Acidified Methanol Extracts Research Articles

The effects of temperature and pH on stability of anthocyanins from red sorghum (Sorghum bicolor) bran

Anthocyanin’s have been suggested as promising dietary compounds with an important role in human health. This study focuses on the effect of temperature and pH on stability of anthocyanins from red sorghum bran. The sorghum anthocyanins were found to be stable at 0°C even when exposed to light and dark conditions. The anthocyanins extracted using acidified methanol showed higher stability than methanol extracts. The study on anthocyanin stability over the pH range (1.1 to 10.5) during 20 days storage was conducted on sorghum anthocyanins compared to methanol and acidified methanol extracts afforded higher colour intensity and higher or similar stability throughout the whole pH range. At pH 8.9, the colour intensity of sorghum anthocyanins was even higher than at the lowest pH values. The sorghum 3-deoxyanthocyanins were a rare class of plant pigments, and were stable to light, heat and change in pH. The use of sorghum anthocyanins as food colourants in slightly alkaline products (bakery, milk, egg, etc.) can therefore be considered. Key words: Sorghum, bran, anthocyanins, temperature, pH stability.

Ergosterol as an indicator of mold resistance in white and colored grain sorghum hybrids

Grain mold is a serious disease of sorghum causing both qualitative and quantitative losses in grain production. There has been a drastic reduction in area and production in India due to the lack of resistant varieties to manage the disease. Genetic improvement for mold resistance has been suggested as a sole criterion for reducing losses associated with grain mold and several morphological and biochemical traits have been employed to aid screening for resistance. However, there is always a bias with visual scoring systems to rate disease severity; thus, ergosterol and flavan-4-ols content within the grain was evaluated as selection criteria. Twenty-five hybrids were selected based on their visual rating for grain mold resistance and grain color from replicated trials at two locations in India during 2004 and 2005 rainy seasons and grouped into colored resistant, white resistant, colored susceptible and white susceptible. The field grade score (FGS) and threshed grade score (TGS) were strongly and positively correlated with ergosterol content and weakly and negatively correlated with flavan-4-ols. Though the resistant hybrids had higher concentration of flavan-4-ols in methanol and acidified methanol extract compared to the susceptible hybrids, the white grained hybrids had negligible amounts indicating flavan-4-ols concentration is not a suitable selection criterion especially in white grained hybrids. The ergosterol content was higher in susceptible hybrids (19.49μg/g) compared to resistant hybrids (9.96μg/g) and it was marginally less in red hybrids (14.0μg/g) compared to white (17.2μg/g) hybrids. The highly significant correlation of ergosterol content with germination percentage, FGS and TGS validates the use of this trait for identification of grain mold resistant genotypes provided there is no delay in the estimation. Also the significant heterosis obtained for this trait indicates the existence of dominance gene action and suggests that further exploitation of this trait would benefit the development of grain mold resistant hybrids.

DNA Damage Protecting Activity and Free Radical Scavenging Activity of Anthocyanins from Red Sorghum (Sorghum bicolor) Bran.

There is increasing interest in natural food colorants like carotenoids and anthocyanins with functional properties. Red sorghum bran is known as a rich source for anthocyanins. The anthocyanin contents extracted from red sorghum bran were evaluated by biochemical analysis. Among the three solvent system used, the acidified methanol extract showed a highest anthocyanin content (4.7 mg/g of sorghum bran) followed by methanol (1.95 mg/g) and acetone (1 mg/g). Similarly, the highest total flavonoids (143 mg/g) and total phenolic contents (0.93 mg/g) were obtained in acidified methanol extracts than methanol and acetone extracts. To study the health benefits of anthocyanin from red sorghum bran, the total antioxidant activity was evaluated by biochemical and molecular methods. The highest antioxidant activity was observed in acidified methanol extracts of anthocyanin in dose-dependent manner. The antioxidant activity of the red sorghum bran was directly related to the total anthocyanin found in red sorghum bran.

