Application of Metabolomics Tools to Determine Possible Biomarker Metabolites Linked to Leaf Blackening in Protea.

The postharvesting disorder leaf blackening is the main cause of product rejection in Protea during export. In this study, we report an investigation into metabolites associated with leaf blackening in Protea species. Methanol extracts of leaf and involucral bract tissue were analyzed by liquid chromatography hyphenated to photodiode array and high-resolution mass spectrometry (LC-PDA-HRMS), where 116 features were annotated. Analytical data obtained from 37 Protea species, selections, and hybrids were investigated using metabolomics tools, which showed that stems susceptible to leaf blackening cluster together and contained features identified as benzenetriol- and/or hydroquinone-derived metabolites. On the other hand, species, selections, and cultivars not prone to blackening were linked to metabolites with known protective properties against biotic and abiotic stressors. During the browning process, susceptible cultivars also produce these protective metabolites, yet at innately low levels, which may render these species and cultivars more vulnerable to blackening. Metabolites that were found to be correlated to the instigation of the browning process, all comprising benzenetriol- and hydroquinone-glycoside derivatives, are highlighted to provide preliminary insights to guide the development of new Protea cultivars not susceptible to leaf blackening.

Invasive Plants: Turning Enemies into Value.

In this review, a brief description of the invasive phenomena associated with plants and its consequences to the ecosystem is presented. Five worldwide invasive plants that are a threat to Portugal were selected as an example, and a brief description of each is presented. A full description of their secondary metabolites and biological activity is given, and a resume of the biological activity of extracts is also included. The chemical and pharmaceutical potential of invasive species sensu lato is thus acknowledged. With this paper, we hope to demonstrate that invasive species have potential positive attributes even though at the same time they might need to be controlled or eradicated. Positive attributes include chemical and pharmaceutical properties and developing these could help mitigate the costs of management and eradication.

Detailed Phenolic Characterization of Protea Pure and Hybrid Cultivars by Liquid Chromatography-Ion Mobility-High Resolution Mass Spectrometry (LC-IM-HR-MS).

In this study we report a detailed investigation of the polyphenol composition of Protea pure (P. cynaroides and P. neriifolia) and hybrid cultivars (Black beauty and Limelight). Aqueous methanol extracts of leaf and bract tissues were analyzed by ultrahigh pressure liquid chromatography hyphenated to photodiode array and ion mobility-high resolution mass spectrometric (UHPLC-PDA-IM-HR-MS) detection. A total of 67 metabolites were characterized based on their relative reversed phase (RP) retention, UV-vis spectra, low and high collision energy HR-MS data, and collisional cross section (CCS) values. These metabolites included 41 phenolic acid esters and 25 flavonoid derivatives, including 5 anthocyanins. In addition, an undescribed hydroxycinnamic acid-polygalatol ester, caffeoyl-O-polygalatol (1,5-anhydro-[6-O-caffeoyl]-sorbitol(glucitol)) was isolated and characterized by 1D and 2D NMR for the first time. This compound and its isomer are shown to be potential chemo-taxonomic markers.

Subpollens delivery of Platanus acerifolia pollen allergen Pla a3 and nucleic acid into lungs and cells.

Pollen allergy is a very serious seasonal respiratory disease. However, there has been a lack of understanding how pollen allergens enter the body and act on cells. This study focused on the release, transport and characteristic of Pla a3 allergen of the Platanus acerifolia pollen. Pla a3 protein was purified by prokaryotic expression system for preparation of polyclonal antibody. The distribution and release of Pla a3 protein in pollen were observed by immunohistochemistry. Mice were immunized with purified Pla a3 protein and SPPs, respectively. The pathological examination of mouse lung tissue proved that SPPs, as a fine particle in the range of 0.1-1µm, can enter the deep part of the lung directly through the respiratory tract and led to inflammation. Furthermore, DAPI staining confirmed a certain amount of nucleic acids in SPPs. After incubation with SPPs for 6 h, the Pla a3 mRNA could be detected in A549 cells by PCR. This suggests that nucleic acid wrapped in SPPs could be delivered into A549 cells. These results could provide a new clue and experimental data accumulation for further study on the mechanism of pollen sensitization.

Investigation of the phytochemical composition and antioxidant properties of chinar (Platanus orientalis L.) leaf infusion against ethanol-induced oxidative stress in rats.

