Increasing P intake had differing effects on plasma FGF21 in rats fed NC and HC vs. rats fed LC at the three sampling times. When compared with the NP groups, FGF21 concentrations decreased at the three sampling points in rats fed NC-HP  and HC-HP . However, FGF21 did not decrease in rats fed LC-HP . In addition, LC groups had a much lower liver FGF21 messenger ribonucleic acid/glyceraldehyde 3-phosphate dehydrogenase (mRNA/GAPDH) ratio (0.51 ± 0.08 and 0.56 ± 0.07) than the NC-NP (0.97 ± 0.14) and HC-NP (0.97 ± 0.22) groups. Increasing P intake reduced liver FGF21 mRNA/GAPDH in rats fed NC and HC to 0.42 ± 0.05 and 0.37 ± 0.04. Liver β-klotho mRNA/GAPDH ratio was lower (p < 0.05) in LC groups (0.66 ± 0.06 and 0.59 ± 0.10) than in NC (1.09 ± 0.17 and 1.03 ± 0.14) and HC (1.19 ± 0.12 and 1.34 ± 0.19) groups. A reduction (p < 0.05) in β-klotho protein/α-tubulin ratio was also observed in LC groups (0.65 ± 0.05 and 0.49 ± 0.08) when compared with NC (1.12 ± 0.11 and 0.91 ± 0.11) and HC (0.93 ± 0.17 and 0.87 ± 0.09) groups. In conclusion β-klotho is potently regulated by caloric restriction but not by increasing P intake while FGF21 is regulated by both caloric restriction and increased P intake. Moreover, increased P intake has a differential effect on FGF21 in calorie repleted and calorie depleted rats.The effect of dietary phosphorus (P) on fibroblast growth factor 21 (FGF21)/β-klotho axis was investigated in rats that were fed diets with: Normal (NP) or high P (HP) and either normal (NC), high (HC) or low calories (LC). Sampling was performed at 1, 4 and 7 months. Plasma FGF21 concentrations were higher ( A growing body of evidence is being gathered about the relationship between energy metabolism and mineral metabolism. This association includes links of obesity with osteoporosis , calciumIn addition to its skeletal functions, phosphorus (P) is closely related to energy metabolism—phosphoproteins are involved in mitochondrial oxidative phosphorylation and energy for essential metabolic processes is stored in high energy phosphate bonds of adenosine triphosphate . Thus, aWithin the superfamily of fibroblast growth factors (FGFs), two hormone-like molecules are key regulators of energy metabolism (FGF21) and mineral metabolism (FGF23). Fibroblast growth factor 21, that is mainly synthesized in the liver, stimulates glucose uptake by adipocytes, increases energy expenditure and improves the lipid profile . FibroblAlthough the main actions of FGF23 are related to P homeostasis, there is evidence of a regulation of FGF23 by energy metabolism. In addition to being regulated by leptin , an incrThe role of the FGF21/β-klotho axis in mineral metabolism is less clear. Based on a recent study that has shown that the tubular load of P modulates the expression of α-klotho in the kidney , we hypoAll experimental protocols were reviewed and approved by the Ethics Committee for Animal Research of the University of Cordoba and by Junta de Andalucia (Spain) . All protocols were carried out in accordance with the approved guidelines. They followed the guiding principle laid down by the Higher Council of Scientific Research of Spain following the normal procedures directing animal welfare and adhered to the recommendations included in the Guide for Care and Use of Laboratory Animals  and European laws  and regulations on protection of animals, under the advice of specialized personnel.Two month-old Wistar rats, provided by the Animal Housing Facilities of the University of Cordoba , were housed with a 12 h/12 h light/dark cycle. Appropriate measures were taken to ensure animal welfare and to address the basic behavioral and physiological needs of rats.Diets with two P concentrations were used in the experiments: Normal P (0.6%) diet (NP) and high P (1.2%) diet (HP). Independent of their P content, diets had a normal energy content (NC diet) providing metabolizable energy = 3528 kcal/kg , a high energy content (HC diet) providing metabolizable energy = 5241 kcal/kg , or a low energy content (LC diet) providing metabolizable energy = 1314 kcal/kg . All diets contained 0.6% of Ca and 500 IU/g of vitamin D.n = 8). Rats in group 1 were fed the NC-NP diet, rats in group 2 were fed the NC-HP diet, rats in group 3 were fed the HC-NP diet, rats in group 4 were fed the HC-HP diet, rats in group 5 were fed the LC-NP diet and rats in group 6 were fed the LC-HP diet. Diets were fed ad libitum for 7 months. Food intake and body weight were measured every week. Blood samples were obtained between 09.00 and 11.00 h from fasted (12 h) rats after the animals had been receiving the experimental diets for 1, 4 and 7 months. Blood sampling at 1 and 4 months was performed on anesthetized  rats and blood was obtained from the jugular vein. Blood samples at 7 months were obtained from the abdominal aorta, at the time of sacrifice. At the end of the experiment (7 months), rats were sacrificed by exsanguination under general anesthesia . In addition to blood, liver samples were also obtained, frozen at −80 °C and stored until processing for ribonucleic acid (RNA) and protein extraction.Rats were allotted to 6 experimental groups . Then, plasma was separated by centrifugation and stored at –20 °C until assayed. Plasma total cholesterol, triglycerides and P were measured by spectrophotometry . ELISA tests were used to quantify plasma FGF21 , leptin and adiponectin .Study of liver β-klotho and FGF21 mRNA was performed by Quantitative Real-Time PCR (RT-PCR). Liver tissue was disrupted using liquid nitrogen and grinded thoroughly with a mortar. Total RNA was extracted using the chloroform and isopropanol precipitation method and a treatment with deoxyribonuclease I (DNAse I) Amplification Grade . Fifty nanograms of total RNA was used to analyze messenger ribonucleic acid (mRNA) expression in the LightCycler thermal cycler system . RT-PCR was performed in one step using the QuantiTect SYBR Green RT-PCR kit  following the manufacturer´s protocol. Primers for β-klotho were designed with the free Oligo 7 software. The FGF21 primer was used based on a previous publication . The expPrimer sequences are listed below:β-klotho: Forward primer: 5′-TCAACCAGGTTCTTCAAGCAATA-3′; Reverse primer: 5′-GGTTTCCTCTCTTTCTGCTCAC-3′.FGF21: Forward primer: 5′-CAAATCCTGGGTGTCAAAGC-3′; Reverse primer: 5′-GCCTCAGACTGGTACACATTG-3′.GAPDH: Forward primer: 5′-AGGGCTGCCTTCTCTTGTGAC-3′; Reverse primer: 5′-TGGGTAGAATCATACTGGAACATGTAG-3′.2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (10 mmol/L), potassium chloride (KCl) (10 mmol/L), ethylenediaminetetraacetic acid (EDTA) (0.1 mmol/L), ethylene glycol tetraacetic acid (EGTA) (0.1 mmol/L), dithiothreitol (DTT) (1mmol/L), phenylmethylsulfonyl fluoride (PMSF) (0.5 mmol/L), protease inhibitor cocktail (70 µg/mL), and I-Gepal CA-630 (0.6%), pH 7.9 . Protein concentration was determined by the Bradford method. For Western blot analysis, 50 µg of protein was electrophoresed on a 10% SDS-polyacrilamide gel  and electrophoretically transferred  from the gels onto nitrocellulose membranes . The following steps were performed with gentle shaking. Membranes were incubated in TBST  solution (20 mM Tris-HCl (pH 7.6), 0.2% Tween 20, 150 mM NaCl) , and 5% nonfat dry milk  at room temperature for 1.5h to avoid nonspecific binding. Membranes were then washed with TBST buffer (the same composition as TBST without nonfat dry milk) and incubated overnight at 4 °C with a rabbit anti-β-klotho antibody . The membranes were then washed with TBST buffer and immunolabeled using a peroxidase-conjugated secondary antibody . Finally, they were revealed on autoradiographic film using ECL Plus Western Blotting Detection System . Alpha tubulin  was used as a housekeeping protein to ensure equal loading of the gels. Protein levels were quantified using ImageJ software .Proteins were isolated from liver tissue by using a lysis buffer containing 4-. The difference between means for two different groups was determined by p < 0.01) in rats fed HC-NP, 63.6 ± 1.3 kcal/day, and markedly decreased (p < 0.0001) in rats fed LC-NP, 35.1 ± 0.1 kcal/day. The phosphorus content of the diet did not influence energy intake . Plasma cholesterol concentrations, which tended to decrease with low caloric intake, increased significantly (p < 0.05) in LC-HP rats when compared with LC-NP rats. Diets with HP significantly decreased plasma glucose both in NC  and LC  rats. Plasma leptin concentrations were lower in LC groups (0.6 ± 0.2 and 0.6 ± 0.1 ng/mL), intermediate in NC groups (4.2 ± 0.5 and 3.1 ± 0.2 ng/mL) and slightly higher in HC groups (5.3 ± 0.8 and 4.1 ± 0.5 ng/mL). When compared with NC, adiponectin tended to be lower in rats fed HC and higher in rats fed LC. Rats fed HP tended to have lower leptin and higher adiponectin concentrations, and differences were significant for adiponectin in LC groups  (Plasma triglycerides were lower in the LC-NP group (22.3 ± 1.7 mg/dL) and higher in the HC-NP group (83.1 ± 11.4 mg/dL) than in NC-NP group (54.8 ± 11.1 mg/dL). High P intake decreased plasma triglycerides in the HC-HP group to 39.6 ± 3.4 mg/dL ( < 0.01) .p < 0.05) in the groups that received NC and HC than in the groups that received LC. Thus, rats in NC-NP and HC-NP groups had mean FGF21 concentrations that ranged between 190 pg/mL, at 1 month, and 434 pg/mL, at 7 months. By contrast, mean values of FGF21 in rats fed LC-NP diet were much lower: 20 pg/mL at 1 month, and 81 pg/mL at 7 months. Interestingly, increasing the P content of the diet had differing effects on plasma FGF21 in rats fed NC and HC vs. rats fed LC. When compared with the NP groups, FGF21 concentrations decreased at the three sampling points in rats fed NC-HP  and HC-HP . However, FGF21 did not decrease in rats fed LC-HP, in fact, FGF21 tended to increase  and the differences were significant (p = 0.007) at 4 months, when compared with the LC-NP group . Increasing the P intake reduced liver FGF21 mRNA/GAPDH in rats fed normal calories  and high calories  to levels comparable to the LC groups. No significant differences were found between LC-NP and LC-HP groups (0.51 ± 0.08 and 0.56 ± 0.07).p < 0.01) in the LC groups (0.66 ± 0.06 and 0.59 ± 0.10) than in the NC groups (1.09 ± 0.17 and 1.03 ± 0.14) and the HC groups (1.19 ± 0.12 and 1.34 ± 0.19). A similar reduction (p < 0.05) in β-klotho/α-tubulin ratio was observed in the LC groups (0.65 ± 0.05 and 0.49 ± 0.08) when compared with the NC groups (1.12 ± 0.11 and 0.91 ± 0.11) and the HC groups (0.93 ± 0.17 and 0.87 ± 0.09). Increasing P intake did not affect either β-klotho mRNA or protein in any  of the diets  is regulated by both caloric deficit and increased P intake. Moreover, increased P intake decreases FGF21 in calorie repleted rats but not in calorie depleted rats.


