Characteristic of the gene candidate SecARS encoding alkylresorcinol synthase in Secale.

BACKGROUND: Alkylresorcinols (ARs) are compounds belonging to the class of phenolic lipids. A rich source of ARs are cereal grains such as rye, wheat, triticale or barley. ARs found in plants are characterized by a variety of biological properties such as antimicrobial, antifungal and cytotoxic activity. Moreover, they are proven to have a positive influence on human health. Here, we aimed to find and characterize the gene with ARs synthase activity in the species Secale cereale. METHODS AND RESULTS: Using BAC library screening, two BAC clones containing the gene candidate were isolated and sequenced. Bioinformatic analyses of the resulting contigs were used to examine the structure and other features of the gene, including promoter, intron, 3'UTR and 5'UTR. Mapping using the FISH procedure located the gene on the 4R chromosome. Comparative analysis showed that the gene is highly similar to sequences coding for type III polyketide synthase. The level of gene expression in various parts of the plant was investigated, and the biochemical function of the gene was confirmed by heterologous expression in yeast. CONCLUSIONS: The conducted analyses contributed to a better understanding of the processes related to ARs synthesis. Although the research concerned the rye model, the knowledge gained may help in understanding the genetic basis of ARs biosynthesis in other species of the Poaceae family as well.

Saponin Fractions from Eryngium planum L. Induce Apoptosis in Ovarian SKOV-3 Cancer Cells.

(1) The cytotoxicity and antioxidant activity of different fractions as well as the pro-apoptotic activity of saponin fractions from Eryngium planum L. in SKOV-3 was investigated. (2) In screening studies, the cytotoxicity of six fractions on SKOV-3 was examined by LDH and SRB assays. The most active fractions-triterpenoid saponins-were selected for further investigation. To determine the mechanism of saponin fractions' cytotoxicity, their ability to induce apoptosis was examined via Annexin V assay. The effect of the saponin fractions on caspase 3 activity was measured using a Caspase 3 Assay Kit. The expression of 84 apoptosis-related genes was investigated in cancer cells exposed to saponin fractions from the roots. The radical scavenging capacity of different fractions was determined via DPPH assay. (3) The pronounced cytotoxic effects in SKOV-3 were demonstrated by saponin fractions from the leaves and roots. Those saponin fractions were chosen for further investigation. The treatment of cancer cell lines with saponins obtained from the roots provoked a significant increase in apoptotic cells. In the SKOV-3 cells, saponins caused upregulation of pro-apoptotic genes and a decrease in anti-apoptotic genes. The activation of caspase 3 was correlated with an increased DFFA expression level in the treated SKOV-3 cells. The most active fractions were phenolic acids from the shoots and roots. (4) To the best of our knowledge, the current study is the first to demonstrate that the barrigenol-type triterpenoid saponin fraction from the roots of E. planum inhibits SKOV-3 cell proliferation and induces apoptosis, which may be regulated by the expression of genes mostly specific to a mitochondria-related pathway.

Anti-inflammatory and antioxidant actions of extracts from Rheum rhaponticum and Rheum rhabarbarum in human blood plasma and cells in vitro.

Rheum rhaponticum L. (rhapontic rhubarb) and Rheum rhabarbarum L. (garden rhubarb) are edible and medicinal rhubarb species used for many centuries in traditional medicine. This work is focused on the biological activity of extracts from petioles and roots of R. rhaponticum and R. rhabarbarum as well as rhapontigenin and rhaponticin, typical stilbenes present in these rhubarbs, in a context of their effects on blood physiology and cardiovascular health. Anti-inflammatory properties of the examined substances were evaluated in human peripheral blood mononuclear cells (PBMCs) and THP1-ASC-GFP inflammasome reporter cells. Due to the coexistence of inflammation and oxidative stress in cardiovascular diseases, the study design included also antioxidant assays. This part of the work involved the assessment of the protective efficiency of the examined substances against the peroxynitrite-triggered damage to human blood plasma components, including fibrinogen, a protein of critical importance for blood clotting and maintaining the haemostatic balance. Pre-incubation of PBMCs with the examined substances (1-50 µg/mL) considerably decreased the synthesis of prostaglandin E2 as well as the release of pro-inflammatory cytokines (IL-2 and TNF-α) and metalloproteinase-9. A reduced level of secreted apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks in the THP-1-ASC-GFP cells was also observed. The examined substances significantly diminished the extent of ONOO‾induced oxidative modifications of blood plasma proteins and lipids and normalized, or even strengthened blood plasma antioxidant capacity. Furthermore, a reduction of oxidative damage to fibrinogen, including modifications of tyrosine and tryptophan residues along with the formation of protein aggregates was found.

