钩藤碱提取工艺研究

对钩藤碱提取工艺进行研究,确定用碱化苯提法提取钩藤碱,经处理后可得到高纯度钩藤碱．该工艺简单可行,适于大规模生产．     

Effect of rhynchophylline on TGF-β1/Smad pathway for processing ventricular remodeling in spontaneously hypertensive rats

AIM: To investigate the effect of rhynchophylline (Rhy) on blood pressure, cardiac hypertrophy and myocardial fibrosis in spontaneously hypertensive rats (SHR). METHODS: Spontaneously hypertensive rats were randomly divided into model group, high dose (10 mg·kg^-1·d^-1) and low dose (2.5 mg·kg^-1·d^-1) group of rhynchophylline, captopril group (17.5 mg·kg^-1·d^-1). Wistar-Kyoto rats were used as normal control. Respectively, systolic blood pressure was measured by tail cuff every 2 weeks. After 10 weeks, heart weight index and left ventricular weight index were calculated. The myocardial hydroxyproline and plasma angiotensin Ⅱ were detected. Moreover, basic myocardial histopathological changes and myocardial collagen fibres were observed by HE staining and Masson staining, respectively. The protein expression of TGF-β₁ and Smad3 in the myocardium was measured by the methods of immunohistochemistry and Western blot. RESULTS: Compared with SHR model group, Rhy significantly reduced blood pressure (P<0.05), the levels of HYP in the myocardium (P<0.05) and the levels of AngⅡ in the plasma (P<0.01). The pathological damages of the myocardial tissues and collagen deposition were attenuated. The protein expression of TGF-β₁ and Smad3 was significantly reduced by the treatment with Rhy (P<0.01). CONCLUSION: Rhynchophylline reduces blood pressure and adjusts to improve ventricular remodeling of SHR. The mechanism may be involved in the TGF-β₁/Smad pathway and reducing AngⅡ content.     

钩藤中钩藤碱的提取和含量测定

[目的]研究钩藤中钩藤碱的提取和含量测定方法。[方法]采用生物碱回流提取方法提取,采用高效液相色谱法对钩藤中钩藤碱的含量进行测定并计算提取率。[结果]专属性考察结果显示,钩藤碱的保留时间约为18.843 min,异钩藤碱的保留时间约在22.323min,两峰分离度良好,且钩藤药材中其他成分不干扰测定;含量测定结果显示,钩藤碱和异钩藤碱的含量分别为23.23、129.17μg/g,加样回收试验平均回收率分别为99.12%、101.33%,RSD分别为1.84%、1.56%。[结论]该方法稳定、有效、准确度高,可用于钩藤中钩藤碱的含量测定。     

Mechanisms for a decrease in mortality of LPS-challenged mice by rhynchophylline

AIM: To observe effect of rhynchophylline （Rhy） on mortality and organ injury in endotoxemic mice and further investigate the mechanisms of its actions. METHODS: Male mice were randomly assigned into control, LPS, Rhy +LPS and Rhy group, and injected subcutaneously with normal saline (0.05 mL/10 g), or rhynchophylline once a day for 3 d, 1 h after subcutaneously treatment on day 3, LPS (20 mg/kg) or normal saline was injected intraperitoneally. Survival rate was recorded every 12 h for 6 d. In another experiment, 12 h after LPS injection, the left lung and intestine tissue sections were prepared for histological analysis and the right lung were used to determine the ratio of wet to dry lung tissue weight (W/D),the serum was collected to detect the concentrations of alanine aminotransferase（ALT）, aspartate aminotransferase (AST ), bloodureanitrogen (BUN) and creatinine (Cr). In addition, the concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β(IL-1β) and interleukin-10 (IL-10) in serum at 2 h after LPS challenge were detected by enzyme-linked immunosorbent assay. The concentration of NO in serum at 8 h was detected by enzymic method. The effect of Rhy on survival rate of mice subjected to cecal ligation and puncture (CLP) was also observed. RESULTS: Mortality of mice challenged with LPS alone was higher significantly than that in control at 24 h after LPS challenge, pretreated with Rhy at a dose of 8 or 16 mg/kg increased markedly the survival rate of LPS-challenged mice. However, Rhy at a dose of 8 mg/kg significantly increased mortality of mice subjected to CLP. In the histological analysis, severe inflammation was observed both in the lung and intestine tissues in the LPS group. LPS elevated lung W/D, the levels of ALT, AST, BUN, Cr, TNF-α, IL-1β, IL-10 and NO in serum. Pretreatment with Rhy had no obvious improvement in the lung and intestine tissue injury, and no significant depression in the lung W/D and the serum levels of ALT, AST, BUN, Cr, IL-1β, IL-10 and NO, but decreased the level of TNF-α in serum significantly in LPS -treated mice. CONCLUSION: Pretreatment with Rhy reduces the mortality in endotoxemic mice, but not decrease the mortality of mice challenged with CLP, at least in part, through inhibiting the synthesis and secretion of TNF-α.     