However, alterations in the concentrations of metabolic intermediates functionally related to the early stage of the salt stress response have not been investigated. The halophilic cyanobacterium Synechococcus sp. PCC 7002 was subjected to salt shock with 0.5 and 1 M NaCl, then we performed metabolomics analysis by capillary electrophoresis/mass spectrometry (CE/MS) and gas chromatography/mass spectrometry (GC/MS) after cultivation for 1, 3, 10, and 24 h. Gene expression profiling using a microarray after 1 h of salt shock was also conducted. We observed suppression of the Calvin cycle and activation of glycolysis at both NaCl concentrations. However, there were several differences in the metabolic changes after salt shock following exposure to 0.5 M and 1 M NaCl: (i): the main compatible solute, glucosylglycerol, accumulated quickly at 0.5 M NaCl after 1 h but increased gradually for 10 h at 1 M NaCl; (ii) the oxidative pentose phosphate pathway and the tricarboxylic acid cycle were activated at 0.5 M NaCl; and (iii) the multi-functional compound spermidine greatly accumulated at 1 M NaCl. Our results show that Synechococcus sp. PCC 7002 acclimated to different levels of salt through a salt stress response involving the activation of different metabolic pathways.In response to salt stress, cyanobacteria increases the gene expression of Na PCC 7942 and of Arabidopsis thaliana, a higher plant [Cyanobacteria are oxygenic photosynthetic prokaryotes that hold promise for producing sustainable fuels and chemicals because of their fast growth compared to higher plants. In addition, some halophilic cyanobacteria can grow under high salinity conditions that are incompatible with plant growth ,2. Advaner plant ,4. In paer plant .Synechocystis sp. and Synechococcus sp. [+ and Cl− ions flow into the cytoplasm [+/H+ antiporters and K+ uptake systems [Salt stress response has been studied in several cyanobacterial species such as ccus sp. . During ccus sp. ; (ii) Naytoplasm ; (iii) t systems ; and  annotated as Na+/H+ antiporters in Synechocystis sp. PCC 6803. NhaS3 is mainly localized to the thylakoid membrane and is essential for cell viability even in low-sodium medium [nhaS4 and nhaS5 genes has no effect on the salt tolerance [nhaS2 deletion mutant is unchanged but the gene is required in low-salt medium for ion homeostasis [+/H+ antiporters are poorly understood, several Na+/H+ antiporters are believed to play a role in sodium-ion export from the cytoplasm in the salt stress response of cyanobacteria .Cyanobacteria have multiple genes encoding Nam medium . In addiolerance . The saleostasis . Althoug+ uptake in the salt stress response of cyanobacteria generally involves a sodium ion-dependent Ktr system  and an ATP dependent Kdp system  [Escherichia coli is responsible for the immediate uptake of K+ after salt shock. However, a mutant of Synechocystis sp. PCC 6803 in which the Kdp system subunit gene kdpA is deleted can grow in 0.8 M NaCl, whereas a mutant lacking the Ktr system subunit gene ktrB is sensitive to 0.1 M NaCl [Knd KdpF) . The Kdp1 M NaCl . Accordi1 M NaCl ,13.Nostoc muscorum, accumulate sucrose and/or trehalose as their major compatible solute. Moderately halophilic strains, such as Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, are characterized by glucosylglycerol as their main compatible solute, whereas halophilic strains such as A. halophytica usually synthesize glycine betaine or glutamate betaine [Compatible solutes in cyanobacteria differ depending on the salt-tolerance of the strain . FreshwaSynechocystis sp. PCC 6803 upregulates 360 genes and downregulates 200 genes [Synechocystis sp. PCC 6803 encode unknown functional proteins [+/H+ antiporters , the potassium-uptake system subunit (ktrB), and genes related to glucosylglycerol biosynthesis  [In the salt stress response process following the addition of 0.68 M NaCl, transcriptomic analysis using microarrays revealed that 00 genes ,15. Apprnd stpA) . Glucosynd stpA) ,18,19,20Synechococcus sp. PCC 7002 from 1 to 24 h after salt shock induced by transferring the cells from freshwater medium (0 M NaCl) to high-salinity medium containing 0.5 or 1 M NaCl. Profiling was performed using capillary electrophoresis/mass spectrometry (CE/MS) and gas chromatography/mass spectrometry (GC/MS). We also analyzed the expression levels of multiple genes after 1 h of salt shock using a microarray to support our metabolomic analysis data.We conducted metabolite profiling of the halophilic cyanobacterium −1 at 10 h, respectively. The cell growth stopped until 10 h upon suddenly increasing the salt concentration to 1 M from 0 M NaCl (Pre-cultured condition), and then they grew again from 10 h . The gene expression level of nhaS3 (A0577) increased 2.4-fold under the 1 M NaCl condition compared with the control  in Synechocystis sp. PCC 6803 after the addition of 684 mM NaCl [Synechocystis sp. PCC 6803 [+ from the cytoplasm. However, we did not observe two-fold higher induction of other Na+/H+ antiporter genes  under both NaCl conditions. Another candidate Na+ transport system in the cytoplasmic membrane of Synechococcus sp. PCC 7002 is the F1F0-type Na+-ATPase homologous operon , atpF-II, atpG-II, and atpH-II), which may be absent in the Synechocystis sp. PCC 6803 genome [1F0-type Na+-ATPase has the broad ion specificity for Na+, H+, or Li+ in an anaerobic bacterium Propionigenium modestum [Synechococcus sp. PCC 7002 after 2 h of salt shock at 0.5 M and 1 M NaCl , glycerol-phosphate synthase (GgpS) and glucosylglycerol-phosphate phosphatase (−1 DW), which is more than 20-fold of that observed at 0.5 M NaCl. In addition, the concentration of ADP-glucose was up to 1 μmol g−1 DW at 1 M NaCl after 1-24 h, which was much lower than that obtained at 0.5 M NaCl. These results show that glucosylglycerol synthesis might be inhibited by a deficiency of ADP-glucose at 1 M NaCl.The moderately halotolerant cyanobacterium e solute . Figure sphatase . The Glpsphatase . HoweverSynechococcus sp. PCC 7002 because its concentration is much lower than that of glucosylglycerol. Sucrose accumulated at both NaCl conditions after 1 h, to a maximum concentration of around 1 μmol g−1 DW at 1 M NaCl , Rubisco small subunit gene (rbcS), and Rubisco chaperone gene (rbcX) were greatly decreased at both NaCl conditions (glpX) and the CO2 concentrating mechanism (CCM) genes ccmK, ccmL, and ccmM were also downregulated at both conditions  carboxylase/oxygenase (Rubisco) is inactivated in the presence of a high concentration of NaCl in both halophytes and glycophytes . In thisnditions . The gennditions . These recreased .The geneondition . HoweverEach value indicates the ratio of the level expression in stressed cells to that in control cells. Values shown are means (±standard deviations) of three independent experiments.−1 DW at 0.5 M NaCl, and those were 7.9, 0.9, and 0.1 μmol g−1 DW at 1 M NaCl , dihydroxyacetone phosphate (DHAP), and glyceraldehyde-3-phosphate (GAP) increased after 1 h at 0.5 M and 1 M NaCl compared to the control. The concentrations of FBP, DHAP, and GAP were 2.5, 0.6, and 0.1 μmol g1 M NaCl . Gene ex1 M NaCl . The gen1 M NaCl . In cont1 M NaCl . GlycolyThe concentration of FBP, DHAP, and GAP increased further after 3 h at 1 M NaCl, but decreased to the same level as the control at 0.5 M NaCl . GlycolyThe ED pathway is also important for sugar catabolism of cyanobacteria in addition to glycolysis and the OPP pathway . The genzwf and fbp were lower at 1 M than 0.5 M NaCl (The OPP pathway metabolite 6-phosphogluconate (6PG) greatly accumulated after 1-10 h at 0.5 M NaCl compared to the control . The gen5 M NaCl . Glycoly+/H+ transporter and ATP production to supplement photosynthetic activity. Synechococcus sp. PCC 7002 may obtain protons and electrons from succinate, in addition to the photosynthetic oxidation of water at 0.5 M NaCl.Isocitrate and malate, the substrates for reducing NAD cofactor in the TCA cycle, accumulated temporarily after 1 h at 0.5 M NaCl, then decreased after 3 h . The genTriticum aestivum [Citrate and isocitrate levels were lower at 1 M NaCl than in the control after 1-10 h . The genaestivum , the pdhSynechocystis sp. PCC 6803 in the presence of 0.5 M NaCl [−1 DW at 0.5 M NaCl and 1 M NaCl after 1 h. The spermidine concentration remained high at 1 M NaCl (above 10 μmol g−1 DW) but decreased gradually at 0.5 M NaCl  and the urea cycle (argG and argH) significantly decreased at both 0.5 M and 1 M NaCl after 1 h  of three independent experiments.6fb complex, F type H+-transporting ATP synthase, and electron carrier proteins  at both 0.5 M and 1 M NaCl conditions. Several of the genes involved in the photosynthetic apparatus are listed in Transcriptomic analysis revealed down-regulation of a large number of genes encoding subunits of photosystem I (PSI), subunits of photosystem II (PSII), phycobilisome (PBS), cytochrome A2164 gene (which encodes PsbA) was up-regulated at both conditions, and the genes encoding phycocyanobilin lyase CpcE and CpcF were up-regulated at 1 M NaCl. Two main isoforms of protein PsbA, D1:1 and D1:2, are found in some cyanobacteria such as Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 [Synechococcus sp. PCC 7942 under optimal growth conditions, whereas D1:2 is upregulated when the cells are exposed to stress conditions such as high illumination or UVB levels [Synechococcus sp. PCC 7002 after acclimating to hyper saline condition [Synechococcus sp. PCC 7002.In contrast, the PCC 7942 . The isoB levels . The A01ondition . We coulEach value indicates the ratio of the level expression in stressed cells to that in control cells. Values shown are means (±standard deviations) of three independent experiments.+ level. Correspondingly, as the glucosylglycerol concentration increased at 1 M NaCl, photosynthesis activity and Rubisco enzyme activity recovered gradually, and cell growth resumed after 10 h . Cells were pre-cultured in hexagonal flat flasks containing 600 mL fresh modified A medium without NaCl (AF medium) under continuous illumination at 400 µmol photons m−2 s−1 at 32 °C and bubbling air containing 2% (v/v) CO2 at 100 mL min−1. AF medium contained 1.5 g L−1 NaNO3, 40 mg L−1 K2HPO4, 75 mg L−1 MgSO4•7H2O, 36 mg L−1 CaCl2•2H2O, 31.5 mg L−1 Na2EDTA•2H2O, 8.0 mg L−1 FeCl3•6H2O, 34.3 mg L−1 H3BO3, 4.3 mg L−1 MnCl2•4H2O, 0.315 mg L−1, ZnCl2, 30 μg L−1 Na2MoO4•2H2O, 3.0 μg L−1 CuSO4•5H2O, 12.2 μg L−1, CoCl2•6H2O, 4.0 μg L−1 cobalamin, and 8.3 mM Tris aminomethane (pH 8.2). All reagents were purchased from Nacalai Tesque, Inc., . After pre-culturing in medium without NaCl for 24 h, the cells were inoculated into three type of medium without added NaCl (defined as control), containing 0.5 M NaCl, and containing 1 M NaCl at 0.32 g DW L−1, and then cultivated at the same culturing conditions as the pre-culture for 48 h.750) measured using a UVmini-1240 . Cell concentration was calculated as DW in medium from the linear correlation between DW and OD750. We determined OD750 of 1 that corresponds to approximately 0.33 g DW L−1.Cell growth was monitored turbidimetrically using the optical density at 750 nm  into a solution of 32.5% (v/v) methanol in water pre-chilled at −30 °C to rapidly terminate metabolism. The cells were harvested by centrifugation at 15,000× g for 5 min using a centrifuge and rotor precooled at −20 °C. The cells were resuspended in 2 mL methanol containing 190 nM (+)-10-camphorsulfonic acid, 31 μM L-methionine sulfone, and 31 μM piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) as internal standards for mass analysis. Intracellular metabolites were extracted with a cold mixture of methanol-chloroform-water , as described previously [g for 5 min at 4 °C. The supernatant cell extract (980 μL) was transferred to a clean tube, 440 μL MilliQ water was added, and the aqueous and organic layers were separated by centrifugation at 22,000× g for 5 min at 4 °C. Two aliquots (450 μL each) of the aqueous layer were transferred to clean tubes for analysis by CE/MS and GC/MS. After removing the solubilized proteins using a 5 kDa cut-off filter , the extracts were evaporated under reduced pressure using a Plus freeze dry system . The dried extracts were stored at –80 °C until used for metabolite analysis.eviously . The mixThe dried extracts were dissolved in 20 μL Milli-Q water and analyzed using an Agilent G7100 CE system, an Agilent G6224AA LC/MSD time-of-flight (TOF) system, and an Agilent 1200 series isocratic high-performance liquid chromatography (HPLC) pump equipped with a 1:100 splitter for delivery of the sheath liquid . Agilent Chem-Station software for CE/MS and MassHunter software for TOFMS were used as the system control and data acquisition software. The CE/MS analytical conditions for cationic and anionic metabolite analyses were as described previously .