Simultaneous analysis of diosgenin and sarsasapogenin in Asparagus officinalis byproduct by thin‐layer chromatography

Introduction – Asparagus officinalis L. has several biological activities including antifungal, antiviral and antitumoral activities due to the steroidal saponins. Normally diosgenin and sarsasapogenin are analysed separately by thin‐layer chromatography or high‐performance liquid chromatography (HPLC‐UV or HPLC‐ELSD), which is time‐consuming and expensive, so we need to find a rapid solution to this problem. Objective – To develop a sensitive, rapid and validated TLC method for simultaneous detection and quantification of diosgenin and sarsasapogenin. Methodology – Samples were prepared by extraction of A. officinalis with 70% aqueous ethanol to get steroidal saponins, and then hydrolysed using 36 mL 2 m hydrochloric acid for 3 h. The hydrolysis product was extracted with chloroform, and then analysed by TLC, the results of which were verified by HPLC and HPLC‐MS. Results – The retention factor (R_f) of diosgenin and sarsasapogenin on TLC plate were 0.49 and 0.6, respectively. After calculation from the regression equation of the standard curve, the contents of diosgenin and sarsasapogenin in the A. officinalis extract were 0.27–0.46 and 0.11–0.32%, respectively. Conclusion – The study showed that thin‐layer chromatography can be applied for the determination of diosgenin and sarsasapogenin in the oldest tissue of A. officinalis, and also can be conducted for screening of sapogenin in other plant or extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Design of experiment assisted concurrent enantioseparation of bupropion and hydroxybupropion by high‐performance thin‐layer chromatography

A simple and efficient high‐performance thin‐layer chromatographic method was developed for chiral separation of rac ‐bupropion (BUP) and its active metabolite rac ‐hydroxybupropion (HBUP). Design of experiment (DoE)‐based optimization was adopted instead of a conventional trial‐and‐error approach. The Box–Behnken design surface response model was used and the operating variables were optimized based on 17 trials design. The optimized method involved impregnation of chiral reagent, L(+)‐tartaric acid, in the stationary phase with simultaneous addition in the mobile phase, which consisted of acetonitrile : methanol : dichloromethane : 0.50% L‐tartaric acid (6.75:1.0:1.0:0.25, v /v /v /v ). Under the optimized conditions, the resolution factor between the enantiomers of BUP and HBUP was 6.30 and 9.26, respectively. The limit of detection and limit of quantitation for (R)‐BUP, (S)‐BUP, (R,R)‐HBUP, and (S,S)‐HBUP were 9.23 and 30.78 ng spot⁻¹, 10.32 and 34.40 ng spot⁻¹, 12.19 and 40.65 ng spot⁻¹, and 14.26 and 47.53 ng spot⁻¹, respectively. The interaction of L‐tartaric acid with analytes and their retention behavior was thermodynamically investigated using van't Hoff's plots. The developed method was validated as per the International Conference on Harmonization guidelines. Finally, the method was successfully applied to resolve and quantify the enantiomeric content from marketed tablets as well as spiked plasma samples.     

Free radical scavenging activity of novel thiazolidine‐2,4‐dione derivatives

Free radical activity towards superoxide anion radical (), hydroxyl radical (HO^?) and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) of a series of novel thiazolidine‐2,4‐dione derivatives (TSs) was examined using chemiluminescence, electron paramagnetic resonance (EPR) and EPR spin trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide (DMPO) was applied as the spin trap. Superoxide radical was produced in the potassium superoxide/18‐crown‐6 ether dissolved in dimethyl sulfoxide. Hydroxyl radical was generated in the Fenton reaction (Fe(II) + H₂O₂. It was found that TSs showed a slight scavenging effect (15–38% reduction at 2.5 mmol/L concentration) of the DPPH radical and a high scavenging effect of (41–88%). The tested compounds showed inhibition of HO^? ‐dependent DMPO‐OH spin adduct formation (the amplitude of EPR signal decrease ranged from 20 to 76% at 2.5 mmol/L concentration. Our findings present new group compounds of relatively high reactivity towards free radicals. Copyright © 2012 John Wiley & Sons, Ltd.     