Chinar (Platanus orientalis L.) is used in folk medicine against tooth and knee pain, wounds, inflammation, and stomach discomfort; however, the effects of P. orientalis leaf (PO-leaf) infusion on the liver and kidney are unknown. The aim of this study was to investigate the phytochemical composition and antioxidant properties of an infusion obtained from dried P. orientalis leaves against ethanol-induced oxidative stress (OS) in rats. After a toxicity test, thirty male Wistar rats were divided into five groups: Control, Ethanol 20%, Ethanol 20% + Silymarin (10 mg/kg), Ethanol 20% + PO-20 mg/mL infusion, and Ethanol 20% + PO-60 mg/mL infusion. The PO-leaf infusion doses were given ad libitum during 28 days to test the biochemical and antioxidant enzyme levels. According to the results, the PO-leaf contained rich compounds such as benzaldehyde, palmitic acid, 2,4-ditert-butylphenol, stearic acid, octadecanoic acid, linoleic acid, linolenic acid, kaempferol, and kaempferol derivatives. In the Ethanol group, AST, ALT, LDH, GGT, UA, and urea in the serum and GST and malondialdehyde (MDA) in the liver and erythrocyte tissues showed a significant increase compared to the Control group. AST, LDH, GGT, UA, and LDL-C levels in the serum and MDA (all tissues) significantly decreased in the Ethanol + PO-60 mg/mL group compared to the Ethanol group. SOD, GPx, and CAT activities in the kidney tissue of the Ethanol group showed a significant decrease compared to the Control group, whereas the GPx activity in kidney tissue in all of the treatment groups increased significantly compared to the Ethanol group. These findings suggest that the administration of the determined PO-leaf infusion doses might have a protective role against ethanol-induced liver and kidney damage in rats.

New arbutin derivatives from the leaves of Heliciopsis lobata with cytotoxicity.

Heliciopsis lobata is a medicinal plant, which is exclusively used to treat tumor in Li folk region. Two new arbutin derivatives, 6'-((E)2-methoxy-5-hydroxycinnamoyl) arbutin (1) and 2'-((E)2, 5-dihydroxycinnamoyl) arbutin (2) along with five known compounds (3-7), were isolated from the leaves of Heliciopsis lobata. Their structures were elucidated on the basis of extensive spectroscopic interpretations. They were evaluated for their potential anticancer activity. Compounds 6 and 7 exhibited cytotoxicity against MGC-803 cells with IC50 values being 44.1 and 11.3 µg·mL-1, respectively. Additionally, compounds 1, 2 and 5-7 exhibited a moderate inhibition of MGC-803 cells invasion; compound 2 at 20 µg·mL-1 inhibited the invasion of MGC-803 cells by 43.0%, compared with the controls.

Expression, purification and identification of Pla a1 in a codon-optimized Platanus pollen allergen.

The present study aimed to express, purify and identify the major allergen gene, Pla a1, in Platanus pollen. According to previous studies, the major gene sequences of the Pla a1 allergen were obtained and codon optimization and synthesis of the genome were performed using DNAStar software. Following binding of the target gene fragment and the pET-44a vector, the JM109 cells were transfected to produce positive clones. The vectors were then transformed into Escherichia coli Rosetta cells to induce the expression of the target protein. The exogenous protein was purified using affinity chromatography and was identified by western blot analysis. Pla a1, the major allergen protein in Platanus pollen, was successfully isolated and this exogenous protein was purified using affinity chromatography. The present study was the first, to the best of our knowledge, to obtain expression of the allergen recombinant protein, Pla a1, fused with a Strep-TagII via codon optimization and provided the basis for the preparation of allergens with high purity, recombinant hypoallergenic allergens and allergen nucleic acid vaccines.

Grevillosides J-Q, arbutin derivatives from the leaves of Grevillea robusta and their melanogenesis inhibitory activity.

From the 1-BuOH-soluble fraction of a MeOH extract of the leaves of Grevillea robusta, new arbutin derivatives and related compounds, named grevillosides J-Q (1-8), together with eight known compounds (9-18) were isolated. Various kind of acyl groups were attached to ß-D-glucose at the 6-position through an ester linkage. Their structures were mainly elucidated from one- and two-dimensional NMR spectroscopic data. For exploitation as skin-lightening and anti-chloasma agents, the inhibitory activities of the isolated compounds as well as ones isolated in previous experiments (19-31) toward tyrosinase and melanin-producing B16 cells were assayed. Several compounds showed promising activity.

