| Most active ingredients of Traditional Chinese Medicines?TCM?are insoluble,whose low solubility leads to a poor oral bioavailability.Nanosuspension technology is an effective preparation method to improve the oral bioavailability of poorly soluble components.However,its application in TCM is still limited to monomer components.This study aimed to explore application of nanosuspension technology in TCM compound formula using Guanxinning as an example.The research methods and results were as follows:?1?Determination of active ingredients of Guanxinning to be investigatedThe network pharmacology was used to explore active ingredients of Guanxinning which was composed of Chuanxiong Rhizoma and Salviae Miltiorrhizae Radix et Rhizoma.70 therapeutic ingredients were selected from 244 Chuanxiong Rhizoma ingredients and 231 Salviae Miltiorrhizae Radix et Rhizoma ingredients,among which30 originated from Chuanxiong Rhizoma and 40 were derived from Salviae Miltiorrhizae Radix et Rhizoma.Finally danshensu,protocatechualdehyde,senkyunolide I,senkyunolide H,salvianolic acid B,rosmarinic acid,ligustrazine,ferulic acid,senkyunolide A,ligustilide,butylidenephthalide,tanshinone?,tanshinone?A,and cryptotanshinone were selected as indicative components for the subsequent research due to their relatively high content in Chuanxiong Rhizoma or Salviae Miltiorrhizae Radix et Rhizoma.And more importance would be attached to senkyunolide A,ligustilide,butylidenephthalide,tanshinone?A,tanshinone?and cryptotanshinone because of their extremely low solubility.?2?Simultaneous determination of the 14 active ingredients in GuanxinningHPLC was used to determine the contents of the 14 aforementioned ingredients in guanxinning simultaneously.The determination was performed on Waters Symmetry C18 column?4.6×250 mm,5?m?with a mobile phase consisted of 0.1%formic acid-methanol?gradient elution?at a flow rate of 1.0 m L·min-1.The detection wavelength was set at 280 nm,and the column temperature was 30?C.Methodological verification was carried out according to the"Guidelines for Validation of 9101Pharmaceutical Quality Standard Analysis Methods"in the Ch.P.The specificity,linearity and range,repeatability,instrument precision,and accuracy all met the requirements of the Pharmacopoeia,confirming that the HPLC method could be used for the simultaneous determination of the 14 components in Guanxinning.?3?Study on preparation of Guanxinning lyophilized nanopowderThe extract of Chuanxiong Rhizoma and the extract of Salviae Miltiorrhizae Radix et Rhizoma on market were used as raw materials and their ratio was set at 8:10according to the ratio of the total content of Salviae Miltiorrhizae Radix et Rhizoma ingredients to Chuanxiong Rhizoma ingredients in Guanxinning tablets.The particle size,yield of each component in suspension,and the content percentage of insoluble components whose size was 100-1 000 nm were used as evaluation indexes.7preparation method,i.e.anti-solvent precipitation,high-pressure homogenization,probe ultrasound,high-pressure homogenization after anti-solvent precipitation,probe ultrasonic after anti-solvent precipitation,anti-solvent precipitation with water bath ultrasound,anti-solvent precipitation with water bath ultrasound followed by probe ultrasound,were compared.The results showed that the last method was more suitable to prepare Guanxinning nanosuspension than others.The Box-Behnken design was then used to optimize technology of anti-solvent precipitation process,including total concentration of the extracts in organic phase,the ratio of organic phase to aqueous phase,and the temperature of water bath.The ultrasonic power and time of the ultrasound process were screened using a single factor experiment.The type and concentration of stabilizers in the nanosuspension,lyophilized protective agents in the freeze-dried powder were also selected.Finally,Guanxinning lyophilized nanopowder with good appearance and redispersibility was obtained.?4?Characterization and properties of Guanxinning nanosuspension and lyophilized nanopowderThe particle size of Guanxinning nanosuspension was?235.40±21.09?nm,PDI was?0.41±0.02?,and Zeta potential was?-13.30±0.10?mV,which was significantly lower than that of the crude suspension,which was?13236.67±132.04?nm,?0.84±0.05?and?-5.55±2.47?mV,respectively.Compared with the crude suspension,the distrubitions of senkyunolide A,ligustilide,butylidenephthalide,cryptotanshinone,tanshinone?and tanshinone?A were transferred from above 1?m to less than 400 nm;but the other 8 ingredients still mainly distributed in the filtrate after 100 nm filtration.Freeze-drying had no significant effect on properties of the nanosuspension.The particle size,PDI,Zeta potential,and distribution of 14 index components were not significantly changed when the lyophilized nanopowder was redispersed in water.SEM and AFM confirmed that senkyunolide A,ligustilide,and butylidenephthalide existed in amorphous form,and tanshinone?,tanshinone?A,and cryptotanshinone existed incrystals of long rods in the nanosuspension.The apparent solubilities of danshensu,protocatechualdehyde,ligustrazine,senkyunolide I,senkyunolide H,rosmarinic acid,and salvianolic acid B in lyophilized nanopowder were similar to those in crude extract materials.However,the apparent solubility of ferulic acid,senkyunolide A,ligustilide,butylidenephthalide,cryptotanshinone,tanshinone?,and tanshinone?A in the lyophilized nanopowder increased significantly.The Ch.P paddle method was used for in vitro dissolution experiments.The cumulative release percentages of danshensu,protocatechualdehyde,ligustrazine,senkyunolide I,senkyunolide H,rosmarinic acid,and salvianolic acid B from crude extract materials and lyophilized nanopowder were all above 90%in simulated gastric juice as well as simulated intestinal fluid,which were much higher than Guanxinning tablets.The cumulative release percentages of senkyunolide A,ligustilide and butylidenephthalide from lyophilized nanopowder in simulated gastric fluid and simulated intestinal fluid were 1.72-2.00,2.48-4.18 and 1.59-2.62 times that of crude extract materials,respectively.For cryptotanshinone,tanshinone?and tanshinone?A they hardly released from crude extract materials,but their cumulative release percentages from the lyophilized nanopowder greatly increased to?54.35±19.37?%,?48.87±0.42?%,?49.44±7.82?%in simulated gastric fluid respectively and to?72.39±8.29?%,?61.68±6.34?%,and?75.43±2.58?%in simulated intestinal fluid,respectively.Guanxinning tablets did not contain these 6 insoluble components.The particle size,PDI,Zeta potential,and contents of each component in lyophilized nanopowder did not changed significantly during 6 months of storage at 4?C,which suggested that the lyophilized nanopowder may have a good stability at a low temperature.The nanosuspension technology was successfully applied to Guanxinning,a TCM compound formula.The optimized lyophilized nanopowder not only kept the properties of danshensu,protocatechualdehyde,ligustrazine,ferulic acid,senkyunolide I,senkyunolide H,salvianolic acid B and rosmarinic acid,but also reduced particle size of the poorly soluble senkyunolide A,ligustilide,butenylphthalide,cryptotanshinone,tanshinone?and tanshinone?A,thus increasing their apparent solubility and improving their in vitro dissolution.The research could provide a reference for the application of nanosuspension technology in TCM compounds,and lay an experimental foundation for the development of new oral preparations of Guanxinning which contain both water-soluble and poorly soluble ingredients. |
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