Identification of Antifungal Substance (Iturin A2) Produced by Bacillus subtilis B47 and Its Effect on Southern Corn Leaf Blight

Bacillus subtilis B47 is an endophytic bacterium of tomato, and produces substance that strongly inhibits the growth of Bipolaris maydis, the pathogen of southern corn leaf blight (SCLB), as well as several other phytopathogenic fungi. The antifungal substance was purified from the broth culture of the bacterium using acid precipitation, methanol extraction, and three-step chromatography. Based on FT-IR spectrometry, amino acid composition, and MALDI-TOF-MS/MS CID analyses, the antifungal substance was identified as iturin A2, a cyclic lipopeptide antibiotic. To evaluate the efficacy of iturin A2 for control of SCLB, partially purified iturin A2 (75%, w/v) was applied under different conditions. At the concentration of 300 mg kg−1, iturin A2 showed efficacy ranging from 100 to 53.1% under in vitro, in plot and in field conditions. This efficacy was higher than or similar to that of the fungicide chlorothalonil. When the concentration of iturin A2 was increased to 500 mg kg−1, the control efficacy was enhanced to 64.2% in field, which was significantly higher than that of chlorothalonil. These results indicate that iturin A2 has potential for SCLB control and could be a substitute to synthetic fungicides. To our knowledge, this is the first report on using partially purified iturin A for control of SCLB under field conditions.

Purification and Structural Analysis of Surfactin Produced by Endophytic Bacillus subtilis EBS05 and its Antagonistic Activity Against Rhizoctonia cerealis

Bacillus subtilis EBS05, an endophytic bacteria strain isolated from a medicinal plant Cinnamomum camphor, can produce antagonistic compounds that effectively inhibit plant pathogenic fungi. The greenhouse experiments showed that wheat sharp eyespot disease (WSED) was reduced by 91.2%, 88.2% and 43.0% after the treatment with fermentation broth, bacteria-free filter and a fungicide fludioxonil, respectively. The culture broth of strain EBS05 can more effectively control WSED than can fludioxonil. The fermentation broth and bacteria-free filter ability to suppress WSED was not significantly different, suggesting that an active secreted substance played a major role in controlling WSED. Separation and purification of the active compounds was carried out by serial processes, including hydrochloric acid (pH 2.0) treatment, methanol extraction and Sephadex LH-20 column chromatography, silica gel column chromatography and reverse-phase high-pressure liquid chromatography (HPLC), respectively. The purified compounds, one of active peaks in the HPLC spectrum, were obtained from the collection. Analysis of the chemical structures by time-of-flight mass spectrometry (TOF-MS) and electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS) showed that the active substances produced by the endophytic bacteria EBS05 are mixture of the <TEX>${\beta}$</TEX>-hydroxy-C12~C15-<TEX>$Leu^7$</TEX> surfactin A isomers with 1035.65 Da, 1021.64 Da, 1007.63 Da and 993.65 Da molecular weights, respectively.

Anthocyanin compositions and biological activities from the red petals of Korean edible rose (Rosa hybrida cv. Noblered)

The objectives of the present study were to investigate anthocyanin profiles and their biological properties, including antioxidant, anticancer, and antiallergic, from the red petals of Korean edible rose (Rosa hybrida cv. Noblered). The acidic methanol extract of this species showed potent biological activities at a concentration of 50μg/mL. Its anthocyanins were characterised as cyanidin 3,5-di-O-glucoside and pelargonidin 3,5-di-O-glucoside using reversed-phase C18 column chromatography, NMR spectroscopy, and HPLC-DAD-ESI/MS analysis. Cyanidin 3,5-di-O-glucoside was the predominant constituent (375mg/100g), representing about 85% of total content. Cyanidin 3,5-di-O-glucoside exhibited good scavenging activity against DPPH radical with IC50 value of 55.2μg/mL; pelargonidin 3,5-di-O-glucoside showed potent anticancer effects against LNCap (human prostate cell line), ACHN (human renal cell line) and MOLT-4F (human leukaemia cell line) cell cultures, with IC50 values of 6.43, 18.3, and 6.78μg/mL, respectively. Antiallergic activities were only moderate.

