Dual amplified ratiometric fluorescence ELISA based on G-quadruplex/hemin DNAzyme using tetrahedral DNA nanostructure as scaffold for ultrasensitive detection of dibutyl phthalate in aquatic system.

Dibutyl phthalate (DBP) is considered as one of the most widely used phthalate esters (PAEs), which has attracted worldwide concerns because of its potential threats to eco-environments and human health. Systematic investigations of DBP environmental occurrence contribute to the further risk assessment, which depends on effective and available analytical methods. In this study, an amplified ratiometric fluorescence ELISA was established for sensitive and high-throughput detection of DBP in the aquatic system based on a novel tetrahedral DNA nanostructure (TDN)-scaffolded-DNAzyme (Tetrazyme). Wherein, Tetrazyme was prepared by the precise folding of G-quadruplex sequence on three vertex angles of the TDN, together with hemin as the horseradish peroxidase (HRP)-mimicking enzyme. The rigid TDN avoided the local overcrowding effect to provide a reasonable spatial spacing on the interface for G-quadruplex sequence, increasing the collision chance between DNAzyme and substrates, improving the catalytic ability of DNAzyme effectively. Besides, streptavidin (SA) and biotin (bio) were used to anchor TDN and antibody, in which the specific binding of SA/bio could make more Tetrazyme conjugate on each signal element, resulting in the dual signal amplification. Meanwhile, the accuracy and precision were enhanced owing to the inherent built-in rectification to the environment from the dual output ratiometric fluorescence assay. Under the optimized conditions, the detection limit of this proposed method was 0.17 ng/mL (16 times lower than that of conventional ELISA using the same antibody) with a satisfactory accuracy (recoveries, 79.0%- 116.2%; CV, 2.1-6.5%). Overall, this platform provides a promising way for accurate, sensitive and rapid determination of DBP from environmental waters.

Overexpression of Capsular Polysaccharide Biosynthesis Protein in Lactobacillus plantarum P1 to Enhance Capsular Polysaccharide Production for Di-n-butyl Phthalate Adsorption.

Exopolysaccharides (EPSs) such as capsular polysaccharide (CPS) are important bioactive carbohydrate compounds and are often used as bioenrichment agents and bioabsorbers to remove environmental pollutants like di-n-butyl phthalate (DBP). Among the EPS-producing bacteria, lactic acid bacteria (LAB) have gained the most attention. As generally recognized as safe (GRAS) microorganisms, LAB can produce EPSs having many different structures and no health risks. However, EPS production by LAB does not meet the needs of large-scale application on an industrial scale. Here, the capA gene (encoding CPS biosynthesis protein) was overexpressed in Lactobacillus plantarum P1 to improve the production of EPSs and further enhance the DBP adsorption capability. Compared with P1, the CPS production in capA overexpressed strain was increased by 11.3 mg/l, and the EPS thickness was increased from 0.0786 ± 0.0224 µm in P1 to 0.1160 ± 0.0480 µm in P1-capA. These increases caused the DBP adsorption ratio of P1-capA to be doubled. Overall, the findings in this study provide a safe method for the adsorption and removal of DBP.

A miniature stainless steel net dumbbell-shaped stir-bar for the extraction of phthalate esters in instant noodle and rice soup samples.

This work reports the development of a very-simple-to-construct stir-bar extraction device so called "a dumbbell-shaped stainless steel stir-bar." The extraction device was assembled from a rolled up stainless steel net filled with an XAD-2 sorbent and a metal rod to allow the use of a magnetic stirrer during extraction. The dumbbell-shaped stainless steel stir-bar was used to extract diethyl phthalate (DEP), dibutyl phthalate (DBP), and di(2-ethylhexyl) phthalate (DEHP) before analysis by a gas chromatograph equipped with an electron capture detector (GD-ECD). Under the optimal conditions, the developed method provided a good linearity from 10.0 to 1,000.0 ng mL-1 for all three compounds. Limits of detection and limits of quantification were 9.37 ± 0.29 ng mL-1 and 31.22 ± 0.95 ng mL-1 for DEP, 5.73 ± 0.31 ng mL-1 and 19.1 ± 1.0 ng mL-1 for DBP and 3.30 ± 0.06 ng mL-1 and 11.0 ± 0.19 ng mL-1 for DEHP, respectively. Good recoveries in the range of 81.89 ± 0.17 to 109.5 ± 2.0% were achieved when the method was used to extract phthalate esters in five instant noodle and two rice soup samples.

