Study On The Effects Of Different Nanoformulations Of Tanshinone ⅡA Combined With Glycyrrhetinic Acid On Acn

ObjectiveAcne is a chronic inflammatory disease which is always attacking people’s face,throracic and dorsal.The disease occurred an adolescence age,and has a long duration.It has serious influence to teenagers in their mental health and social activity.Tanshinone is ether extracts of salvia,it is always used to treat acne systematically in clinical.Tanshinone ⅡA(TSN ⅡA)is one of the effective components in Tanshinone capsule.Glycyrrhetinic acid(GA)is good at anti-inflammatory.Both of TSN ⅡA and GA have poorly water-solubility,which causes a poor oral bioavailability of these two drugs.In this research,TSN ⅡA and GA were embedded together in solid lipid nanoparticles(GT-SLNs)and oral emulsions(GT-Emul)to enhance the bioavailability and explore the better formulation for clinical use.MethodGA-TSN ⅡA solid lipid nanoparticles:GT-SLNs were prepared by high-speed shearingultrasonic emulsification with stearic acid(SA)and glycerol monostearate(GMS)as lipid.Firstly,low-speed centrifugation method was established to detect entrapment rate of GA and TSN ⅡA.And the recovery ration was tested to check method’s accuracy.Particles size and entrapment rate were detected as the index to choose the best temperature,power of ultrasonic,lipid content and ratio between SA and GMS to prepare a nice GT-SLNs by single-factor experiment.Then the central composite design-response surface method was hired to further optimize surfactant’s concentration,the ratio between GA/TSN and lipid.The optimized formula was used to prepared GT-SLNs,and freeze-drying condition was explored to lyophilize SLNs.GA-TSN ⅡA Emulsion:GT-Emul was prepared by the former research.The in vitro and in vivo research were evaluated in this study.Stability of gastrointestinal(GI)tract of GT-SLNs and GT-Emul was proceeded in simulated gastric fluid(SGF)and simulated intestinal fluid(SIF).After incubation in SGF and SIF,the residual content of GA and TSN was detected to evaluate SLNs and Emul’s protecting drugs from degrading by protease in GI tract.Then the drugs’ in vitro release was evaluated by reverse dialysis.The pharmacokinetics of GT-SLNs was carried out in mice,and compared with GA-TSN ⅡA mixed suspension(GT-Mix),to study the effect of GT-SLNs on oral bioavailability of GA and TSN ⅡA.The effect on acne was evaluated after oral administration.The hormone level of mice,sebaceous gland tissue and ear swelling level were selected as index.Mice were divided into five groups,they are blank control group,GT-SLNs group,low dosage GT-Emul group,high dosage GT-Emul group and positive control group.The serum was taken after oral administrated 10 days to detected level of testserone and estradiol by ELISA method.And the glandula sebacea tissues were collected to cut into slices and observe.Ear swelling models were prepared by apply dimethylbenzene on mice’s right ear after oral administrated 10 days.After molding 45min,cut mice’s ear tissues off and weighing to calculate ear swelling level.ResultsThe entrapment rate’s determination is 2000 rpm centrifugal for 10 min.The recovery ration of GA and TSN ⅡA was-0.07%and 2.75%respectively,which was showed the method could desperate free drugs from SLNs.Preparation conditions and recipe selected by single-factor experiment:water base temperature was 85℃,power of ultrasonic was 250 W,lipid content was 2%,SA:GSM=9:1(m/m).The optimized formula by central composite design-response surface method was:surfactant concentration was 6.5%,GA’s dosage was 5.4 mg,TSN ⅡA’s dosage was 3.2 mg.And the lyophilization protector was 15%trehalose and D-mannitol solution(m/m=1:1).The particle size and Zeta potential of GT-SLNs were 250.5 nm and-27.7 mV respectively,and GT-Emul’s were 188.0 nm and-11.7 mV respectively.The in vitro stability of GI tract showed that after incubation,GA and TSN ⅡA’s residual content of GT-SLNs in SGF was 99.24%and 97.36%respectively,in SIF was 95.23%and 95.99%respectively;GA and TSN ⅡA’s residual content of GT-Emul in SGF was 95.61%and 95.84%respectively,in SIF was 93.67%and 94.65%respectively.Although there is no significant difference between GA’s residual content of GT-SLNs,GT-Emul and GT,Mix group,TSN ⅡA’s residual content of SLNs and Emul was higher than Mix group’s significantly,that was indicated that SLNs and Emul could protect loaded-drugs from proteases and gastric acid’s degradation.The cumulative release rate in 24h of GA and TSN in GT-SLNs group was 65.87%and 36.13%respectively,the GT-Emul group was 7.88%and 7.64%respectively.The pharmacokinetic study showed that TSN’s AUC0-t and AUC0-∞ of GT-SLNs were 0.98 time and 3.77 time higher than GT-Mix’s after oral administration.The Cmax of TSN in SLNs was 17.2-fold compared with TSN in GT-Mix.These indicated that SLNs can enhance TSN’s oral absorption.And TSN’s Tmax of SLNs was 6.1 h earlier than GT-Mix’s.But GA’s AUC in SLNs was less than GT-Mix’s.As the pharmacodynamics results showed that after oral administration with GT-SLNs,GT-Emul high and low dose,the testosterone in mice’s serum had decreased obviously.The ratio of testosterone to estradiol in GT-SLNs,GT-Emul high and low dose group had decreased too.This result inferred that oral administration with GT-SLNs,GT-Emul high and low dose can balance mice’s male hormone and female hormone level in a short-term,and the effect was better that Jinhua Xiaocuo Wan.From the observation of sebaceous gland tissue,the glandular body of GT-SLNs,GT-Emul high and low dose group had shrunk,which inferred that there three formulations could inhibit glandula sebacea’s proliferation.Ear swelling models anti-inflammatory study showed that Jinhua Xiaocuo Wan and high dose GT-Emul have the good anti-inflammatory effect on ear swelling caused by dimethylbenzene.