Ononis Spinosa Research Articles

Foraging preferences of bumble bee castes are weakly related to plant species cover on two arable agri-environment habitat types

Arable field margins provide important floral resources for insect foragers. This study assessed the significance of cultivated margins and floristically enhanced margins, both English agri-environment scheme (AES) options, to foraging bumble bees (Bombus species). We examined plant foraging preferences in each habitat according to species and caste. Additionally, detailed botanical surveys were carried out to determine vascular plant densities on the study margins. Overall, our results emphasised the importance of spontaneous (Asteraceae) species emerging from the seed bank in the provision of forage across Bombus species and castes, and highlighted that Bombus foraging preferences appeared to be only weakly related to floral species densities. Although found only occasionally in high densities, the popularity of these dicots was likely due to high nectar sugar mass. Bombus queens were recorded relatively infrequently, implying that these habitats are failing to provide the preferred floral resources of all Bombus spp. queens. Queens that were observed were found to favour earlier-flowering species (e.g. Anchusa arvensis) and species with longer corollas (e.g. Vicia sativa). Worker bees across Bombus spp. showed high overlap in plant preferences (e.g. Cirsium arvense, Ononis spinosa). However, some variability in preferences between castes within a species were noted, for example, only B. terrestris/lucorum drones were found to forage on Crepis vesicaria in cultivated margins. Additionally, bumble bee abundance was only found to increase as dicot cover increased. Overall, our findings highlight the importance of continuing to utilise multiple AES types in order to fully support Bombus and other pollinating insect populations on farmland.

Ononin ameliorates depression-like behaviors by regulating BDNF-TrkB-CREB signaling in vitro and in vivo

Ethnopharmacological relevanceOnonin is a flavonoid compound found in several medicinal plants, including Astragalus membranaceus, Sophora flavescens, and Ononis spinosa. These plants have been traditionally used in various parts of the world for their medicinal properties, including anti-inflammatory, antioxidant, and antitumor effects. Major depression is a common, long-lasting, and recurrent psychiatric disorder with a high suicide rate. Naturally occurring flavonoids treat depression via poorly understood mechanisms. Aim of the studyThe present study aimed to determine whether ononin conferred an antidepressant-like effect in PC12 cell models and chronic mild stress (CMS)-induced depressive rat models and to explore its possible mechanisms. Materials and methodsDepression-related behaviors were measured using sucrose preference, tail suspension and open-field tests. Furthermore, to explore these mechanisms, we employed in vitro and in vivo assay methods, including neurite outgrowth, western blotting, quantitative RT-PCR, and staining methods. ResultsTreatment with ononin or BDNF significantly increased PC12 cells' neuronal growth and differentiation. Furthermore, ononin promotes the activation of TrkB and growth factors and upregulates the PI3K/Akt and BDNF/TrkB/CREB signaling pathways. The in vitro results were consistent with CMS-induced depressive rat models, in which ononin treatment significantly decreased depression-like behaviors and activated TrkB, growth factors, and BDNF/TrkB/CREB signaling pathways in the frontal cortex and hippocampus. Depression-induced microscopic alterations in the frontal cortex and hippocampus of rats with CMS-induced depression were also mitigated following ononin treatment. ConclusionBased on these findings, we suggest that ononin is a promising antidepressant candidate for treating depression.

Evaluation of Antioxidant and Anticancer Activity of Mono- and Polyfloral Moroccan Bee Pollen by Characterizing Phenolic and Volatile Compounds.

