JOURNAL OF COMPLEMENTARY MEDICINE RESEARCH, 2019 VOL 10, NO. 3,PAGE 129-141 10.5455/jcmr.20190513073648 ORIGINAL RESEARCH Open Access Pan-Himalaya ethnomedicine safety: Lithospermeae (Boraginaceae) herbal remedies containing toxic pyrrolizidine alkaloids Latif Ahmad1,2, Yi He1, Andrew J. Semotiuk3, Quan Ru Liu1, Hammad Ahmad Jan4 College of Life Science, Beijing Normal University, Beijing, China Department of Botany, Shaheed Benazir Bhutto University, Dir Upper, Pakistan 3 Department of Botany and Plant Sciences, University of California, Riverside, California 4 Department of Botany, Islamia College University, Peshawar, Pakistan 1 2 ABSTRACT ARTICLE HISTORY Aim/Background: Boraginaceae is famous for the production of pyrrolizidine alkaloids (PAs) and some of these PAs are carcinogenic and also cause liver failure. Therefore, the aim of the present study was to identify the presence or absence of hepatotoxic pyrrolizidine alkaloids in the tribe Lithospermeae (Boraginaceae). If any are found, it may indicate excluding members of this tribe from the herbal formulation or from use on patients with liver problems. Materials and Methods: Plant samples of Onosma hispida Wall. ex G. Don, Onosma paniculatum Bureau & Franch., Onosma hookeri var. longiflorum (Duthie) A.V. Duthie ex Stapf, and Maharanga emodi (Wall.) A. DC. from Boraginaceae—Tribe Lithospermeae were collected from various regions of Pan Himalaya and brought to Beijing Normal University for further experimentation. We used acetonitrile–water gradient with 0.1% formic acid as the mobile phase and Zorbax SB-Aq column to analyze samples. Furthermore, we also searched the literature to find the ethnomedicinal importance of these plants. Results: The results showed that these plants are used orally for the treatment of various human ailments, and, therefore, we further investigated these plants for toxic PAs. High-performance liquid chromatography results showed that leaves of these plants were PA positive, and out of four PA standards, three: Heliotrine (2), Lycopsamine (3), and Echimidine (4) were detected. Conclusions: In this study, we present a new report about the presence of toxic PAs in the leaves of O. hispida, O. paniculatum, O. hookeri var. longiflorum, and M. emodi from the Pan-Himalaya region. These plants are used in traditional medicine mostly in Pakistan, Nepal, and China, and the presence of hepatotoxic PAs limits the use for medicinal purposes. Received May 13, 2018 Accepted July 17, 2018 Published August 05, 2019 Introduction The knowledge of medicinal plants has been accumulated in the course of many centuries based on different medicinal systems, such as Ayurveda, Unani, and Siddha. Often, people of developing countries rely more on traditional medicine, possibly due to low access to modern health services [1]. These ethnomedicinal practices are transmitted from generation-to-generation and still practiced in various communities because of very low expense and good pharmacological results. These valuable medicinal Contact Quan Ru Liu liuquanru@bnu.edu.cn KEYWORDS Ethnomedicine; lithospermeae; toxicity; pyrrolizidine alkaloids; Pan-Himalaya; HPLC plants contain rich bioactive compounds which have various pharmacological activities [2]. Indigenous people around the world depend on plants for their basic healthcare and economic values. These benefits are based on the experience of older native people, need, and observation [3]. Natural resources play a vital role to provide us food, fuel, shelter, clothing, and medications as well as different necessities of sustainable life to the humans [4,5]. Medicinal plants have served mankind by providing local remedies to College of Life Science, Beijing Normal University, Beijing, China. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution NonCommercial ShareAlike 4.0 (https://creativecommons.org/licenses/by-nc-sa/4.0/). Latif Ahmad, Yi He, Andrew J. Semotiuk, Quan Ru Liu, Hammad Ahmad Jan treat ailments. Because of this, people have investigated medicinal properties throughout history [6]. In many developing countries, the safety of herbal medicine is also a major concern [7]. In Western countries, many alternative medicine practitioners claimed that traditional medicines show only benefits and they have no side effects. Therefore, herbal medicines and products may be ingested without taking the necessary precautions and, indeed, several fatal intoxication cases have been reported—the cause being the use of herbal remedies [8]. Unfortunately, the critical and testing role of pharmacovigilance in local ethnomedicinal markets is often lacking to non-existent [9]. These dangers can be magnified with issues of product quality, processing methods, miss identification of taxa, and knowledge of potential physiological effects [10]. Several cases of severe accidental poisoning by medicinal plants used as part of traditional treatment include neurological signs and sometime multi-organ failure [11]. Other poisonous cases due to herbal remedies have also been reported recently [12,13]. Pyrrolizidine alkaloids (PAs) are one of the common sources of herbal remedies to produce toxicity [14]. Pyrrolizidine alkaloids are secondary plant metabolites that are mostly found in the botanical families of Asteraceae, Boraginaceae, and Fabaceae and they form a powerful defense mechanism against herbivores [15,16]. Among 6,000 angiosperm species, approximately 600 have naturally occurring PAs [17]. It is a large group of toxins naturally synthesized by various plant species as secondary metabolites. Several toxic PAs enter into