Aristolochia Species Research Articles

Effects of bioconversion by Battus polydamas on the chemical composition of Aristolochia spp. and evaluation of antimicrobial activity and biocompatibility

Aristolochia plants are emblematic from an ethnopharmacological viewpoint and are know to possess numerous biological properties, including antiseptic. However, the medicinal potential of these species is debatable because of their representative chemical constituents, aristolochic acids (AAs) and aristolactams (ALs), which are associated, for instance, with nephropathy and cancer. These contrasting issues have stimulated the development of approaches intended to detoxification of aristoloquiaceous biomasses, among which is included the bioconversion method using larvae of the specialist phytophagous insect Battus polydamas, previously shown to be viable for chemical diversification and to reduce toxicity. Thus, eleven Aristolochia spp. were bioconverted, and the antimicrobial activities of the plant methanolic extracts and its respective bioconversion products were evaluated. The best results were found for Aristolochia esperanzae, Aristolochia gibertii, and Aristolochia ringens against Bacillus cereus, with MIC ranging from 7.8 to 31.25 μg/mL. These three species were selected for chemical, antioxidant, cytotoxic, hemolytic, and mutagenic analyses. Chemical analysis revealed 65 compounds, 21 of them possible bioconversion products. The extracts showed potential to inhibit the formation and degradation of B. cereus biofilms. Extracts of A. gibertii and its bioconverted biomass showed antioxidant activity comparable to dibutylhydroxytoluene (BHT) standard. Bioconversion decreased the hemolytic activity of A. esperanzae and the cytotoxicities of A. esperanzae and A. gibertii. None of the extracts was found to be mutagenic. The bioactivities of the fecal extracts were maintained, and biocompatibility was improved. Therefore, the results obtained in this study reveal positive expectations about the natural detoxification process of the Aristolochia species.

Two Novel Betarhabdovirins Infecting Ornamental Plants and the Peculiar Intracellular Behavior of the Cytorhabdovirus in the Liana Aristolochia gibertii.

Two novel members of the subfamily Betarhabdovirinae, family Rhabdoviridae, were identified in Brazil. Overall, their genomes have the typical organization 3'-N-P-P3-M-G-L-5' observed in mono-segmented plant-infecting rhabdoviruses. In aristolochia-associated cytorhabdovirus (AaCV), found in the liana aristolochia (Aristolochia gibertii Hook), an additional short orphan ORF encoding a transmembrane helix was detected between P3 and M. The AaCV genome and inferred encoded proteins share the highest identity values, consistently < 60%, with their counterparts of the yerba mate chlorosis-associated virus (Cytorhabdovirus flaviyerbamate). The second virus, false jalap virus (FaJV), was detected in the herbaceous plant false jalap (Mirabilis jalapa L.) and represents together with tomato betanucleorhabdovirus 2, originally found in tomato plants in Slovenia, a tentative new species of the genus Betanucleorhabdovirus. FaJV particles accumulate in the perinuclear space, and electron-lucent viroplasms were observed in the nuclei of the infected cells. Notably, distinct from typical rhabdoviruses, most virions of AaCV were observed to be non-enclosed within membrane-bounded cavities. Instead, they were frequently seen in close association with surfaces of mitochondria or peroxisomes. Unlike FaJV, AaCV was successfully graft-transmitted to healthy plants of three species of the genus Aristolochia, while mechanical and seed transmission proved unsuccessful for both viruses. Data suggest that these viruses belong to two new tentative species within the subfamily Betarhabdovirinae.

