Use of rosmarinic acid and derivatives thereof as tight junction relaxants

Abstract

The invention discloses application of rosmarinic acid and a derivative thereof as a tight connection relaxation agent. The rosmarinic acid and the derivative thereof are competitively combined with the PDZ-1 structural domain of the ZO-1 protein, connection of Claudins protein between epithelial cells is reversibly relaxed, the ability of active substances penetrating epithelial tissues of skin, small intestines and the like is safely and efficiently improved, and the rosmarinic acid and the derivative thereof can be applied to the fields of medicines, foods, cosmetics and the like.

Description

Technical Field

[0001] The present invention relates to new uses of compounds, and specifically to the use of rosmarinic acid and its derivatives as tight junction relaxants, absorption promoters containing rosmarinic acid and its derivatives, and the use of rosmarinic acid and its derivatives in pharmaceutical compositions, food compositions and cosmetic compositions for promoting the passage of active substances through epithelial cells. Background Art

[0002] Tight Junctions are dynamic barrier structures between adjacent epithelial cells, widely distributed in physiological barrier tissues that require precise regulation of substance exchange, such as: intestinal epithelium, blood-brain barrier, renal tubular epithelium, corneal and conjunctival epithelium, as well as respiratory mucosa and skin active epidermis. They play a key role in regulating selective absorption of substances and defending against the invasion of external harmful substances. However, in various active substance delivery scenarios, the barrier function of tight junctions limits the paracellular diffusion behavior of active substances such as drugs, becoming a major obstacle to active substance delivery. Specifically, in the fields of drug delivery, such as oral administration, transdermal administration, nasal mucosa, pulmonary administration, and ocular administration, the tight junction barrier limits drug absorption. In the field of nutrient absorption, tight junctions in the intestinal epithelium directly affect the absorption efficiency of active substances in food by sealing the adjacent cell gaps. The tight junction barrier in the skin active epidermis hinders the transdermal delivery process of active substances such as whitening components and antioxidants.

[0003] Tight junctions are composed of transmembrane proteins and cytoplasmic attachment proteins, and their core protein components include the Claudin protein family, Occludin protein, and ZO-1 (Zonula Occludens-1) cytoplasmic attachment protein. Among them, the Claudin protein affects the permeability and selectivity of the barrier through its extracellular structure, and different subtypes are expressed in different tissues. For example, Claudin-1 is mainly present in the active epidermis of the skin, while Claudin-4 is mainly present in intestinal epithelial cells, and Claudin-5 is mainly expressed at the blood-brain barrier. Through this differential expression, the barrier requirements of different tissues are met. The transmembrane proteins Occludin and Claudin bind to the ZO-1 protein, and the latter further connects to the cytoskeletal actin to form a stable network structure. These proteins form a natural barrier through their interactions with each other, hindering the passage of active substances through epithelial cells.

[0004] To help the active substance better penetrate these epithelial cells, it is necessary to regulate the expression or interaction of tight junction-related proteins (such as Claudin, Occludin, ZO-1) to weaken the epithelial cell barrier. The existing mechanisms for relaxing the tight junction barrier can be summarized into three categories: 1) Reducing the expression of tight junction core proteins: By affecting intracellular signaling pathways (P13K / Akt, MAPK, NF-κB), the expression of tight junction proteins is reduced. This mechanism can enhance barrier permeability for a long time, but it will cause tissue damage. For example, homoharringtonine can relax the tight junction barrier by reducing the expression of Claudin-1 and Claudin-4, but it will also cause side effects (such as diarrhea, nausea, vomiting) due to tissue damage; 2) Indirectly affecting the interaction between tight junction proteins: By affecting the intracellular and extracellular Ca 2+ balance or affecting some kinases (Rho kinase, PKC kinase, myosin light chain kinase, PLC-γ kinase, c-Src and FAK), the interaction between tight junction proteins is indirectly affected. Although this mechanism can relax the tight junction barrier, it will cause side effects due to its overly broad action. For example, 3-nitrocoumarin can relax the tight junction barrier by inhibiting PLC-γ kinase, causing overphosphorylation of tight junction proteins. However, since PLC-γ kinase is involved in regulating cell proliferation and differentiation, inhibiting PLC-γ kinase will inhibit the tissue repair ability; 3) Directly affecting the interaction between tight junction core proteins: By directly binding to tight junction core proteins, the connection between tight junction core proteins can be reversibly disrupted. Therefore, this mechanism has the advantages of safety, reversibility, high specificity, etc. For example, a mutant polypeptide m19 of C-CPE (the C-terminal region of Clostridium enterotoxin) binds to Claudin proteins and directly disrupts the connection of Claudins between adjacent cells, promoting the efficient passage of active substances through intestinal epithelial cells.

