Carbon-ion radiotherapy (CIRT) is a promising treatment for locally advanced non-small-cell lung cancer, especially for patients with inoperable lung cancer. Although the incidence of CIRT-induced radiation pneumonitis (RP) ≥ grade 2 ranges from 2.5 to 9.9%, the association between CIRT-induced RP and dosimetric parameters is not clear. Herein, we identified prognostic factors associated with symptomatic RP after CIRT for patients with non-small-cell lung cancer.Clinical results of 65 patients treated with CIRT between 2000 and 2015 at the National Institute of Radiological Sciences were retrospectively analyzed. Clinical stage II B disease (TNM classification) was the most common stage among the patients (45%). The median radiation dose was 72 Gy (68–76) relative biological effectiveness (RBE) in 16 fractions. In cases involving metastatic lymph nodes, prophylactic irradiation of mediastinal lymph nodes was performed at a median dose of 49.5 Gy (RBE). The median follow-up was 22 months.5), V10, V20 and V30 were determined to be the significant predictive factors for ≥ grade 2 RP. The receiver operating characteristic (ROC) analysis revealed the cutoff values for %VC, FEV1, MLD, V5, V10, V20 and V30 for ≥ grade 2 RP, which were 86.9%, 1.16 L, 12.5 Gy (RBE), 28.8, 29.9, 20.1 and 15.0%, respectively. In addition, the multivariate analysis revealed that %VC <86.9%  and V30 ≥ 15%  were significant risk factors.Grade 2 and grade 3 RP occurred in 6 and 3 patients (9 and 5%), respectively. No patients developed grade 4 or 5 RP. Using univariate analysis, vital capacity as a percentage of predicted (%VC), forced expiratory volume in 1 s (FEV1), mean lung dose (MLD), volume of lung receiving ≥5 Gy (RBE) (VOur study demonstrated the risk factors for ≥ grade 2 RP after carbon-ion radiotherapy for patients with locally advanced lung cancer. Primary lung cancer is one of the most common cancers worldwide, and the most frequent type is non-small-cell lung cancer. Patients with locally advanced non-small-cell lung cancer undergo surgery, chemotherapy, and/or radiotherapy. For elderly patients or patients with serious comorbidities, radiotherapy is often the chosen treatment , 2. CarbRadiation pneumonitis (RP) is a radiation-induced pulmonary injury. RP generally occurs between 1 month and 1 year after radiotherapy. For photon radiotherapy in lung cancer patients, the incidence of symptomatic RP has ranged from 17 to 37% , 9. Symp13, V20, V30 and others [As for predictors associated with ≥ grade 2 RP, many studies regarding photon radiotherapy have already reported that RP risk depends on irradiated lung volume and dose. The predictive dosimetric parameters are mean lung dose (MLD), Vd others , 10. In d others –13. By cd others . As a reth edition, and the N stage was N0, N1 or N2 [This study was performed in accordance with the guidelines approved by the institutional review board of our institution. This study was a retrospective evaluation of all 141 patients who were treated with CIRT at the National Institute of Radiological Sciences (NIRS). Of these patients, 124 individuals who received CIRT between April 2000 and July 2015 were selected for the study. Eligibility criteria were as follows: 1) patients were diagnosed with non-small-cell carcinoma lung cancer by histology or cytology; 2) the clinical stage had been decided by imaging (computed tomography (CT), magnetic resonance imaging (MRI) or positron emission tomography (PET)); 3) the clinical stage ranged from II A to III B according to the Unio Internationalis Contra Cancrum TNM Classification 7The CT images for all patients, which were fixed by an individually tailored immobilization device , were taken in the supine or the prone position, with the respiratory system. The CT images were used to develop a 3-dimensional treatment plan using in-house HIPLAN software  until the end of 2011, and XiO-N  was used beginning in 2012.Carbon-ion beams were generated by the heavy ion medical accelerator in the Chiba (HIMAC) synchrotron and were delivered using the respiratory-gated irradiation system . IrradiaPrimary lesions and metastatic lymph nodes were contoured as gross tumor volume (GTV) on the CT images. The primary lesions with a 10-mm margin and any prophylactic lymph nodes were defined as the clinical target volume (CTV). For N0 cases, irradiation to prophylactic lymph nodes was omitted. Planning target volume (PTV) was defined as the CTV plus a 5-mm safety margin to account for position uncertainty. The dose was prescribed to the isocenter. The PTV was enclosed conformally at a minimum by the 95% isodose line with the prescribed dose. If there were metastatic lymph nodes, prophylactic irradiation to mediastinal lymph nodes was performed at a median dose of 49.5 Gy (RBE). Maximum dose constraints were as follows: main bronchus, 60 Gy (RBE); esophagus, 50 Gy (RBE); and spinal cord, 30 Gy (RBE).The dose calculation algorithm at our hospital was updated from the broad-beam calculation using in-house HIPLAN software to the pencil-beam calculation using the XiO-N software in 2012. Different dose calculation algorithms cause different dose distributions for the same treatment. For the current study, the treatment plans calculated by the HIPLAN software were converted to DICOM format and imported into XiO-N. Their dose distributions were recalculated with XiO-N.5, V10, V20, V30, V40 and V50) were calculated with XiO-N using the bilateral lung volume-GTV as the all-lung volume. Second, clinical parameters (patient characteristics and pulmonary function) and dosimetric parameters associated with ≥ grade 2 RP were identified using univariate analysis. Third, cutoff values of these parameters were determined using the receiver operating characteristic (ROC) curve. Finally, the predictors of ≥ grade 2 RP were identified using multivariate analysis.First, dosimetric parameters  for all statistical analyses.The median follow-up period for all 65 patients was 22.0 months (6.0–145.7 months). The patients consisted of 51 males and 14 females  . The ROC analysis was used to determine the cutoff values for %VC (86.9%) and FEV1 (1.16 L) for ≥ grade 2 RP. Patients were divided into two groups according to the cutoff values, and the actual incidences of ≥ grade 2 RP were 26.5% for %VC <86.9% and 0% for %VC ≥86.9% (p = 0.002). Similarly, the cutoff values were 6.8% for FEV1 ≥ 1.16 and 28.6% for FEV1 < 1.16 (p = 0.048).Univariate analysis of the average pulmonary function was performed to explore potential prognosticators for ≥ grade 2 RP Table . All dat5), V10, V20 and V30 were significant predictive factors for ≥ grade 2 RP. The ROC analysis was used to determine the cutoff values for MLD, V5, V10, V20 and V30 for ≥ grade 2 RP, which were 12.5 Gy (RBE), 28.8, 29.9, 20.1 and 15.0%, respectively. The actual incidences of ≥ grade 2 RP were 35.7% vs. 7.8%  vs. less than 12.5 Gy (RBE), respectively, p = 0.018), 24.1% vs. 5.6% , 26.1% vs. 7.1% , 21.9% vs. 5.9% , and 28.0% vs. 0% .The dose-volume parameters associated with ≥ grade 2 RP were analyzed Table . The resp values from the univariate analysis of the clinical, pulmonary functional or dosimetric factors (Table p = 0.0041) and V30  were significant prognosticators for ≥ grade 2 RP.Multivariate analysis was performed for ≥ grade 2 RP using the two variables with the most significant rs Table . The resCarbon-ion radiotherapy in patients with locally advanced non-small-cell lung cancer is a promising treatment, especially for patients with inoperable lung cancer . GeneralBarriger et al. reported that symptomatic RP developed at a median of 3.5 months (0.5-12 months) after the initiation of treatment . Our stu13, V20, V30 and others were identified [30. While V30 had already been identified as a prognostic factor for RP in photon therapy, in this study, %VC prior to treatment was identified as an independent prognostic factor for RP. This may arise from the fact that our study included patients with very low pulmonary function, and they tended to advance in RP severity. In fact, the average %VC of patients with ≥ grade 2 RP was 67.5%, and that of patients without ≥ grade 2 RP was 89.2%. When patients with a low %VC suffer from RP, dyspnea often appears or the saturation percentage of oxygen in arterial blood deteriorates easily because of low vital capacity. Consequently, they need to be treated with steroids or oxygenation . In the future, our findings may suggest that a dose constraint of V30 < 15% be imposed on treatment planning, and that when patients with %VC < 86.9% are treated, special care must be provided during follow-up.According to the reported studies of photon radiotherapy, several prognosticators such as concurrent chemotherapy, age, MLD, Ventified , 20, 21.Our study had 2 limitations. First, only 9 patients developed ≥ grade 2 RP because CIRT-induced RP is relatively rare . Second,30 less than 15% should be imposed. In the future, when clinical trials of concurrent chemotherapy and CIRT are planned, these results should be informative for treatment decisions.To further reduce the incidence of ≥ grade 2 RP, our results should influence treatment considerations. For example, for CIRT for lung cancer patients whose %VC is less than 86.9%, a dose constraint of V30 ≥ 15% (odds ratio = 6.1) as significant risk factors for ≥ grade 2 RP. Our study was a single institutional retrospective analysis, and further multi-institutional prospective studies are warranted.Our study identified %VC <86.9% (odds ratio = 13.7) and V


Coptotermes formosanus, identified the participants on one of these molecular exchanges. Molecular characterization of several termite-associated actinobacteria strains identified eleven known antimicrobial metabolites that may aid in protecting the C. formosanus colony from pathogenic fungal infections. One particular actinobacterial-derived small molecule, bafilomycin C1, elicited a strong chemical response from Trichoderma harzianum, a common soil saprophyte. Upon purification and structure elucidation, three major fungal metabolites were identified, t22-azaphilone, cryptenol, and homodimericin A. Both t22-azaphilone and homodimericin A are strongly upregulated, 123- and 38-fold, respectively, when exposed to bafilomycin C1, suggesting each play a role in defending T. harzianum from the toxic effect of bafilomycin C1.Bacteria and fungi in shared environments compete with one another for common substrates, and this competition typically involves microbially-produced small molecules. An investigation of one shared environmental niche, the carton material of the Formosan subterranean termite The online version of this article (10.1007/s10886-017-0900-6) contains supplementary material, which is available to authorized users. Throughout their evolutionary history insects have engaged in mutually beneficial associations with microbes, including bacteria belonging to the chemically rich class of actinobacteria , t22-azaphilone (2), and homodimericin A (3) in Trichoderma harzianum  on a Varian Oxford 600 MHz spectrometer equipped with a 5 mm AutoX HCN triple res inverse probe , and on a Varian VNMRS (Varian NMR System) 500 MHz spectrometer equipped with a broadband Cold Probe . LR-LCMS data were obtained using an Agilent 1200 series HPLC system equipped with a photo-diode array detector and a 6130 quadrupole mass spectrometer. HRESIMS was carried out using a Bruker Maxis Impact HD LC-q-TOF Mass Spectrometer equipped with an uHPLC system. HPLC purifications were carried out using Agilent 1100 or 1200 series HPLC systems (Agilent Technologies) equipped with a photo-diode array detector. All solvents were of HPLC quality.Optical rotations were measured on a JASCO P-2000 polarimeter, circular dichroism spectrum