HPLC coupled with quadrupole time of flight tandem mass spectrometry for analysis of glycosylated components from the fresh flowers of two congeneric species: Robinia hispida L. and Robinia pseudoacacia L.

Developing methods for the systematic and rapid identification of the chemical compositions of fresh plant tissues has long attracted the attention of phytochemists and pharmacologists. In the present study, based on highly efficient sample pretreatment and high-throughput analysis of high-performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry data using molecular networks, a method was developed for systematically analyzing the chemical constituents of the fresh flowers of Robinia hispida L. and Robina pseudoacacia L., two congeneric ornamental species that lack prior consideration. A total of 44 glycosylated structures were characterized. And on the basis of establishing of the fragmentation pathways of 11 known flavonoid glycosides, together with the molecular networking analysis, 18 other ions of flavonoid glycosides in five classes were clustered. Moreover, 15 soyasaponins/triterpenoid glycosides were tentatively identified by comparison of their tandem mass spectrometry characteristic ions with those reported in the literature or the online Global Natural Product Social Molecular Networking database. The water extracts were separated by flash chromatography, which resulted in the discovery of one new compound, named rohispidascopolin, along with five known entities. The pharmacological targets were predicted by SwissTargetPrediction.

Biotic and abiotic factors causing the collapse of Robinia pseudoacacia L. veteran trees in urban environments.

Robinia pseudoacacia L. is an interesting example of how one plant species can be considered invasive or useful depending on its environment. In the past this tree species was planted for decorative purposes and for wood in Poland. For many years it was recommended in poor and degraded habitats because it facilitated late-successional plant species. The aim of this study was to verify if black locust can still be regarded as a resistant tree species in urban greenery. The health condition of old tree specimens growing along streets and in parks was compared. The occurrence of pests and pathogens on R. pseudoacacia trees was assessed and the content of mineral elements in leaves was examined. The research results showed that the health of black locust trees growing in the urban environment in Polish cities, especially along streets (in comparison to park sites), deteriorated significantly due to the interaction of harmful biotic and abiotic factors. Increased level of toxic metals (Fe, Zn, Pb, Mn and Cd) in plant tissues and the accumulation of pests and pathogens negatively influenced the health of R. pseudoacacia.

Assessment of the anti-pathogenic effects of condensed tannin extracts using scanning electron microscopy.

Two different types of condensed tannins (CTs), which were extracted and purified from tilia (Tilia L.) and black locust (Robinia pseudoacacia), were studied and tested against two kinds of bacteria, including Gram-negative and Gram-positive, avian pathogenic E. coli (APEC) and Staphylococcus epidermidis (S. epidermidis) respectively, by minimal bactericidal concentrations (MBCs) and scanning electron microscopy (SEM). Both CT extracts were significantly effective (p ≤ 0.05) at MBCs of 5-10 mg CT/ml against APEC (Gram-negative), and at 1.25-5 mg CT/ml on S. epidermidis (Gram-positive). This indicated that the CTs were more potent against the Gram-positive than the Gram-negative bacteria. Further, SEM revealed that CTs caused mainly morphological deformations of the bacterial cells and some conjoined cell growth.

Quantitative and Qualitative Identification of Bioactive Compounds in Edible Flowers of Black and Bristly Locust and Their Antioxidant Activity.

Black and bristly locust flowers are an excellent source of polyphenols, including flavonols, phenolic acids, and anthocyanins. In the present literature, there is a lack of studies showing the quantity and quality of phenolic compounds from different locust flowers. There are a few studies on the status of polyphenols in black locust flowers and their products but not bristly locusts. The aims of this study were to analyze and compare the concentrations of bioactive compounds from Robinia pseudoacacia and Robinia hispida flowers over two years. These two species of plants from six independent locations (parks and green areas) located in Warsaw were assessed in this study. The dry matter and polyphenol contents of the flowers were determined. Black locust flower samples contained significantly more myricetin and luteolin. Only bristly locust flowers contained anthocyanins. Five individual anthocyanins were identified in the pink-colored bristly locust flowers. Pelargonidin-3-O-glucoside and cyanidin-3-O-glucoside were the predominant forms in the pool of total anthocyanins.