Nanomaterials as alternative dispersants for the multiresidue analysis of phthalates in soil samples using matrix solid phase dispersion prior to ultra-high performance liquid chromatography tandem mass spectrometry.

In this study, the application of different nanomaterials as dispersants in matrix solid phase dispersion has been evaluated for the extraction of fifteen phthalates from different environmental samples prior to their separation and quantification by ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry. Within the evaluated nanomaterials, including graphene oxide, multi-walled carbon nanotubes and iron 1,3,5-benzenetricarboxylate metal-organic framework, the last one showed the best results in terms of extraction capacity and sample clean-up. The effects of the different parameters affecting the sample pretreatment efficiency were exhaustively evaluated. The whole methodology was validated for agricultural soil and sand, using dibutyl phthalate-3,4,5,6-d4 as surrogate. Recovery values ranged from 70 to 120% for both matrices with RSD values lower than 20% and the limits of quantification of the method achieved were in the range 0.14-2.7 µg/kg dry weight. Finally, the analysis of soil samples from different locations of Tenerife (Canary Islands, Spain) was carried out finding the presence of BBP, DIBP and DBP in the range 5-52 µg/kg dry weight in agricultural soils, and DIPP, DNOP and DINP in the range 2-101 µg/kg dry weight in sand samples.

Biodegradation of di­n­butyl phthalate by psychrotolerant Sphingobium yanoikuyae strain P4 and protein structural analysis of carboxylesterase involved in the pathway.

A priority pollutant Phthalate Esters (PAEs) are widely used as plasticizers and are responsible mainly for carcinogenicity and endocrine disruption in human. For the bioremediation of PAEs, a psychrotolerant Sphingobium yanoikuyae strain P4, capable of utilizing many phthalates di­methyl phthalate (DMP), di­ethyl phthalate (DEP), di­n­butyl phthalate (DBP), di­isobutyl phthalate (DIBP), butyl benzyl phthalate (BBP), and few Polycyclic Aromatic Hydrocarbons as the sole source of carbon and energy was isolated from Palampur, Kangra, Himachal Pradesh, India. 100% utilization of DBP (1 g L-1) by the strain was observed within 24 h of incubation at 28 °C. Interestingly the strain also degraded DBP completely at 20 °C and 15 °C within 36 h and 60 h, respectively. Esterase involved in DBP degradation was found to be inducible in nature and intracellular. Comparative sequence analysis of carboxylesterase enzyme sequences revealed conserved motifs: G-X-S-X-G and -HGG- which were the characteristic peptide motifs reported in different esterases. Structural analysis showed that the enzyme belongs to serine hydrolase superfamily, which has an α/ß hydrolase fold. Interaction and binding of DBP to a catalytic Ser184 residue in the esterase enzyme were also analysed. In conclusion, carboxylesterase possess the required active site which may be involved in the catabolism of DBP.

Removal efficiency and enzymatic mechanism of dibutyl phthalate (DBP) by constructed wetlands.

