Identification of Flavonoids, Antioxidant and Antiproliferative Activity of Aqueous Infusions of Calendula officinalis L., Chelidonium majus L., Teucrium chamaedrys L. and Alchemilla vulgaris L.

Research background: The current changes in the global economy, characterised by the climate crisis and the economic and health impact of the COVID-19 pandemic, have led to a significant demand for medicinal herbs. This trend is expected to increase significantly by 2050. In this study, we investigated the biopotential of aqueous infusions of four medicinal plants: Calendula officinalis, Chelidonium majus, Teucrium chamaedrys and Alchemilla vulgaris. Experimental approach: The flavonoid analysis of the aqueous infusions of the selected plants was carried out using the RP-HPLC technique. The antiproliferative activity of the prepared aqueous plant infusions was analysed against three human cancer cell lines (MDA-MD-231, T24 and A549), while the antioxidant potential was measured using three antioxidant methods (DPPH, FRAP and Rancimat assay). Results and conclusions: T. chamaedrys had the highest total phenolics (expressed as GAE (2061±42) mg/L), free radical scavenging activity (IC50=1.9 mg/mL) and Fe(III) reducing antioxidant power (expressed as FeCl2 (9798±27) mg/L). At a concentration of 1 mg/mL, the antiproliferation of T24 by C. majus was 96 % and of MDA-MD-231 cells by A. vulgaris was 75 % after 72 h. After principal component analysis, T. chamaedrys and C. majus were grouped together. Quercetin glucoside and antioxidant capacity (DPPH) contributed the most to differentiate these infusions from the other two. Novelty and scientific contribution: This study represents a comparative analysis of the biopotential of four medicinal plants. A new RP-HPLC method was developed to separate the flavonoids in the herbal infusions. This is the first report on the presence of kaempferol-3-O-rutinoside in C. officinalis and isorhamnetin-3-O-rutinoside in A. vulgaris aqueous infusion. For the first time, C. majus has been shown to contribute to the oxidative stability of edible oil. Furthermore, this is the first comparative study on the antiproliferative activity of selected medicinal plants against the cell lines MDA-MD-231, T24 and A549.

Adjustments of the Phytochemical Profile of Broccoli to Low and High Growing Temperatures: Implications for the Bioactivity of Its Extracts.

Climate change causes shifts in temperature patterns, and plants adapt their chemical content in order to survive. We compared the effect of low (LT) and high (HT) growing temperatures on the phytochemical content of broccoli (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.) microgreens and the bioactivity of their extracts. Using different spectrophotometric, LC-MS/MS, GC-MS, and statistical methods, we found that LT increased the total phenolics and tannins in broccoli. The total glucosinolates were also increased by LT; however, they were decreased by HT. Soluble sugars, known osmoprotectants, were increased by both types of stress, considerably more by HT than LT, suggesting that HT causes a more intense osmotic imbalance. Both temperatures were detrimental for chlorophyll, with HT being more impactful than LT. HT increased hormone indole-3-acetic acid, implying an important role in broccoli's defense. Ferulic and sinapic acid showed a trade-off scheme: HT increased ferulic while LT increased sinapic acid. Both stresses decreased the potential of broccoli to act against H2O2 damage in mouse embryonal fibroblasts (MEF), human keratinocytes, and liver cancer cells. Among the tested cell types treated by H2O2, the most significant reduction in ROS (36.61%) was recorded in MEF cells treated with RT extracts. The potential of broccoli extracts to inhibit α-amylase increased following both temperature stresses; however, the inhibition of pancreatic lipase was increased by LT only. From the perspective of nutritional value, and based on the obtained results, we conclude that LT conditions result in more nutritious broccoli microgreens than HT.

Chemometric evaluation of inorganic and organic parameters found in Rosaceae plants proposed as food supplements.

This study discusses the organic and inorganic composition of young inflorescence tissues of seven medical plants from the Prunus, Malus, and Chaenomeles families. These plants contain bioactive compounds with antioxidant and cytotoxic properties, and the study determined 29 elements, including essential and potentially harmful ones, established correlations with inorganic and organic compounds, as well as antioxidative and cytotoxic effects. The elemental patterns show that the plants contribute beneficial essential elements to the human diet. The levels of toxic elements in the plants are within safe limits set by the World Health Organization for medicinal herbs. The results confirmed genus- and species-specific uptake and accumulation. Positive correlations between d-block metals and alkaline earth metals in the inflorescences were found alongside statistically significant differences between analyte categories regarding macro-, micro- and trace elements and bioactive compounds. These correlations need to be considered when giving dietary recommendations or advice for uses as home-remedies.

