A method for testing the permeability of olive leaf extract

Abstract

The present invention discloses a method for testing the permeability of olea europaea leaf extract, including: constructing an initial permeability model, inputting the physical and chemical parameters of oleuropein, and predicting the theoretical permeability; using pre-treated ex vivo animal skin to conduct a transdermal experiment on oleuropein to measure the experimental permeability; based on the difference between the experimental permeability and the theoretical permeability, inversely updating the diffusion coefficient parameter of the initial permeability model through a Bayesian calibration framework to generate a second permeability model; adjusting the skin texture parameters of the second permeability model to construct multiple third permeability models, and calculating the permeability of oleuropein under different skin textures. Respectively conducting the initial permeability model prediction on oleuropein and the transdermal experiment on ex vivo animal skin to obtain the theoretical permeability and the experimental permeability respectively, optimizing the diffusion coefficient parameter of the initial permeability model according to the experimental permeability, and constructing multiple third permeability models corresponding to different skin textures, thus realizing the scientific prediction of the permeability of oleuropein under different skin textures.

Description

Technical Field

[0001] The present invention relates to the technical field of penetration testing, and particularly to a method for testing the penetration rate of olive leaf extract. Background Art

[0002] Olive leaves are the leaves of the olive tree. The olive tree belongs to the Olea genus of the Oleaceae family and is an evergreen small tree. It is native to the Mediterranean region and is now widely planted in subtropical regions around the world. Olive leaves are rich in various active ingredients, mainly including oleuropein, hydroxytyrosol, flavonoid compounds, phenolic compounds, etc. Due to the presence of these active ingredients, olive leaf extract is widely used in cosmetics and has effects such as soothing and anti-allergic, anti-inflammatory, whitening and freckle removal, and antioxidant, and is suitable for various skin types.

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

[0004] Therefore, it is necessary to provide a method for testing the penetration rate of olive leaf extract to solve the above technical problems. Summary of the Invention

[0005] The present invention overcomes the deficiencies of the prior art and provides a method for testing the penetration rate of olive leaf extract.

[0006] To achieve the above object, the technical solution adopted by the present invention is: A method for testing the penetration rate of olive leaf extract, comprising the following steps:

[0007] S1. Construct an initial penetration model, input the physicochemical parameters of oleuropein, and predict the theoretical penetration rate;

[0008] S2. Use pre-treated ex vivo animal skin to conduct a transdermal experiment of oleuropein and measure the experimental penetration rate;

[0009] S3. Based on the difference between the experimental penetration rate and the theoretical penetration rate, inversely update the diffusion coefficient parameter of the initial penetration model through the Bayesian calibration framework to generate a second penetration model;

[0010] S4. Adjust the skin type parameters of the second penetration model, construct multiple third penetration models, input the physicochemical parameters of oleuropein into each third penetration model respectively, and calculate the penetration rate of oleuropein under different skin types.

[0011] In a preferred embodiment of the present invention, the construction of the initial penetration model includes:

[0012] S11. Define the basic permeability coefficient based on the quantitative structure-activity relationship and the skin-PAMPA model :

[0013] ,

[0014] where D is the diffusion coefficient, K partition is the partition coefficient of the stratum corneum / donor solution, and h is the thickness of the stratum corneum;

[0015] ,

[0016] MW is the 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, c2 are the multiple linear regression fitting coefficients (initial values: a1 = -0.006, b1 = -0.0015, c1 = -3.8, a2 = 0.25, b2 = -,0.12, c2 = -1.2).

[0017] S12. Introduce the molecular volume blocking factor , and correct the diffusion coefficient D to D vol :

[0018] , ,

[0019] where is the pore radius, and r is the oleuropein molecular radius;

[0020] S13. Define the polarity blocking coefficient based on the number of polar groups , and correct the partition coefficient K partition .

[0021] In a preferred embodiment of the present invention, in S13, the calculation formula of the polarity blocking coefficient is:

[0022] , where HBD is the number of hydrogen bond donors and HBA is the number of hydrogen bond acceptors;

[0023] The correction formula for the partition coefficient is:

[0024] ,

[0025] where e is the fitting coefficient, .