Four vertical-flow constructed wetland systems were set up in the field in order to study the removal efficiency and possible enzymatic mechanism of the constructed wetlands in treating sewage containing different concentrations of dibutyl phthalate (DBP). Under DBP spiked concentrations of 0.5, 1.0, and 2.0 mg/L, good DBP removal rates of 62.08, 82.17, and 84.17% were achieved, respectively. Meanwhile, certain removal effects of general water quality parameters were observed in all four constructed wetlands: with high average removal rates of nitrate nitrogen (NO3--N) and chemical oxygen demand (COD) of 91.10~93.89 and 82.83~89.17%, respectively, with moderate removal efficiencies of total nitrogen (TN), total phosphorus (TP), ammonia nitrogen (NH4+-N) of 44.59~49.67, 30.58~37.18, and 28.52~37.45%, respectively. Compared to the control, an increase of enzyme activities of urease, phosphatase, dehydrogenase, and nitrate reductase was observed in the treatments with DBP addition. In the presence of 0.5 mg/L of DBP concentration, the urease, phosphatase, and dehydrogenase activities reached the highest levels, with an increase of 350.02, 36.57, and 417.88% compared with the control, respectively. It appeared that the low concentration of DBP might better stimulate the release of enzymes.

Removal mechanism of di-n-butyl phthalate and oxytetracycline from aqueous solutions by nano-manganese dioxide modified biochar.

In this work, nano-manganese dioxide (nMnO2)-modified biochar (BC) was synthesized in order to improve BC's adsorption capacity for di-n-butyl phthalate (DBP) and oxytetracycline (OTC). The results showed that nMnO2 on the BC surface exhibited a poor crystallinity and oxidation state (Mn (IV)). Sorption experiments showed that, compared to BC, DBP sorption capacity of nMnO2-BC (1:20) and OTC sorption capacity of nMnO2-BC (1:10) were 0.0364 and 0.0867 mmol/g, respectively, which are significantly higher than that of BC (0.0141 and 0.0151 mmol/g). Kinetics and isotherm experiments indicated that physical adsorption and chemical interactions have both exerted their impacts on the adsorption process. Further X-ray photoelectron spectroscopy (XPS) analysis showed that part of the Mn (IV) in nMnO2-BC was reduced to Mn (III) and Mn (II) after DBP or OTC adsorption. Therefore, we suggest the nMnO2 also acted as an oxidizer on modified BC, which may accelerate the degradation of DBP and OTC.

Bioassay-guided isolation of an active compound with protein tyrosine phosphatase 1B inhibitory activity from Sargassum fusiforme by high-speed counter-current chromatography.

A rapid and efficient method using high-speed counter-current chromatography was established for the bioassay-guided separation of an active compound with protein tyrosine phosphatase 1B inhibitory activity from Sargassum fusiforme. Under the bioassay guidance, the ethyl acetate extract with the best IC50 value of 0.37 ± 0.07 µg/mL exhibited a potential protein tyrosine phosphatase 1B inhibitory activity, which was further separated by high-speed counter-current chromatography. The separation was performed with a two-phase solvent system composed of n-hexane/methanol/water (5:4:1, v/v). As a result, dibutyl phthalate (19.7 mg) with the purity of 95.3% was obtained from 200 mg of the ethyl acetate extract. Its IC50 was 14.05 ± 0.06 µM, which was further explained by molecular docking. The result of molecular docking showed that dibutyl phthalate enfolded in the catalytic site of protein tyrosine phosphatase 1B. The main force between dibutyl phthalate and protein tyrosine phosphatase 1B was the hydrogen bond interaction with Gln266. In addition, hydrogen bond, van der Waals force and hydrophobic interaction with the amino acids (Ala217, Ile219, and Gly220) were also responsible for the stable protein-ligand complex.

Sorption of four hydrophobic organic contaminants by biochars derived from maize straw, wood dust and swine manure at different pyrolytic temperatures.

Sorption behavior of acetochlor (ACE), dibutyl phthalate (DBP), 17α-Ethynyl estradiol (EE2) and phenanthrene (PHE) with biochars produced from three feedstocks (maize straw (MABs), pine wood dust (WDBs) and swine manure (SWBs)) at seven heat treatment temperatures (HTTs) was evaluated. The bulk polarity of these biochars declined with increasing HTT while the aromaticity and CO2-surface area (CO2-SA) rose. The surface OC contents of biochars were generally higher than bulk OC contents. The organic carbon (OC)-normalized CO2-SA (CO2-SA/OC) of biochars significantly correlated with the sorption coefficients (n and logK(oc)), suggesting that pore filling could dominate the sorption of tested sorbates. SWBs had higher logK(oc) values compared to MABs and WDBs, due to their higher ash contents. Additionally, the logK(oc) values for MABs was relatively greater than that for WDBs at low HTTs (≤400 °C), probably resulting from the higher CO2-SA/OC, ash contents and aromaticity of MABs. Surface polarity and the aliphatic C may dominate the sorption of WDBs obtained at relatively low HTTs (≤400 °C), while aromatic C affects the sorption of biochars at high HTTs. Results of this work aid to deepen our understanding of the sorption mechanisms, which is pivotal to wise utilization of biochars as sorbents for hazardous organic compounds.

