Evaluation of antioxidant capacities of green microalgae

Three strains of green microalgae, Chlorococcum sp.C53, Chlorella sp. E53, and Chlorella sp.ED53 were studied for their antioxidant activities. Crude extracts of these microalgae in hot water and in ethanol were examined for their total phenolic contents and for their antioxidant capacities. In order to determine their phenolic contents, the Folin–Ciocalteu method was used. As for the determination of their antioxidant capacities, four different assays were used: (1) total antioxidant capacity determination; (2) DPPH radical scavenging assay; (3) ferrous ion chelating ability assay; and (4) inhibition of lipid peroxidation (using thiobarbituric acid reactive substance). For all the strains we have studied, their ethanolic extract showed more antioxidant activities than their hot water extract. Categorically, the ethanolic extract of Chlorella sp.E53 exhibited both the highest total phenolic content of 35.5?±?0.14 mg gallic acid equivalent (GAE) g^?1 dry weight and the highest DPPH radical scavenging of 68.18?±?0.38 % at 1.4 mg mL^?1 (IC₅₀ 0.81 mg mL^?1), whereas Chlorella sp.ED53 showed both the highest ferrous ion chelation activity of 42.78?±?1.48 % at 1 mg mL^?1 (IC₅₀ 1.23 mg mL^?1) and the highest inhibition of lipid peroxidation of 87.96?±?0.59 % at 4 mg mL^?1. This high level of inhibition is comparable to 94.42?±?1.39 % of butylated hydroxytoluene, a commercial synthetic antioxidant, at the same concentration.     

Extraction and Antioxidant Activities of Magnolia kwangsiensis Figlar & Noot. Leaf Polyphenols

Microwave‐assisted extraction was employed to extract polyphenols from the leaf of Magnolia kwangsiensis Figlar & Noot . The yield of polyphenols was 2.44±0.02 % under the optimal conditions of RSM: acetone concentration of 70 %, ratio of solvent to material of 21 mL?g^?1 and extraction time of 16 min. The antioxidant activities were evaluated in terms of total antioxidant ability, reducing power, DPPH ^? and ^? OH scavenging activity. Results showed the polyphenols presented potential antioxidant activities, especially the stronger scavenging activity on ^? OH. In term of ^? OH scavenging activity, the IC₅₀ value of NKA‐9 purification was 0.335 mg mL^?1, equivalent to 35.23 % of V_C. The IC₅₀ values of crude extract and ethyl acetate extract were 0.580 and 0.828 mg mL^?1, equivalent to 60.99 % and 87.07 % of V_C. Results indicated that M. kwangsiensis leaf polyphenols present potential antioxidant activities that make it beneficial for human health by preventing or reducing oxidative damage.     

Rapid and direct spectrophotometric method for kinetics studies and routine assay of peroxidase based on aniline diazo substrates

Peroxidases are ubiquitous enzymes that play an important role in living organisms. Current spectrophotometrically based peroxidase assay methods are based on the production of chromophoric substances at the end of the enzymatic reaction. The ambiguity regarding the formation and identity of the final chromophoric product and its possible reactions with other molecules have raised concerns about the accuracy of these methods. This can be of serious concern in inhibition studies. A novel spectrophotometric assay for peroxidase, based on direct measurement of a soluble aniline diazo substrate, is introduced. In addition to the routine assays, this method can be used in comprehensive kinetics studies. 4-[(4-Sulfophenyl)azo]aniline (λ_max?=?390?nm, ??=?32 880 M^?1 cm^?1 at pH 4.5 to 9) was introduced for routine assay of peroxidase. This compound is commercially available and is indexed as a food dye. Using this method, a detection limit of 0.05?nmol mL^?1 was achieved for peroxidase.     

