Studies On Oral Absorption Of Panax Notoginsenoside(PNS) And Preparation Of W/O Microemulsion For Oral Administration

As main ingredients of notoginseng, Panax notoginseng saponins (PNS) proved in recent years to possess pharmacological action in cardiovascular system. But bioavailability for both Rg₁ and Rb₁, which are main ingredients of PNS, is very low after oral administration. In present study, Caco-2 cells and rat models were applied to study the mechanism of absorption after oral administration of PNS, and W/O microemulsion were prepared to enhance bioavailability of PNS.Stability of of both Rb₁ and Rg₁ in PNS in artificial gastric juice, HBSS at different pH value (pH 5.5, 6.0, 6.8, 7.4), contents of different parts of intestine, and homogenates of intestine mucosa were studied. It showed that Rb₁ (and Rg₁) proved to be readily eliminated in stomach, but stable in relatively neutral circumstance. Both Rb₁ and Rg₁ in PNS, especially for Rb₁, degraded significantly in the contents of large intestine. However, both of them kept mainly intact in the contents of small intestine and homogenates of intestinal mucosa.Uptake of both Rb₁ and Rg₁ by Caco-2 cell monolayers was inhibited in low temperature, but not by cyclosporine, and the change in the apical pH showed no pronounced effects. Uptake and transport were non-saturable and increased linearly with rising of concentration of Rb₁ (and Rg₁) over the range of concentration tested, which indicated a passive transport. There was no significant difference between monomer (Rb₁ and Rg₁) and mixture (PNS) for absorption characteristic. Uptake amount of Rg₁(1.07±0.16μg/mg protein) (Co=1mg/ml) in Caco-2 cells was a little higher than that of Rb₁ (0.77±0.03μg/mg protein) (Co=lmg/ml). Meanwhile, apparent permeability coefficient of (5.90±1.02)×10^-8 cm/s(C₀=1mg/ml) for Rb₁ and (2.59±0.17)×l0^-7cm/s (Co=1mg/ml) for Rg₁ from apical compartment to basolateral compartment predicted an incompletely absorption. Transport of both Rb₁ and Rg₁ were not influenced by cyclosporine.The serum concentration-time profiles of Rb₁ afer tail venous (iv), portal venous (pv), intraduodenal (id) and peroral (po) administration to rats were compared to evaluate the the role of stomach, intestine and liver involved in absorption process. The pharmacokinetic behavior of Rb₁ (and Rg₁) after different routes of administration to rats showed that the absolute bioavailability after po (PNS 1500mg/kg), id (PNS 900mg/kg), and pv (PNS 50mg/kg) administration is 0.71%, 2.71% and 65.77% respectively for Rb₁and 3.29%, 3.41% and 50.56% respectivelyfor Rg₁.So, it can be concluded that transport across Caco-2 cell monolayers for PNS (include Rb₁ and Rg₁) is a simple passive diffusion process. No efflux transporters in Caco-2 cells and other components in PNS showed effects on it. The elimination in stomach, large intestine and liver contributed to the low bioavailability of PNS, but the low membrane permeability might be a more important factor dominating the extent of absorption.This thesis studies Plasma protein binding and bile excretion of both Rg₁ and Rb₁ for further analysis of PNS. (61.48±18.30)% dose of Rg₁, and (3.94±1.49)% dose of Rb₁ were respectively excreted into bile 10 hours after iv administration (PNS 50mg/ml), and (0.91±0.51%)% dose of Rg₁ and (0.055±0.02)% dose of Rb, were excreted into bile 12 hours after po administration (PNS 1500mg/ml). Ranging from 20 to 200μg/ml in PNS concentration, the plasma protein binding degrees of Rg₁ and Rb₁ are respectively 6.56-12.74% and 80.11-89.69%.In further study, SP/EtOH was selected as surfactant/co-surfactant for the preparation of W/O microemulsion to enhance the bioavailability of PNS. Studies are mainly focused on the influence of different K_m and oil, and addition of drug (PNS) on the formation and physico-chemical properties of W/O microemulison. When W/O microemulision is composed of 30% SP/EtOH(K_m-1/1), 45%Labrafil M 1944CS and 25%PNS solution (400mg/ml), it shows a transparent system and particle size is 17.8±1.7nm, the content of surfactant is fairly low while that of PNS is relatively high. With the surfactant/co-surfactant of SP/EtOH (K_m=1/1) [or (SQ/O) /EtOH (K_m=1/1) ] and 11 PNS W/O microemulison prepared in different kinds of oils, pharmacokinetic behavior of W/O microemulison was experimented in the rats after intraduodenal administration. The results show that most of microemulison enhances the absorption of Rg₁ and Rb₁ significantly, and the mechanism of the enhanced absorption may be attributed to the effect of surfactant/co-surfactant. Meanwhile, the composition of the oil plays a role that cannot be neglected here and the enhancement of PNS absorption varies with the microemulsion containing different kinds of oil, as follows: BvIWITOR 312^IPM>IPP>2EHP. Accompanying with the rising carbon chain of fatty acid in oil, the rate of absorption enhancement by the long chain glyceride (C> C₁₄) is lower than that by the medium chain glyceride (C₈14).The experiments are also carried out for the analysis of the microemulsion absorption in everted intestine sac, and it is proved by the results that most ofmicroemulison can enhance the absorption of Rb₁ and Rg₁ after diluted with Kreb's solution. Among them, microemulision 3 can significantly enhance the absorption of both Rb₁ and Rg₁.Liposome was used as a model to imitate biological membrane, and then W/O microemulison was found to enhance the membrane fluidity of liposome to different extents. It suggests that the effect of W/O microemulison on the absorption enhancement may be related with its enhancement in the membrane fluidity.PAMPA analysis was first applied in the present study of P_erformulation to explore the effects of different W/O microemulison on the absorption of PNS. The value of P_e and the correlation between P_e and Fr, P_e and R were studied to evaluate the feasibility of PAMPA being an in vitro model and the pharmaceutical dosage.The results show that P_e of Diluted-Microemulsion (D-ME) is mostly higher than that of PNS solution (control), which suggests the components of microemulision can facilitate the membrane P_ermeability of drug. While in the coordinate conditions, P_e of Rg₁ is mainly lower than that of Rb₁, which accords with the results of Caco-2 cell exP_eriments. But for D-ME, neither P_e nor R shows linearity correlation with Fr and this may be attributed to the phase transformation resulted from the separation, precipitation and flocculation of the system after the dilution of the Microemulsion. In some exP_eriments, lipids separated from the system even adhere to PAMPA membrane or intestine mucous membrane, which may also influence the results of the exP_eriment. The linearity correlation between PAMPA and Fr acquired in Microemulison (ME) analysis further indicates that the PAMPA analysis can be brought into not only the investigation of membrane transport of crude drug, but also conditioned preformulation research.