Coreopsis triloba S.F. Blake, collected at Cerro Villonaco in Loja, Ecuador, were investigated with respect to their essential oil (EO). The chemical composition was determined qualitatively through gas chromatography coupled with mass spectrometry (GC-MS) and quantitatively by gas chromatography coupled with flame ionization (GC-FID), using relative response factors (RRF) based on the enthalpy of combustion. The essential oil contained between 92.5% and 93.4% of monoterpene hydrocarbons, with (E)-β-ocimene being the main component (35.2–35.9%), followed by β-phellandrene (24.6–25.0%), α-pinene (15.3–15.9%), myrcene (10.9–11.0%), sabinene (2.2–2.4%), (Z)-β-ocimene (1.5%), and germacrene D (1.2–1.3%). The enantiomeric distribution of α-pinene, β-pinene, limonene, and germacrene D was also determined. The main components responsible for the aroma were identified through aroma extract dilution analysis (AEDA), a gas chromatography-olfactometry (GC-O) based technique, being α-pinene, β-pinene (0.6%), terpinolene (0.1%), α-copaene (0.1–0.3%), β-phellandrene, and (E)-4,8-dimethyl-1,3,7-nonatriene (0.1–0.2%) the main olfactory constituents according to the decreasing factor of dilution (FD) order. The biological tests showed IC50 inhibition values of 42.2 and 6.8 µg/mL for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively.The fresh leaves of  Ecuador is a country of impressive botanical abundance, where the traditional use of many plant species has attracted the curiosity of researchers of natural products. The study of medicinal plants revealed the existence of secondary metabolites, responsible for their biological activity ,2,3,4. AAlzheimer’s disease (AD) is a chronic neurodegenerative illness characterized by a progressive deterioration of memory and other cognitive functions and is currently the focus of scientific research aimed at finding effective therapeutic strategies, mainly related to the inhibition of the enzymes acetylcholinesterase and butyrylcholinesterase, known as cholinesterases (ChEs). These enzymes are involved in the development of the disease, since they catalyze the degradation of the neurotransmitter acetylcholine (ACh) . As summCoreopsis triloba S.F. Blake, also cited as Coreopsis capillacea (Kunth), is a species belonging to the Asteraceae family, the richest angiosperm family. It is a shrub that is 0.5–1.2 m tall and often densely branched. This species is distributed in the countries of Ecuador (1400–2800 m) and Peru (1400–2500 m), being particularly common in the province of Loja (Ecuador). The species is also known with the following Quechua names: Macchia, peña nachic, and caca nachic, and it is traditionally used in medicinal baths for treating inflammation [ammation .Coreopsis tinctoria Nutt., with limonene (11.31%), α-bergamotene (7.31%), and α-phellandrene (3.88%) being the main components [C. tinctoria, however, all of them refer to the non-volatile fraction. C. lanceolata, C. grandiflora, C. mutica and many other species of this genus have also been described in the literature for the chemical composition of their non-volatile fractions [The only study of an essential oil from this genus that reports on the chemical composition and antioxidant activity of the fraction distilled from mponents . Many otractions ,20.C. triloba essential oil and on the chemistry of this species in general.To the best of the author’s knowledge, this is the first article that reports on the chemical composition and the cholinesterase inhibition activity of C. triloba S.F. Blake is 0.1% ± 0.03% (w/w) for both the hydrodistillation and steam distillation on fresh plant material.The essential oil yield of E)-β-ocimene (35.2–35.9%), followed by β-phellandrene (24.6–25.0%), α-pinene (15.3–15.9%), myrcene (10.9–11.0%), sabinene (2.2–2.4%), (Z)-β-ocimene (1.5%), and germacrene D (1.2–1.3%). In addition to the listed components, traces or low amounts (0.1–0.6%) of ten unidentified sesquiterpenes have been detected.The enantiomeric composition of chiral components  were obtC. triloba essential oil aroma were detected. The most important sensory component, determined with the AEDA GC-O analysis, was α-pinene, showing a strong woody odor, followed by β-pinene, terpinolene, and α-copaene. GC-O results were obtained with the AEDA method and are shown in Coreopsis triloba S.F. Blake essential oil against AChE and BChE shows IC50 concentrations of 42.2 and 6.8 μg/mL, respectively -4,8-dimethyl-1,3,7-nonatriene showed a spicy odor.Currently, the enantiomeric ratio of an essential oil is considered the important information obtained from its chemical analysis ,31,32. TAlthough ACh can be hydrolyzed by both AChE and BChE, it has been found that AChE plays the major role in the hydrolysis of ACh in the healthy brain, whereas BChE takes over hydrolysis of ACh in the deficient AChE brain, it has been shown according to tests in mice . HoweverQualitative analyses were run with a GC-MS system consisting of an Agilent Technologies gas chromatograph 6890N and a quadrupole mass spectrometry detector 5973 , operating in SCAN mode and electronic ionization (70 eV), with a mass range of 45–350 m/z. The gas chromatograph was equipped with a non-polar stationary phase capillary column DB-5MS (Agilent Technologies)  and a HP-INNOWAX polar stationary phase column (Agilent Technologies) .9 from BDH, purity 99% and C10–C25 from Fluka, purity 99%).Quantitative analyses were carried out with a GC-FID system consisting of an Agilent Technologies gas chromatograph 6890N  with an Agilent Technologies 7683 series autoinjector . Linear retention indices were obtained using the homologous series of linear alkanes (CThe enantiomeric components and excesses (e.e.) were determined using an enantioselective column diethyl tertbutylsilyl-β-cyclodextrin (25 m × 0.25 mm × film thickness 0.25 µm) from Mega .The GC-O analyses were carried out on an Agilent Technologies gas chromatograph 6890N  coupled with a FID and a GERSTEL ODP 3 olfactory detection port 07615-00113. The analyses were performed with the same DB-5MS column and automatic injector previously described. The GC-O system was configured with a split fraction between sniffing port and detector of 50%.Εlectrophorus electricus acetylcholinesterase , equine serum butyrylcholinesterase , acetylthiocholine iodide (Sigma–Aldrich) and a Varioskan Flash (Thermo Scientific) detection system were used for enzymatic inhibition experiments. Donepezil was taken as positive control of ChE inhibition.All solvents used in this study were of analytical grade (purity > 99%) from Sigma–Aldrich. 5,5′-Dithiobis (2-nitrobenzoic acid), DNTB (Sigma–Aldrich), dimethyl sulfoxide, DMSO (Sigma–Aldrich), Coreopsis triloba S.F. Blake were collected in the province of Loja on Mount Villonaco, at an altitude of 2724 meters above sea level, with geographic coordinates 690644E–9557656N. The plant was collected with permission of the Ministry of Environment of Ecuador (MAE-DBN-2016-0655) and the identification was achieved by Dr. Nixon Cumbicus and Dr. Vladimir Morocho of the Universidad Técnica Particular de Loja (UTPL). A voucher specimen of the species was deposited in the UTPL herbarium with code VMZ_011.The aerial parts of The pure essential oil was obtained by steam distillation for 4 h  from 2 kn-nonane as internal standard (0.7 mg/mL).For the analytical purpose, the essential oil was obtained by hydrodistillation. Four repetitions were carried out using a micro Marcusson-type apparatus ,45. FiveDuring the analyses, all samples were stored in amber vials at −15 °C. The yield was directly calculated by weight in the case of the pure essential oil and analytically, as average and standard deviation, in the case of the micro-scale distillations.The following operative conditions were applied for GC-MS analysis with DB-5MS column: He was the carrier gas, with a flow rate of 1 mL/min; the injection volume was 1 μL. The injector was operated in split mode, with a split ratio of 40:1. The injection temperature was set at 220 °C. The elution was conducted according to the following temperature program: 50 °C for 5 min, a first thermal gradient to 180 °C at a rate of 3 °C/min, then a second gradient to 250 °C at a rate of 15 °C/min. At the end, the oven temperature was kept at 250 °C for 15 min. With the HP-INNOWAX column, the same conditions and thermal program as DB-5MS were applied, with the exception of the final oven temperature that was set at 230 °C.The linear retention indices were obtained according to Van Den Dool and Kratz .The quantitative analyses were carried out with the same instrumental conditions as the qualitative ones. The FID was alimented as follows: hydrogen flow 30 mL/min, air flow 300 mL/min. The temperature of the detector was set at 250 °C. The quantitative composition was obtained by using relative response factors calculated on the basis of the combustion enthalpy ,48. The Enantioselective GC-MS analysis was performed under the following oven thermal program: 50 °C, held for 5 min, rising to 220 °C at a rate of 2 °C/min and kept at this temperature for 5 min. The elution order was established by injecting enantiomerically pure standards, available in one of the authors’ laboratories (C.B.).The system was operated according to the following thermal gradient: 50 °C held for 1 min, rising at 2 °C/min to 100 °C, then to 200 °C (1 min) at a rate of 20 °C/min. The carrier gas (He) and detector were set at the same conditions as for the quantitative analysis. The samples were prepared as solutions of the essential oil in cyclohexane at the concentration of 200 μL/mL, corresponding to a dilution factor (FD) of 1; the injection volume was 1 μL. The AEDA method was applied by acting on the split ratio . The sniffing procedure was divided into 30 min sessions with 20 min intervals to avoid lassitude. The GC-O analyses, executed in duplicate, were performed by two analysts, presenting no anosmia for common terpenes and with previous training and experience in GC-O perception applied to essential oils. The acceptance criteria for the detected odors was that each perception had to be confirmed by at least one panelist in, at least, two following dilutions or, alternatively, once by both panelists at the same dilution.Electrophorus electricus acetylcholinesterase and equine serum butyrylcholinesterase were dissolved in PBS pH 7.4 and were used at 25 mU/mL for the assay. After 10 min of preincubation, the substrate acetylthiocholine iodide  was added to start the reaction. During 30 min of incubation at 30 °C, multiple 96-well microliter sites were read in a Varioskan Flash (Thermo Scientific) detection system. All measurements were made in triplicate. When possible, the IC50 values were calculated using the GNUPLOT package online . Donepezil was used as reference ChE inhibitor with an IC50 = 100 nM for AChE and 8500 nM for BChE. In this assay, the possibility of false positive inhibition results previously described for the high concentration (>100μg/mL) of amine or aldehyde compounds cannot be excluded [The activities of cholinesterase (ChE) were evaluated by following a colorimetric protocol adapted from Ellman et al. . The catCoreopsis triloba S.F. Blake for the first time. Twenty-nine compounds, representing 98.3–99.5% of the EO were identified. The main components were(E)-β-ocimene (35.2–35.9%), followed by β-phellandrene (24.6–25.0%), α-pinene (15.3–15.9%), myrcene (10.9–11.0%), sabinene (2.2–2.4%), (Z)-β-ocimene (1.5%), and germacrene D (1.2–1.3%). The enantioselective analysis indicated that (+)-β-pinene and (−)-germacrene D were present as pure enantiomers, while (S)-α-pinene and (S)-limonene presented the enantiomeric excess of 63.2% and 95.0% respectively. According to GC-O, the enantiomeric mixture of α-pinene was the main component responsible for the aroma of the essential oil, followed by β-pinene (0.6%), terpinolene (0.1%), α-copaene (0.1–0.3%), β-phellandrene, and (E)-4,8-dimethyl-1,3,7-nonatriene (0.1–0.2%). The essential oil showed an IC50 inhibition value of 42.2 µg/mL for AChE and 6.8 µg/mL for BChE.In this work, we determined the chemical composition, enantiomeric distribution, AEDA profile, and enzymatic inhibition of AChE and BChE of the essential oil of C. triloba is a source  of a new monoterpene based EO, characterized by an interesting, quite selective inhibition activity of BChE.We can conclude that The secondary metabolites responsible for the herbaceous/spicy odor of the whole essential oil were also determined.