−1 at constant flow; injector temperature, 230 °C; transfer line, 250 °C; ion source, 200 °C; injection volume, 1 μL; injection mode, split (1:25); oven temperature, 80 °C for 2 min, ramped to 230 °C at a rate of 15 °C min−1, 230 °C for 6 min; solvent delay, 3.5 min; electron voltage: −70 eV; and detection mode, SIM at m/z: 73 and 204. The peak areas of glucosylglycerol and sucrose were determined using GCMS Solution ver. 4.0 (Shimadzu).Dried extracts were derivatized as described previously . The derg for 5 min at 4 °C, then the pellets were washed twice with cold ethanol and dried for 10 min at 60 °C in a heat block. Each dried sample was reconstituted in 100 μL water, centrifuged at 10,000× g for 5 min at 4 °C, and the supernatant was subjected to HPLC analysis as described previously [Glycogen was extracted from cells as described previously . Cells  controlled by a computerized oxygen monitoring system . Oxygen consumption was monitored for 2 min under dark conditions, then oxygen evolution was monitored for 3 min by irradiation at 400 μmol photons mSynechococcus sp. PCC 7002 cultivated under different NaCl conditions were analyzed using a custom 8 × 15K microarray for 3187 Synechococcus sp. PCC 7002 genes (Agilent Technologies). After 1 h cultivation, the cells were harvested by centrifugation at 22,000× g for 5 min at 4 °C and immediately frozen in liquid nitrogen. Total RNA was extracted using a Total RNA Isolation Mini kit (Agilent Technologies) and complementary DNA labeled with cyanine 3 was generated using a Low Input Quick Amp Labeling kit (Agilent Technologies) according to the manufacture’s protocol. After incubation at 65 °C for 17 h, the slides were scanned using an Agilent DNA Microarray scanner (Agilent Technologies). The scanning data were quantified and analyzed using Agilent feature extraction software and GeneSpring GX software (Agilent Technologies). We calculated fold-changes from the transcript level under 0.5 M and 1 M NaCl relative to control condition, and set fold-change threshold to more than 2.Gene expression profiles of


Altogether, 86 new metabolites were identified or tentatively identified, and 23 of them were also new metabolites of mogrosides. In rats, siamenoside I was found to undergo deglycosylation, hydroxylation, dehydrogenation, deoxygenation, isomerization, and glycosylation reactions. Among them, deoxygenation, pentahydroxylation, and didehydrogenation were novel metabolic reactions of mogrosides. The distributions of siamenoside I and its 86 metabolites in rat organs were firstly reported, and they were mainly distributed to intestine, stomach, kidney, and brain. The most widely distributed metabolite was mogroside IIIE. In addition, eight metabolites were bioactive according to literature. These findings would help to understand the metabolism and effective forms of siamenoside I and other mogrosides in vivo.Siamenoside I is the sweetest mogroside that has several kinds of bioactivities, and it is also a constituent of Siraitiae Fructus, a fruit and herb in China. Hitherto the metabolism of siamenoside I in human or animals remains unclear. To reveal its metabolic pathways, a high-performance liquid chromatography-electrospray ionization-ion trap-time of flight-multistage mass spectrometry (HPLC-ESI-IT-TOF-MS Siraitia grosvenorii), a traditional Chinese medicine and an edible fruit − at m/z 1169.59, indicating the molecular formula of C54H92O24, which was the same to siamenoside I. Hence, they were mogroside IV isomers, and M3–M4 were further confirmed as mogroside IVA and mogroside IVE by comparison with reference compounds.M6 was predicted to be C54H90O24 based on its [M + HCOOH − H]− at m/z 1167.5730, which was formed by loss of two hydrogen atoms from siamenoside I, thus M6 was tentatively identified as dehydrogenated siamenoside I.The molecular formula of M7 was C54H92O23 calculated from its [M + HCOOH − H]− at m/z 1153.5957, which has one less oxygen atom than that of siamenoside I. Accordingly, it was tentatively identified as deoxygenated siamenoside I.The molecular formula of M8–M12 showed [M + HCOOH − H]− at m/z 1007.54, implying their molecular formulae of C48H82O19. The formulae had one less glucosyl group (element composition: C6H10O5) than that of siamenoside I, so they were mogroside III isomers. In addition, M8–M10 were unambiguously identified as mogroside III, mogroside IIIE, and mogroside IIIA1 by comparison with reference compounds.M13 had the molecular formula of C48H80O19 predicted by its [M + HCOOH − H]− at m/z 1005.5269. Compared with C48H82O19 of mogroside III isomers, it was tentatively identified as dehydrogenated mogroside III isomer. Moreover, in the MS2 of M13, [M − H − 2Glc]− at m/z 797.4629 (C42H69O14), [M −H − 3Glc]− at m/z 635.4090 (C36H59O9), [aglycon − H]− at m/z 473.3623 (C30H49O4) were observed. Hence, M13 was a triglucoside of dehydrogenated mogrol.M14 was calculated to be C48H82O18 by its [M + HCOOH − H]− at m/z 991.5441, which had one less oxygen atom than C48H82O19 of mogroside III, so it was tentatively identified as deoxygenated mogroside III isomer.The molecular formula of M15–M19 were determined to be C42H72O14 based on their [M + HCOOH − H]− at m/z 845.49, which had one less glucosyl group (element composition: C6H10O5) than C48H82O19 of mogroside III, so they were mogroside II isomers. Furthermore, M15 and M17 were confirmed to be mogroside IIE and mogroside IIA2 by comparison with reference compounds.The molecular formulae of M20–M21 showed [M + HCOOH − H]− at m/z 843.47 in MS, suggesting their molecular formulae of C42H70O14. Additionally, they showed [aglycon − H]− at m/z 473.3623 (C30H49O4) in MS2 spectra. Therefore, they were tentatively identified as dehydrogenated mogroside II isomer, i.e., diglucoside of dehydrogenated mogrol. Further, M20 was unambiguously identified as 11-oxomogroside IIE by comparison with reference compounds.M22 was tentatively identified as deoxygenated mogroside II isomer, since its molecular formula was determined to be C42H72O13 by its [M + HCOOH − H]− at m/z 829.4946, which had one less oxygen atom than C42H72O14 of mogroside II isomers. In addition, M22 showed [M − H]− at m/z 783.4833 (C42H71O13), [M−H−C6H10O4 (deoxyhexosyl)]− at m/z 637.4252 (C36H61O9), and [M−H−C6H10O4−Glc]− at m/z 475.3742 (C30H51O4) in MS2 spectra, which indicated that the deoxygenation occurred in hexose and the aglycone was mogrol. Thus, M22 was identified as a deoxyhexosyl-glucosyl mogrol.M23 was determined to be C42H70O13 according to its [M + HCOOH − H]− at m/z 827.4780, which lost two hydrogen atoms from C42H72O13 of M22. Consequently, M23 was tentatively identified as a dehydrogenated deoxygenated mogroside II isomer.The molecular formula of M24–M25 showed [M + HCOOH − H]− at m/z 683.43 in MS, and [M−H]− at m/z 637.42 (C36H61O9), [aglycone−H]− at m/z 475.37 (C30H51O4) in MS2 spectra, which implied that they were mogrol glucoside. By comparison with the LC-MSn data in literature − at m/z 681.42 in MS, which had two less hydrogen atoms than C36H62O9 of mogroside I isomers. In their negative ion (NI) MS2 spectra, [M − H]− at m/z 635.41 (C36H59O9) and [aglycone−H]− at m/z 473.36 (C30H49O4) were detected. As a result, M26–M27 were tentatively identified as glucosides of dehydrogenated mogrol, i.e., dehydrogenated mogroside I isomers.The molecular formulae of M29 was unambiguously identified as mogrol by comparison with reference compound. M28 had the same molecular formula to mogrol, which showed [M + HCOOH − H]− at m/z 521.38 in MS, so it was a mogrol isomer.M30 and M31 had the molecular formulae of C30H50O4 predicted by their [M + HCOOH − H]− at m/z 519.36, which had two less hydrogen atoms than C30H52O4 of mogrol, thus they were tentatively identified as dehydrogenated mogrols.M32–M35 showed [M + HCOOH − H]− at m/z 553.37, indicating the molecular formula of C30H52O6. Compared to C30H52O4 of mogrol, it had two more oxygen atoms. Accordingly, M32–M35 were tentatively identified as dihydroxylated mogrols.M32 can be deduced by its MS2 data and one possible structure of M32 is shown in In addition, the possible hydroxylation sites of 2 spectra of M32, m/z 433.3011  generated by c,jA cleavage and m/z 349.2406  generated by s,tD cleavage were observed, which indicated that one hydroxylation site was in c,jA, and the other was in c,jABCs,tD , it had one more oxygen atom. Accordingly, M42–M50 were tentatively identified as trihydroxylated mogrols.M51–M61 were determined to be C30H50O7 on the basis of their [M + HCOOH − H]− at m/z 567.35. In comparison with C30H52O7 of M42–M50 (trihydroxylated mogrol), it had two less hydrogen atoms. As a result, M51–M61 were tentatively identified as dehydrogenated trihydroxylated mogrols.The molecular formulae of M53 could be deduced by its MS2 data, and one possible structure of M53 is shown in Further, the possible hydroxylation sites of M53 showed [M + HCOOH − H]− at m/z 567.3524 in MS, and then it was fragmented into [M − H]− at m/z 521.3207 (C30H49O7) in MS2 spectrum. The [M − H]− was further cleaved into product ions at m/z 503.3364 (C30H47O6), 485.3261 (C30H45O5), and 467.3056 (C30H43O4) formed by sequential losses of H2O. It was also cleaved into product ion at m/z 415.2911 (C26H39O4) by losing C4H10O3 (xE+H), which indicated that one hydroxylation site was in xE. Besides, the characteristic fragment ions at m/z 397.2713 (C26H37O3), m/z 379.2621 (C26H35O2), m/z 355.2613 (C24H35O2), and m/z 325.2449 (C22H29O2) were observed in MS2, indicating that the other two hydroxylation sites should be located at a,cABCDuE . Accordingly, they were tentatively identified as dehydrogenated tetrahydroxylated mogrol.The molecular formulae of M74 could be deduced by its MS2 and MS3 data, and one possible structure of M74 is shown in Besides, the possible dehydrogenation and hydroxylation sites of m/z 479.2977  produced by yE cleavage, m/z 419.2762 (C25H39O5) generated by a,cA cleavage, and m/z 195.1347 (C12H19O2) generated by n,pC cleavage indicated that the C24-hydroxyl group of M74 was dehydrogenated, and one of the four tetrahydroxylation sites was at C2, one was in n,pCDyE, and the other two were in ABn,pC  in MS2 spectrum. The [M − H]− was then fragmented into characteristic product ions at m/z 433.2617  and m/z 417.2600  by a,dA cleavage and wE cleavage respectively, which implied that two of the five hydroxylation sites were in a,dA, other two were in wE. Furthermore, the ion at m/z 417.2600  was cleaved into product ions at m/z 399.2515 (C25H35O4) and m/z 203.0987 (C13H15O2) by sequential loss of H2O and l,pCDwE (C12H20O2) in MS3 spectra, which indicated that the last of the five hydroxylation sites was in l,pCDwE.The peak