Activation of M3 muscarinic receptor and Ca²⁺ influx by crude fraction from Crotonis Fructus in isolated rabbit jejunum

Ethnopharmacological relevance

Crotonis Fructus is the mature fruit of Croton tiglium L. (Euphorbiaceae), which has been used in traditional Chinese medicine for the treatment of gastrointestinal (GI) diseases, such as constipation, abdominal pain, peptic ulcer, and intestinal inflammation for thousands of years. The aim of this study was to investigate the pharmacological effect of extracts and fractions from Crotonis Fructus on GI tract.

Materials and methods

The activities of methanol extract and fractions from Crotonis Fructus on the smooth muscle contractions were evaluated using isolated rabbit jejunum model.

Results

The results suggest that the n-BuOH and H₂O fractions showed spasmolytic activity, while the MeOH extract, PE and EtOAc fractions exerted spasmogenic effect. Moreover, bioassay-guided fractionation verified that the EtOAc fraction was more potent than others, followed by PE fraction and methanol extract. Additionally, atropine (10 μM), 4-DAMP (10 μM) and verapamil (0.1 μM) produced a significant inhibition of contractions caused by EtOAc fraction, while either hexamethonium (10 μM) or methoctramine (10 μM) was inactive. Additionally, a HPLC fingerprint of EtOAc fraction was appraised to ensure its chemical consistency and the main component has been identified as phorbol 12-acetate-13-tiglate.

Conclusions

These data indicate that the regulatory effect of EtOAc fraction on GI motility are medicated via the activation of M3 muscarinic receptor and Ca²⁺ influx through L-type Ca²⁺ channel. These provide a scientific basis for the traditional use of Crotonis Fructus in GI disorders.     

Simo decoction promotes contraction of antral circular smooth muscle mainly via muscarinic M3 receptor

Ethnopharmacological relevance

Simo Decoction (SMD), a traditional Chinese medicine, included four elements, such as Fructus aurantii, Radix aucklandiae, Semen arecae and Radix linderae. It has been used to improve gastrointestinal dysmotility in clinical practice for a long history in China. However, the explicit mechanisms are unclear. The aim of this study was to investigate the effect of SMD on contractions of antral circular smooth muscle strips of Sprague-Dawley (SD) rats and its underlying mechanism.

Materials and methods

The antral circular strips were prepared in the organ bath under baseline or to be incubated with muscarinic receptor antagonist atropine (10⁻⁶ M), muscarinic M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (0.4×10⁻⁶ M), muscarinic M2 receptor antagonist gallamine (10⁻⁶ M), adrenergic receptor agonist adrenaline (10⁻⁷ M), exogenous nitric oxide (NO) donor l-arginine (10⁻⁴ M), nicotinic receptor antagonist hexamethonium chloride (10⁻⁴ M) and Ca²⁺ channel antagonist nifedipine (30 nM), and consecutive concentrations of SMD were added to the bath to observe the strip responses. As a control, the responses of strips after administration with the same volume of Krebs solution as SMD were also noted. The strip responses to acetylcholine (10⁻⁷–10⁻³ M) were also noted in organ bath to compare with SMD-induced contraction.

Results

SMD dose-dependently evoked hypercontractility of antral circular strips, and the maximal contractile effect of circular smooth muscle induced by SMD was significantly higher than that induced by acetylcholine (10⁻³ M). The responses of antral circular strips to SMD were completely antagonized by atropine, 4-DAMP or 4-DAMP+gallamine, but partly inhibited by gallamine and partly suppressed by adrenaline, l-arginine, hexamethonium chloride and nifedipine.

Conclusions

SMD promotes contractions of antral circular strips in rats mainly via activation of muscarinic M3 receptor, but partly via activation of muscarinic M2 receptor, Ca²⁺ channel and nicotinic receptor, inhibition of adrenergic receptor and releasing of NO.     

Chemical composition and mechanisms underlying the spasmolytic and bronchodilatory properties of the essential oil of Nepeta cataria L.

Aim of the study

The study was aimed to investigate the chemical composition and pharmacological basis for traditional use of essential oil of Nepeta cataria L. (Limiaceae) (Nc.Oil) in gastrointestinal and respiratory disorders.

Materials and methods

Chemical analysis was carried out through GC-EIMS, ¹³C NMR and Kovats Retention Indices while pharmacological study was carried out in isolated tissues preparations.