Glandular trichomes of Robinia viscosa Vent. var. hartwigii (Koehne) Ashe (Faboideae, Fabaceae)-morphology, histochemistry and ultrastructure.

MAIN CONCLUSION: Permanent glandular trichomes of Robinia viscosa var. hartwigii produce viscous secretion containing several secondary metabolites, as lipids, mucilage, flavonoids, proteins and alkaloids. Robinia viscosa var. hartwigii (Hartweg's locust) is an ornamental tree with high apicultural value. It can be planted in urban greenery and in degraded areas. The shoots, leaves, and inflorescences of this plant are equipped with numerous persistent glandular trichomes producing sticky secretion. The distribution, origin, development, morphology, anatomy, and ultrastructure of glandular trichomes of Hartweg's locust flowers as well as the localisation and composition of their secretory products were investigated for the first time. To this end, light, scanning, and transmission electron microscopy combined with histochemical and fluorescence techniques were used. The massive glandular trichomes differing in the distribution, length, and stage of development were built of a multicellular and multiseriate stalk and a multicellular head. The secretory cells in the stalk and head had large nuclei with nucleoli, numerous chloroplasts with thylakoids and starch grains, mitochondria, endoplasmic reticulum profiles, Golgi apparatus, vesicles, and multivesicular bodies. Many vacuoles contained phenolic compounds dissolved or forming various condensed deposits. The secretion components were transported through symplast elements, and the granulocrine and eccrine modes of nectar secretion were observed. The secretion was accumulated in the subcuticular space at the trichome apex and released through a pore in the cuticle. Histochemical and fluorescence assays showed that the trichomes and secretion contained lipophilic and polyphenol compounds, polysaccharides, proteins, and alkaloids. We suggest that these metabolites may serve an important function in protection of plants against biotic stress conditions and may also be a source of phytopharmaceuticals in the future.

Low-cost field production of biochars and their properties.

Biochar has been intensively investigated for carbon sequestration, soil fertility enhancement, and immobilization of heavy metals and organic pollutants. Large-scale use of biochar in agricultural production and environmental remediation, however, has been constrained by its high cost. Here, we demonstrated the production of low-cost biochar ($20/ton) in the field from Robinia pseudoacacia biowaste via a combined aerobic and oxygen-limited carbonization process and a fire-water-coupled method. It involved aerobic combustion at the outer side of biomass, oxygen-limited pyrolysis in the inner core of biomass, and the termination of the carbonization by water spray. The properties of biochar thus produced were greatly affected by exposure time (the gap between a burning char fell to the ground and being extinguished by water spray). Biochar formed by zero exposure time showed a larger specific surface area (155.77 m2/g), a higher carbon content (67.45%), a lower ash content (15.38%), and a higher content of carboxyl and phenolic-hydroxyl groups (1.74 and 0.86 mol/kg, respectively) than biochars formed with longer exposure times (5-30 min). Fourier-transform infrared spectroscopic (FTIR) spectra indicated that oxygen-containing functional groups of biochar played a role in Cd and oxytetracycline sorption though a quantitative relationship could not be established as the relative contribution of carbon and ash moieties of biochar to the sorption was unknown. Outcomes from this research provide an option for inexpensive production of biochar to support its use as a soil amendment in developing countries.

Effect of Robinia pseudoacacia Leaf Extract on Interleukin-1ß-mediated Tumor Angiogenesis.

BACKGROUND/AIM: Interleukin (IL)-1ß is a pro-inflammatory cytokine that has recently been established as a stimulator of angiogenesis via regulation of proangiogenic factor expression in the tumor microenvironment. This study aimed to demonstrate the inhibitory effects of Robinia pseudoacacia leaf extract (RP) on IL-1ß-mediated tumor angiogenesis. MATERIALS AND METHODS: Secreted embryonic alkaline phosphatase (SEAP) reporter gene assay, ex vivo and in vitro tube formation assay, western blot, and quantitative PCR were used to analyze the inhibitory effect of RP on IL-1ß-mediated angiogenesis. RESULTS: RP inhibited secretion of SEAP, blocked IL-1ß signaling, and inhibited IL-1ß-mediated angiogenesis in ex vivo and in vitro assays. RP inhibited nuclear translocation of NF-ĸB by suppressing phosphorylation of IL-1ß signaling protein kinases and inhibited mRNA expression of IL-1ß-induced pro-angiogenic factors including VEGFA, FGF2, ICAM1, CXCL8, and IL6. CONCLUSION: RP suppressed IL-1ß-mediated angiogenesis and, thus, could be a promising agent in anticancer therapy.