Kalanchoe sp. Extracts-Phytochemistry, Cytotoxic, and Antimicrobial Activities.

Kalanchoe species are succulents occurring in tropical regions. They have many biological and pharmacological properties. In this study, the cytotoxic and antimicrobial activities of water and dichloromethane Kalanchoe fractions obtained from ethanol extracts of three Kalanchoe species-K. daigremontiana, K. pinnata, and K. blossfeldiana were estimated. The cytotoxic effect was assessed on human cancer cell lines-ovarian SKOV-3, cervical HeLa, breast MCF-7, and melanoma A375-by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The antimicrobial activity was estimated on selected Gram-positive and Gram-negative bacteria strains and on Candida albicans. The phytochemical analysis of selected Kalanchoe extracts was conducted by LC-QTOF-MS. The obtained results showed that the water fraction of K. blossfeldiana was active both on the tested cancer cells (IC50 values were 28.28 ± 2.76 and 32.51 ± 0.69 µg/mL on HeLa and SKOV-3, respectively) and bacteria strains (MIC values were 16 and 32 µg/mL on S. epidermidis and S. aureus, respectively). The water fraction of K. pinnata also had a significant effect on S. epidermidis and S. aureus, with MIC values of 32 and 64 µg/mL, respectively. The water fraction of K. blossfeldiana triggered a decrease in mitochondrial membrane potential (MMP) and induced cell cycle arrest in the G2/M phase in the SKOV-3 and HeLa cells. This fraction did not significantly increase cellular oxidative stress level. The DPPH and ABTS assays revealed that the water fraction of K. blossfeldiana had a strong antioxidant effect (IC50 was 9.44 ± 0.06 and 3.17 ± 0.1 µg/mL, respectively). The phytochemical analysis of the extracts of K. blossfeldiana and K. pinnata revealed the presence of at least 218 main components. The most frequently occurring were flavonol glycosides (31 metabolites), phenylpropanoids (13 metabolites), gallic acid derivatives (13 compounds), benzoic acid derived compounds (14 metabolites), and acyclic alcohol glycosides (16 compounds). In addition, proanthocyanidins were detected mainly in K. blossfeldiana. The study indicates that the water fraction of K. blossfeldiana has significant biological potential and can be further investigated towards anticancer and antimicrobial application.

Effects of Rheum rhaponticum and Rheum rhabarbarum extracts on haemostatic activity of blood plasma components and endothelial cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional medicine recommends the use of Rheum rhaponticum L. and R. rhabarbarum L. to treat over thirty complaints, including disorders related to the cardiovascular system such as heartache, pains in the pericardium, epistaxis and other types of haemorrhage, blood purification as well as disorders of venous circulation. AIM OF THE STUDY: This work was dedicated to examining for the first time the effects of extracts from petioles and roots of R. rhaponticum and R. rhabarbarum, as well as two stilbene compounds (rhapontigenin and rhaponticin) on the haemostatic activity of endothelial cells and functionality of blood plasma components of the haemostatic system. MATERIALS AND METHODS: The study was based on three main experimental modules, including the activity of proteins of the human blood plasma coagulation cascade and the fibrinolytic system as well as analyses of the haemostatic activity of human vascular endothelial cells. Additionally, interactions of the main components of the rhubarb extracts with crucial serine proteases of the coagulation cascade and fibrinolysis (i.e. thrombin, the coagulation factor Xa and plasmin) were analyzed in silico. RESULTS: The examined extracts displayed anticoagulant properties and significantly reduced the tissue factor-induced clotting of human blood plasma (by about 40%). Inhibitory effects of the tested extracts on thrombin and the coagulation factor Xa (FXa) were found as well. For the extracts, the IC50 was ranging from 20.26 to 48.11 µg/ml. Modulatory effects on the haemostatic response of endothelial cells, including the release of von Willebrand factor, tissue-type plasminogen activator and the plasminogen activator inhibitor-1, have been also found. CONCLUSIONS: Our results indicated for the first time that the examined Rheum extracts influenced the haemostatic properties of blood plasma proteins and endothelial cells, with the prevalence of the anticoagulant action. The anticoagulant effect of the investigated extracts may be partly attributed to the inhibition of the FXa and thrombin activities, the key serine proteases of the blood coagulation cascade.