on a JASCO J-815, and UV spectra on an Amersham Biosciences Ultrospec 5300-pro UV/Visible spectrophotometer. NMR spectra were recorded with benzene or dimethylsufoxide as internal standards  were both isolated from C. formosanus colonies, though were not found in the same colonies. T. harzianum WC13 was isolated from a dying C. formosanus colony and as the microbial community from the carton nest also collapsed, T. harzianum became the dominant saprophyte in a case of ecological succession  using 2:1 dichloromethane (DCM)—methanol (MeOH), to afford a crude extract. Liquid cultures were centrifuged and the spent media was passed through a column of XAD-16 resin. The resin was subsequently rinsed with deionized water, and washed with MeOH to elute the organic material. The MeOH wash was dried using rotary evaporator, yielding a crude extract. All crude extracts were fractionated by reverse-phase (RP) solid phase extraction (SPE) using a stepwise gradient solvent system of decreasing polarity, starting from 75% water (H2O)/acetonitrile (ACN) to 100% DCM . All SPE fractions were tested for antimicrobial activity against Bacillus subtillis, a gram-positive bacterium and five soil fungi known to opportunistically be associated with termite nests , Phenyl-Hexyl, or Hydro-RP, resulting in the purification of 11 known secondary metabolites (Table The solid agar plates for the actinobacteria and Streptomyces sp. 4231 grown on solid agar (1/5 PDA) induced pigment formation in T. harzianum and exhibited activity against B. subtillis and A. nomius, and consequently was subjected to for further purification by RP HPLC. Further fractionation using a Phenomenex 4 μm Luna C18(2) semi-preparative column, with a gradient from 50% ACN + 0.1% formic acid (FA)/H2O + 0.1% FA to 100% ACN + 0.1% FA over 25 min, yielded pure nataxazole, bafilomycin C1 (34.5 mg/L), B1, and D. The isolated material was confirmed to be bafilomycin C1 (4) via HRLCMS co-injections with authentic bafilomycin C1 (Enzo Scientific) .The D SPE fraction (100% ACN) from Streptomyces sp. 4231 was combined with fresh media  and subsequently used to make CM plates . These plates were then inoculated with refrigerated glycerol spore stocks (in 25% glycerol) from T. harzianum WC13  appeared to be enriched with induced yellow pigments and was thus subjected to further purification by RP HPLC. The pigments were purified using a Phenomenex Kinetex 5 μm Biphenyl 100 Å semi-preparative column, with an isocratic gradient of 35% ACN + 0.1% FA/H2O + 0.1% FA, yielding 4.5 mg/L of culture media of cryptenol (1), 5.8 mg/L of culture media of t22-azaphilone (2), and 4.3 mg/L of culture media of homodimericin A (3). Induction levels for compounds 1, 2, and 3 were quantified from SPE fractions using extracted ion chromatograms (EIC) from LR-LCMS and determined to be 1.2-, 123-, and 38.7-fold increase in production, respectively, when grown in the presence of 30 μM bafilomycin C1 opposed to on 1/5 PDA for the same amount of time. The bafilomycin C1 containing solid agar plates were prepared by the addition of 1 mL of a 3 mM solution of bafilomycin C1 in DMSO to 1 L of freshly autoclaved 1/5 PDA media.The solid CM used in the induction studies was prepared following the previously reported method  was dissolved in 300 μL of ethanol (EtOH) and treated with a small amount of 10% Pd/C and H2 (g) at room temperature (rt) for 4 h. at which point the reaction mixture was filtered through a glass plug containing celite. The reaction product was dried under vaccum, then dissolved in 0.6 mL (3 × 0.2 mL rinses) of dry DCM and transfered to a 10 mL pear flask that was previously charged with a small amount of activated 3 Å molecular sieves. Subsquently, the pear flask was treated with p-toluenesulfonic acid monohydrate  in 0.2 mL of DCM and 2,2-dimethoxypropane (4 μL). The reaction mixture was stirred at room temperature for 16 h. At which point, 1 mL of saturated NaHCO3 (aq) was added and the mixture was extracted three times with DCM. The organic layer was dried using Na2SO4, filtered over celite, and dried under vacuum. The reaction product was then analyzed by 1H NMR using a 3 mm NMR tube (160 μL) in C6D6. The 1H NMR spectrum of the product was compared  to two standards – threo-5,6-dodecanediol and erythro-5,6-dodecanediol – which were synthesized following the above procedure, assigning the relative stereoconfiguration as erythro.An aliquot of cryptenol (Cryptenol (1): pale yellow oil; [α]26D − 16.8 ; UV (MeOH) λmax (log ε) 232 (4.11), 255 (4.20), 265 (4.27), 275 (4.20) nm; NMR  and 13C NMR  see Table +m/z 229.1196 . See supporting inforamtion figs. S1-S8.T22-azaphilone (2): amorphous yellow solid; [α]26D − 70.6 ; UV (MeOH) λmax (log ε) 208 (2.87), 282 (2.80), 355 (2.95) nm; NMR  7.21 , 6.53 , 5.86 , 5.64 , 5.50 , 5.28 , 5.16 , 4.24 , 2.49 , 2.30 , 1.53 , 1.53 , 1.08 ; 13C NMR  192.0 × 2, 171.4, 155.4, 152.5, 141.5, 136.4, 135.3, 130.5, 119.5, 114.2, 109.5, 107.7, 85.1, 65.7, 43.0, 22.3, 21.8, 17.7; HRESIMS [M + H]+m/z 345.1315 . See supporting information figs. S9-S16.B. subtilus assays, 100 μL of an overnight culture (in LB) was added to 10 mL of the soft agar, and was similarly vortexed and poured over a LB plate. Upon solidifying (~10 min), 1.5 μL of each fraction or pure compound resuspended in 50% ACN/H2O at 10 mg/mL, was spotted on the agar along with a solvent control, trying to minimize the surface diffusion. The inoculated assay plates were subsquently incubated at 30 °C for either 24 h or 72 h for the bacteria and fungi, respectively, at which point the zones of inhibition were measured and photgraphed. Determining the induction potential of fractions from Streptomyces sp. 4231 were assessed in a similar manner. See previous publication for detailed method  of the fungal spore stock (in 25% glycerol) was added to a 12 mL conical vial containing 10 mL of 1/5 PDA soft agar . The mixture was vortexed and then poured over a plate of 1/5 PDA . For the Streptomyces sp. 4231 and 2083B and Kitasatospora sp. 2404, adjusted to 1 × 108 cfu/mL . This solution was then further diluted to form six working stock solutions with the final concentrations of 1.5, 0.75, 0.375, 0.190, 0.094, and 0.047 mg/mL. A 0.5 mL aliquot of each of these working solutions was added to a 50 mL conical vial containing 14.5 mL of 1/5 PDA . The solutions were vortexed and dispensed in 1 mL aliquots per well into a 24-well sterile plate with each plate containing three wells per concentration. In addition to the six concentrations of t22-azpahilone, three controls (two wells each) were plated on every plate – DMSO control (0.5 mL of 10% aqueous DMSO), negative control (1/5 PDA), and positive control . Upon solidifiying, each well was inoculated with 1 μL of one of the bacterial suspension, Streptomyces and 2 Kitasatospora strains  exhibited both antibacterial and antifungal activity in spot-on-lawn assays, which agreed with previous literature on each of the secondary metabolites , a well-known antifungal agent, as the bacterial-derived small molecule responsible for inducing pigment formation, but two structural analogs, bafilomycin B1 and D, exhibited no appreciable ability to elicit this response .One particularly interesting binary competition assay revealed a chemical interaction between T. harzianum WC13 on solid media that was conditioned with either sterilized spent liquid media from a 14-day culture of Streptomyces sp. 4231 or with purified bafilomycin C1 (30 μM). In both cases, the growth of T. harzianum WC13 was significantly stunted, with almost no visible mycelia formation or sporulation, though robust pigmentation of the agar was observed after just four days. Extraction of the solid agar plates and subsequent rounds of RP HPLC purification yielded the isolation of three natural products, cryptenol (1), t22-azaphilone (2), and homodimericin A (3). Both t22-azaphilone (2) and homodimercin A (3) have been previously reported  . Subsquently, all three metabolites were found to be consitutively expressed in T. harzianum T22 (ATCC 20847), a strain that is commonly used as a biocontrol agent  was isolated as a pale-yellow oil having a molecular formula C13H18O2 based on ESI-QTOF data and requiring five degrees of unsaturation . The 1H and 13C NMR data suggested the pressence of five olefins and are responsible for all degrees of unsaturation with 10 carbon resonances between 117 and 138 ppm , and 11 proton resonances between 5.0 and 6.4 ppm . Planar structure elucidation was greatly facilitated by the fact that cryptenol contains only one spin-system that could easily be pieced together using gH2BC correlations. Further analysis of the complete 2D NMR dataset [gHSQC, gCOSY, gc2HMBC, and gH2BC , 15.3 (H-4 and H-5), 15.0 (H-8 and H-9), and 14.9 Hz (H-10 and H-11) are all indicative of E configurations for the four internal olefins. The relative configuration of the diol at C-6 and C-7 was determined by preparing the acetonide analog and comparing proton chemical shifts of H-6/H-7, as well as the introduced dimethyl moiety in the acetonide, to two synthetic standards. The acetonide analog of cryptenol was prepared by first reducing all of the olefins with 10% Pd/C and H2 (g), and then reacting with 2,2-dimethoxypropane. 1H NMR analsysis of the derivatived natural product led to the relative assingment of erythro .The configuration of the four internal olefins of 1), homodimericin A (3), and t22-azaphilone (2) were each evaluated for antimicrobial activity against a range of ecologically relevant microbes, including eight strains of termite-associated actinobacteria, and six soil fungi . T22-azaphilone exhibited moderate antibacterial activity against every actinobacteria strain tested, including Streptomyces sp. 4231, the bafilomycin producer with MICs ranging from 5 to 25 μg/mL . No toxicity against any of the fungal pathogens was observed. Cryptenol and homodimericin A were found to be inactive against all microbes screened up to a maximum of 30 μg per spot in a spot-on-lawn assay .Cryptenol , t22-azaphilone (2), and cryptenol (1) by T. harzianum WC13. The prodigious upregulation of homodimericin A and t22-azaphilone production suggests that both have an ecological role in defending T. harzianum from the effects of bafilomycin C1. The bafilomycins are known potent and specific inhibitors of fungal vacuolar-type H+-ATPases, which when inhibited causes increases in both cellular pH and oxidative stress , repression of antibiotic biosynthesis in the producing organism, active efflux of the antibiotic, and/or production of antibiotics to neutralize the chemical defenses