Aplysia genus are recognized as a source of a diverse range of metabolites. 5α,8α-Endoperoxides belong to a group of oxidized sterols commonly found in marine organisms and display several bioactivities, including antimicrobial, anti-tumor, and immunomodulatory properties. Herein we report the isolation of 5α,8α-epidioxycholest-6-en-3β-ol ) from Aplysia depilans Gmelin, based on bioguided fractionation and nuclear magnetic resonance (NMR) analysis, as well as the first disclosure of its anti-inflammatory properties. EnP revealed capacity to decrease cellular nitric oxide (NO) levels in RAW 264.7 macrophages treated with lipopolysaccharide (LPS) by downregulation of the Nos2  gene. Moreover, EnP also inhibited the LPS-induced expression of cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) at the mRNA and protein levels. Mild selective inhibition of COX-2 enzyme activity was also evidenced. Our findings provide evidence of EnP as a potential lead drug molecule for the development of new anti-inflammatory agents.Sea hares of  Aplysia sea hares are herbivorous mollusks that feed on a wide variety of green, red, and brown algae [Aplysia spp. complex digestive gland contributes to its metabolic machinery, leading to the production of innumerable compounds with interesting pharmacological properties [Aplysia metabolites have been extensively explored due to their cytotoxic and antibacterial properties, few reports address their anti-inflammatory activity.wn algae ,2. Indeewn algae ,3. Moreooperties ,3,4. Alt50 values of 4.8, 9.4, and 5.8 µM, respectively [Leishmania donovani, displaying an IC50 of 4.9 µM [During the last decades, marine organisms were shown to be a source of unconventional sterols. In particular, steroidal endoperoxides are a subgroup of steroids which are ubiquitously found in marine invertebrates, such as sponges, and sea hares, among others . These mectively . More ref 4.9 µM .Aplysia depilans Gmelin methanolic extract, and the first disclosure of the molecular mechanisms underlying its anti-inflammatory properties in RAW 264.7 macrophages. Additionally, its capacity to inhibit 5-lipoxygenase (5-LOX), phospholipase A2 (PLA2), and cyclooxygenases (COX-1 and COX-2) was also explored.Herein, we report the isolation of EnP, based on bioguided fractionation and nuclear magnetic resonance (NMR) analysis, from a non-polar fraction of Aplysia metabolites [A. depilans extract  responsible for this effect, a bioguided fractionation of the above-mentioned fraction was performed, based on its capacity to reduce the cellular NO levels.Nitric oxide (NO) is an important signaling molecule synthesized by many cells in response to homeostatic and pathologic stimuli . AlthougA. depilans extract was fractionated, affording 11 fractions (Fr1–Fr11), according to the procedure described in Material and Methods , significant differences being found for all tested concentrations (p < 0.05)  , which ip > 0.05). Of note, in a promonocytic human cell line (THP-1), the structurally related ergosterol peroxide has demonstrated the capacity to dose-dependently (1–20 µM) increase the protection of cells against LPS-induced toxicity [p < 0.001)  can inhibit inducible nitric oxide synthase (iNOS), triggering a reduction in NO and l-citrulline levels; and thirdly, the EnP can modulate several pathways, leading to a decrease on iNOS mRNA and protein expression. In order to clarify the mechanism underlying its activity, EnP was assessed for its capacity to scavenge ●NO and to modulate iNOS action.As can be seen in toxicity . Relevan< 0.001) B. Thus, ●NO [●NO scavenging activity of EnP was investigated, at the same concentrations tested for cellular NO (up to 20.8 µg/mL), which revealed no antioxidant effect (data not shown).Multiple antioxidant molecules have been associated to a decrease on NO production in LPS-stimulated macrophages, by a direct scavenging effect of ●NO . Herewitl-arginine to equimolar amounts of NO and l-citrulline, the levels of these products can be used as markers of iNOS activity. Cells without LPS activation showed negligible activity, as expected (data not shown). As can be seen in N-methyl-l-arginine significantly inhibited iNOS enzyme activity at 25 µM, decreasing to the same extent NO and l-citrulline levels (p < 0.01). On the other hand, no significant differences were found with the pre-incubation with EnP (p > 0.05) (According to our method  and base > 0.05) , indicatp < 0.001).NF-κB has been shown to play an important role in regulating the expression of pro-inflammatory genes, including cytokines, chemokines, and adhesion molecules, which are involved in cell survival, immunity and inflammatory processes . In RAW •NO and/or to inhibit iNOS activity, we performed qPCR and western blotting to determine if the observed effects were related to iNOS expression. In addition and once LPS treatment also increased the mRNA and protein levels of COX-2, IL-6, and TNF-α, the effect of EnP on them was also explored.Since EnP was found to decrease NO production, without any capacity to scavenge p < 0.05)  by EnP at 10.4 µg/mL (25 µM) ( < 0.05) A. A simi < 0.05) , suggest < 0.05) B. Of not < 0.05) . Curious2 (PLA2), cyclooxygenase (COX), and lipoxygenase (LOX). Under inflammatory conditions, AA is released through the action of PLA2 upon the membrane phospholipids, being further metabolized via several different enzymatic machineries, namely the LOX and COX pathways, giving rise to a group of pro-inflammatory mediators called eicosanoids [2 and 5-LOX inhibition, EnP demonstrated no measurable activity at concentrations up to 20.8 µg/mL (50 µM) (data not shown). Curiously, the structurally similar 5α,8α-epidioxy-(24S)-ergosta-6-en-3β-ol, differing by the presence of an additional methyl group in C24, proved also to be ineffective against Apis mellifera bee venom PLA2 [p < 0.01), without notable inhibition of COX-1  pathway: phospholipase Aosanoids . The eicosanoids ,26. In rosanoids ,28. Thernom PLA2 . Recentlnom PLA2 . Howevernom PLA2 . On the of COX-1 . Even wiNoteworthy, a docking study carried out with sterols and triterpenoids proposed key structural requirements for COX binding, namely the presence of a tetracyclic skeleton, such as cholestane or stigmastane, the incorporation of an aliphatic chain in C17, and the presence of hydroxyl groups at C3 or C6, EnP fulfilling these molecular features . DespiteEscherichia coli, sodium pyruvate, sulfanilamide, MTT, Triton X-100, N-(naphth-1-yl)ethylenediamine dihydrochloride, β-nicotinamide adenine dinucleotide reduced form (NADH), sodium deoxycholate, dimethyl sulfoxide (DMSO), trizma hydrochloride, trypan blue, soybean lipoxygenase (LOX) from Glycine max (L.) Merr. , phospholipase A2 (PLA2) from honey bee (Apis mellifera) venom (EC.3.1.1.4), propan-2-ol, butan-1-ol, sodium nitroprusside (SNP), l-arginine monohydrochloride, diacetyl monoxime, antipyrine E, H2SO4, bovine serum albumin (BSA), chloroform, and N-methyl-l-arginine acetate salt were from Sigma-Aldrich . 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DL-PC) was from Larodan . Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS) and Pen Strep solution (penicillin 5000 units/mL and streptomycin 5000 µg/mL) were purchased from GIBCO, Invitrogen . β-Tubulin primary antibody, as well as anti-rabbit secondary antibody, were from Santa Cruz Biotechnology . PureZOL™ reagent was from Bio-Rad . KAPA SYBR® FAST qPCR Kit Master Mix (2X) Universal was from Kapa Biosystems . iNOS primary antibody was from Abcam (UK). COX-2 primary antibody, Qubit™ RNA HS assay kit, Qubit™ RNA IQ assay kit, SuperScript™ IV VILO™ Master Mix and primers were from Invitrogen by Thermo Fisher Scientific . WesternBright ECL HRP substrate was from Advansta . Potassium dihydrogen phosphate was purchased from Merck . Methanol was from Chem-Lab  and hexane from Fisher Chemical . Mouse IL-6 ELISA MAX™ Deluxe and Mouse TNF-α ELISA MAX™ Deluxe were purchased from BioLegend Inc. . COX fluorescent inhibitor screening assay kit was from Cayman chemical .LPS from A. depilans specimens were collected by hand at Praia da Memória , north Portugal, at a depth of 1–3 feet, in June 2015. Specimens were immediately placed on ice and transported to the laboratory. The opisthobranch mollusks were then washed with sea water and kept at −20 °C, until use. Species identification was performed by Prof. Alexandre Lobo-da-Cunha , Porto, Portugal).A. depilans specimens  were ground using an Ultra-Turrax homogenizer. The resulting biomass was exhaustively extracted with methanol, at room temperature. Afterwards, the extracting solution was filtered using a Büchner funnel and concentrated under reduced pressure. The remaining aqueous layer, derived from the water contained in the mollusks, was partitioned with hexane [h hexane  and evapA. depilans extract (2.0 g) was submitted to a flash chromatography on a silica gel column , using a gradient mixture of Hexane-EtOAc with increasing polarity, to afford eleven fractions (Fr1–Fr11). Fr6 (226.5 mg), eluted with 50% of hexane in EtOAc, was further fractionated by flash chromatography on a silica gel column , eluting with an isocratic mixture of hexane:EtOAc (40:60), to give nine sub-fractions (subFr1–subFr9). In order to exclude redundant compounds and/or chemical nullities, 1H-NMR-based dereplication of the sub-fractions allowed the detection of cholesterol and mixtures of common glycerolipids, containing some unsaturated fatty acids, which are extremely explored and recognized for their anti-inflammatory properties. The steroidal endoperoxide was detected in subFr5 and subFr7, and since there are no studies addressing its anti-inflammatory effects, these sub-fractions were selected. SubFr5 (11.9 mg) was separated by HPLC (Agilent HP 1100) (see chromatogram in 2O (98:2) and a flow of 1 mL/min to afford EnP . SubFr7 (22.2 mg) was separated by HPLC (Agilent HP 1100) . Total yield of EnP is 3.54 × 10−4% w/w.A part of the non-polar fraction of 1H-NMR  δ: 6.51 ; 6.24 ; 3.96 ; 2.11/1.90 ; 1.94/1.68 ; 1.83/1.52 ; 1.50/1.21 ; 1.49 ; 1.47 ; 1.21 ; 0.90 ; 0.88 ; 0.86 , 0.86 ; 0.80   δ: 135.4 ; 130.8 ; 82.1 ; 79.4 ; 66.5 ; 56.4 ; 51.6 ; 51.0 ; 44.7 ; 39.4 ; 36.9 ; 36.9 ; 35.9 ; 35.2 ; 34.7 ; 30.1 ; 28.2 ; 28.0 ; 23.8 ; 23.4 ; 22.8 ; 22.5 ; 20.6 ; 18.6 ; 18.2 ; 12.6  (See 13C-NMR spectrum in m/z (rel. int.) 416, ; 398, (7); 384, (100), 334, (80).EIMS (70 ev): 2.RAW 264.7 macrophages were provided by American Type Culture Collection . Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, and were incubated at 37 °C, in a humidified atmosphere of 5% COA. depilans extract/fractions/EnP were added and plates were incubated for 24 h. Afterwards, mitochondrial activity was evaluated by the ability of metabolically active cells to convert yellow tetrazolium MTT to formazan. Results are expressed as percentage of the respective control and correspond to the mean ± SEM of at least three independent experiments performed in triplicate.RAW 264.7 cells were seeded in 96-well plates at a density of 25,000 cells/well. After 24 h of growth, different concentrations of A. depilans extract/fractions/EnP, 20 µL of culture media were removed to a 96-well plate. Then, 25 µL of pyruvate was added to each well, followed by 230 µL of NADH. LDH released was evaluated at 340 nm, in a Multiskan GO plate reader , by monitoring the oxidation of NADH during the conversion of pyruvate to lactate. 