Characterization of two CYP80 enzymes provides insights into aporphine alkaloid skeleton formation in Aristolochia contorta.

Aporphine alkaloids are a large group of natural compounds with extensive pharmaceutical application prospects. The biosynthesis of aporphine alkaloids has been paid attentions in the past decades. Here, we determined the contents of four 1-benzylisoquinoline alkaloids and five aporphine alkaloids in root, stem, leaf, and flower of Aristolochia contorta Bunge, which belongs to magnoliids. Two CYP80 enzymes were identified and characterized from A. contorta. Both of them catalyze the unusual C-C phenol coupling reactions and directly form the aporphine alkaloid skeleton. AcCYP80G7 catalyzed the formation of hexacyclic aporphine corytuberine. AcCYP80Q8 catalyzed the formation of pentacyclic proaporphine glaziovine. Kingdom-wide phylogenetic analysis of the CYP80 family suggested that CYP80 first appeared in Nymphaeales. The functional divergence of hydroxylation and C-C (or C-O) phenol coupling preceded the divergence of magnoliids and eudicots. Probable crucial residues of AcCYP80Q8 were selected through sequence alignment and molecular docking. Site-directed mutagenesis revealed two crucial residues E284 and Y106 for the catalytic reaction. Identification and characterization of two aporphine skeleton-forming enzymes provide insights into the biosynthesis of aporphine alkaloids.

Aristolochia mimics stink bugs to repel vertebrate herbivores via TRPA1 activation.

Mimicry is the phenomenon in which one species (the mimic) closely resembles another (the model), enhancing its own fitness by deceiving a third party into interacting with it as if it were the model. In plants, mimicry is used primarily to gain fitness by withholding rewards from mutualists or deterring herbivores cost-effectively. While extensive work has been documented on putative defence mimicry, limited investigation has been conducted in the field of chemical mimicry. In this study, we used field experiments, chemical analyses, behavioural assays, and electrophysiology, to test the hypothesis that the birthwort Aristolochia delavayi employs chemical mimicry by releasing leaf scent that closely resembles stink bug defensive compounds and repels vertebrate herbivores. We show that A. delavayi leaf scent is chemically and functionally similar to the generalized defensive volatiles of stink bugs and that the scent effectively deters vertebrate herbivores, likely through the activation of TRPA1 channels via (E)-2-alkenal compounds. This study provides an unequivocal example of chemical mimicry in plants, revealing intricate dynamics between plants and vertebrate herbivores. Our study underscores the potency of chemical volatiles in countering vertebrate herbivory, urging further research to uncover their potentially underestimated importance.

The complete chloroplast genome of Aristolochia fangchi provided insights into the phylogeny and species identification of Aristolochia.

Aristolochia fangchi is an important species within the family Aristolochiaceae, most of which contain nephrotoxic aristolochic acid. The inadvertent use of Aristolochiaceae plants as raw ingredients in the manufacturing of patent medicine poses a significant risk warranting considerable attention. In this study, we assembled and analyzed the complete chloroplast genome of Aristolochia fangchi, which is a 159 867 bp long circular molecule. Functional annotation of the A. fangchi plastome unveiled a total of 113 genes, including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Subsequently, a series of genome structure and characteristic evaluations were conducted against the A. fangchi plastome. Further phylogenetic analysis suggested that a plausible phylogenetic relationship among Aristolochiaceae derived from the concatenated sequences of shared conserved genes rather than from the entire chloroplast genome with one IR copy. Finally, a DNA polymorphism assessment against a dozen Aristolochia plastomes yielded multiple potential regions for biomarker designation. Six pairs of primers were generated and underwent both in silico and actual PCR validations. In conclusion, this study identified the unique characteristics of the A. fangchi plastome, providing invaluable insights for further investigations on species identification and the phylogeny evolution between A. fangchi and its related species.

Aristolochia bracteolata flower extract based phytosynthesis and characterization of AgNPs: Antimicrobial, antidiabetic, and antioxidant activities potential assessment.

