预知子提取物的质量控制及其抗抑郁药效—药动学研究
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摘要
目的：提取分离药材预知子(Fructus Akebiae)中抗抑郁活性成分,测定预知子提取物(Fructus Akebiae Extract, FAE)主要成分常春藤皂苷元(hederagenin),齐墩果酸(Oleanolic acid, OA),熊果酸(Ursolic acid, UA)含量,对预知子提取物的溶剂残留及重金属进行检测,初步研究FAE质量控制的标准。建立检测大鼠血浆及脑脊液中常春藤皂苷元的超快速液相色谱-质谱联用(UFLC-MS/MS)方法,初步阐明预知子中抗抑郁活性成分常春藤皂苷元的药代动力学。建立抑郁相关的行为绝望模型,对预知子提取物及常春藤皂苷元抗抑郁活性的药效学筛选,明确预知子中抗抑郁活性的物质基础。
方法：(1)药材预知子中抗抑郁活性成分提取：以预知子药材为原料,先进行脱脂处理,再依次进行80%乙醇、乙酸乙酯和水饱和正丁醇提取后得预知子总皂苷,将所得总皂苷经含酸乙醇降解及纯化处理后得预知子提取物(FAE)。并采用UFLC-MS/MS法,对FAE中的主要化学成分进行系统解析,测定其主要成分常春藤皂苷元,齐墩果酸,熊果酸含量。(2)FAE中有机溶剂的残留量检测：采用自动顶空GC-MS法,用RxiTM-5ms (30mx0.25mm,0.25μm)毛细管色谱柱,优化顶空条件,以叔丁醇为内标。参照中国药典(2010年)中的相关规定,测定了预知子提取过程中使用的三种有机溶剂-乙醇、乙酸乙酯、正丁醇的残留量(3)预知子药材及FAE中5种有害重金属的测定：微波消解,以锗(Ge)、铟(In)铋(Bi)作为内标,补偿基体效应,采用ICP-MS同时测定砷、铅、汞、镉、铜5种元素。(4)①采用UFLC-MS-MS法测定大鼠血浆与脑脊液中常春藤皂苷元的浓度。血浆样品选用甲醇沉淀蛋白处理,而脑脊液用乙酸乙酯萃取,然后N2吹干浓缩,以异戊巴比妥为内标,选用XR-ODSⅡ(75 mm×2.0 mm, i.d.,2.1μm)柱进行分离,流动相为5 mM醋酸铵和乙腈(v/v)梯度洗脱,流速0.35 mL/min。通过电喷雾离子化,负离子检测,优化质谱条件,常春藤皂苷元的定量离子m/z471.5→393.4,内标异戊巴比妥定量离子m/z 225→182。考察了样品在各种储藏条件下,如长期(-20℃,7d)、短期(25℃,4h)、冻融(n=3)以及样品处理后的稳定性,同时也考察了方法的回收率、基质效应、精密度、准确度。②常春藤皂苷元药代动力学探索：SD大鼠,按100mg/kg、400mg/kg两个剂量,单次给药,分别在0,5,10,15,20,30,60,90,120,150,180,210,240,300min采集血样备用。脑脊液样品,分别在给药前与给药后20 mmin采集备用。运用DAS 2.1.1软件计算药代动力学数据。(5)FAE抗抑郁的作用：①建立皮质酮(corticosterone, Cor)损伤的PC12细胞模型,观察常春藤皂苷元对照品和预知子提取物(FAE)对细胞形态、细胞活力的影响。②建立抗抑郁活性的药效学初筛模型,雄性昆明小鼠随机分成5组,即溶剂对照[0.5%羧甲基纤维素钠(CMC-Na)溶液]组、hederagenin (10mg/kg、50mg/kg)组、FAE (100mg/kg)组、草酸-S-西酞普兰(escitalopram, ESC)阳性对照(6.25mg/kg)组,各组动物每日灌胃1次,每次0.2 mmL/10g,连续给药7天。建立小鼠的行为绝望模型,末次给药24小时后分别进行强迫游泳实验(the forced swimming test, FST)和悬尾实验(the tail suspension test, TST)不动时间记录,以及自主活动计数。
结果：(1)FAE中含约70%的常春藤皂苷元及少量的齐墩果酸和熊果酸,在相应的浓度范围内三种化合物对照品的标准曲线线性良好,三批样品中平均含量常春藤皂苷元为70.60%±2.29%；齐墩果酸为2.09%±0.12%,熊果酸为5.32%±0.10%。(2)自动顶空GC-MS法检测FAE中有机溶剂的残留量：结果三批预知子提取物中乙酸乙酯与正丁醇均未检出,乙醇平均百分含量：从0.14%(RSD,4.6%)-0.31%(RSD,3.9%)(n=3)。结果显示预知子提取物中乙醇残留均低于0.5%,符合《中国药典》2010版的溶剂残留标准。(3)ICP-MS法中5种重金属元素的标准曲线线性良好(r>0.9993),平均回收率95.6%～108.0%,RSD<10.3%。