Evaluation of Anthocyanin Content,Antioxidant Potential and Antimicrobial Activity of Black,Purple and Blue Colored Wheat Flour and Wheat-Grass Juice against Common Human Pathogens

The present study aimed to analyze the antioxidant and antimicrobial activity of anthocyanins extracted from colored wheat flour and wheat-grass juice against human pathogens. The total anthocyanin content and antioxidant potential in colored wheat flour and wheat-grass juice extracts were significantly higher than white flour and wheat-grass juice extracts. Ultra-performance liquid chromatography showed the maximum number of anthocyanin peaks in black wheat, with delphinidin-3-o-galactoside chloride, delphinidin-3-o-glucoside chloride, and cyanindin-3-o-glucoside chloride as the major contributors. Among flour extracts, maximum zones of inhibition against Staphylococcus aureus (MTCC 1934), Pseudomonas aeruginosa (MTCC 1434), Escherichia coli, and Candida albicans (MTCC 227) were produced by black flour extract, having the highest anthocyanin content. It exhibited a minimum microbicidal concentration (MMC) of 200 mg/mL against E. coli and C. albicans; and 100 and 150 mg/mL against S. aureus and P. aeruginosa, respectively. Black and purple flour extracts exhibited a minimum inhibitory concentration (MIC) of 50 mg/mL against S. aureus and P. aeruginosa. White flour extracts did not show MMC against E. coli and C. albicans. Among wheat-grass juice extracts, black wheat-grass was most effective and showed an MIC of 100–150 mg/mL against all pathogens. It exhibited an MMC of 200 mg/mL against S. aureus and P. aeruginosa. Hence, anthocyanin-rich colored wheat could be of nutraceutical importance.     

In vitro curcumin delivery and antibacterial activity of RuS2 and RuO2 nanoparticles loaded chitosan biopolymer

In this work, RuS₂ and RuO₂ nanoparticles loaded chitosan (Chitosan was extracted from Lobsters shells of Persian Gulf, IR. Iran) was prepared and characterized via FE‐SEM, EDS and FT‐IR analysis. FESEM showed the formation of spherical nanoparticles in size ranging of 20 to 100 mm. Subsequently, the role of these new materials as curcumin drug carrier and in vitro release of curcumin in simulated body fluid (SBF) solution (pH 7.4) were studied. RuS₂‐NPs‐CS than to RuO₂‐NPs‐CS showed higher drug loading efficiency (>91%) and rapid (<90 min) curcumin drug release in SBF solution. Also, antibacterial activity of RuS₂‐NPs‐CS and RuO₂‐NPs‐CS in presens and absence of Rosemary extracts against the gram negative bacteria Pseudomonas aeruginosa (PAO 1) was evaluated by detection of minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC). MIC of RuS₂‐NPs‐CS, RuO₂‐NPs‐CS and Rosemary extracts on Pseudomonas aeruginosa strains were found to be 50 mg/ml, 50 mg/ml and 1250 mg/ml, respectively. The synergistic effect of these materials for inhibition of PAO 1 growth showed that mixture of RuS₂‐NPs‐CS and Rosemary extracts has a better efficiency than to other mixture materials.     

A Temperature and Salt-Tolerant <Emphasis Type="SmallCaps">l</Emphasis>-Glutaminase from Gangotri Region of Uttarakhand Himalaya: Enzyme Purification and Characterization

Purification and characterization of halotolerant, thermostable alkaline l-glutaminase from a Bacillus sp. LKG-01 (MTCC 10401), isolated from Gangotri region of Uttarakhand Himalaya, is being reported in this paper. Enzyme has been purified 49-fold from cell-free extract with 25% recovery (specific activity 584.2 U/mg protein) by (NH₄)₂SO₄ precipitation followed by anion exchange chromatography and gel filtration. Enzyme has a molecular weight of 66 kDa. l-Glutaminase is most active at pH 11.0 and stable in the pH range 8.0–11.0. Temperature optimum is 70 °C and is completely stable after 3 h pre-incubation at 50 °C. Enzyme reflects more enhanced activity with 1–20% (w/v) NaCl, which is further reduced to 80% when NaCl concentration was increased up to 25%. l-Glutaminase is almost active with K⁺, Zn²⁺, and Ni²⁺ ions and K _m and V _max values of 240 μM and 277.77 ± 1.1 U/mg proteins, respectively. Higher specific activity, purification fold, better halo-tolerance, and thermostability would make this enzyme more attractive for food fermentation with respect to other soil microbe derived l-glutaminase reported so far.     

