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.

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.

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.

Efficacy of Aristolochia rotunda Linn in hyperuricemia: A randomised standard controlled study.

INTRODUCTION: Hyperuricemia (HU) is a health risk that may go undiagnosed and is on the rise in both developed and developing countries. Gout, the most common inflammatory arthritis characterized by painful, disabling acute attack, is widely known to be caused by hyperuricemia. Its prevalence ranges from 2.6% to 36% in different populations. The commonly used drugs for alleviating uric acid and gout have obvious side effects, so herbal therapeutic drugs are in high demand. The aim of the present study was to evaluate the efficacy and safety of Aristolochia rotunda Linn in Hyperuricemia. METHODS: Present study was designed as a single-blind randomized standard controlled trial with 20 patients in each group. Participants in the test group were administered A. rotunda Linn 4 gms in two divided doses in capsule form and the control group was given Febuxostat 40 mg once daily after meals for 28 days. Participants were asked to follow up weekly for the assessment of subjective parameters. The objective parameter was assessed pre- and post-trial. The results were analysed statistically. RESULTS: After the intervention, the test and control groups showed a statistically significant reduction in serum uric acid p = 0.021 and p < 0.01 respectively, while the reduction in the control group was found to be more statistically significant than the test group (p = 0.009). Subjective parameters also showed statistical significance at the end of the trial. CONCLUSION: This study shows that the A. rotunda Linn in a dose of 4 gm for 28 days effectively lowers serum uric acid. The trial was registered in the clinical trial registry of India under CTRI No. CTRI//2020/02/031587.

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.

Efficient separation of aristolochic acid I from Caulis aristolochiae manshuriensis (Guan-mu-tong) with copper mediated magnetic molecularly imprinted polymer.

Screening bioactive compounds from natural products is one of the most effective ways for new drug research and development. However, obtaining a single extract component on a large scale and with high purity from a complex matrix is still an arduous and challenging task. Herein, one metal mediated magnetic molecularly imprinted polymer (mMIP) was rationally designed and prepared for specifically capturing Aristolochic acid I (AAI). The preparation was done with copper(II) as binding pivot, (3-aminopropyl) triethoxysilane as functional monomer, and Fe3O4 as core, by a one-step sol-gel method. Under the optimized conditions, the apparent maximum binding amount of copper mediated mMIP (Cu-mMIP) reaches as high as 349.72 mg g-1, the highest among the reported AAI-MIPs. Moreover, the nanoparticles exhibit excellent specificity and selectivity, good reproducibility and stability, high superparamagnetism (60.32 emu g-1), and high imprinting efficiency (an imprinting factor of 7). By simulating an industrial-scale separation, 16.56 mg AAI (purity of 95.11%) is obtained after six cycles with 100 mg nanoparticles from 20 g Caulis aristolochiae manshuriensis (Guan-mu-tong). Notably, this takes only 3 hours and consumes 50 mL of methanol. The study provides a potent tool for the green, fast, and specific extraction of high-purity ingredients from natural plants in the manufacturing industry and conventional analysis in the lab.

Enhanced Production of Nitrogenated Metabolites with Anticancer Potential in Aristolochia manshuriensis Hairy Root Cultures.

Aristolochia manshuriensis is a relic liana, which is widely used in traditional Chinese herbal medicine and is endemic to the Manchurian floristic region. Since this plant is rare and slow-growing, alternative sources of its valuable compounds could be explored. Herein, we established hairy root cultures of A. manshuriensis transformed with Agrobacterium rhizogenes root oncogenic loci (rol)B and rolC genes. The accumulation of nitrogenous secondary metabolites significantly improved in transgenic cell cultures. Specifically, the production of magnoflorine reached up to 5.72 mg/g of dry weight, which is 5.8 times higher than the control calli and 1.7 times higher than in wild-growing liana. Simultaneously, the amounts of aristolochic acids I and II, responsible for the toxicity of Aristolochia species, decreased by more than 10 fold. Consequently, the hairy root extracts demonstrated pronounced cytotoxicity against human glioblastoma cells (U-87 MG), cervical cancer cells (HeLa CCL-2), and colon carcinoma (RKO) cells. However, they did not exhibit significant activity against triple-negative breast cancer cells (MDA-MB-231). Our findings suggest that hairy root cultures of A. manshuriensis could be considered for the rational production of valuable A. manshuriensis compounds by the modification of secondary metabolism.