Besides, for MSH-1.75, the as-prepared sample with negative charge exhibits ca. 36% higher adsorption quantity compared to the sample at the zero point of charge (pHZPC). Furthermore, magnesium silicate hydrate material with Si/Mg feeding ratio = 1.75 demonstrates the promising removal efficiency of beyond 98% for methylene blue in 10 min, and the maximum adsorption capacity of 374 mg·g−1 calculated from the Langmuir isotherm model.Magnesium silicate as a high-performance adsorption material has attracted increasing attention for the removal of organic dye pollution. Here, we prepared a series of magnesium silicate hydrates (MSH) in a hydrothermal route, and carefully investigated the corresponding adsorption behavior towards methylene blue (MB) as well as the effect of surface charge on adsorption capacity. The results show that surface charge plays a key role in the adsorption performance of MSH for MB, a negative surface charge density follows the increase of Si/Mg feeding ratio from 1.00 to 1.75, and furthermore the higher negative charge favors the improvement of the adsorption capacity. Among four investigated samples , MSH-1.75 has the highest negative surface charge and shows the largest adsorption capacity for MB. For example, the equilibrium adsorption quantity is 307 mg·g Various synthetic dyes have been developed and have played an important role in modern life. During the process of production and application, however, ca. 10% of synthetic dyes are inevitably released to the environment as the main components in effluent wastewaters . The trend in water and wastewater treatment is continuously moving towards more sustainable materials and technologies . RecentlIn this work, we fabricated a series of magnesium silicate hydrate (MSH) adsorbents with different surface charge density by adjusting the Si/Mg feeding ratio in a hydrothermal route, and carefully investigated the corresponding adsorption behavior for methylene blue as well as the relationship between surface charge density and adsorption capacity.3)2·6H2O), sodium silicate nonahydrate (Na2SiO3·9H2O), hydrochloric acid , sodium chloride (NaCl) and methylene blue (MB) were all analytical grade and directly used as received from ALADDIN . The water used in this work was deionized water.The magnesium nitrate hexahydrate (Mg(NO3)2·6H2O and 17.1 g (70 mmol) Na2SiO3·9H2O were separately dissolved in 200 mL of deionized water at 25 °C to produce 0.2 mol·L−1 Mg(NO3)2 solution and 0.35 mol·L−1 Na2SiO3 solution. Both of two solutions were mixed under vigorous stirring for 3 min. Subsequently, the resulting white slurry was transferred into an autoclave (1 L) and then kept at 190 °C for 12 h at the stirring speed of 300 rpm. Finally, the sample was collected after being washed by water, centrifuged, and dried at 60 °C for 12 h. Another three samples were synthesized with the similar procedure and same amount of Mg(NO3)2·6H2O but different Si/Mg feeding ratios. Here, four samples were individually obtained and named as: MSH-1.00 for Si/Mg ratio = 1.00, MSH-1.25 for Si/Mg ratio = 1.25, MSH-1.50 for Si/Mg ratio = 1.50, and MSH-1.75 for Si/Mg ratio = 1.75.Four magnesium silicate samples with different surface charge densities were individually prepared in a hydrothermal route by adjusting the Si/Mg feeding ratio. As for the sample with Si/Mg ratio = 1.75, typically, 10.3 g (40 mmol) Mg. Particle size and size distribution were examined by Malvern Mastersizer 2000-Hydro 2000MU particle size analyzer  by dispersing the powder in water. Elemental analysis was performed using a Shimadzu ICPS-75000  inductively coupled plasma atomic emission spectrometer (ICP-AES). Morphologies were recorded with a Hitachi S-4700 scanning electron microscope (SEM)  at 30 kV and a JEOL JEM-2100F transmission electron microscope (TEM) . Elemental distribution was tested by a EDAX-GENESIS 60 energy-dispersive X-ray spectroscopy (EDX)  coupled with SEM. Specific surface area and pore property were analyzed by low-temperature N2 adsorption-desorption method on Micromeritics ASAP 2390  volumetric adsorption analyzer. Considering the wide range of pore sizes from 0.5~200 nm, the density functional theory (DFT) was used to calculate pore size distributions from adsorption isotherms based on given intermolecular fluid-fluid and fluid-solid potentials [−1. Crystal structure was determined by X-ray Diffraction (XRD) on a Rigaku UItima III X-ray powder diffractometer  in the range of 3°~70°/2θ with 10° mintentials . The pH −1 MB solution was individually added into these flasks to produce a suspension containing 200 mg·L−1 MB. These flasks were sealed using plastic wrap, put in the thermostatic shaker, and shaken for 0~120 min at 25 °C (160 rpm). After a certain time, the suspension was separated by centrifugation , and the rest concentration of MB after adsorption was estimated by a UV/Vis spectrophotometer at λ = 664 nm [tq of the sample at a certain time t was calculated using the following equation [tq (mg·g−1) is the adsorption quantity of the sample for MB at the contact time t (min), C0 (mg·L−1) is the initial concentrations of MB, tC (mg·L−1) is the rest concentration of MB at the contact time t (min), V (L) and m (g) are the volume of MB solution and the mass of adsorbent sample, respectively.The surface charge of the samples was estimated according to the method of potentiometric titration . To furtequation :(1)qt=. Also, the adsorption isotherm experiments were performed in 100 mL flasks where 32.5 mg of the sample was dispersed in 50 mL, 0~300 mg·L−1 MB solution for 120 min. The concentration of MB and the adsorption quantities were determined using a UV/Vis spectrophotometer at λ = 664 nm and calculated following Equation (1). To estimate the regeneration properties, the MSH-1.75 sample after adsorption of MB was collected after being centrifugated, dried at 60 °C, and calcined at 600 °C for 4 h with a rate of 5 °C/min in air [−1 MB solution at 25 °C for 120 min.The adsorption kinetic experiments were carried out in 100 mL flasks with the dispersion of 32.5 mg of the prepared samples in 50 mL of 200 mg·Ln in air , then thAll these experiments were carried out three times, and the average values were used to prepare the graphs in this work.50) is 480, 530, 606, and 615 nm for MSH-1.00, MSH-1.25, MSH-1.50, and MSH-1.75, respectively. 2 adsorption/desorption isotherm and the pore-size distribution of four samples and all the Brunauer-Emmett-Teller (BET) data are listed in 2·g−1 for MSH-1.00 to 283 m2·g−1 for MSH-1.75.−1 are individually attributed to the Mg–O and Si–O stretching vibration while the bands at 1384, 1645 and 3450 cm−1 are due to the O–H vibrations [−1 and the C–N stretching vibrations at 1323 and 1384 cm−1 [−1, in comparison with MB. No similar change is observed for the spectrum of the mixture sample with MSH-1.75 and MB. These results confirm the existence of possible electrostatic interactions between MSH and MB after adsorption.brations . After a384 cm−1  shift toZPC) is 7.86, 7.41, 7.27 and 7.30 for MSH-1.00, -1.25, -1.50, and -1.75, respectively. When the pH value is higher than pHZPC, all the MSH samples exhibit negative charge, resulting from the loss of the protons in the surface silanol groups [Generally, surface charge plays a key role during the adsorption process, especially for the electrostatic attraction mechanism. l groups , and fur−1 respectively, which are much higher than 227 mg·g−1 for MSH-1.00. That is to say, the equilibrium adsorption quantity of MSH-1.75 is 0.35 times higher than that of MSH-1.00 although the surface area of MSH-1.00 (597 m2/g) is 1.1 times higher than MSH-1.75 (283 m2/g). −1 at pH = 10.12, which is much higher than 221 mg·g−1 at pH = 7.39 and 154 mg·g−1 at pH = 4.05. As described in ZPC of MSH-1.75 is 7.30, which is lower that pH = 10.12 and higher than pH = 4.05, and very close to the pH value of 7.39 in this experiment. That is to say, MSH-1.75 with negative surface charge density of −1.16 C·m−2 exhibits ca. 36% higher adsorption capacity related to the same sample at the zero point of charge. The results further provide another proof for the effect of the surface charge on the adsorption capacity.C0 = 200 mg·L−1. As shown in eq (mg·g−1) and tq (mg·g−1) are the adsorbed quantity for MB at equilibrium and at contact time t (min), respectively; k1 (min−1) and k2 (g·mg−1·min−1) is the rate constant for the pseudo-first-order kinetic model and the pseudo-first-order kinetic model. Through the linear relationship, the parameters such as k1, k2 and eq can be determined from the slope and intercept.R2 value, compared to the pseudo-first-order model in qe.cal values from the pseudo-second-order model are very close to the experimental data qe,exp.For MSH-1.75, the adsorption kinetic curves match the pseudo-second-order model well, with a better linear relationship in eq (mg·g−1) and mq (mg·g−1) is equilibrium adsorption quantity and the maximum adsorption capacity, respectively; eC is the equilibrium concentration of MB in solution; LK is the equilibrium constant (L·mg−1); FK is the Freundlich isotherm constant and n presents the adsorption tendency. Through the linear relationship between eC/eq and eC, logeq and logeC, the other parameters: mq, LK, FK and n, can be calculated from the slope and intercept. R2 value as summarized in mq values of MSH-1.50 and MSH-1.75 based on the Langmuir adsorption model are 351 and 374 mg·g−1 respectively, and the mq value of 374 mg·g−1 for MSH-1.75 is the highest one among those reported in the literature as summarized in mq value of 268 mg·g−1 and remains ca. 72% of mq, suggesting promising reusability. Certainly, it still has large space to enhance reusability of the MSH by building a polymer network or porous substrate-supported composites for practical applications, although the reusability efficiency of 72% is higher than that of 60% for mesoporous magnesium silicate in ref [One observes that the adsorption behavior of both MSH-1.50 and MSH-1.75 samples matches the Langmuir adsorption model much better than the Freundlich isotherm with the relatively higher e in ref . The cor−2 and exhibits ca. 36% higher adsorption capacity related to the same sample at the zero point of charge. With the largest adsorption capacity of 374 mg·g−1 calculated from the Langmuir isotherm, furthermore, the MSH materials show good regeneration ability with ca. 72% of reusability efficiency. Therefore, this is an available route to improve the removal capacity of magnesium silicate hydrate for cationic dyes by increasing the corresponding negative surface charge density. Furthermore, magnesium silicate hydrate is one kind of promising material for removing dye pollution from wastewater.In this work, we successfully fabricated magnesium silicate hydrate (MSH) adsorbents with different surface charge by adjusting the Si/Mg feeding ratios, and examined the relationship between surface charge density and adsorption behavior for methylene blue (MB) in aqueous solution. The Si/Mg feeding ratios significantly modify the surface charge density of MSH, and then influence the adsorption capacity for MB in aqueous solution. Among the investigated four samples, MSH-1.75 has the highest negative surface charge density of −1.16 C·m