[0005] When designing or using tight junction relaxants, not only the mechanism of action needs to be considered, but also the physicochemical properties and pharmacotoxicological properties of tight junction relaxants need to be considered. Taking two relaxants whose mechanism of action is to directly affect the interaction between tight junction core proteins as an example. If a polypeptide like m19 is selected as a tight junction relaxant, in practical applications, due to its large molecular weight (about 150 kDa), it is difficult to penetrate tissues such as the skin cutin layer, which will affect its reaching the tight junction to play a role, making it unable to be efficiently applied in the skin field. If a small molecule like diclofenac is selected, although it can directly relax the tight junction barrier by competing with Claudin proteins for binding to ZO-1 protein, due to its pharmacological characteristics (non-selective COX inhibition), while inhibiting the inflammatory response, it may cause a higher risk of gastrointestinal ulcer and bleeding. Summary of the Invention

[0006] Objective of the Invention: The objective of the present invention is to use rosmarinic acid and its derivatives as a tight junction relaxant to relax the tight junctions between epithelial cells and enhance the permeability of active substances in epithelial cells.

[0007] Technical Solution: Use of rosmarinic acid or its derivatives in the preparation of a tight junction relaxant.

[0008] For the above-mentioned use, the rosmarinic acid or its derivatives are selected from one or more of rosmarinic acid or its pharmaceutically acceptable salts, esters, amides, and glycosides.

[0009] For the above-mentioned use, the ester is selected from: the ester formed by the reaction of rosmarinic acid with C 1-10 alkyl-OH; the amide is selected from: the amide formed by the reaction of rosmarinic acid with C 1-10 alkyl-NH2; the glycoside is selected from: the compound formed by the connection of rosmarinic acid and glucoside through the reaction of rosmarinic acid with glucoside.

[0010] For the above-mentioned use, the reaction is an esterification reaction of -OH in C 1-10 alkyl-OH with one of the carboxyl group, the hydroxyl group at the 3-position or the 4-position of the benzene ring in rosmarinic acid, or an amide reaction of -NH2 in C 1-10 alkyl-NH2 with one of the carboxyl group, the hydroxyl group at the 3-position or the 4-position of the benzene ring in rosmarinic acid, or a dehydration condensation reaction of one of the carboxyl group, the hydroxyl group at the 3-position or the 4-position of the benzene ring in rosmarinic acid with glucoside.

[0011] For the above-mentioned use, the rosmarinic acid or its derivatives are derived from chemical synthesis or extraction from traditional Chinese medicine or plants.

[0012] For the above-mentioned use, it acts as a tight junction relaxant by relaxing the tight junctions between cells.

[0013] For the above-mentioned use, the relaxation of the tight junctions between cells specifically refers to the relaxation of the connections between Claudins proteins between epithelial cells.

[0014] Use of rosmarinic acid or its derivatives in the preparation of an absorption promoter or an absorption promoter composition; for enhancing the absorption of active substances by relaxing the tight junctions between cells.

[0015] Use of rosmarinic acid or its derivatives in the preparation of a pharmaceutical excipient or a drug delivery carrier; for promoting the delivery of active substances. Preferably, use of rosmarinic acid or its derivatives in the preparation of a pharmaceutical excipient or a drug delivery carrier for drugs absorbed through the skin and / or through the mucosa.

[0016] Use of rosmarinic acid or its derivatives in the preparation of a penetration enhancer for cosmetics or skin care products; for promoting the skin penetration of active substances.

[0017] Preferably, the rosmarinic acid or its derivative includes: rosmarinic acid, pharmaceutically acceptable salts of rosmarinic acid, rosmarinic acid-C 1～10 esters, rosmarinic acid-3-O-glucoside, rosmarinic acid-4-O-glucoside.

[0018] Preferably, the use of rosmarinic acid or its derivative in preparing an absorption promoter for calcein blue and paeoniflorin.

[0019] The rosmarinic acid and its derivatives enhance the permeability of active substances through epithelial tissues by relaxing the Claudin-mediated junctions between epithelial cells. Its mechanism of action is to directly bind to the ZO-1 protein, reversibly relax the junctions between Claudins by competitively inhibiting the interaction between the ZO-1 protein and the Claudin protein. Since all members of the Claudin protein family can bind to ZO-1, the rosmarinic acid and its derivatives can be applied to the tight junctions of various tissues containing Claudin proteins, such as the gastrointestinal tract, nasal mucosa, skin, etc.

[0020] The use of rosmarinic acid and its derivatives as an absorption promoter and an absorption promoter composition means using one or more of rosmarinic acid and its derivatives as an absorption promoter, or using them in combination with other penetration enhancers to enhance the permeability and absorption efficiency of active substances through biological barriers (such as skin, oral mucosa or nasal cavity).

[0021] The pharmaceutical composition containing rosmarinic acid and its derivatives means combining them as a tight junction relaxant with a drug active ingredient, excipient or carrier material to form a preparation with a specific therapeutic effect. The rosmarinic acid and its derivatives enhance the permeability of biological barriers (such as intestinal mucosa, skin barrier) by relaxing the tight junctions between cells, thereby improving the permeability and absorption efficiency of the drug active ingredient and enhancing the therapeutic effect. According to the treatment needs, the pharmaceutical composition can be made into various dosage forms, such as: cream, gel, patch, tablet, capsule, oral liquid, injection, freeze-dried powder injection, spray, inhalant.