A draft reference genome assembly of the Pipevine Swallowtail butterfly, Battus philenor hirsuta.

The California Pipevine Swallowtail Butterfly, Battus philenor hirsuta, and its host plant, the California Pipevine or Dutchman's Pipe, Aristolochia californica Torr., are an important California endemic species pair. While this species pair is an ideal system to study co-evolution, genomic resources for both are lacking. Here, we report a new, chromosome-level assembly of B. philenor hirsuta 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 109 scaffolds spanning 443 mega base (Mb) pairs, with a contig N50 of 14.6 Mb, a scaffold N50 of 15.2 Mb, and BUSCO complete score of 98.9%. In combination with the forthcoming A. californica reference genome, the B. philenor hirsuta genome will be a powerful tool for documenting landscape genomic diversity and plant-insect co-evolution in a rapidly changing California landscape.

Preliminary population studies of the grassland swallowtail butterfly Euryades corethrus (Lepidoptera, Papilionidae).

Euryades corethrus is a Troidini butterfly (Papilionidae, Papilioninae), endemic to grasslands in southern Brazil, Uruguay, Argentina and Paraguay. Formerly abundant, nowadays it is in the Red list of endangered species for those areas. During its larval stage, it feeds on Aristolochia spp, commonly found in southern grasslands. These native grassland areas are diminishing, being converted to crops and pastures, causing habitat loss for Aristolochia and E. corethrus. This study aimed to assess the genetic diversity, population structure and demographic history of E. corethrus. We sampled eight populations from Rio Grande do Sul, Brazil and based on Cytochrome Oxidase subunit I (COI) molecular marker, our results suggest a low genetic variability between populations, presence of gene flow and, consequently, lack of population structure. A single maternally inherited-genetic marker is insufficient for population-level decisions, but barcoding is a useful tool during early stages of population investigation, bringing out genomic diversity patterns within the target species. Those populations likely faced a bottleneck followed by a rapid expansion during the last glaciation and subsequent stabilization in effective population size. Habitat loss is a threat, which might cause isolation, loss of genetic variability and, ultimately, extinction of E. corethrus if no habitat conservation policy is adopted.

Rapid simultaneous determination of thirteen aristolochic acids analogs in Aristolochiaceae plants by Ultra-High-Performance liquid Chromatography- tandem mass spectrometry in dynamic multiple reaction monitoring mode.

Asarum and Aristolochia are two large genera of Aristolochiaceae plants containing typical toxicant aristolochic acid analogs(AAAs), AAAs can be deemed as toxicity markers of Aristolochiaceae plants. Based on the least AAAs in dry roots and rhizomes of Asarum heterotropoides, Asarum sieboldii Miq and Asarum sieboldii var, all of which are enrolled in the Chinese pharmacopeia up to now. AAAs distribution in Aristolochiaceae plants, especially Asarum L. plants, is still obscure and controversial due to few AAAs measured, unverified species of Asarum, and complicated pretreatment in analytical samples making the results more challenging to reproduce. In the present study, a simple ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method in dynamic multiple reaction monitoring mode for simultaneous determination of thirteen AAAs was developed for evaluating the distribution of toxicity phytochemicals in Aristolochiaceae plants. The sample was prepared by extracting Asarum and Aristolochia powder with methanol, and the supernatant was analyzed using the Agilent 6410 system on an ACQUITY UPLC HSS PFP column with gradient elution of water and acetonitrile, containing 1% v/v formic acid (FA) each, at a flow rate of 0.3 mL/min. The chromatographic condition provided good peak shape and resolution. The method was linear over the specific ranges with the coefficient of determination (R2) > 0.990. Satisfactory intra- and inter-day precisions were achieved with RSD less than 9.79%, and the average recovery factors obtained were in the range of 88.50%～105.49%%. The proposed method was successfully applied for simultaneous quantification of the 13 AAAs in 19 samples from 5 Aristolochiaceae species, especially three Asarum L. species enrolled in the Chinese Pharmacopoeia. Except Asarum heterotropoides, the results supported that the Chinese Pharmacopoeia (2020 Edition) adopting the root with rhizome as medicinal parts of Herba Asari instead of the whole herb for drug safety by providing scientific data.

