Establishment,in Vitro And In Vivo Evaluation Of Andrographolide Nanosuspensions

Andrographolide is a diterpenoid lactone and it is the main active ingredient in the Andrographis Herba,which has a reputation of "natural antibiotic".It is a great potential drug as it shows well anti-inflammatory,anti-tumor and immunomodulatory effects.But its poor water solubility(0.07 mg/mL)and low dissolution resulting in the low bioavailability(2.67%)greatly restricted its development.In this study,andrographolide nanosuspensions were prepared by wet media milling technique.The analysis methods for andrographolide in vitro and in biological samples were established.The characterization,in vitro dissolution,bioavailability and anti-inflammatory effect of andrographolide nanosuspensions were also studied.The main contents of this study include:The paper study the effects of prepared method,the variety of stabilizers,the concentration of drug,the concentration of the stabilizers,the ratio of the stabilizers,milling time and agitator speed on particle size and PDI of andrographolide nanosuspensions.Then the prepared method,the variety of stabilizers and the concentration of drug were confirmed,which were wet media milling technique,SDS,TPGS and 0.5%,respectively.The experiment was based on the basis of single factor investigation,using the Box-Behnken design-Response surface methodology to optimize the preparation technology of andrographolide nanosuspensions.The optimization conditions:Andrographolide coarse powder(1 g)was added to 200 mL solution containing 0.242%TPGS and 0.178%SDS(w/v).Then the solution was shearing at the speed 16000 rpm with 5 min in the homogenizer.The resulted suspension was added to medium milling machine(0.3-0.4 mm zirconia beads 850 g),which was grinded 1.4 h with the agitator speed 1755 rpm.The optimized andrographolide nanosuspensions were obtained.The particle size and the PDI of andrographolide nanosuspensions were 225.3±1.55 nm and 0.179±0.010,respectivelyThe quality of the andrographolide nanosuspensions was evaluated including the particle size and distribution,Zeta potential,differential scanning calorimetry,scanning electron microscopy,X-ray diffraction analysis and dissolution rate in vitro.The results showed that nanosuspensions of size,the dispersion coefficient and Zeta potential were 225.3±1.55 nm,0.179±0.010 and-28.6±1.15mV,respectively.The crystalline nature of andrographolide in the optimized andrographolide nanosuspensions disappeared according to DSC and PXRD.Compared with the unprocessed andrographolide particle size(10-200?m),the particle size of nanosuspensions was much smaller(0.1-0.3 ?m).The dissolution of optimized andrographolide nanosuspensions reach 85%during the first 10 min in each dissolution medium,demonstrating a fast dissolution rate.In pH 1.2 and 60 min,the dissolution of optimized andrographolide nanosuspensions was 3.57,1.43 and 1.37 times of andrographolide,physical mixture of the andrographolide and commercial andrographolide dripping pills.In pH 4.5 and 60 min,the dissolution of optimized andrographolide nanosuspensions was 3.79,1.28 and 1.38 times of the andrographolide,physical mixture of andrographolide and commercial andrographolide dripping pills.In pH 6.8 and 60 min,the dissolution of optimized andrographolide nanosuspensions was 4.76,1.37 and 1.40 times of the andrographolide,physical mixture of andrographolide and commercial andrographolide dripping pills.The in vitro dissolution experimental results showed that andrographolide nanosuspensions had good dissolution in different pH buffer solutions.LC-MS/MS analysis method was established to determinate the andrographolide concentration of biological samples.Twenty-four male SD rats were randomly divided into four groups,each containing six animals.All rats received oral doses of 40 mg/kg including andrographolide,commercial andrographolide dripping pills,the andrographolide nanosuspensions,and physical mixture of andrographolide,TPGS and SDS.The result showed that,after received andrographolide,the Cmax and AUC0-t were 77.74±22.17 ?g/L and 5665.41±1654.43 ?g/L min,respectively.The Cmax of the andrographolide nanosuspensions,physical mixture of andrographolide and commercial andrographolide dripping pills groups were 235.91±53.73,80.25±17.35,168.17±47.28 ?g/L,respectively.The AUC0-t of the andrographolide nanosuspensions,physical mixture of andrographolide and commercial andrographolide dripping pills groups were 21960.14±5392.04,9117.87±2126.20,10273.45±1699.48 ?g/L min,respectively.In compared with andrographolide coarse powder group,the relative bioavailability of the andrographolide nanosuspensions was 408.24%.The bioavailability of andrographolide nanosuspensions groups was 2.41 times of the physical mixture of andrographolide groups and 2.14 times of the commercial andrographolide dripping pills groups.The pharmacokinetic experiment showed that andrographolide nanosuspensions had good absorption in vivo in rats.The rat paw edema models induced by carrageenan were established and the paw swelling,the contents of NO,MDA and activities of NOS,SOD in serum were identified as evaluating indicators to investigate the anti-inflammatory effect of andrographolide nanosuspensions.The results of paw swelling degree of the rats indicated that andrographolide nanosuspensions high dose group could inhibite carrageenan induced paw swelling significantly at different time points.However,andrographolide coarse powder group couldn't inhibite carrageenan induced paw swelling significantly at any time points.The determination results of the NO,MDA contents and NOS,SOD activities in rat serum indicated that,compared with coarse powder group,the NO,MDA contents and NOS,SOD activities in rat serum had significantly changed in andrographolide nanosuspensions medium and high dose groups.Compared with andrographolide coarse powder,andrographolide nanosuspensions had better inhibition effect of rats paw swelling,decrease of the NO,MDA contents and NOS activity and increase of the SOD activity.Andrographolide nanosuspensions had better anti-inflammatory effect then andrographolide coarse powder.In this study,andrographolide nanosuspensions were prepared by wet media milling technique and the characterization,dissolution in vitro,pharmacokinetics and pharmacodynamics in vivo were also undertaken.The results showed that andrographolide nanosuspensions could improve the dissolution and enhance the oral bioavailability,anti-inflammatory effects of andrographolide.