the food chain presenting hazards to humans and animals [18]. In developing and industrialized countries, the use of herbal medicine has become increasingly more common and so PA poisoning is one of the main problems reported within the last 25 years. Those PAs, which possess a 1, 2-double bond in their base moiety, are hepatotoxic, carcinogenic, teratogenic, genotoxic, and sometimes pneumotoxic [19]. The liver of humans can be damaged by acute poisoning with PAs, whereas a sub-acute dose may lead to pulmonary arterial hypertension and liver cirrhosis [20]. The hepatotoxic PAs, particularly 1, 2-unsaturated PAs, are undesirable in herbal products and other foods due to their acute and chronic liver-damaging effects [21]. Oral ingestion of PAscontaining herbal remedy or teas is the main cause of hepatic sinusoidal obstruction syndrome (HSOS). Serious HSOS leads to liver and multi-organ failure, so liver transplantation can be needed and even can 130 result in death. There is still no effective strategy for HSOS treatment in the clinic [22]. PA metabolism occurs mainly in the liver, which is also the main target organ of toxicity [23]. Around the world, thousands of clinical cases of HSOS due to PAs-poisoning have been documented since 1920 [24,25]. In the last few years, PAs, especially in herbal medicinal products became a widely discussed issue and it is the reason that the European Medicinal Agency gives precautions to control PAs contaminating herbal and other food products [26]. Besides many other herbal drugs, Onosma hispida, O. paniculatum, O. hookeri var. longiflorum, and Maharanga emodi (Boraginaceae—Tribe Lithospermeae Dumort.) are also one of the main potential sources of PA toxicity. The genus Onosma L. is a species-rich genus which includes about 150 species all around the world [27]. Onosma hispida Wall. ex G. Don is a perennial herb up to 70 cm tall with a prominent taproot. The plant is distributed in Northern Pakistan Gilgit, Chitral, Swat, and Hazara [28]. Onosma paniculatum Bureau & Franch. is biennial, or rarely perennial herb with the single stem up to 40–80 cm tall [29]. The genus Maharanga is herbaceous, perennial, or biennial with nine species that distribute from middle Himalaya to the southern part of China [30]. The present study is carried out with the aim to determine the presence or absence of hepatotoxic pyrrolizidine alkaloids in O. hispida, O. hookeri var. longiflorum, O. paniculatum, and M. emodi. If these alkaloids are found, it would indicate exclusion from the herbal formulation. Materials and Methods Literature review of ethnomedicinal uses of selected plants We collected data and surveyed relevant literature regarding the herbal remedies O. hispida, O. paniculatum, O. hookeri var. longiflorum, and M. emodi from Pakistan, China, and Nepal. For this, we queried the scientific databases of Web of Science and Google Scholar for the keywords in the search term “ethnomedicinal uses” or “medicinal uses” of O. hispida; “Boraginaceae ethnobotany,” “medicinal uses of M. emodi,” “medicinal uses of O. paniculatum,” and “medicinal uses of O. hookeri.” Plant sample collecting sites—The Pan-Himalayan regions The Pan-Himalayas (the Himalayas and adjacent regions) forms a natural geographic unit from the J Complement Med Res • 2019 • Vol 10 • Issue 3 Lithospermeae herbal remedies containing toxic pyrrolizidine alkaloids Wakhan Corridor and the north-eastern Hindu Kush eastwards to the Hengduan Mountains by way of the Karakorum and the Himalayas. This region covers the north-eastern corner of Afghanistan, Nepal, northern India, northern Pakistan, northern Myanmar, southwest China, and Bhutan [31]. The northern parts of Pan-Himalaya of Pakistan include Azad Kashmir, Chitral, Swat, Dir, Hazara Division, and Gilget-Biltistan. We collected the plant samples of O. hispida Wall. ex G. Don from Bomboret, Chitral in July 2017. Bomboret Valley is located in the south-west of Chitral town district Chitral which lies in north-western Pakistan, and south of the Afghan Wakhan Corridor [32]. Similarly in southwest China, part of PanHimalaya includes SE Gansu, SE Qinghai, NW Yunnan, W Sichuan, and S Tibet [31]. The M. emodi was collected by Lai Wei and Jia-Chen Hao from Geelong county-Tibet, and O. paniculatum was collected from Zhongdian county Yunnan province by Jian-Fei Ye et al. While O. hookeri var. longiflorum was collected by Yi He and Dan-Hui Liu from Ngamring County, Tibet (Supplementary Material). Plant collection, identification, and deposition in herbarium The plant sample of O. hispida collected from northern parts of Pan-Himalaya of Pakistan (Chitral, GilgitBaltistan, Swat, Abbottabad, Azad Kashmir, and Dir). While, M. emodi, O. paniculatum, and O. hookeri var. longiflorum from southwest China, part of Pan-Himalaya along with other Lithospermeae-Boraginaceae members were brought to Beijing Normal University in Beijing, China. The plants were identified, voucher specimen numbers were assigned, and deposited at Herbarium of Beijing Normal University as a ready reference for future studies. The collector number, collector name, voucher specimen number, altitude, and collecting sites are represented in Table 1 (Supplementary Material). Experimental Chemicals, reagents, and standards Methanol and acetonitrile were of high-performance liquid chromatography (HPLC) grade and purchased from Dikma Technologies Inc. (Lake Forest, CA). Formic acid was the product of Aladdin Industrial Corporation (Shanghai, China). Pure water used throughout the experiment was prepared from a Milli-Q water purification system (Millipore Corporation, Billerica, MA). Reference standards of PAs; echimidine, heliotrine, lycopsamine, and europine were purchased from ChemFaces (Hubei, China) and had a purity > 98%. Botanical samples preparation The ethnomedicinal plant samples for HPLC were prepared according to well-known published methods [33,34]. Briefly, dried leaves (500 mg) of mature flowering O. hispida, O. paniculatum, O. hookeri var. longiflorum, and M. emodi were accurately weighed and crushed in a mortar and pestle with liquid nitrogen. The sample was sonicated for 35 minutes with 3 ml of methanol immediately followed by centrifugation for 15 minutes at 4,000 g. After centrifugation, the supernatant was transferred to another clean tube. The above procedure was repeated thrice and the respective supernatants combined into the same tube. The final volume was adjusted to 10 ml with methanol and mixed thoroughly. Prior to injection, 300 μl of the sample was passed through a 13 mm × 0.45 μm FitMax Syringe filter membrane Nylon (Dikma, USA). Standard preparation The stock solutions of the following standards were prepared separately at concentrations of 1.0 mg mL−1: lycopsamine, heliotrine, europine, and echimidine [33–35]. Table 1. Plant samples collected from research sites in Pan-Himalaya regions. Voucher No. BNU0033440 BNU0033442 BNU0033441 BNU0033439 www.jocmr.com Scientific name O. hispida Wall. ex G. Don O.paniculatum Bureau. & Franch O. hookeri var. longiflorum (Duthie) M. emodi (Wall.) A. DC. Collecting site Altitude (m) Bomborait, Chitral, Pakistan 2,151 Zhongdian county; Yunnan; China Ngamring county, Tibet. 4,693 Geelong county; Tibet; China 3,977 131 Latif Ahmad, Yi He, Andrew J. Semotiuk, Quan Ru Liu, Hammad Ahmad Jan Instrumentation and conditions Results The liquid chromatographic system was the Waters Alliance 2695 LC System with 2487 Dual Wavelength UV Detector (Milford, MA) comprised of the following modular components: 4 channel degasser, built-in quaternary pump, auto-injector, and auto-sampler with 120 2 ml vials. We used a Zorbax SB-Aq (4.6 × 250 mm, 5 μm particles) column (Agilent, USA) for separation of the PAs from O. hispida, O. paniculatum, O. hookeri var. longiflorum, and M. emodi. A gradient LC method was developed for the PAs analysis in the leaves of O. hispida, O. paniculatum, O. hookeri var. longiflorum, and M. emodi. The mobile phase compositions of the HPLC system were: (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid at a flow rate of 0.5 ml/minute with the gradient elution system of A and B (87:13; 50:50; 13:87 in 53 minutes). Before moving to the next sample, the system was washed by methanol for 15 minutes. The temperature of the sample tray and column was at room temperature, the injection volume was 10 μl, and the detection wavelength was set at 280 nm. Ethnomedicinal uses of the Onosma species and M. emodi In our previous study, the decoction of the aerial parts was used to treat hypertension [36]. Recently, Sher et al. [32] reported a new traditional remedy for O. hispida. In this remedy, powder of the whole plant is taken with a glass of milk for quick recovery after delivery of a baby. Similarly, M. emodi, known as Marangi in Nepali, is a well-known herb used in Nepalese traditional medicine [37–39]. In traditional Chinese medicine, Zicao is a traditional remedy for the treatment of cancer, and O. paniculatum is one of the main herbs in the preparation of Zicao [40]. Other ethnomedicinal uses of these plants are shown in Table 2. Separation and determination of PAs in Onosma species and M. emodi Four ethnomedicinal plant samples of O. hispida, O. paniculatum, O. hookeri var. longiflorum, and M. emodi, commonly used in traditional medicine in Pakistan, Nepal, and China were phytochemically Table 2. Ethnomedicinal uses of Pan-Himalayan species from Chinese and Pakistani regions. Scientific name Part used Medicinal Uses Literature O. hispida Wall. ex G.Don Whole plant Quick recovery after delivery Sher et al. [32] Aerial Part Hypertension Ahmad et al. [36] Whole plant Jaundice and liver diseases Sher et al. [41] Medicinal use (not mention specific disease) Ahmad et al. [42] Pneumonia and typhoid fever Khan and Khatoon [43] Plant extract Cancer Rinner et al. [40] Root Nettle rash, acute and chronic hepatitis Ning and Cao [44] Root extract O. paniculatum Bureau & Franch. Acute chronic hepatitis, pulmonary tuberculosis, Jiangsu New Medical gynecologic inflammation infant College [45] dermatitis O. hookeri var. longiflorum Root (Duthie) A.V. Duthie ex stapf. M. emodi (Wall.) A. DC. Whole plant Root 132 Pulmonary tuberculosis Gu [46] Pneumonia Luo [47] Anti-inflammation, contraception, antineoplastic Tibet Health Bureau [48] Hypertension, fever, and blood purification Pandey [37] Anti-viral activity Rajbhandari et al. [49] Hair tonic Bhattarai [38] J Complement Med Res • 2019 • Vol 10 • Issue 3 Lithospermeae herbal remedies containing toxic pyrrolizidine alkaloids investigated qualitatively for PAs. These plants were collected from different sites of Pan-Himalaya. In our study, all the samples were processed, using the HPLC conditions optimized for the plant samples and reference standards stated above. HPLC methods are non-destructive and have the advantage of allowing the determination of free bases and N-oxides in a single analytical run without prior reduction of the oxides, thus both the preparation and analysis stages are much reduced [50]. Four reference standards of PAs, namely, europine (1), heliotrine (2), lycopsamine (3), and echimidine (4) were used to identify the possible PAs in