Enhanced extract preparation of native manganese and iron species from brain and liver tissue

To date, no reference method for the extraction of labile Mn species from biological tissues is published which provides sufficient extraction efficiency combined with monitoring speciation. Here, an extraction method is reported using cryogenic conditions (+N) under inert gas atmosphere. Fresh brain and liver tissues were used, then stored either 1 day (+N) or 1 month in N_2liq (+N 1 m) to evaluate degradation effects during long-term storage. Both attempts were compared to a previous extraction method (−N) using neither N_2liq nor storage ability. Mn and Fe concentrations in extracts and pellets were determined with inductively coupled plasma (ICP)-atomic emission spectroscopy (AES) and compared to acid digests of the same sample. Element ratios of extracts/digest indicated the extraction efficiency, which was increased from 17% (−N) to 26% (+N) for Mn in brain or from 28% (−N) to 44% (+N) in liver extracts. For Fe species, the increase was only from 40% (−N) to 44% (+N) in brain but from 64% (−N) to 74% (+N) in liver. Size exclusion chromatography (SEC)-ICP-mass spectrometry (MS) was employed to screen for Mn and Fe species pattern in extracts. In brain, surplus extracted Mn (+N, +N 1 m) was assigned to organic Mn species, mainly from the 0.7–4 kDa fraction, while in the liver, it was seen in the 70–80 kDa fraction. Fe speciation was similar for −N and +N methods in brain extracts. In liver, higher amounts of Fe species were extracted from the 140–160 kDa fraction. Storage at −196 °C for 1 month did neither affect Mn speciation in brain nor in liver extracts. Fe species pattern showed a negligible shift (≤5%) from 140–160 to 70–80 kDa fraction in liver extracts stored 1 month in N_2liq.     

Glucosamine-functionalized silver glyconanoparticles: characterization and antibacterial activity

We report the analytical and in vitro antibacterial activity of glucosamine-functionalized silver glyconanoparticles. Morphological characterization ensured the surface topography and particle size distribution of both silver and glucosamine–silver nanoparticles. Surface plasmon resonance of both types of nanoparticle was determined from UV–visible spectroscopy using four different sample concentrations (10–40 μL). The resulting functionalized glyconanoparticles show maximum absorbance with a red shift of 30 ± 5 nm (390–400 nm) from their initial absorbance (425–430 nm). FT-Raman and ¹H-NMR spectroscopic measurement confirmed the surface functionalization of glucosamine on the silver surface through the carbonyl group of a secondary amide linkage (–NH–CO–), elucidated by the conjugation of N-hydroxysuccinimide (NHS)-terminated silver nanoparticles and the amino group of glucosamine. Antimicrobial experiments with well-characterized silver nanoparticles (AgNPs) and glucosamine-functionalized silver nanoparticles (GlcN-AgNPs) demonstrate that GlcN-AgNPs have similar and enhanced minimum inhibitory concentration (MIC) against eight gram-negative and eight gram-positive bacteria compared with AgNPs. MIC data shows that Klebsiella pneumoniae (ATCC 700603) and Bacillus cereus isolate express high levels of inhibition, with the quantity and magnitude of inhibition being higher in the presence of GlcN-AgNPs.     

