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240527592 | A new class of axial-chiral biisoquinoline N,N′-dioxides was evaluated as catalysts for the enantioselective hydrosilylation of acyl hydrazones with trichlorosilane. While these catalysts provided poor to moderate reactivity and enantioselectivity, this study represents the first example of the organocatalytic asymmetric reduction of acyl hydrazones. In addition, the structures and energies of two possible diastereomeric catalyst–trichlorosilane complexes (2a–HSiCl3) were analyzed using density functional theory calculations. | 1 |
240527592 | Results and Discussion We recently developed the modular method to synthesize axial-chiral 3,3′-triazolyl biisoquinoline N,N′-dioxides from readily available triazoles and optically pure 3,3′dibromo-biisoquinoline N,N′-dioxide [57] as part of our longstanding interests in developing new chiral Lewis-bases [58][59][60][61][62]. Since this new class of catalysts was found capable of activating trichlorosilane at relatively low temperatures, we envisioned that they might be able to catalyze the reduction of acyl hydrazones under conditions where no background reaction would take place. We set out on our investigation by employing benzoyl hydrazone 1a as a model substrate (Scheme 2). To our delight, the background reaction was found negligible at −40 °C, and catalyst 2a provided hydrazine (R)-3a in 48% yield with 53% ee (entries 1 and 2). Next, we looked at several solvents that are commonly used for trichlorosilane-mediated reactions. Chloroform provided the product with a lower yield but with a slightly higher enantioselectivity (34% yield, 66% ee). Acetonitrile gave 3a in a comparable yield but with a lower ee of 32%. The reaction in tetrahydrofuran afforded the opposite enantiomer (S)-3a with a much lower yield and selectivity (entry 5). Overall, dichloromethane was found optimum. We tested with twice as much solvent since benzoyl hydrazone 1a was not fully dissolved under the reaction conditions (entry 6). However, it did not improve the result. Previously, we found that 4 Å molecular sieve was an effective acid scavenger for adventitious HCl in trichlorosilane [63], but its use did not positively impact the outcome in the present case (entry 7). The use of 3.0 equivalent of trichlorosilane did not improve the yield, either (entry 8). As the protecting groups on hydrazones are known to influence their reactivities and enantioselectivities in many cases (e.g., see; [19]), we evaluated the Boc and Cbz protected hydrazones (1b and 1c in entries 9 and 10, respectively). While enantiomeric excesses of the corresponding products were slightly higher than that of the benzoyl counterpart, both Boc and Cbz protecting groups adversely affected the yields. Since the C=O unit of Boc or Cbz group is more Lewis basic than that of the benzoyl counterpart, we tested a less Lewis basic hydrazone (1d). However, the yield decreased to 14% albeit with a slightly higher enantioselectivity (entry 11). Overall, the present method was found to be quite sensitive to reaction solvents and the hydrazone protecting groups. Next, we evaluated different triazolyl groups on the biisoquinoline that are expected to play important roles on the catalyst's reactivity and selectivity (Scheme 3, entries 1-4). Catalyst 2a was clearly superior to the other three catalysts (2b-d) in terms of their reactivity. Catalyst 2c was more enantioselective than others, albeit with a low yield. These results indicate that the reactivity and selectivity of this new class of catalysts can be tuned by changing the triazolyl groups. We also compared these triazolyl catalysts to conventional 3,3′-substituted biisoquinoline N,N′-dioxides (entries 5 and 6). To our surprise, neither 2e nor 2f promoted the reaction although 2f was as reactive as 2b-d for the hydrosilylation of an N-phenyl ketimine with trichlorosilane [57]. Nonetheless, these results clearly demonstrated that this new class of axial-chiral biisoquinolines is indeed complementary to the existing Lewis-base catalysts and bode well for the development of their applications. As we determined the basic reaction parameters, we proceeded to evaluate the extent to which the present catalytic system could enantioselectively promote the hydrosilylation of various benzoyl hydrazones with trichlorosilane (Scheme 4). To our surprise, a paramethyl substitution (3e)-which is a minimal change from the model substrate (3a)-had a detrimental effect on the chemical yield while the corresponding meta-substitution (3f) did not. An ortho-methyl substitution (that is known to push the aromatic ring out of conjugation with a C=N bond) completely shut down the reaction (3g). Eventually, it was gleaned that the para-substitutions have adverse effects on the reactivity but not much on the enantioselectivity regardless of their electronic nature (3e, 3h-m) (these enantioselectivities are approximately the same). A heteroaromatic hydrazone was moderately less reactive and selective than the model substrate (3n). Although an ethyl group at the C=N bond is in general expected to lead to an increased steric demand in the TS, it did not affect the reactivity in the present case (3o). It is noteworthy that a cyclohexyl counterpart provided the opposite sense of enantioselection to the model substrate (3p). Differentiation of the two similar alkyl groups franking the C=N bond was difficult by the present catalytic system (3q). Unreacted hydrazones and corresponding ketones were the major components of the crude reaction mixtures besides the desired products, and no significant amounts of by-products were observed for 1a-1q. An α,β-unsaturated hydrazone was not a viable substrate for this method as the conjugate reduction took place (3r) [64]. We also tested a 1.0 mmol scale reaction with the model substrate. To our delight, it provided essentially the same result (Scheme 5), demonstrating a potential robustness of the method. Furthermore, catalyst 2a was quantitatively recovered after a flash column chromatography on silica gel (see Supplementary Materials for details). The recovered catalyst promoted the model reaction (1a on 0.25 mmol scale) with no loss in reactivity and enantioselectivity. The structure of the active reducing species generated from a chiral catalyst and HSiCl 3 is considered to play a central role for the enantioselectivity of a reaction. Even with notable advances made in this area (selected references; [35][36][37][38][39][40][41][42][43][44][45]), it remains largely elusive and significantly challenging to control the relative populations and reactivities of diastereomeric reducing species that are reversibly produced from a chiral Lewis-base and trichlorosilane. C 2 -symmetric 2a and HSiCl 3 can give rise to two diastereomeric complexes that are expected to have different enantioselectivities (as long as 2a acts as a bidentate Lewis-base). Therefore, the binding geometry of 2a to HSiCl 3 was investigated computationally with the aim of shedding some light on the structure of the active reducing species. To our delight, 2a was found to bind to HSiCl 3 through its two oxygen atoms (i.e., a C 2 -symmetric bidentate ligand), generating two diastereomeric complexes ( Figure 1). Complex 1 was found to be 1.91 kcal/mol lower in energy than the complex 2. The analysis of their electrostatic potentials revealed an anion-π-type interaction between the hydrogen atom in complex 1 or one of the chlorine atoms in complex 2 and the phenyl ring. It should be mentioned that a pileup of electron density occurs at the peripheral atoms of a hypervalent silicon complex of this kind [59,65,66]. This anion-π-type interaction appears to effectively lock the conformation of the benzyl group at least at the ground state, leading to a welldefined chiral pocket around the hypervalent silicon atom. This computationally identified non-covalent attractive interaction could offer a possible basis to rationalize why 2a (benzyl) was as enantioselective as 2d (1-adamantyl), and why 2c (benzhydryl) was substantially more enantioselective than 2d (53% ee, 54% ee and 74% ee, respectively; Scheme 3). | 2 |
240527592 | Data Availability Statement: Data is contained within the article and Supporting Information. Computed structures of the two lowest energy minima for the 2a-HSiCl 3 complex (i.e., two diastereomeric complexes) calculated with PBEh-3c//C-PCM (DCM). Both are shown with balls-and-sticks (left) and space filling (right) models. Molecular electrostatic potentials are also shown in the space filling models. Complex 1 (top) is 1.91 kcal/mol lower in energy than complex 2 (bottom). | 6 |
92113091 | The purpose of this study was to evaluate the influence of squalene (SQ) on plasma and hepatic lipid levels of obese/diabetic KK-Ay mice and wild-type C57BL/6J mice. SQ supplementation significantly increased the HDL cholesterol of KK-Ay mice, which was paralleled with no significant difference in the total and non-HDL cholesterol levels. The increase in HDL cholesterol was also found in the plasma of normal C57BL/6J mice, but the difference was not significant. SQ administration significantly increased neutral lipids (NL) in the liver of KK-Ay mice, while no significant difference was observed in the polar lipids and the total cholesterol levels. The increase in NL was primarily due to the increase in TAG. However, the cholesterol level significantly increased due to SQ intake in the liver of C57BL/6J mice, while no significant difference was found in other lipid levels. The present study suggests that SQ may effectively increase HDL cholesterol level, an important anti-atherosclerotic factor, especially in subjects with metabolic disorders. | 1 |
92113091 | Introduction Squalene (SQ), a naturally occurring triterpenic hydrocarbon, is widespread in nature, especially among shark liver oil and olive oils.The shark Centrophorus squamosus has been reported to contain approximately 14% liver oil, mainly composed of SQ (nearly 80% of the oil) [1].Although shark liver oil remains the richest natural source of SQ, its use is limited by persistent organic pollutants and shark resource protection [1].SQ is also found in many vegetable oils in va-S.Liu rying concentrations.Among vegetable oils, oil from Amaranthus sp. is known to have the highest concentration of SQ (up to 73.0 g/kg oil) [2] [3].The SQ content in olive oil is also high (5.64 g/kg oil) compared to other vegetable oils such as that derived from hazelnuts (0.28 g/kg oil), peanuts (0.27 g/kg oil), corn (0.27 g/kg oil) and soybean (0.10 g/kg oil) [4]. Olive oil intake has shown beneficial health effects [5] [6] [7] [8] and these effects have been recognized to partially derive from olive oil minor compounds, mainly phenolic compounds.In addition, due to the relatively high content in olive oil compared with other vegetable oils, SQ is also regarded as a contributing factor in the reduced risk of diseases associated with olive oil intake.To date, SQ has been reported to show anticancer, anti-inflammatory, antioxidant, skin protection, liver protection, and neuroprotective activities [9] [10] [11] [12].In particular, many studies have been conducted on the relationship between the reduced risk of cancer due to olive oil intake and the role of SQ as a vital dietary cancer chemopreventive agent [9] [10] [11] [12] [13]. Furthermore, the higher intake of olive oil in Mediterranean countries compared to northern European countries is related to the low incidence of cardiovascular disease (CVD) [10].CVD is the leading cause of morbidity and mortality worldwide.In many cases, CVD is caused by atherosclerosis, a chronic vascular disease that generally occurs in the aorta and muscular-type arteries, such as coronary arteries, brain arteries, renal arteries and carotid arteries [14]. Although the exact cause of atherosclerosis is still unknown, modification and deposition of lipids in the vascular wall can induce this event.Among types of lipid deposition, low density lipoprotein (LDL) cholesterol deposition, especially oxidized LDL, is regarded as a main cause.Thus, cholesterolemia is known as a major inducer of atherosclerosis, and much attention has been paid to the hypocholesterolemic activity of olive oil components, such as oleic acid and other minor compounds including SQ [10]. SQ is known as an important intermediate for the biosynthesis of phytosterol or cholesterol in plants, animals and humans [11], and its endogenous synthesis begins with the conversion of acetyl coenzyme A to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), followed by the reduction of HMG-CoA to mevalonate, mediating HMG-CoA reductase.Thus, the involvement of SQ in cholesterol synthesis is easily expected, and the possibility has been demonstrated for the use of SQ as a biomarker to evaluate endogenous cholesterol biosynthesis (Brown et al., 2014).Indeed, numerous studies from this viewpoint have explored this possibility of using several experimental approaches in both animals and humans [11]. Many studies on the effect of dietary SQ on blood cholesterol levels have been [16].The varied effects of SQ are hypothesized to be due to differences in the experimental approaches and sexes [10] [15] [17]. The controversial effect of SQ on blood cholesterol level has been replicated in animal models.Several researchers have reported a reduction of blood lipid levels, including cholesterol in rats after SQ intake [18] [19] [20], while other researchers have found increases in the cholesterol levels in normal animals by SQ feeding [21] [22].In addition, Chmelik et al. [23] found the increase in high density lipoprotein (HDL) cholesterol levels of C57Bl/6J SPF mice fed SQ, while the total cholesterol and LDL cholesterol levels decrease with SQ feeding.This specific increase in HDL cholesterol by SQ intake was also found in three different mouse models (wild-type, Apoa1-and Apoe-deficient) [17]. Many animal and human studies have revealed SQ as a promising agent in CVD prevention [10] [11] [15].The effect of SQ has been explained via several different mechanisms, including the elimination of cholesterol as fecal bile acids [16], inhibition of HMG CoA reductase by dietary SQ due to negative feed-back regulation [24], inhibition of oxidized LDL uptake by macrophages [25], stimulation of reverse cholesterol transport [26], inhibition of isoprenaline-induced lipid peroxidation [18], and attenuation of homocysteine-induced endothelial dysfunction [11].These mechanisms are basically dependent on the antioxidant activity of SQ and the involvement of SQ in cholesterol metabolism [1] [10] [11] [15]. However, SQ's role in plasma lipids is not yet clear, although hyperlipidemia, especially hypocholesterolemia, is regarded as a major risk factor for CVD.In the present study, we assessed the effect of SQ on the plasma lipid content of animal models.For animals, we used C57BL/6J genetic background mice.These mice have been widely used due to their higher predisposition to atherosclerosis development [27].Furthermore, we compared the effect of SQ in normal and obese/diabetes mice.Obesity and diabetes are a major risk factor for CVD [28] [29].Therefore, the comparison may help to elucidate the effect of SQ on the reduction of CVD risk. | 2 |
92113091 | Animals and Diets Obese/diabetic KK-A y mice (male, four weeks old) and wild-type C57BL/6J mice (male, four weeks old) were obtained from the Japan CREA Co., Tokyo, Japan. The mice were housed individually in an air-conditioned room (23˚C ± 1˚C and 50% humidity) with a 12 h light/12 h dark cycle.After acclimation feeding of a normal rodent diet MF (Oriental Yeast Co., Ltd, Tokyo, Japan) for 1 week, the mice were randomly divided into 3 groups of seven and were fed experimental diets for four weeks (Table 1).The body weight, diet and water intake of each mouse was recorded daily. | 3 |
92113091 | Sample Collection Mice were sacrificed under diethyl ether anesthesia after 12 h fasting on day 28.Blood samples were taken from the caudal vena cava of the mice.A portion of blood was used for blood glucose analysis, while the remaining part was stored for lipid analysis.Blood glucose was measured using a blood glucose monitor, namely, the Glutest Neo Sensor (Sanwa Kagaku Kenkyusyo Co. Ltd., Aichi, Japan).This sensor is an amperometric sensor with flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase and ( ) | 5 |
92113091 | Hepatic Lipid Analysis Total lipids (TL) was extracted from the liver with chloroform/methanol (2:1, v/v) [30].The TL (ca.20 mg) was further separated on a Sep-Pak Silica cartridge (Waters Japan, Tokyo, Japan) by elution with chloroform (70 mL) and methanol (50 mL).The neutral lipids (NL) and polar lipids fractions were eluted with chloroform and methanol, respectively.Both lipid contents in the liver (mg/g liver) were calculated from the TL level per liver weight.Total cholesterol and TAG were measured using enzymatic kits (Cholesterol E-test and Triglyceride E-test) obtained from Wako Pure Chemical Industries, Osaka, Japan. The fatty acid composition of the TL was determined by gas chromatography (GC) after conversion of fatty acyl groups in the lipid to their methyl esters.The fatty acid methyl esters (FAME) were prepared according to the method of Prevot and Mordret [31].Briefly, 1 mL of n-hexane and 0.2 mL of 2 N NaOH in methanol were added to an aliquot of total lipid (ca. 10 mg), vortexed and incubated at 50˚C for 30 min.After incubation, 0. In a preliminary experiment, we found that SQ and docosahexaenoic acid (22:6n-3, DHA) could not be separated clearly on the chromatogram using the Omegawax-320 capillary column.Therefore, the prepared FAME sample was | 7 |
92113091 | Quantitative Real-Time PCR Total RNA was extracted from the livers of mice using RNeasy Lipid Tissue Mini Kits (Qiagen, Tokyo, Japan) according to the manufacturer's protocol.The cDNA was synthesized from total RNA using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems Japan Ltd., Tokyo, Japan).Quantitative real-time PCR analyses of individual cDNA were performed with ABI Prism 7500 (Applied Biosystems Japan Ltd., Tokyo, Japan) using TaqMan Gene Expression Assays (Applied Biosystems Japan Ltd., Tokyo, Japan).Gene expression was normalized to the reference gene GAPDH. The mRNA analyses were performed on genes associated with lipid metabolism, which included liver X receptor (LXR), sterol regulatory ele- | 8 |
92113091 | Results 3.1.Food Intake, Water Intake, Weight Gain, Tissue Weights, Blood Glucose Levels, Serum and Hepatic Lipid Parameters The weights of major tissues were not significantly different, except for a significant increase in the liver weight of KK-A y mice fed SQ (1% and 2%) (Table 2).Significant increases in weight gain were also found in KK-A y mice fed SQ (2%) (Table 2).Although a tendency in the decrease in plasma non-HDL cholesterol was found in KK-A y mice, other lipid parameters increased (Table 3).A significant increase in HDL cholesterol was found.However, all plasma cholesterols levels (total cholesterol, HDL cholesterol and non-HDL cholesterol) increased in C57BL/6J mice, but the difference was not significant (Table 3).SQ intake significantly increased TL, NL and TAG in the liver of KK-A y mice, while no significance was observed in the hepatic lipid levels of C57BL/6J mice, except for total cholesterol (Table 4).Total cholesterol level in the liver from C57BL/6J mice significantly increased due to SQ (2%) feeding, but levels in KK-A y mice fed SQ (1% and 2%) were lower than those fed control diet (Table 4). | 11 |
92113091 | Fatty Acid Levels of Liver Lipids SQ (1% and 2%) supplementation significantly all fatty acid contents in the liver from KK-A y mice except for 20:4n − 6, resulting in a significant increase in the total fatty acid contents (Table 5).The increase in total fatty acids presented in Table 5 was consistent with the result in Table 4 showing the increasing effect of SQ on liver TL and NL.However, there was little difference in the fatty acid content in the liver of C57BL/6J mice (Table 5).This was also expected due to the TL and NL levels in the liver of C57BL/6J mice (Table 4).Different letters (a, b) show significant differences at P < 0.05. | 12 |
92113091 | Gene Expression Related to Lipid Metabolism and HMG-CoA Reductase Activity To determine the effect of dietary lipids on liver lipid metabolism, the related gene expressions were analyzed using real-time PCR.Although the analysis showed no significant effect of SQ on the gene expression of C57BL/6J mice (Figure 1), a significant difference was found in the HMGCR and CYP7A1 genes in the liver from KK-A y mice fed SQ compared with the control (Figure 2).Furthermore, SQ (2%) supplementation significantly increased HMG-CoA reductase activity in the liver from KK-A y mice, but not C57BL/6J mice. | 13 |
