Optimization protocol for the extraction of antioxidant components from Origanum vulgare leaves using response surface methodology

In the present work, the response surface methodology (RSM) based on a central composite rotatable design (CCRD), was used to determine optimum conditions for the extraction of antioxidant compounds from Origanum vulgare leaves. Four process variables were evaluated at three levels (31 experimental designs): methanol (70%, 80%, and 90%), the solute:solvent ratio (1:5, 1:12.5, 1:20), the extraction time (4, 10, 16 h), and the solute particle size (20, 65, 110 micron). Using RSM, a quadratic polynomial equation was obtained by multiple regression analysis for predicting optimization of the extraction protocol. Analysis of variance (ANOVA) was applied and the significant effect of the factors and their interactions were tested at 95% confidence interval. The antioxidant extract (AE) yield was significantly influenced by solvent composition, solute to solvent ratio, and time. The maximum AE was obtained at methanol (70%), liquid solid ratio (20), time (16 h), and particle size (20 micron). Predicted values thus obtained were closer to the experimental value indicating suitability of the model. Run 25 (methanol:water 70:30; solute:solvent 1:20; extraction time 16 h and solute particle size 20) showed highest TP contents (18.75 mg/g of dry material, measured as gallic acid equivalents) and DPPH radical scavenging activity (IC₅₀ 5.04 μg/mL). Results of the present study indicated good correlation between TP contents and DPPH radical scavenging activity. Results of the study indicated that phenolic compounds are powerful scavengers of free radical as demonstrated by a good correlation between TP contents and DPPH radical scavenging activity.     

Optimization of extraction method for LC–MS based determination of phenolic acid profiles in different Impatiens species

The main aim of presented study was the comparison of various extraction methods for the quantitative and qualitative analysis (LC-ESI–MS/MS) of phenolic acids present in extracts obtained from leaves, flowers, and roots of Impatiens glandulifera. The accelerated solvent extraction (ASE) at three temperature ranges (80° C, 100° C, and 120° C), ultrasound assisted extraction (USAE) at 60° C, and traditional extraction in Soxhlet apparatus were used. Taking into account the extraction yield, and the diversity of the individual compounds, ultrasound assisted extraction proved to be the most efficient method, and it was used to determine the content of phenolic acids in leaves of four other Impatiens species, including I. balsamina, I. noli-tangere, I. parviflora, and I. walleriana. Eleven phenolic acids were identified in all examined species. These were protocatechuic, gentisic, 4- hydroxybenzoic, vanillic, trans-caffeic, syringic, trans-p-coumaric, trans- and cis-ferulic, salicylic, and 3-hydroxycinnamic acids. In the extract from the leaves of I. balsamina and I. walleriana, gallic and cis-p-coumaric acids were found additionally. The most abundant compounds in all examined extracts were protocatechuic and 3-hydroxycinnamic acids. The latest acid was found in the highest yield in I. noli-tangere (266.12 μg/g DW). In the leaves of I. glandulifera a great amount of 4-hydroxybenzoic (41.44 μg/g DW), vanillic (61.50 μg/g DW), and trans-p-coumaric (58.42 μg/g DW) acids was also observed. Our results indicate that protocatechuic, 4-hydroxybenzoic, vanillic, trans-p-coumaric, trans-ferulic, and 3-hydroxycinnamic acids were most characteristic of Impatiens species.Additionally, various phenolic-rich extracts from leaves, flowers, and roots of Impatiens glandulifera were tested for antioxidant activity. The highest antiradical activity was detected for roots using Soxhlet extraction (EC50 = 0.055 mg [DE/ml]).The study demonstrated that members of the genus Impatiens, and in particular Impatiens glandulifera, and Impatiens noli-tangere, contain significant amounts of phenolic acids. In addition, extracts from various parts of I. glandulifera could be interesting as novel sources of natural antioxidants.     

