Quality Control Of Extract Of Fructus Akebiae And Its Antidepressant Pharmacodynamics And Pharmacokinetics

Objective:1) to determine the contents of hederagenin, ursone and oleanolic acid from the extracts of Fructus Akebiae (FAE) which was extracted by systemic solvent segregation.2) to develope a Head-space GC-MS method for determination of enthanol, ethyl acetate and n-butanol in FAE.3) to establish an ICP-MS method for the determination of heavy metals, including As, Hg, Pb, Cd, Cu, in Fructus Akebiae and Fructus Akebiae extracts.4) to develop a rapid, sensitive, and selective ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method for the quantitative determination of hederagenin in rat plasma and cerebrospinal fluid (CSF) as well as for the pharmacokinetic study of hederagenin accumulation in the central nervous system (CNS).5) to illustrate antidepressant activity of hederagenin by investigating the effect of hederagenin on the morphological features, as well as by using behavioural despair animal models, so as to provide scientific basis for further development of Fructus Akebiae.Methods:(1) FAE was obtained by systemic solvent extraction:Fructus Akebiae was defatted, and then extracted with 80% ethanol, ethyl acetate and H2O-saturated n-butanol, and the general saponin was obtained. Total general saponin was degraded with HC1 in ethanol, resulting in crude crystal. The contents of hederagenin, ursone and oleanolic acid in FAE were determined by UFLC-MS/MS. (2) Multiresidue detection of FAE was performed by a head-space GC-MS. The analysis was carried on RxiTM-5ms (30m×0.25mm,0.25μm) column. The conditions of headspace has been tested and optimized. The tert-butanol was used as an internal standard. (3) The samples of FAE and Fructus Akebiae were digested by closed-vessel Microwave digestion. The five heavy metals were directly analyzed by ICP-MS. Ge,In and Bi was selected as the internal standards to compensate matrix effects. (4) Sample pretreatment of rat plasma and CSF involved a simple protein precipitation with methanol and a one-step extraction with ethyl acetate. Amobarbital was chosen as the internal standard (IS) for the assay. Separation was carried out in a Shim-pack XR-ODS II (75 mm×2.0 mm, i.d.,2.1μm) column with gradient elution at a flow rate of 0.35 mL/min. The mobile phase was 5 mM ammonium acetate and acetonitrile. Detection was performed in a triple-quadruple tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. According to optimization of mass spectrometry conditions, m/z 471.5→393.4 was used for quantification of hederagenin and m/z 225→182 for IS. QC sample were subjected to processed samples kept at 25℃in autosampler for 4 h, to short-term stability kept at ambient temperature (25℃) for 4 h, to long-term (7days) storage conditions (-20℃), and to three freeze-thaw cycles stability studies.The method was applied to determine the plasma and CSF concentrations of hederagenin after oral FAE (100 mg/kg,400 mg/kg). Rat plasma samples were collected before and 0,5,10,15,20,30,60,90, 120,150,180,210,240, and 300 min after oral dosing. Rat CSF samples were only collected before and 20 min post-dosing. DAS 2.1.1 software was performed to deal with pharmacokinetics data. (5)①The differentiated PC 12 cells were cultivated in vitro, pretreated for 4-6 h with 1μg/mL hederagenin,5μg/mL hederagenin,10μg/mL hederagenin,20μg/mL FAE and 12.5μM escitalopram (ESC), respectively, and then injured by 200μM Cor for 48 h. The cellular morphology was examined with light microscopy, and then the cell viability rate in all groups was tested by MTT assay.②Male Kunming mice were randomly divided into five groups:control (0.5% CMC-Na solution),25 mg/kg FAE,50 mg/kg FAE,100 mg/kg FAE, and 6.25 mg/kg ESC. All the drugs were given via the oral route once a day at 8 a.m. for 1 week. The forced swimming test (FST), the tail suspension test (TST) and locomotor activity were conducted 60 min after the first acute treatment 5μg/ml and 24 h after repeated treatment for 7 days with drugs.Result:(1) Hederagenin in FAE have been found about 70% of purity, containing small quantity ursone and oleanolic acid. The standard curves were linear in the range of each consistency. The content of hederagenin was 70.60%±2.29%, ursone 5.32%±0.10%, oleanolic acid 2.09%±0.12%(n=3). (2)Ethyl acetate and n-butanol were not found in FAE. The average content of enthanol ranged from 0.14% (RSD,4.6%) to 0.23% (RSD,3.1%). Multiresidue detection of FAE conformed to the criteria set by Chinese Pharmacopoeia (2010), where content determined for enthanol must not exceed 0.5%. (3)For all of the analyzed heavy metals, the correlative coefficient of the