Identification of the toxic constituents in Rhizoma Coptidis

doi:10.1016/j.jep.2010.01.047

Journal of Ethnopharmacology

Volume 128, Issue 2, 24 March 2010, Pages 357-364

https://doi.org/10.1016/j.jep.2010.01.047 Get rights and content

Abstract

Aim of the study

Rhizoma Coptidis (Huanglian) is a widely used Traditional Chinese Medicine. However, it causes human as well as animal toxicities. In this study, we aimed to ascertain the toxic constituents in Rhizoma Coptidis.

Materials and methods

The acute toxicity of both the total extract and the alkaloid-rich extract of Rhizoma Coptidis were tested in mice. The dose related tissue concentration of the Rhizoma Coptidis alkaloids in mice was determined using high performance liquid chromatography with ultraviolet detection. The influence of phenobarbital sodium [a non-selective hepatic enzyme (P450) inducer] on the acute toxicity of Rhizoma Coptidis as well as the tissue concentration of the alkaloids was investigated. The cytotoxicity of the Rhizoma Coptidis alkaloids was tested in six cell lines using the MTT assay.

Results

The median acute oral lethal dose of the total extract of Rhizoma Coptidis was 2.95 g/kg in mice. The alkaloid-rich extract was much more toxic than the total extract of Rhizoma Coptidis. Four Rhizoma Coptidis alkaloids were detected in brain, heart, and lung tissues of mice that received the oral total extract of Rhizoma Coptidis. Tissue concentration increased nonlinearly with higher doses. Phenobarbital sodium decreased the tissue concentration of every alkaloid as well as the toxicity of Rhizoma Coptidis. All alkaloids, especially berberine, showed dose and time dependent cytotoxicity.

Conclusions

The toxic constituents of Rhizoma Coptidis were the alkaloids, mainly berberine.

Graphical abstract

Rhizoma Coptidis (Huanglian) is a widely used Traditional Chinese Medicine. However, it causes human as well as animal toxicities. In this study, the results of both in vivo and in vitro experiments showed that the toxic constituents in Rhizoma Coptidis were the alkaloids, mainly berberine.

Introduction

Rhizoma Coptidis (Huanglian) is a widely used traditional Chinese medicine (TCM). It has a history of thousands of years for the treatment of dysentery, arrhythmia, diabetes mellitus, and inflammation-related diseases by oral intake of the extract of Rhizoma Coptidis (Xu et al., 2004). However, classical works of TCM, such as Ben Cao Gang Mu (Compendium of Materia Medica) and Ben Cao Yan Yi (Augmented Materia Medica) declared that a short-period of treatment with Rhizoma Coptidis should be recommended for all patients, and this drug was forbidden to use in patients suffering from general debility. Therefore, it is reasonable to speculate that Rhizoma Coptidis is not safe in some cases. But the exact reason is unknown.

The pharmacological properties of Rhizoma Coptidis include antibacterial (Choi et al., 2007), antifungal (Seneviratne et al., 2008), antiviral (H.Y. Kim et al., 2008), antioxidant (Yokozawa et al., 2005), anti-inflammatory (Kim et al., 2007, K.S. Kim et al., 2008), and anticancer (Y. Liu et al., 2009, J. Liu et al., 2009) activities. However, it also has been reported to cause acute toxicity in animals. Calculated by the weight of the dried herbal pieces, the median lethal dose (LD₅₀) of the oral Rhizoma Coptidis in mice was reported to be 4.9 g/kg (Qiu et al., 2004); 8.1 g/kg was 100% lethal (Tan et al., 2004). The clinical conventional dose of Rhizoma Coptidis is 10 g per adult in China. The equivalent surface area dose of mouse is about 1.50 g/kg. The ratio of the LD₅₀ value to the conventional dose is thus calculated to be only 3.3, which suggests that the safety of Rhizoma Coptidis is not high enough. It reportedly can cause respiratory failure, extrapyramidal system reactions, severe arrhythmia, liver function injury and even lead to death in clinic in China (Li et al., 2008). However, some important aspects of the safety of Rhizoma Coptidis are still unknown, such as the toxic constituents, the mechanism of toxicity, and the toxicokinetics, which are the important issues for the modern research and development as well as the clinical application of Rhizoma Coptidis. Therefore, it is necessary to ascertain the toxic constituents at first to guide the further study as well as the clinical application of Rhizoma Coptidis.

