ABSTRACT
OBJECTIVE To prepare hyperoside mixed nanomicelles （Hyp-F127/TPGS） and optimize its preparation technology，and to investigate its intestinal absorption characteristics. METHODS Hyp-F127/TPGS was prepared by thin film dispersion method. Based on single factor test and Plackett-Burman design ，combined with Box-Behnken response surface method ， the preparation process was optimized and validated using entrapped efficiency （EE）and drug loading （DL）as evaluation indexes ， F127-TPGS mass ratio ，hydration time and the amount of Hyp as factors. The appearance and microscopic morphology of Hyp-F127/TPGS obtained by the optimal technology were observed ，and the particle size ，polydispersity index （PDI）and Zeta potential were also determined. The critical micelle concentration （CMC）of blank micelle （F127/TPGS），in vitro release behavior and preliminary stability of Hyp-F 127/TPGS were investigated ，and absorption characteristics of Hyp-F 127/TPGS were investigated by in situ unidirectional intestinal perfusion model. RESULTS The optimal preparation technology of Hyp-F 127/TPGS included F127-TPGS mass ratio of 2∶1，hydration time of 2 h，and Hyp amount of 9 mg. Results of three validation tests showed that the EE of Hyp-F 127/TPGS was （87.20±0.99）％，and the DL was （5.02±1.20）％，deviations from predicted values were 0.92％ and 2.39％. The micelles prepared by optimal technology were yellow ，clear and transparent solution ，with good Tyndall effect ；under transmission electron microscope ，they were spherical ，complete and evenly distributed ；the particle size was （15.02±0.16）nm， the PDI was 0.092±0.031，and the Zeta potential was （－6.67±1.47）mV. The CMC of F 127/TPGS was 21 μg/mL，Hyp-F127/ TPGS was stable after 4 weeks of storage at 4 ℃，and the cumulative release rates of Hyp-F 127/TPGS and Hyp control were （66.30±2.93）％（96 h）and（99.24±0.27）％（60 h），respectively. Hyp-F 127/TPGS and Hyp reference were absorbed in each intestinal segment ，and the main absorption sites were jejunum and duodenum respectively ；drug absorption rate constant andapparent absorption coefficient of the former were significantly higher than those of the latter （P＜0.05 or P＜0.01）. E-mail：zhangyuhangxz@163.com CONCLUSIONS The optimized preparation technology of Hyp-F127/TPGS is stable and feasible ；prepared Hyp-F 127/ TPGS shows a sustained -release effect ，which promotes the intestinal absorption of H yp to a certain extent.
ABSTRACT
The intestinal absorption properties of the main effective components (glycyrrhizic acid, isoliquiritigenin, 6-gingerol, ginsenoside Rb1, atractylode-I) in Lizhong decoction (LZD) extracts were investigated with an in situ single-pass intestinal perfusion model in rats. UPLC-TQ-MS was used to determine the concentration of the five components in the intestinal perfusion. Animal welfare and experimental procedures were in accordance with the regulations of the Animal Ethics Committee of Nanjing University of Chinese Medicine. As evaluation indexes for the intestinal absorption characteristics, the absorption rate constant (Ka) and the apparent permeability coefficient (Peff) of the five main ingredients were analyzed. Results showed that the best absorption sites for glycyrrhizic acid, isoliquiritin and 6-gingerol were the ileum, colon and duodenum, respectively, and the differences between different intestinal segments were statistically significant (P <0.05). There was no notable difference in Ka and Peff between ginsenoside Rb1 and atractylode-I in the different intestinal segments (P > 0.05), suggesting that they were absorbed throughout. The five components were well-absorbed in the whole intestine (Peff > 1.0×10-3 cm·min-1), indicating that LZD is suitable for preparing sustained, controlled release and enteric-coated preparations.
ABSTRACT
OBJECTIVE： To investigate absorption kinetic characteristics of main active components as 4-（glucoseoxy）- glucoseoxybenzyl cinnamate （A1）， 2-isobutyl malic acid （A2）， 1，4-bis [4-（glucoxy） benzyl]-2-isobutyl malic acid ester （A3）， dihydrophenanthrenes 1 （A4） and 1，4-bis [4-（glucosoxy） benzyl]-2-isobutyl malic acid ester-2-（4-O-cinnamoyl-6-O-acetyl） glucoside （A5） from ethanol extract of Bletilla striata in the intestines of rats. METHODS： Using puerarin as internal standard， UPLC-MS/MS was used to determined the concentration of A1-A5 in intestinal circulation fluid. The determination was performed on Acquity UPLC BEH C18 column with mobile phase consisted of acetonitrile （containing 0.1％ formic acid）-water （containing 0.1％ formic acid） （gradient elution） at the flow rate of 0.35 mL/min. The column temperature was 45 ℃， and sample size was 3 μL. The positive ion and negative ion scanning were carried out in the multiple reaction monitoring mode by electrospray ion source. The ion pairs for quantitative analysis were m/z 593.2→431.1 （A1）， m/z 189.0→129.0 （A2）， m/z 725.3→457.2 （A3）， m/z 347.1→332.1 （A4）， m/z 1 059.3→793.1 （A5）， m/z 417.0→267.0 （internal standard）. In the in vivo intestinal circulation perfusion model， using accumulative absorption transfer rate （A） and absorption and transformation rate constant （Ka） as indexes， the effects of different doses of ethanol extract from B. striata （low-， medium-， high-dose were 166， 333，667 μg/mL，respectively）， bile， P-glycoprotein （P-gp） inhibitors （verapamil） and different intestinal segments on the absorption of above 5 components were investigated. RESULTS： The linear range of A1， A2， A3， A4 and A5 were 0.22-14.00， 0.34-21.75， 1.99-127.16， 0.15-9.75， 0.16-10.00 μg/mL（r＞0.99）. The limits of quantitation were 0.22， 0.34， 1.99， 0.15， 0.16 μg/mL， respectively. The lowest detection limits were 0.028， 0.085， 0.251， 0.035 and 0.010 μg/mL. RSDs of inter-day and intra-day were all lower than 10％. The recoveries ranged 83.60％-106.91％. Matrix effect did not affect the determination of the substance to be measured. A and Ka values of A1 in B. striata ethanol extract low-dose and medium-dose groups were significantly higher than high-dose group； A value of A3 in low-dose group was significantly higher than medium-dose and high-dose groups （P＜0.05 or P＜0.01）. A and Ka values of A1 and A3 in non-ligation group were significantly lower than control group， while A and Ka values of A4 were significantly higher than control group （P＜0.05 or P＜0.01）. A and Ka values of A1 and A3 in P-gp inhibitor group were significantly lower than control group （P＜0.05 or P＜0.01）. A values of A1 in jejunum group， ileum group and colon group， Ka value of A1 in colon group， A and Ka values of A2 in colon group， A value of A3 in ileum group， A and Ka values of A4 in ileum group and colon group， A values of A5 in jejunum group and ileum group as well as Ka value of A5 in jejunum group were all significantly lower than duodenum group. Ka values of A3 in jejunum group， ileum group and colon group were significantly higher than duodenum group （P＜0.05 or P＜0.01）. CONCLUSIONS： Established UPLC-MS/MS method is specific， sensitive and simple， and it can be used for quantitative analysis and pharmacokinetic study of A1-A5. The 5 active components in B. striata ethanol extract are absorbed by the whole intestine， and the intestinal segments are different. A1 and A3 are absorbed more in intestinal tract and may be saturated. Bile can inhibit intestinal absorption of A1 and A2， but promoted intestinal absorption of A4. A1-A5 may not be the substrate of P-gp.