Determination of Sinapic Acid Derivatives in Canola Extracts Using High-Performance Liquid Chromatography

A high-performance liquid chromatographic (HPLC) method with diode array detection (DAD) was used to determine the total phenolics, including sinapic acid derivatives in canola. Ten Western Canadian canola seeds, six other commodity canola seeds, their corresponding press cakes and meals were analyzed. Seeds of European 00 rapeseed and Brassica Juncea (Indian mustard) were included for comparison. Phenolic compounds were separated using a gradient elution system of water–methanol-ο-phosphoric acid solution with a flow rate of 0.8 ml/min. In addition to sinapine (SP) and sinapic acid (SA), sinapoyl glucose (SG) is reported in the methanolic extracts. The detection and quantification limits of these compounds were 0.20–0.40 and 0.50–0.80 μg/ml, respectively with recovery values over 98.0%. The content of total phenolics, SP, SA and SG in canola extracts ranged from 9.16 to 16.13, 6.39 to 12.28, 0.11 to 0.59 and 1.36 to 7.50 mg/g, respectively with significant differences among varieties.     

Transformation of 3,5-dimethoxy-4-hydroxy cinnamic acid and its derivatives using enzyme from white-rot fungus Trametes versicolor: Enzyme characteristics and its application

Transformation of 3,5-dimethoxy-4-hydroxy cinnamic acid (sinapic acid), sinapaldehyde, sinapine and sinapoyl in the model system containing an enzyme secreted by the fungus Trametes versicolor was investigated. The affinity of this enzyme was highest for sinapic acid followed by sinapaldehyde and sinapine. The optimum temperature and pH for these transformations were 50°C and pH 3·3, 50°C and pH 4·5, 60°C and pH 4·0 for sinapaldehyde, sinapine, and sinapic acid, respectively. The apparent heat of the enzyme-sinapic acid complex formation is −2557·6 J mol⁻¹. Higher concentrations of sinapine and sinapic acid caused enzyme inhibition. When canola meal was treated with this enzyme the phenolics content in this commodity was decreased by 90%.     

Effect of Operating Parameters on Oil and Phenolic Extraction Using Supercritical CO<Subscript>2</Subscript>

Supercritical fluid extraction (SFE) with carbon dioxide was used to extract oil from canola press cake. Different operating conditions, e.g. pressure, temperature, and co-solvent % were investigated to optimize extraction parameters to yield canola meal with <4% oil. The residual oil content in the extracted canola meal reduced to 2.1–2.9% in our experimental trials. Residues of the optimum conditions based on oil yield were compared for the total phenolic content and the main phenolic compounds. Sinapine (the choline ester of sinapic acid) was the major phenolic constituent in both the SFE and n-hexane extracted canola meals and press cakes. n-Hexane extracted residues showed the retention of the highest sinapic acid, sinapine, sinapoyl glucose and total phenolic contents (mg/g) while the SF-extracted residues showed the lowest values for these compounds.     

Attenuation of sinapic acid and sinapine-derived flavor-active compounds using a factorial-based pressurized high-temperature processing

De-oiled canola meals are sources of protein-containing flavor-active phenolic compounds. Conventional canola oil processing utilizes an excess amount of solvents and is associated with the release of high-intensity bitter flavor-active phenolic compounds, limiting the use of the canola meal. Recent advances in the extraction and isolation of the bitter favor-active phenolic compounds from canola by-products produce protein isolates, however, would benefit the industry by producing a side-stream ingredient rich in phenolics. High temperature and pressure-aided processing, namely the accelerated solvent extraction (ASE) was investigated to extract the flavor-active bitter molecules from the canola meal. The extractability of flavor-active phenolic compounds including the major sinapates, kaempferol derivatives, and other thermo-generative compounds including thomasidioc acid (TA) was evaluated. The effects of temperature, solvent extractant and concentration, and the particle size of the meal were examined on the extraction efficiency of these phenolic compounds. Extraction temperature (180°C) was the primary determinant (p < 0.05) for the attenuation of major sinapates including sinapine and sinapic acid. Both ethanol and methanol extractants at a concentration of 70% (v/v) significantly (p < 0.05) extracted the flavor-active phenolic compounds. The pressurized high temperature through optimized ASE conditions attenuated the bitter undesirable flavor-active phenolic molecules from canola meal, thereby facilitating a potential value-added phenolic-rich by-product.     