Anthocyanin production as a potential visual selection marker during plant transformation

A mutant allele of the transcription factor gene MYB10 from apple induces anthocyanin production throughout the plant. This gene, including its upstream promoter, gene coding region and terminator sequence, was introduced into apple, strawberry and potato plants to determine whether it could be used as a visible selectable marker for plant transformation as an alternative to chemically selectable markers, such as kanamycin resistance. After transformation, red coloured calli, red shoots and red well-growing plants were scored. Red and green shoots were harvested from apple explants and examined for the presence of the MYB10 gene by PCR analysis. Red shoots of apple explants always contained the MYB10 gene but not all MYB10 containing shoots were red. Strawberry plants transformed with the MYB10 gene showed anthocyanin accumulation in leaves and roots. No visible accumulation of anthocyanin could be observed in potato plants grown in vitro, even the ones carrying the MYB10 gene. However, acid methanol extracts of potato shoots or roots carrying the MYB10 gene contained up to four times higher anthocyanin content than control plants. Therefore anthocyanin production as result of the apple MYB10 gene can be used as a selectable marker for apple, strawberry and potato transformation, replacing kanamycin resistance.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-011-9490-1) contains supplementary material, which is available to authorized users.

Identification and characterisation of anthocyanins in the antioxidant activity-containing fraction of Liriope platyphylla fruits

The objective of this study was to investigate antioxidant activities and anthocyanin profiles in the fruits of Liriope platyphylla, where these are considered functional substances in Korea. The acidic methanol extract of this species exhibited potent antioxidant activities, showing 83.9% DPPH scavenging activity and 92.5% ABTS scavenging activity at a concentration of 0.5 mg/ml. Moreover, anthocyanins were identified by reversed-phase C 18 column chromatography, NMR spectroscopy, and HPLC-DAD-ESI/MS analysis. Seven anthocyanins were characterised, including delphinidin-3- O-glucoside ( 1), delphinidin-3- O-rutinoside ( 2), cyanidin-3- O-glucoside ( 3), petunidin-3- O-glucoside ( 4), petunidin-3- O-rutinoside ( 5), malvidin-3- O-glucoside ( 6), and malvidin-3- O-rutinoside ( 7). Among these, petunidin-3- O-rutinoside ( 5) (7302.2 μg/g) and malvidin-3- O-rutinoside ( 7) (5776.1 μg/g) were the predominant anthocyanins, whereas the least prevalent anthocyanin was found to be cyanidin-3- O-glucoside ( 3) (64.9 μg/g). Therefore, our results suggest that strong antioxidant activities of the acidic methanol extract of L. platyphylla fruits are correlated with high anthocyanin contents, particularly the petunidin-3- O-rutinoside ( 5) and malvidin-3- O-rutinoside ( 7).

Polyphenols of Mung Bean (Phaseolus aureusL.) Cultivars Differing in Seed Coat Color: Effect of Dehulling

Polyphenols are bioactive compounds, mainly concentrated in seed coats, which play an important role in human nutrition. Ten samples of mung bean (Phaseolus aureus L.), including three yellow cultivars representing a broad range of varietal characteristics, such as color and seed weight, were analyzed for their polyphenol content. The effect of dehulling on the polyphenol content was studied. The concentration of total polyphenols was found to be in the range of 280 mg to 356 mg/100 g whole seeds, 702 mg to 1296 mg/100 g seed coat, and 172 mg to 286 mg/100 g cotyledons. Most of the polyphenols were concentrated in the seed coat. The average polyphenol content of yellow cultivars was found to be higher than the green cultivars, except in ‘China Mung’. The acidic methanol extract polyphenols constitute about 75% and methanolic extract polyphenols about 25% of the total polyphenols. Dehulling reduced polyphenol content significantly (p < .01) by 14% to 52%. Yellow cultivars contained more seed coat polypheno...

Polyphenol content and antioxidant activity of California almonds depend on cultivar and harvest year

The polyphenol content and antioxidant activity of Nonpareil, Carmel, Butte, Sonora, Fritz, Mission, and Monterey almond cultivars harvested over three seasons in California were examined. LC–MS was employed to quantify 16 flavonoids and two phenolic acids in acidified methanol extracts of almond skins. The 3-year mean polyphenol content of cultivars ranged from 4.0 to 10.7 mg/100 g almonds. Isorhamnetin-3- O-rutinoside was the most abundant flavonoid, present at 28–49% of total polyphenols among cultivars. Almonds from 2006 and 2007 had 13% fewer polyphenols than 2005, but FRAP and total phenols were comparable. Cultivar, but not season, had a differential impact on individual polyphenol synthesis. Using the results of polyphenol, total phenol, and FRAP, multivariate analysis distinguished harvest years and most cultivars with 80% confidence. Flavonoid content and antioxidant activity of almonds may be more dependent on cultivar than on seasonal differences.

