Tu-San-Qi (Gynura japonica): the culprit behind pyrrolizidine alkaloid-induced liver injury in China.

Herbs and dietary supplement-induced liver injury (HILI) is the leading cause of drug-induced liver injury in China. Among different hepatotoxic herbs, the pyrrolizidine alkaloid (PA)-producing herb Gynura japonica contributes significantly to HILI by inducing hepatic sinusoidal obstruction syndrome (HSOS), a liver disorder characterized by hepatomegaly, hyperbilirubinemia, and ascites. In China, G. japonica has been used as one of the plant species for Tu-San-Qi and is often misused with non-PA-producing Tu-San-Qi (Sedum aizoon) or even San-Qi (Panax notoginseng) for self-medication. It has been reported that over 50% of HSOS cases are caused by the intake of PA-producing G. japonica. In this review, we provide comprehensive information to distinguish these Tu-San-Qi-related herbal plant species in terms of plant/medicinal part morphologies, medicinal indications, and chemical profiles. Approximately 2156 Tu-San-Qi-associated HSOS cases reported in China from 1980 to 2019 are systematically reviewed in terms of their clinical manifestation, diagnostic workups, therapeutic interventions, and outcomes. In addition, based on the application of our developed mechanism-based biomarker of PA exposure, our clinical findings on the definitive diagnosis of 58 PA-producing Tu-San-Qi-induced HSOS patients are also elaborated. Therefore, this review article provides the first comprehensive report on 2214 PA-producing Tu-San-Qi (G. japonica)-induced HSOS cases in China, and the information presented will improve public awareness of the significant incidence of PA-producing Tu-San-Qi (G. japonica)-induced HSOS and facilitate future prevention and better clinical management of this severe HILI.

Gynura japonica: A new host of Apple stem grooving virus and Chrysanthemum virus B in China.

Gynura japonica (Thunb.) Juel [Asteraceae; syn: G. segetum (Lour.) Merr] is an important perennial medicinal herb used in China for topical treatment of trauma injuries (Lin et al. 2003). It grows naturally in the southern provinces of China and is also sometimes cultivated. During 2018-2020, wild G. japonica plants exhibiting chlorotic spots and mosaic symptoms were observed in Zhejiang province, China. To identify the possible causal agents of the disease, a single symptomatic leaf sample was collected in August 2019 and sent to Zhejiang Academy of Agricultural Sciences (Hangzhou, China) for next generation sequencing (NGS). Total RNAs extracted with TRIzol (Invitrogen, Carlsbad, USA) were subjected to high throughput sequencing on the Illumina NovaSeq 6000 platform with PE150bp and data analysis was performed by CLC Genomic Workbench 11 with default parameters (QIAGEN, Hilden, Germany). A total of 37,314,080 paired-end reads were obtained, and 11,785 contigs (961 to 10,964 bp) were generated and compared with sequences in GenBank using BLASTn or BLASTx. Of the total of 12 viral-related contigs obtained, one with a length of 6,442 nt mapped to the genomic RNA of ASGV (MN495979), seven contigs with lengths ranging from 1,034 to 2,901 nt mapped to Chrysanthemum virus B (CVB), and four mapped to broad bean wilt virus 2 (BBWV2), a virus which is known to infect G. procumbens (Kwak et al. 2017). To further confirm the presence of ASGV and CVB, primers were designed and the complete nucleotide sequences of both viruses were amplified from the original NGS sample using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) according to the manufacturer's instructions (Tiosbio, Beijing, China). BLASTn analysis revealed that the complete 6,451 nt sequence of ASGV (GenBank accession No. MW259059) shared the highest identity (81.2%) with a Chinese isolate of ASGV from citrus (MN495979). The two isolates grouped with another Chinese isolate (from pear) in phylogenetic analysis. The predicted coat protein of the virus had the highest nt identity of 93.7% (96.2% amino acid sequence identity) with that of the Chinese ASGV isolate XY from apple (KX686100). The complete genomes of two distinct molecular variants of CVB (both 8,987 nt in length) were also obtained from this sample (GenBank accession Nos. MW269552, MW269553). They shared 86.8% nt identity with each other and had 81.1% and 82.1% identity to the only known complete sequence of CVB from chrysanthemum (AB245142). Ten additional wild G. japonica plants with mosaic symptoms were collected randomly during 2019-2020 from Hangzhou (n=6) and Ningbo (n=4) in Zhejiang province and tested by RT-PCR with specific primer pairs to detect BBWV2, ASGV and CVB. RT-PCR and subsequent sequencing revealed that these three viruses were present in all the samples tested, indicating that co-infection of G. japonica by ASGV, CVB and BBWV2 is common. CVB mainly infects chrysanthemum (Singh et al. 2012), while ASGV is known as a pathogen of various fruit trees especially in the family Rosaceae, although there are recent reports that it can also infect some plants in Gramineae, Asparagaceae and Nelumbonaceae (Bhardwaj et al. 2017; Chen et al. 2019; He et al. 2019). Our results provide the first report that Gynura is a natural host of CVB and ASGV. Further surveys and biological studies are underway to evaluate the importance of Gynura as a virus reservoir for epidemics among the various hosts.