Qualitative and quantitative analysis of gallic acid in Alchemilla vulgaris, Allium ursinum, Acorus calamus and Solidago virga-aurea by chip-electrospray ionization mass spectrometry and high performance liquid chromatography

This study presents the results obtained from qualitative and quantitative analysis of gallic acid from hydro-alcoholic extracts (methanol, ethanol) of plants from Plantae regnum. Plant qualitative analysis was performed using a novel mass spectrometric (MS) method based on fully automated chip-nanoelectrospray ionization (nanoESI) high capacity ion trap (HCT) while quantitative analysis was carried out by high performance liquid chromatography (HPLC). These methods were applied to Alchemilla vulgaris — common lady’s-mantle (aerial part), Allium ursinum — bear’s garlic (leaves), Acorus calamus — common sweet flag (roots), Solidago virga-aurea — goldenrod (aerial part). Obtained results indicated that methanol extracts (96%, 80%) have a gallic acid content ranging between 0.0011–0.0576 mg mL⁻¹ extract while the ethanol extracts (96%, 60%) exhibit a gallic acid concentration that varies between 0.0010–0.0182 mg mL⁻¹ extract.      

Semi-solid-state fermentation of Eicchornia crassipes biomass as lignocellulosic biopolymer for cellulase and β-glucosidase production by cocultivation of Aspergillus niger RK3 and Trichoderma reesei MTCC164

An aquatic weed biomass, Eicchornia crassipes, present in abundance and leading to a threatening level of water pollution was used as substrate for cellulase and β-glucosidase production using wild-type strain Aspergillus niger RK3 that was isolated from decomposing substrate. Alkali treatment of the biomass (10%) resulted in a 60–66% increase in endoglucanase, exoglucanase, and β-glucosidase production by the A. niger RK3 strain in semi-solid-state fermentation. Similarly, the alkali-treated biomass led to a 45–54% increase in endo- and exoglucanase and a higher (98%) increase in β-glucosidase production by Trichoderma reesei MTCC164 under similar conditions. However, the cocultivation of A. niger RK3 and T. reesei MTCC164 at a ratio of 3:1 showed a 20–24% increase in endo- and exoglucanase activities and about a 13% increase in the β-glucosidase activity over the maximum enzymatic activities observed under single culture conditions. Multistep physical (ultraviolet) and chemical (N-methyl-N′-nitrosoguanidine, sodium azide, colchicine) mutagenesis of the A. niger RK3 strain resulted in a highly cellulolytic mutant, UNSC-442, having an increase of 136, 138, and 96% in endoglucanase, exoglucanase, and β-glucosidase, activity, respectively. The cocultivation of mutant UNSC-442 along with T. reesei MTCC164 (at a ratio of 3:1) showed a further 10–11% increase in endo- and exoglucanase activities and a 29% increase in β-glucosidase activity in semi-solid-state fermentation.     

Angiotensin I-Converting Enzyme Inhibitory Proteins and Peptides from the Rhizomes of Zingiberaceae Plants

Ammonium sulphate cut protein extracts, and their pepsin hydrolysates, from the rhizomes of 15 plants in the Zingiberaceae family were screened for their in vitro angiotensin I-converting enzyme inhibitory (ACEI) activity. The protein extract from Zingiber ottensii had the highest ACEI activity (IC₅₀ of 7.30 × 10⁻⁷ mg protein/mL) and was enriched for by SP Sepharose chromatography with five NaCl step gradients 0, 0.25, 0.50, 0.75 and 1 M NaCl collecting the corresponding five fractions. The highest ACEI activity was found in the F75 fraction, which appeared to contain a single 20.7-kDa protein, suggesting enrichment to or near to homogeneity. The ACEI activity of the F75 fraction was moderately thermostable (−20–60 °C), showed >80% activity across a broad pH range of 4–12 (optimal at pH 4–5) and appeared as a competitive inhibitor of ACE (K _i of 9.1 × 10⁻⁵ mg protein/mL). For the pepsin hydrolysates, that from Zingiber cassumunar revealed the highest ACEI activity (IC₅₀ of 0.38 ± 0.012 mg/mL), was enriched to a single active hexapeptide by RP-HPLC with a strong ACEI activity (IC₅₀ of 0.011 ± 0.012 mg/mL) and acted as a competitive inhibitor of ACE (K _i of 1.25 × 10⁻⁶ mg protein/mL).     

