Name: a generalized method for determining free soluble phenolic acid composition and antioxidant capacity of cereals and legumes
Uploaded: 2022-06-10T13:00:00.000+00:00
Duration: 10 min 30 s
Description: Watch this Scientific Journal Video about A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes at JoVE.com

The authors gratefully acknowledge the technical support of Ms. Alison Ser and Ms. Hannah Oduro-Obeng, as well as the video editing support by Ms. Janice Fajardo and Mr. Miguel del Rosario.

1. Type of samples
<ol>
	<li>Use five whole grain samples, comprising two cereals (e.g., durum wheat and yellow corn) and three legumes (e.g., Blackeye cowpea bean, soybean, and red kidney bean) for this study.</li>
	<li>Mill 50 g of each grain in triplicates into flour, using a coffee grinder, and pass them through a 500 &#181;m sieve.</li>
	<li>Store them at -20 &#176;C.</li>
</ol>
2. Sample preparation
<ol>
	<li>Determination of dry matter content and expression of dry weight basis 
	NOTE: Determine the dry matter content of each powdered sample according to the method of AOAC (2000)12.
	<ol>
		<li>Turn on a forced-convection oven and set the temperature to 130 &#176;C.</li>
		<li>Dry desiccant (silica gel) in the oven for 30 min to 1 h and transfer the dried silica gel to a desiccator.</li>
		<li>Accurately weigh 2 g of each sample into a clean, pre-dried, and weighed aluminum can.</li>
		<li>Dry the weighed samples at 130 &#176;C for 1 h in a forced convection oven.</li>
		<li>Transfer the dried sample to the desiccator and allow to cool to ambient temperature.</li>
		<li>Weigh the dried, cooled sample and record its weight.</li>
		<li>Calculate the dry matter content of each sample as follows: 
		<img alt="Equation 1" src="/files/ftp_upload/62467/62467eq01v2.jpg" style="margin: auto;" /></li>
		<li>Express each parameter measured in dry weight basis using the following formula: 
		<img alt="Equation 2" src="/files/ftp_upload/62467/62467eq02v2.jpg" style="margin: auto;" /></li>
	</ol>
	</li>
	<li>Phenolic acid extraction 
	NOTE: Extract the soluble free phenolic compounds in the grain samples using a modification of the method of Y. Qiu et al.5 that enables phenolic extraction from milligram quantities of whole grains.
	<ol>
		<li>Accurately weigh 100 mg of the whole grain flour sample directly into an amber-colored 2 mL capacity microcentrifuge tube. The dark color of the tube helps to prevent exposure of the mixture to light.</li>
		<li>Add 1 mL of 80% aqueous HPLC-grade methanol to each of the tubes containing the samples.</li>
		<li>Vortex briefly to mix the methanol solution and the samples.</li>
		<li>Sonicate the samples for 60 min to extract the free soluble phenolic compounds. Put a cover over the samples for the duration of the sonication for added protection from light.</li>
		<li>After sonication, centrifuge the mixture at 20,000 &#215; g for 5 min to sediment the solid residues leaving the supernatant on top. Free phenolic compounds will be present in the supernatant after centrifugation.</li>
		<li>Transfer the supernatant into a clean microcentrifuge tube. 