Seneciphylline, a main pyrrolizidine alkaloid in Gynura japonica, induces hepatotoxicity in mice and primary hepatocytes via activating mitochondria-mediated apoptosis.

Herbal drug-induced liver injury has been reported worldwide and gained global attention. Thousands of hepatic sinusoidal obstruction syndrome (HSOS) cases have been reported after consumption of herbal medicines and preparations containing pyrrolizidine alkaloids (PAs), which are natural phytotoxins globally distributed. And herbal medicines, such as Gynura japonica, are the current leading cause of PA-induced HSOS. The present study aimed to reveal the mechanism underlying the hepatotoxicity of seneciphylline (Seph), a main PA in G. japonica. Results showed that Seph induced severe liver injury through apoptosis in mice (70 mg/kg Seph, orally) and primary mouse and human hepatocytes (5-50 µM Seph). Further research uncovered that Seph induced apoptosis by disrupting mitochondrial homeostasis, inducing mitochondrial depolarization, mitochondrial membrane potential (MMP) loss, and cytochrome c (Cyt c) release and activating c-Jun N-terminal kinase (JNK). The Seph-induced apoptosis in hepatocytes could be alleviated by Mdivi-1 (50 µM, a dynamin-related protein 1 inhibitor), as well as SP600125 (25 µM, a specific JNK inhibitor) and ZVAD-fmk (50 µM, a general caspase inhibitor). Moreover, the Seph-induced MMP loss in hepatocytes was also rescued by Mdivi-1. In conclusion, Seph induced liver toxicity via activating mitochondrial-mediated apoptosis in mice and primary hepatocytes. Our results provide further information on Seph detoxification and herbal medicines containing Seph such as G. japonica.

Mass-spectrometry-directed analysis and purification of pyrrolizidine alkaloid cis/trans isomers in Gynura japonica.

Pyrrolizidine alkaloids are highly hepatotoxic natural chemicals that produce irreversible chronic and acute hepatotoxic effects on human beings. Purification of large amounts of pyrrolizidine alkaloids is necessary for toxicity studies. In this study, an efficient method for targeted analysis and purification of pyrrolizidine alkaloid cis/trans isomers from herbal materials was developed for the first time. Targeted analysis of the hepatotoxic pyrrolizidine alkaloids was performed by liquid chromatography with tandem mass spectrometry (precursor ion scan and daughter ion scan), and the purification of pyrrolizidine alkaloids was achieved with a mass-directed auto purification system. The extraction and preparative liquid chromatography conditions were optimized. The developed method was applied to analysis of Gynura japonica (Thunb.) Juel., a herbal medicine traditionally used for detumescence and relieving pain but is potentially hepatotoxic as it contains pyrrolizidine alkaloids. Twelve pyrrolizidine alkaloids (six cis/trans isomer pairs) were identified with reference compounds or characterized by liquid chromatography with tandem mass spectrometry, and five individual pyrrolizidine alkaloids, including (E)-seneciphylline, seneciphylline, integerrimine, senecionine, and seneciphyllinine, were prepared from G. japonica roots with high efficiency. The results of this work provide a new technique for the preparation of large amounts of pyrrolizidine alkaloid reference substances, which will also benefit toxicological studies of pyrrolizidine alkaloids and treatments for pyrrolizidine alkaloid-induced toxicity.

