Chemical Composition, Antioxidant and Anti-AGEs Activities of a French Poplar Type Propolis

Accumulation in tissues and serum of advanced glycation end-products (AGEs) plays an important role in pathologies such as Alzheimer’s disease or, in the event of complications of diabetes, atherosclerosis or renal failure. Therefore, there is a potential therapeutic interest in compounds able to lower intra and extracellular levels of AGEs. Among them, natural antioxidants (AO) with true anti-AGEs capabilities would represent good candidates for development. The purpose of this study was to evaluate the AO and anti-AGEs potential of a propolis batch and then to identify the main compounds responsible for these effects. In vivo, protein glycation and oxidative stress are closely related. Thus, AO and antiglycation activities were evaluated using both DPPH and ORAC assays, respectively, as well as a newly developed automated anti-AGEs test. Several propolis extracts exhibited very good AO and anti-AGEs activities, and a bioguided fractionation allowed us to identify pinobanksin-3-acetate as the most active component

Correlation coefficients for esterase activity ratios.

<p>Carrot cell suspensions with five different genotypes were tested for esterase relative specific activity in the presence of fungal extracts and toxins. The treatments were as follows: rA: <i>A. dauci</i> (strain FRA017) fungal culture raw extract; rM: uninoculated medium raw extract; aA: <i>A. dauci</i> fungal culture aqueous extract; aM, uninoculated medium aqueous extract; oA: <i>A.dauci</i> fungal culture organic extract; oM: uninoculated medium organic extract; DMSO: DMSO solution at a concentration corresponding to oM, z1, z2 and z3 treatments; z1: 0.025 µM zinniol; z2: 10 µM zinniol; z3: 500 µM zinniol. Correlation coefficients corresponding to significant (α = 0.05) linear regressions are in bold.</p

Correlations between cell suspension reactions to <i>Alternaria dauci</i> raw extracts, organic extracts and low zinniol concentrations.

<p>Five carrot genotypes were tested for their metabolic activity when <i>A. dauci</i> raw (rA) or organic (oA) extract was added to the plant culture medium. The same experiments were conducted while adding uninoculated medium raw (rM) or organic (oM) extract and 0.025 µM zinniol to DMSO (z1) or DMSO. rA/rM denotes plant cell esterase activity variations due to the presence of fungal raw extracts, oA/oM denotes plant cell esterase activity variations due to the presence of fungal organic extracts, and z1/DMSO denotes plant cell esterase activity variations due to the presence of 0.025 µM zinniol in the medium. A: correlation plots of rA/rM, oA/oM and z1/DMSO by pairs. Bars represent standard errors. The three paired correlated activity indices presented here correspond to the most significant r² values (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101008#pone-0101008-t004" target="_blank">Table 4</a>). B: 3D correlation plot of rA/rM, oA/oM and z1/DMSO.</p

Influence of cultivar, fungal exudate fractions and zinniol on cell suspension integrity and somatic embryogenesis.

<p>Carrot cell suspensions with six different genotypes were tested for embryogenesis in the presence of fungal extracts and toxins. Embryogenesis was assessed 3 weeks after treatment.</p>1<p>Treatments were as follows: rA: <i>Alternaria dauci</i> (strain FRA017) fungal culture raw extract; rM: uninoculated medium raw extract; aA: <i>A. dauci</i> fungal culture aqueous extract; aM: uninoculated medium aqueous extract; oA: <i>A. dauci</i> fungal culture organic extract; oM: uninoculated medium organic extract; DMSO: DMSO solution, at a concentration corresponding to oM, z1, z2 and z3 treatments; z1: 0.025 µM zinniol; z2: 10 µM zinniol; z3: 500 µM zinniol. C: no treatment.</p>2<p>The signs are as follows: (−) no embryogenesis was visible and cells were damaged, (+) early-stage embryogenic masses were visible, (++) embryos were present, (+++) embryogenesis was profuse. +/− early-stage embryogenic masses were visible, or no embryogenesis was visible depending on the repetition.</p

Range of symptoms observed on leaves 13 days after inoculation.

<p>The symptom number was assessed at 7, 9 and 13(see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101008#pone-0101008-t001" target="_blank">Table 1</a>). The leaves shown here show a symptom severity representative of the plant partial resistance level. <b>A</b>: H1, <b>B</b>: Presto, <b>C</b>: K3, <b>D</b>: H4, <b>E</b>: Bolero, <b>F</b>: I2. H1, K3, H4 and I2 are breeding lines, while Presto and Bolero are widely cultivated Nantaise type carrot cultivars.</p

Range of embryogenic activity observed in cell suspensions 3 weeks after treatment.

