Dissertation
Dissertation > Medicine, health > Chinese Medicine > Of Pharmacy > Traditional Chinese medicine chemical

Preparation of Extract of Fructus Akebiae and Research for Its Antidepressant Pharmacodynamics

Author ZhouDan
Tutor XuJiangPing
School Southern Medical University,
Course Pharmacology
Keywords Fructus Akebiae Hederagenin Antidepressant Behavioral Despair Chronic Stress Hypothalamus-Pituitary-Adrenal (HPA) Axis
CLC R284
Type Master's thesis
Year 2010
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Objective:To determine the content of hederagenin and identify the substance exerting antidepressant activity from the extracts of Fructus Akebiae (FAE) which was extracted by systemic solvent segregation. To assess pharmacological acute and sub-chronic antidepressant activity of FAE by using behavioural despair animal models. Morever, to indentify its antidepressant effect by using chronic mild stress model of depression. In addition, to illustrate elementarily the pharmacological mechanism of antidepressant activity of FAE by investigating the effect of FAE on the morphological features and viability of PC 12 cells, as well as oxidative stress indicators, so as to provide scientific basis for further development of Fructus Akebiae.Methods:(1) FAE was obtained by systemic solvent segregation: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 HCl in EtOH, resulting in crude crystal. The content of hederagenin in FAE was determined by HPLC. (2) Male 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 escitalopram (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 and 24 h after repeated treatment for 7 days with drugs. (3) Male Sprague-Dawley rats were randomly divided into six groups:vehicle control [0.5% CMC-Na, no chronic unpredicted mild stress (CUMS)], CUMS vehicle (0.5% CMC-Na, with CUMS), CUMS with 6.25 mg/kg FAE, CUMS with 12.5 mg/kg FAE, CUMS with 25 mg/kg FAE, and CUMS with 6.25 mg/kg ESC. All the drugs were administrated via the oral route once a day at 8 a.m. for 3 weeks. The CUMS procedure was simultaneously conducted with drug administration on rats. The CUMS procedure was designed to maximize the unpredictable nature of the stressors. Rats were weighed on day 0 (before experiment, baseline),5,10,15, and 20 during the CUMS experiment. The body weight change was calculated based on the baseline value. The number of ambulation, the number of rears and the frequencies of grooming were counted in the open field test. The sucrose and water intake, as well as total fluid consumption and sucrose preference were tested in the fluid consumption test. After the behavioral tests, the rats were decapitated, and the blood samples were collected to determine Plasma ACTH and serum CORT levels were determined using corresponding radioimmunoassay kits. At the same time, brains were taken out for paraffin processing and stained with HE. The changes of morphological features of granular cells in DG area were observed by phase contrast microscopy. (4) The differentiated PC 12 cells were cultivated in vitro, pretreated for 4-6 h with 5μg/ml FAE,10μg/ml FAE,20μg/ml FAE and 12.5μM ESC, respectively, and then injured by 200 uM 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. Refering to kits of Nanjing Jiancheng biology research institute, the content or activity of superoxide dismutase (SOD), nitrogen monoxidum (NO), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in supernatant preparations from PC 12 cells were detected.Results:(1) The standard curve equation of hederagenin standard was A=13622×C+63.211, R2=0.9999, and the content of total flavonoids was 71.55%. (2) After the first acute treatment, FAE at 25 mg/kg (137.58±49.220 s),50 mg/kg (134.36±70.704 s) and 100 mg/kg (129.42±48.931 s) treated mice had significantly decreased immobility time compared with vehicle-treated mice in the FST. The effect of FAE was similar to that of ESC (114.50±67.766 s). Likewise, FAE at 25 mg/kg (162.50±38.504 s),50 mg/kg (144.38±42.075 s) and 100 mg/kg (119.55±29.453 s), as well as ESC at 6.25 mg/kg (133.75±28.977 s) treated mice had significantly decreased immobility time compared with vehicle-treated mice (219.38±28.495 s) in the TST. However, FAE (dose range:25-100 mg/kg) produced no significant difference (P= 0.738,P=0.668, P=0.916) in the total locomotor activity number compared with vehicle-treated mice in the test. After 7-day administration, FAE at 25 mg/kg (149.88±31.674 s),50 mg/kg (186.63±37.075 s) and 100 mg/kg (178.25±17.044 s) produced a reduction in the immobility time compared with vehicle-treated mice (226.63±27.506 s) in the FST. The effect of FAE was similar to that of ESC (158.63±45.738 s). Likewise, FAE at 25 mg/kg (131.00±33.253 s),50 mg/kg (130.38±38.037 s) and 100 mg/kg (113.89±47.9931 s), as well as ESC at 6.25 mg/kg (133.75±28.977 s) also produced a reduction in the immobility time compared with vehicle-treated mice (226.63±27.506 s) in the