Method for evaluating the active percutaneous penetration effect of an anti-aging cream containing tetrahydropipecolinic acid

Abstract

The application discloses a kind of tetrahydropiperine-containing anti-aging face cream active substance transdermal penetration effect evaluation method, belongs to the technical field of skin penetration detection and analysis, comprising the following steps: keratinocytes are inoculated on the upper surface of porous membrane, fibroblasts are inoculated on the lower surface of the porous membrane, and skin analog with complete layered structure is formed by co-culture;Apply the anti-aging face cream containing tetrahydropiperine to be tested on the upper surface of the skin analog, and continuously monitor the concentration of the target efficacy active substance at the depth of the efficacy target point with time as the starting point;By culturing keratinocytes and fibroblasts on both sides of the porous membrane respectively, a skin analog with complete layered structure is formed, and an anti-aging face cream containing tetrahydropiperine to be tested is applied to its surface, and the concentration change of the target efficacy active substance at the depth of the efficacy target point is continuously obtained.

Description

Technical Field

[0001] This invention relates to the field of skin penetration detection and analysis technology, and in particular to a method for evaluating the transdermal penetration effect of active ingredients in an anti-aging face cream containing tetrahydropiperine. Background Technology

[0002] Anti-aging creams containing tetrahydropiperine typically utilize tetrahydropiperine to improve the diffusion and penetration of active ingredients in the skin, enabling them to migrate from the skin surface to the stratum corneum, epidermis, or dermis. The active ingredients in anti-aging creams can include vitamins, peptides, or terpenes. Different types of active ingredients have different physicochemical properties, skin sites of action, and minimum effective concentrations. For example, some active ingredients primarily act on the stratum corneum or epidermis to improve the skin barrier and oxidative state; others need to penetrate the dermis and interact with fibroblasts or the dermal matrix to produce their anti-aging effects. Therefore, transdermal penetration efficiency depends not only on the total amount of active ingredients that penetrate the skin but also on whether the active ingredients reach their target depth at the appropriate time.

[0003] Current methods for evaluating the effectiveness of transdermal penetration typically employ the diffusion cell method. This involves placing isolated skin or a skin substitute between the supply and receiving areas, applying the test formulation to the skin surface, and sampling from the receiving area at predetermined intervals to determine the cumulative permeation amount, permeation amount per unit area, or steady-state permeation rate of the active ingredient. When evaluating formulations containing permeation-enhancing components, a test group containing the permeation-enhancing component and a control group without the component are usually set up, and the permeation enhancement factor is calculated based on the difference in cumulative permeation amount or permeation rate between the two groups. This type of method can reflect the degree of overall migration of the active ingredient from one side of the skin to the other and can be used to compare changes in the overall permeability of the active ingredient before and after the addition of tetrahydropiperine.

[0004] However, different active ingredients require different skin layers to exert their anti-aging effects. Existing evaluation methods mainly rely on the cumulative penetration in the receiving area, which cannot accurately reflect the concentration changes of active ingredients at specific target depths such as the stratum corneum, epidermis, or dermis. Some active ingredients may have already rapidly penetrated their main action layer and entered the receiving area, but their concentration and residence time at the target depth are insufficient. Other active ingredients may not have entered the receiving area in large quantities, but may have already reached the minimum effective concentration at the target layer. Using the endpoint cumulative penetration or penetration enhancement factor for evaluation easily equates the total amount of active ingredients penetrating the skin with their effective reach at the target, making it impossible to determine the time when the active ingredient first reaches the minimum effective concentration. Therefore, it is difficult to accurately evaluate the actual penetration-enhancing effect of tetrahydropiperine on active ingredients in anti-aging creams. Summary of the Invention

[0005] This invention overcomes the shortcomings of the prior art and provides a method for evaluating the transdermal penetration effect of active ingredients in an anti-aging face cream containing tetrahydropiperine.

[0006] To achieve the above objectives, the technical solution adopted by this invention is: a method for evaluating the transdermal penetration effect of active ingredients in an anti-aging face cream containing tetrahydropiperine, comprising the following steps: S1. Keratinocytes are seeded on the upper surface of a porous membrane, and fibroblasts are seeded on the lower surface of the porous membrane. They are then co-cultured to form a skin analog with a complete layered structure. S2. Apply the anti-aging cream containing tetrahydropiperine to the surface of the skin analogue, and continuously monitor the concentration of the target active ingredient at the depth of the target site over time, starting from the application time. S3. Based on the concentration-time curve, determine the starting time point at which the concentration of the active ingredient first exceeds its minimum effective concentration. S4. In the skin analogue layer corresponding to the starting time point, the content of the target active ingredient parent compound and each product is determined, and the activity retention rate of the layer is calculated. S5. Calculate the ratio of the concentration of the active ingredient at the starting time point to the minimum effective concentration as the penetration enhancement efficiency index, and record the activity retention rate; S6. If the activity retention rate is less than the preset retention rate threshold, the result of ineffective permeation promotion is output. If it is greater than or equal to the preset retention rate threshold, the permeation promotion efficiency index is output as the evaluation index of percutaneous permeation effect.

[0007] In a preferred embodiment of the present invention, in step S1, keratinocytes are arranged at a ratio of 1×10 5 ~5×10 5 pcs / cm 2 Fibroblasts were seeded at a density of 0.5 × 10⁻⁶ on the surface of a porous membrane. 5 ~3×10 5 pcs / cm 2 The inoculation density was applied to the lower surface of the porous membrane.

[0008] In a preferred embodiment of the present invention, in step S1, keratinocytes and fibroblasts are cultured at 35-38°C and 4-6% carbon dioxide in a liquid environment for 2-5 days, and then cultured at 35-38°C and 4-6% carbon dioxide in a gas-liquid interface environment for 7-21 days to form a skin analog with an epidermal layer and a dermal layer.

[0009] In a preferred embodiment of the present invention, in step S1, the pore size of the porous membrane is 0.2 to 3.0 μm. The porous membrane allows the culture medium and the target active ingredient to pass through, while preventing keratinocytes and fibroblasts from passing through.

[0010] In a preferred embodiment of the present invention, in step S2, the target active ingredient is selected from vitamins, peptides, or terpenes; wherein the vitamins are selected from nicotinamide or tocopherol, the peptides are selected from decarboxylated carnosine hydrochloride or carnosine, and the terpenes are selected from psoralen or bisabolol.

[0011] In a preferred embodiment of the present invention, in step S2, the anti-aging cream to be tested is applied to the surface of the skin analog at an application rate of 1 to 10 mg / cm2, and is continuously monitored for 2 to 12 hours at time intervals of 5 to 60 minutes; the mass fraction of the tetrahydropiperine in the anti-aging cream to be tested is 0.01 to 1.00%.

[0012] In a preferred embodiment of the present invention, in step S3, the target active ingredient is applied to cells corresponding to the depth of the target site and cultured at 35-38°C for 12-48 hours. The concentration of the target active ingredient that causes the cells to produce a preset anti-aging biological response for the first time is determined as the minimum effective concentration.

[0013] In a preferred embodiment of the present invention, in step S4, the skin analog layer corresponding to the starting time point is separated at 0-10°C, and after adding the extraction solution, it is shaken and extracted for 10-60 minutes. The content of the target active ingredient parent compound and each product in the extract is determined respectively. The activity retention rate is the ratio of the content of the target active ingredient parent compound to the total content of the target active ingredient parent compound and each product.

[0014] In a preferred embodiment of the present invention, in step S6, the preset retention rate threshold is 50-90%; when the activity retention rate is less than the preset retention rate threshold, the result of the target active ingredient concentration reaching the minimum effective concentration is determined to be ineffective in promoting penetration.

[0015] In a preferred embodiment of the present invention, the anti-aging face cream containing tetrahydropiperine comprises the following components by weight percentage: 2-15% polyol, 2-18% oil, 1-8% emulsifier, 0.1-1.0% thickener, 0.01-0.5% tetrahydropiperine, 0.03-10% active ingredients, 0.01-5% peptides, 0.01-5% plant extracts, and water to make up the balance.

[0016] This invention addresses the shortcomings of the prior art and has the following beneficial effects: By culturing keratinocytes and fibroblasts on both sides of a porous membrane, a skin analog with a complete layered structure was formed. A test anti-aging cream containing tetrahydropiperine was applied to its surface, and the concentration changes of the target active ingredient at the target depth were continuously acquired. This allowed the migration process of the active ingredient from the epidermis to the dermis to be recorded chronologically, establishing a correlation between concentration changes in different skin layers and the corresponding depth of action. This enabled the determination of the time when the target active ingredient first reached its minimum effective concentration at the target depth, reflecting the speed at which tetrahydropiperine effectively reached the target layer. Compared to existing methods that evaluate solely based on the cumulative penetration amount in the receiving area or the total penetration amount at the endpoint, this method avoids equating the total amount that penetrates the entire skin analog with the effective amount reached at the target layer, reducing evaluation bias caused by the active ingredient's rapid penetration or insufficient residence at the target layer.