92113091 | Discussion Epidemiological studies have revealed an inverse correlation between HDL cholesterol levels and the risk of cardiovascular disease and atherosclerosis.HDL cholesterol promotes reverse cholesterol transport and has several atheroprotective functions, such as anti-inflammation, anti-thrombosis, and antioxidation [32] [33].In prospective epidemiologic studies, every 1-mg/dL increase in HDL is associated with a 2% to 3% decrease in CVD risk, independent of LDL cholesterol and TAG levels [34].Furthermore, normal or high HDL levels appear to have anti-atherosclerotic, anti-inflammatory, antioxidant and anti-thrombotic properties, even in the presence of high LDL cholesterol [35].In the present study, we found a significant increase in the plasma HDL cholesterol of obese/diabetic KK-A y mice, with no significant difference in the total and non-HDL cholesterol levels (Table 3).The increase in HDL cholesterol was also results in the effect of SQ on plasma lipid levels, recent studies have demonstrated the effect of SQ on blood HDL cholesterol level as important factor in atherosclerosis protection [11].Administration of SQ for seven weeks (2.1 g/kg) to C57Bl/6J SPF mice showed a 60% increase in HDL cholesterol with no changes in total cholesterol [23].Likewise, SQ administration for 11 weeks at a dose of 1 g/kg caused a specific increase in HDL cholesterol levels in three male mouse models (wild-type, Apoa1and Apoe-deficient) with the C57BL/6J genetic background [17].In a rat model, specific increase in HDL cholesterol has also been reported [18].These studies have demonstrated that high HDL level would be independent of an anti-atherosclerotic factor [35].The present study confirmed that the increase in HDL cholesterol level is a major effects of SQ in atherosclerosis protection.HDL cholesterol biogenesis and its development are involved in various complex metabolic networks, such as upregulation of ATP-binding cassette transporter A1, apoA-I transcription and liver X receptor (LXR) [36].Therefore, the increasing effect of SQ on HDL cholesterol would be related to these events; however, the mechanisms by which SQ elevate plasma HDL-cholesterol levels remain unclear.SQ is known to show a broad repertoire of biological action based on its antioxidant activity [11].This effect could be exerted in HDL to prevent oxidative modifications of the apolipoprotein A-I (ApoA-I), other HDL proteins, and HDL lipids.The prevention by HDL of oxidative stress can make it more fluid and thus more functional.Further study will be needed. In humans, orally administered SQ is well absorbed (60% -85%).This, and the intestinal de novo synthesized SQ, are transported by chylomicrons into circulation and are rapidly taken up by the liver, where it is converted into cholesterol [17].A significant increase in total hepatic cholesterol found in normal C57BL/6J mice may be reflected by the conversion of SQ to cholesterol in the liver (Table 4).However, cholesterol level decreased in the liver of obese/diabetic KK-A y mice (Table 4).However, significant increase in NL by SQ intake was observed in KK-A y mice, while no significant difference was observed in polar lipids (Table 4).The increase in NL resulted in higher TL levels in the liver.SQ administration to KK-A y mice induced its accumulation in the liver.Moreover, SQ is eluted as the NL fraction in the separation of TL with the column chromatography used in the present study.However, the level of SQ measured was less than 20 mg/g liver in both groups.Therefore, the increase in NL found in Table 4 was mainly due to the increase in TAG.The TAG increase due to SQ intake was strongly related to the higher level of total fatty acids found in Table 5. As shown in Table 4 and Table 5, squalene administration to KK-A y mice induces TAG accumulation in the liver.To determine the effect of squalene, gene expression related to fatty acid (FASN and SCD1) and TAG (DGAT1 and DGAT2) synthesis was analyzed (Figure 1).However, no significant difference was found in these gene expressions, together with other kinds of genes related to lipid metabolism, except for HMGCR.It is difficult to explain the discrepancy found between gene expression and TAG content in the liver of KK-A y mice.One possibility is the involvement of TAG that originated from other tissues. HMGCR is a gene related to cholesterol metabolism.Its expression was significantly decreased by squalene (1%) intake, but no significance was observed with administration of squalene (2%) (Figure 1), while a significant increase in HMG-CoA reductase activity was found in the liver of KK-A y mice fed squalene (2%), but with no significance in squalene (1%) (Figure 2).Although HMG-CoA reductase activity and its gene expression, e.g., HMGCR, is known as a key factor in cholesterol synthesis, the changes in these factors would not greatly affect cholesterol concentrations in the liver (Table 4).This may be due to the compensation of other pathways of cholesterol metabolism and/or the effect of cholesterol supplementation from other tissues.CYP7A1 is known to catalyze the cholesterol catabolic pathway.This enzyme expression was decreased by squalene intake (Figure 1).Although the difference was not significant, this may affect the cholesterol level of the liver. | 14 |
92113091 | ( 1975).This procedure is based on the formation of hydroxymate from the reaction of HMG-CoA and mevalonate with hydroxylamine.The resulting hydroxymate can be quantitatively measured using the colorimetric assay.The HMG-CoA and mevalonate concentration in the liver homogenate were collected separately by changing pH to avoid interference by mevalonate in the HMG-CoA analysis.Therefore, liver homogenate was reacted with freshly prepared hydroxylamine reagent (alkaline hydroxylamine reagent in the case of HMG-CoA) at pH 5.5 for HMG-CoA and pH 2.1 for mevalonate, respectively.HMG-CoA reductase activity was calculated from the ratio of HMG-CoA concentration to mevalonate concentration.S. Liu et al.DOI: 10.4236/fns.2018.9121081504 Food and Nutrition Sciences | 16 |
30506728 | Contradictory results has been found on the effects of soybean supplementation and conjugated linoleic acid (CLA) content in milk on feeding systems based on fresh forage The objective of the study was to evaluate the effect of a dietary supplement with different quantities of extruded whole soybean on the production and composition of milk, and CLA concentration or their isomers in Jersey cows under pasture conditions. Twenty-one Jersey cows were randomly assigned into 3 groups of 7 animals each. The cows were supplemented with a dietary concentrate (5 kg d –1 ), and each group received one of the three next treatments: control without soybean (0-SB), with extruded whole soybean at 0.5 kg d –1 (0.5-SB) or at 1 kg d –1 (1-SB). The basic diet was a pasture composed of Lolium perenne (70%), Trifolium repens (25%) and other species. The duration of the study was 75 d. Milk production ( p = 0.706) and protein production ( p = 0.926) were not affected by treatments. Fat ( p = 0.015) and protein ( p = 0.045) content as well as fat production ( p = 0.010) were lower in the 1-SB group. There was no effect of the inclusion of extruded soybean on total CLA content ( p = 0.290) or the content of cis -9, trans -11 ( p = 0.582), trans -10, cis -12 ( p = 0.136) and cis -10, cis -12 ( p = 0.288) isomers. However, concentrations of all isomers were affected by the nutritional quality of the pasture, with low values observed at greater maturity stages of pasture. | 1 |
30506728 | . Although, it has been documented that concentration of CLA in milk from Jersey cows is 18% lower than that observed in Holstein cows (White et al., 2001), apparently few studies have been conducted regarding CLA content in Jersey cows. Whilst functional foods have been considered a promising area for human health (Starling, 2002), it has frequently been observed that consumers expect added-value products without substantial extra cost, suggesting that the development of low-cost approaches will be important (Dewhurst et al., 2006). Considering that soybean is an imported feedstuff in the majority of the countries and given the current prices in the market, the objective of this study was to evaluate the effect of a dietary supplement with different quantities of extruded whole soybean (0.5 and 1.0 kg d -1 ) on milk yield, CLA isomers content and metabolic profile in Jersey cows maintained in pasture-based systems. | 2 |
30506728 | Animals and diets The experiment was carried out in a farm with a Jersey herd based on grazing plus the use of concentrate, located in the Entre Lagos sector (district of Osorno, Chile), approximately 72° 36' 24" W 40° 41' 26" S, 250 m above sea level. The farm lies in the pre-mountain range of the Chilean Andes, Region X, which is characterized by an average rainfall of 2250 mm per year, with an average daily temperature of 21.8°C in January and 3°C in August. The experiment was developed in compliance with the principles and specific guidelines on animal care and welfare as required by Chilean law (SAG, 2010). The duration of the experiment was 75 d, between 15th November 2005 and 25th January 2006 (spring period). The first 15 d were for adaptation to the experimental diets and the experimental period was from day 15 to day 75. Twenty-one healthy cows between 2 and 7 calvings, with a range from 60 to120 days in milk (92 ± 5 DIM) and a body condition score of 2.75 ± 0.7 were used in the study. An average of 18.4 ± 3.73 kg d -1 of milk production was recorded. Cows were selected for the study based on previous milk production in order to make three ho- | 3 |
30506728 | Introduction Conjugated linoleic acid (CLA) represents between 20 and 28 isomers of linoleic acid C18:2 (Lock & Garnsworthy, 2003) that has been indicated as one of the most beneficial fatty acids for human health (Pariza & Park, 2001). Likewise, ruminant products as milk and meat constitute the principal source of CLA for humans. Of all possible isomers, only cis-9, trans-11 and trans-10, cis-12 have shown an interesting biological activity (Wahle et al., 2004). The cis-9, trans-11 isomer, also known as rumenic acid, has been documented to have an anticarcinogenic (Ha et al., 1987;Visonneau et al., 1997;Aro et al., 2000) and antioxidant effect (Devery et al., 2001), whereas the trans-10, cis-12 isomer is capable of decreasing body fat and increasing lean body mass. Diet has a major influence on milk fat CLA and it has been extensively investigated . Several nutritional studies have been addressed to increase CLA content in animal products and to improve their nutritional properties. For instance, it has been reported that fresh forage and oil-rich feeds increase CLA concentration (Khanal et al., 2005;Dewhurst et al., 2006). Soybean is widely used in total mixed ration (TMR) in different proportions, and it has been observed that seed treatment (roasting or extrusion) results in a higher increase on CLA content than that observed with intact seed (Chouinard et al., 2001). However, contradictory results has been found on the effects of soybean supplementation and CLA content in milk on feeding systems based on fresh forage. Some studies (Bartolozzo et al., 2003;Khanal et al., 2005) did not found effects on milk CLA concentration in dairy cows, while others (Lawless et al., 1998;Paradis et al., 2008) observed an increase in the CLA using dairy and beef cattle. On the other hand, the effects of other factors such as breed, lactation and parity on CLA content in milk fat have received less attention. Kelsey et al. (2003) reported that breed (Holstein vs. Brown Swiss), parity, and days in milk accounted for < 0.1, < 0.3, and < 2.0% of the total variation in CLA concentration in milk fat, respectively. The incorporation of Jersey cattle in dairy farms has been increased in the last decade in Chile due to their high level of total milk solids produced CLA content in milk of Jersey cows supplemented with extruded whole soybean on pasture mogenous groups and they were randomly assigned (n=7/per group) to receive a dietary concentrate or treatment with different quantities of extruded whole soybean: T1= control diet without supplementation (0-SB), T2 = 0.5 kg d -1 (0.5-SB) and T3 = 1 kg d -1 (1-SB). Each animal was fed with 5 kg d -1 of isoenergetic dietary concentrate (Table 1), distributed in two visits to the milking parlour, at 06:00 h in the morning and in the afternoon at 16:00 h. Visual observation of feed intake indicated that cows consumed all concentrate offered. The animals grazed in two paddocks of 18 hectares each, and managed with rotational stripgrazing and electric fencing. The pasture was an improved natural pasture with grasses being the predominant species (70% Lolium perenne, 25% Trifolium repens, 3% Bromus sp). The animals were transferred from one strip to the next every 24 h. The diets were formulated according to the animal requirements established by NRC (2001). | 4 |
30506728 | Chemical composition and nutritional value of feedstuff Samples of the pasture and concentrates were taken every 10 days to determine their chemical and nutritional composition (Tables 1 and 2). Representative samples of pasture forage were collected from the paddock before grazing at a height of 8 cm above the ground, using a 1-m 2 quadrant. Dry matter contents of the pasture were determined by forced air oven at 60°C for 48 h. Samples of pasture and concentrates were ground to pass a 1-mm screen in a Willey mill before analysis. Dry matter (method 934.01), ash (method 1 Control without extruded whole soybean (0-SB), with extruded whole soybean at 0.5 kg d -1 (0.5-SB), and with extruded whole soybean at 1 kg d -1 (1-SB). 942.05), ether extract (method 920.39), N (method 984.13) and crude fiber (method 978.10) were determined according to AOAC (2005) methods. The N values determined by the Kjeldahl procedure, and converted to crude protein by multiplying by a factor of 6.25. The analyses of neutral detergent fibre (NDF) and acid detergent fibre (ADF) were carried out according to Van Soest et al. (1991), and both NDF and ADF were expressed exclusive of residual ash. All fiber fractions were analyzed on a Fibertec 1030 Hot Extractor (Tecator, Sweden). The fat content was measured by extraction with petroleum ether (boiling point, 40 to 60°C) on a Soxtec System 1040 Extraction Unit (FOSS Tecator AB, Sweden). The metabolizable energy (ME) of the supplemented dietary concentrates was estimated according to NRC (2001). The in vitro dry matter digestibility (IVDMD) of the pasture was determined according to the procedure described by Tilley & Terry (1963) modified by Van Soest (1991) and the ME was estimated according to the equation (Garrido, 1981): ME = 0.279 + 0.0325 × IVDMD. | 5 |
30506728 | Milk yield and quality Cow milk production was determined using a Waikato® measuring equipment, on days 1, 15, 30, 45, 60 and 75. At each control, a milk sample of 30 ml was taken (to which was added 0.03 g of potassium dichromate at 0.1% as a preservative), and the contents of fat, protein and urea were determined automatically using an infrared spectrophotometer (Foss 4200 Milko-scan; Foss Electric, Denmark). | 6 |
30506728 | CLA content and composition At each milk control, milk samples of 100 mL were taken and sent to the laboratory in thermally insulated containers at 4°C for analysis of CLA isomers (cis-9, trans-11; trans-10, cis-12; cis-10. cis-12). Total lipids were extracted by the method of Folch et al. (1957), using a mixture of chloroform and methanol (2:1, v:v). The methylation of the fatty acids of the samples was done using the method described by Morrison & Smith (1964). Fatty acid methyl esters were analyzed by gas chromatography (HP 6890, Hewlett Packard, Surrey, UK), Flame Ionization Detector (FID), a capillary column SP-2560 (100 m, 0.25 mm i.d. with 0.20 μm thickness in the stationary phase; Supelco Inc., Bellefonte, Pennsylvania, USA) using He as the tracer gas. Gas chromatography conditions were as follow: the injection volume was 0.5 μL, a split injection was used (70:1, v:v); ultrapure hydrogen was the carrier gas; and the injector and detector temperatures were 250 and 300°C, respectively. The initial temperature was 70°C (held for 1 min), increased by 5°C per min to 100°C (held for 3 min), increased by 10°C per min to 175°C (held for 40 min), and then increased by 5°C per min to 220°C (held for 19 min) for a total run time of 86.5 min. Data were then quantified using the HPCHEM Stations software, and expressed as a percentage of area according to the total fatty acids identified. | 7 |
30506728 | Metabolic profile At the beginning and at the end of the experiment blood samples were taken (5 mL animal -1 ) by coccygeal venipuncture flow and placed in tubes with sodium heparin. The samples were then centrifuged for 3 min at 3000 rpm and the plasma was aliquoted and frozen (-18°C) in microtubes of 1.5 mL. For each sample, the following plasma traits were determined: cholesterol (cholesterol-oxidase method, Cholesterol Liquicolor 10028 Human), albumin (Albumin Liquicolor Method BCG-Bromo Cresol), total protein (Total Protein Liquicolor-Biuret Method), calcium (Arsenazo III AA), Mg (Mg-color AA), phosphorus (Fosfataria UV AA), aspartate aminotransferase (IFCC Mod. LiquiUV test) and urea (ureasa/NADH method, UREA LiquiUV 10521 Human). All plasma traits were determined automatically by biochemical analyser (SelectraVitalab, Merk, Darmstdt, Germany). | 8 |
30506728 | Statistical analysis Data of milk production, milk's constituents and metabolic profile were analysed as repeated measures, using the general linear model (GLM) of the SPSS for Windows 18.0 package (SPSS Inc., Chicago, IL, USA). The linear model used for each parameter was as follows: where Y ijk = observations for dependent variables; μ = overall mean; T i = fixed effect of treatment group or CLA content in milk of Jersey cows supplemented with extruded whole soybean on pasture dietary concentrate; A ij = random effect of animal j for the i treatment; W k = fixed effect of the k week of lactation; T × W = interactions among these factors for the i treatment and k week of lactation, and ε ijk = random effect of residual. Pairwise comparisons of means were carried out, where appropriate, using Tukey's honest significant difference tests. The level of significance for the analyses was 5%. The Pearson correlation coefficient between the milk fat concentration and the content of trans-10, cis-12 isomer was also determined. | 9 |
30506728 | Milk yield and quality In the initial day no differences in milk production (p = 0.390) and quality were observed among the three experimental groups (data not shown). In the experimental period (from day 15 to day 75) milk production (p = 0.706) and fat corrected milk production (FCM, kg d -1 ) (p = 0.241) were similar among groups (Table 3). The amounts of milk fat (kg d -1 ) (p = 0.010), as well as protein (p = 0.045) and milk fat (p = 0.015) concentrations were lower in the 1-SB treatment, while the quantities of protein (p = 0.926) and urea (p = 145) were similar among all treatments. The patterns of milk production and basic composition throughout lactation were affected by the lactation day for all the components (Table 3). Milk yield significantly decreased as a function of the week and for the chemical composition, the highest values for these components were found in the last weeks (data not shown). | 10 |
30506728 | CLA content and composition In the initial day no differences in total CLA (p = 0.791) and of each of its isomers were observed among the three experimental groups (data not shown). In the experimental period, there was no effect of the inclusion of extruded soybean on total CLA content (p = 0.290) or the content of cis-9, trans-11 (p = 0.582), trans-10, cis-12 (p = 0.136) and cis-10, cis-12 (p = 0.288) isomers (Table 3). Although the highest values were found for the cis-9, trans-11 isomer (53-59% of total CLA), the trans-10, cis-12 and cis-10, cis-12 isomers presented higher values (17-23% and 20-25% of total CLA, respectively) than normally reported in the literature. The pattern of fatty acid composition throughout lactation was affected by the lactation day for all components (Table 3; Fig. 1). For the content of total CLA and of each of its isomers, a similar trend is observed in all the treatments. The lowest CLA values were obtained in the lasted weeks, when the herbage presented the poorest nutritional quality (see Table 2). The cis-10, cis-12 isomer was the only one that diminished from day 1 to day 45, and increased after day 60. | 11 |
30506728 | Milk yield and quality Although crude protein of supplemented concentrate ranged between 17 and 21%, in all groups the pasture contributed an adequate level of protein and energy in accordance with NRC (2001) recommendations based on milk production and milk urea content (see Table 3). The reduction in the percentage and amount of fat (kg d -1 ) in the group fed with a higher quantity of soybean (1 kg d -1 ) may result from the extrusion process, which breaks up the micelles of fat in the seed, allowing a rapid release of the lipids in the rumen and reducing milk fat content (Mohamed et al., 1988;DePeters & Cant, 1992). Low milk fat syndrome has been recognized for many years, but the exact mechanism is still unclear. Data from several studies revived the theory of trans fatty acids, coming from ruminal biohydrogenations and from desaturation by the mammary gland, as the central mechanism of milk fat depression (Griinari & Bauman, 2003;Loor et al., 2005). In particular, the increase of C18:1 trans-10 and CLA trans-10, cis-12 isomers in the mammary gland has been associated with a reduction in the de novo synthesis of short and medium chain fatty acids (Banks et al., 1980;Grummer, 1991;Baumgard et al., 2000). The CLA trans-10, cis-12 isomer was found in the highest quantity (though such difference was not significant) in the 1-SB treatment. Also, in the present study there were an inverse linear relation (R 2 = 0.11, p = 0.04) between milk fat concentration and the content of this isomer. The milk protein content was lower in the 1-SB diet when compared with 0-SB and 0.5-SB diets. Although the dietary fat and protein were highest in the 1-SB group, Theurer et al. (1995) suggested that increasing the amount of dietary protein within a constant dietary energy level has little effect on milk protein synthesis and whenever dietary protein level increases milk protein yield, the effect seems to be associated with an increase in milk yield. However, in this study no differences between groups were found both in milk protein yield and in milk yield. The decrease in milk protein content might have been due to an increased availability of fat in the rumen in the 1-SB diet (Chouinard et al., 1997). The reduction in the percentage of protein observed in most of studies in animals fed with diets with a high fat content appears to be associated with negative effects on the growth of ruminal micro-organisms and the production of microbial protein (Solomon et al., 2000). In addition, when cows are fed fat, the energetic efficiency of milk synthesis is increased. Cows fed high fat diets required less liters of blood flowing to the mammary gland per kg of milk produced (Cant et al., 1993). Because mammary uptake of amino acids is dependent upon amino acid concentration in the blood and blood flow to the mammary gland, these data suggest that the decrease in blood flow per volume of milk produced would limit the uptake of amino acids for milk protein synthesis. However, there are studies that did not found any effect (Guillaume et al., 1991) or even others that found an increase of protein concentration in milk (Block et al., 1981). The differences in feeding (mainly due to ingestion and nutritional composition of the herbage) and lactational effects can explain the changes on milk production and components across the weeks of the study. However, since total forage ingestion was not monitored in this study this will have to be tested in future studies. | 13 |
