| Astilbin (Ast,3,3',4',5,7-pentahydroxyflavanone 3-(6-deoxy-(L-mannopyranoside))) is a flavanone compound isolated from many plants. In recent years, much attention has been paid to its immunosuppressant activity. But according to the biopharmaceutics classification system, astilbin is an extreme example of a class IV compound due to its low permeability and poor oral absorption, thus it becomes a limiting step in clinical application. This present study aimed to solve the poor solubility of Astilbin, study the effects of dispersion carriers and surfactant on astilbin transport behavior in the caco-2 monolayer model and the pharmacokinetics of astilbin complex dispersion system. The research laid the foundation of further development.1. Extraction and purification research of AstilbinThis systematic research studied the optimum extraction and purification process for Astilbin. The Lg(34) orthogonal experiment was designed with content of flavonoids in Smilax glabra and Astilbin as indicators. The experimental results showed that the influence factors were in the following order:ethanol concentration> extract time> extract number> solvent amount. The optimal extractive conditions obtained was:12 times the amount of solvent,60%ethanol,2 hours for two times. Astilbin content is not stable in Smilax glabra, and differs with different origin, sources and herbs ranging within 1.1～25.13mg/g (0.11%-2.51%).Further, the purification technology was studied by using 6 kinds of macropomus adsorption resin with their Astilbin flavonoids adsorption rate, adsorption rate, desorption rate and product yield and flavonoid content as index. The results showed that resin HPD400 had the largest absorption capacity to Astilbin flavonoids with characters of fast adsorption and easy desorption, and its product had a higer flavonoid content after adsorption. AB-8, a weakly polar resin, is also a good kind of sorbents for Smilax glabra flavonoids, and can be used to enrichment of total flavonoids and Astilbin from Smilax glabra gaining more than 78% content of total flavonoids and 35% above Astilbin.Samples then passes through polyamide column chromatography, eluting with purified water,20%,40%,60% ethanol, obtaining refined products with Astilbin content more than 80%. But with the increase in the adsorption and elution, the adhesive force of polyamide to flavonoids decreased, so it was necessary of the regeneration or replacement of the polyamide after the several uses.Products were further refined through dextran gel column chromatography by 80% methanol elution. The sample was confirmed as the purity compound higher than 95% by determination of physical-chemical and spectral properties and chemical structure identification.The extraction and purification process is stable, easily-operation and suitable for industrial mass production.2. Preparation of astilin solid dispersionIn this paper, the possibility and process of preparation of Astilbin solid dispersion was proved by technicals of improving the solubility of insoluble drugs. In preformulation studies, we first established a sound analysis system, inspected the physical and chemical properties of APIs, and established a complete evaluation indicator of solid dispersion to provide a scientific basis. Researches on the effects of various preparations, carriers, and the ratio between the carriers and drug were carried out. PVPK30/C15 and tweens 80 were used as joint carriers to prepare astilbin solid dispersions by solvent method and Astilin solid dispersion of drug-Tween80-PVP K30 with the proportion of 1:1.5:3 was ideal. The characteristics of astilbin solid dispersions were studied by its in vitro dissolution, solubility, Papp, microscope, X-ray diffraction, and DSC. And experiments on the influence of temperature, humidity and illumination on astilin solid dispersion were also conducted. The results of the microscope, X-ray diffraction, and DSC study showed that astilbin in solid dispersion were molecular or microcrystalline. The content of Astilin in Astilin solid dispersion decreased in high temperature, but which was free from the influence of the humidity and illumination. Solid dispersion can improve the dissolution and solubility of astilin, obviously changed Papp and then promote the in vivo absorption. Astilin content in solid dispersion decreased in high temperature but was free from the influence of the humidity and illumination and it can be kept in normal temperature.The use of Excel software in models fitting of in vitro drug release was discussed, with self-prepared sustained-released Breviscapine tablets as an example. It takes less than lh of time to complete using Excel, containing all of the various models, and the data is accurate and illustrated. The method is easily operated and promoted. This simple data processing method was applied in Astilbin and its solid drug release curve fitting. The results showed that the Weibull equation was highest-fitting with the r of Astilbin, solid dispersions 0.9944, 0.9986 respectively. By mechanism discussion of solubilization, we can see exactly that the solid dispersion carriers suppressed the aggregation trend of highly dispersed particle (suppressing crystalline), and the surface area was expanded. Besides carriers theirselves can promote the dissolution of drug and therefore solid dispersions greatly improved the drug's solubility, and promote the drug absorption and bioavailability.3. Drug transport in caco-2 monolayerThe established Caco-2 cell were observed good and homogenous growth, Clear boundary, and the formation of tight and complete junctions between cells. TEER values