Neuroprotective Effects Of Catalpol On Senescent Mice Induced By D-galactose

Catalpol, one of the main active constituents of Rehmannia glutinosa Libosch, possesses many therapeutic effects such as protection of liver damage, anti-inflammation, reduction of pain and blood sugar. Catalpol has been verified to have neuroprotective effects in vitro and in vivo in our laboratory and be worth further studying. Due to its instable chemical property and complicated seperation technique, it is so difficult to obtain bulk of catalpol that an optimized seperation technique of catalpol becomes urgent. The purpose of this study mainly aims to optimize existed extraction and purification technique of catalpol and obtain enough catalpol to investigate its protective effects on senescent mice induced by D-galactose. The main results are summarized as following.Firstly, an optimized technique for extraction and purification of catalpol had been established, including microwave-assisted extration and column chromatography with macroporpus adsorbent resins.Different extraction methods including extraction at room temperature (ERT), heat reflux extraction, Soxhlet extraction, ultrasonic extraction and microwave-assisted extraction (MAE) to evaluat the percentage extraction of catapol from Rehmannia has been tested. The extracts were analyzed by high performance liquid chromatography (HPLC). The results showed that the percentage extraction of catalpol from Rehmannia by MAE (4 min) was more efficient in short time followed by Soxhlet extraction, heat reflux extraction, ultrasonic and ERT methods. The present results showed that the considerable saving of time by MAE was more competent than the other extraction techniques. Considering its time saving and higher percentage extraction of catalpol, we had choosed MAE technique for catalpol extraction from Rehmannia. Nine different kinds of macroporous adsorbent resins were evaluated by the static capacity of adsorption and desorption. The results showed that D101 resin had higher static adsorption capacity of 76.1mg/g dry resin and desorption ratio of 92.1%. Its isotherm curve can be well described by Langmuir and Freudlich equation and belong to monomolecular layer absorption. D101 resin was choosed for the purification of catalpol from the extraction solution of Rehmannia. After the extract containing 2mg/mL of catalpol was loaded onto the column, resins bed was eluted by 5BV (five folds volume of resin bed) water and different concentrations of ethanol, respectively. The 5% ethanol elution on removal of the solvent under reduced pressure provided a brown powder containing 50% catalpol, which was subjected to an open column chromatography on silica gel eluted with a CHCl₃-MeOH gradient. The fraction eluted with CHCl₃-MeOH (8:2) was identified as catalpol and the purity of the compound was more than 90% purity by HPLC analysis. The yield of this separation technique was 600mg/kg Rehmannia.Secondly, the neuroprotective effects of catalpol on the senescent mice induced by subcutaneous injection of D-galactose were assessed and the mechanisms underlying its protective effects were studied.(1) Effects of catalpol on the cognition ability of senescent mice induced by D-galactose were evaluated by open field test, step-down avoidance test and Morris water maze test. The results showed that injection of D-galactose caused not only degression of autonomic activities and novelty-induced exploratory behaviors but also passive and spacial cognition ability. Administration of catalpol for two weeks was capable of reversing the D-galactose induced behavioral impairment, indicating that catalpol has the effect of ameliorating cognition ability of senescent mice. Hematoxylin and eosin (HE) staining showed that pyramidal neurons either presented a densely stained shrunken appearance with minimal cytoplasm or had disappeared in the brain of D-galactose treated mice. In contrast, majority of the neurons were rescued in mice treated with catalpol. Moreover, catalpol decreased the elevated activity of acetylcholinesterase (AChE) in aging mice brain cortex and hippocampus, increased the positive neurons of choline acetyltransferase (ChAT) in aging mice basal forebrain revealed by immunohistochemical staining and the expression of Acetylcholine M receptor (mAChR) in aging mice brain determined by western blotting method, indicating that catalpol had protective effects on cholinergic impairment in aging model mice so as to maintain the content of acetylcholine (ACh) and eventually ameliorate cognition deficits of senescent mice.(2) Effects of catalpol on the anti-oxidative ability of senescent mice induced by D-galactose were examined by biochemical method. The results showed that catalpol increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GSH-ST), decreased the malondialdehyde (MDA) level in the cerebral cortex, hippocampus, liver and spleen of D-galactose treated mouse. These suggested that the antiaging effects of catalpol were partly mediated via enhancing endogenous antioxidant enzymatic activities, alleviating lipid peroxide and protecting organism from oxidative damage. Significant negative correlation was found between the mean latency to find the platform on fifth day in water maze test and the activities of SOD and GSH-Px in mice cortex and hippocampus. The levels of MDA were positive correlated with the mean latency in the two regions of mice brain. The data indicated that the oxidative damage may play a role in the cognitive decline of the senescent mice induced by D -galactose. (3) Effects of catalpol on the brain neuron apotosis of senescent mice induced by D-galactose were studied utilizing ultraviolet spectrophotometer, fluorospectrophotometer and flow cytometry. The data revealed that catalpol could decreased the activity of lactate dehydrogenase (LDH), increased the activities of creatine kinase (CK), Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in brain cortex and hippocampus of senescent mice induced by D-galactose. Further study found that catalpol could increase the activities of respiratory complex I, II /III, IV and mitochondrial membrane potential level, decrease reactive oxygen species (ROS) production in the brain cortex and hippocampus mitochondria of senescent mice. Flow cytometry analysis revealed that catalpol could inhibit apoptosis in the brain cortex and hippocampus of senescent mice via inhibting the production of ROS and the loss of membrane potential level, up-regulation of Bcl-2 and inactivation of caspase-3. These results suggested that catalpol could exert antiaging effects via maintaining the balance of energy metabolism in the brain of senescent mice by elevating the activities of respiratory complex, inhibting apoptosis and regulating the activities of energy-related enzymes.In conclusion, the results suggest that catalpol may be one of the main antiaging components in the Rehmannia and may be an agent for senescence or neurodegenerative diseases.