30506728 | CLA content and composition As in our study, Khanal et al. (2005) did not found effects of a mixed supplement containing 2.4 kg d -1 of extruded soybean on CLA concentration in the milk of Holstein cows on pasture (1.63 and 1.69% of total FA for groups fed on pasture alone and pasture + supplement mixed with extruded soybean, respectively; these values include the cis-9, trans-11 isomer). The values obtained by these authors are somewhat higher than those found herein for the three CLA isomers together. Bartolozzo et al. (2003), in Friesian cows fed on pasture and fed a mixed supplement containing 2.6 kg of raw soybean, also obtained a high quantity of CLA (0.96%) as compared to TMR diets with raw or extruded soybean (0.52%), which is in agreement with the results of White et al. (2001). All these results indicate a greater influence of the pasture than that of the source or level of soybean incorporated into the diet on the CLA milk content. The different CLA values found in the literature may be related to differences in the nutritional composition of the pasture derived from the different botanical and agronomic characteristics of the herbage used in the various studies (Dewhurst et al., 2006) and, to a lesser degree, to the influence of other factors such as the breed (White et al., 2001;Kelsey et al., 2003). In this respect, White et al. (2001) observed 18% less CLA in milk from Jersey cows compared with milk from Holsteins. On the contrary, other authors observed an increase in the CLA milk content as compared to the control groups, with values up to 2.2% for all the CLA isomers in dairy cows on pasture supplemented with 3.1 kg d -1 roasted soybean (Lawless et al., 1998), and 2.4% for only the cis-9, trans-11 isomer in beef cattle on pasture supplemented with 2 kg d -1 extruded soybean (Paradis et al., 2008). However, the exact influence of breed related to dietary supply, and possible interactions need to be determined in further studies. Rumenic acid is typically the most abundant CLA isomer, with values greater than 80% of total CLA (Palmquist et al., 2005). The cis-10, cis-12 isomer, on the other hand, was found in very low quantities and has no known physiological function (Khanal & Olson, 2004). In the present study, the trans-10, cis-12 and cis-10, cis-12 isomers presented higher values than normally reported in the literature. The regulation of isomer balance is largely unknown. Nevertheless the cis-9, trans-11 isomer is mainly generated from vaccenic acid in the mammary gland (Mosley et al., 2006), while the trans-10, cis-12 is a minor intermediate of rumen biohydrogenation (Walker et al., 2004) and is relatively unaffected by changes in the diet except at very high levels of concentrate feeding (Chilliard et al., 2007). Therefore future studies are necessary to determine its biological function and metabolic production routes. In the temporal pattern ( Fig. 1) for the content of total CLA and of each of its isomers, a similar trend is observed in all the treatments, which would indicate that the influence of the herbage on the CLA content of the milk is greater than that of the different dietary concentrates supplemented. This may be related to differences in the nutritional composition of the herbage, which has also been shown to affect the fatty acid composition of milk (Dewhurst et al., 2006). In this respect, lower CLA contents in milk have been observed with more mature pasture, and this effect has been attributed to the declining quality and quantity of the herbage (Lock & Garnsworthy, 2003;Ward et al., 2003). This is in agreement with the present work, in which the lowest CLA values were obtained in late December and January, when the herbage presented the poorest nutritional quality (see Table 2). The cis-10, cis-12 isomer was the only one that diminished from day 1 to day 45, and increased after day 60. At present, it is difficult to explain both the higher quantity and the evolution of this isomer, as observed in the present study. | 14 |
30506728 | Metabolic profile All the metabolites evaluated, except blood urea at the end of the trial, were found to be within the normal range, in agreement with the values for healthy lactating dairy cows (Bertoni & Piccioli, 1999). Previous studies (Pulido, 2009) have shown an increase in blood urea when diets present high levels of degradable protein, which is the case with animals fed to pasture on grass (L. perenne). Under this conditions, highly soluble protein is associated with low levels of NDF and high leaf/stalk proportions at the beginning of spring (Van Vuuren et al., 1991), resulting in incomplete use of the nitrogen in the rumen and high levels of blood urea during the spring and early summer (Wittwer et al., 1993). These levels may exceed the normal range, especially at the beginning of spring, and this is considered normal in Chile (Wittwer et al., 1993). | 15 |
230524286 | This study aimed to synthesis ZnO, TiO 2 and ZnO–TiO 2 (ratio weight of 1/1 for Zn/Ti) nanoparticles using zinc acetate and titanium isopropoxide through the sol-gel method. Physicochemical and morphological characterization and antifungal properties evaluation like minimum inhibition concentration (MIC) and minimum fungicide concentration (MFC) of nanopowders were investigated against Aspergillus avus at in vitro. All synthesized nanoparticles (50 µg/ml) showed fungal growth inhibition while ZnO-TiO 2 showed higher antifungal activity against A. avus than pure TiO 2 and ZnO. TiO 2 and ZnO-TiO 2 (300 µg/ml) inhibited 100% of spur production. Pure ZnO and TiO 2 showed pyramidal and spherical shapes, respectively whereas ZnO-TiO 2 nanopowders illustrated both spherical and pyramidal shapes with grown particles on the surface. Based on our ndings, low concentration (150 µg/ml) of ZnO-TiO 2 showed higher ROS production and stress oxidative induction thus fungicide effect as compared to alone TiO 2 and ZnO. In conclusion, ZnO-TiO 2 nanostructure can be utilized as an effective antifungal compound but more studies need to be performed to understand the antifungal mechanism of the nanoparticles rather than ROS inducing apoptosis. | 1 |
230524286 | Introduction ZnO and TiO 2 have been used in various biomedical applications due to photocatalytic, antimicrobial and antifungal properties [1,2]. Doping and nanocomposite manufacturing have been previously utilized as the processes for enhancing the antifungal activity of this kind of nanoparticles. Among the various semiconductor nanomaterials, titanium dioxide (TiO 2 ) and zinc oxide (ZnO) have achieved more attention due to their high chemical stability, nontoxicity, relatively low cost and high antimicrobial activity [3,4]. The metal oxide NPs antibacterial and antifungal properties have been previously studied [5][6][7] and the ndings showed the ZnO antibacterial activity as well its capability to increase of induction of reactive oxygen species (ROS) production by decreasing its particle size. The zinc oxide antifungal activity is related to the formation of free radicals on the surface of nanoparticles that damage the fungal cell membrane lipids, which lead to protein leakage through the membrane disruption [8][9][10][11][12]. TiO 2 NPs possess the antimicrobial properties even at low concentrations through the photocatalytic process that causes fatal damage in treated microorganisms [13][14][15]. Based on TiO 2 nanoparticle antimicrobial properties, these nanoparticles in the anatase and rutile phase show the excellent antifungal properties [16]. The titania owned enormous applications because of its high thermal/chemical stability, and high photocatalytic activity. The toxicity of titania nanoparticles originates from its physical properties, not its chemical structure. These nanoparticles can permeate from biological barriers that can damage the cells or even organs. Some methodologies have been previously applied for improving the titania NPs' antimicrobial activities on simple microorganisms such as bacteria and viruses [17][18][19][20][21]. By considering the Aspergillus species as the deadliest opportunistic fungal infections, these fungi are the main threat to human health. Among the 600 species of Aspergillus, the avus, fumigatus, and niger species possess the pathogenicity for humans and growing on crops can cause the occurrence of some disease [22,23]. Upon the previous quantitative reports on ZnO and TiO2 fungal growth inhibition, these nanoparticles possess fungicidal effects on Candida albicans, Aspergillus niger, and Penicillium sp. fungus. We showed previously the increase of ZnO and TiO 2 antibacterial activity by increasing the concentration of dopant in doped ZnO and TiO 2 [24,25]. This study aimed to synthesize the ZnO, TiO 2 , and ZnO-TiO 2 nanostructures using the sol-gel methodology, physicochemical characterization of nanopowders, and antifungal assays against Aspergillus avus to nd the highly effective antifungal concentration at dark condition. | 2 |
230524286 | Nanostructures synthesis ZnO and TiO 2 nanostructures were synthesized using the sol-gel method as described by Najibi [25]. For the preparation of ZnO-TiO 2 nanostructures, separately prepared ZnO and TiO 2 sols were mixed at the same molar ratio of Zn:Ti then the mixture was stirred at ambient temperature for 2 h and the stirred solution was remained for 24 h to obtain a gel. Prepared gel was dried at 100 °C and was calcined at 500 °C for 2.5 hours. | 3 |
230524286 | Material characterization method The XRD pattern and phase identi cation of nanopowders were determined by X-RAY diffraction analysis (Philips-MPD XPERT, λ: CuKα=0.154 nm) and 20-70˚ range of scanned samples were considered as 2Ө. The scanning electron microscopy (SEM), transmission electron microscopy (TEM), particle size analyzer (N5, Backman, USA), and zeta potential analyzer (Malvern Zeta-sizer 3000, Malvern Instrument Inc., London, UK) were utilized for morphological, size, and zeta potential characterization of all samples, respectively. Fourier Transform Infrared (FTIR) Spectroscopy was used to identify organic, polymeric, and in some cases, inorganic materials. Fourier transforms infrared (FTIR) spectra were obtained using a Bruker IFS 48 instrument (Bruker Optik GmbH, Germany). All spectra were taken under air as a function of time with 16 scans at a resolution of 4 cm − 1 and a spectral range of 4000-5000 cm − 1 . | 4 |
230524286 | Antifungal assay A. avus, purchased from the Iranian biological resource center (IBRC), were cultured on Sabouraud dextrose agar (SDA; Merck, Darmstadt, Germany) at 25 °C and the dark condition. The autoclaved SDA media containing ZnO, TiO 2 and ZnO-TiO 2 NPs at concentrations of 0, 37, 75,150 and 300 µg ml − 1 and an NP-free solution were poured onto the 6 cm diameter Petri dishes. To determination of minimum inhibition concentration (MIC) of nanoparticles for each treatment group, the CLSI-M38 standard method was used for the time intervals of 7 days by measuring the diameter of fungal colonies opacity. To determine the minimum fungicide concentration (MFC), the higher concentrations than MIC for each nanostructure were used on SDA medium similar to the MIC determination experiment and the minimum concentration that killed A. avus considered as MFC. To detect the production of ROS after each time point of treatment, 2′-7′-Dichlorodihydro uorescein diacetate (DCFH-DA) solubilized in ethanol (5 µM nal concentration) was added to the cultures and incubated on a shaker at room temperature at the dark condition for 1 h. DCFH-DA, a nonpolar dye, is converted to the non uorescent polar derivative DCFH by cellular esterases. DCFH can switch to highly uorescent DCF through oxidization by intracellular ROS and possessing an excitation wavelength of 485 nm and an emission band between 500 and 600 nm. After incubation time, samples were subjected to uorescence microscopy (Biozero BZ-8000; Keyence, Osaka, Japan) equipped with the following lter set EX 495 nm EM 510 nm, and uorescence spectrophotometric (RF-5000, Shimadzu, Kyoto, Japan) analysis at room temperature. The XRD patterns of ZnO and TiO 2 showed a single high-intensity peak that implies a highly oriented and single-crystalline nature of the samples. As shown in Fig. 1, the intensity of TiO 2 peaks considerably decreased after the addition of TiO 2 into the structure of ZnO in the ZnO-TiO 2 composite that indicates the greater crystallinity of pure TiO 2 NPs compared to ZnO-TiO 2 NPs [27]. Pro le broadening also indicated the small crystalline domain sizes of wurtzite and anatase indicating that the ZnO-TiO 2 composite hinders the growth of particles during calcination. The main peaks of each sample in the range of 2θ = 20-50° speci ed some peaks belonging to anatase (Fig. 1). Table 1 | 5 |
230524286 | PSA and Zeta potential analysis The zeta potential is an important indicator of the stability of dispersed particles in the suspension solution. The zeta potential determines the repulsion of dispersed particles in the solution. Small particles require the high zeta potential for superior stability, and low zeta potential causes to particle accumulation. The zeta potential of a particle alters by the particle surface chemical composition, the pH and ionic strength of the solution. Zeta potential of ZnO, TiO 2 , and ZnO-TiO 2 were − 11.6, -36.4, and − 12 mV, respectively ( Fig. 2 and Table 1). Based on our ndings, TiO 2 and ZnO-TiO 2 showed the highest and lowest stability in aqueous suspension, respectively. Larger particle sizes for ZnO (608 nm), TiO2 (299 nm), and ZnO-TiO2 (983 nm) were determined by PSA analysis showing the agglomeration of nanoparticles. | 6 |
230524286 | SEM and TEM analysis As shown in Fig. 3, the ZnO and TiO 2 nanoparticles illustrated hexagonal-pyramidal and spherical shape with grown articles on surfaces, respectively. The wurtzite-structured ZnOcrystal is described as several alternating planes composed of four-fold tetrahedrally-coordinated O 2− and Zn 2+ ions stacked alternatively along the c-axis [28]. The oppositely-charged ions produce positively-charged Zn (0001) and negatively-charged O(0001 ) surfaces, resulting in a normal dipole moment and spontaneous polarization along the c-axis, as well as a divergence. In the ZnO-TiO 2 nanostructures, the morphology was a mixture of pyramidal and spherical with more agglomeration while the particle sizes were smaller than alone titanium and zinc oxide particles. Upon the EDX analysis, the strong signals of Zn, Ti and Zn-Ti were observed in ZnO, TiO 2 , and ZnO-TiO 2 nanostructures, respectively (Fig. 3). The TEM images of nanostructures clari ed the regular growth of all nanostructures and illuminated the TiO 2 (5 nm) particle size smaller than ZnO (10 nm) and ZnO-TiO 2 (35 nm) nanoparticles with lower agglomeration rate (Fig. 3). related to the hydroxyl groups. Also, water molecules in the bending band at 1630 cm − 1 are visible [31]. The presence of some bands can be associated with the organic phase of solid, despite the use of organic compounds in the synthesis of nanoparticles (Fig. 4). | 7 |
230524286 | Antifungal properties of nanostructures As shown in Table 1, ZnO-TiO 2 nanostructure exhibits better antifungal effects against A. avus than other nanoparticles due to its high speci c surface area. By increasing the speci c surface area, the possibility of chemical reactions and the production of reactive oxygen species on the surface were increased [32]. The MIC for ZnO-TiO 2 , ZnO, and TiO 2 against A. avus was determined 39, 156, and 78 µg/ml, respectively. Because of the small particle size, the best cell internalization, and the ability to produce more reactive oxygen species, TiO 2 showed a higher fungicide than ZnO. The MFC for ZnO, TiO 2 , and ZnO-TiO 2 was 312, 156 and 78 µg/ml, respectively. The particle size of the ZnO-TiO 2 nanostructure possessed a sharp structure with smaller particles than the cell membrane that can inhibit the growth of the fungus by entering the cell membrane and injuring the cell wall thus resulting in the high toxicity. Figure 5 illustrated the inhibition zone of ZnO, TiO 2 , and ZnO-TiO 2 at 37.5, 75, 150, and 300 µg/ml concentrations. By increasing the concentration of nanoparticles, inhibition zone diameter of growth increased and 100% of inhibition was achieved at 300 µg/ml for TiO 2 and ZnO-TiO 2 treated groups. The minimum fungal growth (72%) was obtained at 37.5 µg/ml for ZnO-TiO 2 while for ZnO was 50% at the same concentration showing that the TiO 2 synergistic effect into the mixture [33]. Among all nanoparticles, ZnO nanoparticles showed the lowest fungicide activity compared to others whereas it signi cantly increased the antifungal activity in ZnO-TiO 2 nanocomposite. The destructive changes were observed on the shape and growth of the treated A. avus (at a concentration of 37.5 µg/ml for all samples) compared to the untreated control group. As shown in Fig. 6, the untreated control fungus produced the highest count of fungal spores while treated groups showed a lower count of spores and damaged tubular laments, in instance deformation, smoothness, and noticeably thinner in hyphae compared to the untreated group. Upon the previous reports, increasing the hyphae causes to form whiter medium [34] and our ndings agreed to color changes based on the used nanoparticles (Fig. 6). Among the reactive oxygen species, hydrogen peroxide and hydroxyl radicals as the strong and nonselective ROSs can damage all types of biomolecules including carbohydrates, acids, lipids, proteins, DNA, RNA, and amino acids through inducing the oxidative stress [35]. The production rate of the three [36]. There is a direct dependency between increasing the formation of ROS and the fungicide of nanoparticles. As shown in Fig. 7, all nanoparticles raised the ROS production in treated A. avus compared to untreated control with order ZnO-TiO 2 > TiO 2 > ZnO > untreated control. The production of intracellular ROS was in uenced by the type and speci c surface of nanoparticles. Titania can produce ROS higher than zinc oxide [37], our ndings also con rmed the highest ROS production through stronger uorescence intensity in ZnO-TiO 2 treated group. In ZnO-TiO 2 nanostructures, the speci c surface area is higher than other nanoparticles (TiO 2 and ZnO) and accordingly high ROS generation. Oxidative stress induced by reactive oxygen species generation in ZnO-TiO 2 nanostructures is thought to be the main mechanism of antifungal activity. The suggested mechanism for the antifungal activity of these compounds can be based on the formation of high levels of reactive oxygen species (ROS) that disrupt the integrity of the fungal cell membrane, which assists in the damage of microbial enzyme bodies thus killing the fungi [38]. | 8 |
228834377 | Vacuum freeze-drying is one of the best water removal methods, with final products of the highest quality. The solid-state of water during freeze-drying protects the primary structure and the product's shape with minimal volume reduction. As the leading quality problem of dehydrated green Asparagus, this experiment was to study the technique of improving the rehydration of dehydrated green Asparagus by adding a hydrophilic substance (Maltodextrin, sucrose, salt) and controlling two ways in the process of vacuum freeze-drying. The mixed solution was soaked at the rate of three different concentration ratios, i.e., 1 (10%), 2 (15%), and 3 (20%) for maltodextrin, sucrose, and salts, respectively, using the L9 orthogonal and two-factor comparison experiment. It was concluded that increasing the mass of the Asparagus samples decreased the convective heat transfer coefficient. The evolution of drying months in the range of 1.78 4.74 W/m ̊C was recorded for the mass of Asparagus samples. The results noted that to dry the Asparagus by vacuum freeze-dryer from 09:00 to 18:00 hour decreases the product's drying rate up to 0.011g.(H2O).g (d. m).cm.hr and moisture level up to 8%. The study results noted that the pre-freezing condition was 23°C with the frozen time of 4 hours, which could remarkably improve the vacuum freeze-dried green asparagus rehydration. Finally, from the results, it was recommended that, from the actual production, to save energy, reduce costs; 23C was better for the precooling temperature with the pre-freezing time was 4 hours for drying green Asparagus. | 1 |
