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.

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.

Comparative reproductive ecology of two sister Asarum species (Aristolochiaceae) in relation to the evolution of elongated floral appendage.

Genus Asarum (Aristolochiaceae) shows diverse floral morphology and is hypothesized to have diversified as a result of pollinator-mediated selection. Yet most aspects of their reproductive ecology, including pollinators, remain unclear. This study focuses on A. costatum and A. minamitanianum in Japan, a sister species pair having remarkable differences in calyx lobe length (10-20 mm and 70-180 mm, respectively). The objectives of this study are to elucidate multiple aspects of reproductive ecology of these two species and obtain evolutionary insights into floral organ elongation. We adopted combined approaches, including field observations, molecular analyses and cultivation experiments, such as pollinator observation for 3 years, fine-scale spatial genetic analysis of 769 individuals, paternity analysis based on 566 seeds over 4 years, and control pollination experiments. Both Asarum species had strong spatial genetic structures, indicating limited seed dispersal. Pollinator observation revealed that flies and ground-dwelling insects visited flowers of both species, but that the pollinator fauna differed between the species. The visitation rate of flies was extremely low but was more than twice as high in the species with an elongated floral appendage. Paternity analysis revealed A. minamitanianum was predominantly outcrossing, while A. costatum showed a wide range of selfing rates among fruits. These two Asarum species are likely adapted to fly pollination in the shady forest understorey, where available pollinator fauna is limited. In addition, although its function remains unclear, the elongated calyx lobe of A. minamitanianum could have evolved for effective pollen dispersal by attracting fly visitors.

[Comparison of chloroplast genomes of medicinal plants in Aristolochiaceae].

In this study, the chloroplast genome of Asarum sieboldii f. seoulense was sequenced, analyzed, and compared with chloroplast genomes of other medicinal plants in Aristolochiaceae downloaded from GenBank, aiming to clarify the characteristics of the chloroplast genome of A. sieboldii f. seoulense and the differences in chloroplast genome among medicinal plants of Aristolochiaceae. To be specific, the chloroplast genome of A. sieboldii f. seoulense was sequenced and assembled by high-throughput sequencing, and the general characteristics, repeats, inverted repeat(IR) boundary, and phylogenetic relationship of the chloroplast genomes of 11 medicinal species in Aristolochiaceae were analyzed with REPuter. The result showed that the genome of A. sieboldii f. seoulense was 167 293 bp, with large single-copy(LSC) region of 89 840 bp, small single-copy(SSC) region of 21 415 bp, IR region of 28 019 bp, and GC content of 37.9%. A total of 133 genes were annotated, including 89 protein-coding genes, 36 tRNA genes and 8 rRNA genes. The chloroplast genomes of the 11 medicinal species were 159 308-167 293 bp, with 130-134 genes annotated. Forward(F), reverse(R), complement(C), and palindromic(P) long repeats and simple sequence repeat(SSR) were found in the chloroplast genomes of five species. Among them, A. sieboldii f. seoulense had six types of SSR. In the phylogenetic tree, A. sieboldii f. seoulense and A. heterotropoides were in the same clade. The result is expected to lay a basis for the classification, identification, and phylogeny of medicinal plants in Aristolochiaceae.

Genotoxicity of Asiasari Radix et Rhizoma (Aristolochiaceae) ethanolic extract in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional herbal medicines have diverse efficacy and are increasingly used worldwide. However, some of these herbal medicines have toxicities or side effects, but the scientific understanding of traditional herbal medicine toxicity has not yet been established. Asiasari Radix et Rhizoma (ARE) is known as a herbal medicine used to relieve pain, and recent studies have shown that ARE has anticancer and antimelanogenesis efficacy. AIM OF THE STUDY: Current study was conducted to assess the potential genotoxicity of an ethanolic extract of ARE. MATERIALS AND METHODS: The genotoxixity of ARE was confirmed by the bacterial reverse mutation assay (Ames test), a mammalian chromosomal aberration test, and a micronucleus test in vivo using ICR mice and comet assay using Sprague-Dawley rats. RESULTS: ARE showed no genotoxicity in a micronucleus test up to 2000 mg/kg body weight in vivo. By contrast, the chromosomal aberration test showed that ARE induced an increase in the number of chromosomal aberrations after treatment for 6 h with a metabolic activation system and for 6 and 22 h without the metabolic activation system when compared with vehicle control. In the Ames test, all strains except TA1535, with or without a metabolic activation system, showed an increase in the number of revertant mutant colonies in the ARE-treated group. In comet assay, DNA damage was observed in the stomach when ARE was administered. CONCLUSION: ARE potentially shows genotoxicity by inducing DNA damage.

