Analysis of 3-nitropropionic acid in Fabaceae plants by HPLC-MS/MS.

INTRODUCTION: 3-Nitropropionic acid (3-NPA) is a toxic compound that can accumulate in esterified form in the Fabaceae family. In the Lotae tribe, many species have been identified as 3-NPA producers (e.g., Securigera varia), while some of the genetically close Lotae plants were formerly reported as 3-NPA-free (e.g., Lotus corniculatus and Anthyllis vulneraria). These plants are used as forage and have a tradition in ethnomedicine, also, the extracts of A. vulneraria are used in cosmetics. OBJECTIVES: Our aim was to investigate the 3-NPA content of these selected Fabaceae species and to develop a validated quantitative method to evaluate 3-NPA concentrations in extracts of different herbal parts and cosmetic products. MATERIALS AND METHODS: A UHPLC-ESI-Orbitrap-MS/MS method was applied for detection and identification of 3-NPA derivatives in the form of glucose esters. For the quantitative analysis, an optimized sample processing method was developed. The free 3-NPA content was determined using HPLC-ESI-MS/MS. RESULTS: 3-NPA esters could be detected in all three species, but their quantity showed a high variation. S. varia contained 0.5-1.0 g/100 g of 3-NPA, while in L. corniculatus samples only trace quantities were detectable, below the LOQ (25 ng/ml). Most of the A. vulneraria samples showed similarly low concentrations, but one sample had 3-NPA levels comparable to S. varia. 3-NPA could not be detected in the tested cosmetics containing A. vulneraria extracts. CONCLUSIONS: Using highly sensitive analytical methods, new 3-NPA-containing species were identified. The developed validated quantitative method is suitable for the determination of 3-NPA concentrations in herbal samples.

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

INTRODUCTION: 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. OBJECTIVES: 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. MATERIAL AND METHODS: Quantitative determination was carried out by the means of ultra-performance liquid chromatography coupled with ultraviolet diode-array detection (UPLC-UV-DAD). RESULTS: 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. CONCLUSION: As a result, no characteristic change could be observed between the two species regarding their isoflavonoid pattern.

In vitro and in silico evaluation of Ononis isoflavonoids as molecules targeting the central nervous system.

Isoflavonoids with various structural elements show a promising potential effect on central nervous system activities. Despite their favorable medicinal properties, the pharmacokinetic characteristics of this thoroughly investigated group of natural phenolics have only been described to a limited extent. Regarding the lack of information about the BBB permeability of isoflavones, isoflavanones, and pterocarpans found in Ononis species, the aim of our study was to investigate their physico-chemical properties influencing their absorption and distribution. Furthermore, we aimed to characterize the possible MAO-B inhibiting features of Ononis isoflavonoids in silico. Octanol-water partitioning and BBB-PAMPA permeability of formononetin, calycosin D, onogenin, sativanone, medicarpin and maackiain were assessed for the first time in our study. The log P values ranged from 2.21 to 3.03 and log D7.4 values from 2.48 to 3.03, respectively, indicating optimal polarity for BBB permeation. The results of PAMPA-BBB expressed as log Pe values fell between -5.60 and -4.45, predicting their good permeation capability as well. The effective permeability values showed structure-dependent differences, indicating that the pterocarpan type skeleton was the most preferred type, followed by isoflavanones, then isoflavones. The methoxy or methylenedioxy substitution of the same skeleton did not influence the permeability significantly, contrary to an additional hydroxyl group. Membrane retention showed a similar structure dependent pattern to that of effective permeability, ranging from 16% to 70%. For the identification of volumes of chemical space related to particular biological activities the ChemGPS-NP framework was used. The MAO-B inhibitory potency and selectivity were also predicted and validated. Based on our results, MAO-B inhibitory potency could be predicted with good precision, but in the case of selectivity, only the direction could be concluded (favors MAO-B or MAO-A), not the magnitude. Our finding reflects that Ononis isoflavonoid aglycones show an excellent fit with the suggested parameters for BBB permeability and this is the first study to confirm the highly favorable position of these natural products for MAO-B inhibition.

Enantioseparation and quantitative determination of two homologous beta amino acids found in Fabaceae plants.