Flavescence Dorée Strain-Specific Impact on Phenolic Metabolism Dynamics in Grapevine (Vitis vinifera) throughout the Development of Phytoplasma Infection.

Flavescence dorée phytoplasma (FDp) is a phytopathogenic bacterium associated with Grapevine yellowS disease, which causes heavy damage to viticultural production. Epidemiological data revealed that some FDp strains appear to be more widespread and aggressive. However, there is no data on mechanisms underlying the variable pathogenicity among strains. In this research, we employed chromatographic and spectrophotometric techniques to assess how two strains of FDp influence the levels of grapevine phenolic compounds, which are frequently utilized as indicative markers of stress conditions. The results pointed to the upregulation of all branches of phenolic metabolism through the development of infection, correlating with the increase in antioxidative capacity. The more aggressive strain M54 induced stronger downregulation of phenolics' accumulation at the beginning and higher upregulation by the end of the season than the less aggressive M38 strain. These findings reveal potential targets of FDp effectors and provide the first functional demonstration of variable pathogenicity between FDp strains, suggesting the need for future comparative genomic analyses of FDp strains as an important factor in exploring the management possibilities of FDp.

Invasive Alien Species as a Potential Source of Phytopharmaceuticals: Phenolic Composition and Antimicrobial and Cytotoxic Activity of Robinia pseudoacacia L. Leaf and Flower Extracts.

Black locust (Robinia pseudoacacia L.), an invasive tree in Europe, commonly known for its negative impact on biodiversity, is a rich source of phenolic compounds recognized in traditional medicine. Since the metabolite profile depends on the environment and climate, this study aimed to provide the first LC-MS phytochemical screening of the black locust from the Istria region (Croatia). The compounds were extracted from leaves and flowers with 70% ethanol and 80% methanol. Total phenolics (TP) and flavonoids (TF), as well as antioxidant capacity (AC) measured by ABTS (17.49-146.41 mg TE/g DW), DPPH (24.67-118.49 mg TE/g DW), and FRAP (7.38-77.53 mg TE/g DW) assays, were higher in leaf than in flower extracts. Higher TP and total non-flavonoid (TNF) values were displayed in ethanolic than in methanolic extracts. In total, 64 compounds were identified, of which flavonols (20) and hydroxycinnamic acid derivatives (15) were the most represented. Flavanols such as catechin dominated in leaf extracts, followed by flavonols, with kaempferol glucuronyl rhamnosyl hexosides as the main compound, respectively. Flower extracts had the highest share of flavones, followed by ellagitannins, with luteolin dirhamnosyl hexosides and vescalagin, respectively, being predominant. The extracts had good quorum sensing, biofilm formation prevention, and eradicating capacity. The results provided new insights into the phytochemical properties of R. pseudoacacia as the first step toward its potential pharmaceutical use.

Membrane permeabilizers enhance biofortification of Brassica microgreens by interspecific transfer of metabolites from tea (Camellia sinensis).

Interspecific metabolite transfer (ISMT) is a novel approach for plants biofortification. In this study, the effect of tea (Camellia sinensis; Cs), with or without membrane permeabilizers EDTA and Tween, as a donor plant on broccoli, cauliflower and kale sprouts was investigated. As a result, caffeine- and catechin-enriched broccoli, cauliflower and kale microgreens were produced. Kale sprouts were most permeable for catechins from Cs, while cauliflower was most permeable for caffeine. Cs + EDTA significantly increased vitamin C in broccoli and kale. Among the tested enzymes activity, pancreatic lipase was the most affected by the treatment with broccoli and cauliflower biofortified with Cs or Cs combined with permeabilizers. Broccoli sprouts biofortified with Cs most significantly inhibited α-amylase, while those biofortified with Cs combined with permeabilizers most significantly inhibited α-glucosidase. Results point to ISMT combined with membrane permeabilizers as a promising and eco-friendly biofortification strategy to improve the biopotential of Brassica microgreens.

