Preparation,in Vitro And In Vivo Antioxidant Activity Of Kaempferol-loaded Liposome Hydrogel And Its Preliminary Treatment For Acute Eczema

Objective:The aim of this paper was to select a new dosage form to load kaempferol,to improve its poor water solubility,to enhance drug stability and to better exploit the pharmacological activity of the drug.In this paper,we selected liposome-hydrogel as the carrier of kaempferol drug,studied the prescription and preparation process of kaempferol-loaded liposome hydrogel,investigated its quality characterization and safety,and preliminarily investigated its in vitro and in vivo antioxidant activity and therapeutic effects on acute eczema in mice.Methods:（1）The standard curve of kaempferol was established by UV spectrophotometry,and its precision,reproducibility,stability and recovery were investigated for content determination.（2）The preparation of kaempferol-loaded liposome was screened by the magnitude of liposome encapsulation rate;the morphology of liposome was observed under optical microscope and cryo-electron microscope,and the particle size and potential of liposome were determined;the carbopol concentration was screened by the viscosity magnitude of blank hydrogel.（3）To prepare kaempferol-loaded liposome hydrogel according to the optimal prescription and process of liposome and hydrogel;to observe the appearance morphology of liposome hydrogel with the naked eye and their microstructure under cryo-electron microscope;to investigate the quality characterization of liposome hydrogel by viscosity,p H,spiking recovery and stability;to evaluate the safety of liposome hydrogel by acute and multiple administration skin irritation experiments.（4）In vitro drug release from kaempferol-loaded liposome hydrogel was investigated by Franz diffusion cell as well as kinetic modeling of four drug releases simulated by Origin software to investigate the drug release mechanism.（5）The DPPH method,H₂O₂scavenging assay and homogenization colorimetric method of mouse isolated tissues were used to investigate the antioxidant capacity of kaempferol-loaded liposome hydrogel in vitro and in vivo.（6）2,4-dinitrochlorobenzene was used to induce an acute eczema model in mice,and the result of severe redness and crusting on the back of mice on the fourth day indicated that the modeling was successful.The drug was applied from the 4 day for 12consecutive days.On the 17 day,we observed the skin lesions on the back of mice,measured the swelling of mice’s ears and the rate of swelling inhibition,recorded the number of mice scratching within 20 min,measured the spleen and thymus indices,performed histopathological observation on the skin of mice’s back,and measured the malondialdehyde content in mice’s skin and various tissues.Results:（1）The results of the content methodology experiments showed that kaempferol had a good linear relationship between 0.0125-0.20 mg/m L with R²=0.9995 and the RSD of precision,reproducibility and stability were less than 1%,indicating that the kaempferol solution was more stable and the recoveries were between 99.30%and99.73%.（2）The experimental results showed that the encapsulation rate was50.25±5.17%for the ethanol injection method and 84.33±6.02%for the film dispersion method,so the film dispersion method was chosen for the preparation of kaempferol-loaded liposome,which had the appearance of a yellow mixed-suspension liquid system with a slight soymilk odor.Under optical microscope and cryo-electron microscope,the liposome structure was spherical,the particle size was 161.2±18.5 nm and the zeta potential was-37.2±4.6 m V,the solution was more stable.The experimental results showed that the texture of 1%carbopol hydrogel was more uniform and delicate,colorless and transparent,and the viscosity was moderate,so 1%concentration of carbopol was selected to prepare kaempferol-loaded hydrogel,which had a light-yellow hydrogel appearance,viscosity of 3.2384±0.2967 m Pa·s,and good fluidity.（3）The kaempferol-loaded liposome hydrogel was a light-yellow hydrogel,uniform and fine,without granularity,and the internal structure was a round liposome ball with a regular shape under cryo-electron microscope.The results of characterization experiments showed that the viscosity of kaempferol-loaded liposome hydrogel was3.7075±0.2378 m Pa·s,which was moderate,with relatively good spreadability and easy to apply on skin.p H value was 7.344±0.542,and the spiked recovery was99.30%-99.45%,which was within the range specified in the pharmacopoeia.The results of stability experiments showed that kaempferol-loaded liposome hydrogel was stable under refrigerated conditions at 4°C,with no significant changes in appearance and shape,and little change in encapsulation efficiency.The results of skin irritation experiments showed that kaempferol-loaded liposome hydrogel was not irritating to the skin after acute or multiple transdermal administration,and it was a formulation with high safety.（4）The in vitro drug release results of 27.32±3.49%,70.89±8.91%and87.9±10.13%for kaempferol-loaded hydrogel at p H 3.4,5.4 and 7.4,respectively,indicate that it showed greater dissolution and drug release at higher p H（p H 7.4）,and its drug release mechanism in a weakly acidic environment was more suitable for the Higuchi kinetic model,while in neutral environment it was more suitable for first order kinetic model.The in vitro drug release rate of kaempferol-loaded liposome hydrogel at p H 7.4 was 49.05±6.46%,which was slower and acted as a slow-release control,and its drug release mechanism was also suitable for the first-order kinetic model.（5）The results of antioxidant experiments showed that kaempferol and kaempferol-loaded liposome hydrogel had strong scavenging ability for DPPH radicals and H₂O₂as well as strong inhibition of malondialdehyde production in isolated tissues of mice.The free radical scavenging ability showed a dose-effect relationship with the concentration of kaempferol,which was about 90%for DPPH radicals at 0.01 mg/m L and 50%for H₂O₂radicals at 0.5 mg/m L.The free radical scavenging and inhibition of malondialdehyde production of the blank formulation were weaker than those of the drug-containing formulation.The results of in vitro and in vivo antioxidant experiments show that kaempferol-loaded liposome hydrogel had strong antioxidant ability.（6）The experimental results of acute eczema treatment in mice showed thatcompared with the blank group,the mice in the model group showed obvious eczema pathological changes in the back skin,higher epidermal thickness and scratching times,higher spleen and thymus indices,obvious inflammatory cell infiltration,obvious edema between cells,severe skin damage,higher lesion scores,and high malondialdehyde content in all tissues.Compared with the model group,kaempferol-loaded liposome hydrogel had a therapeutic effect on acute eczema.The drug-treated group reduced the skin thickness and the number of scratching in mice,had lower spleen and thymus indices,reduced skin edema,less inflammatory cell infiltration,and lower levels of malondialdehyde in tissues.The results of all observed indices of acute eczema treatment indicated that kaempferol-loaded liposome hydrogel had strong anti-inflammatory effects.Conclusion:The kaempferol-loaded liposome hydrogel prepared in this paper was homogeneous and delicate with moderate viscosity,which can be used for topical skin administration.It not only had strong antioxidant effect in vivo and in vitro,but also had a therapeutic effect on acute eczema in mice with anti-inflammatory effect.