228834377 | Introduction Green Asparagus is also an extremely perishable vegetable. Freshly harvested Asparagus deteriorates quickly, which results in a short shelf life under normal postharvest handling at room temperature. Drying is one of the most methods used for preservation. The drying of agricultural products has always been of great importance for preserving food by human beings. It is a primary preservation method and applies to a wide range of industrial and agricultural products. Adedeji et al. [1] reported that Asparagus (Asparagus officinalis L.) is commonly grown in temperate climates worldwide perennial plant with 100-150 cm tall, stout stems and soft vegetation. Asparagus's essential ingredients are energy, proteins, vitamins, fats, carbohydrates, etc. necessary in food with high nutritional value in the kitchen [2]. It is not only used to add food palatability, but it is also widely used in medicines, bakery products, wine and meat products, a soap product, etc. [3]. Asparagus is the most important cash crop globally, cultivated in China, Pakistan, Indian, Afghanistan, Uzbekistan, Japan, and Indonesia [4,5]. An et al. [6] stated that the People Republic of Chain producer 17 million tons a year of Asparagus and 45% of the total world's Asparagus contributing. Nearly half of the total production of Asparagus consumed as white and red Asparagus. In contrast, the remaining 30% converted into dry Asparagus for medicinal purposes, and 20% used as seed material [7]. Agricultural product drying has a vital role in preserving and shelf-life improvement after harvesting [8]. In developing countries, sundrying is a popular, effective, and economical method for drying food and herbal products. Sun-drying is a common food preservation technique used to control agricultural products' moisture content [9]. Traditionally, herbs, like Asparagus dried in the open sun, depend on sunshine availability and require ample drying space and long drying time [10]. Green Asparagus is very resistant to the storage; after harvesting of 1-2days, it will lose water, rot, and lose nutritional values [11]. The traditional processing method is to process green Asparagus into canned or frozen products. The research on the drying of green Asparagus is less; the main reason is that the drying of green Asparagus is reduced, which is also the key to dehydrated green quality asparagus [12]. The moisture content of the solar-dried unpeeled Asparagus found to be 7.0 %, unlike that of sun-drying, which could attain only 17.0 % moisture content [13]. Other researchers [14,15] have reported the drying behavior of Asparagus at four different drying air temperatures, i.e., 25 o C, 35 o C, and 45 o C, with the fixed air velocity of 1.3 m/s. The study results concluded that moisture content reduced from 87% to 6%, with a temperature of 45 o C on a wet basis. Blanching is the pre-treatment method used to arrest a few physiological processes. It helps in the inactivation of the enzymes, acceleration of drying rate, and reduced quality loss. It expels intercellular air from the tissues and softens it (16). Generally, the blanching of fruits and vegetables is done by heating in steam or hot water. Drying, a routine food preservation technique, is a crucial aspect of food processing [16,17]. The dried product's shelf life has been demonstrated to extend by reducing the water concentration at which microbiological and physicochemical deterioration is limited [18]. The drying method and processing conditions significantly affect the color, texture, density, porosity, and sorption characteristics of plant materials [19]. Therefore, the same plant raw material may yield a completely different product, depending on the type of drying and extraction methods employed [20]. In the past, it was mainly through controlling the soaking temperature, soaking time, and water consumption to improve the rehydration of dehydrated vegetables. In recent years, researchers have started to develop the ratio of rehydration in the view of pre-treatment. The pore of dehydrated vegetable infiltrated into maltodextrin, sucrose, and salt molecule, improving the rehydration ratio of dehydrated through the immersion of hydrophilic material green asparagus, which was much better than using the physical method alone. | 2 |
228834377 | Material and Methods Description of the experimental procedure We took fresh green asparagus samples from the local market accessible in Nanjing, China, and washed them with distilled water for experimental work. The samples were cut cylindrical tube with a length of 3.6 mm and a 1.4 mm in diameter and placed on the weighing balance. Ullah et al. [21] reported that they are usually tiny and intense, growing 10-20 mm long and 3-7 mm in diameter. Asparagus samples were cut into cylindrical shapes with a length of 4mm, and a diameter of 6mm was also reported [22]. The data was recorded from 9:00 to 18:00 in June, July, and August 2016. The asparagus specimens were put in trays and placed on the digital electronic balance in each drying hour to determine water content discharge. After each hour of drying, the experimental observation data were recorded, as well as the evaporation was scrapped with the attained constant weight of the samples. The literature observed that the Asparagus dried from its average initial moisture content of 89% to the final moisture content of 8% [23]. The data obtained from the measurements of Asparagus weight used for drying kinetics and analysis of Asparagus in terms of moisture removal rate, and the drying was discontinued. The samples' constant weight was achieved. The difference in weight directly gave the quantity of water content evaporated during any time interval. Wet and dried Asparagus samples are shown in (Fig. 1). The moisture removal rate was expressed on a dry basis. Equation 1 was used for the determination of the moisture removal rate of the product. The moisture ratio of Asparagus during the drying can be obtained from equation 2. While the dry matter is the dry weight of the Asparagus can be calculated using equation 3, evaluated [24]. For determining the area of fruits, inch tape was used for recording the diameter before and after each hour of drying with the use of equation 4, reported [25]. Therefore drying rate is the evaporation of water content from the products in unit area unit time. It can be calculated from the dry matter of the product how much moisture was lost during the drying. Equation 5 was used to calculate the product's drying rate each hour studied by [26]. Similarly, the symbols used in the equations, "Dm" is Dry matter (g), "Wt" wet weight (g), "Minitial" is Initial moisture removing rate (%, dry basis), "Ww" is the weight of wet Asparagus (g), "Wd" represents the weight of dry Asparagus (g), "MR" is the moisture ration (%), "Mo" is the initial moisture content (%, dry basis), "Me" is the equilibrium moisture content (%, dry basis), "Ap" is the cross-sectional area of the product (cm 3 ), " " constant term 3.144, "r" radius of the product (cm), "Dr" is the drying rate of the product [(g (H2O).g -1 (d.m).cm -2 .h -1 )] and "Dt" denoted the drying time (hr). In (Table 1) it shows the moisture removal rate data, indoor and outdoor vacuum freezedryer temperature, product temperature, the surrounding temperature of the product, and ambient temperature during the experiment. | 3 |
228834377 | Experimental methods The mixed solution was soaked at the rate of three different concentration ratios, i.e., 1 (10%), 2 (15%), and 3 (20%) for maltodextrin, sucrose, and salts, respectively, using the L9 orthogonal and two-factor comparison experiment. Determination of rehydration of green Asparagus with vacuum freeze-drying process, the fresh green Asparagus was selected for blanching treatment with maltodextrin's robust solution; sucrose, and salt concentrations (sodium Chloride, NaCl) (29) and soaked for 30min. In this experimental work, we used the treatment solution separately for determining the rehydration ratio. Kingsly et al., [29] studied the rapid HPLC method for the separation of isomaltulose (also known as Palatinose) from other common edible carbohydrates such as sucrose, glucose, and maltodextrins, commonly present in food and dietary supplements. After the treatment of blanching, we infiltrate the maltodextrin, sucrose, and salt molecules with the help of the osmotic process to improve the drying of green asparagus water are the best and suitable methods to increase the shelf life of products [30]. In this experiment, the concentration of maltodextrin, the sucrose level, and salt's strength was to select orthogonal analysis quality. Determination of water ratio in drying Asparagus repeated the quality times, using the SAS software reported by Vesali et al. [31] the variance of quality "L9" orthogonal experiment and "LSD," according to the results of multiple comparisons [32]. The experiment chooses the pre-freezing temperature and the precooling time as a factor. The drying ratio product to the measuring index influences its state of dehydrated products and then influences its rehydration factors. Lin & Brewer [33] According to the resistance method, the temperature of the eutectic point of green Asparagus was measured. The temperature of the 5-10 o C was lower than that of the eutectic point, so the highest temperature of green Asparagus was determined to be 23 o C. The pre-freezing time's relevant research data is less, and it needs to choose the wide horizontal range, and according to the result of the preliminary experiment. They used SAS software to analyze the variance of the experimental results and 1/2 multiple comparisons, a better pre-freezing process parameter chosen according to various comparisons. The rehydration capacity was used as a quality characteristic of the dried product [34] expressed in the rehydration rate -RR. Approximately 2g (± 0.01g) of the dried sample was placed in a 250ml laboratory glass (two analyses for each sample), 150ml distilled water was added, and the glass was covered and heated to boil within 3 minutes. The laboratory glass content was then gently boiled for ten (10) min more and then cooled. The cooled content was filtered for 5min under vacuum and weighed. The drying ratio was calculated from equation 6. At the same time, "Wr" is the drained weight (g) of the rehydrated sample, and "Wd" represents the weight of the dry sample used for rehydration T(o,v) is the temperature at vacuum freeze-dryer ( o C), Tc is the product temperature ( o C), Te is the product surrounding temperature ( o C), Mevp is the moisture evaporation (g), and M.removing rat is the moisture removing rat in the products with the unit of (%db). | 4 |
228834377 | Results and Discussion Under natural convection mode, the handpeeled cylindrical shaped (diameter 1.4 mm, length 3.6 mm) mass of Asparagus samples is dry. Rect angular trays were used to conduct drying assessments of Asparagus sp ecimens. (Fig. 2) represents the comparison between the means of solar radiation (MSR) and the mass of the products of asparagus samples for the three months. Jamil et al. [36] studied bean moisture diffusivity and drying kinetics. They reported that the conditions of pre-freezing temperature and time would affect the size and quantity of products. Solar irradiation and product mass data collected for June, July, and August 2016 at a drying time of 60-minute duration under natural heat transfer solar energy drying, as shown in (Fig. 2). It has been observed that green asparagus re-hydration increases from morning to noon and decreases from noon to evening due to swelling and diminishing trend of solar irradiation in one day. The present results are in substantial agreement with the study's previous results reported by Deshmukh et al. [37]. They said that different products' rehydration process, i.e., apples, banana, green chili, red chili, green Asparagus, etc., started increasing from morning to noon. The study results agreed with Ismail [38]. They studied that the rehydration process increased with increasing solar irradiance. The data given in (Table 2) show the moisture removing rate, indoor and outdoor collector temperature, product and product surrounding temperature, and ambient temperature during the experiment. Table 2 shows the moisture removal rate is dependent on the total moisture present in the product mass. Hence, it has been observed that the moisture removal rate increases with an increase in green Asparagus samples mass and decreases significantly with the progression of drying days [39]. However, the moisture removal rate is also dependent on the ease of heat transfer [40]. (Fig. 3) shows the moisture lost and drying rate in Asparagus during dry. The products (Green Asparagus) were dried in the vacuum freezedryer with the process of rehydration, moisture loss, and drying rate was determined. Moisture lost in each hour of drying by a vacuum freeze-dryer is correlated with drying time. The drying rate is correlated with the change in percent moisture content to find the vacuum freeze dryer's promising performance as a drier for Asparagus's rehydration. The results show that moisture was lost with the higher temperature of the dryer. Before drying, 89% moisture was noted, and dried the product up to 8% moisture level with a dryer. Ullah and Kang [24] reported that the moisture content of the product was decreased with the highest temperature of the dryer. Similarly, the drying rate of the apples was starting to decrease from 0.032 g(H2O).g -1 (d.m).cm -2 .h -1 to 0.011 g(H2O).g -1 (d.m).cm -2 .h -1 with the increasing temperature of the dryer after 10hr of the drying process. Kohli et al., [15] stated that the drying rate of the product was reduced with the highest temperature of the dryer. The results are similar to [41], who found that the drying is directly related to the product's moisture content. Similarly, when the 15% (2) concentration of maltodextrin was applied, the water recovery was not significant. The second level of concentration of 15% (2) could be used as a balanced choice for other factors. The results agree with the findings [43]. They reported that decreasing the maltodextrin's concentration ratio in the products' rehydration process, the effect on the water recovery rate was not significant. The results showed that the difference between the first 10% (1) and the third 20% (3) levels of concentration is substantial, and the effect on the rehydration is not apparent. Table 2 shows the multiple comparisons of maltodextrin, sucrose, and salt concentration. When the sucrose concentration was at 20% (3), which improved the asparagus' rehydration, so optimum absorption of sucrose was 20%. The results are similar to the results [44]. The static results show in (Table 3) that when the sucrose concentration is the 10% (1) level, the difference between the water and the 10% (1) level is not significant, theoretically chosen as the object. Still, the influence of the other two factors should be considered. Serratosa et al., [45] conducted experimental results that are nearly matched with the present study results. At the 20% (3) level, the ratio of rehydration was significantly different, and the effect on the water recovery was not noticeable. The multiple salt concentration comparisons from ( Table 2) show that salt's frequency is 15% (2). The ratio of green Asparagus is higher than the average value, so, to improve the rehydration of dehydrated products, a better salt concentration should selected as 15%. These results are in agreement with the findings [46]. They reported that the concentration ratio of salt for the rehydration process of different products was 17%. Therefore, when the salt concentration was 10% (1) level, the proportion of water to the (2) level was not significant and could be used in balance with the other two factors. Singh et al., [47] reported that study results contradict the present study results. When the salt concentration was at the (3) level 20%, the rehydration effect was not significant, as shown in (Table 3). Determination of the concentration of hydrophilic substances found in preliminary experiments, when the frequency of maltodextrin was low, mostly under 10% concentration, there was little effect on the drying green Asparagus [48]. The main reason was that the low concentration maltodextrin's osmotic pressure was not enough to spread the maltodextrin to the inside of the material, only on the paste's surface. As a result, when the green Asparagus is dried and reused, the dextrin's surface first absorbs water to expand, instead of preventing it from spreading to the inside [49]. When the concentration of maltodextrin is more significant, especially when it is greater than 45%, the material is easily absorbed and melted during the dry process, resulting in shrinkage and deformation. Also, the dry products in the maltodextrin are very easy to moisture absorption, so that the storage becomes difficult, and there is too sticky feeling [50]. So in the appropriate concentration range, you can choose a higher concentration of maltodextrin, according to the results of multiple comparisons, the final determination of maltodextrin concentration of 20% [51]. Sucrose and salt are added to the maltodextrin to increase the solution's osmotic pressure [17]. However, the addition of sucrose and salt will affect the taste of dehydrated green Asparagus, after the experiment found that the sweet and salty ratio of 4:3 is more appropriate, so sucrose concentration of 20%, the salt concentration of 15% [52]. It is more suitable to be considered the final process parameter, whether from improving the rehydration or from a sweet-salty angle. | 5 |
228834377 | Determination of pre-freezing process conditions The independence of the Eutectic Point of the pre-freezing cooling curve of green Asparagus is shown in (Fig. 4). As the temperature drops, the figure shows that the resistance changes very little; when the temperature drops to -11 o C to -13 o C, the resistance value suddenly increased. Due to the early freeze, green Asparagus inside there is a lot of water present, more charged ions can be moved freely, and with the temperature [53]. Most of the green asparagus water is converted to ice crystals, and when the temperature drops to -11 o C to -13 o C, the green Asparagus is frozen, and the resistance value suddenly increases [54]. Therefore, from the experimental result, the eutectic point temperature range for the green Asparagus is noted up to -11 o C to -13 o C. The results and analysis of the two factors of the water recovery ratio shown in (Table 4). They are using SAS software to analyze the variance of water recovery effects, shown in (Table 4). From the analysis of variance results in (Table 4), it can be seen that the Pvalue of the precooling temperature is less than 0.05, the P-value of the pre-freezing time is less than 0.01, and these two factors have a significant or significant effect on the rehydration ratio of the dehydrated green Asparagus [23]. Among them, pre-time is an essential factor, and the precooling temperature is the secondary factor. They are using the method of LSD to compare the prefreezing temperature and the pre-freezing time, taking the rehydration ratio of the dehydrated green Asparagus as the object of study, the proper conditions of precooling determined according to the results [14]. The multiple comparisons of the pre-freezing temperature ( Table 5) show that when the pre-freezing temperature -20 ( o C) and -30 ( o C), the green asparagus ratio is higher than the average is no significant difference between the two. So the precooling temperature is 23 o C and -30 o C, which is beneficial to improving the drying ratio of the green Asparagus [44]. When the pre-freezing temperature is the second level, the water recovery ratio's mean value is lower. There is a significant difference with the other two levels, which has no significant effect on improving the rehydration of the dehydrated green Asparagus. The multiple comparisons of pre-freezing time are shown in (Table 5). When the prefreezing time is 6 hours and 4 hours, the green asparagus ratio is higher, and the difference between them is not significant. . 6 hour is the first choice of parameters, the remaining three levels and above two levels of water ratio difference is significant, to improve the drying of green Asparagus. For analyzing the above factors, 6 hours and 4 hours is the pre-freeze time [20]. | 6 |
214702650 | In this work, a new photocatalytic system consisting of iron oxide nanoparticles (IONPs), coated with a catechol-flavin conjugate (DAFL), is synthesized and explored for use in water remediation. In order to test the efficiency of the catalyst, the photodegradation of amaranth (AMT), an azo dye water pollutant, was performed under aerobic and anaerobic conditions, using either ethylenediaminetetraacetic acid (EDTA) or 2-(N-morpholino)ethanesulfonic acid (MES) as electron donors. Depending on the conditions, either dye photoreduction or photooxidation were observed, indicating that flavin-coated iron-oxide nanoparticles can be used as a versatile enzyme-inspired photocatalysts. | 1 |
214702650 | Synthesis of γ-Fe2O3 Maghemite Iron Oxide Nanoparticles (IONPs) The γ-Fe2O3 IONPs were prepared using an oil in water (o/w) reverse micro-emulsion protocol, as reported by Benyettou et al. [36] using ferrous dodecyl sulfate (Fe(DS)2). Prepared IONPs were first characterized using Attenuated total reflection Fourier transform IR (ATR-FTIR ) and TEM ( Figure 1). Figure 1A shows bands at 962 cm −1 and 1619 cm −1 in the FTIR fingerprint region of the Fe(DS)2 spectrum that correspond to the S=O stretching vibrations of the sulfonic acid [37]. The sharp peaks at ~2919 cm −1 relate to the C-H stretching mode. The band at 3415 is a characteristic of the O-H bond vibration from the residual water that remained in the dried sample. The FTIR spectrum of the IONP shows characteristic bands at 556 cm −1 and 3270 cm −1 that, respectively, correspond to the Fe-O stretching vibration and the O-H stretching bond vibrations from the residual water molecules coordinated to the particle surfaces [38]. The absence of the strong, sharp peaks in the fingerprint region of the IONP spectrum indicates that only trace amounts of surfactant molecules are present [39]. Some examples of the flavin-conjugated metal-oxide surface such as TiO 2 and BiOCl have been reported, but not applied to the degradation of complex molecules, such as AMT, and to the best of our knowledge, none demonstrated both the reduction and the oxidation of the substrates as presented within this paper [33][34][35]. | 3 |
