Molecular Structure And Pharmacological Studies On Aucubin And Its Derivatives

Aucubin (AU), which is the main iridoid component from the seeds of Eucommia ulmoides Oliv., has been found to have various pharmacological activities such as high liver-protective activity, antiviral activity, antitumor, anti-inflammatory activity and so on. These actions require the presence of aβ-glucosidase to hydrolyse aucubin to its aglycone, aucubigenin (AUG).In our studies, the chemistry and efficacy of AU and its derivatives were investigated, including molecular structure, chemical synthesis, antioxidant and antimicrobial activities, pharmacokinetics, acute toxicity and long-term toxicity. The detailed descriptions are listed below.Part one:The crystallographic study on AU, AUG and isoeucommiol (ISE).We obtained AU from the seeds of Eucommia ulmoides Oliv. by a procedure of extraction with ethanol. We also have improved the procedure for the extraction of AUG from theβ-glucosidase hydrolysis of AU at 37℃. ISE was obtained through NaBH4 reduction of AUG. The crystal structures of the three compounds were determined by a single-crystal X-ray diffraction analysis. It has been found that crystals of AU are orthorhombic, crystallize in the P212121 space group, and have four molecules in the crystal unit cell. While the crystals of AUG are monoclinic, space group P21, and have two molecules in the unit cell. The crystals of ISE adopts an envelope conformation, presenting a triclinic system, space group P1 with one molecule in the unit cell. Moreover, intensive strength O-H…O hydrogen bonds in all crystal lattices can be observed.Part two:The bioactivities study on AU and AUG.AU and its aglycone AUG have been investigated for their in vitro free radical scavenging and antibacterial activities. Various methods, such as the scavenging activities towards DPPH radical, superoxide anion radical, and hydroxyl radical, were established. The antibacterial activities of AU and AUG were also determined by the disc diffusion method and micro-well dilution assay with spectrophotometric method. AU and AUG were individually tested against Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and Salmonella enterica (ATCC 14028), respectively. The results showed for the first time that the two compounds exhibited significant differences in free radical scavenging activities. AU showed little DPPH radical scavenging activity. However, a dose response relationship is found in DPPH scavenging activity of AUG. Both AU and AUG showed a moderate scavenging activity against superoxide anion radical. AUG appeared to have little scavenging activity on hydroxyl radical scavenging effects but AU exhibited moderate scavenging activity in a dose dependent manner. The results also indicated that AUG exhibited considerable antibacterial activity against all tested strains, and was particularly more effective against Staphylococcus aureus. As expected, AU had no inhibitory activity against the up growth of all tested strains.Part three:The pharmacokinetic study on AU.A simple and accurate high performance liquid chromatographic method was developed and validated for the determination of AU in rabbit plasma. For the experimental group, a dose of 500 mg·kg-1 oral administration of AU and 100 mg·kg-1 intravenous injection of AU were finished. The result showed that both drug concentration-time data were fitted to a two-compartment model with first order absorption. This method could determine the AU level in plasma and are suitable for its pharmacological study.Part four:The toxicity study on AU.The acute toxicology experiment study on AU showed that MTD (maximal tolerance dose) administration to mice was 40 g·kg-1. Results of 6 months' long-term toxicity studies indicate that AU has no effect on weight, hair, activity, diet and general condition of rats. Hematologic parameters and blood biochemical indices were within normal physiological range. Heart, liver, spleen, lung, kidney, stomach, intestines and other organs and tissues do not contain any visible pathological changes.Every organ has no change of histopathology. In the recovery period, the rats showed no significant chronic toxicity or delayed toxicity.Part five:Effect of AU on bone microarchitecture and biomechanics in rats.To our knowledge, the direct effect of AU on bone structure in vivo has not been investigated yet. Therefore we became interested to study the effect of AU on bone structural and biomechanical analysis in rats after 6 months'long-term toxicity studies. Then the rats were killed and the femurs were dissected for measurement of trabecular microarchitecture and biomechanical analysis. Micro-CT analysis of the distal femur showed that AU significantly increased in bone volume/tissue volume, connect density, trabecular number and trabecular thickness, and decrease in trabecular separation and structure model index in normal rats. In the three-point bending test, the maximum load (ultimate strength) was determined until the specimen was broken. Daily oral administration of AU was found to be able to significantly increase biomechanical quality of femur. The mechanical changes were associated with the bone mineral density increase or even with some improvements in microarchitecture. We demonstrated for the first time that AU taken orally increased bone density and bone strength of rats at the femur. It might be a potential alternative medicine to be used for the treatment of bone deficient diseases such as osteoporosis.