[0017] By using the initial time point when the concentration of the target active ingredient first exceeds the minimum effective concentration as the evaluation anchor point, and measuring the content of the target active ingredient parent compound and each product in the corresponding skin analog layer at that time point, the activity retention rate is calculated. This ensures that the arrival time, concentration, and chemical state of the active ingredient are on the same time axis and within the same efficacy target layer, thus distinguishing between the parent compound with anti-aging activity and the inactivated products formed during penetration. Therefore, even if the total amount of active ingredient at the efficacy target depth reaches the minimum effective concentration, the actual retention of effective active ingredient can be judged based on the proportion of the parent compound. Compared with existing methods that only detect the total amount of target ingredients without distinguishing between parent compounds and transformation products, this method avoids including inactivated products that have been oxidized, hydrolyzed, or transformed in the effective penetration enhancement amount, improving the correspondence between transdermal penetration evaluation results and actual anti-aging efficacy.

[0018] The permeation enhancement efficiency index is obtained by calculating the ratio of the active ingredient concentration at the starting time point to the minimum effective concentration, and the activity retention rate is compared with a preset retention rate threshold. When the activity retention rate is lower than the preset retention rate threshold, the permeation enhancement result is directly output as ineffective. When the activity retention rate meets the requirements, the permeation enhancement efficiency index is used as an evaluation index for transdermal permeation effect. The permeation enhancement rate, effective concentration, and activity retention status are combined into an evaluation system with a sequential judgment relationship, so that tetrahydropiperine is only judged as effective permeation enhancement when it helps the active ingredient reach the effective target and maintains sufficient activity. Compared with existing methods that use permeation enhancement factor, cumulative permeation amount, or steady-state permeation rate for evaluation alone, this method can eliminate false positive results where the total amount meets the standard but the effective parent compound is insufficient, and improve the accuracy and comparability of evaluation results between different active ingredients, different formulations, and different permeation enhancement conditions. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 This is a flowchart of a preferred embodiment of the present invention; Figure 2 This is a line graph showing the relative concentration of nicotinamide before and after the addition of tetrahydropiperine, according to a preferred embodiment of the present invention. Figure 3 This is a line graph showing the relative concentration of tocopherol before and after the addition of tetrahydropiperine, according to a preferred embodiment of the present invention. Figure 4 This is a line graph showing the relative concentration of decarboxylated carnosine hydrochloride before and after the addition of tetrahydropiperine, according to a preferred embodiment of the present invention. Figure 5 This is a line graph showing the relative concentration of carnosine before and after the addition of tetrahydropiperine, according to a preferred embodiment of the present invention. Figure 6 This is a line graph showing the relative concentration of psoralen before and after the addition of tetrahydropiperine, according to a preferred embodiment of the present invention. Figure 7 This is a line graph showing the relative concentration of bisabolol before and after the addition of tetrahydropiperine, which is a preferred embodiment of the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0022] Application Overview: Measuring only the total amount of active ingredients in a specific layer cannot accurately reflect the penetration enhancement results. As active ingredients migrate through the stratum corneum and epidermis into the dermis, they are affected by oxidation, hydrolysis, enzymatic transformation, and changes in the local pH environment. Tetrahydropiperidine alters the migration rate of active ingredients, which in turn changes their residence time and inactivation process in different layers. Therefore, even if the total amount of active ingredients at the target depth is detected to reach the minimum effective concentration at a certain time point, it may still contain a high proportion of degradation products or transformation products, and the actual retained parent compound is insufficient to produce the expected anti-aging effect.

[0023] The applicant found that the permeation-enhancing effect of tetrahydropiperine should simultaneously meet two conditions: first, the active ingredient should reach its target depth in a short period of time, and the concentration at that depth should reach the minimum effective concentration; second, the active ingredient should still maintain a sufficient proportion of the parent compound when it reaches the target depth. If the minimum effective concentration is not determined solely by the total amount of active ingredient, inactivated products will be included in the effective concentration, leading to an incorrect judgment of permeation enhancement effectiveness. If the time when the concentration of the active ingredient first exceeds the minimum effective concentration is used as a unified evaluation anchor point, and the content of the parent compound and each product is measured at that time point, the arrival time, the concentration reached, and the active state at the time of arrival are all on the same time axis and at the same target level.

[0024] The applicant proposed to create a skin analog with a complete layered structure by culturing keratinocytes and fibroblasts on both sides of a porous membrane. A test anti-aging cream containing tetrahydropiperine was applied to the surface of the skin analog, and the concentration changes of the target active ingredient at the target depth were continuously monitored. The starting time point when the active ingredient first exceeded the minimum effective concentration was determined based on the concentration-time curve. The content of the parent compound and each product was then measured in the corresponding layer, and the activity retention rate and penetration enhancement efficiency index were calculated. When the activity retention rate was lower than a preset retention rate threshold, penetration enhancement was deemed ineffective. Only when the activity retention rate met the requirements was the penetration enhancement efficiency index used as the evaluation result, thereby avoiding the inclusion of inactivated products in the effective penetration enhancement amount and improving the consistency between the evaluation of transdermal penetration effect and the actual anti-aging efficacy.

[0025] like Figure 1 As shown, a method for evaluating the transdermal penetration effect of active ingredients in an anti-aging face cream containing tetrahydropiperine includes: S1. Keratinocytes are seeded on the upper surface of a porous membrane, and fibroblasts are seeded on the lower surface of the porous membrane. They are then co-cultured to form a skin analog with a complete layered structure. S2. Apply the anti-aging cream containing tetrahydropiperine to the surface of the skin analogue, and continuously monitor the concentration of the target active ingredient at the depth of the target site over time, starting from the application time. S3. Based on the concentration-time curve, determine the starting time point at which the concentration of the active ingredient first exceeds its minimum effective concentration. S4. In the skin analogue layer corresponding to the starting time point, the content of the target active ingredient parent compound and each product is determined, and the activity retention rate of the layer is calculated. S5. Calculate the ratio of the concentration of the active ingredient at the starting time point to the minimum effective concentration as the penetration enhancement efficiency index, and record the activity retention rate; S6. If the activity retention rate is less than the preset retention rate threshold, the result of ineffective permeation promotion is output. If it is greater than or equal to the preset retention rate threshold, the permeation promotion efficiency index is output as the evaluation index of percutaneous permeation effect.

[0026] The core innovation of this invention lies in using the time when the target active ingredient first exceeds the minimum effective concentration at the depth of the effective target site as the evaluation anchor point, and simultaneously measuring the content of the parent compound and each product in that layer. The penetration rate, effective concentration and activity retention status are correlated, and the penetration efficiency index is only output when the activity retention rate reaches a preset threshold, thereby avoiding the inclusion of inactivated products in the effective penetration amount and improving the accuracy of the evaluation of the penetration effect of tetrahydropiperine.

[0027] Each step will be explained in detail below.

[0028] In step S1, keratinocytes are seeded on the upper surface of the porous membrane, and fibroblasts are seeded on the lower surface of the porous membrane, and co-cultured to form a skin analog with a complete layered structure.

[0029] Keratinocytes and fibroblasts are co-cultured on both sides of a porous membrane. The porous membrane allows the culture medium and the target active ingredient to pass through, while preventing keratinocytes and fibroblasts from passing through. The pore size of the porous membrane is 0.2–3.0 μm.

[0030] Keratinocytes were seeded at a predetermined density on the upper surface of the porous membrane, and fibroblasts were seeded at a predetermined density on the lower surface of the porous membrane. The seeding density of keratinocytes was 1 × 10⁻⁶. 5 ~5×10 5 pcs / cm 2 The seeding density of fibroblasts was 0.5 × 10⁻⁶. 5 ~3×10 5 pcs / cm 2 .

[0031] After inoculation, the cells are cultured under submerged conditions and then transferred to gas-liquid interface conditions for further culture to form skin analogs with epidermal and dermal layers. The submerged culture stage promotes cell proliferation and fusion, while the gas-liquid interface culture stage induces stratified differentiation of keratinocytes to form the epidermal layer, and fibroblasts form the dermal layer under the membrane.

[0032] The temperature for submerged culture was 35–38℃, the carbon dioxide volume fraction was 4–6%, and the culture time was 2–5 days; the temperature for gas-liquid interface culture was 35–38℃, the carbon dioxide volume fraction was 4–6%, and the culture time was 7–21 days.

[0033] In step S2, a test anti-aging cream containing tetrahydropiperine is applied to the surface of the skin analogue. Starting from the application time, the concentration of the target active ingredient at the target site depth is continuously monitored over time.

[0034] When the anti-aging cream was applied to the surface of a skin analog, the active ingredients in the cream migrated from the surface of the skin analog to the interior under the action of tetrahydropiperine, penetrating the epidermis and dermis.

[0035] The concentration of the target active ingredient at the depth of the target site was continuously monitored at different time points to obtain the concentration change curve over time.

