Mechanism Of Interaction Between Berberine And Paeoniflorin By Percutaneous Absorption And Pharmaceutical Studies

Objective:In this paper,we intend to study the interaction law and mechanism of percutaneous absorption of berberine hydrochloride and paeoniflorin,and select microemulsion gel as a carrier for formulation design and systematic evaluation.Methods:1.Study on the interaction law of percutaneous absorption of paeoniflorin and berberine hydrochloride:The percutaneous permeation kinetic parameters of paeoniflorin and berberine hydrochloride in two-layer skin model were investigated by the ex vivo method,and the rate-limiting factors of percutaneous absorption were clarified;by measuring the solubility of drugs in different solvents and selecting the acceptor solution that meets the conditions of leakage tank,while using the abdominal skin of isolated mice as the transdermal barrier,the modified Franz diffusion cell method was applied,the transdermal permeability of berberine hydrochloride and paeoniflorin in different p H solutions was investigated,and the effects of combining berberine hydrochloride and paeoniflorin at different concentrations on the Q₂₄and ER of berberine hydrochloride and paeoniflorin were examined under the optimal p H conditions to determine the optimal ratio and explore the law of permeation promotion.2.The mechanism of percutaneous absorption interaction between berberine hydrochloride and paeoniflorin was studied:the change of oil-water partition coefficient（Log P）before and after the combination of paeoniflorin and berberine hydrochloride was determined by shaking flask method;the effect of berberine-paeoniflorin hydrochloride on the structure of skin stratum corneum was determined by scanning electron microscopy（SEM）;laser confocal microscopy（CLSM）was used to observe the penetration depth and fluorescence intensity of hydrophilic sodium fluorescein and lipophilic Nile red dye in mouse;fourier transform infrared spectroscopy（FT-IR）was used to confirm whether paeoniflorin and berberine hydrochloride formed ionic pairs after pairing;the interaction mechanism between paeoniflorin,berberine hydrochloride and lipid components of the stratum corneum was studied with the aid of Auto Dock software.3.Study on the preparation process and quality evaluation of paeoniflorin-berberine hydrochloride microemulsion:Based on the equilibrium solubility of berberine hydrochloride in the microemulsion components under the conditions of primary screening,each phase of the microemulsion and its approximate range were determined by inter-component pairing experiments and the size of the area of the pseudo-ternary phase diagram,and the Box-Behnken effect surface method was used with the percent mass ratio of the oil phase（X1）,the percent mass ratio of the mixed emulsifier（X2）,the mass ratio of emulsifier and coemulsifier（Km）（X3）as independent variables,and the particle size（Y1）and PDI（Y2）as investigation indexes,the three-factor and three-level design was carried out to optimize the optimal excipients and the ratio;The pharmacological characteristics such as morphological characteristics and particle size of paeoniflorin-berberine hydrochloride microemulsion were studied by using nano particle size meter,p H meter,transmission electron microscope,conductivity meter,UV spectrophotometer and other instruments.4.Preparation of paeoniflorin-berberine hydrochloride microemulsion gel and its in vitro evaluation:the gel matrix was screened for formability and spreadability,the microemulsion and gel were mixed by three different methods to select the system that could form a clear and transparent homogeneous system,the concentration of the gel matrix was selected according to the required fluidity of the preparation,and the optimal prescription was determined;The Q₂₄and Q_Sof saturated aqueous solution,microemulsion,microemulsion gel and cream with the same drug load were compared by using Franz diffusion cell;SD rats were taken as the study subjects.After back hair removal,the prepared blank microemulsion gel group and paeonidin-berberine hydrochloride microemulsion gel group were administered transdermal,and the safety of their skin medication was observed by visual scoring and HE staining as the evaluation index.Results:1.The Q₂₄of berberine hydrochloride in the cuticle was 507.66±59.9418μg·cm^-2,and the Q₂₄in the whole skin was 256.06±54.1682μg·cm^-2,showing a significant difference（P<0.001）.The cuticle is the percutaneous rate-limiting barrier of paeoniflorin,which has long been confirmed in the literature.With 20%ethanol-p H 7.4 phosphate buffer solution（PBS）as the receiving solution,the best transdermal effect of berberine-paeoniflorin hydrochloride was achieved under the medium condition of p H 5.0,and under this condition,the Q₂₄of paeoniflorin increased by 78.03%,44.73%,and 35.63%in the high,medium,and low concentrations of paeoniflorin paired with berberine hydrochloride,respectively,with the Q₂₄of paeoniflorin in the high concentration paired group Q₂₄of paeoniflorin was significantly different（P<0.05）compared with the paeoniflorin alone administration group,and the transdermal rate was also significantly increased（ER=1.74）,while the Q₂₄of berberine hydrochloride was also increased after the combination of berberine hydrochloride with different concentrations of paeoniflorin.2.The results of the shaking