Rheum rhaponticum and Rheum rhabarbarum Extracts as Modulators of Endothelial Cell Inflammatory Response.

BACKGROUND: Inflammation, endothelial dysfunction, and alterations in blood physiology are key factors contributing to atherosclerosis and other cardiovascular disorders. Hence, modulation of endothelial function and reducing its pro-inflammatory and pro-thrombotic activity is considered one of the most important cardioprotective strategies. This study aimed to evaluate the anti-inflammatory potential of rhubarb extracts isolated from petioles and underground organs of Rheum rhabarbarum L. (garden rhubarb) and R. rhaponticum L. (rhapontic rhubarb) as well as two stilbenoids, typically found in these plants, i.e., rhapontigenin (RHPG) and its glycoside, rhaponticin (RHPT). METHODS: Analysis of the anti-inflammatory effects of the indicated rhubarb-derived substances involved different aspects of the endothelial cells' (HUVECs) response: release of the inflammatory mediators; cyclooxygenase (COX-2) and 5-lipoxygenase (5-LOX) expression as well as the recruitment of leukocytes to the activated HUVECs. The ability of the rhubarb-derived extracts to inhibit COX-2 and 5-LOX activities was examined as well. The study was supplemented with the in silico analysis of major components of the analyzed extracts' interactions with COX-2 and 5-LOX. RESULTS: The obtained results indicated that the examined plant extracts and stilbenes possess anti-inflammatory properties and influence the inflammatory response of endothelial cells. Biochemical and in silico tests revealed significant inhibition of COX-2, with special importance of rhaponticin, as a compound abundant in both plant species. In addition to the reduction in COX-2 gene expression and enzyme activity, a decrease in the cytokine level and leukocyte influx was observed. Biochemical tests and computational analyses indicate that some components of rhubarb extracts may act as COX-2 inhibitors, with marginal inhibitory effect on 5-LOX.

Pioneering Metabolomic Studies on Diaporthe eres Species Complex from Fruit Trees in the South-Eastern Poland.

Fungi from the genus Diaporthe have been reported as plant pathogens, endophytes, and saprophytes on a wide range of host plants worldwide. Their precise identification is problematic since many Diaporthe species can colonize a single host plant, whereas the same Diaporthe species can inhabit many hosts. Recently, Diaporthe has been proven to be a rich source of bioactive secondary metabolites. In our initial study, 40 Diaporthe isolates were analyzed for their metabolite production. A total of 153 compounds were identified based on their spectroscopic properties-Ultraviolet-visible and mass spectrometry. From these, 43 fungal metabolites were recognized as potential chemotaxonomic markers, mostly belonging to the drimane sesquiterpenoid-phthalide hybrid class. This group included mainly phytotoxic compounds such as cyclopaldic acid, altiloxin A, B, and their derivatives. To the best of our knowledge, this is the first report on the metabolomic studies on Diaporthe eres species complex from fruit trees in the South-Eastern Poland. The results from our study may provide the basis for the future research on the isolation of identified metabolites and on their bioactive potential for agricultural applications as biopesticides or biofertilizers.