calibration curves was over 0.9993. The recovery rates of the procedure were 95.6%-108.0%, and its RSD was lower than 10.3%. Heavy metal elements Pb (2.11±0.09)μg/g, As(0.26±0.03)μg/g, Cd(0.038±0.001)μg/g, Hg(0.037±0.010)μg/g and Cu(14.46±0.50)μg/g were found in Fructus Akebiae. The average contents of heavy metal elements in FAE were Pb (1.48±0.02)μg/g, As(0.22±0.02)μg/g, Cd (0.017±0.002)μg/g, Hg (0.026±0.007)μg/g and Cu (14.45±0.29)μg/g. The results indicated that all of the concents heavy metals conformed to the criteria set by Chinese Pharmacopoeia (2010). (4)A linear calibration curve for hederagenin was obtained over a concentration range of 0.406(lower limit of quantification, LLOQ) to 200 ng/mL (r2>0.99) for both plasma and CSF. The intra-day and inter-day precision (relative standard deviation, RSD) values were less than 15% and the accuracy (relative error, RE) was within-9.0% and 11.1% for plasma and CSF at all quality control (QC) levels. The extraction recoveries to determine hederagenin in rat plasma and CSF are both more than 85%. No interference from endogenous substances was observed at the retention times of hederagenin and IS. Results from all stability tests demonstrated good stability of hederagenin. This method conformed to the criteria for the analysis of biological samples set by the USFDA. It was also successfully applied to the pharmacokinetic study of hederagenin accumulation following oral administration of Fructus akebiae extract in rats. After administration of a single dose of 100 mg/kg FAE, the Cmax and Tmax were 11.21±0.50 ng/mL and 20 min, respectively. Plasma concentration declined with a t1/2 of 49.90±1.85 minin. The AUC0-t and AUC0-∞values were 668.44±20.21 ng/mL-min and 701.42±28.62 ng/mL·min, respectively. The Ke was 0.014±0.001. After administration of a single dose of 400 mg/kg FAE (approximately 70% hederagenin or about 280mg), the Cmax and Tmax were 47.73±1.39 ng/mL and 20 min, respectively. Plasma concentration declined with a t1/2 of 42.52±1.94 min. The AUCo-t and AUC0-∞values were 3023.18±254.23 ng/mL-min and 3090.47±241.52 ng/mL-min, respectively. The Ke was 0.016±0.001. At 20 min, 6.17±0.22 ng/mL (RSD,3.5%) of hederagenin was detected in rat CSF after administration of a single 400 mg/kg dose of FAE. The pharmacokinetics results indicated that hederagenin can pass through the blood-brain barrier. (5)①In PC 12 cell experiments, the A value of model group is 0.686±0.058, significantly lower compared to the control group (0.983±0.054), but with hederagenin(1,5, 10μg/mL) and FAE(20μg/mL) (0.780±0.018,0.874±0.030,0.942±0.021,0.957±0.039) or 5μg/mL ESC (0.956±0.036) pretreated cells, the A values were significantly Increased (P=0.000). Therefor, hederagenin and FAE had potencial protected effect to oxidative damaged PC 12 cells caused by Cor.②After repeated hederagenin administration for 7 days, the immobility time of groups of 10mg/kg,50mg/kg Hederagenin or 100 mg/kg FAE or positive control of ESC was 115.9±30.49 s,110.1±26.49 s,97.4±22.24 s and 100.25±20.63 s, respectively, significantly reduced compared to the control group (198.4±43.96 s) in the FST; Simily to FST, thle immobility time of TST was significantly different between groups (P=0.000). Compared with the control group (228.13±40.34 s), 10mg/kg (197.89±30.65 s),50 mg/kg (128.38±36.49 s) Hederagenin,100 mg/kg (119.67±23.64 s) FAE and (109.89±22.75 s) ESC groups had a significantly reduced immobility time in the air, The results of spontaneous activity test did not have significantly different tendecy between the drug intervented groups and the untreatment group by the level of activity counts (P= 0.799).Conclusion:Studies reveal that hederagenin from the extracts of Fructus akebiae was enriched to approximately 70% purity. ICP-MS arid GC-MS are both suitable for quality control of FAE. The new UFLC-MS-MS developed for the quantitative determination of hederagenin in rat plasma and CSF demonstrated high sensitivity, selectivity, and speed of analysis, as well as fulfilling FDA guidelines for bioanalysis. Furthermore, the method has been successfully applied in a pharmacokinetic study of hederagenin in rat plasma and CSF. Results of this study show that hederagenin can be distributed rapidly in plasma and CSF, which indicated that hederagenin could be quickly gastrointestinal absorbed. Moreover, hederagenin can pass through the blood-brain barrier. In addition, hederagenin and FAE can remarkably improve depressive behaviors in behavioral despair animal models. These results suggest both hederagenin and rude FAE possess potent antidepressant properties. Therefore, hederagenin could be substance used in treating depression.