1% Triton X-100 was used as positive control to assure cell lysis (30 min). All the results correspond to the fold-increase of absorbance in treated vs. untreated cells of, at least, three independent experiments performed in triplicate.LDH release into the media is usually used as a marker for loss of membrane integrity. To assess the release of this cytosolic enzyme, a protocol described by Silva et al. was followed . Briefly2. Then, the medium of each well was replaced by different concentrations of A. depilans extract/fractions/EnP or media only, followed by a stimulation with 1 µg/mL of LPS 2 h later. After 22 h challenge with LPS, the quantity of NO in cell culture medium was measured according to a previously described method [RAW 264.7 cells were cultured in 96-well plates (35 000 cells/well) and incubated for 24 h at 37 °C and 5% COd method . Briefly●NO generated from SNP was measured according to a previously described method [●NO scavenging capacity was determined adding 75 µL of Griess reagent and the absorbance was read at 560 nm after 10 min incubation in the dark. Three independent experiments were performed in triplicate.d method . Brieflyl-citrulline, after its action on l-arginine. RAW 264.7 cells (90000 cells/well) were seeded in 48-well plates and treated for 22 h with LPS (1 µg/mL). After this period of activation, which lead to an increased expression of iNOS enzyme, the culture medium was removed and cells were washed with HBSS and pre-incubated with different concentrations of EnP in HBSS  for 1 h. Simultaneously, N-methyl-l-arginine  was used as positive control. Then, l-arginine was added to each well, at a final concentration of 50 µM, and allowed to react during 2 h. Afterwards, the levels of NO and l-citrulline were determined using the Griess reagent and a previously described method [iNOS activity was evaluated based on the measurement of the two bio-products, NO and d method . The resg for 15 min at 4 °C. Following centrifugation, the aqueous phase containing the RNA was immediately transferred to a new RNase-free tube and 0.5 mL of isopropyl alcohol were added, and mixture was incubated at room temperature for 5 min. Afterwards, the tubes were centrifuged at 12,000× g for 10 min at 4 °C, the RNA appearing as a white pellet on the side and bottom of the tube. Supernatant was carefully discarded and the RNA pellet was washed with 1 mL of 75% of ethanol. After vortexing, the mixture was centrifuged at 7500× g for 5 min at 4 °C and the supernatant was carefully discarded. Then, the RNA pellet was air-dried for about 5 min and reconstituted in 50 µL of PCR grade water. Subsequently, the RNA was quantified in a Qubit 4 fluorometer , using the Qubit™ RNA HS assay kit. The RNA quality and integrity was then evaluated using the QubitTM RNA IQ assay kit. In order to obtain the complementary DNA, 1 µg of RNA was mixed with 4 µL SuperScript™ IV VILO™ Master Mix in a 20-µL reaction. The reverse-transcribed reaction involved three steps: incubation at 25 °C for 10 min, incubation at 50 °C for 10 min, and incubation at 85 °C for 5 min.RAW 264.7 cells (500000 cells/well) were seeded in 6-well plates and treated for 4 h with LPS (1 µg/mL), with or without pre-incubation with EnP for 2 h. Afterwards, the supernatant was removed and the cells were disrupted in 1 mL of PureZOL™ reagent. Then, the samples were transferred to a RNase-free tube and 0.2 mL of chloroform were added. The mixture was shaken vigorously for 15 s. After 5 min incubation at room temperature, the samples were centrifuged at 12,000× qPCR analysis were conducted on multiple genes, namely iNOS, COX-2, IL-6, and TNF-α . β-Actin® FAST qPCR Kit Master Mix (2X) Universal. The thermal cycling conditions were as follows: 3 min at 95 °C, followed by 40 cycles of denaturation at 95 °C for 3 s, specific annealing temperatures for each gene , and supernatants were frozen at −80 °C. BSA was used as reference in order to quantify the total protein in cell lysates by the Bradford method [® Turbo . Membranes were blocked for 1 h at room temperature with a solution of 5% skimmed milk powder in PBS 0.1% Triton X-100 and subsequently incubated overnight at 4 °C with specific antibodies against iNOS (1:200), COX-2 (1:2000) and β-tubulin (1:200). After washing, membranes were incubated with secondary antibody (1:1750) at room temperature for 1 h. Immunoreactive bands were visualized by adding WesternBright ECL HRP substrate and detecting the luminescent signal in a ChemiDoc™ Imaging System . The relative optical density of bands was quantified by densitometry and normalized with respect to β-tubulin (loading control). The results correspond to the mean ± SEM of three independent experiments.Western blot analysis was performed according to a previously described method . Brieflyd method . ProteinRAW 264.7 macrophages were seeded and treated for 16 h with LPS (1 µg/mL), with or without pre-incubation with EnP, as described in 2 inhibition assay was based on an indirect enzymatic method, which uses 5-LOX as coupling enzyme [2 (1.75 µg/mL) and 20 µL of 5-LOX (1.61 µg/mL) in 3 mM deoxycholate dissolved in 50 mM Tris-HCL buffer (pH 8.5) were mixed with 50 µL of different concentrations of EnP dissolved in the same buffer (3.125–50 µM). The reaction was initiated with the addition of 20 µL of DL-PC (PLA2 substrate) at 455 µM in 10 mM deoxycholate dissolved in 50 mM Tris-HCL buffer (pH 8.5). Then, the linoleic acid released through PLA2 activity was oxidized by 5-LOX at 37 °C, the increase in absorbance at 234 nm being followed spectrophotometrically in a Multiskan GO plate reader . Three independent experiments were performed in triplicate.The PLAg enzyme . BrieflyThe capacity of EnP to inhibit 5-lipoxygenase was assessed according to a previously described method . Briefly2 (PGG2) (product of COX activity) and 10-acetyl-3,7-dihydroxyphenoxazine (ADHP), which produces a highly fluorescent compound, resorufin. Briefly, 60 µL of assay buffer , 5 µL of hemin, 5 µL of enzyme (either COX-1 or COX-2), and 5 µL of EnP were added in a black 96-well plate. After 5 min of incubation at room temperature, 5 µL of ADHP and 20 µL of a solution containing arachidonic acid (0.5 mM) and KOH (2.5 mM) were added to each well. After 2 min at room temperature, the fluorescence of resorufin was monitored with an excitation wavelength between 530–540 nm and an emission wavelength between 585–595 nm. SC-560 and DuP-697 inhibitors were used as positive controls to COX-1 and COX-2 assay, respectively. The results correspond to the mean ± SEM of at least three independent experiments performed in duplicate.The assay was performed using the COX fluorescent inhibitor screening assay kit , with some modifications. It utilizes the peroxidase component of COXs and is based on the reaction between prostaglandin Gt-test where all columns of treatments were compared to the control. All data were expressed as mean ± SEM. Values of p < 0.05 were considered statistically significant.Data analysis was performed using GraphPad Prism 6.01 Software . Grubbs’ test was used to detect and exclude outliers. Prior to the analysis, the data set was checked for normality of distribution using the Shapiro–Wilk test, ensuring that all data followed a normal distribution. Levels of significance were determined using unpaired Student’s A. depilans, and performed the bioguided isolation of the steroidal endoperoxide, EnP. In addition to the purified extract, the EnP isolated from the most active sub-fraction demonstrated the ability to decrease the cellular NO levels in LPS-stimulated RAW 264.7 macrophages. Mechanistic studies evidenced that this decrease was due to a reduction in iNOS mRNA and protein expression. Moreover, EnP also decreased the mRNA and protein expression of other inflammation mediators involved in the NF-κB pathway, namely COX-2, IL-6, and TNF-α. Even with a non-significant decrease in COX-2 protein levels, the overall results suggest that EnP may act by an inhibitory effect upon the NF-κB activation. Furthermore, enzymatic assays carried out with multiples enzymes involved in the AA pathway showed a selective inhibition of COX-2 by the same molecule. The above-mentioned findings provided further clues on the potential of EnP as an anti-inflammatory agent.In this work we evaluated the anti-inflammatory activity of a purified extract of


To rapidly increase the total PD content, the β-glucosidase from Caldicellulosiruptor bescii was used for the deglucosylation of the PD precursors platycoside E (PE) and platycodin D3 (PD3) in the root extract into PD. Under the optimized reaction conditions, the enzyme completely converted the PD precursors into PD with the highest productivity reported so far, increasing the total PD content to 48% (w/w). In the biotransformation process, the platycosides in Platycodi radix were hydrolyzed by four pathways: deapiosylated (deapi)-PE → deapi-PD3 → deapi-PD, PE → PD3 → PD, polygalacin D3 → polygalacin D, and 3″-O-acetyl polygalacin D3 → 3″-O-acetyl polygalacin D.Platycodin D (PD), a major saponin (platycoside) in Platycodi radix , has higher pharmacological activity than the other major platycosides; however, its content in the plant root is only approximately 10% ( Platycodon grandiflorum, a common vegetable commonly known as “balloon flower”, has been used as a health food to make side dishes, desserts, teas, and flavored liquors in Northeast Asia. It has also been used as a traditional herbal medicine for its antibacterial activity ..9].Aspergillus niger [Aspergillus usamii [Deglycosylated saponins are better absorbed into the body and have a better biological effect than their glycosylated counterparts ,10. The us niger  converteus niger , snailasus niger , and lamus niger , converts usamii  also conCaldicellulosiruptor bescii was applied for the complete biotransformation of PE and PD3 in Platycodi radix extract into the bioactive PD.In this study, the enzymatic reaction conditions, such as pH, temperature, and enzyme concentration, were optimized for the effective production of PD. Under optimized conditions, a thermostable β-glucosidase from C. bescii has been used for the complete biotransformation of protopanaxadiol-type saponins into 20-O-β-glucopyranosyl-20(S)-protopanaxadiol (compound K) by deglycosylation because of its high hydrolytic activity. This enzyme is a non-specific enzyme that can hydrolyze not only glucose but also other monosaccharides such as galactose, arabinose, and xylose [C. bescii was cloned and expressed in Escherichia coli as described previously [C. bescii β-glucosidase hydrolyzed only the outer two glucose molecules at C-3 of the platycosides but not the inner glucose at C-3 and the other sugar moiety at C-28.