[0022] The food composition containing rosmarinic acid and its derivatives means a food product formed by combining them as a tight junction relaxant with nutritional components, flavoring agents and additives. The rosmarinic acid and its derivatives enhance the intestinal permeability by relaxing the tight junctions between intestinal epithelial cells, promote the absorption and utilization of nutrients, and thus improve the nutritional value and health benefits of the food. The food composition can be of types such as foods for special medical purposes, functional beverages, nutritional supplements and dairy products.

[0023] A cosmetic composition containing rosmarinic acid and its derivatives refers to a skin care or beauty product with multiple effects formed by combining it as a tight junction relaxant with active ingredients, matrix components, and functional additives. Rosmarinic acid and its derivatives enhance skin permeability and improve the permeability and absorption efficiency of active ingredients by relaxing the tight junctions between cells in the stratum granulosum of the skin, thereby enhancing the overall efficacy of the product. According to requirements, the cosmetic composition can be made into various dosage forms, such as emulsions, microemulsions, gels, solutions, tinctures, films, ointments, creams, and patches.

[0024] Through long-term series of research, the applicant of this patent discovered the use of rosmarinic acid and its derivatives as tight junction relaxants from more than a thousand natural products, and demonstrated the following characteristics: 1) It can reversibly and directly affect the interaction between the core proteins Claudins of tight junctions, achieving safe and efficient relaxation of the tight junction barrier; 2) Rosmarinic acid is a natural polyphenolic compound widely present in edible plants such as rosemary, perilla, and sage. It can effectively scavenge free radicals and protect cells from oxidative stress damage. Therefore, it has good pharmacological and toxicological properties and extremely high safety; 3) The molecular weights of rosmarinic acid and its derivatives are only about 400 Da, and they can penetrate tissues such as the skin stratum corneum without restricting their access to the tight junctions to play their roles. Therefore, rosmarinic acid and its derivatives can not only be widely applied to oral, nasal mucosa, inhalation, ophthalmic administration and other routes, but also have high application potential in the skin field.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) Strong specificity: Rosmarinic acid and its derivatives can specifically bind only to the ZO-1 protein, and have no significant effect on the expression levels of Occludin protein and ZO-1 protein, indicating that their action has strong specificity. (2) Higher safety: Rosmarinic acid and its derivatives are widely present in plants such as rosemary, perilla, and sage. These plants are all edible plants, so they have extremely high safety. More importantly, their mechanism of relaxing tight junctions is reversible, which can improve permeability without damaging the structural integrity of tight junctions, significantly reducing the risk of adverse reactions. (3) Can be efficiently applied to the skin field: Since rosmarinic acid and its derivatives have good ability to penetrate the skin stratum corneum to reach the tight junctions of the viable epidermis, they can effectively play their roles at the skin tight junctions. Description of the Drawings

[0026] Figure 1 Shows the effects of rosmarinic acid, sodium rosmarinate, and methyl rosmarinate on the cell viability of in vitro HaCaT monolayers;

[0027] Figure 2Effects of rosmarinic acid, sodium rosmarinate, and methyl rosmarinate on the TEER of HaCaT monolayers in vitro;

[0028] Figure 3 Effects of sodium rosmarinate on tight junction proteins;

[0029] Figure 4 Enhancement effect of 5% sodium rosmarinate on the permeation of the model drug calcein blue at skin tight junctions;

[0030] Figure 5 Enhancement effect of 5% sodium rosmarinate on the permeation of paeoniflorin at skin tight junctions;

[0031] Figure 6 Enhancement effect of 5% sodium rosmarinate on the permeation of paeoniflorin. Specific Embodiments

[0032] The following will further clarify the content of the present invention in conjunction with test examples, but these examples do not limit the scope of the invention protected by the present invention.

[0033] Example 1

[0034] Preparation of Sodium Rosmarinate

[0035] Rosmarinic acid was mixed with a 0.1 mol / L sodium hydroxide standard solution in a 1:1 stoichiometric ratio. The resulting mixture was dissolved, and then the water was evaporated by heating in a water bath. The obtained precipitate was dried at 1 at 105°C for 24 h to obtain sodium rosmarinate.

[0036] Example 2

[0037] Preparation of Methyl Rosmarinate

[0038] To synthesize methyl rosmarinate, 1 g of rosmarinic acid was dissolved in 42 ml of methanol containing 1% HCl. The reaction was carried out at room temperature for 1 day. Then the solution was diluted with 83 ml of water and extracted three times with 83 ml of ether in a separating funnel. The combined ether solutions were washed in the order of water, 5% NaHCO3, and saturated brine. After drying the extract with anhydrous sodium sulfate overnight, the solution was evaporated to dryness to obtain methyl rosmarinate.

