Preparation Of Florfenicol Nanocrystals And Preliminary Study On Bacteriostasis Of Its Combination With Total Flavonoids From Canarium Pimela Leenh Leaves

Florfenicol is a broad-spectrum antibiotic of chloramphenicol.It is widely used in the treatment of bacterial diseases in animal husbandry.However,due to its low solubility in the body and slow dissolution,the bioavailability of florfenicol is low,large doses in clinical applications,which are easy to cause florfenicol residue in the body and environmental pollution.Therefore,increasing the solubility of the drug to improving the bioavailability in animals is of great significance.On the other hand,bacteria are becoming more resistant to flufenicol,and using florfenicol at high concentrations for a long time can easily increase the resistance of bacteria.The combined application of florfenicol with natural plants with antibacterial effects is expected to improve the efficacy and reduce the dosage of florfenicol.Canarium pimela Leenh is a tree belonging to the Olive family,Olive genus.Its leaves are rich in resources,and polyphenols,flavonoids and other substances.The study on its antibacterial effect and its combined use with florfenicol has practical application value.This current research uses nanocrystalline technology to improve the solubility and bioavailability of florfenicol,and conducts preliminary studies on the in vitro antibacterial and in vivo pharmacokinetics of its combined application with Canarium pimela Leenh,in order to enhance the antibacterial effect and reduce the dosage of florfenicol.The methods and results of the main research content are as follows:1.The florfenicol nanocrystals were prepared by media grinding.The florfenicol nanocrystals were prepared by media grinding.The types and combination of stabilizers added in the process,the ratio of the additives to the drug,grinding speed,grinding time,diameter and dosage ratio of grinding zirconium beads were investigated by a single-factor experiment,and orthogonal test was used to optimize the prescription process.The physical and chemical properties of florfenicol nanocrystals were evaluated.The results showed that the saturated solubility of florfenicol nanocrystals in water was（2.01±0.13）mg·m L^-1,which was1.7 times higher than that of florfenicol raw drugs;the average particle size was between 203.70 and 209.10 nm,the Zeta potential was-（22.80±2.31）m V.Florfenicol nanocrystalline was white powder with loose texture and uniform color,which was in needle-like form when observed under a transmission electron microscope.2.The AB-8 macroporous adsorption resin was used to enrich and purify the extract of total flavonoids from Canarium pimela Leenh leaves and the content of total flavonoids was determined and the extract chemical composition was analyzed.The contents of total flavonoids in the enrich extract from Canarium pimela Leenh leaves was（665.49±16.89）mg·g^-1,which was 1.8 times of crude extract from Canarium pimela Leenh leaves,achieving a good purification effect.The chemical constituents of the extract from Canarium pimela Leenh leaves were identified,11 phenolic compounds and 11 flavonoids were identified,among which chlorogenic acid,kaempferol,quercetin,dihydromyricetin,luteolin and other compounds were commonly used antibacterial active ingredients.This results provided material basic information for the antibacterial activity of Canarium pimela Leenh leaves and its antibacterial combination with florfenicol.3.The minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）experiments were used to evaluate the difference in antibacterial activity of florfenicol raw drugs and florfenicol nanocrystals in vitro.The results showed that the MIC of the florfenicol nanocrystals was less than that of the florfenicol raw drugs,the antibacterial effect was enhanced.Inhibition zone,MIC and MBC were used to initially explore the antibacterial activity of total flavonoids from Canarium pimela Leenh leaves,and the results showed that it had good antibacterial activities,and the MIC range was 4.16～12.50mg·m L^-1.The micro-checkerboard method was used to determine the combined antibacterial effect of florfenicol nanocrystals and the total flavonoids from Canarium pimela Leenh leaves.The results showed that the FICI index was between 0.31 and 0.63 after combined administration,showing a synergistic and additive antibacterial effect.When used in combination,the MIC of florfenicol nanocrystals against Escherichia coli,Klebsiella pneumoniae,and Salmonella was 1/4 times that of florfenicol nanocrystals alone,and it is 1/2 times to Pseudomonas aeruginosa,Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus.The Preliminary experimental results showed that the combined use of florfenicol and the total flavonoids from Canarium pimela Leenh leaves enhanced antibacterial effect and reduced the dosage of florfenicol.4.The pharmacokinetic behaviors of florfenicol nanocrystals and florfenicol raw materials in rats were compared by intragastric administration method.The experimental results show that the AUC _0-∞(36.08±6.85mg·h·L^-1)、C_max(11.15±2.62mg·L^-1)、t_1/2α（1.95±0.33h）were 3.0 times、1.5 times and 1.8 times of the florfenicol raw drug,indicating that florfenicol nanocrystals had the effect of promoting absorption and higher oral bioavailability.The AUC_0-∞and C_maxparameters of florfenicol combined with total flavonoids from Canarium pimela Leenh leaves were shown as florfenicol nanocrystals combined with total flavonoids from Canarium pimela Leenh>florfenicol nanocrystals>florfenicol raw drugs combined with total flavonoids from Canarium pimela Leenh>florfenicol raw drugs,showed that thetotal flavonoids from Canarium pimela Leenh leaves can promote absorption of florfenicol and florfenicol nanocrystals in the body and improve their oral bioavailability.The AUC_0-∞and C_maxof florfenicol nanocrystals combined with total flavonoids from Canarium pimela Leenh leaves were 2.6,2.0imes that of florfenicol raw drugs alone.This result indicated that the preparation of florfenicol into drug nanocrystals,and combination with total flavonoids from Canarium pimela Leenh leaves could significantly improve the oral bioavailability of florfenicol.In this study,florfenicol nanocrystals were prepared to improve the solubility and bioavailability of florfenicol,and it was preliminary studied that the combination of florfenicol with total flavonoids from Canarium pimela Leenh leaves could enhance the antibacterial effect and reduce the dosage of florfenicol in vitro,and could promote the absorption of florfenicol in the body.It laid the foundation for reducing the resistance of florfenicol and provided a basis for the development of antibacterial effect of Canarium pimela Leenh leaves resources.