leaves of the investigated plant species and their chemical structures shown in the Figure 1. In our previous study of PAs from Arnebia benthamii tribe Lithospermeae (Boraginaceae), we used the same HPLC machines conditions for these new samples [14]. Using the above stated HPLC conditions, the four standards of europine, heliotrine, lycopsamine, and echimidine were separated with retention times of 10.21, 10.77, 37.16, and 46.64 minutes, respectively. After identification of the retention time of standards, 10 μl of each plant samples was injected and the retention times were compared to those of reference standards. The qualitative analyses of four PA compounds in O. hookeri var. longiflorum and O. hispida show positive for three PAs, i.e., heliotrine, lycopsamine, and echimidine. Similarly, O. paniculatum were positively detected for the two PAs lycopsamine and echimidine. While two PAs lycopsamine and echimidine were found in M. emodi. Figure 2 represents the chromatogram of the mixture and of the four samples. Discussion In the present study, we have investigated the pyrrolizidine alkaloid profile of the leaves of O. hispida, O. paniculatum, O. hookeri var. longiflorum, and M. emodi from Pan-Himalaya and, for the first time, reported this in the literature. However, PAs have been previously reported from some other Onosma spp. such as 3′-O-acetylechinatine N-oxide from O. kaheirei Teppner [51], intermedine and lycopsamine in O. alborosea, 7-acetylintermedine, lycopsamine, and intermedine in O. arenaria subsp. pennina [52], 7-Acetyllycopsamine, 5, uplandicine, 7-acetylretronecine etc. in O. arenaria [53], viridinatine and onosmerectine in O. erecta [54], heliotridine in O. hetrophyllum [55], eptanthine and echihumiline in O. leptantha [56], and lycopsamine, leptanthine, echimidine, heliospathuline, and 7-viridiflorylretronecine in O. stellulatum [57,58]. To the best of authors’ knowledge, genus Maharanga is investigated for the first time to determine PAs. In the past, Roeder and Wiedenfeld [39] alluded to this by proposing that the genus Maharanga is similar to Onosma and that plants of this genus have toxic alkaloids. Many PA-containing plants and individual PA compounds have been tested in animal models and Figure 1. Structure of authentic standards of pyrrolizidine alkaloids. www.jocmr.com 133 Latif Ahmad, Yi He, Andrew J. Semotiuk, Quan Ru Liu, Hammad Ahmad Jan Figure 2. Base-peak chromatograms of standard mix and leaves of M. emodi, O. hispida, O. hookeri var. longiflorium, and O. paniculatum. 134 J Complement Med Res • 2019 • Vol 10 • Issue 3 Lithospermeae herbal remedies containing toxic pyrrolizidine alkaloids shown to be carcinogenic in various tissues. The liver is the main carcinogenic target [17]. Lycopsamine is one of the heptotoxic PAs and it damages the liver [59]. Additionally, heliotrine is classified as a heliotridine-type PA; these have been shown to induce mutagenesis and liver tumors, be carcinogenic to the liver, and also damage chromosomes [8,17,60]. Echimidine is a hepatotoxic pyrrolizidine alkaloid [61]. Unfortunately, in our study, these two toxic PAs are found in all four of the investigated species. Pyrrolizidine alkaloids are typical metabolites of the family Boraginaceae present usually in the form of their N-oxides that are hydrophilic because of polar compounds [62]. These N-oxides cannot be directly converted to the hydroxy-PAs, but whenever humans or livestock intake PA containing plant parts, they are reduced by the gut enzymes or the liver microsomes and NADH or NADPH to the free bases and metabolic activation forms hepatotoxic pyrroles. Therefore, they show equal toxicity to that of the free bases and cause acute and chronic effects in man and livestock [63–65]. Unfortunately, in our study, all four investigated species O. hispida, O. paniculatum, O. hookeri var. longiflorum, and M. emodi were reported in the literature to be used orally for the treatment of various human ailments. In our previous study, the decoction of the leaves of O. hispida was used by the traditional people in Dir, Pakistan, for the treatment of hypertension [36]. Similarly, for another three species, oral ingestion is also reported as shown in Table 2. There are a large numbers of reports on PA poisoning and intoxication in humans. Previously, it has been well-established that PA containing plants and contaminated food affect both developing and developed countries. In the 1920s in South Africa, liver disease was widespread and it was caused by the consumption of bread contaminated with PAs from seeds from Senecio species [66]. Similarly in 1968, in the same country, 15 children had the veno-occlusive disease (VOD) by using bush-teas with Crotalaria spp., out of 15, 10 children died [67]. In 1954, 23 adults had VOD in Jamaica be caused by bush-teas with Crotalaria fulva [68]. In 1970 in Iraq, 9 children have VOD because of food contaminated by a Senecio spp. [69]. In Afghanistan in 1970–1972, contaminated wheat with Heliotropium popovii, ssp. gillianum made approximately 7,200 people to suffer from VOD [70]. Similarly, 3,906 people suffered abdominal pain, hepatomegaly, ascites, alteration of consciousness, and were hospitalized in Tajikistan because of Heliotropium lasiocarpum contamination of grain in 1992 [71]. In India, in 1974–1977, www.jocmr.com six people had VOD because of contaminated food with Heliotropium eichwaldii [72]. Intoxication in humans due to PA containing plants and contaminated food were also established in well-developed countries like the USA, the UK, Switzerland, China, Argentina, and