Valorisation, Green Extraction Development, and Metabolomic Analysis of Wild Artichoke By-Product Using Pressurised Liquid Extraction UPLC-HRMS and Multivariate Data Analysis.

Valorisation of food by-products has recently attracted considerable attention due to the opportunities to improve the economic and environmental sustainability of the food production chain. Large quantities of non-edible parts of the artichoke plant (Cynara cardunculus L.) comprising leaves, stems, roots, bracts, and seeds are discarded annually during industrial processing. These by-products contain many phytochemicals such as dietary fibres, phenolic acids, and flavonoids, whereby the most challenging issue concerns about the recovery of high-added value components from these by-products. The aim of this work is to develop a novel valorisation strategy for the sustainable utilisation of artichoke leaves' waste, combining green pressurised-liquid extraction (PLE), spectrophotometric assays and UPLC-HRMS phytochemical characterization, to obtain bioactive-rich extract with high antioxidant capacity. Multivariate analysis of the major selected metabolites was used to compare different solvent extraction used in PLE.

Towards a Cardoon (Cynara cardunculus var. altilis)-Based Biorefinery: A Case Study of Improved Cell Cultures via Genetic Modulation of the Phenylpropanoid Pathway.

Cultivated cardoon (Cynara cardunculus var. altilis L.) is a promising candidate species for the development of plant cell cultures suitable for large-scale biomass production and recovery of nutraceuticals. We set up a protocol for Agrobacterium tumefaciens-mediated transformation, which can be used for the improvement of cardoon cell cultures in a frame of biorefinery. As high lignin content determines lower saccharification yields for the biomass, we opted for a biotechnological approach, with the purpose of reducing lignin content; we generated transgenic lines overexpressing the Arabidopsis thaliana MYB4 transcription factor, a known repressor of lignin/flavonoid biosynthesis. Here, we report a comprehensive characterization, including metabolic and transcriptomic analyses of AtMYB4 overexpression cardoon lines, in comparison to wild type, underlining favorable traits for their use in biorefinery. Among these, the improved accessibility of the lignocellulosic biomass to degrading enzymes due to depletion of lignin content, the unexpected increased growth rates, and the valuable nutraceutical profiles, in particular for hydroxycinnamic/caffeoylquinic and fatty acids profiles.

Seasonal variation in bioactive properties and phenolic composition of cardoon (Cynara cardunculus var. altilis) bracts.

Cardoon (Cynara cardunculus L.) bracts were collected at different maturation stages to investigate seasonal changes in the phenolic compounds profile and in vitro bioactivities. Among the 12 phenolic compounds tentatively identified, 3,5-O-dicaffeoylquinic acid (21.83 mg/g extract) and apigenin-7-O-glucuronide (10.6 mg/g extract) were the most abundant. Immature bracts (C1: principal growth stage (PGS) 5) had the highest phenolic compounds content, and anti-inflammatory (IC50 = 72 µg/mL) and cytotoxic (GI50 of 30-79 µg/mL) activities. Moreover, extract C1 inhibited efficiently the formation of thiobarbituric acid reactive substances (TBARS; IC50 = 26.8 µg/mL), while extract C8 (PGS 8/9) was more effective against oxidative haemolysis (IC50 38 and 75 µg/mL). The highest antibacterial and antifungal activities were attributed to samples C1 and C6 (PGS 7/8) and samples C2 (PGS 5/6) and C4 (PGS 6/7), respectively. Overall, the obtained results suggest the seasonal changes of polyphenolic composition and bioactivity of cardoon bracts of variable maturity.

Effect of Shading on the Sesquiterpene Lactone Content and Phytotoxicity of Cultivated Cardoon Leaf Extracts.

The work described here follows on from a previous study focused on the influence of the genotype and harvest time on the sesquiterpene lactone (STL) profile of Cynara cardunculus L. leaf extracts. The aim of this study was to investigate the effect that 60% plant shading in cultivated cardoon (C. cardunculus var. altilis) leaf extracts harvested in winter and spring had on the composition of STLs and the phytotoxicity. The phytotoxicity of leaf extracts was evaluated by assessing wheat coleoptile elongation along with seed germination and the root and shoot length of the weeds Amaranthus retroflexus L. and Portulaca oleracea L. Shading increased the production of STLs in spring, and this effect correlated positively with the phytotoxic activity. The induction of shading can therefore be used to modulate STL concentrations and their phytotoxic potential in cultivated cardoon leaves for industrial applications.

