| Silymarin?SM?is flavonoids components isolated from the milk thistle plant silybum marianum.It is mixture including silybin,silychristin,silydianin and et al.It has been widely used for the treatment of hepatitis and liver damage.SM has extensive sources,low toxicity and good curative effect.It is a kind of excellent liver protective agent.But its low aqueous solubility,poor oral absorption and low bioavailability,which clinical role is limited.This experiment studies a kind of emulsion solvent evaporation method?ESE?,which prepares silymarin nanoparticles?SMNs?.It improves the solubility of the drugs,bioavailability and pharmic content in target organs.The optimum condition of SMNs is study by single-factor experiments.Eight nain factors,i.e.,drug concentration,the proportion of water to organic phase,the concentration of poloxamer 188,homogenate speed and time,homogenization pressure?HP?and times,the proportion of SM to mannitol,were picked out for the preparation optimization of SMNs.The physical and chemical properties of SMNs,content,stability,solvent residual determination,dissolving capability test,equilibrium solubility,the antioxidant activities,bioavailability,nano drug distribution in the orsianization are studied.The research results are as follows:1?The optimum conditions of SMNs is determined by single-factor experiments,the results are as followed:15 mg/mL of SM solution,6:1 of volume ratio of water to organic phase,0.5 mg/mL of poloxamer 188,3000 rpm of homogenate speed for 0.5 min,a homogenization pressure of 600 bar for 6 times and proportion of SM to mannitol of 1:7.Under the optimum conditions,SMNs with an MPS of 107.1 nm are obtained.2?The research results of characteristics of SMNs are as follows:SMNs have a smaller particle size about 100 nm as seen through the morphology.SMNs were nearly spherical with uniform particle size distribution.The FTIR analyse demonstrated that SM has been entirely wrapped up inside poloxamer 188 and mannitol.The SMNs have less crystalline by XRD detection and it might be present in the nearly desired amorphous form.The DSC analyse demonstrated that SMNs prepared by ESE have a lower crystallinity compared with SM.The TGA anal\yse demonstrate that SMNs have easier vaporization and a faster thermal decomposition rate.It may be because SMNs have the smaller particle size and higher specific surface than raw SM and physical mixture.3?Solvent residue analysis:The residual ethanol content in SMNs is 0.0017%and the residual chloroform content in SMNs is 0.0010%.The ICH limit for ethanol is 5000 ppm or 0.5%in class ? solvents and the ICH limit for chloroform is 60 ppm or 0.006%in class ?solvents.So,the solvent residues of SMNs obtained in this study met the ICH requirements,and they are suitable for pharmaceutical use.4?The content of the drug:The content test of silybin of SM and SMNs is performed by HPLC.Silybin have two peaks about silybin and isosilybin.The regression equation is 7=30920x-23464?R2=0.9999?.According to the result of regression equation,the content of silybin is 33.00%in SM,the content of silybin is 3.00%in SMNs.5?Stability:SMNs have good stability.The MPS of aqueous dispersion of SMNs are less than 150 nm and had a good dispersion during 90 days.Dispersion coefficient is in the range of 0.164-0.260,the sample is uniformly dispersed.The content of the sample has no obvious change.The colour has not changed and the lustre is uniform.It has not the phenomenon of collapse or drying shrinkage.6?Equilibrium solubility:The equilibrium solubility of SMNs is far greater than raw SM.The equilibrium solubilities of raw SM and SMNs are 28.01 and 62.66 ?g/mL in SGF,and 29.50 and 52.13 ?g/mL in SIF,respectively.The equilibrium solubilities of SMNs in SGF are 2.23-and 1.76-fold to those of raw SM in SIF,respectively.7?Solubility in vitro:SMNs have a rapid solubility and dissolution rate than raw SM.18.94%of the silybin in SM and 92.33%of the silybin in SMNs are dissolved within 60 min in SGF.Thus,the dissolution rate of SMNs is 4.87 times as high as that of the raw SM.And 20.95%of the silybin in SM and 77.66%of the silybin in SMNs are dissolved within 60 min in SIF.Hence,the dissolution rate of SMNs is 3.70 times as high as that of raw SM.The smaller particle size and the amorphous SMNs have a higher dissolution rate and solubility and played a very good effect in a short time.8?Antioxidant activity in vitro:Scavenging effect on DPPH radical and reducing power of SMNs have been demonstrated.The experimental results show that,both of raw SM and SMNs have scavenging effects on the DPPH radical,and the effects increased with increasing concentrations in the range of 0.0156-1.0 mg/mL.The IC50 value of SMNs in DPPH radical scavenging assay is found to be 0.27 mg/mL,while that of raw SM is 0.71 mg/mL.The results show that SMNs had a higher DPPH-scavenging activity than raw SM.The reducing power of SMNs was found to be significantly more pronounced than that of raw SM.At a concentration of 1 mg/mL,SMNs showed high reducing power values of 0.685 and raw SM only exhibited a low reducing power value of 0.390.Hence,SMNs are a powerful oxidation resistance which might be used in reducing free radicals?inhibiting liver lipid peroxidation and preventing HSC activation as an antioxidant.It is important for the treatment of liver fibrosis and improving liver detoxification function.It provides strong scientific data for the clinical application of milk thistle.9?In vivo bioavailability studies of SMNs in rats:The experimental results show that the silybin concentration in rat plasma of the SMNs group was also higher than that of the raw SM.This is because the particle size reduction could increase the efficiency of SMNrs absorption.Distribution half-life t1/2a and biological half-life t1/2? of SMNs were 0.105h and 4.455h in rats,respectively.Distribution half-life t1/2a and biological half-life t1/2? of SM are 0.147h and 9.12h in rats,respectively.The average residence time?MRT?of SMNs and SM are 3.979 h and 4592h in rats,respectively.This is because the particle size reduction could increase the efficiency of SMNs absorption,decreasing the blood concentration rapidly.The AUC value of SMNs and SM are 7.511 mg/L*h and 2.023 mg/L*h in rats,respectively.The results demonstrate that the AUC value of the SMNs is 3.71 times as high as that of the raw SM.The oral bioavailability of the SMNs is improved significantly compared with the raw SM.10?In vivo tissue distribution studies of SMNs in rats:the silybin concentrations in the organization after administration of raw SM and SMNs are studied at 0.5?1?2?4?8?12h.The results indicate that the absorption of SMNs in the organization is higher than that of raw SM.The particle size reduction is beneficial to improve the efficiency of SMNs absorption in the organization.The experimental group has a very high drug concentration in liver tissue and drugs had a long time left in the liver.It has been clear about the SM had the characteristics of the liver targeting property.The average residence time?MRT?of SMNs and SM in liver is 4.332h and 3.345h,respectively.The MRT value of the SMNs is 1.29 times as high as that of the raw SM.The AUC value of SMNs and SM is 11.424 mg/L*h and 2.21 mg/L*h in liver,respectively.The results demonstrate that the AUC value of the SMNs is 5.16 times as high as that of the raw SM.Therefore,the obtained SMNs could be abounded in liver that is beneficial to therapy of liver disease.From the above results,SMNs have a small and uniform particle size distribution.SMNs have less crystalline and SM has been entirely wrapped up inside poloxamer 188 and mannitol.So its solubility,bioavailability and the drug content in the target organs have been improved.It may have a potential application value for developing new dosage forms of drugs in clinical liver disease. |
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