M3 muscarinic receptor- and Ca2+ influx-mediated muscle contractions induced by croton oil in isolated rabbit jejunum

doi:10.1016/j.jep.2010.04.020

Journal of Ethnopharmacology

Volume 129, Issue 3, 16 June 2010, Pages 377-380

https://doi.org/10.1016/j.jep.2010.04.020 Get rights and content

Abstract

Aim of study

Croton oil is the fruit oil of Croton tiglium L., which is well known in folk medicine for the treatment of gastrointestinal (GI) diseases, including constipation, abdominal pain, peptic ulcer, and intestinal inflammation for a long period. This study was to investigate the pharmacological effect of croton oil on GI tract.

Materials and methods

The effect of croton oil on the smooth muscle contractions was investigated in vitro using the isolated rabbit jejunum model.

Results

Croton oil has a biphasic action contracting and relaxing intestinal tissue. At the concentrations of 20–80 μg/mL, croton oil produced a concentration-dependent increase in the amplitude and tension of muscle contractions, whereas at high concentrations (>200 μg/mL) it decreased the contractile amplitude and had no impact on the tension. Moreover, croton oil was less effective in increasing muscle amplitude and tension than Ach, confirming that the effect of croton oil on muscle contractions is not a simply stimulatory or inhibitory action, but a unique modulatory process depending on the concentration of croton oil. In addition, croton oil concentration-dependently suppressed the frequency of muscle contractions. On the other hand, atropine (10 μM) and 4-DAMP (10 μM) produced a significant inhibition of contractions caused by croton oil, while either hexamethonium (10 μM) or methoctramine (10 μM) was inactive, implying that the regulatory effects of croton oil on GI motility are mediated via the activation of M3 muscarinic receptor. Furthermore, muscle contractions induced by croton oil were dramatically reduced by verapamil (0.1 μM) but not by NE (1 μM), suggesting that the action of croton oil on GI motility is also mediated by Ca²⁺ influx through L-type Ca²⁺ channel.

Conclusions

The results suggest that croton oil possesses spasmogenic and spasmolytic properties and the regulatory effects of croton oil on GI motility are mediated via the activation of M3 muscarinic receptor and Ca²⁺ influx through L-type Ca²⁺ channel.

Graphical abstract

Introduction

Croton tiglium L. (Euphorbiaceae) is one of the most popular medicinal plants distributed in tropical and subtropical regions (Qiu, 1996, Salatino et al., 2007). The fruit of Croton tiglium L. is Crotonis Fructus, which has been used in traditional Chinese medicine for the treatment of gastrointestinal (GI) diseases, including constipation, abdominal pain, peptic ulcer, and intestinal inflammation for thousands of years (Villena et al., 1999, Editorial Board of Zhonghua Bencao, 2000, Tsai et al., 2004, Committee of National Pharmacopoeia, 2010).

Croton oil, the fatty oil of Crotonis Fructus, has been reported to have a direct effect on guinea pig colonic smooth muscle cell, regulate the GI transit in mice, and affect inflammatory and immunological milieu (Wang et al., 2002, Wang et al., 2008). However, the pharmacological effect of croton oil on GI motility, and particularly the underlying mechanisms are less explored.

Thus, the aim of the present study was to investigate the effect of croton oil on spontaneous smooth muscle contractions in isolated rabbit jejunum. An additional objective was to determine the mechanisms by which croton oil mediates regulation of GI motility.

Section snippets

Plant material and preparation of croton oil

Croton oil was extracted from the fruit of Croton tiglium L. as previously described (Wang et al., 2002). Croton tiglium L. was provided by Tianjin Zhongxin Pharmaceutical Group Co., Ltd. (Tianjin, China) and identified by Professor Wenyuan Gao from School of Pharmaceutical Science and Technology, Tianjin University, China. The voucher specimens (voucher No. BD070701) are available in the herbarium of Research Center of Tianjin Zhongxin Pharmaceuticals.

Drugs and chemicals

Acetylcholine perchlorate (Ach),

Effect of croton oil on spontaneous smooth muscle contractions

As shown in Fig. 1, the vehicle (0.5% tween-80), in a volume equivalent to that of croton oil (200 μL), did not change spontaneous smooth muscle contractions of isolated rabbit jejunum. We observed that croton oil, at the concentrations of 20–80 μg/mL, concentration-dependently increased the amplitude and tension of muscle contractions, whereas at high concentrations (>200 μg/mL) it decreased the amplitude and had no impact on the tension of muscle contractions. In addition, croton oil suppressed

Discussion and conclusions

In the present study, we investigate the effects of croton oil on spontaneous smooth muscle contractions of isolated rabbit jejunum and those induced by antagonistic and agonistic agents.