		NOTE: The supernatant needs to be filtered prior to injecting into the HPLC instrument. To filter the supernatant, remove the plunger of a 3 mL syringe and attach a syringe filter. The filter should have a pore size of no larger than 0.22 &#181;m.</li>
		<li>Pipette approximately 0.4 mL of the supernatant into the top of the syringe. Re-insert the plunger and push the liquid through the filter into an HPLC vial containing a vial insert.</li>
		<li>Once the instrument has been set up to run the method outlined in the manuscript for HPLC analysis, load the vials into the carousel to correspond with the sample list.</li>
		<li>Obtain HPLC chromatograms at 320 nm and 280 nm showing distinct peaks representing different phenolic compounds.</li>
		<li>Using appropriate standard curves, quantify hydroxycinnamic acids at 320 nm since they have maximum absorbance at this wavelength. By the same principle, quantify hydroxybenzoic acids at 280 nm.</li>
		<li>Store the remaining extracts at -20 &#176;C for other analyses.</li>
	</ol>
	</li>
</ol>
3. Phenolic composition
<ol>
	<li>Use a high-pressure liquid chromatograph (Table of Materials) to identify and quantify the extracted phenolic compounds in the samples, based on the method of J. Xiang, F. B. Apea-Bah, V. U. Ndolo, M. C. Katundu, and T. Beta 4.</li>
	<li>Prepare phenolic acid standards (vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid) to identify and quantify constituent phenolic compounds in the extracts.</li>
	<li>To do this, weigh 1 mg of each standard and dissolve in 1 mL of 50% aqueous methanol to produce 1,000 &#181;g/mL of each standard.</li>
	<li>Mix equal volumes of all five standards in a 2 mL amber centrifuge tube to produce a cocktail of standards, each having a concentration of 200 &#181;g/mL.</li>
	<li>Prepare serial dilutions of the standard cocktail by taking a volume into a new tube and diluting with equal volume of the aqueous methanol solvent.</li>
	<li>Repeat the serial dilutions down to a concentration of 3.125 &#181;g/mL.</li>
	<li>Also, separately dilute each standard 40 times with the solvent to obtain 25 &#181;g/mL concentration of each standard.</li>
	<li>Set the column temperature to 35 &#176;C and the sample oven temperature to 15 &#176;C.</li>
	<li>To prepare mobile phase A (0.1% aqueous formic acid), transfer 1 mL of formic acid into a 1 L capacity volumetric flask and add HPLC-grade water to the 1 L mark. Shake well to mix.</li>
	<li>To prepare mobile phase B (0.1% formic acid in methanol), transfer 1 mL of formic acid into a 1 L capacity volumetric flask and add HPLC-grade methanol to the 1 L mark. Shake well to mix.</li>
	<li>Adjust the volumes to the 1 L mark if necessary.</li>
	<li>Filter both mobile phases through a 0.45 &#181;m hydrophilic filter paper.</li>
	<li>For the analysis, inject 10 &#181;L of each extract or standard (25 &#181;g/mL standards and the standard cocktails) on to the reverse phase column.</li>
	<li>Elute with the mobile phases according to the linear gradient program for a 25 min run as follows: 0-3.81 min, 9%-14% B; 3.81-4.85 min, 14%-15% B; 4.85-5.89 min, 15%-15% B, 5.89-8.32 min, 15%-17% B; 8.32-9.71 min, 17%-19% B; 9.71-10.40 min, 19%-19% B; 10.40-12.48 min, 19%-26% B; 12.48-13.17 min, 16%-28% B; 13.17-14.21 min, 28%-35% B; 14.21-15.95 min, 35%-40% B; 15.95-16.64 min, 40%-48% B; 16.64-18.37 min, 48%-53% B; 18.37-22.53 min, 53%-70% B; 22.53-22.88 min, 70%-90% B; 22.88-25.00 min, 90% B.</li>
	<li>Compare the retention times of chromatographic peaks obtained for the authentic standards of concentrations 25 &#181;g/mL, at 280 nm and 320 nm, with those of the extracts, in order to identify the constituent phenolic compounds in the samples.</li>
	<li>Plot calibration curves for the phenolic acid standard cocktails, with concentration of the standards on the horizontal axis, and the peak area on the vertical axis</li>
	<li>Use the calibration curves to estimate the concentrations of the identified phenolic compounds, by comparing the peak areas on the plot with that of the identified compounds at 280 nm and 320 nm as mentioned in the earlier section.</li>
</ol>
4. Total phenolic content
NOTE: Determine the total phenolic content of the extracts using the Folin-Ciocalteu method described by F. B. Apea-Bah et al.13.