Bee pollen is frequently characterized as a natural source of bioactive components, such as phenolic compounds, which are responsible for its pharmaceutical potential and nutritional properties. In this study, we evaluated the bioactive compound contents of mono- and polyfloral bee pollen samples using spectroscopic and chromatographic methods and established links with their antioxidant and antitumor activity. The findings demonstrated that the botanical origin of bee pollen has a remarkable impact on its phenolic (3-17 mg GAE/g) and flavonoid (0.5-3.2 mg QE/g) contents. Liquid chromatography-mass spectrometry analysis revealed the presence of 35 phenolic and 13 phenylamide compounds in bee pollen, while gas chromatography-mass spectrometry showed its richness in volatiles, such as hydrocarbons, fatty acids, alcohols, ketones, etc. The concentration of bioactive compounds in each sample resulted in a substantial distinction in their antioxidant activity, DPPH (EC50: 0.3-0.7 mg/mL), ABTS (0.8-1.3 mM Trolox/mg), and reducing power (0.03-0.05 mg GAE/g), with the most bioactive pollens being the monofloral samples from Olea europaea and Ononis spinosa. Complementarily, some samples revealed a moderate effect on cervical carcinoma (GI50: 495 μg/mL) and breast adenocarcinoma (GI50: 734 μg/mL) cell lines. This may be associated with compounds such as quercetin-O-diglucoside and kaempferol-3-O-rhamnoside, which are present in pollens from Olea europaea and Coriandrum, respectively. Overall, the results highlighted the potentiality of bee pollen to serve health-promoting formulations in the future.

Chromosome numbers in 31 taxa of Asteraceae, Brassicaceae, Poaceae, Hypericaceae, Lamiaceae, Fabaceae and Plantaginaceae from wetlands of Hamadan region in Iran

In this study, we report the somatic chromosome numbers (2n) of 31 taxa belonging to Asteraceae, Brassicaceae, Poaceae, Hypericaceae, Lamiaceae, Fabaceae and Plantaginaceae families from wetlands of Hamadan region in Iran. The chromosome numbers of about half of these species have not been reported in the previous literature. Chromosome number of Ononis spinosa counted chromosomally for the first time at the world level and the chromosome numbers of Erigeron acer, Pulicaria dysenterica, Arctium lappa, Xanthium strumarium, X. spinosum, Barbarea plantaginea, Brachypodium sylvaticum, Polypogon fugax, Prunella vulgaris and Lathyrus chloranthus, are here reported for the first time from Iran. In all taxa 2n was 12, 14, 16, 18, 20, 24, 28, 30, 32 and 42, as a diploid, tetraploid and hexaploid levels. These newly available data are further compared with those previously published for the same or related species.

Anti-adipogenic activity of maackiain and ononin is mediated via inhibition of PPARγ in human adipocytes

Obesity is a global health burden for which we do not yet have effective treatments for prevention or therapy. Plants are an invaluable source of bioactive leads possessing anti-adipogenic potential. Ethnopharmacological use of Ononis spinosa L. roots (OSR) for treatment of obesity and metabolic disorders requires а scientific rationale. The current study examined the anti-adipogenic capacity of OSR and its secondary metabolites ononin (ONON) and maackiain (MACK) in human adipocytes as an in vitro model of obesity. Both ONON and MACK diminished lipid accumulation during adipocyte differentiation. Molecular docking analysis exposed the potential interactions between MACK or ONON and target regulatory adipogenic proteins. Furthermore, results from an RT-qPCR analysis disclosed significant upregulation of AMPK by MACK and ONON treatment. In addition, ONON increased SIRT1, PI3K and ACC mRNA expression, while MACK notably downregulated CEBPA, AKT, SREBP1, ACC and ADIPOQ. The protein level of PI3K, C/EBPα, PPARγ and adiponectin was reduced upon MACK treatment in a concentration-dependent manner. Similarly, ONON suppressed PI3K, PPARγ and adiponectin protein abundance. Finally, our study provides evidence that ONON exerts anti-adipogenic effect by upregulation of SIRT1 and inhibition of PI3K, PPARγ and adiponectin, while MACK induced strong inhibitory effect on adipogenesis via hampering PI3K, PPARγ/C/EBPα signaling and anti-lipogenic effect through downregulation of SREBP1 and ACC. Even though OSR does not hamper adipogenic differentiation, it could be exploited as a source of natural leads with anti-adipogenic potential. The multidirectional mechanism of action of MACK warrant further validation in the context of in vivo obesity models.

