Puerarin, Jasminoidin Concentration And Pharmacokinetics In Human Saliva Drug ELISA Method For The Determination

Objective1To develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for concentration measurement and pharmacokinetic analysis of puerarin and geniposide in human saliva using monoclonal antibodies (MAb) against PRR (anti-PRR MAb) and monoclonal antibodies (MAb) against GEN (anti-GEN MAb), validating icELISA method to determine PRR and GEN concentration in human saliva, providing an new method and an new idea of medicine concentration analysis and pharmacokinetic studies of traditional Chinese medicine in human saliva.2To study pharmacokinetic characteristics of different doses of puerarin and geniposide in human saliva, observing dynamic changes of concentrations in saliva in different time, providing research foundation and scientific basis of saliva-based medicines monitoring of various clinical therapeutic substances and the body metabolism analysis.Methods1Indirect competition ELISA (ic-ELISA) methods were developed and validated for the measurements and pharmacokinetic analysis of puerarin in human saliva, standard curve were prepared and its methodology including assay linearity and sensitivity of detection, precision and accuracy, recovery, stability and specificity of anti-PRR MAb were validated. Determination of puerarin concentration in saliva using ic-ELISA compare with HPLC.2Pharmacokinetic analyses of orally administered pueraria capsules in human saliva using indirect competition ELISA:Thirty healthy volunteers were randomly divided into three groups, and each group of ten subjects received an oral administration of20,40or60mg/kg of pueraria capsules in the morning. The saliva from each subject was collected30min before drug administration, followed by15,30,50,60,70,80,90,100,110,125,140,155,170,240,300,360and420min after drug administration using Salivette(?) cotton swab tubes. After processing saliva samples, a standard curve comparing concentration of puerarin with its absorbance measured by ic-ELISA was plotted. These pharmacokinetic parameters were calculated in Kinetica version4.4and SPSS17.0.3Indirect competition ELISA methods were developed and validated for the measurements and pharmacokinetic analysis of geniposide in human saliva, standard curve were prepared and its methodology including assay linearity and sensitivity of detection, precision and accuracy, recovery, stability and specificity of anti-GEN MAb were validated.4Correlation between saliva concentration and plasma concentration of geniposide after two female healthy volunteers orally administered gardenia capsules using indirect competition ELISA:Two healthy volunteers received an oral administration12mg/kg of gardenia capsules in the morning. Saliva samples and plasma samples were collected from each subject at30min before oral administration, and then at15,30,45,60and120min after oral administration. After processing saliva samples and plasma samples, a standard curve comparing concentration of geniposide with its absorbance measured by icELISA was plotted.5Pharmacokinetic analyses of orally administered gardenia capsules in human saliva using indirect competition ELISA:Twelve healthy volunteers were randomly divided into two groups, and each group of six subjects received an oral administration of20mg/kg or12mg/kg of gardenia capsules in the morning, respectively. Saliva samples (approximately1mL) were collected from each subject at30min before oral administration, and then at5,15,30,40,50,60,70,80,100,125,140,155,170,200,240,300,360and420min after oral administration using Saliverte(?) cotton swab tubes. After processing saliva samples, a standard curve comparing concentration of geniposide with its absorbance measured by icELISA was plotted. These pharmacokinetic parameters were calculated in Kinetica version4.4and SPSS17.0.Results1An indirect competition ELISA method was developed and validated for the measurements and pharmacokinetic analysis of puerarin in human saliva. We obtained a linear regression coefficient of0.9939, and the linear regression equation, y=-0.1781n(x)+1.4562. A linear relationship was found within the range of5ng/ml-1280ng/ml. The sensitivity (IC50) of the assay was determined as140ng/ml. The limit of detection was2.47ng/ml (mean±S.D×3, n=20). Both intra-day and inter-day repeatability and precision was achieved, with relative standard deviation (RSD) lower than15%. A mean recovery of95-115%was obtained, with RSD lower than15%. Stability studies indicated that puerarin is stable during sample preparation and analysis. We did not detect cross-reactivity against any of the other related compounds. Idirect competitive ELISA method for the determination of puerarin content in saliva was good correlation with HPLC method and with a linear regression coefficient of0.9916. 