Phenolic Compounds in Poorly Represented Mediterranean Plants in Istria: Health Impacts and Food Authentication.

Phenolic compounds are well-known bioactive compounds in plants that can have a protective role against cancers, cardiovascular diseases and many other diseases. To promote local food development, a comprehensive overview of the phenolic compounds' composition and their impact on human health from typical Mediterranean plants such as Punica granatum L., Ziziphus jujuba Mill., Arbutus unedo L., Celtis australis L., Ficus carica L., Cynara cardunculus var. Scolymus L. is provided. Moreover, the potential use of these data for authenticity determination is discussed. Some of the plants' phenolic compounds and their impact to human health are very well determined, while for others, the data are scarce. However, in all cases, more data should be available about the content, profile and health impacts due to a high variation of phenolic compounds depending on genetic and environmental factors. Quantifying variation in phenolic compounds in plants relative to genetic and environmental factors could be a useful tool in food authentication control. More comprehensive studies should be conducted to better understand the importance of phenolic compounds on human health and their variation in certain plants.

Toward alternative sources of milk coagulants for cheese manufacturing: establishment of hairy roots culture and protease characterization from Cynara cardunculus L.

KEY MESSAGE: Extracts from hairy root cultures of Cynara cardunculus L. contain proteases and show milk-clotting activity. Cynara cardunculus L. or cardoon is often used as rennet in traditional cheese manufacturing, due to the presence of specific proteases in the flower. However, the flower extracts are variable depending on the provenance and quality of the flowers as well as high genetic variability among cardoon populations, and this affects the quality of the final product. In search for alternative sources of milk-clotting enzymes, hairy root cultures from cardoon were obtained and characterized regarding their protease content and proteolytic activity toward milk proteins. Aspartic, serine and cysteine proteases were identified in hairy roots by mass spectrometry analysis and an azocasein assay combined with specific inhibitors. RT-PCR analysis revealed the expression of cardosin A and D, and immunoblotting analysis suggested the presence of cardosin A or cardosin A-like enzyme in its mature form, supporting this system as an alternative source of cardosins. Hairy root protein extracts showed activity over caseins, supporting its use as milk coagulant, which was further tested by milk-clotting assays. This is also the first report on the establishment of hairy root cultures from cardoon, which paves the way for future work on controlled platforms for production of valuable metabolites which are known to be present in this species.

Cardoon as a Sustainable Crop for Biomass and Bioactive Compounds Production.

Cardoon is a multi-purpose and versatile Mediterranean crop, adapted to climate change, with a wide spectrum of potential applications due its added value as a rich source of fibers, oils and bioactive compounds. The Cynara species are a component of the Mediterranean diet and have been used as food and medicine since ancient times. The important role of cardoon in human nutrition, as a functional food, is due to its high content of nutraceutical and bioactive compounds such as oligofructose inulin, caffeoylquinic acids, flavonoids, anthocyanins, sesquiterpenes lactones, triterpenes, fatty acids and aspartic proteases. The present review highlights the characteristics and functions of cardoon biomass which permits the development of innovative products in food and nutrition, pharmaceutics and cosmetics, plant protection and biocides, oils and energy, lignocellulose materials, and healthcare industries following the actual trends of a circular economy.

Influence of Genotype and Harvest Time on the Cynara cardunculus L. Sesquiterpene Lactone Profile.

The excessive and inappropriate application of herbicides has caused environmental pollution. The use of allelochemicals as bioherbicides could provide a solution to this problem. The allelopathic activity of Cynara cardunculus L. has been studied previously, and sesquiterpene lactones (STLs) were identified as the most relevant allelochemicals. The goal of the study reported here was to investigate the effect of six genotypes and three harvest times on the qualitative and quantitative composition of STLs in C. cardunculus leaves through a new ultra-high-performance liquid chromatography-tandem mass spectrometry analysis method and, thus, the effect on phytotoxicity. Overall, wild cardoon contained the highest levels of STLs of the three botanical varieties studied. Nevertheless, climatic conditions had a marked influence on the presence of STLs among the six genotypes, which was higher in the April harvest. Cynaropicrin was the most abundant STL detected. A close relationship was found between the STL profiles and the allelopathic activity, expressed as inhibition of wheat coleoptile elongation. The data provide a new and important contribution to our understanding of C. cardunculus allelopathy.