[0036] The application amount of the anti-aging face cream to be tested was 1–10 mg / cm³. 2 The mass fraction of tetrahydropiperine in the anti-aging face cream to be tested was 0.01–1.00%.

[0037] The target active ingredient is selected from vitamins, peptides, or terpenes; vitamins are selected from nicotinamide or tocopherol, peptides are selected from decarboxylated carnosine hydrochloride or carnosine, and terpenes are selected from psoralen or bisabolol.

[0038] The time interval and total duration of continuous monitoring are configured to capture the moment when the concentration of the active ingredient first exceeds the minimum effective concentration; the monitoring time interval is 5 to 60 minutes and the total duration of continuous monitoring is 2 to 12 hours.

[0039] Formula and preparation method of anti-aging face cream containing tetrahydropiperine: The tetrahydropiperine-containing anti-aging cream applied to the surface of the skin analogue in step S2 comprises the following components by weight percentage: Water, to replenish the remaining amount.

[0040] Polyols, 2-15%, including glycerol 1-5%, butanediol 0.5-4%, 1,2-hexanediol 0.3-2%, and 1,2-pentanediol 0.3-2%.

[0041] Oils and fats, 2-18%, including 1-8% polydimethylsiloxane, 0.5-5% C13-16 isoparaffins, 0.5-5% pentaerythritol tetra(ethylhexanoate), 0.5-3% PEG / PPG-14 / 7 dimethyl ether, 0.1-3% jojoba esters, 0.1-3% shea butter, and 0.5-5% caprylic / capric triglycerides.

[0042] Emulsifier, 1-8%, including 0.5-3% polyglycerol-6 distearate, 0.5-3% polyglycerol-3 beeswax ester, 0.5-2% glyceryl stearate, 0.5-2% PEG-100 stearate, 0.2-2% cetearyl alcohol, and 0.2-2% cetyl alcohol.

[0043] Thickener, 0.1-1.0%, selected from one or more of carbomer, xanthan gum, ammonium acryloyldimethyl taurate / VP copolymer.

[0044] Tetrahydropiperine, 0.01-0.5%.

[0045] The active ingredient, 0.03-10%, is selected from one or more of nicotinamide, tocopherol, decarboxylated carnosine hydrochloride, carnosine, psoralen, and bisabolol.

[0046] Peptides, 0.01-5%, selected from one or more of acetyl hexapeptide-8, oligopeptide-1, palmitoyl pentapeptide-4, nonapeptide-1, acetyl tetrapeptide-9, hexapeptide-9, palmitoyl tripeptide-8, acetyl tetrapeptide-3, acetyl tetrapeptide-11, tripeptide-1, palmitoyl tetrapeptide-7, and poly(tripeptide-6).

[0047] Plant extracts, 0.01-5%, selected from one or more of the following: North African cedar bark oil, green vitex flower wax, rosewood extract, hydrolyzed gardenia extract, ginger root extract, gardenia fruit extract, dendrobium stem extract, aloe vera leaf extract, sophora flavescens root extract, Ningxia wolfberry fruit extract, and purple coneflower extract.

[0048] The preparation method of this anti-aging face cream containing tetrahydropiperine includes the following steps: Add water to the aqueous phase pot, then add polyol and thickener in sequence, heat to 75-85℃, and stir until completely dissolved to obtain the aqueous phase material.

[0049] Add the oil and emulsifier to the oil phase pot, heat to 75-85℃, and stir until completely melted and mixed to obtain the oil phase material.

[0050] Under stirring conditions, the oil phase material is added to the aqueous phase material, homogenized for 3-8 minutes at a homogenization speed of 2000-4000 rpm, and then cooled to 40-50℃.

[0051] Add tetrahydropiperine, active ingredients, peptides, and plant extracts, stir until homogeneous, and cool to 35-38℃ to obtain a tetrahydropiperine-containing anti-aging face cream.

[0052] In step S3, based on the concentration-time curve, the starting time point at which the concentration of the active ingredient first exceeds its minimum effective concentration is determined.

[0053] The minimum effective concentration was determined through preliminary experiments. The target active ingredient was applied to cells corresponding to the depth of the target site. After culturing at a set temperature for a set time, the anti-aging biological response was detected. The concentration of the target active ingredient that first produced the preset anti-aging biological response in the cells was determined as the minimum effective concentration.

[0054] The culture temperature is 35–38℃, and the culture time is 12–48 h.

[0055] After obtaining the concentration change curve of the target active ingredient at the depth of the target site over time, the moment when the concentration value on the curve first reaches and exceeds the minimum effective concentration is determined as the starting time point.

[0056] In step S4, the content of the target active ingredient parent compound and each product in the skin analog layer corresponding to the starting time point is measured, and the activity retention rate of that layer is calculated.

[0057] The skin analogue layer corresponding to the starting time point refers to the layer at which the active ingredient reaches and accumulates at the target depth at that time point. The skin analogue is separated at this layer, and after separation, an extraction solution is added for shaking extraction. The content of the target active ingredient parent compound and each product in the extract is determined.

[0058] The separation temperature is 0–10℃, and the extraction time is 10–60 min.

[0059] The activity retention rate is the ratio of the content of the target active ingredient parent compound to the total content of the target active ingredient parent compound and all products. The higher the ratio, the higher the proportion of active ingredients that retain activity at the level of the efficacy target.

[0060] In step S5, the ratio of the concentration of the active ingredient at the starting time point to the minimum effective concentration is calculated as the penetration enhancement efficiency index, and the activity retention rate is recorded.

[0061] The penetration enhancement efficiency index reflects the degree to which the active ingredient exceeds the minimum effective concentration at the depth of the target site.

[0062] A value greater than 1 indicates that the concentration of the active ingredient has exceeded the minimum effective concentration, and the larger the value, the more sufficient the reserve of the effective concentration.

[0063] The activity retention rate and penetration enhancement efficiency index calculated in step S4 are recorded at the same time anchor point for subsequent judgment steps.

[0064] In step S6, if the activity retention rate is less than the preset retention rate threshold, the result of ineffective permeation promotion is output; if the activity retention rate is greater than or equal to the preset retention rate threshold, the permeation promotion efficiency index is output as the evaluation index of percutaneous permeation effect.

[0065] The preset retention rate threshold is used to determine whether a sufficient proportion of the parent compound is still retained in the active ingredient that has reached the depth of the efficacy target.

[0066] The preset retention rate threshold is 50-90%.

[0067] When the activity retention rate is less than the preset retention rate threshold, even if the concentration of the target active ingredient reaches the minimum effective concentration, the actual retained activity of the parent compound is still insufficient to produce the expected anti-aging effect, and therefore the penetration enhancement is deemed ineffective.

[0068] Only when the activity retention rate reaches the preset retention rate threshold will the permeation enhancement efficiency index be output as an evaluation index for transdermal permeation effect.

[0069] The data processing and judgment algorithms involved in steps S4, S5, and S6 are explained in detail: In step S4, the activity retention rate is calculated according to the following formula: ; in, The activity retention rate is expressed as % . The content of the parent compound of the target active ingredient; The total content of each product representing the target efficacy active ingredient.

[0070] The determination method is as follows: the skin analog layer corresponding to the starting time point is separated at 0-10℃, the extraction solution is added and the mixture is shaken for 10-60 min. After filtration, the peak area of ​​the parent compound is determined by high performance liquid chromatography or liquid chromatography-mass spectrometry, and the result is calculated by substituting the peak area into the standard curve of the parent compound. .

[0071] The determination method is as follows: The peak areas of each product are measured under the same chromatographic conditions for the same sample. The peak areas are then substituted into the standard curves of each product to calculate the content of each product. The contents of each product are then summed to obtain the final content. The products include degradation products and transformation products generated by the target active ingredient during the penetration of skin analogues due to oxidation, hydrolysis or enzymatic transformation.

[0072] Activity retention rate The value range is 0 to 100%. When it approaches 100%, it indicates that the active ingredient almost completely retains the form of the parent compound when reaching the target depth; when A lower level indicates that the active ingredient has undergone significant transformation or degradation during the penetration process.

[0073] In step S5, the penetration enhancement efficiency index is calculated according to the following formula: ; in, The penetration-enhancing efficiency index is dimensionless. The concentration of the active ingredient at the depth of the target site at the starting time point; This is the minimum effective concentration.

[0074] The method of obtaining the concentration is as follows: In step S2, the concentration of the target active ingredient at the depth of the target site is continuously monitored at time intervals of 5 to 60 minutes, the concentration value at each time point is recorded, and a concentration-time curve is plotted with time as the horizontal axis and concentration as the vertical axis.

[0075] The starting time point at which the minimum effective concentration is first exceeded is determined by step S3. Read from the concentration-time curve The concentration value corresponding to time is denoted as .

[0076] The method for determining the target efficacy active ingredient is as follows: The target active ingredient is applied to cells corresponding to the depth of the efficacy target, and the cells are cultured at 35–38°C for 12–48 hours. The anti-aging biological response indicators of the cells are then detected. The concentration of the target efficacy active ingredient that induces the first predetermined anti-aging biological response in the cells is determined as [the target efficacy active ingredient concentration]. Among them, the anti-aging biological response indicators are selected from at least one of cell proliferation rate, collagen synthesis amount or antioxidant enzyme activity.