N) and a receptor-binding domain (HC). Here we report the crystal structure of HC/FA, complementing an existing structure through the modelling of a previously unresolved loop which is important for receptor-binding. Our HC/FA structure also contains a previously unidentified disulphide bond, which we have also observed in one of two crystal forms of HC/A1. This may have implications for receptor-binding and future recombinant toxin production.The binding specificity of botulinum neurotoxins (BoNTs) is primarily a consequence of their ability to bind to multiple receptors at the same time. BoNTs consist of three distinct domains, a metalloprotease light chain (LC), a translocation domain (H C) domain and a translocation (HN) domain  protein which consequently inhibits vesicle release. Many immunologically distinct BoNTs have been discovered over the years—serotypes /A through to /G; although, only serotypes /A, /B, /E and /F have been reported to cause botulism in humans (Botulinum neurotoxin (BoNT) is the active agent that causes the deadly condition botulism. It is expressed as a single polypeptide chain of approximately 150 kDa and is cleaved post-translationally to yield an active di-chain molecule held together by a single disulphide bond. The smaller 50 kDa light chain (LC) possesses a single zinc-endopeptidase domain whereas the larger 100 kDa heavy chain is comprised of two domains—a target cell receptor binding (H) domain . BoNTs a) domain  which al) domain . The HN n humans . Each sen humans .C domain is responsible for targeting the toxin to a specific cell type and the specific receptors involved have been identified for most serotypes. For example, serotype /A binds to a glycosylated luminal domain of the synaptic vesicle 2 (SV2) protein, preferentially to the C isoform (SV2C) , as well as electrostatic interactions between charged surfaces (The BoNT Hm (SV2C) , as wellm (SV2C) . X-ray csurfaces . This issurfaces .BoNTs have been exploited for therapeutic use in many neurological indications such as dystonia and overactive bladder . More reClostridium botulinum, IBCA10-7060, was reported to produce BoNT/B2 and a previously unknown BoNT serotype—“BoNT/H”  differing by the presence of this disulphide bond.Recently, a new bivalent strain of “BoNT/H” . This no BoNT/A1 . The cry BoNT/A1 . Here, wC/A1) and BoNT/FA (HC/FA) were provided by Ipsen Bioinnovation Ltd. Each was cloned into the pJ401 expression vector  with an N-terminal 6× histidine tag using standard molecular biology techniques and confirmed by sequencing .The genes encoding the binding domain of BoNT/A1 (H6-HC/A1 and His6-HC/FA were expressed in E. coli strain BL21(DE3)  using the following protocol. A glycerol stock was used to inoculate 100 mL TB medium containing 50 µg/mL kanamycin, and grown at 37 °C for 16 h. From this, 10 mL of culture was used to inoculate 1 L TB medium containing 50 µg/mL kanamycin and grown at 37 °C to an OD600 of 0.6. The temperature was reduced to 16 °C and the cells grown to an OD600 of 1.0 at which point 0.5 mM isopropyl-d-1-thiogalactopyranoside (IPTG) was added to induce expression. Cells were grown for an additional 16 h at 16 °C and then harvested by centrifugation at 4,000× g for 30 min.His6-HC/A1 and His6-HC/FA were further purified by SEC using a Superdex 200 16/60 column  and 0.5 M NaCl, 50 mM Tris-HCl pH 7.4. Purified samples were concentrated to 10 mg/mL using a 10 kDa MWCO centrifugal filter .Expression cell pastes were resuspended in 0.5 M NaCl, 50 mM Tris–HCl pH 7.4, 20 mM imidazole and lysed using a Constant Systems cell disruptor at 20 kPSI. Cell debris were removed by centrifugation at 80,000× g for 30 min and the supernatant was filtered through a 0.22 µm membrane syringe filter. The clarified lysate was loaded onto a 5 mL HisTrap column , washed, and the target protein eluted with 0.5 M imidazole. His6-HC/FA and His6-HC/A1 were grown at 16 °C using a 1:1 ratio of protein solution (10 mg/mL) to well solution using the sitting-drop vapour-diffusion method—4 M sodium formate, 0.1 M sodium acetate pH 5.5 for the former, and 0.1 M MIB pH 4.0, 25% w/v PEG 1500 for the latter. Crystals were soaked in cryoprotectant  before vitrification in liquid nitrogen. Complete datasets were collected on beamline I03 and I04, respectively, at the Diamond Light Source . Diffraction images were processed using DIALS . A high-multiplicity dataset was collected containing 360 degrees of data over 3,600 images. No significant radiation damage was observed over the course of data collection and thus all data were used. The CC1/2 value for the outer shell was 0.832, indicating there was still very usable data at this resolution (C/FA is shown in 2 and 26.84 Å2 respectively) with a corresponding overall Wilson B-factor of 24.55 Å2. As expected, our structure is highly similar to PDB: 5V38 with an RMSD value for combinations of chains between structures ranges from 0.54 Å to 0.36 Å  that diffracted to a resolution of 1.45 Å. No disulphide bond was observed in this structure  that diffracted to 1.8 Å resolution. Inspection of this structure  from BoNT/FA and BoNT/A1 are reported here. The former complements a recently published structure (C/A1 structures differed from one another by the presence or absence of a disulphide bond. This bond was also observed in our structure of HC/FA. Considering their location near the ganglioside-binding pocket, and conservation across BoNT subtypes, the redox status of these conserved cysteines may have implications in BoNT stability and manufacture. Botulinum neurotoxins are used therapeutically for many indications and their production is currently from the native host Clostridium botulinum. However, considering the safety implications and the advent of engineered BoNT derivatives, such as TSIs, production from a recombinant source would be highly desirable. The importance of these two cysteine residues is being further investigated.The high-resolution crystal structures of the binding domains (Htructure  and reso10.7717/peerj.4552/supp-1Supplemental Information 1Click here for additional data file.


In vitro antimalarial activity and cytotoxicity of novel derivatives were evaluated and fundamental information to facilitate the discovery of more promising antimalarials was obtained.Divergent synthesis of antimalarial troponoids, including naturally occurring compounds, some of which were identified and isolated by our group, has been achieved utilizing the total synthetic route of puberulic acid. Structure-activity relationships of natural products and simple troponoids inspired us to explore more detailed properties of this class of compounds. Access to new derivatives was facilitated through intermediate compounds generated during the total synthesis of puberulic acid by a stepwise oxidation-aromatization sequence to provide 7-hydroxytropolones and bromination for conversion of the carboxylic acid moiety. The first total synthesis of viticolin A, as well as the synthesis of different methyl-substituted derivatives, has also been achieved.  In spite of various traditional antimalarial drugs such as quinine2, chloroquine3 and sulfadoxine/pyrimethamine4 having been developed for malaria, plus current artemisinin-based combination therapy (ACT)5, drug-resistant parasites and multi-drug resistance against ACT have been rapidly and continually emerging6. Development of antimalarial drugs with novel structures and new modes of action is, therefore, incessantly and urgently required.As one of the most critical infectious diseases in the world, particularly in sub-Saharan Africa, malaria remains a serious global health problem. The World Health Organization (WHO) estimated that there were 212 million clinical cases of malaria and 429 000 deaths in 2015 and has been warning that it puts 3.2 billion people, about half of the world’s population, at risk1)7, stipitatic acid (2)8 and viticolins A and B  as novel natural products have been isolated from a culture broth of Penicillium viticola9 FKI-4410 through our screening system and found to have promising antimalarial activity  parasite strain (IC50 = 0.050 µM), as well as in vivo efficacy with 69% inhibition for a dose of 2 mg/kg × 4 through subcutaneous (s.c.) administration in 4-day suppressive test using a P. berghei-infected mouse model12. However, 1 exhibits toxicity in vivo, four out of five mice dying by day 3, after a s.c. dose of 5 mg/kg × 2 (day 0 and 1). While structually simple compounds such as tropone (5), tropolone (6) and hinokitiol (8), and natural 2 and 3 showed weaker activity than that of 1, 7-hydroxytropolone (7)13 was much more potent, exhibiting a >18-fold stronger IC50 value of 6.44 µM than that of 5. This observation suggested that the presence of more than three contiguous oxygen atoms in a compound might significantly affect antimalarial activity. These results stimulated us to undertake a structure-activity relationship (SAR) study based on the establishment of a total synthetic route, aiming to create new antimalarial candidates which retained potency but which were non-toxic. Furthermore, we expected that these compounds’ properties, especially low molecular weight and simple planar structures, could be invaluable for antimalarial drug leads with respect to ease of supply14, enabled by efficient and practical synthesis. Herein, we report the divergent synthesis of several related troponoids, including natural products, via utilization of the established total synthetic route of 115, and biological evaluation of their in vitro antimalarial activity and cytotoxicity.Puberulic acid 7, stipit1)17, we proposed an original divergent synthetic route to efficiently produce various analogues and novel derivatives to help clarify the SAR -galactose (13) containing the C-C and C-O bonds in the backbone of the target compound by a mild Barbier type addition of allyl chloride19. With an efficient supply of compounds established, we overlapped the characteristic highly-oxygenated structure of 1, with a sugar as one of the cheapest and unlimited natural sources, and chose 13 as a starting material. Although sugars are generally used in syntheses of complex molecules in the chiral pool method from 3-dimensional information21, in this synthesis we focused on structural information to utilize the sugar as a “framework source”. This should lead to the target compound using minimal bond-forming reactions23.In contrast with synthetic strategy for puberulic acid  under high dilution condition (0.01 M), afforded the cyclic compound 17 in excellent yield. Using the major diastereomer (6S)-17, deprotection of the acetonide group under an acidic condition produced the desired tetraol 18  or undergo oxidative cleavage (e.g. DDQ or CAN), directly providing 1 or the corresponding aldehyde. However, efforts were unsuccessful (see Supplementary Table 27) were attempted with expectations that the PMB ether moiety would remain. In some of these reaction conditions, the loss of two molecules of the hydroxyl group from the desired compound 19 was observed, as determined by mass spectrometry analysis, suggesting that aromatization by β-elimination of oxygen functions occurred when the allylic hydroxyl group on the 7-membered ring was oxidized. Although we considered that stepwise oxidation of diol 17 might be beneficial to obtain clues about multi-oxidation, this transformation was unsuccessful under any conditions  in 8 steps, with 52% overall yield  could not be monitored and purified by silica gel, it was found that these compounds could be monitored using LC-UV analysis and purified by reverse-phase chromatography, enabling reliable supply of this class of compounds. We envisioned that the methylated compounds would be valuable to clarify more detailed SAR induced by the information from natural products 3 and 4, and exploited them for further derivatization. Hydrolysis of the methyl ester in both 25 and 26 afforded two methoxy carboxylic acids 27 and 28, respectively. Interestingly, removal of the acetonide group of 28 proceeded with 80% TFA at 120 °C in a sealed tube to provide iso-viticolin A (29) in 94% yield over 2 steps. However, we failed to obtain the natural product, viticolin A (3), because the methyl group of its regioisomer 27 was removed faster than the acetonide group under any acidic conditions. We therefore decided to produce natural products 3 and 4 from puberulic acid (1) as the starting point. Treatment of 1 with MeOH in the presence of TsOH, followed by careful methylation with TMSCHN2, afforded the mono-methylated methylester 30 in 47% yield as a single regioisomer, along with the recovered starting material. We supposed that the inner hydroxyl group of 1 is partly anionic and thus more nucleophilic than the two outer hydroxyl groups due to delocalization of the tropolone framework. It, hence, selectively underwent the methylation allowing access to the natural product. Finally, total synthesis of 3 was accomplished by hydrolysis of 30 in 73% yield. On the other hand, direct methylation of 1 using two equivalents of TMSCHN2 gave a complex mixture of the starting material, mono-, di-, and possibly tri-methylated products, as well as stepwise methylation of 30. In these reactions, viticolin B was detected by LC-UV analysis and1H NMR but no selectivity was observed, and thus the given mixture was unable to