β-Glucosidase from the hyperthermophilic bacterium d xylose . The β-geviously . Using hw/v) Platycodi radix extract was 2.51 mg/mL, whereas the concentrations of PE, polygalacin D, PD, platycodin A, and 3″-O-acetyl polygalacin D3 (as the main compounds) were 1.00, 0.73, 0.27, 0.17, and 0.16 mg/mL, corresponding to 39.74%, 29.14%, 10.60%, 6.77%, and 6.18% (w/w) of the total platycosides, respectively , PE (2), polygalacin D3 (5), and 3″-O-acetyl polygalacin D3 (9) at only the outer one or two glucose molecules at C-3. Thus, the enzyme-catalyzed biotransformation pathways of the platycosides in Platycodi radix were determined  → 3″-O-acetyl polygalacin D (11)  . To the C. bescii β-glucosidase for PE biotransformation at 70, 75, 80, 85, and 90 °C were 136, 30, 4.5, 0.07, and 0.03 h, respectively. The optimal reaction temperature was determined as 80 °C because the half-life of the enzyme at this temperature (4.5 h) was longer than the reaction time (2.3 h) for the biotransformation of platycosides in Platycodi radix.The hydrolytic activity of the purified β-glucosidase was measured over a pH range of 4.5–6.5 at 80 °C, using PE as the substrate. The maximum activity was observed at pH 5.5 , whereasA. usamii β-glucosidase for the conversion of PE and PD3 into PD were pH 6.0 and 40 °C, respectively [C. bescii β-glucosidase for ginsenoside Rb1 as a substrate occurred at pH 5.5 and 80 °C [C. bescii β-glucosidase was constant regardless of whether ginsenosides or platycosides were used as the substrate.The optimal pH and temperature of ectively . The optectively  and snaiectively  for simind 80 °C , which wnd 80 °C . TherefoTo investigate the effect of the enzyme concentration on the production of PD from reagent-grade PE, the concentration of β-glucosidase was varied from 0.01 to 0.2 mg/mL . PD prodThe purified β-glucosidase at 0.07 mg/mL completely converted 1 mg/mL reagent-grade PE to 0.79 mg/mL PD via PD3 within 1.7 h, with a molar yield of 100%, a total productivity of 465 mg/L/h, and a specific productivity of 6639 mg/g/h a. The enO-acetyl polygalacin D3 (9), and platycodin A (10)  , PE (2), deapi-PD3 (3), PD3 (4), polygalacin D3 (5), deapi-PD (6), PD (7), polygalacin D (8), 3″-n A (10) a. After n D (11) b.C. bescii β-glucosidase in this study was 160-fold higher than that of deapi-PD plus PD by snailase. The commercially available cellulase from Trichoderma reesei completely converted PE and PD3 in a Platycodi radix extract into 0.2 mg/mL PD after 24 h, with a total productivity of 0.85 mg/L/h [Cyberlindnera fabianii produced 0.17 mg/mL PD from platycosides in Platycodi radix extract after 72 h, with a total productivity of 2.42 mg/L/h and a molar yield of 43% [A. usamii β-glucosidase converted approximately 0.2 mM of PE and PD3 in Platycodi radix extract into 0.2 mM (0.24 mg/mL) PD within 2 h, with a total productivity of 122 mg/L/h and a specific productivity of 40.7 mg/g/h [C. bescii β-glucosidase were 3.0-fold and 18-fold higher than those of the extracellular A. usamii β-glucosidase , E. coli ER2566 , and pET-28a(+)  were used as the DNA template source of the β-glucosidase gene (GenBank accession number ACM59590), host cells, and expression vector, respectively [C. bescii DSM 6725 was cloned using the one-step isothermal assembly method as described previously.Genomic DNA from ectively . The β-gO-acetyl polygalacin D3 (90% purity) standards were purified from the Platycodi radix extract. Deapi-PD (89% purity) and 3″-O-acetyl polygalacin D (90% purity) were purified from the deglucosylated platycosides obtained from the Platycodi radix extract that had been biotransformed by C. bescii β-glucosidase. Preparation of the platycosides was carried out by preparative high-performance liquid chromatography (Prep-HPLC)  on an instrument equipped with a Hydrosphere C18 prep column , as described previously [The platycoside standards PE, deapi-PE, PD3, deapi-PD3, PD, polygalacin D3, and platyconic acid A were purchased from Ambo Laboratories . Polygalacin D was provided by Dr. Dae Young Lee of the National Institute of Horticultural and Herbal Science . The platycodin A 89% purity) and 3″-% purity E. coli expressing the β-glucosidase gene from C. bescii was cultured at 37 °C in a 2 L flask containing 500 mL of Luria-Bertani medium mixed with 100 μg/mL kanamycin, with agitation at 200 rpm on a shaker. Once the optical density of the culture had reached 0.8 at 600 nm, 0.1 mM isopropyl-β-d-thiogalactopyranoside was added to induce β-glucosidase expression. The cells were then further cultured for 14 h at 16 °C, with shaking at 150 rpm.Recombinant w/v) Platycodi radix extract was produced. The concentration of this extract was further diluted to 7.4% (w/v) in order to adjust the concentrations of PE to 1 mg/mL.Platycodi radix was prepared using the method of ginseng root extraction as described previously . One litE. coli cells expressing the β-glucosidase gene from C. bescii DSM 6725 were harvested from culture, washed with 0.85% NaCl, suspended in 50 mM citrate/phosphate buffer (pH 5.5), and disrupted by sonication on ice for 20 min. The unbroken cells and cell debris were eliminated by centrifugation at 13,000 ×g for 10 min at 4 °C, and the supernatant was incubated at 70 °C for 10 min. The heat-precipitated proteins were eliminated by centrifugation at 13,000 ×g for 10 min at 4 °C, and the supernatant was filtered using a 0.45-μm filter. The filtered solution was used as the purified enzyme.The recombinant C. bescii β-glucosidase toward PE, deapi-PE, PD3, deapi-PD3, PD, polygalacin D3, polygalacin D, and platyconic acid A was measured by reacting various concentrations of the enzyme (0.0003–0.07 mg/mL) with 0.4 mg/mL of each platycoside for 10 min at 80 °C and pH 5.5.Unless otherwise stated, the reaction was performed at 80 °C in 50 mM citrate/phosphate buffer (pH 5.5) containing 0.05 mg/mL β-glucosidase and 0.4 mg/mL platycoside for 10 min. The specific activity of C. bescii β-glucosidase for PE were examined by varying the pH from 4.5 to 6.5 at 80 °C, and by varying the temperature from 70 to 90 °C at a pH of 5.5, respectively. The effect of temperature on the stability of the enzyme was examined by pre-incubating the enzyme solutions at five different temperatures  for up to 20 h. Samples were withdrawn at regular time intervals and assayed by adding 0.05 mg/mL of the enzyme to citrate/phosphate buffer (pH 5.5) containing 0.4 mg/mL PE. The optimal enzyme concentration for the production of PD from platycosides in Platycodi radix extract was determined by varying the β-glucosidase concentration from 0.1 to 1.2 mg/mL. The reactions were carried out at 80 °C in 50 mM citrate/phosphate buffer (pH 5.5) containing β-glucosidase and 1 mg/mL PE and 0.04 mg/mL PD3 in 7.4% (w/v) Platycodi radix extract for 30 min.The effects of pH and temperature on the activity of C. bescii β-glucosidase was carried out for 3 h at 80 °C in 50 mM citrate/phosphate buffer (pH 5.5) containing 0.07 mg/mL β-glucosidase and 1 mg/mL PE. The biotransformation of PE and PD3 in the Platycodi radix extract was carried out for 4 h at 80 °C in 50 mM citrate/phosphate buffer (pH 5.5) containing 0.5 mg/mL β-glucosidase and 7.4% (w/v) Platycodi radix extract.Biotransformation of reagent-grade PE into PD catalyzed by v/v): from 10: 90 to 40: 60 for 30 min, from 40: 60 to 90: 10 for 15 min, from 90: 10 to 10: 90 for 5 min, and isocratic at 10: 90 for 10 min.To extract platycosides from Platycodi radix for HPLC analysis, an equal volume of n-butanol was added to the Platycodi radix powder solution containing digoxin as an internal standard. The n-butanol fraction of the extracted solution was evaporated to complete dryness, and methanol was added to the residue for HPLC analysis. The platycosides were analyzed using an HPLC system (Agilent 1100) equipped with a Hydrosphere C18 column  and an evaporation light scattering detector. The column was eluted for 55 min at 30 °C, at a flow rate of 1 mL/min, with the following gradient of acetonitrile: water (C. bescii hydrolyzed only the two outer glucose molecules, but not the inner glucose molecule at C-3 of platycosides and the other sugar moiety at C-28. Thus, the enzyme was applied to the increase the total PD content by the deglucosylation of Platycodi radix saponins. The reaction pH, temperature, and enzyme concentration were optimized for the production of PD. Under the optimized conditions, the enzyme completely converted PE and PD3 in Platycodi radix into PD with the highest productivity reported to date, suggesting that C. bescii β-glucosidase is an effective enzyme in increasing the PD content rapidly. Our results may contribute to an improvement in the industrial biotransformation of the bioactive PD from Platycodi radix extract.In this paper, β-glucosidase from


Lycium chinense, an important traditional medicinal plant. Sequence analyses showed that these genes exhibited high identities with their orthologous genes as well as having the same common features related to plant riboflavin biosynthetic genes. LcRIBA, like other plant RIBAs, contained a DHBPS region in its N terminus and a GCHII region in its C-terminal part. LcLSs and LcRS carried an N-terminal extension found in plant riboflavin biosynthetic genes unlike the orthologous microbial genes. Quantitative real-time polymerase chain reaction analysis showed that 4 riboflavin biosynthetic genes were constitutively expressed in all organs examined of L. chinense plants with the highest expression levels found in the leaves or red fruits. LcRIBA, which catalyzes 2 initial reactions in riboflavin biosynthetic pathway, was the highest transcript in the leaves, and hence, the richest content of riboflavin was detected in this organ. Our study might provide the basis for investigating the contribution of riboflavin in diverse biological activities of L. chinense and may facilitate the metabolic engineering of vitamin B2 in crop plants.Riboflavin (vitamin B2) is the precursor of flavin mononucleotide and flavin adenine dinucleotide—essential cofactors for a wide variety of enzymes involving in numerous metabolic processes. In this study, a partial-length cDNA encoding bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase (LcRIBA), 2 full-length cDNAs encoding lumazine synthase (LcLS1 and LcLS2), and a full-length cDNA encoding riboflavin synthase (LcRS) were isolated from  H)-pyrimidinone-5'-phosphate, which is then converted to 5-amino-6-ribitylamino-2,4-pyrimidinedione by several reactions involving deamination, side chain reduction, and dephosphorylation  × 6.022 × 1023. Real-time PCR was conducted using a 20-μL reaction mix that contained 5 μL of template cDNA, 10 μL of 1× SYBR Green Real-time PCR Master Mix , 0.5 μL of each primer (10 μM), and diethylpyrocarbonate water. Thermal cycling conditions were as follows: 95 °C for 5 min, and 40 cycles of 95 °C for 15 s, 56 °C for 15 s, and 72 °C for 20 s. Each run contained a series of standards and a negative control (containing water instead of cDNA). PCR products were analyzed using Bio-Rad CFX Manager 2.0 software . Three replications for each sample were used for the real-time analysis.On the basis of the sequences of LcRIBA, LcLS1, LcLS2, and LcRS, we designed qRT-PCR primers using the Primer3 website  Table 1Table 1.  et al. [L. chinense samples (0.1 g) by adding 0.5 mL of 0.1 N hydrochloric acid, vortex mixing for 20 s, and placing in a water bath at 80 °C for 30 min. After cooling, the pH was adjusted to 4–4.5 using 2 M sodium acetate, and then, 0.1 mL of freshly prepared 10% (w/v) takadiastase solution in water was added. The mixtures were placed in a water bath at 50 °C for 3 h and then heated at 80 °C for 5 min. After cooling, 0.3 mL of water was added, and the extracts were filtered. Riboflavin was separated on a C18 column  by using an HPLC instrument  equipped with a fluorometric detector . The compound was detected on the basis of the λex/λem at 422/515 nm. Elution was performed using a binary gradient of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B), according to the following program: 0 min, 95% A/5% B; 25 min, 65% A/35% B; 27 min, 5% A/95% B; 37 min, 5% A/95% B; 41 min, 95% A/5% B; and 51 min, 95% A/5% B. The flow rate was 1.0 mL/min, and the column temperature was 40 °C.Riboflavin was extracted according to the method of Esteve et al. , with a The data for gene expression and riboflavin content were analyzed using the computer software Statistical Analysis System . Treatment means were compared using Duncan’s multiple range test.To date, riboflavin is mainly produced by biotechnological fermentation using bacteria or yeasts for over-production ,34; ther