Carob extract induces spermatogenesis in an infertile mouse model via upregulation of Prm1, Plzf, Bcl-6b, Dazl, Ngn3, Stra8, and Smc1b.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnopharmacological studies for drug discovery from natural compounds play an important role for developing current therapeutical platforms. Plants are a group of natural sources which have been served as the basis in the treatment of many diseases for centuries. In this regard, Ceratonia siliqua (carob) is one of the herbal medicine which is traditionally used for male infertility treatments. But so far the main mechanisms for effects of carob are unknown. Here, we intend to investigate the ability of carob extract to induce spermatogenesis in an azoospermia mouse model and determine the mechanisms that underlie its function. AIM OF THE STUDY: This is a pre-clinical animal model study to evaluate the effect of carob extract in spermatogenesis recovery. METHODS: We established an infertile mouse model with the intent to examine the ability of carob extract as a potential herbal medicine for restoration of male fertility. Sperm parameters, as well as gene expression dynamics and levels of spermatogenesis hormones, were evaluated 35 days after carob administration. RESULTS: Significant enhanced sperm parameters (P < 0.05) showed that the carob extract could induce spermatogenesis in the infertile mouse model. Our data suggested an anti-apototic and inducer role in the expressions of cell cycle regulating genes. Carob extract improved the spermatogenesis niche by considerable affecting Sertoli and Leydig cells (P < 0.05). The carob-treated mice were fertile and contributed to healthy offspring that matured. Our data confirmed that this extract triggered the hormonal system, the spermatogenesis-related gene expression network, and signaling pathways to induce and promote sperm production with notable level (P < 0.05). We found that the aqueous extract consisted of a polar and mainly well water-soluble substance. Carob extract might upregulate spermatogenesis hormones via its amino acid components, which were detected in the extract by liquid chromatography-mass spectrometry (LC-MS). CONCLUSION: Our results strongly suggest that carob extract might be a promising future treatment option for male infertility. This finding could pave the way for clinical trials in infertile men. This is the first study that has provided reliable, strong pre-clinical evidence for carob extract as an effective candidate for fertility recovery in cancer-related azoospermia.

Fingerprinting of two an acylated polyoxypregnane glycosides from Caralluma quadrangula (Forssk.) N.E.Br. using UPLC-ESI-Q-TOF and computational study.

The present study has tentatively elucidated the structure of two acylated polyoxypregnane glycosides from Caralluma quadrangula (Forssk.) N.E.Br. (CQ). The analyses were performed using an electrospray-ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer in positive ionization modes to explore fragmentation pathways. The used ionization mode provided consistent and/or complementary information for most of the pregnane glycosides, their fragmentation sequences, and their aglycones. Presumably, this is due to higher efficiency, sensitivity, and better selectivity of the mass spectrometry-based method. The present experimental and theoretical report deals with the characteristic fragmentation behaviors of two acylated polyoxypregnane glycosides CQ1 and CQ2 from the aerial parts of C. quadrangular. A DFT Study was performed to elucidate the position of ikemoyl, and benzoyl residues in compounds CQ1 and CQ2, respectively.

Comprehensive polyoxypregnane glycosides report in Caralluma quadrangula using UPLC-ESI-Q-TOF and their antioxidant effects in human plasma.

ETHNOPHARMACOLOGICAL RELEVANCE: Caralluma quadrangula (Forssk.) N.E.Br. (Syns: = Stapelia quadrangula Forssk. = Monolluma quadrangula Forssk.) is an indigenous member of the genus Caralluma and it is a rather common species on rocky hillsides in the southwestern part of Saudi Arabia. Several members of this genus have found medicinal uses in the treatment of rheumatism, diabetes, leprosy and as antiseptics and disinfectants. All parts are edible but rather more bitter and can cause diarrhea. AIM OF THE STUDY: The present report was tentatively elucidated the structure of acylated and non-acylated polyoxypregnane glycosides from Caralluma quadrangula. MATERIALS AND METHODS: The analyses were performed using an electrospray-ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer in both positive and negative ionization modes to explore fragmentation pathways. The antioxidant and prooxidant properties of the different mobility portions of human plasma were evaluated in vitro using thiobarbituric acid reactive substance assay (TBARS). RESULTS: The analyses showed sixty-five characteristic ion peaks which could be more efficient to assignment the aglycones and fragmentation sequences of sugar moieties. The used ionization modes provided consistent and/or complementary information for most of the pregnane glycosides, their fragmentation sequences, and their aglycones. A DFT Study was performed to elucidate the neutral loss of H2O molecules sequences from aglycones and the esterification linkage. CONCLUSIONS: This report could be useful to reduce material consuming and time in phytochemistry analysis of the different medicinal plants. The two portions significantly depleted TBARS were subjected to autoperoxidation assay in the presence of hydrogen peroxide.

