Identification of Antioxidative Hydrolyzable Tannins in Water Chestnut.

Despite the various biological activities exhibited by water chestnut (the fruit of the Trapa genus), the phenolic compounds present in its extract require comprehensive characterization. Accordingly, we analyzed a 80% methanol extract of commercially available water chestnut and identified a new hydrolyzable tannin dimer termed trapadin A. Additionally, 22 known compounds, including 10 hydrolyzable tannin monomers and 2 dimers, were also detected in the extract. Spectroscopic and chemical methods were used to elucidate the structure of trapadin A, revealing it to be a hydrolyzable tannin dimer formed from units of tellimagrandin II and 1,2,3,6-tetra-O-galloyl-ß-d-glucose. Moreover, the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity assay used to determine the half-maximal effective concentration values for the 23 compounds isolated from water chestnut indicated significant radical scavenging activity associated with hydrolyzable tannins. Notably, trapadin A, the new hydrolyzable tannin dimer, exhibited the highest activity value among the tested compounds.

Potent insecticidal activity of Eleocharis dulcis (Burm. f.) Trin peel extract and its main components against aphids.

BACKGROUND: Aphids are significant pests of cash crops and food farm crops. Botanical insecticides are safe for aphid control, especially for organic farming. In this study, Eleocharis dulcis (Burm. f.) Trin. peel extract (EDPE), a new botanical insecticide, was investigated for its active compositions against several agricultural aphids. RESULTS: The results showed that the EDPE had high insecticidal activity against Sitobion avenae Fabricius, Aphis gossypii Glover, Megoura crassicauda Mordvilko, and Acyrthosiphon pisum Harris, with half-lethal concentration (LC50 ) values of 95.92, 81.04, 140.31, and 255.73 mg/L after 48 h of treatment. In the pot culture assay, the aphicidal effects of 25% EDPE soluble liquid (SL) at a concentration of 0.016% were 68.98 ± 5.61%, 79.33 ± 8.27%, and 88.82 ± 3.91% after the first, third, and seventh days of treatment, respectively. Nine compounds were identified by bioactivity-directed fractionation: 4',5'-dimethoxy-6,6-dimethylpyranoisoflavone (1), 3-methoxy-4-hydroxylonchocarpin (2), 4-hydroxylonchocarpin (3), 4-methoxylonchocarpin (4), barbigerone (5), lonchocarpusone (6), 6a,12a-dehydrodeguelin (7), 13-homo-13-oxa-6a, 12a-dehydrodeguelin (8) and deguelin (9). Among them, 4-hydroxylonchocarpin (3) showed the highest aphidicidal activity against M. crassicauda, S. avenae, and A. pisum, with LC50 values of 97.24, 140.63, and 112.31 mg/L, respectively. CONCLUSION: These data contribute to a better understanding of the aphicidal activity of EDPE and its main component, 4-hydroxylonchocarpin. This will help to develop new botanical insecticides to contro aphids. © 2022 Society of Chemical Industry.

Physiological and microbiological hormesis in sedge Eleocharis palustris induced by crude oil in phytoremediation of flooded clay soil.

Soil contamination with petroleum hydrocarbons affects plants and rhizospheric microorganisms. Microbial activity participates in important biochemical processes that stimulate, together with plants, the modification of toxic compounds for organisms. A nine-month experiment was set up to study the effect over time of oil on plant height (cm), formation of new plants, plant matter production (gravimetry), and population of rhizospheric microorganisms (serial dilution) in the sedge Eleocharis palustris. Removal of total petroleum hydrocarbons (soxhlet and gravimetry) from the soil was also evaluated. The means of the evaluated variables registered significant statistical differences (Duncan, p < 0.05) regarding the age of the plant and the amount of crude oil. There was a high correlation between oil and plant height (0.848) and with new plants (0.994). 60 mg oil dose promoted the greatest statistical difference in the amounts of roots and plant biomass (p < 0.05). E. palustris exposed to 60 and 75 mg of oil stimulated high densities of microalgae, actinomycetes, fungi, hydrocarbonoclastic bacteria and Pseudomonas spp; the overall ratio was 2:1 relative to natural attenuation. Plant and microorganism variables evaluated registered physiological and microbiological hormetic indices ≥1, showing a positive linear relationship. Natural attenuation was more efficient in removing crude oil. We conclude that E. palustris is tolerant to oil exposure. It is suggested to combine it with natural attenuation for the optimization of soils contaminated with crude oil.