Toxicity study of dibutyl phthalate of Rubia cordifolia fruits: in vivo and in silico analysis.

Natural toxins from plant sources with wide ranges of biological activities reflect the upswing of drug design in the pharmaceutical industry. Rubia cordifolia L. is one of the most important red dye yielding plants. Most of the former researches have focused on the bioactive compounds from the roots of R. cordifolia, while no attention was paid towards the fruits. For the first time, here we report the presence of dibutyl phthalate in the fruits of R. cordifolia. Structural characterization was carried out using Ultraviolet-Visible spectrophotometer (UV-Vis), Fourier transform infrared (FTIR), gas chromatography-mass spectrophotometer (GC-MS), Nuclear magnetic resonance (NMR). Acute toxicity of the crude ethanolic extracts of the R. cordifolia fruits was examined in Swiss albino mice. No mortality was observed in all treated mice with 100, 500, 1000 mg/kg body weight of crude extract of R. cordifolia fruit and it indicates that the LD50 value is higher than 1000 mg/kg body weight. This study exhibited a significant change in the body weight. Alanine transaminase (ALT), total protein, triglycerides, glucose, and also the histopathological analysis of liver for all treated mice showed difference from the control group. The dibutyl phthalate was further evaluated for the toxicity study through in silico analysis. Together, the results highlighted that the toxic potential of R. cordifolia fruits extracts and also the toxicity profile of the fruit should be essential for the future studies dealing with the long term effect in animals. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1059-1067, 2016.

Ortho-phthalic acid esters in lipophilic extract from the cell culture of Aconitum baicalense Turcz ex Rapaics 1907.

Optically active bis-2R(-)ethylhexyl o-phthalate was obtained with 0.18% yield from dry cultured cells of Aconitum baicalense Turcz ex Rapaics 1907 by extraction with petroleum ether followed by silica gel column chromatography. Its structure was confirmed by the analysis of 13C and 1H NMR spectra. Seasonal fluctuations of quantitative phthalate content in A. baicalense cells were identified. The tests were performed under conditions excluding the presence of phthalates in reagents, materials, and laboratory dishes. The same substance was shown to be produced by cultivated cells of other plants. Biosynthesis of esters of ortho-phthalic acid by cultivated plant cells was discovered for the first time.

Chemical Composition of the Essential Oil of the Boreal Relict of Pyrola rotundifolia L. from Northern Kazakhstan.

In Kazakhstan Pyrola rotundifolia L. is the plant-relict in the flora of insular pine forests of the region of low hillocks and declivities in Kazakhstan - a group of insular pine forests of Kokshetau, Bayanaul and Karkaralinsk. In this study, the essential oils from dried aerial parts of P. rotundifolia, collected in natural habitats of the State National Natural Park "Burabay" (Akmola oblast, Northern Kazakhstan), were extracted by hydrodistillation and analyzed by gas chromatography - mass spectrometry. The yield of the essential oil amounted to 0.057 % in relation to the mass of the air-dry raw material. The major components in dried plant oil were 2,6-dimethyl-1,4-naphthoquinone (12.99-93.49%) and dibutyl phthalate (4.42-40.48%), depending on the growth conditions.

Nanostructured copper-coated solid-phase microextraction fiber for gas chromatographic analysis of dibutyl phthalate and diethylhexyl phthalate environmental estrogens.

