【作者】 王文凯；

【导师】 李廷利；

【作者基本信息】 黑龙江中医药大学 ， 中药学， 2014， 博士

【摘要】 目的：明确生、炒酸枣仁(1：1)相伍的抗抑郁作用及单胺能系统作用机制。阐明生、炒酸枣仁相伍后化学成分变化。方法：1.生、炒酸枣仁相伍抗抑郁作用研究1.1生、炒酸枣仁相伍对行为绝望模型的作用将ICR小鼠随机分为5组,每组10只,分别为模型组、氟西汀组、生枣仁组、炒枣仁组和相伍组。各给药组灌胃给予相应药物,模型组给予同体积蒸馏水。于末次给药后1h,进行小鼠游泳应激实验和小鼠悬尾应激实验,记录小鼠6min内后4min的累计不动时间。1.2生、炒酸枣仁相伍对氟西汀的抗抑郁协同作用将ICR小鼠随机分为6组,每组10只,分别为模型组、氟西汀组、生枣仁联合组、炒枣仁联合组、相伍联合组。各给药组灌胃给予相应药物,模型组给予同体积蒸馏水。于末次给药后1h,进行小鼠游泳应激实验和小鼠悬尾应激实验,记录小鼠6min内后4min的累计不动时间。1.3生、炒酸枣仁相伍抗抑郁作用的量效关系与时效关系研究将ICR小鼠随机分为5个给药剂量组(2.5g/kg,5g/kg,10g/kg,20g/kg,40g/kg),灌胃途径给药,于末次给药后1h,进行小鼠游泳应激实验,记录小鼠6min内后4min的累计不动时间。将ICR小鼠随机分为5个给药天数组(5天,7天,9天,11天,14天),灌胃途径给药,于末次给药后1h,进行小鼠游泳应激实验,观察6min,记录小鼠6min内后4min的累计不动时间。1.4生、炒酸枣仁相伍对小鼠慢性应激抑郁模型的作用将ICR小鼠随机分为4组,每组10只,分别为空白组、模型组、氟西汀组和相伍组。除空白组外,其余各组采用孤养结合慢性轻度不可预见性刺激的方法,进行连续21天的刺激,复制小鼠慢性应激抑郁模型,从造模第11天开始,各给药组于每次刺激前1h灌胃给予相应药物,模型组和空白组灌胃给予同体积蒸馏水,连续11天。末次刺激结束后,记录各组小鼠体质量变化,糖水消耗百分比和旷场行为学参数。1.5生、炒酸枣仁相伍对小鼠利血平拮抗模型的作用将ICR小鼠随机分为3组,每组10只,分别为模型组、丙咪嗪组和相伍组。各给药组灌胃给予相应药物,模型组给予同体积蒸馏水,连续11天。于末次给药1h后,腹腔注射利血平(2.5mg/kg)。观察小鼠体温下降、睁眼不能和运动不能情况,记录体温下降值、睁眼不能的百分率和运动不能的百分率。1.6生、炒酸枣仁相伍对5-HTP引起的小鼠甩头模型的作用将ICR小鼠随机分为3组,每组10只,分别为模型组、氟西汀组和相伍组。各给药组灌胃给予相应药物,模型组给予同体积蒸馏水,连续11天。于末次给药1h后,腹腔注射5-HTP(300mg/kg),观察小鼠甩头行为,记录15min内的甩头次数。1.7生、炒酸枣仁相伍对小鼠育亨宾毒性增强模型的作用将ICR小鼠随机分为3组,每组10只,分别为模型组、丙咪嗪组和相伍组。各给药组灌胃给予相应药物,模型组给予同体积蒸馏水,连续11天。于末次给药1h后,皮下注射育亨宾(30mg/kg),空白组皮下注射同体积生理盐水,观察记录各组24h内死亡只数,计算死亡百分率。2.生、炒酸枣仁相伍抗抑郁作用机制研究2.1生、炒酸枣仁相伍对慢性应激抑郁模型小鼠海马和额叶皮质5-HT含量及生物代谢的影响将ICR小鼠随机分为4组,每组10只,分别为空白组、模型组、氟西汀组和相伍组。除空白组外,其余各组采用孤养结合慢性轻度不可预见性刺激的方法,进行连续21天的刺激,复制小鼠慢性应激抑郁模型,从造模第11天开始,各给药组于每次刺激前1h灌胃给予相应药物,模型组和空白组灌胃给予同体积蒸馏水,连续11天,末次刺激结束后,断头取脑,分离海马和额叶皮质,ELISA法检测5-HT、5-HTIAA和TPH含量,并推算5-HTIAA/5-HT比值。2.2生、炒酸枣仁相伍对慢性应激抑郁模型小鼠海马5-HT转运体编码基因(S1c6a4)和5-HTlA受体编码基因(Htrla)表达的影响将ICR小鼠随机分为4组,每组10只,分别为空白组、模型组、氟西汀组和相伍组。除空白组外,其余各组采用孤养结合慢性轻度不可预见性刺激的方法,进行连续21天的刺激,复制小鼠慢性应激抑郁模型,从造模第11天开始,各给药组于每次刺激前1h灌胃给予相应药物,模型组和空白组灌胃给予同体积蒸馏水,连续11天,末次刺激结束后,断头取脑,冰上迅速分离海马,随机选择5个样本,采用荧光定量PCR技术,检测海马部位Slc6a4和Htrla的基因表达量。2.3生、炒酸枣仁相伍对慢性应激抑郁模型小鼠海马和额叶皮质DA含量及生物代谢的影响将ICR小鼠随机分为4组,每组10只,分别为空白组、模型组、氟西汀组和相伍组。除空白组外,其余各组采用孤养结合慢性轻度不可预见性刺激的方法,进行连续21天的刺激,复制小鼠慢性应激抑郁模型,从造模第11天开始,各给药组于每次刺激前1h灌胃给予相应药物,模型组和空白组灌胃给予同体积蒸馏水,连续11天,末次刺激结束后,断头取脑,分离海马和额叶皮质,ELISA法检测DA、HVA和TH含量,并推算HVA/DA比值。2.4生、炒酸枣仁相伍对慢性应激抑郁模型小鼠海马DA转运体编码基因(Slc6a3)表达的影响将ICR小鼠随机分为4组,每组10只,分别为空白组、模型组、氟西汀组和相伍组。除空白组外,其余各组采用孤养结合慢性轻度不可预见性刺激的方法,进行连续21天的刺激,复制小鼠慢性应激抑郁模型,造模第11天起,各给药组于每次刺激前1h灌胃给予相应药物,模型组和空白组灌胃给予同体积蒸馏水,末次刺激结束后,断头取脑,冰上迅速分离海马,随机选择5个样本,采用荧光定量PCR技术,检测小鼠海马部位Slc6a3基因表达量。3.生、炒酸枣仁相伍后化学成分变化比较生酸枣仁水煎提取物(简称生品水煎物)、炒酸枣仁水煎提取物(简称炒品水煎物)、生、炒酸枣仁合煎提取物(简称合煎物)的HPLC-ELSD指纹图谱,分析生、炒酸枣仁相伍后化学成分变化；采用HPLC-ELSD法同时检测生品水煎物、炒品水煎物、合煎物中三种成分斯皮诺素、酸枣仁皂苷A和酸枣仁皂苷B的含量,分析生、炒酸枣仁相伍后抗抑郁有效成分含量变化结果：1.生、炒酸枣仁相伍对行为绝望模型的作用与模型组相比,相伍组和氟西汀组小鼠游泳不动时间显著缩短(P<0.01),作用优于生枣仁组(P<0.05)。相伍组和氟西汀组小鼠悬尾不动时间显著缩短(P<0.01),作用优于生枣仁组(P<0.05)。2.生、炒酸枣仁相伍对氟西汀的抗抑郁协同作用与氟西汀组相比,相伍联合组小鼠游泳不动时间和悬尾不动时间显著缩短(P<0.05),表现出对氟西汀的抗抑郁协同作用。生枣仁联合组和炒枣仁联合组小鼠游泳不动时间和悬尾不动时间没有显著缩短(P<0.05),未表现出对氟西汀的抗抑郁协同作用。3.生、炒酸枣仁相伍抗抑郁作用的量效关系与时效关系在2.5g/kg-10g/kg剂量范围内,生、炒酸枣仁相伍抗抑郁作用存在剂量剂量依赖性,超出此范围,抗抑郁作用不再继续加强,10g/kg抗抑郁作用最佳。在5-11天给药天数范围内,生、炒酸枣仁相伍抗抑郁作用存在时间依赖性,超出此范围,抗抑郁作用不再增加。连续给药11天抗抑郁作用最佳。4.生、炒酸枣仁相伍对小鼠利血平拮抗模型的作用生、炒酸枣仁相伍能对抗利血平拮抗模型的抑郁症状,表现为注射利血平2h后体温下降显著减少(P<0.01)；运动不能的百分率显著降低(P<0.05,P<0.01)。5.生、炒酸枣仁相伍对5-HTP引起的甩头模型的作用生、炒酸枣仁相伍能增加5-HTP引起的小鼠甩头次数,表现腹腔注射5-HTP后15min内甩头次数显著提高(P<0.05)。6.生、炒酸枣仁相伍对小鼠育亨宾毒性增强模型的的作用生、炒酸枣仁相伍不能增强育亨宾的毒性反应,表现为小鼠24h内死亡百分率未见显著提高(P>0.05)。7.生、炒酸枣仁相伍对小鼠慢性应激抑郁模型的作用生、炒酸枣仁相伍能改善慢性应激抑郁模型的抑郁症状,表现为显著提高慢性应激抑郁小鼠体质量增长速度(P<0.01)；显著提高糖水消耗百分比(P<0.05)；显著增加小鼠穿格数、直立次数、理毛次数(P<0.01,P<0.05),减少排便次数(P<0.05)。