Ononis spinosa alleviated capsaicin-induced mechanical allodynia in a rat model through transient receptor potential vanilloid 1 modulation

BackgroundTransient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel implicated in pain sensation in response to heat, protons, and capsaicin (CAPS). It is well established that TRPV1 is involved in mechanical allodynia. This study investigates the effect of Ononis spinosa (Fabaceae) in CAPS-induced mechanical allodynia and its mechanism of action.MethodsMechanical allodynia was induced by the intraplantar (ipl) injection of 40 µg CAPS into the left hind paw of male Wistar rats. Animals received an ipl injection of 100 µg O. spinosa methanolic leaf extract or 2.5% diclofenac sodium 20 minutes before CAPS injection. Paw withdrawal threshold (PWT) was measured using von Frey filament 30, 90, and 150 minutes after CAPS injection. A molecular docking tool, AutoDock 4.2, was used to study the binding energies and intermolecular interactions between O. spinosa constituents and TRPV1 receptor.ResultsThe ipsilateral ipl injection of O. spinosa before CAPS injection increased PWT in rats at all time points. O. spinosa decreased mechanical allodynia by 5.35-fold compared to a 3.59-fold decrease produced by diclofenac sodium. The ipsilateral pretreatment with TRPV1 antagonist (300 µg 4-[3-Chloro-2-pyridinyl]-N-[4-[1,1-dimethylethyl] phenyl]-1-piperazinecarboxamide [BCTC]) as well as the β2-adrenoreceptor antagonist (150 µg butoxamine) attenuated the action of O. spinosa. Depending on molecular docking results, the activity of the extract could be attributed to the bindings of campesterol, stigmasterol, and ononin compounds to TRPV1.ConclusionsO. spinosa alleviated CAPS-induced mechanical allodynia through 2 mechanisms the direct modulation of TRPV1 and the involvement of β2 adrenoreceptor signaling.

Phytochemical Profile and Microbiological Activity of Some Plants Belonging to the Fabaceae Family.

This study aimed to investigate the chemical composition and the activity against Staphylococcus aureus (S. aureus) (ATCC 25923), Streptococcus pyogenes (S. pyogenes) (ATCC 19615), Escherichia coli (E. coli) (ATCC 25922), Pseudomonas aeruginosa (P. aeruginosa) (ATCC 27853), Shigella flexneri (S. flexneri) (ATCC 12022), Salmonella typhimurium (S. typhimurium) (ATCC 14028), Haemophillus influenzae (H. influenza) type B (ATCC 10211) and two fungal strains: Candida albicans (C. albicans) (ATCC 10231) and Candida parapsilopsis (C. parapsilopsis) (ATCC 22019) of the extracts obtained from Melilotus officinalis (MO), Coronilla varia (CV); Ononis spinosa (OS) and Robinia pseudoacacia (RP) (Fabaceae), and to identify the chemical compounds responsible for the antimicrobial effect against the tested strains. The extracts were obtained by conventional hydroalcoholic extraction and analyzed in terms of total polyphenols using the spectrophotometric method and by liquid chromatography (LC). The results have shown that the highest polyphenols content was recorded in the RP sample (16.21 mg gallic acid equivalent GAE/g), followed by the CV (15.06 mg GAE/g), the OS (13.17 mg GAE/g), the lowest value being recorded for the MO sample (11.94 mg GAE/g). The antimicrobial testing of plant extracts was carried out using the microdilution method. The most sensitive strains identified were: E. coli, S. typhimurium, P. aeruginosa and S. pyogenes, while protocatechuic acid, gallic acid, caffeic acid, quercetin, rutin, and kaempferol were identified as the chemical compounds responsible for the antibacterial effect. The analysis of the correlation between the chemical composition and the antimicrobial effect proved a moderate (r > 0.5) positive correlation between rosmarinic acid and S. pyogenes (r = 0.526), rosmarinic acid and S. typhimurium (r = 0.568), quercetin and C. albicans (r = 0.553), quercetin and S. pyogenes (r = 0.605). Therefore, it suggested possible antimicrobial activity generated by these chemical components. The results recommend the Fabaceae plants as promising candidates for further research to develop novel natural antimicrobial drugs.