<p>In order to assess carrot cell resistance to fungal toxins, carrot cell suspensions were tested for embryogenesis in the presence of fungal extracts and toxins. Embryogenesis was assessed 3 weeks after treatment, and compared to negative controls. Four levels of embryogenic activity were noted. <b>A</b>: (−) no embryogenesis was visible, cells were damaged, <b>B</b>: (+) early-stage embryogenic masses were visible, <b>C</b>: same as B, but after 6 weeks. <b>D</b>: (++) embryos were present, and <b>E</b>: (+++) embryogenesis was profuse.</p

UHPLC detection of zinniol in fungal extracts.

<p>UHPLC chromatograms were obtained from different FRA017 <i>Alternaria dauci</i> fungal extracts and compared with an UHPLC chromatogram of pure synthetic zinniol. Retention times corresponding to main peaks are indicated <b>A</b>: UHPLC chromatogram of 10 µg synthetic zinniol. Observed zinniol retention time is 8.38 minutes <b>B</b>: UHPLC chromatogram of 13.4 µg organic extract of an <i>A. dauci</i> culture after 48 h under shaking conditions in carrot juice medium. Zinniol expected retention time of 8.38 minutes is indicated. <b>C</b>: UHPLC chromatogram of 13.4 µg organic extract of an <i>A. dauci</i> culture after 12 days without shaking (anoxic conditions) in V8 medium. A strong peak is visible, corresponding to zinniol retention time. <b>D</b>: UHPLC chromatogram of 13.4 µg organic extract of an <i>A. dauci</i> culture after 48 h under shaking conditions in V8 medium. Zinniol expected retention time of 8.38 minutes is indicated. Chromatograms C and D have the same scale. uAU: micro Absorption Units (optical density) at 233 nm.</p

Stability of zinniol over time.

<p>Synthetic zinniol was added to Gamborg medium in order to check its stability over time under our experimental conditions (dark, 22°C, shaking). HPLC was used to measure variations in the zinniol concentration over a time course. Three different HPLC analyses were performed for each time. Zinniol concentrations were divided by the initial zinniol concentration in the medium, giving a relative zinniol concentration (noted % t_o). Except for small (less than 2%) random variations, the zinniol concentration did not vary over time, indicating stability. Standard errors are not represented because they were smaller than the dots we used.</p

Toxicity and resistance evaluations using fluorescence microscopy.

<p>Liquid cell cultures from two carrot genotypes were tested for mortality and metabolic activity when <i>Alternaria dauci</i> organic extract (oA) was added to the plant culture medium. The same experiments were conducted while adding uninoculated medium organic extract (oM). Seven and 14 days after adding extracts, membrane integrity and cell viability were evaluated by microscopy using a double staining method with fluoresceine diacetate (FDA) and propidium iodide (IP). The images shown are representative of results obtained from three independent experiments. oA treated K3 cell esterase activity, survival and embryogenesis could not be differentiated from oM treated cells. At 7 days, mortality was somewhat higher and esterase activity lower in oA- than oM-treated H1 cells. At 14 days, much greater observed differences followed a similar trend. High mortality was visible in oA treated H1 cells compared to oM-treated cells. Moreover, proembryogenic masses were visible in oM-treated H1 cultures, and not in oA treated cultures.</p

Comparison of two different carrot <i>A. dauci</i> colonization evaluation methods, symptom number assessment and qPCR-based fungal biomass evaluation.

<p>Carrot plants of six different genotypes were tested for <i>Alternaria dauci</i> resistance using two different methods simultaneously. Plants were grown in greenhouse conditions. The third leaf was inoculated after it was isolated in an incubation chamber without detaching it from the plant. The symptom number was assessed at 7, 9 and 13 dpi. At 13 dpi, leaves were detached and then subjected to DNA extraction and qPCR for fungal biomass evaluation. Log(AUDPC) was calculated from the visual assessments, log(I+1) from the qPCR experiments. Both were subjected to variance analysis followed by a Waller-Duncan multiple comparison. As could be expected, the two parameters were closely correlated (r² = 0.793). Interestingly, log(AUDPC) seemed to show a higher resolution, as the homogeneity groups appeared to be more numerous (4 vs 2).</p>1<p>Homogeneity goups were calculated using the Waller-Duncan multiple comparison following an ANOVA analysis.</p