GROWTH CHARACTERISTICS AND ANTIOXIDANT PROPERTIES OF THE BENTHIC DIATOM NAVICULA INCERTA (BACILLARIOPHYCEAE) FROM JEJU ISLAND,KOREA1

Benthic diatoms are a commonly used food source in shellfish aquaculture. Diatoms of the genus Navicula are the most abundant benthic diatoms occurring year‐round on the coast of Jeju Island, Korea. We isolated an axenic strain of N. incerta Grunow; estimated its growth characteristics under 27 different combinations of temperature, salinity, and nutrients; and determined its biochemical composition and antioxidant activities. The maximum specific growth rate (μ_max), defined as the increase in cell density per unit time, was 0.81–1.04 · d^?1, and the maximum cell density, 7.99 × 10⁵ cells · mL^?1, was reached at 0.88 · d^?1 μ_max, 20°C, 30 psu salinity, and F/2 nutrient concentration on day 12 of the culture period. The approximate cellular composition was as follows: 7.0 ± 0.04% protein, 1.7 ± 0.28% lipid, 12.8 ± 0.85% carbohydrate, 68.4 ± 0.09% ash, and 10.1 ± 0.44% moisture. The antioxidant properties of N. incerta were determined for various extracts. The rates of 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) free‐radical scavenging for Neutrase and methanol extracts were 81.6% and 62.8%, respectively. Flavourzyme extract had a superoxide‐scavenging rate of 57.7%. Kojizyme and Ultraflo extracts had nitric‐oxide‐scavenging rates of 42.2% and 40.6%, respectively, significantly higher than commercial antioxidants, such as α‐tocopherol and butylated hydroxytoluene (BHT). The metal‐chelating activities of the methanol, Neutrase, and Termamyl extracts were 68.5%, 45.2%, and 41.2%, respectively, four to six times higher than commercial antioxidants. The Termamyl extract showed the highest linoleic acid peroxidation inhibition, exceeding α‐tocopherol and on par with BHT.     

OPTIMAL GROWTH CONDITIONS AND ANTIOXIDATIVE ACTIVITIES OF CYLINDROTHECA CLOSTERIUM (BACILLARIOPHYCEAE)1

We isolated the unialgal strain of Cylindotheca closterium (Ehrenb.) Reimann et J. C. Lewin and produced an axenic strain using an antibiotic cocktail of enriched f/2 artificial seawater medium. The optimal growth conditions were estimated under 27 different combinations of temperature, salinity, and nutrients, and mass culture was performed based on the best specific growth conditions. Its antioxidant activities were determined from the extracts of methanol, water, and enzymes (proteases and carbohydrases). The maximum specific growth rate (μ_max) varied from 0.63 to 0.97 · d^?1. The maximum cell density was 7.20 × 10⁴ cells · mL^?1, while the μ_max was 0.82 · d^?1 in culture conditions of 20°C, 30 psu (practical salinity unit), and “F” nutrient concentrations on day 10 of the culture period. The scavenging rates for 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) free radical were 72.5% and 69.4% from Viscozyme and methanol extracts, respectively. The enzymatic extracts of C. closterium prepared by the hydrolyses of Amyloglucosidase (AMG) and Viscozyme showed 45.8% and 45.5% nitric‐oxide‐scavenging rates, slightly lower than the activity of alpha‐tocopherol (α‐tocopherol) but similar to butylated hydroxytoluene (BHT). The extract from methanol and water showed 44.8% and 44.4% scavenging rates, statistically similar with BHT. The metal‐chelating activities of the Kojizyme, Alcalase, methanol, Viscozyme, and Neutrase extracts were 67.1, 53.9, 53.2, 52.1, and 50.2 %, respectively, five to six times higher than the commercial antioxidants. The AMG, Viscozyme, and Neutrase extracts showed a remarkable linoleic acid peroxidation inhibition, which was higher than BHT and statistically similar with α‐tocopherol.     

Oxidative degradation of Amaranth dye by a new genus Kerstersia sp.

A dye-decolorizing bacterium was isolated from a coconut coir sample and identified as a new genus Kerstersia sp. by various biochemical tests and 16S rRNA gene sequencing. This bacterium was capable of degrading sulfonated azo dye Amaranth aerobically at 40?°C and pH 7.0. Tests conducted on intracellular crude enzyme extract identified an oxygen insensitive azoreductase. The optimum dye-decolorizing activity at pH 7.0 and 40?°C for the decolorization of dye was 0.091?U mL^?1 (μ_max 0.522?mg h^?1). The K_s 104.51?μM^?1 has been evaluated by plotting Lineweaver–Burk plot for the Amaranth dye. The dye degraded products were extracted and characterized by TLC, diazotization and Carbylamines test, which indicated that Amaranth was biotransformed into non-toxic aromatic metabolite without amine group.     