A new cyclic polyketide and other constituents from Ononis spinosa growing wildly in Jordan and their antioxidant activity

The new cyclic polyketide 7,9,11-trihydroxytetracos-2-eneoic acid δ-lactone (1), together with other eleven known compounds, were isolated from Ononis spinosa, growing wildly in Jordan. All isolated compounds were identified by thorough investigation of their spectral data including NMR and HRESIMS. Antioxidant activity testing of puerol B, specionin and the new cyclic polyketide revealed that puerol B had the highest DPPH radical scavenging activity (IC50 0.09 ± 0.006 mg/ml) as compared to α-tocopherol (IC50 0.039 ± 0.0006 mg/ml), while specionin had the highest ABTS radical scavenging power (IC50 0.013 ± 0.0008 mg/ml) as compared to α-tocopherol and ascorbic acid (IC50 0.042 ± 0.0004; 0.026 ± 0 .0007 mg/ml; respectively).

Protective effects induced by the food supplement Fluxonorm® in the lower urinary tract.

Fluxonorm® is a dietary supplement that includes water-soluble extracts of Solidago virga-aurea, Phyllantus niruri, Epilobium angustifolium, Peumus boldus and Ononis spinosa. The aim of the present study was to evaluate the tolerability and efficacy of Fluxonorm® in improving lower urinary tract symptoms in patients with benign prostatic hyperplasia (BPH) in combination with standard of care. Lower urinary tract symptoms can be improved by a marked anti-inflammatory action on the lower urinary tract (irritative symptoms) and/or by an anti-proliferative action (obstructive symptoms) on the prostate. Thirty patients were enrolled to evaluate the effect of Fluxonorm® on improving lower urinary tract symptoms. All patients complained of lower urinary tract symptoms (LUTS), such as hesitancy, poor flow, intermittent flow, incomplete voiding (obstructive symptoms), as well as increased frequency, nocturia and urgency (storage symptoms). All patients were treated with one tablet of Fluxonorm® (1200 mg) daily for 30 days to corroborate the results of our observation in which the food supplement (800 µg/mL) was also studied on the human prostate cancer PC3 cell line (antiproliferative activity) and on prostaglandin (PG)E2 production (anti-inflammatory activity). In addition, the effect of this compound on cyclooxygenase-2 (COX-2) gene expression was investigated. Finally, a bioinformatic analysis was conducted with the aim of unravelling the mechanism of action underlying the observed bio-pharmacological effects. As hypothesized in our preclinical research, adding Fluxonorm® to the therapy of enrolled patients improved all studied clinical parameters, including maximum flow (Qmax), after one month of treatment. In the preclinical evaluation, this formulation reduced PC3 cell viability and PGE2 production. The effects were also paralleled by reduced COX-2 gene expression and Fluxonorm®'s partly related content of catechin. While docking studies pointed out to the putative inhibition of matrix metalloproteinse-2 by gallic acid, as a further mechanism underlying the observed anti-proliferative effects, in PC3 cells exposed to Fluxonorm®. Fluxonorm® improved the efficacy of standard therapy, in terms of antioxidant/anti-inflammatory effects, for the management of lower urinary tract symptoms (LUTS). This could be related, albeit partially, to the blunting effect of this compound on PGE2 production.

Qualitative and Quantitative Phytochemical Analysis of Ononis Hairy Root Cultures.

Hairy root cultures are genetically and biochemically stable, and they regularly possess the same or better biosynthetic capabilities for specialized (secondary) metabolite production compared to the intact plant. Ononis species are well-known herbal remedies in ethnopharmacology and rich sources of isoflavonoids. Besides isoflavones, less prevalent isoflavones and pterocarpans with valuable biological effects can be found in Ononis species as well. As these plants are only collected but not cultivated, biotechnological methods could play a role in the larger-scale extraction of Ononis isoflavonoids. Regarding this information, we aimed to establish Ononis spinosa and Ononis arvensis hairy root cultures (HRCs) and analyze the isoflavonoid profile of hairy root cultures qualitatively and quantitatively, in order to define their capacity to produce biologically valuable isoflavonoids. During the qualitative description, beside isoflavonoids, two new phenolic lactones, namely, bulatlactone 2″-O-β-D-glucoside and ononilactone, were isolated, and their structures were characterized for the first time. Altogether, 29 compounds were identified by the means of UPLC-Orbitrap-MS/MS. Based on UHPLC-UV-DAD measurements, the isoflavonoid spectrum of the Ononis HRCs differed markedly from wild-grown samples, as they produce a limited range of the scaffolds. The most abundant compounds in the HRCs were medicarpin glucoside and sativanone glucoside. The overall isoflavonoid production of the cultures was comparable to wild-grown O. arvensis and approximately twice as high as in wild-grown O. spinosa samples. As the overall content of wild-grown samples include more isoflavonoid derivatives, the HRCs contain structurally less divergent isoflavonoids but in higher quantity.