Optimization of ultrasound-assisted extraction conditions for total phenols with anti-hyperglycemic activity from Psidium guajava leaves

The high concentration of total phenolic compounds (TPC) in Psidium guajava leaf extracts (GvEx) is correlated to its anti-hyperglycemic activity. In this study, we established the optimum ultrasound extraction conditions for maximizing TPC yield. The response surface methodology (RSM) was employed for empirical model building. The maximum value of TPC (26.12%) was obtained at solvent to solid ratio (v/w) of 12.1, extraction temperature of 59.8 °C, and extraction time of 5.1 min. The anti-hyperglycemic activity of GvEx was compared to the commonly used diabetic drug acarbose. The IC₅₀ of GvEx for α-amylase and α-glucosidase inhibition was 50.5 μg/mL and 34.6 μg/mL, respectively. However, the IC₅₀ of acarbose for α-amylase and α-glucosidase inhibition was 95.3 μg/mL and 1075.2 μg/mL, respectively. In conclusion, GvEx obtained under optimum extraction conditions had higher anti-hyperglycemic activity than acarbose. In addition, the recommended extraction procedures for GvEx save time and are environmentally friendly.     

Retinoid compounds associated with water blooms dominated by Microcystis species

Retinoic acids play a critical role in vital physiological processes and vertebrate development, and their derivatives can be produced by some cyanobacterial species into surface waters. This study presents important environmentally-relevant information on total retinoid-like activity of field cyanobacterial biomasses and their surrounding waters. Intracellular and extracellular levels of total retinoid-like activity and retinoic acids have been investigated at a set of independent sites with the occurrence of water bloom dominated by widespread species Microcystis aeruginosa. Twelve samples of biomass and surrounding water from seven localities affected by blooms were studied in comparison with samples from M. aeruginosa laboratory cultures. The method for biomass extraction was optimized and final extracts and samples of surrounding water concentrated by solid phase extraction were assessed using in vitro reporter gene bioassay and chemical analyses for all-trans-retinoic acid (ATRA), 9-cis retinoic acid (9-cis RA) and microcystins RR, LR and YR. Methanol was the most efficient solvent for the extraction of compounds with retinoid-like activity. An in vitro bioassay with the P19/A15 transgenic cell line revealed retinoid-like activity in all cyanobacterial biomasses in the range of 356–2838 ng of retinoid acid equivalents (REQ)/g dry mass (dm), while only three of surrounding water samples exhibited detectable retinoid-like activity, in the range of 12.8–28.7 ng REQ/L. Microcystins were detected in all samples, but they elicited no detectable retinoid-like activity up to 10 mg/L. Chemical analyses detected concentrations up to 340 ng/g dm of all-trans-retinoic acid (ATRA) and 84 ng/g dm 9-cis retinoic acid (9-cis RA) in bloom extracts, and up to 19 ng/L ATRA and 2.2 ng/L 9-cis RA in surrounding water. In most samples, ATRA and 9-cis RA contributed relatively little to the total REQs, which indicates the presence of significant amounts of other compounds with retinoic acid receptor-mediated modes of action. The impact of retinoid-like cyanobacterial metabolites could be of importance namely in smaller water bodies with dense water blooms and low dilution.     

Novel extraction method of antioxidant compounds from Sasa palmata (Bean) Nakai using steam explosion

Antioxidant compounds were extracted from various parts of Sasa palmata (Bean) Nakai, a bamboo plant whose leaves are commonly used to wrap foodstuffs such as Sushi in Japan. Highest concentrations of antioxidant compounds existed in the leaf part of S. palmata. Steam explosion treatment followed by hot water and methanol extractions was used for separating the antioxidant compounds from S. palmata leaf. The steam explosion treatment is the physical–chemical treatment which crushes a sample by sudden reduction of the pressure in reactor to atmospheric pressure after steaming the sample at high temperature and pressures. Sasa palmata leaf was hydrolyzed by steaming and crushed by the rapid decompression. The optimal condition of steam explosion for the effective extraction of antioxidant compounds from S. palmata was determined as a steam of temperature of 250 °C and a steaming time of 1 min. In these conditions 217.41 mg/(g-Sasa leaf) of phenolic compounds and 142.81 mg/(g-Sasa leaf) of radical scavenging activity, that was expressed as butylated hydroxyanisole (BHA), were obtained.     

