Microwave-assisted extraction of embelin from <Emphasis Type="Italic">Embelia ribes</Emphasis>

A rapid and efficient microwave-assisted extraction (MAE) process for the selective extraction of embelin from Embelia ribes was developed. Solvent selection, microwave energy input and solid loading were optimized. The rate of extraction and purity of embelin depended upon the solvent used and exposure time to microwaves. Maximum MAE was achieved in acetone with total yield of 92% (w/w) embelin with 90% (w/w) purity with 1% (w/v) raw material loading at 150 W power level in 80 s. Non-polar solvents, such as hexane and dichloromethane, were not effective for the selective extraction of embelin.     

Organic solvent tolerance of an alkaline protease from salt-tolerant alkaliphilic <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> strain Mit-1

A salt-tolerant alkaliphilic actinomycete, Mit-1 was isolated from Mithapur, coastal region of Gujarat, India. The strain was identified as Streptomyces clavuligerus and based on 16S rRNA gene sequence (EU146061) homology; it was related to Streptomyces sp. (AY641538.1). The organism could grow with up to 15% salt and pH 11, optimally at 5% and pH 9. It was able to tolerate and secrete alkaline protease in the presence of a number of organic solvents including xylene, ethanol, acetone, butanol, benzene and chloroform. Besides, it could also utilize these solvents as the sole source of carbon with significant enzyme production. However, the organism produced spongy cell mass with all solvents and an orange brown soluble pigment was evident with benzene and xylene. Further, the enzyme secretion increased by 50-fold in the presence of butanol. With acetone and ethanol; the enzyme was highly active at 60–80°C and displayed optimum activity at 70°C. The protease was significantly stable and catalyzed the reaction in the presence of xylene, acetone and butanol. However, ethanol and benzene affected the catalysis of the enzyme adversely. Crude enzyme preparation was more stable at 37°C in solvents as compared to partially purified and purified enzymes. The study holds significance as only few salt-tolerant alkaliphilic actinomycetes are explored and information on their enzymatic potential is still scares. To the best of our knowledge this is the first report on organic solvent tolerant protease from salt-tolerant alkaliphilic actinomycetes.     

Chemical composition and antibacterial activity of Cordia verbenacea extracts obtained by different methods

The present study describes the chemical composition and the antibacterial activity of extracts from Cordia verbenacea DC (Borraginaceae), a traditional medicinal plant that grows widely along the southeastern coast of Brazil. The extracts were obtained using different extraction techniques: high-pressure operations and low-pressure methods. The high-pressure technique was applied to obtain C. verbenacea extracts using pure CO₂ and CO₂ with co-solvent at pressures up to 30 MPa and temperatures of 30, 40 and 50 °C. Organic solvents such as n-hexane, ethyl acetate, ethanol, acetone and dichloromethane were used to obtain extracts by low-pressure processes. The antibacterial activity of the extracts was also subjected to screening against four strains of bacteria using the agar dilution method. The extraction yields were up to 5.0% w/w and up to 8.6% w/w for supercritical fluid extraction with pure CO₂ and with ethyl acetate as co-solvent, respectively, while the low-pressure extraction indicates yields up to 24.0% w/w in the soxhlet extraction using water and aqueous mixture with 50% ethanol as solvents. The inhibitory activity of the extracts in Gram-positive bacteria was significantly higher than in Gram-negative. The quantification and the identification of the extracts recovered were accomplished using GC/MS analysis. The most important components identified in the extract were artemetin, β-sitosterol, α-humulene and β-caryophyllene, among others.     