Herbal Nephropathy and Balkan Endemic Nephropathy: The Pathogenic Role of Aristolochic Acid

Aristolochic acid nephropathy is a worldwide iatrogenic disease affecting individuals consuming herbal remedies derived from Aristolochia and Asarum species. The disease is characterized by prominent renal atrophy and extensive interstitial fibrosis. A considerable proportion of patients, about 30-45 % develop also transitional cell carcinoma mainly of the upper urinary tract. A special attention to the disease was paid in early 90’s because of an epidemic of rapidly progressive interstitial nephropathy affecting young women consuming slimming pills, containing Chinese herbs, supplied from the same clinic in Brussels, Belgium. Detailed investigation of slimming pills showed that they inadvertently contained Aristolochic acid considered as the causative factor of the disease. Soon thereafter it was showed that another devastating chronic interstitial nephropathy accompanied also with increased incidence of upper urothelial cancer, known as Balkan Endemic Nephropathy, is the result of chronic intoxication of affected individuals with low doses of Aristolochic acid derived from the soil contamination from the plant Aristolochia clematitis. Although Aristolochic acid has already characterized as class I carcinogen for humans, Aristolochia species continues to be used as herbal remedies especially in Asia countries and China as well as in westernized communities. It is noteworthy that although Aristolochia species are used as herbal remedies since antiquity there are no references for nephrotoxicity and carcinogenicity until the Belgium epidemic. In this review article we attempt to elucidate the historical evolution of our knowledge upon the etiology of Aristolochic acid nephropathy as well as the underlying mechanisms of Aristolochic acid cytotoxicity.

Aristolochia manshuriensis and Aristolochia contorta in the collection of the Botanical Garden-Institute FEB RAS

Ex situ is a reliable way to conservation rare plants of native flora outside of populations. There are two species of Aristolochia (Aristolochiaceae) in the collection of the Botanical Garden-Institute of the Far East Branch RAS. Aristolochia manshuriensis is included in the Red Data Book of Russian Federation and the Red Data Book of the Primorsky Krai. Aristolochia contorta is included in the Red Data Book of the Primorsky Krai and the Red Data Book of the Jewish Autonomous Region. To assess the introduction success we studied individual and seasonal development, flowering, productivity, and reproduction. A. manshuriensis begins to bloom and bear the fruit in 6-8 years, A. contorta in 3-4 years after sowing the seeds. Seasonal development cycle is terminated by fructification. Fruits are produced each year. Life duration of individual flower is 7-9 days. The long life of the flower and the long flowering period of the individual increase the chance of cross-pollination. The high factual seed set on the fruit has A. manshuriensis - from 76 ± 14 to 134 ± 10, more 80% of them are great vitality, freshly-harvested seed germination is 80-85 %. The factual seed set on the fruit of A. contorta is 129 ± 22 seeds of which intact and capable to germinate about 50 %. When sowing seeds in autumn, the germination is 40 ± 3 %. The studied species are perspective for reproduction and conservation in culture.

Assessment of drought resistance in plants of the genus Aristolochia L.

The examination of plant resistance to drought is becoming increasingly relevant due to global warming. All species of the genus Aristolochia L. possess high decorative qualities and play an important role in natural ecosystems. However, under conditions of temperature imbalance, their ornamental value may decrease due to leaf turgor loss and even leaf shedding. The purpose of the paper is to highlight the results of studies on the drought resistance assessment of Aristolochia L. plant species to identify the most promising taxa suitable for cultivation in the city of Kyiv. The study was conducted in 2022 on three species of vines: Aristolochia macrophylla Lam., A. tomentosa Sims., A. manshuriensis Kom., which grow in the M.M. Gryshko National Botanical Garden (Kyiv). The study presented results on leaf hydration parameters, water deficit, water-holding capacity of leaves, electrical conductivity, and specific leaf area conducted in the Plant Physiology and Microbiology Laboratory of the Institute of Horticulture, National Academy of Agrarian Sciences of Ukraine. Field assessment of Aristolochia L. plant drought resistance was also conducted. It was established that Aristolochia macrophylla Lam. is the most drought-resistant species, exhibiting low transpiration rates and high water-holding capacity. Aristolochia tomentosa Sims. it is the least drought-resistant species, which therefore needs additional care. In field conditions, significant signs of wilting were not observed, indicating that leaves reduce turgor during the day and recover at night. It is also important to preserve the environment in which these plants grow, as its changes affect the drought tolerance and life cycle of lianas. The results of the drought resistance research on Aristolochia L. plants point to the potential of these species. They can be used for selecting plant assortments for creating various types of plantations in urban environments

Generation of a high-affinity DNA aptamer for on-site screening of toxic aristolochic acid I in herbal medicines and botanical products