Effect of rhynchophylline on central neurotransmitter levels in amphetamine-induced conditioned place preference rat brain

The effects of rhynchophylline on expression of amphetamine reward using a conditioned place preference (CPP) paradigm and central neurotransmitter levels in rat brain was investigated. Rats were injected with amphetamine (2 mg/kg, per day for 4 consecutive days) and treated with rhynchophylline (60 mg/kg, per day for the later 3 days). Control rats were administered with rhynchophylline (60 mg/kg) instead of amphetamine to evaluate whether rhynchophylline by itself produced CPP. Glutamic acid, γ-aminobutyric acid, endorphin, acetylcholine, norepinephrine, dopamine, and 5-hydroxytryptamine contents were examined by encephalofluogram technology. Rhynchophylline reversed the expression of amphetamine-induced CPP and itself did not produce a CPP. Glutamic acid, dopamine, and norepinephrine contents in amphetamine-CPP rat brain were significantly higher; while γ-aminobutyric acid, endorphin, and acetylcholine contents were significantly lower than those of control rats. Rhynchophylline reversed those central neurotransmitter levels induced by amphetamine to control levels; rhynchophylline by itself had no effect on central neurotransmitter in control rats. These findings show that rhynchophylline reverses the expression of amphetamine-induced rewarding effect which is partly mediated by regulation of central neurotransmitter levels in the rat brain.     

Effects of rhynchophylline on adriamycin-induced renal injury in rats

AIM: To explore the effects of rhynchophylline (RHL) on rat renal injury induced by adriamycin. METHODS: Fifty-two female SD rats were randomly divided into normal saline group (NSG, n=10), model group (MG, n=14), low-dose RHL treatment group (RHL-LG, n=14) and high-dose RHL treatment group (RHL-HG, n=14). The animals in the latter 3 groups were injected with adriamycin at a dose of 5 mg/kg through the tail vein. The animals in RHL-LG and RHL-HG were treated with RHL at doses of 5 mg/kg and 15 mg/kg, respectively, by intraperitoneal injection for 8 weeks. The animals in NSG and MG were treated with normal saline only. Urine and blood samples were collected to detect the urine protein, blood urea nitrogen (BUN) and serum creatinine (SCr). The renal tissues of the animals were collected for detection of malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, pathological changes and mRNA expression of angiotension Ⅱreceptors 1,2 (AT_1,2-R), angiotensin-converting enzyme (ACE) and angiotensinogen (AGT). RESULTS: The urine protein, BUN and SCr in RHL-LG were significantly lower than those in MG, but higher than those in RHL-HG (P<0.05). The SOD activity in MG was significantly lower than that in RHL-LG in the renal tissues. The SOD activity in RHL-LG was significantly lower than that in RHL-HG (P<0.05), but the content of MDA was on the contrary. The renal pathological damages in RHL-LG were weaker than that in MG, and that in RHL-HG were weaker than that in RHL-LG. The mRNA expression of AT₁-R, ACE and AGT in MG was significantly higher than that in RHL-LG in the renal tissues, and that in RHL-LG was higher than that in RHL-HG (P<0.05). The mRNA expression of AT₂-R in MG was significantly lower than that in RHL-LG, and that in RHL-LG was significantly lower than that in RHL-HG (P<0.05). CONCLUSION: RHL reduces adriamycin-induced renal injury in rats by attenuating the injury of lipid peroxidation in renal tissue, regulating the mRNA expression of AT_{1, 2}-R, ACE and AGT, and increasing renal blood flow.     

HPLC法测定钩藤提取物中钩藤碱的含量

[目的]测定钩藤提取物中有效成分钩藤碱的含量。[方法]采用HPLC法测定钩藤提取物中钩藤碱的含量,色谱柱为DiamonsilC18(250 mm×4.6 mm,5μm);流动相为无水甲醇-水(60∶40,V/V)并含10 mmol/L三乙胺,冰醋酸调pH 7.5;柱温为30℃;流速为1.0ml/min;紫外检测波长为254 nm。[结果]钩藤碱浓度在11.0～110.0μg/ml范围内与峰面积的线性关系良好(R=0.999 6),平均回收率为96.84%,RSD为1.46%。[结论]该方法简单、快速、准确,可用于钩藤提取物中钩藤碱的含量测定。