Production and characterization of a C 15-surfactin-O-methyl ester by a lipopeptide producing strain Bacillus subtilis HSO121

A surfactin mono-methyl ester was isolated from the cell broth of Bacillus subtilis HSO121 by acid precipitation, methanol extraction and two-step chromatographic methods. The structure was analyzed by GC/MS, HPLC, ESI Q-TOF MS/MS and NMR. It indicates that this ester is a C 15-surfactin-O-methyl ester of which the fatty acid portions are composed of iso-C 15 and anteiso-C 15 β-hydroxy fatty acids. Based on the critical micelle concentration (CMC) in phosphate buffered saline (PBS, pH 8.0) and the IC 50 value on Hela cell lines, the C 15-surfactin-O-methyl ester has a stronger surface activity and lower antitumoral effect than C 15-surfactin does. It is found that the Glu residues of surfactin-like lipopeptides play a role in their anti-proliferative effects on Hela cells. The possible antitumoral activity of surfactin is discussed in relation to their structures.

Rapid detection of ketamine and norketamine in rat hair using micropulverized extraction and ultra‐performance liquid chromatography–electrospray ionization mass spectrometry

A new method for the rapid and simultaneous detection of ketamine and its major metabolite, norketamine, in rat hair has been developed by combining micropulverized extraction and ultraperformance liquid chromatography-electrospray ionization mass spectrometry. By using reversed-phase UPLC, ketamine and norketamine were well separated within 2 min. Using ketamine-dosed rat hair, the conditions for micropulverized extraction were optimized, and the limits of detection and quantification of the developed method were found to be 1.7 and 5.7 pg/mg hair for ketamine, respectively. The precisions achieved with this method were slightly better than that obtained with conventional acidic methanol extraction method. Using this proposed method, analysis of the washed rat hair could be completed within 16-17 min. This method is expected to be applied for the analysis of the hair samples of not only rats but also ketamine abusers.

Evaluation of antioxidant and antimicrobial properties of finger millet polyphenols ( Eleusine coracana)

Phenolic acids from finger millet ( Eleusine coracana) milled fractions (whole flour, seed coat, 3%, 5% and 7%) were isolated and their antioxidant and antimicrobial properties were evaluated. Acidic methanol extracts from seed coat to whole flour were rich in polyphenol content and were found to be stable up to 48 h at pH 4, 7, and 9 as studied by ultraviolet spectroscopy. Diadzene, gallic, coumaric, syringic and vanillic acids were identified as major phenolic acids from the extracted phenolics. Diadzene content was highest in concentration in the 5% flour. The reducing power of seed coat extract was significantly ( p < 0.05) higher than that of whole flour extract. Antioxidant activity (AA) as determined by the β-carotene–linoleic acid assay indicated that the AA was highest in seed coat extract (86%), whilst at the same concentration it was only 27% in the whole flour extract. The seed coat extract showed higher antimicrobial activity against Bacillus cereus and Aspergillus flavus compared to whole flour extract. From these observations, it can be inferred that the polyphenols are responsible for the microbial activity of the millet and the results indicate that potential exists to utilise finger millet seed coat as an alternative natural antioxidant and food preservative.

Determination of lasalocid sodium in animal feeds and premixes by reversed-phase liquid chromatography: collaborative study.