Endophytic fungus from Gynura japonica: phytochemical screening, biological activities, and characterisation of its bioactive compound.

The research aims to identify the chemical constituents of endophytic fungi associated with Gynura japonica and their biological activities. Two endophytic fungi, labelled as GS-1 and GS-2, have been isolated from the leaves of G. japonica. They were cultivated on white rice media for their optimum cultivation time. Cultivated fungi were extracted with ethyl acetate and concentrated to give a crude extract. All crude extracts were evaluated for their phytochemical screening and assayed for their antibacterial and antioxidant activities. Based on the results, fungal GS-1 was the most potential fungus to produce bioactive secondary metabolites and identified as Dimorphiseta acuta. A bioactive compound was isolated from crude extract of fungal D. acuta and identified as emodin. To the best of our knowledge, the study of secondary metabolite and its biological activity of endophytic fungus colonised with the leaves of G. japonica is reported here for the first time.

Exposure to Gynura japonica (Thunb.) Juel plants induces hepatoxicity in rats and Buffalo rat liver cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Gynura japonica (Thunb.) Juel is often confused with the non-pyrrolizidine alkaloid-producing herbs, Tu-San-Qi (Sedum aizoon L.) and San-Qi (Panax notoginseng L.), due to similarities in name, appearance, and medicinal use. It contains pyrrolizidine alkaloids, which cause over 50% of cases of hepatic sinus obstruction syndrome. However, the mechanisms underlying G. japonica-induced hepatotoxicity remain poorly understood. AIM OF THE STUDY: In this study, we aimed to investigate the toxic effects of a G. japonica decoction on liver and Buffalo rat liver (BRL) cells and elucidate the associated mechanisms. MATERIALS AND METHODS: This study employed G. japonica decoction and examined its effects on liver function and tissue damage in Sprague-Dawley rats. Bioinformatics analysis was employed to identify gene expression and enriched pathways related to hepatotoxicity. Laser scanning confocal microscopy and flow cytometric annexin V/PI labeling assays were utilized to observe apoptosis in BRL cells induced by G. japonica. Transmission electron microscopy and JC-1 staining were used to determine the effects of G. japonica on mitochondrial ultrastructure and membrane potential in BRL cells. The bicinchoninic acid method and enzyme-linked immunosorbent assays were used to detect the expression of apoptosis-related proteins and caspase-3 activity, respectively. RESULTS: Comparisons of body weight, liver histopathology, and serum liver function-related indices in rats, t showed that exposure to G. japonica may cause liver damage. Bioinformatics analysis indicated that hepatotoxicity might be related to apoptotic signaling pathways, the positive regulation of programmed cell death, and responses to toxic substances. BRL cells exposed to the G. japonica decoction exhibited mid-to late-stage apoptosis and necrosis, along with alterations in mitochondrial morphology and membrane potential. Furthermore, expression of cytochrome C (Cyt C) and pro-apoptotic proteins was increased, anti-apoptotic proteins decreased, and caspase-3 activity elevated. CONCLUSIONS: These findings indicate that G. japonica-induced hepatotoxicity involves the activation of mitochondria-mediated apoptosis. Our research enhances the scientific and theoretical foundation for clinical therapy and improves public awareness of the potential toxicity of herbal remedies.

The complete chloroplast genome of Gynura japonica and its phylogenetic implications.

Gynura japonica (Asteraceae) is a folk herbal medicine with multi-pharmacological functions involving analgesic, hemostatic and antiangiogenic activities. The study was conducted to assemble the complete chloroplast (cp) genome of G. japonica through a genome-skimming approach. The assembled cp genome was 151,023 bp in size, with 62.8% AT content, consisting of a large single copy (LSC) of 83,185 bp, two copies of inverted repeat (IRa and IRb) of 24,847 bp, and a small single copy (SSC) of 18,144 bp. The cp genome of G. japonica contained 133 genes, including eight ribosomal RNA genes (rRNAs), 37 transfer RNA genes (tRNAs), 86 protein-coding genes (PCGs), and two pseudogenes (ψycf1 and ψrps19). Our phylogenomic analysis based on whole plastid genomes strongly supports G. japonica is a sister to the clade including Crassocephalum crepidioides and Jacobaea vulgaris.