Aristolochic Acid I (AAI) is an environmental and foodborne toxin found in the Aristolochia and Asarum species of plants that are widespread all over the world. Therefore, there is an urgent need to develop a sensitive and specific biosensor for identifying AAI. Aptamers as a powerful biorecognition element provide the most viable options for solving this problem. In this study, we used library-immobilized SELEX to isolate an AAI-specific aptamer with a KD value of 86 ± 13 nM. To verify the practicability of the selected aptamer, a label-free colorimetric aptasensor was designed. This aptasensor exhibited a low detection limit of 225 nM. Besides, it had been further applied for the determination of AAI in real samples and the recoveries ranged from 97.9% to 102.4%. In the future, AAI aptamer will provide a promising tool for safety evaluation in various fields of agriculture, food, and medication.

Critical Appraisal of Īśvarī (Aristolochia indica L.) in Viṣa Chikitsa with Respect to Classical Ayurveda and Traditional Texts on Toxicology

Abstract Background: Īśvarī, Aristolochia indica, belonging to the Aristolochiaceae family is a big climber. Aristolochia species have been used widely in traditional viṣa vaidya. The herb is known for its therapeutic efficacy in snake bite cases. Studies indicate that Īśvarī possesses antioxidant, anti-inflammatory, abortifacient, analgesic, antidiarrheal activity, cytotoxic activity, hypoglycemic activity, antibacterial activity, pesticidal activity, mast cell-stabilizing, and antipruritic properties. Studies also proved the anti-ophidian, anti-scorpion, and antimalarial properties of Īśvarī. Objective: This review is aimed at providing comprehensive information regarding Īśvarī in classical Ayurveda books and traditional viṣa vaidya texts. Materials and Methods: Data on Īśvarī were collected from 11 Nighaṇḍu (lexicons), 10 Saṃhita and Chikitsa grantha, and 4 Viṣa chikitsa grantha. In addition, the information is gathered from indexed databases (Google Scholar and PubMed) for evaluating the efficacy of Īśvarī in poisoning cases. Results: In saṃhitas, Īśvarī is mentioned as an ingredient in only six formulations. However, in the Viṣa chikitsa grantha, we can see a wide application of this plant as a single medicine or in combination. Īśvarī is an ingredient in 32 main formulations in Viṣa chikitsa grantha. Conclusion: In Viṣa chikitsa, the herbal medicine Īśvarī has a miraculous healing role. This herb was widely implemented by the ancient viṣa vaidyas for healing the effects of poisoning.

De novo assembly of a chromosome-level reference genome of the ornamental butterfly Sericinus montelus based on nanopore sequencing and Hi-C analysis.

Sericinus montelus (Lepidoptera, Papilionidae, Parnassiinae) is a high-value ornamental swallowtail butterfly species widely distributed in Northern and Central China, Japan, Korea, and Russia. The larval stage of this species feeds exclusively on Aristolochia plants. The Aristolochia species is well known for its high levels of aristolochic acids (AAs), which have been found to be carcinogenic for numerous animals. The swallowtail butterfly is among the few that can feed on these toxic host plants. However, the genetic adaptation of S. montelus to confer new abilities for AA tolerance has not yet been well explored, largely due to the limited genomic resources of this species. This study aimed to present a chromosome-level reference genome for S. montelus using the Oxford Nanopore long-read sequencing, Illumina short-read sequencing, and Hi-C technology. The final assembly was composed of 581.44Mb with an expected genome size of 619.27Mb. Further, 99.98% of the bases could be anchored onto 30 chromosomes. The N50 of contigs and scaffolds was 5.74 and 19.12Mb, respectively. Approximately 48.86% of the assembled genome was suggested to be repeat elements, and 13,720 protein-coding genes were predicted in the current assembly. The phylogenetic analysis indicated that S. montelus diverged from the common ancestor of swallowtails about 58.57-80.46 million years ago. Compared with related species, S. montelus showed a significant expansion of P450 gene family members, and positive selections on eloa, heatr1, and aph1a resulted in the AA tolerance for S. montelus larva. The de novo assembly of a high-quality reference genome for S. montelus provided a fundamental genomic tool for future research on evolution, genome genetics, and toxicology of the swallowtail butterflies.