AN IN VITRO NITRATE REDUCTASE ASSAY FOR MARINE MACROALGAE: OPTIMIZATION AND CHARACTERIZATION OF THE ENZYME FOR FUCUS GARDNERI (PHAEOPHYTA)1

Measurement of the activity of the enzyme nitrate reductase (NR) may provide a useful index of nitrogen metabolism in marine macroalgae. In several species, including Fucus gardneri P. C. Silva, in vitro assays previously failed to detect NR activity, necessitating the use of in situ (or so-called“in vivo”) assays, which are more loosely controlled and lead to dafficulties in assessing enzyme characteristics such as the half-saturation constant (K_m). In this paper, we describe an in vitro NR assay developed for F. gardneri, in which tissue was homogenized using liquid nitrogen prior to the assay. In contrast to previous studies, enzyme activity was always detectable in F. gardneri collected directly from the field at levels up to 30 nmol nitrate converted to nitrite·min^?1·g^?1 wet weight. The effect of a variety of compounds, commonly added to NR extraction buffers, were tested. Additions of protease inhibitors, bovine serum albumin, and ethylenediamine tetraacetic acid had no consistent effects on NR activity, while polyvinyl pyrrolidone, potassium ferricyanide, and flavin adenine dinucleotide significantly decreased activity. The half-saturation constant (K_m) for NADH was 0.18 (± 0.05) mM and for nitrate, K_m=0.99 (±0.41) mM. Significant NR activity was detected without the addition of nitrate, suggesting that internal pools of nitrate averaging approximately 20 μmol NO₃^?·g^?1 wet weight were present in F. gardneri in February. The distribution of NR activity within the plant was highly variable between individuals, but activities were approximately 5-fold lower in the stipe than in midregions. In plants freshly sampled from the field, NR activity increased 7-fold from February to March, then fell to near-February levels by April. These changes in activity may correspond to seasonal changes in growth rate. The assay, optimized for F. gardneri, was used in several different macroalgal species from different taxa: Porphyra sp., Coralina vancouveriensis Yendo, Ulva sp., Enteromorpha intestinalis (Linnaeus) Nees, Macrocystis integrifolia Bory; and Costaria costatum (C. Agardh) Saunders. For all species tested, NR activity was detectable and, except for one species (Porphya sp.), was equal to or greater than activities measured by other workers using in vivo or in vitro assays for plants under similar conditions.     

Purification and kinetic studies on a porphobilinogen deaminase from the unicellular green alga Scenedesmus obliquus

Porphobilinogen deaminase, the enzyme condensing four molecules of porphobilinogen, was isolated and purified from light grown Scenedesmus obliquus (wild type). The purification procedure included heat treatment, ammonium sulphate fractionation, gel filtration, high-resolution anion-exchange chromatography and hydrophobic interaction chromatography. The enzyme was purified 1368-fold, compared to the initial crude extract. Its final specific activity was 6812 units · (mg · protein)^?1 at pH 7.4 with a recovery of 44%. The relative molecular mass was 33000, as determined by Sephadex G-100 gel filtration, and 35900 by lithium dodecyl sulfate-polyacrylamide-gel electrophoresis, indicating that the enzyme is a monomer. Studies of initial reaction velocities showed a linear progress curve for hydroxymethylbilane formation and a hyperbolic dependence of the initial reaction rate on substrate concentration, consistent with a sequential displacement mechanism. Apparent kinetic constants (K _m and V _max) for the conversion of porphobilinogen to hydroxymethylbilane at 37 ° C, pH 7.4, were 79 μM and 176 pmol · min^?1, respectively. Variation of both V _max and K _max with pH indicated the presence of ionizable groups in the enzyme-substrate complex(es), showing a single ionization (pK 7.15) in V _max/K _m plots. A sharp pH-profile for V _max was interpreted as a positive cooperative proton dissociation. In spite of the two pathways existing for 5-aminolevulinate biosynthesis in Scenedesmus, currently there is no indication of the existence of two porphobilinogen deaminases or even of isoenzymes.     