Study on the difference and correlation between the contents and toxicity of aristolochic acid analogues in Aristolochia plants.

ETHNOPHARMACOLOGICAL RELEVANCE: The nephrotoxicity and carcinogenicity induced by traditional Chinese medicines (TCMs) containing aristolochic acids (AAs) and related compound preparations have greatly limited their clinical application. While the toxicity of AA-I and AA-II is relatively clear, there are marked differences in the toxic effects of different types of aristolochic acid analogues (AAAs). Thus, the toxicity of TCMs containing AAAs cannot be evaluated based on the toxicity of a single compound. AIM OF THE STUDY: To systematically investigate the toxicity induced by Zhushalian (ZSL), Madouling (MDL) and Tianxianteng (TXT) as representative TCMs derived from Aristolochia. MATERIALS AND METHODS: AAA contents in ZSL, MDL and TXT were determined using HPLC. Subsequently, mice were treated for 2 weeks with high (H) and low (L) dosages of TCMs containing total AAA contents of 3 mg/kg and 1.5 mg/kg, respectively. Toxicity was evaluated using biochemical and pathological examination and was based on organ indices. Correlations between AAA contents and induced toxicity were analysed using multiple methods. RESULTS: Of the total AAA content, ZSL contained mainly AA-I and AA-II (>90%, of which AA-I accounted for 49.55%). AA-I accounted for 35.45% in MDL. TXT mainly contained AA-IVa (76.84%) and other AAAs accounted for <10%. Short-term toxicity tests indicated that ZSL and high-dose MDL induced obvious renal interstitial fibrosis and gastric injury, whereas TXT (high and low dosages) caused only slight toxicity. Correlation analysis suggested that AA-I might be the critical hazard factor for toxicity. CONCLUSIONS: The toxicity of TCMs containing AAAs cannot be generalised. The toxicity of TXT is relatively low compared with those of ZSL and MDL. The toxicity of Aristolochia depends mainly on the AA-I content; therefore, control of AA-I levels in TCMs and related compound preparations is required to reduce the risk of toxicity associated with the use of Aristolochia herbs in clinical settings.

Effects of human activities on Sericinus montela and its host plant Aristolochia contorta.

Sericinus montela, a globally threatened butterfly species, feeds exclusively on Aristolochia contorta (Northern pipevine). Field surveys and glasshouse experiments were conducted to obtain a better understanding of the relationship between the two species. Interviews with the persons concerned with A. contorta were conducted to collect information about the site management measures. We found that management practices to control invasive species and manage the riverine areas might reduce the coverage of A. contorta and the number of eggs and larvae of S. montela. Our results indicated that the degraded quality of A. contorta may result in a decrease in S. montela populations by diminishing their food source and spawning sites. This study implies that ecological management in the riverine area should be set up to protect rare species and biodiversity.

Characterization of two putative norlaudanosoline methyltransferases from Aristolochia debilis.