Effect of Hot- and Cold-Water Treatment on Broccoli Bioactive Compounds, Oxidative Stress Parameters and Biological Effects of Their Extracts.

The goal of this work was to define resistant and susceptible variables of young broccoli (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.) plants treated with cold and hot water. Additionally, we wanted to single out variables that could potentially be used as biomarkers of cold/hot-water stress in broccoli. Hot water changed more variables (72%) of young broccoli than cold water (24%) treatment. Hot water increased the concentration of vitamin C for 33%, hydrogen peroxide for 10%, malondialdehyde for 28%, and proline for 147%. Extracts of broccoli stressed with hot water were significantly more efficient in the inhibition of α-glucosidase (65.85 ± 4.85% compared to 52.00 ± 5.16% of control plants), while those of cold-water-stressed broccoli were more efficient in the inhibition of α-amylase (19.85 ± 2.70% compared to 13.26 ± 2.36% of control plants). Total glucosinolates and soluble sugars were affected by hot and cold water in an opposite way, which is why they could be used as biomarkers of hot/cold-water stress in broccoli. The possibility of using temperature stress to grow broccoli enriched with compounds of interest to human health should be further investigated.

High Growing Temperature Changes Nutritional Value of Broccoli (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.) Seedlings.

High temperature (HT) causes physiological and biochemical changes in plants, which may influence their nutritional potential. This study aimed to evaluate the nutritional value of broccoli seedlings grown at HT on the level of phytochemicals, macro- and microelements, antioxidant capacity, and their extracts' in vitro cytotoxicity. Total phenols, soluble sugars, carotenoids, quercetin, sinapic, ferulic, p-coumaric, and gallic acid were induced by HT. Contrarily, total flavonoids, flavonols, phenolic acids, hydroxycinnamic acids, proteins, glucosinolates, chlorophyll a and b, and porphyrins were reduced. Minerals As, Co, Cr, Hg, K, Na, Ni, Pb, Se, and Sn increased at HT, while Ca, Cd, Cu, Mg, Mn, and P decreased. ABTS, FRAP, and ß-carotene bleaching assay showed higher antioxidant potential of seedlings grown at HT, while DPPH showed the opposite. Hepatocellular carcinoma cells were the most sensitive toward broccoli seedling extracts. The significant difference between control and HT-grown broccoli seedling extracts was recorded in mouse embryonal fibroblasts and colorectal carcinoma cells. These results show that the temperature of seedling growth is a critical factor for their nutritional value and the biological effects of their extracts and should definitely be taken into account when growing seedlings for food purposes.

Lemon Juice Formulations Modulate In Vitro Digestive Recovery of Spinach Phytochemicals.

Research background: Citrus limon (L.) Burm lemon juice is rich in many important natural chemical components (flavonoids, citric acid and vitamin C) and its use in traditional medicine is well known. Formulations of lemon juice with fruit polyphenols in beverages have been investigated, but there is very little information about their ability to modulate the digestive behaviour of polyphenols. The goal of this study is to determine the stability and digestive availability of spinach (Spinacia oleracea L.) polyphenols by adding different volume fractions of lemon juice (0, 2, 5, 10 and 20%) during in vitro digestion. Experimental approach: The content of polyphenols and other abundant compounds including nitrates, oxalic acid and l-ascorbic acid in spinach formulation with various volume fractions of lemon juice were measured in predigested and digested samples using in vitro human digestion model. Antioxidant and α-amylase inhibitory activities of spinach lemon juice formulation were also measured. Results and conclusions: The highest increases in total polyphenols, total flavonoids, total phenolic acids, oxalic acid and nitrate content were noted in predigested and almost all digested spinach samples formulated with the highest volume fraction of lemon juice. In the same sample, the content of individual compounds significantly increased after salivary (l-ascorbic acid), initial (p-coumaric acid) and intestinal (quercetin) phase of digestion. High bioaccessibility of polyphenols and l-ascorbic acid in all phases of digestion was observed in almost all spinach lemon juice formulations, with the exception of nitrates in gastric and intestinal phases and oxalic acid in the intestinal phase, which had moderate bioaccessibility. Novelty and scientific contribution: For the first time the stability and digestive availability of spinach polyphenols, oxalic acid, nitrates and l-ascorbic acid were tested with the addition of different volume fractions of lemon juice. The pH of lemon juice and its l-ascorbic acid content increase the stability and availability of polyphenols in spinach lemon juice formulation during in vitro digestion. Antioxidant and α-amylase inhibitory activities increase in dose-dependent manner after lemon juice addition. Accordingly, spinach formulated with 20% of lemon juice appears as the best source of dietary polyphenols with antioxidant and antidiabetic activities and nitrates that may be used as a functional drink.

