Multiomics Reveals Mechanisms of Alternaria oxytropis Inhibiting Pathogenic Fungi in Oxytropis ochrocephala.

Endophytic fungi can benefit the host plant and increase the plant resistance. Now, there is no in-depth study of how Alternaria oxytropis (A. oxytropis) is enhancing the ability of inhibiting pathogenic fungi in Oxytropis ochrocephala (O. ochrocephala). In this study, the fungal community and metabolites associated with endophyte-infected (EI) and endophyte-free (EF) O. ochrocephala were compared by multiomics. The fungal community indicated that there was more A. oxytropis, less phylum Ascomycota, and less genera Leptosphaeria, Colletotrichum, and Comoclathris in the EI group. As metabolic biomarkers, the levels of swainsonine and apigenin-7-O-glucoside-4-O-rutinoside were significantly increased in the EI group. Through in vitro validation experiments, swainsonine and apigenin-7-O-glucoside-4-O-rutinoside can dramatically suppress the growth of pathogenic fungi Leptosphaeria sclerotioides and Colletotrichum americae-borealis by increasing the level of oxidative stress. This work suggested that O. ochrocephala containing A. oxytropis could increase the resistance to fungal diseases by markedly enhancing the content of metabolites inhibiting pathogenic fungi.

Identification of azukisapogenol triterpenoid saponins from Oxytropis hirta Bunge and their aphicidal activities against pea aphid Acyrthosiphon pisum Harris.

BACKGROUND: Acyrthosiphon pisum Harris is the most destructive pest worldwide because of its ability to feed on plants directly and transmit plant viruses as a vector. This study aims to identify triterpenoid saponins from Oxytropis hirta Bunge as biopesticides to control aphids. RESULTS: Three new azukisapogenol triterpenoid saponins (1-3), a new pinoresinol lignan glycoside (8), and four known saponins (4-7) were identified from the root of O. hirta. Compounds 4-7 displayed significant aphicidal activities against A. pisum with oral toxicities (LC50  = 51.10-147.43 µg/mL, 72 h), deterrent effects (deterrence index = 1.00, 100-200 µg/mL, 24 h), and aphid reproduction inhibitory effects (inhibition rates = 75.91-86.73%, 400 µg/mL, 24 h), respectively. The carboxyl groups at C-3 GlcA and C-30 were functional groups for their aphicidal activities. The toxic symptoms caused by the optimal 5 involved insect body-color changes from light green to dark or gray-green, and then brown until death. The intestinal cavity, apical microvilli, nuclei, mitochondria, and electron dense granules in the midgut tissues of A. pisum were the target sites showing aphicidal activity. The suppression of pepsin and α-amylase, and the activation of lipase and trypsin could be the signs of organelle damage in the midgut tissues. CONCLUSION: Azukisapogenol triterpenoid saponins from O. hirta could be used as biopesticides to control aphids for their multiple efficacies, including oral toxicity, deterrent activity, and reproduction inhibitory activity. The toxic symptoms involved insect body-color changes. Midgut tissues and their related enzymes were the targets for saponins showing aphicidal activities. © 2022 Society of Chemical Industry.

Assembly of high-quality genomes of the locoweed Oxytropis ochrocephala and its endophyte Alternaria oxytropis provides new evidence for their symbiotic relationship and swainsonine biosynthesis.