Insights into taxonomy and phylogenetic relationships of eleven Aristolochia species based on chloroplast genome.

The Aristolochia, as an important genus comprised of over 400 species, has attracted much interest because of its unique chemical and pharmacological properties. However, the intrageneric taxonomy and species identification within Aristolochia have long been difficult because of the complexity of their morphological variations and lack of high-resolution molecular markers. In this study, we sampled 11 species of Aristolochia collected from distinct habitats in China, and sequenced their complete chloroplast (cp) genomes. The 11 cp genomes of Aristolochia ranged in size from 159,375bp (A. tagala) to 160,626 bp (A. tubiflora), each containing a large single-copy (LSC) region (88,914-90,251 bp), a small single-copy (SSC) region (19,311-19,917 bp), and a pair of inverted repeats (IR) (25,175-25,698 bp). These cp genomes contained 130-131 genes each, including 85 protein-coding genes (CDS), 8 ribosomal RNA genes, and 37-38 transfer RNA genes. In addition, the four types of repeats (forward, palindromic, reverse, and complement repeats) were examined in Aristolochia species. A. littoralis had the highest number of repeats (168), while A. tagala had the lowest number (42). The total number of simple sequence repeats (SSRs) is at least 99 in A. kwangsiensis, and, at most, 161 in A. gigantea. Interestingly, we detected eleven highly mutational hotspot regions, including six gene regions (clpP, matK, ndhF, psbT, rps16, trnK-UUU) and five intergenic spacer regions (ccsA-ndhD, psbZ-trnG-GCC, rpl33-rps18, rps16-trnQ-UUG, trnS-GCU-trnG-UCC). The phylogenetic analysis based on the 72 protein-coding genes showed that 11 Aristolochia species were divided into two clades which strongly supported the generic segregates of the subgenus Aristolochia and Siphisia. This research will provide the basis for the classification, identification, and phylogeny of medicinal plants of Aristolochiaceae.

Pollination biology of Aristolochia bianorii Sennen & Pau: promoting cross-pollination but assuring reproductive success in island ecosystems.

Deceptive pollination has been reported in the genus Aristolochia, but the floral biology and pollination strategy of A. bianorii, an endemic of the Balearic Islands, have not yet been studied. Here, we investigated floral anthesis, mating system, pollinators and volatile organic compounds (VOCs) emitted by its flowers. Flower buds were marked and monitored daily to define floral stages and their duration. Experimental bagging and hand-pollination were performed to test for autonomous self-pollination, induced self-pollination and cross-pollination. Flowers were collected to analyse the presence of entrapped pollinators. VOCs emitted by flowers were evaluated by means of solid phase microextraction followed by immediate GC-MS. Anthesis lasted between 63 and 96 h, and the species exhibited autonomous self-pollination with moderate inbreeding depression. Pollinators were mainly females of Oscinomorpha longirostris (Diptera; Chloropidae). The number of pollinators inside flowers was affected by floral stage and time of flowering. The most common VOCs were alkanes, oximes, esters, alkenes, cyclic unsaturated hydrocarbons, isocyanates, amides and carboxylic acids. Aristolochia bianorii can set seed by autonomous self-pollination, in contrast to other Aristolochia species, in which both protogyny and herkogamy prevent autonomous self-pollination. However, the species may encourage cross-pollination by attracting female chloropid flies though emission of floral scents that may mimic an oviposition site and, possibly, freshly killed true bugs (i.e. Heteroptera). In conclusion, A. bianorii promotes cross-pollination, but delayed autonomous self-pollination assures reproductive success in the putative absence of pollinators.

Assessment of the Cytotoxicity, Mutagenicity, and Genotoxicity of Two Traditional Chinese Herbs: Aristolochia baetica and Magnolia officinalis.