Production and Characterization of an Alkaline Thermostable Crude Lipase from an Isolated Strain of <Emphasis Type="Italic">Bacillus cereus</Emphasis> C<Subscript>7</Subscript>

A bacterial strain isolated from spoiled coconut and identified as Bacillus cereus was found capable of producing alkaline thermostable extracellular lipase. Optimum temperature, time, and pH for enzyme substrate reaction were found to be 60 °C, 10 min, and 8.0 respectively. Common surfactants except Triton X 100 and cetyltrimethylammonium bromide have no or very little inhibitory effects on enzyme activity. The enzyme was found to be stable in presence of oxidizing agents and protease enzyme. The maximum lipase production was achieved at 30–33 °C, pH 8.0 on 24 h of fermentation using 50 ml medium in a 250-ml Erlenmeyer flask. The superior carbon and nitrogen sources for lipase production were starch (2%) and ammonium sulfate (nitrogen level 21.2 mg/100 ml), peptone (nitrogen level 297 mg/100 ml), and urea (nitrogen level 46.62 mg/100 ml) in combination, respectively. The maximum enzyme activity obtained was 33 ± 0.567 IU/ml.     

Quantitative estimation of riluzole in human plasma by LC-ESI-MS/MS and its application to a bioequivalence study

A novel simple, sensitive, selective, and rapid high-performance liquid chromatography coupled with tandem mass spectrometry method was developed and validated for quantification of riluzole in human plasma. The chromatography was performed by using a Zorbax-SB-C18 (4.6 × 75 mm, 3.5 μm) column , isocratic mobile phase 0.1% formic acid/acetonitrile (10:90 v/v), and an isotope-labeled internal standard (IS), [¹³C,¹⁵N₂]riluzole. The extraction of drug and internal standard was performed by liquid–liquid extraction and analyzed by MS in the multiple reaction monitoring (MRM) mode using the respective [M+H]⁺ ions, m/z 235.0/165.9 for riluzole and m/z 238.1/169.0 for the IS. The calibration curve was linear over the concentration range 0.5–500.0 ng/ml for riluzole in human plasma. The limit of quantification (LOQ) was demonstrated at 0.5 ng/ml. The within-batch and between-batch precision were 0.6–2.3% and 1.4–5.7%, and accuracy was 97.1–101.1% and 98.8–101.2% for riluzole respectively. Drug and IS were eluted within 3.0 min. The validated method was successfully applied in a bioequivalence study of riluzole in human plasma.     

Preparation,characterization, and evaluation of cytotoxicity,antioxidant, cutaneous wound healing,antibacterial, and antifungal effects of gold nanoparticles using the aqueous extract of Falcaria vulgaris leaves

The new age drugs are nanoparticles of metals, which can combat conditions like wounds and fight human pathogens like bacteria. The aim of the experiment was preparation, characterization, and assessment of cytotoxicity, antioxidant, cutaneous wound healing, antibacterial, and antifungal potentials of gold nanoparticles using the aqueous extract of Falcaria vulgaris leaves (AuNPs@F. vulgaris) under in vitro and in vivo condition. These nanoparticles were characterized by FT‐IR, UV, XRD, FE‐SEM, TEM, and AFM. The synthesized AuNPs@F. vulgaris had great cell viability dose‐dependently (Investigating the effect of the nanoparticles on HUVEC cell line) and indicated these nanoparticles were nontoxic. DPPH free radical scavenging test was done to evaluate the antioxidant potentials, which showed similar antioxidant potentials for AuNPs@F. vulgaris and butylated hydroxytoluene. In part of cutaneous wound healing effect of F. vulgaris, after creating the cutaneous wound, the rats were randomly divided into six groups: untreated control, treatment with Eucerin basal ointment, treatment with 3% tetracycline ointment, treatment with 0.2% HAuCl₄ × H₂O ointment, treatment with 0.2% F. vulgaris ointment, and treatment with 0.2% AuNPs@F. vulgaris ointment. These groups were treated for 10 days. Use of AuNPs@F. vulgaris ointment in the treatment groups substantially decreased (p ≤ 0.01) the wound area, total cells, neutrophil, and lymphocyte and remarkably raised (p ≤ 0.01) the wound contracture, hydroxyl proline, hexuronic acid, fibrocyte, and fibrocytes/fibroblast rate compared to other groups. In antimicrobial part, MIC, MBC, and MFC were specified by macro‐broth dilution assay. AuNPs@F. vulgaris revealed higher antibacterial and antifungal properties than many standard antibiotics (p ≤ 0.01). Also, AuNPs@F. vulgaris prevented the growth of all bacteria at 2‐8 mg/ml concentrations and removed them at 2‐16 mg/ml concentrations (p ≤ 0.01). In case of antifungal potentials of AuNPs@F. vulgaris, they inhibited the growth of all fungi at 2‐4 mg/ml concentrations and destroyed them at 2‐8 mg/ml concentrations (p ≤ 0.01). In conclusion, synthesized AuNPs@F. vulgaris revealed non‐cytotoxicity, antioxidant, cutaneous wound healing, antibacterial, and antifungal activities.     