Results

Four major components; 1,8-cineol (21.00%), α-humulene (14.44%), α-pinene (10.43%) and geranyl acetate (8.21%) were identified among the 27 compounds in Nc.Oil. In isolated rabbit jejunum, Nc.Oil, papaverine and verapamil inhibited spontaneous and high K⁺(80 mM) precontractions, as well as shifted the Ca⁺⁺ concentration–response curves (CRCs) to right, indicating calcium channel blocking activity. In isolated guinea-pig trachea, Nc.Oil and papaverine inhibited carbachol (1 μM) and K⁺ precontractions with similar potency, while verapamil was more potent against K⁺. Nc.Oil also potentiated isoprenaline inhibitory CRCs, similar to papaverine, indicating papaverine-like PDE inhibitor activity. In isolated guinea-pig atria, Nc.Oil caused cardiodepression at around 25–80 times higher concentrations, similar to papaverine.

Conclusions

These data indicate that Nepeta cataria possesses spasmolytic and myorelaxant activities mediated possibly through dual inhibition of calcium channels and PDE, which may explain its traditional use in colic, diarrhea, cough and asthma.     

Vasorelaxation by extracts of Casimiroa spp. in rat resistance vessels and pharmacological study of cellular mechanisms

Aim of the study

Casimiroa spp. are Mexican plants traditionally used for treatment of hypertension. To study their antihypertensive action, we determined the arterial dilatation induced by extracts from leaves and seeds of Casimiroa calderoniae F. Chiang & Medrano, Casimiroa edulis Llave et Lex, and Casimiroa pubescens Ramirez.

Materials and methods

The vascular effects of Casimiroa spp. extracts were investigated on rat caudal and aortic arteries. In addition, the extracts were characterized by HPLC using heraclenol, isopimpinellin, heraclenin and phellopterin as external standards. The methanolic extract of Casimiroa pubescens seeds (Cp12) was also studied by H-NMR and LC-MS (ESI-TOF) for the determination of casimiroin and zapotin.

Results

The hexanic and methanolic extracts of Casimiroa spp. (20 μg/ml) showed vasorelaxation in arterial tissues precontracted by phenylephrine (0.5 μM); the extracts from seeds always caused a greater relaxation in comparison to those from leaves. The most active were the methanolic seed extracts of Casimiroa edulis (Ce8) and Casimiroa pubescens (Cp12). To study the pharmacological mechanisms of vasodilatation we used various inhibitors selective to different receptor subtypes or intracellular enzymes. The vasorelaxant effect of Ce8 (20 μg/ml) remained unaffected by the pretreatment with pyrilamine (10 μM), an antagonist of histamine H₁ receptors, but was inhibited by atropine (0.1 μM), a muscarinic receptor antagonist. Therefore, to determine muscarinic receptor subtypes, we used pirenzepine (1 μM), a selective inhibitor of M₁ receptor, and 4-diphenylacetoxyl-N-methylpiperidine methiodide (DAMP, 0.01 μM), a selective inhibitor of M₃ receptor. Only the latter reduced the vasodilatation by Ce8 and Cp12. To investigate the role of the nitric oxide synthase (NOS), we used N^G-nitro-l-arginine methyl ester (l-NAME, 10 μM), a selective NOS inhibitor, which decreased the dilatation induced by Ce8 and Cp12. Finally, we studied the action of (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) (ODQ, 3 μM), a selective guanylyl cyclase inhibitor, which inhibited the dilatation by Casimiroa extracts.

Conclusion

The data show that methanolic seed extracts of Casimiroa edulis (Ce8) and Casimiroa pubescens (Cp12) induce vasorelaxation by M₃ receptor through the activation of cGMP-dependent NO signaling. These results support the traditional use of Casimiroa decoctions for antihypertensive treatments in the Mexican ethnomedicine.     

广西产龙眼叶及花挥发油成分气质联用分析

目的:采用GC-MS分析广西产龙眼叶及花挥发油的成分。方法:采用水蒸气蒸馏法分别提取广西产龙眼叶和龙眼花中的挥发油,并通过GC-MS技术对龙眼叶和龙眼花的挥发油成分进行分析。结果:龙眼叶和龙眼花挥发油得率分别为0.35%和0.20%,龙眼叶挥发油共鉴定出了39个化合物,占挥发油总成分的96.2%;龙眼花挥发油共鉴定出了32个化合物,占挥发油总成分的97.01%。结论:该法简便、快速、灵敏度高,为合理使用龙眼叶和花提供一定的科学依据。     

响应曲面法优化黄花败酱总皂苷提取工艺及体外促进肠平滑肌收缩活性

目的 响应曲面法优化黄花败酱总皂苷(TSPS)的提取工艺及研究TSPS促进大鼠离体肠平滑肌收缩活性。方法以齐墩果酸为对照品,采用香草醛.冰醋酸显色,利用紫外分光光度计在550 nm处测定TSPS的量。以TSPS提取率作为响应值,在单因素试验的基础上采用响应曲面法考察提取溶剂、料液比、提取时间及其交互作用对TSPS提取效果的影响。并且测定了优化提取工艺过程中获取的不同TSPS提取物的促肠平滑肌收缩活性,结合生物活性全面评价TSPS提取工艺。结果TSPS的最佳提取工艺条件为提取溶剂75%乙醇,料液比1:12,提取时间为1.5 h,提取温度90℃、提取2次。在此工艺条件下TSPS的提取率达到10.96 mg/g,其对大鼠离体肠平滑肌的收缩作用强弱表现为结肠回肠空肠十二指肠,质量浓度达0.32 mg/mL时促进离体肠收缩效果最佳。结论经优化后的TSPS提取工艺条件具有提取率高,提取工艺高效、稳定等特点,且TSPS具有显著的促进大鼠离体肠平滑肌收缩功能,其功效与提取率呈正相关。     

