Preparation Of Ligustrazine Hydrochloride-loaded Liposome-gels And Evaluation Of Its Transdermal Administration For The Treatment Of Skin Photoaging

Objective:As people’s quality of life improves,they are increasingly concerned about the problems of oxidative damage and aging.Oxidative damage not only ages the skin but also causes diseases;therefore,antioxidant is especially important.Ligustrazine hydrochloride was used as a raw material to study the antioxidant activity of its preparation.After a certain route,ligustrazine hydrochloride was prepared into liposome-gels,the best prescription of ligustrazine hydrochloride-loaded liposome-gels was screened,and a complete in vitro and ex vivo antioxidant activity examination system was established.The properties of stability,skin irritation,drug‘s release characteristics of transdermal administration and in vitro antioxidant activity of ligustrazine hydrochloride-loaded liposome-gels were examined to explore the feasibility of preparing ligustrazine hydrochloride into liposome-gels for transdermal administration to exert preventive and treatment of skin photoaging,and to provide a new basis for transdermal administration of ligustrazine hydrochloride-loaded liposome-gels for the treatment of skin oxidative diseases.Methods:Solutions of different concentrations of ligustrazine hydrochloride were selected,their absorbance was recorded at the characteristic wavelength of 295 nm with an UV spectrophotometer,and established a standard curve with the concentration as the horizontal coordinate and the absorbance as the vertical coordinate.Ligustrazine hydrochloride-loaded liposomes were prepared by film dispersion method using soybean particulate phospholipids and cholesterol,and the stability of liposomes was investigated.Morphological features of liposomes were viewed with optical microscopes and cryo-electron microscopy,measured the particle size and zeta potential by a zeta potential and particle size analyser,and measured the encapsulation efficiency of the liposomes by ultracentrifugation.By using sodium carboxymethyl cellulose as the gels matrix,the natural swelling method was used to prepare ligustrazine hydrochloride-loaded gels,the appearance characteristics of gels with different matrix contents were examined and their viscosities were measured by an ostwald-viscosimeter to select the gels formulation with the best sodium carboxymethyl cellulose content.A combination of film dispersion and natural swelling methods was used to prepare ligustrazine hydrochloride-loaded liposome-gels,and their morphology was observed using cryo-electron microscopy,and their skin irritation properties were investigated.The absorbance values of the drug released at different times were measured using Franz diffusion cell with dialysis membrane and mice skin as the barrier and phosphate buffer as the dissolution medium,calculated the amount of drug released to investigate the release rate（R%）of ligustrazine hydrochloride,liposomes,gels and liposome-gels,the R%is the ratio of the actual amount of drug released to the theoretical amount of drug released,which was used to examine the penetration effect of drug through bio-dialysis membranes and mice skin,and to provide a basis for the transdermal administration of ligustrazine hydrochloride-loaded liposome-gels.Different concentrations of ligustrazine hydrochloride solution and its preparations were reacted with DPPH solution and H₂O₂ solution to observe the changes of color and absorbance value,and the absorbance value was used to calculate the elimination rate（E%）and to investigate the antioxidant effect of the drug.It used the red colouring principle of malondialdehyde and thiobarbituric acid in high temperature acidic conditions,the supernatant of liver homogenate of mice and the low-density lipoprotein in the blood of mice were extracted,which were made to react with the drug,after which its absorbance was measured at the specific absorption wavelength of 532 nm of the reaction product and the inhibition rate（I%）of malondialdehyde was calculated to investigate the antioxidant effect of the drug ex vivo.The experimental mice were irradiated with a home-made UV light box for a long period of time.The experiment was started with a minimum erythematous dose and then increased by one minimum erythematous dose per week.The drug was applied daily before the mice were exposed to UV light and the skin thickness of the back of the mice was measured once a week for a total of eight weeks.At the end of the experiment,the skin condition of the mice was observed,the skin of the mice was sectioned and stained with HE,the spleen and thymus indices of the mice were calculated,the content of malondialdehyde in the liver homogenate and low-density lipoprotein extract of the mice was measured,the activity value of superoxide dismutase and the content of malondialdehyde in the skin of the mice were measured,and the therapeutic effect of ligustrazine hydrochloride-loaded liposome-gels on skin photoaging was preliminarily evaluated.Result:（1）Six concentrations in the range of 3.0–15.0μg/m L were selected to prepare the standard curves.The correlation coefficient R² was 0.9999,and the curve equation was A=0.0367C+0.0009.