Non-protein amino acids are important metabolites of the Fabaceae family, possessing valuable biological effects in addition to their toxic properties. We have previously identified two non-protein amino acids homoproline and homopipecolic acid in Ononis species for the first time, and herein the study was extended to investigate further Fabaceae species (O. spinosa, O. arvensis, M. sativa, A. vulneraria) with medicinal, food or cosmetic uses. As the enantiomers of these beta amino acids can carry different activity or toxicity, our aim was to develop a chiral separation method for homoproline and homopipecolic acid enantiomers and apply it to plant samples. For this purpose, dansylated derivatives were prepared and a cyclodextrin-modified capillary electrophoresis in addition to a chiral HPLC method were developed. Although baseline separation was achieved by CE applying mono-(6-N-pyrrolidine-6-deoxy)-ß-CD, mono-(6-N-piperidine-6-deoxy)-ß-CD or sulfated-gamma-cyclodextrin at pH 6.0, the HPLC method was found to be more suitable for the analysis of the plant samples. Both homoproline and homopipecolic acid were confirmed in plant samples as racemates. The quantitative determination of homoproline and homopipecolic acid in several Fabaceae species were also aimed. Since these molecules can be found in the plants as esters, sample preparation was optimized to liberate the target molecules. Several SPE methods were tested for sample purification and a HPLC-MS/MS method using C8 stationary phase was developed and validated. The presence of homoproline and homopipecolic acid could be confirmed in all species ranging from 1 µg/g through 500 µg/g homopipecolic acid and 6 µg/g to 60 µg/g homoproline and significant changes could be observed between species, geographical origins, and botanical parts. Generally O. spinosa root samples were found to be the richest sources of the two amino acids, but a high variance could be observed between species.

Stability Study of Alpinia galanga Constituents and Investigation of Their Membrane Permeability by ChemGPS-NP and the Parallel Artificial Membrane Permeability Assay.

Alpinia galanga Willd., greater galangal, has been used for thousands of years as a spice as well as in traditional medicine. Its central nervous system (CNS) stimulant activity and neuroprotective effects have been proved both in animal models and human trials. However, the compounds responsible for these effects have not been identified yet. Therefore, the main constituents (p-OH-benzaldehyde (1), trans-p-coumaryl-alcohol (2), p-coumaryl-aldehyde (4), galanganol A (5), galanganol B (6), trans-p-acetoxycinnamyl alcohol (7), 1'S-1'-acetoxychavicol acetate (ACA, 9), and 1'S-1'-acetoxyeugenol acetate (AEA, 10)) were isolated to investigate their aqueous stability and passive diffusion across the gastro-intestinal tract (GIT) membrane and the blood-brain barrier (BBB) by the parallel artificial membrane permeability assay (PAMPA). Our positive results for compounds 1, 2, 4, 7, 9, and 10 suggest good permeability, thus potential contribution to the effects of greater galangal in the CNS. The results of the PAMPA-BBB were corroborated by in silico chemography-based ChemGPS-NP framework experiments. In addition, examination of the chemical space position of galangal compounds in relation to known psychostimulants revealed that all the molecules in proximity are NET/SERT inhibitors. As ACA and AEA did not show much proximity to either compound, the importance of further investigation of their degradation products becomes more pronounced.

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-ß-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.

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.

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.

Characterization and identification of isoflavonoid glycosides in the root of Spiny restharrow (Ononis spinosa L.) by HPLC-QTOF-MS, HPLC-MS/MS and NMR.

Restharrow root has been used in traditional medicine for thousands of years; however, the active ingredients responsible for the diuretic effect are still unknown. Previous studies have proved that the root extract contains isoflavonoids, however only few derivatives were identified, mostly relying on retention times or UV data. The aim of our work was to perform a detailed structural characterization of the complete isoflavonoid profile in the aqueous-methanolic extract of Ononis spinosa root by high-performance liquid chromatography coupled with electrospray ionization accurate-mass quadrupole time-of-flight and tandem mass spectrometry in positive ionization mode (HPLC-ESI-QTOF-MS, HPLC-ESI-MS/MS) and nuclear magnetic resonance spectroscopy (NMR). On the basis of the accurate masses and fragmentation patterns isoflavones (formononetin, calycosin and pseudobaptigenin) and pterocarpans (maackiain and medicarpin) were identified. Two further dihydroisoflavone aglycones, namely onogenin and sativanone and a unique glucoside were isolated and their structures were elucidated by NMR experiments. Calycosin, onogenin and sativanone were detected in this plant for the first time. In contrast to previous works, the presence of biochanin A could not be confirmed, however its regioisomer calycosin and its derivatives were identified. Similarly, neither tectorigenin derivatives could be detected, however the isobar compound sativanone and its various glucosides were elucidated. The presence of genistein and daidzein could not be confirmed in the extract. Fragmentation pathways for onogenin and sativanone are presented. In the aqueous-methanolic extract 9 glucosides, 6 minor and 8 major glucoside malonates, 4 glucoside acetates and 7 aglycones were found. In total, 34 compounds were successfully identified.