be separated even by HPLC.We synthesized compounds 1, 2) and simple troponoids (5–8)11. Since the SAR of 1, 2, 6 and 7 indicated that more than three contiguous oxygen atoms on the tropolone ring seem to increase antimalarial activity, we planned to synthesize 7-hydroxytropolone-type derivatives and envisioned that stepwise oxidation of a total synthetic intermediate might allow access to different substituted-type derivatives from 2.With respect to bioactivity, we first intended to explore the SAR of hydroxyl groups on the tropolone ring which might possibly intensify previously reported bioactivity of the natural compounds -17 was converted from the acetonide group to the methyl groups in a 2-step manipulation, methylation of the hydroxyl groups and removal of the acetonide group under an acidic condition, providing (6S)-31 in 99% yield. Finally, oxidation of the vicinal diol with IBX proceeded smoothly to afford the diketone (6S)-32, which was then treated with DBU occurring aromatization by β-elimination to provide the 7-methoxytropolone 33. For purification, the hydroxyl group of 33 was protected with a methyl group by TMSCHN2 to give tropones 34 and 35 as a 1:1.4 separable mixture of the regioisomers in 94% yield over 3 steps. The structure of these compounds was determined by NOE analysis. Using the minor isomer 34, simultaneous removal of the methyl groups and bromination of the PMB-ether moiety was found to occur following treatment with 33% HBr/AcOH in the presence of H2O, providing the bromide 36, which was then converted to the corresponding alcohol 37 in 94% yield, or methyl ether 38 in 80% yield, by working-up with H2O or MeOH, respectively. Conversely, when the major isomer 35 was treated with DDQ, concurrent deprotection of the PMB group and oxidation occurred to provide the aldehyde 39 in a single step. Subsequent oxidation to the carboxylic acid under the Pinnick condition, followed by removal of the methyl groups with HBr/AcOH, afforded the desired iso-stipitatic acid (40) in a quantitative yield over 2 steps. In this way, we successfully prepared three non-natural 7-hydroxytropolones.With the observation of stepwise oxidation of the total synthetic intermediate, diol ide Fig. . As aforin vitro antimalarial activity against the Plasmodium falciparum K1 (chloroquine-resistant) parasite strain and for cytotoxicity against a human lung fibroblast cell line MRC-5 (Table 1) showed a similar IC50 value (0.044 µM) to that of natural 1. All intermediates possessing the acetonide group, such as 23–28, did not show any antiparasitic activity, whereas 7-hydroxytropolones, such as 37, 38, and 40, were active , indicating that free hydroxyl groups on the 7-membered ring seem to bestow potency. Although introduction of the methylene hydroxyl group to the C-4 position of 7 did not affect the activity, IC50 values of compounds 38 and 40 were approximately 2-fold better than those of 7 and 37. However, compounds 37 and 38 possessing the methyleneoxy group showed strong cytotoxicity , suggesting that substituents at the C-4 position might critically affect cytotoxicity. Remarkably, comparing 40 with stipitatic acid (2), the substitution pattern on the tropolone ring seems to be very important. In addition, from the SAR of 7-hydroxytropolones 37, 38 and 40, it was found that the carboxyl group is not indispensable for activity. Iso-viticolin A (29) showed approximately 100-fold stronger activity than viticolin A (3). The SAR among natural products 3, 4 and 29 suggests that three contiguous oxygen atoms with no substitutions increase antiparasitic activity.Synthesized derivatives, including intermediate compounds, were evaluated for Since the SAR information suggested that tetra-substituted tropones with at least three consecutive free-hydroxyl groups and a carbonyl group might have more potent antiparasitic activity, in addition to the convertible carboxylic acid moiety, we next planned to synthesize 6,7-dihydroxytropolones via conversion of the carboxyl group.1, we envisioned that the same method to synthesize derivatives 30 and 31 could be applicable for transformation of the carboxylic acid moiety . ODS column chromatography was carried out with Sep-Pak® Plus C18 Short Cartridge  or CHROMATOREX® . 1H NMR spectra were recorded on JEOL JNM-ECA-500 (500 MHz) and 13C NMR spectra were recorded on JEOL JNM-ECA-500 (125 MHz). Chemical shifts are expressed in ppm downfield from the internal solvent peaks for CD3OD  and J values are given in Hertz. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, and br = broad. High- and Low-resolution mass spectra were measured on JEOL JMS-AX505 HA, JEOL JMS-700 MStation and JEOL JMS-T100LP.Unless otherwise noted, reagents and solvents were purchased at the highest commercial quality and used without further purification. LC-UV analysis was carried out with Agilent 1100 system  under the following condition; column, Symmetry C18 ; UV detection, 210 nm; flow rate, 0.2 mL/min; mobile phase, MeCN-H30  in THF (1.55 mL) and H2O (1.55 mL) was added K2CO3  at room temperature. After being stirred at 50 °C for 1 d, H2O (20 mL) was added to the reaction mixture. The resulting mixture was extracted with CHCl3 (20 mL × 2). The aqueous layer was acidified by 1 M HCl, and extracted with CHCl3:i-PrOH = 10:1 (40 mL × 4). The combined organic layer was dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by Sep-pak® Plus C18 Short Cartridge to afford 3  as a yellow solid.To a solution of 1H NMR  δ7.82 , 7.72 , 3.95 ; 13C NMR  δ172.0, 168.7, 163.3, 160.6, 150.0, 134.4, 123.4, 112.0, 59.6; HRMS-ESI (m/z) [M–H]− calcd for C9H7O6 211.0243, found 211.0234; mp 227 °C.26  in MeOH (0.40 mL), H2O (0.40 mL), and THF (0.40 mL) was added K2CO3  at room temperature. After being stirred at room temperature for 5 h, H2O (20 mL) was added to the reaction mixture. The resulting mixture was extracted with CHCl3 (20 mL) The aqueous layer was acidified by 1 M HCl, and extracted with CHCl3:i-PrOH = 10:1 (40 mL × 3). The combined organic layer was dried over sodium sulfate, and concentrated under reduced pressure to yield the crude product as a yellow solid. This crude product was used in the next reaction without further purification.To a solution of ® Plus C18 Short Cartridge to afford 29  as a yellow solid.To the crude product in a sealed tube was added 80% TFA aq. (0.40 mL) at room temperature. After being stirred at 120 °C for 6 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by Sep-pak1H NMR  δ8.01 , 7.96 , 4.03 ; 13C NMR  δ169.6, 162.7, 160.7, 157.4, 156.7, 128.8, 121.6, 116.4, 57.3; HRMS-ESI (m/z) [M–H]− calcd for C9H7O6 211.0243, found 211.0240; mp 183 °C (decomp.).34  in a sealed tube was added a 4:1 mixture of 33% HBr/AcOH and H2O (0.19 mL) at room temperature. After being stirred at 120 °C for 2 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by Sep-pak® Plus C18 Short Cartridge using acetone/H2O to afford 37  as a yellow solid.To 1H NMR  δ7.73 , 7.60 , 7.42 , 4.64 ; 13C NMR  δ168.9, 161.5, 160.8, 144.5, 127.9, 122.0, 121.2, 67.2; HRMS-EI (m/z) [M+] calcd for C8H8O4 168.0423, found 168.0426; mp 144 °C (decomp.).34  in a sealed tube was added a 4:1 mixture of 33% HBr/AcOH and H2O (0.16 mL) at room temperature. After being stirred at 120 °C for 2 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by Sep-pak® Plus C18 Short Cartridge using MeOH/H2O to afford 38  as a yellow oil.To 1H NMR  δ7.48 , 7.41 , 7.18 , 4.42 , 3.40 ; 13C NMR  δ169.5, 161.4, 161.2, 141.1, 129.0, 121.73, 121.68, 77.7, 58.5; HRMS-EI (m/z) [M+] calcd for C9H10O4 182.0579, found 182.0572.39  and 2-methyl-2-butene  in THF (0.21 mL) and t-BuOH (0.21 mL) was added the mixture of NaClO2  and NaH2PO4  in H2O (0.21 mL) dropwise by a Pasteur pipette at room temperature. After being stirred at room temperature for 30 min, the reaction mixture was directly filtrated by silica gel, eluted with CHCl3/MeOH = 10:1, and concentrated under reduced pressure to afford the crude product as a yellow solid. This crude product was used in the next reaction without further purification.To a solution of 2O (0.42 mL) at room temperature. After being stirred at 120 °C for 2 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by Sep-pak® Plus C18 Short Cartridge to afford 40  as a yellow solid.To the crude product in a sealed tube was added a 4:1 mixture of 33% HBr/AcOH and H1H NMR  δ8.15 , 8.06 , 7.45 ; 13C NMR  δ171.7, 169.2, 164.3, 159.9, 132.3, 130.3, 120.5, 119.5; HRMS-ESI (m/z) [M–H]− calcd for C8H5O5 181.0137, found 181.0128; mp 170 °C (decomp.).37, 42  was obtained from 22  as a yellow solid.With the same procedure for the synthesis of 1H NMR  δ 7.15 , 4.52 ; 13C NMR  δ 158.4, 157.2, 143.8, 117.2, 67.3; HRMS-EI (m/z) [M+] calcd for C8H8O5 184.0372, found 184.0388; mp 162 °C.38, 43  was obtained from 22  as a yellow solid.With the same procedure for the synthesis of 1H NMR  δ7.10 , 4.38 , 3.40 ; 13C NMR  δ158.4, 157.2, 140.1, 117.8, 77.6, 58.3; HRMS-ESI (m/z) [M–H]− calcd for C9H9O5 197.0450, found 197.0450; mp 120 °C.42 without purification, 41 was obtained as a intermediate from (6S)-17  as a dark brown amorphous. This crude product was used in the next reaction without further purification.With the same procedure for the synthesis of 41 in DMF (0.93 mL) was added NaN3  at room temperature. After being stirred at room temperature for 2.5 h, the reaction mixture was quenched with H2O (10 mL). The organic layer was separated, and the aqueous layer was extracted with EtOAc (10 mL × 3). The combined organic layer was dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by Sep-pak® Plus C18 Short Cartridge to afford 44  as a yellow solid.To a solution of the crude 1H NMR  δ7.09 , 4.35 ; 13C NMR  δ158.0, 157.6, 137.3, 118.8, 58.8; HRMS-ESI (m/z) [M–H]− calcd for C8H6O4N3 208.0358, found 208.0358; mp 159 °C.*Experimental details of other new compounds and additional information are given in the Supplementary Information.In vitro cultivation and antimalarial activity against the Plasmodium falciparum K1 (chloroquine-resistant) parasite strain were measured using the method described previously30. Briefly, the P. falciparum K1 strain was cultured in human erythrocytes in RPMI medium supplemented with 10% human plasma at 37 °C, under 93% N2, 4% CO2, and 3% O2. Asynchronous parasites (2% hematocrit and 0.5 or 1% parasitaemia) were seeded in a 96-well microtiter plate and serially diluted test compounds were added. Positive controls, such as chloroquine and artemisinin, were added in a similar fashion. After 72-hours incubation, parasite lactate dehydrogenase (p-LDH) was assayed using a slight modification of the procedure reported by Makler et al.31 and Vivas et al.32. The 50% inhibitory concentration (IC50) value was calculated from a dose response curve. This study was approved by “Kitasato Institute Hospital Research Ethics Committee (No12102)” using human erythrocytes donated by volunteers.33.Measurement of cytotoxicity against human fetal lung fibroblast MRC-5 cells was carried out as described previouslySupplementary Information