flask method showed that the Log P tended to increase after the combination of berberine hydrochloride and paeoniflorin,resulting in enhanced lipid solubility,which may be one of the reasons for the small promotion of transdermal absorption of berberine hydrochloride after the combination of berberine hydrochloride and paeoniflorin;SEM results showed that after drug treatment,the imbriculate structure of the stratum corneum was loosened to a certain extent,forming some small interstitial cracks,and the surface folds were significantly increased and deepened,similar to the skin soaked with 3%azone;LCSM results showed that the combination of berberine hydrochloride and paeoniflorin promoted the greater penetration of hydrophilic sodium fluorescein and lipophilic nile red in the skin,which made the fluorescent dye transport to deeper skin tissues and indirectly fed back the effect of berberine hydrochloride on skin permeability;FT-IR results showed that the absorption peak of paeoniflorin combined with berberine hydrochloride at 3430cm^-1was stronger than that of Paeoniflorin alone,and a wider absorption band was not very obvious in the range of 2900-1700cm^-1,which may be caused by the oscillation of protons in the stronger hydrogen bond between the two species that form hydrogen bonds.This feature indicates that hydrogen bonds are over-polarized,that is,hydrogen bonded ion pairs may be formed;using Auto Dock Vina software,the intrinsic mechanism of the more significant interaction between berberine hydrochloride and paeoniflorin was investigated at the molecular mechanism level,where berberine hydrochloride could competitively interact with the polar head of the lipids of the stratum corneum,while freeing paeoniflorin from the hydrogen-bonding network,thus promoting the transdermal absorption of paeoniflorin.3.Optimal prescription:Peceol:MCT（1:1,4.188%）,RH-40:anhydrous ethanol（1.349:1,27.184%）,water（68.628%）,the maximum loading of berberine hydrochloride was（2.33±0.06）mg·ml^-1,the loading of paeoniflorin was 30 mg·ml^-1,the appearance of the resulting microemulsion was clarified.The particle size was（21.54±0.14）nm,PDI was（0.217±0.03）,zeta potential was（-0.168±0.016）m V,p H was（5.33±0.06）,spherical shape under transmission electron microscope,transmittance was 93.11±0.3（%）,good stability,no precipitation and delamination after three months.4.The optimal prescription was:1%carbomer 940 was evenly spread on the surface of the prepared drug-laden microemulsion,swelling overnight,stirred evenly and then triethanolamine was added to adjust the p H to 6-7,which resulted in the drug-laden microemulsion gel.The prepared paeoniflorin-berberine hydrochloride microemulsion gel had a yellow clarified and transparent appearance,p H（6.44±0.13）,viscosity of 91.025 Pa·s,sphere-like shape under transmission electron microscopy,and good rheological properties.The Q₂₄of berberine hydrochloride in microemulsion and microemulsion gel was 1.99 and 1.35 times of saturated aqueous solution,respectively,and the content of berberine hydrochloride in cream was lower than that of saturated aqueous solution.The Q₂₄of paeoniflorin in microemulsion and microemulsion gel was 1.04 and 1.02times of saturated aqueous solution,respectively,and the transdermal permeability of paeoniflorin in the cream was better than the most.Berberine hydrochloride Qs in microemulsion and microemulsion gel were 2.40 and 1.73 times more than saturated aqueous solution,respectively,and paeoniflorin Qs were 1.71 and 2.23 times more than saturated aqueous solution,respectively,indicating that the prepared microemulsions and their gels enhanced the reservoir effect of the skin.The skin irritation response score showed no skin irritation by microemulsion gel either by single administration or multiple administrations,and the pathological histological results showed no significant changes in the skin tissue sections of the blank microemulsion gel group and the paeoniflorin-berberine hydrochloride microemulsion gel group compared with normal skin,both of which exhibited basically normal overall skin tissue structure,showing intact keratinized epidermis and follicular structure of the dermis,no destruction of skin accessory structures,no significant thickening of the stratum corneum,tightly arranged and normal morphology of collagen fibers in the dermis,and no obvious inflammatory cell infiltration.Conclusion:The stratum corneum is the percutaneous rate-limiting barrier of paeoniflorin and berberine hydrochloride.The optimal p H of paeoniflorin and berberine hydrochloride transdermal administration is 5.0.In p H 5.0 medium,paeoniflorin combined with berberine hydrochloride transdermal administration can promote the transdermal absorption of paeoniflorin,and it is concentration dependent,and also has a certain promoting effect on the transdermal absorption of berberine hydrochloride.The interaction mechanism was confirmed by shaker method,SEM,CLSM,FT-IR and molecular docking techniques.On this basis,paeoniflorin and berberine hydrochloride were prepared into microemulsion and its gel.Combined with the cumulative penetration and skin retention,the percutaneous penetration and skin reservoir effect of paeoniflorin and berberine hydrochloride in microemulsion gel were more superior and could be considered as safe and non-irritating,and could be used for transdermal drug delivery system.