An In Vitro Anticancer, Antioxidant, and Phytochemical Study on Water Extract of Kalanchoe daigremontiana Raym.-Hamet and H. Perrier.

Kalanchoe species are succulents with anti-inflammatory, antioxidant, and analgesic properties, as well as cytotoxic activity. One of the most popular species cultivated in Europe is Kalanchoe daigremontiana Raym.-Hamet and H. Perrier. In our study, we analyzed the phytochemical composition of K. daigremontiana water extract using UHPLC-QTOF-MS and estimated the cytotoxic activity of the extract on human ovarian cancer SKOV-3 cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, flow cytometry, luminometric, and fluorescent microscopy techniques. The expression levels of 92 genes associated with cell death were estimated via real-time PCR. The antioxidant activity was assessed via flow cytometry on human keratinocyte HaCaT cell line. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and FRAP (ferric-reducing antioxidant power) assays were also applied. We identified twenty bufadienolide compounds in the water extract and quantified eleven. Bersaldegenin-1,3,5-orthoacetate and bryophyllin A were present in the highest amounts (757.4 ± 18.7 and 573.5 ± 27.2 ng/mg dry weight, respectively). The extract showed significant antiproliferative and cytotoxic activity, induced depolarization of the mitochondrial membrane, and significantly arrested cell cycle in the S and G2/M phases of SKOV-3 cells. Caspases-3, 7, 8, and 9 were not activated during the treatment, which indicated non-apoptotic cell death triggered by the extract. Additionally, the extract increased the level of oxidative stress in the cancer cell line. In keratinocytes treated with menadione, the extract moderately reduced the level of oxidative stress. This antioxidant activity was confirmed by the DPPH and FRAP assays, where the obtained IC50 values were 1750 ± 140 and 1271.82 ± 53.25 µg/mL, respectively. The real-time PCR analysis revealed that the extract may induce cell death via TNF receptor (tumor necrosis factor receptor) superfamily members 6 and 10.

Multifunctional compounds in the extract from mature seeds of Vicia faba var. minor: Phytochemical profiling, antioxidant activity and cellular safety in human selected blood cells in in vitro trials.

BACKGROUND: The field bean (Vicia faba) is a valuable fodder plant of the Fabaceae family, grown as a main crop for its seed yield. Its phytochemical profile is characterized by the presence of a range of compounds with various biological activities. PURPOSE: The present study investigates the phytochemical profile of the extract from mature seeds of Vicia faba var. minor and examines its impact on preventing oxidative damage to various lipids, protein and DNA molecules in vitro. METHODS: Human plasma was treated with H2O2/Fe (an OH. donor) to induce oxidative damage to lipids and proteins, and the plant extract was then added. As oxidative stress may influence the biological activity of plasma, e.g. coagulation, and influence cardiovascular disease, the study also examined the effect of the plant extract on coagulation and monoamine oxidase activity (MAO, EC 1.4.3.4). RESULTS: The tested extract exerted a protective effect on plasma lipids and proteins treated with H2O2/Fe. However, while it appears to effectively protect the DNA in peripheral blood mononuclear cells from oxidative damage, it did not induce changes in the coagulation process, and significantly reduced MAO activity when applied at 1, 5, and 10 µg/mL. It is possible that the observed antioxidant potential may be due to the complex chemical composition of the extract: the phytochemical profile demonstrated a range of phenolic compounds, including catechins. CONCLUSION: Our findings indicate that extract from mature seeds of V. faba var. minor may be a promising source of antioxidants in multiple applications, including diseases associated with oxidative stress; however, more studies based on in vitro and in vivo models are needed to determine its biological properties.