Development of easy, practical, and low-cost spectrophotometric methods is required for the selective determination of phenolic antioxidants in the presence of other similar substances. As electron transfer (ET)-based total antioxidant capacity (TAC) assays generally measure the reducing ability of antioxidant compounds, thiols and phenols cannot be differentiated since they are both responsive to the probe reagent. In this study, three of the most common TAC determination methods, namely cupric ion reducing antioxidant capacity (CUPRAC), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt/trolox equivalent antioxidant capacity (ABTS/TEAC), and ferric reducing antioxidant power (FRAP), were tested for the assay of phenolics in the presence of selected thiol and protein compounds. Although the FRAP method is almost non-responsive to thiol compounds individually, surprising overoxidations with large positive deviations from additivity were observed when using this method for (phenols + thiols) mixtures. Among the tested TAC methods, CUPRAC gave the most additive results for all studied (phenol + thiol) and (phenol + protein) mixtures with minimal relative error. As ABTS/TEAC and FRAP methods gave small and large deviations, respectively, from additivity of absorbances arising from these components in mixtures, mercury(II) compounds were added to stabilize the thiol components in the form of Hg(II)-thiol complexes so as to enable selective spectrophotometric determination of phenolic components. This error compensation was most efficient for the FRAP method in testing (thiols + phenols) mixtures. There is a critical balance between reactive oxygen/nitrogen species (ROS/RNS) and antioxidants (AOx) in the human body. Although there are some important endogenous AOx sources, such as small molecule antioxidants  and enzymes , substantial amounts of AOx are taken by foods. Polyphenols have a special importance among dietary antioxidants as being the most consumed phytochemicals. Plenty of foodstuffs and beverages can be mentioned as dietary polyphenol sources such as vegetables, fruits, juices, tea and coffee. Polyphenol intake is generally accepted to be effective on the prevention of oxidative stress-originated diseases, such as cardiovascular and neurodegenerative diseases and cancer. Polyphenols are usually accepted as antioxidants serving cell survival and contributing to the regulation of cellular redox status; they may interfere at the initiation, development, and progression of cancer through the modulation of certain cellular processes and signaling pathways . HoweverAn antioxidant can be defined as a substance  that, when added to an oxidizable molecule in small amounts, is able to protect such molecules by delaying, retarding, or inhibiting their autoxidation  . Though Total antioxidant capacity (TAC) is a useful parameter reflecting the cumulative effect of several antioxidants in complex matrices rather than individual antioxidative properties of relevant components. By definition, TAC should be equal to the sum of the antioxidant capacities of components constituting a mixture. TAC is usually measured by several spectrophotometric tests, but absorbances of mixture components is not always additive, and synergistic or antagonistic effects may frequently emerge as a result of interactions among several components. These different interactions can be explained by AOx structure and reactivity. Knowing the initial antioxidant capacity of a single component can give useful information to solve this interaction problem . Wang et•), and oxygen radical absorbance capacity (ORAC) methods. The authors reported some synergistic and antagonistic interactions, and more than half of these combinations gave additive results. Among the tested samples, mixtures of raspberry and adzuki bean extracts showed synergistic interactions in all three TAC determination methods | × 100 formula was used . The results are shown in For selected thiol compounds (homocysteine (HCYS), 2+ salts, the experimental absorbance values for binary mixtures were very close to the theoretical ones, and the resulting RE values were roughly between 1% and 9%.As can be seen from 2+, all RE values were reduced to <5%. The absorbance differences in the absence and presence of Hg2+ salts are shown in Similar binary mixtures were tested for all mentioned polyphenols. The biggest RE (378%) was calculated for the (0.1 mL CFA + 0.1 mL CYS) mixture. On the other hand, when the experiments were repeated in the presence of HgHg(II) optimization experiments were performed as stated in •+ reference reading, and for the Hg(II):CYS mol ratio ≥5, the obtained absorbances were nearly the same—within experimental error—as the reference absorbance. We can conclude that Hg(II) could complex all CYS in solution and prevent the oxidation of thiols with ABTS•+ . For further experiments, 1:10 mol ratio was selected as optimal.As can be seen from •+ chromogenic radical accepts an electron from antioxidants to convert into the colorless ABTS form, where the absorbance decrease at 734 nm is related to AOx concentration. ABTS/TEAC method studies were realized as described in ref. and Asample (∆A = Aref. − Asample). The relative errors were calculated by using the equation: RE% = |[(∆Aexp. − ∆Atheo)/∆Atheo]| × 100. For GSH and CAT mixtures, the RE values were calculated between 0.1% and 5.7% even in the absence of Hg2+, so these experiments were not repeated in the presence of Hg2+. For binary mixtures of 0.1 mL of 1.0 × 10−4 M HCYS (or NAC) and 0.05–0.25 mL volumes of 5.0 × 10−5 CAT, the results are depicted in −4 thiol compounds and 0.05–0.25 mL EC mixtures, the lowest RE was calculated as 0.5% for HCYS + 0.1 mL EC and the highest RE was 23% for NAC + 0.1 mL EC mixtures; however, the RE was brought down to 2.4% for the latter mixture in the presence of Hg2+. For mixtures consisting of 0.1 mL of 1.0 × 10−4 M thiol compound and 2.0 × 10−4 M CFA, the lowest RE was calculated as 0.8% for CYS + 0.3 mL CFA and the highest RE was calculated to be 33.6% for HCYS + 0.2 mL of the CFA mixture; however, the RE was brought down to 5.4% in the presence of Hg2+. All results are summarized in The ABTS2+ provided better RE results, i.e., Hg(II) addition brought relative errors up to 22% down to <10%.As can be seen from −4 M GSH + 0.1 mL of 4.0 × 10−5 M GA + 5.0 × 10−5 M CAT and 0.1 mL of 0.1 × 10−4 M CYS + 0.1 mL of 2.0 × 10−4 M CFA + 0.1 mL of 5.0 × 10−5 M EC, respectively. When the same experiments were repeated in the presence of Hg2+, the RE for CYS + CFA + EC was calculated as 1.6%, again confirming the thiol-stabilizing effect of added Hg(II).For ternary mixtures, the RE values ranged between 1.6%–14.3% for 0.1 mL of 1.0 × 10−3 M Hg(II) but, unfortunately, the results were not better than those in the absence of Hg(II). For CAT and CFA mixtures with 0.3 mL of 1:20 diluted egg white protein solutions, the RE% values were between 3.5 and 12.6 for CAT + egg white protein solution and 0.6–19.3 for CFA + egg white protein solution. The experiments were repeated in the presence of 0.1 mL of 1.0 × 10−3 M Hg2+, with no significant improvement.ABTS/TEAC method experiments for polyphenol–protein mixtures were applied as stated in exp. − Atheo)/Atheo]| × 100. Among all tested TAC methods, CUPRAC gave the most additive results with RE < 5%, without requiring Hg(II) correction. The obtained results for ternary mixtures are shown in The CUPRAC reagent, i.e., Cu(II)-neocuproine complex, accepts an electron from antioxidants to form the orange-yellow colored chromophore (Cu(I)-neocuproine chelate), and the absorbance increase at 450 nm is related to AOx concentration. To investigate the effects of thiols on determination of polyphenols using the CUPRAC method, different binary and ternary thiol-polyphenol mixtures were prepared as stated in −4 M CAT were determined in the presence of 0.2 mL of 1.0 × 10−3 M GSH or NAC. These mixtures gave perfectly additive results with CUPRAC, and the observed absorbances at 450 nm arose from the Cu(II)-neocuproine oxidation of thiol and polyphenol components in an additive manner. The same experiments were repeated in the presence of 0.1 mL of 1.0 × 10−2 M Hg(Ac)2 added as a stabilization agent for the thiol component. During the experiments, the thiol compound, phenolic AOx, and Hg2+ were mixed together, and this mixture was added as sample into the CUPRAC reagent mixture. After incorporating Hg(II), only the polyphenol component (CAT) responded to the CUPRAC assay, confirming that the polyphenol component of a mixture could be selectively determined with CUPRAC in the presence of a thiol.For CUPRAC measurements of polyphenols in the presence of thiols, different volumes between 0.1 and 0.5 mL of 1.0 × 10Related experiments were conducted as mentioned in Since polyphenols may exist together with thiol- or protein-type antioxidants in natural and functional food formulations to extend the shelf-life, accurate and precise measurement of the antioxidant capacity of such food mixtures is of vital importance. The definition of TAC usually necessitates the additivity of individual antioxidant capacities of mixture components, but synergistic or antagonistic interactions may occur among various antioxidants giving rise to large deviations from additivity. In this study, three of the most common spectrophotometric TAC determination methods  were applied to different thiol-polyphenol and protein-polyphenol mixtures. The most additive results were obtained for the CUPRAC method, and the largest deviations were seen for the FRAP method, whereas ABTS/TEAC exhibited medium level deviations.•) [−) were directly involved in the oxidation reaction [The additivity of CUPRAC antioxidant capacities of thiol-containing proteins in admixture with polyphenols was previously shown by the authors’ laboratory . The CUP•)  which ar•) . As anotreaction . Thus, ireaction . Thapa areaction . It shoureaction . Even a reaction ,36.In cases when phenols are oxidized faster than thiols , the Michael addition of nucleophilic thiols to quinones  may rapidly produce new products (thiol-quinone adducts), depending on the nature, number, and position of substituents on the quinone, and this addition may also give rise to overoxidation of phenols and thiols via redox cycling involving molecular oxygen and ROS ,42. Over•) by a one-electron transition, sulfenic acid (–SOH), and disulfide (–S–S–) by a two-electron transition, sulfinic acid (–SO2H) by a four-electron transition, and eventually sulfonic acid (–SO3H) by a six-electron transition [Cysteine can be oxidized to form several different products. These include the thiyl radical  correction for thiol components.The formation of stable Hg(II)-thiol complexes proved to be successful for independent determination of the polyphenolic content of thiol-phenol mixtures using all three TAC assay methods, because the strongly-complexed thiol components of the mentioned mixtures did not respond to these spectrophometric tests. Addition of Hg(II) to protein-phenol mixtures was basically unsuccessful in achieving additivity of TAC values, probably because of the fact that most protein thiols are not freely available for metal complexation and are buried within the macromolecular backbone, and that phenolic oxidation products (quinones) may bind site-specifically to proteins through thiol addition and addition-elimination reactions . Thus, tChemicals and samples were weighed using a Radwag AS 220/C/2 analytical balance ; pH measurements were performed using a HANNA HI 221 pH-meter , and an Elmasonic ultrasonic water bath was used to dissolve chemicals. A Varian Cary 100 Bio UV-VIS spectrophotometer  with matched HELLMA quartz cuvettes (light path = 10 mm) was used for optical absorption measurements.N-acetyl-l-cysteine, 2,4,6-Tris(2-pyridyl)-S-triazine (TPTZ) were from Fluka ; caffeic acid, (±)-catechinhydrate, epicatechin, dl-homocysteine, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), neocuproine  hydrochloride, casein from bovine milk, trizma, and glycine were obtained from Sigma ; gallic acid, l-glutathione (reduced), acetic acid, ethanol, potassium persulfate, urea, and trichloroacetic acid (TCA) were purchased from Sigma-Aldrich ; l-cysteine from Aldrich and sodium acetate trihydrate, hydrochloric acid, sodium hydroxide, sodium chloride, and ammonium acetate were from Riedel de Haen .All reagents were of analytical grade and