Hepatoprotective Glucosyloxybenzyl 2-Hydroxy-2-isobutylsuccinates from Pleione yunnanensis.

Nine new glucosyloxybenzyl 2-hydroxy-2-isobutylsuccinates, pleionosides M-U (1-9), and 12 known compounds (10-21) were isolated from the pseudobulbs of Pleione yunnanensis. Their structures and absolute configurations were established through a combination of HRESIMS and NMR data and supported by physical and chemical methods. Compounds 5, 6, 10, and 15 showed significant in vitro hepatoprotective activity against d-galactosamine (d-GalN)-induced toxicity in HL-7702 cells with increasing cell viability by 27%, 22%, 19%, and 31% compared to the model group (cf. bicyclol, 14%) at 10 µM, respectively. Compounds 4, 9, and 11 exhibited moderate hepatoprotective activity against N-acetyl-p-aminophenol (APAP)-induced toxicity in HepG2 cells with increasing cell viability by 9%, 16%, and 12% compared to the model group (cf. bicyclol, 9%) at 10 µM, respectively.

Chemical Diversity of Bastard Balm (Melittis melisophyllum L.) as Affected by Plant Development.

The phytochemical diversity of Melittis melissophyllum was investigated in terms of seasonal changes and age of plants including plant organs diversity. The content of phenolics, namely: coumarin; 3,4-dihydroxycoumarin; o-coumaric acid 2-O-glucoside; verbascoside; apiin; luteolin-7-O-glucoside; and o-coumaric; p-coumaric; chlorogenic; caffeic; ferulic; cichoric acids, was determined using HPLC-DAD. Among these, luteolin-7-O-glucoside, verbascoside, chlorogenic acid, and coumarin were the dominants. The highest content of flavonoids and phenolic acids was observed in 2-year-old plants, while coumarin in 4-year-old plants (272.06 mg 100 g-1 DW). When considering seasonal changes, the highest content of luteolin-7-O-glucoside was observed at the full flowering, whereas verbascoside and chlorogenic acid were observed at the seed-setting stage. Among plant organs, the content of coumarin and phenolic acids was the highest in leaves, whereas verbascoside and luteolin-7-O-glucoside were observed in flowers. The composition of essential oil was determined using GC-MS/GC-FID. In the essential oil from leaves, the dominant was 1-octen-3-ol, whilst from flowers, the dominant was α-pinene.

Optimized Bioproduction of Itaconic and Fumaric Acids Based on Solid-State Fermentation of Lignocellulosic Biomass.

The bioproduction of high-value chemicals such as itaconic and fumaric acids (IA and FA, respectively) from renewable resources via solid-state fermentation (SSF) represents an alternative to the current bioprocesses of submerged fermentation using refined sugars. Both acids are excellent platform chemicals with a wide range of applications in different market, such as plastics, coating, or cosmetics. The use of lignocellulosic biomass instead of food resources (starch or grains) in the frame of a sustainable development for IA and FA bioproduction is of prime importance. Filamentous fungi, especially belonging to the Aspergillus genus, have shown a great capacity to produce these organic dicarboxylic acids. This study attempts to develop and optimize the SSF conditions with lignocellulosic biomasses using A. terreus and A. oryzae to produce IA and FA. First, a kinetic study of SSF was performed with non-food resources (wheat bran and corn cobs) and a panel of pH and moisture conditions was studied during fermentation. Next, a new process using an enzymatic cocktail simultaneously with SSF was investigated in order to facilitate the use of the biomass as microbial substrate. Finally, a large-scale fermentation process was developed for SSF using corn cobs with A. oryzae; this specific condition showed the best yield in acid production. The yields achieved were 0.05 mg of IA and 0.16 mg of FA per gram of biomass after 48 h. These values currently represent the highest reported productions for SSF from raw lignocellulosic biomass.

DISCOVER: A facile structure-based screening method for vinyl compound producing microbes.