We have demonstrated that croton oil has a biphasic action contracting and relaxing intestinal tissue. It increased jejunal contractions at low concentrations, whereas it exerted inhibitory effect at high concentrations. A previous study has also reported that low concentrations of croton oil enhance the phasic

Acknowledgements

The work was supported by a grant from Modernization of Traditional Chinese Herbs of Tianjin, China (No. 05ZHGCGX01000).

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Crotonis Fructus (CF), the seeds of Croton tiglium L., have been commonly used in the treatment of constipation for more than two thousand years in traditional Chinese medicine (TCM). CF needs to be processed before clinical use and Crotonis Semen Pulveratum (CP) is the processed cream of CF, which could reduce the drastic purgative action and gastrointestinal damages. However, the mechanism of CF and CP in the treatment of constipation is still unclear.
This study was to evaluate the effects of CF and CP on loperamide-induced constipation and the underlying mechanism.
The chemical compositions of CF and CP were analyzed by UPLC-Q-TOF-MS. Constipated mouse model was established by loperamide (9.6 mg/kg, b.w., i.g.) for two weeks. After successful modeling, the mice were treated with CF or CP (45.5 and 136.5 mg/kg, b.w., i.g.) once a day for seven days. The physiological status, defecation indices, defecation time, and intestinal propulsion rate in mice were measured. Histopathologic examination and serum biochemical parameters were further estimated. 16S rDNA gene sequencing was carried out to characterize the effects of CF and CP on intestinal microbiome structure. Spearman correlation analysis was also performed to explore the association between gut microbiotic abundance and serum indices.
The results verified the therapeutic effects of CF and CP on loperamide-induced constipation. CF and CP could significantly ameliorate the reduction of fecal number, fecal weight, fecal water content, and intestinal propulsion rate in mice with constipation, and the first stool defecation time was also obviously reduced. Moreover, CF and CP could regulate the secretion of gastrointestinal hormones and inflammatory factors induced by constipation. Histopathologic examination showed that CP was superior to CF in relieving pathological injury and inflammatory cell infiltration. According to 16S rDNA sequencing, CF and CP treatment could improve gut microbiota disturbance in mice with constipation and the abundance of opportunistic pathogens such as Parabacteroides, Parasutterella and Bacillus remarkably declined, while the levels of beneficial bacterial such as Candidatus_Arthromitus significantly increased. Besides, CP may play a better role in correcting the intestinal flora disorder than CF, which was more obvious in the high-dose group. In addition, phytochemical analysis revealed the presence of diterpenoids and alkaloids in CF and CP.
CF and CP could ameliorate loperamide-induced constipation by regulating gastrointestinal hormones secretion, reducing the levels of inflammatory cytokines and improving the disturbance of gut microbiota. Moreover, CP was superior to CF in the enrichment of beneficial bacteria and reduction of harmful bacteria and histopathological damage induced by constipation, which may be related to the changes in the species and content of diterpenoids after processing. The study provides new evidence for the processing mechanism and clinical application of CF and CP.
Impact of Shodhana an Ayurvedic purification process on cytotoxicity and mutagenicity of Croton tiglium Linn.
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Croton tiglium Linn. (CT) which is commonly called Jaypal is used in Ayurvedic preparations like Ichhabhedi Ras, Asvakancuki Rasa. Due to its toxic contents, seeds of Croton tiglium are purified before use, by the process mentioned in classical Ayurvedic texts called Shodhana meaning purification.
The objective of the present study is to study the impact of Ayurvedic Purification process on cytotoxicity and genotoxicity of Croton tiglium Linn.
Croton tiglium Linn. Seeds were processed for Shodhana by soaking in water, heating with milk (Snehan) and later grinding in Lemon Juice (Bhavana). Aqueous and Hydroalcoholic extracts were prepared before and after purification i.e. Shodhana. Cytotoxicity of the Croton tiglium was studied against Chinese Hamster Ovary cell line by MTT assay. Ames test was performed to study the mutagenicity of the extracts in Salmonella typhi TA 98, 100 and 102 strains. Phytoconstituents were studied by using LCMS analysis.
