coefficient and distribution coefficient to obtain the initial permeability coefficient: .

[0040] Through the above steps, the basic permeation model is corrected by molecular volume and polar groups to obtain the initial permeation model.

[0041] It is worth mentioning that the physical and chemical parameters of oleuropein include: molecular weight (MW), LogP, polar surface area (PSA), total number of hydrogen bond donors / acceptors (HB) and molecular volume (V m ).

[0042] Further, the theoretical permeability of oleuropein was calculated using the constructed initial permeation model, including the following steps: S14. Substitute the physical and chemical parameters of oleuropein into the initial permeability model to calculate the initial permeability coefficient .

[0043] S15. Based on the steady-state diffusion model of Fick's first law and the initial permeability coefficient, the cumulative permeability Q is calculated: , where C0 is the initial concentration of oleuropein, t is the penetration time, and A is the skin contact area.

[0044] S16. Divide the cumulative permeation amount Q by the permeation time t to obtain the permeability per hour.

[0045] Through the above steps, an initial permeation model for the characteristics of oleuropein is obtained. By inputting the physical and chemical parameters of oleuropein, the permeability of oleuropein can be predicted, which is convenient and quick.

[0046] Step S2: using the pretreated ex vivo animal skin to conduct a transdermal experiment of oleuropein and determine the experimental permeability of oleuropein.

[0047] Compared with the thickness of human skin (forearm: stratum corneum 17μm, epidermis 36μm, full thickness skin 1.5mm), the ex vivo animal skin commonly used in transdermal experiments includes: pig (back: stratum corneum 26μm, epidermis 66μm, full thickness skin 3.4mm), pig (ear: stratum corneum 10μm, epidermis 50μm, full thickness skin 1.3mm), mouse (back: stratum corneum 5μm, epidermis 13μm, full thickness skin 0.8mm), rat (stratum corneum 18μm, epidermis 32μm, full thickness skin 2.09mm), etc. Among them, pig skin is not only rich in skin blood vessels compared with human skin, but also has a higher fat content in the body. The distribution of fat-soluble compounds in the body is different from that of humans, and the transdermal permeability of some compounds is higher than that of human skin; while the structure of rat skin is quite different from that of human skin, with more skin appendages, thinner stratum corneum and epidermis, and higher transdermal absorption of drugs than humans.

[0048] In this embodiment, the ex vivo animal skin is preferably pig back skin, which is pretreated before conducting the oleuropein transdermal experiment.

[0049] It is worth noting that if Figure 2As shown, the pretreatment process of pig back skin includes the following steps: S21. Take the skin 5 cm on both sides of the spine of an adult white pig (60-80 kg), remove the hair on the surface, apply 8% sodium sulfide solution evenly, rinse with water after 5 minutes, then soak in 0.1% benzalkonium chloride solution for 10 minutes, and wipe dry with sterile gauze; The fat was carefully cut off along the dermis-subcutaneous fat junction, leaving the full-thickness skin with a thickness of 1.5±0.2mm and trimmed into 3 3cm 2 Square leather pieces with smooth edges.

[0050] S22. Preparation of treatment solution: Dissolve 0.6-1.2 U / mL collagenase type IV, 2.0-3.0 mg / mL saponin, 80-120 mg / mL glycerol, 1.5-2.5 mg / mL BSA and 0.8-1.2 mg / mL trehalose in HBSS solution. The specific preparation process includes: 1) Take 800 mL of pre-cooled HBSS (containing 0.02% gentamicin, i.e. 0.2 mg / mL), pH 7.4.

[0051] 2) Gradient addition of corresponding ingredients: Glycerol: Add 64-96 mL of glycerol and stir to mix; BSA: weigh 1.5-2.5g BSA and add slowly, avoiding vortexing and foaming; Trehalose: Dissolve 0.8-1.2g trehalose and stir magnetically until transparent; Saponin: add 2.0-3.0g saponin and dissolve in 40℃ water bath for 10 minutes; Collagenase: Weigh the enzyme powder according to the measured specific activity, dissolve it, filter and sterilize it, then add it.