Here we report a novel structure-based microbial screening method for vinyl compound discovery, DISCOVER (direct screening method based on coupling reactions for vinyl compound producers). Through a two-step screening procedure based on selective coupling reactions of terminal alkenes, the thiol-ene reaction (1st step of screening) and Mizoroki-Heck reaction, followed by iodine test (2nd step of screening), microbes producing vinyl compounds like itaconic acid (IA) can be isolated from soil samples. In the 1st step of screening, soil sources are plated on agar medium supplemented with an antimicrobial agent, α-thioglycerol (TG), and a radical initiator, VA-044 (VA). In the 2nd step of screening, vinyl compounds produced in the cultures are labelled with iodobenzene via the Mizoroki-Heck reaction, followed by an iodine test, leading to the detection and characterisation of labelled products. We evaluated the validity of DISCOVER using IA and its producer Aspergillus terreus. Experimental data supported our hypothesis that IA reacts with TG in the medium via the thiol-ene reaction and consequently, A. terreus rapidly forms colonies on the agar medium because of the loss of the antimicrobial activity of TG. Using DISCOVER, high throughput and selective isolation of A. terreus strains producing IA was possible from soils.

Dibutyl succinate, produced by an insect-pathogenic fungus, Isaria javanica pf185, is a metabolite that controls of aphids and a fungal disease, anthracnose.

BACKGROUND: An entomopathogenic fungus, Isaria javanica pf185, causes mortality in nymphs of the green peach aphid and inhibits the growth of fungal plant pathogens. However, the metabolites of pf185 involved in these antifungal and aphicidal activities are unknown. This study was performed to identify the metabolites with these activities. RESULTS: An antifungal metabolite was purified by repetitive column chromatography and preparative high-performance liquid chromatography. Based on data from mass spectrometry and nuclear magnetic resonance, the active metabolite was identified as dibutyl succinate. The minimum concentration of dibutyl succinate to inhibit germination of conidia of the cause of anthracnose, Colletotrichum acutatum, was 4 mg mL-1 . Dibutyl succinate at 2 µg mL-1 inhibited mycelial growth. It also had strong aphicidal activity against the nymphs of the green peach aphid, Myzus persicae, median lethal concentration (LC50) of 306 mg L-1 at 24 h exposure, and median lethal time (LT50) of 14.5 h at 388 mg L-1 exposure. CONCLUSION: This is the first report indicating that a single metabolite, dibutyl succinate, from the beneficial fungus I. javanica, has potential for use in integrated pest management to inhibit both insect and fungal plant pathogens. © 2018 Society of Chemical Industry.

Highly selective separation and purification of chicoric acid from Echinacea purpurea by quality control methods in macroporous adsorption resin column chromatography.

Chicoric acid is the main phenolic active ingredient in Echinacea purpurea (Asteraceae), best known for its immune-enhancing ability, as well as used as a herbal medicine. To achieve further utilization of medicinal ingredients from E. purpurea, an efficient preparative separation of chicoric acid was developed based on macroporous adsorption resin chromatography. The separation characteristics of several different typical macroporous adsorption resins were evaluated by adsorption/desorption column experiments, and HPD100 was revealed as the optimal one, which exhibited that the adsorbents fitted well to the pseudo-second-order kinetics model and Langmuir isotherm model, and the optimal process parameters were obtained. The breakthrough curves could be predicted and end-point could be determined early. Besides, the optimal elution conditions of chicoric acid can be achieved using the quality control methods. As a result, the purity of chicoric acid was increased 15.8-fold (from 4 to 63%) after the treatment with HPD100. The process of the enrichment and separation of chicoric acid is considerate, because of its high efficiency and simple operation. The established separation and purification method of chicoric acid is expected to be valuable for further utilization of E. purpurea according to product application in pharmaceutical fields in the future.

Xylarianins A-D from the endophytic fungus Xylaria sp. SYPF 8246 as natural inhibitors of human carboxylesterase 2.