Antigenotoxicity of Roupala montana extract in the mouse micronucleus and comet assays.

Roupala montana Aubl. (Proteaceae) is a typical savannah species and native to tropical South America that has a moderate mortality for adult forms of Schistossoma mansoni. Because this species has been little studied, the aim of this investigation was to evaluate the influence of R. montana extract on DNA damage induced by methyl methanesulfonate (MMS) in peripheral blood cells and liver of Swiss mice using the micronucleus and comet assay, respectively. R. montana dichloromethane extract was prepared from a stock solution (0.5 mg/mL) in 5% dimethyl sulfoxide in water. Animals received a single dose of different concentrations of R. montana (5, 10 and 20 mg/kg body weight) by gavage (0.5 mL/animal). For antigenotoxicity assessment, different concentrations of R. montana were administered simultaneously with MMS diluted in water (40 mg/kg, intraperitoneally; 0.3 mL/animal). Peripheral blood and hepatocyte samples were obtained 48 and 24 h after treatment, respectively. Results showed that R. montana administered alone indicated the absence of genotoxicity in the mouse micronucleus or comet assay. On the other hand, administration of different doses of R. montana concomitantly with MMS led to a significant reduction in frequency of micronucleated polychromatic erythrocytes and DNA damage, when compared to the group treated only with MMS. Further, for the micronucleus assay, the gradual increase of R. montana concentration led to a proportional increase in the reduction of genotoxicity induced by MMS, indicating a dose-response relationship.

An alkenylresorcinol derivative from Hakea sericea fruits and their antimicrobial activity.

Hakea sericea has been introduced to Portugal for ornamental purposes. The phytochemical composition and the antioxidant, antibacterial, antibiofilm and cytotoxic properties of this shrub species have been previously reported. The present work describes the bioassay-guided fractionation of the crude methanolic extract of H. sericea fruits and the isolation of 9-(3,5-dihydroxy-4-methylphenyl)nona-3(Z)-enoic acid. The structure of this new compound was established by one- and two-dimensional NMR and IR spectroscopy, and high-resolution mass spectrometry. The antibacterial properties of the new alkenylresorcinolwere studied by determining its MIC values against several strains of Gram-positive and Gram-negative bacteria using the resazurin microtiter assay. The new alkenylresorcinol inhibited the growth of Enterococcus faecalis, Listeria monocytogenes and Bacillus cereus with MIC values of 0.31, 0.02 and 0.16 mg/mL, respectively. Good MIC values were obtained against Staphylococcus aureus strains (0.005 - 0.16 mg/mL), including the clinical isolates (SA 01/10, SA 02/10 and SA 03/10) and MRSA strains.

Actividad antioxidante y antihiperglucemiante de la especie medicinal Oreocallis grandiflora (Lam.) R. Br., al sur del Ecuador / Antioxidant and antihyperglycemic activity of the medicinal specie Oreocallis grandiflora (Lam.) R. Br., in southern Ecuador

In this study we evaluated the antioxidant and antihyperglycemic activity in vitro of the extracts obtained with solvents: hexane, ethyl acetate and methanol, of the medicinal plant Oreocallis grandiflora (cucharillo), collected in the Saraguro indian community of the province Loja, southern Ecuador. The antioxidant activity was evaluated by the tests: DPPH, FOLIN-CIOCALTEU and beta-CLAMS, while the antihyperglycemic activity was determined by inhibition assay á-amylase and alpha-glucosidase. The samples were diluted to different concentrations and the reading was performed in a UV spectrophotometer, using as positive control á-tocopherol for DPPH and Folin-ciocalteu test, trolox for beta-CLAMS test, and Glucobay® for testing alpha-amylase and alpha-glucosidase.The results are expressed as IC50, these show that the methanol extract of Oreocallis grandiflora has inhibitory effect on alpha-amylase, the IC50 is 109 ug/ml, compared to 126 ug/ ml of Glucobay®. It also shows inhibitory effect on á-glucosidase, the IC50 is 3 ug/ml compared to 1316 ug/ml of Glucobay®. It also shows antioxidant activity, its IC50 is 15 ug/ml compared to 5 ug/ml of á-tocopherol.