The hydrolysate of barley straw containing inhibitors can be used to produce cephalosporin C by solvent extraction using ethyl acetate

When the solvent extraction of the hydrolysate from barley straw was performed using ethyl acetate (EA), the logarithm of the partition coefficient (log P) of the phenols and furans for EA was found to be more than 1.00, which means that more than 90% of the inhibitors were removed from the hydrolysate layer. Cephalosporin C (CPC) was produced from the hydrolysate of dilute acid pretreatment (DAP) by Acremonium chrysogenum M35. A. chrysogenum M35 was cultured using the hydrolysate and the amount of CPC produced was found to be 10.35 g/L at 144 h. Also, the dry cell weight was about 101.5 g/L at 120 h. The utilization of the hydrolysate for CPC production was effective and the solvent extraction method for the removal of inhibitory substances could contribute to the biorefinery process.     

Effects of fermented rooibos (Aspalathus linearis) on adipocyte differentiation

Rooibos (Aspalathus linearis) contains a rich complement of polyphenols, including flavonoids, considered to be largely responsible for its health promoting effects, including combatting obesity. The purpose of this study was to examine the effect of fermented rooibos hot water soluble solids on in vitro adipocyte differentiation by using differentiating 3T3-L1 adipocytes. Hot water soluble solids were obtained when preparing an infusion of fermented rooibos at “cup-of-tea” strength. The major phenolic compounds (>5 mg/g) were isoorientin, orientin, quercetin-3-O-robinobioside and enolic phenylpyruvic acid-2-O-β-d-glucoside. Treatment of 3T3-L1 adipocytes with 10 μg/ml and 100 μg/ml of the rooibos soluble solids inhibited intracellular lipid accumulation by 22% (p < 0.01) and 15% (p < 0.05), respectively. Inhibition of adipogenesis was accompanied by decreased messenger RNA (mRNA) expression of PPARγ, PPARα, SREBF1 and FASN. Western blot analysis exhibited decreased PPARα, SREBF1 and AMPK protein expression. Impeded glycerol release into the culture medium was observed after rooibos treatment. None of the concentrations of rooibos hot water soluble solids was cytotoxic, in terms of ATP content. Interestingly, the higher concentration of hot water soluble solids increased ATP concentrations which were associated with increased basal glucose uptake. Decreased leptin secretion was observed after rooibos treatment. Our data show that hot water soluble solids from fermented rooibos inhibit adipogenesis and affect adipocyte metabolism, suggesting its potential in preventing obesity.     

Hollow fiber liquid phase microextraction followed by high performance liquid chromatography for determination of ultra-trace levels of Se(IV) after derivatization in urine,plasma and natural water samples

In the present work, a simple and high sensitive method based on hollow fiber liquid phase microextraction (HF-LPME) was developed followed by high performance liquid chromatography (HPLC) for determination of ultra-trace amounts of Se(IV) after derivatization in biological and natural water samples. Se(IV) was complexed with o-phenylenediamine to form piazselenol. The formed piazselenol was extracted into 20 μL of 1-octanol located in the lumen of a hollow fiber and the solution was injected into HPLC-UV for analysis. Using the Taguchi method, an orthogonal array design (OAD), OA₁₆ (4⁵) was employed to optimize the HF-LPME of piazselenol. The effect of five experimental factors (each factor at four levels) including the volume of the organic phase, extraction time, pH of the solution, stirring rate and ionic strength on the extraction efficiency of piazselenol was studied and optimized. The maximum extraction efficiency of piazselenol was obtained at 20 μL of 1-octanol as the extracting solvent, 30 min extraction time, pH 2, stirring rate of 500 rpm and 30% (w/v) NaCl. Under the optimum conditions, preconcentration factors up to 130 were achieved and the relative standard deviation (%RSD) of the method was <3.7% for different concentrations of Se(IV). The calibration curves were obtained in the ranges of 0.2–100 and 0.05–10 μg L^?1 for the 11 and 50 mL of the sample volumes with reasonable linearity, respectively (r² > 0.995). The limits of detection (LOD) were 0.1 and 0.02 μg L^?1 for the 11 and 50 mL sample volumes, respectively (S/N = 3). Finally, the applicability of the proposed method was evaluated by the extraction and determination of Se(IV) in the plasma, urine and water samples.     