214702650 | Synthesis of γ-Fe 2 O 3 Maghemite Iron Oxide Nanoparticles (IONPs) The γ-Fe 2 O 3 IONPs were prepared using an oil in water (o/w) reverse micro-emulsion protocol, as reported by Benyettou et al. [36] using ferrous dodecyl sulfate (Fe(DS) 2 ). Prepared IONPs were first characterized using Attenuated total reflection Fourier transform IR (ATR-FTIR ) and TEM ( Figure 1). Figure 1A shows bands at 962 cm −1 and 1619 cm −1 in the FTIR fingerprint region of the Fe(DS) 2 spectrum that correspond to the S=O stretching vibrations of the sulfonic acid [37]. The sharp peaks at~2919 cm −1 relate to the C-H stretching mode. The band at 3415 is a characteristic of the O-H bond vibration from the residual water that remained in the dried sample. The FTIR spectrum of the IONP shows characteristic bands at 556 cm −1 and 3270 cm −1 that, respectively, correspond to the Fe-O stretching vibration and the O-H stretching bond vibrations from the residual water molecules coordinated to the particle surfaces [38]. The absence of the strong, sharp peaks in the fingerprint region of the IONP spectrum indicates that only trace amounts of surfactant molecules are present [39]. The zeta potential and hydrodynamic size of the nanoparticles were investigated using a Zetasizer Nano Range instrument (see Table S1). At a pH ~7, the IONPs have a surface charge of −19.25 ± 4.51 mv. The negative charges, due to hydroxyl groups on the nanoparticle surfaces, stabilize the suspension and stop the particles from aggregating. The IONPs displayed an average hydrodynamic size of 152.3 ± 2.45 nm, with a polydispersity index (PDI) of 0.211 ± 0.014 from dynamic light scattering (DLS) measurements. Transmission electron microscopy (TEM) was also used to study the IONPs size, distribution and morphology ( Figure 1B,C). The data shows rough spherical particles with an average diameter of 12.38 ± 1.12 nm. Particle diameters range from 10 to 15 nm with 12 nm, particles being the most frequently occurring. This disparity in particle size from DLS and TEM techniques is likely due to hydrate layers present on the IONPs in an aqueous medium [40] and the nanoparticle agglomeration in water due to magnetic attraction [41]. | 4 |
214702650 | Surface Modification of γ-Fe2O3 Nanoparticles Although the functionalization of IONPs is well documented in the literature [44,45], the use of a catechol linker such as dopamine for functionalization, while avoiding the generation of the polydopamine coating, which could interfere with the electron transfer, has been reported less frequently. Herewith, a modified protocol described by Geiseler et al. [46] was used for the functionalization of the IONP surfaces with dopamine (DA) or dopamine-flavin (DAFL, see electronic supplementary information (ESI) for details on the synthesis). The dopamine-based linker was added to an IONP suspension at 23 °C (0.1 mg of DAFL for every 1 mg of IONP). The zeta potential and hydrodynamic size of the nanoparticles were investigated using a Zetasizer Nano Range instrument (see Table S1). At a pH~7, the IONPs have a surface charge of −19.25 ± 4.51 mv. The negative charges, due to hydroxyl groups on the nanoparticle surfaces, stabilize the suspension and stop the particles from aggregating. The IONPs displayed an average hydrodynamic size of 152.3 ± 2.45 nm, with a polydispersity index (PDI) of 0.211 ± 0.014 from dynamic light scattering (DLS) measurements. Transmission electron microscopy (TEM) was also used to study the IONPs size, distribution and morphology ( Figure 1B,C). The data shows rough spherical particles with an average diameter of 12.38 ± 1.12 nm. Particle diameters range from 10 to 15 nm with 12 nm, particles being the most frequently occurring. This disparity in particle size from DLS and TEM techniques is likely due to hydrate layers present on the IONPs in an aqueous medium [40] and the nanoparticle agglomeration in water due to magnetic attraction [41]. | 5 |
214702650 | Surface Modification of γ-Fe 2 O 3 Nanoparticles Although the functionalization of IONPs is well documented in the literature [44,45], the use of a catechol linker such as dopamine for functionalization, while avoiding the generation of the polydopamine coating, which could interfere with the electron transfer, has been reported less frequently. Herewith, a modified protocol described by Geiseler et al. [46] was used for the functionalization of the IONP surfaces with dopamine (DA) or dopamine-flavin (DAFL, see electronic supplementary information (ESI) for details on the synthesis). The dopamine-based linker was added to an IONP suspension at 23 • C (0.1 mg of DAFL for every 1 mg of IONP). Since DA polymerizes into polydopamine (PDA) in the presence of O 2 , light and at basic pH [47], the linker coordination process was conducted in an Ar atmosphere, in the dark, and at a pH of~7. The coated IONPs were kept in the dark at room temperature in air. ATR-FTIR and TEM were used to characterize IONP-DAFL ( Figure 2) and the IONP-DA control ( Figures S2 and S3). The ATR-FTIR spectrum of DAFL ( Figure 2A) shows a sharp peak at 1535 cm −1 that is characteristic of flavin C=N modes of the isoalloxazine ring [48]. Moreover, combined bond vibrations of C=N and C=C are present at 1258 cm −1 [20]. These bond vibrations are also present in the IONP-DAFL FTIR spectrum, indicating a successful coordination of the linker on the particle surfaces. Furthermore, the band at 1388 cm −1 corresponds to the stretching vibration of the C-N bond, connecting the catechol to the flavin moiety. The vibrations at 647 cm −1 and 928 cm −1 correspond to the aromatic C-H bond vibrations [49]. Lastly, the peaks at 3241 cm −1 and 559 cm −1 in the IONP-DAFL spectrum correspond to the O-H and Fe-O bond vibrations, respectively. Catalysts 2020, 10, x FOR PEER REVIEW 5 of 17 Since DA polymerizes into polydopamine (PDA) in the presence of O2, light and at basic pH [47], the linker coordination process was conducted in an Ar atmosphere, in the dark, and at a pH of ~7. The coated IONPs were kept in the dark at room temperature in air. ATR-FTIR and TEM were used to characterize IONP-DAFL ( Figure 2) and the IONP-DA control ( Figures S2 and S3). The ATR-FTIR spectrum of DAFL ( Figure 2A) shows a sharp peak at 1535 cm −1 that is characteristic of flavin C=N modes of the isoalloxazine ring [48]. Moreover, combined bond vibrations of C=N and C=C are present at 1258 cm −1 [20]. These bond vibrations are also present in the IONP-DAFL FTIR spectrum, indicating a successful coordination of the linker on the particle surfaces. Furthermore, the band at 1388 cm −1 corresponds to the stretching vibration of the C-N bond, connecting the catechol to the flavin moiety. The vibrations at 647 cm −1 and 928 cm −1 correspond to the aromatic C-H bond vibrations [49]. Lastly, the peaks at 3241 cm −1 and 559 cm −1 in the IONP-DAFL spectrum correspond to the O-H and Fe-O bond vibrations, respectively. After functionalization with DA and DAFL, the zeta potential of the IONPs increases from −19.25 ± 4.51 mv to 27.33 ± 0.22 mV and 17.93 ± 1.04 Mv, respectively (Table S1). The shift from a negative to positive charge for both modified IONPs indicates successful surface functionalization, due to the introduction of amino moieties within the linker molecules. The size of the IONPs also increases from 152.3 ± 2.5 nm (PDI = 0.211) to 196.4 ± 4.7 nm (PDI = 0.320) for IONP-DA and 209.5 ± 1.4 nm (PDI = 0.320) for IONP-DAFL. This increase of hydrodynamic radius provides further evidence of successful catechol coating. Sizes obtained from DLS are compared to TEM images of the IONP-DAFL ( Figure 2B) and IONP-DA ( Figure S3). IONP-DAFL particles are spherical in shape, and have an average diameter of 16.17 ± 0.86 nm. They range in size from 14 to 19 nm, with 16 nm particles being the most frequently occurring. IONP-DA nanoparticles, on the other hand, have an average size of 17.69 ± 0.94 nm, with particles ranging from 15 to 20 nm in diameter. Thus, the size of both surface-modified IONPs is roughly the same. In order to confirm the presence and determine the loading percentage of DAFL on the IONP surface, XPS analysis was utilized ( Figure S4). The XPS survey spectrum ( Figure S4A) shows the presence of Fe, O, N and C, as expected. The atomic % of N was used to calculate the % loading of DAFL, as there are 5 N atoms per DAFL molecule, we can therefore assume the loading to be 1%. The high-resolution Fe 2p spectrum ( Figure S4B) shows two distinct peaks with binding energies of After functionalization with DA and DAFL, the zeta potential of the IONPs increases from −19.25 ± 4.51 mv to 27.33 ± 0.22 mV and 17.93 ± 1.04 Mv, respectively (Table S1). The shift from a negative to positive charge for both modified IONPs indicates successful surface functionalization, due to the introduction of amino moieties within the linker molecules. The size of the IONPs also increases from 152.3 ± 2.5 nm (PDI = 0.211) to 196.4 ± 4.7 nm (PDI = 0.320) for IONP-DA and 209.5 ± 1.4 nm (PDI = 0.320) for IONP-DAFL. This increase of hydrodynamic radius provides further evidence of successful catechol coating. Sizes obtained from DLS are compared to TEM images of the IONP-DAFL ( Figure 2B) and IONP-DA ( Figure S3). IONP-DAFL particles are spherical in shape, and have an average diameter of 16.17 ± 0.86 nm. They range in size from 14 to 19 nm, with 16 nm particles being the most frequently occurring. IONP-DA nanoparticles, on the other hand, have an average size of 17.69 ± 0.94 nm, with particles ranging from 15 to 20 nm in diameter. Thus, the size of both surface-modified IONPs is roughly the same. In order to confirm the presence and determine the loading percentage of DAFL on the IONP surface, XPS analysis was utilized ( Figure S4). The XPS survey spectrum ( Figure S4A) shows the presence of Fe, O, N and C, as expected. The atomic % of N was used to calculate the % loading of DAFL, as there are 5 N atoms per DAFL molecule, we can therefore assume the loading to be 1%. The high-resolution Fe 2p spectrum ( Figure S4B) shows two distinct peaks with binding energies of 710.9 eV for Fe 2p 3/2 and 724.5 eV for Fe 2p 1/2 [50]. The satellite peaks present at 718.8 eV and 732.7 eV are characteristic for Fe 3+ ions in Fe 2 O 3 [51]. The fitting also gives more detail as to the IONP composition, which shows both Fe 3+ and Fe 2+ ions present with the larger amount of Fe 3+ . This has been observed for other Fe 2 O 3 nanostructures containing both αand γ-phase Fe 2 O 3 [52]. The O 1s spectrum clearly shows the characteristic signals one would expect for the IONP-DAFL hybrid, including lattice Fe 2 O 3 at 530.1 eV, carbonyl C=O at 531.1 eV, a lattice hydroxyl signal at 532.7 eV that is commonly observed in Fe 2 O 3 , and another peak at around 536.6 eV, which could be ascribed to the catechol Fe-O bond [53]. The nitrogen 1s spectrum clearly shows a main C-N/C=N bond signal at 400.0 eV. Finally, the C 1s spectrum displays all characteristic signals associated that correspond to DAFL, including C-C at 285.0 eV, C-O bonding at 286.3 eV, carbonyl C=O at 288.3 eV, and the π-π* satellite can be observed at 290.4 eV. | 6 |
214702650 | Amaranth Photooxidation (Aerobic Activity) The photocatalytic activity of IONP-DAFL towards AMT degradation was first carried out in an aerobic environment. In this condition, dye degradation is dependent upon the formation of reactive oxygen species (ROS) during the irradiation process [8]. These ROS are produced by semiconductors, such as IONP, through electron-hole (e-h) pair generation upon irradiation with photons with energy equal to or greater than the band gap energy of the material [54]. If the charge separation is maintained and the electron and holes do not recombine, electrons migrate to the conduction band, leaving a vacancy (a hole) in the valence band. The electron and hole can then recombine with O 2 and H 2 O, resulting in the production of the superoxide and hydroxyl radicals, as shown in Scheme 3. This process is limited by the rate of electron-hole recombination, which has been shown to be high for IONPs [29], so that a suitable sacrificial electron donor (ED) needs to be employed to quench generated holes, and an additional photoactive 'sensitizer' is required to improve activity. We envisioned that the addition of flavin-catechol, DAFL, would act as an IONP stabilizer and improve the visible-light IONPs catalysis. This is due to the direct charge transfer of electrons from flavin to the particle, as well as the enhanced ROS production by intermolecular charge and energy transfer to molecular oxygen, which results in the production of superoxide and singlet oxygen (Scheme 3). The direct charge transfer from flavin to NP through the catechol anchoring group could be expected as we observed a large degree of fluorescence quenching in dopamine-bound flavin (DAFL) in comparison to the NBoc-protected flavin, 3 ( Figure S6). The same phenomena was observed for other flavin-catechol conjugates that we have prepared [21]. In order to explore the efficiency of our system we chose two different classes of sacrificial electron donors: 2-(N-morpholino)ethanesulfonic acid (MES) at pH 6, a zwitterionic buffer recently shown to have favorable electron donor properties with flavins [55], and ethylenediaminetetraacetic acid (EDTA) at pH 6, which is one of the most widely used electron donors for flavin photoreduction [56]. The electron donor was used in excess (0.1 M) to AMT and IONP-DAFL, since the rate of the reaction has been shown to be proportional to the concentration of the donor present [57]. | 7 |
214702650 | Amaranth Photoreduction (Anaerobic Activity) The photocatalytic degradation of AMT was also investigated under anaerobic conditions in an Ar atmosphere. In this case, we hoped to take advantage of the flavin photoredox catalysis via formation of the hydroquinone capable of reducing the azo substrate by hydride transfer [56], as well as a flavin-sensitized charge transfer to IONP and surface interaction of absorbed AMT [61] (see Scheme 4). Similarly, the reaction was conducted in the presence of either MES or EDTA solution as the electron donor. The addition of TEMPO, DABCO and mannitol leads to a 2.5%, 14.09% and 20.17% photodegradation of AMT after 1 h of irradiation in the presence of IONP-DAFL ( Figure 4B). This is significantly less than the 91% degradation after 1 h of irradiation without any scavenger ( Figure 3A). Therefore, it could be concluded that hydroxyl radicals, superoxide and singlet oxygen, are all formed during the aerobic photodegradation of AMT. However, the photoreactions with both mannitol and DABCO proceed to near completion, while the addition of TEMPO only leads to a 24.76% degradation of AMT in 3 h. Therefore, superoxide radicals are the predominant reactive oxygen species. | 9 |
214702650 | Amaranth Photoreduction (Anaerobic Activity) The photocatalytic degradation of AMT was also investigated under anaerobic conditions in an Ar atmosphere. In this case, we hoped to take advantage of the flavin photoredox catalysis via formation of the hydroquinone capable of reducing the azo substrate by hydride transfer [56], as well as a flavin-sensitized charge transfer to IONP and surface interaction of absorbed AMT [61] (see Scheme 4). Similarly, the reaction was conducted in the presence of either MES or EDTA solution as the electron donor. After 3 h of irradiation, both bare IONPs and IONP-DA (both 0.333 mg/mL) show negligible activity in MES and EDTA, as seen in previous aerobic experiments. In MES, after 1 h of irradiation, the C/C 0 of AMT decreases by 33.4% in the presence of IONP-DAFL (0.333 mg/mL) and by 5.3% in the presence of DAFL (0.003 mg/mL) on its own ( Figure 5A). After 3 h of irradiation, the total AMT degradation is 39.5% and 5.7% in the presence of IONP-DAFL and DAFL, respectively (for UV-vis absorption spectra at each time point refer to Figure S11). However, after 1 h of irradiation in the presence of EDTA ( Figure 5B) the C/C 0 of AMT decreases by 92.2% in the presence of IONP-DAFL, and by 77.2% in the presence of DAFL (for UV-vis absorption spectra at each time point refer to Figure S12). After 2 h of irradiation, the photodegradation process in the presence of both IONP-DAFL and DAFL is complete. EDTA is therefore a much better electron donor than MES in anaerobic conditions, which could be attributed to the radical EDTA oxidation products that facilitate further AMT degradation [59]. Moreover, it is apparent that, irrespective of the electron donor, IONP-DAFL is a far more efficient photocatalyst than homogenous flavin, clearly demonstrating the benefit of our synergistic photocatalytic system. Refer to the 'Quantum Efficiency' section of the ESI for the rate of the reaction and quantum efficiency calculation. Catalysts 2020, 10, x FOR PEER REVIEW 10 of 17 After 3 h of irradiation, the total AMT degradation is 39.5% and 5.7% in the presence of IONP-DAFL and DAFL, respectively (for UV-vis absorption spectra at each time point refer to Figure S11). However, after 1 h of irradiation in the presence of EDTA ( Figure 5B) the C/C0 of AMT decreases by 92.2% in the presence of IONP-DAFL, and by 77.2% in the presence of DAFL (for UV-vis absorption spectra at each time point refer to Figure S12). After 2 h of irradiation, the photodegradation process in the presence of both IONP-DAFL and DAFL is complete. EDTA is therefore a much better electron donor than MES in anaerobic conditions, which could be attributed to the radical EDTA oxidation products that facilitate further AMT degradation [59]. Moreover, it is apparent that, irrespective of the electron donor, IONP-DAFL is a far more efficient photocatalyst than homogenous flavin, clearly demonstrating the benefit of our synergistic photocatalytic system. Refer to the 'Quantum Efficiency' section of the ESI for the rate of the reaction and quantum efficiency calculation. Figure S14A). IONP-DAFL, on the other hand, showed 34.02% AMT degradation in O2 ( Figure S14A) and a 25.05% degradation in Ar ( Figure S14B) after 3 h, which is most likely the result of electron donation from AMT itself, which then leads to its degradation, rather than water acting as the donor. | 10 |
214702650 | Reusability of the Catalyst The reusability of the IONP-DAFL in the anaerobic photodegradation of AMT using EDTA ( Figure 6A) and the aerobic degradation of AMT with MES ( Figure 6B) were investigated. AMT (0.05mg/mL) was irradiated in the presence of IONP-DAFL (0.333 mg/mL) and EDTA or MES (0.1 M) for 1 h under Ar or O2, respectively. The IONP-DAFL was then removed from the solution with a magnet, washed, and re-used in another run. Heterogenous flavin-based photocatalysts have often shown poor recyclability in the literature [62], which is also confirmed in this study. Although the IONP-DAFL system can be easily removed from the solution with the use of a magnet, IONP-DAFL is not recyclable in either aerobic or anaerobic conditions. For the UV-vis absorption spectra for each run in aerobic and anaerobic conditions, refer to Figure S15A and S15B, respectively Figure S14A) and a 25.05% degradation in Ar ( Figure S14B) after 3 h, which is most likely the result of electron donation from AMT itself, which then leads to its degradation, rather than water acting as the donor. | 11 |
214702650 | Reusability of the Catalyst The reusability of the IONP-DAFL in the anaerobic photodegradation of AMT using EDTA ( Figure 6A) and the aerobic degradation of AMT with MES ( Figure 6B) were investigated. AMT (0.05mg/mL) was irradiated in the presence of IONP-DAFL (0.333 mg/mL) and EDTA or MES (0.1 M) for 1 h under Ar or O 2 , respectively. The IONP-DAFL was then removed from the solution with a magnet, washed, and re-used in another run. Heterogenous flavin-based photocatalysts have often shown poor recyclability in the literature [62], which is also confirmed in this study. Although the IONP-DAFL system can be easily removed from the solution with the use of a magnet, IONP-DAFL is not recyclable in either aerobic or anaerobic conditions. For the UV-vis absorption spectra for each run in aerobic and anaerobic conditions, refer to Figure S15A,B, respectively. A significant loss in the catalyst's activity is observed after one run. The low recyclability of the material is most likely due to particle and flavin instability. Flavin photodealkylation at the N10 position has been reported in the literature [33]. In the case of IONP-DAFL, this would release the flavin moiety into the supernatant. To test this hypothesis, two samples of 1.0 mg/mL IONP-DAFL in H2O were irradiated with a 450 nm blue light in O2 and Ar for 1 h. The particles were then removed, and the fluorescence intensity of the supernatants of the samples were measured ( Figure S16). A strong fluorescence signal was obtained from both supernatants after excitation at 450 nm, indicating that flavin species were present in solution and no longer conjugated to the IONP surface after irradiation. Furthermore, a stronger fluorescence signal was obtained from the supernatant of the sample irradiated in O2, thus indicating that IONP-DAFL is less stable when irradiated in an O2-rich environment. Lastly, Figure S6C indicates that the fluorescence signal of DAFL is weak in comparison to NBoc-protected flavin, 3 due to quenching by the catechol moiety. Therefore, the fluorescence signals in Figure S16 can be attributed to lumiflavin that is a product of flavin photodegradation [63]. The regeneration of the catalyst was therefore not possible. For this reason, our current work looks at addressing this issue through investigating different anchoring groups, conjugation strategies and immobilization for flavin IONP hybrids with a wider range of sacrificial electron donors [59] that, unlike MES and EDTA, are stable in nonacid environments and at different temperatures. | 12 |
214702650 | General Commercially available reagents were purchased in the highest purity from Acros Organics A significant loss in the catalyst's activity is observed after one run. The low recyclability of the material is most likely due to particle and flavin instability. Flavin photodealkylation at the N10 position has been reported in the literature [33]. In the case of IONP-DAFL, this would release the flavin moiety into the supernatant. To test this hypothesis, two samples of 1.0 mg/mL IONP-DAFL in H 2 O were irradiated with a 450 nm blue light in O 2 and Ar for 1 h. The particles were then removed, and the fluorescence intensity of the supernatants of the samples were measured ( Figure S16). A strong fluorescence signal was obtained from both supernatants after excitation at 450 nm, indicating that flavin species were present in solution and no longer conjugated to the IONP surface after irradiation. Furthermore, a stronger fluorescence signal was obtained from the supernatant of the sample irradiated in O 2 , thus indicating that IONP-DAFL is less stable when irradiated in an O 2 -rich environment. Lastly, Figure S6C indicates that the fluorescence signal of DAFL is weak in comparison to NBoc-protected flavin, 3 due to quenching by the catechol moiety. Therefore, the fluorescence signals in Figure S16 can be attributed to lumiflavin that is a product of flavin photodegradation [63]. The regeneration of the catalyst was therefore not possible. For this reason, our current work looks at addressing this issue through investigating different anchoring groups, conjugation strategies and immobilization for flavin IONP hybrids with a wider range of sacrificial electron donors [59] that, unlike MES and EDTA, are stable in nonacid environments and at different temperatures. | 13 |
