摘要
目的：(1)研究1,3,7,9-四甲基尿酸(TC)、咖啡因(CAF)、可可碱(TB)、茶碱(TP)等茶叶嘌呤生物碱对抑郁的影响,并探讨可能的作用机制。
(2)比较TC提取方法并优化TC提取分离的工艺条件。
方法：(1)采用回流法、微波法及超声波法对苦茶TC成分进行提取；采用真空升华法来分离纯化TC；实验设计方法采用正交设计来获取各因素的影响及优化最佳工艺条件。
(2)采用小鼠自主活动实验、悬尾实验、强迫游泳实验、利血平实验、育亨宾毒性实验、5-HTP诱导甩头实验及大鼠慢性不可预知温和应激(CUMS)实验来评价茶叶嘌呤生物碱对抑郁的影响。
(3)采用HPLC-ECD来分析检测茶叶嘌呤生物碱对·CUMS大鼠皮质、海马、下丘脑等部位DA、5-HT、NE、DOPA、HVA和5-HIAA等单胺类神经递质及衍生物含量的影响。
(4)采用全细胞膜片钳技术来评价茶叶嘌呤生物碱对低渗激活的容积敏感性氯电流的影响。
(5)采用HPLC法测定茶叶嘌呤生物碱对PDE4体外抑制的影响。
(6)采用RT-PCR技术检测茶叶嘌呤生物碱对PC12细胞腺苷受体A1、A2A、CREB、BDNF和TrkB mRNA表达的变化。
结果：(1)对于回流法提取TC,其影响因素的顺序为回流温度>回流时间>pH,其中回流温度具有非常显著的影响(p<0.01)；最佳工艺条件为：回流时间为50 min,回流温度为90℃,pH为7.5。对于微波法提取TC,其影响因素的顺序为pH>微波时间>微波功率,其中pH具有显著的影响(P<0.05)；其最佳工艺条件为：微波时间为10 min,微波功率为480 W,pH为7.5。对于超声波法提取TC,其影响因素的顺序为pH>超声波时间>超声波功率,其中pH具有非常显著的影响(P<0.01)；其最佳工艺条件为：超声波时间为10 min,超声波功率为450 W,pH为7.5。对于真空升华分离纯化TC,其影响因素的顺序为真空度>升华时间>升华温度,其中升华时间和真空度有显著性影响(p<0.05)；其最佳工艺条件为：升华温度为175℃,真空度为0.10×105 Pa,升华时间为1 min。
(2)小鼠的自主活动实验结果表明,CAF具有十分明显的兴奋作用(P<0.01),TB和TP也有显著性作用(P<0.05),而TC虽然可增加自主活动的总路程,但无统计学意义。小鼠悬尾实验、强迫游泳实验结果表明,TC可显著缩短行为绝望小鼠的不动时间用(P<0.01),CAF对行为绝望小鼠的不动时间缩短非常显著(P<0.001),TB只对强迫游泳小鼠不动时间有显著缩短(P<0.05),TP虽然对行为绝望小鼠不动时间有缩短的趋势,但无统计学意义。CUMS实验结果表明,10 mg·kg-1和30 mg·kg-1 TC可非常显著地增加CUMS大鼠的体重(P<0.001),并显著改善大鼠蔗糖偏嗜度(P<0.05)；CAF对CUMS大鼠体重和蔗糖偏嗜度都有改善的趋势,但无统计学意义；TP和TB只可十分显著地增加CUMS大鼠的体重(P<0.05)。利血平实验、5-HTP诱导小鼠甩头实验结果表明10 mg·kg-1和30mg·kg-1TC对于抑郁特征具有良好的改善作用(P<0.05),CAF、TB和TP只对利血平实验有非常显著的改善(P<0.001)。四种茶叶嘌呤生物碱对育亨宾毒性增强的趋势都无统计学意义。
(3)茶叶嘌呤生物碱对CUMS大鼠皮质、海马、下丘脑等部位的DA、5-HT、NE、DOPAC、HVA和5-HIAA等单胺类神经递质或其衍生物含量都有一定程度改善,其中TC在几乎所有的神经递质的紊乱中都表现出了显著性改善作用,并对5-HT和DA等神经递质代谢异常有良好的改善。TP及TB对CUMS大鼠脑部单胺类神经递质及其衍生物的紊乱及代谢异常的改善作用较弱。CAF对CUMS大鼠脑部单胺类神经递质及其衍生物的紊乱及代谢异常的改善作用基本无统计学意义。
(4)茶叶嘌呤生物碱对低渗激活的容积敏感性氯电流都有一定程度的抑制作用,其中TC与CAF有显著性抑制作用(P<0.05),抑制作用强弱顺序为：TC>CAF>TB≈TP。
(5)茶叶嘌呤生物碱对体外cGMP为底物的PDE4基本无抑制效果；对cAMP为底物的PDE4有较强的抑制作用,抑制作用强弱顺序为：TC≈CAF>TB≈TP。
(6)茶叶嘌呤生物碱对PC12细胞的腺苷A1、A2A mRNA都的表达有一定程度的抑制作用,其中TC与CAF都有较强的显著性作用(P<0.05)；对CREB mRNA表达都有一定的增强作用,但无统计学意义；对BDNF和TrkB mRNA表达有都显著的增强作用(P<0.05),且TC效果最为显著(P<0.01)。
结论：(1)相对于常规的回流法,微波法、超声波法都能高效、经济及环保地提取TC；真空升华法分离TC时,具有快速、高效,且环境条件要求低,成本十分低廉等优点；正交设计不仅可以有效地分析TC制备的各影响因素,而且可以较好地获得TC制备的最佳工艺条件。
(2)TC具有较强的抗抑郁作用,可能是源于对实验动物单胺类神经递质含量紊乱的改善和神经元的保护功能；CAF对行为绝望动物的行为学影响可能是中枢兴奋的结果；TP与TB可能具有较弱的抗抑郁作用。
(3)TC等茶叶嘌呤生物碱对抗抑郁的作用机制主要涉及：1)腺苷受体-AC-cAMP/PKA-CREB-BDNF-TrkB通路；2)PDE-cAMP/PKA-CREB-BDNF-TrkB途径。3)氯通道的GABA等嘌呤受体的补充途径；4)神经递质对离子通道的修饰作用等几个信号转导通路。
创新点：(1)本论文首次提出了TC抗抑郁的假说,并通过各种行为学实验证实了TC抗抑郁的效果；而初步阐明了TC抗抑郁作用的主要机制。此研究不仅有助于苦茶及TC的深入开发,对于推动我国天然抗抑郁药物现代化和国际化的进程也具有重要意义。
(2)首次题出氯通道对中枢神经系统抑郁等方面作用。
(3)本论文首次系统比较了TC、CAF、TB和TP四个典型的茶叶生物碱对抑郁作用特点。
(4)本论文通过逆向排除CAF的思路,首次采用了真空升华法制备了纯度和产率较高的TC。