Chemical profiling of Aristolochia tagala Cham. leaf extracts by GC-MS analysis and evaluation of its antibacterial activity

Aristolochia tagala Cham. (Aristolochiaceae) is an underexplored medicinal plant traditionally used to treat snakebites, stomachaches, and poisonous bites. In this study, chemical profiling of the petroleum ether, chloroform, ethyl acetate, methanol, and hydro-alcoholic extracts of the plant was investigated by gas chromatography-mass spectrometry. The antibacterial activity of the plant was tested against ten bacterial strains using the agar disc diffusion and microdilution method. In total, forty two compounds were identified from the extracts with neophytadiene, palmitic acid, phytol, trans-δ9-octadecenoic acid, phytyl palmitate, phytyl tetradecanoate, ergost-5-en-3-ol, (3beta,24r)-,z,z-8,10-hexadecadien-1-ol, stigmasterol, and tetrapentacontane as major phytoconstituents. The hydro-alcoholic extract possessed maximum total phenolics (52.58 ± 06 mg GAE/g), total flavonoids (48.66 ± 91 QRE/g), total flavanols (67.20 ± 64 QRE/g) and vitamin E content (31.26 ± 0.05 mg ATE/g). For antibacterial activity, hydro-alcoholic extract of Aristolochia tagala effectively controlled the growth of bacterial strains such as Proteus valgaris (26.3 mm) and Pseudomonas aeruginosa (19.33 mm) and the same extract showed notable minimum inhibitory concentration (MIC) against the growth of bacteria like Escherichia coli (10.93 μg/ml) and Enterobacter aerogenes (43.7 μg/ml). It was determined that, hydro-alcoholic and methanolic extracts Aristolochia tagala leaf found to have a number of bioactive compounds with significant antibacterial activity against the pathogenic bacteria. Further investigations are necessary to isolate and characterize bioactives and to evaluate its therapeutic potential.     

An insight into antimicrobial activity of the freshwater bryozoan Pectinatella magnifica

The antimicrobial activity of five crude extracts of the freshwater bryozoan Pectinatella magnifica (Leidy, 1851) was evaluated in vitro for the first time. P. magnifica acetone extract exhibited the highest antibacterial activity (minimum inhibitory concentrations (MIC) 0.004–0.350 mg/mL and MBC 0.007–0.500 mg/mL), while its methanol extract showed the most promising antifungal activity (MIC 0.03–0.12 mg/mL and MFC 0.06–0.25 mg/mL). Furthermore, at a concentration of 0.25 MIC, the methanol extract reduced biofilm formation of the bacterial strain Pseudomonas aeruginosa PAO1 in a considerable extent (59.14%). FTIR spectra of the most active extracts indicate the presence of carbonyl compounds, long-chain alcohols and/or sterols. According to the experimental data obtained, P. magnifica methanol extract may be considered as a good resource of novel natural products with potent antibiofilm activity against the bacterium well known for its resistance.     