LC-DAD-MS Phenolic Characterisation of Six Invasive Plant Species in Croatia and Determination of Their Antimicrobial and Cytotoxic Activity.

Invasive plants' phytochemicals are important for their invasiveness, enabling them to spread in new environments. However, these chemicals could offer many pharmaceutical compounds or active ingredients for herbal preparations. This study provides the first LC-MS phytochemical screening of six invasive alien plant species (IAPS) in the Istria region (Croatia): Ailanthus altissima, Ambrosia artemisiifolia, Conyza canadensis, Dittrichia viscosa, Erigeron annuus, and Xanthium strumarium. The study aims to identify and quantify the phenolic content of their leaf extracts and assess their antimicrobial and cytotoxic potential. A total of 32 species-specific compounds were recorded. Neochlorogenic, chlorogenic, and 5-p-coumaroylquinic acids, quercetin-3-glucoside, and kaempferol hexoside were detected in all the tested IAPS. Hydroxycinnamic acid derivatives were the main components in all the tested IAPS, except in E. annuus, where flavanones dominated with a share of 70%. X. strumarium extract had the best activity against the tested bacteria, with an average MIC value of 0.11 mg/mL, while A. altissima and X. strumarium extracts had the best activity against the tested fungi, with an average MIC value of 0.21 mg/mL in both cases. All the plant extracts studied, except X. strumarium, were less cytotoxic than the positive control. The results provided additional information on the phytochemical properties of IAPS and their potential for use as antimicrobial agents.

Proteome changes in human bladder T24 cells induced by hydroquinone derived from Arctostaphylos uva-ursi herbal preparation.

ETHNOPHARMACOLOGICAL RELEVANCE: Arctostaphylos uva-ursi (L.) Spreng. (bearberry) is a well-known traditional herbal plant used as a urinary tract disinfectant. Its antiseptic and diuretic properties can be attributed to hydroquinone, obtained by hydrolysis of arbutin. AIM OF THE STUDY: This study aimed to determine the toxic profile of free hydroquinone on urinary bladder cells (T24) as a target of therapeutic action. MATERIALS AND METHODS: Quantitative and qualitative analysis of the extract and the digestive stability and bioavailability of arbutin and hydroquinone were performed by HPLC assay and simulated in vitro digestion, respectively. Cytotoxic effect, reactive oxygen species induction and proteome changes in T24 cells after hydroquinone treatment were determined using Neutral red assay, 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay and mass spectrometry, respectively. RESULTS: Through in vitro digestion, arbutin was stable, but hydroquinone increased after pepsin treatment (109.6%) and then decreased after the small intestine phase (65.38%). The recommended doses of Uva-ursi had a cytotoxic effect on T24 cells only when all hydroquinone conjugates were converted to free hydroquinone (320 and 900 µg/mL) and the toxic effect was enhanced by recovery. One cup of the therapeutic dose had a prooxidative effect after 4 h of incubation. Shorter time of cell exposure (2 h) to hydroquinone did not have any impact on reactive oxygen species induction. Proteomic analysis found 17 significantly up-regulated proteins compared to control. Hydroquinone activated proteins related to oxidative stress response, stress-adaptive signalling, heat shock response and initiation of translation. CONCLUSIONS: Despite the therapeutic properties of bearberry, up-regulated T24 cell proteins are evidence that plant compounds, although from a natural source, may exhibit negative properties.

Biopotential of Underutilized Rosaceae Inflorescences: LC-DAD-MS Phytochemical Profiles Associated with Antioxidant, Antidiabetic, Anti-Inflammatory and Antiproliferative Activity In Vitro.