purchased from the corresponding sources: iron(III) chloride hexahydrate, copper(II) chloride dihydrate, potassium dihydrogen phosphate, potassium monohydrogen phosphate, mercury(II) acetate, dodecyl sulfate sodium salt, tri-sodium citrate-5,5-hydrate, and bovine serum albumin were purchased from E. Merck ; −5 M, CFA 2.0 × 10−4 M, CAT and EC 5.0 × 10−5 M, thiol-type antioxidants were at 1.0 × 10−4 M. For the CUPRAC method, working solutions of CAT, EC, CFA were at 1.0 × 10−4 M, GA was at 2.0 × 10−4 M and thiol-type AOxs at 1.0 × 10−3 M.Stock solutions of phenolic  and thiol-type antioxidants were prepared at 10 mM concentration. GSH, NAC were dissolved and diluted to volume with distilled water. HCYS CYS were dissolved with 0.5 mL of 1.0 M HCl and diluted to volume with distilled water. Solutions of phenolic AOxs were prepared in ethyl alcohol. For preparing working solutions of FRAP method, stock solutions of phenolic AOxs were diluted 100 times with ethanol and stock solutions of thiol-type AOxs were diluted 50 times with distilled water. For use in the ABTS method, AOx working solutions were diluted to the following concentrations: GA 4.0 × 10w/v) for ABTS method and at 2.0% (w/v) for the CUPRAC method, BSA was dissolved with distilled water.To prepare a solution at 0.125% (w/v) for ABTS/TEAC method and 1.0% (w/v) for the CUPRAC method, suitable amounts of casein were weighed, 0.4 mL of 1.0 M NaOH, distilled water was added, and the mixture was slightly heated. The pH of the solution was adjusted to 8.0 with 1.0 M HCl, and diluted to volume with distilled water.To obtain a solution at 0.5% (w/v) sodium dodecyl sulfate and 8.0 M urea solutions were mixed, and the pH was adjusted to 6.8 with 6.0 M HCl.For preparation of protein dissolution buffer, 50 mM tris (hydroxymethyl)aminomethane, 2% (w/v) was added to precipitate the proteins, and the mixture was centrifuged at 5000 rpm for 5 min. The separated precipitate was washed with distilled water 3–4 times and redissolved with protein dissolution buffer. Egg white protein solution was diluted 20 times with distilled water prior to use for the ABTS method, and it was used directly for the CUPRAC method.Egg white and yolk were separated; egg white was suspended in distilled water using an ultrasonic water bath, and then filtered. Five milliliters of an aliquot was withdrawn, 5.0% TCA (−2 M concentration was prepared by dissolving a suitable amount of Hg(Ac)2 with 0.5 mL of 1.0 M HCl and distilled water. The working solutions at 5.0 × 10−4 M for use in the FRAP method and at 1.0 × 10−3 M for use in the ABTS method were prepared from the stock solution by suitable dilution with distilled water.A stock solution at 1.0 × 10Buffer solution at pH 3.6 (0.3 M): A weight of 3.1 g of sodium acetate was dissolved in distilled water, 16.0 mL acetic acid was added to the NaAc solution, and diluted to 1.0 L.3·6H2O solution (2.0 × 10−2 M): A suitable amount of FeCl3·6H2O was dissolved using 0.5 mL of 1.0 M HCl and diluted to 25 mL with distilled water.FeCl−2 M concentration was prepared by dissolving the suitable weight of TPTZ in ethyl alcohol.2,4,6-Tris(2-pyridyl)-S-triazine (TPTZ) solution: TPTZ solution at 1.0 × 103·6H2O:TPTZ solutions were mixed at 10:1:1 volume ratio.FRAP reagent: pH 3.6 buffer solution:FeCl2S2O8) and let to stand for 12–16 h in the dark; then it was diluted 40 times with 5 mM phosphate buffered saline (PBS) solution at pH 7.4.ABTS/persulfate solution: 7.0 mM ABTS solution in distilled water was mixed with 2.45 mM potassium persulfate  solution (pH 7.4): 100 mM KH2·2H2O solution: Prepared from CuCl2·2H2O in distilled water at 1.0 × 10−2 M concentration.CuCl−3 M concentration.Neocuproine (Nc) solution: Prepared from neocuproine hydrochloride in ethyl alcohol at 7.5 × 104Ac) solution: Prepared from NH4Ac in distilled water at 1.0 M concentration.Ammonium acetate (NHUrea buffer at pH 7: Tris (hydroxymethyl)aminomethane, glycine, sodium citrate, and urea at 0.086 M, 0.09 M, 4 mM, and 8.0 M final concentrations, respectively, were mixed and pH was adjusted to 7.0 with 6.0 M HCl.Standard tris buffer at pH 8: Tris (hydroxymethyl)aminomethane, glycine, and sodium citrate at 0.086 M, 0.09 M, and 4 mM final concentrations, respectively, were mixed and pH was adjusted to 8.0 with 6.0 M HCl solution.−4 M Hg(Ac)2) and 3.0 mL of FRAP reagent solution were mixed respectively. The reaction mixture was let to stand at room temperature for six minutes and absorbance was read at 595 nm against reagent blank.Application of FRAP to a phenolic AOx and/or thiol compound: 0.1 mL thiol solution + x mL of phenolic antioxidant solution + (0.4 − x) mL ethyl alcohol + 0.1 mL distilled water  salt was added to thiol + polyphenol mixtures. GA was used as phenolic and CYS as thiol type AOx, and a series of thiol:Hg(Ac)2 mol ratios were investigated. For this purpose, three types of solution were prepared, namely GA alone, GA + CYS and GA + CYS + Hg2+ mixtures. The amount of (GA + CYS) was kept constant while Hg2+ amount was varied. A series of CYS:Hg2+ solutions were prepared at 1:0.5, 1:1, 1:2.5, 1:5, and 1:10 mol ratios, and the FRAP method was applied.Optimization of the Hg(Ac)−4 M CYS, GSH, NAC, and HCYS were taken and different volumes of 1.0 × 10−4 M polyphenols were added to each thiol solution individually. For this purpose, different volumes ranging between 0.05–0.3 mL of GA, 0.1–0.4 mL of CFA, CAT, and EC were mixed and the FRAP method was applied. Each mentioned AOx compound (thiol or phenol) was tested individually and absorbance values were recorded. For binary mixtures to control additivity, the mathematical sum of these values were calculated and compared with experimental findings. Then all experiments were repeated in the presence of 0.1 mL of 5.0 × 10−4 M Hg(Ac)2. Since proteins precipitated in the presence of ethanol, protein mixtures could not be tested.FRAP method experiments: Different binary mixtures of thiols and phenolic AOx were tested with the FRAP method. To obtain binary mixtures, 0.1 mL of 2.0 × 10+ were mixed in this order. After the reaction mixture was let to stand for six minutes at room temperature, absorbance was read against PBS at 734 nm.Application of ABTS/TEAC to a phenolic AOx and/or thiol compound: 0.1 mL thiol solution + 0.4 mL distilled water, x mL phenolic antioxidant + (0.5 − x) mL ethanol + 2.0 mL ABTS2) + 1.0 mL ABTS/persulfate (1:20 diluted with PBS) were mixed, and absorbance at 734 nm was read after 6 min standing.Application of ABTS/TEAC to a protein-phenol antioxidant mixture: 1.0 mL PBS + 0.3 mL protein sample solution + 0.1 mL distilled water + x mL phenolic antioxidant + (0.5 − x) mL ethanol + (0.1 mL Hg(Ac)2 for the ABTS/TEAC Method: For 0.1 mL of 1.0 × 10−4 M CYS, Hg(II) salt was added at different mol ratios between 1:1–1:10 and ABTS/TEAC method was applied. The same amount of Hg(II) was also added to the reference (blank) solution. Both reference and CYS-Hg(II) samples were read against PBS, and the difference calculated (∆A = Aref. − Asample).Optimization of the amount of Hg(Ac)−4 M GSH, CYS, NAC, HCYS, and each aliquot was mixed with 0.1–0.4 mL of 2.0 × 10−4 M CFA, 0.1–0.4 mL of 4.0 × 10−5 M GA and 0.05–0.25 mL of 5.0 × 10−5 M CAT and EC separately in the presence or absence of 0.1 mL of 1.0 × 10−3 M Hg2+. Using 0.15 mL CAT and 0.1 mL EC, CFA, CYS, and GSH; CYS + EC + GA; CYS + CFA + CAT; CYS + GA + CAT; GSH + EC + GA; GSH + CFA + CAT; GSH + GA + CAT; GSH + CFA + EC ternary mixtures were tested in the presence or absence of Hg(II) as stated earlier.To investigate the effect of thiols on the determination of phenolic AOx compounds, different binary and ternary mixtures were prepared. To prepare binary mixtures, 0.1 mL volumes were taken from 1.0 × 10−4 M CFA were added to 0.2 mL of casein solution, and the ABTS/TEAC method was applied. Then, different volumes between 0.1–0.5 mL of 2.5 × 10−5 M CAT were added to 0.2 mL of 0.5% casein. These phenolic AOx compounds were also added to 0.1 mL of 0.125% BSA. The same antioxidant mixture was also prepared with 0.3 mL of 1:20 diluted egg white protein solution. Experiments were repeated in the presence and absence of 0.1 mL of 1.0 × 10−3 M Hg2+.To investigate polyphenol-protein interactions in the ABTS/TEAC method, three types of proteins were used: 0.125% BSA, 0.5% casein, and 1:20 diluted egg white protein solution. Different volumes between 0.1–0.4 mL of 2.0 × 102·2H2O + 1.0 mL Nc + 1.0 mL NH4Ac, x mL phenolic antioxidant + (1 − x) mL ethanol + 0.2 mL thiol solution, and 0.8 mL distilled water were mixed in this order. The mixture was let to stand for 30 min at room temperature. Absorbance was read at 450 nm against a reagent blank.Application of CUPRAC to a phenolic AOx and/or thiol compound: 1.0 mL CuCl2·2H2O + 1.0 mL Nc, 2 mL pH 7 urea buffer + 1.0 mL standard tris pH 8.0 buffer + x mL phenolic antioxidant + (0.5 − x) mL ethanol + 0.4 mL protein and 0.1 mL distilled water were mixed, and the measurement was also made after 30 min standing. Here, NH4Ac buffer was replaced with urea buffer to prevent protein precipitation [Application of CUPRAC to a protein-phenolic antioxidant mixture: 1.0 mL CuClpitation .−3 M thiol compounds , 2.0 × 10−4 M GA, 1.0 × 10−4 M CAT, EC, and CFA were used. To prepare binary mixtures (i) 0.1–0.4 mL of GA; (ii) 0.2–0.6 mL of CFA; and (iii) 0.1–0.5 mL CAT (or EC) were added to 0.2 mL of thiol solution  individually. For ternary mixtures, 0.2 mL aliquots of the corresponding components of mixtures CYS + EC + GA; CYS + CFA + CAT; CYS + GA + CAT; CYS + CFA + EC were mixed; then, the same phenolic AOx compounds were mixed with 0.2 mL of GSH instead of CYS.To prepare binary and ternary mixtures, 1.0 × 10−4 M CFA, 0.1–0.5 mL of 1.0 × 10−4 M CAT, EC, and 0.1–0.4 mL of 2.0 × 10−4 M GA individually. The effect of Hg+ was also investigated for the CUPRAC method, and in the presence of Hg(II), the results were not significantly different.Aliquots of 0.4 mL of 1.0% casein, 2.0% BSA, and egg white protein solution (without any dilution) were mixed with 0.1–0.5 mL of 1.0 × 10Accurate and precise measurement of the antioxidant capacity of thiol-phenol mixtures is a priority challenge in food analysis since any synergistic or antagonistic interactions between the mixture components may affect the shelf-life of food. Synergism between antioxidants is defined as the more effective behavior of mixtures compared to single compounds, and usually used in the sense of protection of one antioxidant by the other against lipid peroxidation. However, in non-competitive spectrophotometric TAC assays, a chromogenic redox probe oxidizes the tested antioxidants and changes color, where the additivity of TAC values is a consequence of Beer’s law applied to mixtures. Three of the most common spectrophotometric TAC determination methods  were applied to different thiol-polyphenol and protein-polyphenol mixtures, where the most additive results were obtained for the CUPRAC method; the largest deviations were seen for the FRAP method, whereas ABTS/TEAC exhibited medium level deviations. Thus, overoxidation of thiol-phenol mixtures primarily observed in the FRAP method (and to a minor extent in the ABTS method) seems to arise from systematic methodological errors. The best way to correct this error  was to add Hg(II) salts to strongly complex the thiol components so that the true TAC values manifested by phenols in such mixtures could be observed. Applying the same measure  complexation) to protein-phenol mixtures was not as effective, possibly due to the fact that most protein thiols are buried within the macromolecular backbone. The CUPRAC method worths to be recommended for the TAC determination of thiol-phenol mixtures to avoid systematic errors and get additive results.