Their structures were elucidated by extensive analysis of spectroscopic data  and a molecular modeling study. The new isolates showed weak anti-bacterial activity, mild cytotoxicity against HCT116 cells, and anti-inflammatory effect in LPS/IFN-γ-stimulated RAW 264.7 macrophage cells.A new rare casbane-type diterpenoid  Lobophytum soft coral  in 1 with those of the known cis-fused casbane diterpenes + .Compound 2: Colorless oil; c 0.05 CH3OH); FT/IR νmax (film) 3481, 2932, 1295 and 1252 cm−1; 1H and 13C-NMR (CDCl3) data are listed in m/z 321.2418 [M + H]+ .Compound 3: Colorless oil; c 0.06 CH3OH); FT/IR νmax (film) 3465, 2930, 1254 and 1166 cm−1; 1H and 13C-NMR (CDCl3) data are listed in m/z 321.2419 [M + H]+ .Compound Implementation of the MM2 force field  in ChemB1–5, using the bacterial strains Staphylococcus aureus, Salmonella enterica and Escherichia coli. The strains were received from the Biological Resource Center , Japan and cultured in an agar medium containing polypeptone (10 g/L distilled water), yeast (2 g/L distilled), MgSO4·7H2O (1 g/L distilled) and agar (15 g/L distilled). The medium was autoclaved and transferred into petri dishes. The bacterial inoculum was evenly spread on the above agar medium. Each methanolic solution of the test compounds was perfused (25 µg/25 µL) to a sterilized disc . After the removal of the solvent, the disks containing test compounds were placed on seeded bacterial lawn on the agar surface. The plate was incubated for 2 days at 30 °C and then the inhibition zone sizes were measured.The paper disk diffusion method  was used2 atmosphere.HCT116 human colon cancer cells  and RAW 264.7 cells  were cultured in DMEM  medium  at 37 °C in a 5% CO1–3. Briefly, HCT116 cells were seeded at a density of 5.0 × 105 cells/mL in 96-well plate and cultured for 24 h with or without the test compound. After the culture, MTT (0.05%) was added to each well and incubated for 2 h, and then suspension was removed. Extraction with DMSO (50 μL) was measured at 540 nm with the reference at 655 nm using a microplate reader .The MTT assay was used to examine the cytotoxity of compounds 6 cells /mL) were treated with the compounds 1–3 in the presence of LPS (100 ng/mL), L-arginine (2 mM), and IFN-γ (100 U/mL) in 96-well microplate. Cells with or without LPS, IFN-γ and L-arginine were used as the positive control and the control, respectively. After culturing for 17 h, the nitrite concentrations in the medium were determined by previously reported method [The RAW 264.7 cells (2.5 × 10d method .p < 0.01) was analyzed by Student’s t-tests.Data were expressed as mean ± SD. Statistical significance  and comparisons with similar reported metabolites. The new isolates showed weak antibacterial activity, mild cytotoxicity against human colon cancer cells and showed anti-inflammatory effect in LPS/IFN-γ-stimulated RAW 264.7 macrophage cells.Seven diterpenoids


Bacillus amyloliquefaciens has been widely used as a probiotic in the field of biological control,and its antibacterial compounds plays an important role in the prevention and control of plant, livestock and poultry diseases. It has the advantages of green, safe and efficiency. This study aims to separate and purify active ingredient from Bacillus amyloliquefaciens GN59 and study its antibacterial activity. A novel compound was isolated from GN59 by column chromatography on silica gel and HPLC purification. The chemical structure was identified as α-d-glucopyranosyl-(1 → 1′)-3′-amino-3′-deoxy-β-d-glucopyranoside  on the basis of spectroscopic analysis. This is the first report about a,β-3-trehalosamine isolated from biological resources on an antibiotic activity against pathogenic bacterium. The 3′-neotrehalosamine displayed antibacterial activity across a broad spectrum of microorganisms, including different gram-positive and gram-negative bacteria, with minimal inhibitory concentration (MIC) values ranging from 0.5 to 0.7 mg/mL. The results indicated that the 3′-neotrehalosamine from GN59 might be a potential candidate for bactericide. Staphylococcus aureus -3′-amino-3′-deoxy-β-d-glucopyranoside  on the basis of spectroscopic analysis. This is the first isolated a,β-3-trehalosamine that has antibacterial activity from biological resources. Further, we characterized the antimicrobial activity properties of a,β-3-trehalosamine. The minimal inhibitory concentration (MIC) to eight selected strains were between 0.5 and 0.7 mg/mL.In the present study, we isolated a novel bactericidal compound from the fermentation liquid of Staphylococcus aureus ATCC6538, Micrococcus luteus CMCC280010, Escherichia coli CMCC44102, Salmonella pullorum CVCC533, C79-13, Salmonella gallinarum CVCC79201, C79-1, Salmonella gallinarum CVCC79207, C79-7, Pasteurella multocida CVCC474, C48-7 and Salmonella enteritidis ATCC13076. The strain of B. amyloliquefaciens was got from China General Microbiological Culture Collection Center. It was numbered as CGMCC 1.936, and named GN59 in this paper (http://www.cgmcc.net/index.html). GN59 was cultured in specific fermentation medium 2SO4, 0.5 g trisodium citrate, 0.1 g MgSO4·7H2O in 1 L distilled water, pH 7.0) and the media was autoclaved at 121 °C for 20 min. Liquid cultures were shaken at 180 rpm at 37 °C for 42 h. All other strains were grown on lysogeny broth agar (LB) plates at 28 °C and cultured at 37 °C in LB medium.Different pathogenic bacterial strains were used to test the antimicrobial activity of metabolites, including All the chemicals and reagents were analytical grade or highest available purity, and purchased from Sigma Chemical Co.7 CFU/mL of suspensions in 100 μL and wells of 2.5-mm diameter were formed by a sterile cork borer. 20 μL of the separated fractions were added into the wells. Specific solvents were the negative controls and the antibiotic ampicillin (15 mg/mL) for gram-negative bacteria and vancomycin (50 mg/mL) for gram-positive bacteria were used as the positive controls. The plates were incubated at 37 °C for 12 h and the activities were determined by measuring the diameter of the inhibition zone. The experiments were performed in triplicate.The agar-well diffusion method according to the National Committee for Clinical Laboratory Standards (NCCLS) was used to measured the antimicrobial activity of each fraction  was extracted with methanol  for three times (each time for 12 h) at room temperature to extract the active constituent. The combined solution was concentrated by rotary evaporator at 45 °C. Then the subfractions BA1–BA5 were isolated using silica column chromatography with petroleum ether/ethyl acetate  and the subfractions BA6–BA9 were isolated with ethyl acetate/methanol . The active fraction BA8 was further purified by semi-preparative High Performance Liquid Chromatography  using a YMC-Pack ODS-A column (250*20.0 mm) at a flow rate of 2.0 mL/min at 254 nm. Methanol  and H2O  were used as the mobile phase in a isocratic elution mode. All peak fractions were collected individually and concentrated via lyophilization to get six components, a , b  and c , d , e . Component c, d and e were found to contain glycoside components by TLC analyses (data are not shown). Then their antibacterial activity was detected to make sure the active constituent.The bacterial precipitate was removed by centrifugation at 80001H NMR 400 MHz and 13C NMR 100 MHz) on Bruker AMX400MHz Spectrometer  using deuterated chloroform (CDCl3) as solvent. Mass spectrum was carried out using Varian 1200 L Mass Spectrometer .The active compound of fermentation supernatant of GN59 was subjected to NMR and LC–MS analysis to obtain the chromatogram and the prospective mass spectra of the separated compound. NMR spectra was recorded  and minimum bactericidal concentration (MBC) of the antimicrobial compound from GN59 against eight tested pathogenic bacterium were determined by micro-broth dilution .S. aureus ATCC6538 and E. coli CMCC44102. The highest activity was observed at 42 h with the inhibition zone of 18.3 and 20.9 mm separately  showed high antibacterial activity. Other fractions didn’t have an antibacterial effect : δ 4.95 , 4.37 , 3.68 , 3.66–3.61 , 3.57–3.53 , 3.40–3.20 , 2.83  : δ 97.1 (C-1), 92.5 (C-1′), 76.9 (C-5), 76.9 (C-5), 75.1 (C-3), 73.3 (C-5′), 72.6 (C-3′), 72.1 (C-4), 0.8 (C-4′), 70.5 (C-2), 61.4 (C-6), 61.1 (C-6′) -3′-amino-3′-deoxy-β-d-glucopyranoside . It is identified as a new trehalosamine, which has the potential to be safe and environmentally friendly bactericide. This is the first report on antibacterial activity of a new trehalosamine from biological resources. The biologically sourced a,β-3-trehalosamine with antibacterial activity that we discovered represents a new antibacterial compound resource.The results revealed that the antibacterial compound of GN59 was proposed to be α-S. aureus ATCC6538, M. luteus CMCC280010, E. coli CMCC44102, S. pullorum CVCC533, C79-13, S. gallinarum CVCC79201, C79-1, S. gallinarum CVCC79207, C79-7, P. multocida CVCC474, C48-7 and S. enteritidis ATCC13076 -3′-amino-3′-deoxy-β-d-glucopyranoside ) from B. amyloliquefaciens GN59 fermentation broth. This component displayed strongly antimicrobial activity against most of the pathogenic bacterium. This is the first report on antibacterial activity of a new trehalosamine from biological resources. The biologically sourced trehalosamine with antibacterial activity that we discovered represents a new antibacterial component resource. It showed that the metabolites of probiotics are a potential source for the discovery of new beneficial substances. In addition, the a,β-3-trehalosamine from GN59 has the potential to be safe and environmentally friendly. Therefore, it may facilitate the potential application in the livestock and poultry farming.Bacillus are presented with different proteins and lipopeptides (Abriouel et al. Studies have found that a number of metabolites from microbial strains including lipopeptide, macrolide, surfactin (Santos et al. B. amyloliquefaciens produces a number of bioactive substances which have antibacterial activity, immunocompetence, antioxidant activity. So it is generally applied in animal husbandry and aquaculture. At the same time, it is expected to provide information for further exploration and application. The a,β-3-trehalosamine we found has strong activity and is easy to synthesize (Shazia et al. During the metabolic process, Additional file 1. Fig S1. Positive HR-ESI-MS data for active substance. Fig S2. 1H NMR spectrum active subsatnce in D2O. Fig S3.13C NMR spectrum of active substance in D2O.