En el presente trabajo se evaluó la actividad antioxidante y antihiperglucemiante in vitro de los extractos obtenidos con los solventes: hexano, acetato de etilo y metanol, de la planta medicinal Oreocallis grandiflora (cucharillo), recolectada en la comunidad indígena de Saraguro en la provincia de Loja, al sur del Ecuador. La actividad antioxidante fue evaluada a través de los ensayos: DPPH, FOLIN-CIOCALTEU y beta-CLAMS, mientras que la actividad antihiperglucemiante fue determinada por el ensayo de inhibición de alfa- amilasa y alfa-glucosidasa. El extracto metanólico de Oreocallis grandiflora presenta efecto inhibitorio sobre la enzima alfa-amilasa, su concentración inhibitoria (CI50) es de 109 ug/ml, frente a 126 ug/ml del control positivo Glucobay®. Además, muestra efecto inhibitorio sobre la enzima alfa-glucosidasa, su concentración inhibitoria (CI50) es de 3 ug/ml, frente a 1316 ug/ml del Glucobay®. Muestra también actividad antioxidante, su concentración inhibitoria (CI50) es de 15 ug/ml, frente a 5 ug/ml del alfa-tocoferol.

Chemical constituents and biological studies of the leaves of Grevillea robusta.

Three new compounds: Graviquinone (1), cis-3-hydroxy-5-pentadecylcyclohexanone (2), and methyl 5-ethoxy-2-hydroxycinnamate (3), and thirty-eight known compounds were isolated and identified from the leaves of Grevillea robusta. The structures of these compounds were determined by spectroscopic and chemical transformation methods. Graviquinone (1) showed the strongest cytotoxicity against MCF-7, NCI-H460, and SF-268 cell lines. Methyl 2,5-dihydroxycinnamate (4), graviphane (13), and dehydrograviphane (14) exhibited very potent DPPH scavenging activity compared with α-tocopherol. Methyl 2,5-dihydroxycinnamate (4) and bis-norstriatol (17) demonstrated strong inhibition of L-DOPA oxidation by mushroom tyrosinase compared with kojic acid.

Prenylated alkylbisphenols from Grevillea whiteana.

Eleven new bisresorcinols including four mixtures each of two isomers and one resorcinol/phloroglucinol derivative, together with five known resorcinols have been isolated from the ethyl acetate extract of stems of Grevillea whiteana. The new compounds were identified as 4-(3-hydroxy-3-methylbutyl)grebustol-B (10a), 4'-(3-hydroxy-3-methylbutyl)grebustol-B (10b), 4-(4-hydroxy-3-methylbutyl)grebustol-B (2a) and 4'-(4-hydroxy-3-methylbutyl) rebustol-B (2b), 2,2-dimethyldihydropyrano grebustol-B (11a) and iso-2,2-dimethyldihydropyranogrebustol-B (11b), 2,2-dimethyl-3xi-hydroxydihydropyranogrebustol-B (7a) and iso-2,2-dimethyl-3xi-hydroxydihydropyranogrebustol-B (7b), 15-(2-(4-hydroxy-3-methylbutyl)-resorcinol-5-yl)-1-(phloroglucinolyl)-9(Z)pentadecen-one (whiteanone) (4), 5,5'-(hexadecan-diyl)bisresorcinol (12) and 2-methyl-5,5'-(8(Z)-hexadecen-1,16-diyl)bisresorcinol (9). This is the first record of pyranobisresorcinols in the genus and the first report of a phloroglucinol terminal phenolic unit in any Grevillea species.

A new phenolic glucoside from the leaves of Heliciopsis lobata.

A new phenolic glucoside ester, 6'-E-(2''-methyl-2''-butenoyl) arbutin (1), was isolated from the leaves of Heliciopsis lobata. Its structure was elucidated by spectral analysis.

[Studies on chemical constituents of Heliciopsis lobata II].

OBJECTIVE: To study the constituents of the leaves of Heliciopsis lobata. METHOD: Compounds were isolated by column chromatography on silica gel, neutral aluminum oxide and Sephadex LH -20, and their structures were identified by spectroscopic and physicochemical data. RESULT: Seven known compounds were isolated and identified as: myricetin (1), myricitrin (2), syringetin-3-O-beta-D-glucopyranoside (3), medioresinol (4), D-1-O-methyl-myo-inositol (5), hydroquinone (6), and beta-sitosterol (7). CONCLUSION: All of these compounds were isolated from genus Heliciopsis for the first time.