8.生、炒酸枣仁相伍对慢性应激抑郁模型小鼠海马和额叶皮质部位5-HT含量及生物代谢的影响生、炒酸枣仁相伍能显著增加慢性应激抑郁小鼠海马和皮质部位的5-HT含量(P<0.05),显著增加海马部位5-HIAA含量(P<0.01),提高海马部位5-HIAA/5-HT比值(P<0.05)。9.生、炒酸枣仁相伍对慢性应激抑郁模型小鼠海马5-HT转运体编码基因(Slc6a4)和5-HTlA受体编码基因(Htrla)表达量的影响生、炒酸枣仁相伍能够下调慢性应激抑郁小鼠海马部位Slc6a4基因表达,上调海马部位Htr1a基因表达。10.生、炒酸枣仁相伍对慢性应激抑郁模型小鼠海马和额叶皮质部位DA含量及生物代谢的影响生、炒酸枣仁相伍能显著增加慢性应激抑郁小鼠海马部位DA含量(P<0.05)。11.生、炒酸枣仁相伍对慢性应激抑郁模型小鼠海马DA转运体编码基因(Slc6a3)的影响生、炒酸枣仁相伍能够下调慢性应激抑郁小鼠海马部位Slc6a3基因表达。12.生、炒酸枣仁相伍后化学成分变化通过对比合煎物、生品水煎物和炒品水煎物的HPLC-ELSD指纹图谱,三种水煎物的峰数量一致,合煎物中未发现新成分。合煎物中一些峰的峰面积百分比发生变化,表明生、炒酸枣仁相伍后一些成分比例发生变化。13.生、炒酸枣仁相伍后斯皮诺素、酸枣仁皂苷A和酸枣仁皂苷B含量变化合煎物斯皮诺素含量为3.7898mg,酸枣仁皂苷A含量为2.2849mg,在三种水煎物中含量最高；酸枣仁皂苷B含量为1.1823mg,居于生品水煎物和炒品水煎物之间。结论：1.生、炒酸枣仁相伍抗抑郁作用显著,优于生、炒酸枣仁单用。2.生、酸枣仁相伍抗抑郁作用机制为：抑制突触间隙5-HT的重摄取,增加突触后膜5-HT1A受体,提高中枢5-HT能系统的功能和兴奋性；抑制突触间隙DA的重摄取,提高中枢部分脑区DA含量。3.生、炒酸枣仁相伍后没有化学成分没有显著变化,一些成分比例产生较大变化。斯皮诺素含量和酸枣仁皂苷A含量增加。更多还原

【Abstract】 Objective:To detect the changes of chemical components after the compatibility of SZS with its processing product, and to observe the antidepressant effects of the compatibility of crude SZS with SZS (1:1), and then to elucidate the mechanism of the central monoaminergic nerve system.Method:1. The research of antidepressant effect of the compatibility of SZS with its processing product.1.1The antidepressent effect of the the compatibility of SZS with its processing product on the despair model.The ICR mice were randomly divided into5groups,10rats in each group, respectively, model group, fluoxetine group,SZS crude, processing product group and combination group. After the last administration of1H, the mouse swimming test and mice tail suspension stress experiment, observe them for6min, record different mice group after4min motionless time.1.2The antidepressent combination effect of the the compatibility of SZS with its processing product on the fluoxetine.The ICR mice were randomly divided into6groups,10rats in each group, respectively, model group, fluoxetine group, szs combined group, fried combined group, compatibility combined group. After the last administration of1H, the mouse swimming test and mice tail suspension stress experiment, observe them for6min, record the mice’s motionless time after4min.1.3Study on the dose effect relationship and time effect relationship antidepressant effect of the the compatibility of SZS with its processing product.The ICR mice were randomly divided into5dosage groups (2.5g/kg,5g/kg,10g/kg,20g/kg,40g/kg), after the last administration of1H, the mouse swimming stress experiment, observe them for6min, record the mice’s motionless time after4min.The ICR mice were randomly divided into5dosage day array (5days,7days,9days,11days,14days), after the last administration of1H, the mouse swimming stress experiment, observe them for6min, record the mice’s motionless time after4min.1.4The effects of the the compatibility of SZS with its processing product on mouse reserpine antagonistic model. The ICR mice were randomly divided into3groups,10rats in each group, respectively, model group, imipramine group and compatibility group. In the1h after the last administration, intraperitoneal injection of reserpine (2.5mg/kg). Mice were observed for hypothermia, eyes openless and motionless, recording body temperature decrease, eyes openless percentage and percentage of motionless.1.5The effect of the compatibility of SZS with its processing product on mouse reserpine antagonistic model of5-HTP induced head