| Genistein(GEN)is a kind of active isoflavone extracted from Pueraria lobata.It has various biological activities such as anti-inflammatory,antioxidant and hypolipidemic,etc.However,GEN belongs to class Ⅱof biopharmaceutical classification system,its water solubility and bioavailability are very low.Tetramethylpyrazine(TMP)is a kind of active isoflavone extracted from Rhizoma chuanxiong and it has various biological activities.We prepared the GEN-TMP cocrystal by solution suspension method,it can not only improve the defect of easy sublimation of TMP,but also improve the solubility of GEN in water.However,it is unclear that the co-crystals exert the pharmacological effect in the form of"physical mixture(PM)of API and CCF" or a "new entity based on weak force".In this work,established a Caco-2 cell absorption model and verified by transepithelial electrical resistance and sodium fluorescein flux through the monolayer et al.The toxicity of GEN,TMP,GEN-TMP and GEN+TMP to Caco-2 cells were detected by CCK-8 method,and the safe concentrations were determined.The cumulative amount of transport,the permeability coefficient(Papp)and efflux ratio(ER)were determined by high-performance liquid chromatography(HPLC).And added efflux inhibitors such as P-glycoprotein(P-gp)inhibitor Verapamil,multidrug resistance protein 1/2(MRP 1/2)inhibitor MK571,and breast cancer resistance protein(BCRP)inhibitor KO143 to explore the transport mechanism.In addition,the SPIP model was successfully established,and the drug concentration in the intestinal perfusion fluid was determined by HPLC method.Furthermore,the powder dissolution and oral pharmacokinetics of GEN,TMP,GEN-TMP and GEN+TMP in male SPF rats were studied by HPLC to determine the drug concentration in water and plasma,and the pharmacokinetic parameters were calculated.The results showed that the Caco-2 cell absorption model was successfully established.TEER decreased to 820 Ω·cm2 at day 21.The cell viability of GEN,TMP,GEN-TMP and GEN+TMP were 90.06%,84.21%,97.60%and 89.37%respectively;The Papp of GEN,TMP,GEN-TMP and GEN+TMP were all higher than 1.0×10-6 cm/s,which were drugs with good absorption.The cumulative amount of transport and Papp of GEN were(1.92±0.15)μg and(5.96±0.47)×10-6 cm/s,and GEN in GEN-TMP were(2.78±0.11)μg and(8.61±0.33)×10-6 cm/s.Compared with GEN,GEN in GEN-TMP were increased 44.79%and 44.46%respectively.There was no significant difference of the cumulative transport and Papp value between GEN in GEN+TMP and GEN;GEN was affected by the efflux of BCRP,GEN in GEN-TMP was affected by the efflux of P-gp and BCRP;The transport of TMP,TMP in GEN-TMP and TMP in GEN+TMP were all influenced by MRP2.The studies in SPIP showed that the effective permeability coefficient(Peff)of GEN,GEN in GEN-TMP and GEN in GEN+TMP were(3.18±0.40),(10.60±1.22)and(2.89±0.43)×10-5 cm/s,GEN in GEN-TMP was 3.34-fold and 3.67-fold higher than GEN and GEN in GEN+TMP.The Peff of TMP,TMP in GEN-TMP and TMP in GEN+TMP were(2.49±0.29),(4.40±0.48)and(2.39±0.27)×10-5 cm/s.TMP in GEN-TMP was 1.77-fold and 1.84-fold higher than TMP and TMP in GEN+TMP.The solubility of the GEN from cocrystal GEN-TMP was 5.25 times higher than that of GEN.The pharmacokinetics results showed that AUC0～12h of GEN in co-crystal GEN-TMP was(6.85±0.33)mg·h/L,which was 2.07 times of GEN alone,and Cmax of GEN in co-crystal GEN-TMP was(3.08±0.43)μg/mL,which was 4.50 times of GEN alone.The GEN+TMP of GEN-TMP showed that no significant mechanism diffrence with GEN and TMP.The results show that the cumulative amount of transport and Papp of GEN-TMP in the Caco-2 cell model and Peff in the SPIP model were different from those of GEN and GEN+TMP.This indicating a different transport characteristic between the co-crystals and physical mixture,which provides a reference for the intestinal absorption mechanism and characteristics of the co-crystals. |
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