Blood microRNA Signatures Serve as Potential Diagnostic Biomarkers for Hepatic Sinusoidal Obstruction Syndrome Caused by Gynura japonica Containing Pyrrolizidine Alkaloids.

Background and Aims: The Gynura japonica-induced hepatic sinusoidal obstruction syndrome (HSOS) is closely related to pyrrolizidine alkaloids (PAs), and its prevalence has been increasing worldwide in recent years. However, no effective therapy for PA-induced HSOS in clinics is available, partially due to the failure of quick diagnosis. This study aims to identify blood microRNA (miRNA) signatures as potential biomarkers for PA-induced HSOS in clinics. Methods: The microarray-based miRNA profiling was performed on blood samples of the discovery cohort, which consisted of nine patients with HSOS and nine healthy donors. Differentially expressed miRNAs were further confirmed using a validation cohort, which consisted of 20 independent patients with HSOS. In addition, the rat model was established through the oral administration of the total alkaloid extract from G. japonica to investigate the association of miRNA biomarkers with the progression of HSOS. Bioinformatic analyses, including GO and KEGG enrichment, receiver operating characteristics curve, and correlation analyses were conducted to evaluate the accuracy of the potential miRNA biomarkers. Results: Three miRNAs, namely miR-148a-3p, miR-362-5p, and miR-194-5p, were overexpressed in patients and rats with PA-induced HSOS. These miRNAs were positively related to the severity of liver injury and displayed considerable diagnostic accuracy for patients with HSOS with areas under the curve over 0.87. Conclusion: In summary, this study demonstrated that three miRNAs, hsa-miR-148a-3p, hsa-miR-362-5p, and hsa-miR-194-5p, might serve as potential biomarkers for PA-induced HSOS in clinics.

Rapid identification and determination of pyrrolizidine alkaloids in herbal and food samples via direct analysis in real-time mass spectrometry.

Pyrrolizidine alkaloids (PAs) are naturally occurring plant toxins associated with severe liver damage if excessive ingestion. Herein, a novel analytical strategy on utilizing direct analysis in real-time mass spectrometry (DART-MS) was developed, and applied in analysis of six representative PAs. The calibration curves in the range of 10-1000 ng·mL-1 were established, and relative standard deviations (RSDs) were less than 10%. The limits of detection (LODs) and limits of quantitation (LOQs) were 0.55-0.85 ng·mL-1 and 1.83-2.82 ng·mL-1, respectively. The feasibility of method was indicated by analysing real samples including Gynura japonica, drug tablets, granules, and fresh cow's milk. Moreover, the results of DART-MS were in good agreement with those observed by high performance liquid chromatography mass spectrometry (HPLC-MS), but consumed less time without chromatographic separation. This research provides a facile fashion for safety assessment of herbal and food products containing PAs and presents promising applications in food, pharmaceutical and clinical analysis.

Pyrrolizidine Alkaloid-Induced Hepatotoxicity Associated with the Formation of Reactive Metabolite-Derived Pyrrole-Protein Adducts.

Pyrrolizidine alkaloids (PAs) with 1,2-unsaturated necine base are hepatotoxic phytotoxins. Acute PA intoxication is initiated by the formation of adducts between PA-derived reactive pyrrolic metabolites with cellular proteins. The present study aimed to investigate the correlation between the formation of hepatic pyrrole-protein adducts and occurrence of PA-induced liver injury (PA-ILI), and to further explore the use of such adducts for rapidly screening the hepatotoxic potency of natural products which contain PAs. Aqueous extracts of Crotalaria sessiliflora (containing one PA: monocrotaline) and Gynura japonica (containing two PAs: senecionine and seneciphylline) were orally administered to rats at different doses for 24 h to investigate PA-ILI. Serum alanine aminotransferase (ALT) activity, hepatic glutathione (GSH) level, and liver histological changes of the treated rats were evaluated to assess the severity of PA-ILI. The levels of pyrrole-protein adducts formed in the rats' livers were determined by a well-established spectrophotometric method. The biological and histological results showed a dose-dependent hepatotoxicity with significantly different toxic severity among groups of rats treated with herbal extracts containing different PAs. Both serum ALT activity and the amount of hepatic pyrrole-protein adducts increased in a dose-dependent manner. Moreover, the elevation of ALT activity correlated well with the formation of hepatic pyrrole-protein adducts, regardless of the structures of different PAs. The findings revealed that the formation of hepatic pyrrole-protein adducts-which directly correlated with the elevation of serum ALT activity-was a common insult leading to PA-ILI, suggesting a potential for using pyrrole-protein adducts to screen hepatotoxicity and rank PA-containing natural products, which generally contain multiple PAs with different structures.