<ol>
	<li>Prepare ferulic acid and gallic acid standards within the concentration range of 0.025 to 0.150 mg/mL to plot calibration curves in comparison, for the estimation of total phenolic content.</li>
	<li>To do this, accurately weigh 1 mg each of ferulic acid and gallic acid standards into 2 mL capacity centrifuge tubes.</li>
	<li>Add 1 mL of 50% aqueous methanol to each standard and vortex to dissolve, producing 1 mg/mL stock of each standard.</li>
	<li>Prepare a series of dilutions from each stock solution in a total of 500 &#181;L.</li>
	<li>Pipette 18.2 &#181;L of each extract or standard into a separate well on a 96-well microplate.</li>
	<li>Add 36.4 &#181;L of 10% (v/v) aqueous Folin-Ciocalteu reagent to each extract or standard.</li>
	<li>Then, add 145.4 &#181;L of 700 mM sodium carbonate to each reaction mixture.</li>
	<li>Incubate the reaction mixtures in the dark at room temperature (20-25 &#176;C) for 2 h.</li>
	<li>Read the absorbance on a microplate reader at 750 nm.</li>
	<li>Plot calibration curves of change in absorbance against concentration of the phenolic acid standards and use them to estimate the total phenolic contents.</li>
	<li>Express the results as milligram ferulic acid equivalents per gram of milled sample (mg FAE/g) and milligram gallic acid equivalents per gram of milled sample (mg GAE/g) on dry weight basis.</li>
</ol>
5. Antioxidant assays
NOTE: Determine the antioxidant capacity of the grain extracts using the following three assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity; 2,2&#39;-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging capacity, which is also called the Trolox equivalent antioxidant capacity (TEAC); and oxygen radical absorbance capacity (ORAC).
<ol>
	<li>Preparation of Trolox standards for standard curves
	<ol>
		<li>Use Trolox, a water-soluble analogue of vitamin E, as a standard to estimate the in vitro antioxidant capacity of the whole grain extracts.</li>
		<li>Accurately weigh 1 mg of Trolox into a 15 mL tube. Dissolve in 4 mL of 50% aqueous methanol. Vortex to dissolve to prepare a stock solution of 1 mM (1000 &#181;M).</li>
		<li>Prepare six concentrations of Trolox, that is, 50, 100, 200, 400, 600, and 800 &#181;M to plot standard curves for the estimation of DPPH radical scavenging capacity and Trolox equivalent antioxidant capacity (TEAC). Similarly, prepare 6.25, 12.5, 25, and 50 &#181;M concentrations of Trolox for estimating oxygen radical absorbance capacity (ORAC). Make up the total volume of each concentration to 500 &#181;L as shown in Table 1.</li>
	</ol>
	</li>
	<li>Dilution of sample extracts
	<ol>
		<li>Dilute the sample extracts with methanol prior to analysis. Here, yellow corn and cowpea extracts were diluted two times, the wheat and kidney bean were diluted five times while the soybean extract was diluted 10 times with methanol.</li>
	</ol>
	</li>
	<li>Trolox equivalent antioxidant capacity (TEAC) assay
	<ol>
		<li>Measure the Trolox equivalent antioxidant capacity (TEAC) of the samples using the method described previously by F. B. Apea-Bah et al.14.</li>
		<li>Weigh 8.23 mg of ABTS into a clean 2 mL capacity amber centrifuge tube.</li>
		<li>Next, weigh 1.62 mg of potassium persulfate into another clean 2 mL capacity amber centrifuge tube.</li>
		<li>Add 1 mL distilled water to each of them and vortex to dissolve them. 
		NOTE: This results in 16 mM ABTS stock solution with 6 mM aqueous potassium persulfate solution.</li>
		<li>Prepare the ABTS stock solution by mixing the ABTS and potassium persulfate solutions in equal volumes. The solution will immediately change to a dark color.</li>
		<li>Incubate the reagent mixture in the dark for 12-16 h.</li>
		<li>Dilute the ABTS stock solution 30 times with 200 mM phosphate buffered saline (PBS), to form the ABTS working solution. To do this, add 58 mL of 200 mM PBS to 2 mL of the ABTS stock solution. The working solution will contain 0.27 mM ABTS and 0.1 mM potassium persulfate.</li>
		<li>For the analysis, place 10 &#181;L of each diluted extract or Trolox into a 96-well microplate.</li>
		<li>Add 190 &#181;L of ABTS working solution to each well and incubate the reaction mixtures for 60 min.</li>
		<li>Measure the absorbance of the reaction mixtures at 750 nm in a microplate reader.</li>
		<li>Use the Trolox standards in a concentration ranging from 100 to 800 &#181;mol/L to plot a calibration curve.</li>
		<li>Estimate the ABTS radical scavenging capacity from the calibration curve.</li>
		<li>Express the results as micromole Trolox equivalents per gram (&#181;mol TE/g) sample on dry weight basis.</li>
	</ol>
	</li>
	<li>DPPH assay 
	NOTE: Determine the DPPH radical scavenging capacity of the samples using the method described previously by F. B. Apea-Bah et al.13. The DPPH antioxidant assay requires a radical-generating compound, DPPH (2,2-diphenyl-1-picrylhydrazyl).