twitches.The ICR mice were randomly divided into3groups,10rats in each group, respectively, model group, fluoxetine group and compatibility group. In the1h after the last administration, intraperitoneal injection of5-HTP (300mg/kg), and observe the behavior of mice head shakes, recorded head shake times in15min.1.6The compatibility of SZS with its processing product of effect on the mice yohimbine increased toxicity model.The ICR mice were randomly divided into3groups,10rats in each group, respectively, model group, imipramine group and compatibility group. In the1h after the last administration, subcutaneous injection of yohimbine (30mg/kg), control group were injected with the same volume of physiological saline, observed the died in24h, calculate the mortality percentage.1.7The compatibility of SZS with its processing product of effects on mice with chronicstress depression model. The ICR mice were randomly divided into4groups,10rats in each group, respectively, into blank group, model group, fluoxetine group and compatibility group. Except the blank group, other groups with solitary condition with chronic mild not foreseen by method of stimulation, stimulation for21consecutive days.The last time after stimulation, record the body mass of mice changes, and the sugar consumption percentage and open field behavior parameters.2. Study on antidepressent effect mechanism of the compatibility of SZS with its processing product2.1The study on the effects of the compatibility of SZS with its processing product on chronic stress depression model mice in hippocampus and frontal cortex of5-HT biological metabolism.The ICR mice were randomly divided into4groups,10rats in each group, respectively, into blank group, model group, fluoxetine group and compatibility group. Except the blank group, other groups with solitary condition with chronic mild not foreseen by method of stimulation, stimulation for21consecutive days, The last time after stimulation, the brains were removed, the cortex of frontal lobe and hippocampus in separation, ELISA detection of5-HT,5-HTIAA and TPH contents, and calculate the5-HTIAA/5-HT ratio.2.2Study on the effects of the compatibility of SZS with its processing product on the chronic stress depression model mice hippocampus Slc6a4and Htrla gene expression.The ICR mice were randomly divided into4groups,10rats in each group, respectively, into blank group, model group, fluoxetine group and compatibility group. The last time after stimulation, the brains were removed, the ice quickly isolated hippocampal, randomly selected5samples, using fluorescence quantitative PCR technology, detect the expression quantity of Slc6a4and Htrla genes in the hippocampus.2.3Study on the effects of the compatibility of SZS with its processing product on chronic stress depression model mice in hippocampus and frontal cortex of DA biological metabolismThe ICR mice were randomly divided into4groups,10rats in each group, respectively, into blank group, model group, fluoxetine group and compatibility group. Except the blank group, other groups with solitary condition with chronic mild not foreseen by method of stimulation, stimulation for21consecutive days.The last time after stimulation, the brains were removed, the cortex of frontal lobe and hippocampus in separation, ELISA detection of DA, HVA and TH contents, and calculate the HVA/DA ratio.2.4Study on the effect