Journal of Ethnopharmacology 193 (2016) 566–573

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Journal of Ethnopharmacology

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Review Toxic polyacetylenes in the genus () – Distribution, toxicity, molecular mechanism and analysis crossmark ⁎ Meiyu Lin, Weidong Zhang, Juan Su

School of Pharmacy, Second Military Medical University, Shanghai 200433, China

ARTICLE INFO ABSTRACT

Keywords: Ethnopharmacological relevance: The genus Bupleurum includes approximately 200 species that are widely Polyacetylenes distributed in the Northern Hemisphere, Eurasia and North Africa. Certain species of this genus have long been Bupleurum used as antiphlogistic, antipyretic and analgesic agents in traditional folk medicine. As described in the Chinese Toxicity Pharmacopoeia, the roots of DC. and B. scorzonerifolium Willd. are the herbal materials Mechanism that compose Chaihu (Radix Bupleuri), a well-known TCM herb. Quality analyses Aim of the review: This review aims to provide up-to-date and comprehensive information regarding the distribution, toxicity, molecular mechanism and relatively new methods for the qualitative and quantitative determination of polyacetylenes in different Bupleurum species. Method: The information needed for this paper were sourced from publishing sites such as Elsevier, science Direct, PubMed; electronic search engines such as Scopus and Web of Science, Google scholar; other scientific database sites for chemicals such as ChemSpider, PubChem, SciFinder, and also from on line books. Results: Polyacetylenes, which are widely distributed in genus Bupleurum of the Apiaceae family, have high toxicity. Among polyacetylenes, bupleurotoxin, acetylbupleurotoxin and have strong neurotoxi- city. Through previous research, it was found that the toxicity of Bupleurum polyacetylenes manifested as epileptic seizures, with the target of toxicity being the brain. The neurotoxicity of polyacetylenes exhibits a relationship with the γ-aminobutyric acid (GABA) receptor pathway, and polyacetylenes have been shown to

inhibit GABA-induced currents (IGABA) in a competitive manner. Conclusions: The of genus Bupleurum have been used in traditional medicine for thousands of years. However, certain species of this genus are poisonous, and it was attributed to the high content of polyacetylenes. The present review indicates that certain polyacetylenes in the genus Bupleurum have highly neurotoxic effects. The major challenge with regard to toxic polyacetylenes is to test their neurotoxic effects in vivo as well as in further preclinical studies, which will require large amounts of purified polyacetylenes. More reference substances should be prepared, and sophisticated analytical technologies should be developed to comprehen- sively assess the quality of Radix Bupleuri herbs. These investigations will be helpful for further utilization of the plants of genus Bupleurum.

1. Introduction undesirable due to their toxic properties. Certain polyacetylenes are known to be potent skin sensitizers and have a pronounced selective Polyacetylenes are widely distributed among the families cytotoxic activity against cancer cells (Machado et al., 2002; Murdoch Umbelliferae, Araliaceae, and Asteraceae. They form a distinct group and Dempster, 2000; Ahn and Kim, 1988; Matsunaga et al., 1989; of relatively reactive natural products, and more than 1400 different Matsunaga et al., 1990). Some studies have focused on the neurotoxi- polyacetylenes and related compounds have been isolated from plants city of polyacetylenes. Polyacetylenes isolated from the roots of poison (Christensen and Brandt, 2006). Some of the polyacetylenes isolated hemlock and Oenanthe fistulosa have high neurotoxicity (Anet et al., from food plants such as carrot, celery and parsley have received much 1953; Wittstock et al., 1995; Uwai et al., 2000), and some species of the attention due to their extensive physiological impact, including anti- genus of Bupleurum have also been shown to be neurotoxic at high inflammatory, antibacterial, antifungal, anticancer, antiplatelet aggre- concentrations (Vetter, 2004; Wittstock et al., 1997). gation activities. Still, some polyacetylenes have been considered The genus Bupleurum (Family: Apiaceae) includes approximately

⁎ Corresponding author. E-mail addresses: [email protected] (W. Zhang), [email protected] (J. Su). http://dx.doi.org/10.1016/j.jep.2016.09.052 Received 12 July 2016; Received in revised form 28 September 2016; Accepted 28 September 2016 Available online 30 September 2016 0378-8741/ © 2016 Elsevier Ireland Ltd. All rights reserved. M. Lin et al. Journal of Ethnopharmacology 193 (2016) 566–573