[0039] Example 3

[0040] Preparation of Decyl Rosmarinate

[0041] In a 100 mL dry ground round-bottom flask, 0.365 g of rosmarinic acid, 0.790 g of n-decanol, 43 mL of 1,4-dioxane solvent, and 0.430 g of p-toluenesulfonic acid as a catalyst were successively added. The molecular sieve was used as a water absorbent and was fully shaken and dissolved. After adding a reflux device, it was heated to boiling on an electric heating mantle. After a reaction time of 48 h, the molecular sieve was filtered off, and the filtrate was collected and vacuum concentrated until no solvent flowed out to obtain a concentrate. The concentrate was dissolved in ethyl acetate, and the organic phase was washed several times with distilled water. Then, 0.43 g of anhydrous magnesium sulfate was added to the ethyl acetate phase to dry and remove the trace water in the ethyl acetate phase. After drying for 12 h, the magnesium sulfate was removed by filtration, and the residue was washed 3 times with anhydrous ethyl acetate. The combined filtrates were vacuum concentrated to a small volume. After cooling, the ethyl acetate solution was slowly added to a triangular flask containing 30 mL of petroleum ether while stirring. After addition, it was allowed to stand for 24 h and then filtered. The crystals were washed three times with an appropriate amount of petroleum ether and then dried in vacuo to obtain a pale yellow solid, which was decyl rosmarinate.

[0042] Example 4

[0043] Molecular docking of rosmarinic acid and its derivatives with the PDZ-1 domain of ZO-1

[0044] Molecular structure preparation: The 2D structure file of rosmarinic acid was downloaded from the PubChem database and imported into the MOE software. In the main window of MOE, the rosmarinic acid molecule was selected and entered into the editing mode. The carboxyl group (-COOH) was located, the hydrogen atom in the carboxyl group was deleted, and a sodium ion was added through the software function to ensure the formation of an ionic bond between the sodium ion and the carboxyl oxygen atom, and the three-dimensional molecular structure of sodium rosmarinate was constructed and saved. The structures of sodium rosmarinate and decyl rosmarinate drawn, as well as the structures of rosmarinic acid, methyl rosmarinate, rosmarinic acid-3-O-glucoside, and rosmarinic acid-4-O-glucoside downloaded from the PubChem database, were imported into the molecular docking database. Receptor protein preparation: The crystal structure of the ZO-1 protein was downloaded from the RCSB Protein Data Bank, and the water molecules and redundant protein chains were deleted using the MOE software. The receptor protein was optimized through the "QuickPrep" function, including adding hydrogen atoms, assigning charges, and energy minimization. Molecular docking: In the docking program, the C-terminal structure of the Claudin protein was used as a reference to manually define the binding site. The database containing rosmarinic acid, sodium rosmarinate, and methyl rosmarinate just saved was selected as the ligand, and the docking parameters were set: the Triangle Matcher algorithm was selected, and LondondG and GBVI / WSAdD were used as the scoring functions, and 100 docking conformations were generated. The molecular docking program was run, and the docking scores and optimal conformations of each ligand were recorded. The results are shown in Table 1.

[0045] Table 1 Molecular docking scoring results of rosmarinic acid, sodium rosmarinate, methyl rosmarinate, etc.

[0046]

[0047] As can be seen from Table 1, the docking scores of rosmarinic acid, sodium rosmarinate, methyl rosmarinate, decyl rosmarinate, rosmarinic acid-3-O-glucoside, and rosmarinic acid-4-O-glucoside are <0, indicating that these ligands can spontaneously bind to the PDZ-1 domain of ZO-1 without external energy input. And the docking scores ≤ -5 indicate that the binding of these ligands to the PDZ-1 domain of ZO-1 is stable. From the molecular docking results, rosmarinic acid, sodium rosmarinate, methyl rosmarinate, decyl rosmarinate, rosmarinic acid-3-O-glucoside, and rosmarinic acid-4-O-glucoside can compete with Claudin protein for binding to ZO-1.

[0048] Example 5

[0049] Determination of the cytotoxicity of rosmarinic acid and its derivatives on skin cells

[0050] Human keratinocytes (HaCaT cells) were inoculated in DMEM medium (containing 10% fetal bovine serum) and cultured in an incubator at 37°C with 5% CO2. HaCaT cells in the logarithmic growth phase were taken to prepare a single-cell suspension, which was inoculated into a 96-well culture plate and cultured for 24 hours under the conditions of 5% CO2 and 37°C. The original medium was aspirated, and fresh serum-free medium and serum-free medium containing different concentrations (0 - 1200 μM) of rosmarinic acid, sodium rosmarinate, methyl rosmarinate, decyl rosmarinate, rosmarinic acid-3-O-glucoside, and rosmarinic acid-4-O-glucoside were added. Each concentration was set with 5 replicates, and the cells were continued to be cultured for 24 h under the conditions of 5% CO2 and 37°C. The medium was removed, 10% MTT solution (5 mg / mL) was added to each well, and the cells were incubated in the cell culture incubator for 4 h. The MTT solution was aspirated, 150 μL of dimethyl sulfoxide (DMSO) was added to each well, and the mixture was shaken in the dark on an oscillator for 20 min. The absorbance of each well was measured at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated. The absorbance of each well was measured in the same operation method after applying homoharringtonine (0 - 0.32 μM) as a positive control. The results are shown in Table 2 and Table 3. Figure 1 as shown.