Development of an efficient and scalable synthesis of 6-formylindolocarbazole (FICZ), a naturally-occurring aryl hydrocarbon receptor (AhR) ligand, allowed its biological and physical properties to be studied. FICZ was shown to be the most potent among a series of 6-substituted indolocarbazoles for activation of AhR in cells. Photostability studies of FICZ revealed a non-enzymatic mechanism for its conversion to a biologically active quinone. These results further support the hypothesis that FICZ is a light-dependent hormone that links sun exposure to regulation of biological pathways in peripheral tissues. Strong evidence for its formation in humans by the action of light on the skin and its metabolism by the AhR-inducible CYP1A have been presented4. As such, FICZ (1) fulfills many of the requirements for a hormone that links sunlight to regulation of peripheral biological rhythms5. Conversion of tryptophan to FICZ (1) in cells requires the action of visible or UV-light and is accelerated by the presence of hydrogen peroxide6. Notably, FICZ (1) in combination with UV-light also leads to the induction of oxidative stress and dermal toxicity7.6-Formylindolocarbazole FICZ,  is an im1) as light-dependent hormone with potential immune-modulating activity9, it is available in only limited quantities and at high cost from commercial vendors. The chemical synthesis of FICZ (1) was first described by Bergman using a low yielding route that required purification of the final product by sublimation under reduced pressure10, although subsequent improvements were made to facilitate the synthesis of several related carbazoles12. More recent syntheses were also completed on scales yielding only a few milligrams of product14. A key challenge in the synthesis is the difficult purification of the carbazole intermediates due to their poor solubility. To address the issue that no efficient large-scale synthesis FICZ (1) has been reported to date15, we developed an optimized procedure to produce it in gram quantities. We also determined the biological activity of several intermediate 6-substituted indolocarbazoles that illuminated the critical role of the 6-formyl group in activation of AhR. Finally, the availability of gram quantities of FICZ (1) led to the discovery of a previously unknown photo-decomposition pathway that may contribute to its biological role as a light-dependent hormone -1H-indole-3-carbaldehyde (4) afforded the desired bis-indole intermediate 5 in a good yield following chromatography.The readily available 1-(phenylsulfonyl)-1H-indole , Fig. 2 6 at 80% purity. The limited stability of 611 required use of the crude material without further purification. Reaction of 6 with excess ethyl oxalyl chloride did not yield the desired 3-acylated indole 7 but instead produced the double-acylated product 8 due to the NH group reacting as a vinylogous amide. Saponification of the crude 8 in 4N NaOH followed by heating in aqueous HCl gave a low yield of the known carboxylic acid CICZ (9). Fortunately, reaction of crude 6 with 0.95 equivalents of ethyl oxalyl chloride yielded pure mono-carboxy 7 in good yield following crystallization from acetonitrile. Ring closure and carbazole formation was achieved by heating 7 in dioxane with methanesulfonic acid followed by work up and crystallization to deliver pure carbazole ester 10. Borane-methyl sulfide complex was found to be the optimal reagent for reduction of the ester 10 due to the homogenous reaction mixture and simple work up to produce alcohol 11. Oxidation of 11 by DDQ yielded aldehyde 1, which was isolated by filtration. Following extensive washing with water, analytically pure FICZ (1) was produced as a yellow solid in high yield. The synthetic procedure . In this assay, the standard AhR ligand TCDD demonstrated an EC50 = 0.3 nM (data not shown). Using the cyp1a1 gene reporter assay, we determined the AhR agonist activity of FICZ (1) and several of its 6-substituted analogs (Table 1) was the most potent analog with an average EC50 of 10 nM, consistent with its reported potency for induction of IL-22 and inhibition of IL-17 in immune cells19. The unsubstituted carbazole ICZ (12), a known AhR ligand that is generated in the gut from dietary sources20, was only slightly less potent. The other three 6-substituted analogs (9–11) were much less potent than FICZ (1). Notably, CICZ (9) a naturally-occurring oxidation product of FICZ (1)2 was 40-times less potent as an AhR agonist.Although several indolocarbazoles have been reported as AhR ligands1) was stable as a pure solid, we observed decomposition of DMSO solutions upon standing under laboratory lighting. To more accurately measure the photostability of 1 by 1H NMR, a deuterated DMSO solution was placed in a clear Flint bottle inside a closed chamber and subjected to a light intensity of 11000 lux . Maleic acid was employed as an internal NMR reference. FICZ (1) decomposed rapidly in the presence of air and light with a half-life of around 3 h  by MS, 1H and 13C NMR  was included as a comparator, decomposition was at least 10-times slower under identical conditions  was formed as supported by 1H NMR and LCMS analysis. When the experiment was repeated in direct sunlight at a light intensity of 63000 lux, decomposition of FICZ (1) was even more rapid with a half-life of ~30 min  and explore the mechanism of photo-decomposition, DMSO solutions were subjected to 11000 lux over 28 d under different storage conditions  was completely decomposed within 1 day (Condition A). When the bottle was flushed with nitrogen to minimize the presence of air, decomposition of FICZ (1) was much slower (Condition B). In contrast, protection of the bottle from light exposure with aluminum foil further slowed, but did not prevent the decomposition (Condition C). Finally, removal of air and protection from light (Condition D) yielded the slowest rate of decomposition. Since the photo-reaction was highly dependent on the presence of air and light, we propose that the formation of quinone (13) from FICZ (1) proceeds by a radical chain reaction through an intermediate carbazole 6-carboperoxy radical   showed a wide range of stability as a solution in DMSO. Decomposition required the presence of air and was greatly accelerated by bright light. Importantly, we observed that air or light alone was sufficient to promote significant decomposition of FICZ (1) as a DMSO solution upon prolonged storage.Although FICZ  showed an EC50 value of 70 nM (Table 1) to the quinone (13) results in a ligand with at least 7-fold decreased potency for the activation of AhR (Table Using the hR Table . It rema1) demonstrates many of the properties of a light-dependent hormone that could entrain peripheral tissues through exposure to sunlight. FICZ (1) is formed by UV or light irradiation of tryptophan22 and, following activation of AhR, can increase expression of CYP1A1 to induce its own metabolism23. Our photostability experiments now reveal an alternative pathway to limit exposure of peripheral tissues to high levels of FICZ (1) through a non-enzymatic light and air-induced oxidation of the indolocarbazole.FICZ (1) was much more rapid than the unsubstituted analog ICZ (12), an alternate AhR ligand that is formed in the gut from dietary precursors but is not produced by light15. Although both naturally-occurring indolocarbazoles decomposed to the identical quinone (13), the presence of the aldehyde in FICZ (1) clearly accelerated photo-induced oxidation of the carbazole core. Aromatic aldehydes undergo photo-oxidation much more rapidly than their corresponding unsubstituted analogs likely due to the formation of intermediate benzoylperoxy radicals24. Photo-oxidation of FICZ (1) may proceed by an analogous radical chain process, given its dependency on light and air , whereas radical initiation from ICZ (12) would be much slower. However, this mechanism is only speculative given the absence of stable intermediates that were isolated in our studies.Notably, photodecomposition of FICZ  should be prepared immediately prior to biological testing and be shielded from air and light to ensure accurate results.Our stability experiments were conducted using light intensities comparable to normal daylight as well as in direct bright sunlight. We observed that photodecomposition was faster in the brighter light corresponding to direct sun exposure. Although regular laboratory lighting is generally 20–100 times less intense than daylight, our results dictate that solutions of FICZ (1) was first identified as a potent AhR ligand over 20 years ago1, there have been no systematic studies of its structure-activity for activation of the receptor. The reason may be due, in part, to a lack of an efficient synthesis of FICZ analogs. With access to a series of 6-substituted analogs, generated as intermediate compounds in our improved synthesis, we employed a cell-based reporter assay to explore the role of this key substituent. Cyp1a1 is a primary target gene of AhR in cells  and the unsubstituted carbazole ICZ (12) showed similar potency in cells. In contrast, the 6-substituted acid (9), ester (10), and alcohol (11) were each less active. Thus, the 6-formyl group in FICZ (1) appears to be the only one of these substituents that is well tolerated by the receptor. Although the quinone (13) was slightly less active than FICZ (1), it was still more potent as an AhR agonist than the other 6-substituted carbazoles.Although FICZ  was firs1) is a naturally-occurring AhR ligand that is formed in the skin25 and may be an important physiological regulator of the transcription factor9. Many dermatological conditions, such as atopic dermatitis and psoriasis, can be alleviated though judicious sun exposure26. In fact, therapeutic UV-B dosing is a common treatment for both conditions. FICZ (1) has demonstrated immune modulating effects through activation of AhR and is effective in treating animal models of psoriasis27. Pharmacological treatment of atopic dermatitis and psoriasis with topical AhR ligands has also been shown to be effective in human clinical trials29. Thus, the daily light-regulated level of FICZ (1) may have an important role in maintaining healthy skin30. Our results suggest the possibility that the combination of light exposure with free oxygen in cells provides an additional non-enzymatic mechanism for limiting the total levels of FICZ (1) in peripheral tissues.FICZ  to quinone (13) is part of a feedback system to protect skin from photoaging due to oxidative stress. It is notable that FICZ (1) is a photosensitizer that increases ROS levels in cells exposed to UVA7. Blue light has also been shown to increase H2O2 levels in cells and activate AhR signaling32. Our results raise the possibility that formation of quinone (13) may contribute to this process. FICZ (1) and its photoproduct (13) merit further research to explore their roles in the photobiology of skin health and aging due to sunlight.Quinones are highly redox active molecules that redox cycle with semiquinone radical anions resulting in the formation of hydrogen peroxide and reactive oxygen species (ROS). Electron rich quinones with extended ring systems, such as ing Fig. 31. It is1), which will allow investigators ready access to this important tool for the study of AhR biology in pharmacological models of disease. Our results provide support for the proposed role of FICZ (1) as a light-dependent switch for a ligand-dependent transcription factor in peripheral tissues, such as the skin. It is remarkable that nature has non-enzymatic light-facilitated mechanisms to both generate2 and remove this signaling molecule. Whether this process functions in humans to limit the concentration of FICZ (1) in skin under conditions of bright sunlight or protect the skin from the detrimental effects of ROS through formation of quinone (13) remains to be explored.In conclusion, we have developed the first efficient gram-scale synthesis of FICZ  and were used in accordance with the protocol described at https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2Fgeneblazer_cyp1a1blals0180_man.pdf, but adapted to run in 384-well plates.Starting materials, reagents, and solvents were obtained from commercial sources and used as received. ICZ was purchased from AstaTech. Intestinal human colon adenocarcinoma LS180 cells stably transfected with a β-lactamase reporter gene downstream of the 1H NMR and proton decoupled 13C NMR spectra were recorded on a Bruker 400-MHz spectrometer in CDCl3 or DMSO-d6 with TMS as the internal reference. Chemical shifts (δ) are reported in parts per million (ppm) relative to deuterated solvent as the internal standard , coupling constants (J) are in hertz (Hz). Peak multiplicities are expressed as follows: singlet (s), doublet (d), doublet of doublets (dd), triplet (t), quartet (q), multiplet (m), and broad singlet (br s). Melting points were recorded with a Büchi 520 apparatus and are uncorrected. Purification of intermediates and final products, if mentioned, was carried out on normal phase using an ISCO CombiFlash system and prepacked SiO2 cartridges eluted with optimized gradients of heptane−ethyl acetate mixture. Progress of the reactions was monitored by thin-layer chromatography (TLC) analysis  or by LCMS (described below). The purity of all products was established on a LCMS method:LC conditions: UPLC analysis was conducted on a Phenomenex Kinetex 1.7 um XB-C18 column at 40 °C. 0.2 uL of sample was injected using a partial loop with needle overfill injection mode. The gradient employed was: mobile phase A – water +0.2% v/v formic acid., mobile phase B – acetonitrile +0.15% v/v formic acid. UV detection was provided by summed absorbance signal from 210 to 350 nm scanning at 40 Hz. MS detection by Waters SQD LBA555 using alternating positive/negative electrospray with scan range = 125–1200 amu, scan time = 110 msec, and interscan delay = 50 msec.n-butyllithium in hexane  slowly over 15 min. The mixture was warmed to room temperature and stirred for 0.5–1 h. The reaction mixture became a slurry. It was cooled back to −78 °C and a solution of 1-(phenylsulfonyl)1-(phenylsulfonyl)-1H-indole-3-carbaldehyde  in 110 mL of THF was added dropwise over 20 min. The reaction mixture became a homogeneous solution. After stirring for 30 min with continued cooling by a dry ice-acetone bath, LCMS revealed the aldehyde had been consumed. The reaction was quenched by slowly adding 1 N HCl aq. solution (100 mL) and then allowed to warm to room temperature and stirred overnight. The reaction mixture was diluted with EtOAc (200 mL) and brine (200 mL). The organic was separated, and the aqueous phase was further extracted with EtOAc (100 mL). The combined organic (volume ~600 mL) was washed with brine, dried over MgSO4 and concentrated under reduced pressure to yield a crude brown liquid (81% purity) that was purified by chromatography on a 120 g ISCO silica gel column (20–40% EtOAc/heptane) to afford the title compound as a yellow foam . 1H NMR  δ: 8.17 , 8.03 , 7.92–7.97 , 7.79–7.85 , 7.74 , 7.55–7.62 , 7.43–7.51 , 7.30–7.38 , 7.21–7.27 , 7.07 , 6.94 , 6.55 , 6.23 , 3.71 . LCMS m/z: [M-OH]+  = 525.53, rt = 1.02 min.To a stirred solution of 1-(phenylsulfonyl)-1H-indole  in dry 2-methyltetrahydrofuran (200 mL) at −78 °C was added 1.6 M 4 and concentrated under reduced pressure to an amber oil that was used without further purification. A solution of the resulting crude 3-((13-((1H-indol-2-yl)methyl)-1H-indole  and pyridine  in dry THF (100 mL) was covered with aluminum foil and cooled to 0 °C. Ethyl oxalyl chloride  was dissolved in 25 mL of THF and added dropwise over a period of 60 min. The mixture was stirred at room temperature overnight and then partitioned between CH2Cl2 (250 mL) and 5% sodium bicarbonate (100 mL). The organic layer was separated, washed with brine, dried over MgSO4 and concentrated under reduced pressure. The residue was recrystallized in acetonitrile (~75 mL) to afford the title compound as a pale yellow solid (3.65 g). The filtrate was concentrated to half volume to provide a second batch (0.95 g). Total = 4.60 g, 52% yield. 1H NMR  δ: 8.44 , 8.28 , 7.97 , 7.50 , 7.24–7.33 , 7.13–7.23 , 4.69 , 4.51 , 1.46 . LCMS m/z: [M + H]+  = 347.34, rt = 0.85 min.To a stirred suspension of lithium aluminum hydride  in dry 2-methyltetrahydrofuran (250 mL) at −20 °C was added slowly a solution of (1-(phenylsulfonyl)-1H-indol-2-yl)(1-(phenylsulfonyl)-1H-indol-3-yl)methanol  in 100 mL of 2-methyltetrahydrofuran. The flask was covered with aluminum-foil, heated under gentle reflux for 6.5 h and then stirred at room temperature overnight. LCMS revealed the starting material (rt = 1.02 min) was nearly consumed and the product at rt = 0.89 min was a major peak. The reaction mixture was cooled to 0 °C, quenched by adding slowly 9.4 mL of water, 9.4 mL of 15% NaOH and 28.2 mL of water sequentially, stirred for 1 h and filtered through a Celite pad. The filter cake was washed with EtOAc (~500 mL). The filtrate was washed with brine two times, quickly dried over MgSO1H NMR  δ 13.53 , 11.48 , 10.99 , 8.93 , 8.46 , 8.26 , 7.72 , 7.53 , 7.42 , 7.05–7.25 . LCMS m/z: [M-H]+  = 299.24, rt = 0.79 min.Ethyl 2-(3-((3-(2-ethoxy-2-oxoacetyl)-1H-indol-2-yl)methyl)-1H-indol-1-yl)-2-oxoacetate  was dissolved in 1,4-dioxane (20 mL). 4 N sodium hydroxide  was added. The resulting mixture was heated at 90 °C for 3 h and cooled to room temperature. The organic solvent was removed in vacuo. The insoluble solids were removed by filtration. The filtrate was adjusted to pH 3–4 with 1 N HCl. A large amount of solid precipitated. LCMS revealed the cyclization had occurred, but was not complete. The suspension was heated at 90–100 °C for 1 h and cooled in an ice-water bath. The tan solid was collected by filtration, washed with water and dried to afford the crude title product. The crude material was dissolved in EtOAc and purified on a silica gel chromatography column (50% EtOAc/heptane). The solid obtained was suspended in hot MeOH, cooled, filtered, washed with MeOH and dried to afford the pure title compound as a yellow solid . 3 (250 mL) and stirred for 1 h. The solid was collected by filtration, washed with EtOAc and water, and dried in air flow to afford the title compound as a yellow solid (3.72 g). The organic was separated from the filtrate, washed with brine, dried over MgSO4 and concentrated under reduced pressure to give a second batch of the title compound (1.0 g). Total = 4.72 g, 99% yield. mp 175–177 °C (decomp.). 1H NMR  δ: 11.51 , 10.95 , 8.72 , 8.49 , 8.28 , 7.69 , 7.54 , 7.45 , 7.19 , 4.69 , 1.53 . 13C NMR  δ: 167.5, 142.1, 141.6, 136.2, 135.6, 126.7, 126.6, 125.4, 123.8, 122.1, 121.7, 120.8, 120.5, 119.1, 118.2, 112.0, 111.2, 107.2, 105.6, 61.5, 14.5. LCMS m/z: [M + H]+  = 329.52, rt = 0.99 min.Into a solution of ethyl 2-(2-((1H-indol-3-yl)methyl)-1H-indol-3-yl)-2-oxoacetate  was added slowly methanesulfonic acid . The resulting mixture was heated at 105 °C under gentle reflux for 30 min. LCMS revealed complete conversion to a single product. The mixture was cooled and concentrated under reduced pressure to a minimal volume. The residue was suspended in 5% NaHCO4 and concentrated under reduced pressure. The residue was suspended in hot EtOAc (20 mL), sonicated, cooled to room temperature and filtered to afford the title compound as a light yellow solid (0.63 g). The filtrate was concentrated, taken up in EtOAc (15 mL), sonicated, heated to boiling, cooled to room temperature, filtered and washed with 50% EtOAc/heptane to give a second batch of the title compound (0.26 g). Total = 0.89 g, 89% yield. mp >225 °C. 1H NMR  δ: 11.07 , 10.94 , 8.30 , 8.19 , 8.06 , 7.75 , 7.38 , 7.13 , 5.43 , 5.33 . 