Austria [73–78]. Based on reports about diseases and intoxications to human as well as livestock from PAs around the globe, the European Medicines Agency (EMA) has implemented a limit of intake of PAs from herbal medicinal products (i.e., 1 μg/day) as a transitional measure for 3 years, after which the threshold will be set to 0.007 μg of 1, 2-unsaturated PA/kg body weight [79,80]. In the European Union, the so-called “zero-tolerance principle” can be applied; this principle is used in cases where no safe or tolerable level can be determined based on available, valid scientific data, or if insufficient toxicological data are available. Due to their genotoxic and carcinogenic potential, this principle can be applied for PA in food and fodder [81]. In our study, we have limited information about safe or tolerable levels of the studied species containing PAs. We also have insufficient data about toxicity and quantitative analysis of O. hispida, O. paniculatum, O. hookeri var. longiflorum, and M. emodi. So as for the suggested principle by Bundesinstitut für Risikobewertung [81], we also suggest the ‘‘zero-tolerance principle’’ be applied for the investigated species before the quantitative analysis of PAs. Conclusion This study identified, for the first time, PAs in the leaves of O. hispida, O. paniculatum, O. hookeri var. longiflorum, and M. emodi from Pan-Himalaya region. The selected plants were found to be positive of hepatotoxic Pas, such as heliotrine, lycopsamine, and echimidine. Our results show that besides their ethnomedicinal value, the species is also a source of hepatoxic PAs. Because of insufficient data in the literature about the toxicity of these plants, quantity of PAs, and non-availability of tolerable or safe level, we suggest that the “zero-tolerance principle” should be applied. Furthermore, we recommend that these plants be excluded from local markets and their herbal formulations should not be sold before PA safe levels or tolerable levels are determined. Acknowledgments The authors would like to thank Professor Xiao-Bo Qiu’s lab and Professor Chang-Qi Zhao’s lab at the College of Life Science, Beijing Normal University for support of this research project. The authors 135 Latif Ahmad, Yi He, Andrew J. Semotiuk, Quan Ru Liu, Hammad Ahmad Jan highly acknowledged Dr. Kai Liu, from the experimental technology center of the College of Life Science for supporting us through all experimental processes. We are also thankful to UC MEXUS for support of one author. Funding This work was supported by the National Natural Science Foundation of China (grant numbers 31770213), and Science and Technology Basic work (2013FY112100). Conflict of interest The authors declare that they have no competing interest. References [1] [2] [3] [4] [5] [6]. [7] [8] [9] 136 Singh A, Nautiyal MC, Kunwar RM, Bussmann RW. Ethnomedicinal plants used by local inhabitants of Jakholi block, Rudraprayag district, western Himalaya, India. J Ethnobiol Ethnomed 2017; 13:49. Chekole G. Ethnobotanical study of medicinal plants used against human ailments in Gubalafto District, Northern Ethiopia. J Ethnobiol Ethnomed 2017; 13:55. Kunwar RM, Baral K, Paudel P, Acharya RP, ThapaMagar KB, Cameron M. Land use and socioeconomic change, medicinal plant selection and biodiversity resilience in far western Nepal. PLoS One 2016;11:e0167812; doi:10.1371/journal. pone.0167812.s López-Sánchez A, San Miguel A, Dirzo R, Roig S. Scattered trees and livestock grazing as keystones organisms for sustainable use and conservation of Mediterranean dehesas. J Nat Conserv 2016;33:58–67. Asadbeigi M, Mohammadi T, Rafieian-Kopaei M, Saki K, Bahmani M, Delfan M. Traditional effects of medicinal plants in the treatment of respiratory diseases and disorders: an ethnobotanical study in the Urmia. Asian Pac J Trop Med 2014; 7:364–8. Akan H, Korkut MM, Balos MM. An ethnobotanical study around Arat Mountain and its surroundings (Birecik, Sanlıurfa). Fırat Univ J Sci Eng 2008; 20:67–81. Popat A, Shear NH, Malkiewicza I, Stewartc MJ, Steenkamp V, Thomson S, Neuman M. The toxicity of Callilepis laureola, a South African traditional herbal medicine. Clin Biochem 2001; 34:229–36. Wiedenfeld H, Edgar J. Toxicity of pyrrolizidine alkaloids to humans and ruminants. Phytochem Rev 2011; 10:137–51. Debbie S, Graeme L, Pierre D, Elizabeth W, et al. Pharmacovigilance of herbal medicine. J Ethnopharmacol 2012; 140:513–8. [10] Shaw D. Toxicological risks of Chinese herbs. Planta Med 2010; 76:2012–8. [11] Durasnel P, Blondé R, Lion F, Galas T, MoussetHovaere M, Balaÿ I, et al. Severe poisoning by plants used for traditional medicine in Mayotte. Bull Soc Pathol Exot 2014; 107:306–11. [12] Gimenez N, Magro N, Cortes N, Guitart R. Poisoning after Ingestion of Spartium junceum seeds: dose-dependent effects in three boys. J Emerg Med 2017; 53:e41–4. [13] Oulmaati A, Hmami F, Achour S, Bouharrou A. Severe poisoning by traditional medication in the newborn. Arch Pediatr 2017; 24:833–6. [14] Ahmad L, He Y, Hao JC, Semotiuk A, Liu QR, Mazari P. Toxic pyrrolizidine alkaloids provide a warning sign to overuse of the ethnomedicine Arnebia benthamii. J Ethnopharmacol 2018; 210:88–94. [15] Mulder PJ, López P, Castelari M, Bodi D, Ronczka S, Preiss-Weigert A, et al. Occurrence of pyrrolizidine alkaloids in animal- and plant-derived food: results of a survey across Europe. Food Addit Contam Part A 2017; doi: 10.1080/19440049.2017.1382726. [16] Schoning V, Hammann F, Peinl M, Drewe J. Identification of any structure-specific epatotoxic potential of different pyrrolizidine alkaloids using random