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•Conventional solid–liquid and ultrasound-assisted extraction methods were performed.•Folin–Ciocalteu assay was used to determine the content of phenolic ...compounds.•Ultrasound-assisted extraction (ethanol:water 50:50) was the best extraction method.•Bioactive compounds from Moringa leaves were characterized by HPLC–ESI–QTOF–MS.•30 bioactive compounds were characterized for the first time in M. oleifera leaves.
Moringa oleifera Lam is considered one of the most useful tree in the world because every part of the Moringa tree can be used such as nutritional supplement, for medication, and industrial purposes.
Conventional solid–liquid extraction and ultrasound-assisted extraction (UAE) were performed using different solvents and mixtures of solvents with water. The total phenolic content was determined using Folin–Ciocalteu assay. UAE using ethanol:water (50:50) was the best extraction procedure, which allowed 47±4mg gallic acid equivalents (GAE)/g dry leaf to be obtained. In addition, high-performance liquid chromatography coupled to electrospray ionization quadropole-time of flight mass spectrometry (HPLC–ESI–QTOF–MS) was used to characterize the bioactive compounds in the resulting extract. Consequently, 59 compounds were tentatively characterized, phenolic acid derivatives and flavonoids being the most abundant. Furthermore, 30 of these compounds were tentatively identified for the first time in M. oleifera leaves.
This study shows that leaves from M. oleifera are a good nutritional resource used as a nutritional supplement and may carry additional opportunities for food ingredient innovations, pharmaceutical and cosmetics products.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
•Spectrophotometric assays to assess the major phenolic classes were discussed.•Chemical reactions, pros and cons of these quantitative methods were debated.•A criticism was made on some methods that ...tentatively estimate the content of phenolics, o-diphenols, flavonoids, anthocyanins, flavonols, flavanols.•Experimental protocols for determination of those phenolic classes were described in details.
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Consumers have sought for functional foods aiming to decrease the risk of various non-communicable diseases, such as diabetes, hypertension, overweight/obesity and principal cardiovascular diseases. Epidemiological and clinical studies are continuously corroborating the fact that individuals with higher intake of (poly)phenolic compounds present lower risk of incidence of non-communicable diseases, especially those related to the cardiovascular system. In this sense, food companies and regulatory health agencies together with academic bodies are investing in analytical methods to screen and typify the phenolic content of food extracts and beverages. In this scenario, this review focuses on the chemical perspective of some methods used to assess the total content of phenolic compounds, flavonoids, anthocyanins, ortho-diphenols, flavonols, and tannins (flavanols), by UV-Vis spectrophotometry, giving emphasis on the structures, reactions, overall positive aspects and limitations of the most used quantitative assays.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
A new combination among time, temperature, alkali and alcohol is described for the spectrophotometric determination of small concentrations of phenolics in methanol extracts from plant. It is a ...variation of the classical Folin–Ciocalteu (F–C) method, but the reaction conditions are optimized in order to eliminate methanol interferences in the assay. Alcohol concentration and reaction time limits have been evaluated as 4% methanol (v/v) and 20 min at 40 °C, using a 5% (w/v) sodium carbonate solution. This F–C micro-method is reproducible, quick, inexpensive and particularly helpful if it works with numerous samples or on a small scale, such as during the setting up of an experimental procedure of alcoholic extractions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
A validated silver nanoparticle assay (SNaP-C) for quantitation of Vitamin C, as ascorbic acid (AA) and total AA (TAA), was applied to 31 beverages. SNaP-C assay results (LOD of 2.2 mg/L AA) were ...compared to AA and TAA determined by high-performance liquid chromatography with UV/Vis (LOD = 0.4 mg/L AA), and two well-known assays. All approaches were calibrated using meta-phosphoric acid stabilized AA, where the reducing agent tris(2-carboxyethyl) phosphine hydrochloride was added to convert dehydroascorbic acid to AA for determination of TAA. Statistical comparisons of these four resulting datasets were completed. SNaP-C and HPLC were not statistically significantly different (P > 0.05) for comparison of AA and TAA (mg/L) in these samples, whereas the CUPRAC and Folin-Ciocalteu assays statistically significantly overestimated values of AA and TAA content, respectively. The SNaP-C method is a novel assay that has high specificity for AA capable of quantifying TAA with addition of TCEP.