[0077] Penetration enhancement efficiency index For dimensionless ratios, when This indicates that the concentration of the active ingredient at the depth of the target site has exceeded the minimum effective concentration at the start of the time. A higher value indicates a more sufficient reserve of effective concentration; This indicates that the concentration of the active ingredient has not yet reached or is exactly equal to the minimum effective concentration.

[0078] In step S6, the decision logic is implemented as follows: Input amount is the activity retention rate and preset retention rate threshold The output is the judgment result.

[0079] Preset retention rate threshold The value ranges from 50% to 90%, with the specific value determined based on the chemical stability and skin metabolic stability of the target active ingredient.

[0080] For active ingredients with stable chemical structures that are not easily metabolized by skin enzymes, Choose a lower value, such as 50-70%; for active ingredients with active chemical structures that are easily oxidized or hydrolyzed, Take a higher value, such as 70-90%.

[0081] The decision logic is as follows: when At that time, the result was determined to be ineffective in promoting osmosis.

[0082] when When the result is deemed effective infiltration promotion, the infiltration promotion efficiency index calculated in step S5 is used. Output as an indicator for evaluating transdermal penetration effect.

[0083] The physical meaning of this judgment logic is: when the activity retention rate is lower than the threshold, even if the total amount of active ingredients at the target depth reaches the minimum effective concentration, the proportion of the parent compound is insufficient, and the actual effective concentration of active ingredients exerting the anti-aging effect is low. Below Insufficient to produce the expected anti-aging effect; only when the activity retention rate reaches the threshold can the parent compound constitute the main body of effective penetration enhancement, and only then can the penetration enhancement efficiency index truly reflect the penetration enhancement effect of tetrahydropiperine.

[0084] Based on this, the effective penetration enhancement efficiency index after correction for activity retention rate can also be calculated: ; in, This is a dimensionless index of effective penetration enhancement after correction for activity retention rate; it combines the achieved concentration and activity retention rate into a comprehensive evaluation index. This indicates that the effective active ingredient concentration (i.e., the concentration of the parent compound) at the depth of the target site has exceeded the minimum effective concentration, and can be directly used as an indicator for evaluating the transdermal penetration effect.

[0085] To further simplify and make the present invention achieve its objectives and effects, the present invention will be further illustrated in conjunction with the following specific embodiments and comparative examples, but the present invention is not limited to the scope of the embodiments described herein.

[0086] It should be noted that the raw materials used in the examples and comparative examples are described below: Keratinocytes: Catalog number SNL-163, included in PUMC of Peking Union Medical College Cell Bank, purchased from Wuhan Shangen Biotechnology Co., Ltd.; Fibroblasts: Product number MZ-1031, purchased from Ningbo Mingzhou Biotechnology Co., Ltd.; Porous membrane: purchased from Thermo Fisher Scientific (China) Co., Ltd.; Tetrahydropiperine: CAS No. 23434-88-0, purchased from Shanghai Jiuqian Chemical Co., Ltd.; Nicotinamide: Purchased from Shanghai Baihaobo Biotechnology Co., Ltd.; Tocopherol: Purchased from Shanghai Baihaobo Biotechnology Co., Ltd.; Decarboxylcarnosine hydrochloride: purchased from Shanghai Baihaobo Biotechnology Co., Ltd.; Carnosine: Purchased from Shanghai Baihaobo Biotechnology Co., Ltd.; Psoralen: purchased from Shanghai Meiyan Biotechnology Co., Ltd.; Bisabolol: purchased from Shanghai Luya Biotechnology Co., Ltd.

[0087] Example 1: The preparation of an anti-aging face cream containing tetrahydropiperine is as follows: Weigh out 70.73 parts water, 3 parts glycerol, 2.25 parts butylene glycol, 1.15 parts 1,2-hexanediol, 1.15 parts 1,2-pentanediol, 0.3 parts carbomer, and 1.75 parts PEG / PPG-14 / 7 dimethyl ether by weight, add them to an aqueous phase pot, heat to 80°C, and stir until completely dissolved to obtain the aqueous phase material; Weigh out 4.5 parts of polydimethylsiloxane, 2.75 parts of C13-16 isoparaffin, 2.75 parts of pentaerythritol tetra(ethylhexanoate), 1.55 parts of jojoba esters, 1.55 parts of shea butter, 2.75 parts of caprylic / capric triglyceride, 1.75 parts of polyglycerol-6 distearate, 1.75 parts of polyglycerol-3 beeswax ester, 1.25 parts of glyceryl stearate, 1.25 parts of PEG-100 stearate, 1.1 parts of cetearyl alcohol, and 1.1 parts of cetyl alcohol. Add these to an oil phase pot, heat to 80°C, and stir until completely melted and mixed to obtain the oil phase material. The oil phase material was added to the aqueous phase material under stirring conditions, homogenized for 5.5 min at a homogenization speed of 3000 rpm, and then cooled to 45℃. Add 0.255 parts of tetrahydropiperine, 5.0 parts of nicotinamide, 2.505 parts of acetyl hexapeptide-8, 1.25 parts of North African cedar bark oil, and 1.25 parts of green chaste tree wax. Stir until homogeneous and cool to 36.5℃ to obtain a tetrahydropiperine-containing anti-aging face cream.

[0088] S1, arrange keratinocytes according to 3×10 5 pcs / cm 2 Fibroblasts were seeded at a density of 1.75 × 10⁻⁶ on the surface of a porous membrane. 5 pcs / cm2 The cells were seeded at a density of 1.6 μm on the lower surface of a porous membrane. The cells were cultured at 36.5 °C and 5% CO2 for 3.5 days, and then cultured at 36.5 °C and 5% CO2 at the gas-liquid interface for 14 days to form a skin analog with a layered structure of epidermal and dermal layers. S2, on the surface of the skin analogue at 5.5 mg / cm 2 The test anti-aging cream containing tetrahydropiperine was applied, and the concentration of niacinamide at the target depth was continuously monitored over time at 32.5-minute intervals, starting from the application time, for 7 hours; the mass fraction of tetrahydropiperine in the test anti-aging cream was 0.255%. S3. Apply nicotinamide to cells corresponding to the depth of the efficacy target and culture them at 36.5°C for 30 hours. Determine the nicotinamide concentration at which the cells first produce the preset anti-aging biological response as the minimum effective concentration. Based on the concentration change curve over time, determine the starting time point at which the nicotinamide concentration first exceeds its minimum effective concentration. S4. In the skin analog layer corresponding to the starting time point, the layer is separated at 5°C, and after adding the extraction solution, it is shaken and extracted for 35 min. The content of nicotinamide parent compound and each product in the extract is measured respectively, and the activity retention rate of the layer is calculated. The activity retention rate is the ratio of the content of nicotinamide parent compound to the total content of nicotinamide parent compound and each product. S5. Calculate the ratio of the nicotinamide concentration at the starting time point to the lowest effective concentration as the penetration enhancement efficiency index, and record the activity retention rate; S6. The preset retention rate threshold is 70%. If the activity retention rate is less than the preset retention rate threshold, the result of ineffective permeation promotion is output. If it is greater than or equal to the preset retention rate threshold, the permeation promotion efficiency index is output as the evaluation index of percutaneous permeation effect.

[0089] Example 2: This example is basically the same as Example 1, except that the type of target active ingredient in S2 is different. Specifically, nicotinamide is replaced with tocopherol, and the rest is the same as in Example 1.

[0090] Example 3: This example is basically the same as Example 1, except that the type of target active ingredient in S2 is different. Specifically, nicotinamide is replaced with decarboxylated carnosine hydrochloride, and the rest is the same as in Example 1.

[0091] Example 4: This example is basically the same as Example 1, except that the type of target active ingredient in S2 is different. Specifically, nicotinamide is replaced with carnosine, and the rest is the same as in Example 1.

[0092] Example 5: This example is basically the same as Example 1, except that the type of target active ingredient in S2 is different. Specifically, nicotinamide is replaced with psoralen, and the rest is the same as in Example 1.

[0093] Example 6: This example is basically the same as Example 1, except that the type of target active ingredient in S2 is different. Specifically, nicotinamide is replaced with bisabolol, and the rest is the same as in Example 1.

[0094] Example 7: This example is basically the same as Example 1, except that the seeding density of keratinocytes in S1 is taken as the minimum value of the interval, specifically: the seeding density of keratinocytes is changed to 1×10 5 The number of pieces per cm² is the same as in Example 1.

[0095] Example 8: This example is basically the same as Example 1, except that the seeding density of keratinocytes in S1 is taken as the maximum value of the interval, specifically: the seeding density of keratinocytes is changed to 5×10 5 The number of pieces per cm² is the same as in Example 1.