Chalcones are the biogenetic precursors of all known flavonoids, which play an essential role in various metabolic processes in photosynthesizing organisms. The use of whole cyanobacteria cells in a two-step, light-catalysed regioselective bio-reduction of chalcone, leading to the formation of the corresponding dihydrochalcone, is reported. The prokaryotic microalgae cyanobacteria are known to produce phenolic compounds, including flavonoids, as natural components of cells. It seems logical that organisms producing such compounds possess a suitable “enzymatic apparatus” to carry out their biotransformation. Therefore, determination of the ability of whole cells of selected cyanobacteria to carry out biocatalytic transformations of chalcone, the biogenetic precursor of all known flavonoids, was the aim of our study.Anabaena laxa, Aphanizomenon klebahnii, Nodularia moravica, Synechocystis aquatilis (>99% yield) and Merismopedia glauca (92% yield). The strains Anabaena sp. and Chroococcus minutus transformed chalcone in more than one way, forming a few products; however, dihydrochalcone was the dominant product. The course of biotransformation shed light on the pathway of chalcone conversion, indicating that the process proceeds through the intermediate cis-chalcone. The scaled-up process, conducted on a preparative scale and by using a mini-pilot photobioreactor, fully confirmed the high effectiveness of this bioconversion. Moreover, in the case of the mini-pilot photobioreactor batch cultures, the optimization of culturing conditions allowed the shortening of the process conducted by A. klebahnii by 50% (from 8 to 4 days), maintaining its >99% yield.Chalcone was found to be converted to dihydrochalcone by all examined cyanobacterial strains; however, the effectiveness of this process depends on the strain with biotransformation yields ranging from 3% to >99%. The most effective biocatalysts are This is the first report related to the use of whole cells of halophilic and freshwater cyanobacteria strains in a two-step, light-catalysed regioselective bio-reduction of chalcone, leading to the formation of the corresponding dihydrochalcone. The total bioconversion of chalcone in analytical, preparative, and mini-pilot scales of this process creates the possibility of its use in the food industry for the production of natural sweeteners. Angelica, Sophora, Glycyrrhiza, Humulus, Scutellaria, Parartocarpus, Ficus, Dorstenia, Morus, Artocarpus, and so forth—many of which have been used for centuries in traditional herbal medicine , or in freshwater ecosystems, such as 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]. Axenic culture of the S. platensis strain was obtained from the Pasteur Culture Collection (PCC) , whereas all other cyanobacterial species were purchased from the Culture Collection of Autotrophic Organisms (CCALA) .Cyanobacteria used in our experiments occur either naturally in hypersaline ponds, such as To make the inoculates, which were used to initiate the experimental cultures, all tested microorganisms were pre-grown in standard media: MSp (ATCC 1679) medium for the halophilic strain, and BG11 (ATCC 616) or Z8 medium for freshwater strains.Subcultures of cyanobacteria were revitalized every 3 weeks by transferring 10-mL aliquots to 50 mL of fresh adequate media . The culScreening  scale biotransformations of chalcone were arranged by transferring the appropriate volumes of aliquots from 21-day-old subcultures to adequate, fresh media. The volumes of the inoculates were established experimentally by considering the final concentration of chlorophyll, which was 1 mg/L at the beginning of culturing for all tested species. Chlorophyll content was measured in methanolic extracts, according to a previously described procedure . The inoCyanobacterial cultures were incubated at 24 ± 1 °C under constant adjusted light conditions with a photoperiod  of 14 days. The stability of substrate under these conditions was demonstrated by preparing and incubating control samples consisting of the solution of tested flavonoid in sterile cultivation medium, the substrate control. The culture controls consisted of the microbial colonies (inocula) in fermentation medium cultivated without the chalcone. All experiments, including controls, were performed at least in triplicate.g for 1 min. Next, all the transformation medium obtained in each repetition was removed separately and was extracted three times with 10 mL of ethyl acetate. Then, these three extracts (from each separate repetition) were combined and dried over anhydrous magnesium sulphate, and the solvent was evaporated to dryness using a vacuum evaporator. The remaining residue was dissolved in 200 µL of ethyl acetate and subjected to analysis with the use of planar chromatography (TLC) and gas chromatography (GC–MS), using the appropriate standards. The effectiveness of the biotransformations was calculated based on the quantification of the products obtained with respect to the areas of relevant peaks, while the total peak area was treated as 100%. The resulting biotransformation yield is the average of the values obtained.After fourteen (14) days of incubation, the cells and culture medium were separated by filtration followed by centrifugation at 5.000×A. klebahnii, the entire transformation mixture was removed every 24 h, prepared as described above and analysed by GC–MS. The determination of the growth of A. klebahnii in experimental cultures was performed by time-course measurements of total chlorophyll content  of 2,5 ± 0,3 klux intensity with a defined photoperiod . The temperature of the culture was set at 25 °C, and the stirring speed was 100 rpm. Circulating, filtered air was supplied through a syringe filter to the bottom part of the vessel for uniform mixing of the culture and to prevent stagnation. This setup was left undisturbed for 7 days, the entirety of the batch experiment. A photobioreactor was connected to the additional unit, a gas analyser (produced by Solaris), which allowed for the measurement of oxygen and carbon dioxide concentrations in the headspace of the culture in the real-time mode. Those data were recorded, and the respiration coefficient was calculated.The 2 L volume mini-pilot scale of The same parameters were applied to the sterile Z8 medium containing chalcone at a concentration of 20 mg/L but without cyanobacterial cells (without inoculum), which acted as a substrate control. The same parameters were also applied to the sterile Z8 medium inoculated with the biota under examination but without the chalcone that was treated as the culture control.4)2 and 20 g of phosphomolybdic acid (H3[P(Mo3O10)4]) dissolved in 1 L of 10% H2SO4 to verify the presence of flavonoids, which give yellow to brown-orange spots. After spraying, the plates were gently heated until coloured spots appeared, whereupon the plates were inspected again under the UV light at 254 and 366 nm wavelengths and under white light in the reflectance mode. The retention coefficients (Rf) and colours of the spots were recorded using CAMAG VisionCats software.The biotransformations were carried out by TLC and GC. In the case of thin layer chromatography, the 10 µL volumes of ethyl acetate extracts from experimental cultures, substrate and culture controls, and the solution of chalcone standard were applied on TLC aluminium plates  using a CAMAG Linomat 5 applicator . Plates were developed in a dedicated glass chamber previously saturated with a hexane:acetone mobile phase mixture . Developed plates were visualised under 254 and 366 nm wavelengths of UV-light and under white light in a CAMAG TLC Visualizer 2 . Then, the plates were sprayed with a solution of 10 g of Ce equipped with an HP-5 fused silica capillary column . To determine the composition of the products the following temperature program was used: 90 °C/0 min, gradient 3 °C/min to 200 °C/5 min, gradient 10 °C/min to 280 °C/5 min. The injector temperature was set at 250 °C. The flow rate of the carrier gas (helium) was set at 1.5 mL/min. The split flow was 30 mL/min. A split ratio of 20:1 was applied. The chromatographic data were recorded and processed using the XCalibur software.1H NMR, 13C NMR, HSQC and COSY. Spectra were recorded using the NMR Bruker UltraShield 400 MHz spectrometer with tetramethylsilane (TMS) as an internal reference. The NMR spectra were measured in DMSO-d6. To confirm the presence of characteristic absorption bands, the obtained products were incorporated in KBr pellets and were subjected to FT-IR measurements using a Thermo Scientific NICOLET 6700 spectrometer . The structures of the known compounds were confirmed by comparing their spectroscopic properties with spectra of appropriate standards and with data published in the literature.The products of biotransformations performed in preparative and mini-pilot scale experiments were separated by PTLC by the use of a CAMAG system and silica gel glass plates without a fluorescent indicator  using a hexane:acetone  mixture as the eluent. Separated products were extracted from the silica bed with ethyl acetate, which was then evaporated under a stream of nitrogen. Then, the structures of isolated products were confirmed by MS, FTIR and nuclear magnetic resonance techniques: S. platensis) and seven freshwater strains , were screened. These species are known to be capable of transforming monoterpenes and organophosphonic compounds +, 106.0 (12), 105.0 (100), 91.2 (10), 77.1 (39), 51.0 (10); the retention times: 1–32.80, 2 –28.40 min.Chalcone (1) in the Anabaena sp. culture yielded compound 3 (white solid); mp. 55–57 °C; 1H NMR (400 MHz) (CDCl3) δ (ppm): 7.26–7.40 , 6.66 , 6.40 , 5.14 , 1.61 ; 13C NMR (100 MHz) (CDCl3) δ (ppm): 142.8, 136.5, 131.5, 130.6, 128.6, 128.5, 127.8, 127.7, 126.6, 126.3, 75.1; IR (KBr) νmax/cm−1 3345, 3027, 1599, 1493, 1450, 1010, 966, 746, 696; GC–MS (EI) m/z (%) = 208.1 (42) [M]+, 121.1 (21), 105.1 (100), 91.1 (57), 77.1 (61), 51.1 (15); the retention time: 3–30.20 min.Transformation of chalcone (1) in the culture Anabaena sp. yielded compound 4 ; mp. 52–54 °C; 1H NMR (400 MHz) (CDCl3) δ (ppm): 7.37–7.21 , 4.70 , 2.83–2.61 , 2.22–1.97 ; 13C NMR (100 MHz) (CDCl3) δ (ppm): 144.6, 141.9, 128.6, 128.5, 128.4, 127.7, 126.0, 125.9, 73.9, 40.5, 32.1; IR (KBr) νmax/cm−1 3395, 3027, 2940, 1603, 1495, 1454, 1058, 747, 699; GC–MS (EI) m/z (%) = 208.1 (4) [M]+, 107.1 (80), 92.1 (61), 79.0 (100), 65.1 (13), 51.0 (12); the retention time: 4–28.20 min.Transformation of chalcone (1) in the C. minutus culture yielded compound 5 ; mp. 132–133 °C; 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 12.41 , 7.69–7.67 , 7.60 , 7.42–7.40 , 6.54 ; 13C NMR (100 MHz) (DMSO-d6) δ (ppm): 168.4, 144.7, 135.1, 133.1, 129.7, 129.1, 120.1.; IR (KBr) νmax/cm−1 3230–2485, 1692, 1625, 1450, 1288, 982, 769, 712; GC–MS (EI) m/z (%) = 148.1 (58), 147.1 (100) [M]+, 131.1 (15), 119.1 (4), 103.1 (45), 91.1 (18), 77.1 (22), 51.2 (14); the retention times: 5–13.60 min.Transformation of chalcone (1) in the C. minutus culture yielded compound 6 (colourless to faintly yellow); mp. 45–48 °C; 1H NMR (400 MHz) (CDCl3) δ (ppm): 7.21–7.33 , 2.97 , 2.70 ; 13C NMR (25 MHz) (CDCl3) δ (ppm): 179.6, 140.2, 128.6, 128.3, 126.4, 35.7, 30.6; IR (KBr) νmax/cm−1 3060–2633, 1699, 1603, 1409, 1304, 1221, 933, 703; GC–MS (EI) m/z (%) = 150.0 (45) [M]+, 131.1 (4), 104.1 (100), 91.2 (81), 78.1 (18), 65.1 (14), 51.1 (10); the retention times: 5–10.60 min.Transformation of chalcone  was obtained. The use of blue-green algae as biocatalysts in the biotransformation of chalcones seems to be an important biotechnological strategy to obtain dihydrochalcone with high chemoselectivity, using low-cost reagents. Moreover, the scaling-up of this process to preparative and mini-pilot scales fully corroborates the results obtained with the analytical scale. Moreover, >99% effectiveness of bioconversion on analytical, preparative, and mini-pilot scales creates the possibility of rescaling this process to a semi-technical or technical scale. Therefore, we propose a relatively simple way of obtaining dihydrochalcone. Due to its sweet taste and growing interest in its use, this method may find an application in the food industry that is still eager for the production of new, natural sweeteners.