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•Ascorbic acid is selectively quantified using silver nanoparticles (SNaP-C).•SNaP-C assay results were compared to HPLC, CUPRAC, and Folin-Ciocalteu.•SNaP-C assay results were statistically comparable to HPLC results.•Low LOD and LOQ were observed using SNaP-C for AA and DHAA.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Multiple analytical methods are used for quantification of total polyphenols and total flavanols in fruit juices and beverages. Four methods were evaluated in this study: Folin‐Ciocalteu (F‐C), ...Lowenthal permanganate (L‐P), 4‐dimethylaminocinnamaldehyde (DMAC), and the bovine serum albumin (BSA) precipitation method. Method validation parameters, including working range, limit of detection, limit of quantitation, precision (repeatability), accuracy, and specificity, were assessed and compared. The F‐C method was not specific to polyphenols, and the L‐P method had the widest working range but lacked accuracy. The DMAC method was the most specific to flavanols, and the BSA method was not suitable for quantification of smaller flavanols, such as catechin and epicatechin. Quantitative performance was evaluated using commercial fruit juice samples (n = 14), apple juice samples of different cultivars (n = 22), and commercial ciders (n = 17). In general, the L‐P titration method and DMAC method resulted in higher quantitative values than the F‐C method and BSA precipitation method, respectively. However, ratios of results obtained by the L‐P and F‐C method ranged from 1 to 28, and ratios of results obtained by the DMAC and BSA precipitation method ranged from <1 to 280. This tremendous variation is likely due to variation in polyphenol composition and sample matrix. This information provides perspective for comparison of results obtained through these different methods, and a basis for choosing the most appropriate analytical method for quantification of polyphenols to address a specific research question when working with commercial fruit juice, apple juice from different apple cultivars, and commercial ciders.
Practical Application
This study compared results obtained when four common polyphenol quantification methods were applied to a diverse selection of fruit juices and beverages with distinct polyphenol composition and sample matrix. The matrix and polyphenol composition of the samples significantly influenced the results. Our findings can help manufacturers of fruit‐based products choose the most appropriate analytical method for polyphenol quantification as part of a quality assurance program or to convey information on dietary polyphenol content to consumers. An assessment of analytical method validation parameters is provided for each of the four methods, which will help users of these methods to understand their limitations.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
•We evaluate the Folin–Ciocalteu method used for quantifying total phenols in wine to determine total phenols in bio-oil.•We examine accuracy relative to interferences by use of positive and negative ...controls.•Total phenolics (wt% gallic acid equivalent) detected by the Folin–Ciocalteu method was comparable to that of liquid–liquid extraction of phenols.•Uncertainty of measurement was ±1.1% at the 95% confidence level.
Bio-oil from fast pyrolysis of biomass contains phenolics derived from the lignin portion of the biomass. Traditional testing for total phenolics in bio-oil is based on either a rough estimate of the weight percent water-insolubles in bio-oil or on tedious liquid–liquid extraction methods. We have evaluated the Folin–Ciocalteu (FC) colorimetry method used for quantifying total phenols in wine to determine total phenols in bio-oil. This method, based on the oxidation of phenolic compounds by the FC reagent, is fast and easy to perform. This study evaluated its accuracy relative to interferents by the use of positive and negative controls. Positive controls included phenol, 4-methylphenol, 3-ethylphenol, guaiacol, 2,6-dimethoxyphenol and eugenol. The negative controls included sugars, furfural, and acids. Potential interferents with the quantification of total phenols by the FC method was calculated for all positive and negative controls by using data obtained when adding the contributor (positive controls) and the interferent (negative controls) into bio-oil using typical concentrations found in bio-oil. The positive and several of the negative controls produced strongly correlated linear relationships between the indicated phenolic content of the bio-oil and the amount of contributor or interferent added. However, the slopes of these relationships for the negative controls were much smaller than those for the positive controls, indicating that the error in the prediction of phenolic content was small even for large concentrations of interferent compounds. For typical concentrations of non-phenolic compounds in bio-oil, the error in predicted phenolic content as a result of their presence was ≤5.8%. Total phenolic content in bio-oil detected by the FC method was comparable to the quantity of total phenolics obtained by liquid–liquid extraction. All results fell within the margin of error and the uncertainty of the measurement by the FC method indicating there was no significant difference in the results between the two methods. The FC method uncertainty of measurement was ±1.1% at the 95% confidence level.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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•Folin-Ciocalteu reagent and AlCl3 are used to assess the total phenolics and total flavonoids.•ABTS, CUPRAC, ORAC, DPPH and many other assays are used to assess the antioxidant ...activity.•Colorimetric methods present many pitfalls but can be used as screening tools.•LC–MS is recommended to quantify antioxidant compounds in food matrices.•In vivo tests and clinical studies are required to attest functionality of foods.