The aim of this work was to assess the biopotential of the young inflorescence tissues of Prunus, Malus and Chaenomeles in order to evaluate the possibility of their application in the food industry, and to provide a polyphenolic fingerprint for their quality control. The contents of different bioactive compounds and their antioxidant capacities were spectrophotometrically measured, the main phenolic compounds were identified and quantified using LC-DAD-MS, the antidiabetic potential was determined using α-amylase and α-glucosidase inhibition assays, the anti-inflammatory potential was determined using a 5-lipoxygenase inhibition assay, and the cytotoxicity was determined by MTT assay. Using one-way ANOVA, principal component analysis, hierarchical clustering and Pearson's correlation coefficient, the relations between the samples, and between the samples and the measured parameters, were revealed. In total, 77 compounds were identified. The concentration of sugars was low in M. purpurea, at 1.56 ± 0.08 mg/g DW. The most effective sample in the inhibition of antidiabetic enzymes and anti-inflammatory 5-lipoxygenase was C. japonica. The inhibition of α-glucosidase was strongly positively correlated with the total and condensed tannins, procyanidin dimers and procyanidin tetramer, and was very strongly correlated with chlorogenic acid. In α-amylase inhibition, C. japonica and P. serrulata 'Kiku Shidare Zakura' were equally efficient to the standard inhibitor, maltose. The most effective in the growth and proliferation inhibition of HepG2, HCT116 and HaCaT cells was P. avium. The results suggest Prunus, Malus and Chaenomeles inflorescences as functional food ingredients.

Matcha and Sencha green tea extracts with regard to their phenolics pattern and antioxidant and antidiabetic activity during in vitro digestion.

The purpose of this study was to compare the polyphenol, antioxidant and antidiabetic potential of powdered Matcha and bagged Sencha tea during in vitro digestion. Total phenols (TP), flavonoids (TF), flavanols (TFLA), antioxidant and antidiabetic (α-glucosidase inhibition) activity were higher in Matcha tea before and in most in vitro digestion phases. Upon gastric digestion, in Matcha tea TP, TF, TFLA were 2.6, 1.4 and 1.2 times significantly higher (p ≤ 0.05), respectively; gallic acid, gallocatechin, epigallocatechin, quercetin and kaempferol 1.5, 1.6, 1.8, 1.7, 1.2 times, respectively; whereas antioxidant activity was significantly (p ≤ 0.05) higher 3.2 and 1.1 times with ABTS and FRAP and α-glucosidase inhibition 1.8 times. After the intestinal phase, TP and TFLA were 3.4 and 1.7 times significantly (p ≤ 0.05) higher, respectively, antioxidant activity was significantly (p ≤ 0.05) higher 2.4 and 2.0 times with ABTS and FRAP, respectively, while inhibition of α-glucosidase was 1.7 time significantly (p ≤ 0.05) higher in Matcha tea, but the differences in TF, TP and identified phenolics (with the exception of gallic acid) between Matcha and Sencha tea were neutralized. Our results are the first to demonstrate that, during digestion of Matcha powder together with its water extract, Matcha polyphenols are more bioavailable and possess higher antioxidant and antidiabetic activity compared to Sencha. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13197-021-05086-5.

The Role of Polyphenols in Abiotic Stress Response: The Influence of Molecular Structure.

Abiotic stressors such as extreme temperatures, drought, flood, light, salt, and heavy metals alter biological diversity and crop production worldwide. Therefore, it is important to know the mechanisms by which plants cope with stress conditions. Polyphenols, which are the largest group of plant-specialized metabolites, are generally recognized as molecules involved in stress protection in plants. This diverse group of metabolites contains various structures, from simple forms consisting of one aromatic ring to more complex ones consisting of large number of polymerized molecules. Consequently, all these molecules, depending on their structure, may show different roles in plant growth, development, and stress protection. In the present review, we aimed to summarize data on how different polyphenol structures influence their biological activity and their roles in abiotic stress responses. We focused our review on phenolic acids, flavonoids, stilbenoids, and lignans.

Improving the phytochemical profile and bioactivity of Chinese cabbage sprouts by interspecific transfer of metabolites.