In view of the nephrotoxicity, hepatotoxicity, and carcinogenicity of aristolochic acids (AAs), the removal of AAs from plants becomes an urgent priority for ensuring the safety of Aristolochia herbal materials. In this study, based on the root-predominant distribution of aristolochic acid I (AAI) in Aristolochia debilis, transcriptome sequencing, in combination with phylogenetic analyses, and gene expression pattern analysis together provided five candidate genes for investigating AAI biosynthesis. Comprehensive in vitro and in vivo enzymatic assays revealed that Ab6OMT1 (6-O-methyltransferase) and AbNMT1 (N-methyltransferase) exhibit promiscuity in substrate recognition, and they could act in a cooperative fashion to achieve conversion of norlaudanosoline, a predicted intermediate in AAI biosynthetic route, into 3'-hydroxy-N-methylcoclaurine through two different methylation reaction sequences. These results shed light on the molecular basis for AAI biosynthesis in Aristolochia herbs. More importantly, Ab6OMT1 and AbNMT1 may be employed as targets for the metabolic engineering of AAI biosynthesis to produce AAs-free Aristolochia herbal materials.

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.

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.

Anti-hyperglycemic potential and chemical constituents of Aristolochia triangularis Cham. leaves - A medicinal species native to Brazilian forests.

ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochia triangularis Cham. has been used in Brazilian traditional medicine for various therapeutic purposes, including as a leaf-based infusion for diabetes management. AIM OF THE STUDY: This study was designed to chemically characterize an infusion of in natura A. triangularis leaves and evaluate the in vivo anti-hyperglycemic properties of this infusion. MATERIALS AND METHODS: Chemical composition was examined using liquid-liquid extraction procedure, chromatographic methods, NMR, and LC-MS/MS. The in vivo anti-hyperglycemic activity of the freeze-dried infusion of A. triangularis leaves (Inf-L-At) was assessed using oral glucose tolerance test (OGTT). Initially, normoglycemic male rats were pre-treated with orally administered Inf-L-At at doses of 62.5, 125, and 250 mg/kg for two consecutive days. On the day of the OGTT, fasting animals received a glucose load (4 g/kg) 30 min after treatment with Inf-L-At, and the blood glucose levels were verified at 15, 30, 60, and 180 min. Intestinal maltase, lactase, and sucrase activities and muscle and liver glycogen contents were also assessed after the OGTT. RESULTS: Inf-L-At extract led to glycemic reduction with no dose-response at 15, 30, and 60 min comparable to that of the antidiabetic drug glibenclamide and was accompanied by an increase in hepatic and muscle glycogen contents. Additionally, there was a significant statistically decrease in the in vitro activity of disaccharidases. Maltase and sucrase activities were inhibited at all doses, whereas lactase activity was inhibited only at 62.5 and 250 mg/kg. In total, 75 compounds were found in the infusion, including seven new ones, (7S*,8S*,7ꞌS*,8ꞌR*)-4,4ꞌ-dihydroxy-3,3ꞌ-dimethoxy-7,9ꞌ-epoxylignan-7ꞌ-ol; 4ꞌ-hydroxy-3ꞌ-methoxy-3,4-methylenedioxy-7,9ꞌ-epoxylignan-9,7ꞌ-diol; triangularisines A, B, and C; N-ethyl-N-methyl-affineine; and N-methyl pachyconfine, and one previously not described as a natural product, epi-secoisolariciresinol monomethyl ether. CONCLUSION: The results demonstrated the anti-hyperglycemic activity of the infusion from A. triangularis leaves and showed that it is a rich source of lignoids, alkaloids, and glycosylated flavonoids, which are known to exhibit antidiabetic effects and other biological properties that can be beneficial for patients with chronic hyperglycemia, thus certifying the popular use of this herbal drink.

Studies on antioxidant, anti-diabetic and GC-MS analyses of methanol extract of Aristolochia bracteolata root bark.