areas and distributions of siamenoside I and the 86 identified metabolites in different biological samples are shown in n data.The metabolism of siamenoside I in rats was firstly investigated in the present work. In total, 86 new metabolites of siamenoside I were detected in different biological samples from rats, and nine of them were unambiguously identified by comparison with reference compounds, and the others were tentatively identified by careful interpretation of their LC-MSM1–M86), the metabolic pathways of siamenoside I in rats are proposed and shown in Based on the structures of the metabolites  are formed by oxidation reactions such as hydroxylation and dehydrogenation, and 79 metabolites (M8–M86) are formed by deglycosylation, which indicate that deglycosylation and oxidation  are the major metabolic reactions of siamenoside I.Among 86 metabolites, 63 metabolites  are formed by reduction reaction (deoxygenation). The deoxygenation reaction might occur in hexose, not in aglycone, which is inferred from the identification of M22  are firstly reported as new metabolites of mogrosides.Furthermore, 23 metabolites  are only detected in feces; M2 is only detected in urine and the brain; M7 is only detected in urine and feces; M51 is only detected in kidneys; M85 is only detected in kidneys and feces; M17, M31, M32 are only detected in stomach and feces; M65 and M81 are only detected in intestines and feces; M83 is only detected in brain and feces. Besides, siamenoside I and 14 metabolites  are detected in the brain for the first time.We also could find the specific metabolites detected in different biosamples. For example, 50 metabolites (M9 (mogroside IIIE) is detected in all biosamples except muscle, indicating that it is the most widely distributed metabolite.M1–M29 seem to be the main metabolites, and most of the other metabolites are only detected in feces. We think the reasons for this result may be: (1) the first pass effect of these metabolites may be very high, i.e., their hepatic extraction ratios are very high, which leads to their very low contents in plasma, organs, and urine samples. As a result, they are not easily detected in these samples; (2) these metabolites are mainly excreted into bile and then to feces, which make their contents in feces high and detectable.From in vivo process of siamenoside I in rats may be as follows.Based on our research results and general metabolic knowledge, we hypothesize that the 2, IA1, IE1, etc.) and its aglycone (mogrol) or dehydrogenated aglycone (dehydrogenated mogrol) by gastric juice, intestinal juice, intestinal enzymes, or intestinal microflora. Then, mogrol or dehydrogenated mogrol permeate across intestinal mucosa and enter the liver, where they undergo extensive oxidative metabolic reactions to form lots of hydroxylation and/or dehydrogenation metabolites. These polar oxidative metabolites may be largely excreted into the bile and then to the feces, and only a limited amount of them enter general circulation. Besides, some of them may also undergo hepatoenteral circulation and are absorbed into general circulation, and then distributed to different organs, and finally excreted into feces or/and urine.After oral administration, siamenoside I is degraded into its secondary glycosides  of siamenoside I can be regarded as bioactive metabolites.On the basis of literature retrieval, eight metabolites  are able to inhibit the induction of Epstein-Barr virus early antigen by 12-O-tetradecanoylphorbol-13-acetate in Raji cells  at 9:00, and all 72-h urine and feces were collected as drug-containing urine and feces samples, respectively. The rats of blank group were orally administrated with the same volume of NS. On day 6 at 9:00, the test and the blank group were treated with siamenoside I and NS again, respectively. After 1 h, blood sample was collected into a vacuum tube with sodium citrate as anticoagulant  from rat heart under anesthesia. Then, the organs  and skeletal muscles of rats were collected and washed with NS, separately. All samples were kept at −80 °C before further pretreatment.The blood samples were centrifuged at 3000 rpm for 20 min at 4 °C, and the supernatant plasma samples were collected. Afterward, 8 mL methanol was added to 2 mL of plasma sample, and then was mixed and centrifuged at 9000 rpm for 30 min. The supernatant was collected and evaporated to dryness at 55 °C by a rotatory evaporator. The 15 mg residue was reconstituted in 1.00 mL methanol, filtrated through 0.45 μm filter membrane, and stored at 4 °C before analysis.Urine samples were filtered and then evaporated to dryness under vacuum at 55 °C by a rotatory evaporator. Subsequently, the residue was ultrasonically extracted with 10 mL methanol for 30 min, and the extract was then centrifuged at 9000 rpm for 30 min. The supernatant was transferred to another tube and evaporated to dryness at 55 °C by a rotatory evaporator. Next, a 100 mg residue was dissolved in 1.00 mL methanol, filtered through 0.45 μm membranes, and stored at 4 °C before analysis.Feces samples were dried at 55 °C and pulverized. Subsequently, the 10 g powder was ultrasonically extracted with 50 mL methanol for 30 min for three times, and the three supernatants were combined and evaporated to dryness. Next, the residue was mixed with 20 mL methanol and centrifuged at 9000 rpm for 30 min. The supernatant was evaporated to dryness again at 55 °C and the residue was collected. Then, 15 mg of residue was dissolved in 1.00 mL methanol, 0.45 μm membranes, and stored at 4 °C before analysis.Each organ was weighed, minced, and homogenized in 4 times volume (mL/g) of 4 °C NS by a homogenizer . Then, 10 mL homogenate was mixed with 9 times volume (mL/mL) of acetonitrile, ultrasonically treated for 30 min, and centrifuged at 12,000 rpm for 30 min at 4 °C. Afterward, the supernatant was collected and evaporated to dryness at 50 °C. The residue was dissolved in 1.00 mL methanol, filtered through 0.45 μm filter membrane, and stored at 4 °C before analysis. The skeletal muscle samples were treated by the same method.18   protected with a Phenomenex Security Guard column  . The column temperature was 35 °C. The injection volume of all samples was 20 μL. The mobile phases were water-formic acid  (A) and acetonitrile (B).The flow rate was 1.0000 mL/min. A gradient elution program was used: 0.01–10.00 min, 10%–18% B; 10.00–20.00 min, 18%–28% B; 20.00–35.00 min, 28%–45% B; 35.00–60.00 min, 45%–90% B; 60.00–70.00 min, 90%–100% B; 70.00–80.00 min, 100% B. The UV spectrum was recorded from 195 nm to 400 nm.The column used was Inertsil ODS-3 Cn instrument were: (1) flow rate: 0.2000 mL/min (split from HPLC effluent); (2) positive ion (PI) and negative ion (NI) alternate detection; (3) mass range: MS, m/z 300–2000; MS2 and MS3, m/z 50–2000; (4) temperature of heat block and curved desolvation line (CDL): 250 °C; (5) nebulizing nitrogen gas flow: 1.5 L/min; interface voltage: (+), 4.5 kV; (−), −3.5 kV; detector voltage: 1.70 kV; (6) ion accumulation time: MS, 30 ms; MS2 and MS3, 20 ms; relative collision-induced dissociation (CID) energy: 50%; (7) data-dependent MS2 and MS3 fragmentation; (8) All data were recorded and analyzed by LCMS solution Version 3.60, Formula Predictor Version 1.01, and Accurate Mass Calculator . The mass range of 50–3000 Da was calibrated by a trifluoroacetic acid sodium solution (2.5 mM).The parameters of the ESI-IT-TOF-MSn data with those of reference compounds, or tentatively identified by interpretation of their MSn data.The strategy  previousTo preliminarily estimate the relative contents of siamenoside I and its metabolites in biosamples, the peak area of each metabolite calculated from its NI EIC was used. In the present study, only the data of different organ samples were comparable, since they were prepared and analyzed by the same method.n technique. The metabolic pathways of siamenoside I in rats were proposed based on the structures of metabolites. The metabolic reactions of siamenoside I were found to be deglycosylation, hydroxylation, dehydrogenation, deoxygenation, isomerization, and glycosylation, among which deoxygenation, pentahydroxylation, and didehydrogenation were novel metabolic reactions of mogrosides. In addition, 23 metabolites were new metabolites of mogrosides. The distributions of siamenoside I and its 86 metabolites in rat organs were firstly reported, and they were mainly distributed to intestines, the stomach, kidneys, and the brain. Mogroside IIIE was the most widely distributed metabolite. Eight metabolites had bioactivities, indicating that they might contribute to the bioactivities of siamenoside I. These findings not only provide valuable information on the metabolism and disposition of siamenoside I and mogrosides in rats but also provide useful information on the chemical basis of the pharmacological effects of siamenoside I and mogrosides in vivo.The metabolism of siamenoside I in rats was studied for the first time. In total, 86 new metabolites were detected. Nine of them were unambiguously identified by comparison with reference compounds, and the other 77 were tentatively identified by HPLC-ESI-IT-TOF-MS


Galinsoga parviflora. A strain SYPF 7336, isolated from the rhizospheric soil, was identified as Seltsamia galinsogisoli sp. nov., by morphological and molecular analyses, which displayed high antibacterial activity. In order to study the secondary metabolites of Seltsamia galinsogisoli sp. nov., nine compounds were successfully seperated from the strain fermentation broth, including two new compounds and seven known compounds. Their structures were elucidated based on spectral analysis including 1D and 2D NMR. All the seperated compounds were evaluated for their antimicrobial activities. Compounds 2, 5 and 1 displayed antimicrobial activities against Staphylococcus aureus with MIC values of 25, 32 and 75 μg/mL, respectively. Moreover, morphological observation showed the coccoid cells of S. aureus to be swollen to a volume of 1.4 to 1.7-fold after treatment with compounds 1, 2 and 5, respectively. Molecular docking was carried out to investigate interactions of filamentous temperature-sensitive protein Z (FtsZ) with compounds 1, 2 and 5.A total amount of 116 fungal strains, belonging to 30 genera, were acquired from the rhizosphere soil and plant of  Currently, due to the increase of antibiotic resistance, there is an urgent need for novel classes of lead compounds and novel mechanisms to confront the antibiotic crises7.Secondary metabolites coming from microorganism represent a large number of diverse components which have been treated as potential candidates for drugs9. The inhibitors of FtsZ prevent the cellular fission of bacteria, which lead to apoptosis of bacteria12. Therefore, morphological observation of microbial fission and molecular docking between the lead compound and FtsZ were accomplished to explore the possible mechanism14. Up to now, the discovered FtsZ inhibitors are divided into natural products  and synthetic small molecules 19. At present, PC190723 has been the candidate drugs to enter the clinical trials, which inspires scientists to make more effort to find potential FtsZ inhibitors as antibacterial agents22.Microbial filamentous temperature-sensitive protein Z (FtsZ) is a novel target for drug discovery, which plays a key role in cell divisionThe endophytic fungi are known as a source of abundant secondary metabolites for functional bioactive substances. As we know, only a small amount of microbes have been studied so far, and a vast number of new taxa waiting for discovery, especially those separated from medicinal plants. This leads us to isolate and evaluate the potential pharmacological activity of bioactive compounds produced