Antioxidative effect of the main sinapic acid derivatives from rapeseed and mustard oil by‐products

Amongst oilseeds, rapeseed and mustard are rich sources of phenolic compounds, which also prominent in the by‐products of their respective oil processing or in commercial rapeseed and mustard press cakes. These cakes are rich sources of sinapic acid derivatives, which could be extracted as free sinapic acid or sinapine, the choline ester of sinapic acid. Sinapic acid is a widely investigated antioxidative compound. However, the main compound in the press cakes is present as sinapine. Investigations on the free‐radical‐scavenging activity of sinapic acid and sinapine indicate that sinapine had a significant but lower activity as compared to sinapic acid. Apart from this, sinapic acid, sinapine and different tocopherols were compared as antioxidants for inhibition of the formation of lipid oxidation products in purified rapeseed oils. The oxidation at 40 °C was monitored by the formation of hydroperoxides and propanal. The experiments indicate that in contrast to tocopherol mixtures addition of sinapic acid causes increasing inhibition of hydroperoxide formation when enhancing the concentration from 50 to 500 μmol/kg oil. Sinapine was not able to inhibit the formation of hydroperoxides, compared to sinapic acid. This indicates that sinapic acid‐rich extracts, as compared to sinapine‐rich fractions, could better inhibit the lipid oxidation in bulk lipid systems.     

Antioxidant activity of rapeseed phenolics and their interactions with tocopherols during lipid oxidation

Commercial rapeseed press cakes are rich sources of phenolic compounds, namely, sinapic acid derivatives, which can be extracted as free sinapic acid and its bound forms (such as sinapine, the choline ester of sinapic acid). Fractionated rapeseed extracts rich in sinapic acid and sinapine were compared for their capacity to inhibit the formation of lipid oxidation products. Oxidation at 40°C was monitored by the formation of hydroperoxides (indicating primary oxidation products) and propanal (secondary oxidation products). The 70% methanolic extract of rapeseed meal, added as an equivalent of 500 μmol/kg oil (based on sinapic acid equivalent for sinapic acid-rich extracts or sinapine equivalent for sinapinerich extracts) showed good antioxidative activity compared with the addition of 500 μmol/kg oil sinapic acid. Apart from this, the interaction between a mixture of α-/γ-tocopherol and sinapic acid was investigated using response surface methodology for the experimental design. The experiments indicated that the addition of sinapic acid (concentration dependent) caused inhibition of peroxide formation, complementing further lower endogenous tocopherol concentration in oils. This paper was initially presented at the 95th AOCS Annual Meeting and Expo in Cincinnati, Ohio, May 1–4, 2004.     

Primary Metabolites and Polyphenols in Rapeseed (Brassica napus L.) Cultivars in China

Defatted meals of 10 rapeseed (Brassica napus L.) varieties were investigated for their total phenolic, phenolic acid (free, esterified, and insoluble-bound forms), and tannin contents. The antioxidant capacities (AC) of methanol extracts from samples were assessed using the 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH•), Folin–Ciocalteu method and ferric reducing antioxidant power (FRAP), and β-carotene–linoleic acid tests. In the fraction of free phenolic acids, sinapic, caffeic, ferulic, syringic, gallic, and p-coumaric acids were identified. In the fraction of esterified phenolic acids, sinapine, sinapoyl glucoside, and disinapoyl gentiobiose were identified. After basic hydrolysis, sinapic, ferulic, cinnamic, and 4-hydroxybenzoic acids were identified, and sinapic acid (SA) constituted 98.3% to 99.6% of the total esterified phenolic acids. Eleven components (sinapic, protocatechuic, p-coumaric, syringic, vanillic, gallic, caffeic, ferulic, salicylic, cinnamic, and 4-hydroxybenzoic acids) in the fraction of insoluble-bound phenolic acids were identified. The AC of the samples correlated with the total phenolic content. Overall, the total phenolics showed a better correlation with AC than the individual phenolic compounds. Moreover, SA, sinapoyl glucoside, and disinapoyl gentiobiose showed a highly significant and strong positive correlation with the AC of rapeseed meals, and the derivatives of cinnamic acid showed a higher correlation with AC than the derivatives of benzoic acid. The change in the canolol content in rapeseeds under microwave irradiation is discussed. The correlation of the canolol formed with SA and its derivatives is discussed.     