[0052] 3) Add HBSS to 1 L and filter through a 0.22 μm filter membrane.

[0053] S23, fix the pig skin epidermis side up on the customized bracket, add pre-cooled mixed treatment solution (0-6°C), the liquid surface completely covers the skin, and perform stage temperature control; Enzyme activation stage: incubate at 35-40°C with shaking (30 rpm) for 15 minutes to activate collagenase to degrade stratum corneum collagen; Lipid removal stage: raise the temperature to 41-45°C and maintain for 10 minutes to accelerate lipid dissolution; Glycerol penetration stage: cool to 4°C and let stand for 20 minutes to promote glycerol penetration into the loosened lipid layer.

[0054] The treated pig skin was transferred to pre-cooled HBSS (containing 5% fetal bovine serum) and rinsed twice at 2-5°C to remove residual saponin and enzyme.

[0055] S24, cool to -70℃ at a rate of -1℃ / min to prevent ice crystals from destroying the lipid structure protected by glycerol, then thaw in a water bath at 25-40℃ and immerse in HBSS for 30 minutes. It is worth noting that the resistance value after thawing needs to be greater than 19kΩ·cm².

[0056] By pre-treating the pig skin, specifically: collagenase-saponin combined treatment makes the porosity and lipid composition close to those of human skin, while 10% glycerol protective agent maintains the integrity of the lipid membrane. The resistance value after thawing is stable at >19kΩ·cm². Through the above treatment, the permeation behavior of pig skin can be systematically calibrated to a level close to that of human skin, significantly improving the in vitro and in vivo correlation of in vitro test data, providing more reliable raw material support for subsequent oleuropein transdermal experiments.

[0057] Further, the transdermal experiment of oleuropein specifically includes the following steps: S25, fixing the pretreated pig skin sample in a Franz diffusion cell, with the skin stratum corneum facing upward and the dermis side facing the receiving cell, ensuring that the skin is in full contact with the receiving solution; Add 5 mL of PBS (pH 7.4) as the receiving solution to the receiving cell of the Franz diffusion cell, making sure there are no bubbles in the receiving solution; Different concentrations of oleuropein solutions (10 μg / mL, 50 μg / mL, and 100 μg / mL) were added to the donor pool, and the initial time was recorded.

[0058] S26. Place the Franz diffusion cell on a constant temperature magnetic stirrer, maintain the temperature at 31-32°C and the stirring speed at 300 r / min to simulate the flow of blood and tissue fluid under human skin; At predetermined time points (0, 10, 20, 30 min and 1, 2, 3, 4, 6, 8, 10, 12 h), 0.5 mL of sample was taken from the receiving pool, and then, an equal amount of fresh receiving solution was immediately added; The sampled receiving solution was filtered through a 0.45 μm microporous filter membrane, and the concentration of oleuropein was determined using HPLC.

[0059] S27. Calculate the cumulative penetration of oleuropein per unit area Q n and the cumulative penetration percentage Q e : , , Among them, C n is the oleuropein concentration in the receiving pool at the nth time point, A is the effective skin area for penetration, and W is the total amount of oleuropein; With time t as the horizontal axis, Q n and Qe As the vertical axis, draw the penetration curve.

[0060] S28. Calculate the permeability of oleuropein according to the permeation curve drawn in step S27: , where J is the permeability, Q n The slope of the -t curve is recorded as the experimental permeability.

[0061] The present invention aims to adjust the barrier properties of ex vivo pig skin to a level close to that of human skin by subjecting it to a series of pretreatment steps. Specifically, collagenase treatment is used during the treatment process to accurately calibrate the stratum corneum porosity of ex vivo pig skin; at the same time, saponin is used to effectively remove excess lipids. On this basis, the transepidermal water loss value of ex vivo pig skin is further reduced by the application of glycerol penetration and cryoprotection technology, making it close to the actual level of human skin. This series of treatment measures ensures that the deviation between the permeability measurement results of oleuropein and the actual value of the human body is less than 10%, thereby significantly improving the accuracy of the transdermal experiment.