The study was carried out to evaluate the effectiveness of the Aristolochia bracteolata water flower extract-mediated AgNPs synthesis and assess their antimicrobial potential. According to the experimental and analytical results, A. bracteolata flower extract can produce valuable AgNPs. The characteristic features of these AgNPs were assessed with UV-visible spectrophotometer, Fourier transform-infrared spectroscopy, Transmission Electron Microscope, Scanning Electron Microscopy, as well as. Under UV-vis. spectrum results, showed major peak at 430 nm and recorded essential functional groups responsible for reducing, capping, and stabilizing AgNPs by FT-IR analysis. In addition, the size and shape of the synthesized AgNPs were found as 21.11-25.17 nm and spherical/octahedral shape. The A. bracteolata fabricated NPs showed remarkable antimicrobial activity against fish bacterial pathogens (V. parahaemolytics, Serratia sp., B. subtilis, and E. coli) as well as common fungal pathogens (A. niger, C. albicans, A. flavus, and A. terreus) at the quantity of 100 µg mL-1 than positive controls. Nevertheless, it was not effective against human bacterial pathogens. It concludes that AgNPs synthesized from A. bracteolata aqueous flower extract have excellent antimicrobial activity and may have a variety of biomedical applications.

A draft reference genome assembly of California Pipevine, Aristolochia californica Torr.

The California Pipevine, Aristolochia californica Torr., is the only endemic California species within the cosmopolitan birthwort family Aristolochiaceae. It occurs as an understory vine in riparian and chaparral areas and in forest edges and windrows. The geographic range of this plant species almost entirely overlaps with that of its major specialized herbivore, the California Pipevine Swallowtail Butterfly Battus philenor hirsuta. While this species pair is a useful, ecologically well-understood system to study co-evolution, until recently, genomic resources for both have been lacking. Here, we report a new, chromosome-level assembly of A. californica as part of the California Conservation Genomics Project (CCGP). Following the sequencing and assembly strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin proximity sequencing technology to produce a de novo assembled genome. Our genome assembly, the first for any species in the genus, contains 531 scaffolds spanning 661 megabase (Mb) pairs, with a contig N50 of 6.53 Mb, a scaffold N50 of 42.2 Mb, and BUSCO complete score of 98%. In combination with the recently published B. philenor hirsuta reference genome assembly, the A. californica reference genome assembly will be a powerful tool for studying co-evolution in a rapidly changing California landscape.

Genome-Wide Identification and Characterization of miRNAs and Natural Antisense Transcripts Show the Complexity of Gene Regulatory Networks for Secondary Metabolism in Aristolochia contorta.

Aristolochia contorta Bunge is an academically and medicinally important plant species. It belongs to the magnoliids, with an uncertain phylogenetic position, and is one of the few plant species lacking a whole-genome duplication (WGD) event after the angiosperm-wide WGD. A. contorta has been an important traditional Chinese medicine material. Since it contains aristolochic acids (AAs), chemical compounds with nephrotoxity and carcinogenicity, the utilization of this plant has attracted widespread attention. Great efforts are being made to increase its bioactive compounds and reduce or completely remove toxic compounds. MicroRNAs (miRNAs) and natural antisense transcripts (NATs) are two classes of regulators potentially involved in metabolism regulation. Here, we report the identification and characterization of 223 miRNAs and 363 miRNA targets. The identified miRNAs include 51 known miRNAs belonging to 20 families and 172 novel miRNAs belonging to 107 families. A negative correlation between the expression of miRNAs and their targets was observed. In addition, we identified 441 A. contorta NATs and 560 NAT-sense transcript (ST) pairs, of which 12 NATs were targets of 13 miRNAs, forming 18 miRNA-NAT-ST modules. Various miRNAs and NATs potentially regulated secondary metabolism through the modes of miRNA-target gene-enzyme genes, NAT-STs, and NAT-miRNA-target gene-enzyme genes, suggesting the complexity of gene regulatory networks in A. contorta. The results lay a solid foundation for further manipulating the production of its bioactive and toxic compounds.

Comparative morphoanatomy and transcriptomic analyses reveal key factors controlling floral trichome development in Aristolochia (Aristolochiaceae).