Evaluation of antiviral effect and toxicity of total flavonoids extracted from Robinia pseudoacacia cv. idaho.

In this study, we aimed to evaluate the antiviral effect of total flavonoids extracted from Robinia pseudoacacia cv. idaho (RPTF) in vivo and its toxicity on rats with oral gavage. RPTF was prepared by percolation with 70% ethanol for 24 h and its antiviral effect on different kinds of viruses was evaluated in vitro by MTT staining. The long-term toxicity of RPTF on rats was evaluated through the detection of general behavior, body weight, food intake and related organ tissue sections of experimental animals. We found that RPTF produced significantly inhibitory effects on HSV-1 and EV-71 viruses with the therapeutic index TI values 113.8 and 46.2, respectively. Moreover, toxicity evaluation in vivo showed no significantly adverse effects in rats, indicating that RPTF was safe in use. In conclusion, we demonstrated that RPTF, natural compounds in the Chinese traditional medicine, could act as promising and effective antiviral therapeutics with relative safety in use.

Chemical and thermal analysis of lignin streams from Robinia pseudoacacia L. generated during organosolv and acid hydrolysis pre-treatments and subsequent enzymatic hydrolysis.

Lignin streams produced in biorefineries are commonly used to obtain energy. In order to increase the competitiveness of this industry, new lignin valorization routes are necessary, for which a depth characterization of this biological macromolecule is essential. In this context, this study analyzed lignin streams of Robinia pseudoacacia L. generated during organosolv and acid hydrolysis pre-treatments and during the subsequent enzymatic hydrolysis. These lignins included dissolved lignins from pre-treatment liquors and saccharification lignins from pre-treated materials. Chemical composition and structural features were analyzed by analytical standard methods and Fourier Transform Infrared spectroscopy (FTIR), size exclusion chromatography (SEC), 13C solid state nuclear magnetic resonance (13C NMR) and 1H-13C two-dimensional nuclear magnetic resonance (2D NMR); while thermal characterization included thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). In general, all studied lignins contained a predominance of ß-O-4' aryl ether linkages, followed by resinol (ß-ß') and phenylcoumaran (ß-5'), with a predominance of syringyl over guaiacyl and hydroxyphenyl units. Nevertheless, the dissolved lignins revealed a removal of linkages, especially ß-O-4', leading to an enrichment of phenolic groups. Moreover, high thermal stability and good thermoplasticity were characteristics of these lignins. Contrary, the saccharification lignins exhibited a more intact structure, but with an important remaining carbohydrates content.

Development of a Female-Targeted Lure for the Box Tree Moth Cydalima perspectalis (Lepidoptera: Crambidae): a Preliminary Report.

The box tree moth, Cydalima perspectalis, is an invasive pest in Europe causing damage on Buxus species. In this study, we aimed to develop a "bisexual" lure to attract both female and male moths. Based on a previous screening bioassay we tested methyl salicylate, phenylacetaldehyde and eugenol as potential attractants in different combinations. The trapping results showed that both binary and ternary blends attracted male and female moths. Catches with these blends were comparable to catches with the synthetic pheromone. Subsequently we carried out single sensillum recordings, which proved the peripheral detection of the above-mentioned compounds on male and female antennae. To identify synergistic flower volatiles, which can be also attractive and can increase the trap capture, we collected flower headspace volatiles from 12 different flowering plant species. Several components of the floral scents evoked good responses from antennae of both females and males in gas chromatography-electroantennographic detection. The most active components were tentatively identified by gas chromatography coupled mass spectrometry as benzaldehyde, cis-ß-ocimene, (±)-linalool and phenethyl alcohol. These selected compounds in combination did not increase significantly the trap capture compared to the methyl salicylate- phenyacetaldehyde blend. Based on these results we discovered the first attractive blend, which was able to attract both adult male and female C. perspectalis in field conditions. These results will yield a good basis for the optimization and development of a practically usable bisexual lure against this invasive pest.