Production of Canolol from Canola Meal Phenolics via Hydrolysis and Microwave-Induced Decarboxylation

A potent antioxidant, anti-inflammatory and anti-mutagenic agent; 4-vinyl-2,6-dimethoxyphenol (canolol) was obtained from canola meal in a significant yield via alkaline (NaOH)/enzymatic (ferulic acid esterase) hydrolysis followed by microwave-assisted decarboxylation. The hydrolysis was carried out either through using canola meal directly as a substrate or by using the 70 % aqueous methanolic extract filtrates. The hydrolyzed extracts underwent RP-HPLC analysis which showed that 81.0 and 94.8 % of the total phenolics were hydrolyzed to sinapic acid after the alkaline hydrolysis of the meal and the methanolic extracts, respectively. The enzymatic hydrolysis showed lower conversion rates (49.5 and 58.3 %). The hydrolyzed extracts were consequently decarboxylated using 8-diazabicyclo[5.4.0]undec-7-ene under microwave irradiation at different conditions. The HPLC profiling of decarboxylated extracts showed that using microwave at 300 W of microwave power for 12 min brought the highest sinapic acid conversion to canolol (58.3 %) yielding 4.2 mg canolol from each gram of canola meal suggesting that the process could be commercially economical.     

Radical scavenging activity of canola hull phenolics

Possible use of canola hulls as a source of natural anti-oxidants was explored. Cyclone canola hulls were extracted with methanol (30 to 80%, vol/vol) and acetone (30 to 80%, vol/vol). The free radical-scavenging activity of phenolic extracts so prepared was evaluated using the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical ion (ABTS^o−), 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and chemiluminescence assays. The total content of phenolics in prepared extracts from canola hulls ranged from 15 to 136 mg sinapic acid equivalents per gram of extract. Higher levels of condensed tannins were detected in the acetone extracts than in the corresponding methanolic counterparts. Seventy and 80% (vol/vol) acetone extracts displayed markedly stronger antioxidant activity than any of the other extracts investigated. Statistically significant linear correlations were found between TEAC (Trolox equivalent antioxidant capacity) values (expressed in mM of Trolox equivalents per gram of extract) and total pehnolics, TEAC and total condensed tannins (i.e., determined using the modified vanillin and pronthocyanidin assays), as well as TEAC and protein precipitation activity of phenolic extracts (i.e., measured using the dye-labeled assay). The antioxidant activities of extracts as determined by the ABTS^o− radical ion assay correlated highly with those of the chemiluminescence and DPPH radical assays.     

Effect of Microwave Treatment on Sinapic Acid Derivatives in Rapeseed and Rapeseed Meal

Microwave treatment is a new method for rapeseed processing. To better understand the benefits of microwave pretreatment on rapeseed processing, qualitative and quantitative analysis of sinapic acid derivatives is needed. In this study, the effect of microwave treatment on the content of sinapic acid derivatives in rapeseed and the effect of cold-press and solvent extraction on the content of sinapic acid derivatives in rapeseed meal were evaluated using ultrahigh pressure liquid chromatography. It was found that during the microwave treatment, the content of sinapine and sinapic acid in the rapeseed increased slightly at the outset of treatment and significantly decreased thereafter. Following 7 min of microwave treatment, the content of sinapine in rapeseed exhibited its largest decrease of 16.7 %. By contrast, microwave heating of the rapeseed increased the canolol content significantly. We employed two sample paired t tests as a means to analyze the difference between meal and defatted cold-pressed cake. The results indicated that the content of sinapine and sinapic acid in defatted cake was significantly higher than that in meal, while the different extraction methods (solvent extraction and cold-press) did not appear to have a significant influence on the content of canolol in meal (p ≤ 0.05).     