Degradation of alkali lignin by two ascomycetes and free radical scavenging activity of the products

Biodegradation and bioconversion of extracted alkali lignin was performed under varying concentrations of carbon and nitrogen sources, by two potential Ascomycetes ligninolytic fungus isolated from soil. Fungus, F10 was identified as Aspergillus flavus, while APF4 as Emericella nidulans based upon closed similarity with their morphology and high homology in 18S rRNA gene sequences. The alkali lignin degradation was checked in term of disappearance of lignin content and colority. Selected fungus, degraded 19–41.6% of alkali lignin (0.25%, w/v) within 21 days of incubation and reduced the colority up to 14.4–21%. The activity of ligninolytic enzymes was periodically checked. During alkali lignin degradation manganese peroxidase (13.31?U/ml), lignin peroxidase (13.73?U/ml) and laccase (0.05?U/ml) activities were observed (at highest level). The alkali lignin degradation products and functional group changes in degraded lignin were analysed through gas chromatography-mass spectroscopy (GC-MS) and solid state ¹³C-NMR spectroscopy, respectively. The functional group modifications in alkali lignin moiety, alter its biochemical property, thus fungal mediated modified alkali lignin was further tested for reactive free radical scavenging potential with respect to hydroxyl, nitric oxide and superoxide radicals. Results demonstrate that the alkali lignin undergo degradation in studied nutritional conditions (high-carbon low nitrogen) and consequently increase its free radical scavenging activity up to 1–18%.     

Hydroxyl and superoxide radical scavenging abilities of chromonyl‐thiazolidine‐2,4‐dione compounds

The oxygen free radical scavenging activities of 15 chromonyl‐thiazolidine‐2,4‐dione compounds (CTDs) were examined in chemical systems producing superoxide anion radicals, O (potasium superoxide–18‐crown‐6 ether–DMSO), and hydroxyl radicals, HO^? (a Fenton reaction: Fe(II)–H₂O₂–sodium trifluoroacetate, pH 6.15). Chemiluminescence and electron spin resonance (ESR) spectroscopy using 5,5‐dimethyl‐1‐pyrroline‐1‐oxide (DMPO) as spin trap were applied to evaluate antioxidant behaviour of CTDs towards the oxygen radicals. The results indicated that 11 of the 15 tested compounds showed a significant inhibitory effect on the chemiluminescence generated from the O‐generating system, ranging from 41 to 86%, and 13 CTDs quenched the ESR signal of the DMPO–OH spin adduct by 33–86%, at a concentration of 1 mmol L^?1. Our findings demonstrate that CTDs could be good free radical scavengers. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of polyphenolics in extracts of Potentilla species by high‐performance thin‐layer chromatography photodensitometry method

Introduction – Separation of polyphenolics in different plant materials using high‐performance thin‐layer chromatography (HPTLC) represents an effective method for their detection and quantification. Objective – To develop a simple, specific, precise, sensitive and accurate method for the simultaneous quantification of tiliroside (TRS), methyl brevifolincarboxylate (MBR) and ellagic acid (EA) in a plant extract using the HPTLC‐photodensitometry method. Methodology – Aerial parts of the selected Potentilla species, P. anserina, P. erecta, P. grandiflora and P. nepalensis var. ‘Miss Willmott’, were extracted with methanol. After solvent evaporation, the methanolic extracts were diluted with water and successively partitioned between chloroform and then diethyl ether. The diethyl ether extracts from each sample were used for quantification. The analyses were performed on HPTLC precoated silica gel 60F₂₅₄ plates with toluene–ethyl formate–formic acid (6 : 4 : 1 v/v/v) as the mobile phase (distance of 7.5 cm). Densitometric detections of TRS, MBR and EA were performed at 320, 287 and 280 nm, respectively. The amounts of these compounds were calculated using the regression equations of the calibration curves, which were linear within a range of 0.05–0.5 μg/spot (R² = 0.9957) for TRS, 0.05–0.525 μg/spot (R² = 0.9965) for MBR and 0.0525–0.5 μg/spot (R² = 0.9998) for EA. Results – The amounts of marker compounds measured by the method developed are expressed in mg/g of dry extracts. TRS ranged from 20.3 ± 0.3 mg/g for P. erecta herbs to 197.7 ± 2.9 mg/g for P. grandiflora herbs; MBR ranged from 5.0 ± 0.6 mg/g for P. erecta herbs to 68.5 ± 3.4 mg/g for P. nepalensis flowers; and EA ranged from 24.0 ± 0.6 mg/g for P. erecta herbs to 216.2 ± 3.2 mg/g for P. anserina leaves. Conclusion – The proposed method was found to be relatively simple, specific, precise, sensitive and accurate and may be used for the routine assay of simultaneous determination of TRS, MBR and EA in other extracts and phytomedicines containing Potentilla species. Copyright © 2009 John Wiley & Sons, Ltd.     