Phenolic Content and Antimicrobial and Anti-Inflammatory Effects of Solidago virga-aurea, Phyllanthus niruri, Epilobium angustifolium, Peumus boldus, and Ononis spinosa Extracts

Prostatitis is an inflammatory condition that is related to multiple infectious agents, including bacteria and fungi. Traditional herbal extracts proved efficacious in controlling clinical symptoms associated with prostatitis. In this context, the aim of the present study was to explore the efficacy of extracts from Solidago virga-aurea, Ononis spinosa, Peumus boldus, Epilobium angustifolium, and Phyllanthus niruri against bacterial (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus) and fungi strains (Candida albicans; C. tropicalis) involved in prostatitis. Additionally, anti-mycotic effects were tested against multiple species of dermatophytes (Trichophyton rubrum, T. tonsurans, T. erinacei, Arthroderma crocatum, A. quadrifidum, A. gypseum, A. currey, and A. insingulare). Antioxidant effects were also evaluated in isolated rat prostates challenged with lipopolysaccharide (LPS), and phytochemical analyses were conducted to identify and quantify selected phenolic compounds, in the extracts. Finally, a bioinformatics analysis was conducted to predict putative human and microbial enzymes targeted by extracts’ phytocompounds and underlying the observed bio-pharmacological effects. The phytochemical analysis highlighted that rutin levels could be crucial for explaining the highest antibacterial activity of P. boldus extract, especially against E. coli and B. cereus. On the other hand, in the E. angustifolium extract, catechin concentration could partially explain the highest efficacy of this extract in reducing lipid peroxidation, in isolated rat prostates stimulated with LPS. Concluding, the results of the present study showed moderate antimicrobial and anti-inflammatory effects induced by water extracts of S. virga-aurea, P. boldus, E. angustifolium, P. niruri, and O. spinosa that could be related, at least partially, to the phenolic composition of the phytocomplex.

Quantitative determination of isoflavonoids in Ononis species by UPLC-UV-DAD.

The root of the Ononis species has been used internally and externally in ethnomedicine for centuries and contains biologically valuable isoflavonoid compounds. Therefore, it is important to obtain quantitative information about the isoflavonoid profile of these plants. In this article we aimed to develop an optimised sample preparation protocol alongside a validated method for the quantitative measurement of isoflavones, isoflavanones and pterocarpans in the form of glucosides and aglycones, in order to compare the specialised metabolites of Ononis spinosa L. and O. arvensis L. Quantitative determination was carried out by the means of ultra-performance liquid chromatography coupled with ultraviolet diode-array detection (UPLC-UV-DAD). An optimised sample preparation method was developed to transform malonyl glucosides to their glucosidic forms. Chromatographic methods were created for the baseline separation of isoflavones, isoflavanones and pterocarpans alongside with their glucosides. Altogether 12 compounds were evaluated quantitatively in samples of O. spinosa and O. arvensis. As a result, no characteristic change could be observed between the two species regarding their isoflavonoid pattern.

Root Extracts From Ononis spinosa Inhibit IL-8 Release via Interactions With Toll-Like Receptor 4 and Lipopolysaccharide.