Trichomes are specialized epidermal cells in aerial plant parts. Trichome development proceeds in three stages, determination of cell fate, specification, and morphogenesis. Most genes responsible for these processes have been identified in the unicellular branched leaf trichomes from the model Arabidopsis thaliana. Less is known about the molecular basis of multicellular trichome formation across flowering plants, especially those formed in floral organs of early diverging angiosperms. Here, we aim to identify the genetic regulatory network (GRN) underlying multicellular trichome development in the kettle-shaped trap flowers of Aristolochia (Aristolochiaceae). We selected two taxa for comparison, A. fimbriata, with trichomes inside the perianth, which play critical roles in pollination, and A. macrophylla, lacking specialized trichomes in the perianth. A detailed morphoanatomical characterization of floral epidermis is presented for the two species. We compared transcriptomic profiling at two different developmental stages in the different perianth portions (limb, tube, and utricle) of the two species. Moreover, we present a comprehensive expression map for positive regulators and repressors of trichome development, as well as cell cycle regulators. Our data point to extensive modifications in gene composition, expression, and putative roles in all functional categories when compared with model species. We also record novel differentially expressed genes (DEGs) linked to epidermis patterning and trichome development. We thus propose the first hypothetical genetic regulatory network (GRN) underlying floral multicellular trichome development in Aristolochia, and pinpoint key factors responsible for the presence and specialization of floral trichomes in phylogenetically distant species of the genus.

Aristolochic acid-containing Chinese herbal medicine and upper urinary tract urothelial carcinoma in Taiwan: a narrative review.

PURPOSE: The high incidence of upper urinary tract urothelial carcinoma (UTUC) in Taiwan is largely due to exposure to aristolochic acid (AA), a principal component of Aristolochia-based herbal medicines. Here we systematically review the molecular epidemiology, clinical presentation and biomarkers associated with AA-induced UTUC. METHODS: This is a narrative review. Medline, Embase, and Web of Science were searched from inception to December 31, 2021. Studies evaluating the association, detection, and clinical characteristics of AA and UTUC were included. RESULTS: A nationwide database revealed 39% of the Taiwanese population had been exposed to AA-containing herbs between 1997 and 2003. Epidemiological reports revealed AA posed a significantly higher hazard for renal failure and UTUC in herbalists and the general population who ingested AA-containing herbs. The presence of aristolactam-DNA adducts and a distinctive signature mutation, A:T to T:A transversions, located predominantly on the non-transcribed DNA strand, with a strong preference for deoxyadenosine in a consensus sequence (CAG), was observed in many UTUC patients. Clinically, AA-related UTUC patients were characterized by a younger age, female gender, impaired renal function and recurrence of contralateral UTUC. To date, there are no preventive measures, except prophylactic nephrectomy, for subjects at risk of AA nephropathy or AA-related UTUC. CONCLUSION: AA exposure via Aristolochia-based herbal medicines is a problem throughout Taiwan, resulting in a high incidence of UTUC. Aristolactam-DNA adducts and a distinctive signature mutation, A:T to T:A transversions, can be used as biomarkers to identify AA-related UTUC. AA-related UTUC is associated with a high recurrence rate of contralateral UTUC.

Paradigm Shift in Gastric Cancer Prevention: Harnessing the Potential of Aristolochia olivieri Extract.

Gastric cancer, particularly adenocarcinoma, is a significant global health concern. Environmental risk factors, such as Helicobacter pylori infection and diet, play a role in its development. This study aimed to characterize the chemical composition and evaluate the in vitro antibacterial and antitumor activities of an Aristolochia olivieri Colleg. ex Boiss. Leaves' methanolic extract (AOME). Additionally, morphological changes in gastric cancer cell lines were analyzed. AOME was analyzed using HPLC-MS/MS, and its antibacterial activity against H. pylori was assessed using the broth microdilution method. MIC and MBC values were determined, and positive and negative controls were included in the evaluation. Anticancer effects were assessed through in vitro experiments using AGS, KATO-III, and SNU-1 cancer cell lines. The morphological changes were examined through SEM and TEM analyses. AOME contained several compounds, including caffeic acid, rutin, and hyperoside. The extract displayed significant antimicrobial effects against H. pylori, with consistent MIC and MBC values of 3.70 ± 0.09 mg/mL. AOME reduced cell viability in all gastric cancer cells in a dose- and time-dependent manner. Morphological analyses revealed significant ultrastructural changes in all tumor cell lines, suggesting the occurrence of cellular apoptosis. This study demonstrated that AOME possesses antimicrobial activity against H. pylori and potent antineoplastic properties in gastric cancer cell lines. AOME holds promise as a natural resource for innovative nutraceutical approaches in gastric cancer management. Further research and in vivo studies are warranted to validate its potential clinical applications.

