Investigation Of Salvianolic Acids Multiple Emulsion Drop Pills

Salvianolic acids are an effective and safe drug for the treatment of cerebral apoplexy.However,owing to the low oral bioavailability and rapid metabolism of the water-soluble components,salvianolic acids have to date only been administered intravenously in clinical treatments.Nonetheless,the intravenous administration route possesses certain drawbacks,such as that it is painful for the patient and time consuming for the medical personnel and can only be used in a hospital setting.Therefore,this paper reports on the preparation of an oral formulation,salvianolic acid multiple emulsion drop pills,and the investigation of their preparation technology,quality standard,oral bioavailability,pharmacodynamics,and mechanism of protecting endothelial cells.This study provides a theoretical and practical basis for the development of salvianolic acids,a key component of the traditional Chinese medicine Salvia miltiorrhiza.?1?Optimization of the preparation conditions for the salvianolic acid W/O/W multiple emulsionPreliminary research into the salvianolic acid W/O/W multiple emulsion was conducted first,including the development of methods for determining the salvianolic acid B content in vitro,entrapment efficiency,and centrifuge retention.Determination of the salvianolic acid B content was performed by HPLC,which afforded good selectivity,recovery,precision,and linearity in the range of 0.31–70.98?g/mL.The results revealed that salvianolic acid B was the most abundant component,representing 40.00%of the total salvianolic acid content.Determination of the entrapment efficiency and centrifuge retention of the salvianolic acid W/O/W multiple emulsion following a high-speed centrifugation method was achieved with good precision.According to the comprehensive scores of entrapment efficiency and centrifuge retention,the optimal preparation method for the salvianolic acid W/O/W multiple emulsion was determined using a single-factor test and an L₉?3⁴?orthogonal test,and the quality was also controlled.In the optimal preparation method for the multiple emulsion,the internal water phase contained 12%salvianolic acids,the hydrophile–lipophile balance?HLB?value of the lipophilic emulgator was4.3,the percentage of the lipophilic emulgator in the oil phase was 20%,the weight ratio of oil and water was 1:0.8,the ratio of the pre-emulsion and external water phase was 1:1,the HLB value of the hydrophilic emulgator was 8.1,and the percentage of the hydrophilic emulgator in the external phase was 10%.Under these conditions,the entrapment efficiency and centrifuge retention of the salvianolic acid W/O/W multiple emulsion were 81.73%and 83.37%,respectively.?2?Quality evaluation of the salvianolic acid W/O/W multiple emulsionThe optimal preparation procedure for the salvianolic acid W/O/W multiple emulsion according to the results of the orthogonal test was scaled up and three batch samples were prepared.The physical properties such as morphology and particle size were then investigated.The entrapment efficiency and centrifugal retention after applying a high-speed centrifugation method were measured,and the stability was also evaluated.The salvianolic acid W/O/W multiple emulsion was a white to light yellow uniform emulsion without any particular smell or taste.The median particle diameter was 0.622?m and the polydispersion index was 0.279.The mean content of salvianolic acid B in the salvianolic acid W/O/W multiple emulsion was233.51 mg/g,with a relative standard deviation?RSD?of 1.11%.Determination of the release rate showed that 90.56%of the salvianolic acid B was released in 12 hours,which was clearly slower than that of the free drug.The stability of the salvianolic acid W/O/W multiple emulsion was confirmed using a six-month accelerated stability study,and the salvianolic acid B content,entrapment efficiency,and centrifugal retention were all found to be stable.?3?Preparation process for the salvianolic acid multiple emulsion drop pillsBy using a single-factor test and an L₉?3⁴?orthogonal test,a comprehensive score including appearance dissolution time,and pill-weight variation was used to assess and optimize the preparation conditions.In the optimal preparation method,PEG6000 was used as the matrix?the drug:matrix ratio was 1:6?,methyl silicone oil was used as the condensing agent,the melting temperature was 70?,and the dripping distance was 5 cm.?4?Quality evaluation of the salvianolic acid multiple-emulsion drop pillsTo control the quality of the salvianolic acid multiple-emulsion drop pills,three batch samples were evaluated.All of the pills were yellow with a glossy surface and no particular smell.The average dissolution time was 3.07 min,the pill-weight variation coefficient was2.68%,the comprehensive score was 66.12%with an RSD value of 0.86%,and the weight differences conformed to the requirements of the Chinese Pharmacopoeia.According to the Chinese Pharmacopoeia,the quality standard of the salvianolic acid multiple-emulsion drop pills was formulated,which provided a good basis for further development of the preparation.?5?Investigation of bioavailability and pharmaceutical effects for salvianolic acid multiple emulsion drop pillsWe established an LC-MS/MS method to determine the salvianolic acid B concentration in rat plasma,which provided the advantages of rapid analysis,low detection limit,and quantitative accuracy.This method has the additional advantage of a simple pre-treatment procedure,that is,after protein precipitation using methanol and subsequent centrifugation,the supernatant was directly injected,leading to a high extraction recovery and meeting the testing requirements.This method afforded good linearity,precision,and extraction recovery,and the RSD value was less than 15%.The RSD value was also less than 15%for freeze–thawed samples and the stability was good.The free drug was administered both intravenously and orally,and the multiple-emulsion drop pills containing20 mg/kg salvianolic acid B were administered orally.The bioavailability of the orally administered free drug was only 0.8%,whereas those of the multiple emulsion and drop pills were 19.48%and 22.44%,respectively.Therefore,compared with the orally administered free drug,the multiple emulsion and drop pills exhibited dramatically improved bioavailabilities,and the half-life(t_1/2)and mean residence time(MRT_0–?)were also slightly extended.To evaluate the pharmaceutical effects of the salvianolic acid multiple emulsion drop pills,we used acute blood stasis rats to assess the coagulation function and hemorheology.Compared with the Salvianolic Acids for Injection preparation used clinically,the salvianolic acid multiple emulsion and drop pills were found to improve the coagulation function by reducing the blood viscosity under different shear values and prolonging the prothrombin time?PT?and activated partial thromboplastin time?APTT?.Based on these results,salvianolic acid multiple emulsions drop pills showed a good effect in terms of improving blood viscosity and coagulation function.?6?Mechanism of salvianolic acid protect against high-glucose-induced cellular dysfunction in HUVECsIn the present study,we also showed that salvianolic acids protect against high-glucose-induced cellular dysfunction in human umbilical vein endothelial cells?HUVECs?.Our results indicated that the pre-administration of salvianolic acids significantly reverses the apoptosis of HUVEC cells induced by high glucose and increases cell viability.At the same time,salvianolic acids were found to lead to upregulation of the NO levels and clearly decrease the generation of reactive oxygen species.Salvianolic acids inhibited the shear activation of the apoptotic proteins cysteinylaspartate specific proteinase-3/9?caspase-3/9?in HUVEC cells.Moreover,salvianolic acids upregulated the expression levels of B-cell lymphoma-2?Bcl-2?and decreased the levels of Bcl-2 associated X protein?Bax?,contributing to reversing the apoptosis of HUVECs.Furthermore,the expression levels of silent information regulator 1?Sirt1?and endothelial nitric oxide synthase?eNOS?were markedly increased in response to salvianolic acid treatment.These results indicate that salvianolic acids protect HUVECs from high glucose via the Sirt 1–eNOS pathway.