Study On Transportation Characteristics Of Flavonoids From ZiziphiSpinosae Semen And Its Self-microemulsion Of Phospholipid Complex

Flavonoids,derived from the seed of Ziziphous jujuba(medicinal and food homology),are believed to be the main constituents of Zizyphi Spinosae Semen(ZSS)responsible for a variety of pharmacological activities,such as sedative-hypnosis,depression,anxiolytic and antioxidant.The intestinal absorption characteristics of flavonoids from ZSS are crucial for evaluating its activity and efficacy.Studying the intestinal absorption properties of flavonoids from ZSS can not only guide people to choose the appropriate dosage form to increase the absorption and utilization,but also reduce the investment risk when it is used as a functional food research and development.The structure and function of the Caco-2cell monolayer model are very similar to that of the human small intestine epithelial cells,so it has been widely used in the study of in vitro intestinal absorption of nutrients.Flavonoids from ZSS are flavone-based carbon glycoside compounds,which generally have good water solubility,poor fat solubility and low bioavailability.Therefore,it is particularly vital to select a suitable dosage form for improving the membrane permeability.This paper firstly used shake flask method to determine the oil-water partition coefficient of six flavonoids(Isovitexin,Swertisin,Isovitexin-2''-O-?-D-glucopyranoside,Spinosin,6'''-p-coumaroylspinosin and 6'''-feruloylspinosin).The values of Log P were used to examine the lipophilicity and water-solubility of the six flavonoids,so as to predict its absorption in the intestine.Subsequently,the classical Caco-2 cell model was used to study the intestinal absorption characteristics and mechanism of flavonoids.The influencing factors of intestinal absorption were investigated at the same time to predict the in vivo absorption process of active ingredients.Finally,Spinosin self-microemulsion of phospholipid complex(SPI-PLC-SMEDDS)was prepared based on the structural characteristics of Spinosin.And then the quality of SPI-PLC-SMEDDS was evaluated mainly through the methods of morphological observation,particle size distribution measurement,in vitro dispersibility investigation,intestinal absorption characteristics research and oral bioavailability measurement.The main findings of this paper were as follows:(1)The results of oil-water partition coefficients concluded that the six flavonoids have small fat-solubility,and their membrane permeability are expected to be poor,which may lead to low oral bioavailability.Flavonoids had good fat-soluble properties under the condition of weakly acidic,and it could be presumed that flavonoids might be absorbed well in weak acid environment such as duodenum.(2)The integrity of the constructed Caco-2 cell monolayer model was evaluated by the observation of cell morphology,transmembrane resistance(TEER)assay and determinationof fluorescence yellow permeability.Transport experiments were performed when the cells were pooled into a complete dense monolayer.The results of the two-way transport experiment showed that the six flavonoids belonged to better absorbed drugs.The order of intestinal absorption capacity were as follows: 6'''-feruispinosin >6'''-p-coumaroylspinosin > Isovitexin-2''-O-?-D-glucopyranoside > Spinosin > Isovitetin >Swertisin.(3)The results of effects of pH and temperature on the transmembrane transport showed that neutral(pH=7.4)and 37 °C environments are conducive to transmembrane transport of Spinosin and 6'''-feruispinosin.The results of the intestinal absorption mechanism study showed that both Spinosin and 6'''-feruispinosin are mediated by energy-dependent active transport.Among them,monocarboxylate transporter(MCT)and organic anion transporter(OATP)are involved in the absorption of Spinosin,while6'''-feruisporosporin has only the participation of monocarboxylate transporter(MCT).In addition to carrier protein-mediated active transport,there is a paracellular transport pathway in the absorption process of Spinosin.The results of the efflux mechanism showed that P-glycoprotein(P-gp)and multidrug resistance protein(MRP)mediated the efflux process of Spinosin and 6'''-feruispinosin.In addition,breast cancer resistance protein(BCRP)also participated in the efflux of Spinosin.(4)Spinosin phospholipid complex(SPI-PLC)was prepared by solvent evaporation method.The results showed that solvent evaporation preparation was set up and its best preparation conditions were acetone as reaction dissolvants,the ratio of drug and phospholpid was 1:14.5,the rate of agitation,reaction temperature and time were 850 r/min,40 °C and 3 h,respectively,its average yield ratios were(91.01±0.79)%;UV and IR showed that the new SPI-PLC were formatted and its solubility was improved.(5)Based on SPI-PLC,self-microemulsion technology was used to prepare more stable Spinosin self-microemulsion of phospholipid complex(SPI-PLC-SMEDDS).The results showed that SPI-PLC-SMEDDS has smaller particle size,more uniform dispersion,higher in vitro release property.And SPI-PLC-SMEDDS can significantly increase the intestinal absorption and bioavailability of Spinosin.Therefore,it has a good application prospect.