[0026] In a preferred embodiment of the present invention, the constructed initial penetration model is used to calculate the theoretical permeability of oleuropein, including the following steps:

[0027] S14. Substitute the physicochemical parameters of oleuropein into the initial penetration model to calculate the initial permeability coefficient. .

[0028] S15. Based on the steady-state diffusion model of Fick's first law and the initial permeability coefficient, calculate the cumulative penetration amount Q: , where C0 is the initial concentration of oleuropein, t is the penetration time, and A is the skin contact area.

[0029] S16. Divide the cumulative penetration amount Q by the penetration time t to obtain the permeability per hour.

[0030] 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.

[0031] In a preferred embodiment of the present invention, in S2, the ex vivo animal skin is porcine back skin, and its pretreatment steps include:

[0032] S21. After removing the hair, use a sodium sulfide solution for hair removal and soak in benzalkonium chloride for disinfection.

[0033] S22. Cut off the subcutaneous fat and retain the skin thickness of 1.5 ± 0.2 mm.

[0034] S23. Use a treatment solution containing type IV collagenase, saponin, and glycerol to perform temperature-controlled treatment on the skin in stages.

[0035] In a preferred embodiment of the present invention, in S23, the temperature control stages include:

[0036] Enzyme activation stage: Oscillate and incubate at 35 - 40 °C for 10 - 20 min.

[0037] Lipid removal stage: Maintain at 41 - 45 °C for 5 - 15 min.

[0038] Glycerol penetration stage: Let it stand at 3 - 5 °C for 10 - 30 minutes.

[0039] In a preferred embodiment of the present invention, the preparation of the treatment solution: Dissolve 0.6 - 1.2 U / mL type IV collagenase, 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 into HBSS solution. The specific preparation process includes:

[0040] 1) Take 800 mL of pre-cooled HBSS (containing 0.02% gentamicin, i.e., 0.2 mg / mL), pH 7.4.

[0041] 2) Gradually add the corresponding components:

[0042] Glycerol: Add 64 - 96 mL of glycerol and stir well to mix evenly;

[0043] BSA: Weigh 1.5 - 2.5 g of BSA and slowly add it, avoiding vortexing and foaming;

[0044] Trehalose: Dissolve 0.8 - 1.2 g of trehalose and stir magnetically until transparent;

[0045] Saponin: Add 2.0 - 3.0 g of saponin and assist in dissolving it in a 40°C water bath for 10 minutes;

[0046] Collagenase: Weigh the enzyme powder according to the measured specific activity, dissolve it, filter it through a bacteria - proof filter, and then add it.

[0047] 3) Make up the volume to 1 L with HBSS and filter it through a 0.22 - μm filter membrane.

[0048] In a preferred embodiment of the present invention, the transdermal experiment uses a Franz diffusion cell, which specifically includes:

[0049] The receiving solution is PBS, and the receiving solution is kept at a constant temperature of 31 - 32°C with a stirring speed of 100 - 300 rpm;

[0050] The concentration of oleuropein in the donor cell is 10 - 100 μg / mL;

[0051] The sampling time points are 0, 1, 2, 4, 6, 8, 10, 12, and 24 hours.

[0052] In a preferred embodiment of the present invention, the calculation of the experimental permeability includes:

[0053] S27. According to the concentration C of oleuropein in the receiving solution n , calculate the cumulative permeation amount Q n :

[0054] ,

[0055] where A is the effective permeation area;

[0056] S28. Obtain the permeability by linearly regressing and fitting the slope of the Q n -t curve.

[0057] In a preferred embodiment of the present invention, the Bayesian calibration in S3 specifically includes:

[0058] S31. Define the log - normal prior distribution of the diffusion coefficient D: ;

[0059] S32. Construct a likelihood function , where the experimental data follows a heteroscedastic normal distribution;

[0060] S33. Sample the posterior distribution through Markov chain Monte Carlo , and take the posterior mean to update the diffusion coefficient.