Free radical scavenging activity of Lactobacillus fermentum in vitro and its antioxidative effect on growing–finishing pigs

Aims:  To evaluate the free radical-scavenging capacity of Lactobacillus fermentum and its effects on antioxidant enzyme levels in finishing pigs.
Methods and Results:  The free radical-scavenging activity of Lact. fermentum was analysed in vitro . The tested Lactobacillus showed a high scavenging ability against DPPH (1,1-diphenyl-2-picrylhydrazyl), superoxide and hydroxyl radicals which was dose dependent. Subsequently, 108 crossbred pigs weighing 20·67 BW, were allotted to dietary treatments including a basal diet or the basal diet supplemented with either aureomycin or 10·2 × 10⁷  Lact. fermentum CFU g⁻¹ diet. Supplementation of Lact. fermentum increased total antioxidant capacity ( P  < 0·01) in serum from 50 kg pigs, while serum superoxide dismutase ( P  = 0·01) and glutathione peroxidase ( P  < 0·01) increased, and malondialdehyde levels decreased ( P  < 0·01) in 90 kg pigs. Hepatic catalase ( P  = 0·04), muscle superoxide dismutase ( P  < 0·01) and copper–zinc-superoxide dismutase were enhanced ( P  = 0·01), whereas malondialdehyde levels were reduced ( P  = 0·05) by Lact. fermentum .
Conclusions:  The free radical-scavenging capacity of Lact. fermentum was dose dependent and its supplementation improved the antioxidant status of pigs.
Significance and Impact of the Study:  Lactobacillus fermentum could be used to alleviate oxidative stress and increase pig performance and improve pork quality.     

Scavenging of superoxide anion radical and hydroxyl radical by novel thiazolyl‐thiazolidine‐2,4‐dione compounds

The antioxidant behavior of a series of new synthesized substituted thiazolyl‐thiazolidine‐2,4‐dione compounds (TZDs) was examined using chemiluminescence and electron paramagnetic resonance spin trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide (DMPO) was used as the spin trap. The reactivity of TZDs with superoxide anion radical (O) and hydroxyl radical (HO^?) was evaluated using potassium superoxide/18‐crown‐6 ether dissolved in dimethylsulfoxide, and the Fenton‐like reaction (Fe²⁺ + H₂O₂), respectively. The results showed that TZDs efficiently inhibited light emission from the O generating system at a concentration of 0.05–1 mmol L^?1 (5–94% reductions were found at 1 mmol L^?1 concentration). The TZD compounds showed inhibition of HO^?‐dependent DMPO–OH spin adduct formation from DMPO (the amplitude decrease ranged from 8 to 82% at 1 mmol L^?1 concentration). The findings showed that examined TZDs had effective activities as radical scavengers. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of Tanshinones in Danshen (Salvia miltiorrhiza) by High‐Performance Liquid Chromatography with Fluorescence Detection after pre‐Column Derivatisation

Introduction

Tanshinones are a major class of bioactive ingredients in the traditional herbal medicines, Danshen (Salvia miltiorrhiza). A sensitive and reliable determination method for tanshinones is useful to ensure the quality of Danshen.

Objective

To develop a sensitive and selective analytical method for tanshinones by high‐performance liquid chromatography (HPLC) with fluorescence detection after pre‐column derivatisation.