Production and characterization of Penicillium brasilianum pectinases with regard to industrial application

The objective of this study was to evaluate the production of pectinase by an isolated strain of Penicillium brasilianum in a bioreactor and to consider its potential for industrial applications (i.e. fruit juice). The optimization of production was achieved through experimental design. The maximum exo-polygalacturonase (Exo-PG) production in the bioreactor was 53.8?U mL^?1 under the conditions of 180?rpm, an aeration rate of 1.5 vvm, 30?°C, pH_initial of 5.5, 5?×?10⁶ spores mL^?1, 32?g L^?1 pectin, 10?g L^?1 of yeast extract and 0.5?g L^?1 magnesium sulfate and bioproduction for 36?h. The production of Exo-PG in the bioreactor was 1.3 times higher than that obtained in shake flasks, with aeration (1.5 vvm) and agitation (180?rpm) control. The crude enzyme complex, beyond the pectinolytic activity of Exo-PG (53.8?U mL^?1), also contained activity pectin methylesterase (6.0?U mL^?1) and pectin lyase (6.61?U mL^?1). At a crude enzyme complex with a concentration of 0.5% (v/v), viscosity of peach juice was reduced by 11.66%, turbidity was reduced by 13.71% and clarification was increased by 26.92%. Based on the present results, we can conclude that the new strain of isolated P. brasilianum produced high amounts of pectinases in a bioreactor with mechanical agitation, and has the potential to be applied to in the clarification of juices.     

EXTRACTION,ASSAY AND SOME PROPERTIES OF FLORIDOSIDE PHOSPHATE SYNTHASE FROM PORPHYRA PERFORATA (RHODOPHYTA)1

A suitable method for extraction of floridoside phosphate synthase (FPS, UDP-galactose: sn-3-glycerol phosphate: 1→2′α-D-galactosyl transferase)from Porphyra perforata J. Ag. was developed. Two assay methods for enzyme activity were utilized, one measuring the amount of floridoside formed by using gas-liquid chromatography, the other measuring the sn-3-glycerol phosphate-dependent formation of UDP; both assays gave similar results. FPS is a soluble protein, and FPS activity in the extract as determined by the amount of product formed in vitro compared well with the in vivo rate of floridoside synthesis (4–7 μMmol product formed·h^?1·g^?1 fresh wt). The rate of product formation in vitro was linear up to 45 min and proportional to protein concentration in the assay mixture. The temperature optimum was 30–35° C. FPS was active over a range of pH values from 7.0–8.5. It was stable in concentrated solutions in the presence of 0.3 M ammonium sulfate, but activity was lost in diluted solution (protein concentration below 0.2 mg·mL^?1) or below 0.2 M ion strength. The data suggest that FPS may be an oligomeric protein which occurs free in the cytoplasm or loosely bound to a membrane. It may also be a regulatory protein controlling the overall rate of synthesis of floridoside in vivo.     

Effect of Mg2+ on the Structure and Function of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Mg²⁺ in various concentrations was added to purified Rubisco in vitro to gain insight into the mechanism of molecular interactions between Mg²⁺ and Rubisco. The enzyme activity assays showed that the reaction between Rubisco and Mg²⁺ was two order, which means that the enhancement of Rubisco activity was accelerated by low concentration of Mg²⁺ and slowed by high concentration of Mg²⁺. The kinetics constant (K _m) and V _max was 1.91 μM and 1.13 μmol CO₂ mg⁻¹ protein∙min⁻¹, respectively, at a low concentration of Mg²⁺, and 3.45 μM and 0.32 μmol CO₂∙mg⁻¹ protein∙min⁻¹, respectively, at a high concentration of Mg²⁺. By UV absorption and fluorescence spectroscopy assays, the Mg²⁺ was determined to be directly bound to Rubisco; the binding site of Mg²⁺ to Rubisco was 0.275, the binding constants (K _A) of the binding site were 6.33 × 10⁴ and 5.5 × 10⁴ l·mol⁻¹. Based on the analysis of the circular dichroism (CD) spectra, it was concluded that the binding of Mg²⁺ did not alter the secondary structure of Rubisco, suggesting that the observed enhancement of Rubisco carboxylase activity was caused by a subtle structural change in the active site through the formation of the complex with Mg²⁺.     

