Johanna Michl1, Olusheyi Bello1, Geoffrey C. Kite2, Monique S. J. Simmonds2 and Michael Heinrich1*
1Research Cluster Biodiversity and Medicines, UCL School of Pharmacy, London, UK
2Royal Botanic Gardens, Richmond, UK
Species of Asarum are used in traditional Chinese medicine and, similar to members of the genus Aristolochia, they contain aristolochic acid analogs (AAAs). These compounds are known for their nephrotoxic and carcinogenic effects. So far, the phytochemistry and nephrotoxicity of species of Asarum is not well studied. A high-resolution LC-MS-based metabolomic approach was used to study the phytochemical variation in medicinally used Asarum species. The cytotoxicity of the samples was assessed using human kidney (HK-2) cells. The majority of samples contained potentially nephrotoxic AAAs, including 9-methoxy aristolactam (AL) IV, AL I, and AL IV. These compounds were present in methanol as well as water extracts. AAAs were detected in all parts of the plant. The majority of the extracts were not cytotoxic to HK-2 cells at the doses tested. However, other mechanisms relating to aristolochic acid nephropathy and cancer development, such as DNA adduct formation may occur. The results of this study provide a model for assessing lesser-known plant species for toxicity.
Introduction
Species of the genus Asarum are used as herbal medicines in many parts of the world, including Europe and Asia. The Chinese Pharmacopeia lists the roots and rhizomes of Asarum heteropoides f. mandshuricum (Maxim.) Kitag, and Asarum sieboldii Miq. under the Pin Yin name Xixin (Achenbach and Fischer, 1997). In Europe Asarum europaeum L. is used in homeopathic tinctures (Nitzsche et al., 2013) and in Canada and the USA Asarum canadense L. was used by Native Americans (Moermon, 2017).
Like the related genus Aristolochia (which is also listed in the Chinese Pharmacopeia), Asarum contain aristolochic acids and aristolactams (Mix et al., 1982; Kumar et al., 2003). These nitrophenanthrene derivates have nephrotoxic and carcinogenic effects (Michl et al., 2014). Species of Aristolochia have become a key concern in healthcare as they are associated with aristolochic acid nephropathy (AAN), a renal fibrosis often associated with upper urothelial cancer (UUC; Chen et al., 2012). It is estimated that in China alone 100 million people may be at risk of developing AAN (Hu et al., 2004; Grollman, 2013).
Species of Asarum are generally considered to be less toxic than species of Aristolochia. However, a few cases of Asarum-related AAN have been reported. In one case report a male patient displayed subacute renal failure after ingesting a herbal powder containing Xixin (Yang et al., 2006). A case of acute poisoning due to the intake of A. europaeum has been reported in Switzerland (Jaspersen-Schib et al., 1996). Surprisingly, only eight cases of Asarum-related AAN have been reported in the last 45 years (Kim et al., 2013). Like Aristolochia-related AAN, it is likely that health practitioners failed to identify the link between nephropathy or tumor development and the exposure to these plants.
Aristolochic acid I (AA I) and aristolochic acid II (AA II) are considered to be the cause of these nephrotoxic and carcinogenic effects (Nortier et al., 2000; Balachandran et al., 2005; Jelakovic et al., 2012). After reductive metabolic activation into aristolactams (ALs), AA I and AA II form DNA adducts, which were found in renal tissues of patients. A large number of in vitro and in vivo studies showed that AA I and AA II are toxic (Mengs, 1988; Arlt et al., 2011; Yang et al., 2011; Michl et al., 2014). However, they are not necessarily the only (or most potent) toxins present in Aristolochia and related genera (Michl et al., 2016). At least 178 aristolochic acid analogs (AAAs) exist, many of which are aristolactams. It is unclear whether these compounds are also able to form DNA adducts. Their possible implications in AAN may have been overlooked (Michl et al., 2014). Apart from AA I and AA II, other compounds may contribute to processes that lead to renal damage (Li et al., 2004; Wen et al., 2006) and carcinogenesis.