by endophytic fungi.Galinsoga parviflora was surveyed and 116 fungal strains were isolated from the whole plant  which attributed to 30 genera. Forty-three percent of the strains revealed antimicrobial abilities against at least one kind of human pathogenic microorganisms. Strain SYPF 7336, presented the strongest antibacterial activity, could not be affiliated to any known taxon, so it was identified as a novel species of genus Seltsamia by phylogenetic analyses, given the name Seltsamia galinsogisoli sp. nov. The genus Seltsamia, (Cucurbitariaceae) was first proposed in 2018, and the only strain Seltsamia ulmi CBS 143002 was isolated on corticated Ulmus glabra in Norway23. Seltsamia ulmi produces pyriform, black ascomata and cylindrical asci. Asci contain 8 uni- to partly biseriately arranged ascospores and ascospores are fusoid to subclavate with 3 main septa. Due to none of the secondary metabolites of the genus Seltsamia having been recorded, the compounds and bioactivities of the novel species, strain SYPF 7336, were explored in the present study. Further, the molecular docking analysis was carried out for mechanism investigation.In the present study, the endophytic fungi diversity of the traditional Chinese herb, Galinsoga parviflora, which were divided into 30 groups according to morphological characteristics and phylogenetic analyses. Twelve genera, Acremonium, Beauveria, Trichoderma, Periconia, Gibberella, Discosi, Scolecobasidium, Sporobolomyces, Pyrenochaeta, Peyronellaea, Chaetopyrena and Seltsamia, were found only in the soil. Twelve genera, Sarocladium, Talaromyces, Botryotinia, Cadophora, Phomopsis, Volutella, Epicoccum, Mucor, Plectosphaerella, Cylindrocarpon, Clonostachys, and Phialophora just survived in the plant. Six genera, Cladosporium, Paraphoma, Penicillium, Alternaria, Fusarium, and Phoma could simultaneously survive in the soil and plant of G. parviflora  to clarify the taxon status of strain SYPF 7336. In the MP analysis, 1938 characters were constant, 87 were parsimony-uninformative, and 152 were parsimony-informative. After phylogenetic analysis, the best MP tree was shown in Fig. Seltsamia and formed a sister clade together with S. ulmi. The ITS and LSU sequences were deposited in GenBank with accession numbers KU759584 and KU759581, respectively .Vegetative hyphae hyaline, smooth walled. Pycnidia subglobose to globose, brown to dark brown, 70–125 × 45–96 μm. Surface roughened by colourless hyphal appendages Fig. . ConidioColonies on PDA attaining 36.6 mm diameter after 11 d at 26 °C, surface floccose, grey to dark grey, reverse grey to dark grey Fig. . ColonieG. parviflora. Ex-type culture CBS 140956 = CGMCC 3.17981 = SYPF 7336.China, Liaoning province, Huludao city, 40°82′26.5″N, 119°78′52.0″E, Sep 2014, from the rhizosphere of 1)  Fig.  was isolies Figs –S6. The 1 ,6.50 d (J = 2.2 Hz)] and one methoxy . The spectra of 1 were in part very similar to those of alter lactone except for the absence of a methylene group24. The HMBC spectra indicated the presence of long-range correlations  was seperated as a brown powder, −1 in IR spectrum suggested the presence of hydroxyl carboxylic acid carbonyl, and carbonyl functionalities , indicating the molecular formula of C19H32O4. The 1H NMR spectrum of 2 , 0.97 , 0.849 , 0.877 , 1.59 , one methoxyl groups at δH 3.68  . It also showed four methylene protons at δH 4.05 , 2.66 , 1.95  and 1.75 , five methine protons at 5.85 , 4.83 , 3.6 , 3.6 , 2.92 ; The 13C NMR spectra (Table δC 174.3 and 193.0), a pair of olefinic carbons (δC 174.3 and 193.0), five methyl carbons , one oxygenated methylene proton carbon (δC 58.9), three methylene carbons . The 1H and 13C NMR spectra of 2 were similar to those of 4-methyl-5,6-dihydro-2H-pyran-2-one except for the absence of two side chains26. The planar structure of 2 , including 1,3-Benzenediol,5-(2-hydroxypropyl) (3)27, 3,4-Dihydroxy-2-methyl-7-[prop-1-enyl]-3,4-dihydro-2H-pyranopyran-5-one (4)30, 1H-2-Benzopyran-1-one,6,8-dihydroxy-3-(2-hydroxypropyl) (5)32, 2,5-dimethyl-7-hydroxyl chromone (6)33, 3-methyl-6,8-dihydroxyisocoumarin (7)34, Curvulapyrone (8)34, and 3,4-dihydro-8-hydroxyisocoumarin-3-carboxylic methyl ether (9)35.Additionally, the discovery metabolites of S. aureus, B. subtilis, P. aeruginosa, K. pneumonia and Bacillus cereus. The results were shown in Table All the seperated metabolites were tested for antimicrobial effects against five common pathogenic bacteria, 2, 5 and 1 showed antimicrobial activities against S. aureus with MIC values of 25, 32 and 75 μg/mL, respectively. Compounds 3–4, 6–7 and 8–9 showed weak antimicrobial effects.Compounds S. aureus treated with compounds were observed carefully , 2 (1.7 fold) and 5 (1.6 fold), respectively. In order to explain the possible mechanism, FtsZ, key protein of cell division35, was explored for molecular docking study.The cells of lly Fig. . Interes1, 2 and 5 to FtsZ from S. aureus (PDB:ID 3VOB)  Fig.  resultedOB) Fig. . Thus, c 5 Table . Compoun 1 Table . Five hyG. parviflora were fermented and the crude extracts of each strain were tested for the microbial activities  and seven known compounds (3–9)  Fig.  were pur36. In bacterial cytokinesis, FtsZ protein is the earliest known step to build a contractile ring on the inner surface of the cytoplasmic membrane36. The inhibitors of FtsZ might prevent the cellular fission of bacteria, which lead to apoptosis of bacteria. Therefore, morphological observation and molecular docking were carried out to search the possible interactions between the active compounds and FtsZ.FtsZ is a pop target for drug discovery in recent years. The gene of FtsZ has the ability of high conservation and presented almost in all bacteriaS. aureus treated with active compounds 1, 2 and 5 were observed to further study the possible antibacterial mechanism. Interestingly, As Fig. S. aureus were swollen to 1.4 to 1.7-fold volume after treatment with compound 1 (1.4 fold), 2 (1.7 fold) and 5 (1.6 fold), respectively. In order to explain this interesting appearance, FtsZ, the key protein of cell division9, was explored to illustrate the mechanism of cells that became swollen. Thus, a molecular docking study was carried out to verify the deduction.Thus, the cells of S. aureus (PDB:ID 3VOB) displayed the docking score of the ligand  was the lowest. Compound 5  displayed three hydrophobic bonds with THR309, ASN263, LEU209 residues but no hydrogen bonds with neighbouring amino acid residues. Compound 2  formed four hydrogen bonds with the ILE228*2, THR309, VAL297 residues and four hydrophobic bonds with the ASN263*2,THR309 and THR265 residues. Although compound 1 formed two hydrogen bonds and five hydrophobic bonds with the residues, one unfavorable interactions between compound 1 and the active region or intramolecular of FtsZ were existed. Thus, compound 1 showed a weak ability to combine with the docking score of −109 kcal/mol. The docking scores −125 kcal/mol (2) −113 kcal/mol (5) and -109 kcal/mol (1), indicated that compounds 2 and 5 might form lower potential energies and more stable binding sites with the target protein FtsZ compared to coumpound 1 which validated the observed antimicrobial activities. Based on the antimicrobial activities, phenotypic consequences and docking studies, compounds 2 and 5 were identified as promising antimicrobial lead molecules.The docking results are shown in Fig. 37. IR spectra were measured on an Equinox55 spectrophotometer in KBr discs . The 1D- and 2D-NMR spectra were recorded at 600 for 1H and 150 MHz for 13C . HR-ESI-MS data were acquired on a Bruker Customer micrOTOF-Q 125 mass spectrometer . Solvents were purchased from Tianjin Kemiou Chemical Reagent Company , MeOH and CH3CN for HPLC analysis were chromatographic grades . Silica gel  were used for column chromatography.Optical rotations were recorded using a P-2000 Digital Polarimeter G. parviflora in Huludao city, Liaoning province, northeast of China . The samples were conducted as described previously39. All plates were incubated at 26 °C and examined daily. Single colonies were picked and transferred to freshly prepared PDA plates. The single spore-origin strain was stored at 4 °C and/or conidia suspension in 20% glycerol for further study.Samples of soil and plant were collected from the field of a traditional Chinese medical herb 37. Mycelium structure was observed under an optical microscope Olympus BX53 .The colony morphology of the isolate was studied on PDA, pine-needle agar (PNA), corn meal agar (CMA), malt extract agar (MEA) and oatmeal agar (OA) plates and incubated at 26 °C41. The internal transcribed spacer (ITS) region was amplified with primers ITS4 and ITS542. The partial 28S ribosomal RNA (LSU) gene region was amplified with primers LROR and LR744. The thermocycling conditions for amplifications had an initial denaturing step of 94 °C for 5 min, 32 cycles of 94 °C for 60 s, 55 °C for 30 s, 72 °C for 90 s, followed by a final elongation step at 72 °C for 7 min. A G1000 Thermal Cycler  was used for PCR amplification. Amplicons were verified with 1% agarose electrophoresis gel, and the expected bands were excised and purified with an AxyPrepTM gel purification kit . These fragments were cloned into the pEASY-T5 zero cloning kit  and followed by sequencing . The nucleotide sequences of the genes have been deposited in GenBank  and Mega 7.046. Datasets were analyzed using both maximum parsimony (MP) and Bayesian tree inference (BI). MP analyses were performed using PAUP* 4.0b10, a heuristic search option was chosen with random addition of sequences as 1,000 replications; gaps were treated as missing data47. BI analyses were run with MrBayes 3.2.4 using the GTR substitution model with gamma-distributed rate variation across sites and a proportion of invariable sites48. Two sets of four chains were executed until the standard deviation of split frequencies reached 0.01. Sample frequency was set at 100 and 25% of trees removed as burn-in.All the sequences Table  were ali37. Briefy, sterile water (53 ml) and rice (40 g) were mixed to an Erlenmeyer flask (250 ml), which were autoclaved at 121 °C for 30 min. The strain of SYPF 7336 (1 ml) was inoculated in each Erlenmeyer flask (250 ml × 120), which were cultivated at 28 °C for 30 days. The fermented material was extracted using ethyl acetate (12 L × 3) to give the crude extract (127 g). Then it was dissolved in 90% MeOH–H2O (1 L), and extracted by hexane (1 L × 3) to obtain the residue (62 g).This assay was performed according to our previous method2Cl2/CH3OH (v/v 80:1–3:1), and yielding four fractions (A-D). Fraction B (10.2 g) was separated by ODS eluting with MeOH-H2O (v/v 10:90 to 90:10) to give another eight fractions (B1-B7). Fraction B1 (1.6 g) was further subjected to semipreparative HPLC , eluted with CH3CN-H2O  to yield compounds 1 (10 mg), 3 (12 mg) and 8 (10 mg). Subfraction B 3–4 (0.8 g) was further purified by semipreparative HPLC  to produce compounds 2 (10 mg), 4 (13 mg) and 9 (11 mg). Similarly, subfraction B7 (1.2 g) was subjected to semipreparative HPLC, eluted with CH3CN-H2O  to afford compounds 5 (13.6 mg), 6 (11.2 mg) and 7 (17 mg).Silica gel chromatography was used to separate the extract (41.44 g) eluting with CH1: white flakes; 1H  and 13C NMR  data, see Table m/z 325.2738 [M + Na]+ .Compound 2: yellowish oil; 1H  and 13C NMR  data, see Table m/z 307.2551 [M + Na]+ .Compound B. subtilis (CMCC63501), S. aureus (CMCC26003), P. aeruginosa (CMCC10104), K. pneumonia (CMCC46117), and E. coli (CMCC44102)50. The antimicrobial assay was performed according to the procedure of Zhu et al.50. Briefly, DMSO was used to dissolved the positive control and compounds 1–9 at an initial concentration of 10 mg/ml, respectively. Luria-Bertani (LB) media was used for the five pathogenic bacteria for 24 h at 37 °C until the absorbance OD600 = 0.2. The bacterial solution was diluted with LB medium (4%). 