200 species, a number of which have been pharmaceutically used for authentic Chaihu samples (Huang et al., 2011). These results indicated thousands of years, mainly in Asia and Europe (Pan, 2006; Chang and that the distribution and the contents of the tested polyacetylenes in But, 1987). Preparations containing the roots of Bupleurum species different Bupleurum species vary significantly, and they were much have been prescribed for more than 2000 years in China, where the first higher in the B. longiradiatum than in the other samples. The toxicity record about their use appeared in Shen-Nong's Herbal (Yao et al., of B. longiradiatum could readily be distinguished from other medic- 2013). Certain Bupleurum species have been used in traditional inal Bupleurum species based on polyacetylene profile. medicine in Asia for the treatment of the common cold, inflammatory Thus, in order to ensure its safety clinical use, the commercially disorders, hepatitis, cancer and fever. Radix Bupleuri, with the Chinese prepared crude drugs of Chaihu in herb markets should be labeled name Chaihu, has been widely used to treat influenza, fever, inflam- before circulation, and the qualitative and quantitative determination mation, malaria as well as menstrual disorders and is recorded as being of polyacetylenes is useful for the assessment of the toxicity of crude prepared from the roots of Bupleurum Chinense and B. scorzoner- herbal material. ifolium (Family: Apiaceae) in the Chinese Pharmacopoeia (Ikegami et al., 2006; National Commission of Chinese Pharmacopoeia, 2015). 3. Toxicity of polyacetylenes However, there are at least ten species of the genus Bupleurum (Apiaceae) that are also used locally under the name Chaihu (Pan, In general, polyacetylenes which distributed in B. falcatum, B. 2006). Even worse, B. longiradiatum, widely distributed in north- spinosum, B. salicifolium, and B. acutifolium exhibit antibacterial, eastern mainland China, is a poisonous that has been found in anti-inflammatory, antifungal, and anti-platelet aggregatory effects herb markets (National Commission of Chinese Pharmacopoeia, 2015). (Alanko et al., 1994; Kobaisy et al., 1997; Fujimoto et al., 1998). The misuse of B. longiradiatum as Chaihu has caused several cases of However, some polyacetylenes have been considered undesirable in human poisoning, and at least three people died after administration of Bupleurum species to their toxicant properties. Polyacetylenes are this plant, showing symptoms such as serious nausea, vomiting, known to be neurotoxic in high concentrations (Anet et al., 1953), and twitching, and opisthotonus (Pan, 2006). they have also been shown to have a pronounced selective cytotoxic The investigation of the acute toxicity of B. longiradiatum extract activity against cancer cells (Matsunaga et al., 1990). uncovered strong toxicity in mice, which was attributed to its high Radix Bupleuri represents one of the most successful and widely content of polyacetylenes (Chen, 1981; You et al., 2002). This review used herbal drugs in Asia for treatment of many diseases over the past highlights the present state of knowledge on polyacetylenes in the 2000 years. There are many adulterants of Radix Bupleuri in the herb genus Bupleurum, including their distribution, toxicity, mechanism, market (Yao, 2006; Li, et al., 2012). Among them, B. longiradiatum is quality analysis and possible relevance to human health. a representative poisonous plant which contains high concentrations of polyacetylenes. And B. longiradiatum is prohibited to be used in herb 2. Distribution of polyacetylenes in the genus Bupleurum market according to the Chinese Pharmacopoeia (2015 Edition). The misuse of B. longiradiatum as Chaihu had caused several cases of The genus Bupleurum (Family: Apiaceae) includes approximately human poisoning, and at least three people died after administration of 200 species that are widely distributed in the Northern Hemisphere, this plant, which showed symptoms such as serious nausea, vomiting, Eurasia and North Africa (Su et al., 1998). In addition to the South twitching, opisthotonus, and so on (Pan, 2006; Zhao et al., 1987). The Bupleurum and the North Bupleurum