[0061] In a preferred embodiment of the present invention, in S4, the skin quality parameters include:

[0062] The stratum corneum thickness h, with an adjustment range of 10 - 30 μm;

[0063] The lipid content L, with an adjustment range of 4 - 17%;

[0064] Among them, the further adjustment of the partition coefficient based on the lipid content is: ,

[0065] Among them, , L 默认 = 10%, and β is the lipid sensitivity coefficient.

[0066] In a preferred embodiment of the present invention, the skin quality types include: dry skin, neutral skin, oily skin, and sensitive skin.

[0067] In a preferred embodiment of the present invention, the parameter combinations of different skin quality types include:

[0068] Dry skin: stratum corneum thickness 18 - 22 μm, lipid content 4 - 6%;

[0069] Neutral skin: stratum corneum thickness 14 - 16 μm, lipid content 9 - 11%;

[0070] Oily skin: stratum corneum thickness 10 - 12 μm, lipid content 14 - 17%;

[0071] Sensitive skin: stratum corneum thickness 16 - 19 μm, lipid content 7 - 9%.

[0072] In a preferred embodiment of the present invention, the method is applicable to the development of cosmetics or drugs, and adapts to the requirements of different skin qualities for the penetration rate of oleuropein by adjusting the concentration of the penetration enhancer or the type of carrier in the formula.

[0073] The present invention solves the defects in the background technology, and the present invention has the following beneficial effects:

[0074] (1) The present invention provides a method for testing the permeability of olive leaf extract. The initial penetration model of oleuropein is predicted and the transdermal experiment on ex vivo animal skin is carried out respectively to obtain the theoretical permeability and the experimental permeability. The diffusion coefficient parameter of the initial penetration model is optimized according to the experimental permeability, and multiple third penetration models corresponding to different skin types are constructed, realizing the scientific prediction of the permeability of oleuropein under different skin types. It not only solves the problem that the existing data model is not applicable to the permeability test of oleuropein with a large molecular weight and multiple polar groups, but also can quickly and comprehensively evaluate the penetration of oleuropein under different skin types.

[0075] (2) By applying the Bayesian calibration framework, the present invention maps the penetration data obtained from experiments back to the diffusion coefficient in the initial penetration model. This process closely combines the initial model that originally relied only on data prediction with the actual transdermal experiment verification data, thus generating a second penetration model that integrates theory and practice, significantly improving the prediction accuracy of the penetration model, and solving the problem that the existing mathematical model is not applicable to the permeability test of oleuropein.

[0076] (3) By implementing a series of pretreatment steps on ex vivo pig skin, the present invention aims to adjust its barrier properties to a level similar to that of human skin. Specifically, during the treatment process, collagenase treatment is used to accurately calibrate the stratum corneum porosity of ex vivo pig skin; at the same time, saponins are used to effectively remove excess lipids. On this basis, by applying glycerol penetration and cryoprotection techniques, the transdermal water loss value of ex vivo pig skin is further reduced to be close to the actual level of human skin. This series of treatment measures ensures that the deviation between the measured result of the permeability of oleuropein and the true value of the human body is less than 10%, thus significantly improving the accuracy of the transdermal experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings;

[0078] Figure 1 It is a flow chart of the permeability test method of the preferred embodiment of the present invention;

[0079] Figure 2 It is a diagram of the pretreatment method of ex vivo animal skin of the preferred embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0080] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0081] Many specific details are set forth in the following description in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited by the specific embodiments disclosed below.

[0082] In addition, terms such as "first" and "second" are used only for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Therefore, features defined with "first", "second", etc. may explicitly or implicitly include one or more of such features. In the description of the present invention, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0083] As Figure 1 shown, the present invention provides a method for testing the permeability of olea europaea leaf extract, including the following steps:

[0084] S1. Construct an initial permeability model, input the physical and chemical parameters of oleuropein, and predict the theoretical permeability of oleuropein;

[0085] S2. Use the pretreated ex vivo animal skin to conduct a transdermal experiment on oleuropein, and measure the experimental permeability of oleuropein;

[0086] S3. Based on the difference between the experimental permeability and the theoretical permeability, inversely update the diffusion coefficient parameter of the initial permeability model through a Bayesian calibration framework to generate a second permeability model;

[0087] S4. Adjust the skin texture parameters in the second permeability model, construct multiple third permeability models, input the physical and chemical parameters of oleuropein into each third permeability model respectively, and calculate the permeability of oleuropein under different skin textures.