Anatomical element localization by EDXS in Grevillea exul var. exul under nickel stress.

Grevillea exul var. exul, an endemic serpentinic Proteaceae of New Caledonia, was chosen to study the spatial distribution of Ni because this species supports strong content of metals, which can allow important absorptions thus detectable by microanalysis. Fine transversal sections of axenic G. exul var. exul plants grown during 15 days on nickel sulphate medium were examined by EDXS microanalysis. It showed that in Ni treated plants, Ni was concentrated mostly in the phloem compared to the xylem and the epidermis, either in roots or in the basal part of the stems and was mostly in the epidermis in the upper part of the stems and not detectable in the leaves. This metal took the place of P and K in the treated plants whereas the localization of these macroelements was quite uniform in control sections. We assume that a mechanism of phloem loading is implicated to restrict Ni accumulation in G. exul var. exul.

Cytotoxic 5-alkylresorcinol metabolites from the leaves of Grevillea robusta.

Bioassay-guided fractionation of the MeOH extract of the leaves of Grevillea robusta led to the isolation of six new 5-alkylresorcinols, gravicycle (1), dehydrogravicycle (2), bisgravillol (3), dehydrobisgravillol (4), dehydrograviphane (5), and methyldehydrograviphane (6), as well as eight known compounds. The structures of these compounds were determined by spectroscopic and chemical methods. Graviphane (7) and methylgraviphane (8) were isolated in the pure form for the first time from a natural source. The compounds all showed marginal cytotoxicity against MCF-7, NCI-H460, and SF-268 cell lines.

Ethnopharmacological evaluation of radal (leaves of Lomatia hirsuta) and isolation of 2-methoxyjuglone.

BACKGROUND: Leaves of Lomatia hirsuta are used in traditional medicine in Chile under the common name of "radal". A tea of radal is traditionally used for treatment of cough, bronchial troubles, and asthma. In a preliminary screening, extracts of the leaves revealed antifungal activity, and the present phytochemical study was undertaken to explain this activity and support the traditional use. METHODS: Along with the traditional tea, extracts of the leaves were screened for antifungal and toxic activities. The profile of secondary constituents was obtained using GC-MS. RESULTS: 2-Methoxyjuglone was isolated from the leaves of Lomatia hirsuta and found to be active against the pathogenic fungus Candida albicans (MIC = 8 microg/mL). Cinnamic acid and vanillic acid were identified as major constituents in the tea by GC-MS. The tea was found not to be toxic against Artemia salina. CONCLUSION: The presence of phenolic acids with antimicrobial properties supports the traditional use of Radal, and encourages further studies.

5-O-glucosyldihydroflavones from the leaves of Helicia cochinchinensis.

From the leaves of Helicia cochinchinensis, collected on Okinawa Island, seven phenolic glucosides and two terpenic glucosides were isolated. Five of the phenolic glucosides were previously known, being identified with p-coumaric and ferulic acids glucosyl esters, rhodioloside, helicidiol, and naringenin 5-O-beta-D-glucopyranoside. The structures of two other phenolic glucosides, named heliciosides A and B, were elucidated to be 5-O-beta-D-glucosides of 3-hydroxyflavanone, namely aromadendrin and taxifolin, by means of spectroscopic analyses. The two terpenic glucosides were identified with ampelopsisionoside and icariside C1.

[Study on chemical constituents in seeds of Helicia nilagirica (II)].

OBJECTIVE: To study the chemical constituents in seeds of Helicia nilagirica. METHOD: The ethanol extract was seperated by petroleum ether, dichloromethane, n-butanol in sequence, then isolated by silica gel column chromatography. The structures were identified and elucidated by physicochemical properties and spectral analysis. RESULT: Five compounds were isolated from the dichloromethane and n-butanol extracts, identified as p-hydroxybenzaldehyde (1), p-hydroxybenzoic acid (2), gallic acid (3), helicide (4), 4-formylpymyl-O-beta-D-glucopyranoside (5). CONCLUSION: All the compounds except IV were isolated from the plant for the first time. The compounds I, II and III were isolated from the genus Helicia for the first time.