An efficient method for the application of PHA‐poor solvents to extract polyhydroxybutyrate from Cupriavidus necator

There are many published studies presenting ethanol and acetone as PHAs‐poor solvents, where these two solvents are shown to dissolve <2% (w/v) of PHAs at low temperatures. In this study, the suitability of ethanol and acetone for the recovery of PHB at different temperatures (from room temperature to near boiling point) in Cupriavidus necator was investigated. Experiments were performed using response surface methodology to examine the effects of different temperatures and heating incubation times on recovery percentage using the two solvents. The highest recovery percentage (92.3%) and product purity (up to 99%) were obtained with ethanol‐assisted extraction at 76°C for 32 min of incubation time. Under these conditions the extracted PHB exhibited a molecular mass of 1.2 × 10⁶. The present strategy showed that at temperatures near its boiling point, ethanol, as a nonhalogenated solvent, represents a good alternative to halogenated solvents, like chloroform, when PHB recovery is concerned. DSC analysis showed good thermal properties for ethanol‐ and acetone‐extracted biopolymers. GC and ¹H NMR analysis confirmed the extracted biopolymer to be polyhydroxybutyrate of good purity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1480–1486, 2016     

Degradation of ribonucleic acid in Candida lipolytica: extraction of ribonucleic acid degrading enzymes

We developed an efficient and simple method for RNase extraction from Candida lipolytica cells which consists of predrying the cells with solvents and incubating them for 8 to 15 hr at 37 to 45°C in a slightly acid buffer which contains EDTA or salts. This method is called Solvent Dehydration Buffer Extraction (SDBE) procedure. Predrying with acetone or ethanol, or by lyophilization, followed by washing with acetone or ethylacetate gives the most efficient RNase extraction. The yield and specific activity obtained by this extraction procedure are higher than by any other method examined. An apparent 1.5- to 2.0-fold activation of RNase occurred during the SDBE process. Activation of RNase in homogenates obtained by grinding fresh cells is also observed with EDTA or acetate buffer. The SDBE procedure works efficiently regardless of growth phase for Candida lipolytica, and works also with other Candida yeasts.     

Chemical analysis of phenolic glycosides: art,facts, and artifacts

Summary Phenolic glycosides have been the subject of considerable interest in recent ecological and systematic studies, especially those involving the Salicaceae. But these compounds are markedly labile in aqueous media, and the consequences of spontaneous degradation for valid interpretation of results have been largely ignored by researchers. We found that freeze-drying and oven-drying of leaf samples from several Populus and Salix species produced dramatic changes in the total and relative concentrations of specific phenolic glycosides, when compared to analyses of fresh material. Extraction in aqueous and alcoholic media for extended (24 h) periods also effected changes in glycoside concentrations. Alterations in phenolic glycoside concentrations, interconversions among glycosides, and production of artifactual glycosides result from a series of hydrolytic reactions. These deleterious effects can be best (but not entirely) avoided by the use of fresh plant material, cold, nonaqueous extraction solvents, and short extraction times. Because individual phenolic glycosides exhibit very different biological activities against herbivores, we caution ecologists to use utmost care in the performance and interpretation of phenolic glycoside assays.     

Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis

Alexandrium is a wide-spread genus of dinoflagellate causing harmful algal blooms and paralytic shellfish poisoning around the world. Proteomics has been introduced to the study of Alexandrium, but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of dinoflagellates. In this study we compared four protein preparation methods for the two-dimensional electrophoresis (2DE) analysis of A. tamarense: (1) urea/Triton X-100 buffer extraction with trichloroacetic acid (TCA)/acetone precipitation; (2) direct precipitation with TCA/acetone; (3) 40 mM Tris (hydroxymethyl) aminomethane (Tris) buffer extraction; and (4) 50 mM Tris/5% glycerol buffer extraction. The results showed that, among the four protein preparation methods, the method combining the urea/Triton X-100 buffer extraction and TCA/acetone precipitation allowed detection of the highest number and quality of protein spots with a clear background. Although the direct TCA/acetone precipitation method also detected a high number of protein spots with a clear background, the spot number, separation and intensity were not as good as those obtained from the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method. The 40 mM Tris buffer and 50 mM Tris/5% glycerol buffer methods allowed the detection of fewer protein spots and a pH range only from 4 to 7. Subsequently, the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method was successfully applied to profiling protein expression in A. catenella under light stress conditions and the differential expression proteins were identified using MALDI TOF–TOF mass spectrometry. The method developed here appears to be promising for further proteomic studies of this organism and related species.     