[0088] It should be noted that oleuropein in the olive leaf extract is a secoiridoid glycoside compound with a relatively large molecular weight and a complex glycoside structure. Specifically, it has a relatively large molecular weight and contains multiple polar groups. Existing mathematical models are usually established based on small molecule compounds with a molecular weight less than 500 Da (such as traditional drug or cosmetic active ingredients), while the molecular weight of oleuropein is as high as 540 Da, and its diffusion rate across the stratum corneum is significantly reduced, resulting in systematic errors in the diffusion coefficient of existing mathematical models due to the failure to consider the steric hindrance effect of macromolecules. That is to say, it will lead to a higher prediction result and cannot be applied to the permeability test of oleuropein.

[0089] In the present invention, the initial permeation model of oleuropein is predicted and the transdermal experiment on ex vivo animal skin is carried out respectively to obtain the theoretical permeability and the experimental permeability. The diffusion coefficient parameter of the initial permeation model is optimized according to the experimental permeability, and multiple third permeation models corresponding to different skin types are constructed, realizing the scientific prediction of the permeability of oleuropein under different skin types. It not only solves the problem that the existing data model is not applicable to the permeability test of oleuropein with a relatively large molecular weight and containing multiple polar groups, but also can quickly and comprehensively evaluate the permeation situation of oleuropein under different skin types. These prediction results provide important references for the application of olive leaf extract in the fields of cosmetics, pharmaceuticals, etc., help developers more accurately formulate product formulas and usage plans, meet the needs of people with different skin types, and promote the in-depth research and wide application of olive leaf extract.

[0090] The above steps will be described in detail below.

[0091] Step S1: Construct an initial permeation model, input the physicochemical parameters of oleuropein, and predict the theoretical permeability of oleuropein.

[0092] The construction of the initial permeation model specifically includes:

[0093] S11: Taking the quantitative structure-activity relationship (QSAR) model as the core framework and combining the skin permeation prediction algorithm of the skin-PAMPA model, construct a basic permeation model:

[0094] ,

[0095] ,

[0096] Among them, is the basic permeation coefficient, with the unit of cm / h, representing the permeation rate under a unit concentration gradient per unit time; D is the diffusion coefficient, K partition is the partition coefficient of the stratum corneum / donor solution, h is the thickness of the stratum corneum;

[0097] MW is the molecular weight (unit: Da), logP Kow 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, c2 are the coefficients of multiple linear regression fitting (initial values: a1 = -0.006, b1 = -0.0015, c1 = -3.8, a2 = 0.25, b2 = -0.12, c2 = -1.2).

[0098] S12. In view of the large molecular weight characteristics of oleuropein, the basic permeation model is corrected;

[0099] The molecular volume directly affects the diffusion coefficient through steric hindrance. The basic permeation model assumes that the compound freely passes through the pores of the stratum corneum (default pore radius ), but when the molecular radius r of oleuropein approaches the pore size, a blocking factor needs to be introduced:

[0100] , which quantifies the degree of inhibition of the molecular size on the diffusion rate (when , the blocking factor is 0 and the permeation stops);

[0101] Based on the molecular volume V m（ obtained through molecular dynamics simulation): , in nm;

[0102] For oleuropein (V m = 620 Å 3 = 0.62 nm 3 ): ;

[0103] The blocking factor is introduced into the diffusion coefficient D to obtain the diffusion coefficient D vol after correction by molecular volume:

[0104] ;

[0105] S13. According to the number of hydroxyl groups and glycosidic bonds of oleuropein, the polar retardation coefficient is defined:

[0106] , where HBD is the number of hydrogen bond donors and HBA is the number of hydrogen bond acceptors;

[0107] The partition coefficient is corrected according to the polar retardation coefficient:

[0108] , where e is the fitting coefficient (e = -2.1), is the experimentally calibrated coefficient, obtained by fitting the permeation data of oleuropein and glycoside compounds with similar structures (such as paeoniflorin, geniposide);

[0109] S14. Integrate the corrected diffusion coefficient and partition coefficient to obtain the initial permeability coefficient:

[0110] .