2 μl sample solution and 198 μl bacterial culture were mixed into 96-well plate. The 96-well plate was determined by a microplate reader (OD600) after 24 h of cultivation at 37 °C. Growth inhibition (%) was calculated as [1 − (OD600 of treatment/OD600 of control)] × 100. Every experiment was performed in triplicate to validate the biological activities.Bacterial strains used in our anti-bacterial studies are from National Center for Medical Culture Collections (CMCC), and the CMCC numbers are listed as below: 1, 2 and 5 were assessed against bacteria S. aureus (CMCC26003). The MIC values of the isolated compounds against human pathogenic bacteria were determined by the modified CLSI M38-A method50. Briefly, compounds 1, 2 and 5 were dissolved using DMSO with the final concentrations of 100, 50, 25, 12.5, and 6.25 ug/mL, respectivly for the MIC determination. The dosage-response curve was drawed according to different concentrations of compound to S. aureus cells growth inhibition rates. The MIC values were calculated from the dosage-response curves. DMSO and Ampicillin were used as the negative control and positive control, respectively.Minimal inhibitory concentration (MIC) of compounds 37. Briefy, the crystal structure of protein obtained from RCSB Protein Data Bank (PDB Code 3VOB) were used for docking51. The 3D structures of the compounds 1–2 and 5 were prepared and Gasteiger-Hückel charges were added using Sybyl software . The ligand, guanosine-5′-diphosphate, was subjected to energy minimization with Tripos force filed parameters51. Blind docking was carried out using Molegro Virtual Docker 4.0  program. The 3D docking grid was sufficiently large to cover the protein.This assay was performed according to our previous methodS. aureus after treated with compounds 1–2 and 5, observations under a transmission electron microscope  were performed. S. aureus cells were grown at 37 °C on an agar plate, then diluted by LB broth to an OD600 of 0.2, and 25 μg/ml compounds 1–2 and 5 were added to the suspensions. Samples of treated cells and controls were further cultivated at 37 °C for 3 h. Then, the bacterial suspensions were dyed with 2% phosphotungstic acid (v/v = 1:1 pH 6.5) for 3–5 min, and transmission scan was performed as previously described20.To identify whether there are changes in morphology of Supporting information


Modern agricultural practises rely on surfactant-based spray applications to eliminate weeds in crops. The wide spread and indiscriminate use of surfactants may result in a number of deleterious effects that are not limited to impacts on the crop and surrounding farm eco-system but include effects on human health. To provide a safer alternative to the use of surfactant-based formulations, we have synthesised a novel, self-assembling herbicide conjugate for the delivery of a broad leaf herbicide, picloram.Arabidopsis thaliana and found that when tested at agriculturally relevant doses between 0.58 and 11.70 mM a dose-dependent herbicidal effect with comparable kill rates was evident.The synthesized self-assembling amphiphile–picloram (SAP) conjugate has three extending arms: a lipophilic lauryl chain, a hydrophilic polyethylene glycol chain and the amphiphobic agrochemical active picloram. We propose that the SAP conjugate maintains its colloidal stability by quickly transitioning between micellar and inverse micellar phases in hydrophilic and lipophilic environments respectively. The SAP conjugate provides the advantage of a phase structure that enables enhanced interaction with the hydrophobic epicuticular wax surface of the leaf. We have investigated the herbicidal efficiency of the SAP conjugate compared against that of commercial picloram formulations using the model plant Though self-assembling drug carriers are not new to the pharmaceutical industry their use for the delivery of agrochemicals shows great promise but is largely unexplored. We have shown that SAP may be used as an alternative to current surfactant-based agrochemical formulations and has the potential to shift present practises towards a more sustainable approach.The online version of this article (10.1186/s12951-018-0423-5) contains supplementary material, which is available to authorized users. Arabidopsis thaliana, showed that mesoporous silica nanoparticles (MSNs), can deliver the phytohormone and disease resistance inducer, salicylic acid, in-planta only when redox stress is high  − 4.14 × 10−1 mM [1.0 × 10−2 % (w/v)] and mixed with one of the commercial surfactants empimin, empigen or pluronic F127 at 0.01% v/v , 1.17 mM [0.1% (w/v)], 2.34 mM [0.2% (w/v)], 5.85 mM [0.5% (w/v)] and 11.70 mM [1.0% (w/v)] and spray applied to a plants , 17. Blia plants . For ourTo calculate the critical micelle concentration (CMC) of the conjugate, the fluorescence spectrum of pyrene was used. The intensity ratio of the first and third fluorescence emission peaks of pyrene is very sensitive to aggregation of pyrene, and is indicative of the formation of micelles. An obvious trend change in intensity ratio of the first and third fluorescence emission peaks of pyrene, found at 0.13 mM, confirmed the formation of micelles Fig. .Fig. 2FlThe SAP conjugate aggregated into small particles around 200 nm at low concentration in water and had a narrow polydispersity index (PDI) of 0.3. The SAP conjugate aggregated into bigger particles and had a wide PDI when the concentration of the conjugate was above 2 mM  measurement of the cast film of the SAP conjugate indicated that the SAP conjugate had excellent amphiphilicity and quickly adjusted to a new environment. For water, the CA of the SAP conjugate decreased from 21.8 ± 2.6° to 3.7 ± 1.8° in 4 min, indicating that the SAP conjugate had good hydrophilicity and capability to form a colloidal-stable dispersion in water. For oil, the CA was close to zero degrees demonstrating the super-lipophilic nature of SAP formulations and a potential to adsorb and interact with the hydrophobic wax layers on a leaf surface.To quantify the herbicidal effect after spray application of various formulations each collected image of the treated plants was assessed on a five-point scale ranging between 0 and 5 by blind assessment carried out by three independent assessors (n = 3 for each replicate). The 0–5 scale was as follows; 0 = healthy plant with no visible signs of leaf curling or necrosis  applied to 3-week-old −3 mM a dose-dependent trend in herbicidal effect was observed for picloram (in water) treatments between 4.14 × 10−5 mM and 4.14 × 10−1 mM. The herbicidal effect at a dose of 4.14 × 10−1 mM was statistically different from other picloram (in water) treatments  mM. Before the spray application of the amphiphile–picloram (SAP) conjugate to the plants, its solubility was tested by mixing the SAP conjugate in water at concentrations of 0.585 (or 5.85 × 10−1) mM [equal to 0.05% (w/v)], 1.17 mM [0.1% (w/v)], 2.34 mM [0.2% (w/v)], 5.85 mM [0.5% (w/v)] and 11.70 mM [1.0% (w/v)]. The SAP conjugate formed an emulsion at all the concentrations tested while for picloram-oleyl (PO) and picloram , a precipitate formed at the bottom of the tube at the same concentrations of that for the SAP conjugate , the herbicidal effect recorded was 2.56 ± 0.17 which then gradually increased to 4.11 ± 0.25 with each increment in the applied dosage of SAP to 11.70 mM Fig. a. The hePhytotoxicity observations at 7 days after spray application of SAP conjugate, PO and picloram were broadly consistent with the day three observations Fig. b. There A. thaliana grown in vitro was sprayed with agriculturally relevant concentrations of the SAP conjugate containing the herbicide picloram that the nanoformulation was as equally effective for killing target plants as current surfactant-based formulations. This approach to agrochemical delivery has several clear advantages over surfactant-based delivery that include the prevention of crop and off target toxicity, reduction in chemical load and use of less water which, together, benefits the environment and has the potential to increase crop productivity. The three radiating arms that formed the SAP conjugate were the key features of this nanomaterial that enabled the conjugate to form a stable emulsion in water and, when applied, to interact directly with the target hydrophobic plant leaf surface.The use of self-assembling lipids for the delivery of therapeutics such as antibiotics  and antiPicloram has a pKa value of 2.30 and is poorly soluble in water at 430 mg per litre . SurfactTherefore, we synthesised SAP conjugate whose arms reduce the free energy barrier and help its aqueous formulation to attain colloidal stability, leading to a quick transition between micellar and inverse micellar structures either in a hydrophilic or lipophilic environment respectively. The low water contact angle of the SAP conjugate provided a very good indication of the hydrophobic switch and the natural ability of the conjugate to attain better colloidal stability . Such a 7)phenol (component 5) is very unlikely to freely disassociate from the conjugate. After adsorption the SAP conjugate acts like a lipophilic non-electrolyte in that it diffuses through the amorphous regions of the cuticle to elicit the herbicidal effect [7) phenol.When the aqueous formulation of the self-assembling conjugate was spray applied to hydrophobic leaf surfaces, the extending arms of the SAP conjugate enabled its quick transition upon the evaporation of the water. For the activity of the SAP conjugate, it is inaccurate to assume that herbicidal activity will occur after the disassociation of picloram from the conjugate. We predict that the entire SAP conjugate is adsorbed onto the plant cuticular surface. Picloram, because of its strong covalent bond with 3-(dodecyloxy)-5-, wild mustard (Sinapis arvensis) and wild turnip (Myagrum rugosum) in monocot crops such as wheat (Triticum aestivum), barley (Hordeum vulgare) and oats (Avena sativa) [2 assimilation and that ultimately leads to plant death [Picloram like 2,4-D is an amphiphobic phenoxy herbicides (or auxin mimic) that is routinely used to eliminate dicot weeds such as wild radish ( sativa) . Epinast sativa) . Auxin mnt death , 41.In our laboratory experiments, for all the formulations, a dose range that was agriculturally relevant was tested. It is likely that the diffusion of SAP conjugate is slower than picloram by itself due to its larger size that in turn limits the rate of diffusion of the conjugate into the amorphous regions of the cuticle, that are usually available for the diffusion of lipophilic non-electrolytes . At the A. thaliana in our proof of concept study but clearly agriculturally relevant crop and weed species must now be examined for their interactions with self-assembling conjugates and with a variety of actives. We expect that self-assembling conjugates will find broad use in agriculture for the delivery of actives such as herbicides, pesticides and fungicides and that their use will strengthen existing practises for sustainable agriculture.It was recently shown that agrochemicals, such as pesticides, contribute to the largest fraction of volatile organic compound sources that not only damage the environment but also increasingly impact human health . In addiAdditional file 1: Figure S1. Component (2) 3-(dodecyloxy)-5-(hydroxymethyl)phenol. 1H NMR : 0.79–0.82 ; 1.15–1.26 ; 1.30–1.37 ; 1.66–1.73 ; 3.77–3.81 ; 4.46 ; 6.24–6.25 ; 6.32–6.33 ; 6.35–6.36 ; 7.89 .Additional file 2: Figure S2. Component (4) Ots-EO7-Me. 1H NMR : 2.37 ; 3.3 ; 3.46–3.48 ; 3.5 ; 3.54–3.58 ; 3.6–3.62 ; 4.07–4.09 ; 7.26–7.28 ; 7.71–7.73 . The 1H NMR spectrum of component (4) Ots-EO7-Me.Additional file 3: Figure S3. Component (5) 3-(dodecyloxy)-5-(EO7)phenol. 