Eighteen secondary metabolites were isolated from the fermentation broth of the endophytic fungus Xylaria sp. SYPF 8246, including four new compounds, xylarianins A-D (1-4), three new natural products, 6-methoxycarbonyl-2'-methyl-3,5,4',6'-tetramethoxy-diphenyl ether (5), 2-chlor-6-methoxycarbonyl-2'-rnethyl-3,5,4',6'-tetramethoxy-diphenyl ether (6), and 2-chlor-4'-hydroxy-6-methoxy carbonyl-2'-methyl-3,5,6'-trimethoxy-diphenyl ether (7), and eleven known compounds (8-18). Their structural elucidations were conducted by using 1D and 2D NMR, HRESIMS, and Rh2(OCOCF3)4-induced electronic circular dichroism (ECD) spectra analyses. The integrated 1H and 13C NMR data of three new natural products 5-7 were reported for the first time. All the isolated compounds were assayed for their inhibitory activities against human carboxylesterase 2 (hCE 2). Compounds 1, 5-9, and 18 displayed significant inhibitory activities against hCE 2 with IC50 values of 10.43 ±â€¯0.51, 6.69 ±â€¯0.85, 12.36 ±â€¯1.27, 18.25 ±â€¯1.78, 29.78 ±â€¯0.48, 18.86 ±â€¯1.87, and 20.72 ±â€¯1.51 µM, respectively. The interactions between compounds 1 and 5 with hCE 2 were anaylzed by molecular docking.

Optimization of a multiple headspace sorptive extraction method coupled to gas chromatography-mass spectrometry for the determination of volatile compounds in macroalgae.

A novel extraction technique is proposed in which the Multiple Headspace Extraction (MHE) approach is used in conjunction with Headspace Sorptive Extraction (HSSE) and Gas Chromatography-Mass Spectrometry (GC-MS) detection. The extraction method was developed to determine volatile compounds in macroalgae. Optimization of the extraction parameters was carried out using design of experiments to identify factors that affect the extraction: extraction time, temperature, twister length and amount of sample. The results of the optimization led to an extraction of 2 g of sample using a 20 mm Twister® at 66 °C for 180 min. The progression constants (ß) were calculated for 43 volatile compounds, 29 of which could be quantified using the method. Linearity was attained with a determination coefficient higher than 0.99 for all studied compounds. Inter-day and inter-twister precisions ranged from 0.22% to 19.01% and from 0.69% to 14.76% respectively, and values below 10% were obtained for the majority of compounds. LOD and LOQ values ranged from the values obtained for diethyl succinate (0.012 µg/L and 0.088 µg/L, respectively) and those obtained for dimethyl sulfide (5.544 µg/L and 40.286 µg/L, respectively). However, for the majority of compounds values obtained were below 1 µg/L (LOD) and 5 µg/L (LOQ). Compounds such as ethyl acetate, hexanal, heptadecane, 2-hexenal, 6-methyl-5-hepten-2-one, dimethyl sulfide, benzyl alcohol, beta ionone, or beta cyclocitral, among others were correctly determined in three species of macroalgae: Ulva sp., Gracillaria sp. and Enteromorpha sp.

Characterization of Polyphenolic Content in the Aquatic Plants Ruppia cirrhosa and Ruppia maritima -A Source of Nutritional Natural Products.

Herein, the polyphenolic content in extracts of Ruppia cirrhosa (Petagna) Grande and Ruppia maritima L.was fully characterized for the first time. High amounts of the main compound chicoric acid (CA) (≤30.2 ± 4.3 mg/g) were found in both Ruppia species. In addition, eight flavonoids, namely the 3-O-glucopyranosides and 3-O-galactopyranosides, as well as malonylated 3-O-glycosides of quercetin and isorhamnetin, were isolated and identified. The antioxidant activity of Ruppia cirrhosa extracts and isolated compounds was investigated spectrophotometrically by a 1,1-diphenyl-2-picrylhydrazyl (DPPH·) radical scavenging assay. IC50 values were 31.8-175.7 µg/mL for Ruppia cirrhosa extracts and 12.1-88.4 µg/mL for isolated flavonoids. Both individual and total phenolic and flavonoid content were quantified in crude extracts using analytical HPLC. The relative high amount of total flavonoids ranged from 5.9 to 14.7 mg/g in both species, with concentrations of individual flavonoids ranging from 0.4 to 2.9 mg/g dry weight. The content of chicoric acid was twofold more in Ruppia maritima than in Ruppia cirrhosa. Seasonal variation of the quantitative content in Ruppia cirrhosa was examined. Total flavonoid content ranged from 8.4 mg/g in October to 14.7 mg/g in August, whereas the highest concentration of chicoric acid was observed in March (29.2 mg/g).