described in the Chinese surveys on B. longiradiatum demonstrated that polyacetylenes were of Pharmacopoeia, there are also other species of this genus, such as B. particular abundance in root of this plant and proved to be responsible smithivar. parvifolium, B. marginatum vat. stenophyllum, B. margin- for the toxicity of B. longiradiatum (Huang et al., 2009). As major toxic atum, B. bicaule, B.smithii, B. longiradiatum Turcz, and others. ingredients of B. longiradiatum, bupleurotoxin (1), acetylbupleurotox- Certain species of this genus, such as B. chinense, B. scorzonerifolium in (2) and oenanthotoxin (5) attracted the most attention. and B. falcatum, have been used as antiphlogistic, antipyretic, and Investigation on the acute toxicity of B. longiradiatum showed it is analgesic agents in traditional folk medicine preparations (Chang and capable of poisoning mice, with an LD50 value of 3.0 g/kg (Pan, 2006). But, 1987). However, B. longiradiatum, widely distributed in north- In order to study the toxicity of B. longiradiatum and its constituents, eastern mainland China, is a poisonous plant that has sometimes been Huang et al. (2011) examined the acute toxicity of B. longiradiatum found in herb markets (Pan, 2006). As the characteristic components of extract and its fractions. The results indicated that the CH2Cl2 fraction the genus Bupleurum, polyacetylenes are widely distributed in some and the ethanol extract exhibited much higher toxicity, with LD50 species of this genus, such as B. longiradiatum, B. falcatum (Uwai values of 37.5 mg/kg (95% CI: 32.8–42.9 mg/kg) and 77.7 mg/kg et al., 2000), B. acutifolium (Bohlmann et al., 1973), B. spinosum (95% CI: 67.7–89.3 mg/kg), respectively, and toxicity was correlated (Huang et al., 2009), B. ranunculoides (Barrero et al., 1999; Bohlmann with the high content of polyacetylenes in this herb (You et al., 2002). and Rode, 2006), B. smithii, and B. bicaule. Altogether, approximately Histopathological results demonstrated that bupleurotoxin can induce 27 polyacetylenes have been isolated from the genus Bupleurum internal brain injury in healthy mice. In addition, there were no (Table 1). obvious lesions in the heart, liver, or kidney, which suggests that the A total of 27 Bupleurum samples were examined by High-perfor- nervous system may be a specific target of bupleurotoxin (Zhang et al., mance liquid chromatography method coupled with diode array 2014). Based on the above results and the available data in the detector and mass spectrometry (HPLC-DAD-MS) in a previous study, literature, polyacetylenes are suggested to be neurotoxic constituents which revealed great variety in the distribution and contents of that are responsible for the toxicity of B. longiradiatum. polyacetylenes across species (Huang et al., 2011). It was found that Another investigation on the acute toxicity of B. longiradiatum polyacetylenes 1–6 and 18–27 were the main ingredients in B. long- extracts also found severe toxicity in mice, which was attributed to its iradiatum, and polyacetylenes 7–17 were the major ingredients in B. high content of polyacetylenes, among which bupleurotoxin (1) and acutifolium, B. ranunculoides, B. falcatum and B. spinosum. Only a acetylbupleurotoxin (2) are extremely poisonous with LD50 values of few types of polyacetylenes were identified in B. smithii, B. smithii var. 3.03 mg/kg and 3.13 mg/kg, respectively (Zhao et al., 1987); bupleur- parvifolium, B. bicaule and B.angustissimum. otoxin and acetylbupleurotoxin at these doses caused violent convul- Among the tested polyacetylenes, bupleurotoxin (1) and acetylbu- sions and death. Oenanthotoxin (5), which is a toxin extracted from pleurotoxin (2) were determined as the main compounds at concentra- water-hemlock and other plants of the genus Oenanthe, was also tions of about 2.25–0.18 mg/g and 3.91–0.02 mg/g respectively. isolated from B. longiradiatum. This toxin is extremely poisonous and Comparatively, they were not detectable from the other species of also causes violent convulsions and death (Anet et al., 1953). Bupleurum genus. And very few polyacetylenes were detected in Oenanthetol (6), which was found in B. longiradiatum, B. acutifolium,