214702650 | General Commercially available reagents were purchased in the highest purity from Acros Organics (Pittsburgh, PA, USA), Alfa Aeser (Haverhill, MA, USA), Sigma-Aldrich (St. Louis, MO, USA), and TCI Chemicals (Tokyo, Kanto region, JPN) [20]. A Bruker (Billerica, MA, USA) 500 MHz DCH Cryoprobe Spectrometer was used for the 13 C and 1 H Nuclear Magnetic Resonance (NMR) spectroscopy measurements. UV-vis absorption spectra were obtained using an Agilent (Santa Clara, CA, USA) Cary 300 Spectrophotometer. Attenuated Total Reflection Fourier-Transform Infra-Red (ATR-FTIR) spectroscopy data was acquired from powder samples using a Perkin Elmer (Waltham, MA, USA) Spectrum One FT-IR Spectrometer. Fluorescence intensity measurements were done using an Agilent Technologies Cary Eclipse Fluorescence Spectrophotometer. Dynamic Light Scattering (DLS) and Zeta potential measurements were obtained using a Zetasizer Nano Range instrument from Malvern Panalytical (Malvern, Worcs, UK). | 14 |
214702650 | Synthesis of Ferrous dodecyl Sulfate (Fe(DS) 2 ) Briefly, a 100 mL solution containing 1 M sodium dodecyl sulfate (SDS) and 1 M iron (II) chloride (FeCl 2 ) in Milli-Q ® water is prepared and stored at 2 • C for 1 h. The resulting Fe(DS) 2 precipitate is then washed with 2 • C Milli-Q ® several times, and allowed to re-crystallize overnight. | 15 |
214702650 | Synthesis of γ-Fe 2 O 3 Maghemite Iron Oxide Nanoparticles (IONPs) The γ-Fe 2 O 3 maghemite IONPs were prepared using a modified "one emulsion plus reactant" [64] micro-emulsion synthesis protocol described by Benyettou et al. [36]. 3.05 g of Fe(DS) 2 is dissolved in a 500 mL round-bottom flask containing 345 mL of Milli-Q ® water at 32 • C. Once the iron salt is dissolved, the temperature is reduced to 28 • C. 50 mL (40 wt%) dimethylamine, heated to the same temperature, is then added dropwise. The solution is left shaking for 2 h at the same temperature without the use of a magnetic stirrer. The flask is then placed on ice. The nanoparticles are then cleaned 10 times with Milli-Q ® water using a magnet. 4 mL of 4 M HCl is added before each wash for the first fourwashes. The IONPs are then stored in water at a pH of 7. | 16 |
214702650 | Surface Modification of γ-Fe 2 O 3 Nanoparticles Briefly, 5 mg of IONPs are suspended in a 5 mL solution of dopamine (DA) or dopamine-flavin (DAFL) (0.5 mg/mL in H 2 O) in an argon atmosphere at 23 • C. The solution is sonicated for 5 min. A vortex mixer is then used to shake the suspension for 2 h, while being protected from direct sunlight. The suspension is then washed 3-4 times on a magnet using Milli-Q ® water. The coated IONPs are kept in the dark at room temperature in air. | 18 |
214702650 | General Procedure for Photoreduction and Photooxidation of Amaranth AMT (0.05 mg/mL) and an IONP sample (0.333 mg/mL) or DAFL (0.003 mg/mL) were added to a solution of either MES or EDTA buffer (0.1 M, pH 6, 3 mL), and then saturated with either O 2 gas or Ar for 15 min before being irradiated with a 450 nm blue LED (18 W; 34 mW/cm 2 ) in a HepatoChem (Beverly, MA, USA) EvoluChem™ PhotoRedOx Box photoreactor, equipped with a cooling fan to keep the temperature at~23 • C. 100 µL aliquots were removed from the reaction at specific time points and diluted to 1 mL with 0.1 M MES/EDTA. Any nanoparticles were removed by centrifugation or using a magnet before monitoring the degradation of AMT by UV-vis absorption spectroscopy, using λ max = 520 nm of AMT [8] to observe the relative concentration of remaining AMT in solution (C/C 0 ), where C 0 represents the initial concentration of AMT before irradiation. | 19 |
214702650 | General Procedure for ROS Scavenging Experiments Mannitol, TEMPO or DABCO (1.5 equiv) were added to a solution of AMT (0.1 mg/mL), IONP-DAFL or IONP (0.333 mg/mL) in MES buffer (0.1 M, pH 6, 3 mL). The mixture was then saturated with O 2 gas and irradiated with a 450 nm blue LED (18 W; 34 mW/cm 2 ) in an EvoluChem™ PhotoRedOx Box photoreactor equipped with a cooling fan to keep the temperature at~23 • C. | 20 |
214702650 | General Procedure for Recyclability Measurements The recyclability of IONP-DAFL was investigated using the general procedure described in Section 3.6 using MES buffer (0.1 M, pH 6) in an aerobic environment and with EDTA buffer (0.1 M, pH 6) under anaerobic conditions. One time point is taken at 1 h for each run before the catalyst was removed from the reaction via magnetic separation and washed with Milli-Q ® H 2 O for reuse. Four cycles for both samples were completed. | 21 |
99953786 | The composite membranes were prepared by sol-gel process, and the membrane potential has been measured for characterizing the ion-transport phenomena across a charged membrane using electrolytes (KCl, NaCl and LiCl). The membrane potential offered by the electrolytes is in the order of LiCl>NaCl>KCl. The results have been used to estimate fixed-charge density, distribution coefficient, charge effectiveness and transport properties of electrolytes of this membrane. The fixed-charge density is the most important parameter, governing transport phenomena in membranes. It is estimated by the TMS method; it is dependent on the feed composition due to the preferential adsorption of some ions. The results indicate that the applied pressure is also an important variable to modify the charge density and, in turn, the performance of membrane. The experimental results for membrane potential are quite consistent with the theoretical prediction. The morphology of the membrane surface is studied by Scanning Electron Micrographs (SEM). | 1 |
99953786 | Introduction Ion-exchange membranes (IEM) carry the fixed positive or negative charges (called anion-exchange membranes, AEM or cation-exchange membranes, CEM, respectively). They are generally used in the treatment of ionic aqueous solutions, e.g., electrodialytic concentration of seawater, desalination of saline water, demineralization process, acid and alkali recovery and others [1][2][3][4]. Ion-exchange charged membranes, which are now extensively utilized in industries, have attracted considerable attentions due to their extraordinary properties and practical demands and thus a large number of researchers have concentrated on these investigations for many years [5]. With the rapid development of industry and population explosion throughout the world, the demand for fresh water has become increasingly urgent due to the scarcity of drinking water resource and the contamination of industry to environment. Thus, the treatment of industrial wastewater is becoming imperative; while innovative technologies, which are used to prepare fresh water such as the desalination of brackish water and to treat the industrial refuses, have attracted numerous researchers. Among these novel methods, ion-exchange membrane-based technologies have been regarded as both effective and economical due to its lower operation expense and secure process, etc. [6][7][8]. Composite membranes have high thermal and chemical stability, long life and good defouling properties in their application, and they can have catalytic properties [9]. These properties have made these membranes desirable for industrial applications in the food, pharmaceutical and electronic industries. The sol-gel technique is an extremely flexible method to produce inorganic materials with highly homogeneous and controlled morphology [10][11][12]. Recently, due to the mild reaction conditions that can be used, the great potential of sol-gel processes, both hydrolytic and non-hydrolytic, has been extensively investigated for the synthesis of organic/inorganic materials [13]. A potential difference can be observed and measured, at least partly ionically perm selective, membrane in contact with two solutions at following cases: (1) same electrolyte of different concentration; and (2) same ionic strength but different counter-ions or co-ions. The former is called concentration potential and the latter bi-co-ionic/bicounter-ionic potential [14,15]. These potentials are of great interest in connection with the analysis of effective charge density, ionic transport number, and selectivity as well as interaction between charged species and membranes in both single charged membrane and bipolar membranes and thus caused great attention for many years [16][17][18]. For this purpose, a potential model correlating the intrinsic parameters of the membrane and ionic species with transport properties is actually needed and a body of such models has been obtained for single charged membranes and bipolar membranes [19][20][21][22]. It is now recognized that the electrical charge on the pore wall of membranes plays an important role in its separation performance and fouling behavior [23][24][25]. The choice of a membrane with suitable charge or electrical potential property can lead to optimization of existing processes or allow selective separations. For these reasons there is much interest in characterizing the charge or potential property of membranes. The electrical potential difference which is generated when an electrolyte solution flows across a charged membrane under a concentration gradient is among the most convenient experimental techniques for studying such electrical potential properties of porous membranes [26]. In the present investigation, a composite titanium-vanadium phosphate membrane is developed by sol-gel process using polystyrene as a binder. Fixed-charge density, the most effective parameter, has been evaluated and utilized to calculate membrane potentials for different electrolyte concentrations using TMS method [27][28][29][30][31]. In addition to the fixed-charge density, distribution coefficient, transport numbers, mobility, charge effectiveness and other related parameters were calculated for characterizing the composite membrane. | 2 |
99953786 | Preparation of membrane Titanium-Vanadium phosphate precipitate was prepared by mixing a 0.2 mol titanium (III) chloride (Otto Kemi, India with 99.989% purity) and vanadium (III) chloride (Merck, Germany with 99.989% purity) with 0.2 mol tri-sodium phosphate (E. Merck, India with 99.90% purity) solutions. The precipitate was washed properly with deionized water to remove free electrolytes and then dried at 100°C. The precipitate was ground into fine powder and was sieved through 200 mesh (granule size <0.07 mm). Pure crystalline polystyrene (Otto Kemi, India, AR) was also ground and sieved through 200 mesh. The titanium-vanadium phosphate along with appropriate amount of polystyrene powder was mixed thoroughly using mortar and pestle. The mixture was then kept into a cast die having a diameter of 2.45 cm and placed in an oven maintained at 300°C for about an hour to equilibrate the reaction mixture [32]. The die containing the mixture was then transferred to a pressure device (SL-89, UK), and various pressures such as 80, 100, 120, 140 and 160 MPa were applied during the fabrication of the membranes. As a result titanium-vanadium phosphate membrane of approximate thicknesses 0.095, 0.090, 0.085, 0.080 and 0.075 cm were obtained, respectively. The membranes prepared by embedding 25% of polystyrene by weight were suitable, and the greater or lesser than this weight did not show reproducible results and appeared to be unstable. Membranes prepared in this way were stable and further subjected to microscopic and electrochemical examinations for cracks and homogeneity of the surface. | 3 |
99953786 | Scanning electron microscopy (SEM) The prepared samples at various pressures was heated in the tabular furnace for 3 hours and then cooled. A very thin transparent polymer glue tape was applied on the sample and then placed on an aluminum stub of 15 mm diameter. Thereafter, the sample was kept in a chamber at a very low pressure where the entire plastic foil containing the sample was coated with gold (60 µm thickness) for 5 minutes. The scanning electron micrograph of gold coated specimen was recorded, operating at an accelerating voltage of 10 kV using the scanning electron microscope (GEOL JSM-840). | 4 |
99953786 | Measurement of membrane potential The freshly prepared charged membrane was installed at the center of the measuring cell, which had two glass containers, one on either side of the membrane. Both collared glass containers are having a hole for introducing the electrolyte solution and Saturated Calomel Electrodes (SCEs). The half-cell contained 40 ml of the electrolyte solutions. Electrochemical cells of the type C 1 SCE Solution and C 2 Membrane Solution SCE were used for measuring membrane potential using Osaw Vernier Potentiometer. In all measurements, the electrolyte concentration ratio across the membrane was taken as C 2 /C 1 =10. All solutions were prepared by using Analytical Reagent (AR) grade chemicals and ultra-pure distilled water. The electrodes used were saturated calomel electrode and were connected to a galvanometer. The solutions in both containers were stirred by a magnetic stirrer to minimize the effects of boundary layers on the membrane potential. The pressure and temperature were kept constant throughout the experiment and the potentials were measured at 25°C. | 5 |
99953786 | Results and Discussion The composite membranes using polystyrene as a binder were prepared by sol-gel process. The membranes were found to have the following properties: • They were thermally stable up to 500°C. • They were resistant to compaction. • They were inert to harsh chemical (K 2 Cr 2 O 7 , H 2 O 2 , HNO 3 , H 2 SO 4 , etc.) as they did not decompose in their presence. | 6 |
99953786 | • They did not show any swelling. • They were stable after long usage, i.e., they were durable. The characterization of membrane morphology has been studied by using SEM [33]. The information obtained from SEM images have provided guidance in the preparation of well-ordered precipitates, composite pore structure, micro/macro porosity, homogeneity, thickness, surface texture and crack-free membranes [34]. The SEM surface images of the composite membranes were taken at different applied pressure and are presented in Figure 1. Inorganic composite membranes have the ability to generate potential when two electrolyte solutions of unequal concentration are separated by a membrane and driven by different chemical potential acting across the membrane [35]. The electrical character of the membrane regulates the migration of charged species, and diffusion of electrolytes from higher to lower concentration takes place through the charged membrane. The values of membrane potential m Ψ ∆ measured across membranes in contact with various 1:1 electrolytes (KCl, NaCl and LiCl) were dependent on concentration of electrolytes present on both sides of the membrane at 25 ± 1°C are given in Table 1. The observed potential was low (mV, +ve). It was found to increase on decreasing the concentration of electrolytes (KCl, NaCl and LiCl), which is a usual behavior of inorganic membranes. The selectivity character of ion-exchange membranes were reported on the basis of membrane potential values, performed on uni-uni and multi-uni valents electrolytes as 1:1, 2:1 and 3:1. The reversal in sign from positive to negative values of membrane potential occurred with the 2:1 and 3:1 electrolytes. This is evidently due to the adsorption of multivalent ions, which led to a state where the net positive charge left on the membrane surface made the anion selective with 2:1 or 3:1 electrolytes. The membrane potential was also seen to be largely dependent on the pressure applied during the membrane fabrication. Application of higher pressure at composite membranes led to reduction in their thicknesses, contraction in pore volume and consequently offered a progressively higher fixed-charge density [36]. The charge property of the membrane matrix greatly influences the counter-ion than co-ion as well as the transport phenomena in the solutions. The surface charge concept of the TMS model for charged membrane is an appropriate starting point for the investigations of actual mechanisms of ionic or molecular processes which occur in membrane phase [27][28][29][30][31]. The TMS model assumes uniform distribution of surface charge and consists of Donnan potential and diffusion potential. According to the TMS, the membrane potential m Ψ ∆ is applicable to an idealized system and is given by Where v and v are the ionic mobilities (m 2 /V/s), of cation and anion, respectively, in the membrane phase. The charge densities of inorganic membranes were estimated from the membrane potential measurement and can also be estimated from the transport number. From the plots in Figure 2, the charge density parameters can be evaluated for a membrane carrying various charge densities, D ≤ 1 for different 1:1 electrolytes systems. The theoretical and observed potentials were plotted as a function of -logC 2 as shown in Figure 2. Thus, the coinciding curve for various electrolytes system gave the value for the charge density D within the membrane phase. Therefore, the increase in the values of D with higher applied pressure is due to successive increase of charge per unit volume as well as the modification in the surface microstructure of the membrane. The plot of charge density D of the membrane for 1:1 electrolytes (KCl, NaCl and LiCl) versus pressures is shown in Figure 3. The order of charge density of various electrolytes is found to be KCl>NaCl>LiCl throughout the range of applied pressure at which the membranes were prepared. The surface charge model may work as a tool to improve the performance of the membrane filtration process. Since, the charge density is an important parameter governing transport phenomena and the charge property of the membrane dominates the electrostatics interaction between the membrane and particles in the feed solution due to the prefential adsorption of some ions. Therefore, by controlling the solution physico-chemistry, the optimum charge property of the membrane can be obtained as desired. The TMS equation (1) The R, T and F have their usual significance; ± ′ ′ γ and ± ′ ′ γ are the mean ionic activity coefficients; v u = ω is the mobility ratio of the cation to the anion in the membrane phase and + 2 C and + 2 C are the cation concentrations in the membrane phase first and second, respectively. The cation concentration is given by the equation Here V k and V x refer to the valency of cation and fixed-charge group on the membrane matrix, q is the charge effectiveness of the membrane and is defined by the equation Where K ± is the distribution coefficient. It is expressed as Where i C the i th ion concentration in the membrane is phase and C i is the i th ion concentration of the external solution. The transport properties of the membrane in various electrolyte solutions are important parameters to further investigate the membrane phenomena as shown in Eq. (7) Equation (7) was first used to calculate the values of transport numbers t + , mobility ratio v u = ω and finally Ū as given in Table 2. The values of mobility ω of the electrolytes in the membrane phase were found to be high at lower concentration of all the electrolytes (KCl, NaCl and LiCl). Further increase in concentration of the electrolytes led to a sharp drop in the values of ω as given in Table 2. The high mobility is attributed to higher transport number of comparatively free cations of electrolytes and also be similar trend as the mobility in least concentrated solution. The values of the parameters K + , q and + C derived for the system have also been included in Table 2. Using Eq. (6) it was found that the values of distribution coefficients increased at lower concentration of electrolytes. As the concentration of electrolytes increased, the values of distribution coefficients sharply dropped and, thereafter, a stable trend was observed as shown in Figure 4. The large deviation in the value of K ± at the lower concentration of electrolytes was attributed to the high mobility of comparatively free charges of the strong electrolyte and thus, reached into the membrane phase easily compared to higher concentrated electrolytes solution. In order to interpret the variation of the charge effectiveness depending on those values, that the ion-pairing effect causes the difference between the effective charge density and the fixed-charge density in membrane phase. In our membrane, counter ion Cl − is same for 1-1 electrolytes therefore, the variation in the q values are follow the similar trend and the order is LiCl>NaCl>KCl up to the C 2 =0.01 mol/l and then drop in the q values were analyzed from Figure 5. When, the external electrolyte concentration is higher or lower, a number of counter ions go into the membrane due to imbalance in the counter ion concentration of external electrolyte and fixed charged group in the membrane phase. Therefore, the ion association with the fixed charged group and counter ions in the membrane is enhanced as a result the charge effectiveness has a lower value whereas in the moderate concentration region the counter ion concentration in the external electrolyte and the fixed-charge density in the membrane are comparable. Therefore, a less number of ion pair formation and consequently higher values of the charged effectiveness, the optimum value of charge effectiveness are obtained at C 2 =0.01 mol/l and then decreased steeply. The order of the charge effectiveness of 1-1 electrolytes may depend on increasing ionic charge density of co-ion adsorption on the charged membranes. The membrane potential derived in this way (theoretical) and the experimentally obtained membrane potentials at different concentrations for various electrolytes systems have been compared and provided in Figure 6. It may be noted that the experimental data follow the theoretical curve quite well. However, some deviations may be due to various non ideal effects, such as swelling effect and osmotic effects. These effects are often simultaneously present in the charged membranes. | 7 |