Effects of water chestnut (Tarpa bispinosa Roxb.) extract/lutein on fingertip-measured advanced glycation endproduct/carotenoid levels.

This pilot study tested the effects of the supplements containing water chestnut extract and carotenoids on antiglycation and carotenoid levels. Twenty Japanese subjects (mean age, 67 ± 7 years; 13 men) ingested 200 mg of Tarpa bispinosa Roxb. extract (containing >50 mg of polyphenols), 20 mg of lutein, and 3 mg of zeaxanthin daily for 3 months. Advanced glycation end product (AGEs) levels were estimated by fingertip skin autofluorescence using the AGEs Sensor; carotenoid levels were estimated by pressure-mediated reflection spectroscopy of the fingertips using the Veggie Meter. Compared to baseline, the mean AGEs score decreased significantly (0.55 ± 0.04 arbitrary units (AU) vs. 0.52 ± 0.07 AU, p = 0.03); the mean carotenoid score increased significantly (256 ± 68 optical density (OD) vs. 302 ± 109 OD, p = 0.02) at 3 months. Blood pressure, body weight, visual acuity, refractive error, and intraocular pressure were equivalent between baseline and 3 months. Compared to baseline, 13 (65%) patients had decreased AGEs scores, and 14 (70%) had increased carotenoid scores at 3 months; 9 (45%) subjects had both decreased AGEs scores and increased carotenoid scores, and two (10%) subjects had an inverse response. Co-administration of water chestnut extract and lutein for 3 months decreased the AGEs and increased the carotenoids estimated in the fingertip skin of humans.

[Studies on phenylpropanoids from Eleocharis dulcis and their hepatoprotective activities].

To study phenylpropanoids from Eleocharis dulcis and their hepatoprotective activities. The compounds were separated and purified from ethyl acetate part by conventional column chromatography and preparative liquid chromatography, and their structures were identified by various spectral techniques. The HL-7702 cells damage model of hepatocytes induced by APAP was used to screen and evaluate the hepatoprotective activities of these compounds. Sixteen compounds were isolated from ethyl acetate part of E. dulcis, and their structures were identified as 6'-(4″-hydroxy-3″-methoxy-phenylpropenyl)-1-(10-methoxy-phenylacetone)-1'-O-ß-D-glucopy-ranoside(1), susaroyside A(2), clausenaglycoside B(3), clausenaglycoside C(4), clausenaglycoside D(5), emarginone A(6), emarginone B(7), thoreliin B(8), 4-O-(1',3'-dihydroxypropan-2'-yl)-dihydroconiferyl alcohol 9-O-ß-D-glucopyranoside(9), 2-[4-(3-methoxy-1-propenyl)-2-methoxy-phenoxy]-propane-1,3-diol(10), 6'-O-(E-cinnamoyl)-coniferin(11), methyl 3-(2-O-ß-D-glucopyranosyl-3,4,5,6-tetramethoxyphenyl) propanoate(12), clausenaglycoside A(13), 9-O-(E-cinnamoyl)-coniferin(14), 6'-O-(E-cinnamoyl)-syringin(15), 2'-O-(E-cinnamoyl)-syringin(16). Among them, compound 1 was a new compound. Compounds 2-16 were isolated from this plant for the first time. Among them, compounds 2 and 8 showed certain hepatoprotective activities.