Objective:(1)To study the effects of theacrine (TC), caffeine (CAF), theobromine (TB) and theophylline (TP) on depression, and discuss the possible mechanisms.
(2) To compare the various extraction and purification methods of TC, and study their optimum preparation.
Method:(1) TC was prepared by reflux extraction, microwave extraction and supersonic wave extraction, and it was purified by vacuous sublimation method. In order to assess the effects of all the factors and acquire the optimum technology, orthogonal design was used for the experiment.
(2) The antidepressant effects of tea purine alkaloids were examined by ambulatory activity test, suspension test, forced swimming test, yohimbine induced toxicity test, reserpine test, 5-HTP induced head-twitching test and chronic unpredictable mild stress (CUMS) test.
(3) The effects of tea purine alkaloids on concent of DA,5-HT, NE, DOPAC, HVA and 5-HIAA in cortex, hypothalamus and hippocampus of CUMS rats were analyzed with HPLC-ECD.
(4) The inhibitory effects of tea purine alkaloids on volume-activated chloride currents under hypotonic conditions were recorded by the whole-cell patch clamp technique.
(5) The inhibitory effects of tea purine alkaloids on PDE4 in vitro were evaluated with HPLC.
(6) The effects of tea purine alkaloids on the expression A1, A2A, CREB, BDNF and TrkB mRNA in PC 12 cell were examined by RT-PCR methods.
Result:(1) As for the reflux extraction, the sequence of influential factors were reflux temperature> reflux time> pH, while reflux temperature could markedly affect the extraction(P < 0.01); The optimum technology was shown to require 50 min of reflux time,90℃of reflux temperature and pH 7.5. As for the microwave extraction, the sequence of influential factors were pH> microwave time> microwave power, while pH could significantly affect the extraction (P< 0.05); The optimum technology was shown to require 10 min of microwave time, 480 W of microwave power and pH 7.5. As for the supersonic wave extraction, the sequence of influential factors were pH> supersonic wave time> supersonic wave power, while pH could markedly affect the extraction (P< 0.01); The optimum technology was shown to require 10 min of supersonic wave time,450 W of supersonic wave power, and pH 7.5. As for the TC purification by vacuous sublimation method, the sequence of influential factors were vacuity> sublimation time> sublimation temperature, while sublimation time and vacuity could significantly affect the extraction (P<0.05); The optimum technology was shown to require 175℃of sublimation temperature,0.10×105 Pa of vacuity, and 1 min of sublimation time.