Locoweeds are perennial forbs poisonous to livestock and cause extreme losses to animal husbandry. Locoweed toxicity is attributed to the symbiotic endophytes in Alternaria sect. Undifilum, which produce a mycotoxin swainsonine (SW). We performed a de novo whole genome sequencing of the most common locoweed in China, Oxytropis ochrocephala (2n = 16), and assembled a high-quality, chromosome-level reference genome. Its genome size is 958.83 Mb with 930.94 Mb (97.09%) anchored and oriented onto eight chromosomes, and 31,700 protein-coding genes were annotated. Phylogenetic and collinearity analysis showed it is closely related to Medicago truncatula with a pair of large interchromosomal rearrangements, and both species underwent a whole-genome duplication event. We also derived the genome of A. oxytropis at 74.48 Mb with a contig N50 of 8.87 Mb and 10,657 protein-coding genes, and refined the genes of SW biosynthesis. Multiple Alternaria species containing the swnK gene were grouped into a single clade, but in other genera, swnK's homologues are diverse. Resequencing of 41 A. oxytropis strains revealed one SNP in the SWN cluster causing changes in SW concentration. Comparing the transcriptomes of symbiotic and nonsymbiotic interactions identified differentially expressed genes (DEGs) linked to defence and secondary metabolism in the host. Within the endophyte DEGs were linked to cell wall degradation, fatty acids and nitrogen metabolism. Symbiosis induced the upregulation of most of the SW biosynthetic genes. These two genomes and relevant sequencing data should provide valuable genetic resources for the study of the evolution, interaction, and SW biosynthesis in the symbiont.

Chemical Components of Oxytropis pseudoglandulosa Induce Apoptotic-Type Cell Death of Caco-2 Cells.

Oxytropis pseudoglandulosa plant is used in traditional Mongolian medicine. However, its chemical composition and biological properties are poorly explored. In this study, the total content of polyphenols and flavonoids as well as antioxidant activity were verified in plant extract. The total phenolic and flavonoid contents were determined by spectrometric (6.62 mg GAE/g and 10.32 mg QE/g) and chromatographic (17,598 mg/kg and 17,467 mg/kg) assays. The antioxidant potential was investigated by DPPH assay and yielded IC50 at 18.76 µg/mL. Twelve phenolic compounds were identified as components of O. pseudoglandulosa extract. Kaempferol-3-O-robinosyl-7-O-rhamnoside and kaempferol-3-(p-coumaroyl)-rutinosyl-7-rhamnoside made up 80% of determined components and were found to be the major polyphenolic compounds. The biological properties of O. pseudoglandulosa extracts were determined in vitro using human epithelial adenocarcinoma Caco-2 cell line. Low concentrations of extract (0-30 µg/mL) exhibited protective effects against cell damage caused by chemically induced oxidative stress. Elevated concentrations, on the other hand, resulted in apoptotic-type cell death induction. Metabolic failure, ROS elevation and membrane permeabilization observed in cells upon incubation with extract dosages above 50 µg/mL allowed us to conclude on O. pseudoglandulosa being predominantly a necrosis inducer.

Host-Species Variation and Environment Influence Endophyte Symbiosis and Mycotoxin Levels in Chinese Oxytropis Species.

Oxytropis plants are widely distributed in the grasslands in northern China. Some Oxytropis species have been reported to contain the mycotoxin swainsonine, an alkaloid which causes poisoning in livestock, referred to as locoism. Previous studies showed that endophytic fungi (Alternaria oxytropis) symbiotically associate with these Oxytropis species to produce swainsonine. However, the influence of variation within the Oxytropis genus on the fixation or loss of symbiosis and toxicity is poorly understood, as is the influence of environmental factors. Here we used a collection of 17 common Oxytropis species sampled in northern China to assess genetic diversity using genotyping by sequencing which was compared with the levels of the endophyte and swainsonine. Results showed that nine Oxytropis species have detectable A. oxytropis colonisation, and seven Oxytropis species contain sufficient swainsonine to be considered poisonous, whereas the rest may be non-toxic. Species variation rather than the genetic lineage was associated with the fixation or loss of endophyte and swainsonine production, which appears to have resulted from genetic drift. Genotype × Environment (G × E) effects were also found to influence endophyte and swainsonine levels amongst species of the Oxytropis genus. Our study will provide a better understanding about the evolutionary basis of A. oxytropis symbiosis and swainsonine biosynthesis in locoweeds.

Mechanism of Action of Flavonoids of Oxytropis falcata on the Alleviation of Myocardial Ischemia-Reperfusion Injury.