Silver nanoparticles (AgNPs) fabricating potential of aqueous shoot extract of Aristolochia bracteolata and assessed their antioxidant efficiency.

This research was performed to evaluate the silver nanoparticles (AgNPs) fabricating potential of aqueous shoot extract of Aristolochia bracteolata and also assess the free radicals scavenging potential of synthesized AgNPs. The results obtained from this study showed that the aqueous shoot extract of A. bracteolata has the potential to synthesize the AgNPs and it was initially confirmed by color change in the reaction blend as yellow to dark brownish. Subsequently, a clear absorbance peak was found at 425 nm in UV-visible spectrum analysis. The functional groups involved in the capping and stabilization of AgNPs were confirmed by Fourier Transform-Infrared spectroscopy (FTIR) analysis and recorded about 10 sharp peaks 3688, 3401, 2980, 2370, 1948, 1642, 1480, 1280, 782, and 628 cm-1. The Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) observations revealed that the predominant shape of the AgNPs was spherical and size ranged from 41.43 to 60.51 nm. Interestingly, the green fabricated AgNPs showed significant free radicals scavenging activity and were confirmed with ferric reducing assay, 1, 1-diphenyl-2-picryl-hydrazyl (DPPH), H2O2 radicals, and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals scavenging activity. Thus, after a few in-vivo antioxidant studies, Aristolochia bracteolata-mediated AgNPs can be considered as an antioxidant agent.

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.

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.

Gene expression underlying floral epidermal specialization in Aristolochia fimbriata (Aristolochiaceae).

BACKGROUND AND AIMS: The epidermis constitutes the outermost tissue of the plant body. Although it plays major structural, physiological and ecological roles in embryophytes, the molecular mechanisms controlling epidermal cell fate, differentiation and trichome development have been scarcely studied across angiosperms, and remain almost unexplored in floral organs. METHODS: In this study, we assess the spatio-temporal expression patterns of GL2, GL3, TTG1, TRY, MYB5, MYB6, HDG2, MYB106-like, WIN1 and RAV1-like homologues in the magnoliid Aristolochia fimbriata (Aristolochiaceae) by using comparative RNA-sequencing and in situ hybridization assays. KEY RESULTS: Genes involved in Aristolochia fimbriata trichome development vary depending on the organ where they are formed. Stem, leaf and pedicel trichomes recruit most of the transcription factors (TFs) described above. Conversely, floral trichomes only use a small subset of genes including AfimGL2, AfimRAV1-like, AfimWIN1, AfimMYB106-like and AfimHDG2. The remaining TFs, AfimTTG1, AfimGL3, AfimTRY, AfimMYB5 and AfimMYB6, are restricted to the abaxial (outer) and the adaxial (inner) pavement epidermal cells. CONCLUSIONS: We re-evaluate the core genetic network shaping trichome fate in flowers of an early-divergent angiosperm lineage and show a morphologically diverse output with a simpler genetic mechanism in place when compared to the models Arabidopsis thaliana and Cucumis sativus. In turn, our results strongly suggest that the canonical trichome gene expression appears to be more conserved in vegetative than in floral tissues across angiosperms.

Evolution of Class II TCP genes in perianth bearing Piperales and their contribution to the bilateral calyx in Aristolochia.