The high demand for healthy food in recent years has led to an increasing need for highly bioactive plant materials. One such plant, Chinese cabbage, possesses flavonoids with antioxidant and antidiabetic properties, but they appear in low quantities. The interspecific transfer of metabolites is a promising technique that could contribute to the increase of the beneficial properties of food. The objective of the study was to determine how an interspecific source-sink phytochemical transfer from donor extracts to the Chinese cabbage affects its phenolic and vitamin C profile and intestinal bioaccessibility, hypoglycemic potential and antioxidant capacity. In addition, sprouts treated with Rosa sp. and Hypericum perforatum extracts showed better antiproliferative effect towards human breast adenocarcinoma cells than untreated sprouts. The results suggest that treatment of plants with donor extracts is a promising approach to improve the nutritional and phytochemical profile and bioactive properties of acceptor plants.

Developmentally-related changes in phenolic and L-ascorbic acid content and antioxidant capacity of Chinese cabbage sprouts.

The phytochemical and antioxidant properties of mature (head stage) Chinese cabbage (Brassica rapa ssp. pekinensis) are known; however, data on the phenolic profile, vitamin C (L-ascorbic acid) content and antioxidant capacity of its fresh sprouts are lacking. Since the human consumption of fresh cruciferous sprouts has significantly increased in recent years, their nutritional characterization has become a somewhat urgent matter. Therefore, in this study the contents of total phenolics, flavonols and hydroxycinnamic acids were measured spectrophotometrically, whereas individual flavonoids, phenolic acids and vitamin C were identified and quantified using a newly-developed high performance liquid chromatography method. Also, the antioxidant capacity of five Chinese cabbage sprout growth stages was determined. These stages contained either cotyledons only (seedlings), cotyledons and two leaves, four leaves, six leaves, or ten leaves. Principal component analysis (PCA) and hierarchical clustering (HC) were implemented in order to visualize the classification trend between the stages. Seedlings contained more sinapic acid and vitamin C than older plants. Plants containing six or ten leaves had more ferulic acid and isorhamnetin than younger ones. Total phenolics, flavonols, hydroxycinnamic acids, quercetin and antioxidant capacity did not statistically differ between seedlings and stages with six or ten leaves and their concentrations were significantly higher than in stages with two or four leaves. PCA and HC confirmed the higher phytochemical similarity between seedlings and plants with six or ten leaves than plants with two or four leaves. Therefore, Chinese cabbage seedlings and plants with six or ten leaves should be preferred over plants with two or four leaves, which were ultimately shown to be of lesser nutritional quality.

Antiproliferative and pro-apoptotic activities of wild asparagus (Asparagus acutifolius L.), black bryony (Tamus communis L.) and butcher's broom (Ruscus aculeatus L.) aqueous extracts against T24 and A549 cancer cell lines.

The aim of this study was to determine the phenolic profile, antiproliferative, and pro-apoptotic activities of Asparagus acutifolius, Tamus communis, and Ruscus aculeatus aqueous extracts against human bladder (T24) and lung cancer (A549) cell lines. Antiproliferative activity of the extracts at the concentration range from 0.5 to 4 mg/ml was measured by MTT test, after the incubation period of 4, 24, 48, or 72 hr. Pro-apoptotic activity of selected plant extracts against T24 and A549 cells was analyzed by flow cytometry at the concentrations of 0.5, 1, and 2 mg/ml. The highest antiproliferative activity against both cancer cell lines was detected by T. communis aqueous extract. The highest pro-apoptotic effect was achieved by A. acutifolius aqueous extract and its effect was similar against A549 and T24 cells. PRACTICAL APPLICATIONS: Wild edible plants, such as wild asparagus, black bryony, and butcher's broom represent important aspect of the Mediterranean diet. They are rich source of various natural bioactive compounds which express different biological activities (antioxidative, antiproliferative, antibacterial, anti-inflammatory…). The current trend for natural and healthy diet, such as the Mediterranean diet, is in relation to the rapidly growing problem of chronic diseases in a modern society. Wild edible plants have a high direct-use value using them in dishes containing mixed vegetables, in soup preparation, as natural additives in fermented natural products containing cheese or fish, in water infusions (natural drinks).

Comparative Analysis of Native Crocus Taxa as a Great Source of Flavonoids with High Antioxidant Activity.