[0062] S3. Based on the difference between the experimental permeability and the theoretical permeability, the diffusion coefficient parameters of the initial permeability model are updated through the Bayesian calibration framework to generate a second permeability model.

[0063] Specifically, the posterior distribution of the diffusion coefficient D is updated by Bayesian inference: S31. Assume that the diffusion coefficient D obeys the log-normal distribution and define the prior distribution: ; The lognormal distribution ensures that D>0, which is consistent with physical meaning (the diffusion coefficient is positive); μ=logD 初始 It means that the initial model prediction value is the center; Reflects the uncertainty in the initial value (approximately ±50% variation).

[0064] S32, assuming experimental penetration data Q n (t) obeys heteroscedastic normal distribution (the error increases with the increase of penetration), and constructs the likelihood function: , in, is the initial infiltration model prediction value, is the standard deviation of the transdermal experiment, estimated by repeated experiments.

[0065] S33. Use the Markov Chain Monte Carlo (MCMC) method to sample the posterior distribution: , Specifically: Bayes' theorem converts the posterior distribution of the diffusion coefficient D into Decomposed into likelihood function and the prior distribution The product of Prior distribution : The diffusion coefficient D setting of the initial permeability model; Likelihood function : When the diffusion coefficient D is given, the experimental data Q n Probability of occurrence; Posterior distribution : Updated probability distribution of the diffusion coefficient D after combining the experimental data.

[0066] S34, take the mean μ of the posterior distribution post As the updated diffusion coefficient D new =exp(μ post ), and then generate the second penetration model.

[0067] By using the Bayesian calibration framework, the experimental permeation data were reverse mapped to the diffusion coefficient D in the initial permeation model. This process closely combined the initial model, which originally relied solely on data prediction, with the actual transdermal experimental verification data, thereby generating a second permeation model that integrates theory and practice. This significantly improved the prediction accuracy of the permeation model and solved the problem of the existing mathematical model's inapplicability to oleuropein permeability testing.

[0068] S4. Adjust the skin quality parameters in the second permeation model to construct multiple third permeation models, input the physicochemical parameters of oleuropein into each of the third permeation models, and calculate the permeability of oleuropein under different skin qualities.

[0069] Skin quality parameters include: stratum corneum thickness and lipid content. Lipids are the main component of the stratum corneum barrier. The higher the lipid content, the greater the penetration resistance of polar molecules.

[0070] The stratum corneum thickness h is directly replaced by the set value of the corresponding skin type.

[0071] Based on the lipid content L, the partition coefficient is adjusted: , in, , L 默认 =10%, β is the lipid sensitivity coefficient, β=0.3.

[0072] Human skin types include dry skin, oily skin, neutral skin and sensitive skin. Different skin types have differences in stratum corneum thickness, water content, sebum secretion, etc. These factors will affect the penetration effect of cosmetics / drugs.

[0073] According to the target application scenario, different skin quality parameter combinations are set, as shown in Table 1: Table 1. Combination of skin quality parameters

[0074] For each skin type, the corresponding parameters of the second permeation model are adjusted respectively to obtain the third permeation model.

[0075] The physical and chemical parameters of oleuropein are input into each third permeation model respectively, and the permeability of oleuropein on different skin types can be calculated.

[0076] Step S4 constructed a multi-skin permeation model (third permeation model) by adjusting the stratum corneum thickness and lipid content, and quantified the effect of different skin types on the permeation behavior of oleuropein.

[0077] Next, the effect of the third permeation model constructed by the method of the present invention is verified.

[0078] 1) The skin type of the volunteers was determined by dermatologist diagnosis or standardized questionnaire, and was divided into dry, neutral, oily and sensitive skin. The inner forearm was taken and the thickness and barrier function were standardized. At least 6 ex vivo skin samples were collected in each group.