The long persistence of pyrrolizidine alkaloid-derived pyrrole-protein adducts in vivo: Kinetic study following multiple exposures of a pyrrolizidine alkaloid containing extract of Gynura japonica.

Gynura japonica (also named Tusanqi in Chinese) is used as a folk herbal medicine for treating blood stasis or traumatic injury. However, hundreds of hepatic sinusoidal obstruction syndrome (HSOS) cases have been reported after consumption of preparations made from G. japonica because it contains large amounts of hepatotoxic pyrrolizidine alkaloids (PAs). To date, blood pyrrole-protein adducts (PPAs) are suggested as biomarkers for the diagnosis of PA-induced HSOS in clinics. However, the concentration of PPAs in the blood is greatly affected by several factors including the amount of PA exposure, herb intake period, and blood sampling time after the last exposure. In present study, the kinetic characters of PPAs in serum and liver as well as other potential target organs were studied systematically and comprehensively following multiple exposures of PAs in G. japonica extract (GJE). As results, PPAs content reached to a plateau both in serum and liver after the mice were treated with GJE for 2 weeks on daily basis. PPAs cleared significantly slower in liver (T1/2ke∼184.6 h, ∼7.7 days) than in serum (T1/2ke∼95.8 h, ∼4.0 days). Although more than 90 % PPAs were removed 2 weeks after the last dosing, PPAs still persisted in the liver until the end of the experiment, i.e. 8 weeks after the last dosing. The results would be of great help for understanding the importance of PPAs for PA-induced toxicity and its detoxification.

Comparative analysis of toxic components in different medicinal parts of Gynura japonica and its toxicity assessment on mice.

BACKGROUND: The roots of Gynura japonica are used as traditional medicine for treating blood stasis or traumatic injury even though hundreds of hepatic sinusoidal obstruction syndrome cases have been reported after consumption of the roots, which contain large amounts of hepatotoxic pyrrolizidine alkaloids (HPAs). However, no information is available about the toxic compounds in the aerial parts of G. japonica, which are also used as herbal medicines and even vegetables in several areas. Thus, the toxic chemicals in the aerial parts of G. japonica, i.e., HPAs, must be urgently identified. PURPOSE: In this study, we aimed to 1) identify the toxic compounds in different medicinal parts and 2) examine the hepatotoxicity of G. japonica. STUDY DESIGN: A total of 35 batches of the roots and aerial parts of G. japonica were collected from different sources and analyzed for HPAs. The hepatotoxicity of different extracts (i.e., total extracts [TE] and total alkaloids [TA]) and a single compound (i.e., senecionine) was evaluated on mice. METHODS: Qualitative analysis of HPAs was performed using an ultra-performance liquid chromatography (UPLC)-mass spectrometry (MS)-parent ion scan approach, whereas a quantitative assay was performed by a UPLC-MS-selected ion monitoring approach. Male C57BL mice were orally administered the different extracts or the single compound at dosages equivalent to 50  mg HPAs/kg body weight. The sera and the livers were collected at 48  h after treatment and used to evaluate the hepatotoxicity through serum clinical biomarkers assay, liver histology, and bile acid profiling. RESULTS: A total of 21 HPAs were identified in the roots and the aerial parts. The roots contained higher levels of HPAs (4.90  mg/g) than did the aerial parts (2.21 mg/g). TE and TA induced similar acute liver injuries, but senecionine was considerably more toxic than these extracts. Mice treated with TE showed significantly impaired bile acid homeostasis in the sera and the livers. CONCLUSION: The roots and aerial parts of G. japonica contained large amounts of HPAs, including senecionine, which were responsible for the hepatotoxicity of G. japonica. Bile acid homeostasis was uniquely impaired after exposure to the plant. Therefore, neither the roots nor the aerial parts of G. japonica should be consumed as medicines or vegetables.