Herbal remedies used in traditional medicine often contain several compounds combined in order to potentiate their own intrinsic properties. However, herbs can sometimes cause serious health troubles. In Belgium, patients who developed severe aristolochic acid nephropathy ingested slimming pills containing root extracts of an Aristolochia species, as well as the bark of Magnolia officinalis. The goal of the study was to evaluate, on a human renal cell line, Aristolochia and Magnolia extracts for their cytotoxicity by a resazurin cell viability assay, and their genotoxicity by immunodetection and quantification of the phosphorylated histone γ-H2AX. The present study also sought to assess the mutagenicity of these extracts, employing an OECD recognized test, the Ames test, using four Salmonella typhimurium strains with and without a microsomial fraction. Based on our results, it has been demonstrated that the Aristolochia-Magnolia combination (aqueous extracts) was more genotoxic to human kidney cells, and that this combination (aqueous and methanolic extracts) was more cytotoxic to human kidney cells after 24 and 48 h. Interestingly, it has also been shown that the Aristolochia-Magnolia combination (aqueous extracts) was mutagenic with a TA98 Salmonella typhimurium strain in the presence of a microsomial liver S9 fraction. This mutagenic effect appears to be dose-dependent.

Aristolochia stevechurchillii, a new pseudostipule-bearing Aristolochiaceae (Piperales) from Bolivia, with a key to identify the species of the ´Pseudostipulosae´ group in the country

A new pseudostipule-bearing species of Aristolochia, A. stevechurchillii, is here described and illustrated, along with a key to identify all the species of the informal ´Pseudostipulosae´ group naturally occurring in Bolivia. The new species differs from its closely related species by the formation of two serial flowers per axil, the base of the utricle with six retrorse digitiform processes, and the perianth limb narrowly ovate, with acute apex. Along with A. andina and A. lozaniana, A. stevechurchillii is the third species of the genus endemic from mid-elevation (1600-2500 m) Tucumano-Bolivian forests of the departments of Cochabamba, Chuquisaca and Tarija (Bolivia). The new species is morphologically similar to A. melanoglossa, from northern Argentina, which provides additional evidence to floristic affinities previously proposed between the Tucumano-Bolivian forests and the Chaco vegetation.

Xylem Development and Xylem Conductivity of Furrowed Xylem in Stem Terminals of Five Species of Aristolochia (Aristolochiaceae)

Xylem cells are responsible for water transport and mechanical support in most plants. Since vines depend upon other plants for mechanical support, the main responsibility of xylem cells in vines is primarily water transport and not mechanical support. The purpose of the current study was to study xylem characteristics in stems of five species of Aristolochia. Tissue samples from about 1.0 to 5.5 mm in diameter were processed with standard histological techniques. Anatomical characteristics were similar among all five species. Results show: (1) interfascicular cambia were not present, so stems had only furrowed xylem, (2) numbers of vascular bundles in stems were specific for each species and did not increase as stems enlarged, (3) radii of vessels were not dimorphic for any species, (4) numbers of vessels were linearly related with stem diameters, and (5) the largest half of all vessels supplied 95% of total xylem conductivity. To our knowledge, this is the first publication to document the development of furrowed xylem, describing both vessel characteristics and xylem conductivities in stems of Aristolochia species.

AGRYPON POLYXENAE (SZÉPLIGETI, 1899) (HYMENOPTERA: ICHNEUMONIDAE: ANOMALONINAE); NEWLY RECORDED PARASITOID OF ZERYNTHIA POLYXENA (DENIS &amp; SCHIFFERMÜLLER, 1775) (LEPIDOPTERA: PAPILIONIDAE: PARNASSIINAE) IN THE FAUNA OF SERBIA

Ichneumonid Agrypon polyxenae is a solitary larval-pupal endoparasitoid developing within the caterpillars of the genus Zerynthia and distributed in the western Palearctic. Z. polyxena is the most common host, with its caterpillars feeding on highly poisonous Aristolochia species. Here we report A. polyxenae parasitising Z. polyxena for the first time in Serbia and provide a short species description. This parasitoid has also been recorded on Z. rumina in Spain. We suggest some evidence for its presence in the newly described Z. cassandra in Italy.

Genetic diversity assessment and biotechnological aspects in Aristolochia spp.