of Caco-2 cell monolayer increased with the incubation time with the value> 500Ω·cm-2 after 21 days of cell culture, indicating the formation of integrate monolayer. The activity of ALP in the AP and BL side were 46.371±5.902,5.286±0.86 respectively with a significant difference (P<0.01). The activity of ALP of the AP side is 8.77 times of the BL side indicating cell polarity had been formed and ALPs were mostly concentrated in the AP side. Papp value of propranolol was (2.193±0.170)×10-5 cm/s with the reference value (2.397±0.630)×10-5 cm/s. The established Caco-2 cell monolayer model was in full compliance with the requirements of drug transport experiments, and could be used to simulate characteristics of intestinal absorption in vitro.For astilbin and its solid dispersion, the cell viability gradually decreased with the increase of the drug concentration, and the cell proliferation was significantly enhanced in a dose dependent manner. The cell viability of both astilbin and its solid dispersion were more than 80% in the concentration range 5～400μg/mL, and latter was lower than that of astilbin with no significant difference. The safety concentration range of astilbin in Caco-2 cell was 5-400μg/mL.This study explored the effect of PVP-tween 80 combined solid dispersion system on the astilbin transportation in Caco-2 cell model. Papp of the drug and drug in solid dispersion increased with dose, and Papp of the latter was significantly different (P=0.005). As to the effects of carriers to promote drug penetration (P=0.017) PVP k30 was best, while PEG4000 and F68 not significant. The effects of drug/carrier ratio on the Papp of astilbin were of significant difference (P=0.006), and the Papp of astilbin increased with the amount of the carrier in a certain range, in which the ratio of drug/carrier 1:3 was best and the ratio of 1:1 worst. Carrier and surfactant combined system can significantly improve drug apparent permeability coefficient (P=0.008), and the effect of surfactant amount on the Papp was of significantly different, in which the carrier/surfactant ratio 1:1.5 best. The carrier and the surfactant in solid dispersion system were the key factors in astilbin transporting behavior in the caco-2 monolayer model. The apparent permeability coefficient of astilbin in the caco-2 monolayer model was improved, suggesting the improved drug penetration and enhanced drug absorption in vivo by solid dispersion system.4. Pharmacokinetics and oral bioavailability in beagle dogThis report describes a successful development of LC/MS/MS assay for astilbin in plasma. [M-H]-proton peak was m/z 449.0 for astilbin and m/z 414.5 for internal standard puerarin. The main fragments were m/z 284.9 for astilbin and m/z 266.9 for internal standard as the product ion for quantitative analysis. The retention time of astilbin and internal standard puerarin were 1.7 min and 1.8 min respectively and the method shows a good separation of astilbin and internal standard to the plasma constituents. The Standard profile of astilbin in plasma was linear over the concentration range of 2.5-1000|-ig/ml examined. The regression equation for astilbin was Y=-0.0023578+0.000887034*X (r= 0.9816).The precision was determined at three different concentrations (5.0,50.0,500.0 ng/mL). The relative standard deviation (R.S.D.) for every studied concentration was less than 7.7%, relative deviation (RE) less than 12.0%, indicating the precision of the method for routine purposes. The mean extraction recovery was 81.6±9.3%,82.2±3.6%,80.0±4.5% and 86.9±4.5% for astilbin at 5.0,50.0,500.0ng/mL and internal standard respectively. The medium effect was 93.4±5.8%,96.1±3.4%,97.8±7.9% and 102.1±3.2% for astilbin at 5.0,50.0,500.0ng/mL and internal standard respectively. Based on a signal-to-noise ratio (S/N)=10 (R.S.D.<20%), the limit of quantization of astilbin was 2.5 ng/m.L in plasma and the precision of LLOQ concentration of (RSD) was 3.18% (n=6). The method has been applied to pharmacokinetic studies after oral administration of the compound and its solid dispersion to beagle dogs. The sensitivity of this method appeared satisfactory for monitoring the plasma concentration.This is the first study on the pharmacokinetics and bioavailability of astilbin and its solid dispersions in Beagle dogs. The non-compartmental calculation indicated that the rmax of astilbin solid dispersion (2.583±0.665 h) appeared to be longer than that for API (1.667±0.258 h) of significantly different (P< 0.05). The Ke of astilbin solid dispersion (0.65±0.12 h"1) was significantly different from that of astilbin (0.31±0.16 h'1) (P< 0.01). The Cmax of astilbin solid dispersion (122.550±52.740 ng-mL"1) was significantly different from that of astilbin (71.050±34.251 ng-mL-1) (P< 0.05). Astilbin solid dispersion had a faster elimination rate with a smaller tm (1.106±0.218 h) than astilbin (3.380±3.190 h) (P< 0.05). The AUCo-t of astilbin solid dispersion (298.642±173.673 ng-mLT1) was significantly different from that of astilbin (133.186±49.071 ng-h-mL-1) (P< 0.05), while the AUC0-∞of astilbin solid dispersion (305.994±70.640 ng·h·mL-1) and the AUC0-∞of astilbin (160.048±86.874 ng·h·mL-1) was of no significantly difference (P> 0.05). Indeed, calculated on the basis of the AUC0-∞of each formulation, the oral bioavailability of astilbin solid dispersions evaluated by Cmax,AUC(0-t),AUC(0-∞)was about 1.8,212.4%,192.0% reapectively as compared to astilbin. Comparing the pharmacokinetic parameters and oral bioavailability data indicates the effects of solid dispersions on the oral absorption of astilbin.
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