Application of genetic algorithm for determination of the effective diffusivity of andrographolide in plant materials

The effective diffusivity of natural medicinal components is an important property for a detailed pharmaceutical extraction study and a proper design of the extraction process and unit. A mass transfer mathematical model for solvent extraction of andrographolide was developed and numerically solved taking into account particle geometries, solvent concentration, mass fraction of two different geometries of plant materials, external mass transfer resistance and solid–liquid equilibrium concentration. Using inverse simulation approach with genetic algorithm, unknown four key parameters in the developed model, effective diffusivity, partition coefficient, average particle size and mass fraction of leaf particles were determined at different extraction temperatures and solvent concentrations. The simulated data through the genetic algorithm-numerical model (GA-NM) and experimental data showed a good agreement (r² > 0.97 and MSE < 0.0016) indicating that the scheme with genetic algorithm can be effectively used for determination of the effective diffusivity of andrographolide in plant materials.     

Magnetic molecularly imprinted polymers for improved extraction of tanshinones from herbs via integrated extraction and cleanup system

A novel separation technology at room temperature for traditional Chinese medicines was proposed in this work by adding magnetic molecularly imprinted polymers (M-MIPs) into extraction solution and sample matrix. The M-MIPs show a more adsorption capacities and higher selectivity for the template than magnetic non-molecularly imprinted polymers (M-NMIPs) without the specific binding sites. Addition of the M-MIPs to the extraction solution provides one-step extraction and cleanup, the improvement of extraction rate and extraction yields of three tanshinones (from 0.40, 0.23 and 0.12 mg g⁻¹ in 240 min by solvent extraction to 0.52, 0.27 and 0.19 mg g⁻¹ in 5 min by 200 mg sorbent), and reusability of extraction solvent. The extraction yields of three tanshinones by this technology at room temperature in 5 min were higher than those by ultrasonic extraction in 30 min, by heat reflux extraction in 45 min and by solvent extraction at room temperature in 4 h. The integrated technology has the advantages of one-step extraction and cleanup, high extraction efficiency, low solvent consumption and room temperature.     

Extraction,purification and characterization of galactomannans from non-traditional sources

This work presents a methodology for the extraction of galactomannans from seeds of four different species of Leguminosae (Adenanthera pavonina, Caesalpinia pulcherrima, Gleditsia triacanthos and Sophora japonica) to be used e.g. in the food and biomedical industries. The galactomannans were obtained by aqueous extraction followed by a precipitation with ethanol. This methodology is simpler and easier to perform than other existing extraction and purification methodologies, and because it avoids the use of organic solvents (other than ethanol), it is able to generate food grade substances and is environmentally friendlier. The yield of extraction in different stages of the process, monosaccharide composition, as well as physical and chemical parameters of the isolated galactomannans were determined and compared with previously published results. The mannose/galactose ratio of the extracted galactomannans ranged from 1.35 (A. pavonina) to 5.75 (S. japonica). The intrinsic viscosity ranged from 11.34 dL/g (C. pulcherrima) to 8.74 dL/g (S. japonica), while the viscosity average molecular mass ranged between 1.81 × 10⁶ Da and 1.17 × 10⁶ Da (A. pavonina > C. pulcherrima > G. triacanthos > S. japonica). The results confirm the suitability of the extraction and purification procedure to obtain galactomannans from non-traditional sources.     