forests and artificial neural networks. Toxicol Sci 2017; 160:361–70. [17] Chen T, Mei N, Fu PP. Genotoxicity of pyrrolizidine alkaloids. J Appl Toxicol 2010; 30:183–96. [18] EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), H.K. Knutsen J. Alexander Barregard L, et al. Statement on the risks for human health related to the presence of pyrrolizidine alkaloids in honey, tea, herbal infusions and food supplements. EFSA J 2017; 15:4908, 34; https://doi. org/10.2903/j.efsa. 2017.4908. [19] Wiedenfeld H. Plants containing pyrrolizidine alkaloids: toxicity and problems. Food Addit Contam 2011; 28:282–92. [20] EFSA Panel on Contaminants in the Food Chain (CONTAM). Scientific Opinion on Pyrrolizidine alkaloids in food and feed. EFSA J 2011; 9(2406):134; https://doi.org/10.2903/j.efsa.2011.2406. Available via www.efsa.europa.eu/efsajournal [21] Stegelmeier BL, Colegat SM, Brown AW. Dehydropyrrolizidine alkaloid toxicity, cytotoxicity, and carcinogenicity. Toxins 2016; 8(12):356; doi: 10.3390/toxins8120356 [22] Huang Z, Chen M, Zhang J, Sheng Y, Ji L. Integrative analysis of hepatic microRNA and mRNA to identify potential biological pathways associated with monocrotaline-induced liver injury in mice. Toxicol Appl Pharmacol 2017; 333:35–42. [23] Neumann MG, Cohen LB, Opris M, Nanau RM, Hyunjin J. Hepatotoxicity of pyrrolizidine alkaloids. J Pharm Pharm Sci 2015; 18:825–43. [24] Lin G, Wang YJ, Li N, Gao H, Ji Y, Zhang F, et al. Hepatic sinusoidal obstruction syndrome associated with J Complement Med Res • 2019 • Vol 10 • Issue 3 Lithospermeae herbal remedies containing toxic pyrrolizidine alkaloids [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] consumption of Gynura segetum. J Hepatol 2011; 54(4) 666–73. Dai N, Yu YC, Ren TH, Wu JG, Jiang Y, Shen LG, et al. Gynura root induces hepatic veno-occlusive disease: a case report and review of the literature. World J Gastroenterol 2007; 13:1628–31. EMA. EMA/HMPC/328782/2016. Public statement on contamination of herbal medicinal products/ traditional herbal medicinal products with pyrrolizidine alkaloids, pp 1–11, 2016. Cecchi L, Selvi F. Phylogenetic relationships of the monotypic genera Halascya and Paramoltkia and the origins of serpentine adaptation in circummediterranean Lithospermeae (Boraginaceae): insights from ITS and matK DNA sequences. Taxon 2009; 58:700–14. FOP. Flora of Pakistan, 1989. Available via http:// www.tropicos.org/Name/40001957?Projectid=32 FOC. Flora of China. Available via http://www.efloras. org/florataxon.aspx?flora_id=2&taxon_id=10115. Johnston IM. Studies in the Boraginaceae, XXVI: further revaluations of the genera of the Lithospermeae. J Arn Arb 1954; 35:1–81. FOPH. Flora of the Pan-Himalayas: General guidelines. J Syst Evol 2011; 49:617–24. Sher H, Bussmann RW, Hart R, de Boer RJ. Traditional use of medicinal plants among Kalasha, Ismaeli and Sunni groups in Chitral District, Khyber Pakhtunkhwa province, Pakistan. J Ethnopharmacol 2016; 188:57–69. Avula B, Wang YH, Wang M, Smillie TJ, Khan IA. Simultaneous determination of sesquiterpenes and pyrrolizidine alkaloids from the rhizomes of Petasites hybridus (L.) GM et Sch. and dietary supplements using UPLC-UV and HPLC-TOF-MS methods. J Pharm Biomed 2012; 70:53–63. Avula B, Sagi S, Wang YH, Zweigenbaum J, Wang M, Khan IA. Characterization and screening of pyrrolizidine alkaloids and N-oxides from botanicals and dietary supplements using UHPLC-high resolution mass spectrometry. Food Chem 2015; 178:136–48. Griffin CT, Danaher M, Elliott CT, Glenn Kennedy D, Furey A. Detection of pyrrolizidine alkaloids in commercial honey using liquid chromatography– ion trap mass spectrometry. Food Chem 2013; 136:1577–83. Ahmad L, Semotiuk A, Zafar M, Ahmad M, Sultana S, Liu QR, et al. Ethnopharmacological documentation of medicinal plants used for hypertension among the local communities of DIR Lower, Pakistan. J Ethnopharmacol 2015; 175:138–46. Pandey MR. Use of medicinal plants in traditional Tibetan therapy system in upper Mustang, Nepal. Our Nature 2006; 4:69–82. Bhattarai NK. Medical ethnobotany in the Karnali Zone, Nepal. Econ Bot 1992; 46:257–61. www.jocmr.com [39] Roeder E, Wiedenfeld H, Pyrrolizidine alkaloids in medicinal plants of Mongolia, Nepal and Tibet. Pharmazie 2009; 64:699–716. [40] Rinner B, Kretschmer N, Knausz H, Mayer A, Boechzelt H, Hao XJ, et al. A petrol ether extract of the roots of Onosma paniculatum induces cell death in a caspase dependent manner. J Ethnopharmacol 2010; 129:182–8. [41] Sher H. Ethnoecological evaluation of some medicinal and aromatic plants of Kot Malakand Agency, Pakistan. Sci Res Essay 2011; 6:2164–73. [42] Ahmad H, Khan SM, Ghafoor S, Ali N. Ethnobotanical study of upper siran. Herbs Spices Med Plants 2009; 15:86–97. [43] Khan SW, Khatoon S. Ethnobotanical studies on some useful herbs of Haramosh and Bugrote valleys in Gilgit, northern areas of Pakistan. Pak J Bot 2008; 40:43–58. [44] Ning W, Cao RQ. Onosma paniculatum: in vitro culture and the production of purple-red pigment. In: Bajaj YPS (ed.). Medicinal and aromatic plants IX. Biotechnology in Agriculture and Forestry, Springer, Berlin, Heidelberg, 37 p, 1996. [45] Jiangsu New Medical College. The Dictionary of Chinese Medicinal Plant. Shanghai Science and Technology Press, Shanghai, China, 1986. [46] Gu J, Traditional Tibetan medicine. Nationalities Publishing House, Beijing, China, 2016. [47] Luo DS. Tibetan Materia Medica. Nationalities Publishing House, Beijing, China, 1997. [48] Tibet Health Bureau et al. Standard of Tibetan medicine. Qinghai People’s Publishing House, Xining, China, 1979. [49] Rajbhandari