Study on quality standard of Yi medicine Gynura japonica and quality evaluation of medicinal materials from different producing areas / 中国药房

OBJECTIVE To improve the quality standard of Yi medicine Gynura japonica， and to evaluate its quality. METHODS Using 15 batches of G. japonica from different producing areas as samples， the contents of water， total ash， acid- insoluble ash and water-soluble extract were determined according to the method stated in part Ⅳ of Chinese Pharmacopoeia （2020 edition）. The contents of total alkaloid （calculated by senecionine） was determined by UV spectrophotometry. The contents of senecionine and seneciphylline were determined by HPLC. Using above 7 indexes as evaluation indexes， cluster heat map analysis， principal component analysis （PCA） and entropy weight approximation ideal ranking （TOPSIS） were used to evaluate the quality of medicinal material comprehensively. RESULTS Among 15 batches of G. japonica， the moisture contents were 8.88%-12.60%， the total ash contents were 4.43%-11.02%， the acid-insoluble ash contents were 0.56%-3.45%， the water-soluble extract contents were 21.71%-53.91%， the total alkaloid contents （calculated by senecionine） were 0.15%-0.39%， and the contents of senecionine and seneciphylline were 0.01% -0.05% and 0.01%-0.06% respectively. According to the results of various indicators， it was preliminarily proposed that the water content in the sample of G. japonica should not exceed 13.00%， the total ash content should not exceed 11.50%， the acid-insoluble ash content should not exceed 3.70%， the water-soluble extract should not be less than 20.70%， the total alkaloid content should not be less than 0.15%， the contents of senecionine and seneciphylline should not be less than 0.01% both. The results of cluster heat map analysis showed that the 15 batches of samples could be divided into four categories； the results of PCA and TOPSIS showed that the samples with high-quality ranking were jsq-2， jsq-5， jsq-6 and jsq-10， and the samples with low-quality ranking were jsq-4， jsq-13 and jsq-14. CONCLUSIONS In this study， the quantitative analysis method of total alkaloids （calculated by senecionine）， senecionine and seneciphylline in G. japonica is established， and the limits of each index are preliminarily determined. Among 15 batches of samples， the qualities of medicinal material collected from Linza Village of Ganluo County of Liangshan Yi Autonomous Prefecture， Machangping Village of Luojishan Town of Puge County of Liangshan Yi Autonomous Prefecture and other places are better.

Kinetic study of pyrrolizidine alkaloid-derived pyrrole-protein adducts in rats after intragastric administration of Gynura japonica / 药学学报

Recently, hepatic sinusoidal obstruction syndrome (HSOS) induced by misuse of Gynura japonica has increased and gained global attention. Large amounts of pyrrolizidine alkaloids (PAs) are present in G. japonica; these PAs are metabolically activated to generate pyrrole-protein adducts (PPAs). In this study, male SD rats were treated orally with a single dose of G. japonica extract (GJE) at 0.062 5, 0.25, 0.5, 1, and 2 g·kg-1. Blood was collected from the orbital venous plexus at 2, 12, 24 and 48 h, and at 48 h after treatment the rats were anesthetized with isoflurane and livers were collected for hematoxylin & eosin staining. The kinetics of PPAs at different doses were studied at 10, 20, 30 min, 1, 2, 4, 6, 12, 24 h, and 48 h, after a single gavage of GJE. The experimental scheme was approved by the ethics committee of animal experiments of Shanghai University of Traditional Chinese Medicine (PZSHUTCM190912019). The concentration of PPAs in serum was determined by liquid chromatography-mass spectrometry (LC-MS). Kinetic data were processed by using the non-compartmental pharmacokinetics data analysis software program PK solutions 2™. The results demonstrate that the concentration of PPAs increased with the dose of GJE and positively correlated with the severity of liver injury. The elimination rate of PPAs in rats was significantly prolonged at higher doses. The level of PPAs and their clearance rate may serve as useful references for the detoxification of PAs-induced injuries.