[0111] Through the above steps, the basic permeability model is corrected for molecular volume and polar groups to obtain the initial permeability model.

[0112] It should be noted that the physicochemical parameters of oleuropein specifically include: molecular weight (MW), LogP, polar surface area (PSA), total number of hydrogen bond donors / acceptors (HB), and molecular volume (V m ).

[0113] Furthermore, using the constructed initial permeability model to calculate the theoretical permeability of oleuropein includes the following steps:

[0114] S14. Substitute the physicochemical parameters of oleuropein into the initial permeability model to calculate the initial permeability coefficient .

[0115] S15. Based on the steady-state diffusion model of Fick's first law and the initial permeability coefficient, calculate the cumulative permeation amount Q: , where C0 is the initial concentration of oleuropein, t is the permeation time, and A is the skin contact area.

[0116] S16. Divide the cumulative permeation amount Q by the permeation time t to obtain the permeability per hour.

[0117] Through the above steps, an initial permeability model for the characteristics of oleuropein is obtained. By inputting the physicochemical parameters of oleuropein, the permeability of oleuropein can be predicted conveniently and quickly.

[0118] Step S2. Use the pretreated ex vivo animal skin to conduct a transdermal experiment on oleuropein and measure the experimental permeability of oleuropein.

[0119] Relative to the thickness of the human skin layer (forearm: stratum corneum 17μm, epidermis 36μm, full-thickness skin 1.5mm), the commonly used ex vivo animal skins in transdermal experiments include: 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, compared with human skin, pig skin not only has richer skin blood vessels and a higher body fat content, but also 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, there are more skin appendages, the stratum corneum and epidermis are thinner, and the percutaneous absorption of drugs is higher than that of humans.

[0120] In this embodiment, the ex vivo animal skin is preferably the back skin of a pig, which is pretreated and then the transdermal experiment of oleuropein is carried out.

[0121] It should be noted that as Figure 2 shown, the pretreatment process of the back skin of a pig includes the following steps:

[0122] S21: Take the skin at 5 cm on both sides of the spine on the back of an adult white pig (60 - 80 kg), remove the surface hair, evenly apply an 8% sodium sulfide solution, rinse with water after 5 minutes, then soak in a 0.1% benzalkonium chloride solution for 10 minutes, and dry with sterile gauze;

[0123] Carefully cut off the fat along the dermo - subcutaneous fat junction, retain the full - thickness skin thickness of 1.5 ± 0.2 mm, and trim it into a 3 2 3 cm square skin piece with smooth - trimmed edges.

[0124] S22: Preparation of the treatment solution: Dissolve 0.6 - 1.2 U / mL type IV collagenase, 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 into HBSS solution. The specific preparation process includes:

[0125] 1) Take 800 mL of pre - cooled HBSS (containing 0.02% gentamicin, i.e., 0.2 mg / mL) with pH 7.4.

[0126] 2) Gradually add the corresponding components:

[0127] Glycerol: Add 64 - 96 mL of glycerol and stir well;

[0128] BSA: Slowly add 1.5 - 2.5 g of BSA, avoiding vortexing and foaming.

[0129] Trehalose: Dissolve 0.8 - 1.2 g of trehalose and stir magnetically until transparent.

[0130] Saponin: Add 2.0 - 3.0 g of saponin and assist in dissolving in a 40 °C water bath for 10 minutes.

[0131] Collagenase: Weigh the enzyme powder according to the measured specific activity, dissolve it, filter it through a bacteria - proof filter, and then add it.

[0132] 3) Make up HBSS to 1 L and filter it through a 0.22 μm filter membrane.