Oxytropis falcata Bunge is a plant used in traditional Tibetan medicine, with reported anti-inflammatory and antioxidants effects and alleviation of myocardial ischemia reperfusion injury (MIRI). However, the underlying mechanism against MIRI and the phytochemical composition of O. falcata are vague. One fraction named OFF1 with anti-MIRI activity was obtained from O. falcata, and the chemical constituents were identified by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS). The potential targets and signaling pathways involved in the action of O. falcata against MIRI were predicted by network pharmacology analysis, and its molecular mechanism on MIRI was determined by in vitro assays. The results revealed that flavonoids are the dominant constituents of OFF1. A total of 92 flavonoids reported in O. falcata targeted 213 potential MIRI-associated factors, including tumor necrosis factor (TNF), prostaglandin-endoperoxide synthase 2 (PTGS2), and the NF-κB signaling pathway. The in vitro assay on H9c2 cardiomyocytes subjected to hypoxia/reoxygenation injury confirmed that the flavonoids in OFF1 reduced myocardial marker levels, apoptotic rate, and the inflammatory response triggered by oxidative stress. Moreover, OFF1 attenuated MIRI by downregulating the ROS-mediated JNK/p38MAPK/NF-κB pathway. Collectively, these findings provide novel insights into the molecular mechanism of O. falcata in alleviating MIRI, being a potential therapeutic candidate.

The plastome of Arctic Oxytropis arctobia (Fabaceae) is significantly different from that of O. splendens and other related species.

Anatomical and physiological specializations for plant adaptation to harsh climates result from molecular mechanisms that can be encoded in the nucleus or organelle. In this study, the complete plastomes of an arctic species, Oxytropis arctobia Bunge (Fabaceae), and a closely related temperate species, O. splendens Douglas ex Hook., were assembled, annotated, and analyzed to identify differences that might help explain their adaptation to different environments. This is consistent with the previously sequenced O. bicolor DC. and O. glabra plastomes, O. arctobia and O. splendens plastomes both have the common features of the inverted repeat-lacking clade (IRLC), as well as atpF intron loss, which is unique to the genus. However, significant differences were observed between the plastomes of O. arctobia and O. splendens and other closely related species (Oxytropis spp. and Astragalus spp.), including a 3 kb inversion, two large insertions (>1 kb), significant modifications of the accD gene, and an overall larger size.

Quercetin promotes cutaneous wound healing in mice through Wnt/ß-catenin signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxytropis falcata Bunge is a legume distributed in Northwest China, which is mainly used to treat knife wounds and inflammation. Quercetin is a bioactive flavonoid in O. falcata and becomes a promising healing compound for its angiogenic and anti-inflammatory activities. However, the healing mechanism of quercetin in cutaneous wound remains elusive. AIM OF THE STUDY: The purpose of this study was to evaluate the healing effect of quercetin on cutaneous wound models in vivo and in vitro, and to reveal the Wnt/ß-catenin pathway and Telomerase reverse transcriptase (TERT) involved mechanisms. MATERIALS AND METHODS: The effects of quercetin on the proliferation and migration of 4 kinds of skin cells were determined by CCK-8 and scratch assay. The wound-healing capacity of quercetin was evaluated in cutaneous wound model of C57BL/6 mice and the wound healing degree was observed by histological staining. The expressions of inflammatory factors, growth factors and the related proteins were detected via Western blot and RT-qPCR analyses. The molecular docking was adopted to evaluate the binding ability of quercetin and TERT. RESULTS: Quercetin could promote both proliferation and migration of fibroblasts, and enhance cutaneous wound healing capacity in mice. Compared to the control group, the wound healing rates in low (1.5 mg/mL), medium (3.0 mg/mL) and high dose (6.0 mg/mL) quercetin groups reached 94.67%, 97.31% and 98.42%, respectively. Moreover, the dermal structure in quercetin treated mice restored normal and the content of collagen fiber became abundant after administration. The levels of inflammatory factors, including tumor necrosis factor-α, interleukin-1ß and interleukin-6 were significantly reduced after quercetin administration. Among which, the level of IL-1ß in cutaneous wound was 0.007 times higher than that of the control group when treated with quercetin of high dose (6.0 mg/mL). The improved level of GSH in quercetin treated cutaneous wounds also indicated its higher antioxidant ability. In addition, dose-dependent positive associations were found in the expression levels of vascular endothelial growth factor, fibroblast growth factor and alpha smooth muscle actin in quercetin treated cutaneous wounds. The significantly upregulated protein levels of Wnt and ß-catenin further indicated the important role of quercetin in promoting wound healing in mice. According to molecular docking analysis, the formed hydrogen bonds between quercetin and Ala195, Gln308, Asn369 and Lys372 residues of TERT also indicated the indispensable role of TERT in improving wound healing capacity. CONCLUSION: Quercetin effectively promoted cutaneous wound healing by enhancing the proliferation and migration of fibroblasts, as well as inhibiting inflammation and increasing the expression of growth factors in mice via Wnt/ß-catenin signaling pathway and TERT. It provides a basis for a more thorough understanding of mechanism of action of O. falcata Bunge in the treatment of knife wounds and burns.