	<ol>
		<li>Accurately weigh 1.2 mg of DPPH into an empty 50 mL capacity centrifuge tube. Dissolve the DPPH in 30 mL of methanol to prepare a 60 &#181;mol/L methanolic solution. 
		NOTE: The DPPH assay tests the ability of the sample extracts to scavenge free radicals produced by DPPH.</li>
		<li>For the analysis, add 5 &#181;L of the sample extracts or Trolox solution into the microplate wells.</li>
		<li>Next, add 195 &#181;L of the 60 &#181;mol/L DPPH methanolic solution and incubate for 60 min.</li>
		<li>Measure the absorbance of the reaction mixture at 515 nm.</li>
		<li>Use the Trolox standards (50-800 &#181;mol/L) to plot a calibration curve, with the change in absorbance on the vertical axis and the Trolox concentrations on the horizontal axis.</li>
		<li>Estimate the DPPH scavenging capacity from the calibration curve.</li>
		<li>Express the results as micromole Trolox equivalents per gram (&#181;mol TE/g) sample on dry weight basis.</li>
	</ol>
	</li>
	<li>Oxygen radical absorbance capacity
	<ol>
		<li>Determine the oxygen radical absorbance capacity (ORAC) of the samples based on the method of F. B. Apea-Bah et al.13.</li>
		<li>To begin, prepare Trolox standards of concentrations 6.25, 12.5, 25, and 50 &#181;M from a 1,000 &#181;M Trolox standard stock solution in 75 mM potassium phosphate (K2HPO4/KH2PO4) buffer.</li>
		<li>To do this, weigh 1 mg Trolox powder and dissolve in 4 mL of the buffer solution under sonication to prepare a 1,000 &#181;M stock solution.</li>
		<li>Then, take 50 &#181;L of the stock solution into a 2 mL capacity centrifuge tube and dilute with 950 &#181;L of buffer solution to obtain 1,000 &#181;L of 50 &#181;M Trolox solution.</li>
		<li>Pipette 500 &#181;L of the 50 &#181;M solution into a new tube and dilute with equal volume of buffer to obtain a 25 &#181;M Trolox solution.</li>
		<li>Repeat the serial dilution of the 25 &#181;M to obtain 12.5 &#181;M, and then similarly repeat the dilution of 12.5 &#181;M to obtain 6.25 &#181;M.</li>
		<li>Prepare appropriate dilutions of the sample extracts.</li>
		<li>Dilute the yellow corn and cowpea extracts 20 times, the wheat and kidney bean extracts 50 times, and the soybean extract 100 times with the buffer solution.</li>
		<li>Transfer 200 &#181;L of each sample or Trolox standard into the wells of a black 96-well microplate for automatic pipetting.</li>
		<li>Then, fill the three reagent tanks provided with the ORAC equipment, with the following: (1) the buffer solution; (2) 0.816 nM fluorescein in the buffer; and (3) 153 mM of 2,2&#8242;-azobis(2-amidinopropane) dihydrochloride (AAPH) in buffer.</li>
		<li>Place them in their receptacles for automatic pipetting.</li>
		<li>Set up the ORAC machine and automatic pipetting system for analysis, based on the standard operating procedure.</li>
		<li>For the analysis, set up the automated pipetting system to transfer 25 &#181;L of each diluted extract or standard to the wells of a clear bottom black 96-well microplate.</li>
		<li>Then, automatically add 150 &#181;L of 0.816 nM fluorescein in buffer.</li>
		<li>Incubate the reaction mixture at 37 &#176;C for 15 min in the ORAC machine.</li>
		<li>Afterwards, automatically add 25 &#181;L of the AAPH to each reaction mixture.</li>
		<li>Incubate them at 37 &#176;C for 50 min in the ORAC machine.</li>
		<li>Set up the ORAC machine to measure the fluorescence decay, over the incubation period, at excitation and emission wavelengths of 485 nm and 520 nm, respectively.</li>
		<li>After the measurements, plot a Trolox standard curve, with Trolox (6.25-50 &#181;M) on the horizontal axis and fluorescence decay on the vertical axis.</li>
		<li>Estimate the oxygen radical absorbance capacity of the extracts from the Trolox standard curve.</li>
		<li>Express the results as &#181;mol TE/g sample on dry weight basis.</li>
	</ol>
	</li>
	<li>Statistical analysis
	<ol>
		<li>Present all the results as means &#177; standard deviation of at least triplicates.</li>
		<li>Perform analysis of variance (ANOVA) to determine the effect of grain type on the response variables.</li>
		<li>Where significant differences exist at p &#60; 0.05, use least significant difference (LSD) to compare the means.</li>
		<li>Perform Pearson correlation analysis to estimate the relationship between the phenolic content and antioxidant capacities.</li>
	</ol>
	</li>
</ol>

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B., Serem, J. C., Bester, M. J., Duodu, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenolic+composition+and+antioxidant+properties+of+Koose,+a+deep-fat+fried+cowpea+cake.">Phenolic composition and antioxidant properties of Koose, a deep-fat fried cowpea cake.</a> Food Chemistry. 237, 247-256 (2017).</li></ol>

The authors declare no conflicts of interest.