567 M. Lin et al. Journal of Ethnopharmacology 193 (2016) 566–573

Table 1 Polyacetylenes isolated from species of the genus Bupleurum.

No. Chemical component Structure Species References

1 Bupleurotoxin B. longiradiatum Huang et al. (2009) Zhao et al. (1987) B. OH

OH

2 Acetylbupleurotoxin B. longiradiatum Huang et al. (2009) Zhao et al. (1987) B. OH

OAc

3 Bupleuonol B. longiradiatum Huang et al. (2009) Zhao et al. (1987) B. OH

O

4 Bupleurynol B.longiradiatum Barrero et al. (1999) B.longir B.acutifolium Zhao et al. (1987)

OH B.acutifol

5 Oenanthotoxin B. longiradiatum Zhao et al. (1987) OH B.

OH

6 Oenanthetol/(2E,8E,10E)-Heptadeca-2,8,10-triene- B. longiradiatum Zhao et al. (1987) 4,6-diyn-1-ol B. B.acutifolium Barrero et al. (1999) OH B.ranunculoides Bohlmann et al. (1971) B.acutifol

B.ranunc

7(2E,8E,10E)-Heptadeca-2,8,10-triene-4,6-diyn-1-yl B.acutifolium Wu (2005) acetate B.acutifol OAc B.ranunculoides Bohlmann et al. (1973) Barrero et al. (1999) B.ranunc Bohlmann and Rode (2006)

8(2E,8E,10E)-Heptadeca-2,8,10-triene-4,6-diyn-1-al B.acutifolium Huang et al. (2009) B.acutifol B.ranunculoides Barrero et al. (1999) O Bohlmann and Rode (2006) H B.ranunc

9(2E,9Z)-Octadeca-2,9-diene-4,6-diyne-1,18-diol B.acutifolium Huang et al. (2009) (continued on next page)

568 M. Lin et al. Journal of Ethnopharmacology 193 (2016) 566–573

Table 1 (continued)

No. Chemical component Structure Species References

Huang B.acutifol OH

HO

10 (2E,9Z)-18-Hydroxyoctadeca-2,9-diene-4,6-diyn-1-yl B.acutifolium Bohlmann et al. (1973) Bohlm acetate B.acutifol OAc

HO

11 (2E,9Z)-1-Hydroxyoctadeca-2,9-diene-4,6-diyn-18-yl B.acutifolium Huang et al. (2009) Huang acetate B.acutifol OH

AcO

12 (2Z,8E)-Pentadeca-2,8-diene-4,6-diyne-1,10-diol B.falcatum Morita et al. (1991)

B.falcatu OH

OH

13 10-Hydroxy-(2Z,8E)-diene-4,6-diyne-pentadecanal B.falcatum Morita et al. (1991) O

H B.falcatu

OH

14 (2Z,9Z)-Pentadeca-2,9-diene-4,6-diyn-1-ol B.falcatum Morita et al. (1991) B.falcatu OH

15 (2Z,9Z)-Pentadeca-2,9-diene-4,6-diyn-1-yl-acetate B.falcatum Morita et al. (1991) B.falcatu OAc

16 (5E,7E)-Pentadeca-5,7-diene-9,11,13-triyn-2-one B. spinosum Barrero et al. (1998)

B.

O

17 (8Z)-Decene-4,6-diyn-1-ol B. spinosum Barrero et al. (1998) B. OH

18 (2E,4E,8E,10E)-Heptadecatetraene-6-yn-1-yl acetate B. longiradiatum Huang et al. (2009) OAc B. longirad iatum

(continued on next page)

569 M. Lin et al. Journal of Ethnopharmacology 193 (2016) 566–573

Table 1 (continued)

No. Chemical component Structure Species References

19 (2E,4E,9Z)-Heptadecatriene-6-yn-1-yl acetate B. longiradiatum Huang et al. (2009) OAc B. longirad iatum

20 (2E,4E,9Z)-Octadecatriene-6-yne-1,18-diyl diacetate B. longiradiatum Huang et al. (2009) OAc B. longirad AcO iatum

21 (2E,4E,9Z)−1-Hydroxyoctadecatriene-6-yn-18-yl B. longiradiatum Huang et al. (2009) acetate OH B. longirad AcO iatum

22 (2E,4E,9Z)-Octadecatriene-6-yne-1,18-diol B. longiradiatum Huang et al. (2009) OH B. longirad HO iatum

23 (2Z,8E,10E)-Pentadecatriene-4,6-diyn-1-ol B. longiradiatum Huang et al. (2009) B. OH longirad iatum

24 (2Z,9Z)-Octadecadiene-4,6-diyne-1,18-diol B. longiradiatum Huang et al. (2009) B.