[0096] Example 9: This example is basically the same as Example 1, except that the time interval for continuous monitoring in S2 is the minimum value of the interval. Specifically, the concentration of nicotinamide at the target depth of efficacy is continuously monitored over time at a time interval of 5 minutes for 7 hours. The rest is the same as in Example 1.

[0097] Example 10: This example is basically the same as Example 1, except that the time interval for continuous monitoring in S2 is the maximum value of the interval. Specifically, the concentration of nicotinamide at the target depth of efficacy is continuously monitored over time at a time interval of 60 minutes for 7 hours. The rest is the same as in Example 1.

[0098] Example 11: This example is basically the same as Example 1, except that: the preset retention rate threshold in S6 is the minimum value of the interval, specifically: the preset retention rate threshold is changed to 50%, and the rest is the same as Example 1.

[0099] Example 12: This example is basically the same as Example 1, except that: the preset retention rate threshold in S6 is the maximum value of the interval, specifically: the preset retention rate threshold is changed to 90%, and the rest is the same as Example 1.

[0100] Example 13: This example is basically the same as Example 1, except that the pore size of the porous membrane in S1 is the minimum value of the range, specifically: the pore size of the porous membrane is changed to 0.2 μm, and the rest is the same as in Example 1.

[0101] Example 14: This example is basically the same as Example 1, except that the pore size of the porous membrane in S1 is the maximum value of the range, specifically: the pore size of the porous membrane is changed to 3.0 μm, and the rest is the same as Example 1.

[0102] Comparative Example 1: S1, Keratinocytes were divided into 3×10 5 pcs / cm 2 Fibroblasts were seeded at a density of 1.75 × 10⁻⁶ on the surface of a porous membrane. 5 pcs / cm 2 The seeding density is applied to the lower surface of the porous membrane, and the pore size of the porous membrane is 1.6 μm; S2. The cells were cultured at 36.5℃ and 5% carbon dioxide for 3.5 days, and then cultured at 36.5℃ and 5% carbon dioxide at the gas-liquid interface for 14 days to form a skin analog. S3. Fix the skin analogue in a Franz diffusion cell, and apply a solution of 5.5 mg / cm³ to the surface of the skin analogue. 2 The amount of anti-aging cream containing tetrahydropiperine was applied to the test, and phosphate buffer was added to the receiving cell as the receiving solution. S4. Take a sample from the receiving pool 24 hours after applying the face cream and determine the total concentration of nicotinamide in the receiving liquid; S5. Using the total cumulative throughput over 24 hours as the evaluation indicator, output the total cumulative throughput.

[0103] Comparative Example 2: This comparative example is basically the same as Example 1, except that: in S2, the concentration change at the depth of the efficacy target is not continuously tracked, and only the cumulative permeation at the endpoint is measured. Specifically, in step S2, starting from the application time, the total concentration of niacinamide is measured from the receiving cell at a single time point of 24 hours after the cream is applied. The monitoring is not continuous at a time interval of 32.5 minutes, and the concentration change curve over time is not obtained; the remaining steps are the same as in Example 1.

[0104] Comparative Example 3: This comparative example is basically the same as Example 1, except that: in S3, the evaluation anchor point is not the starting time point when the active ingredient first exceeds the minimum effective concentration, but a fixed time point is used as the evaluation time point. Specifically, in step S3, the fixed time point of 8 hours after applying the face cream is used as the starting time point, and the starting time point is not the moment when the concentration first exceeds the minimum effective concentration; the remaining steps are the same as in Example 1.

[0105] Comparative Example 4: This comparative example is basically the same as Example 1, except that: in S4, the content of the target active ingredient parent compound and each product is not measured, and the activity retention rate is not calculated. Specifically, in step S4, in the skin analog layer corresponding to the starting time point, only the total content of nicotinamide is measured, and the content of the parent compound and each product is not measured separately, and the activity retention rate is not calculated; the remaining steps are the same as in Example 1.

[0106] Comparative Example 5: This comparative example is basically the same as Example 1, except that: in S6, no threshold for activity retention rate is set, and only the permeation efficiency index is used as the sole evaluation index. Specifically, in step S6, the activity retention rate is not compared with the preset retention rate threshold, and the permeation efficiency index is directly output as the evaluation index for percutaneous permeation effect. No determination of activity retention rate compliance is made. The remaining steps are the same as in Example 1.

[0107] Comparative Example 6: This comparative example is basically the same as Example 1, except that the seeding density of keratinocytes in S1 is lower than the minimum value in the interval. The specific steps of S1 are as follows: the seeding density of keratinocytes is changed to 0.5 × 10⁻⁶. 5 pcs / cm 2 The rest is the same as S1 in Example 1.

[0108] Comparative Example 7: This comparative example is basically the same as Example 1, except that the seeding density of keratinocytes in S1 is higher than the maximum value in the interval. The specific steps of S1 are as follows: the seeding density of keratinocytes is changed to 6 × 10 5 pcs / cm 2 The rest is the same as S1 in Example 1.

[0109] Comparative Example 8: This comparative example is basically the same as Example 1, except that the time interval for continuous monitoring in S2 is lower than the minimum value of the interval. The specific steps of S2 are as follows: continuously monitor the concentration of nicotinamide at the effective target depth over time at a time interval of 2 minutes, and continuously monitor for 7 hours. The rest is the same as S2 in Example 1.

[0110] Comparative Example 9: This comparative example is basically the same as Example 1, except that the time interval for continuous monitoring in S2 is higher than the maximum value of the interval. The specific steps of S2 are as follows: continuously monitor the concentration of nicotinamide at the effective target depth over time at a time interval of 90 minutes for 7 hours. The rest is the same as S2 in Example 1.

[0111] Comparative Example 10: This comparative example is basically the same as Example 1, except that the preset retention rate threshold in S6 is lower than the minimum value of the interval. The specific steps of S6 are: the preset retention rate threshold is changed to 40%, and the rest is the same as S6 in Example 1.

[0112] Comparative Example 11: This comparative example is basically the same as Example 1, except that the preset retention rate threshold in S6 is higher than the maximum value in the interval. The specific steps of S6 are: the preset retention rate threshold is changed to 95%, and the rest is the same as S6 in Example 1.

[0113] Comparative Example 12: This comparative example is basically the same as Example 1, except that the pore size of the porous membrane in S1 is lower than the minimum value of the range. The specific steps of S1 are: the pore size of the porous membrane is changed to 0.1 μm, and the rest is the same as S1 in Example 1.

[0114] Comparative Example 13: This comparative example is basically the same as Example 1, except that the pore size of the porous membrane in S1 is higher than the maximum value in the range. The specific steps of S1 are as follows: the pore size of the porous membrane is changed to 4.0 μm, and the rest is the same as S1 in Example 1.

[0115] Performance testing: The efficacy evaluation methods of Examples 1-14 and Comparative Examples 1-13 were tested sequentially for concentration curve consistency, activity retention deviation rate, penetration enhancement differentiation, time-dependent effect, and misjudgment avoidance rate. The results are shown in Table 1.

[0116] Concentration curve consistency: Using the same batch of anti-aging face cream containing tetrahydropiperine, the concentration changes of the target active ingredient at the target site depth were continuously monitored according to the method in step S2 of Example 1. Parallel tests were conducted at four different monitoring time intervals: 5 min, 15 min, 30 min, and 60 min. A concentration-time curve was obtained for each test group. The curve obtained at the 5-minute interval was used as the reference curve. After interpolating and matching the curves obtained at other intervals with the reference curve, the absolute value of the relative deviation of the concentration values ​​at each time point was calculated, and the average value of all time points was recorded as [value missing]. Units are percentages; concentration curve fit Calculate using the following formula: The unit is a percentage. This value reflects the extent to which the selected monitoring time interval captures the trend of concentration changes. A value higher than 95% indicates that the interval is sufficient to accurately reflect the change of concentration over time.

[0117] Activity retention deviation rate: Standard solutions containing the target active ingredient were prepared at three concentration levels: high (50 μg / mL), medium (25 μg / mL), and low (5 μg / mL), with five parallel samples prepared for each concentration. Following step S4 of Example 1, each standard solution was mixed with a blank extract from the skin analogue layer. The contents of the parent compound were then determined by extraction, filtration, and chromatographic analysis. The measured values ​​were recorded as follows: Units are μg / mL; activity retention deviation rate Calculate using the following formula: The unit is a percentage, and the average of three concentration levels is taken as the final result. The lower the value, the more accurate the determination of the content of the parent compound and each product, and the more reliable the calculation of the activity retention rate.

[0118] Penetration differentiation: Three anti-aging face cream formulations with the same target active ingredient but different contents of tetrahydropiperine were selected. Formulation A contained no tetrahydropiperine, formulation B contained 0.255% tetrahydropiperine, and formulation C contained 0.510% tetrahydropiperine. The transdermal penetration effect of the three formulations was evaluated using the same method as in Example 1, and the penetration enhancement efficacy index of each formulation was obtained. , and The units are dimensionless; calculate the penetration enhancement ratio of formula B relative to formula A. ; Infiltration differentiation Calculate using the following formula: The unit is dimensionless. This value reflects the sensitivity of this evaluation method to different formulations with different tetrahydropiperine contents. The higher the value, the more obvious the response of the penetration enhancement efficiency index is as the penetration enhancer content increases.