预知子药材与FAE中5种重金属含量分别为：Pb(2.11±0.09)μg/g,(1.48±0.02)μg/g；As(0.26±0.03)μg/g,(0.22±0.02)μg/g；Cd(0.038±0.001)μg/g,(0.017±0.002)μg/g；Hg(0.037±0.010)μg/g,(0.026±0.007)μg/g；Cu(14.46±0.50)μg/g,(14.45±0.29)μg/g。结果表明预知子药材及FAE中5种有害重金属元素的含量均低于《中国药典》2010版的限量标准。(4)首次建立了检测血浆和脑脊液的中常春藤皂苷元的UFLC-MS-MS法,常春藤皂苷元在0.406-200 ng/mL范围内：血浆中：y=0.0245x+0.00671,r=0.9981,脑脊液中：少=0.0520x+0.0529, r=0.9960.日间差2.4%～8.9%,日内差1.1%～13.0%,准确度在-9.0%～11.1%之间。常春藤皂苷元在血浆及脑脊液中的回收率均大于85%,且血浆及脑脊液中内源性物质无干扰,基质无显著影响。常春藤皂苷元在血浆及脑脊液中冻融(n=3)、长期(-20℃,7d)、短期和样品处理后(25℃,4h)稳定性良好。表明方法的有效性符合美国食品药品管理局对生物样品的分析检测要求。药代动力学研究结果显示,大鼠灌胃单次给药100 mg/kg、400 mg/kg FAE:Cmax分别为11.21±0.50 ng/mL,47.73±1.39 ng/mL；Tmax分别为20 min,18.33±2.58 min。血药浓度消除半衰期t1/2为49.90±1.85 min,44.06±2.98 min。而AUC0-t分别为668.44±20.21 ng/mL*min,3066.46±243.07 ng/mL*min,AUC0-∞为701.42±28.62 ng/mL*min,3131.47±236.83 ng/mL*min。消除速率Ke为0.014±0.001和0.016±0.001。在单次给药(400 mg/kg FAE)20 min时,脑脊液中常春藤皂苷元含量6.17±0.22 ng/mL(RSD,3.5％),表明常春藤皂苷元能穿透血脑屏障。(5)①细胞实验中,常春藤皂苷元对照品和FAE可显著对抗Cor造成的PC12细胞损伤,模型组细胞A值为0.686±0.058,与溶剂对照组(0.983±0.054)相比较显著降低,但经常春藤皂苷元(1,5,10μg/mL)和FAE(20μg/mL)(0.780±0.018、0.874±0.030、0.942±0.021；0.957±0.039)或5.0μg/mL ESC(0.956±0.036)预处理过的细胞,其A值较之于模型组细胞具有显著升高(P=0.000)。常春藤皂苷元和FAE可显著对抗Cor所致的细胞氧化损伤。②常春藤皂苷元连续7天重复给药后,在FST中与溶剂对照组(198.4±43.96s)比较,给予剂量为10 mg/kg、50 mg/kg常春藤皂昔元,100 mg/kg FAE或ESC阳性对照组的小鼠游泳不动时间分别为115.9±30.49、110.1±26.49、97.4±22.24、100.25±20.63s,不动时间显著减少；TST中各实验组组间差异显著(P=0.000),不同给药剂量10mg/kg(197.89±30.65 s)、50 mg/kg(128.38±36.49 s)常春藤皂苷元,100 mg/kg(119.67±23.64 s)FAE以及ESC阳性对照组(109.89±22.75s)小鼠,与溶剂对照组(228.13±40.34s)相比,均显著缩短了空中不动时间；小鼠自主活动检测实验中,常春藤皂苷元和FAE给药组及ESC组小鼠与溶剂对照组相比较,水平活动计数值均无显著性差异(P=0.799)。
结论：FAE中常春藤皂苷元含量达68%,有机溶剂残留量及重金属检测,符合《中国药典》2010版的相关标准,能够满足二类中药新药的要求。检测血浆及脑脊液中常春藤皂苷元的UFLC-MS/MS法,具有高灵敏度,高选择性,可有效用于药代动力学的研究,药代动力学结果显示,常春藤皂苷元可经肠胃快速吸收分布到血液及脑脊液中,表明常春藤皂苷元能穿透血脑屏障。常春藤皂苷元和FAE均对Cor诱导的PC12细胞损伤模型具有明显的保护作用；并可显著改善行为绝望模型小鼠的抑郁样行为。因此,可以初步确定预知子提取物产生抗抑郁药效的物质基础为常春藤皂苷元。
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.