Green synthesis and chemical characterization of copper nanoparticles using Allium saralicum leaves and assessment of their cytotoxicity,antioxidant, antimicrobial,and cutaneous wound healing properties

In recent decades, nanotechnology is growing rapidly owing to its widespread application in science and industry. The aim of the experiment was the green synthesis of copper nanoparticles using Allium saralicum R.M. Fritsch aqueous extract and assessment of their cytotoxicity, antioxidant, antibacterial, antifungal, and cutaneous wound healing effects under in vitro and in vivo conditions. These nanoparticles were characterized by Fourier transformed infrared spectroscopy (FT‐IR), UV–visible spectroscopy, field emission scanning electron microscopy (FE‐SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). DPPH free radical scavenging test was done to assess the antioxidant properties, which indicated similar antioxidant potentials for CuNPs@Allium and butylated hydroxytoluene. Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and Minimum Fungicidal Concentration (MFC) were specified by macro‐broth dilution assay. CuNPs@Allium indicated higher antibacterial and antifungal effects than all standard antibiotics (p ≤ 0.01). Also, CuNPs@Allium inhibited the growth of all bacteria at 1–8 mg/ml concentrations and removed them at 2–8 mg/ml concentrations (p ≤ 0.01). In the case of antifungal properties of CuNPs@Allium, they prevented the growth of all fungi at 1–4 mg/ml concentrations and destroyed them at 2–8 mg/ml concentrations (p ≤ 0.01). In vivo experiment, after creating the cutaneous wound, the rats were randomly divided into six groups (n = 10): untreated control, treatment with Eucerin basal ointment, treatment with 3% tetracycline ointment, treatment with 0.2% CuSO₄ ointment, treatment with 0.2% A. saralicum ointment, and treatment with 0.2% CuNPs@Allium ointment. Use of CuNPs@Allium ointment in the treatment groups substantially reduced (p ≤ 0.01) the wound area, total cells, neutrophil, macrophage, and lymphocyte and remarkably raised (p ≤ 0.01) the wound contracture, hydroxyl proline, hexosamine, hexuronic acid, fibrocyte, and fibrocytes/fibroblast rate compared to other groups. The synthesized CuNPs@Allium had high cell viability dose‐dependently (Investigating the effect of the plant on HUVEC cell line) and revealed this method was nontoxic. The results revealed the useful non‐cytotoxic, antioxidant, antibacterial, antifungal, and cutaneous wound healing effects of CuNPs@Allium.     

Determination of fatty acid compositions and cholesterol levels of some freshwater fish living in Porsuk Dam,Turkey

We determined the oil content, fatty acid composition, and cholesterol content of common carp (Cyprinus carpio), crucian carp (Carassius carassius), chub (Leusiscus cephalus), and tench (Tinca tinca) by GLC. The saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) levels were found to be 36.49%, 31.92%, 31.59% in common carp; 32.92%, 32.21%, and 34.87% in crucian carp; 36.19%, 32.91%, and 30.90% in chub; and 32.86%, 30.77%, and 36.37% in tench, respectively. The cholesterol (mg/100 g oil) levels of common carp, crucian carp, chub, and tench were determined by GLC methods as 119 ± 2.64 mg, 170.37 ± 2.36 mg, 94.68 ± 3.13 mg, and 179.84 ± 6.75 mg, respectively. Thus, the cholesterol contents of the analyzed freshwater fish species were low but their PUFA contents and nutritional values were high. Published in Khimiya Prirodnykh Soedinenii, No. 1, pp. 15–17, January–February, 2009.     

Purification and Characterization of Pectin Lyase Produced by Aspergillus terricola and its Application in Retting of Natural Fibers

An indigenously isolated fungal strain identified as Aspergillus terricola with assigned fungal strain number MTCC 7588 has been used as source for pectin lyase production. The extracellular pectin lyase was purified to homogeneity from the culture filtrate of A. terricola by ion exchange and gel filtration chromatography. The determined molecular weight was 35 ± 01 kDa. The K _m and k _cat (turnover) values of the purified enzyme at 37 °C using citrus pectin as the substrate were found to be 1.0 mg/ml and 110.0 s⁻¹, respectively. The pH and temperature optima of the enzyme were 8.0 and 50 °C, respectively. The retting ability of the purified pectin lyase for natural fibers viz. Cannabis sativa and Linum usitatissimum has been demonstrated for the first time.     