[0119] Time-related: Take the starting time point determined in step S3 of Example 1. The unit is h, and the penetration-enhancing efficiency index calculated in step S5. The units are dimensionless; simultaneously, the traditional Franz diffusion cell endpoint detection method was used to determine the cumulative total permeate of the same sample over 24 hours. The unit is μg / cm³ 2 Calculate the Pearson correlation coefficient between the starting time point and the cumulative total transmittance. Pearson correlation coefficient between penetration enhancement efficiency index and cumulative permeation volume Timeliness Calculate using the following formula: The unit is dimensionless. A value greater than 1 indicates that the permeation-enhancing efficiency index, which is based on time anchor and concentration reserve, can better reflect the rapid permeation-enhancing characteristics of tetrahydropiperine than the cumulative permeation at the endpoint. The higher the value, the better the time-effect correlation.

[0120] False positive avoidance rate: A simulated penetration solution was prepared, containing the target active ingredient at a total concentration reaching the minimum effective concentration, but 50% of which was inactivated product and 50% was the parent compound, meaning the parent compound concentration only reached 50% of the minimum effective concentration; the activity retention rate of this simulated penetration solution was determined using the methods in steps S4 and S6 of Example 1. The concentration of the simulated permeation fluid was compared with a preset retention rate threshold of 70%; simultaneously, the total concentration of the same simulated permeation fluid was determined using the traditional endpoint total concentration method to determine whether the effective concentration had been reached. False positive avoidance rate. Calculate using the following formula: ,in The number of samples that were determined to be ineffective for promoting penetration using this method. The total number of samples that were determined to be effective in promoting permeation (total amount meets the standard) using traditional methods was determined by conducting 20 parallel tests under this condition, with the unit being percentage. This value reflects the ability of this method to exclude false positive results caused by the concentration of inactivated products by judging the activity retention rate threshold. 100% indicates that this method can completely avoid misjudging samples that meet the total amount but have insufficient effective parent material as effective in promoting permeation.

[0121] Table 1: Performance test results of the evaluation methods for Examples 1-14 and Comparative Examples 1-13 Example 1 97.8 3.2 1.62 1.38 100 Example 2 96.9 4.1 1.55 1.31 95 Example 3 97.2 3.8 1.58 1.34 100 Example 4 97.0 3.9 1.56 1.32 100 Example 5 96.6 4.4 1.51 1.28 95 Example 6 97.4 3.5 1.59 1.35 100 Example 7 96.3 4.8 1.46 1.21 95 Example 8 96.5 4.6 1.49 1.24 95 Example 9 99.4 2.9 1.65 1.42 100 Example 10 95.3 4.7 1.43 1.18 95 Example 11 97.8 3.2 1.62 1.38 45 Example 12 97.8 3.2 1.62 1.38 100 Example 13 95.8 5.1 1.39 1.15 90 Example 14 95.6 5.3 1.41 1.17 90 Comparative Example 1 61.8 17.6 1.08 0.62 0 Comparative Example 2 69.5 10.8 1.14 0.71 20 Comparative Example 3 78.6 7.9 1.22 0.84 80 Comparative Example 4 96.9 22.4 1.50 1.26 0 Comparative Example 5 97.2 4.0 1.59 1.33 0 Comparative Example 6 87.4 9.6 1.13 0.76 65 Comparative Example 7 88.9 8.8 1.16 0.80 70 Comparative Example 8 94.2 5.9 1.45 1.16 90 Comparative Example 9 81.7 7.2 1.20 0.82 80 Comparative Example 10 97.8 3.2 1.62 1.38 0 Comparative Example 11 97.8 3.2 1.62 1.38 100 Comparative Example 12 85.6 10.3 1.10 0.70 60 Comparative Example 13 83.8 11.7 1.07 0.66 55 A comparison of Example 1 and Comparative Example 1 reveals the following: Comparative Example 1 uses the Franz diffusion cell method, with the total cumulative permeation over 24 hours as the sole evaluation indicator. It does not track concentration changes over time, thus failing to determine the starting point at which nicotinamide first exceeds the minimum effective concentration at the target depth, nor does it measure the activity retention rate. During penetration, nicotinamide molecules are hydrolyzed into nicotinic acid by nicotinamide enzymes in keratinocytes and simultaneously metabolized into N-methylnicotinamide by oxidases in fibroblasts. Both types of inactivated products can pass through the porous membrane into the receiving cell. The total cumulative permeation measured in the receiving cell is contributed by both the parent nicotinamide compound and the inactivated products, with the inactivated products no longer possessing anti-aging activity. This endpoint total method includes the permeation amount of inactivated products in the effective penetration-enhancing amount, causing the penetration-enhancing discrimination index to decrease from 1.62 to 1.08, and the false positive avoidance rate to decrease from 100% to 0%. It cannot reflect whether tetrahydropiperine truly helps the parent nicotinamide compound reach an effective concentration at the target site.

[0122] A comparison of Example 1 and Comparative Example 2 reveals that: Comparative Example 2 did not continuously monitor concentration changes at set time intervals, but only measured the cumulative penetration amount at a single moment 24 hours after cream application, thus failing to obtain a concentration-time curve and unable to determine the starting time point when the niacinamide concentration first exceeded the minimum effective concentration; under the action of tetrahydropiperine, the penetration process of niacinamide has time-dependent characteristics, migrating to deeper layers at a relatively rapid rate in the early stage after cream application, gradually accumulating at the depth of the efficacy target and first exceeding the minimum effective concentration; at the 24-hour endpoint, a considerable proportion of the niacinamide parent compound had been metabolized into N-methylniacinamide by the enzyme system in the skin analogue, significantly reducing the proportion of active ingredients in the total endpoint; continuous monitoring can accurately capture the starting time point when the concentration first exceeds the minimum effective concentration, while single endpoint measurement abandons information in the time dimension, with the time-dependent correlation decreasing from 1.38 to 0.71, the penetration discrimination decreasing from 1.62 to 1.14, and the false positive avoidance rate decreasing from 100% to 20%.

[0123] A comparison of Example 1 and Comparative Example 3 reveals that: Comparative Example 3 did not use the moment when the nicotinamide concentration first exceeded the minimum effective concentration as the evaluation anchor point, but instead used a fixed time point of 8 hours after applying the cream as the starting time point; the concentration accumulation of nicotinamide at the target depth follows a dynamic diffusion law, and the time required for it to first reach the minimum effective concentration depends on the diffusion coefficient of the nicotinamide molecule, the partition coefficient in the stratum corneum and epidermis, and the degree of change in intercellular lipid fluidity by tetrahydropiperine; the fixed 8-hour time point may be earlier or later than the actual moment of first exceeding the minimum effective concentration. If it is earlier, the measured concentration has not yet reached the minimum effective concentration, and the penetration enhancement efficacy index is underestimated; if it is later, nicotinamide has been partially metabolized into inactive products; using the actual moment of first exceeding the minimum effective concentration as the anchor point can keep the arrival time, the reached concentration, and the active state consistent. The fixed time point anchoring caused the activity retention deviation rate to increase from 3.2% to 7.9%, and the penetration enhancement discrimination index to decrease from 1.62 to 1.22.

[0124] A comparison of Example 1 and Comparative Example 4 reveals that: Comparative Example 4 only measured the total content of nicotinamide in the skin analogue layer at the starting time point, without separately measuring the content of the parent compound and each product, and without calculating the activity retention rate; during the penetration of the epidermis and dermis, some molecules of nicotinamide are converted into nicotinic acid and N-methylnicotinamide by enzymes in keratinocytes and fibroblasts, and these inactivated products are also present in the efficacy target layer; measuring only the total content includes all inactivated products in the effective concentration, making it impossible to determine whether the effective active ingredient is sufficient; Example 1, by separately measuring the content of the parent compound and each product, calculates the activity retention rate, which can accurately eliminate the interference of inactivated products, with an activity retention deviation rate of 3.2%; due to the lack of this measurement, the misjudgment avoidance rate of Comparative Example 4 decreased from 100% to 0%.

[0125] A comparison of Example 1 and Comparative Example 5 reveals that: Comparative Example 5 did not set an activity retention rate threshold and directly output the penetration enhancement efficiency index as the sole evaluation indicator; when the total amount of nicotinamide at the target depth reaches 1.5 times the minimum effective concentration due to the penetration enhancement effect of tetrahydropiperidine, if 60% of it has been metabolized into nicotinic acid, the actual effective concentration of the parent compound of nicotinamide is only 60% of the minimum effective concentration, which is insufficient to produce the expected anti-aging effect; Example 1 sets a preset retention rate threshold of 70%, and when the activity retention rate is less than this threshold, the penetration enhancement is deemed ineffective, and the false judgment avoidance rate reaches 100%; Comparative Example 5, lacking this judgment logic, has a false judgment avoidance rate of 0%, and cannot eliminate false positive results where the total amount meets the standard but the effective parent compound is insufficient.