A novel nanostructured copper-based solid-phase microextraction fiber was developed and applied for determining the two most common types of phthalate environmental estrogens (dibutyl phthalate and diethylhexyl phthalate) in aqueous samples, coupled to gas chromatography with flame ionization detection. The copper film was coated onto a stainless-steel wire via an electroless plating process, which involved a surface activation process to improve the surface properties of the fiber. Several parameters affecting extraction efficiency such as extraction time, extraction temperature, ionic strength, desorption temperature, and desorption time were optimized by a factor-by-factor procedure to obtain the highest extraction efficiency. The as-established method showed wide linear ranges (0.05-250 µg/L). Precision of single fiber repeatability was <7.0%, and fiber-to-fiber repeatability was <10%. Limits of detection were 0.01 µg/L. The proposed method exhibited better or comparable extraction performance compared with commercial and other lab-made fibers, and excellent thermal stability and durability. The proposed method was applied successfully for the determination of model analytes in plastic soaking water.

[A new phthalide from angelicae sinensis radix].

Angelicae Sinensis Radix, dried roots of Angelicae sinensis, is a well-known traditional Chinese medicine. Six compounds were isolated and purified by silica gel and preparative HPLC. According to physicochemical properties and spectral data, the compounds were identified as senkyunolide H-7-acetate (1), o-phthalic acid (2), diisobutyl phthalate (3), p-hydroxyphenylethanol ferulate (4), ferulic acid (5) and coniferylferulate (6). Compound 1 was a new one.

Sorption of phthalic acid esters in two kinds of landfill leachates by the carbonaceous sorbents.

Sorption of phthalic acid esters (PAEs: diethyl phthalate, DEP: dibutyl phthalate, DBP as model compounds) in landfill leachates by activated carbon (AC), carbon nanotubes (CNTs), and biochars, were examined. The young leachate (YL) and old leachate (OL) were synthesized to imitate acetogenic and methanogenic phases, respectively, and glucose (GLU) and fulvic acid (FA) were selected to represent dissolved organic matter (DOM). GLU in leachates generally facilitated the sorption of PAEs while FA restrained sorption of PAEs, suggesting the type of DOM associated with leachates possibly regulated the removal efficiency of PAEs from leachates. The pores and organic carbon of carbonaceous sorbents should be major factors in influencing the sorption of PAEs in leachates. The data showed PAEs in acetogenic leachates was removed more easily than those in methanogenic leachates and CNTs have the less advantage to remove PAEs from methanogenic leachates compared to AC.

A dibutyl phthalate sensor based on a nanofiber polyaniline coated quartz crystal monitor.

Dibutyl phthalate (DBP) is a commonly used plasticizer and additive to adhesives, printing inks and nail polishes. Because it has been found to be a powerful reproductive and developmental toxicant, a sensor to monitor DBP in some working spaces and the environment is required. In this work polyaniline nanofibers were deposited on the electrode of a quartz crystal oscillator to form a Quartz Crystal Microbalance gas sensor. The coated quartz crystal and a non-coated quartz crystal were mounted in a sealed chamber, and their frequency difference was monitored. When DBP vapor was injected into the chamber, gas adsorption decreased the frequency of the coated quartz crystal oscillator and thereby caused an increase in the frequency difference between the two crystals. The change of the frequency difference was recorded as the sensor response. The sensor was extremely sensitive to DBP and could be easily recovered by N2 purging. A low measurement limit of 20 ppb was achieved. The morphologies of the polyaniline films prepared by different approaches have been studied by SEM and BET. How the nanofiber-structure can improve the sensitivity and stability is discussed, while its selectivity and long-term stability were investigated.

[Study on constituents of the aerial parts of Pogostemon cablin].