13C NMR  δ: 141.6, 141.6, 135.8, 134.3, 125.9, 125.4, 123.8, 123.0, 122.9, 122.9, 121.2, 120.6, 118.1, 118.0, 117.0, 111.1, 110.6, 100.1, 58.2. LCMS m/z: [M + H]+  = 287.29, rt = 0.78 min.Into a solution of ethyl 5,11-dihydroindolocarbazole-6-carboxylate  in THF (75 mL) was slowly added 2 M borane-methyl sulfide complex in THF  at 0 °C. The resulting homogenous solution was heated under reflux overnight. LCMS showed complete conversion to a single product. The reaction mixture was cooled to 0 °C, slowly quenched with water and then diluted with EtOAc. The organic layer was washed with brine, dried over MgSO3 and stirred overnight. The organic layer was separated and the aqueous was extracted with EtOAc two times. The combined organic was washed with 5% NaHCO3 and brine, dried over MgSO4 and concentrated. The residue was suspended in hot EtOAc, sonicated, cooled to room temperature, filtered, washed with fresh EtOAc and dried under reduced pressure to obtain a second batch of product (0.18 g). Total yield = 0.57 g, 90%. mp > 225 °C. 1H NMR  δ: 11.75 , 11.63 , 11.37 , 8.61 , 8.57 , 8.31 , 7.75 , 7.61 , 7.47 , 7.22 . 13C NMR  δ: 190.5, 142.1, 142.1, 135.8, 135.2, 126.9, 126.7, 125.1, 123.8, 122.1, 121.8, 121.5, 120.9, 119.7, 119.3, 112.8, 112.5, 111.9, 110.4. LCMS m/z: [M + H]+  = 285.25, rt = 0.91 min. HRMS calcd for C19H12N2O: 284.0950; Found: 284.0954.Into a solution of methanol  in 1,4-dioxane (50 mL) was added DDQ . The resulting mixture was stirred at room temperature overnight. The solid was collected by filtration, washed with dioxane, suspended in water (125 mL) and saturated sodium bicarbonate (75 mL) and stirred at room temperature for 2 h. The mixture was sonicated to break up any large blocks of solid. The solid was collected by filtration, washed with water and dried in a vacuum oven (~60 °C) to afford the first batch of FICZ as a yellow solid (0.39 g). The filtrate from the first collection was concentrated under reduced pressure, partitioned between EtOAc and 5% NaHCO3, stirred and filtered. The residue that was insoluble in CHCl3 was collected, washed with CHCl3, and dried to yield the title compound as a yellow solid . 1H NMR  δ: 12.92 , 8.07 , 7.54 , 7.38–7.28 . 13C NMR  δ: 176.3, 140.1, 137.9, 125.9, 124.5, 124.4, 121.8, 115.3, 114.5. LCMS m/z: [M + H]+  = 287.16, rt = 0.80 min.6-Formylindolocarbazole  was suspended in MeOH (15 mL) in a 500 mL Erlenmeyer flask and covered with plastic film to prevent evaporation. The flask was irradiated in a chamber with illuminance at 11000 lux with occasional shaking. LCMS indicated very slow decomposition due to the low solubility of FICZ in MeOH. After 10 days, LCMS revealed no residual FICZ was present. The suspension was concentrated under reduced pressure. The residue was suspended in CHCld6. The solution was analyzed by 1H NMR as time point zero. 0.5 mL of the solution was placed into separate ICH 20 mL Flint clear glass bottles that were irradiated in a photostability chamber with illuminance set to 11000 lux as measured by a digital light meter. The contents of separate bottles were checked by 1H NMR at 1 h, 2 h, 3 h, 4 h, 5 h, 7 h, 10 h, 16 h, and 24 h. Residual FICZ was quantified by the peak height of the CHO proton  and maleic acid  were dissolved in 5 mL of DMSO-d6. The solution was analyzed by 1H NMR as time point zero. 0.5 mL of the solution was placed into separate ICH 20 mL Flint clear glass bottles that were placed in direct sunlight with illuminance of 63000 lux as measured by a digital light meter . The contents of separate bottles were checked by 1H NMR at 1.5 min, 3 min, 10 min, 30 min, and 93 min. Residual FICZ was quantified by the peak height of the CHO proton (11.37 ppm) compared to the height of the vinyl proton of maleic acid (6.27 ppm).Method 2: FICZ (8.5 mg) and maleic acid (3.5 mg) were dissolved in 4 mL of DMSO-d6. The solution was analyzed by NMR as time point zero. The solution was then divided into four ICH 20 ml Flint clear glass bottles: Bottle A was exposed to air and was unprotected from the bright light; Bottle B: was flushed with nitrogen and left unprotected from the bright light; Bottle C was exposed to air but covered by aluminum-foil; and Bottle D was flushed with nitrogen and covered by aluminum foil. All bottles were placed in a photostability chamber with illuminance set to 11100 lux. The contents of each bottle were checked by 1H NMR at 1 d, 4 d, 8 d, 14 d and 21 d using the quantification procedure described in Method 1.Method 3: FICZ (14.4 mg) and maleic acid  were dissolved in 5 mL of DMSO-2. The plate was equilibrated at room temperature for 30 min before adding 10 μL of the LiveBLAzer™ FRET B/G substrate solution (Life Technologies) to each well. The plate was incubated for 90 min at room temperature in the dark before being read on a Perkin Elmer Envision using the bottom read fluorescence program with excitation at 400 nm and detection at 460/535 nm. Results for each test well were expressed as % inhibition using DMSO as the 0% control and 1 µM FICZ as the 100% control. In control experiments the standard AhR agonist TCDD demonstrated an EC50 = 0.31 nM, consistent with the vendor’s protocol.CYP1A1-bla-LS180 cells  were suspended in assay media  at a density of 20000 cells/mL. The compounds were dissolved in DMSO at a concentration of 10 mM. 0.5 mL of the compound solutions were added to a 384-well black, clear bottom, cell-coat assay plate (Griener Bio-one). A serial dilution at a ratio of 1:3 was made across the plate using assay media. A 50 μL aliquot of cell suspension was added to each well, and the compound plate was incubated overnight at 37 °C in 5% COSupplementary Information


The most relevant results were observed for Compounds 5 and 6–8 at 150 μM, which decreased dry biomass by 20% and 23%, respectively. These are significant results since they presented similar values with the positive control, commercial herbicide 3--1,1-dimethylurea (DCMU). Based on the performed assays, Compound 5 (graveoline) is classified as an electron-transport inhibitor during the light phase of photosynthesis, as well as a plant-growth regulator. On the other hand, Compounds 6–8 inhibited electron and energy transfers, and are also plant-growth inhibitors. These phytotoxic behaviors based on acridone and quinolone alkaloids may serve as a valuable tool in the further development of a new class of herbicides since natural products represent an interesting alternative to replace commercial herbicides, potentially due their low toxicity.Eight alkaloids ( Ruta graveolens L. (Rutaceae) is a medicinal plant whose roots and aerial parts contain more than 120 special metabolites as coumarins, flavonoids, acridones, and furoquinoline alkaloids  and 6 mM NH4Cl [Electron-flow activities were monitored by an oxygen monitor yellow spring instrument model 5300A using a Clark-type electrode. All reaction mixtures were illuminated with filtered light , then chloroplasts (20 μg Chl/mL) were added and illuminated for 1 min . 4Cl [Uncoupled PSI electron transport from the reduced DCPIP with sodium ascorbate to MV was determined in a similar form in a basal noncyclic electron-transport medium. However, the following reagents were added: 10 μM DCMU, 100 µM DCPIP, 50 μM MV, 300 μM sodium ascorbate, and 6 mM NHS. oleracea L. were resuspended in a solution of 0.35 M sorbitol, 2 mM EDTA, 1 mM MgCl2.6H2O, 1 mM MnCl2, and 50 mM Hepes medium at pH 7.6. H+-ATPase activity was measured as reported [4Cl and DMSO were employed as positive and negative controls, respectively. Pi was quantified using a UV spectrophotometer with measurements in λ = 660 nm.Intact chloroplasts isolated from reported . NH4Cl a2, 1.2 mM KH2PO4, 1.2 mM Na2SO4, 2.5 mM CaCl2, and 25 mM NaHCO3 (pH 7.4). The Petri dishes were maintained in orbital stirring for 12 h at room temperature. All alkaloids, 1–8, were added to the system for a new period of stirring (12 h). The discs were dark-adapted for 30 min and chlorophyll a fluorescence was measured at room temperature through a Hansatech Fluorescence Handy PEA  [Ten 7 mm leaf discs were placed in Petri dishes with 10 mL of a modified Krebs medium containing 115 mM NaCl, 5.9 mM KCl, 1.2 mM MgClynn, UK) ,25.Lolium perenne seeds prepared with 10% sodium hypochlorite solution was kept in an orbital shaker for 15 min. Then, the sodium hypochlorite solution was removed and the seeds were washed 3 times with distilled water; 100 seeds were placed in 12 cm diameter pots containing a mixture of 50:25:25 (w/w/w) soil/peat-moss/agrolite. All pots were watered daily and maintained in a greenhouse at 25–30 °C under normal day/night illumination (12/12 h). L. perenne plants were selected by uniformity after being 15 days old. The plants were separated in 3 groups: negative control (DMSO), positive control (50 µM of DCMU), and plants treated with each alkaloid at 150 and 300 µM [A suspension of a fluorescence was measured in leaves from the control plants and those treated with alkaloids 1–8 at 150 and 300 µM. After 24, 48, and 72 h of spraying, the leaves that adapted to the dark for 15 min were excited by light from an array of 3 light-emitting diodes delivering 3000 µmol m−2 s−1 of red light (650 nm). The Chl a fluorescence induction curves were measured at room temperature with a portable Hansatech Fluorescence Handy PEA apparatus. Photosynthetic parameters like as PIabs, dV/dt0, Sm, ABS/RC, TR0/RC, ET0/RC, TR0/ABS, ET0/TR0, ET0/ABS, PHI(D0), ABS/CS0, TR0/CS0, ET0/CS0, kp, kn, and Sumk were represented in a radar plot [L. perenne plants treated with alkaloids 1–8 at 150 and 300 µM were dried in an oven at 65 °C to reach a constant weight. Then, the dry biomass was measured using analytical balance [Chl dar plot . For the balance .