Species of Asarum generally contain lower amounts of AA I and AA II than Aristolochia species (Hashimoto et al., 1999; Chan et al., 2003, 2006; Yuan et al., 2007). Yet, high amounts of AA I (3376.9 ng/mg) were reported in Asarum crispulatum C.Y. Cheng and C.S. Yang (Jong et al., 2003). According to Zhao et al. (2008) aerial parts of Xixin herbs contained higher levels of AA I than the roots. Methanol extracts typically contained more AA I than water extracts. A second study by Hsu et al. (2009) found that the amounts of AA I in leaves were the highest followed by petioles, rhizomes and roots.
While a number of studies assessed the amounts of AA I and AA II in Asarum spp., little is known about the effects of the entire (small molecule) metabolome and specifically other AAAs. For example, although other compounds, such as AL I are often found in higher amounts in Asarum than in Aristolochia (Yuan et al., 2008), the Chinese Pharmacopoeia still lists roots and rhizomes of Asarum for medicinal use. Furthermore, only the decoction of the root portion is recommended for usage. However, it is questionable as to whether current recommendations for the medicinal uses of Asarum species are justified.
The aim of this work is to assess the metabolomic profile and in vitro toxicity of medicinally used species of Asarum and to evaluate whether current recommendations for their use are appropriate. Therefore, we utilized a systems biology approach to establish the full range of AAAs in a series of Asarum species. We carried out a LC-MS-based metabolomic study to compare the secondary metabolites of Asarum samples originating from different species, different plant parts, as well as obtained through different extraction techniques. We assessed the cytotoxicity of these extracts in HK-2 kidney cells and studied the relationship between the plants’ metabolic profiles and their in vitro toxicity using statistical approaches. In a wider context, the current work can be used as a model for assessing toxicity of medicinal plant species, and for elucidating bioactive principles of medicinal plants.
Discussion
Asarum species have been used as medicinal plants in China and in other parts of the world. However, so far little is known about their content in aristolochic acids and aristolactams or their potential kidney toxicity. A comprehensive metabolomic approach has shown that the majority of Asarum samples contain AAAs considered to be nephrotoxic.
In TCM, plants known to contain toxic compounds are often regarded as safer after preparation of the plant material in a specific way, or only those plant parts known not to contain the toxins are used. Furthermore, monographs on species in national pharmacopeias require time to change and often do not take into account recent advances in the knowledge about the chemistry of the species. This could result in misleading recommendations about the use of some species with potential hazards for public health. The plant genus Asarum is one example of this, especially since its true level of usage is poorly known (e.g., in China and Southern Europe). In addition to changing guidelines in national pharmacopeias, more awareness needs to be raised about potential health risks associated with the use of herbal medicine. This will in time lead to changes in the traditional practices.
Previous reports have shown that levels of aristolochic acids were low in decoction of roots and rhizomes of Herba Asari (Zhao et al., 2008) thus it was retained in the Chinese Pharmacopoeia. However, in this study, no significant difference in the levels of AA I was detected in aerial parts of Asarum spp. compared to root samples. Relative levels of AL I were found to be even higher in root samples compared to aerial parts, and AL I was also detected in root decoctions. Little is known about the toxicity and carcinogenicity of AL I.
Although most Xixin samples included in this study contained potentially nephrotoxic AAAs, some samples did not contain detectable amounts of these compounds. This could indicate that Xixin samples purchased from markets may not originate from Asarum sp., and were either misidentified or replaced with other medicinal plants. The lack of detectable AAAs could also be due to natural variation (genetic, seasonal or ecological) and requires further investigation. Most samples were non-cytotoxic to kidney cells in vitro. Interestingly, no correlation was found between the amounts of AA I and their toxicity. However, other mechanisms relating to aristolochic acid nephropathy, such as DNA adduct formation may occur and deserve further research.
The present study has important implications on whether and how the genus Asarum should be used medicinally in the future. In contrast to previous studies, where only AA I and AA II were taken into account, we detected other AAAs in root and rhizome samples of Asarum. Since little is known about the toxicity of these compounds, we cannot conclude that root and rhizome samples are safe to use. The study also demonstrates that systematic assessment of a group of species’ metabolomic profile can provide a basis for a broader assessment of associated risks. Not only are there other compounds than aristolochic acids that need to be taken into consideration, but the entire composition of some of the most active extracts needs to be understood. The study thus serves as a model for assessing closely related species used as traditional medicines. More broadly, the strategy presented here can also be used in identifying new drug leads from medicinal plants (and fungi).
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Source: Frontiers in Pharmacology