Extracts from the roots of Ononis spinosa L. (restharrow roots) are traditionally used for the treatment of patients with urinary tract infections due to its mild diuretic activity, caused by the inhibition of renal human hyaluronidase-1 by isoflavonoids. Preliminary studies also indicated anti-inflammatory effects. The following study aimed at investigating potential anti-inflammatory effects of restharrow extracts, prepared with solvents of different polarity. A dichloromethane extract (OS1), mainly composed of isoflavonoids and triterpenes as characterized by LC-MS, showed a concentration-dependent (25–100 μg/ml) inhibition of IL-8 and TNF-α release from LPS-stimulated human neutrophils. Significant inhibition was also found for the triterpene α-onocerin and the norneolignan clitorienolactone B, isolated from OS1. Further, OS1 and both compounds significantly decreased the expression of the adhesion molecules CD11b/CD18 and conversely increased the expression of CD62L in LPS-stimulated human neutrophils. This finding corresponds to a reduced inflammatory response by the inhibition of adhesion and migration of immune cells. As all of the observed effects are potentially mediated via Toll-like receptor 4 (TLR4) signaling, TLR4 transfected HEK293 cells were incubated with OS1. LPS-induced IL-8 secretion was significantly inhibited in a concentration-dependent manner, confirming TLR4 antagonism. This inhibition, however, was in part caused by an interaction of OS1 with LPS. In addition, also an aqueous extract containing high amounts of isoflavonoid glycosides and saponins from the roots of O. spinosa showed anti-inflammatory effects by interacting with the TLR4 signaling pathway. This study rationalizes the traditional use of extracts from O. spinosa for therapy of urinary tract infections, due to its potential anti-inflammatory effects that are mediated via TLR4 receptor antagonism.

Methanolic Extract of the Herb Ononis spinosa L. Is an Antifungal Agent with no Cytotoxicity to Primary Human Cells.

Ononis spinosa L. is a plant traditionally used as folk remedy. There are numerous studies regarding chemical constituents and health beneficial properties of Ononidis Radix. The following study was designed to investigate chemical composition and antifungal potential of the methanolic extract obtained from the O. spinosa L. herb. Chemical analyses regarding phenolic compounds of O. spinosa were performed by liquid chromatography with mass spectrometry (LC-DAD-ESI/MSn). Antifungal activity, antibiofilm properties and antifungal mode of action of the extract were evaluated, as well as cytotoxicity. Chemical analyses revealed the presence of flavonoids, isoflavonoids and phenolic acids in O. spinosa, with kaempherol-O-hexoside-pentoside being the most abundant compound (5.1 mg/g extract). Methanolic extract was active against all of the tested microfungi with Penicillium aurantiogriseum being the most sensitive to the extract inhibitory effect at 0.02 mg/mL; and effectively inhibited biofilms formed by Candida strains. Minimum fungicidal concentrations of extract rose in the presence of ergosterol and leakage of cellular components was detected. The extract showed no cytotoxicity to human gingival fibroblast (HGF-1) cells. This study significantly contributes to overall knowledge about medicinal potential of O. spinosa herbal extract and enlightens previously unrevealed properties. O. spinosa aerial parts seem to be an interesting candidate for the development of antifungal preparations, non-toxic to human cells.

The possible litholytic effect of Ononis Spinosa L. on various human kidney stones—An in vitro experimental evaluation

IntroductionUrolithiasis is a widespread health problem with a high recurrence and is a clinical condition that can progress to renal failure. This study aimed to investigate whether Ononis spinosa L. has a litholytic effect on kidney stones with different chemical patterns. Materials and methodsUrinary stones extracted from seven patients over 18 years of age during surgery were used as the study material. Each urinary stone was divided into two parts, one of which was left in a solution prepared with O. spinosa L. and the other constituted the control sample. The amount of calcium, phosphor and uric acid passing through each solution was detected by spectrophotometric analysis to assess its efficiency and determine whether or not the urinary stones were dissolved in the solution. The chemical compositions of the urinary stones were analyzed using reflectance Fourier transform infrared spectroscopy. To the best of our knowledge, this is the first study to investigate the litholytic effect of O. spinosa L. on kidney stones. ResultsThe passage of calcium, phosphate and uric acid through the solution containing O. spinosa L. was found to be statistically significant by spectrophotometry (p < 0.05) for all seven kidney stone samples left in the solution, and it was observed that the dissolution rates of the four stones with uric acid were higher than others. ConclusionThe results of this study showed that O. spinosa L. may have a litholytic effect on kidney stones.