OH HO longirad iatum

25 (2Z,8E,10E)-14S-Hydroxyheptadecatriene-4,6-diyn- B. longiradiatum Huang et al. (2009) 1-yl acetate B. OAc longirad OH iatum

26 (2Z,8Z,10E)-Heptadecatriene-4,6-diyne-1,14-diol B. longiradiatum Huang et al. (2009)

OH B. longirad OH iatum

27 (2Z,8Z,10E)-1-Hydroxyheptadecatriene-4,6-diyn-14- B. longiradiatum Huang et al. (2009) yl acetate (continued on next page)

570 M. Lin et al. Journal of Ethnopharmacology 193 (2016) 566–573

Table 1 (continued)

No. Chemical component Structure Species References

OH B. longirad OAc iatum

and B. ranunculoides (Table1), is structurally closely related to OH oenanthotoxin (5); however, a comparative study on the mechanism terminal hydroxyl group of convulsive action (Wittstock et al., 1997) indicated that the toxic required for activity effects require a very specific distance between the two OH-groups of OH allylic the molecule, indicating that oenanthetol (6) does not have similar polyunsaturated moiety toxic effects to oenanthotoxin (5). critical for activity E-geometry is required: The acetylenes oenanthotoxin and isolated from water- a longer conjugation system is more eff ective hemlock (Cicuta virosa L.), hemlock water dropwort (Oenanthe crocata L.), and from the spotted water hemlock (C. maculata L.) Fig. 1. Structural requirements for toxicity. are extremely poisonous causing violent convulsions and death (Anet et al., 1953). Polyacetylenes of the falcarinol type such as falcarinol and longiradiatum. Bupleurotoxin has been shown to induce internal brain falcarindiol, are widely distributed in the Apiaceae (Hansen and Boll, injury in healthy mice based on histopathological observations. A 1986). The neurotoxicity of falcarinol has been demonstrated poiso- comprehensive and holistic approach based on metabolomics was nous with LD values of 100 mg/kg whereas no acute effects have been 50 performed to reveal urine metabolic changes and identify potential demonstrated for the related falcarindiol (Crosby and Aharonson, biomarkers of toxicity induced by bupleurotoxin (Zhang et al., 2015). 1967). The type of neurotoxic symptoms produced by falcarinol is This study identified 11 potential biomarkers of bupleurotoxin toxicity similar to those of oenanthotoxin and cicutoxin, although a much in urine and provided new insights into the toxicity of bupleurotoxin in higher dose is required, and no poisoning of mammals has been vivo. This investigation revealed an effect of bupleurotoxin on both reported from voluntary ingestion of natural sources. fatty acid metabolism and the cell's ability to provide energy through Polyacetylenes have also been shown to have a pronounced the mitochondrial beta-oxidation pathway. These findings suggested selective cytotoxic activity against cancer cells (Matsunaga et al., that oxidative stress, mitochondrial dysfunction and neuronal inflam- 1990). It has been reported that certain polyacetylenes possess mation were the underlying mechanisms behind bupleurotoxin-in- cytotoxic activity against cancer cell lines (Siddiq and Dembitsky, duced toxicity. 2008). Bioassay tests showed that (2Z,8E,10E)-14S-hydroxy-heptade- Zhang et al. (2014) used a metabolomics approach to screen serum catriene-4,6-diyn-1-yl acetate (25) and mTPA esters of bupleurotoxin samples from control and bupleurotoxin-treated groups . First, male (1) were cytotoxic compounds with IC values of 9.4 and 4.9 μM, 50 mice were intragastrically administered 2.5 mg/kg of bodyweight respectively, and they also possessed this cytotoxic activity against bupleurotoxin once a day for seven consecutive days. The histopatho- cancer cell lines. Compounds Acetylbupleurotoxin (2), Bupleuronol (3), logical results showed that bupleurotoxin could induce severe morpho- Bupleurynol (4), (2Z,9Z)-Pentadeca-2,9-diene-4,6-diyn-1-ol (14), (2E, logical damages in the brain hippocampus. Then used metabolomics 4E,8E,10E)-Heptadecatetraene-6-yn-1-yl acetate (18), (2E,4E,9Z)- approaches to screen serum samples from the control and bupleur- Heptadecatriene-6-yn-1-yl acetate (19), and (2Z,8E,10E)- otoxin-treated groups. 17 toxicity biomarkers were identified using Pentadecatriene-4,6-diyn-1-ol(23) exhibited IC values in the 11– 50 global metabolomics, four of them were then verified by targeted 18 μM range (Huang et al., 2009). Acetylbupleurotoxin (2) and metabolomics. Bioinformatics analysis using the Ingenuity Pathway bupleurotoxin (1) are found to completely inhibit the tube-like forma- Analysis (IPA) software found a strong correlation between the GABA tion of human umbilical venous endothelial cells, but they do not receptor and these metabolites. exhibit antitumor activity in BDF1 mice bearing Lewis lung carcinoma That the altered metabolites belonged to the GABA receptor cells (You et al., 2002). signaling pathway may be a distinctive feature of bupleurotoxin toxicity The toxicity of polyacetylenes has a relationship with their chemical and reflect the abnormal GABA receptor signaling status in mice after structure. In order to understand the properties of polyacetylene bupleurotoxin treatment. On the basis of these results, a validation test compounds, the structure-activity relationships involved in the acute using a rat hippocampal neuron cell line was performed, and the results toxicity of polyacetylenes in mice were investigated. One study confirmed that bupleurotoxin inhibited GABA-induced currents (I ) demonstrated that the length of the π-bond conjugation system and GABA in a competitive manner. These results were also verified in the the geometry of the double bonds are critical for toxicity. Moreover, the metabolomics study on the toxicity of polyacetylenes which illustrated terminal O-functional group and the allylic alcohol are essential for the molecular mechanism of the toxicity of polyacetylenes (Zhang, toxicity (Anet et al., 1953; Starreveld and Hope, 1975; Ohta et al., 2014). 1999; Uwai et al., 1999)(Fig.1). Uwai et al. examined the binding of the polyacetylenic compounds − to GABA-gated Cl channels of GABAA receptors in rat brain cortex. 4. Molecular mechanism underlying the toxicity of Polyacetylene are believed to act like biological analogues polyacetylenes of the sesquiterpene lactone , binding to the site