M, Mentel R, Jha PK, Chaudhary RP, Bhattarai S, Gewali MB, et al. Antiviral activity of some plants used in Nepalese traditional medicine. eCAM 2009; 6(4):517–22. [50] Crews C, Berthiller F, Krska R. Update on analytical methods for toxic pyrrolizidine alkaloids. Anal Bioanal Chem 2010; 396:327–38. [51] Orfanou IM, Damianakos H, Bazos I, Graikou K, Chinou I. Pyrrolizidine alkaloids from Onosma kaheirei Teppner (Boraginaceae). Rec Nat Prod 2016; 10:221–7. [52] Roeder E, Wiedenfeld H, Kroeger R, Teppner H. Pyrrolizidine alkaloids of three taxa of Onosma (Boraginaceae-Lithospermeae). Phyton 1993; 33:41–9. [53] El-Shazly A, Adel-Ghani A, Wink M. Pyrrolizidine alkaloids from Onosma arenaria Waldst. and Kit (Boraginaceae). Biochem Syst Ecol 2003; 31:477–85. [54] Damianakos H, Sotiroudis G, Chinou I. Pyrrolizidine alkaloids from Onosma erecta. J Nat Prod 2013; 76:1892–35. [55] Mellidis AS, Papageorgiou VP. Pyrrolizidine alkaloids of the plant Onosma heterophylla. Chem Chron 1988; 17:67–73. 137 Latif Ahmad, Yi He, Andrew J. Semotiuk, Quan Ru Liu, Hammad Ahmad Jan [56] Kretsi O, Aligiannis N, Skaltsounis AL, Chinou IB. Pyrrolizidine alkaloids from Onosma leptantha, Helv. Chim Acta 2003; 86:3136–40. [57] Mroczek T, Baj S, Chrobok A, Glowniak K. Screening for pyrrolizidine alkaloids in plant materials by electron ionization RP-HPLC-MS with thermabeam interface. Bio Med Chromatogr 2004; 18:745–51. [58] Mroczek T, Ndjoko K, Glowniak K, Hostetmann K. On-line structure characterization of pyrrolizidine alkaloids in Onosma stellulatum and Emilia coccinea by liquid chromatography-ion-trap mass spectrometry. J Chromatogr A 2004; 1056:91–7. [59] Liu F, Wan SY, Jiang Z, Li SF, Ong ES, Osorio JC. Determination of pyrrolizidine alkaloids in comfrey by liquid chromatography electrospray ionization mass spectrometry. Talanta 2009; 80:916–23. [60] Xia Q, Y. Zhao Y, Tungeln LV, Von Tungeln LS, Doerge DR, Lin G, Cai L, et al. Pyrrolizidine alkaloid-derived DNA adducts as a common biological biomarker of pyrrolizidine alkaloid-induced tumorigenicity. Chem Res Toxicol 2013; 26:1384−96. [61] Bach N, Thung SN, Schaffner F. Comfrey herb tea-induced hepatic veno-occlusive disease. Am J Med 1989; 87:97–9. [62] Damianakos H, Jeziorek M, Pietrosiuk A, Chinou I. The chemical profile of pyrrolizidine alkaloids from selected Greek endemic boraginaceae plants determined by gas chromatography/mass spectrometry. J AOAC Int 2014; 97:1244−9. [63] Chou MW, Wang YP, Yan J, Yang YC, Beger RD, Williams LD, et al. Riddelliine N-oxide is a phytochemical and mammalian metabolite with genotoxic activity that is comparable to the parent pyrrolizidine alkaloid riddelliine. Toxicol Lett 2003; 145:239–47. [64] Wang X, Yan J, Fu PP, Chou MW. Metabolic activation of the tumorigenic pyrrolizidine alkaloid, retrorsine, leading to DNA adduct formation in vivo. Int J Environ Res Public Health 2005; 2:74–9. [65] Fu PP, Xia QS, Lin G, Chou MW. Pyrrolizidine alkaloids—genotoxicity, metabolism enzymes, metabolic activation, and mechanisms. Drug Metab Rev 2004; 36:1–55. [66] Willmott FC, Robertson GW. Senecio disease or cirrhosis of the liver due to Senecio poisoning. Lancet 1920; 196:848–9. [67] Freiman I, Schmaman A, Zamit R, Appleberg M. Venoocclusivev disease of the liver—some new aspects. S Afr Med J 1968; 42:126–9. [68] Bras G, Brooks SEH, Watler DC. Cirrhosis of liver in Jamaica. J Pathol Bacteriol 1961; 82:503–11. [69] Al-Hasany M, Mohamed AS. Veno-occlusive of the liver in Iraq. Arch Dis Childhood 1970; 45:722–4. [70] Mohabbat O, Younos MS, Merzad AA, Srivastava RN, Sediq GG, Aram GN. An outbreak of hepatic 138 [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] venoocclusive disease in north-western Afghanista. Lancet 1976; 308:269–71. Chauvin P, Dillon JC, Moren AE. ´ pide´mie d’ intoxication alimentaire a´ l’ he´liotrope, Tadjikistan, Novembre 1992-Mars 1993. Cahiers Sante´ 1994; 4:263–8. Datta DV, Khuroo MS, Mattocks AR, Aikat BK, Chhuttani PN. Herbal medicines and veno-occlusive disease in India. Postgrad Med J 1978; 54:511–5. Stillman AE, Huxtablen RJ, Fox DW, et al. Poisoning associated with herbal teas—Arizona, Washington. Morb Mortal Wkly Rep 1977; 26:257–9. Weston CFM, Cooper BT, Davies JD, Levine DF. Venoocclusive disease of the liver secondary to ingestion of comfrey. Brit Med J 1987; 295:183. Roulet M, Laurini R, Rivier L, Calarme A. Hepatic veno-occlusive disease in newborn infant of a woman drinking herbal tea. J Pediatr 1988; 112:433–6. Culvenor CJ, Edgar JA, Smith W, Kumana CR, Lin HJ. Heliotropium lasiocarpum Fisch and Mey identified as cause of veno-occlusive disease due to a herbal tea. Lancet 1986; 1:978. Vilar JH, Garcia M, Cabrera P. Enfermedad venooclusiva hepa´tica de causa To´xica por Senecio vulgaris. Gastroenterol Hepatol 2000; 23:285–6. Sperl W, Stuppner H, Gassner I, Judmaier W, Dietze O, Vogel W. Reversible hepatic veno-occlusive disease in an infant after consumption of pyrrolizidine-containing herbal tea. Eur J Pedriatr 1995; 154:112–6. BfR, Bundesamt für Risikobewertung/Federal Institute of Risk Assessment. Chemical analysis and toxicity of pyrrolizidine alkaloids and assessment of the health risks posed by their occurrence in honey. BfR Opinion No. 038/2011. Berlin, Germany, 2011. Available via http://www.bfr.bund.de/cm/349/ chemical-analysis-and-toxicity-of-pyrrolizidinealkaloids-and-assessment-of-the-health-risksposed-by-their-occurence-in-honey.pdf (Accessed 27 October, 2017). EMA, European Medicines Agency. Committee on Herbal Medicinal Products (HMPC) Public statement on contamination of herbal medicinal products/traditional