[0079] 2) Transdermal experiments on ex vivo skin samples: Donor solution: 2% (w / v) oleuropein solution (solvent: 30% ethanol-water, pH 5.5); Franz Diffusion Cell Setup: Effective diffusion area: 0.64cm²; Receiving solution: pH 7.4 PBS (containing 0.01% NaN3 for antibacterial effect); Temperature: 37±0.5℃; Magnetic stirring speed: 600rpm; At 1, 2, 4, 6, 8, and 24 hours after the start of the test, 200 μL of sample was taken each time (with an equal amount of fresh receiving solution added).

[0080] 3) Perform HPLC analysis on the sampled receiving liquid: Chromatographic conditions: C18 column, mobile phase acetonitrile-water (25:75), flow rate 1.0 mL / min, detection wavelength 280 nm; Limit of quantitation (LOQ): 0.1 μg / mL; intra-day precision RSD < 5%.

[0081] 4) Obtain the input parameters of the third penetration model, including: Physicochemical parameters of oleuropein: molecular weight (540 Da), logP (1.2), molecular volume (620 ų), number of polar groups (7); The measured values ​​of skin quality parameters are shown in Table 2: Table 2. Measured skin quality parameters

[0082] 5) Input the parameters obtained in step (4) into the third permeation model and calculate the 24-hour permeation volume as shown in Table 3: Table 3. Comparison of penetration

[0083] The average relative error between the predicted value using the third permeability model and the measured value of the transdermal experiment was -5.1%, with no significant difference. Through in vitro experiments, the prediction error of the third permeability model for oleuropein permeability of different skin types was <8%, which was significantly better than the traditional QSAR model (error> 20%), proving the high accuracy and practicality of the method of the present invention.

[0084] The present invention can be used in the formulation development of cosmetics (such as anti-inflammatory essences, moisturizing creams) and transdermal drugs (such as local analgesic patches). By predicting the penetration behavior of active ingredients under different skin types, it can guide the addition amount of penetration enhancers and the selection of carrier types (such as liposomes, nanoemulsions), thereby improving product efficacy and safety.

[0085] The method of the present invention is not only applicable to oleuropein, the main component of olive leaf extract, but can also be extended to the permeability test of other active ingredients in the extract (such as hydroxytyrosol, flavonoids, phenolic compounds, etc.). According to the physicochemical properties of different components (such as molecular weight, LogP, polar surface area, number of hydrogen bond donors / acceptors and molecular volume, etc.), by adjusting the input parameters in the initial permeation model and using the same Bayesian calibration framework and transdermal experimental process, the accurate prediction of multi-component permeation behavior can be achieved. For example: Hydroxytyrosol (molecular weight 154Da, LogP0.8): Its small molecule characteristics can reduce the steric hindrance effect. The molecular volume blocking factor correction needs to be turned off in the model, and the distribution coefficient is optimized only by the polar retardation coefficient.

[0086] Flavonoids (such as luteolin, molecular weight 286Da, LogP2.5): It is necessary to introduce molecular shape factors in the diffusion coefficient correction in combination with their planar molecular structure characteristics.

[0087] Phenolic compounds (such as tyrosol, molecular weight 138Da, LogP1.2): Due to their high polarity, the influence of the polar retardation coefficient needs to be strengthened in the distribution coefficient correction.

[0088] By inputting the physicochemical parameters of different ingredients and calibrating the model based on experimental data, this method can comprehensively evaluate the permeation behavior of multiple ingredients in olive leaf extract and provide data support for the synergistic effect analysis of compound formulas.

[0089] The above is based on the ideal embodiment of the present invention. Through the above description, relevant personnel can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of the present invention is not limited to the content in the specification, and the technical scope must be determined according to the scope of the claims.