[0133] S23. Fix the pig skin with the epidermal side up on a customized support, add the pre - cooled mixed treatment solution (0 - 6 °C), ensure that the liquid surface completely covers the skin, and perform stage - wise temperature control.

[0134] Enzyme activation stage: Incubate at 35 - 40 °C with shaking (30 rpm) for 15 minutes to activate the collagenase to degrade the collagen in the stratum corneum.

[0135] Lipid removal stage: Raise the temperature to 41 - 45 °C and maintain for 10 minutes to accelerate lipid dissolution.

[0136] Glycerol penetration stage: Lower the temperature to 4 °C and let it stand for 20 minutes to promote the penetration of glycerol into the loosened lipid layer.

[0137] Transfer the treated pig skin to pre - cooled HBSS (containing 5% fetal bovine serum), rinse it twice at 2 - 5 °C to remove the residual saponin and enzyme.

[0138] S24. Cool it to - 70 °C at a rate of - 1 °C / min, avoiding ice crystal damage to the lipid structure protected by glycerol. Then, thaw it in a 25 - 40 °C water bath and immerse it in HBSS for equilibration for 30 minutes. It should be noted that the resistance value after thawing should be > 19 kΩ·cm².

[0139] By pre - treating the pig skin, specifically: the combined treatment of collagenase - saponin makes the porosity and lipid composition close to that of human skin, while the 10% glycerol protectant maintains the integrity of the lipid membrane, and the resistance value after thawing is stably > 19 kΩ·cm². Through the above - mentioned treatment, the permeation behavior of pig skin can be systematically calibrated to be close to the level of human skin, significantly improving the in - vitro - in - vivo correlation of in - vitro test data, and providing more reliable raw material support for subsequent oleuropein transdermal experiments.

[0140] Furthermore, the transdermal experiment of oleuropein specifically includes the following steps:

[0141] S25. Fix the pre - treated pig skin sample in a Franz diffusion cell with the stratum corneum of the skin facing up and the dermal side facing the receiving cell, ensuring sufficient contact between the skin and the receiving solution.

[0142] Add 5 mL of PBS with a pH of 7.4 to the receiving pool of the Franz diffusion cell as the receiving solution, and ensure that there are no bubbles in the receiving solution;

[0143] Add oleuropein solutions with different concentrations (10 μg / mL, 50 μg / mL, 100 μg / mL) to the donor pool respectively, and record the initial time.

[0144] S26. Place the Franz diffusion cell on a thermostatic 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 the human skin;

[0145] At predetermined time points (0, 10, 20, 30 min and 1, 2, 3, 4, 6, 8, 10, 12 h), take 0.5 mL of samples from the receiving pool, and then immediately supplement an equal volume of fresh receiving solution;

[0146] Filter the sampled receiving solution through a 0.45 - μm microporous membrane, and use HPLC to determine the concentration of oleuropein.

[0147] S27. Calculate the cumulative permeation amount Q of oleuropein per unit area n and the cumulative permeation percentage Q e :

[0148] ,

[0149] ,

[0150] where C n is the concentration of oleuropein in the receiving pool at the nth time point, A is the effective skin area for permeation, and W is the total amount of oleuropein;

[0151] With time t as the abscissa, and Q n and Q e as the ordinates, plot the permeation curve.

[0152] S28. According to the permeation curve plotted in step S27, calculate the permeability of oleuropein: , where J is the permeability, is the slope of the Q n -t curve, denoted as the experimental permeability.

[0153] By implementing a series of pretreatment steps on ex vivo porcine skin, the present invention aims to adjust its barrier properties to a level similar to that of human skin. Specifically, during the treatment process, collagenase treatment is employed to precisely calibrate the stratum corneum porosity of ex vivo porcine skin; meanwhile, saponins are utilized to effectively remove excessive lipids. On this basis, through the application of glycerol permeation and cryoprotection techniques, the transdermal water loss value of ex vivo porcine skin is further reduced to approach the actual level of human skin. This series of treatment measures ensures that the deviation between the measured permeability of oleuropein and the true human value is less than 10%, thereby significantly improving the accuracy of the transdermal experiment.

