| In the present thesis, the chemical constituents, quality control method, improvement on learning and memory of AD rats, pharmacokintics of the active constituents and effects on catecholamines of AD rats of Xanthoceras sorbifolia Bunge (yellowhorn) were studied.Phytochemical studies on the n-butanol extract of yellowhorn husks with vacuum flash chromatography on silica gel/ODS and semi-preparative HPLC led to the isolation and purification of seven metabolites. On the basis of physicochemical properties and spectroscopic analysis including ID,2D NMR, MS, their structures were identified as follows: 3-O-(3-O-angeloyl-6-O-β-D-glucopyranosyl)-β-D-glucopyranosyl-28-O-(2-a-L-rhamnopyranosl-6-0-β-D-glucopyranosyl)-β-D-glucopyranosyl-16-deoxybarringtogenol C (1, WGA),3-O-(3-O-angeoyl-6-O-β-D-glucopyranosyl)-β-D-glucopyranosyl-28-O-(2-a-L-rhamnopyranosyl-6-O-β-D-glucopyranosyl)-β-D-glucopyranosyl-21-O-acetyl-16-deoxybarringtogenol C (2, sorbifoside A), 3-O-(3-O-angeloyl-4-O-acetyl-6-O-β-D-glucopyranosyl)-β-D-glucopyranosyl-28-O-(2-a-L-rhamnopyranosyl-6-O-β-D-glucopyranosyl)-β-D-glucopyranosyl-16-deoxybarringtogenol C (3, sorbifoside B), R1-barrigenol (4),3-O-(3-O-α-L-arabinofuranosyl-2-O-β-D-galactopyranosyl)-β-D-glucuronopyranosyl-21,22-di-O-angeloyl-R1-barrigenol (5),21,22-di-O-angeloyl-R1-barrigenol (6), rutin (7). Among them, compounds 2 and 3 were new compounds.A simple and sensitive assay based on high performance liquid chromatography-mass spectrometry (HPLC-MS) coupled to an electrospray ionization (ESI) interface for the simultaneous determination of 6 triterpenoids (1-6, including two triterpenoid aglycones) in different parts of X. sorbifolia. Separations were carried out with a Diamonsil C18 reversed-phase column (250 mm×4.6 mm,5μm). The mobile phase consisted of methanol and 0.05%(v/v) aqueous formic acid. A gradient elution program was used. The flow rate was set at 0.8 mL·min-1 and aliquots of 5μL were injected into the HPLC system for analysis. Mass data were acquired using a mono-quadrupole mass spectrometer coupled with an electrospray ionization source (ESI) in the positive ion mode with a curved desolvation line (CDL) temperature of 250℃, a heat block temperature of 200℃, a CDL voltage of 10.0 kV and a detector voltage of 1.75 kV. Target ions were monitored at m/z 698.40 for compound 1 [M+2Na]2+, m/z 719.35 for compound 2 and 3 [M+2Na]2+, m/z 529.20 for compound 4 [M+Na]+, m/z 1163.60 for compound 5 [M+Na]+ and m/z 693.40 for compound 6 [M+Na]+using the selected ion monitoring (SIM) mode. All the analytes showed good linearity (r2> 0.9980) in a relatively wide concentration range. The precisions were evaluated by intra-and inter-day tests, and the relative standard deviation (RSD) values were within the range of 2.0-2.8% and 1.7-2.9%, respectively. The recoveries for the quantified compounds were observed over the range of 95.3-104.7% with RSD values less than 4.6%. The method developed was successfully applied for simultaneous quantification of the six triterpenoids in X. sorbifolia, and our results showed that husks contained all the six triterpenoids, five (2-6) of which were in their highest concentrations and the contents of triterpenoids in different parts of X. sorbifolia varied significantly. This method was validated for good accuracy, repeatability and precision, and can be used as a valid analytical method to evaluate the intrinsic quality of yellowhorn.Improvement of extract of the husks of yellowhorn on learning and memory was studied with AD model rats induced with D-Gal and amyloid peptide fragment 25-35 (Aβ25-35).Learning and memory of rats were evaluated with Y maze and Morris water maze and the hippocampus slices were stained with hematoxylin-eosin (HE). The results showed that the extract of the husks of yellowhorn could significantly (P< 0.05) improve percent alternation in Y maze task and decrease escape latency in the directional swimming test in AD rats. And it could also prolong the swimming distance and time in the target quadrant and around the platform 10 cm, and increase the number of across the platform in rats with D-Gal and Aβ25-35 in probe test. Morphological observation on hippocampal slices showed extract of the husks of yellowhorn improved pathological changes of nerve cells in CA1 area.An accurate and sensitive HPLC-MS method was developed for the determination of WGA in rat plasma. And the pharmacokinetics of WGA was systematically investigated after oral administration of the 70% ethanolic extract of the husks of yellowhorn. The ESI source and the selected ion monitoring with positive ion mode was chosen. With digoxin as internal standard, the plasma samples were extracted with ethyl acetate-islpropanol (1:1, v/v). The method was validated with the concentration range of 10.03-1003 ng·mL-1 in rat plasma for WGA. The intra-and inter-day precision (RSD) values were below 11.1% and accuracy (RE) was from-8.1% to 9.3%. The mean plasma concentration-time profiles of WGA were drafted and the pharmacokinetic parameters were as follow:T1/2 was 2.9 h, Cmax was 0.5182μg·mL-1, rmax was 1.5 h, AUC-t was 1.570μg·h-mL-1.Effects of the extract of the husks of yellowhorn on catecholamines of AD rats were also investigated. An accurate and sensitive HPLC-FL method was developed for simultaneous determination of NE, DA, E, L-DOPA, DOPAC in rat plasma and urine. The results showed that the levels of NE, DA, E, L-DOPA, DOPAC in the plasma and urine of AD model group were significantly (P<0.05) lower than control group. And their levels in rat plasma and urine tent to increase in WG group after oral administration of the yellowhorn husks extract and were different significantly (P<0.05) from model group (except for plasma E). However, WG group showed no statistically (P>0.05) significant difference from the control and Hup-A groups. The levels of NE, DA, E, L-DOPA, DOPAC in rat plasma and urine of Hup-A group showed not significant difference from the control and WG group but were significantly (P<0.05) different from the model group (except for urine L-DOPA). |
PDF Full Text Request