The Roots of Rye (Secale cereale L.) Are Capable of Synthesizing Benzoxazinoids.

According to current opinion, the first step of benzoxazinoids (BXs) synthesis, that is, the conversion of indole-3-glycerol phosphate to indole, occurs exclusively in the photosynthesising parts of plants. However, the results of our previous work and some other studies suggest that this process may also occur in the roots. In this study, we provide evidence that the first step of BXs synthesis does indeed occur in the roots of rye seedlings. We detected ScBx1 transcripts, BX1 enzyme, and six BXs (2-hydroxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-1,4-benzoxazin-3-one, (2R)-2-O-ß-d-glucopyranosyl-4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one glucoside, 2,4-dihydroxy- 7-methoxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside, and 6-methoxy-2-benzoxazolinone) in the roots developed from seeds deprived of the coleoptile at 2 days after sowing (i.e., roots without contact with aerial parts). In roots regenerated in vitro, both ScBx1 transcripts and BX1 enzyme were detected at a low but still measurable levels. Thus, BXs are able to be synthesised in both the roots and above-ground parts of rye plants.

Pulmonaria obscura and Pulmonaria officinalis Extracts as Mitigators of Peroxynitrite-Induced Oxidative Stress and Cyclooxygenase-2 Inhibitors-In Vitro and In Silico Studies.

The Pulmonaria species (lungwort) are edible plants and traditional remedies for different disorders of the respiratory system. Our work covers a comparative study on biological actions in human blood plasma and cyclooxygenase-2 (COX-2) -inhibitory properties of plant extracts (i.e., phenolic-rich fractions) originated from aerial parts of P. obscura Dumort. and P. officinalis L. Phytochemical profiling demonstrated the abundance of phenolic acids and their derivatives (over 80% of the isolated fractions). Danshensu conjugates with caffeic acid, i.e., rosmarinic, lithospermic, salvianolic, monardic, shimobashiric and yunnaneic acids were identified as predominant components. The examined extracts (1-100 µg/mL) partly prevented harmful effects of the peroxynitrite-induced oxidative stress in blood plasma (decreased oxidative damage to blood plasma components and improved its non-enzymatic antioxidant capacity). The cellular safety of the extracts was confirmed in experimental models of blood platelets and peripheral blood mononuclear cells. COX-2 inhibitor screening evidently suggested a stronger activity of P. officinalis (IC50 of 13.28 and 7.24 µg/mL, in reaction with synthetic chromogen and physiological substrate (arachidonic acid), respectively). In silico studies on interactions of main components of the Pulmonaria extracts with the COX-2 demonstrated the abilities of ten compounds to bind with the enzyme, including rosmarinic acid, menisdaurin, globoidnan A and salvianolic acid H.

Phytochemical Screening, Phenolic Compounds and Antioxidant Activity of Biomass from Lychnis flos-cuculi L. In Vitro Cultures and Intact Plants.

Lychnis flos-cuculi L., a species with potential medicinal value, contains flavonoids, phenolic acids, triterpenoid saponins and ecdysteroids. In this study, the antioxidant activity of plant material of L. flos-cuculi obtained from in vitro cultures compared to that of intact plants from the natural site has been evaluated for the first time. Phytochemical screening of the in-vitro-derived material by ultra-high-performance liquid chromatography mass spectrometry (UHPLC-MS) confirmed the presence of the aforementioned metabolite classes. The aqueous methanolic extracts from in-vitro-derived plant material and the organs of intact plants were analyzed using spectrophotometric methods to quantify total phenolics, phenolic acids and flavonoids, and determine the preliminary antioxidant activity by ferric reducing antioxidant potential (FRAP) and DPPH radical scavenging activity assays. The results showed that the inflorescence (Ns-F), and flowering herb of both plants gathered from natural habitat (Ns-H) and in-vitro-derived plants from the experimental plot (ExV-H) are the materials richest in polyphenols (195.4, 113.47, 112.1 mg GAE g-1 d.w., respectively), and demonstrate the highest antioxidant activity (20.14, 11.24, and 11.46 mg AAE g-1 d.w.). The extract from callus exhibited the lowest polyphenol content and antioxidant potential. The contents of total phenolics, flavonoids and phenolic acids correlate with the results of the antioxidant capacity of L. flos-cuculi extracts.