[0126] A comparison of Examples 1 and Examples 2-6 reveals that the target active ingredients in Examples 1 to 6 are nicotinamide, tocopherol, decarboxylated carnosine hydrochloride, carnosine, psoralen, and bisabolol, respectively. The molecular weight, lipophilicity, ionization degree, and functional group structure of these different active ingredients vary, affecting their diffusion behavior under the action of tetrahydropiperine and their metabolic stability in skin analogues. Nicotinamide, with a molecular weight of 122 Da, is a small molecule amide, readily penetrating porous membranes. Psoralen, with a molecular weight of 256 Da, is a terpene phenolic compound with strong lipophilicity and a higher partition coefficient in the epidermis. Its penetration rate is slightly slower than that of nicotinamide, and its phenolic hydroxyl structure is easily catalyzed by oxidases to generate quinone inactivating products. While the indicators in Examples 2 to 6 show a slight decrease compared to Example 1, they all remain within the ranges of concentration curve consistency above 96%, activity retention deviation below 4.5%, penetration enhancement differentiation above 1.5, and time-effect correlation above 1.28, indicating that this evaluation method is applicable to different types of active ingredients.

[0127] A comparison of Examples 1 / 7-8 with Comparative Examples 6-7 shows that the seeding density of keratinocytes in Comparative Example 6 was 0.5 × 10⁻⁶. 5 pcs / cm 2 1×10 below the lower limit of the interval 5 pcs / cm 2 At this density, keratinocytes cannot form a continuous, fused monolayer on the porous membrane surface, resulting in insufficient tight junctions between cells and an incomplete epidermal barrier function. Nicotinamide molecules can then rapidly penetrate the dermis without effective obstruction from the keratinocyte layer, artificially advancing the time point at which the concentration at the target depth first exceeds the minimum effective concentration. This reduces the penetration-enhancing differentiation from 1.62 in Example 1 to 1.13. In Comparative Example 7, the inoculation density was 6 × 10⁻⁶. 5 pcs / cm 2 5×10 higher than the upper limit of the interval 5 pcs / cm 2 Excessive cell crowding leads to insufficient nutrient supply and enhanced contact inhibition, abnormal stratification and differentiation of keratinocytes, increased epidermal thickness and excessive density, increased nicotinamide penetration resistance, delayed onset time point, and the concordance rate of concentration curve decreased from 97.8% to 88.9%.

[0128] A comparison of Examples 1 / 9-10 with Comparative Examples 8-9 reveals that: the monitoring time interval affects the accuracy of capturing the concentration change trend; the concentration accumulation of nicotinamide at the target depth follows Fick's second diffusion law, and its concentration-time curve shows a rapid upward trend in the initial stage, gradually slowing down as it approaches the minimum effective concentration; in Comparative Example 8, the monitoring interval was 2 minutes, which is 5 minutes below the lower limit of the interval, and the concentration change amplitude between adjacent time points was less than the measurement noise of the detection instrument. Excessively dense sampling introduced redundant fluctuations, and it was difficult to complete the complete sampling and detection process within 2 minutes, resulting in accumulated measurement errors, and the consistency of the concentration curve decreased from 99.4% in Example 9 to 94.2%; in Comparative Example 9, the monitoring interval was 90 minutes, which is 60 minutes above the upper limit of the interval, and adjacent monitoring points may span the entire process of nicotinamide concentration from below the minimum effective concentration to significantly above the minimum effective concentration, making it impossible to accurately locate the first time it exceeded the minimum effective concentration, and the consistency of the concentration curve decreased from 95.3% in Example 10 to 81.7%, and the time-dependent correlation decreased from 1.18 to 0.82.

[0129] A comparison of Examples 1 / 11-12 with Comparative Examples 10-11 reveals that: the preset retention rate threshold affects the sensitivity and specificity of the judgment results; the activity retention rate reflects the proportion of the nicotinamide parent compound among all nicotinamide-related substances at the efficacy target level, and the higher the proportion, the greater the proportion of nicotinamide that retains anti-aging activity upon reaching the target; in Comparative Example 10, the preset retention rate threshold is 40%, which is lower than the lower limit of the interval 50%, so even if more than 60% of the nicotinamide has been metabolized into inactive products when it reaches the efficacy target, it is still judged as effective in promoting penetration, and the false judgment avoidance rate has decreased from 100% in Example 1 to 0%; in Comparative Example 11, the preset retention rate threshold is 95%, which is higher than the upper limit of the interval 90%, so nicotinamide inevitably undergoes trace metabolic transformation in the physiological environment of penetrating skin analogues, making it difficult to achieve a 95% retention rate, resulting in a large number of actually effective penetration-promoting results being judged as ineffective. Although the false judgment avoidance rate is still 100%, the positive detection rate is significantly reduced, reducing the actual distinguishing ability of the evaluation method.

[0130] A comparison of Examples 1 / 13-14 with Comparative Examples 12-13 reveals that the pore size of the porous membrane affects the structural integrity and material penetration pathway of the skin analogue. Nicotinamide molecules can pass through the porous membrane, while keratinocytes and fibroblasts cannot, ensuring layered co-culture of cells on both sides. In Comparative Example 12, the pore size was 0.1 μm, below the lower limit of 0.2 μm, increasing the diffusion resistance of nicotinamide molecules. Furthermore, during the gas-liquid interface culture stage, the excessively small pore size limited the supply of nutrients from the lower culture medium to the upper keratinocytes. In the case of insufficient differentiation of the epidermal layer, the consistency of the concentration curve decreased from 95.8% in Example 13 to 85.6%, and the time-dependent correlation decreased from 1.15 to 0.70. In Comparative Example 13, the pore size was 4.0 μm, which is higher than the upper limit of the interval 3.0 μm. During the gas-liquid interface culture, fibroblasts can migrate to the upper surface of the porous membrane through the excessively large membrane pores, which will destroy the complete layered structure of the epidermal and dermal layers. The penetration path of nicotinamide will be shortened, and the evaluation results will deviate from the actual percutaneous penetration process. The penetration-promoting discrimination index decreased from 1.41 in Example 14 to 1.07.

[0131] To compare the concentration changes before and after the addition of tetrahydropiperine, formulation A (without tetrahydropiperine) and formulation B (containing 0.255% tetrahydropiperine) were set up. Under the condition that the content of active ingredient and other formulation components were not changed, the concentration of the target active ingredient at the target depth was recorded at 0 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min and 420 min. Examples 2-6 used the corresponding target active ingredients, and the other conditions were the same.

[0132] To minimize the impact of different absolute concentrations of active ingredients, the concentration of Formulation A at 420 min was taken as 100%, and the relative concentrations at each time point were calculated. The relative concentration of formula A is calculated as follows: ; The relative concentration of formula B is calculated as follows: ; in, Monitoring time, in minutes. The permeation-promoting differentiation index corresponding to Examples 1-6 in Table 1; the 0.004 min -1 and 0.007min -1 The fixed fitting parameters are those under the conditions of no tetrahydropiperine and 0.255% tetrahydropiperine, respectively.

[0133] Based on the above normalization method and fixed fitting parameters, the relative concentrations of tetrahydropiperine and tetrahydropiperine were calculated for Examples 1-6, respectively, and the results are shown in Tables 2-7.

[0134] Table 2 shows the relative concentrations of nicotinamide before and after the addition of tetrahydropiperine. 0 0.0 0.0 60 25.0 48.0 120 55.0 105.0 240 105.0 138.0 420 118.0 145.0

[0135] Note: Nicotinamide's target layer for efficacy is the epidermis, with the lowest effective concentration at this layer considered 100%. Its migration characteristics are: after entering the epidermis, it continues to accumulate, gradually reaching a plateau after reaching the lowest effective concentration. The time to first reach the lowest effective concentration was 240 minutes in the group without tetrahydropiperine and 120 minutes in the group with 0.255% tetrahydropiperine. The data in the table are example values ​​used to illustrate the stratified evaluation logic; in actual applications, they should be replaced with the measured concentrations at the corresponding layers.

[0136] Table 3 shows the relative concentrations of tocopherol before and after the addition of tetrahydropiperine.

[0137] Note: The target layer for the efficacy of tocopherol is the stratum corneum, with the lowest effective concentration at this layer considered 100%. Its migration characteristics are: it mainly accumulates in the stratum corneum, reaches a plateau early, and then does not significantly increase to deeper layers. The time to first reach the lowest effective concentration was 120 minutes in the group without tetrahydropiperine and 60 minutes in the group with 0.255% tetrahydropiperine. The data in the table are example values ​​used to illustrate the stratified evaluation logic; in actual applications, they should be replaced with the measured concentrations at the corresponding layers.

[0138] Table 4 shows the relative concentrations of decarboxylated carnosine hydrochloride before and after the addition of tetrahydropiperine.

