Investigation On The Absorption And Metabolism Of Soyasaponin Bb By Using In Vivo And In Vitro Methods

BackgroundSoyasaponin Bb is a saponin found in soybeans.It has various biological effects.such as cholesterol reduction and liver protection.However,there are still many problems in absorption,metabolism and excretion,trans-intestinal epithelial absorption and others that need to be clarified on soyasaponin Bb in vivo.This is also the theoretical basis for explaining the biological activity of soysaponin Bb in vivo.ObjectivesThis study mainly took soyasaponin Bb as the research object to explore its absorption,metabolism and excretion in SD rats,and established an in vitro Caco-2 cell model to further study the absorption characteristics of soyasaponin Bb in the intestine.It provided a theoretical basis for studying the mechanism of biological characteristics in soysaponin Bb.Methods1.Establish the HPLC-MS/MS analytical method for detecting soyasaponin Bb and its metabolite soyasapogenol B in plasma,urine and feces in rats,and soyasaponin Bb in HBSS.2.After oral gavage of soyasaponin Bb(100 mg/kg)in rats,plasma,urine and feces were collected at different times,then determined their content of soyasaponin Bb and soyasapogenol B.3.Establish an in vitro Caco-2 cell model,observe the morphology of the cell and changes in transmembrane resistance during cell culture,measure the activity of alkaline phosphate and the amount of permeation of phenol red across the membrane,these were all used to evaluate the integrity of the model.Determine the concentration of soyasaponin Bb applied to Caco-2 cells by CCK-8 method,and the two-way transport experiment was further carried out.Results1.The HPLC-MS/MS method for the detection of soyasaponin Bb and soyasapogenol B in rat plasma,urine and feces,and soyasaponin Bb in HBSS had been successfully established.2.After the rats were administrated,the plasma concentration of soyasaponin Bb reached a maximum value of 19.8 ng/mL at an average of 1.5 h,and there would be second peak at an average of 10.7 h,while soyasapogenol B was not detected in plasma.The cumulative excretion rate of soyasaponin Bb in urine at 168 h was(0.0022±0.0006)%,the cumulative excretion rate of feces at 168 h was(0.3 6±0.21)%;Soyasapogenol B was not detected in urine,while the cumulative excretion rate of feces at 168 h was(0.45±0.29)%.3.A Caco-2 cell model was established after 21 days of in vitro cell culture,the cells could be observed to form tight junctions under microscope.The transmembrane resistance of the cells was greater than 900 Ω·cm2,the alkaline phosphatase activity ratio reached 2(p<0.001)and the Papp value of phenol red was less than 1.0×10-6 cm/s,which verified the integrity of the Caco-2 cell model.Soyasaponin Bb with a concentration of 9.43 mg/L was selected for the transport experiment.The permeation amount of soysaponin Bb was positively correlated with the incubation time.The Papp values in the transport directions on both sides were less than 1.0×10-6 cm/s,and the ER value was less than 1.Conclusions1.The optimized HPLC-MS/MS method had good recovery and precision,and was suitable for the detection of soysaponin Bb and soyasapogenol B in rat and cell samples.2.Soysaponin Bb was poorly absorbed in rats,and the total excretion in urine and feces was less than 1%,indicating that there was a certain biotransformation in the body.The soyasapogenol B was not detected in rat plasma and urine,the excretion in feces was less than 1%,suggesting that soysaponin Bb might also have other metabolites.3.Soyasaponin Bb was poorly absorbed in the intestine.On the one hand,soyasaponin Bb was absorbed by passive transport and also affected by efflux transporters;On the other hand,Caco-2 cells uptook the soyasaponin Bb.