方法：(1)药材预知子中抗抑郁活性成分提取：以预知子药材为原料,先进行脱脂处理,再依次进行80%乙醇、乙酸乙酯和水饱和正丁醇提取后得预知子总皂苷,将所得总皂苷经含酸乙醇降解及纯化处理后得预知子提取物(FAE)。并采用UFLC-MS/MS法,对FAE中的主要化学成分进行系统解析,测定其主要成分常春藤皂苷元,齐墩果酸,熊果酸含量。(2)FAE中有机溶剂的残留量检测：采用自动顶空GC-MS法,用RxiTM-5ms (30mx0.25mm,0.25μm)毛细管色谱柱,优化顶空条件,以叔丁醇为内标。参照中国药典(2010年)中的相关规定,测定了预知子提取过程中使用的三种有机溶剂-乙醇、乙酸乙酯、正丁醇的残留量(3)预知子药材及FAE中5种有害重金属的测定：微波消解,以锗(Ge)、铟(In)铋(Bi)作为内标,补偿基体效应,采用ICP-MS同时测定砷、铅、汞、镉、铜5种元素。(4)①采用UFLC-MS-MS法测定大鼠血浆与脑脊液中常春藤皂苷元的浓度。血浆样品选用甲醇沉淀蛋白处理,而脑脊液用乙酸乙酯萃取,然后N2吹干浓缩,以异戊巴比妥为内标,选用XR-ODSⅡ(75 mm×2.0 mm, i.d.,2.1μm)柱进行分离,流动相为5 mM醋酸铵和乙腈(v/v)梯度洗脱,流速0.35 mL/min。通过电喷雾离子化,负离子检测,优化质谱条件,常春藤皂苷元的定量离子m/z471.5→393.4,内标异戊巴比妥定量离子m/z 225→182。考察了样品在各种储藏条件下,如长期(-20℃,7d)、短期(25℃,4h)、冻融(n=3)以及样品处理后的稳定性,同时也考察了方法的回收率、基质效应、精密度、准确度。②常春藤皂苷元药代动力学探索：SD大鼠,按100mg/kg、400mg/kg两个剂量,单次给药,分别在0,5,10,15,20,30,60,90,120,150,180,210,240,300min采集血样备用。脑脊液样品,分别在给药前与给药后20 mmin采集备用。运用DAS 2.1.1软件计算药代动力学数据。(5)FAE抗抑郁的作用：①建立皮质酮(corticosterone, Cor)损伤的PC12细胞模型,观察常春藤皂苷元对照品和预知子提取物(FAE)对细胞形态、细胞活力的影响。②建立抗抑郁活性的药效学初筛模型,雄性昆明小鼠随机分成5组,即溶剂对照[0.5%羧甲基纤维素钠(CMC-Na)溶液]组、hederagenin (10mg/kg、50mg/kg)组、FAE (100mg/kg)组、草酸-S-西酞普兰(escitalopram, ESC)阳性对照(6.25mg/kg)组,各组动物每日灌胃1次,每次0.2 mmL/10g,连续给药7天。建立小鼠的行为绝望模型,末次给药24小时后分别进行强迫游泳实验(the forced swimming test, FST)和悬尾实验(the tail suspension test, TST)不动时间记录,以及自主活动计数。
结果：(1)FAE中含约70%的常春藤皂苷元及少量的齐墩果酸和熊果酸,在相应的浓度范围内三种化合物对照品的标准曲线线性良好,三批样品中平均含量常春藤皂苷元为70.60%±2.29%；齐墩果酸为2.09%±0.12%,熊果酸为5.32%±0.10%。(2)自动顶空GC-MS法检测FAE中有机溶剂的残留量：结果三批预知子提取物中乙酸乙酯与正丁醇均未检出,乙醇平均百分含量：从0.14%(RSD,4.6%)-0.31%(RSD,3.9%)(n=3)。结果显示预知子提取物中乙醇残留均低于0.5%,符合《中国药典》2010版的溶剂残留标准。(3)ICP-MS法中5种重金属元素的标准曲线线性良好(r>0.9993),平均回收率95.6%～108.0%,RSD<10.3%。预知子药材与FAE中5种重金属含量分别为：Pb(2.11±0.09)μg/g,(1.48±0.02)μg/g；As(0.26±0.03)μg/g,(0.22±0.02)μg/g；Cd(0.038±0.001)μg/g,(0.017±0.002)μg/g；Hg(0.037±0.010)μg/g,(0.026±0.007)μg/g；Cu(14.46±0.50)μg/g,(14.45±0.29)μg/g。