Polyphenol Characterization,Antioxidant and Skin Whitening Properties of Alnus cordata Stem Bark

In this study, we investigated the phenolic composition of the crude extract (MeOH 80 %) of Alnus cordata (Loisel .) Duby stem bark (ACE) and its antioxidant and skin whitening properties. RP‐LC‐DAD analysis showed a high content of hydroxycinnamic acids (47.64 %), flavanones (26.74 %) and diarylheptanoids (17.69 %). Furthermore, ACE exhibited a dose‐dependent antioxidant and free‐radical scavenging activity, expressed as half‐maximal inhibitory concentration (IC₅₀): Oxygen radical absorbance capacity (ORAC, IC₅₀ 1.78 μg mL^?1)>Trolox equivalent antioxidant capacity (TEAC, IC₅₀ 3.47 μg mL^?1)>2,2‐Diphenyl‐1‐picrylhydrazyl (DPPH, IC₅₀ 5.83 μg mL^?1)>β‐carotene bleaching (IC₅₀ 11.58 μg mL^?1)>Ferric reducing antioxidant power (FRAP, IC₅₀ 17.28 μg mL^?1). Moreover, ACE was able to inhibit in vitro tyrosinase activity (IC₅₀ 77.44 μg mL^?1), l ‐DOPA auto‐oxidation (IC₅₀ 39.58 μg mL^?1) and in an in vivo model it exhibited bleaching effects on the pigmentation of zebrafish embryos (72 h post fertilization) without affecting their development and survival. In conclusion, results show that A. cordata stem bark may be considered a potential source of agents for the treatment of skin disorders due to its bleaching properties and favorable safety profiles, associated to a good antioxidant power.     

Distribution of N, Rubisco and photosynthesis in Pinus pinaster and acclimation to light

The light–nitrogen hypothesis suggests canopy photosynthesis is maximized when there is a positive relationship between irradiance received by foliage, its nitrogen content (per unit area N_area), and maximum rate of photosynthesis (A_max). Relationships among relative irradiance and N_area, allocation of nitrogen within the photosynthetic apparatus to Rubisco and chlorophyll, and A_max were examined in Pinus pinaster Ait. needles up to 6 years of age. Measurements were made before bud break in August 1998, and in May 1999 after the first ‘winter’ rains. In August, N_area in P. pinaster needles decreased from 5·1 to 5·7 g m^?2 in sunlit 1‐year‐old needles to 2·3 g m^?2 in shaded 6‐year‐old needles. In May, N_area was 5–40% less but spatial trends were the same. At both sampling dates, A_max was less in old shaded needles compared with young sunlit needles, and was thus consistent with the light–nitrogen hypothesis. Relationships between N_area and A_max were positive at both dates yet varied in strength and form. Allocation of nitrogen within the photosynthetic apparatus was qualitatively consistent with acclimation to light (i.e. Rubisco/Chl decreased with shading), but quantitatively suboptimal with respect to photosynthesis owing to consistent over‐investment in Rubisco. This over‐investment increased with height in the canopy and was greater in May than in August.     

Macrocystis angustifolia is a potential source of enzyme inhibitors linked to type 2 diabetes and dementia

In continuation of the screening of South African seaweeds to identify potential candidates for the development of pharmaceutically active functional foods, we investigated the inhibitory effects of a crude 80 % methanol extract, solvent fractions and isolated compounds from the kelp Macrocystis angustifolia against enzymes involved in type 2 diabetes and dementia. Repeated column fractionation of the ethyl acetate fraction of the crude extract of M. angustifolia afforded two phenol derivatives identified by spectroscopic analyses (1D and 2D NMR): 4-(2-hydroxyethyl)phenol (tyrosol) (1) and 4-(1,2-dihydroxyethyl)phenol (2). These compounds were isolated from a marine alga for the first time. The ethyl acetate (IC₅₀?=?14.08?±?1.21 μg mL^?1) and butanol (IC₅₀?=?77.94?±?11.69 μg mL^?1) fractions exhibited potent inhibition against α-glucosidase and acetylcholinesterase (AChE) enzymes, respectively. Tyrosol (1) and its derivative, 4-(1,2-dihydroxyethyl)phenol (2), showed potent inhibition against both α-glucosidase and AChE enzymes. Based on in silico evaluation, these two compounds are anticipated to possess sufficient oral bioavailability in accordance to the Lipinski Rule of Five without any toxicity risk. Natural α-glucosidase and AChE inhibitors from M. angustifolia offer a novel approach to control type 2 diabetes and dementia.     