Studies on phenylpropanoids from Eleocharis dulcis and their hepatoprotective activities / 中国中药杂志

To study phenylpropanoids from Eleocharis dulcis and their hepatoprotective activities. The compounds were separated and purified from ethyl acetate part by conventional column chromatography and preparative liquid chromatography, and their structures were identified by various spectral techniques. The HL-7702 cells damage model of hepatocytes induced by APAP was used to screen and evaluate the hepatoprotective activities of these compounds. Sixteen compounds were isolated from ethyl acetate part of E. dulcis, and their structures were identified as 6'-(4″-hydroxy-3″-methoxy-phenylpropenyl)-1-(10-methoxy-phenylacetone)-1'-O-β-D-glucopy-ranoside(1), susaroyside A(2), clausenaglycoside B(3), clausenaglycoside C(4), clausenaglycoside D(5), emarginone A(6), emarginone B(7), thoreliin B(8), 4-O-(1',3'-dihydroxypropan-2'-yl)-dihydroconiferyl alcohol 9-O-β-D-glucopyranoside(9), 2-[4-(3-methoxy-1-propenyl)-2-methoxy-phenoxy]-propane-1,3-diol(10), 6'-O-(E-cinnamoyl)-coniferin(11), methyl 3-(2-O-β-D-glucopyranosyl-3,4,5,6-tetramethoxyphenyl) propanoate(12), clausenaglycoside A(13), 9-O-(E-cinnamoyl)-coniferin(14), 6'-O-(E-cinnamoyl)-syringin(15), 2'-O-(E-cinnamoyl)-syringin(16). Among them, compound 1 was a new compound. Compounds 2-16 were isolated from this plant for the first time. Among them, compounds 2 and 8 showed certain hepatoprotective activities.

Preventive Effects of Three Polysaccharides on the Oxidative Stress Induced by Acrylamide in a Saccharomyces cerevisiae Model.

Saccharomyces cerevisiae was used as a model to explore the preventive effect of two marine polysaccharides separately derived from Sepia esculenta ink (SIP) and Laminaria japonica (FL) as well as one terrestrial polysaccharides from Eleocharis tuberosa peel (WCPP) on toxic injury induced by acrylamide (AA). The growth of yeast was evaluated by kinetics indexes including doubling time, lag phase and maximum proliferation density. Meanwhile, intracellular redox state was determined by contents of MDA and GSH, and SOD activity. The results showed that AA inhibited yeast growth and destroyed the antioxidant defense system. Supplement with polysaccharides, the oxidative damage of cells was alleviated. According to the growth recovery of yeast, FL and WCPP had similar degree of capacity against AA associated cytotoxicity, while SIP was 1.5~2 folds as strong as FL and WCPP. SIP and FL significantly reduced production of MDA by AA administration. Moreover, SIP, FL and WCPP increased SOD activity and repressed GSH depletion caused by AA.

Structural characterization of polysaccharides with potential antioxidant and immunomodulatory activities from Chinese water chestnut peels.

Chinese water chestnut peels are a kind of vegetable processing waste containing many active components such as polysaccharides, the structure of which remains unknown. To elucidate the structure of polysaccharides from Chinese water chestnut peels, two polysaccharides named WVP-1 and WVP-2 were isolated. WVP-1 (3.16 kDa) consisted of mannose (1.75 %), glucose (84.69 %), galactose (6.32 %), and arabinose (7.24 %), while WVP-2 (56.97 kDa) was composed of mannose (3.18 %), rhamnose (1.52 %), glucuronic acid (1.42 %), galacturonic acid (4.83 %), glucose (11.51 %), galactose (36.02 %), and arabinose (41.53 %). Linkage and NMR data indicated that WVP-1 was composed mainly of →4)-α-d-Glcp(1→ and a certain proportion of →3)-ß-d-Glcp-(1→, including linear and branched polysaccharides simultaneously. WVP-2 was a pectin-like polysaccharide with →4)-α-d-GalpA6Me-(1→ units and the branch points of →3,4)-α-l-Arap-(1→, →3,6)-ß-d-Galp-(1→. WVP-2 exhibited stronger potential antioxidant and immunomodulatory activities than WVP-1 in vitro. These results provide a foundation for the further study of polysaccharides from Chinese water chestnut peels.

Development, characterization, and biocompatibility of zinc oxide coupled starch nanocomposites from different botanical sources.