Evaluation and improvement of spectrophotometric assays of TTC reduction: maize (Zea mays) embryo as an example

The triphenyl tetrazolium chloride (TTC) reduction assay was evaluated and improved with maize seed (Zea mays cv. Zhengdan958). The reduced TTC in embryo was extracted with three kinds of organic solvents: trichloroacetic acid (TCA)/acetone, ethanol, and acetone. The absorbance spectra of the three extracts were similar, with a maximum at 485 nm. The efficiency of TCA/acetone in extracting the reduced TTC was higher than that of acetone and ethanol. A negative correlation between TTC reduction and malondialdehyde content in embryo was demonstrated. The TCA/acetone extraction may be used as a routine protocol for TTC reduction assay of seed vigor in cereal (e.g. maize, rice, wheat and barley) seeds.     

西花蓟马蛋白的提取及双向电泳体系的建立

【目的】蛋白样品的制备是获得良好双向凝胶电泳(2-DE)图谱的前提,建立合理的西花蓟马蛋白的双向电泳体系,获得分辨率较高、重复性较好的图谱,能够为后续的研究提供有力支撑。【方法】实验以西花蓟马成虫为实验材料,对比了饱和酚法、TCA/丙酮法和直接裂解法3种蛋白提取方法,从中选出最适宜双向电泳分析的一种蛋白提取方法。【结果】3种方法蛋白提取率差异显著,直接裂解法蛋白提取率最高,饱和酚法的蛋白提取率最低;3种方法的SDS-PAGE条带数差异不明显;TCA/丙酮法的双向凝胶图谱效果最好,蛋白点最多。【结论】TCA/丙酮法能够有效去除西花蓟马蛋白中的干扰物质,是最适合西花蓟马双向凝胶电泳的蛋白提取方法,为后续西花蓟马在蛋白组学方面的研究奠定了基础。     

Extractability of polyphenols of sunflower seed in various solvents

The extractability of chlorogenic acid from defatted sunflower seed flour in water and salt solutions at different pH values and also in aqueous organic solvents was determined. It increased with increase in pH and at pH 8 in water nearly 70% chlorogenic acid was removed in a single extraction, while NaCl did not increase the extraction, and, MgCl₂ and CaCl₂ increased it, especially at higher concentrations. Methanol, ethanol, isopropanol and acetone, at 20% concentration in water, caused the maximum extraction of polyphenol. These organic solvents without added water were poor solvents for the extraction of polyphenol from the flour.     

Extraction of crystal violet-iodine complex from gram positive bacteria by different solvents and its implication on gram differentiation

Summary Crystal violet from Gram stained S. aureus can be extracted completely by 95% ethanol if the stained bacteria is pre-treated with dilute sodium thiosulphate solution. Thiosulphate removes iodine form the cell component-dye-iodine complex instantaneously and renders the dye extractable by the differentiating medium. 11 alcoholic solutions of aniline, dimethyl aniline, nitro-benzene, benzene, toluene or xylol can also extract the color from the stained S. aureus; the extraction with the first three solvents is almost exhaustive while with the latter solvents extraction is appreciable but incomplete. These solvents can form charge-transfer complexes with iodine. The findings indicate that the stability of the cell component-dye-iodine complex determines the Gram-character of the cell. A model hasbeen presented for the Gram cell component-dye-iodine complex.     

Multiple allelopathic activity of the crustose coralline algaLithophyllum yessoenseagainst settlement and germination of seaweed spores

A study was made to investigate possible formation by the crustose coralline algaLithophyllum yessoenseof multiple allelopathic-related substances against the settlement and germination of spores of various seaweeds. Seven different solvents (n-hexane, diethyl ether, acetone, ethyl acetate, acetonitrile, methanol, distilled water) and seawater were used to obtain crude extracts and secretory exudates from the coralline alga. The extracts and the algal conditioned seawater were tested for inhibitory activity against the settlement and germination of spores from 17 species representing 15 genera. Spore settlement of 14 species was inhibited over 90% by one or more extracts of the six organic solvents and conditioned seawater. The germination of spores from 13 species was inhibited by one or more extracts of all seven solvents and conditioned seawater. The species where spore settlement was not significantly affected showed strong inhibition of germination, andvice versa.     