236570447 | Purpose: To investigate the effects of (-)-epigallocatechin gallate (EGCG) and quercetin on the activity and structure of α-amylase. Methods: The inhibitory effects of 7 functional factors were compared by measuring half maximal inhibitory concentration (IC50) values. Lineweaver-Burk plots were used to determine the type of inhibition exerted by EGCG and quercetin against α-amylase. The effect of EGCG and quercetin on the conformation of α-amylase was investigated using fluorescence spectroscopy. Results: Quercetin and EGCG inhibited α-amylase with IC50 values of 1.36 and 0.31 mg/mL, respectively, which were much lower than the IC50 values of the other compounds (puerarin, paeonol, konjac glucomannan and polygonatum odoratum polysaccharide). The Lineweaver−Burk plots indicated that EGCG and quercetin inhibited α-amylase competitively, with ki values of 0.23 and 1.28 mg/mL, respectively. Fluorescence spectroscopy revealed that treatment with EGCG and quercetin led to formation of a loosely-structured hydrophobic hydration layer. Conclusion: This study has unraveled the mechanism underlying the inhibition of α-amylase activity by EGCG and quercetin in vitro. This should make for better understanding of the mechanisms that underlie the antidiabetic effects of EGCG and quercetin in vivo. | 1 |
236570447 | INTRODUCTION Diabetes mellitus is a chronic metabolic disorder characterized by high level of fasting blood glucose. One therapeutic approach for diabetes is to decrease postprandial hyperglycemia by the inhibition of carbohydrate-hydrolyzing enzymes such as α-amylase and α-glucosidase [1]. α-Amylase (α-1,4-glucan-4-glucanohydrolase) catalyzes the hydrolysis of internal α-1,4-glucosidic linkage in starch, releasing glucose, maltose and maltotriose [2]. The control of carbohydrate digestion and monosaccharide absorption is beneficial for avoiding complications of diabetes. Acarbose, a fermentation product of actinoplanes species, has been shown to inhibit α-amylase competitively [3]. Studies have been carried out to identify inhibitors of α-amylase from natural sources so as to develop physiologically functional foods for treating diabetes [4,5]. Studies have shown that tea polyphenols and flavonoids effectively inhibit the activity of αamylase [6,7]. Tea catechins include EGCG, (-)epigallocatechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epicatechin (EC). In recent studies, it was shown that EGCG treatment ameliorated free fatty acid-induced peripheral insulin resistance through decrease in oxidative stress, activation of the AMPK pathway and improvement of the insulin signaling pathway in vivo [8]. Although the prevention and treatment of type 2 diabetes mellitus have been investigated using EGCG supplementation [9], the effect of EGCG on the secondary and tertiary structures of α-amylase have not been investigated. Based on previous reports, dietary polyphenols have considerable potential for reducing the risk of diabetes. Epidemiological studies have also shown that the intake of certain types of flavonoids, including quercetin and myricetin is inversely associated with the risk of type 2 diabetes [10]. Flavonoids are beneficial for reducing the risk of metabolic syndrome. In addition to their antioxidant effects, flavonoids have been reported to prevent diabetes in vivo [11]. Studies on the inhibitory effects of isolated flavonoid compounds against α-glucosidase and α-amylase revealed that quercetin inhibited αamylase with IC 50 of 4.8mM [6,7]. However, the effect of quercetin on α-amylase conformation has not been demonstrated. The objectives of the present study were to evaluate in vitro pancreatic α-amylase-inhibitory activities of 7 functional factors, and the mechanism underlying the inhibition of αamylase by EGCG and quercetin. Furthermore, fluorescence measurements were applied to analyze changes in the tertiary structure of αamylase due to interaction of the enzyme with EGCG and quercetin. EXPERIMENTAL Materials α-Amylase was purchased from Sigma Aldrich (St. Louis, MO, USA). (-)-Epigallocatechin gallate (EGCG), quercetin, puerarin, paeonol, sulfated konjac glucomannan (SKGM) and Polygonatum odoratum polysaccharide (PoPs) (> 98 % purity) were purchased from Jingzhu Biotechnology Co. Ltd (Nanjing, China). Enzymatic assays were carried out using a UNIC-2100 visible spectrum. | 2 |
236570447 | α-Amylase inhibition assay The inhibition of α-amylase was assayed according to the procedure of Song Liu [8]. Sample solution (50 µL) and 50 µL of 20 mM phosphate buffer (pH 6.9) containing 0.006 M sodium chloride and α-amylase solution (15 u/mL) were incubated at 37 °C for 10 min. The reaction was initiated by adding 600 µL of 1.5 % starch solution in 0.02 M sodium phosphate buffer, pH 6.9, and the mixture was incubated for 5 min at 37 °C, followed by the addition of 1 mL dinitrosalicylic acid. The reaction mixture was then placed in a boiling water bath for 5 min, and thereafter cooled to room temperature. The absorbance was measured at 540 nm in a UVvisible spectrophotometer (Shimadzu UV-1700, Japan). Acarbose was used as a positive control. Inhibition was calculated using Eq 1. (1) where Abs1 and Abs2 represent absorbance at 540 nm without and with inhibitor, respectively. | 3 |
236570447 | Determination of inhibition mechanism and V max and K m values The mechanisms of the inhibitory effect of EGCG and quercetin against α-amylase, and values of maximum velocity (V max ) and Michaelis constant (K m ) were determined using the Lineweaver-Burk plot [11]. Substrate solutions at concentrations of 6.0, 8.0, 10.0, 12.0, 14.0, and 16.0 mg/mL were reacted with α-amylase, with or without inhibitor. The concentrations of α-amylase and inhibitor were 0.4 and 0.2 mg/mL, respectively, while distilled water was used as control. The V max and K m values were obtained from the least-squares regression lines of the double reciprocal plots of the tested sample (inhibitor) concentration (1/[S]) against the reciprocal of reaction rate (1/v). Half -maximal inhibitory concentration (IC 50 ) was calculated from inhibition curve. V max and K m values were obtained from the least-squares regression lines of the double reciprocal plots of the tested sample (inhibitor) concentration versus the reciprocal of reaction rate. | 4 |
236570447 | Fluorescence measurements All fluorescent spectra measurements on the potential interaction between α-amylase, EGCG and quercetin were carried out on an F-7000 fluorescence spectrophotometer (HITACHI, F-7000, Japan). To each of a series of 5-mL test tubes was successively added 0.3 mL buffer solution (pH 7.4), 0.2 mL α-amylase (1 mg/mL), and varying amounts of EGCG and quercetin. After equilibration for 5 min, fluorescence spectra were measured at excitation wavelength of 280 nm, and emission wavelengths of 300 -480 nm. The slit width was set at 3 nm, and the scan speed was 12000 nm/min. | 5 |
236570447 | The inhibitory effects of seven functional factors on α-amylase activities In this study, the inhibitory effects of seven functional factors against α-amylase were evaluated, with acarbose as control. As shown in Figure 1, the IC 50 values for α-amylase inhibition by EGCG, quercetin and acarbose (as the positive control) were 0.31, 1.36, 0.45 mg/mL, respectively. The IC 50 value of EGCG (0.31mg/mL) was much lower than that of acarbose (0.45 mg/mL), indicating that EGCG strongly suppressed α-amylase activity, indicating that it could possibly be utilized for controlling postprandial hyperglycemia. Quercetin (IC 50 =1.36 mg/mL) had a stronger inhibitory effect on α-amylase activity than puerarin, paeonol, SKGM, and PoPs. It has been reported that quercetin significantly and dose-dependently decreased plasma glucose level of streptozotocin-induced diabetic rats [12]. In this study, quercetin inhibited αamylase activity in a dose-dependent manner, indicating that quercetin inhibition may effectively reduce plasma glucose level. Puerarin and paeonol showed weaker α-amylase inhibitory activities, while PoPS and SKGM had little inhibitory activities against α-amylase. | 7 |
236570447 | Determination of inhibition types and V max and K m Values To investigate the inhibition characteristics of EGCG and quercetin against α-amylase, the kinetics of α-amylase reaction was investigated at different substrate concentrations. The Lineweaver -Burk plots for EGCG (Figure 2 A) and quercetin (Figure 2 B) showed the same intersection on Y-axis, indicating that the mode of inhibition of α-amylase by EGCG and quercetin was competitive. As the dose of EGCG increased in Figure 2 A, the K m value for α-amylase increased, while the value of V max remained unchanged. Such results are consistent with competitive inhibition characteristics. The K i values for EGCG and quercetin were 0.23 and 1.28 mg/mL, respectively. The smaller the K i , the higher the affinity of the inhibitor for α-amylase and the higher is the inhibition. It appears therefore that the inhibition of starch hydrolysis was significantly higher with EGCG than with quercetin. | 8 |
236570447 | Effects of EGCG and quercetin on the tertiary structure of α-amylase To monitor the changes in the microenvironment of aromatic amino acid residues of α-amylase in response to EGCG and quercetin treatment, intrinsic fluorescence spectra of the enzyme were recorded in the range of 300 -500 nm. As shown in Figure 3, the relative fluorescence quantum yields of EGCG-and quercetin-treated α-amylase exhibited obvious decreases. A blue shift in the maximum peak wavelength was observed with increasing concentrations of EGCG and quercetin. The intrinsic fluorescence of α-amylase was quenched by EGCG and quercetin. Compared to quercetin, the addition of increasing concentrations of EGCG caused more progressive reductions in fluorescence intensity. The reduction in fluorescence intensity indicated that EGCG and quercetin treatment induced disruption of hydrophobic bonds, thereby exposing the nonpolar amino acid residues (e.g., tryptophan) to a more polar environment. It also caused the formation of a loosely structured hydrophobic hydration layer, and the fluorescence was quenched by that environment. | 9 |
236570447 | DISCUSSION It has been suggested that the inhibition of αamylase and other carbohydrate-hydrolyzing enzymes is a potential way of controlling postprandial blood glucose levels. Thus, the search for effective and non-toxic inhibitors of αamylase has important significance for the prevention and treatment of diabetes. Radovanović has assessed the antioxidant and antimicrobial activities of polyphenolic extracts of three wild berry fruit species from Southeast Serbia [13]. The anti-glycemic and hypolipidemic potential of polyphenols from Zingiber officinale in streptozotocin-induced diabetic rats have been reported [14]. Previous studies have shown that polyphenols and flavonoids inhibit or activate enzymes in vitro [15]. In a study by Kalita et al, it was reported that potato polyphenolic compounds inhibited pancreatic α-amylase in vitro [16]. Radovanović have assessed the antioxidant and antimicrobial activities of polyphenolic extracts of three wild berry fruit species from Southeast Serbia [13]. The antiglycation and hypolipidemic potential of polyphenols from Zingiber officinale in streptozotocin-induced diabetic rats have been verified [14]. Previous research have shown that polyphenol and flavonoids have the ability to inhibit or activate enzymes in vitro [15]; Kalita discovered that potato polyphenolic compounds have the ability to inhibit pancreatic α-amylase in vitro [16]. Recent findings showed that Qingzhuan tea extracts exerted potent inhibitory effects on αamylase [17]. In addition, tea polyphenols composed of EGCG, EGC, ECG and EC inhibited α-amylase with an IC 50 of 0.41 mg/mL. In the study, EGCG which appeared to be one of the main components of tea polyphenols, exhibited the most effective inhibition of αamylase, with IC 50 value of 0.31 mg/mL. It has been reported that quercetin significantly and dose-dependently decreased the plasma glucose level of streptozotocin-induced diabetic rats [12]. In this study, the inhibition of α-amylase by quercetin was dose-dependent, with IC 50 value 1.36 mg/mL, indicating that the inhibition may be an effective approach towards decreasing plasma glucose level. Overall, the findings suggest that EGCG and quercetin may limit the release of simple sugars from the gut, thereby alleviating postprandial hyperglycemia. The fluorescence spectrum was associated with polarity of the environment of the tryptophan and tyrosine residues. The decreases in fluorescence quantum yield may be due to the interaction of chromophores with quenching agents. Changes in intrinsic fluorescence emission have been attributed to the changes in protein tertiary structure [18]. Molecular interactions between pancreatic lipase and EGCG have been studied [19]. It has been shown that the α-helix content of pancreatic lipase secondary structure decreased as a function of EGCG concentration, and that static fluorescence quenching occurred as a result of EGCG treatment [20]. Tryptophan fluorescence is considered a very reliable index of conformational changes in proteins [21]. Thus, it was used to investigate the effect of EGCG and quercetin on the tertiary structure of α-amylase in this study. The fluorescence intensity of α-amylase decreased with increasing concentrations of EGCG and quercetin. This implies that the binding of EGCG and quercetin to α-amylase caused microenvironment changes in α-amylase. Inhibitors of α-amylase may directly interact with the side chains of Asp197, Glu233, and Asp300: substitution of these residues lead to a considerable drop in catalytic activity of the enzyme [22]. The inhibitory activity of EGCG on α-amylase led to the formation of soluble or insoluble complexes. The hydrogen bonds between the hydroxyl groups of EGCG and the catalytic residues of the binding site stabilized the interaction with active site [23]. Some researchers have used molecular docking to study the structure-activity relationship in the binding of flavonols to α-amylase and the possible mechanisms involved. Molecular modeling studies revealed that salivary αamylase inhibitors occupied a docking mode that allowed for H-bonds between the enzyme Asp197 side chain carboxyl oxygen atom and the hydroxyl groups in ring B of the flavonoid skeleton [24]. Thus, the hydrogen bond formed between the quercetin hydroxyl groups and the binding site of the catalytic residue accounts for the inhibition of α-amylase by quercetin. | 10 |
86848539 | This study includes the photocatalytic degradation of Rhodamine B (Rh.B) employing a heterogeneous photocatalytic process by using ZnO nanoparticles that was prepared by green sol-gel process. The structural, morphological, and its optical properties of ZnO Photocatalyst was studied using different characterization techniques such as Xray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), The influencing factors studied were the amount of the catalyst, the concentration of dye and pH on photocatalytic degradation of Rhodamine B. The experiments were carried out by irradiating the aqueous solutions of dyes containing photocatalysts with Sunlight. The rate of decolorization was estimated from residual concentration spectrophotometrically. Similar experiments were carried out by varying pH (3–11), amount of catalyst (0.25–2.0 g/L) and initial concentration of dye (5–50 mg/L). The experimental results indicated that the maximum degradation (71%) of dyes was achieved using ZnO photocatalyst at pH 10 after 240 min. | 1 |
86848539 | INTRODUCTION Pollution problem is getting worse day by day, so Researchers are looking for ways to get rid of this problem.Among the most common contaminants are dissolved dyes in industrial wastewater from textile [1] and paper mills [2].Synthetic dyes are extensively used for dyeing and printing in textile industries.Over 10,000 dyes with an once a year production over 10 5 metric tons worldwide are commercially available and about 50% among them are azo dyes [3].It is estimated that approximately 15% of the dyestuffs are lost in the industrial effluents during manufacturing and processing operations [4].Color is usually the first contaminant to be recognized in wastewater.Many dyes may be decomposed into potential oncogenic amines under anaerobic conditions in the environment [5].There are many ways for pollutant elimination such as adsorption on activated carbon [6], reverse osmosis [7], ultrafiltration [8], and ozonation [9] etc. Photodegradation is also one of the most important technologies used in the disposal of pollutants in industrial wastewater.Researchers want to use natural resources available to obtain the energy needed for the degradation of dyes in industrial wastewater, the most important source of natural energy is sunlight that consists of about 5-7% UV light, 46% visible light and 47% infrared radiation [10].Photocatalytic oxidation of various harmful organic dyes in industrial wastewater has been carried over ZnO semiconductor oxides under UV light irradiation [11].Research is now focused on to achieve high photocatalytic efficiency with ZnO [12] especially with sunlight.Zinc oxide can be prepared in more than one way, each method has its own conditions that determine product characteristics, preferably use environmentally friendly methods.Green chemistry is generally accepted as "the design, development, and implementation of chemical processes and products to reduce or eliminate substances hazardous to human health and the environment" [13].There has been an explosive growth in the field of green chemistry both in preparing green Nanocatalysts [14] as well as green conditions during catalysis of industrially important reactions.Preparing green Nanocatalysts refers to manufacturing the nanocatalysts using green solvents or processing the nanocatalysts so that they are finally Al-Bedairy & Alshamsi / Environmentally Friendly Preparation of Zinc Oxide, Study Catalytic Performance … 2 / 9 dispersed in green solvents.Green nanocatalysis refers to doing the catalytic reaction in green solvents and rather by the use of green nanocatalysts for these reactions [15].According to the fourth principle of Anastas' and Warner's 12 principles of green chemistry "Chemical products should be designed to preserve efficacy of function while reducing toxicity," [16], should of course also be applied to the synthesis of Nanocatalysts [17].This is typified by the synthesis of nontoxic ZnO nanoparticle catalysts [18,19]. | 2 |
86848539 | Instruments UV-vis spectrophotometer double beam PC 1650 SHIMADZU, UV-vis spectrophotometer 780 Sunny China, The crystalline character of the solid has been identified by X-ray diffraction (XRD) analysis using a D/Max 2,550 V diffractometer with Cu Kα radiation (λ = 1.54056Å) ( Japan), and the XRD data were collected at a scanning rate of 0.03 s -1 for 2θ in a range from 10° to 80°, The morphology of prepared materials was noted by field emission scanning electron microscopy (FE-SEM ) with (MIRA3 TESCAN -Czech), The optical band gap Eg was projected from the UV-Vis-NIR diffuse reflectance spectroscopic (UV-Vis-NIR DRS) determined in a wavelength range from (200 -1100) nm with UV-1800 UV-VIS Spectrophotometer from SHIMADZU, pH meter (Sartorius, Germany), 100 mL Teflon-lined autoclave. | 4 |
86848539 | Preparation of ZnO In a typical synthesis, of ZnO nanoparticles is carried out by sol-gel process, at 80-90C o .Solution of zinc acetate Zn(CH3COO)2 was prepared by dissolving 2.195 g of zinc acetate in 100ml distilled water, and stirred in ambient atmosphere.Potassium hydroxide KOH 1.122g is dissolved in 10 ml distilled water and was added to the above solution drop wise under continuous stirring.After few minutes solution turn into jelly form and a milky white solution was obtained, the mixture was then further heated for 3 h at 80-90C o without stirring.The resulting suspension was centrifuged to retrieve the product, and the mixture was washed with distilled water and then the powder was dried at 70 C o overnight and determined in terms of their structural, morphology and optical properties [20,21]. | 5 |
86848539 | Photo-catalytic Degradation Experiment ZnO was added to 100 ml of Rhodamine-B dye solution and it is undergo irradiation by sunlight.After adding the catalyst and stirrer it at a constant speed.The samples were taken at different times.Before the irradiation, the dye catalyst suspension was kept in the dark with steering for 90 min to ensure an adsorption-desorption equilibrium.The solution was separated from the catalyst by the centrifuge.Measure the absorbance of each sample, absorption spectra were recorded and rate of decolorization was seen as far as change in power at λmax of the colors.The decolonization efficiency has been ascertained as equation 1: Where Co is the initial concentration of dye and C is the concentration of dye after photo irradiation.Similar experiments were carried out by varying the pH of the solution (pH 3-11), concentration of dye (10 -100 mg/L) and catalyst loading (0.25-2.0 g/L), pH of aqueous solution was adjusted with 0.1M H2SO4 or 0.1M NaOH. | 6 |
86848539 | UV-vis Diffuse Reflectance Spectra (DRS) The properties of semiconductor nanoparticles are strongly size dependent.It is well known that the nano-scale systems show interesting properties, for example, increasing of the semiconductor band gap due to electron confinement [24].The UV-vis diffuse reflectance, (Tauc's plot) of synthesized ZnO was shown in Figure 3, the calculated band gap energy for synthesized ZnO is 3.39 eV, the determination of optical band gap is obtained by Tauc's equation [25]. | 8 |