Antioxidant, Anti-Inflammatory and Anticancer Activities of the Methanolic Extract of Thottea siliquosa: An In Vitro and In Silico Study.

BACKGROUND: Oxidative stress and inflammation are the predominant cause of chronic diseases, including multiple forms of cancers. Prevention of oxidative stress and inflammation is considered to be a target for preventing these disorders due to their significant roles in various degenerative diseases. Various natural products and plant extracts prevent the process of free radical- induced damages. OBJECTIVE: The present study evaluated the biological properties of Thottea siliquosa, belonging to the family Aristolochiaceae, which is a traditionally used Ayurvedic plant. METHODS: Antioxidant assays carried out were DPPH, FRAP, hydrogen peroxide scavenging, and hemolysis inhibition assay; nitric oxide and lipoxygenase inhibition assays were used for anti-inflammatory studies. Anticancer activity was evaluated using human endometrial and breast cancer cells by MTT assay. Bioactive compounds present in T. siliquosa were identified by LCMS and each was docked with various cancer targets, including EGFR, VEGFR, GST, COX2, and Lipooxygenase. RESULTS: The results of the present study showed antioxidant properties of the methanolic crude extract of T. siliquosa as DPPH radical scavenging (110.40 ± 4.5 µg/mL), FRAP capacity (41.1 ± 6.2), and peroxide scavenging (233.4 ± 14.2 µg/mL). Besides, anti-inflammatory properties were also evident in terms of nitric oxide radical scavenging (28.76± 3.9 µg/mL) and lipoxygenase inhibition (39.2 ± 3.2 µg/mL) assays. In silico analysis confirmed the inhibitory potential of the bioactive compounds of T. siliquosa against cancer drug targets such as EGFR, VEGFR, and inflammatory enzymes cyclooxygenase as well as lipooxygenase. Further, the anticancer activity of the extract has been identified against human endometrial and breast cancer cells. The possible mechanism of anticancer action of the extract is mediated through the apoptosis induction mechanism acting through increased caspase and APAF-1 expressions. CONCLUSION: The study thus concludes that T. siliquosa showed significant antioxidant, anti-inflammatory and anticancer properties. Further studies together with a bioassay-guided fractionation may identify possible bioactive compounds.

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.

Cytotoxicity and the bioconversion strategy of Aristolochia spp

Aristolochia plants are notable from an ethnopharmacological viewpoint, but the relevance of these species for medicinal purposes has been debated because of their inherent toxicity. The convergence of these contrasting realities can be readily achieved using bioconversion methods, which have been shown to be useful tools for numerous applications, including the detoxification of biomass. In this context, methanolic extracts of leaves from Aristolochia triangularis and Aristolochia gibertii, as well as the feces of Battus polydamas larvae fed with leaves from these plants, were prepared, and their cytotoxic activities were evaluated on a human fibroblast cell line (GM07492). The leaf extracts were found to be cytotoxic, leading to reductions of 42.1 and 33.8% on cell viability, respectively, while the fecal extracts were considered inactive. In addition to evidencing the cytotoxicity of A. triangularis and A. gibertii, these findings demonstrated a potential bioconversion strategy for obtaining aristolochiaceous extracts with reduced toxicity using the larvae of a specialist phytophagous insect, thus renewing expectations in relation to the pharmacological importance of Aristolochia spp. The results were also ecologically relevant, as B. polydamas larvae were found to be able to detoxify compounds from host plants.(AU)

Purification and characterization of the major protein isolated from Semen Armeniacae Amarum and the properties of its thermally induced nanoparticles.

From the aqueous extract of Semen Armeniacae Amarum, a major protein isolate was purified and characterized as a novel member of the 11S globulin family, which is composed of three polypeptides linked by disulfide bond. Furthermore, the feasibility of using the isolated protein for fabricating nanocarriers was investigated. The results indicate that thermal treatment of the globulin induced the rearrangement of the disulfide bond to form homodimers of acid polypeptides during the formation of nanoparticles. The harvested nanoparticles produced by heat-induced assembly are spherical in shape, with an average size of 92 nm and exhibited low cytotoxicity to L-02 and MDCK cell lines. These nanoparticles are capable to encapsulate paclitaxel, estimated the maximum encapsulation efficiency of paclitaxel loaded to the nanoparticles was 92.6% and the maximum release of paclitaxel was 57.4%. This research suggests that the screening of traditional herbal extracts could provide a novel source of protein nanocarriers.