1H NMR : 0.86–0.9 ; 1.26–1.35 ; 1.39–1.46 ; 1.72–1.79 ; 3.37 ; 3.53–3.55 ; 3.63–3.68 ; 3.69–3.72 ; 3.82–3.85 ; 3.9–3.93 ; 4.1–4.12 ; 4.6 ; 6.37–6.38 ; 6.51–6.53 . The 1H NMR spectrum of component (5) 3-(dodecyloxy)-5-(EO7)phenol.Additional file 4: Figure S4. Component (6) 3-(dodecyloxy)-5-(EO7)benzyl-PIC. 1H NMR : 0.85–0.89 ; 1.25–1.34 ; 1.38–1.45 ; 1.71–1.78 ; 3.36 ; 3.52–3.55 ; 3.63–3.68 ; 3.71–3.73 ; 3.82–3.84 ; 3.89–3.92 ; 4.09–4.11 ; 5.31 ; 5.53 ; 6.41–6.42 ; 6.56–6.57 . The 1H NMR spectrum of component (6) 3-(dodecyloxy)-5-(EO7)benzyl-PIC.Additional file 5: Figure S5. Component (6) 3-(dodecyloxy)-5-(EO7)benzyl-PIC. 13C NMR : 14.13 (s); 22.68 (s); 26.03 (s); 29.21 (s); 29.34–29.39 (d); 29.58–29.66 (m); 31.91 (s); 59.02 (s); 67.49–68.12 (t); 69.63 (s); 70.49–70.79 (t); 71.91 (s); 76.79–77.42 (t); 101.57 (s); 106.32–106.83 (d); 114.06–114.94 (d); 137.06 (s); 144.39 (s); 146.74 (s); 148.96 (s); 160.04–160.41 (d); 163.33 (s).Additional file 6: Figure S6. Size distribution of the SAP molecules. From the TEM images the length and width of SAP particle was estimated to be 82 ± 30 nm and 55 ± 17 nm respectively. Though TEM images are an indicative of smaller SAP conjugates, a through sampling regime can confirm the larger size of the aggregates.Additional file 7: Figure S7. Surfactant-assisted delivery of picloram to A. thaliana. The image on the left hand side is labelled with relevant surfactant whose concentration was kept constant at 0.01% (w/v) while the respective concentrations of picloram were given on the top. (a) Control A. thaliana plant three days after water treatment. (b to f) The herbicidal effect intensified with the increase in the concentration of picloram (mixed in water) spray application and are marked by the presence of leaf curling (c to f), chlorosis (d to f) and petiole curling (f). (g to l) F127 based picloram treatments were similar to picloram (in water) treatments. (m to r) A stronger phytotoxicity effect on images (m to r) was evident for the treatments containing incremental doses of picloram in empigen. (s to x) For picloram delivered with empimin the herbicidal effect was similar to the same effect earlier observed for F127 based picloram delivery. Scale bar approximately equals to 1.5 cm.Additional file 8: Figure S8. Herbicidal effect on A. thaliana after surfactant assisted picloram delivery. Three days after the application of picloram separately with water, F127, empimin and empigen, maximum herbicidal effect was observed for the highest concentration of picloram (10−1 mM) applied. Note that in the figure all x-axis concentrations are multiplied by 4.14, Ctrl is the control for the relevant picloram solvents. Ctrl = control, Pic = picloram. The bars ± SE labelled with the same letters are statistically similar (p < 0.05), Duncan’s posthoc test.Additional file 9: Figure S9. Solubility of SAP, PO and picloram in water. Self-assembly picloram when mixed in water formed a cloudy emulsion while PO and picloram at similar concentrations formed a precipitate.Additional file 10: Figure S10. Effects of SAP delivery to plants. The images are labelled with relevant formulations on the left hand side with which they were spray applied while on the top are their respective concentrations. (a) Water treated healthy control plant three days after the spray treatments. The herbicidal effect of the SAP on plants was evident by the presence of leaf curling that elevated to petiole curling with the increase in dosage of SAP from (b) 0.58 mM through (c) 1.17 mM to (d) 2.34 mM. In addition to leaf and petiole curling, further increase in the concentration of SAP from 2.34 mM to (e) 5.85 mM and (f) 11.70 mM caused the leaves to twist along the main axis of the plant. (g) Water treated control for PO treatments. For PO applications at low concentrations, the phytotoxicity was limited to leaf curling (h to l) irrespective of the concentration. (m) A. thaliana plant treated with water. On the contrary, picloram applications induced a strong phytotoxicity marked by (n to p) the presence of chlorosis, petiole and leaf curling, (q and r) tissue necrosis and death. Scale bar equal to one cm.


Based on those results, we believe that the very efficient biohydrogenation of hydroxychalcones, which may easy be scaled up for biotechnological purposes, reflects the natural activity of the cyanobacterial defence system, because hydroxydihydrochalcones were less active inhibitors of the growth of cyanobacterial cultures than the corresponding substrates.To verify the hypothesis that cyanobacteria naturally biosynthesising polyphenolic compounds possess an active enzymatic system that enables them to transform these substances, such an ability of the biocatalytic systems of whole cells of these biota was assessed for the first time. One halophilic strain and seven freshwater strains of cyanobacteria representing four of the five taxonomic orders of Cyanophyta were examined to determine the following: (i) whether they contain polyphenols, including flavonoids; (ii) the resistance of their cultures when suppressed by the presence of exogenous hydroxychalcones—precursors of flavonoid biosynthesis and (iii) whether these photoautotrophs can transform hydroxylated chalcones. All examined strains were found to contain polyphenols and flavonoids, and the growth of their cultures was inhibited in the presence of 2′-hydroxychalcone, 2″-hydroxychalcone and 4″-hydroxychalcone. We also confirmed that the examined cyanobacteria transformed hydroxychalcones via hydrogenative bio-reduction and formed the corresponding hydroxydihydro derivatives with yields above 90% whenever the substrates were bioavailable for such a conversion. Moreover, we observed that the routes and efficiency of biohydrogenation (and hydroxylation) of chalcones were dependent on the location of the hydroxyl substituent. The final products obtained as the results of biotransformations were extracted from the media and identified by mass spectrometry (LC-MS/MS) and nuclear magnetic resonance ( Chalcones are a group of plant-derived compounds belonging to the flavonoid family that are synthesised through the phenylpropanoid pathway and play a vital role in different metabolic pathways in photosynthesising organisms. Structurally, chalcones are composed of two aryl groups (A- and B-rings) linked by an open-chain three-carbon unit α,β-unsaturated carbonyl system. Naturally occurring chalcones are broadly distributed in various plant species and possess a conspicuously yellow colour ], and seven freshwater strains, including Anabaena sp. [strain CCALA 007], Anabaena laxa [strain CCALA 805], Aphanizomenon klebahnii [CCALA 009], Nodularia moravica [strain CCALA 797], Chroococcus minutus [strain CCALA 055], Merismopedia glauca [strain CCALA 099] and Synechocystis aquatilis [strain CCALA 190], were used in our experiments. The axenic strain of Spirulina platensis was purchased from the Pasteur Culture Collection (PCC) , whereas all freshwater strains were obtained from the Culture Collection of Autotrophic Organisms (CCALA) .One halophilic strain of cyanobacteria, Subcultures of cyanobacteria were revitalised every 3 weeks by transferring 10-mL aliquots to 50 mL of fresh suitable media  by centrifugation at 5000×g for 1 min, and the supernatants were filtered using Whatman No. 1 filter paper, pooled, concentrated to 1 mL and sterilised by filtration using a 0.22-μm Millipore filter. The resultant extracts were stored at 4 °C in the dark for 24 h before determination of total phenolic and flavonoid content.For the efficient extraction of polyphenols and flavonoids, 100 mg of lyophilized cells of each strain was sonicated for 30 min in 3 mL of pure methanol using an ultrasonic bath  and for a further 30 min using a Hielscher UP200HT ultrasonic homogeniser . The homogenates were centrifuged at 5000×2O was added to the mixture and shaken vigorously. After a 2-min incubation at room temperature, 1.5 mL of Na2CO3  solution was added, and the volume was then made up to the mark with distilled water. The samples were incubated for 2 h in the dark. The mixture was allowed to stand for 2 h with intermittent shaking. After incubation for 120 min at room temperature in the darkness, the absorbance against distilled water was determined at 760 nm with a UV-Visible spectrophotometer. TPC was expressed as milligram gallic acid equivalents/g dry-extract weight (mg GAE/g DW). A reagent blank was prepared using distilled water.The total phenolic content (TPC) was determined according to a modified Folin-Ciocalteu method .The total flavonoid content (TFC) was determined by the aluminium chloride colorimetric assay . After conditioning the column with 5 mL of methanol, the concentrated cell-free extract was loaded, and the analytes of interest were selectively eluted using 5 mL of methanol  was used for chemical screening of extracts of tested cyanobacterial cells. For this purpose, 500 mg of lyophilized cells of each strain was homogenised in a ceramic mortar and sonicated for 15 min in pure methanol (5 mL) using an Ultrasonic sonicator . Then, the samples were centrifuged for 3 min at 3000×g). The samples were then subjected to UHPLC-UV-ESI-MS analysis employing a Dionex UltiMate 3000 UHPLC system  coupled to a micrOTOF-QII mass spectrometer . Chromatographic separation was carried out on a Gemini-NX C18 column  thermostated at 30° and protected by a Gemini NX pre-column (4 × 3 mm). The mobile phase consisted of acetonitrile with 0.1% formic acid v/v (eluent A) and water containing 0.1% formic acid v/v (eluent B) delivered by a gradient at a flow rate 250 μL min−1. The program for gradient elution was 40% A/60% B (0 min), 100% A/0%B (15 min), 100% A/0% B (20 min), 40% A/60% B (25 min) and 40% A/60% B (30 min). The injection volume was 20 μl. The autosampler temperature was set at 4 °C, and the presence of polyphenols was monitored at 254 nm.The cell-free extracts were dried under a stream of nitrogen, redissolved in a mixture of 600 μL of methanol and 400 μL of formate buffer (4 g of ammonium formate and 300 μL of formic acid per litre of buffer) and centrifuged .In order to determine the highest concentration of hydroxylated chalcones that did not induce the sudden death of cyanobacterial cells  to obtain a final concentration of 20 mg/L of hydroxylated chalcone. The stability of the tested chalcones was positively verified in appropriate substrate control samples consisting of the solution of the tested flavonoid in sterile cultivation medium, whereas the culture controls were established as consisted of cyanobacterial cells in medium cultivated without the hydroxylated chalcone. All experiments, including the controls, were performed at least in triplicate and were incubated under adjusted conditions of light and temperature for 14 days. After this time, the cells and culture media were separated by filtration followed by centrifugation, and each repetition was extracted three times with 10 mL of ethyl acetate. Then, these extracts were combined, dried over anhydrous magnesium sulphate, and the solvent was removed using a vacuum evaporator. The remaining residue dissolved in 200 μL of methanol was subjected to analysis by high-performance planar chromatography (HPTLC) and liquid chromatography-mass spectrometry (LC-MS). The resulting biotransformation yield was established as the average of the values calculated based on the quantification of the products obtained with respect to the areas of relevant peaks.Screening  scale biotransformations of all hydroxylated chalcones were studied according to previously described methodology  and colours of the spots