川芎当归提取物对血管平滑肌细胞MAPK信号通路与周期蛋白的调控效应

目的揭示川芎当归提取物(ELCAS)抑制血管平滑肌细胞增生的分子机制。方法以体外培养的原代大鼠血管平滑肌细胞为研究对象,应用Westernblot方法检测不同质量浓度ELCAS对血管平滑肌细胞内的ERK、JNK、p38磷酸化水平的影响,以及周期蛋白CyclinD1和细胞周期抑制子p21的表达情况。结果ELCAS能显著抑制ERK、JNK和p38的磷酸化以及血清诱导的CyclinD1蛋白的表达,并表现出良好的浓度依赖性;同时ELCAS能剂量依赖性地促进细胞内周期蛋白抑制子p21过表达,并抑制pRb磷酸化水平和CyclinD1蛋白表达。结论ELCAS可通过抑制MAPK信号通路以及对周期蛋白的调控来抑制血管平滑肌细胞增生。     

基于NLRP3/细胞焦亡通路研究荆芥挥发油的抗抑郁作用及作用机制

目的 观察荆芥Schizonepeta tenuifolia挥发油对慢性温和不可预知应激（chronic unpredictable mild stress,CUMS）模型小鼠抑郁样行为的干预作用,探究其调控NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor pyrin domain containing 3,NLRP3)炎症小体/细胞焦亡从而抑制神经炎症的作用机制。方法 雄性ICR小鼠制备CUMS抑郁样模型,依据糖水偏好率将造模成功的小鼠随机分为模型组、氟西汀（10 mg/kg）组和荆芥挥发油高、低剂量（100、50 mg/kg）组,同时设置对照组。给予药物干预5周后,进行小鼠糖水偏好实验、强迫游泳实验、悬尾实验与飞溅实验。行为学测试结束后,取血并分离血清,取海马组织。ELISA法测定血清中白细胞介素-1β(interleukin-1β,IL-1β)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平;尼氏染色法检测海马CA3区神经元尼氏小体平均吸光度值;免疫荧光法检测海马CA3区NLRP3、活化的半胱氨酸天冬氨酸蛋白酶-1(cle...     

Anti-diabetic activity and potential mechanism of total flavonoids of Selaginella tamariscina (Beauv.) Spring in rats induced by high fat diet and low dose STZ

Aim of the study

To evaluate the anti-diabetic effects of the total flavonoids of Selaginella tamariscina (Beauv.) Spring (TFST), and to explore the pertinent mechanism.

Materials and methods

High fat diet and STZ (35 mg/kg) induced diabetic rats were administered with TFST at graded oral doses (100, 200 and 400 mg/kg/day, ig.) for 8 weeks. A range of parameters, including blood glucose and lipid, serum insulin and glucagon, glucose tolerance, were tested to evaluate its anti-diabetic effects. The determination of protein expression of peroxisome proliferator activated receptor γ (PPAR-γ) in adipose tissue and insulin receptor substrate 1 (IRS-1) in hepatic and skeletal muscle tissues was used to study the mechanism of TFST. Moreover, the preliminary study of TFST on the antioxidant activity was performed.

Results

The TFST possessed anti-diabetic activities as shown by the decreased serum levels of fast blood glucose (FBG), glycosylated hemoglobulin A1C (HbA1c), triglyceride (TG), total cholesterol (TC), free fatty acid (FFA), low density lipoprotein-cholesterol (LDL-C) and glucagon, as well as increased serum levels of high density lipoprotein-cholesterol (HDL-C), insulin and C-peptide. TFST also improved the oral glucose tolerance test (OGTT) to a certain degree. Furthermore, TFST increased the protein expression of PPAR-γ in adipose tissue, and increased the protein expressions of IRS-1 in hepatic and skeletal muscle tissues. These benefits were associated with increased superoxide dismutase (SOD) and decreased malondialdehyde (MDA) in serum.

Conclusions

TFST exert beneficial effects on hyperglycosemia and hyperlipoidemia in diabetic rats possibly through regulating the levers of PPAR-γ in adipose tissue and IRS-1 in hepatic and skeletal muscle tissues.