AN ALTERNATE GROWTH PATTERN FOR LAMINARIA LONGICRURIS1,2

A population of Laminaria longicruris de la Pylaie was followed for a year at Bic Island, Quebec, Canada where nutrient levels in the seawater were elevated throughout the year. Tagged kelp were measured each month for growth and analyzed for alginic acid, laminaran, mannitol, carbon, nitrogen, and nitrate. Maximum growth (3.5 cm · d^?1) was observed in June, and minimal growth (0.18 cm · d^?1) from December to February, when ice cover limited light levels. No reserves of carbon or nitrate were formed. Laminaran levels remained below 2.7% dry weight while tissue nitrate did not exceed 0.75 μmol · g^?1 dry weight. Total carbon produced per plant was 40 g C · yr^?1. Nutrient availability enables the kelp to take advantage of summer light and temperature conditions to grow rapidly.     

Characterization of a novel xylanase from Armillaria gemina and its immobilization onto SiO2 nanoparticles

Enhanced catalytic activities of different lignocellulases were obtained from Armillaria gemina under statistically optimized parameters using a jar fermenter. This strain showed maximum xylanase, endoglucanase, cellobiohydrolase, and β-glucosidase activities of 1,270, 146, 34, and 15 U mL^?1, respectively. Purified A. gemina xylanase (AgXyl) has the highest catalytic efficiency (k _cat/K _m?=?1,440 mg?mL^?1?s^?1) ever reported for any fungal xylanase, highlighting the significance of the current study. We covalently immobilized the crude xylanase preparation onto functionalized silicon oxide nanoparticles, achieving 117 % immobilization efficiency. Further immobilization caused a shift in the optimal pH and temperature, along with a fourfold improvement in the half-life of crude AgXyl. Immobilized AgXyl gave 37.8 % higher production of xylooligosaccharides compared to free enzyme. After 17 cycles, the immobilized enzyme retained 92 % of the original activity, demonstrating its potential for the synthesis of xylooligosaccharides in industrial applications.     

Antioxidant activity of brown alga Saccharina bongardiana from Kamchatka (Pacific coast of Russia). A methodological approach

The present study aims to investigate the levels of polyphenols and antioxidant activity in one of the most important commercial species of seaweeds in Kamchatka, an edible brown seaweed Saccharina bongardiana. Six extracts of S. bongardiana, acetone, methanol, ethanol, and the respective 70 % aqueous solutions, were assessed for total phenol content in order to determine the most efficient extracting solvent. The total phenol content was measured by the Folin–Ciocalteu method and expressed as phloroglucinol equivalents (PGE). The antioxidant tests used were 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, linoleic acid-β carotene oxidation inhibiting assay, and Fe²⁺ ion chelating method. Higher phenolic contents were obtained using aqueous organic solvents, as compared to the respective absolute solvents; 70 % acetone was found to be the most efficient solvent (1.039 mg PGE 100 mg^?1 dry algal powder). High significant correlations were noted between total phenol content and the tested antioxidant activities; so the aqueous organic extracts exhibited the highest antioxidant activities versus DPPH radicals (EC₅₀ values of 0.6–1.1 mg dry weight (DW) mL^?1), linoleic acid-β carotene oxidation (74–78 % at 0.8 mg DW mL^?1), as well as ferrous ions (EC₅₀ values of 5.0–7.9 mg DW mL^?1). Some methodological recommendations regarding the assays used and the expression of results are proposed.     