The whole grains were selected as representative cereal grains and legumes that find wide food applications worldwide. While variations may exist among cultivars of each grain, the focus of this study was to demonstrate a generalized method for free phenolic acid extraction and analysis for whole grains. The extraction method was modified by substantially reducing the amounts of samples and solvents, in order to reduce the amount of chemicals that would be released into the environment when such experiments are conducted. The modification also enables phenolic extraction from milligram quantities of whole grains.
HPLC analysis produces a chromatogram of the constituent phenolic acids in the sample. Each chromatographic peak representing a phenolic acid, can be identified by comparing its retention time, at a predefined wavelength, with that of authentic phenolic acid standards. Ultraviolet (UV) detectors are normally used to identify phenolic acids within the UV wavelength range. Hydroxybenzoic acids are usually identified between 254-280 nm while hydroxycinnamic acids are usually identified between 300-330 nm15. Photodiode array detectors can be used to scan the entire UV-visible wavelength range, and are particularly useful in determining the UV-visible spectra of compounds present in samples that have never been studied. R. J. Robbins and S. R. Bean15 reported the following wavelengths for maximum absorption, for vanillic acid, caffeic acid, p-coumaric acid, and ferulic acid, respectively: 260 nm; 325 nm; 310 nm; 325 nm. The HPLC method of identification is normally acceptable for samples of known phenolic acid composition, whose retention times and UV spectral data have been previously established. This is important because some compounds can co-elute when good separation is not achieved on the column. For samples of unknown phenolic acid composition, a mass spectrometer is used in addition to the authentic standards for identification. Published literature provides useful information about phenolic compounds that have been previously identified in the sample under study or in similar samples3,9,14. It is worth noting that during HPLC analysis, phenolic acid peaks in the chromatogram are identifiable only when authentic standards are available for comparison. There may, therefore, be other peaks that will not be identifiable without their corresponding standards. This is a limitation with HPLC analysis which can be overcome when the HPLC is coupled to a mass spectrometer. Therefore, the total concentration of quantified phenolic acids will depend on the number of phenolic acids identified.
The Folin-Ciocalteu method, which was first published by V. L. Singleton and J. A. Rossi (1965)16 is an electron transfer-based assay that is used to estimate TPC of an analyte17. It is based on the ability of the analyte to reduce phosphomolybdate-phosphotungstate in an alkaline medium, thereby converting it from yellow to a dark blue solution18. The absorbance of the resulting solution can then be measured at 765 nm19. The reagent is not specific for phenolic compounds alone but can react with several other compounds with reducing properties such as thiols, reducing sugars and some amino acids (e.g., tyrosine). Therefore, it is suggested that TPC could be used to estimate the reducing property of a sample or analyte17. During the phenolic extraction, most interfering compounds are excluded and since phenolic compounds are the most ubiquitous phytochemicals, Folin-Ciocalteu assay remains a useful method to estimate the TPC of a sample. In most samples whose phenolic composition is not known, gallic acid is used as a standard to draw a calibration curve for estimating TPC. However, not all samples will contain gallic acid. In the current study, we used ferulic acid to compare its results to that of gallic acid. A two-sample T-test showed that TPC determined as ferulic acid equivalent had significantly higher values than TPC expressed as a gallic acid equivalent. Since we confirmed ferulic acid to be present in all the samples based on our HPLC analysis and other reported results, we would lean more toward expressing the TPC results as a ferulic acid equivalent. It is useful to express TPC relative to a predominant constituent phenolic compound in the sample.