Structural changes of sinapic acid and sinapine bisulfate during autoclaving with respect to the development of colored substances

Structural changes in sinapic acid during autoclaving were studied using spectral analysis, thin-layer chromatography, high-performance liquid chromatography, nuclear magnetic resonance (NMR), and mass spectroscopy. Color properties of sinapic acid and its derivatives were studied by determining the transmittance spectrum, calculating the Commission Internationale de l’Eclairage 1931 tristimulus values and converting to Hunter L a b values. It was found that the colorless sinapic acid aqueous solution (100 μg/mL) turned yellow after 15 min in an autoclave at 121°C and 0.1 MPa. Filtering the yellow aqueous solution through a 0.45-μm filter removed a brown solid consisting of at least three undetermined colored substances and left a yellow liquid. A newly developed yellow substance, syringaldehyde, was identified in the liquid phase by comparing the NMR and mass spectra of the unknown with those of authentic syringaldehyde. Thomasidioic acid was also found in the liquid phase. Under the same autoclaving conditions, sinapine bisulfate showed no evidence of any structural or color changes.     

Identification of phenolic compounds and antioxidant activity of guava dehydrated by different drying methods

Abstract

The influence of different drying techniques on guava was investigated, including phenolic components and antioxidant activities. Through drying processes, total phenolic content (TPC) increased and formation of small molecular phenolic acids (multi-methoxy benzoic acid and sinapic acid) was promoted. UPLC-ESI-QTOF-MS determination showed flavanol compounds, hydrolyzable tannins, ellagic acid conjugates and cinnamic acid derivatives were four predominant phenolics of guava. Drying treatments caused degradation of catechin and its derivatives. Contrarily, drying treatments contributed to higher contents of procyanidin trimers. Moreover, thermal drying treatments led to degradation of macromolecular tannins and formation of smaller molecular tannins and ellagic acid conjugates, while simultaneously reduced the stabilities of most intrinsic ellagic acid conjugates. Furthermore, drying processes increased the yield of cinnamic acid dihexose, probably generating from lignin or phenolics–carbohydrate complex. Freeze drying and hot air drying showed better performance on retention of TPC and enhancement of antioxidant activity (AA).     

Antioxidative and Bactericidal Properties of Phenolic Compounds in Rapeseeds

Antioxidative and bactericidal properties were investigated in isolated and fractionated phenolic compounds of rapeseeds. Among the investigated groups of compounds (phenolic acids, sinapine and the products of its hydrolysis) phenolic acids, and especially sinapic acid, were found to be the most active. Due to valuable properties of these compounds they can be considered to be applied for food and fodder preserving.     

Antioxidant activity of crude tannins of canola and rapeseed hulls

The antioxidant activity of crude tannins of canola and rapeseed hulls was evaluated by β-carotene-linoleate, α,α-diphenyl-β-picrylhydrazyl (DPPH) radical, and reducing power assays. Crude tannins were extracted from three samples of Cyclone canola (high-tannin) hulls and Kolner, Ligaret, and Leo Polish rapeseed (low-tannin) hulls with 70% (vol/vol) acetone. The total phenolic content in crude tannin extracts ranged between 128 and 296 mg of sinapic acid equivalents per 1 g of extract. The ultraviolet spectra of methanolic solution of canola extracts showed two absorption maxima (282 and 309 nm), whereas those of rapeseed extracts exhibited a single maximum (326 nm). Crude tannins isolated from canola hulls exerted significantly (P<0.025) greater antioxidant activity than those from rapeseed in all three assays. The scavenging effect of all crude tannins, at a dose of 1 mg, on the DPPH radical ranged from 35.2 to 50.5%. The reducing power of Cyclone canola hull extracts on potassium ferricyanide was significantly (P≤0.0025) greater than that of rapeseed hull extracts, and the observed data correlated well (r=0.966; P=0.002) with the total content of phenolics present.     