Methodology

The proposed method depends on derivatisation reaction of tanshinones with 4‐carbomethoxybenzaldehyde and ammonium acetate forming intensely fluorescent imidazole derivative.

Results

The proposed method provided excellent sensitivity with the detection limits of 3.3 nM (66 fmol/injection), 3.2 nM (64 fmol/injection) and 2.0 nM (40 fmol/injection) for cryptotanshinone, tanshinone I and tanshinone IIA, respectively, without the necessity of complicated instrumentations. The developed method is successfully applied to quantify the contents of tanshinones in Danshen.

Conclusion

The developed method is the first analytical method for tanshinones by fluorescence detection. Since the derivatisation reaction is selective for the o‐quinone structure of tanshinone, the developed method will become a suitable mean for the discovering of tanshinone type diterpenoids from herbal samples. Copyright © 2017 John Wiley & Sons, Ltd.     

Optimization of enzyme-assisted improvement of polyphenols and free radical scavenging activity in red rice bran: A statistical and neural network-based approach

The current study is focused on optimizing the parameters involved in enzymatic processing of red rice bran for maximizing total polyphenol (TP) and free radical scavenging activity (FRSA). The sequential optimization strategies using central composite design (CCD) and artificial neural network (ANN) modeling linked with genetic algorithm (GA) was performed to study the effect of incubation time (60–90?min), xylanase concentration (5–10?mg/g), cellulase concentration (5–10?mg/g) on the response, i.e., total polyphenol and FRSA. The result showed that incubation time has a negative effect on the response, while the square effect of xylanase and cellulase showed positive effect on the response. A maximum TP of 2,761?mg ferulic acid Eq/100?g bran and FRSA of 778.4?mg Catechin Eq/100?g bran was achieved with incubation time (min)?=?60.491; xylanase (mg/g)?=?5.4633; cellulase (mg/g)?=?11.5825. Furthermore, ANN-GA-based optimization showed better predicting capabilities as compared to CCD.     

Real‐time measurement of cytosolic free calcium concentration in DEM‐treated HL‐60 cells during static magnetic field exposure and activation by ATP

This study investigated whether glutathione depletion affected the sensitivity of HL‐60 cells to static magnetic fields. The effect of Diethylmaleate (DEM) on static magnetic field induced changes in cytosolic free calcium concentration ([Ca²⁺]_c) was examined. Cells were loaded with a fluorescent dye and exposed to a uniform static magnetic field at a strength of 0 mT (sham) or 100 mT. [Ca²⁺]_c was monitored during field and sham exposure using a ratiometric fluorescence spectroscopy system. Cells were activated by the addition of ATP. Metrics extracted from the [Ca²⁺]_c time series included: average [Ca²⁺]_c during the Pre‐Field and Field Conditions, peak [Ca²⁺]_c following ATP activation and the full width at half maximum (FWHM) of the peak ATP response. Comparison of each calcium metric between the sham and 100 mT experiments revealed the following results: average [Ca²⁺]_c measured during the Field condition was 53 ± 2 nM and 58 ± 2 nM for sham and 100 mT groups, respectively. Average FWHM was 51 ± 3 s and 54 ± 3 s for sham and 100 mT groups, respectively. An effect of experimental order on the peak [Ca²⁺]_c response to ATP in sham/sham experiments complicated the statistical analysis and did not allow pooling of the first and second order experiments. No statistically significant difference between the sham and 100 mT groups was observed for any of the calcium metrics. These data suggested that manipulation of free radical buffering capacity in HL‐60 cells did not affect the sensitivity of the cells to a 100 mT static magnetic field. Bioelectromagnetics 30:213–221, 2009. © 2008 Wiley‐Liss, Inc.     