Using three different antioxidant assays, it was observed that the different grains responded differently in their abilities to scavenge free radicals. DPPH radical scavenging capacity and TEAC assay are based on electron transfer (ET) mechanism while the ORAC assay is based on hydrogen atom transfer (HAT) mechanism20. Therefore, combining the three assays gives a better indication of the antioxidant capacity of a sample. It is worth noting that, the ORAC assay measures the ability of a sample to scavenge the physiologically-relevant peroxyl radical, which, in a physiological system, can cause lipid peroxidation that is related to foam cell formation and atherogenesis21,22. On the other hand, DPPH and TEAC assays use radicals that are not of physiological relevance. However, their ease of use makes them useful as rapid methods to estimate the radical scavenging capacity of a sample. ORAC assay also compare well with other in vitro or ex vivo antioxidant assays that use cells and DNA as biomarkers23.
All the methods described above for the extraction and identification of free phenolic acids, estimation of total phenolic content, and determination of antioxidant properties, are not standardized. Different manufacturers of liquid chromatographic columns recommend different gradient elution systems of solvents to separate phenolic acids for their identification. Although the Folin-Ciocalteu method is the most common for estimating total phenolic content of samples, different laboratories perform this assay differently. The same can be said about the antioxidant methods. There is, therefore, the need for harmonization of these methods to promote inter-laboratory comparison of results. There is also the need for development of robust predictive models that can relate phenolic acid composition to the antioxidant properties measured by these important methods of analysis.
It is concluded from this study that the solvent extraction method used is effective in extracting free soluble phenolic acids from the flour of whole grain cereals and legumes, thereby facilitating their identification and quantification. The HPLC method enables identification and quantification of free soluble phenolic acids in whole grain extracts, provided there are authentic standards for comparison. Using the three different antioxidant methods: DPPH, TEAC, and ORAC, the antioxidant capacities of free soluble phenolic extracts from whole grains can be effectively determined based on the hydrogen atom transfer and electron transfer mechanisms. This study further confirms that whole grains differ in their phenolic acid composition and antioxidant capacities. The correlation analysis demonstrates that the antioxidant capacities measured are related to the constituent phenolic acids in the whole grain free soluble extracts. Although both TEAC and DPPH scavenge free radicals by electron transfer mechanism, they correlated differently with TPC. The difference in behaviors of different antioxidant assays necessitate the need to determine antioxidant capacity of samples using a variety of assays. Among the five whole grains studied, soybean has the highest phenolic acid concentration and correspondingly highest antioxidant capacity. The study also demonstrates that whole grain free soluble phenolic acids can be extracted and their antioxidant capacities studied in as little as 1 mL of aqueous methanolic solution, thereby reducing the amount of laboratory chemicals released into the environment.

Phenolic acids are among the most important phytochemicals studied in plants due to the vital role they play in plant defense against herbivory and fungal infection, as well as maintaining structural support and integrity in plant tissues1,2. They are abundant in the bran of cereals and seed coat of legumes3. Structurally, they are divided into two groups: the hydroxybenzoic acids (Figure 1) and hydroxycinnamic acids (Figure 2). The common hydroxybenzoic acids in cereals and legumes include gallic, p-hydroxybenzoic, 2,4-dihydroxybenzoic, protocatechuic, vanillic, and syringic acids, while the common hydroxycinnamic acids include caffeic, p-coumaric, ferulic, and sinapic acids3. Phenolic acids also possess antioxidant properties since they are able to scavenge free radicals, which cause oxidative rancidity in fats, and initiate and propagate radical-induced oxidative stress in physiological systems4,5. Due to this vital physiological role as antioxidants, they are the subject of recent research. This is because when consumed as components of plant foods, they can exert antioxidant protection.
