Fluorescent Labeling And Pharmacokinetics Of Codonopsis Polysaccharide CPA

Objective:Codonopsis pilosula is a kind of edible herbs and commonly used in many Asian countries such as China,Japan,and Korea.Codonopsis polysaccharide is rich in Codonopsis pilosula,and has various biological activities such as immunity-modifying,anti-oxidative,anti-tumor and anti-inflammatory.Studies have found that Codonopsis polysaccharide CPA has good effect on gastric ulcer,ulcerative colitis and other diseases,with low toxicity and good biocompatibility.It was considered a promising natural drug candidate.However,due to the complex chemical structure of polysaccharides and the lack of ultraviolet absorption or fluorescent groups,it is extremely difficult to detect the concentrations of polysaccharides in vivo.This phenomenon leads to few reports on the pharmacokinetic information of CPA,which seriously limits the further development of CPA as a new drug candidate.To clarify the pharmacokinetic characteristics and in vivo process of CPA can make a preliminary judgment on whether it can be used as an expected drug candidate,which is crucial for determining the route of administration,dosage and dosage form.Therefore,the aim of this work has been to establish a simple and efficient method to detect the concentration of inulin-type fructan CPA in biosamples,and then applied it to evaluate the pharmacokinetics behavior and distribution character in tissues and excretion in mice.Methods:1.Preparation and characterizations of fluorescently labeled products of CPA（FCPA and F-NH-CPA）In this part,fluorescein isothiocyanate（FITC）was used to label CPA by direct labeling method and indirect labeling method.The FCPA and F-NH-CPA were characterized by infrared spectrometer,ultraviolet-visible spectrophotometer and high performance gel permeation chromatography.The labeling efficiency was determined using multifunctional microplate reader.The stability of FCPA and F-NH-CPA both in vitro and in vivo were also investigated.Finally,F-NH-CPA was confirmed for pharmacokinetic study through structure identification and stability investigation.2.Establishment of quantitative detection methods for fluorescently labeled product F-NH-CPA in biological samplesBlood,liver,spleen,lung,kidney,stomach,large intestine,small intestine,feces,and urine samples were collected from male healthy BALB/c mice.The calibration curves of F-NH-CPA in different biological samples were established,and the intra-day precision,inter-day precision,accuracy and stability were investigated.3.Pharmacokinetics study after oral administration of CPAMale healthy BALB/c mice were randomly divided into 3 groups,and each group was given 28,56 and 112 mg kg^-1 F-NH-CPA by oral administration,respectively.Blood,liver,lung,kidney,stomach,large intestine and small intestine were collected at 30,60,120,240,360,and 480 min,respectively.The concentration of F-NH-CPA in biological samples was measured by multifunctional microplate reader.For the elimination study,eighteen mice were randomly assigned to three groups（n=6 in each group）.The mice were placed separately in metabolism cages and orally administered three different doses of F-NH-CPA(28,56 and 112 mg kg^-1)after an overnight fast.Urine and feces were collected during periods of 0–1,1–2,2–4,4–6,6–8,8–12 and 12–24 h after administration.After processing the feces and urine,the fluorescence intensity of F-NH-CPA was detected by multifunctional microplate reader.4.Pharmacokinetics of CPA after intravenous administrationMale healthy BALB/c mice were randomly divided into 3 groups,and 7,14 and 28mg kg^-1 F-NH-CPA were injected through the tail vein,respectively.Blood,liver,spleen,lung,kidney,stomach,large intestine and small intestine were collected at 10,30,60,120,240,360,and 480 min,respectively.The F-NH-CPA content in biological samples was determined by multifunctional microplate reader.Results:1.Preparation and characterizations of fluorescently labeled products of CPA（FCPA and F-NH-CPA）CPA was fluorescently labeled with FITC by direct and indirect synthesis.The results of high performance gel permeation chromatography,infrared spectra and ultraviolet-visible spectra showed that FITC labelling reactions were all successful,and the labelling efficacy of FITC in FCPA and F-NH-CPA were 0.88%and 1.07%,respectively.The stability studies have found that the fluorescently labeled products synthesized by two methods had good stability in different biological media in vitro,such as plasma,artificial gastric juice and artificial intestinal juice.In vivo studies have found that FCPA was mainly exists in the prototype form in plasma after intravenous administration,and some FITC is shed.While the F-NH-CPA can be degraded in vivo,but no free FITC can be detected,indicating that the indirect fluorescent labeling product was relatively stable.Therefore,this experiment believed that the F-NH-CPA synthesized by the indirect method has better stability and is more suitable for the pharmacokinetic study of CPA.2.Establishment of quantitative detection methods for fluorescently labeled product F-NH-CPA in biological samplesThe calibration curves,intra-day precision,inter-day precision,accuracy and stability of F-NH-CPA in biological samples were investigated.The linear relationship was good in the range of 0.10～25.00μg m L^-1 and 0.78～50.00μg m L^-1.The accuracy,intra-day and inter-day precision were all in the acceptable range.F-NH-CPA was stable in biosamples under different storage conditions.These proved that this method is suitable for the determination of F-NH-CPA in mice plasma,tissue,excrement and other biological samples.3.Pharmacokinetics study after oral administration of CPAAfter oral administration of F-NH-CPA,the concentration of F-NH-CPA in plasma gradually increased,and then decreased gradually after reaching a peak at 1 h.The average elimination half-life of the three oral doses in mice was long,and elimination was slow.The area under curve（AUC（0→∞））and peak concentration(C_max)was positively correlated with the dosage,indicating that CPA exhibits linear metabolic kinetics feature in mice.The absolute bioavailability of F-NH-CPA was a little poor.F-NH-CPA was widely distributed in tissue,especially in the gastrointestinal tract,and mainly excreted through feces.4.Pharmacokinetics of CPA after intravenous administrationAfter intravenous administration,the concentration of F-NH-CPA in the circulatory system decreased slowly.The half life(T_1/2)and mean residence time（MRT）were long,the clearance（CL）was low,and the elimination in the body was slow.The area under curve（AUC（0→∞））and peak concentration(C_max)was positively correlated with the dosage,indicating that CPA exhibits linear metabolic kinetics feature in mice.F-NH-CPA mainly accumulated in the stomach,kidney,and spleen,and the concentration in the lung was low.The F-NH-CPA concentration decreased rapidly in the liver and slowly in the small intestine and large intestine.Conclusion:In this study,a selective and sensitive fluorescence labeling method was developed to detect the concentration of inulin type fructan CPA in biosamples.Then,the validated method was applied to evaluate the pharmacokinetic behavior after oral and intravenous administration to mice.The results indicated that the valid method was suitable to detect the polysaccharide concentration.In both intravenous and oral administration,the concentration of F-NH-CPA declined slowly in the circulatory system with a much longer T_1/2 and MRT.F-NH-CPA could be absorbed into blood circulation after oral administration with low bioavailability.It was also proved to be excreted by urine and feces.This study could provide comprehensive knowledge on the in vivo behavior of CPA and may reveal important implications for the development and application of CPA oral formulations.