herbal medicinal products with pyrrolizidine alkaloids. Transit Recomm Risk Manag Qual Control 2016. Available via http://www.ema. europa.eu/docs/en_GB/document_library/Public_ statement/2016/06/WC500208195.pdf (Accessed 27 October, 2017). BfR, Bundesinstitut für Risikobewertung. Nulltoleranzen in Lebens- und Futtermitteln— Positionspapier des BFR vom 12.Ma¨rz. Berlin, Germany, 2007. J Complement Med Res • 2019 • Vol 10 • Issue 3 Lithospermeae herbal remedies containing toxic pyrrolizidine alkaloids e-Supplementary Material For the last few years, our lab has been working on the taxonomic revision and importance of the family Boraginaceae for the “Flora of Pan-Himalaya”. In July 2016–2018, a plant collection trip was arranged to collect members of Boraginaceae in the Pakistani Pan-Himalayan regions. In addition, samples from southwest China, part of Pan-Himalaya, from last 5 years for the project of the “Flora of Pan-Himalaya” were used (Fig. S1). July to August were selected because it is the peak flowering and fruiting season for Boraginaceae members. In the field, whole plants were collected and pressed for herbarium specimens, and in a paper bag, fresh plant material was collected in silica gel for molecular and phytochemical analysis. Dr. Latif Ahmad is of Pakistani nationality so we decided to have him cover the Pakistani Pan-Himalaya region in Northern Pakistan, while Chinese students were assigned to investigate Chinese Himalayan regions. Figure S1. Map of the plants collected sites (Pan-Himalaya part taken from source: www.flph.org). www.jocmr.com 139 Latif Ahmad, Yi He, Andrew J. Semotiuk, Quan Ru Liu, Hammad Ahmad Jan From literature searches, we know that Boraginaceae is famous for Pyrrolizidine alkaloids (PAs) with some of them being carcinogenic and also causing liver failure. Our interest lies in those species which are used in herbal remedies. For this, we also asked during interviews, about their ethnomedicinal value in addition to that presented in the studied literature. The tribe Lithospermeae of Boraginaceae has many ethnomedicine which are used for a plethora of herbal remedies. We collected 26 Lithospermeae members from Chinese and Pakistani Pan-Himalaya regions and brought them to Beijing Normal University for identification and experimental work. We targeted those species which have ethnomedicinal value and are not being studied for toxic pyrrolizidine alkaloids. Among 26 species of Lithospermeae, we select four ethnomedicines O. hispida Wall. ex G. Don, O. paniculatum Bureau & Franch., O. hookeri var. longiflorum (Duthie) A.V. Duthie ex Stapf, and M. emodi (Wall.). Onsoma hispida is a well-known herbal remedy in Northern Pakistan, while Maharanga has medicinal value in Nepal, O. paniculatum and O. hookeri var. longiflorum are widely used in traditional Chinese medicine (Fig. S2). After reviewing the literature on the above species that we selected, O. paniculatum, O. hookeri var. longiflorum, M. emodi, O. hispida, for investigation of toxic PAs, we organized them into a table for clear reference. Members of Lithospermeae are presented in Table S1. Figure S2. (a)–(b) Onosma hookeri var. longiflorum (Duthie) A.V. Duthie ex Stapf, (c)–(d) Maharanga emodi (Wall.) A. DC., (e)–(f) Onosma hispida Wall. ex G. Don, and (g)–(h) Onosma paniculatum Bureau & Franch. 140 J Complement Med Res • 2019 • Vol 10 • Issue 3 Lithospermeae herbal remedies containing toxic pyrrolizidine alkaloids Table S1. List of Lithospermeae members collected from different Pan-Himalaya regions. Collector No. Plant name Collection sites Altitude (m) LA-95BNU O. hispida Wall. ex G. Don Bomborait, Chitral, Pakistan 2,151 CH-03 BNU Onosma dichroantha Boiss. Chitral Goal, Chitral, Pakistan 1,642 LA-29BNU Onosma hypoleucum I.M. Johnst. Toli pir, Azad Kashmir, Pakistan 2,446 LA-87BNU Onosma griffithii Vatke Astore, Gilgit-Biltistan, Pakistan 3,384 LA-64BNU Onosma thomsonii Clarke Malam Jaba, Swat, KPK, Pakistan 2,521 CH-06 BNU Onosma chitralicum I.M. Johnst. Chitral, Khyber Puktunkhwa, Pakistan 1,620 BNU2017XZ052 O. hookeri C.B. Clarke Angren county, Tibet, China 4,693 20110804040 O. paniculatum Bureau & Franch. Zhongdian county; Yunnan; China BNU2017XJ383 Onosma apiculatum Riedl Zhaosu county, Xinjiang , China Unknown 2,178 BNU2017XJ153 Onosma gmelinii Ledeb. Altai City, Xinjiang , China XMLY12019 Onosma maaikangense W.T. Wang Maerkang county, Sichuan ; China 2,650 15107 Onosma confertum W.W. Sm. Daocheng county; Sichuan ; China 3,579 XMLY12033 Onosma liui Kamelin & T.N. Popova Rangtang county; Sichuan ; China 2,938 815 HY2017015 Onosma sinicum Diels Wenxian county, Gansu, China 860 15576 Onosma multiramosum Hand.-Mazz. Zuogong county, Tibet, China 4,000 15583 Onosma adenopus I.M. Johnst. Mangkang county, Tibet, China 3,508 BNU2017XZ326 Onosma waddellii Duthie Qushui county, Tibet, China 3,720 LA-49BNU A. benthamii (Wall. ex) G. Don . I.M. Johnst. Taiobat, Nellum, Azad Kashmir, Pakistan 2,415 BNU2017XJ125 Arnebia guttata Bunge Fuxun county, Xinjiang , China 1,140 LA-82BNU Arnebia euchroma (Royle) I.M. Johnst. Below Deosai Top, Skardu, Gilgit-Biltistan, Pakistan 4,037 LA-67BNU Arnebia hispidissima (Lehm.) A. DC. Tooq, Mustooj, Chitral, Pakistan 2,321 15361 M. emodi (Wall.) A. DC. Geelong county; Tibet; China 3,977 15431 Maharanga bicolor (Wall. ex G. Don) A. DC. Geelong county; Tibet; China 3,500 LA-51BNU Lithospermum officinale L. Taiobat, Azad Kashmir, Pakistan 2,315 LA-53BNU Lithospermum arvense L. Swat, Khyber Puktunkhwa, Pakistan 1,241 LA-85BNU Lithospermum tenuiflorum L.f. Swat, Khyber Puktunkhwa, Pakistan 1,175 www.jocmr.com 141