Aristolochia, belonging to the family Aristolochiaceae, has immense ecological significance due to its large size and huge geographic distribution. In the context of dealing with a genus with a huge number of species like Aristolochia, these markers come in handy to precisely identify a particular species and enumerate the genetic diversity. Also, certain species of Aristolochia are economically important due to the presence of secondary metabolites and vast use in traditional and modern medicine. But, the presence of profitable biochemical constituents in Aristolochia is very low and the breeding process of the plant is highly dependable on pollinators. Hence, identifying different biotechnological approaches to fasten the reproductive cycle of Aristolochia and increase the secondary metabolites is of great interest to the researchers. In this study, a comprehensive review has been established on different types of morphological/anatomical markers (starch grains with "Maltese cross"), phytochemical markers (aristolochic acid, triterpenoid, aristolactam etc.) and genetic markers (ISSR, SSR, DNA bar-coding) for various Aristolochia spp. We have also discussed the applications of different biotechnological tools in Aristolochia spp. which include discrete approaches to promote in vitro germination, in vitro shooting, root induction, somatic embryogenesis, synthetic seed production, acclimatization and hardening and sustainable production of secondary metabolites. In a nutshell, the present review is a first of kind approach to comprehensively demonstrate the genetic diversity studies and biotechnological aspects in Aristolochia spp. KEY POINTS: • Insights into the in vitro propagation of Aristolochia spp. • In vitro production and optimization of secondary metabolites. • Assessment of genetic diversity by molecular markers.

A PCR-lateral flow immunochromatographic assay (PCR-LFA) for detecting Aristolochia species, the plants responsible for aristolochic acid nephropathy

Aristolochic acids (AAs), which are strong carcinogens, have caused dietary supplements with Aristolochia plants to be discontinued worldwide. Therefore, the development of a method to identify these herbs is critical for customer safety. To support the regulation of Aristolochia-free products, a PCR coupled with lateral flow immunochromatographic assay (PCR-LFA) that is specific to the nucleotide signature in plastid rbcL gene region of Aristolochia species was developed to detect Aristolochia plants and related herbal products. Triplex primers (A397F, C357F and R502) were designed based on specific nucleotides observed exclusively in the rbcL sequences of Aristolochia. Positive results for Aristolochia occur when the three pink lines are clearly developed on the developed lateral flow strip and can be seen by the naked eye. In this study, the lateral flow strip has sensitivity for detecting amplicons amplified from genomic DNA at the concentrations as low as 0.01 ng. Various kinds of samples, including purchased crude drugs and polyherbal samples, have been investigated, and the results showed that Aristolochia crude drugs and Aristolochia-containing products are still present in dispensaries. In conclusion, with the goal of protecting consumers from the health risks associated with Aristolochia contamination, PCR-LFA was developed and demonstrated to be efficient for detecting plants belonging to Aristolochia in various kinds of samples.

Biological Activities of Organic Extracts of the Genus Aristolochia: A Review from 2005 to 2021.

Different ethnomedicinal studies have investigated the relationship between various phytochemicals as well as organic extracts and their bioactive aspects. Studies on biological effects are attributed to secondary metabolites such as alkaloids, phenolic compounds, and terpenes. Since there have been no reviews in the literature on the traditional, phytochemical, and ethnomedicinal uses of the genus Aristolochia so far, this article systematically reviews 141 published studies that analyze the associations between secondary metabolites present in organic extracts and their beneficial effects. Most studies found associations between individual secondary metabolites and beneficial effects such as anticancer activity, antibacterial, antioxidant activity, snake anti-venom and anti-inflammatory activity. The aim of this review was to analyze studies carried out in the period 2005–2021 to update the existing knowledge on different species of the genus Aristolochia for ethnomedicinal uses, as well as pharmacological aspects and therapeutic uses.