of GABAA receptors and causing death by inducing convulsions and Certain polyacetylenes that are found in high concentrations in respiratory paralysis. The potency in inhibition of the specific binding Bupleurum species are known to be neurotoxic, among which bupleur- of the noncompetitive GABA antagonist, [3H]EBOB, to GABA-gated Cl− otoxin (1) is a representative component that can be isolated from B. channels of GABA receptors in rat brain cortex was found to be

571 M. Lin et al. Journal of Ethnopharmacology 193 (2016) 566–573

lished an HPLC-DAD-MS method for the simultaneous determination of nine polyacetylenes in the roots of B. longiradiatum. This work focuses on developing a simple, effective and reliable method to analyze polyacetylenes in B. longiradiatum and related Bupleurum species. A high-performance liquid chromatography (HPLC) method coupled with diode array detection (DAD) and mass spectrometry (MS) was established to the qualitative and quantitative determination of poly- acetylenes (Huang et al., 2011). Liu et al. developed an ultra-performance liquid chromatography coupled with photodiode array detector (UPLC-PDA) method for the determination of four polyacetylenes from Radix Bupleuri. The analy-

sis was performed on a Waters BEH C18 column (1.7 µm, 100×2.1 mm i.d.) using a gradient system of acetonitrile and 0.03% trifluoroacetic acid water, with a detector wavelength of 315 nm (Liu et al., 2015). Zhao et al. established a double wavelength TLC scanning method for Fig. 2. Identification of the GABA receptor signaling pathway by IPA. the determination of bupleurotoxin and acetylbupleurotoxin in B. longiradiatum. The results showed that the content measured was correlated with acute toxicity, indicating that the ability to bind to these 0.49% and 0.22% for bupleurotoxin and acetylbupleurotoxin respec- channels plays an important role in the acute toxicity of these tively. According to the integral value, the RSD measured was 1.4% and compounds (Uwai et al., 2000). 2.6% respectively (Zhao et al., 1993). Appendino et al. found that both oenanthotoxin and dihydrooe- Up until now, the quality control of Chaihu and related Bupleurum nanthotoxin could potently block the GABAergic responses in neuronal species mainly emphasized on the determination of bioactive saponin compounds (Tian et al., 2009; Huang, et al., 2009; Liau, et al., 2007; cell cultures, with an EC50 value in the low micromolar range for both compounds (Appendino et al., 2009), while others showed that it Bao, et al., 2004; Hsieh and Huang, 1997), but not considered the scrutiny of various toxic ingredients in Bupleurum. As to the clinic induced an open channel block and modulated GABAA receptors (Wyrembek et al., 2010, 2012). In addition, oenanthotoxin has also security, it is crucial to determine the toxic compounds, and to confirm been reported to dramatically affect cationic currents in excitable the absence or presence of toxic ingredients in any drug products. A + 2+ systematic study on the authentication and assessment on related membranes. It has been found to change [K ]0 and [Ca ]0 in oenanthotoxin-induced epilepsy (Louvel et al., 1982; Louvel and Bupleurum species should be well defined. Analysis of the major Heinemann, 1983; Dubois and Schneider, 1981). Next, the mechanism polyacetylenes, such as bupleurotoxin, acetylbupleurotoxin and bu- behind oenanthotoxin toxicity has been attributed to how oenantho- pleurynol should be applied to the quality control of Chaihu. toxin affected receptors in cultured hippocampal neurons by exerting a strong and complex effect including an open channel block and allosteric