TST. However, FAE (dose range: 25-100 mg/kg) also produced no significant difference (P=0.794, P=0.943, P= 0.485) in the total locomotor activity number compared with vehicle-treated mice in the test. (3) A repeated measures ANOVA was performed for the analysis of weight change. Rats in the vehicle group(11.19±2.565%,22.62±5.245%,40.19±8.102 %and 37.38±7.726%) without stress treatment showed a significant increase in body weight on any of the 4 sampling days and also exhibited significant difference compared with any other stressed groups, while CUMS-treated rats (7.97±2.315%, 15.90±4.170%,20.65±5.256%and 26.85±5.150%) exhibited a relatively small increases in body weight throughout the 5th,10th,15th and 20th days. The magnitude of increases in body weight of rats was similar in all CUMS-treated groups. In the open field test, there was significant difference in ambulation, rearing and grooming among experimental groups (P=0.001, P= 0.010, P=0.016). Compared to the vehicles (66.83±43.906,19.17±15.613 and 4.83±5.565), CUMS-treated rats showed a significant reduction in ambulation (4.83±4.167), rearing (2.00±1.549), and grooming (0.00±0.000) activities. Compared to the CUMS-treated rats,21-day treatment with FAE at various dosagesor with ESC at 6.25 mg/kg did not significantly affect ambulatory, rearing, and grooming activities, though they all showed an increasing tendency on locomotor activity. In the fluid consumption test, the sucrose and water intake volumn, as well as sucrose preference of rats in the vehicle group were 10.68±2.409 ml,5.58±1.855 ml and 66.67±9.214%, while there were significant changes in sucrose intake (2.60±1.463 ml), water intake (12.97±3.034 ml) and sucrose preference (20.67±12.213%) in the vehicle group. After 21-day administration, both FAE at all dosages used (7.11±2.638 ml,7.39±3.153 ml and 51.04±15.029%; 8.07±2.388 ml,6.07±2.169 ml and 57.16±14.481%; 12.57±1.718 ml,5.14±2.035 ml and 71.68±9.237%) and ESC (8.14±1.547 ml,6.71±1.286 ml and 54.66±8.927%) significantly increased rats’ sucrose preference at all dosages used. However, there was no significant difference in total fluid consumption among experimental groups (P=0.149). CUMS procedure markedly induced increase in plasma ACTH and serum CORT levels into 28.05±2.985 pg/ml and 8.53±2.614 ng/ml compared to rats in the vehicle control group (13.04±3.650 pg/ml and 3.63±1.533 ng/ml). After 21 days of treatment with FAE at 12.5 (21.04±7.423 pg/ml and 5.31±1.507 ng/ml) and 25 mg/kg (6.05±4.138 pg/ml and 4.56±2.050 ng/ml), as well as ESC at 6.25 mg/kg (5.50±2.080 pg/ml and 3.83±1.190 ng/ml), the levels of plasma ACTH and serum CORT both decreased significantly. Under phase contrast microscopy, the normal granular cells and neurons treated by FAE or ESC in hippocampus DG areas, were globular or oval with clear nucleoli, homogeneous staining in nuclear heterochromatin, while the cells in CUMS-treated rats presented characteristic morphological changes, including pyknotic (shrunken and dark) nuclei, strong acidophil staining. (4) In the cell test, FAE could produce the protective effect against on PC 12 cells injuries induced by Cor. Compared with the control group (1.138±0.081), the absorbance value (A 570) of MTT in the model group (0.745±0.051) decreased obviously. After pretreatment with FAE at 5μg/ml (1.083±0.107), 10μg/ml (1.013±0.082) and 5μg/ml (0.958±0.063), as well as ESC at 12.5μM (1.022±0.082), the A values all increased significantly. FAE could also resist oxidative damage on PC 12 cells induced by Cor. Compared with the control group (4.29±0.198 U/ml,6.04±0.958μM,3.96±3.816 nM), the level of SOD activity (3.51±0.363 U/ml) decreased, while the content of NO (9.05±0.117μM) and MDA (15.81±2.306 nM) both increased in model group. FAE at 10μg/ml (4.41±0.242 U/ml) and 20μg/ml (4.28±0.146 U/ml), as well as ESC at 12.5μM (4.29±0.122 U/ml) increased the activity of SOD in supernatant preparations from PC 12 cells. However, FAE at 5μg/ml (6.67±1.134 Mm,3.39±1.690 nM),10μg/ml (6.80±0.815μM,3.70±1.737 nM) and 20μg/ml (5.98±1.160μM,1.59±3.022 nM), as well as ESC at 12.5μM (7.86±0.639μM,4.48±2.121 nM) decreased the content of NO and MDA. There was no significant difference in the content of GSH among experimental groups (P=0.146).Conclusion:The content of hederagenin was 71.55% in FAE. Hederagenin is its substance foundation for use in treating depression. FAE can remarkably improve depressive behaviors in behavioral despair animal models, and reverse CUMS-induced anhedonia-like behaviors in rats. Moreover, FAE can result in normal HPA function, and ameliorate the pathological changes in DG areas after chronic stress. These results suggest both crude FAE and hederagenin possess potent antidepressant properties. The mechanisms probably are related to its neuroprotective effect, which may be due to its activity of scavenging free radicals and improvement of anti-oxidases.

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