Aristolochia bracteolatais utilized in confronting multiple and complicated disease conditions such as cancer, lung inflammation, dysentery, syphilis, gonorrhea, arthritis, skindiseases, snake bite and oxidative stress relating to humans due to their acceptability, affordability and proximity. This investigation seeks to determine the antioxidant and anti-diabetic effects of methanol extract of A. bracteolate root bark in vitro. The phytochemical screening, antioxidant, and enzymes inhibitory (alpha-amylase and alpha-glucosidase) properties of root bark extract were evaluated by standard procedures. The methanol extract indicated the presence of diverse phytochemicals (tannins, saponins, flavonoids, alkaloids, phenols, glycosides and terpenoids) and contained a remarkable amount of saponins (8.20±0.03%), phenols (6.82±0.01%), alkaloids (4.71±0.03%) and flavonoids (3.50±0.12%). The extract showed not only strong antioxidant properties against DPPH, FRAP and TBARS radicals with IC50 value of 57.87, 54.64 and 47.54 mg/ml, respectively but also anti-diabetic activity by inhibiting alpha-amylase (IC50=53.70 mg/ml) and alpha-glucosidase (IC50=49.18 mg/ml). GC-MS chromatogram identified a diverse array of active metabolites in the methanol extract of A. bracteolate root bark. This study suggested that the methanol extract of A. bracteolate root bark possessed anti-oxidative and anti-diabetic activities.

Comparative Analysis of Aristolochic Acids in Aristolochia Medicinal Herbs and Evaluation of Their Toxicities.

Aristolochic acids (AAs) are a group of nitrophenanthrene carboxylic acids present in many medicinal herbs of the Aristolochia genus that may cause irreversible hepatotoxicity, nephrotoxicity, genotoxicity and carcinogenicity. However, the specific profile of AAs and their toxicity in Aristolochia plants, except for AAs Ι and ΙΙ, still remain unclear. In this study, a total of 52 batches of three medicinal herbs belonging to the Aristolochia family were analyzed for their AA composition profiles and AA contents using the UPLC-QTOF-MS/MS approach. The studied herbs were A. mollissima Hance (AMH), A. debilis Sieb.etZucc (ADS), and A. cinnabaria C.Y.Cheng (ACY). Chemometrics methods, including PCA and OPLS-DA, were used for the evaluation of the Aristolochia medicinal herbs. Additionally, cytotoxicity and genotoxicity of the selected AAs and the extracts of AMH and ADS were evaluated in a HepG2 cell line using the MTT method and a Comet assay, respectively. A total of 44 AAs, including 23 aristolochic acids and 21 aristolactams (ALs), were detected in A. mollissima. Moreover, 41 AAs (23 AAs and 18 ALs) were identified from A. debilis Sieb, and 45 AAs (29 AAs and 16 ALs) were identified in A. cinnabaria. Chemometrics results showed that 16, 19, and 22 AAs identified in AMH, ADS, and ACY, respectively, had statistical significance for distinguishing the three medicinal herbs of different origins. In the cytotoxicity assay, compounds AL-BΙΙ, AAΙ and the extract of AMH exhibited significant cytotoxicities against the HepG2 cell line with the IC50 values of 0.2, 9.7 and 50.2 µM, respectively. The results of the Comet assay showed that AAΙ caused relatively higher damage to cellular DNA (TDNA 40-95%) at 50 µM, while AAΙΙ, AMH and ADS extracts (ranged from 10 to 131 µM) caused relatively lower damage to cellular DNA (TDNA 5-20%).

Uptake, Translocation, and Fate of Carcinogenic Aristolochic Acid in Typical Vegetables in Soil-Plant Systems.

When Aristolochia plants wilt and decay, aristolochic acids (AAs) are released into the soil, causing soil contamination. It has been demonstrated that aristolochic acid can be accumulated and enriched in crops through plant uptake. However, there is a lack of systematic studies on the migration and accumulation of AAs in a realistic simulated soil environment. In this study, Aristolochia herbal extracts were mixed with soil for growing three typical vegetables: lettuce, celery, and tomato. The contents of AAs in the above-mentioned plants were determined by an established highly sensitive LC-MS/MS method to study the migration and accumulation of AAs. We found that AAs in the soil can be transferred and accumulated in plants. AAs first entered the roots, which were more likely to accumulate AAs, and partially entered the above-ground parts. This further confirms that AAs can enter the food chain through plants and can have serious effects on human health. It was also shown that plants with vigorous growth and a large size absorbed AAs from the soil at a faster rate. The more AAs present in the soil, the more they accumulated in the plant.

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.