Photocatalytic processes assisted by artificial solar light for soil washing effluent treatment.

Contaminated soil has become a growing issue in recent years. The most common technique used to remove contaminants (such as metals) from the soil is the soil washing process. However, this process produces a final effluent containing chelating agents (i.e., ethylenediaminedisuccinic acid, also known as EDDS) and extracted metals (i.e., Cu, Fe, and Zn) at concentrations higher than discharge limits allowed by the Italian and Brazilian environmental law. Therefore, it is necessary to develop further treatments before its proper disposal or reuse. In the present study, soil washing tests were carried out through two sequential paths. Moreover, different artificial sunlight-driven photocatalytic treatments were used to remove Cu, Zn, Fe, and EDDS from soil washing effluents. Metal concentrations after the additional treatment were within the Brazilian and Italian regulatory limits for discharging in public sewers. The combined TiO2-photocatalytic processes applied were enough to decontaminate the effluents, allowing their reuse in soil washing treatment. Ecotoxicological assessment using different living organisms was carried out to assess the impact of the proposed two-step photocatalytic process on the effluent ecotoxicity. Graphical Abstract ᅟ.

Enhanced Production of Itaconic Acid through Development of Transformed Fungal Strains of Aspergillus terreus.

Metabolic engineering with a high-yielding mutant, A. terreus AN37, was performed to enhance the production of itaconic acid (IA). Reportedly, the gene cluster for IA biosynthesis is composed of four genes: reg (regulator), mtt (mitochondrial transporter), cad (cis-aconitate decarboxylase), and mfs (membrane transporter). By overexpressing each gene of the IA gene cluster in A. terreus AN37 transformed by the restriction enzyme-mediated integration method, several transformants showing high productivity of IA were successfully obtained. One of the AN37/cad transformants could produce a very high amount of IA (75 g/l) in shake-flask cultivations, showing an average of 5% higher IA titer compared with the high-yielding control strain. Notably, in the case of the mfs transformants, a maximal increase of 18.3% in IA production was observed relative to the control strain under the identical fermentation conditions. Meanwhile, the overexpression of reg and mtt genes showed no significant improvements in IA production. In summary, the overexpressed cis-aconitate decarboxylase (CAD) and putative membrane transporter (MFS) appeared to have positive influences on the enhanced IA productivity of the respective transformant. The maximal increases of 13.6~18.3% in IA productivity of the transformed strains should be noted, since the parallel mother strain used in this study is indeed a very high-performance mutant that has been obtained through intensive rational screening programs in our laboratory.

Downstream process development in biotechnological itaconic acid manufacturing.

Itaconic acid is a promising chemical that has a wide range of applications and can be obtained in large scale using fermentation processes. One of the most important uses of this biomonomer is the environmentally sustainable production of biopolymers. Separation of itaconic acid from the fermented broth has a considerable impact in the total production cost. Therefore, optimization and high efficiency downstream processes are technological challenges to make biorefineries sustainable and economically viable. This review describes the current state of the art in recovery and purification for itaconic acid production via bioprocesses. Previous studies on the separation of itaconic acid relying on operations such as crystallization, precipitation, extraction, electrodialysis, diafiltration, pertraction, and adsorption. Although crystallization is a typical method of itaconic acid separation from fermented broth, other methods such as membrane separation and reactive extraction are promising as a recovery steps coupled to the fermentation, potentially enhancing the overall process yield. Another approach is adsorption in fixed bed columns, which efficiently separates itaconic acid. Despite recent advances in separation and recovery methods, there is still space for improvement in IA recovery and purification.

Genetic and biochemical insights into the itaconate pathway of Ustilago maydis enable enhanced production.