The species of Oxytropis DC. of section Gloeocephala Bunge (Fabaceae) from Northeast Asia: genetic diversity and relationships based on sequencing of the intergenic spacers of cpDNA and ITS nrDNA.

Phylogenetic relationships within Oxytropis DC. sect. Gloeocephala Bunge from Northeast Asia were studied using plastid intergenic spacers (psbA-trnH + trnL-trnF + trnS-trnG) and ITS nrDNA. Populations of O. anadyrensis Vass., O. borealis DC., O. middendorffii Trautv., O. trautvetteri Meinsh., and O. vasskovskyi Jurtz. were monomorphic or characterised by a low level of chloroplast genetic diversity (h varied from 0.143 to 0.692, and π from 0.0001 to 0.0005). Presumably, the low genetic diversity was a result of the severe bottlenecks during Pleistocene glaciation-interglacial cycles. Twenty chlorotypes were identified; species studied had no shared chlorotypes. Chlorotypes of O. anadyrensis, O. borealis, and O. middendorffii formed two lineages each, while the chlorotypes of O. trautvetteri and O. vasskovskyi formed one separate lineage each in the phylogenetic network. There were specific diagnostic markers of cpDNA in each lineage, excluding O. vasskovskyi. The presence of a species-specific diagnostic marker in O. trautvetteri and specific markers in two lineages of O. anadyrensis support circumscribing these taxa as independent species. Regarding ITS nrDNA polymorphism, five ribotypes were detected. The differences revealed in plastid and nuclear genomes of Oxytropis sect. Gloeocephala confirmed that the Asian sector of Megaberingia was the main centre of diversification of arctic legumes.

UPLC-Q-TOF-MS/MS analysis of the guaiane sesquiterpenoids oxytropiols A-J and detection of undescribed analogues from the locoweed endophytic fungus Alternaria oxytropis (Pleosporaceae).

INTRODUCTION: In addition to the mycotoxin swainsonine, the locoweed endophytic fungus Alternaria oxytropis (Pleosporaceae) also produces a series of rarely reported, highly oxygenated bicyclic guaiane sesquiterpenoids. Few investigations on the electrospray tandem mass fragmentation pattern of this sesquiterpenoid have been reported. OBJECTIVES: We aimed to analyze and detect new guaiane sesquiterpenoid analogues from crude extracts of the locoweed endophytic fungus A. oxytropis by UPLC-Q-TOF-MS/MS experiments. MATERIALS AND METHODS: Oxytropiols A-J (1-10) and the extract of the locoweed endophytic fungus A. oxytropis were analyzed by UPLC-Q-TOF-MS/MS in positive mode. RESULTS: Typical neutral losses, McLafferty rearrangement, 1,2-rearrangement, and 1,3-rearrangement were considered to be the main fragmentation patterns for the [M + H]+ /[M + Na]+ ions of 1-10 by UPLC-Q-TOF-MS/MS experiments, and possible fragmentation pathways of 1-10 were suggested. A unique and undescribed analogue named oxytropiol K (11) was found in the extract based on UPLC-Q-TOF-MS/MS analysis. Compound 11 was isolated and elucidated by NMR spectrometry, and its UPLC-Q-TOF-MS/MS analysis was consistent with the fragmentation pathways of 1-10. CONCLUSION: The results further support that UPLC-Q-TOF-MS/MS is a powerful and sensitive tool for the characterization of known compounds (dereplication) and the detection of new analogues from crude extracts and imply that the locoweed endophytic fungus A. oxytropis, with few chemical investigations, is an important resource for undescribed metabolites.

Total flavonoids of Oxytropis falcata Bunge have a positive effect on idiopathic pulmonary fibrosis by inhibiting the TGF-ß1/Smad signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with unknown etiology. Oxytropis falcata Bunge (O. falcata) is a 1-35 cm high perennial clustered herb, also known as edaxia, has viscosity and a special smell, and is mainly distributed in the western areas of China. The root of O. falcata has a diameter of 6 mm, is straight and deep, dark red and its stems are shortened, woody and multibranched. O. falcata has heat-clearing, detoxification, analgesic, anti-inflammatory, antibacterial, hemostatic and antitumor activities. Furthermore, O. falcata has excellent anti-inflammatory and analgesic effects, and it is one of the three major anti-inflammatory drugs in Tibetan medicine, known as "the king of herbs". Total flavonoids of Oxytropis falcata Bunge (FOFB) were previously extracted, and their pharmacological activities are consistent with those of the whole herb. In this study, FOFB was extracted from O. falcata by ethanol extraction, and the mechanism of FOFB on IPF was verified by in vivo and in vitro experiments. AIM OF THE STUDY: In this study, we aimed to observe the effects of FOFB on idiopathic pulmonary fibrosis. MATERIALS AND METHODS: In in vivo experiments, an IPF rat model was established by bleomycin induction. The rats were treated with FOFB (100, 200, 400 mg kg-1·d-1) for 4 weeks. Masson staining and the expression of TGF-ß, p-Smad2, p-Smad3 and Smad7 in the lung tissue of rats were detected. In in vitro experiments, we perfused normal rats with FOFB (100, 200, 400 mg kg-1·d-1) and obtained the corresponding drug-containing serum. The HFL-1 cell model induced by TGF-ß1 was used to detect the corresponding indices through intervention with drug-containing serum. The best intervention time for drug-containing serum was detected by the CCK-8 method. Changes in apoptosis, cytoskeleton and rough endoplasmic reticulum structure were detected. Finally, the expression of TGF-ß, p-Smad2, p-Smad3 and Smad7 in cells was examined. RESULTS: In vivo, Masson staining indicated that the degree of pulmonary fibrosis increased significantly, the expression of TGF-ß, p-smad2 and p-Smad3 increased significantly, and the expression of Smad7 decreased in the model group. We found that the degree of pulmonary fibrosis gradually decreased and that the inhibition of the TGF-ß/Smad signaling pathway became more obvious with increasing FOFB dose. FOFB (400 mg kg-1·d-1) significantly improved the degree of pulmonary fibrosis in rats. In in vitro experiments, the CCK-8 results showed that 120 h was the best intervention time for drug-containing serum. In the model group, there was no obvious apoptosis or changes in microfilaments and microtubules, the number of rough endoplasmic reticulum increased, and the expression of TGF-ß, p-Smad2 and p-Smad3 increased significantly, while the expression of Smad7 decreased significantly. We found that with the increase in drug-containing serum concentration, the apoptosis, cytoskeleton and degree of destruction of the rough endoplasmic reticulum in the HFL-1 cell model also increased, and the inhibition of the TGF-ß/Smad signaling pathway became more pronounced; the effect of the drug-containing serum administered with FOFB (400 mg kg-1·d-1) was the most significant. CONCLUSIONS: The results suggest that FOFB can improve the occurrence and development of IPF. The effect of FOFB on IPF may be mediated by inhibition of the TGF-ß1/Smad signaling pathway.

Chemical and Biological Characteristics of Oxytropis pseudoglandulosa Plant of Mongolian Origin.