提取方法和溶剂对薏苡仁油提取率的影响

为了提高薏苡仁油的提取效率,考察5种不同极性的溶剂（无水乙醇、丙酮、二氯甲烷、乙酸乙酯、环己烷）在3种不同的提取方法中对薏苡仁油提取率的影响,并分析其原因。结果表明,丙酮在加热回流提取法中提取率最高（8.17%）,无水乙醇在超声辅助提取和微波辅助提取法中得率均较高。3种提取方法中,超声辅助提取法的得油率最高（10.79%）,而微波法虽然提取效率高（0.0678g/min）,但得油率太低（6.78%）,不适于薏苡仁油的提取。     

EVALUATION OF PIGMENT EXTRACTION METHODS AND A RECOMMENDED PROTOCOL FOR PERIPHYTON CHLOROPHYLL a DETERMINATION AND CHEMOTAXONOMIC ASSESSMENT1

Many methods have been proposed to extract and quantify algal pigments. Comparative studies have found that pigment extraction efficiency varies among solvent and mechanical disruption protocols due to differential cellular resistance, thereby, leading to potential misinterpretation of pigment data. When the type or resistance of algae are unknown, a method is required that efficiently extract pigments from all taxonomic groups. The objective of this study was to develop a simple and efficient one stage periphyton pigment extraction protocol by comparing the extractability of four solvents (acetone, methanol, methanol/acetone, and methanol/acetone/N,N‐dimethylformamide), the effects of grinding, and the effects of freeze‐drying. The best overall extraction was obtained using freeze‐dried samples extracted with methanol/acetone/DMF/water (MAD). Eighty‐six percent more chlorophyll was extracted when the sample was freeze‐dried relative to fresh/frozen samples extracted with 90% acetone. Freeze‐drying greatly improved the extraction of both polar and non‐polar (lipophilic/hydrophobic) pigments while MAD increased the extractability of polar pigments and improved peak resolution of all pigments. Chemotaxonomic assessment differed between samples that were fresh/frozen or freeze‐dried before extraction. The relative abundance of cyanobacteria was greater for freeze‐dried material compared with fresh/frozen due to the improved extractability of cyanobacterial pigments. Based on the results of this study, the traditional approach of 90% acetone as a solvent is not recommended for periphyton samples containing cyanobacteria or when the composition of the mat is unknown. The combination of freeze‐drying and MAD was sufficient for the extraction of pigments from a periphyton mat containing filamentous cyanobacteria, green algae, and diatoms.     

Host plant constituents as oviposition stimulants for a generalist herbivore: European corn borer

Oviposition response of the polyphagous European corn borer,Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae), to chemical constituents in host plants was investigated in the laboratory using two-choice bioassays. Foliar extracts of corn (Zea mays L.), pepper (Capsicum annuum L.) and potato (Solanum tuberosum L.) were prepared using the solvents pentane, acetone and methanol. In all three host plants, chemicals soluble in pentane stimulated oviposition. In potato, chemicals extractable in acetone also elicited a positive oviposition response. When presented with a choice between pentane extracts of corn and pepper, females preferred corn. No preferences were exhibited between pentane extracts of corn and potato or pepper and potato. Pentane extracts of corn husks, tassels, silk, and corn leaves from plants at early whorl and tassel (pre-pollen shed) stages of development also stimulated oviposition. Similar extracts from plants at 2-leaf and blister (when kernels resemble blisters) stages were not stimulatory. This indicates that plant phenology affects chemically mediated oviposition response in European corn borer. The potential use of plant chemicals for management ofO. nubilalis in the field is suggested.     

Chlorophyll <Emphasis Type="Italic">a</Emphasis> extraction from freshwater algae — a reevaluation

In this study, two extracting methods (sonication and dispersing) and three solvents (90% acetone, N,N′-dimethylformamide and methanol) were compared for their ability to extract chlorophyll a of freshwater phytoplankton. Measurements were performed with both spectrophotometry and high-performance liquid chromatography. Results showed that (i) cell disruption is essential and that (ii) the method of cell disruption and solvent applied differed significantly. Dispersing in acetone surpassed all other combinations. Sonication in N,N′-dimethylformamide was found less effective. N,N′-dimethylformamide and methanol seem to promote the formation of degradation products (chlorophyllide a, allomer, epimer and phaeophytin a) which lead to overestimates of chlorophyll a of about 10% by means of spectrophotometry.     