Radiation history of Asian Asarum (sect. Heterotropa, Aristolochiaceae) resolved using a phylogenomic approach based on double-digested RAD-seq data.

BACKGROUND AND AIMS: The genus Asarum sect. Heterotropa (Aristolochiaceae) probably experienced rapid diversification into 62 species centred on the Japanese Archipelago and Taiwan, providing an ideal model for studying island adaptive radiation. However, resolving the phylogeny of this plant group using Sanger sequencing-based approaches has been challenging. To uncover the radiation history of Heterotropa, we employed a phylogenomic approach using double-digested RAD-seq (ddRAD-seq) to yield a sufficient number of phylogenetic signals and compared its utility with that of the Sanger sequencing-based approach. METHODS: We first compared the performance of phylogenetic analysis based on the plastid matK and trnL-F regions and nuclear ribosomal internal transcribed spacer (nrITS), and phylogenomic analysis based on ddRAD-seq using a reduced set of the plant materials (83 plant accessions consisting of 50 species, one subspecies and six varieties). We also conducted more thorough phylogenomic analyses including the reconstruction of biogeographic history using comprehensive samples of 135 plant accessions consisting of 54 species, one subspecies, nine varieties of Heterotropa and six outgroup species. KEY RESULTS: Phylogenomic analyses of Heterotropa based on ddRAD-seq were superior to Sanger sequencing-based approaches and resulted in a fully resolved phylogenetic tree with strong support for 72.0-84.8 % (depending on the tree reconstruction methods) of the branches. We clarified the history of Heterotropa radiation and found that A. forbesii, the only deciduous Heterotropa species native to mainland China, is sister to the evergreen species (core Heterotropa) mostly distributed across the Japanese Archipelago and Taiwan. CONCLUSIONS: The core Heterotropa group was divided into nine subclades, each of which had a narrow geographic distribution. Moreover, most estimated dispersal events (22 out of 24) were between adjacent areas, indicating that the range expansion has been geographically restricted throughout the radiation history. The findings enhance our understanding of the remarkable diversification of plant lineages in the Japanese Archipelago and Taiwan.

Protective effects of Herpetospermum caudigerum extracts against liver injury induced by carbon tetrachloride in mouse.

Herpetospermum caudigerum (H. caudigerum; HC), popularly known as “Sejimeiduo” in Tibet, it is widely used in Tibetan traditional medicine for the treatment of dyspepsia, liver and colic diseases. This study was designed to evaluate the effect of H. caudigerum extract (HCE) on suppressing liver injury induced by carbon tetra chloride (CCl4). For this purpose, we used CCl4 to induce acute liver injury in mouse model. The protective effects of HCE against liver injury were evaluated by biochemical parameters, histopathological and immunohistochemical staining. The results showed that the superoxide dismutase (SOD) activity was significantly increased with the increasing dose of HCE as compared to the CCl4-treated group (p less than 0.01); while AST and ALT levels in serum, MDA and MPO in liver were reduced in a dose-dependent manner. The histopathology showed that HCE treatment promoted the recovery of histopathological changes in liver in a dose-dependent way. Meanwhile, there was a higher expression of caspase-3 and NF-κB in the nucleus of several liver cells in the CCl4-induced group, and a low expression of caspase-3 and NF-κB were found with the increasing dose of HCE. Therefore, the present study suggests that HCE is a potent hepatoprotective agent that can treat acute liver injury and this ability may be attributed towards its anti-inflammatory and antioxidant potential.

The potential of three different PCR-related approaches for the authentication of mixtures of herbal substances and finished herbal medicinal products.