Claims

1. A method for testing the permeability of olive leaf extract, characterized in that: The following steps are involved: S1. Construct an initial permeation model, input the physical and chemical parameters of oleuropein, and predict the theoretical permeability; S2. Using the pretreated ex vivo animal skin, conduct a transdermal experiment of oleuropein to determine the experimental permeability; S3, based on the difference between the experimental permeability and the theoretical permeability, the diffusion coefficient parameters of the initial permeability model are updated through the Bayesian calibration framework to generate a second permeability model; S4. Adjust the skin quality parameters of the second permeation model, construct multiple third permeation models, input the physicochemical parameters of oleuropein into each of the third permeation models, and calculate the permeability of oleuropein under different skin qualities.

2. A method for testing the permeability of olive leaf extract according to claim 1, characterized in that: The construction of the initial penetration model includes: S11. Define the basic permeability coefficient based on quantitative structure-activity relationship and skin-PAMPA model : ， Where D is the diffusion coefficient, K partition is the partition coefficient of stratum corneum / donor solution, and h is the thickness of stratum corneum; S12, introduction of molecular volume blocking factor , the corrected diffusion coefficient D is D vol : ， ， in, is the pore radius, r is the oleuropein molecular radius; S13, defining polar retardation coefficient based on the number of polar groups , modified distribution coefficient K partition .

3. A method for testing the permeability of olive leaf extract according to claim 2, characterized in that: In S13, the polarity retardation coefficient The calculation formula is: , where HBD is the number of hydrogen bond donors and HBA is the number of hydrogen bond acceptors; The correction formula of the distribution coefficient is: ， Among them, e is the fitting coefficient, .

4. A method for testing the permeability of olive leaf extract according to claim 1, characterized in that: The physicochemical parameters of oleuropein include: molecular weight, LogP, polar surface area, total number of hydrogen bond donors / acceptors and molecular volume.

5. A method for testing the permeability of olive leaf extract according to claim 1, characterized in that: In S2, the ex vivo animal skin is pig back skin, and the pretreatment steps include: S21. After removing the hair, use sodium sulfide solution to remove the hair, and soak and disinfect with benzalkonium chloride; S22, trim subcutaneous fat, leaving skin thickness of 1.5±0.2mm; S23. Use a treatment solution containing collagenase type IV, saponin, and glycerin to treat the skin in stages with controlled temperature.

6. A method for testing the permeability of olive leaf extract according to claim 5, characterized in that: In S23, the temperature control stage includes: Enzyme activation stage: incubate at 35-40℃ with shaking for 10-20min; Lipid removal stage: 41-45°C for 5-15 min; Glycerol infiltration stage: incubate at 3-5°C for 10-30 minutes.

7. A method for testing the permeability of olive leaf extract according to claim 1, characterized in that: The transdermal experiment uses a Franz diffusion cell, which specifically includes: The receiving solution is PBS, and the receiving solution is kept at a constant temperature of 31-32°C and a stirring speed of 100-300rpm; The donor pool oleuropein concentration was 10-100 μg / mL; The sampling time points were 0, 1, 2, 4, 6, 8, 10, 12, and 24 h.

8. The method for testing the permeability of olive leaf extract according to claim 1, characterized in that: The calculation of the experimental permeability includes: S27, according to the concentration of oleuropein in the receiving solution C n , calculate the cumulative permeability Q n : ， Where A is the effective penetration area; S28. Fitting Q by linear regression n The slope of the -t curve gives the permeability.

9. The method for testing the permeability of olive leaf extract according to claim 1, characterized in that: The Bayesian calibration in S3 specifically includes: S31. Define the lognormal prior distribution of the diffusion coefficient D: ; S32. Constructing likelihood function , where the experimental data follows a heteroskedastic normal distribution; S33. Sampling the posterior distribution via Markov Chain Monte Carlo , take the posterior mean to update the diffusion coefficient.

10. The method for testing the permeability of olive leaf extract according to claim 1, characterized in that: In S4, the skin quality parameters include: The thickness of the stratum corneum, h, can be adjusted in the range of 10-30 μm; Lipid content L, adjustment range is 4-17%; Among them, the partition coefficient is further adjusted based on lipid content: , in, , L 默认 =10%, β is the lipid sensitivity coefficient.

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