Evaluation of antioxidant and anticancer activities of chemical constituents of the Saururus chinensis root extracts

Evaluation of antioxidant and anticancer activities were screened by various Saururus chinensis root extracts. Four solvents (ethyl acetate, methanol, ethanol, and water) extracts were investigated for their total flavonoids, phenol contents and their antioxidant activity of DPPH (2,2-diphenyl-1-picrylhydrazyl), NO (nitric oxide), H₂O₂ (hydrogen peroxide), ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)diammonium assays, FRAP (ferric reducing ability of plasma) assays and anticancer activity. The total phenolic and flavonoid content of extracts were determined by using FC (Folin–Ciocalteu) and AlCl₃ colorimetric assay method. Total flavonoid content in these plants ranged from 24.7 to 72.1 mg g^?1 and amount of free phenolic compounds was between 11.2 and 67.1 mg g^?1 extract. The all extracts have significant levels of phenolics and flavonoids content. Anticancer activity was screened for MCF-7 breast cancer cell line. Ethanol extract shows significant of antioxidant activity and water extract shows significant of anticancer activity compared with standard (BHT) butylated hydroxy toluene. These ethanol and water extracts could be considered as a natural source for using antioxidant, and anticancer agents compared to commercial available synthetic drugs.     

Hybrid On-Line Optimal Control Strategy for Producing α-Amylase by Bacillus subtilis

The combined effect of macronutrients in the extraction medium on α-amylase produced by Bacillus subtilis were studied by using response surface methodology in shaken flask cultures. The production of amylase was significantly affected by the interaction between wheat bran and the cotton seed extract in the extraction medium and by the interaction between the cotton seed extract and starch. The optimal combination in the extraction medium for maximum α-amylase production was determined as 10.80 g·L^?1 of wheat bran, 9.90 g·L^?1 of cotton seed extract, 0.5 g·L^?1 of starch, 2.0 g·L^?1 of yeast extract, 5.00 g·L^?1 of NaCl and 2.00 g·L^?1 of CaCl₂. A 12.55-fold increase of enzyme activity was recorded in the optimized medium compared to the result acquired in a minimum essential medium. The optimized medium was used to compare different cultivation strategies in fermenters. The pH-stat strategy for reducing cellular stress response and the substrate concentration-stat strategy for reducing substrate inhibition were independently investigated. The temperature-limited strategy has been proposed to solve the proteolytic digestion problem, although the high-pressure strategy resulted in high productivity. A hybrid strategy simultaneously controlling pH, temperature, substrate concentration and pO₂ was finally investigated to enhance the efficiency of the process. This hybrid strategy resulted in high activity of α-amylase, increasing the productivity almost three-fold as compared to an ordinary fed-batch culture.     

Antitumor and antioxidant activity of the freshwater macroalga <Emphasis Type="Italic">Cladophora surera</Emphasis>

Macroalgae are currently being explored as novel and sustainable sources of bioactive compounds for both pharmaceutical and nutraceutical applications arising from their antioxidant, anticancer, and antimicrobial activity. In the present study, the antitumoral and antioxidant activities of crude methanolic extracts of the freshwater macroalga Cladophora surera Parodi & Cáceres, harvested from Napostá Creek (Argentina), were investigated in vitro. The antioxidant activity was assessed by DPPH method and polyphenol content using Folin-Ciocalteu phenol reagent. Antitumoral activity was evaluated on the human breast adenocarcinoma cell line MCF-7 by measuring proliferation, migration, and cell adhesion. The algal extract (AE) showed a total phenol content of 1.62?±?0.17 μg GAE mg^?1 dry alga and DPPH scavenging activity of 25.03?±?1.99% (10 mg)^?1 dry alga. The trypan blue assay after 48 h of treatment indicated that the AE significantly inhibits proliferation in a dose-dependent manner (1–100 μg mL^?1), being more effective the highest dose employed, with a concomitant increment in dead cells. However, the colorimetric MTS assay only showed a significant decrease in cell viability at 100 μg mL^?1 AE. Using the wound healing assay, we demonstrated that AE inhibits cell migration. Through a cell adhesion assay, we found that AE affects considerably the cell adhesion capacity at all doses probed. Analysis of cell spreading indicated that cell morphology was also affected by AE treatment. These results indicate that C. surera could be a source of valuable bioactive compounds usable as antitumoral preventive therapy for their effects on the regulation of processes involved in metastasis in cells derived from human mammary cancer.