BACKGROUND: Herbal substances and preparations thereof play an important role in healthcare systems worldwide. Due to the variety of these products regarding origin, composition and processing procedures, appropriate methodologies for quality assessment need to be considered. A majority of herbal substances is administered as multicomponent mixtures, especially in the field of Traditional Chinese Medicine and ayurvedic medicine, but also in finished medicinal products. Quality assessment of complex mixtures of herbal substances with conventional methods is challenging. Thus, emphasis of the present work was directed on the development of complementary methods to elucidate the composition of mixtures of herbal substances and finished herbal medicinal products. HYPOTHESIS/PURPOSE: An indispensable prerequisite for the safe and effective use of herbal medicines is the unequivocal authentication of the medicinal plants used therein. In this context, we investigated the potential of three different PCR-related methods in the characterization and authentication of herbal substances. METHODS: A multiplex PCR assay and a quantitative PCR (qPCR) assay were established to analyze defined mixtures of the herbal substances Quercus cortex, Juglandis folium, Aristolochiae herba, Matricariae flos and Salviae miltiorrhizae radix et rhizoma and a finished herbal medicinal product. Furthermore, a standard cloning approach using universal primers targeting the ITS region was established in order to allow the investigation of herbal mixtures with unknown content. RESULTS: The cloning approach had some limitations regarding the detection/recovery of the components in defined mixtures of herbal substances, but the complementary use of two sets of universal primer pairs increased the detection of components out of the mixture. While the multiplex PCR did not retrace all components in the defined mixtures of herbal substances, the established qPCR resulted in simultaneous and specific detection of the five target sequences in all defined mixtures. CONCLUSION: These data indicate that for authentication purposes, complementary PCR-related methods are highly recommendable for the analysis of herbal mixtures in parallel.

Hybrid-type carbon microcoil-chitosan composite for selective extraction of aristolochic acid I from Aristolochiaceae medicinal plants.

Aristolochic acid I is a nephrotoxic compound widely existing in many kinds of traditional Chinese medicines, especially in Aristolochiaceae medicinal plants. In this study, chitosan modified carbon microcoils were designed and prepared for the selective separation of aristolochic acid I from medicinal herbs. Successful modification of carbon microcoils was confirmed by scanning electron microscopy, Fourier-transfer infrared spectroscopy, elemental analysis, X-ray photoelectron spectroscopy, and thermogravimetric analyses. The effects of adsorption conditions were investigated and it was determined that the adsorption of aristolochic acid I was controlled by pH. Adsorption isotherms, kinetics, and selectivity tests were performed to evaluate the adsorption capacity and selectivity of the modified carbon microcoils. The chitosan modified carbon microcoils exhibited excellent binding ability (77.72 mg g-1) and satisfactory selectivity. Finally, this material was used in solid phase extraction combined with HPLC to enrich and detect aristolochic acid I from medicinal plants. The detector response for aristolochic acid I was linear from 0.5 to 150 mg L-1, and the recoveries of aristolochic acid I ranged from 73.61 to 77.73% with the relative standard deviations of less than 5%. Thus, chitosan modified carbon microcoils were ideal adsorbents for the selective extraction of aristolochic acid I from Aristolochiaceae plants.

Total duplication of the small single copy region in the angiosperm plastome: Rearrangement and inverted repeat instability in Asarum.

PREMISE OF THE STUDY: As more plastomes are assembled, it is evident that rearrangements, losses, intergenic spacer expansion and contraction, and syntenic breaks within otherwise functioning plastids are more common than was thought previously, and such changes have developed independently in disparate lineages. However, to date, the magnoliids remain characterized by their highly conserved plastid genomes (plastomes). METHODS: Illumina HiSeq and MiSeq platforms were used to sequence the plastomes of Saruma henryi and those of representative species from each of the six taxonomic sections of Asarum. Sequenced plastomes were compared in a phylogenetic context provided by maximum likelihood and parsimony inferences made using an additional 18 publicly available plastomes from early-diverging angiosperm lineages. KEY RESULTS: In contrast to previously published magnoliid plastomes and the newly sequenced Saruma henryi plastome published here, Asarum plastomes have undergone extensive disruption and contain extremely lengthy AT-repeat regions. The entirety of the small single copy region (SSC) of A. canadense and A. sieboldii var. sieboldii has been incorporated into the inverted repeat regions (IR), and the SSC of A. delavayi is only 14 bp long. All sampled Asarum plastomes share an inversion of a large portion of the large single copy region (LSC) such that trnE-UUC is adjacent to the LSC-IR boundary. CONCLUSIONS: Plastome divergence in Asarum appears to be consistent with trends seen in highly rearranged plastomes of the monocots and eudicots. We propose that plastome instability in Asarum is due to repetitive motifs that serve as recombinatory substrates and reduce genome stability.

Pollen ultrastructure in Aristolochia manshuriensis and A. contorta (Aristolochiaceae).