A liquid chromatographic (LC) method for the analysis of lasalocid sodium in premixes, complete animal feeds, and trace-level feeds was collaboratively studied. The method employs a 0.5% HCI acidified methanol extraction followed by 20 min sonication in a water bath heated to 40 degrees C. Samples are then shaken on a mechanical shaker for 1 h and stored overnight, followed by an additional 10 min shaking the following morning. Sample extracts are diluted if necessary with extractant, filtered, and injected onto an LC system. Determination of all lasalocid homologs is by reversed-phase LC with fluorescence detection at 314 nm excitation and 418 nm emission. Eight samples of drug premixes, medicated feeds, and mineral supplements, along with 2 samples for trace-level analysis were sent to 20 collaborators in the United States, Canada, and The Netherlands. Study data were returned by 17 laboratories. Two additional supplemental trace-level samples and a blank feed were provided to 15 of the collaborating laboratories, and test data were received from all 15 participants. For the drug premixes, medicated feeds, and mineral supplements, RSDr values (within-laboratory repeatability) ranged from 1.2 to 19.9%, RSDR values (among-laboratory reproducibility) ranged from 3.4 to 32.3%, and HorRat values ranged from 0.35 to 3.73. For the trace-level samples, only lasalocid A, the predominant homolog comprising > 90% of the sum of all homolog peak area, was quantified. All laboratories correctly identified the analyte. Although some instrument response was reported by a number of laboratories for the blank feed, all but one laboratory's results were well below the 1 mg/kg limit of quantification. RSDr values for the initial 2 trace-level samples were excessive, ranging from 51.6 to 64.4%. RSDR values ranged from 51.6 to 75.7%, and HorRat values ranged from 3.6 to 4.0. Data for the initial trace-level samples indicated that the test samples were improperly prepared to ensure homogeneity, and a new set of supplemental samples was provided to collaborators, with significantly improved results. RSDr values for the 2 supplemental trace-level samples ranged from 1.6 to 2.5%, RSDR values ranged from 5.6 to 9.2%, and HorRat values ranged from 0.43 to 0.62.

Occurrence of tetrodotoxin and paralytic shellfish poisons in a gastropod implicated in food poisoning in southern Taiwan

The toxicity of the gastropod Nassarius papillosus implicated in a food paralytic poisoning incident in Liuchiu Island, Taiwan, in October 2005 is reported. The symptoms of a victim (67 years old) were featured by general paresthesia, paralysis of phalanges and extremities, paralysis, coma, and aphasia. The remaining specimens of shell were assayed for toxicity. The range of specimen toxicity was found to be 63–474 mouse units (MU) per specimen for N. papillosus by a tetrodotoxin (TTX) bioassay. The mean (SD) toxicity of the digestive gland and other portions were 296 ± 120 and 382 ± 156 MU in N. papillosus. The toxin was partially purified from the acidic methanol extract of the gastropod by using a C18 solid-phase extraction column. The eluate was then filtered through a 3000 MW cut-off ultrafree microcentrifuge filter. It was shown that the toxin purified from gastropods analysed by high-performance liquid chromatography and liquid chromatography/mass spectrometry contained TTX 42–60 µg g−1 (about 90%), whereas along with minor paralytic shellfish poisons (PSP) it was 3–6 µg g−1 (about 10%).

Study of the Antifungal Ability of &amp;lt;italic&amp;gt;Bacillus subtilis&amp;lt;/italic&amp;gt; Strain PY-1 &amp;lt;italic&amp;gt;in Vitro&amp;lt;/italic&amp;gt; and Identification of its Antifungal Substance (Iturin A)

A Bacillus strain, denoted as PY-1, was isolated from the vascular bundle of cotton. Biochemical, physiological and 16S rDNA sequence analysis proved that it should belong to Bacillus subtilis. The PY-1 strain showed strong ability against many common plant fungal pathogens in vitro. The antibiotics produced by this strain were stable in neutral and basic conditions, and not sensitive to high temperature. From the culture broth of PY-1 strain, five antifungal compounds were isolated by acidic precipitation, methanol extraction, gel filtration and reverse-phase HPLC. Advanced identification was performed by mass spectrometry and nuclear magnetic resonance spectroscopy. These five antifungal compounds were proved to be the isomers of iturin A: A2, A3, A4, A6 and A7. In fast atom bombardment mass spectrometry/mass spectrometry collision-induced dissociation spectra, fragmentation ions from two prior linear acylium ions were observed, and the prior ion, Tyr-Asn-Gln-Pro-Asn-Ser-betaAA-Asn-CO+, was first reported.

Use ofTerminalia catappafruit extract as an indicator in acid-base titrations

Terminalia catappa L. (tropical almond), family Combretaceae, is a large deciduous tree, originally from India. It thrives as an ornamental tree in many tropical cities of the world. The present work highlights the use of the acidified methanol extract of the outer cover of Terminalia catappa fruits as an acid-base indicator in different types of acid-base titrations. It is found to be a very useful, economical, simple, and accurate indicator for said titrations.