Native Crocus taxa are abundant in regions with moderate and dry climate; however, their potential in the food industry has thus far been neglected. The objective of this study was to compare the quantitative and qualitative flavonoid content and antioxidant and cytotoxic activity in organs of several populations of Croatian native taxa and assess their potential for application in the food industry. The dominant flavonoids in the native Crocus taxa were kaempferol and quercetin. Tepals contained similar or higher concentrations of flavonoid-glycosides than other organs. Tepals from Cvv1 and Cvv2 populations contained more flavonoid-glycosides than the commonly used spice saffron (C. sativus stigmas). The FRAP antioxidant activity of Cvv1 and Cvv5 tepals was similar to that of standard Trolox. DPPH inhibition of Cvv1 and Cvv3 tepals was within the range of that recorded for saffron. Cvv1 tepals significantly reduced reactive oxygen species in the broadest concentration range (50-1000 µg/ml), and showed considerable antioxidant activity in the ABTS assay, equal to 82% of standard Trolox antioxidant activity. A significantly higher concentration of kaempferol-rutinoside was recorded in this than in other taxa. The flavonoid showed a very strong or strong correlation with antioxidant assays results, and a negative correlation with cellular reactive oxygen species concentration. We therefore presumed that kaempferol-rutinoside is one of the main antioxidant phenolics in Crocus tepals. None of the tested extracts showed cytotoxicity toward Caco-2 cells. The results revealed that Cvv tepals have potential as a food supplement and are a promising material for further food safety tests.

Analysis of salicylic acid-dependent pathways in Arabidopsis thaliana following infection with Plasmodiophora brassicae and the influence of salicylic acid on disease.

Salicylic acid (SA) biosynthesis, the expression of SA-related genes and the effect of SA on the Arabidopsis-Plasmodiophora brassicae interaction were examined. Biochemical analyses revealed that, in P. brassicae-infected Arabidopsis, the majority of SA is synthesized from chorismate. Real-time monitored expression of a gene for isochorismate synthase was induced on infection. SA can be modified after accumulation, either by methylation, improving its mobility, or by glycosylation, as one possible reaction for inactivation. Quantitative reverse transcription-polymerase chain reaction (qPCR) confirmed the induction of an SA methyltransferase gene, whereas SA glucosyltransferase expression was not changed after infection. Col-0 wild-type (wt) did not provide a visible phenotypic resistance response, whereas the Arabidopsis mutant dnd1, which constitutively activates the immune system, showed reduced gall scores. As dnd1 showed control of the pathogen, exogenous SA was applied to Arabidopsis in order to test whether it could suppress clubroot. In wt, sid2 (SA biosynthesis), NahG (SA-deficient) and npr1 (SA signalling-impaired) mutants, SA treatment did not alter the gall score, but positively affected the shoot weight. This suggests that SA alone is not sufficient for Arabidopsis resistance against P. brassicae. Semi-quantitative PCR revealed that wt, cpr1, dnd1 and sid2 showed elevated PR-1 expression on P. brassicae and SA + P. brassicae inoculation at 2 and 3 weeks post-inoculation (wpi), whereas NahG and npr1 showed no expression. This work contributes to the understanding of SA involvement in the Arabidopsis-P. brassicae interaction.

Physcomitrella patens auxin conjugate synthetase (GH3) double knockout mutants are more resistant to Pythium infection than wild type.

Auxin homeostasis is involved in many different plant developmental and stress responses. The auxin amino acid conjugate synthetases belonging to the GH3 family play major roles in the regulation of free indole-3-acetic acid (IAA) levels and the moss Physcomitrella patens has two GH3 genes in its genome. A role for IAA in several angiosperm--pathogen interactions was reported, however, in a moss--oomycete pathosystem it had not been published so far. Using GH3 double knockout lines we have investigated the role of auxin homeostasis during the infection of P. patens with the two oomycete species, Pythium debaryanum and Pythium irregulare. We show that infection with P. debaryanum caused stronger disease symptoms than with P. irregulare. Also, P. patens lines harboring fusion constructs of an auxin-inducible promoter from soybean (GmGH3) with a reporter (ß-glucuronidase) showed higher promoter induction after P. debaryanum infection than after P. irregulare, indicating a differential induction of the auxin response. Free IAA was induced upon P. debaryanum infection in wild type by 1.6-fold and in two GH3 double knockout (GH3-doKO) mutants by 4- to 5-fold. All GH3-doKO lines showed a reduced disease symptom progression compared to wild type. Since P. debaryanum can be inhibited in growth on medium containing IAA, these data might indicate that endogenous high auxin levels in P. patens GH3-doKO mutants lead to higher resistance against the oomycete.