OBJECTIVE: To investigate the chemical constituents of the aerial parts of Pogostemon cablin. METHODS: The constituents were isolated by column chromatography over silica gel, Sephadex LH-20 and C8. Structures were identified by spectroscopic data analysis. RESULTS: Thirteen compounds were obtained and elucidated as patchouli alcohol (1), pogostone (2), friedelin (3), epifriedelinol (4), oleanolic acid (5), methyl oleanolate (6), 5alpha-stigmast-3,6-dione (7), stigmast-4-ene-3-one (8), beta-sitosterol (9), pachypodol (10), retusin (11), (-)-guaiacylglycerol (12) and dibutyl phthalate (13). CONCLUSION: Compounds 6, 7, 8, 12 and 13 are isolated from this genus for the first time.

Biodegradation of di-n-butyl phthalate by a stable bacterial consortium, HD-1, enriched from activated sludge.

HD-1, a stable microbial consortium capable of mineralizing di-n-butyl phthalate (DBP), was developed from activated sludge. The dominant microorganisms in the consortium, Gordonia sp., Burkholderia sp. and Achromobacter sp., were identified by denaturing gradient gel electrophoresis (DGGE). The consortium could mineralize approximately 90% of 1200 mg/L DBP after 48 h of cultivation. The optimal DBP degradation conditions were 25-30 °C and pH 8.0-9.0. The addition of yeast (0.5 g/L), sodium acetate (0.5 g/L, 1.0 g/L), Brij 35 (0.2%, 0.5%, 1.0%), or Triton X-100 (0.2%) enhanced DBP degradation. The DBP degradation rate was influenced by the presence of dimethyl phthalate (DMP) and diethyl phthalate (DEP). Only one main intermediate, phthalic acid, could be monitored by gas chromatography-mass spectrometry (GC-MS) during the degradation process. The HD-1 consortium also utilized phenol, o-dihydroxybenzene as the sole carbon and energy source. The results indicate the consortium may represent a promising application for DBP bioremediation.

Determination of phthalic acid diesters in human milk at low ppb levels.

Phthalic acid diesters (PAE) are omnipresent in the human environment and food is a major contributor to the overall human exposure towards these chemicals. Due to developmental effects, PAE infants' exposure via human milk has been subjected to a number of analytical studies. These previous studies, however, revealed that normal laboratory blank values are in the range of or even higher than human milk levels due to the presence of PAE in laboratory environments. In order to provide more reliable data on PAE exposure via human milk, the aim of this study was to develop and validate a robust and sensitive analytical method. This should be capable of removing matrix components efficiently and guarantee limits of quantification in the low ppb range. The method development took into account liquid-liquid extraction and selective pressurised fluid extraction (sPFE) as well as chromatography-based clean-up steps. The final method consisted of a liquid-liquid extraction followed by an automated chromatographic clean-up by an sPFE device. After volume reduction the cleaned extracts were analysed by quadrupole GC/MS. Quantification was based on internal standards. An extensive quality assurance and method test programme demonstrated conservatively determined limits of detection and quantification from 0.3 to 10 ng g⁻¹ in human milk, with recoveries of internal standards from 50% to 101%. Thus, the method allowed the quality-assured detection of di-isobutyl phthalate (DiBP), di-n-butyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), di-allyl phthalate (DAP), benzylbutyl phthalate (BBP) and di-cyclohexyl phthalate (DcHP) in 30 human milk samples provided by 30 volunteers from southern Germany. DiBP, DBP and DEHP were the most commonly detected PAE, with median levels of 1.0, 0.6 and 2.3 ng g⁻¹, respectively.

[Study on chemical constituents of the ethyl acetate extract from Blumea aromatica].

OBJECTIVE: To study the chemical constituents of the ethyl acetate extract from Blumea aromatica. METHODS: Column chromatographic techniques were used for the isolation and purification. Spectroscopic techniques were used for the identification of structures. RESULTS: Seven compounds were isolated from the ethyl acetate extract of 80% alcohol extract of the whole plant, and their structures were identified as xanthoxylin(1), dibutyl phthalate(2), luteolin-3',4',7-trimethyl ether(3), rubiadin(4), eriodictyol(5), kaempherol(6), luteolin 7,3'-dimethyl ether(7). CONCLUSION: All the compounds are isolated from this plant for the first time. Among them, compounds 2,4 and 7 are isolated from this genus for the first time.