The results indicated decrease in cytotoxic concentration (IC₅₀) of Croton tiglium seeds after purificationa from 3.03 mg/mL to 0.99 mg/mL in aqueous extract and 18.56 mg/mL to 5.45 mg/mL. Genotoxicity study by Ames test indicated Croton tiglium Linn. Croton tiglium Linn. Seeds are non-genotoxic in strains like S. typhi, TA 98, 100 and 102. There was change in Phytochemical profile before and after shodhana.
Although both the concentrations are practically non-toxic, the decrease in cytotoxic concentration indicates Purification process as described in classical ayurvedic texts i.e. Shodhana has definitely increased the potency of the seeds of Croton tiglium Linn.
Aster tataricus alleviates constipation by antagonizing the binding of acetylcholine to muscarinic receptor and inhibiting Ca<sup>2+</sup> influx
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Citation Excerpt :
Moreover, the laxative activity of ATE could be either produced as a result of a non-specific membrane mediated effect or a more specific receptor mediated effect [53]. The release of the neurotransmitter Ach activates muscarinic receptors on smooth muscle cells, which in turn stimulate spontaneous muscle contractions [32,54]. In our study, we investigated the effects of single and cumulative concentration of ATE on Ach-induced duodenal contraction in rats.
The dried root and rhizome of Aster tataricus (RA), is a traditional Chinese medicine has been used for more than 2000 years with the function of antitussive, expectorant and antiasthmatic. Ancient books and modern pharmacological researches demonstrated that RA may have the function of moistening intestines and relieving constipation, but there was a lack of systematic evidence. The aim of this study was to comprehensively evaluate the efficacy and possible mechanisms of ethanol extract of Aster tataricus (ATE) in treating constipation from in vivo to in vitro.
In vivo, the ATE was studied in loperamide-induced constipation of mice. In vitro, different concentrations of ATE was tested separately or cumulatively on spontaneous and agonists-induced contractions of isolated rat duodenum strips.
In vivo, at doses of 0.16, 0.8 g/mL, ATE showed significantly promotion of the small intestinal charcoal transit, decrease of the amount of remnant fecal, and increase of the content of fecal water in colon. In addition, ATE could effectively relieve colonic pathological damage caused by loperamide as well. In vitro, with the cumulative concentration increase of ATE from 0.8 to 6.4 mg/mL, it could significantly decrease the contraction caused by KCl or Ach, and gradually restore to near base tension value.Meanwhile, it could also partially but significantly inhibit the contractions induced by Ach and CaCl₂ on rat duodenum in a concentration related manner.
Taking all these findings together, it could be speculated that ATE may attenuate constipation mainly through antagonizing the binding of acetylcholine to muscarinic receptor, inhibiting Ca²⁺ influx and anti-inflammation.
Effects of the aqueous extract of Pittosporum mannii Hook. f. (Pittosporaceae) stem barks on spontaneous and spasmogen-induced contractile activity of isolated rat duodenum
2015, Journal of Ethnopharmacology
Citation Excerpt :
The most important finding in the present study was that PMAE elicited its relaxant effect only within a very short range of concentrations (10–40 µg/mL) whereas high concentrations (80 µg/mL and above) further induced a slight or a potent transient increase of the tonus (biphasic effect). A similar biphasic effect was observed in several studies with other plant extracts which exhibited a biphasic action on contractility of intestinal smooth muscle cells (Udia et al., 2009; Hu et al., 2010). Taken together, these findings suggest that the effect of PMAE on gastrointestinal motility is not a simply inhibitory or stimulatory action, but a unique modulatory process depending on the concentration of the extract (Hu et al., 2010).
Pittosporum mannii Hook. f. (Pittosporaceae) is a plant widely used in traditional medicine in Cameroon for the treatment of many gastrointestinal disorders including diarrhea. To date, no pharmacological study on the antidiarrheal and the antispasmodic properties of this plant has been reported. The aim of the present study was to evaluate in vitro the relaxant activity of the aqueous extract of stem barks of P. mannii (PMAE) on rat duodenum.
Different concentrations of PMAE were tested separately (10–80 µg/mL) or cumulatively (5–80 µg/mL) on spontaneous and spasmogen (carbachol, histamine and KCl)-induced contractions of isolated rat duodenum strips.