Indirect synchronous fluorescence spectroscopy and direct high‐performance thin‐layer chromatographic methods for enantioseperation of zopiclone and determination of chiral‐switching eszopiclone: Evaluation of thermodynamic quantities of chromatographic separation

Economic and enantioselective synchronous fluorescence spectroscopy and high‐performance thin‐layer chromatography methods have been developed and validated as per ICH guidelines for the separation of zopiclone enantiomers using L‐(+)‐tartaric acid as a chiral selector, followed by determination of the chiral‐switching eszopiclone. Synchronous fluorescence spectroscopy was successfully applied for chiral recognition of R & S enantiomers of zopiclone at ?λ = 110 nm based on creating of diastereomeric complexes with 0.06M tartaric acid in an aqueous medium containing 0.2M disodium hydrogen orthophosphate. Synchronous fluorescence intensities of eszopiclone were recorded at 296 nm in concentration range 0.2‐ to 4‐μg/mL eszopiclone. High‐performance thin‐layer chromatography method depends on resolution of zopiclone enantiomers on achiral HPTLC silica‐gel plates using acetonitrile:methanol:water (8:2:0.25, v/v/v) containing L‐(+)‐tartaric acid as a chiral mobile‐phase additive followed by densitometric measurements at 304 nm in concentration range of 1 to 10 μg/band of eszopiclone. The effect of chiral‐selector concentration, pH, and temperature on the resolution have been studied and optimized for the proposed methods. The cited procedures were successfully applied to determine eszopiclone in commercial tablets of pure and racemic forms. Enantiomeric excess was evaluated using optical purity test and integrated peak area to describe the enantiomeric ratio. Thermodynamics of chromatographic separation, enthalpy, and entropy were evaluated using the Van't Hoff equation. The proposed methods were found to be selective for identification and determination of the eutomer in drug substances and products.     

Phytochemical analysis of Moringa Oleifera leaves extracts by GC-MS and free radical scavenging potency for industrial applications

Natural extracts have been of very high interest since ancient time due to their enormous medicinal use and researcher’s attention have further gone up recently to explore their phytochemical compositions, properties, potential applications in the areas such as, cosmetics, foods etc. In this present study phytochemical analysis have been done on the aqueous and methanolic Moringa leaves extracts using Gas Chromatography-Mass spectrometry (GCMS) and their free radical scavenging potency (FRSP) studied using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical for further applications. GCMS analysis revealed an extraction of range of phytochemicals in aqueous and methanolic extracts. In aqueous, extract constituents found with high percent peak area are Carbonic acid, butyl 2-pentyl ester (20.64%), 2-Isopropoxyethyl propionate (16.87%), Butanedioic acid, 2-hydroxy-2-methyl-, (3.14%) (also known as Citramalic acid that has been rarely detected in plant extracts) and many other phytochemicals were detected. Similarly, fifty-four bio components detected in methanolic extract of Moringa leaves, which were relatively higher than the aqueous extract. Few major compounds found with high percent peak area are 1,3-Propanediol, 2-ethyl-2- (hydroxymethyl)- (21.19%), Propionic acid, 2-methyl-, octyl ester (15.02%), Ethanamine, N-ethyl-N-nitroso- (5.21%), and 9,12,15-Octadecatrienoic acid etc. FRSP for methanolic extract was also recorded much higher than aqueous extract. The half-maximal inhibitory concentration (IC₅₀) of Moringa aqueous extract observed is 4.65 µl/ml and for methanolic extract 1.83 µl/ml. These extracts can act as very powerful antioxidants, anti-inflammatory ingredient for various applications in diverse field of food, cosmetics, medicine etc.     

A flow‐injection chemiluminescent method for the evaluation of the antioxidant activity of 5′‐nucleotides

In the present work, a simple chemiluminescence (CL) method coupled with flow‐injection analysis for the evaluation of antioxidant activity of 5′‐nucleotides (5′‐AMP, 5′‐CMP, 5′‐GMP, 5′‐UMP) was proposed. It is based on inhibition effect of the studied substances on CL emission of luminol–potassium ferricyanide–pyrogallol. Experiments were performed to evaluate the nature of the inhibition by 5′‐nucleotides of the CL reaction and their antioxidant activities. Based on the experimental results, it was observed that 5′‐nucleotides are available antioxidants that could effectively scavenge superoxide anion free radicals in a concentration‐dependent way. This will provide a basis for further development of the use of nucleotides. Copyright © 2009 John Wiley & Sons, Ltd.