Starch nanoparticles (SNP) were prepared from different botanical sources (wheat, potato, mung bean, water chestnut and mango kernels) and these were further coupled with zinc oxide (ZnO) to form starch nanocomposites. The nanocomposites were characterized for their particle size, morphological properties, energy dispersive X-ray spectroscopy (EDX) and their biocompatibility was analyzed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, using HeLa cells. From the morphological results, it was observed that ZnO forms super molecules with SNP. Further, EDX studies also confirmed the presence of zinc in coupled molecules. The size distribution of ZnO coupled SNP from different botanical sources revealed that the average diameter of nanocomposites ranged between 506 and 1209 nm. ZnO coupled starch nanocomposites were found to be biocompatible with 77-90% cell viability up to 24 h on HeLa cells. Among all botanical sources studied, ZnO coupled mung bean starch nanocomposite showed the highest cell viability (75% up to 50 h) while ZnO coupled potato starch nanocomposite showed the lowest cell viability (65% up to 50 h).

Structural characterization, neuroprotective and hepatoprotective activities of flavonoids from the bulbs of Heleocharis dulcis.

Chinese water chestnut, the bulb of Heleocharis dulcis, has been widely consumed as fruit or vegetable in China since ancient times. It exhibits health-promoting properties that leads to an extensive study of their active components. Successive chromatography of active fragments of H. dulcis resulted in isolation of five new chalcone-flavonone heterodimers (1-3, 6, 9), four new diverse flavonoids (4, 5, 7, 8), and sixteen known flavonoids derivatives (10-25) were elucidated on the basis of their IR, UV, NMR, MS spectrometry data analysis and references from H. dulcis for the first time. Among these isolates, compounds 4, 7, 9, 12, 13, and 17 showed moderate neuroprotective activity, which increased the cell survival rate from 49.23 ± 3.68% for the model to 67.75 ± 2.75%, 57.83 ± 2.46%, 67.98 ± 2.74%, 58.65 ± 3.43%, 56.14 ± 1.99%, and 56.70 ± 1.38% at 10 µM, respectively. Moreover, compounds 1-3, 15, 16, 18, and 20 were found to moderately improve the HepG2 cell survival rates from 39.53% (APAP, 10 mM) to 45.53-53.44%. The outcome of the study provided crucial information regarding the structural diversity and health benefits of the edible bulbs of H. dulcis.

Reintroducing plant coverage in a tropical wetland contaminated with oil and sulfate: rhizosphere effects on Desulfovibrio populations / Reintroducción de cobertura vegetal en un humedal tropical contaminado con petróleo y sulfato: efecto de la rizosfera sobre poblaciones de Desulfovibrio

Abstract The Mexican tropical wetland is a coastal system with capacity to support the contamination derived from the extractive industry and the transformation of crude oil, due to its high plant biodiversity and the presence of rhizospheric reducing sulphate bacteria from plants tolerant to crude oil. A field experiment was carried out for nine months to evaluate the adaptation of aquatic plants Leersia hexandra grass and Eleocharis palustris spikerush reintroduced in a wetland contaminated with 75 560 to 118 789 mg kg-1 of weathered petroleum and also with sulfate, derived from oil and gas pipeline leaks, and gaseous emissions. The effect of the weathered oil and sulfate on the dry matter production and the population density of the bacterium Desulfovibrio spp, isolated from the rhizosphere and soil of both plants, were evaluated. The means of the variables had statistical differences (p< 0.05). Weathered oil inhibited dry matter production of L. hexandra but not E. palustris; the effect of petroleum on Desulfovibrio density was very significant negative in the rhizosphere and in the soil of both plants. Sulfate reduced the dry matter of grass. The exposure of Desulfovibrio to sulfate significantly reduced its density in rhizosphere and soil (p< 0.01). We recommend the use of E. palustris for the decontamination of flooded soils contaminated with weathered oil and sulfate. Rev. Biol. Trop. 66(2): 908-917. Epub 2018 June 01.