[0051] Cell survival rate formula:

[0052] Among them, OD represents the absorbance value after the action of different concentrations of rosmarinic acid (sodium rosmarinate, methyl rosmarinate, decyl rosmarinate, rosmarinic acid-3-O-glucoside, rosmarinic acid-4-O-glucoside, homoharringtonine), and OD0 represents the absorbance value when the concentration of rosmarinic acid (rosmarinic acid, sodium rosmarinate, methyl rosmarinate, decyl rosmarinate, rosmarinic acid-3-O-glucoside, rosmarinic acid-4-O-glucoside, homoharringtonine) is 0.

[0053] Table 2 Results of HaCaT cell cytotoxicity assay ( n = 5)

[0054]

[0055]

[0056] Continued table of the results of HaCaT cell cytotoxicity assay in Table 2

[0057]

[0058] Note: Each group was compared with the cell viability (%) at a concentration of 0, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0059] It can be seen from Table 2 that between 0 and 200 μM, with the increase of the concentrations of rosmarinic acid, sodium rosmarinate, rosmarinic acid-3-O-glucoside and rosmarinic acid-4-O-glucoside, the viability of Hacat cells did not show a significant decrease. When the concentration reached 400 μM, the cell viability began to decrease significantly; between 0 and 50 μM, with the increase of the concentrations of methyl rosmarinate and decyl rosmarinate, the viability of Hacat cells did not show a significant decrease. When the concentration reached 100 μM, the cell viability began to decrease; between 0 and 0.16 μM, with the increase of the concentration of homoharringtonine, the cell viability did not change significantly. When the concentration continued to increase to 0.32 μM, the cell viability decreased significantly. According to the above results, it was determined that in the next experiment to verify the effects of these compounds on the permeability of monolayer cells and on tight junction proteins, the concentrations of rosmarinic acid, sodium rosmarinate, rosmarinic acid-3-O-glucoside and rosmarinic acid-4-O-glucoside should be less than 400 μM, and the concentrations of methyl rosmarinate and decyl rosmarinate should be less than 50 μM. And by comparing the experimental results of rosmarinic acid and its derivatives with those of homoharringtonine, it can be obtained that the safety of rosmarinic acid, sodium rosmarinate, methyl rosmarinate, decyl rosmarinate, rosmarinic acid-3-O-glucoside and rosmarinic acid-4-O-glucoside is much higher than that of homoharringtonine.

[0060] Example 6

[0061] Measurement of the resistance value during the action of rosmarinic acid and its derivatives on the Hacat cell layer

[0062] The cultured Hacat cells were inoculated on a Transwell culture plate at a density of 300,000 cells / well. 600 μl of DMEM medium was added to the basal side of each well, and 200 μl of cell suspension was added to the apical side. Then it was transferred to an incubator at 37 °C and 5% CO2 for incubation until the resistance of Hacat cells was greater than 150 Ω. The original culture medium was aspirated, and fresh serum-free medium and serum-free media containing 20 μM rosmarinic acid, 20 μM sodium rosmarinate, 20 μM methyl rosmarinate, 20 μM decyl rosmarinate, 20 μM rosmarinic acid-3-O-glucoside, and 20 μM rosmarinic acid-4-O-glucoside were added. Three replicate wells were set for each group. The trans-epithelial electrical resistance value (TEER value) of Hacat cells was measured at 4 h, 8 h, and 24 h of co-incubation at 37 °C and 5% CO2. After measuring the trans-epithelial electrical resistance value of Hacat cells at 24 h, the small pores were washed with PBS solution, and blank medium was added. The trans-epithelial electrical resistance value (TEER value) of Hacat cells was measured at 32 h and 48 h. The resistance value of the Hacat cell layer after administration of homoharringtonine (0.16 μM) was measured using the same operation method as a positive control. The results are shown in Table 3, Figure 2 as shown

[0063] Table 3 Resistance values during the action of rosmarinic acid and its derivatives on the Hacat cell layer ( n = 3)

[0064]

[0065] Table 3 Continued table of resistance values during the action of rosmarinic acid and its derivatives on the Hacat cell layer

[0066]