（2）It was found that the best liposomes encapsulation rate was 75.05±6.37%when the ratio of phospholipid to cholesterol was 3:1;particle size of liposomes was118±10.61 nm,and zeta potential of liposomes was-39.3±3.7 m V.When screening gels with different matrix contents,it was found that 2%sodium carboxymethylcellulose prepared the best liposome-gels with the most suitable flow and viscosity,uniform and fine state,which can be well applied on the skin without irritation.（3）The results of release curves showed that the R%of the preparations were less than that of the raw material,the R%of the ligustrazine hydrochloride-loaded liposome-gels was the lowest,R%_LTH>R%_LTH-L≥R%_LTH-G>R%_LTH-LG;It indicated that the liposome-gels exerted a double effect of slow and controlled release of the drug,compared to the raw material and other preparations,ligustrazine hydrochloride encapsulated in the liposome-gels released slowly,so the R%was lower for the same time.（4）The E%of ligustrazine hydrochloride eliminated DPPH free radical was enhanced with an increase concentration.The increase of reaction time will also increase the E%;the liposomes with the same concentration as the raw material had a greater DPPH elimination effect,and the gels almost had no elimination effect on DPPH radical,E%_LTH>E%_LTH-L>E%_LTH-LG>E%_LTH-G.The reaction of ligustrazine hydrochloride with H₂O₂ solution showed the E%was enhanced with increase concentration,and the E%for the same concentration of raw material and preparations were as follows,E%_LTH-L>E%_LTH-LG>E%_LTH-G>E%_LTH.The mice liver homogenate was taken as the experimental object.The ligustrazine hydrochloride and its preparations had a certain inhibitory effect on the production of malondialdehyde,and the I%was in the order of I%_LTH>I%_LTH-L>I%_LTH-LG>I%_LTH-G.The blood of mice was taken as the experimental object,and it was found that the raw material and preparations could inhibit the oxidation of low density lipoprotein extracted from blood to malondialdehyde,and the I%as follows,I%_LTH>I%_LTH-L>I%_LTH-LG>I%_LTH-G.（5）The results of the photoaging experiment showed that the liposome-gels of ligustrazine hydrochloride had certain therapeutic effects on the photoaging of mice skin;the HE staining,the increase of skin thickness,the degree of skin damage,the superoxide dismutase activity value and malondialdehyde content in the skin,and the malondialdehyde content in the liver homogenate and low density lipoprotein extract were measured,and it was concluded that the mice in the model group had the most serious skin damage,and compared to these,other group mice skin damage symptoms were reduced,and the increase of skin thickness was not obvious.HE staining showed that the thickening of the stratum corneum was the most obvious in the model group,and that the thickening of the stratum corneum was the least in the mice coated with ligustrazine hydrochloride-loaded liposome-gels;the superoxide dismutase activity value and malondialdehyde content were not as high as those in the model group.The results of all the experiments together can be tentatively concluded that the ligustrazine hydrochloride-loaded liposome-gels had the best therapeutic effect on skin photoaging in mice,and it was feasible to prepare ligustrazine hydrochloride in the form of liposome-gels for the prevention and treatment of skin oxidation through dermal administration.Conclusion:The evaluation indexes of the ligustrazine hydrochloride-loaded liposome-gels were all in accordance with the standards,and it was not irritating to the skin,and the transdermal release of the drug was also good;Ligustrazine hydrochloride had good antioxidant effect,and prepared it into liposome-gels could also exert certain antioxidant effect,and the liposome-gels also had the effect of slow and controlled release,which can release the drug slowly and continuously,which preliminary indicated that it was feasible to prepare liposome-gels of ligustrazine hydrochloride and administration through the skin to exert antioxidant effects for the treatment of skin photoaging.