Resumen El humedal tropical mexicano es un sistema costero con capacidad para resistir la contaminación derivada de la industria extractiva y la transformación del petróleo crudo, debido a su alta biodiversidad de plantas y la presencia de bacterias rizosféricas reductoras de sulfato de plantas tolerantes al petróleo crudo. Se realizó un experimento en campo durante nueve meses para evaluar la adaptación de las plantas acuáticas Leersia hexandra y Eleocharis palustris reintroducidas en un humedal contaminado con 75 560 a 118 789 mg kg-1 de petróleo intemperizado y también con sulfato, derivados de fugas de oleoductos y de emisiones gaseosas crónicas. Se evaluó el efecto del petróleo intemperizado y del sulfato en la producción de materia seca vegetal y en la densidad poblacional de la bacteria Desulfovibrio spp, aislada de la rizosfera y del rizoplano de ambas especies vegetales. Las medias de las variables tuvieron diferencias estadísticas (p< 0.05). El petróleo intemperizado inhibió la producción de materia seca de L. hexandra pero no de E. palustris; el efecto del petróleo en la densidad de Desulfovibrio fue negativo muy significativo en la rizosfera y en el rizoplano de ambas plantas. El sulfato redujo la materia seca de L. hexandra. La exposición de Desulfovibrio a sulfato redujo muy significativamente (p< 0.01) su densidad en rizosfera y en rizoplano. Recomendamos el uso de E. palustris para la descontaminación de suelos inundables afectados con petróleo intemperizado y con sulfato.

A systematic comparative study on morphological, crystallinity, pasting, thermal and functional characteristics of starches resources utilized in China.

In this study, starches from diverse botanical sources (arrowroot, cassava, Chinese yam, fern root, kidney beans, lotus seed, taro, and water chestnut) were isolated and examined for morphological, pasting, thermal, and physico-chemical characteristics in order to distinguish their end use potential. Among isolated starches significant differences (p < 0.05) were observed in gelatinization temperatures, morphology, color, pasting, and functional properties. Amylose content of isolated starches varied between 17.12% and 35.62%. X-ray diffraction pattern of isolated starches displayed A-type (arrowroot, cassava, corn, and kidney bean), B-type (potato) and C-type (water chestnut, taro, Chinese yam, fern root, and lotus seed) crystalline pattern. The FT-IR spectra of isolated starches confirmed their carbohydrate nature. Furthermore, the current study affords information for the exploitation of isolated starches from the diverse botanical sources cultivated in China that would be convenient for commercial applications.

Antitumor, Antioxidant, and Nitrite Scavenging Effects of Chinese Water Chestnut (Eleocharis dulcis) Peel Flavonoids.

The preparation, quantification, and characterization of flavonoid compounds from Chinese water chestnut peel (CWCP) flavonoid extract and ethyl acetate fraction (EF), n-butanol fraction, and water fraction were studied. Among these, EF showed the maximum free radical levels (IC50 values of 0.36, 0.40, and 0.37 mg/mL for DPPH•, ABTS•+ , and •OH, respectively), nitrite scavenging effects (IC50 = 1.89 mg/mL), and A549 cell inhibitory activities (IC50 = 776.12 µg/mL) with the highest value of total flavonoid content (TFC, 421.32 mg/g). Moreover, the contents of 8 flavonoids in this fraction were quantified using high-performance liquid chromatography, and fisetin, diosmetin, luteolin, and tectorigenin were the 4 major flavonoids with levels of 31.66, 29.91, 13.69, and 12.41 mg/g, respectively. Luteolin produced a greater inhibition of human lung cancer A549 cells (IC50 = 59.60 µg/mL) than did fisetin, diosmetin, and tectorigenin. Flow cytometry revealed that the cellular mechanisms of luteolin inhibition of A549 cells were achieved via the induction of cell proliferation arrest at G1 phase and apoptosis/necrosis. Our findings suggest that flavonoids are closely associated with antitumor, antioxidant, and nitrite scavenging effects of CWCP.

Natural attenuation of weathered oil using aquatic plants in a farm in Southeast Mexico.