Hierarchically porous and heteroatom self-doped graphitic biomass carbon for supercapacitors.

Here we report a simple, low-cost and environment friendly method, in which Black locust seed dregs and potassium ferrate (K2FeO4) are used as starting raw materials and activation agent. The hierarchically porous carbons (BDPC) with high special surface area and abundant mesopores (SBET = 2010.1 m2 g-1 and Vmeso = 1.457 cm3 g-1) are obtained through hydrothermal treatment and chemical activation. The BDPC electrode exhibits excellent electrochemical performances by virtue of unique architecture and heteroatoms pseudocapacitance contribution. In the three-electrode system, the optimized carbon material (BDPC-2) achieves a high specific capacitance of 333 F g-1 at 1 A g-1 and displays the high rate capability (81.1% capacitance retention at 100 A g-1) in 6 M KOH electrolyte. The symmetric supercapacitor based BDPC-2 exhibits energy density as high as 26.2 Wh kg-1 (at a power density of 0.79 kW kg-1) and excellent long-term cycling stability (only 8% decrease after 10,000 cycles) in 6 M KOH.

From Robinia pseudoacacia L. nectar to Acacia monofloral honey: biochemical changes and variation of biological properties.

BACKGROUND: Robinia pseudoacacia L. nectar and its derivative monofloral honey were systematically compared in this study, to understand how much the starting solution reflected the final product, after re-elaboration by Apis mellifera ligustica Spinola. RESULTS: Subjected to dehydration in the hive, nectar changed in its water and sugar content when transformed into honey, as physicochemical and gas chromatographic-mass spectrometric analyses revealed. Spectrophotometric measurements and characterization by high-performance liquid chromatography-diode array detection of 18 plant molecules demonstrated honey to be richer than nectar in secondary metabolites. For the first time, the hypothesis of the existence of a nectar redox cycle in R. pseudoacacia was reported, as previously described for Nicotiana sp., based on 1D-protein profiles, western blot analysis and detection of H2 O2 and ascorbate. The bioactivity of both matrices was also investigated. Antiradical in vitro tests showed that Acacia honey was more antioxidant than nectar, which was even able to induce oxidative stress directly in a eukaryotic cell system. Antimicrobial assays demonstrated that nectar was bacteriostatic, due to H2 O2 activity, whereas honey was even bactericidal. CONCLUSION: All these data support the ecological role of nectar and honey in nature: protection of the gynoecium from pathogens and preservation from degradative processes, respectively. © 2018 Society of Chemical Industry.

Volatile-Compound Fingerprinting by Headspace-Gas-Chromatography Ion-Mobility Spectrometry (HS-GC-IMS) as a Benchtop Alternative to 1H NMR Profiling for Assessment of the Authenticity of Honey.

This work describes a simple approach for the untargeted profiling of volatile compounds for the authentication of the botanical origins of honey based on resolution-optimized HS-GC-IMS combined with optimized chemometric techniques, namely PCA, LDA, and kNN. A direct comparison of the PCA-LDA models between the HS-GC-IMS and 1H NMR data demonstrated that HS-GC-IMS profiling could be used as a complementary tool to NMR-based profiling of honey samples. Whereas NMR profiling still requires comparatively precise sample preparation, pH adjustment in particular, HS-GC-IMS fingerprinting may be considered an alternative approach for a truly fully automatable, cost-efficient, and in particular highly sensitive method. It was demonstrated that all tested honey samples could be distinguished on the basis of their botanical origins. Loading plots revealed the volatile compounds responsible for the differences among the monofloral honeys. The HS-GC-IMS-based PCA-LDA model was composed of two linear functions of discrimination and 10 selected PCs that discriminated canola, acacia, and honeydew honeys with a predictive accuracy of 98.6%. Application of the LDA model to an external test set of 10 authentic honeys clearly proved the high predictive ability of the model by correctly classifying them into three variety groups with 100% correct classifications. The constructed model presents a simple and efficient method of analysis and may serve as a basis for the authentication of other food types.

The Taishan Robinia pseudoacacia polysaccharides enhance immune effects of rabbit haemorrhagic disease virus inactivated vaccines.