Pre-treatment of corn stover, Cynara cardunculus L. stems and wheat straw by ethanol-water and diluted sulfuric acid: Comparison under different energy input conditions.

Ethanol-water (EW) and diluted sulfuric acid (DSA) pre-treatment have been studied for lignocellulosic biomass (corn stover, Cynara cardunculus L. stems and wheat straw). Both pre-treatments have been compared taken into account: solids recovery, glucans recovery, xylans removed, delignification and glucose yield. In all cases, the amount of energy involved has been taken as a criterion for sustainability. In general terms, EW is more efficient to remove lignin and DSA more appropriate to hydrolysate xylans. The combined effect of delignification and xylans removal is responsible for the improvement in the enzymatic cellulose hydrolysis. Under conditions of moderate-low energy inputs, EW pre-treatment yields better results than DSA with glucose yields in the range of 50-60% for EW pre-treated corn stover and cardoon stems; while wheat straw pulps reach up to 80%. So, multiple raw materials biorefinery needs a previous study to fit the type and conditions of the pre-treatment to each feedstock.

Performance of three cardoon cultivars in an industrial heavy metal-contaminated soil: Effects on morphology, cytology and photosynthesis.

In the present work the cytomorphological and physiological effects on three cardoon cultivars - Sardo, Siciliano, Spagnolo - grown in a metal-polluted soil, were investigated, to assess the traits concurring to the high tolerance to metal stress observed in cv. Spagnolo compared to the other two cultivars. The plants were grown for one month on a real polluted soil collected at a dismantling battery plant, highly enriched by heavy metals, especially Cd and Pb, and their leaves were analyzed by a multidisciplinary approach. TEM observations highlighted severe ultrastructural damage in Sardo and Siciliano, and preserved cytological traits in Spagnolo. Both pigment content and photochemistry indicated a decline in photosynthesis in Sardo and Sicilano and a substantial stability of the same parameters in Spagnolo. Protein analysis indicated a decrease in D1 level in all cultivars; in Spagnolo the D1 decrease was more pronounced and associated to a significant increase in Rubisco, a pattern likely preserving photosynthetic efficiency and high biomass production. In conclusion, Spagnolo cardoon was able to face metal stress through a prompt, multiple response balancing structural and functional traits.

Haplotype analysis of the germacrene A synthase gene and association with cynaropicrin content and biological activities in Cynara cardunculus.

Cynara cardunculus: L. represents a natural source of terpenic compounds, with the predominant molecule being cynaropicrin. Cynaropicrin is gaining interest since it has been correlated to anti-hyperlipidaemia, antispasmodic and cytotoxicity activity against leukocyte cancer cells. The objective of this work was to screen a collection of C. cardunculus, from different origins, for new allelic variants in germacrene A synthase (GAS) gene involved in the cynaropicrin biosynthesis and correlate them with improved cynaropicrin content and biological activities. Using high-resolution melting, nine haplotypes were identified. The putative impact of the identified allelic variants in GAS protein was evaluated by bioinformatic tools and polymorphisms that putatively lead to protein conformational changes were described. Additionally, cynaropicrin and main pentacyclic triterpenes contents, and antithrombin, antimicrobial and antiproliferative activities were also determined in C. cardunculus leaf lipophilic-derived extracts. In this work we identified allelic variants with putative impact on GAS protein, which are significantly associated with cynaropicrin content and antiproliferative activity. The results obtained suggest that the identified polymorphisms should be explored as putative genetic markers correlated with biological properties in Cynara cardunculus.

Ultrastructural, protein and photosynthetic alterations induced by Pb and Cd in Cynara cardunculus L., and its potential for phytoremediation.