An experiment was conducted in field for three years to assess the sustainability of aquatic plants Leersia hexandra, Cyperus articulatus, and Eleocharis palustris for use in the removal of total hydrocarbons of weathered oil in four areas contaminated with 60916-119373 mg/kg of hydrocarbons. The variables evaluated were coverage of plant, dry matter, density of plant growth-promoting rhizobacteria, and the removal of total weathered oil. The variables showed statistical differences (p = 0.05) due to the effects of time and the amount of oil in the soil. The three aquatic plants survived on the farm during the 36-month evaluation. The grass L. hexandra yielded the greatest coverage of plant but was inhibited by the toxicity of the oil, which, in contrast, stimulated the coverage of C. articulatus. The rhizosphere of L. hexandra in control soil was more densely colonized by N-fixing bacteria, while the density of phosphate and potassium solubilizing rhizobacteria was stimulated by exposure to oil. C. articulatus coverage showed positive relationship with the removal of weathered oil; positive effect between rhizosphere and L. hexandra grass coverage was also identified. These results contributed to the removal of weathered oil in Gleysols flooded and affected by chronic discharges of crude oil.

Isolation, purification and identification of etiolation substrate from fresh-cut Chinese water-chestnut (Eleocharis tuberosa).

Fresh cut Chinese water-chestnut is a popular ready-to-eat fresh-cut fruit in China. However, it is prone to etiolation and the chemicals responsible for this process are not known yet. To address this problem, we extracted phytochemicals from etiolated Chinese water-chestnut and separated them using MPLC and column chromatography. Four compounds were obtained and their structures were determined by interpretation of UV, TLC, HPLC and NMR spectral data and by comparison with reported data. We identified these compounds as eriodictyol, naringenin, sucrose and ethyl D-glucoside. Among those, eriodictyol and naringenin were both isolated for the first time in fresh-cut Chinese water-chestnut and are responsible for the yellowing of this fruit cutting.

Arbuscular mycorrhizal fungal diversity associated with Eleocharis obtusa and Panicum capillare growing in an extreme petroleum hydrocarbon-polluted sedimentation basin.

Arbuscular mycorrhizal fungi (AMF) have been extensively studied in natural and agricultural ecosystems, but little is known about their diversity and community structure in highly petroleum-polluted soils. In this study, we described an unexpected diversity of AMF in a sedimentation basin of a former petrochemical plant, in which petroleum hydrocarbon (PH) wastes were dumped for many decades. We used high-throughput PCR, cloning and sequencing of 18S rDNA to assess the molecular diversity of AMF associated with Eleocharis obtusa and Panicum capillare spontaneously inhabiting extremely PH-contaminated sediments. The analyses of rhizosphere and root samples over two years showed a remarkable AMF richness comparable with that found in temperate natural ecosystems. Twenty-one taxa, encompassing the major families within Glomeromycota, were detected. The most abundant OTUs belong to genera Claroideoglomus, Diversispora, Rhizophagus and Paraglomus. Both plants had very similar overall community structures and OTU abundances; however, AMF community structure differed when comparing the overall OTU distribution across the two years of sampling. This could be likely explained by variations in precipitations between 2011 and 2012. Our study provides the first view of AMF molecular diversity in soils extremely polluted by PH, and demonstrated the ability of AMF to colonize and establish in harsh environments.

Isolation, characterisation, and antioxidant activities of flavonoids from chufa (Eleocharis tuberosa) peels.

In this paper, chufa peels (Eleocharis tuberosa) were researched for the flavonoid profile for the first time. Twenty flavonoids were isolated and identified, including six new ones, named eleocharins A-F (1-6). Their structures were characterised by spectroscopic methods and compared with published data. The antioxidant activity of the acetone extract, EtOAc fraction, and nBuOH fraction of chufa peels as well as the isolated flavonoids were assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical bioassay. The results showed that chufa peels can be regarded as an excellent source of natural antioxidants (mainly flavonoids) and a good additive in the beverage and canning.

Compositional analysis of Chinese water chestnut (Eleocharis dulcis) cell-wall material from parenchyma, epidermis, and subepidermal tissues.