Pollen ultrastructure has been studied in two relict and rare species of the genus Aristolochia, A. contorta Bunge and A. manshuriensis Kom. (Aristolochiaceae). Both species have inaperturate, spheroidal, sometimes distally monocolpate or distally bicolpate pollen grains. The equatorial and polar axes of pollen grain in A. manshuriensis are 48.5 and 44.0 µm, respectively. The percentage of defective pollen grains in A. manshuriensis is 3.4%. The fossulate, perforated exine is up to 2.3 µm in thickness; the sexine and the nexine are almost equal in thickness. In A. contorta, the equatorial axis of pollen grain is 36.6 µm: the defectiveness percentage, 24.5%. The exine is verrucate, up to 0.3 µm in thickness, while the sexine is two to three times thicker than the nexine. The pollen germination experiments have shown that pollen of A. manshuriensis, in contrast to A. contorta, can germinate in 10-20% sucrose at 22°Ð¡. These data and the high percentage of pollen defectiveness in A. contorta indicate that the androecium function in this species is reduced. The reduction of the androecium function is evidenced by a small amount of pollen grains in anthers or empty anthers and a high percentage of defective pollen grains.

Relative contributions of neutral and non-neutral processes to clinal variation in calyx lobe length in the series Sakawanum (Asarum: Aristolochiaceae).

Background and Aims: Clines, the gradual variation in measurable traits along a geographical axis, play a major role in evolution and can contribute to our understanding of the relative roles of selective and neutral process in trait variation. Using genetic and morphological analyses, the relative contributions of neutral and non-neutral processes were explored to infer the evolutionary history of species of the series Sakawanum (genus Asarum), which shows significant clinal variation in calyx lobe length. Methods: A total of 27 populations covering the natural geographical distribution of the series Sakawanum were sampled. Six nuclear microsatellite markers were used to investigate genetic structure and genetic diversity. The lengths of calyx lobes of multiple populations were measured to quantify their geographical and taxonomic differentiation. To detect the potential impact of selective pressure, morphological differentiation was compared with genetic differentiation (QCT-FST comparison). Key Results: Average calyx lobe length of A. minamitanianum was 124.11 mm, while that of A. costatum was 13.80 mm. Though gradually changing along the geographical axis within series, calyx lobe lengths were significantly differentiated among the taxa. Genetic differentiation between taxa was low (FST = 0.099), but a significant geographical structure along the morphological cline was detected. Except for one taxon pair, pairwise QCT values were significantly higher than the neutral genetic measures of FST and G'ST. Conclusions: Divergent selection may have driven the calyx lobe length variation in series Sakawanum taxa, although the underlying mechanism is still not clear. The low genetic differentiation indicates recent divergence and/or gene flows between geographically close taxa. These neutral processes would also affect the clinal variation in calyx lobe lengths. Overall, this study implies the roles of population history and divergent selection in shaping the current cline of a flower trait in the series Sakawanum.

Urotensin II enhances transforming growth factor­ß1 expression and secretion in the kidney during aristolochic acid nephropathy.

Aristolochic acid is a component of many types of Chinese medicine, which are commonly used to treat almost all human diseases. However, aristolochic acid may cause nephropathy. Urotensin II (UII) and transforming growth factor (TGF)­ß1 are important signaling factors, which are expressed at elevated levels during the development of nephropathy. However, the association between UII and TGF­ß1 expression remains unclear. In the current study, the regulatory association between UII and TGF­ß1 expression was investigated using a rat aristolochic acid nephropathy model and the NRK­52E cell line. The expression levels of UII and TGF­ß1 were identified to be constantly increased in the rat aristolochic acid nephropathy model, even 10 days after administration of Aristolochiae manshuriensis decoction was terminated. Notably, increases in the TGF­ß1 expression levels occurred later than those of UII. Furthermore, UII enhanced TGF­ß1 expression in, and secretion from, NRK­52E cells. These data indicate that UII and TGF­ß1 are important in the development of aristolochic acid nephropathy, and UII enhances TGF­ß1 expression levels and secretion during aristolochic acid nephropathy. However, the underlying mechanisms for the precise roles of UII and TGF­ß1 as well as the method by which UII regulates the expression TGF­ß1 in aristolochic acid nephropathy remain to be elucidated in future studies.

Development of validated high-performance thin layer chromatography for quantification of aristolochic acid in different species of the Aristolochiaceae family.