decreases in the binding and desensitization rates 6. Conclusions (Wyrembek et al., 2010)(Fig. 2). In this review, we have summarized the distribution, toxicity, molecular mechanism behind the toxicity and methods to ensure 5. Analyses of polyacetylenes in Bupleurum species quality analysis of polyacetylenes in the genus Bupleurum. Polyacetylenes, which are widely distributed in genus Bupleurum of Among the medicinal plants of the genus Bupleurum, the roots of the Apiaceae family, have high toxicity. Among polyacetylenes, bu- Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. are pleurotoxin, acetylbupleurotoxin and oenanthotoxin have strong neu- the herbal materials that compose Chaihu (Radix Bupleuri). Because of rotoxicity. Through previous research, it was found that the toxicity of the complex constituents in Radix Bupleuri, it is necessary to study the Bupleurum polyacetylenes manifested as epileptic seizures, with the quality control and quantitative analysis of the active components in it. target of toxicity being the brain. The neurotoxicity of polyacetylenes Due to its various pharmacological properties, saikosaponins have been exhibits a relationship with the γ-aminobutyric acid (GABA) receptor regarded as the important quality indexes. In the Chinese pathway, and polyacetylenes have been shown to inhibit GABA- Pharmacopoeia (2015 Edition), saikosaponin is used as an indicator induced currents (I ) in a competitive manner. to control the quality of Radix Bupleuri, and the saikosaponin content GABA Consequently, the plants of genus Bupleurum have been used in in the roots should be more than 0.30% as determined by HPLC traditional medicine for thousands of years. However, certain species of analysis. But there is no method for the determination of polyacety- this genus are poisonous, and it was attributed to the high content of lenes in the Chinese Pharmacopoeia. polyacetylenes. The present review indicates that certain polyacety- Besides Bupleurum chinense DC. and Bupleurum scorzonerifolium lenes in the genus Bupleurum have highly neurotoxic effects. The major Willd., there are at least ten species of the genus Bupleurum that are challenge with regard to toxic polyacetylenes is to test their neurotoxic also used under the name of Chaihu. Among them, some species are effects in vivo as well as in further preclinical studies, which will require poisonous plants but sometimes have been found in herb markets large amounts of purified polyacetylenes. More reference substances locally (Pan, 2006). The surveys on B. longiradiatum demonstrated should be prepared, and sophisticated analytical technologies should that polyacetylenes were of particular abundance in root of this plant be developed to comprehensively assess the quality of Radix Bupleuri and proved to be responsible for the toxicity of B. longiradiatum herbs. These investigations will be helpful for further utilization of the (Huang et al., 2009). So, it is necessary that polyacetylenes should be plants of genus Bupleurum. analyzed. HPLC-DAD-MS shows its wide application in TCM (Traditional Chinese Medicine) research because the combination of DAD and MS can provide online UV and MS information at the same time for each Acknowledgments individual peak in a chromatogram, while HPLC-UV is a convenient and effective method to control the quality of TCM for its rapid This study was supported by National Natural Science Foundation separation and quantitation (Liang et al., 2009). Huang et al. estab- of China (NSFC 81402823).

572 M. Lin et al. Journal of Ethnopharmacology 193 (2016) 566–573

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