The effects of cadmium and lead were investigated in Cynara cardunculus L. Plant uptake by root and shoot, changes in cell ultrastructure and photosynthetic efficiency, photosynthetic key protein levels, as well as regulation of stress-induced Hsp70 were examined. Cynara cardunculus accumulated Cd and Pb in their tissue, with a different trend for the two metals. The prompt translocation of Cd to the shoot may justify the ultrastructural injuries, especially observed in chloroplasts. However, Cd- treated plants did not show any decline in photochemistry; it is likely that Cd in shoot tissue triggers defense mechanisms, increasing the level of proteins involved in photosynthesis (i.e., Rubisco and D1 increased 7 and 4.5 fold respectively) as a compensatory response to neutralize chloroplast damage. The accumulation of Pb mainly in root, can explain the increase in Hsp70 level (23 folds) in this tissue. Pb reached the shoots, even at low amounts, causing an overall significant change in some photochemical parameters (QY and NPQ decreases and increases of 25%, respectively). The results suggest a higher sensitivity of C. cardunculus to Pb than Cd, although maximal photochemical efficiency suggests that this species seems to tolerate Pb and Cd and hence, it is a suitable candidate for phytoremediation.

Impact of novel SNPs identified in Cynara cardunculus genes on functionality of proteins regulating phenylpropanoid pathway and their association with biological activities.

BACKGROUND: Cynara cardunculus L. offers a natural source of phenolic compounds with the predominant molecule being chlorogenic acid. Chlorogenic acid is gaining interest due to its involvement in various biological properties such as, antibacterial, antifungal, antioxidant, hepatoprotective, and anticarcinogenic activities. RESULTS: In this work we screened a Cynara cardunculus collection for new allelic variants in key genes involved in the chlorogenic acid biosynthesis pathway. The target genes encode p-coumaroyl ester 3'-hydroxylase (C3'H) and hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase (HQT), both participating in the synthesis of chlorogenic acid. Using high-resolution melting, the C3'H gene proved to be highly conserved with only 4 haplotypes while, for HQT, 17 haplotypes were identified de novo. The putative influence of the identified polymorphisms in C3'H and HQT proteins was further evaluated using bioinformatics tools. We could identify some polymorphisms that may lead to protein conformational changes. Chlorogenic acid content, antioxidant and antithrombin activities were also evaluated in Cc leaf extracts and an association analysis was performed to assess a putative correlation between these traits and the identified polymorphisms. CONCLUSION: In this work we identified allelic variants with putative impact on C3'H and HQT proteins which are significantly associated with chlorogenic acid content and antioxidant activity. Further study of these alleles should be explored to assess putative relevance as genetic markers correlating with Cynara cardunculus biological properties with further confirmation by functional analysis.

Enzymatic saccharification and bioethanol production from Cynara cardunculus pretreated by steam explosion.

The correct choice of the specific lignocellulosic biomass pretreatment allows obtaining high biomass conversions for biorefinery implementations and cellulosic bioethanol production from renewable resources. Cynara cardunculus (cardoon) pretreated by steam explosion (SE) was involved in second-generation bioethanol production using separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) processes. Steam explosion pretreatment led to partial solubilisation of hemicelluloses and increased the accessibility of residual polysaccharides towards enzymatic hydrolysis revealing 64% of sugars yield against 11% from untreated plant material. Alkaline extraction after SE pretreatment of cardoon (CSEOH) promoted partial removal of degraded lignin, tannins, extractives and hemicelluloses thus allowing to double glucose concentration upon saccharification step. Bioethanol fermentation in SSF mode was faster than SHF process providing the best results: ethanol concentration 18.7 g L(-1), fermentation efficiency of 66.6% and a yield of 26.6g ethanol/100 g CSEOH or 10.1 g ethanol/100 g untreated cardoon.

Effects of saline stress on mineral composition, phenolic acids and flavonoids in leaves of artichoke and cardoon genotypes grown in floating system.