结果表明预知子药材及FAE中5种有害重金属元素的含量均低于《中国药典》2010版的限量标准。(4)首次建立了检测血浆和脑脊液的中常春藤皂苷元的UFLC-MS-MS法,常春藤皂苷元在0.406-200 ng/mL范围内：血浆中：y=0.0245x+0.00671,r=0.9981,脑脊液中：少=0.0520x+0.0529, r=0.9960.日间差2.4%～8.9%,日内差1.1%～13.0%,准确度在-9.0%～11.1%之间。常春藤皂苷元在血浆及脑脊液中的回收率均大于85%,且血浆及脑脊液中内源性物质无干扰,基质无显著影响。常春藤皂苷元在血浆及脑脊液中冻融(n=3)、长期(-20℃,7d)、短期和样品处理后(25℃,4h)稳定性良好。表明方法的有效性符合美国食品药品管理局对生物样品的分析检测要求。药代动力学研究结果显示,大鼠灌胃单次给药100 mg/kg、400 mg/kg FAE:Cmax分别为11.21±0.50 ng/mL,47.73±1.39 ng/mL；Tmax分别为20 min,18.33±2.58 min。血药浓度消除半衰期t1/2为49.90±1.85 min,44.06±2.98 min。而AUC0-t分别为668.44±20.21 ng/mL*min,3066.46±243.07 ng/mL*min,AUC0-∞为701.42±28.62 ng/mL*min,3131.47±236.83 ng/mL*min。消除速率Ke为0.014±0.001和0.016±0.001。在单次给药(400 mg/kg FAE)20 min时,脑脊液中常春藤皂苷元含量6.17±0.22 ng/mL(RSD,3.5％),表明常春藤皂苷元能穿透血脑屏障。(5)①细胞实验中,常春藤皂苷元对照品和FAE可显著对抗Cor造成的PC12细胞损伤,模型组细胞A值为0.686±0.058,与溶剂对照组(0.983±0.054)相比较显著降低,但经常春藤皂苷元(1,5,10μg/mL)和FAE(20μg/mL)(0.780±0.018、0.874±0.030、0.942±0.021；0.957±0.039)或5.0μg/mL ESC(0.956±0.036)预处理过的细胞,其A值较之于模型组细胞具有显著升高(P=0.000)。常春藤皂苷元和FAE可显著对抗Cor所致的细胞氧化损伤。②常春藤皂苷元连续7天重复给药后,在FST中与溶剂对照组(198.4±43.96s)比较,给予剂量为10 mg/kg、50 mg/kg常春藤皂昔元,100 mg/kg FAE或ESC阳性对照组的小鼠游泳不动时间分别为115.9±30.49、110.1±26.49、97.4±22.24、100.25±20.63s,不动时间显著减少；TST中各实验组组间差异显著(P=0.000),不同给药剂量10mg/kg(197.89±30.65 s)、50 mg/kg(128.38±36.49 s)常春藤皂苷元,100 mg/kg(119.67±23.64 s)FAE以及ESC阳性对照组(109.89±22.75s)小鼠,与溶剂对照组(228.13±40.34s)相比,均显著缩短了空中不动时间；小鼠自主活动检测实验中,常春藤皂苷元和FAE给药组及ESC组小鼠与溶剂对照组相比较,水平活动计数值均无显著性差异(P=0.799)。
结论：FAE中常春藤皂苷元含量达68%,有机溶剂残留量及重金属检测,符合《中国药典》2010版的相关标准,能够满足二类中药新药的要求。检测血浆及脑脊液中常春藤皂苷元的UFLC-MS/MS法,具有高灵敏度,高选择性,可有效用于药代动力学的研究,药代动力学结果显示,常春藤皂苷元可经肠胃快速吸收分布到血液及脑脊液中,表明常春藤皂苷元能穿透血脑屏障。常春藤皂苷元和FAE均对Cor诱导的PC12细胞损伤模型具有明显的保护作用；并可显著改善行为绝望模型小鼠的抑郁样行为。因此,可以初步确定预知子提取物产生抗抑郁药效的物质基础为常春藤皂苷元。
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.
引文
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