The Ustilaginaceae family of smut fungi, especially Ustilago maydis, gained biotechnological interest over the last years, amongst others due to its ability to naturally produce the versatile bio-based building block itaconate. Along with itaconate, U. maydis also produces 2-hydroxyparaconate. The latter was proposed to be derived from itaconate, but the underlying biochemistry and associated genes were thus far unknown. Here, we confirm that 2-hydroxyparaconate is a secondary metabolite of U. maydis and propose an extension of U. maydis' itaconate pathway from itaconate to 2-hydroxyparaconate. This conversion is catalyzed by the P450 monooxygenase Cyp3, encoded by cyp3, a gene, which is adjacent to the itaconate gene cluster of U. maydis. By deletion of cyp3 and simultaneous overexpression of the gene cluster regulator ria1, it was possible to generate an itaconate hyper producer strain, which produced up to 4.5-fold more itaconate in comparison to the wildtype without the by-product 2-hydroxyparaconate. By adjusting culture conditions in controlled pulsed fed-batch fermentations, a product to substrate yield of 67% of the theoretical maximum was achieved. In all, the titer, rate and yield of itaconate produced by U. maydis was considerably increased, thus contributing to the industrial application of this unicellular fungus for the biotechnological production of this valuable biomass derived chemical.

A New Succinate Derivative from Ajuga decumbens.

A new succinate derivative, ethyl (5-formylfuran-2-yl)methyl succinate (1), along with three known compounds (2-4) have been isolated from the whole plants of Ajuga decumbens Thunb. Their structures were elucidated by extensive spectroscopic (1D and 2D NMR) and HR-ESI-MS data analysis, and literature values. Compound 1 was isolated as a new succinate derivative, and compounds 2 and 3 were for the first time separated from A. decumbens.

Enantioselective extraction of phenylsuccinic acid in aqueous two-phase systems based on acetone and ß-cyclodextrin derivative: Modeling and optimization through response surface methodology.

A novel aqueous two-phase system (ATPS) composed of ß-cyclodextrin (ß-CD) derivative and acetone was developed for enantioselective extraction of racemic phenylsuccinic acid (PSA). Binodal curves, tie-lines, and critical points for the investigated ATPS were determined and the experimental tie-lines data were successfully correlated by Othmer-Tobias, Bancroft, and Setschenow-type equations. ATPS containing sulfobutyl ether-ß-CD (SBE-ß-CD) exhibited better enantioselectivity than that using carboxymethyl-ß-CD (CM-ß-CD). To optimize enantioselective partitioning conditions of PSA in acetone/SBE-ß-CD ATPS, three factors (PSA concentration, pH, and equilibrium temperature) were analyzed by using central composite design in response surface methodology. The calculated equilibrium constants of inclusion complexation are 1638.64M-1 for SBE-ß-CD-(R)-PSA and 835.84M-1 for SBE-ß-CD-(S)-PSA, respectively. Under the optimized conditions, the separation factor of 3.14 and high enrichment efficiency (ER=98.06%, ES=99.25%) were simultaneously achieved in a single step.

Model-based metabolic engineering enables high yield itaconic acid production by Escherichia coli.

Itaconic acid is a high potential platform chemical which is currently industrially produced by Aspergillus terreus. Heterologous production of itaconic acid with Escherichia coli could help to overcome limitations of A. terreus regarding slow growth and high sensitivity to oxygen supply. However, the performance achieved so far with E. coli strains is still low. We introduced a plasmid (pCadCS) carrying genes for itaconic acid production into E. coli and applied a model-based approach to construct a high yield production strain. Based on the concept of minimal cut sets, we identified intervention strategies that guarantee high itaconic acid yield while still allowing growth. One cut set was selected and the corresponding genes were iteratively knocked-out. As a conceptual novelty, we pursued an adaptive approach allowing changes in the model and initially calculated intervention strategy if a genetic modification induces changes in byproduct formation. Using this approach, we iteratively implemented five interventions leading to high yield itaconic acid production in minimal medium with glucose as substrate supplemented with small amounts of glutamic acid. The derived E. coli strain (ita23: MG1655 ∆aceA ∆sucCD ∆pykA ∆pykF ∆pta ∆Picd::cam_BBa_J23115 pCadCS) synthesized 2.27g/l itaconic acid with an excellent yield of 0.77mol/(mol glucose). In a fed-batch cultivation, this strain produced 32g/l itaconic acid with an overall yield of 0.68mol/(mol glucose) and a peak productivity of 0.45g/l/h. These values are by far the highest that have ever been achieved for heterologous itaconic acid production and indicate that realistic applications come into reach.