As many studies are exploring the association between ingestion of bioactive compounds and decreased risk of non-communicable diseases, the scientific community continues to show considerable interest in these compounds. In addition, as many non-nutrients with putative health benefits are reducing agents, hydrogen donors, singlet oxygen quenchers or metal chelators, measurement of antioxidant activity using in vitro assays has become very popular over recent decades. Measuring concentrations of total phenolics, flavonoids, and other compound (sub)classes using UV/Vis spectrophotometry offers a rapid chemical index, but chromatographic techniques are necessary to establish structure-activity. For bioactive purposes, in vivo models are required or, at the very least, methods that employ distinct mechanisms of action (i.e., single electron transfer, transition metal chelating ability, and hydrogen atom transfer). In this regard, better understanding and application of in vitro screening methods should help design of future research studies on ‘bioactive compounds’.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The purpose of this work was to find a simple, cheap, and suitable method, among the most widely employed, able to guarantee a proper determination and quantification of the phenolic content of extra ...virgin olive oils (EVOOs), in order to satisfy the requirements of the specific health claim (EU Reg. 432/2012). Total phenolic content by Folin–Ciocalteu (FC) was used and compared versus phenolic profile by HPLC‐UV, considering this latter as the most sensitive and specific method for evaluating the phenolic content. Both protocols were performed before and after an acid hydrolysis of the polar phenolic fraction that involves a break of the bound forms of hydroxytyrosol (HTyr) and tyrosol (Tyr), with a simplification of the phenolic profile, and quantification of their total free forms. Results of the phenolic compounds of twelve EVOOs, determined by the different analytical approaches, were statistically compared by means of two‐tailed paired t‐tests: data obtained by the FC assay (expressed as HTyr) before and/or after acid hydrolysis were statistically comparable with results obtained by acid hydrolysis‐HPLC (as sum of HTyr and Tyr).
Practical applications: The promising results obtained in this study show that the simple and cheap colorimetric assay based on the use of the FC reagent, commonly used for the evaluation of phenolic compounds in hydro‐alcoholic extracts of EVOO, can be also efficiently applied, without acid hydrolysis of extracts and HPLC analysis, to verify the compliance to the polyphenols health claim introduced by EU Reg. 432/2012. In fact, in order to preserve the positive image of EVOO due to its healthy properties, it is necessary i) to share an analytical protocol to determine the amount of hydroxytyrosol and its derivatives having a demonstrated effect of protection of blood lipids from oxidative stress ii) to check by this protocol if EVOOs satisfy the EU requirement for including the specific health claim on the oil label.
The simple and cheap Folin–Ciocalteu colorimetric assay, commonly used for the evaluation of phenolic compounds in hydro‐alcoholic extracts of EVOO, can also be efficiently applied to verify the compliance to the health claim introduced by EU Reg. 432/2012.
The simple and cheap Folin–Ciocalteu colorimetric assay, commonly used for the evaluation of phenolic compounds in hydro‐alcoholic extracts of EVOO, can also be efficiently applied to verify the compliance to the health claim introduced by EU Reg. 432/2012.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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