In vitro anti-cancer activities of Job’s tears (Coix lachryma-jobi Linn.) extracts on human colon adenocarcinoma

The whole seed (W), endosperm (E) and hull (H) of five cultivars of Job’s tears (Coix lachryma-jobi Linn. var. ma-yuen Stapf) including Thai Black Phayao, Thai Black Loei, Laos Black Loei, Laos White Loei and Laos Black Luang Phra Bang were processed before solvent extraction by non-cooking, roasting, boiling and steaming Each part of the Job’s tears was extracted by the cold and hot process by refluxing with methanol and hexane. The total of 330 extracts included 150 methanol extracts and 180 hexane extracts were investigated for anti-proliferative activity on human colon adenocarcinoma cell line (HT-29) by the sulforhodamine B (SRB) assay. The extracts which gave high anti-proliferative activity were tested for apoptotic activity by acridine orange and ethidium bromide double staining and anti-oxidative activities including free radical scavenging and lipid peroxidation inhibition activities. The extract from the hull of Thai Black Loei roasted before extracting by hot methanol (M-HTBL-R2) showed the highest anti-proliferative activity on HT-29 with the IC₅₀ values of 11.61 ± 0.95 μg/ml, while the extract from the non-cooked hull of Thai Black Loei by cold methanol extraction (M-HTBL-N1) gave the highest apoptosis (8.17 ± 1.18%) with no necrosis. In addition, M-HTBL-R2 and M-HTBL-N1 indicated free radical scavenging activity at the SC₅₀ values of 0.48 ± 0.12 and 2.47 ± 1.15 mg/ml, respectively. This study has demonstrated the anti-colorectal cancer potential of the M-HTBL-R2 and M-HTBL-N1 extracts.     

Extraction of peroxidase from bitter gourd (Momordica charantia) by three phase partitioning with dimethyl carbonate (DMC) as organic phase

Three phase partitioning (TPP) is most renowned technique used for extraction and purification of natural products. In previous studies of TPP, t-butanol is mainly used as an organic phase. This is the first report that explores ability of dimethyl carbonate (DMC) in the field of TPP as an alternate solvent for t-butanol. In the present study TPP process with t-butanol and DMC as organic phase along with different salts was applied to waste bitter gourd powder to obtained peroxidase enzyme. DMC was found to be compatible with most of salts such as ammonium sulphate and sodium citrate and explored as more efficient solvent than t-butanol. This TPP system provides 4.84 fold purity of peroxidase enzyme at optimum source concentration of 0.15 g/mL, with a system comprising DMC as organic phase, sodium citrate (20%) as salt, agitation speed 120 rpm, pH 7, temperature 30 °C and extraction time of 3 h. Present study has aimed for extraction and separation of peroxidase from bitter gourd waste with TPP technique and ensures the scope of carbonated solvents in extraction and purification of proteins.     

Free radical scavenging activity from different extracts of leaves of Bauhinia vahlii Wight & Arn.

The objectives of this study were to determine phenolic content and antioxidant activities of chloroform, acetone, methanol and hot water extracts of Bauhinia vahlii leaves. The hot water extract afforded the highest yield (6.3%) while the lowest yield was obtained from the chloroform extract (2.1%). The methanol extract contains higher levels of total phenolics (48.7 ± 0.7 g GAE/100 g extract), tannins (21.7 ± 0.7 g GAE/100 g extract) and flavonoids (10.3 ± 0.2 RE/100 g extract). The extracts were subjected to assess their antioxidant potential using various in vitro systems such as DPPH, ABTS⁺, FRAP, OH, β-carotene linoleic acid bleaching system, phosphomolybdenum reduction and Fe²⁺ chelation. It is concluded that the methanolic extract of B. vahlii leaves have strong antioxidant potential. Further study is necessary for isolation and characterization of the active antioxidants, which may serve as a potential source of natural antioxidants.     