BACKGROUND: Cynara cardunculus is a species native to the Mediterranean basin. It includes globe artichoke and cultivated cardoon as well as their progenitor wild cardoon. The species is a source of biophenols, and its leaf extracts have been widely used in herbal medicine as hepatoprotectors and choleretics since ancient times. The aim of this study was to determine the effect of increasing the level of salinity in the nutrient solution (1 or 30 mmol L⁻¹ NaCl) on biomass production, mineral composition, radical-scavenging activity, caffeoylquinic acids and flavonoids in three artichoke ('Romolo', 'Violetto di Provenza' and 'Violetto di Romagna') and three cultivated cardoon ('Bianco Avorio', 'Bianco Gigante Inerme' and 'Gigante di Romagna') cultivars grown in a floating system. RESULTS: Increased salinity in the nutrient solution decreased the leaf dry biomass and leaf number of artichoke and cultivated cardoon cultivars. Salinity reduced macro- and microelement accumulation in leaves (e.g. N, K, Ca, Mg, Fe, Mn and B) but improved their antioxidant activity, total polyphenols, chlorogenic acid, cynarin and luteolin. The cultivated cardoons, especially 'Bianco Avorio' and 'Gigante di Romagna', showed higher biomass and leaf number than those observed in artichoke genotypes. 'Violetto di Provenza' exhibited the highest content of chlorogenic acid, closely followed by 'Violetto di Romagna', whereas for cynarin content the highest values were recorded in 'Violetto di Provenza', 'Bianco Avorio' and 'Gigante di Romagna'. The highest content of luteolin was recorded in 'Gigante di Romagna' and 'Bianco Avorio', while the highest content of apigenin was observed in 'Gigante di Romagna'. CONCLUSION: The results showed that the floating system could be considered an effective tool to improve quality aspects through proper management of the salt concentration in the nutrient solution. They also suggest that specific cultivars should be selected to obtain the desired profile of bioactive compounds.

Cyclopentyl methyl ether: a green co-solvent for the selective dehydration of lignocellulosic pentoses to furfural.

The effects of cyclopentyl methyl ether (CPME) addition during the aqueous xylose dehydration reaction to furfural are reported here. These investigations were conducted by using pure xylose and Cynara cardunculus (cardoon) lignocellulose as sugar source and H(2)SO(4) as catalyst. The research was also applied to aqueous solutions containing NaCl, since it has been previously demonstrated that NaCl incorporation to these reaction mixtures remarkably increases the furfural formation rate. It has been found that CPME incorporation inhibits the formation of undesired products (resins, condensation products and humins). Thus, cardoon lignocellulosic pentoses were selectively transformed into furfural (near 100%) at the following reaction conditions: 1 wt.% H(2)SO(4), 4 wt.% biomass referred to aqueous solution, 30 min reaction, 443 K, CPME/aqueous phase mass ratio equals to 2.33, and NaCl/aqueous solution mass ratio of 0.4. In contrast, no effect was observed for cellulosic glucose transformation into hydroxymethylfurfural and levulinic acid at identical reaction conditions.

In-silico and in-vivo analyses of EST databases unveil conserved miRNAs from Carthamus tinctorius and Cynara cardunculus.

BACKGROUND: MicroRNAs (miRNAs) are small RNAs (21-24 bp) providing an RNA-based system of gene regulation highly conserved in plants and animals. In plants, miRNAs control mRNA degradation or restrain translation, affecting development and responses to stresses. Plant miRNAs show imperfect but extensive complementarity to mRNA targets, making their computational prediction possible, useful when data mining is applied on different species. In this study we used a comparative approach to identify both miRNAs and their targets, in artichoke and safflower. RESULTS: Two complete expressed sequence tags (ESTs) datasets from artichoke (3.6 · 10(4) entries) and safflower (4.2 · 10(4)), were analysed with a bioinformatic pipeline and in vitro experiments, identifying 17 potential miRNAs. For each EST, using RNAhybrid program and 953 non redundant miRNA mature sequences, available in mirBase as reference, we searched matching putative targets. 8730 out of 42011 ESTs from safflower and 7145 of 36323 ESTs from artichoke showed at least one predicted miRNA target. BLAST analysis showed that 75% of all ESTs shared at least a common homologous region (E-value < 10(-4)) and about 50% of these displayed 400 bp or longer aligned sequences as conserved homologous/orthologous (COS) regions. 960 and 890 ESTs of safflower and artichoke organized in COS shared 79 different miRNA targets, considered functionally conserved, and statistically significant when compared with random sequences (signal to noise ratio > 2 and specificity ≥ 0.85). Four highly significant miRNAs selected from in silico data were experimentally validated in globe artichoke leaves. CONCLUSIONS: Mature miRNAs and targets were predicted within EST sequences of safflower and artichoke. Most of the miRNA targets appeared highly/moderately conserved, highlighting an important and conserved function. In this study we introduce a stringent parameter for the comparative sequence analysis, represented by the identification of the same target in the COS region. After statistical analysis 79 targets, found on the COS regions and belonging to 60 miRNA families, have a signal to noise ratio > 2, with ≥ 0.85 specificity. The putative miRNAs identified belong to 55 dicotyledon plants and to 24 families only in monocotyledon.