Robinia pseudoacacia flower, a common component in traditional Chinese medicine, has long been well-known for its high pharmaceutical value. This study aimed to assess the immunopotentiating effects of Taishan Robinia Pseudoacacia polysaccharides (TRPPS) in rabbits inoculated with a rabbit haemorrhagic disease virus (RHDV) inactivated vaccine. The rabbits were administered with the RHDV vaccine in conjunction with varying concentrations of TRPPS, and their blood samples were collected at different time points to analyze the ratio and number of blood lymphocytes. In addition, sera were prepared and analyzed to determine the overall antibody titer and the level of IL-2, a cytokine commonly used as an indicator of immune activity. The various TRPPS-supplemented vaccines were shown to be more effective in enhancing the immune functions of the inoculated rabbits compared to their polysaccharide-free counterpart, with 200 mg/mL of TRPPS exhibiting the most pronounced benefits that were comparable to those of propolis. In addition, the TRPPS-supplemented RHDV inactivated vaccines could significantly improve the survival rates of the immunized rabbits against RHDV infection. Our studies offered convincing experimental evidence for the development of TRPPS as a new type of plant-derived immunopotentiator.

Air pollution tolerance index and heavy metal bioaccumulation in selected plant species from urban biotopes.

This research was carried out on plants Taraxacum officinale, Plantago lanceolata, Betula pendula and Robinia pseudoacacia growing in urban biotopes with different levels of heavy metal contamination in the city of Dabrowa Górnicza (southern Poland). Based on the pollution index, the highest heavy metal contamination was determined in the site 4 (connected with industry emitters) and 6 (high traffic). The metal accumulation index (MAI) values ranged within the biotopes in Dabrowa Górnicza between 7.3 and 20.6 for R. pseudoacacia, 4.71-23.1 for P. lanceolata, 4.68-28.1 for T. officinale and 10.5-27.2 for B. pendula. Increasing tendency in proline content in biotopes connected with high traffic was found in the leaves of investigated plants (except R. pseudoacacia). Similar tendency was observed for ascorbic acid content in the foliage of the plants as well as in T. officinalle in stands connected industrial emission. Non-protein thiols content increased especially in the leaves of R. pseudoacacia in biotopes with high traffic emissions as well as in T. officinale in stands connected with industry. The mean values of APTI (Air Pollution Tolerance Index) within the city of Dabrowa Górnicza for investigated plants were found in the following ascending order P. lanceolata < R. pseudoacacia < B. pendula < T. officinale. Among the investigated plants B. pendula and T. officinale may be postulated as appropriate plants in urban areas with considerable soil and air contamination, especially with heavy metals. The results indicate that species deemed tolerant according to APTI are suitable plants in barriers areas to combat atmospheric pollution.

Rational approach to solvent system selection for liquid-liquid extraction-assisted sample pretreatment in counter-current chromatography.

A rational liquid-liquid extraction approach was established to pre-treat samples for high-speed counter-current chromatography (HSCCC). n-Hexane-ethyl acetate-methanol-water (4:5:4:5, v/v) and (1:5:1:5, v/v) were selected as solvent systems for liquid-liquid extraction by systematically screening K of target compounds to remove low- and high-polarity impurities in the sample, respectively. After liquid-liquid extraction was performed, 1.4g of crude sample II was obtained from 18.5g of crude sample I which was extracted from the flowers of Robinia pseudoacacia L., and then separated with HSCCC by using a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:2:1:2, v/v). As a result, 31mg of robinin and 37mg of kaempferol 7-O-α-l-rhamnopyranoside were isolated from 200mg of crude sample II in a single run of HSCCC. A scale-up separation was also performed, and 160mg of robinin with 95% purity and 188mg of kaempferol 7-O-α-l-rhamnopyranoside with 97% purity were produced from 1.2g of crude sample II.

Enzymatic activities for lignin monomer intermediates highlight the biosynthetic pathway of syringyl monomers in Robinia pseudoacacia.