Phytochemical Analysis and Antioxidant Potential of the Phytonutrient-Rich Decoction of Cichorium spinosum and C. intybus.

The Cretan diet, as the basis of the Mediterranean diet, has provided traditional remedies for the general well being of people through the long-established consumption of cooked wild greens and vegetables. The intake of the water decoctions of Cichorium spinosum and Cichorium intybus in the context of the daily dietary regime in Greece has been long associated with "liver detoxifying" properties. In the current study, we performed an in-depth investigation of the water decoctions traditionally prepared from C. spinosum and C. intybus through qualitative UHPLC-HRMS profiling and direct quantification of cichoric and caftaric acid as major antioxidant components of the decoction. In addition, we developed a one-step countercurrent chromatography method for the isolation of the two phenolic acids, along with a sulfoconjugate sesquiterpene lactone present only in the Cretan C. spinosum. All water decoctions were found not to be cytotoxic in human fibroblasts, whereas they all significantly reduced the intracellular reactive oxygen species, which is consistent with the major presence of strong antioxidant compounds such as cichoric acid. This work demonstrates that the intake of these decoctions in doses suggested by Greek traditional use is comparable to the ingestion of a phytomedical preparation of antioxidants. These results contribute to our current knowledge on the beneficial health effect of the Cretan diet.

Sustainable production of valuable compound 3-succinoyl-pyridine by genetically engineering Pseudomonas putida using the tobacco waste.

Treatment of solid and liquid tobacco wastes with high nicotine content remains a longstanding challenge. Here, we explored an environmentally friendly approach to replace tobacco waste disposal with resource recovery by genetically engineering Pseudomonas putida. The biosynthesis of 3-succinoyl-pyridine (SP), a precursor in the production of hypotensive agents, from the tobacco waste was developed using whole cells of the engineered Pseudomonas strain, S16dspm. Under optimal conditions in fed-batch biotransformation, the final concentrations of product SP reached 9.8 g/L and 8.9 g/L from aqueous nicotine solution and crude suspension of the tobacco waste, respectively. In addition, the crystal compound SP produced from aqueous nicotine of the tobacco waste in batch biotransformation was of high purity and its isolation yield on nicotine was 54.2%. This study shows a promising route for processing environmental wastes as raw materials in order to produce valuable compounds.

Challenges in the production of itaconic acid by metabolically engineered Escherichia coli.

Metabolic engineering allows the production of a variety of high-value chemicals in heterologous hosts. For example, itaconic acid (IA) has been produced in several microorganisms, such as Escherichia coli, Aspergillus niger, and Synechocystis sp. through the expression of cis-aconitate decarboxylase gene (cad) from Aspergillus terreus. Recently, we showed that inactivation of the isocitrate dehydrogenase gene and overexpression of the aconitase gene dramatically enhanced the production levels of IA in E. coli expressing cad. Furthermore, we demonstrated that it is possible to produce IA directly from starch by engineered E. coli that additionally expresses the α-amylase gene from Streptococcus bovis. In this study, we sum up our findings regarding the challenges of IA production in E. coli.

Preparation and application of molecularly imprinted polymer for isolation of chicoric acid from Chicorium intybus L. medicinal plant.

Molecularly imprinted polymer (MIP) was synthesized and applied for the extraction of chicoric acid from Chicory herb (Chicorium intybus L.). A computational study was developed to find a suitable template to functional monomer molar ratio for MIP preparations. The molar ratio was chosen based on the comparison of the binding energy of the complexes between the template and functional monomers. Based on the computational results, eight different polymers were prepared using chicoric acid as the template. The MIPs were synthesized in a non-covalent approach via thermal free-radical polymerization, using two different polymerization methods, bulk and suspension. Batch rebinding experiments were performed to evaluate the binding properties of the imprinted polymers. The best results were obtained with a MIP prepared using bulk polymerization with 4-vinylpyridine (4-VP) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the crosslinker with a molar ratio of 1:4:20. The best MIP showed selective binding ability toward chicoric acid in the presence of the template's structural analogues, caffeic acid, caftaric acid and chlorogenic acid.