Analytical approach and effects of condensed tannins in carob pods ( Ceratonia siliqua) on feed intake, digestive and metabolic responses of kids

There are contradicting data in the literature regarding condensed tannins content in carobs. In this study, urea (0.5 g/ml) under reflux conditions increased dramatically the yield of condensed tannins to 17% of dry matter, as compared to 3.5% in acidic methanol extraction. This shows that carob pods are rich source of nonextractable condensed tannins. The effect of PEG in diet rich in carob pods on voluntary feed intake, digestibility and growth in weaning kids was studied during 15 days of balance trials in 24 kids distributed into three dietary treatments. The corn grain and wheat bran components in the control diet (diet 1) were replaced by carob in treatments 2 and 3; treatment 3 was supplemented by 3.3% of PEG of molecular weight-4000. Food intake, diet utilization and growth rate of weaning kids fed a diet containing 52% carob pods were decreased considerably in comparison to kids fed the control commercial diet. Supplementing the kids fed the carob-based diet with PEG increased feed intake, crude protein digestibility and growth to the rate obtained with the control diet. The nutritional experiment highlights the following aspects relevant to all mammals consuming carob pods rich diets: (i) tannins increased the variability in feed intake between days, (ii) hypocholesterolemic effect of carob pods is related to lipid binding capacity of nonextractable condensed tannins in the digestive tract, (iii) supplementing high level of carob pods to animals with normal blood cholesterol level may induce hypocholesterolemia.

Effect of pretreatment of barley seedlings with different salts on the level of UV-B induced and UV-B absorbing compounds

The relationship between the level of UV-B-induced and/or UV-B-absorbing compounds and stress tolerance of barley seedlings (Hordeum vulgare L. cv. Alfa) was investigated. A physiological response to UV-B irradiation was evaluated by measuring the oxygen evolution rate and chlorophyll fluorescence. UV-B irradiation led to an increase of the amount of UV-B absorbing compounds, including flavonoids, measured in acidified methanol extract at 300 nm and of UV-B induced compounds, with maximum absorbance at 438 nm, extracted in 0.1% trichloracetic acid. The content of free proline, malondialdehide and H 2O 2 increased as a result of 4 days treatment with 150 mM NaCl, KCl or NaNO 3. Salt pretreatment resulted in considerable decrease of the level of UV-induced and UV-B absorbing compounds measured 24 h after UV-B irradiation. In the meantime chlorophyll fluorescence parameters and oxygen evolution in salt pretreated seedlings were less affected by UV-B in comparison to the control. Damaging effect of UV-B measured by the MDA and H 2O 2 generation and electron transport activity corresponded to the increased levels of UV-B induced and UV-B absorbing compounds. We do not necessarily exclude UV-inducing compounds from an important role in overall UV-B protection but the data presented here showed that the accumulation of these compounds could be a consequence of stress-induced damage to the cells and probably they may serve as stress markers.

Quantitative analysis of phytoestrogens in kudzu-root, soy and spiked serum samples by high-pressure liquid chromatography.

A sensitive and reliable HPLC method that allows simultaneous quantification of phytoestrogens extracted from kudzu-root and soy preparations, and serum samples has been developed. Kudzu-root and soy preparations were mixed with 5 microg flavone and 15 microg rutin (internal standards) and the phytoestrogens were extracted by using solid-phase (C18) extraction cartridges. Blank or spiked serum samples were extracted by using either C18 cartridges or trichloroacetic acid-methanol extraction. The extracts were analyzed by the HPLC equipped with a reverse-phase (250 x 4 mm, C18) column and UV, diode-array or MS detector. A linear gradient of acetic acid and acetonitrile provided excellent separation of glycoside and aglycone-phytoestrogens from kudzu root and soy preparations. The C18 cartridge extraction of serum yielded excellent recovery of both glycoside- and aglycone-phytoestrogens, while the trichloroacetic acid-methanol extraction yielded excellent recovery of glycoside but poor recovery of aglycone compounds. UV and MS detectors were suitable for phytoestrogen analysis in plant and serum samples, while the diode-array detector was suitable for generating the UV absorbance curve for phytoestrogens.