Controlled spatiotemporal cell division and expansion are responsible for floral bilateral symmetry. Genetic studies have pointed to class II TCP genes as major regulators of cell division and floral patterning in model core eudicots. Here we study their evolution in perianth-bearing Piperales and their expression in Aristolochia, a rare occurrence of bilateral perianth outside eudicots and monocots. The evolution of class II TCP genes reveals single-copy CYCLOIDEA-like genes and three paralogs of CINCINNATA (CIN) in early diverging angiosperms. All class II TCP genes have independently duplicated in Aristolochia subgenus Siphisia. Also CIN2 genes duplicated before the diversification of Saruma and Asarum. Sequence analysis shows that CIN1 and CIN3 share motifs with Cyclin proteins and CIN2 genes have lost the miRNA319a binding site. Expression analyses of all paralogs of class II TCP genes in Aristolochia fimbriata point to a role of CYC and CIN genes in maintaining differential perianth expansion during mid- and late flower developmental stages by promoting cell division in the distal and ventral portion of the limb. It is likely that class II TCP genes also contribute to cell division in the leaf, the gynoecium and the ovules in A. fimbriata.

Two New Aristolochic Acid Analogues from the Roots of Aristolochia contorta with Significant Cytotoxic Activity.

Twelve compounds, including two new aristolochic acid analogues with a formyloxy moiety (9-10) and 10 known aristolochic acid derivates (1-8 and 11-12), were obtained from the roots of Aristolochiacontorta. Their structures were elucidated using extensive spectroscopic methods. Their cytotoxic activity in human proximal tubular cells HK-2 was evaluated by the MTT method, which has been widely used to assess cell viability. Among these molecules, compounds 3 and 9 were found to be more cytotoxic. Furthermore, molecular modeling was used to evaluate, for the first time, the interactions of compounds 3 and 9 with the target protein organic anionic transporter 1 (OAT1) that plays a key role in mediating aristolochic acid nephropathy. Structure-activity relationships are briefly discussed.

Antinociceptive Effect of Hinokinin and Kaurenoic Acid Isolated from Aristolochia odoratissima L.

Aristolochia odoratissima L. is employed for the treatment of pain and as an antidote against the poison of venomous animals in traditional medicine. However, reports have not been found, to our knowledge, about the evaluation of the antinociceptive activity of extracts nor about the presence of compounds associated with this activity. Thus, the purpose of this work was to evaluate the antinociceptive activity of extracts and compounds isolated from the stems of Artistolochia odoratissima L. The extracts were obtained with solvents of increasing polarity and the compounds were isolated and characterized by column chromatography, HPLC, and NMR. The antinociceptive activity was carried out by the formalin test in mice. Ethyl acetate (AoEA) and methanolic (AoM) extracts decreased the paw licking in both phases of the formalin test. The isolated compounds (kaurenoic acid and hinokinin) from AoEA showed the highest antinociceptive activity in both phases of the formalin test. These results confirmed the analgesic effect of this specie described in traditional medicine and provided a base for a novel analgesic agent. They also allowed an approach for the development of standardized plant extracts with isolated metabolites.

Aristolochia Herbs and Iatrogenic Disease: The Case of Portland's Powders.

Aristolochia herbals have a 2500-year history of medicinal use. We focused this article on Portland's Powders, an 18th-century British gout medicine containing Aristolochia herbs. The powders constitute an 18th-century iteration of an herbal remedy, which was used, with variations, since at least the fifth century BCE. The use of Portland's Powders in Great Britain may appear to be an unusual choice for investigating a public health problem currently widespread in Asia. Yet it exemplifies long-term medicinal use of Aristolochia herbs, reflecting our argument that aristolochic acid nephropathy (AAN) is a historically persistent iatrogenic disease. Moreover, we provide compelling evidence that individuals taking Portland's Powders for gout would have ingested toxic quantities of aristolochic acid, which causes AAN and cancer. Several factors, including long history of use, latency of toxic effects, and lack of effective regulation, perpetuate usage of Aristolochia herbals to the present day.

(+)-Isobicyclogermacrenal and spathulenol from Aristolochia yunnanensis alleviate cardiac fibrosis by inhibiting transforming growth factor ß/small mother against decapentaplegic signaling pathway.