Rhizoma Coptidis yields eight major alkaloids (berberine, coptisine, palmatine, jatrorrhizine, epiberberine, worenine, columbamine and magnoflorine) and some non-alkaloid constituents such as ferulic acid (Chen et al., 2008) and chlorogenic acid. Berberine, coptisine, palmatine and jatrorrhizine (Fig. 1) are considered the primary active constituents. Berberine is an important and typical constituent in Rhizoma Coptidis. Berberine posses a variety of activities including anti-inflammatory (Jeong et al., 2009), antimicrobial (Yan et al., 2008), anti-diabetic (Lu et al., 2009), cholesterol lowering (Jia et al., 2008), anticancer (Pandey et al., 2008), and post angioplasty restenosis inhibition (Lee et al., 2006). The other constituents of Rhizoma Coptidis including coptisine, palmatine and jatrorrhizine were also reported to present some pharmacological effects such as anti-diabetes (Jung et al., 2008), antimicrobial (Yan et al., 2008) and anti-proliferation (Tanabe et al., 2005). But it is still unclear whether these constituents (berberine, coptisine, palmatine and jatrorrhizine) are responsible for the acute toxicity of Rhizoma Coptidis.

In this study, both in vivo and in vitro experiments were carried out to ascertain the toxic constituents in Rhizoma Coptidis.

Section snippets

Materials

Rhizoma Coptidis (Coptis chinensis Franch) was purchased from the Shanghai Kang Qiao herbal pieces Co. Ltd. The authentication of this herb was performed by Prof. Zhi-Li Zhao, Department of Botany, Shanghai University of Traditional Chinese Medicine. The authentication was performed by comparison with appropriate voucher specimens at the herbaria and by performing both physical and chemical properties identifications according to The Pharmacopoeia of People's Republic of China (2005 edition).

Acute toxicity assay of TERC

The oral administration of TERC caused death of the mice in a dose dependent manner. The oral administration of 4.00, 3.20, 2.56, 2.05 and 1.64 g/kg of TERC caused death of 85%, 80%, 75%, 30% and 25% of the mice, respectively. The LD₅₀ value was 2.95 g/kg; the lower and upper limits of 95% confidence were 2.60 and 3.51 g/kg, respectively. The LD₅₀ value was equal to 7.95 g/kg of the herb per kilogram of the body weight according to the yield of the extract (37.1%).

Comparison of the acute toxicity of TERC and ARP

The oral administration of ARP

Discussions and conclusions

The results showed that the acute toxicity of ARP was much more toxic than TERC. This suggested that the toxicity of Rhizoma Coptidis might be caused mainly by the alkaloids. We proved this assumption by the following experiments.

Firstly, four Rhizoma Coptidis alkaloids were detected in the brains, hearts and lungs. A common assumption was that berberine is very difficult to be absorbed because of its relative low plasma concentration. However, we ascertained that berberine as well as the other

Acknowledgements

This work was financially supported by the Construction Program for Innovative Research Team in Shanghai Institutions of Higher Education, the National Natural Science Foundation of China (No. 30873231) and the Shanghai Special Fund to Select and Train the Outstanding Young College Teachers (No. 2006A-35).

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Identification of the toxic constituents in Rhizoma Coptidis

Abstract

Aim of the study

Materials and methods

Results

Conclusions

Graphical abstract

Introduction

Section snippets

Materials

Acute toxicity assay of TERC

Comparison of the acute toxicity of TERC and ARP

Discussions and conclusions

Acknowledgements

Toxicology In Vitro

Atherosclerosis

Journal of Clinical Virology

Atherosclerosis

Journal of Pharmaceutical Sciences

Biochemical and Biophysical Research Communications

Toxicology In Vitro

Toxicology In Vitro

Biochemical Pharmacology

Life Sciences

Journal of Biochemical and Biophysical Methods

Analysis of major alkaloids in Rhizoma coptidis by capillary electrophoresis-electrospray-time of flight mass spectrometry with different background electrolytes

Electrophoresis

Optimization of antibacterial activity by Gold-Thread (Coptidis Rhizoma Franch) against Streptococcus mutans using evolutionary operation-factorial design technique

Journal of Microbiology and Biotechnology

Screening of toxic compounds in mammalian cell cultures

Annals of the New York Academy of Sciences

Berberine suppresses proinflammatory responses through AMPK activation in macrophages

American Journal of Physiology. Endocrinology and Metabolism