This study was undertaken to isolate and quantify aristolochic acid in Aristolochia indica stem and Apama siliquosa root. Aristolochic acid is an important biomarker component present in the Aristolochiaceae family. The isolation method involved simple solvent extraction, precipitation and further purification, using recrystallization. The structure of the compound was confirmed using infrared spectroscopy, mass spectrometry and nuclear magnetic resonance. A specific and rapid high-performance thin layer chromatography (HPTLC) method was developed for analysis of aristolochic acid. The method involved separation on the silica gel 60 F254 plates using the single solvent system of n-hexane: chloroform: methanol. The method showed good linear relationship in the range 0.4-2.0 µg/spot with r2 = 0.998. The limit of detection and limit of quantification were 62.841 ng/spot and 209.47 ng/spot, respectively. The proposed validated HPTLC method was found to be an easy to use, accurate and convenient method that could be successfully used for standardization and quality assessment of herbal material as well as formulations containing different species of the Aristolochiaceae family.

Analysis of aristolochic acids, aristololactams and their analogues using liquid chromatography tandem mass spectrometry.

More than 80 aristolochic acids (AAs) and aristololactams (ALs) have been found in plants of the Aristolochiaceae family, but relatively few have been fully studied. The present study aimed at developing and validating a liquid chromatography tandem mass spectrometry (LC/MS(n)) for the analysis of these compounds. We characterized the fragmentation behaviors of 31 AAs, ALs, and their analogues via high performance liquid chromatography coupled with electrospray ionization mass spectrometry. We summarized their fragmentation rules and used these rules to identify the constituents contained in Aristolochia contorta, Ar. debilis, Ar. manshurensis, Ar. fangchi, Ar. cinnabarina, and Ar. mollissima. The AAs and ALs showed very different MS behaviors. In MS(1) of AAs, the characteristic pseudomolecular ions were [M + NH4](+), [M + H](+), and [M + H - H2O](+). However, only [M + H](+) was found in the MS(1) of ALs, which was simpler than that of AAs. Distinct MS(n)fragmentation patterns were found for AAs and ALs, showing the same skeleton among the different substituent groups. The distribution of the 31 constituents in the 6 species of Aristolochia genus was reported for the first time. 25 Analogues of AAs and ALs were detected in this genus. A hierarchical schemes and a calculating formula of the molecular formula of these nitrophenanthrene carboxylic acids and their lactams were proposed. In conclusion, this method could be applied to identification of similar unknown constituents in other plants.

Effect of Asiasarum Root Extract and Its Constituents on Interleukin-1ß-Stimulated Matrix Metalloproteinase-1 Secretion from Human Gingival Fibroblasts.

Asiasarum root (roots and rhizome of Asiasarum sieboldii or A. heterotropoides var. mandshuricum) has been frequently used in traditional Chinese medicinal formulas for the management of oral malodor syndrome caused by periodontal disease. However, there are no scientific reports concerning these effects and the mechanism of action. The objective of this study was to examine the inhibitory effects of Asiasarum root and its constituents on oral malodor syndrome and periodontal disease. A 50% ethanolic extract of Asiasarum root (AR-ext) showed L-methionine γ-lyase (METase) inhibitory activity at a concentration of 200 µg/mL, and inhibited interleukin (IL)-1ß-stimulated matrix metalloproteinase (MMP)-1 secretion from human gingival fibroblasts (HGFs) at a concentration of 10 and 50 µg/mL without cytotoxic effects. Activity-guided fractionation of the AR-ext suggested that METase inhibitory activity was attributable to a mixture of linoleic and oleic acid, because these unsaturated fatty acids showed weak METase inhibitory activities. Similar fractionation using MMP-1 secretion inhibitory activity led to the isolation of two unsaturated fatty acid amides, (2E,4E,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide (1) and (2E,4E,8Z,10Z)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide (2), as active constituents with inhibitory activity on MMP-1 secretion from HGFs. To elucidate the inhibition mechanism on MMP-1 secretion, the effect of 2 on mitogen-activated protein kinase (MAPK) phosphorylation was examined. Western blotting analysis revealed that 2 (10 µM) reduced the phosphorylation of p38 and c-Jun-N-terminal kinase. These results suggested that 2 suppresses intracellular MMP-1 expression and MMP-1 secretion from IL-1ß-stimulated HGFs by down-regulation of MAPK phosphorylation.