(2) In the ambulatory activity test, CAF could markedly affect the central excitation (P< 0.01); TB and TP could significantly affect the central excitation (P<0.05); TC could increase the route length and stand frequency but with no significance. In the suspension test and forced swimming test, TC could markedly shorten the immobility time (P<0.01); CAF could notably shorten the immobility time (P<0.001); TB could only significantly shorten the immobility time of forced swimming test (P<0.05); TP could shorten the immobility time but with no significance. In the CUMS test, compared with model group,10 mg·kg-1 and 30 mg·kg-1TC could notably increase the body weight (P< 0.001) and significantly decrease sucrose preference (P< 0.05); CAF could change body weight and sucrose preference but with no significance; TB and TP could only significantly increase the body weight (P< 0.05). In the yohimbine induced toxicity test and reserpine test,10 mg·kg-1 and 30 mg·kg-1TC could significantly improve the depressant symptoms (P< 0.05); CAF, TB and TP could only notably improve the depressant symptoms induced by reserpine (P< 0.001). All the tea purine alkaloids could increase the toxicity induced by yohimbine but with no significance.
(3) All of the tea purine alkaloids could change the contents of DA,5-HT, NE, DOPAC, HVA and 5-HIAA in cortex, hypothalamus and hippocampus of CUMS rats. TC could significantly improve the contents of DA,5-HTand NE, and notably retrieve the metabolism of DA and 5-HT. TP and TB could weakly recuperate the contents of monoamine neurotransmitters and metabolisms. CAF could recuperate the contents of monoamine neurotransmitters and metabolisms but with no significance.
(4) All of the tea purine alkaloids could suppress the effects of volume-activated chloride currents under hypotonic conditions with different levels, during which TC and CAF could significantly suppress the chloride current in volume-activated chloride currents under hypotonic conditions(p<0.01), and the sequence of inhibitory effects were TC> CAF> TB≈TP.
(5) All of the tea purine alkaloids could hardly suppress the PDE4 with cGMP substrate, but could significantly suppress the PDE4 with cAMP substrate, and the sequence of inhibitory effects were TC≈CAF>TB≈TP。
(6) All of the tea purine alkaloids could suppress the expression of A1 and A2A mRNA with different levels, during which TC and CAF could significant inhibitory effects (P<0.05). All of the tea alkaloids could enhance the expression of CREB mRNA but with no significance. All of the tea alkaloids could significantly enhance the expression of BDNF and TrkB mRNA with different levels (P< 0.05), and TC was the most significant (P< 0.01).
Conclusion:(1) Compared with traditional reflux extraction, microwave extraction and supersonic wave extraction could extract TC more efficiently, economically and environment friendly. Vacuous sublimation method could purify TC quickly, efficiently, conveniently and economically. Orthogonal design was not only a method to analyze the factors of TC extraction and purification, but also an easy method to acquire the optimum preparation conditions of TC.
(2) TC had antidepressant effects in various depression models, which may be contributed to its influence on monoamine neurotransmitter and neuron protection.The ethology effects of CAF was the result of its central excitation, moreover, the antidepressant effects of TB and TP were not very significant.
(3) The mechanisms of tea alkaloids on anti-depression were involved the following signal pathways:1) The signal pathway of AR-AC-cAMP/PKA-CREB-BDNF-TrkB; 2) The signal pathway of PDE-cAMP/PKA-CREB-BDNF-TrkB; 3) The complementary pathway of chloride current induced by purine receptor; 4) The modification of neurotransmitter to ion channel.
Novel Points:(1) The antidepressant effect of TC was firstly proposed, which was verified through various ethology studies. Their preliminary mechanisms of TC were also studied in the first time. This study are expected to promote the development of Kucha and TC, and to promote the modernization and internationalization of antidepressant medicine of Chinese natural products.
(2) The depressant effect of chloride channel was proposed for the first time.
(3) The antidepressant characteristics of TC, CAF, TB and TP were systematically compared for the first time.
(4) According to the thought of eliminating CAF convereely, vacuous sublimation method was firstly used to prepare highly purified and high productive TC.
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