Antioxidant capacity of rapeseed meal and rapeseed oils enriched with meal extract

Response surface methodology (RSM) was used to evaluate the quantitative effects of two independent variables: solvent polarity and temperature of the extraction process on the antioxidant capacity (AC) and total phenolics content (TPC) in meal rapeseed extracts. The mean AC and TPC results for meal ranged between 1181–9974 µmol TE/100 g and 73.8–814 mg sinapic acid/100 g of meal. The experimental results of AC and TPC were close to the predicted values calculated from the polynomial response surface models equations (R² = 0.9758 and 0.9603, respectively). The effect of solvent polarity on AC and TPC in the examined extracts was about 3.6 and 2.6 times greater, respectively, than the effect of processing temperature. The predicted optimum solvent polarity of ε = 78.3 and 63.8, and temperature of 89.4 and 74.2°C resulted in an AC of 10 014 µmol TE/100 g and TPC of 863 mg SAE/100 g meal, respectively. The phenolic profile of rapeseed meal was determined by an HPLC method. The main phenolics in rapeseed meal were sinapine and sinapic acid. Refined rapeseed oils were fortified with an extract – rich in polyphenols – obtained from rapeseed meal. The supplemented rapeseed oil had higher AC and TPC than the refined oil without addition of meal extracts. However, AC and TPC in the enriched oils decreased during storage. The TPC in the studied meal extracts and rapeseed oils correlated significantly (p<0.0000001) positively with their AC (R² = 0.9387). Practical applications: Many bioactive compounds extracted from rapeseed meal provide health benefits and have antioxidative properties. Therefore, it seems worth to consider the application of antioxidants extracted from the rapeseed meal for the production of rapeseed oils with potent AC. Moreover, antioxidants extracted from the rapeseed meal were added to refined rapeseed oil in order to enhance its AC. AC was then tested by FRAP assay. FRAP method is based on the reduction of the ferric tripyridyltriazine (Fe³⁺‐TPTZ) complex to the ferrous tripyridyltriazine (Fe²⁺‐TPTZ), and it is simple, fast, low cost, and robust method. FRAP method does not require specialized equipment and can be performed using automated, semi‐automatic, or manual methods. Therefore the proposed FRAP method can be employed by the fat industry laboratories to asses the AC of rapeseed oils and meal.     

Sulfated Phenolic Substances: Preparation and Optimized HPLC Analysis

Sulfation is an important reaction in nature, and sulfated phenolic compounds are of interest as standards of mammalian phase II metabolites or pro-drugs. Such standards can be prepared using chemoenzymatic methods with aryl sulfotransferases. The aim of the present work was to obtain a large library of sulfated phenols, phenolic acids, flavonoids, and flavonolignans and optimize their HPLC (high performance liquid chromatography) analysis. Four new sulfates of 2,3,4-trihydroxybenzoic acid, catechol, 4-methylcatechol, and phloroglucinol were prepared and fully characterized using MS (mass spectrometry), ¹H, and ¹³C NMR. The separation was investigated using HPLC with PDA (photodiode-array) detection and a total of 38 standards of phenolics and their sulfates. Different stationary (monolithic C18, C18 Polar, pentafluorophenyl, ZICpHILIC) and mobile phases with or without ammonium acetate buffer were compared. The separation results were strongly dependent on the pH and buffer capacity of the mobile phase. The developed robust HPLC method is suitable for the separation of enzymatic sulfation reaction mixtures of flavonoids, flavonolignans, 2,3-dehydroflavonolignans, phenolic acids, and phenols with PDA detection. Moreover, the method is directly applicable in conjunction with mass detection due to the low flow rate and the absence of phosphate buffer and/or ion-pairing reagents in the mobile phase.     