Aristolochia thotteaeformis, a distinct new species from southern Vietnam

Aristolochia thotteaeformis, a distinct new species from southern Vietnam, is described here. The new species clearly differs from all the known Aristolochia species in Vietnam by its erect shrubby habit and zig-zag young branches. The vegetative phenotype resembles the genus Thottea (Aristolochiaceae). Morphological characters such as the 1-lipped limb, the 6-lobed gynostemium, and the flower with a stipe between the utricle and ovary place this new species in Aristolochia subgenus Aristolochia section Diplolobus subsection Podanthemum. Morphologically, Aristolochia thotteaeformis is most similar to A. philippinensis and A. humilis by sharing a cuneate to rounded leaf base, a straight perianth in pre-anthesis, then slightly curved upward, an ovoid to ellipsoid utricle, and a 5–10 mm long, cylindrical tube. However, the new species clearly differs from these two species by some morphological characters of young branches, leaves, petiole, inflorescence axis, bracteole, perianth, limb, and seed. In addition to the detailed description, illustrations, information on ecology, conservation status, and comparison with its similar species, a key to all the known species of Aristolochia subsection Podanthemum from Vietnam is provided.

Exposure to aristolochic acid I is associated with poor prognosis of liver cancer patients.

The aristolochic acids (AAs), derived from Aristolochia and Asarum species used widely in herbal medicines, are closely associated with liver cancer. The major AA derivatives are aristolochic acid I (AAI) and II (AAII), which can bind DNA covalently to form AA-DNA adducts after metabolic activation in vivo. Among all these AA-DNA adducts, 7-(deoxyadenosine-N6-yl) aristolactam I (dA-AL-I) is the most abundant and persistent DNA lesion in patients. However, the direct evidence indicating AA exposure in human liver cancer is still missing. Here, we analyzed dA-AL-I adduct, the direct biomarker of AAI exposure, by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-TQ/MS) in 209 liver cancer patients. Also, DNA samples from mice treated with/without AAI were used as positive and negative controls. dA-AL-I adduct was present in 110 of 209 (52.6%) patients, indicating that these patients were exposed to AAI prior to their clinical investigations and also had a worse prognosis. The relative high AA exposure rate and worse prognosis in our cohort of patients emphasize the significance to increase public awareness to avoid the use of herbal medicine containing AAs or their derivatives.

Quantitative Determination and Toxicity Evaluation of Aristolochic Acid Analogues in Asarum heterotropoides F. Schmidt (Xixin) and Traditional Chinese Patent Medicines.

Asarum (Xixin), which contains analogues of aristolochic acid (AA), is the only species of the genus Aristolochia included in the Chinese Pharmacopoeia 2020. However, the contents and nephrotoxic effects of AA analogs in Asarum (Xixin) and its formulations have not been clarified. An automatic, effective solid phase extraction process and UPLC-MS/MS method were established for the pretreatment and quantitative detection of AA analogues in commercially available traditional Chinese patent medicines. The cytotoxicity and DNA damage induced by five analogues of AA were evaluated by CCK8 using human kidney cells (HK-2) and comet assays. HPLC was used to detect the analogues of AA in Asarum heterotropoides F. Schmidt (Xixin). The results showed that the contents of AA I, AA II, and AA IIIa were below the detection limit, while AA IVa and AL I presented relatively high contents of Asarum heterotropoides F. Schmidt (Xixin), within the range of 66.50–121.03 μg/g and 19.73–43.75 μg/g, respectively. The levels of AA analogues were in the nanogram-per-gram level in the main traditional Chinese patent medicines. AA I and AL I exhibited relatively high cytotoxicity at 48 h in CCK8 assays, while AA II, AA IIIa, and AA IVa showed weak cytotoxicity even at 800–1,000 μM. AA I induced significant pathological alterations and direct DNA damage at 40 mg/kg and 20 mg/kg, respectively. No distinct nephrotoxicity or hepatotoxicity was observed in mice treated with AA II, AA IIIa, AA IVa, or AL I at 40 mg/kg in this study. Consumption of Asarum heterotropoides F. Schmidt (Xixin) with controlled doses and periods is relatively safe as the contents of AA analogues in Asarum heterotropoides F. Schmidt (Xixin) and its formulations were far below those causing acute toxicity in this study. But, the long-term toxicity of Asarum heterotropoides F. Schmidt (Xixin) still needs further study.