[0067] Note: Each group was compared with the resistance value at 0 h, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0068] As can be seen from Table 3, rosmarinic acid, sodium rosmarinate, methyl rosmarinate, decyl rosmarinate, rosmarinic acid-3-O-glucoside and rosmarinic acid-4-O-glucoside at safe concentrations can all reduce the trans-epithelial resistance value of Hacat cells. When the medium containing rosmarinic acid, sodium rosmarinate, methyl rosmarinate, decyl rosmarinate, rosmarinic acid-3-O-glucoside and rosmarinic acid-4-O-glucoside was removed and replaced with blank medium, the resistance value of Hacat cells increased rapidly. This proves that rosmarinic acid, sodium rosmarinate, methyl rosmarinate, decyl rosmarinate, rosmarinic acid-3-O-glucoside and rosmarinic acid-4-O-glucoside can enhance the permeability of Hacat cells, and the effects of rosmarinic acid, sodium rosmarinate, methyl rosmarinate, decyl rosmarinate, rosmarinic acid-3-O-glucoside and rosmarinic acid-4-O-glucoside on Hacat cells are recoverable. By comparing the resistance change processes of the three with that of homoharringtonine, the reversibility of the action mechanism of rosmarinic acid, sodium rosmarinate, methyl rosmarinate, decyl rosmarinate, rosmarinic acid-3-O-glucoside and rosmarinic acid-4-O-glucoside is better than that of homoharringtonine.

[0069] Example 7

[0070] Immunofluorescence staining experiment of Hacat cells

[0071] Hacat cells were inoculated into a 24-well culture plate at a density of 80,000 cells per coverslip, transferred to an incubator at 37 °C and 5% CO2, and incubated until the cells grew confluent. Then, 800 μL of the medium mixture containing sodium rosmarinate (20 μM) was added to each well and transferred to an incubator at 37 °C and 5% CO2 for incubation. After 12 h, the coverslips were collected, washed 3 times with PBS, fixed with methanol for 20 min, washed with PBS, and then blocked with goat serum at room temperature for 30 min. After adding the primary antibodies (1:500 polyclonal anti-Claudin-1 antibody, 1:500 polyclonal anti-ZO-1 antibody, and 1:500 polyclonal anti-Occludin antibody) for the cells, they were placed at 4 °C for 16 h. After washing with PBS, they were stained with the secondary antibodies (goat anti-rabbit 594, goat anti-mouse 488, and goat anti-mouse 555) and incubated in the dark at room temperature for 2 h. After washing with PBS, DAPI staining solution was added and incubated in the dark at room temperature for 10 min. After washing with PBS and waiting for the coverslips to dry, they were placed under a laser confocal microscope STELLARIS5 for observation. The results are as Figure 3 shown.

[0072] From Figure 3It can be seen that, compared with the blank control group without sodium rosmarinate, the change of Claudin-1 protein in the group with 20 μM sodium rosmarinate was significant. The distribution of Claudin-1 protein was shifted between cells, while the effects on ZO-1 protein and Occludin protein were not significant. This proves that the mechanism of rosmarinic acid and its derivatives in relaxing tight junctions is highly specific and will not affect other tight junction proteins.

[0073] Example 8

[0074] Promoting effect of sodium rosmarinate on the permeation of model drug calcein blue through tight junctions

[0075] Three rabbits were taken. After anesthesia with 20% urethane solution, the hair on the abdomen of the rabbits was removed with a hair clipper. Six pieces of skin with a size of 3 cm × 3 cm were drawn. The stratum corneum was removed with tape (since tight junctions in the skin mainly exist in the viable epidermis under the stratum corneum of the skin, in order to directly investigate the effect of sodium rosmarinate on skin tight junctions, the stratum corneum needs to be removed first before drug administration). Three of the six pieces of skin were randomly selected and treated with an OP-10 solution containing 5% sodium rosmarinate at a dose of 47 μl per piece of skin for 2 h; the remaining three pieces were treated with OP-10 solution in the same way. After the pretreatment, the residual drug on the skin was gently wiped off with a cotton swab. The rabbits were sacrificed, the abdominal skin was cut, the subcutaneous fat was removed, and it was rinsed clean with normal saline, put into a sealed bag, and stored in an ice bath for later use.

[0076] The excised abdominal skin of the rabbit was taken out from the ice bath and fixed on a Franz diffusion cell, with the viable epidermis layer facing the supply pool and the dermis layer facing the receiving pool. For the skin treated with an OP-10 solution containing 5% sodium rosmarinate, 600 μL of an OP-10 solution containing 5% sodium rosmarinate and 0.13 mM calcein blue was added as the supply solution on the side of the viable epidermis layer of the skin; for the skin treated with OP-10 solution, 600 μL of 0.13 mM calcein blue OP-10 solution was added as the supply solution on the side of the viable epidermis layer of the skin. Then, 7 ml of PBS was added to the receiving pool as the receiving solution, so that the dermis layer just contacted the liquid surface without bubbles. It was placed in a transdermal diffusion instrument and carried out under continuous stirring at 32 °C and 600 rpm. Samples of 1 mL were taken from the receiving pool at 1, 2, 4, 5, and 6 hours. Using a fluorescence spectrophotometer, the samples were detected under the conditions of an excitation wavelength of 354 nm and an emission wavelength of 441 nm. The fluorescence intensity of each sample was obtained and recorded, and the concentration of the sample was calculated according to its standard curve, and then the cumulative permeation amount of the drug was calculated. The results are shown in Table 4. Figure 4 as shown

[0077] Formula for calculating the cumulative permeation amount of the drug:

[0078] where: Q nCumulative permeation amount at the nth time point (μg / cm 2 ), V is the volume of the transdermal receiving solution (mL), A is the effective area of drug permeation (cm 2 ), C n is the concentration of calcein blue measured at the nth sampling time point (μg / mL), is the sum of the cumulative permeation amounts at the previous n - 1 time points.