of the compatibility of SZS with its processing product on chronic stress depression model mice hippocampus Slc6a3gene expression.The ICR mice were randomly divided into4groups,10rats in each group, respectively, into blank group, model group, fluoxetine group and compatibility group. the last after stimulation, the brains were removed, the ice quickly isolated hippocampal, randomly selected5samples, using fluorescence quantitative PCR, expression of Slc6a3gene in mice hippocampus detection.3. The changes of components after the compatibility of SZS with its processing product.To use the Hyphenated techniques of HPLC-ELSD, I compare the SZS decoction,its processing produce decoction, and mixed decoction,the I detect the content of Jujuboside A, Jujuboside B and spinosin.I analysis the changes of the components in the index components of the compatibility of SZS with its processing product. Results:1. The antidepressent effect of the the compatibility of SZS with its processing product on the despair model.Compared with the model group, compatibility group and fluoxetine group mice immobility time was significantly shortened (P<0.01), the function is better than SZS crude and processing product group (P<0.05). compatibility group and fluoxetine group immobility time in tail suspension was significantly shortened (P<0.01), the function is better than SZS (P<0.05).2. The anti depression combination effects of the compatibility of SZS with its processing product on fluoxetine.Compared with fluoxetine in the I group, compatibility group mice non swimming time and immobility time in tail suspension were significantly shortened (P<0.05), it is showed that the anti depression combination effects of fluoxetine. SZS crude and processing product group mice immobility time and immobility time in tail suspension were not significantly shortened (P<0.05), it showed no effect of antidepressant on fluoxetine.3. The dose effect relationship and time effect relationship antidepressant effect of the compatibility of SZS with its processing product.In the range of2.5g/kg-10g/kg,there is dose dependent of antidepressant effects of the the compatibility of SZS with its processing product, beyond this range, the antidepressant effect no longer increases. Antidepressant effect is best in10g/kg.In5-11days delivery time range, there is time dependent of antidepressant effects of the compatibility of SZS with its processing product,beyond this range, the antidepressant effect no longer increases. Continuous administration for11days is the best for antidepressant effect.4. The influence of the compatibility of SZS with its processing product on mouse reserpine antagonistic model.The compatibility of SZS with its processing productcan antagonize the reserpine antagonistic model of depressive symptoms,showed that the injection of reserpine hypothermia significantly reduced after2H (P<0.01); motionless percentage significantly reduced th (P<0.05, P<0.01).5. The influence of the the compatibility of SZS with its processing product on mice model of5-HTP induced head twitches. The compatibility of SZS with its processing product can improve the mice5-HTP induced head twitches, it showed that after intraperitoneal injection of5-HTP15min significantly increased (P<0.05) the number of head twitches.6. The influence of the compatibility of SZS with its processing product on the mice model of