Development, validation, and application of capillary zone electrophoresis method for determination of pyrimidine glucosides in seeds of Vicia faba L. var. minor.

INTRODUCTION: Consumption of fava beans seeds can be harmful to both humans and animals due to the presence of pyrimidine glucosides: vicine and convicine. Therefore, seeds, feed supplements, and protein extracts derived from fava beans should be monitored for the contents of pyrimidine derivatives. OBJECTIVES: The main objective of this work was the development and validation of an inexpensive, uncomplicated, quick, and robust method for the quantitation of vicine and convicine in seeds of fava beans. METHODOLOGY: Plant material was extracted using a dual-phase extraction system to remove lipophilic contaminants and deactivate residual enzymes. Pyrimidine glucosides together with the internal standard: 5-bromouridine were separated using capillary zone electrophoresis (CZE) on an uncoated fused-silica capillary with alkaline, tetraborate-based electrolyte. Linearity, limits of detection and quantitation, precision, accuracy, recovery, and robustness of the method were investigated. The method was applied for quantitation of pyrimidine glucosides in seeds. RESULTS: Validation results suggest that the method is suitable for quantitation of pyrimidine glucosides, although it may not be sensitive enough for low-concentrated samples. Application of the method showed that seeds of the investigated variety of fava beans contain both vicine at 10-15 mg/g of dry weight and convicine at 3-5 mg/g of dry weight. CONCLUSION: Developed and validated CZE-ultraviolet (UV) method is suitable for analysis of pyrimidine glucosides in seeds of Vicia faba L. var. minor. It can be used as an inexpensive, greener alternative to more advanced methods while providing equally reliable and accurate results.

Biological activities of leaf extracts from selected Kalanchoe species and their relationship with bufadienolides content.

CONTEXT: Kalanchoe species (Crassulaceae) are widely used in traditional medicine as remedies in infectious diseases and cancer treatment. OBJECTIVE: Cytotoxic and antimicrobial activities of Kalanchoe daigremontiana Raym.-Hamet & H. Perrier, K. pinnata (Lam.) Pers., and K. blossfeldiana Poelln. extracts were determined. The relationship between biological activities and the extracts bufadienolides content was also investigated. MATERIALS AND METHODS: Fresh leaves of Kalanchoe species were macerated with 95% ethanol or water. The quantitative analysis of bufadienolides in the extracts was carried out with mass spectrometry. Cytotoxicity tests were performed on human cancer cell lines - HeLa, SKOV-3, MCF-7, and A375 by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and Real-Time Cell Analysis system. The microbiological study was done using a few bacteria strains (ß-hemolytic Streptococcus, Corynebacterium diphtheriae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus hirae, Escherichia coli) and Candida albicans. RESULTS: The K. blossfeldiana ethanol extract and K. daigremontiana water extract exhibited the most potent cytotoxic activity (IC50 < 19 µg/mL for HeLa and SKOV-3 cells). The strongest antibacterial effects showed ethanol extract of K. blossfeldiana and K. pinnata (MIC values were 8.45, 8.45, 0.25 and <33.75 µg/mL for S. aureus, S. epidermidis, and E. hirae, respectively). The highest total amount of bufadienolides was in K. daigremontiana ethanol extract. In contrast, K. blossfeldiana ethanol extract did not show the presence of these compounds. CONCLUSIONS: Kalanchoe blossfeldiana ethanol extract is a potential candidate for cancer and bacterial infection treatment. Additionally, the biological effects of Kalanchoe extracts are not dependent on the presence and amount of bufadienolides in the plant extracts.