Oxytropis pseudoglandulosa is used in Mongolian traditional medicine due to its numerous reported health-promoting effects. To date, there are very few scientific reports that describe this species. In this article, its volatile oil composition, lipid extract composition, total phenolic and flavonoid content, antibacterial and allergenic properties are elucidated for the first time. Hexadecanoic acid, fokienol and tricosane were determined as the most notable components of the volatile oil, at 13.13, 11.46 and 5.55%, respectively. Methyl benzoate was shown to be the most abundant component of lipid extract at 40.69, followed by (E)-prop-2-enoic acid, 3-phenyl- and benzenepropanoic acid, at 18.55 and 9.97%. With a TPC of 6.620 mg GAE g-1 and TFC of 10.316 mg QE g-1, the plant extract of O. pseudoglandulosa indicated good antioxidant activity measured by IC50 at 18.761 µg mL-1. Of the 12 tested microorganisms, B. subtilis and S. cerevisiae were the shown to be most susceptible to the plant extract, with MIC at 2.081 and 0.260% (v/v), respectively. Bet v 1-a major birch pollen allergen found in plant-based foods-was determined to be at 192.02 ng g-1 with ELISA. Such a wide spectrum of biological activity indicated by O. pseudoglandulosa lends credence for its application in food industry. Its exerted antioxidant and antimicrobial effects could improve preservation of low-processed food dedicated for consumers afflicted with allergies. Hexadecanoic acid supplemented in foods with dietary plant extracts could add to the potential anti-inflammatory impact. The analysis of lipid makeup suggests O. pseudoglandulosa extract could also be considered as natural pesticide in organic farming.

Genetic diversity of Oxytropis section Xerobia (Fabaceae) in one of the centres of speciation.

The genetic diversity and phylogenetic relationships of Oxytropis caespitosa, O. grandiflora, O. eriocarpa, O. mixotriche, O. nitens, O. peschkovae and O. triphylla, section Xerobia subgenus Oxytropis, in one of the main speciation centres of the genus Oxytropis (Baikal Siberia and adjacent territories of Northeastern Mongolia) were studied based on sequence analysis of the psbA-trnH, trnL-trnF and trnS-trnG intergenic spacers of cpDNA, as well as the ITS nrDNA. Most populations are characterized by a high level of chloroplast genetic diversity (h varied from 0.327 to 1.000 and π from 0.0001 to 0.0090) due to the ancient origin for some species and to hybridization and polyploidy for others. 67 haplotypes were identified, of which six were shared. Phylogenetic relationships among species could not be satisfactorily resolved. Only the haplotypes of O. triphylla formed a group with rather high support. Probably, O. caespitosa, O. grandiflora, O. mixotriche and O. nitens constitute a single genetic complex. As regards the ITS nrDNA polymorphism, we detected only two ribotypes (RX1, RX2). Both were found in O. caespitosa, O. eriocarpa, O. mixotriche and O. peschkovae, while RX1 was present in O. nitens and O. triphylla, RX2 in O. grandiflora. The absence of diagnostic species-specific variants for the markers studied, together with the sharing of cpDNA haplotypes and nrDNA ribotypes between species, and the resulting polytomies on the phylogenetic trees, confirm the hypothesis on the hybrid origin of some of them. Obviously, the reproductive barriers within the sect. Xerobia are weak. However, morphological differences between the species of the sect. Xerobia are clearly pronounced, even when they grow in sympatry.

Preparation, characterization, and pharmacodynamic study on deep second degree burns of total flavonoids composite phospholipids liposome gel of Oxytropis falcata Bunge.