Antioxidant activity and mineral composition of three Mediterranean common seaweeds from Abu-Qir Bay,Egypt

Antioxidant activity and mineral composition were evaluated seasonally from spring to autumn 2010 in the three common seaweeds Ulva lactuca Linnaeus (Chlorophyta), Jania rubens (Linnaeus) J.V. Lamouroux and Pterocladia capillacea (S.G. Gmelin) Bornet (Rhodophyta). The antioxidant activity was measured with β-carotene, total phenol content and DPPH (2,2-diphenyl-1-picrylhydrazyl). Seaweeds were collected from the rocky site near Boughaz El-Maadya Abu-Qir Bay of Alexandria, Egypt. The results showed maximum increase of β-carotene in P. capillacea during summer. A significant increase in total phenolic content at P ⩽ 0.05 was found in the red alga (J. rubens) during summer. Also, U. lactuca showed the maximum antioxidant scavenging activity especially during summer. Minerals in all investigated samples were higher than those in conventional edible vegetables. Na/K ratio ranged between 0.78 and 2.4 mg/100 g, which is a favorable value. All trace metals exceeded the recommended doses by Reference Nutrient Intake (RNI). During summer season, it was found that Cu = 2.02 ± 0.13 and Cr = 0.46 ± 0.14 mg/100 g in U. lactuca and Fe had a suitable concentration (18.37 ± 0.5 mg/100 g) in P. capillacea. The studied species were rich in carotenoids, phenolic compounds, DPPH free radicals and minerals, therefore, they can be used as potential source of health food in human diets and may be of use to food industry.     

Extraction and purification of a lectin from small black kidney bean (Phaseolus vulgaris) using a reversed micellar system

Lectin from crude extract of small black kidney bean (Phaseolus vulgaris) was successfully extracted using the reversed micellar extraction (RME). The effects of water content of organic phase (W_o), ionic strength, pH, Aerosol-OT (AOT) concentration and extraction time on the forward extraction and the pH and ionic strength in the backward extraction were studied to optimize the extraction efficiency and purification factor. Forward extraction of lectin was found to be maximum after 15 min of contact using 50 mM AOT in organic phase with W_o 27 and 10 mM citrate-phosphate buffer at pH 5.5 containing 100 mM NaCl in the aqueous phase. Lectin was backward extracted into a fresh aqueous phase using sodium-phosphate buffer (10 mM, pH 7.0) containing 500 mM KCl. The overall yield of the process was 53.28% for protein recovery and 8.2-fold for purification factor. The efficiency of the process was confirmed by gel electrophoresis analysis.     

Total phenolics,flavonoids and antioxidant properties of three Ceropegia species from Western Ghats of India

The aim of present work was to assess the total phenolic content (TPC), total flavonoid content (TFC), phenolic compounds and antioxidant properties of various extracts of three Ceropegia spp.: Ceropegia spiralis, Ceropegia panchganiensis and Ceropegia evansii from Western Ghats of India. TPC of the samples varied from 0.3 ± 0.2 to 28.5 ± 0.3 mg TAE/g FW, whereas, TFC of the samples ranged between 0.1 ± 0.1 and 15.3 ± 0.3 mg RE/g FW. The major phenolic compounds identified were gallic acid, vanillin, cathechol and ferulic acid. All the extracts possess 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP) as well as metal chelating ability and this was also supported by significant correlation with TPC and TFC. To the best of our knowledge, this is the first paper presenting comprehensive data on TPC, TFC, phenolic profile and antioxidant properties of the Ceropegia spp.     

Extraction of trace amounts of pioglitazone as an anti-diabetic drug with hollow fiber liquid phase microextraction and determination by high-performance liquid chromatography-ultraviolet detection in biological fluids