Responses of Cynara cardunculus L. to single and combined cadmium and nickel treatment conditions.

A greenhouse pot experiment was carried out to study the responses of Cynara cardunculus L. (cardoon) to cadmium and nickel. Three groups of 12 pots each were planted with cardoon plants and spiked with single and combined cadmium and nickel aqueous solutions. The bioavailable metal concentrations, measured in soil, ranged widely and were up to 246.7 mg kg⁻¹ for Cd and 61.1 mg kg⁻¹ for Ni. Under Cd treatment, cardoon growth remained unaffected, while increased Ni soil concentrations inhibited plant growth and were lethal to the highly treated plants. In the combined Cd and Ni treatments, an antagonistic effect was observed between the two metals. Cadmium and nickel concentrations in cardoon tissues rose with increasing metal concentrations in the soil. Cadmium and nickel contents in shoots reached 169.3 and 342.3 mg kg⁻¹ in the single treatments while, under the combined Cd and Ni treatments, they were up to 235.0 and 440.7 mg kg⁻¹, respectively. Generally, mean contents of both metals in the shoots were higher than in the roots and the translocation factor was greater than 1. A possible enhancing effect of nickel on cadmium uptake was observed. Cardoon showed characteristics of a Cd accumulator.

Effects of chelates on plants and soil microbial community: comparison of EDTA and EDDS for lead phytoextraction.

Most studies on chelate-induced phytoextraction have focused on EDTA-mediated Pb phytoextraction. But EDTA and the formed EDTA-Pb complexes have low biodegradability and high solubility in soil, resulting in an elevated risk of adverse environmental effects. EDDS is an easily biodegradable chelating agent that has recently been proposed as an environmentally sound alternative to EDTA. Consequently, a greenhouse experiment, using a completely randomized factorial design with four replications, was carried out to compare the potential of EDTA and EDDS for chelate-induced Pb phytoextraction with Cynara cardunculus, as well as to investigate the toxicity of these two chelates to both cardoon plants and soil microorganisms. The effects of chelate addition on soil microbial communities were studied through the determination of a variety of biological indicators of soil quality such as soil enzyme activities, basal and substrate-induced respiration, potentially mineralizable nitrogen, and community level physiological profiles. EDTA was much more efficient than EDDS for the enhancement of root Pb uptake and root-to-shoot Pb translocation. In a soil polluted with 5000 mg Pb kg(-1), as a result of the addition of 1 g EDTA kg(-1) soil, a value of 1332 mg Pb kg(-1) DW shoot was obtained. EDDS application resulted in a shoot Pb accumulation of only 310 mg kg(-1)DW. Plants treated with EDDS showed lower values of biomass than those treated with EDTA. EDDS proved to be rapidly degraded, and less toxic to the soil microbial community in control non-polluted soils. Pb-polluted EDDS-treated soils showed significantly higher values of basal and substrate-induced respiration than those treated with EDTA. Although EDDS had a lower capacity to enhance Pb phytoextraction than EDTA, it has the advantage of rapid biodegradation.

Phenolic composition of Cynara cardunculus L. organs, and their biological activities.

Polyphenols are bioactive molecules exhibiting a lot of scientific attention due to their multiple biological activities. This study compared phenolic contents and antioxidant activity in Cynara cardunculus L. organs and focus on leaf phenolic compounds identification by RP-HPLC and their antibacterial activity. The analyzed organs exhibited different total polyphenol contents (7-14.8 mg GAE g(-1) DW). Leaf and seed phenolic contents were similar and two times higher than those in flowers. The same tendency was observed for the amount of flavonoids and tannins. However, seed extracts displayed the highest DPPH. scavenging ability with the lowest IC50 value (23 microg ml(-1)), followed by leaves and flowers (over 50 microg ml(-1)). In contrast, leaves showed the highest capacity to quench superoxide (IC50: 1 microg ml(-1)) as compared to seeds (6 microg ml(-1)). In addition, cardoon leaves were efficient to inhibit growth of pathogenic bacteria mainly against Staphylococcus aureus and Escherichia coli. The identification of phenolic compounds from leaves revealed that syringic and trans-cinnamic acids were the major molecules.