2Pharmacokinetics of puerarin in human saliva:Our data show that puerarin in saliva reached maximum concentration first at48.89±7.81min after oral administration of60mg/kg pueraria capsules, with an average Cmax of152.55±78.45ng/mL. An AUC0-t of41252.50±25224.71ng/mL· min, T1/2of173.78±75.24and MRT of311.49±95.42min, was measured. Puerarin in saliva first reached maximum concentration at49.00±7.38min after oral administration of40mg/kg pueraria capsules with an average Cmax of90.85±66.56ng/ml. An AUC0-t of32018.40±14502.25ng/mL· min, T1/2of191.91±78.70and MRT of333.10±121.11min, was obtained. Following oral administration of20mg/kg pueraria capsules, puerarin in saliva first reached maximum concentration at49.00±11.01min, with an average Cmax of47.04±22.53ng/mL. An AUC0-t of23844.10±13352.90ng/mL±min, T1/2of280.43±82.44MRT of378.08±150.68min, was obtained.Puerarin can be detected after oral administration in10minutes. Our results indicate the occurrence of bimodal phenomena in the mean puerarin concentration versus time profiles of puerarin in saliva all dosage groups. Maximum concentration was measured at two different time-points following administration, the first at about49min and the second at about110min. This assay also shows that the mean puerarin concentration versus time profile of all three dosage groups is very similar. However, puerarin concentration in saliva of10healthy volunteers in each group at the same time point had large individual differences. We found significant differences in Cmax and AUCo-t between the three dosage groups (P<0.05) and good correlation between doses and AUC-t.This suggested that the linear pharmacokinetics of puerarin in saliva. We found no significant difference in Tmax and MRT between the three dosage groups (P>0.05). This shows that Tmax, MRT and the change in drug half-life, is not dose-dependent.3An indirect competition ELISA method was developed and validated for the measurements and pharmacokinetic analysis of geniposide in human saliva. Method validation in human saliva revealed a linear correlation in the range of0.78-12.50μg/mL. The linear regression equation was y=-0.252ln(x)+0.9937with a linear regression coefficient of0.9972. Both intra-day and inter-day repeatability and precision were associated with relative standard deviations (RSD) of less than15%. A mean recovery of80%to120%was obtained, with RSD of less than15%. Stability studies indicated that GEN is stable during sample preparation and analysis.4Preliminary study analysis geniposide saliva concentration correlated with blood concentration after two female healthy volunteers orally administered gardenia capsules: Geniposide average concentration in saliva and blood-time curve consistent with the overall trend, and the two subjects those with blood concentrations in saliva concentrations have reached a peak30min. Saliva concentrations lower than the concentration in blood, ratio of saliva and plasma concentration (S/P ratio) less than1. A preliminary study on the correlation between the concentration in saliva and plasma concentration showed that R2=0.7953, indicated a relative good correlation between saliva concentration and plasma concentration of geniposide.5Statistical and pharmacokinetic analysis of geniposide in human saliva:20mg/kg gardenia capsules:Cmax=31.93±8.12μg/mL, Tmax=56.67±12.11min, AUCo-t=21249.08±9332.28μg/mL-min, T1/2=433.66±188.26min, MRT=677.30±249.27min;12mg/kg gardenia capsules:Cmax=22.81±20.85μg/mL, Tmax=98.33±27.14min, AUC0-t=4794.65±2885.74μg/mL-min, T1/2=141.52±38.25min, MRT=285.26±47.66min.Our results indicate the occurrence of bimodal phenomena in the mean GEN concentration versus time profiles of GEN in saliva among two groups. Maximum concentration was measured at two different time-points following administration-20mg/kg dose group:50min and155min (approximately);12mg/kg dose group:70min and140min (approximately). The mean GEN concentration versus time profile was found to be similar among two groups. However, the time-point corresponding to the GEN concentration in saliva showed large variations among individuals within each group. We found significant differences in Cmax and AUC0-t between the two dosage groups (P<0.05). The concentration of GEN in saliva not to a minimum after420min but still decreased slowly in two groups and a longer MRT.Conclusions1Quick, specific and sensitive indirect competitive ELISA assay methods for the measurement and pharmacokinetic analysis of puerarin and geniposide in human saliva were developed for the first time. Method validations in human saliva were satisfactory and meet the criteria specified by the United States Food and Drug Administration (US FDA) for the analysis of biological samples. Stability studies indicated that puerarin and geniposide are stable during sample preparation and analysis. The validated ic-ELISA method can be successfully applied to the investigation of the pharmacokinetics of puerarin and geniposide in human saliva, providing a new method and a new idea of medicine concentration analysis and pharmacokinetic studies of traditional Chinese medicine in human saliva. 2Saliva is an important body fluid suited to the investigation of the pharmacokinetics of puerarin and geniposide in humans, due to advantages such as non-invasiveness and ease of sample collection. This assay was applied to the measurement and pharmacokinetic analysis of puerarin and geniposide in human saliva samples from healthy volunteers, following oral administration of different doses of pueraria capsules and gardenia capsules. This method can be used as a tool to conduct pharmacokinetic studies and saliva-based medicines monitoring of various clinical therapeutic substances.