The applicability of hollow fiber liquid phase microextraction (HF-LPME) for extraction and preconcentration of trace amounts of pioglitazone (PGL) as an anti-diabetic drug in biological fluids, prior to determination by high-performance liquid chromatography (HPLC), was evaluated. In this technique, the target drug was extracted into di-n-hexyl ether immobilized in the wall pores of a porous hollow fiber from 10 mL of the aqueous sample (source phase, SP) with pH 8.0, and then back extracted into the receiving phase (RP) with pH 2.2 located in the lumen of the hollow fiber. The extraction occurred due to a pH gradient between the two sides of the hollow fiber. After extracting for a prescribed time, 24 μL of the RP solution was taken back into the syringe and injected directly into a HPLC instrument for quantification. The Taguchi orthogonal array (OAD) experimental design with an OA₁₆ (4⁵) matrix was employed to optimize the HF-LPME conditions. Different factors affecting the HF-LPME efficiency such as the nature of organic solvent used to impregnate the membrane, pH of the SP and RP, stirring speed, extraction time and ionic strength were studied and optimized. Under the optimum conditions (di-n-hexyl ether as membrane impregnation solvent, pHs of the SP and RP equal to 8.0 and 2.2, respectively, extraction time of 30 min, stirring speed of 500 rpm and 10% (w/v) NaCl for adjusting the ionic strength), preconcentration factor of 180, linear dynamic range (LDR) of 2.5–250 μg L^?1 with good correlation of determination (r² > 0.998) and limit of detection (LOD) of 1.0 μg L^?1 were obtained for the target drug. The percent relative intra-day and inter-day standard deviations (RSDs%) based on five replicate determinations were 4.7 and 15%, respectively. Once LPME was optimized, the performance of the proposed technique was evaluated for the determination of PGL in different types of biological fluids such as plasma and urine samples. The results showed that the proposed HF-LPME method could be successfully applied to determine trace amounts of PGL in biological samples.     

Purification and characterization of solvent stable,alkaline protease from Bacillus firmus CAS 7 by microbial conversion of marine wastes and molecular mechanism underlying solvent stability

A marine bacterium Bacillus firmus CAS 7 produced protease in the medium supplemented with 3:1 shrimp and crab shell powder at 55 °C and was purified with the specific activity of 473.4 U/mg. The purified protease was highly stable up to 70 °C, pH 11.0 and 30% NaCl. The protease purified was quite stable in the presence of anionic and non-ionic surfactants and organic solvents. The molecular dynamics simulation confirmed that the competition between organic solvent and water for the enzyme surface was comparatively higher in water–miscible organic solvent which is responsible for organic solvent stability. The purified protease from B. firmus CAS 7 could be greatly useful to develop industrial processes performed under harsh conditions or with denaturants and organic solvents. The protease production by microbial conversion of marine wastes suggested its potential utilization to generate high value-added products.     

Development of a highly sensitive ELISA for aldosterone in mouse urine: Validation in physiological and pathophysiological states of aldosterone excess and depletion

BackgroundClinical studies have established aldosterone as a critical physiological and pathophysiological factor in salt and water homeostasis, blood pressure control and in heart failure. Genetic and physiological studies of mice are used to model these processes. A sensitive and specific assay for aldosterone is therefore needed to monitor adrenocortical activity in murine studies of renal function and cardiovascular diseases.MethodsAntibodies against aldosterone were raised in sheep as previously described. HRP-Donkey-anti-sheep IgG enzyme tracer was produced in our laboratory using the Lightning-Link HRP technique. Aldosterone ELISA protocol was validated and optimised to achieve the best sensitivity. The assay was validated by analysing the urine of mice collected under various experimental conditions designed to stimulate or suppress aldosterone in the presence of other potentially interfering steroid hormones.ResultsCross-reactivity with the steroids most likely to interfere was minimal: corticosterone = 0.0028%, cortisol = 0.0006%, DOC = 0.0048% except for 5α-dihydro-aldosterone = 1.65%. Minimum detection limit of this ELISA was 5.2 pmole/L (1.5 pg/mL). The validity of urinary aldosterone ELISA was confirmed by the excellent correlation between results obtained before and after solvent extraction and HPLC separation step (Y = 1.092X + 0.03, R² = 0.995, n = 54). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Using this assay, mean urinary aldosterone levels were (i) approximately 60-fold higher in females than males mice; (ii) increased 6-fold by dietary sodium restriction; (iii) increased 10-fold by ACTH infusion and (iv) reduced by >60% in Cyp11b1 null mice.ConclusionWe describe an ELISA for urinary aldosterone that is suitable for repeated non-invasive measurements in mice. Female aldosterone levels are higher than males. Unlike humans, most aldosterone in mouse urine is not conjugated. Increased levels were noted in response to dietary sodium restriction and ACTH treatment. The sensitivity of the assay is sufficient to detect suppressed levels in mouse models of congenital adrenal hyperplasia.