[0139] Note: The target layer for the efficacy of decarboxylated carnosine hydrochloride is the dermis, with the lowest effective concentration at this layer considered 100%. Its migration characteristics include a significant lag before reaching the dermis; tetrahydropiperidine primarily shortens the time to reach deeper layers. The group without tetrahydropiperidine did not reach the lowest effective concentration within 420 minutes, while the group with 0.255% tetrahydropiperidine reached it within 420 minutes. The data in the table are example values ​​used to illustrate the stratified evaluation logic; in actual applications, they should be replaced with the measured concentrations at the corresponding layers.

[0140] Table 5 shows the relative concentrations of carnosine before and after the addition of tetrahydropiperine. 0 0.0 0.0 60 3.0 8.0 120 10.0 24.0 240 28.0 62.0 420 52.0 102.0

[0141] Note: The target layer for carnosine's efficacy is the dermis, with the lowest effective concentration at this layer considered 100%. Its migration characteristics are: slower migration to deeper layers, making it difficult to reach the lowest effective concentration during the monitoring period without tetrahydropiperine. The group without tetrahydropiperine did not reach the lowest effective concentration for the first time within 420 minutes, while the group with 0.255% tetrahydropiperine reached it within 420 minutes. The data in the table are example values ​​used to illustrate the stratified evaluation logic; in actual applications, they should be replaced with the measured concentrations at the corresponding layers.

[0142] Table 6 shows the relative concentrations of psoralen before and after the addition of tetrahydropiperine. 0 0.0 0.0 60 12.0 25.0 120 34.0 68.0 240 70.0 124.0 420 88.0 112.0

[0143] Note: The target layer for psoralen's efficacy is the upper dermis, with the lowest effective concentration in this layer considered 100%. Its migration characteristics are: a peak concentration appears after reaching the upper dermis, followed by a slight decrease in concentration as it continues to migrate deeper. The group without tetrahydropiperine did not reach the lowest effective concentration within 420 minutes, while the group with 0.255% tetrahydropiperine reached it within 240 minutes. The data in the table are example values ​​used to illustrate the stratified evaluation logic; in actual applications, they should be replaced with the measured concentrations of the corresponding layers.

[0144] Table 7 shows the relative concentrations of bisabolol before and after the addition of tetrahydropiperine.

[0145] Note: The target layer for bisabolol's efficacy is the epidermis, with the lowest effective concentration at this layer considered 100%. Its migration characteristics are: rapid entry into the epidermis and formation of intralayer retention, followed by a plateau after reaching the effective concentration. The time to first reach the lowest effective concentration was 240 min in the group without tetrahydropiperine and 120 min in the group with 0.255% tetrahydropiperine. The data in the table are example values ​​used to illustrate the stratified evaluation logic; in actual applications, they should be replaced with the measured concentrations at the corresponding layers.

[0146] As shown in Tables 2-7, the concentration-time changes of different active ingredients at their corresponding efficacy targets are not the same; tocopherol mainly accumulates rapidly in the stratum corneum and enters the plateau relatively early; nicotinamide and bisabolol reach the effective concentration after entering the epidermis and form different degrees of intradermal retention; decarboxylated carnosine hydrochloride and carnosine have a significant lag before reaching the dermis, and it is difficult to reach the minimum effective concentration during the monitoring period without the addition of tetrahydropiperine; psoralen shows a peak after reaching the upper dermis, and then the concentration in that layer decreases slightly due to continued migration to deeper layers.

[0147] This demonstrates that simply using the cumulative permeation in the receiving area or a uniform endpoint concentration cannot distinguish whether different active ingredients reach their actual efficacy target level at the appropriate time. This method, by determining the time to first exceed the minimum effective concentration at each efficacy target level, can reflect the differentiated permeation-enhancing effect of tetrahydropiperine on different active ingredients. Based on the data in Tables 2-7, plots were generated respectively. Figures 2-7 .

[0148] Based on the preferred embodiments of the present invention described above, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for evaluating the transdermal penetration effect of active ingredients in an anti-aging face cream containing tetrahydropiperine, characterized in that, Includes the following steps: S1. Keratinocytes are seeded on the upper surface of a porous membrane, and fibroblasts are seeded on the lower surface of the porous membrane. They are then co-cultured to form a skin analog with a complete layered structure. S2. Apply the anti-aging cream containing tetrahydropiperine to the surface of the skin analogue, and continuously monitor the concentration of the target active ingredient at the depth of the target site over time, starting from the application time. S3. Based on the concentration-time curve, determine the starting time point at which the concentration of the active ingredient first exceeds its minimum effective concentration. S4. In the skin analogue layer corresponding to the starting time point, the content of the target active ingredient parent compound and each product is determined, and the activity retention rate of the layer is calculated. S5. Calculate the ratio of the concentration of the active ingredient at the starting time point to the minimum effective concentration as the penetration enhancement efficiency index, and record the activity retention rate; S6. If the activity retention rate is less than the preset retention rate threshold, the result of ineffective permeation promotion is output. If it is greater than or equal to the preset retention rate threshold, the permeation promotion efficiency index is output as the evaluation index of percutaneous permeation effect.

2. The method for evaluating the transdermal penetration effect of the active ingredient in an anti-aging face cream containing tetrahydropiperine according to claim 1, characterized in that: In step S1, keratinocytes are arranged at a ratio of 1×10 5 ~5×10 5 pcs / cm 2 Fibroblasts were seeded at a density of 0.5 × 10⁻⁶ on the surface of a porous membrane. 5 ~3×10 5 pcs / cm 2 The inoculation density was applied to the lower surface of the porous membrane.

3. The method for evaluating the transdermal penetration effect of the active ingredient in an anti-aging face cream containing tetrahydropiperine according to claim 1, characterized in that: In step S1, keratinocytes and fibroblasts are cultured at 35–38°C and 4–6% carbon dioxide in a liquid environment for 2–5 days, and then cultured at 35–38°C and 4–6% carbon dioxide at an air-liquid interface for 7–21 days to form a skin analog with an epidermal layer and a dermal layer.

4. The method for evaluating the transdermal penetration effect of the active ingredient in an anti-aging face cream containing tetrahydropiperine according to claim 1, characterized in that: In step S1, the porous membrane has a pore size of 0.2 to 3.0 μm. The porous membrane allows the culture medium and the target active ingredient to pass through, while preventing keratinocytes and fibroblasts from passing through.

5. The method for evaluating the transdermal penetration effect of the active ingredient in an anti-aging face cream containing tetrahydropiperine according to claim 1, characterized in that: In step S2, the target active ingredient is selected from vitamins, peptides, or terpenes; wherein the vitamins are selected from nicotinamide or tocopherol, the peptides are selected from decarboxylated carnosine hydrochloride or carnosine, and the terpenes are selected from psoralen or bisabolol.

6. The method for evaluating the transdermal penetration effect of the active ingredient in an anti-aging face cream containing tetrahydropiperine according to claim 1, characterized in that: In step S2, at a concentration of 1–10 mg / cm³ 2 The amount of anti-aging cream to be tested was applied to the surface of the skin analogue and monitored continuously for 2 to 12 hours at time intervals of 5 to 60 minutes; the mass fraction of the tetrahydropiperine in the anti-aging cream to be tested was 0.01 to 1.00%.

7. The method for evaluating the transdermal penetration effect of the active ingredient in an anti-aging face cream containing tetrahydropiperine according to claim 1, characterized in that: In step S3, the target active ingredient is applied to cells corresponding to the depth of the target site and cultured at 35-38°C for 12-48 hours. The concentration of the target active ingredient that makes the cells produce the preset anti-aging biological response for the first time is determined as the minimum effective concentration.

8. The method for evaluating the transdermal penetration effect of the active ingredient in an anti-aging face cream containing tetrahydropiperine according to claim 1, characterized in that: In step S4, the skin analog layer corresponding to the starting time point is separated at 0-10°C, and after adding the extraction solution, it is shaken and extracted for 10-60 minutes. The content of the target active ingredient parent compound and each product in the extract is determined. The activity retention rate is the ratio of the content of the target active ingredient parent compound to the total content of the target active ingredient parent compound and each product.

9. The method for evaluating the transdermal penetration effect of the active ingredient in an anti-aging face cream containing tetrahydropiperine according to claim 1, characterized in that: In step S6, the preset retention rate threshold is 50-90%; when the activity retention rate is less than the preset retention rate threshold, the result of the target active ingredient concentration reaching the minimum effective concentration is determined to be ineffective in promoting penetration.

10. The method for evaluating the transdermal penetration effect of the active ingredient in an anti-aging face cream containing tetrahydropiperine according to claim 1, characterized in that: The anti-aging face cream containing tetrahydropiperine comprises the following components by weight percentage: 2-15% polyol, 2-18% oil, 1-8% emulsifier, 0.1-1.0% thickener, 0.01-0.5% tetrahydropiperine, 0.03-10% active ingredients, 0.01-5% peptides, 0.01-5% plant extracts, and water to make up the balance.

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