86848539 | Adsorption of the dyes on ZnO-photocatalyst Degradation of the dyes occurs predominantly on the photocatalyst surface [26].In order to investigate the adsorption behavior of Rhodamine B, the suspension was prepared by mixing 100 ml of dye solutions 20 mg/L with fixed photocatalyst ZnO amount (1 g/L) at 35 C°, natural pH of Rhodamine B. The suspensions were kept for different times in the dark under shaking for 120 min.The absorbance measured at the max553 nm to determine the concentration of dyes.The experimental results are shown in Figure 4 from the results, it was noticed that the adsorption equilibrium under 20 mg/L initial concentration was reached at about 60 min of equilibration time. | 9 |
86848539 | Photocatalyst loading The experiments were carried out by varying ZnO photocatalyst amount from 0.25 to 2.0 g/L for dye solutions of 20 mg/L at natural pH of Rhodamine B. The decolorization efficiency for various photocatalysts amount for Rhodamine B has been depicted in Figure 5.It is observed that rate increases with increase in catalyst amount and becomes constant above a certain level then will be decreased.The optimum photocatalyst amount for Eurasian J Anal Chem 5 / 9 decolorization efficiency of dye is 0.8 g/L.The reasons for this decrease in decolorization efficiency at given time it aggregation of catalyst particles at high concentrations causing a decrease in the number of surface active sites [27,28] and increase in light scattering of catalyst particles to decrease in the passage of irradiation through the sample [29,30]. | 10 |
86848539 | Effect of pH Wastewater containing dyes at different pH therefore it is important to study the effect of pH on degradation efficiency of dye [29], the amphoteric behavior of most semiconductor oxides influences the surface charge of the photocatalyst [31].The effect of pH values on the degradation efficiency is studied in the pH range 3-11 at the dye concentration 20 mg/L and catalyst amount 0.8 g/L, 35C°.Figure 6 illustrated the results of the decolorization efficiency after 160 min irradiation with different pH values (3, 5, 7, 9 and 11) It can be observe increase in the decolorization efficiency of Rhodamine B with increase of the pH value from 3 up to 11, exhibiting maximum efficiency at pH 10, This behavior could be explained by pHpzc of the material, as well as the molecular nature of the dyes, the zero point charge of ZnO equal to 9.0, Therefore photocatalysts surface is positively charged below pHzpc, whereas it is negatively charged when pH > pHzp. | 11 |
86848539 | Effect of initial dye concentration The results reported that the initial dye concentration effects the degradation efficiency strictly.With the increase of initial dye concentration, the degradation efficiency decreases remarkably [27].The negative effects of the initial dye concentration are attributed to the competency between dye and OH − ion adsorption on the surface of catalyst.The adsorption of dye reduces the OH − ion adsorption, which results in the reduction on the formation of hydroxyl radicals [32].The rate of degradation relates to formation of OH radicals, which is the critical species in the degradation process.At the same time, as the initial dye concentration increases, the path length of photons entering the solution decreases [33].Hence in the solution with constant catalyst concentration, the formation of hydroxyl radicals that can attack the pollutants decreases, thus leading to the lower decolorization efficiency.Figure 7 shows the effect of initial dye concentration on degradation efficiency by varying the initial concentration from 5 to 50 mg/L with the constant ZnO catalyst loading (0.8 g/L) and pH 10. | 12 |
86848539 | Kinetic of photodegradation Figure 8 shows a typical UV-Vis spectrum of Rhodamine B solution during photo-irradiation time (dye conc.10 ppm, pH10, catalyst amount 0.8 g/L, T= 308K°).The absorption peaks, corresponding to dye, diminished and finally disappeared under reaction which indicated that the dye had been degraded.No new absorption bands appear in the visible region.The spectrum of Rhodamine B in the visible region exhibits a main band with a maximum at 553 nm. Figure 9 shows the kinetics of disappearance of Rhodamine B for an initial concentration of 20 ppm at pH10, catalyst amount 0.8 g/L.The results show that the photodegradation of the dye in aqueous ZnO can be described by the first order kinetic model, ln(C0/C) = kt, where C0 is the initial concentration and C is the concentration at any time, t.The semi logarithmic plots of the concentration data give a straight line. | 13 |
55193349 | Corresponding Author: Yuanda Song Colin Ratledge Center for Microbial Lipids, School of Agriculture Engineering and Food Science, Shandong University of Technology, Zibo, Shandong, China Email: ysong@sdut.edu.cn Abstract: In this paper, the optimum extraction conditions of total flavonoids extracted from walnut leaves subjected to Ultrasonic Assisted Extraction (UAE) were optimized by Response Surface Methodology (RSM). The mathematical model showed the high coefficient of measurement (R = 0.9938) which indicated that this model could be used to guide the response surface methodology. The optimum extraction parameters for extracting flavonoids from walnut leaves determined in this study were extraction temperature 47.73°C, extraction time 30.79 min, ethanol concentration 72.89% (v/v). Under the optimal extraction conditions, the flavonoids yield was about 3.5315%. Statistical analysis of the results showed that extraction temperature, extraction time and ethanol concentration significantly affected the extraction yield of total flavonoids. In addition, the antibacterial activity assays of the flavonoids were carried out and it was demonstrated that the total flavonoids extracted at the optimum conditions had pronounced antibacterial effects against the four bacterial species. Therefore, this study suggested that walnut leaves are promising resources with antibacterial properties for the development of phytomedicines. | 1 |
55193349 | Introduction Walnut (Juglans regia L.), which belongs to the Juglandaceae family, is a local deciduous tree in northwestern Chain (2012;Moser, 2012). Walnut leaves are known to possess many biological properties and are easily available in abundant amounts (Derebecka et al., 2012). They have been used as a traditional medicine in China and have shown various health benefits for the treatment of skin inflammations, venous insufficiency and ulcers (Cheniany et al., 2013). Moreover, researches in pharmacology and therapeutics have shown that walnut leaves have hypoglycemic, antioxidative, antimicrobial and antihypertensive effects (Gîrzu et al., 1988). In recent years, there has been an in-depth study on substances having considerable antimicrobial properties. It is well known that certain chemicals produced by plants are naturally toxic to bacteria and fungi. Various medicinal plant extracts containing flavonoids are reported to have antimicrobial activity (Basile et al., 1999). Walnut leaves are good sources of flavonoids (Zhao et al., 2014). As natural products, flavonoids exert an extensive biochemical and pharmacological properties. They are described as dietary supplements that promote health, prevention of disease and active cancer preventive agents (Duarte et al., 1993;Hodek et al., 2002). Flavonoids are present in photosynthetic cells and are therefore widespread in the plant kingdom (Manthey et al., 2001). They are common ingredient in the human diet and are found in vegetables and fruits (Xie et al., 2007;Harborne and Baxter, 1999). Flavonoids have been shown to possess a series of important biological activities, including antifungal and antibacterial activities (Galeotti et al., 2008;Kabir et al., 2015;Alarcón et al., 2008). Flavonoids compounds can form complexes with soluble proteins and extracellular matrix and bacterial cell walls, which probably lead to their antibacterial activities (Cushnie and Lamb, 2005). Presence of flavonoids in plants might have some or significant contribution to the antimicrobial activity of plants. Up-to now, several traditional extraction methods have been applied to the extraction of flavonoids from walnut leaves such as Maceration Extraction (ME) (Djozan and Assadi, 1995), Heat Reflux Extraction (HRE) (Zhang and Liu, 2004), soxhlet extraction (Shang and Yuan, 2003) and Microwave-Assisted Extraction (MAE) (Xia et al., 2006). This extraction process usually takes several hours or even days and requires a large amount of solvents, which may result in the damage of flavonoids due to hydrolysis and oxidation (Camel, 2000). Ultrasonic Assisted Extraction (UAE) method can extract bioactive molecules at lower temperature, shorter time and also can relatively reduces the structural damage of compounds in plants than using other traditional extraction (Yuan et al., 2015). Response Surface Methodology (RSM) is a collection of improvement method of optimization mathematical and statistical process (Talebpour et al., 2009). This is a useful tool for studying the mutual effect between various factors on their measurement and quantification of the influence of reaction parameters (Al-Matani et al., 2015;Teng and Choi, 2014). Box-Behnken Design (BBD) is a commonly used process of RSM which make it easier to arrange the experiments result (Borges et al., 2009). Therefore, BBD technology was employed to analyze the influence of various process variables, including extraction temperature, extraction time and ethanol concentration on the yield of the flavonoids extracted from walnut leaves. In the current study, UAE was used to extract flavonoids from walnut leaves using one factor and RSM experimental design to optimize extraction conditions. Furthermore, the antibacterial effects of the total flavonoids extracted at the optimum conditions were determined by using the diffusion methods of agar well. The purpose of this study was to determine the best extraction process parameters for flavonoids extraction from walnut leaves by ultrasonic assisted method and to explore its potential antibacterial properties, so as to establish a scientific basis for the development and utilization of flavonoids. | 2 |
55193349 | The Total Flavonoids Extraction from Walnut Leaves The powders of walnut leaves (1g) were placed in 50 mL −1 centrifuge tubes and mixed with ethanol. After ultrasonic extraction, the samples were centrifuged at 5000 rpm and the supernatant was collected 15 min later. The residue continued to be extracted twice according to the above mentioned conditions, then all supernatants were mixed up and concentrated by a rotary evaporator, then, flavonoids were separated and purified used large hole resin, finally, the collected fractions were freeze dried to powder. | 5 |
55193349 | Determination of the Content of Total Flavonoids The total flavonoids content in the extracted solution was measured by an aluminum-chloride-colorimetric method (Qadir et al., 2015). In brief, the Rutin standard and the extracted solution with different concentrations were appropriately diluted by 30% ethanol to 5 mL and added 0.3 mL of 5% sodium nitrite solution, placed 6 min then added 0.3 ml of 10% alchlor solution, placed 6 min, then added 4 mL of 5% sodium hydroxide solution. Finally, adjusted the volume of the mixture to 10 mL by 30% ethanol and placed for 15 min. The absorbance of the mixture was measured at 510 nm and distilled water was used as a blank control. The reference standard was Rutin, while the contents of total flavonoids in extracts were presented as Rutin equivalents. All determinations were performed in triplicate. In this work, the total flavonoids of the total extract obtained from the walnut leaves were calculated from the equation of the standard plots ( Fig. 1) as follows: Absorbance = 8.66×total flavonoids + 0.0004 (R 2 = 0.9997) | 6 |
55193349 | Single Factor Experiments Total flavonoids extraction yield of walnut leaves was influenced by many factors. Therefore, choosing appropriate extraction solvent and extraction method is an important consideration. Based on the preliminary experiments results ethanol and UAE were selected as reasonable options. The experiment used ethanol as the extraction solvent and UAE as extraction method, respectively. The maximum total flavonoids content were determined by single factor experiments. Before RSM analysis, an initial experiment was performed to screen for important factors affecting the experimental responses. | 7 |
55193349 | Box-Behnken Design (BBD) Optimized UAE Conditions Box-Behnken Design (BBD) is a frequently used method of RSM that is composed of several intermediate points and a central point (Saniah and Samsiah, 2012). BBD was employed to design the experiments, optimize the extraction conditions and analyze the interactions between the above-mentioned parameters. In the present study, three main factors to RSM were used to describe the relationship between responses and variables to obtain the best extraction conditions. Therefore, the influences of three variables X 1 (ethanol concentration, 60 to 80%), X 2 (extraction temperature, 30°C to 50°C) and X 3 (extraction time, 20 to 40 min) were considered (Table 1). The BBD method was consisted of three factors and levels of 17 experimental operations. In the observed response, the experiment was randomized to maximize the effect of unexplained variability. A quadratic equation was used for this model as follows: The leves of independent variable and the term X i , X j and X i 2 represent the interaction and quadratic terms, respectively | 8 |
55193349 | Bacterial Strains and Cultures Pure bacterial strains used in this study, including Staphylococcus aureus, Escherichia coli, Salmonella typhi and Bacillus subtilis, were obtained from the Department of Microbiology, Agriculture Culture Collection of China. Separate sterile nutrient agar slants were prepared and the bacterial strains were individually inoculated under aseptic conditions and incubated at 37°C for 24 h. Colonies were harvested separately under aseptic condition from the slants and individually inoculated into sterile nutrient broths in separate test tubes and kept in refrigerated condition (Channabasava et al., 2014). Active cultures were achieved by dispensing a tube of cells into 100 mL of nutrient broth and incubating at 37°C for 10 h. The turbidity of the cell suspension was adjusted to the initial concentration 10 8 CFU /mL according to the McFarland standard (Lv et al., 2011). | 9 |
55193349 | Antimicrobial Assay The effective antibacterial capacity of total flavonoids against bacterial strains was determined by diffusion method of agar well and further confirmed by analyzing the Minimal Inhibitory Concentration (MIC) (Vutuc and Holzer, 2014). The walnut leaves extract was diluted with sterile water to 100 mg/mL. Then pour 100 µL bacteria suspension (108 CFU mL) on the solid medium, evenly distributed. Oxford cup of 5 mm diameter were sterilized, then the cups were set on the medium and different concentration of the extraction (100 µL) were filled respectively. | 10 |
55193349 | Minimum Inhibitory Concentration (MIC) Determination Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of antibacterial agents that inhibits the proliferation of the bacteria (Doss et al., 2011). For the determination of MIC, 100µL bacteria suspension (10 8 CFU /mL) was added on the medium evenly distributed. Sterilized oxford cup of 5 mm diameter were prepared, then added with 100 µL different concentrations of the extract, which were 100, 50, 25, 12.5, 6.25, 3.125 mg/mL, respectively and kept at 37°C for 24 h. For each microorganism, at least three replicated experiments were carried out for date analysis. | 11 |
55193349 | Extraction of Total Flavonoids Influenced by Ethanol Concentration A fundamental aspect of solvent selection is 'similar dissolution', which indicated that there is a high degree of solubility in the selected solvents (Mustafa and Turner, 2011). A mixture of ethanol and water is usually used to extract flavonoids from different herbs (Garcia-Castello et al., 2015;Luthria et al., 2007). The main reason is that a large amount of phenolic and flavonoids compounds could be dissolved in water and ethanol mixture (Alothman et al., 2009). In addition, the important aspects of solvent selection are economy, security and sustainability. Due to the volatility of ethanol, it is a better fit polar modifier in the choice of extraction solvent. In addition, ethanol has been considered as one of the most safe and environmentally friendly solvents (Otero-Pareja et al., 2015). To study the influence of ethanol concentration on the total flavonoids extraction from walnut leaves, ethanol concentrations of 30, 40, 50, 60, 70 and 80% were used. Figure 3 indicated the influence of the ethanol concentration on the extraction yield of flavonoids, the extraction yield of flavonoids was not significantly affected by 30-60% ethanol; however, peak extraction of total flavonoids was achieved when the alcohol concentration reached 70%, then the extraction yield decreased with ethanol concentration higher than 70%. Different products are extracted under different conditions. Because of the different chemical structure and polarity of the extracts, solvent has different extraction capacities. Existing studies have shown that the binary solvent system is better than the single-solvent system in extracting flavonoids. It was observed in this study that the optimum yield of flavonoids was obtained at 70% ethanol, which suggested that the flavonoids in walnut leaves were highly soluble in ethanol-water mixture and the yield difference of walnut leaves could be due to different polar and chemical constituents of flavonoids. | 14 |
55193349 | Extraction of Total Flavonoids Influenced by Extraction Temperature Extraction temperature affects the movement of molecule and heat could promote the dissolution of a large number of compounds (Pompeu et al., 2009). In the present study, the temperatures of 30, 40, 50, 60, 70 and 80°C were selected to study temperature influence on total flavonoids extraction from walnut leaves. Figure 4 presented the influence of the extraction temperature on the extraction yield of flavonoids. When the temperature was increased from 30 to 45°C, the extraction yield increased and then the extraction yield decreased when the extraction temperature was over 45°C. The increase in molecular motion is caused by the increase in temperature, so it accelerating the dissolution of flavonoids from plant cells (Lai et al., 2014). As in this study, the appropriate temperature increase in plant cell decomposition and solubility helping to release flavonoids from the substances. But the temperature was too high; it may also cause the damage of the flavonoids. Similar results have also been reported for total flavonoids extraction from alfalfa (Jing et al., 2015). Therefore, 45°C is selected as the optimal extraction temperature. | 15 |
55193349 | Extraction of Total Flavonoids Influenced by Extraction Time The time range required of ultrasonic extraction was the third factor investigated, when the other two factors were fixed, e.g., extraction temperature was set at 45°C and ethanol concentration was set at 70%, respectively. As indicated in Fig. 5, the extraction time has a significant effect on total flavonoids and the yield increased with the increase of time and then decreased at long extraction time. The maximum yield was achieved at 30 min. The presence of different degrees of flavonoids polymerization and their interaction, may have caused this phenomenon, as the equilibrium between the bulk solution and the solution in the material being reached at different times) (Lissi et al., 1999). Therefore, the optimum extraction time is 30 min. From the above analysis, we can find that the ethanol concentration, extraction time, extraction temperature are the main factors of the preparation technology and the best extraction conditions were ethanol concentration 70%, extraction time 30 min and extraction temperature 45°C. | 16 |
55193349 | Data Analysis and Evaluation of RSM Model The experiments for RSM model were conducted based on the design matrix under the defined conditions and the responses from the experimental runs were obtained by using 'design expert' (Table 2). A total of 17 runs of experiments were carried out and three individual parameters that affect the flavonoids extraction yield were optimized. Analysis of variance (ANOVE) and the resulting model regression coefficients were presented in Table 3 which demonstrated the contribution of the variable to the quadratic model. A multivariate regression equation was established and the response variable coding level of the independent variable was analyzed. The quadratic polynomial model of walnut leaves flavonoids was predicted by the least square method and the multiple regression coefficients were determined. The responses of flavonoids extraction ratio of walnut leaves were considered in studying the influence of process variable. The extraction yield of total flavonoids and independent variables of walnut leaves were studied and an empirical model was proposed (Equation 1): Y%=3.7-0.069X 1 +0.059X 2 +0.11X 3 +0.025X 1 X 2 +0.11X 1 X 3 -0.10X 2 X 3 -0.25X 1 The variance analysis of the extraction yields of the total flavonoids from the walnut leaves using Box-Behnken design was shown in Table 3. The determination coefficient (R 2 ) was 0.9938, which is greater than 0.8, indicating a very high correlation (Mirhosseini et al., 2009). The F value and P value were 124.75 and 0.9679, respectively, which indicated the suitability of model that can accurately predict the change of variations. Based on this, the model was used to predict the response. The regression equation coefficients and p-values coefficients for total flavonoids extraction were shown in Table 3. The second-order terms of extraction time, extraction temperature and ethanol concentrations (X 1 2 , X 2 2 , X 3 2 ), one interaction parameters (X 1 X 3 , X 2 X 3 ) and the first-order term of extraction time, extraction temperature and ethanol concentrations(X 1 ,X 2 ,X 3 )were extremely significant with a small P value (p<0.01), whereas parameters (X 1 X 2 ) model term were significant (p<0.05). | 17 |
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