were recorded using CAMAG VisionCats software.The products of biotransformation were identified by HPTLC and UHPLC-UV-ESI-MS techniques similarly as it was reported for dihydrochalcone  using a CAMAG system and silica gel glass plates without a fluorescent indicator and were extracted with ethyl acetate, which was then evaporated under a stream of nitrogen. Then, the structures of the isolated products were determined and confirmed by mass spectrometry (MS) and nuclear magnetic resonance techniques: 1H NMR and 13C NMR, including COSY and HSQC correlation experiments. The NMR spectra were measured in DMSO-d6 using a Bruker UltraShield 400 MHz spectrometer with tetramethylsilane (TMS) as an internal reference.Eight repetitions of the same experiment were performed in 1000-mL Erlenmeyer flasks containing 250 mL of the cultures to scale-up the biotransformation process. After 14 days of incubation, the mixtures were extracted with ethyl acetate , dried over anhydrous MgSOS. platensis) and seven freshwater strains , were evaluated for the total content of phenolics (TPC) and flavonoids (TFC). The total amounts of phenolic compounds, including flavonoids, were determined spectrophotometrically, and the results showed that the phenolic composition differed significantly among the strains , with higher contents in N. moravica and S. aquatilis, which contained 7.90 ± 0.40 and 6.94 ± 0.35 mg/g QE, respectively. The lowest TFC among the tested strains was found in cells of M. glauca. The UHPLC-MS/MS analysis allowed us to determine the chemical nature of the phenolic compounds extracted from the cyanobacterial biomass. Using the set of standards to fortify appropriate samples, we confirmed the natural presence of phenolic acids, including protocatechuic, gallic, ferulic and sinapic acid, as well as the presence of flavonoids, including apigenin, genistein, naringenin, acacetin, biochanin A, quercetin and dihydroquercetin, in all the studied microalgal strains. Therefore, we surmised that the examined photoautotrophic microorganisms also possess a pool of adequate enzymes required for the biosynthesis and transformation of these compounds with prominent antioxidant properties.We tested the hypothesis that cyanobacterial cells naturally contain phenolic compounds and therefore possess the enzymatic apparatus enabling them to transform these compounds. Cell-free extracts of eight strains of cyanobacteria belonging to different genera, including one halophilic strain  affected cyanobacterial growth in a species-specific manner , 2″-hydroxychalcone (2) and 4″-hydroxychalcone (3), which all contain the three-carbon enone moiety 1,3-diphenyl-2-propen-1-one, in biocatalytic transformations conducted by the suppressed cyanobacteria was studied.Two products of transformation of 2′-hydroxychalcone were obtained in quantifiable amounts. According to concentration, the first was flavanone 4), whereas 2′-hydroxydihydrochalcone (5) was second  and strongly favours the formation of flavanone, since this substance was detected at a ratio of 20:1 over the initial 2′-hydroxychalcone. This conversion seems to be natural for 2′-hydroxychalcone because the presence of flavanone was even confirmed in the purchased standard.Independent of this chemical rearrangement, all tested  cyanobacterial species were able to biotransform 2′-hydroxychalcone during the 14 days of incubation to form the same product, albeit with moderate efficiency. The MS/MS spectra of the product, the mass of the molecular ion, its fragmentation pathway and the specific differences compared to the spectrum of 2′-hydroxychalcone clearly suggested the bio-reduction of the propenyl chain and thus the formation of 2′-hydroxydihydrochalcone. The structures of the MS/MS fragment ions in positive ion mode for 2′-hydroxychalcone and 2′-hydroxydihydrochalcone are presented in Fig. A. laxa after 14 days of bio-catalytic interactions and the adequate MS/MS spectra of the analysed compounds.The retention time, the high agreement with the pattern of specific fragmentation and the compatibility with spectroscopic data published in the literature confirmed that 2′-hydroxydihydrochalcone was obtained as the product of cyanobacterial biotransformation of 2′-hydroxychalcone  performed following preparative (PTLC) separation of the product undoubtedly confirmed the structures of flavanone and 2′-hydroxydihydrochalcone.The results of the scaled-up transformation experiments with 2′-hydroxychalcone fully confirmed those obtained at the analytical scale, both with respect to the chemical nature of the products and the dynamics and effectiveness of conversion. Moreover, the experiments performed on the preparative scale yielded sufficient amounts (15–30 mg) of the products for separation by PTLC and for fully credible confirmation of their structures by MS and NMR. The results of NMR experiments , the B-ring substituted 2″-hydroxychalcone (2) was stable in each of the microbial media used. Therefore, its transformation was the result of biocatalytic processes during the 14 days of cyanobacterial culture. Notably, these conversions, independent of the strain, were completely chemoselective, producing only the corresponding 2″-hydroxydihydrochalcone, as shown in Fig. The LC-MS profiles of the extracted fractions showed that the fraction containing the product after incubation was more homogenous than the corresponding fraction extracted from the substrate control Fig. . We supp1H NMR, 13C NMR and MS spectra of the product with those of the substrate fully confirmed the lack of the double C=C bond of the enone moiety and the presence of the unchanged carbonyl group C=O, thus verifying biocatalytic reduction of this bond as the only observed route of transformation by the examined cyanobacteria.Similar to substrate 1, to confirm the efficiency of conversion and to isolate the product in an amount permitting spectroscopic analysis, a 14-day biotransformation of substrate 2 was conducted on a larger scale. The comparison of the A. laxa, Anabaena sp., A. klebahnii, N. moravica, Ch. minutus, and S. aquatilis, converted this substrate into its dihydro-derivative in nearly 100% yield  resulted in the increased abundance of three metabolites: 4″-hydroxydihydrochalcone (7), 4″-hydroxy-1,3-diphenylpropan-1-ol 8) and 4″,x-dihydroxydihydrochalcone (9) , and 4″,x″-dihydroxydihydrochalcone (9)  cultures. The structures of all products were determined by MS, S. platensis, A. laxa, Ch. minutus and S. aquatilis) of the five cyanobacterial strains transformed the substrate in the most typical way (in this study)—by regiospecific hydrogenation of the enone subunit to form 4″-hydroxydihydrochalcone with > 99% yield  (DMSO-d6) δ (ppm)—7.81 , 7.39–7.62 , 7.10 , 5.68 , 3.26 , 2.84 ; 13C NMR (100 MHz) (DMSO-d6) δ (ppm)—191.63 (C-4), 161.11 (C-8a), 138.95 (C-1), 136.34 (C-7), 128.59 , 128.57 (C-4′), 126.67 (C-5), 126.36 , 121.51 (C-6), 120.68 (C-4a), 118.09 (C-8), 78.85 (C-2), 43.54 (C-3); ESI-MS m/z 225.0865 [M + H]+ ; the retention time 19.84 min.(1) (80 mg) in the A. laxa culture yielded 17 mg of compound 5; 1H NMR (400 MHz) (DMSO-d6) δ (ppm)—11.87 , 7.95 , 7.51 , 7.29 , 7.28 , 7.18 , 6.96 , 6.93 , 3.36 , 2.95 ; 13C NMR (100 MHz) (DMSO-d6) δ (ppm)—205.21 (C-1), 160.61 (C-2′), 141.03 (C-1″), 136.04 (C-4′), 130.79 (C-6′), 128.44 , 128.34 , 125.98 (C-4″), 120.38 (C-1′), 119.26 (C-5′), 117.65 (C-3′), 40.07 (C-3), 29.41 (C-2); ESI-MS m/z 227.0948 [M + H]+ ; the retention times 1–22.26, 4–21.91 min.As an example, 14-day transformation of 2′-hydroxychalcone (2) (40 mg) in the A. klebahnii culture yielded 38 mg of compound 6; 1H NMR (400 MHz) (DMSO-d6) δ (ppm)—9.39 , 7.97 , 7.63 , 7.52 , 7.12 , 7.01 , 6.78 , 6.70 , 3.27 , 2.86 ; 13C NMR (100 MHz) (DMSO-d6) δ (ppm)—199.64 (C-1), 155.21 (C-2″), 136.61 (C-1″), 133.13 (C-4′), 129.89 (C-6″), 128.75 , 127.93 , 127.14 (C-1′), 127.08 (C-4″), 118.91 (C-5″), 114.85 (C-3″), 38.10 (C-3), 24.84 (C-2); ESI-MS m/z 227.0948 [M + H]+ ; the retention times 2–17.31, 5–17.80 min.As an example, transformation of 2″-hydroxychalcone (3) (40 mg) in the Ch. minutus culture yielded 37 mg of compound 7; 1H NMR (400 MHz) (DMSO-d6) δ (ppm)—9.14 , 7.95 , 7.62 , 7.51 , 7.05 , 6.65 , 3.30 , 2.82 ; 13C NMR (100 MHz) (DMSO-d6) δ (ppm)—199.46 (C-1), 155.46 (C-4″), 136.50 (C-1″), 133.32 (C-4′), 131.15 (C-1′), 129.28 , 128.76 , 127.96 , 115.19 , 39.50 (C-3), 28.76 (C-2); ESI-MS m/z 227.0948 [M + H]+ ; the retention times 3–16.22, 6–16.35 min.As an example, transformation of 4″-hydroxychalcone (3) (80 mg) in the Anabaena sp. culture yielded 21 mg of compound 8; 1H NMR (400 MHz) (DMSO-d6) δ (ppm)—9.14 , 7.30 , 7.21 , 6.94 , 6.64 , 4.48 , 2.44 , 1.81 , 1.23 ; 13C NMR (100 MHz) (DMSO-d6) δ (ppm)—155.21 (C-4″), 146.40 (C-1″), 132.21 (C-1′), 129.06 , 128.00 , 126.64 (C-4′), 125.81 , 115.05 , 71.98 (C-1), 41.47 (C-3), 30.69 (C-2); ESI-MS m/z 229.1103 [M + H]+ ; the retention times 7–9.68 min.As an example, transformation of 4″-hydroxychalcone (3) (80 mg) in the Anabaena sp. culture yielded 14 mg of compound 9; 1H NMR (400 MHz) (DMSO-d6) δ (ppm)—9.23 ), 9.08 ), 7.96 , 7.62 , 7.52 , 7.30 , 7.08 , 6.68 , 3.27 , 2.86 ; 13C NMR (100 MHz) (DMSO-d6) δ (ppm)—200.72 (C-1), 158.38 (C-3″ or C-5″), 155.65 (C-4″), 136.52 (C-1″), 133.35 (C-4′), 131.18 (C-1′), 129.25 , 128.76 (C-2″or C-6″), 128.58 (C-2″or C-6″), 127.93 , 115.34 (C-3″or C-5″), 39.548 (C-3), 28.72 (C-2); ESI-MS m/z 243.0903 [M + H]+ ; the retention times 8–12.89 min.As an example, transformation of 4″-hydroxychalcone The production of a wide range of secondary metabolites is a common adaptation of cyanobacteria to compete successfully in different ecosystems. Because of their phototrophic lifestyle and constant exposure to high oxygen and radical stresses, these biota have a high capability for producing plentiful efficient protective chemicals against oxidative and radical stressors and exhibit adaptive responses to oxidative stresses by stimulating their intrinsic antioxidant defence systems based on mediator compounds, including polyphenolic substances  in 84% yield hydrogenation of the C=C bond in the three-carbon enone subunit of the hydroxylated chalcones were regiospecific and mainly proceeded by cyanobacterial transformation. This finding fully corresponds with the results of our previous study on the bio-reduction of chalcones  and thus may be considered a fully ‘green’ process. This rearrangement, however, was not the only side process with unexpected benefits. All examined strains turned out to have a tendency to purify, probably by utilisable absorption, the biotransformation media of small amounts of impurities that accompanied the substrates. In such circumstances, a simplified extraction of pure product together with the mild conditions of the process significantly supports the use of cyanobacterial biocatalysis in the hydrogenation of hydroxychalcones.A. laxa and S. aquatilis performed this transformation with high efficiencies similar to those of the corresponding conversion of 4″-hydroxychalcone. In the case of this compound, the routes and efficiency of biohydrogenation and hydroxylation were dependent on the presence and location of the hydroxyl substituent. This information strengthens our conclusions on the structural dependence of the biotransformations of chalcones and their derivatives by cyanobacteria. In this way, these results support the usefulness of these substances as molecular probes for more detailed studies of the enzymes responsible for these transformations. The existence of such enzymes in cyanobacterial cells and their catalytic mechanism has only been postulated (Fu et al. The results presented have important implications when compared to those obtained during biotransformations of chalcone published previously (Żyszka et al.