Enhanced Production of an Extracellular β-<Emphasis Type="SmallCaps">d</Emphasis>-Fructofuranosidase Fructohydrolase from a 2-Deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-glucose Stabilized Mutant of <Emphasis Type="Italic">Candida utilis</Emphasis>

The enzyme β-d-fructofuranosidase fructohydrolase (FFH) cleaves the α-1,4 glycosidic linkage between α-d-glucose and β-d-fructose molecules of sucrose, releasing monosaccharides by hydrolysis. In the present study, FFH production in Candida utilis GC-46, a lipolytic wild yeast strain was improved by exposure to N-methyl N-nitro N-nitroso guanidine (NG) and 2-deoxy-d-glucose (2dg) at various levels. The mutant strain NG-5 was obtained after exposure to 0.06 mg/ml of NG for 20 min. NG-5 offers improved extracellular FFH production (34 ± 2.6 U/ml/min) when compared to the wild strain (1.15 ± 0.01 U/ml/min). A 40-fold increase of FFH (45.65 ± 2.0 U/ml/min) was achieved when the process parameters, including incubation period (48 h), sucrose concentration (5.0 g/l), initial pH (6.0), inoculum size (2.0% v/v, 16 h old), and urea concentration (0.2%, w/v) were identified using Plackett–Burman design. The kinetic parameters viz. Q _p (0.723 U/g/h), Y _p/s (2.036 U/g), and q _p (0.091 U/g yeast cells/h) indicate that NG-5 is a hyperproducer of extracellular FFH with a concomitant increase in growth rate. The volumetric productivity of NG-5 was over sixfold improved over the parental strain. The enzyme production improvement is highly significant (HS, LSD 0.042, p ≤ 0.05), indicating commercial utility.     

A sensitive liquid chromatography–mass spectrometry method for simultaneous determination of alisol A and alisol A 24-acetate from <Emphasis Type="Italic">Alisma orientale</Emphasis> (Sam.) Juz. in rat plasma

A liquid chromatography–mass spectrometry (LC-MS) method was developed and validated for the simultaneous determination of alisol A and alisol A 24-acetate from Alisma orientale (Sam.) Juz. in rat plasma using diazepam as an internal standard. A 200-μl plasma sample was extracted by methyl tert-butyl ether and the separation was performed on Kromasil C₁₈ column (150 × 4.6 mm, 5 μm) with the mobile phase of acetonitrile (containing 0.1% of formic acid)–water (73:27, v/v) at a flow rate of 0.8 ml/min in a run time of 10 min. The two analytes were monitored with positive electrospray ionization by selected ion monitoring mode. The lower limit of quantitation for both alisol A and alisol A 24-acetate were 10 ng/ml. The calibration curves were linear in the measured range 10–1,000 ng/ml for alisol A and 10–500 ng/ml for alisol A 24-acetate. The mean extraction recoveries were above 74.7% for alisol A and above 72.4% for alisol A 24-acetate from biological matrixes. The intra- and inter-day precision for all concentrations of quality controls was lower than 14.1% (RSD %) for each analyte. The accuracy ranged from −12.3% to 9.8% (RE %) for alisol A, and −8.6% to 14.2% (RE %) for alisol A 24-acetate. The method was successfully applied to the study on the pharmacokinetics of alisol A and alisol A 24-acetate in rat plasma.     