[0079] Table 4 Cumulative permeation amount of calcein blue drug ( n = 3)

[0080]

[0081] Note: Compared with the blank control group, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0082] [[ID= (30)]]As can be seen from Table 4, the cumulative permeation amount of the sodium rosmarinate group at 6 h is 3.11 times that of the blank control group, indicating that the addition of 5% sodium rosmarinate can significantly increase the permeation amount of calcein blue at the skin tight junctions.

[0083] Example 9

[0084] ' Promoting effect of sodium rosmarinate on the permeation of paeoniflorin through tight junctions

[0085] Three rabbits were taken. After anesthesia with 20% urethane solution, the abdominal hair of the rabbits was removed with a hair clipper. Six pieces of skin of 3 cm × 3 cm were drawn. The stratum corneum was removed with tape (since the skin tight junctions mainly exist in the viable epidermis under the stratum corneum of the skin, in order to directly investigate the effect of sodium rosmarinate on the skin tight junctions, the stratum corneum needs to be removed first before drug administration). Three of the skins were randomly selected and treated with an OP - 10 solution containing 5% sodium rosmarinate at a dose of 100 μL per skin for 2 h; then the remaining three pieces were smeared with the OP - 10 solution in the same way. After the pretreatment, the residual drug on the skin was gently wiped off with a cotton swab. The rabbits were sacrificed, the abdominal skin was cut, the subcutaneous fat was removed, rinsed thoroughly with normal saline, placed in a sealed bag, and stored in an ice bath for standby.

[0086] Remove excised rabbit abdominal skin from the ice bath and mount it on a Franz diffusion cell, with the viable epidermis facing the donor cell and the dermis facing the receiver cell. For skin treated with OP-10 solution containing 5% sodium rosmarinate, add 600 μL of a 0.5% paeoniflorin solution in OP-10 containing 5% sodium rosmarinate to the viable epidermis side of the skin as the donor solution. For skin treated with OP-10 solution, add 600 μL of a 0.5% paeoniflorin solution in OP-10 to the viable epidermis side of the skin as the donor solution. Then, add 7 mL of PBS to the receiver cell, ensuring that the dermis is just in contact with the liquid surface and that there are no bubbles. The cells were placed in a transdermal diffusion instrument at 32°C and under continuous stirring at 600 rpm. Samples of 1 mL were taken from the receiver cell after 1, 2, 4, 5, and 6 hours. High-performance liquid chromatography (HPLC) was used under the following conditions: Luna C18 column (150 x 4.6 mm, 5 μm); mobile phase: methanol: 0.1% aqueous phosphoric acid (35:65, v / v); flow rate: 1.0 mL / min; detection wavelength: 230 nm; temperature: 30°C; injection volume: 20 μL. An external standard calibration curve method was used. Standard solutions ranging from 0.08 to 20 μg / mL were prepared and injected before transdermal injection. An external standard calibration curve was constructed, and sample concentrations were calculated based on this calibration curve. The cumulative permeation of each sample at different times was then calculated.

[0087] The formula for calculating the cumulative drug permeation amount is:

[0088] Where: Q n The cumulative permeation amount at the nth time point (μg / cm 2 ), V is the volume of transdermal receiving fluid (mL), A is the effective area of drug penetration (cm 2 ), C n is the concentration of paeoniflorin measured at the nth sampling time point (μg / mL), is the sum of the cumulative permeation at the first n-1 time points. The results are shown in Table 5. Figure 5 shown.

[0089] Table 5 Cumulative permeation of paeoniflorin ( n=3)

[0090]

[0091] Note: Compared with the cumulative permeation of blank control, * P<0.05, ** P < 0.01, *** P < 0.001, **** P<0.0001.

[0092] As can be seen from Table 5, the cumulative penetration amount of sodium rosmarinate group at 6 h is 5.36 times that of the blank control group, indicating that 5% sodium rosmarinate can significantly increase the permeation amount of paeoniflorin at the skin tight junctions.

[0093] Example 10

[0094] Promoting effect of sodium rosmarinate on percutaneous absorption of paeoniflorin

[0095] Three rabbits were taken. After anesthesia with 20% urethane solution, the abdominal hair of the rabbits was removed with a hair clipper, and 9 pieces of skin of 3 cm×3 cm were drawn. Randomly, 3 pieces of skin were treated with OP-10 solution containing 5% sodium rosmarinate and 5% azone at a dose of 100 μL per piece of skin for 2 h; another 3 pieces were smeared with OP-10 solution containing 5% azone in the same way, and finally the remaining 3 pieces were smeared with OP-10 solution in the same way. After 2 h of treatment, the residual drug on the skin was gently wiped off with a cotton swab, the rabbits were sacrificed, the abdominal skin was cut, the subcutaneous fat was removed, rinsed thoroughly with normal saline, put into a sealed bag, and stored in an ice bath for standby.