Wound healing is the treatment problem after deep second degree (II°) burns. The p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-κB/inhibitory factor-κB (NF-κB/IκB) signal pathways play significant role in angiogenesis and wound repair after burns.This study aimed to investigate the preparation, characterization and pharmacodynamics of the total flavonoids composite phospholipids liposome of Oxytropis falcata Bunge (TFOFB-CPL) on deep II° burns to research its biological activity and underlying mechanism. The TFOFB-CPL was prepared by thin-film dispersion method and the preparation process was optimized via central composite design. The TFOFB-CPL was then characterized by using particle size, polydispersity indexes (PDIs), zeta potential, encapsulation efficiency (EE) and morphology. Moerover, in vitro transdermal test and in vivo pharmacodynamic study included wound healing rate, hematoxylin-eosin (HE) staining, masson staning, western blotting and RT-PCR. The results showed that the therapeutic effects of TFOFB-CPL gel on deep II° burns, especially during wound healing were significant. TFOFB-CPL gel has a sustained-release effect during the treatment of deep II° burns with forming drug depot in the dermis layer. The wound healing rate of TFOFB-CPL gel group was near positive group and better than the other groups. TFOFB-CPL gel could promote the growth of epidermis, skin appendages, fibrovascular and collagen fibers, and had obvious anti-inflammatory effects. Moreover, TFOFB-CPL gel inhibited the activation of p38MAPK and the degradation of IκBα, and promoted the neonatal wounds during the early stage. Therefore, TFOFB-CPL gel could be considered as a novel preparation for treating deep II° burns.

Rhizobial Microsymbionts of Kamchatka Oxytropis Species Possess Genes of the Type III and VI Secretion Systems, Which Can Affect the Development of Symbiosis.

A collection of rhizobial strains isolated from root nodules of the narrowly endemic legume species Oxytropis erecta, O. anadyrensis, O. kamtschatica, and O. pumilio originating from the Kamchatka Peninsula (Russian Federation) was obtained. Analysis of the 16S ribosomal RNA gene sequence showed a significant diversity of isolates belonging to families Rhizobiaceae (genus Rhizobium), Phyllobacteriaceae (genera Mesorhizobium, Phyllobacterium), and Bradyrhizobiaceae (genera Bosea, Tardiphaga). A plant nodulation assay showed that only strains belonging to genus Mesorhizobium could form nitrogen-fixing nodules on Oxytropis plants. The strains M. loti 582 and M. huakuii 583, in addition to symbiotic clusters, possessed genes of the type III and type VI secretion systems (T3SS and T6SS, respectively), which can influence the host specificity of strains. These strains formed nodules of two types (elongated and rounded) on O. kamtschatica roots. We suggest this phenomenon may result from Nod factor-dependent and -independent nodulation strategies. The obtained strains are of interest for further study of the T3SS and T6SS gene function and their role in the development of rhizobium-legume symbiosis. The prospects of using rhizobia having both gene systems related to symbiotic and nonsymbiotic nodulation strategies to enhance the efficiency of plant-microbe interactions by expanding the host specificity and increasing nodulation efficiency are discussed.

Hyaluronidase inhibitory saponins and a trypanocidal isoflavonoid from the aerial parts of Oxytropis lanata.

A chemical examination of an extract from the aerial part of Oxytropis lanata led to the isolation and identification of 36 compounds, including saponins, isoflavonoids, oxazoles, and glycosides. The three among them were previously unreported oleanane-type saponins. In trypanocidal screening, 5,7,4'-trihydroxyisoflavone showed inhibitory activity against Trypanosoma congolense (IC50 = 10.5 µM), the causative agent of African trypanosomosis in animals; this activity was similar to that of active compounds from the roots of this plant. O. lanata is known to be a traditional medicinal plant in Mongolia for the treatment of inflammatory diseases. The anti-hyaluronidase effect of saponins 3, 5, 8, and 9, (IC50 = 0.15-0.22 mM) was stronger than that of sodium cromoglicate, which was used as a reference drug (IC50 = 0.37 mM). The chemical structures of the new saponins were determined based on HRFABMS, 1H and 13C NMR, 1H-1H COSY, HMQC, HMBC, and ROESY spectroscopic data along with chemical procedures.

Analysis of chemical constituents in the extract and rat serum from the chloroform extract of Oxytropis falcata Bunge by HPLC-MS.