Changes in benzoxazinoid contents and the expression of the associated genes in rye (Secale cereale L.) due to brown rust and the inoculation procedure.

Benzoxazinoids (BXs) are secondary metabolites with diverse functions, but are primarily involved in protecting plants, mainly from the family Poaceae, against insects and fungal pathogens. Rye is a cereal crop that is highly resistant to biotic stresses. However, its susceptibility to brown rust caused by Puccinia recondita f. sp. secalis (Prs) is still a major problem affecting its commercial production. Additionally, the genetic and metabolic factors related to this disease remain poorly characterized. In this study, we investigated whether and to what extent the brown rust infection and the inoculation procedure affect the contents of specific BXs (HBOA, GDIBOA, DIBOA, GDIMBOA, DIMBOA, and MBOA) and the expression of genes related to BX (ScBx1-5, ScIgl, and Scglu). We revealed that treatments with water and a urediniospore suspension usually downregulate gene expression levels. Moreover, HBOA and DIBOA contents decreased, whereas the contents of the remaining metabolites increased. Specifically, the MBOA content increased more after the mock treatment than after the Prs treatment, whereas the increase in GDIBOA and GDIMBOA levels was usually due to the Prs infection, especially at two of the most critical time-points, 17 and 24 h post-treatment. Therefore, GDIBOA and GDIMBOA are glucosides that are important components of rye defence responses to brown rust. Furthermore, along with MBOA, they protect rye against the stress associated with the inoculation procedure used in this study.

Genes ScBx1 and ScIgl-Competitors or Cooperators?

Two genes, Bx1 and Igl, both encoding indole-3-glycerol phosphate lyase (IGL), are believed to control the conversion of indole-3-glycerol phosphate (IGP) to indole. The first of these has generally been supposed to be regulated developmentally, being expressed at early stages of plant development with the indole being used in the benzoxazinoid (BX) biosynthesis pathway. In contrast, it has been proposed that the second one is regulated by stresses and that the associated free indole is secreted as a volatile. However, our previous results contradicted this. In the present study, we show that the ScIgl gene takes over the role of ScBx1 at later developmental stages, between the 42nd and 70th days after germination. In the majority of plants with silenced ScBx1 expression, ScIgl was either expressed at a significantly higher level than ScBx1 or it was the only gene with detectable expression. Therefore, we postulate that the synthesis of indole used in BX biosynthesis in rye is controlled by both ScBx1 and ScIgl, which are both regulated developmentally and by stresses. In silico and in vivo analyses of the promoter sequences further confirmed our hypothesis that the roles and modes of regulation of the ScBx1 and ScIgl genes are similar.

Electrospray ionization mass spectrometry characterization of ubiquitous minor lipids and oligosaccharides in milk of the camel (Camelus dromedarius) and their inhibition of oxidative stress in human plasma.

The aim of this study was to characterize minor lipids in methanol fraction extracted from raw camel milk after loading it on a water-preconditioned short C18 open column and fractionating with a gradient of methanol/water. The C18 column showed high fractionation efficiency of minor lipids, such as glycosphingolipids, lipopolysaccharides, or oligosaccharides, when compared with other constituents, in particular polysaccharides, proteins, and free fatty acids. Liquid chromatography electrospray ionization tandem mass spectrometry in negative ion mode was used to identify 21 new glycosphingolipids, lipopolysaccharides, and oligosaccharides. Electrospray ionization tandem mass spectrometry was qualified to provide relevant data for recognizing the molecular mass, glycosylation sequences, and structure of saccharide moieties for the revealed compounds. The sequence of combinations of one selected lipopolysaccharide, which was considered the backbone of the remaining lipopolysaccharides, was confirmed in a density functional theory study. The obtained results showed that the tested fraction is a rich source of glycosphingolipids, lipopolysaccharides, and oligosaccharides with antioxidant activity.