[0096] The excised abdominal skin of rabbits was taken out from the ice bath and fixed on a Franz diffusion cell, with the active epidermal layer facing the supply pool and the dermal layer facing the receiving pool. For the skin treated with OP-10 solution containing 5% sodium rosmarinate and 5% azone, 600 μL of 0.5% paeoniflorin OP-10 solution containing 5% sodium rosmarinate and 5% azone was added to the active epidermal layer side of the skin as the supply solution; for the skin treated with OP-10 solution containing 5% azone, 600 μL of 0.5% paeoniflorin OP-10 solution containing 5% azone was added to the active epidermal layer side of the skin as the supply solution; for the skin treated with OP-10 solution, 600 μL of 0.5% paeoniflorin OP-10 solution was added to the active epidermal layer side of the skin as the supply solution. Then 7 mL of PBS was added to the receiving pool as the receiving solution, so that the dermal layer just contacted the liquid surface without bubbles. It was placed in a transdermal diffusion instrument and carried out under continuous stirring at 32 °C and 600 rpm. 1 mL of sample was taken from the receiving pool at 1, 2, 4, 5, and 6 h. It was measured using a high performance liquid chromatograph under the following conditions: chromatographic column: Luna C18 column (150*4.6 mm, 5 μm); mobile phase: methanol: 0.1% phosphoric acid aqueous solution (35:65, V / V); flow rate: 1.0 mL / min; detection wavelength: 230 nm; temperature: 30 °C; injection volume: 20 μL.

[0097] The external standard standard curve method was adopted. Before injecting the transdermal sample, a standard solution of 0.08 - 20 μg / mL was prepared and injected, and the external standard standard curve was drawn. The concentration of the sample was calculated according to its standard curve, and then the cumulative penetration amount of each sample at different times was calculated.

[0098] Formula for calculating drug cumulative penetration amount:

[0099] Where: Q n The cumulative permeation amount at the nth time point (μg / cm 2 ), V is the volume of the transdermal receiving solution (mL), A is the effective area of drug permeation (cm 2 ), C n is the concentration of paeoniflorin measured at the nth sampling time point (μg / mL), is the sum of the cumulative permeation amounts at the previous n - 1 time points. The results are shown in Table 6, Figure 6 as follows.

[0100] Table 6 Cumulative permeation amount of paeoniflorin ( n = 3)

[0101]

[0102] Note: Compared with the cumulative permeation amount of the blank control, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0103] It can be seen from Table 6 that there is a significant difference between the cumulative permeation amount of paeoniflorin in the skin after applying azone and sodium rosmarinate for 6 h and that of the blank control group for 6 h, and it is 5.136 times that of the blank control group. The cumulative permeation amount of paeoniflorin in the skin after applying only azone for 6 h has a significant difference from that of the blank control group and is 2.681 times that of the blank control group. The experimental results prove that sodium rosmarinate can further increase the skin permeation amount of paeoniflorin.

Claims

1. Use of rosmarinic acid or its derivatives in the preparation of a tight junction relaxant.

2. The use according to claim 1, characterized in that, The rosmarinic acid or its derivatives are selected from one or more of rosmarinic acid or its pharmaceutically acceptable salts, esters, amides, and glycosides.

3. The use according to claim 2, characterized in that, The esters are selected from: esters formed by the reaction of rosmarinic acid with C 1-10 alkyl-OH; the amides are selected from: amides formed by the reaction of rosmarinic acid with C 1-10 alkyl-NH2; the glycosides are selected from: compounds in which rosmarinic acid is linked to glucoside formed by the reaction of rosmarinic acid with glucoside.

4. The use according to claim 3, characterized in that, The reaction is an esterification reaction of one of the carboxyl group in rosmarinic acid, the hydroxyl group at the 3-position or 4-position on the benzene ring with the -OH in C 1-10 alkyl-OH, or an amide reaction of the -NH2 in C 1-10 alkyl-NH2, or a dehydration condensation reaction of one of the carboxyl group in rosmarinic acid, the hydroxyl group at the 3-position or 4-position on the benzene ring with glucoside.

5. The use according to claim 1, characterized in that, The rosmarinic acid or its derivatives are derived from chemical synthesis or extraction from traditional Chinese medicine or plants.

6. The use according to claim 1, characterized in that, It acts as a tight junction relaxant by relaxing the tight junctions between cells.

7. The use according to claim 6, characterized in that, The relaxation of the tight junctions between cells specifically refers to the relaxation of the connections between Claudins proteins between epithelial cells.

8. Use of rosmarinic acid or its derivatives in the preparation of an absorption enhancer or an absorption enhancer composition.

9. Use of rosmarinic acid or its derivatives in the preparation of a pharmaceutical excipient or a drug delivery carrier.

10. Use of rosmarinic acid or its derivatives in the preparation of a penetration enhancer for cosmetics or skin care products.

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