The Fatty Acid Profile and Phenolic Composition of Descurainia sophia Seeds Extracted by Supercritical CO2

Oil and phenolics were extracted from Descurainia sophia (Sophia) seeds by a supercritical CO₂ system. Extractions were conducted in two sequential steps, first using 100 % CO₂ and then adding 10 % ethanol as co‐solvent. The extracts were collected in each step using two separate collectors operating at different pressures. The extraction run was 3 and 4 h for the first period, and 2 h for the second period. The majority of the oil was collected in the first extraction period while phenolic compounds were obtained in the second extraction period. A combined mode of static/dynamic extraction (3 h running and 1 h soaking in CO₂) was also used in the first extraction period, which enhanced the total extraction yield (29.3 ± 0.5 %) and was comparable to the 4 h extraction yield (31.4 ± 0.1 %). The total fatty acid (FA) content of oil in collector 1 (0.94 g) was nearly twice that in collector 2 (0.60 g). The oil contained 14 FAs with α‐linolenic being predominant (48.5 %), with a total 91.1 % unsaturated FAs, a ω3/ω6 ratio of 2.7, and an erucic acid content of 6.2 %. More than 10 phenolic compounds were detected by HPLC in the Sophia seed extracts of which sinapic acid was the dominant compound. Sophia seed extracts showed high levels of antioxidant activity. These results suggest that Sophia seed oil and phenolics have the potential for functional food and pharmaceutical applications.     

Application of Silver Ion High-Performance Liquid Chromatography for Quantitative Analysis of Selected n-3 and n-6 PUFA in Oil Supplements

The aim of this study was to develop a simple method for simultaneous determination of selected cis/cis PUFA–LNA (18:2), ALA (18:3), GLA (18:3), EPA (20:5), and DHA (22:6) by silver ion high‐performance liquid chromatography coupled to a diode array detector (Ag‐HPLC‐DAD). The separation was performed on three Luna SCX Silver Loaded columns connected in series maintained at 10 °C with isocratic elution by 1 % acetonitrile in n‐hexane. The applied chromatographic system allowed a baseline separation of standard mixture of n‐3 and n‐6 fatty acid methyl esters containing LNA, DHA, and EPA and partial separation of ALA and GLA positional isomers. The method was validated by means of linearity, precision, stability, and recovery. Limits of detection (LOD) for considered PUFA standard solutions ranged from 0.27 to 0.43 mg L^?1. The developed method was used to evaluate of n‐3 and n‐6 fatty acids contents in plant and fish softgel oil capsules, results were compared with reference GC‐FID based method.     

Organoleptic and nutritional effects of phenolic compounds on oilseed protein products: A review

In many new oilseed protein sources, phenolic compounds are as important as unsaturated lipids, carbonyl compounds, and nonenzymatic browning in the development of adverse flavors and colors in food products. The free phenolic acids are of particular concern because of enzymatic oxidation too-quinones and subsequent binding to lysine and methionine in the proteins. Numerous free phenolic acids have been identified in all oilseed flours with syringic, ferulic and vanillic being the major components in cottonseed, peanut and soybean flours. Gossypol in cottonseed, chlorogenic acid in sunflower, and sinapine in rapeseed are microconstituent phenolics which cause unique problems in the utilization of these defatted flours and their protein isolates in food applications. The roles of bound phenolics and tannins in the binding of essential nutrients or altering chemical and functional properties require further investigation.     

Anti-Nutritional Components, Fibre, Sinapine and Glucosinolate Content, in Australian Canola (Brassica napus L.) Meal

Canola meal is highly regarded as a component of animal feed with a high protein content and a desirable amino acid profile. The presence of some components, in particular glucosinolates, sinapine and fibre, affects the value of the meal and reduces the amount that can be used in animal feed formulations. Glucosinolates in traditional cultivars (rapeseed) had very high amounts and this severely limited the usefulness of the meal. Canola breeding programs have successfully reduced glucosinolate content to trace amounts. However sinapine remains at levels sufficiently high to cause problems, particularly in poultry feed. The relatively high fibre level in canola also reduces the value of the product for animal feed. This study has determined the level of sinapine, glucosinolates and fibre in current cultivars of canola in Australia to illustrate advances made by breeding programs and limitations which still remain to raise the usefulness of a potentially valuable feedstock. Although glucosinolate levels in meal were shown to have been reduced to 11 μmol/g in some cases, sinapine remained at traditional levels of about 12–15 g/kg and neutral detergent fibre levels were about 30–40%. These issues are important priorities for canola breeders.