[0154] S3. Based on the difference between the experimental permeability and the theoretical permeability, the diffusion coefficient parameter of the initial permeability model is inversely updated through a Bayesian calibration framework to generate a second permeability model.

[0155] Specifically, the posterior distribution of the diffusion coefficient D is updated through Bayesian inference:

[0156] S31. Assume that the diffusion coefficient D follows a lognormal distribution, and define the prior distribution:

[0157] ;

[0158] The lognormal distribution ensures that D > 0, which conforms to the physical meaning (the diffusion coefficient is a positive value);

[0159] μ = logD 初始 represents centered on the predicted value of the initial model;

[0160] reflects the uncertainty about the initial value (a variation range of approximately ±50%).

[0161] S32. Assume that the experimental permeation data Q n (t) follows a heteroscedastic normal distribution (the error increases with the increase in the permeation amount), and construct the likelihood function:

[0162] ,

[0163] where is the predicted value of the initial permeability model, is the standard deviation of the transdermal experiment error, estimated through repeated experiments.

[0164] S33. Use the Markov chain Monte Carlo (MCMC) method to sample the posterior distribution:

[0165] ,

[0166] Specifically: Bayes' theorem decomposes the posterior distribution of the diffusion coefficient D into the likelihood function and the prior distribution Product;

[0167] Prior distribution : Setting the diffusion coefficient D of the initial penetration model;

[0168] Likelihood function : Probability of the experimental data Q n appearing when the diffusion coefficient D is given;

[0169] Posterior distribution : Updated probability distribution of the diffusion coefficient D after combining the experimental data.

[0170] 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.

[0171] By applying the Bayesian calibration framework, the experimentally obtained penetration data is inversely mapped to the diffusion coefficient D in the initial penetration model. This process closely combines the initial model that originally relied only on data prediction with the actual transdermal experimental verification data, thereby generating a second penetration model that integrates theory and practice, significantly improving the prediction accuracy of the penetration model, and solving the problem of inapplicability of existing mathematical models to the permeability test of oleuropein.

[0172] S4. Adjust the skin texture parameters in the second penetration model to construct multiple third penetration models, input the physicochemical parameters of oleuropein into each third penetration model, and calculate the permeability of oleuropein under different skin textures.

[0173] Skin texture parameters include: stratum corneum thickness and lipid content. Among them, lipids are the main components of the stratum corneum barrier. The higher the lipid content, the greater the penetration resistance of polar molecules.

[0174] The stratum corneum thickness h is directly replaced with the set value of the corresponding skin texture.

[0175] Based on the lipid content L, adjust the partition coefficient:

[0176] ,

[0177] where , L 默认 = 10%, and β is the lipid sensitivity coefficient, β = 0.3.

[0178] Human skin textures include: dry skin, oily skin, neutral skin, and sensitive skin. Different human skin textures have differences in stratum corneum thickness, water content, sebum secretion, etc. These factors will all affect the penetration effect of cosmetics / drugs.

[0179] According to the target application scenario, different combinations of skin texture parameters are set as shown in Table 1:

[0180] Table 1. Combinations of skin texture parameters

[0181]

[0182] For each skin texture type, the corresponding parameters of the second penetration model are adjusted respectively to obtain the third penetration model.

[0183] The physical and chemical parameters of oleuropein are input into each of the third penetration models respectively, and the permeability of oleuropein under different skin textures can be calculated.

[0184] In step S4, a multi-skin-texture penetration model (the third penetration model) is constructed by adjusting the stratum corneum thickness and lipid content, quantifying the influence of different skin textures on the penetration behavior of oleuropein.

[0185] Next, the effectiveness of the third penetration model constructed by the method of the present invention is verified.

[0186] 1) Determine the skin texture type of the volunteers through dermatologist diagnosis or a standardized questionnaire, divided into dry, neutral, oily, and sensitive skin. Take the inner forearm, and standardize the thickness and barrier function. Each group has at least 6 ex vivo skin samples.