Analysis of Tn916-induced mutants ofClostridium acetobutylicum altered in solventogenesis and sporulation

Summary The conjugative transposon Tn916 was used for mutagenesis ofClostridium acetobutylicum ATCC 824. Tetracycline-resistant mutants were screened for loss of granulose synthesis and five classes of granulose mutants, that contained single transposon insertions, were identified on the basis of altered solvent production. Class 1 mutants did not make acetone or butanol, lacked activity of enzymes induced during solventogenesis, and did not sporulate, indicating that they are regulatory mutants. The class 2 mutant strains also did not produce acetone but did form small amounts of butanol and ethanol while the class 3 mutants produced low amounts of all solvents. Class 4 and 5 mutants produced essentially the same or higher amounts of solvents than the parent strain. Transposon insertions in the class 1 mutants were used as markers for in vitro synthesis of flanking chromosomal DNA using Tn916-specific primers. The DNA fragments were labeled to produce specific probes. Transposon insertion sites in the chromosomes of 13 different class 1 regulatory mutants were compared by hybridization of the specific probes to Southern blots of restriction endonuclease-digested parental chromosomal DNA. Insertions in two mutants appeared to be, in the same region of the chromosome. These results predict, that multiple regulatory elements are required to induce solvent production and sporulation.     

Enzymatic synthesis of α-2-deoxyglucosyl derivatives catalyzed by organic solvent-resistant α-glucosidase

Organic solvent-resistant Aspergillus niger α-glucosidase (ANGase) can synthesize α-2-deoxyglucosyl derivatives (2DDs) in water-organic solvent media by a trans-addition reaction from d-glucal to various acceptors. Herein, we studied the influence of four different solvents on ANGase stability and activity. ANGase exhibited 47 or 43% residual activity following incubation in 50% (v/v) or in 70% (v/v) acetone for 4 h, respectively. When various carbohydrates were used as acceptor molecules, ANGase catalyzed the addition reaction of four different sugar alcohols, glucose, sucrose, or trehalose to d-glucal. Among the acceptor molecules tested, xylitol was the best acceptor by producing the highest yield (87% addition). The concentration of acetone/acceptor influenced the formation of 2DDs and the yields. We confirmed the molecular weight of five kinds of products by mass spectrometry and enzymatic hydrolysis. Current method is useful for the production of carbohydrates containing 2-deoxyglucose moiety.     

Production of antibacterials from the freshwater alga Euglena viridis (Ehren)

The antibacterial properties of Euglena viridis, collected from a freshwater pond at the Central Institute of Freshwater Aquaculture (CIFA), Bhubaneshwar, India, were tested against various strains of virulent pathogens viz. Pseudomonas putida(PP1, PP2),P. aeruginosa (PA1, PA2, PA3, PA4), P. fluorescens (PF1, PF2, PF3, PF4), Aeromonas hydrophila (AH30, AH31, AH32, AH34), Edwardsiella tarda, Vibrio alginolyticus (VA1),V. anguillarum(VN1, VN2 & VN3), V. fluvialis (VF1), V. parahemolyticus (VP1) and V. harveyi (VH1) andEscherichia coli(O115, O1, O156, O164, O111 & O109). Four organic extracts viz. methanolic, ethanolic, acetone and acetone/ethanol of theE. viridis showed moderate to high antibacterial activity to all the bacterial pathogens. Rotavapor extraction products showed higher sensitivity in comparison to cold and hot extractions.     

Zur Gewinnung von Ubichinon-10 aus Acetobacter methanolicus IMET B 346

Mixtures of acetone/water (93 : 7 ··· 90 : 10, v/v) are favoured solvents for the extraction of ubiquinone-10 from Acetobacter methanolicus IMET B 346. Using these solvent mixtures ubiquinone-10 was extracted nearly completely. Other lipids were extracted partially. Extracts were obtained by optimal conditions with a ubiquinone content > 5%.