| 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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