[0187] 2) Conduct a transdermal experiment on the ex vivo skin samples:

[0188] Donor solution: 2% (w / v) oleuropein solution (solvent: 30% ethanol-water, pH 5.5);

[0189] Franz diffusion cell settings:

[0190] Effective diffusion area: 0.64 cm²; receptor solution: pH 7.4 PBS (containing 0.01% NaN3 for antibacterial);

[0191] Temperature: 37 ± 0.5 °C; magnetic stirring speed: 600 rpm;

[0192] At the 1st, 2nd, 4th, 6th, 8th, and 24th hours after the start of the test, 200 μL of samples are taken each time (equal amount of fresh receptor solution is supplemented).

[0193] 3) Conduct HPLC analysis on the sampled receptor solution:

[0194] Chromatographic conditions: C18 column, mobile phase acetonitrile-water (25:75), flow rate 1.0 mL / min, detection wavelength 280 nm;

[0195] Limit of quantitation (LOQ): 0.1 μg / mL; intra-day precision RSD < 5%.

[0196] 4) Obtain the input parameters of the third penetration model, including:

[0197] Physicochemical parameters of oleuropein: molecular weight (540 Da), logP (1.2), molecular volume (620 ų), number of polar groups (7);

[0198] The measured values of skin texture parameters are shown in Table 2:

[0199] Table 2. Measured skin texture parameters

[0200]

[0201] 5) Input the parameter values obtained in step (4) into the third penetration model, and calculate the 24-hour penetration amount as shown in Table 3:

[0202] Table 3. Comparison of penetration amounts

[0203]

[0204] The average relative error between the predicted value of the third penetration model and the measured value of the transdermal experiment is -5.1%, with no significant difference. Verified by in vitro experiments, the prediction error of the third penetration model for the oleuropein penetration rate of different skin textures is less than 8%, which is significantly better than the traditional QSAR model (error > 20%), proving the high precision and practicability of the method of the present invention.

[0205] The present invention can be used for the formulation development of cosmetics (such as anti-inflammatory essence, moisturizing cream) and transdermal drugs (such as local analgesic patches). By predicting the penetration behavior of active ingredients under different skin textures, it guides the addition amount of penetration enhancers and the selection of carrier types (such as liposomes, nanoemulsions), and improves the efficacy and safety of products.

[0206] The method described in the present invention is not only applicable to oleuropein, the main component in olive leaf extract, but also can be extended to the permeability test of other active ingredients (such as hydroxytyrosol, flavonoids, phenolic compounds, etc.) in the extract. For 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 penetration model and adopting the same Bayesian calibration framework and transdermal experiment process, accurate prediction of the penetration behavior of multiple components can be achieved. For example:

[0207] Hydroxytyrosol (molecular weight 154 Da, LogP 0.8): Its small molecule characteristics can reduce the steric hindrance effect. In the model, it is necessary to turn off the modification of the molecular volume blocking factor and only optimize the partition coefficient through the polar blocking coefficient.

[0208] Flavonoids (such as luteolin, molecular weight 286 Da, LogP 2.5): Considering its planar molecular structure characteristics, a molecular shape factor needs to be introduced in the diffusion coefficient correction.

[0209] Phenolic compounds (such as tyrosol, molecular weight 138 Da, LogP 1.2): Due to its high polarity, the influence of the polarity retardation coefficient needs to be strengthened in the partition coefficient correction.

[0210] By inputting the physicochemical parameters of different components and calibrating the model based on experimental data, this method can comprehensively evaluate the penetration behavior of multiple components in olive leaf extract, providing data support for the analysis of the synergistic effect of compound formulations.

[0211] Based on the inspiration of the ideal embodiments of the present invention, through the above description, relevant personnel can make various changes and modifications within the scope not deviating from the technical idea of this invention. The technical scope of this invention is not limited to the content in the specification, and 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 between the stratum corneum and the donor solution, and h is the thickness of the 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 and 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: , where D>0, μ=logD 初始 It means that the initial model prediction value is the center. Reflects uncertainty about the initial value; 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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