Analysis of the constituents of the chloroform extract of Oxytropis falcata Bunge (CEOF), a traditional Tibetan medicine, in rat's serum after oral administration, has been performed by HPLC-MS. We have identified 10 compounds in CEOF and 11 bioactive ingredients from rat's serum after given CEOF. Six bioactive ingredients from rat's serum are matched with original form of the compounds of CEOF. Other five bioactive ingredients were seemed to be respectively metabolites. HPLC-MS is rapid, sensitive method and suitable for identification of bioactive components absorbed into blood of CEOF providing information for further research of pharmacological mechanism.

Oxytropis glabra DC. Inhibits Biofilm Formation of Staphylococcus epidermidis by Down-Regulating ica Operon Expression.

Staphylococcus epidermidis is one of the main causes of medical device-related infections and bovine mastitis owing to its biofilm-forming abilities. Oxytropis glabra DC. is one of the most widespread Fabaceae species and used as a Chinese herbal formulation in Western China. Our research investigated the effects of O. glabra on the biofilm formation of S. epidermidis and the possible inhibiting mechanism. The biofilm-forming reference strain, S. epidermidis SE-1 (ATCC 35,984), was employed as a model and semi-quantitative biofilm assay was performed to evaluate the antibiofilm activity of O. glabra. The exopolysaccharides (EPS) production and expression of ica operon were studied to explore the possible antibiofilm mechanism using thin-layer chromatography and quantitative real-time PCR assay, respectively. The results obtained indicated that O. glabra decoction at 7.5 mg mL-1 significantly inhibited biofilm formation by about 95% without affecting cell growth of S. epidermidis. Two hydrolysis productions of EPS were significantly decreased by 64% and 54% with the addition of 7.5 mg mL-1O. glabra and the expression of icaR was significantly up-regulated 2.2-times, whereas icaB was significantly down-regulated more than 50% by 7.5 mg mL-1O. glabra. These findings suggest a potential application for O. glabra as a promising candidate for the exploration of new drugs against S. epidermidis biofilm-associated infections.

Separation of 3-Hydroxy-3-Methylglutaryl Flavonoid Glycosides from Oxytropis falcata Using 2D Preparative Chromatography.

Oxytropis falcata Bunge, known as "the King of Herbs" in Tibetan medicine, is used for treatment of hyperpyrexia, pain, wounds, inflammation and anthrax. However, it is difficult to isolate compound with high-purity from O. falcata because of its complexity. In this work, an efficient method was successfully established for the separation of 3-hydroxy-3-methylglutaryl (HMG) flavonoid glycosides from O. falcata by 2D preparative chromatography (2D-HPLC). An ODS C18 preparative column was used for the first-dimension preparation, and the XCharge C18 preparative column with different separation selectivity to the ODS C18 stationary phase was used for the second-dimension preparation. Four HMG flavonoid glycosides (oxytroflavosides A-D) and two flavonoid glycosides (oxytroflavosides F and G) were separated with purities over 98%. Their structures were elucidated by nuclear magnetic resonance spectroscopy. The 2D-HPLC method used in this study is effective for preparative separation of HMG flavonoid glycosides from O. falcata. Additionally, this method shows great potential for the separation of flavonoid glycosides from other plant extracts.

Flavonoids as key bioactive components of Oxytropis falcata bunge, a traditional anti-inflammatory and analgesic Tibetan medicine.

The traditional Tibetan medicine Oxytropis falcata Bunge, in the Leguminosae family, is widely used in the west area owing to its significant anti-inflammatory and analgesic activities. O. falcata is rich in flavonoids, which are the main secondary metabolites and key bioactive components of this plant. Up to now, 91 flavonoids have been isolated from O. falcata, including isoflavone, flavone, flavonone, flavonol, homoisoflavonoid, chalcone, dihydrochalcone, chalcone dimers, and pterocarpans. The flavonoids in O. falcata have good anti-inflammatory and analgesic activities, which are comparable to those of a positive drug control (indomethacin). Furthermore, these flavonoids exhibit antibacterial, antioxidant, antitumour, anti-cardiovascular disease, and haemostatic activities. However, to date, O. falcata has not been reviewed comprehensively. Herein, the main secondary metabolites, biosynthetic pathways, and bioactivities of O. falcata are discussed.