yohimbine increased toxicity.The compatibility of SZS with its processing product does not increase the toxicity of yohimbine,it showed that the mice dieth percentage within24h was significantly increased (P>0.05).7. The influence of the compatibility of SZS with its processing product on mice with chronic stress depression model.The compatibility of SZS with its processing product can improve chronic stress depression model of depressive symptoms, were significantly increased in chronic stress depression mice body weight growth rate (P<0.01); increase sucrose consumption percentage (P<0.05); mice induced a significant increase in cell number, the number of wear upright, grooming (P<0.01, P<0.05), reduce the frequency of defecation (P<0.05).8. The influence of the compatibility of SZS with its processing product on chronic stress depression model mice in hippocampus and frontal cortex of5-HT biological metabolism.The compatibility of SZS with its processing product could significantly I ncrease the5-HT content in chronic stress depression mice hippocampus and cortical sites (P<0.05), significantly increased the content of5-HIAA in hippocampus (P<0.01), improve the ratio of5-HIAA/5-HT (P<0.05) in the hippocampus.9. The influence of the compatibility of SZS with its processing product on the chronic stress depression model mice hippocampus Slc6a4and Htrla gene expression.The compatibility of SZS with its processing product can regulate the expression of chronic stress depression mice hippocampus Slc6a4gene down, Htrl a gene expression in the hippocampus up.10. The influence of the compatibility of SZS with its processing product on chronic stress depression model mice in hippocampus and frontal cortex of DA biological metabolism.The compatibility of SZS with its processing product could significantly increase the DA content in hippocampus of chronic stress depression mice (P<0.05). 11. The influence of the compatibility of SZS with its processing product on the chronic stress depression model mice hippocampus Slc6a3gene.The compatibility of SZS with its processing product can regulate the expression of Slc6a3in hippocampus of chronic stress depression mice gene down.12. The changes of the components of the after compatibility of SZS with its processing productBy analyzing,the HPLC-ELSD chromatogram peaks of mixed decoction, SZS decoction and its processing product decoction, In the decoction of individual peak area percentage changes.13. The changes of three kinds of index components after compatibility of SZS with its processing productThe mixed decoction spinosin was3.7898mg, Jujuboside A in mixed decoction for2.2849mg,the highest in the content of three kinds of water decoction; the content of Jujuboside B content for1.1823mg.Conclusions:1. The compatibility of SZS with its processing product has obvious antidepressant effect,better than in crude and fried alone.2. Antidepressive mechanism of the compatibility of SZS with its processing product can uptake through the inhibition of central5-HT, increased central5-HT system function and excitability. At the same time can inhibit the reuptake of central DA, increase the central content of DA.3. The compatibility of SZS with its processing product has no new components, each component of HPLC-ELSD had no significant change. In the three index components, spinosin content and Jujuboside A content increases.更多还原