Aqueous Root Extract from Ononis spinosa Exerts Anti-adhesive Activity against Uropathogenic Escherichia coli.

Extracts from Ononis spinosa are traditionally used for urinary tract infections due to diuretic and anti-inflammatory activity. A potential influence on the virulence of uropathogenic Escherichia coli has not been investigated until now. The following study aimed to investigate the influence of an aqueous O. spinosa extract on uropathogenic E. coli and uropathogenic E. coli host cell interaction. A hot water extract from the roots of O.spinosa (O. spinosa extract) was characterized by LC-qTOF-MS. The influence of O. spinosa extract on the proliferation of uropathogenic E. coli UTI89 and on cell viability against human T24 bladder cells was investigated. Anti-adhesive activity of O. spinosa extract was assessed by flow cytometry, evaluating the adhesion of fluorescent-labelled UTI89 to T24 bladder cells. Internalization of uropathogenic E. coli into T24 cells was monitored by an invasion assay. O. spinosa extract was characterized by the presence of isoflavones, isoflavanones, licoagrosides, pterocarpans, tartaric acid derivatives, and saponines. O. spinosa extract had no influence on the proliferation of uropathogenic E. coli (125 - 1000 µg/mL) and did not influence the cell viability of T24 cells. Bacterial adhesion to T24 cells was significantly (p > 0.001) inhibited by O. spinosa extract in a concentration-dependent manner (125 - 1000 µg/mL) during coincubation. Preincubation of uropathogenic E.coli or T24 cells with O. spinosa extract reduced bacterial adhesion, but to a lower extent than during coincubation. Consequently, the reduced bacterial adhesion also leads to a reduced internalization of uropathogenic E. coli uropathogenic E. coli into the host cell. O. spinosa extract does not interact with FimH-mediated uropathogenic E. coli adhesion to host cells. From these data, the traditional use of O. spinosa extracts for urinary tract infections seems to be rationalized.

Ononis spinosa L., an edible and medicinal plant: UHPLC-LTQ-Orbitrap/MS chemical profiling and biological activities of the herbal extract

This study explored the chemical profile of the aerial parts of Ononis spinosa and further investigated its biological activities. Chemical profiling of the extract revealed the presence of 63 different compounds: phenolic acids, flavonoid glycosides and aglycones, isoflavonoid glycosides and aglycones, and other related compounds. Our results revealed that the extract was active against 8 strains of free floating bacteria. It showed anti-biofilm potential against Staphylococcus aureus and was able to supress the production of staphyloxanthin in S. aureus at sub-minimal inhibitory concentrations. Its antioxidant activity was evaluated by using several assays (phosphomolybdenum, DPPH, ABTS, CUPRAC, FRAP, and metal chelating assay), which showed that the extract exhibited a dose dependent activity. Inhibition of AChE, BChE, amylase, glucosidase and tyrosinase was achieved by the extract, demonstrating its anti-enzymatic activity. The antiproliferative potential of the extract towards human cancer cell lines (HepG2, MCF-7, SiHa and A172) was determined by using the crystal violet assay. Ki67, a marker of proliferation was downregulated in the A172 glioblastoma cell line.

Isoflavonoids with inhibiting effects on human hyaluronidase-1 and norneolignan clitorienolactone B from Ononis spinosa L. root extract