Most of the known 4-coumarate:coenzyme A ligase (4CL) isoforms lack CoA-ligation activity for sinapic acid. Therefore, there is some doubt as to whether sinapic acid contributes to sinapyl alcohol biosynthesis. In this study, we characterized the enzyme activity of a protein mixture extracted from the developing xylem of Robinia pseudoacacia. The crude protein mixture contained at least two 4CLs with sinapic acid 4-CoA ligation activity. The crude enzyme preparation displayed negligible sinapaldehyde dehydrogenase activity, but showed ferulic acid 5-hydroxylation activity and 5-hydroxyferulic acid O-methyltransferase activity; these activities were retained in the presence of competitive substrates (coniferaldehyde and 5-hydroxyconiferaldehyde, respectively). 5-Hydroxyferulic acid and sinapic acid accumulated in the developing xylem of R. pseudoacacia, suggesting, in part at least, sinapic acid is a sinapyl alcohol precursor in this species.

Characterisation of Antimicrobial Properties of Extracts of Selected Medicinal Plants.

The scope of the experiments included analysis of the antimicrobial activity of ethanolic, methanolic and aqueous extracts against bacterial and fungal cultures and determination of the minimum inhibitory concentration of plant extracts tested microbial growth. Analysis of the antifungal and antibacterial activity was carried out by the disc diffusion method using paper discs. In the experiment 11 species of microorganisms - 8 bacterial and 3 fungal strains were used. The highest antimicrobial activity against the tested strains was demonstrated by black elder (Sambucus nigra L.), black locust (Robinia pseudoacacia L.) and lingonberry (Vaccinium vitis-idaea L.) extracts. The study showed the diverse morphological activity of specific parts of elderberry and quince, which is the effect of different polyphenolic profile of these plants. The yeast Saccharomyces cerevisiae, Escherichia coli, Pseudomonas putida and Bacillus subtilis showed the highest sensitivity to the effect of extracts of the analysed plants. As a positive control three antibiotics - amphotericin B, vancomycin and amoxicillin with clavulanic acid were used.

Evaluation of genotoxic variations in plant model systems in a case of metal stressors.

This study was designed to assess the effects of long term, high metal exposition (cadmium, lead, copper, nickel and zinc) on DNA damage in four plant model systems [Taraxacum officinale (Asteraceae), Matricaria recutita L. (Asteraceae), Robinia pseudoacacia L. (Fabaceae), and Urtica dioica (Urticaceae)]. DNA stability was investigated by a Random Amplified Polymorphic DNA (RAPD) technique. Agarose-gel electrophoresis revealed total of 37 bands with different molecular weights ranging from 1250 to 5000 bp. It generated distinctive polymorphism value of 72.97% (27 bands) total in four plant species investigated. The dendrogram constructed using NTSYSpc programme showed that there is grouping in separate clusters of the same plant model collected from two different areas (metal-exposed and control samples). The study concluded that the long term metal-exposing periods had genotoxic stress on macromolecules of plant model systems investigated and biomarkers used should be augmented for reliable estimates of genotoxicity after exposure of plants to metal stressors.

Hydroponic screening of black locust families for heavy metal tolerance and accumulation.

Present work examines phytoextraction potential of four black locust families (half-sibs 54, 56, 115, and 135) grown hydroponically. Plants were treated with 6 ppm of cadmium (Cd), 100 ppm of nickel (Ni), and 40 ppm of lead (Pb) added in Hoagland nutrient solution, accompanying with simultaneously applied all three metals. Responses to metals exposure among families were different, ranging from severe to slight reduction of root and shoot biomass production of treated plants. Calculated tolerance indices are indicating tested families as highly tolerant (Ti > 60). Family 135 had the lowest tolerance index, pointing that it was highly susceptible to applied metals. Comparing photosynthetic activities of tested families it has been noticed that they were highly sensitive to stress induced by heavy metals. Net photosynthetic rate of nickel treated plants was the most affected by applied concentration. Cadmium and nickel concentrations in stems and leaves of black locust families exceeded 100 mg Cd kg(-1) and 1000 mg Ni kg(-1), in both single and multipollution context. On the contrary, accumulation of lead in above ground biomass was highly affected by multipollution treatment. Tf and BCF significantly varied between investigated treatments and families of black locust. Concerning obtained results of heavy metals accumulation and tolerance of black locust families can be concluded that tested families might be a promising tool for phytoextraction purposes, but it takes to be further confirmed in field trials.