Chinese water chestnut (Eleocharis dulcis (Burman f.) Trin ex Henschel) is a corm consumed globally in Oriental-style cuisine. The corm consists of three main tissues, the epidermis, subepidermis, and parenchyma; the cell walls of which were analyzed for sugar, phenolic, and lignin content. Sugar content, measured by gas chromatography, was higher in the parenchyma cell walls (931 µg/mg) than in the subepidermis (775 µg/mg) or epidermis (685 µg/mg). The alkali-extractable phenolic content, measured by high-performance liquid chromatography, was greater in the epidermal (32.4 µg/mg) and subepidermal cell walls (21.7 µg/mg) than in the cell walls of the parenchyma (12.3 µg/mg). The proportion of diferulic acids was higher in the parenchyma. The Klason lignin content of epidermal and subepidermal cell walls was ~15%. Methylation analysis of Chinese water chestnut cell-wall polysaccharides identified xyloglucan as the predominant hemicellulose in the parenchyma for the first time, and also a significant pectin component, similar to other nongraminaceous monocots.

Δ(1)-pyrroline-5-carboxylate/glutamate biogenesis is required for fungal virulence and sporulation.

Proline dehydrogenase (Prodh) and Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5Cdh) are two key enzymes in the cellular biogenesis of glutamate. Recombinant Prodh and P5Cdh proteins of the chestnut blight fungus Cryphonectria parasitica were investigated and showed activity in in vitro assays. Additionally, the C. parasitica Prodh and P5Cdh genes were able to complement the Saccharomyces cerevisiae put1 and put2 null mutants, respectively, to allow these proline auxotrophic yeast mutants to grow on media with proline as the sole source of nitrogen. Deletion of the Prodh gene in C. parasitica resulted in hypovirulence and a lower level of sporulation, whereas deletion of P5Cdh resulted in hypovirulence though no effect on sporulation; both Δprodh and Δp5cdh mutants were unable to grow on minimal medium with proline as the sole nitrogen source. In a wild-type strain, the intracellular level of proline and the activity of Prodh and P5Cdh increased after supplementation of exogenous proline, though the intracellular Δ(1)-pyrroline-5-carboxylate (P5C) content remained unchanged. Prodh and P5Cdh were both transcriptionally down-regulated in cells infected with hypovirus. The disruption of other genes with products involved in the conversion of arginine to ornithine, ornithine and glutamate to P5C, and P5C to proline in the cytosol did not appear to affect virulence; however, asexual sporulation was reduced in the Δpro1 and Δpro2 mutants. Taken together, our results showed that Prodh, P5Cdh and related mitochondrial functions are essential for virulence and that proline/glutamate pathway components may represent down-stream targets of hypovirus regulation in C. parasitica.

Cascade utilization of water chestnut: recovery of phenolics, phosphorus, and sugars.

Overgrowth of aquatic plants, such as water chestnut, has been reported as a regional problem in various areas. We proposed cascade utilization of water chestnut through the recovery of phenolics, phosphorus, and sugars. Phenolics were extracted using 50 g (wet weight) of biomass with 300 mL of acetone, methanol, or hot water, and the yields of total phenolics were 80.2, 56.2, and 49.7 mg g(-1) dry weight of native biomass, respectively. The rate of eluted phosphorus in the phenolic extraction step was 8.6, 14.8, and 45.3 % of that in the native biomass, respectively, indicating that the use of polar organic solvents suppressed phosphorus elution at the phenolic extraction step. Extraction of phosphorus following the phenolic extraction was combined with alkaline pretreatment (1 % NaOH solution) of biomass for saccharification; 64.1 and 51.0 % of phosphorus in the native biomass were extracted using acetone and methanol for the phenolic extraction, respectively. Saccharification following the alkaline pretreatment showed that the glucose recovery rates were significantly increased (p<0.05) with the phenolic extraction step compared to alkaline pretreatment alone. This finding indicates that extraction of phenolics not only provides another useful material but also facilitates enzymatic saccharification.