Cardiac fibrosis contributes to both systolic and diastolic dysfunction in many cardiac pathophysiologic conditions. Antifibrotic therapies are likely to be a crucial strategy in curbing many fibrosis-related cardiac diseases. In our previous study, an ethyl acetate extract of a traditional Chinese medicine Aristolochia yunnanensis Franch. was found to have a therapeutic effect on myocardial fibrosis in vitro and in vivo. However, the exact chemicals and their mechanisms responsible for the activity of the crude extract have not been illustrated yet. In the current study, 10 sesquiterpenoids (1-10) were isolated from the active extract, and their antifibrotic effects were systematically evaluated in transforming growth factor ß 1 (TGFß1)-stimulated cardiac fibroblasts and NIH3T3 fibrosis models. (+)-Isobicyclogermacrenal (1) and spathulenol (2) were identified as the main active components, being more potent than the well-known natural antifibrotic agent oxymatrine. Compounds 1 and 2 could inhibit the TGFß1-induced cardiac fibroblasts proliferation and suppress the expression of the fibrosis biomarkers fibronectin and α-smooth muscle actin via down-regulation of their mRNA levels. The mechanism study revealed that 1 and 2 could inhibit the phosphorylation of TGFß type I receptor, leading to the decrease of the phosphorylation levels of downstream Smad2/3, then consequently blocking the nuclear translocation of Smad2/3 in the TGFß/Smad signaling pathway. These findings suggest that 1 and 2 may serve as promising natural leads for the development of anticardiac fibrosis drugs.

Complete Chloroplast Genomes and Comparative Analysis of Sequences Evolution among Seven Aristolochia (Aristolochiaceae) Medicinal Species.

Aristolochiaceae, comprising about 600 species, is a unique plant family containing aristolochic acids (AAs). In this study, we sequenced seven species of Aristolochia, and retrieved eleven chloroplast (cp) genomes published for comparative genomics analysis and phylogenetic constructions. The results show that the cp genomes had a typical quadripartite structure with conserved genome arrangement and moderate divergence. The cp genomes range from 159,308 bp to 160,520 bp in length and have a similar GC content of 38.5%⁻38.9%. A total number of 113 genes were identified, including 79 protein-coding genes, 30 tRNAs and four rRNAs. Although genomic structure and size were highly conserved, the IR-SC boundary regions were variable between these seven cp genomes. The trnH-GUG genes, are one of major differences between the plastomes of the two subgenera Siphisia and Aristolochia. We analyzed the features of nucleotide substitutions, distribution of repeat sequences and simple sequences repeats (SSRs), positive selections in the cp genomes, and identified 16 hotspot regions for genomes divergence that could be utilized as potential markers for phylogeny reconstruction. Phylogenetic relationships of the family Aristolochiaceae inferred from the 18 cp genome sequences were consistent and robust, using maximum parsimony (MP), maximum likelihood (ML), and Bayesian analysis (BI) methods.

Balkan endemic nephropathy and aristolochic acid I: an investigation into the role of soil and soil organic matter contamination, as a potential natural exposure pathway.

Aristolochic acids (AAs) are carcinogenic and nephrotoxic plant alkaloids present in Aristolochia species, used in traditional medicine. Recent biomolecular and environmental studies have incriminated these toxins as an etiological agent in Balkan endemic nephropathy (BEN), a severe kidney disease occurring in the Balkan Peninsula. The questions on how the susceptible populations are exposed to these toxins have not yet been clearly answered. Exposure to AAs through the food chain, and environmental pollution (soil/dust), could provide an explanation for the presence of BEN in the countries where no folkloric use of the plant has been documented (Bulgaria, Croatia). Additional exposure pathways are likely to occur, and we have shown previously that AAs can contaminate crop plants through absorption from soil, under controlled laboratory environment. Here, we attempt to provide additional support to this potential exposure pathway, by revealing the presence of AAI in soil and soil organic matter samples collected from BEN and non-BEN areas. The samples were processed in order to be analyzed by high-pressure liquid chromatography, and ion trap mass spectrometry. Our results showed the presence of AAI in small concentrations, both in BEN and non-BEN soils, especially where Aristolochia plants and seeds were present.