Comparison of different sample treatments for the analysis of ochratoxin A in wine by capillary HPLC with laser-induced fluorescence detection

Ochratoxin A (OTA) is a mycotoxin naturally found in various foods, including wine. As OTA is considered as a possible human carcinogen, the maximum concentration for this compound has been established at 2 μg kg⁻¹ in wine by the EU (Directive (CE) No 1881/2006). Typically, immunoaffinity columns have been used for its extraction. However, simpler, more efficient and less contaminant extraction systems are demanding. In this work, dispersive liquid–liquid microextraction using ionic liquid as extractant solvent (IL-DLLME) and the QuEChERS procedure, have been evaluated and compared for extraction of OTA in wine samples. Laser-induced fluorescence (LIF, He–Cd Laser excitation at 325 nm) coupled with capillary HPLC has been used for the determination of OTA, using a sodium dodecyl sulfate micellar solution in the mobile phase to increase the fluorescence intensity. Matrix-matched calibration curves were established for both methods, obtaining LODs (3× S/N) of 5.2 ng·L⁻¹ and 85.7 ng·L⁻¹ for IL-DLLME and QuEChERS, respectively. Clean extracts were obtained for white, rose and red wines with both methods, with recoveries between 88.7–94.2% for IL-DLLME and between 82.6–86.2% for QuEChERS. The precision was evaluated in terms of repeatability (n = 9) and intermediate precision (n = 15), being ≤ 8.5% for IL-DLLME and ≤ 5.4% for QuEChERS.     

Preparation,characterization, and assessment of cytotoxicity,antioxidant, antibacterial,antifungal, and cutaneous wound healing properties of titanium nanoparticles using aqueous extract of Ziziphora clinopodioides Lam leaves

In recent decades, nanotechnology is growing rapidly owing to its widespread application in science and industry. The aim of the experiment was chemical characterization and evaluation of cytotoxicity, antioxidant, antibacterial, antifungal, and cutaneous wound healing activities of titanium nanoparticles using aqueous extract of Ziziphora clinopodioides Lam leaves (TiNPs@Ziziphora). These nanoparticles were characterized by fourier transformed infrared spectroscopy (FT‐IR), field emission scanning electron microscopy (FE‐SEM), energy dispersive X‐ray spectroscopy (EDS), and UV–visible spectroscopy. The synthesized TiNPs@Ziziphora had great cell viability dose‐dependently (Investigating the effect of the plant on human umbilical vein endothelial cells (HUVECs) cell line) and revealed this method was nontoxic. Then, 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) free radical scavenging test was done to assess the antioxidant properties, which indicated similar antioxidant potentials for TiNPs@Ziziphora and butylated hydroxytoluene. Agar diffusion tests were applied to determine the antibacterial and antifungal characteristics. Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and Minimum Fungicidal Concentration (MFC) were specified by macro‐broth dilution assay. The data were analyzed by SPSS 21 software (Duncan post‐hoc test). TiNPs@Ziziphora indicated higher antibacterial and antifungal effects than all standard antibiotics (p ≤ 0.01). Also, TiNPs@Ziziphora inhibited the growth of all bacteria at 2‐16 mg/ml concentrations and removed them at 2‐32 mg/ml concentrations (p ≤ 0.01). In case of antifungal properties of TiNPs@Ziziphora, they prevented the growth of all fungi at 2‐8 mg/ml concentrations and destroyed them at 2‐16 mg/ml concentrations (p ≤ 0.01). In vivo experiment, after creating the cutaneous wound, the rats were randomly divided into six groups: untreated control, treatment with Eucerin basal ointment, treatment with 3% tetracycline ointment, treatment with 0.2% TiO₂ ointment, treatment with 0.2% Z. clinopodioides ointment, and treatment with 0.2% TiNPs@Ziziphora ointment. These groups were treated for 10 days. For histopathological and biochemical analysis of the healing trend, a 3 × 3 cm section was prepared from all dermal thicknesses at day 10. Use of TiNPs@Ziziphora ointment in the treatment groups substantially reduced (p ≤ 0.01) the wound area, total cells, neutrophil, and lymphocyte and remarkably raised (p ≤ 0.01) the wound contracture, hydroxyl proline, hexosamine, hexuronic acid, fibrocyte, and fibrocytes/fibroblast rate compared to other groups. In conclusion, the results revealed the useful non‐cytotoxic, antioxidant, antibacterial, antifungal, and cutaneous wound healing effects of TiNPs@Ziziphora.