Human hyaluronidase-1 (Hyal-1) is one of the main enzymes in the homeostasis of hyaluronic acid (HA), the main polysaccharide of extracellular matrix. Development of specific Hyal-1 inhibitors might be a promising target for improved wound healing, tissue regeneration, and looking at renal function for diuresis. By using surface-displayed Hyal-1 on Escherichia coli F470 cells, HA as substrate and stains-all method for quantification of undegraded HA, the respective enzyme activity can be determined easily. Based on the traditional use of extracts from the roots from Ononis spinosa L. (Restharrow root) as a weak diuretic to achieve flushing of the urinary tract and as an adjuvant in minor urinary complaints the herbal material was selected for bioactivity guided fractionation for compounds with Hyal-1 inhibition activity. Hot water and hydroalcoholic extracts showed moderate inhibiting effects (IC50 1.36 resp. 0.73 mg/mL) while dichloromethane extract exerted an IC50 of 190 μg/mL. Bioassay guided fractionation of the dichloromethane extract yielded four isoflavonoids with anti Hyal-1 activity: onogenin 1, sativanone 2, medicarpin 3 and calycosin-D 4 with inhibition rates of 25.4, 61.2, 22.4 and 23.0%, respectively at test concentration level of 250 μM. The norneolignan clitorienolactone B 5, the first time described for the genus Ononis, was inactive. The IC50 of sativanone, the most active compound was determined with 1501 μM, which was better than that of the positive control glycyrrhizinic acid (177 μM). Thus, a possible explanation for diuretic properties of Ononis spinosa L. root extract may be postulated from the results so far obtained.

Separation and characterization of homopipecolic acid isoflavonoid ester derivatives isolated from Ononis spinosa L. root

Spiny restharrow root (Ononis spinosa L.) and its preparations are mainly used for the treatment of urinary infections or bladder stones in numerous countries. Spiny restharrow root is rich in isoflavonoids (formononetin, calycosin and pseudobaptigenin), pterocarpans (medicarpin and maackiain) and dihydroisoflavonoids (onogenin and sativanone), which metabolites are present as glucosides, glucoside malonates, glucoside acetates and free aglycones in the root. The in-depth analysis of tandem mass spectrometric (MS) and high-resolution MS (HR-MS) data revealed the presence of nitrogen-containing compounds in the root extracts. An ion-exchange-based purification and a preparative-scale reversed phase chromatographic isolation procedure was developed for the characterization of these new natural products. For the unambiguous identification of the isolated compounds NMR experiments were carried out. The thorough characterization confirmed the presence of six piperidin-2-yl-acetic acid (homopipecolic acid) esters of isoflavonoid glucosides. This is the first report of homopipecolic acid esters isolated from higher plants.

A Single Oxidosqualene Cyclase Produces the Seco-Triterpenoid α-Onocerin.

8,14-seco-Triterpenoids are characterized by their unusual open C-ring. Their distribution in nature is rare and scattered in taxonomically unrelated plants. The 8,14-seco-triterpenoid α-onocerin is only known from the evolutionarily distant clubmoss genus Lycopodium and the leguminous genus Ononis, which makes the biosynthesis of this seco-triterpenoid intriguing from an evolutionary standpoint. In our experiments with Ononis spinosa, α-onocerin was detected only in the roots. Through transcriptome analysis of the roots, an oxidosqualene cyclase, OsONS1, was identified that produces α-onocerin from squalene-2,3;22,23-dioxide when transiently expressed in Nicotiana bethamiana In contrast, in Lycopodium clavatum, two sequential cyclases, LcLCC and LcLCD, are required to produce α-onocerin in the N. benthamiana transient expression system. Expression of OsONS1 in the lanosterol synthase knockout yeast strain GIL77, which accumulates squalene-2,3;22,23-dioxide, verified the α-onocerin production. A phylogenetic analysis predicts that OsONS1 branches off from specific lupeol synthases and does not group with the known L. clavatum α-onocerin cyclases. Both the biochemical and phylogenetic analyses of OsONS1 suggest convergent evolution of the α-onocerin pathways. When OsONS1 was coexpressed in N. benthamiana leaves with either of the two O. spinosa squalene epoxidases, OsSQE1 or OsSQE2, α-onocerin production was boosted, most likely because the epoxidases produce higher amounts of squalene-2,3;22,23-dioxide. Fluorescence lifetime imaging microscopy analysis demonstrated specific protein-protein interactions between OsONS1 and both O. spinosa squalene epoxidases. Coexpression of OsONS1 with the two OsSQEs suggests that OsSQE2 is the preferred partner of OsONS1 in planta. Our results provide an example of the convergent evolution of plant specialized metabolism.