At concentrations ranging from 10 to 80 µg/mL, PMAE significantly decreased the tonus and the amplitude of spontaneous contractions. However, at high concentration (80 µg/mL), the extract elicited a transient relaxation was followed by a slight increase of tonus, while the amplitude remained lower compared to the normal spontaneous activity. The relaxant effect of the extract was not significantly affected in the presence of atropine (0.713 µg/mL) and promethazine (0.5 µg/mL). In addition, PMAE (20, 40, and 80 µg/mL) partially but significantly inhibited in a concentration related manner the contractions induced by carbachol (10⁻⁹–10⁻⁴ M) and histamine (10⁻⁹–10⁻⁴M) on rat duodenum. PMAE (10–80 µg/mL) also significantly induced a concentration-dependent relaxation on KCl (20 mM, 50 mM, 10⁻³–6.10⁻³ M)-induced contraction of rat duodenum.
These results show that the aqueous extract of P. mannii stem barks possesses antispasmodic and spasmolytic effects at lower concentrations; therefore, supporting the use of the stem barks of this plant in the folk medicine for the treatment of diarrhea. However, caution should be paid while using higher concentrations that instead might produce spasmogenic effect and might worsen the diarrheal condition. The relaxant effect of PMAE appears to be non-specific of muscarinic or histaminic receptors, but may involve at least in part a mechanism of inhibition of the Ca²⁺ influx into the smooth muscle cells through voltage-operated Ca²⁺ channels.
The influence of compatibility of traditional Chinese medicine on the pharmacokinetic of main components in Fructus Aurantii
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Citation Excerpt :
Crotonis Fructus, the fruit of Croton tiglium L., has been used in traditional Chinese medicine to treat of gastrointestinal disease, including constipation, abdominal pain, intestinal inflammation for thousands years (Tsai et al., 2004; Villena et al., 1999). In the reports, Croton oil has a biphasic action contracting and relaxing intestinal tissue Hu et al., 2012, 2010. In this study, the processed produce of Crotonis Fructus, Semen Crotonis Pulveratum, was used according to the composing prescriptions of WCA pill.
Fructus Aurantii is one of the commonly used traditional Chinese medicines and the compatibility of Fructus Aurantii, Radix et Rhizoma Rhei and Semen Crotonis Pulveratum were the main components in a Chinese traditional patent medicine Weichang'an pill. Naringin and neohesperidin are two main flavonoid glycosides in the extract of Fructus Aurantii. The pharmacokinetics of naringin and neohesperidin, as the main marker constituents were analysis by a simple and accurate method.
In the present study, pharmacokinetic differences of two flavonoid glycosides were detected after oral administration of the extract of single herb and different compatibility of Fructus Aurantii, Radix et Rhizoma Rhei and Semen Crotonis Pulveratum which at doses of approximately 700 mg/kg naringin and 300 mg/kg neohesperidin.
It indicated that the pharmacokinetic process of naringin and neohesperidin in four groups were demonstrated bimodal phenomenon after oral administration. Comparing to pharmacokinetic parameters of the single herbs administration, there were no significant difference after the compatibility of Fructus Aurantii and Radix et Rhizoma Rhei. However, the absorption of two flavonoid glycosides was increased dramatically after the compatibility of Fructus Aurantii or Semen Crotonis Pulveratum. The same results were attained after co-administration of the extract of three herbs.
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M3 muscarinic receptor- and Ca2+ influx-mediated muscle contractions induced by croton oil in isolated rabbit jejunum

Abstract

Aim of study

Materials and methods

Results

Conclusions

Graphical abstract

Introduction

Section snippets

Plant material and preparation of croton oil

Drugs and chemicals

Effect of croton oil on spontaneous smooth muscle contractions

Discussion and conclusions

Acknowledgements

Life Sciences

Journal of Ethnopharmacology

Journal of Surgical Research

European Journal of Pharmacology

Journal of Chromatography A

Journal of Ethnopharmacology

Anti-HIV phorbol esters from the seeds of Croton tiglium

Phytochemistry

The role of Ca2+ in the contractility of rabbit small intestine in vitro

Journal of Physiology and Pharmacology

Ion channels in gastrointestinal smooth muscle and interstitial cells of cajal

Current Opinion in Pharmacology

The enteric nervous system. II: Gastrointestinal functions

Pharmacology & Toxicology

M3 muscarinic receptor- and Ca²⁺ influx-mediated muscle contractions induced by croton oil in isolated rabbit jejunum

The role of Ca²⁺ in the contractility of rabbit small intestine in vitro