Cereals and cereal products are major carbohydrate food sources for humans and animals worldwide6. Cereals include wheat, rice, corn (maize), barley, triticale, millets, and sorghum. Among them, corn is the most utilized, with an estimated global utilization of 1,135.7 million tonnes in 2019/2020, followed by wheat with an estimated global utilization of 757.5 million tonnes during the same period7. Cereal foods are great sources of energy to consumers since they are rich sources of carbohydrates. They also provide some protein, fat, fiber, vitamins and minerals6. In addition to their nutritional value, cereals are good sources of phytochemical antioxidants, particularly phenolic acids, which have the potential to protect the physiological system from radical-induced oxidative damage3. Legumes are also good sources of nutrients and are generally higher in protein than cereals. They also contain vitamins and minerals and are used in the preparation of various foods8. Additionally, legumes are good sources of a variety of phytochemical antioxidants, including phenolic acids, flavonoids, anthocyanins, and proanthocyanidins9,10. Different varieties of cereals and legumes may have a different phenolic acid composition. There is therefore the need to study the phenolic acid composition of cereals and legumes and their varieties, in order to know their potential health benefits with respect to phenolic antioxidants.
A number of assays have been reported for measuring the quantity of phenolic acids in cereal and legume grains, and determining their antioxidant activities. The most common methods of analysis for whole grain phenolic acids are spectrophotometry and liquid chromatography11. The aim of this work was to demonstrate a generalized high pressure liquid chromatographic method for determining free soluble phenolic acid composition, and spectrophotometric methods for determining total phenolic content and antioxidant capacity of some whole grain cereals and legumes.

<table><tbody><tr><td>15 mL Falcon conical centrifuge tubes</td><td>Fisher Scientific</td><td>05-527-90</td><td></td></tr><tr><td>2 mL Amber glass ID Surestop vial</td><td>Thermo Scientific</td><td>C5000-2W</td><td></td></tr><tr><td>2 mL Amber microcentrifuge tubes</td><td>VWR</td><td>20170-084</td><td></td></tr><tr><td>2,2&amp;prime;-Azobis(2-amidinopropane) dihydrochloride (AAPH)</td><td>Sigma-Aldrich</td><td>440914-100G</td><td></td></tr><tr><td>2,2&#039;-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (C&lt;sub&gt;18&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;N&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;S&lt;sub&gt;4&lt;/sub&gt;) &amp;ge;98%,</td><td>Sigma Aldrich</td><td>A1888-2G</td><td></td></tr><tr><td>2,2-Diphenyl-1pikrylhydrazyl (DPPH) (C&lt;sub&gt;18&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;N&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>D913-2</td><td></td></tr><tr><td>6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (C&lt;sub&gt;14&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;), &amp;ge;98%</td><td>Fluka Chemika</td><td>56510</td><td></td></tr><tr><td>9 mm Autosampler Vial Screw Thread Caps</td><td>Thermo Scientific</td><td>60180-670</td><td></td></tr><tr><td>96 well flat bottom plates</td><td>Fisher Scientific</td><td>12565501</td><td></td></tr><tr><td>Agilent BioTek ELx800 microplate reader</td><td>Fisher Scientific</td><td>BT-ELX800NB</td><td></td></tr><tr><td>Agilent BioTek Precision 2000 96/384 Automated Microplate Pipetting System</td><td>Fisher Scientific</td><td>N/A</td><td></td></tr><tr><td>Agilent BioTek FLx800 Microplate Fluorescence Reader</td><td>Fisher Scientific</td><td>N/A</td><td></td></tr><tr><td>Analytical balance SI-114</td><td>Denver Instrument</td><td>SI-114.1</td><td></td></tr><tr><td>Autosampler, Waters 717 Plus</td><td>Waters</td><td>WAT078900</td><td></td></tr><tr><td>BD 3 mL syringe Luer-Lok Tip</td><td>BD</td><td>309657</td><td></td></tr><tr><td>Bransonic ultrasonic cleaner, Branson 5510</td><td>Millipore Sigma</td><td>Z245143</td><td></td></tr><tr><td>Corning LSE Vortex Mixer</td><td>Corning</td><td>6775</td><td></td></tr><tr><td>Durapore Filter (0.45 &amp;micro;m PVDF Membrane)</td><td>Merck Millipore Ltd</td><td>HVLP04700&amp;nbsp;</td><td></td></tr><tr><td>Durapore Membrane Filters (0.45 &amp;micro;m HV)</td><td>Merck Millipore Ltd</td><td>HVHP04700</td><td></td></tr><tr><td>Eppendorf Research&amp;nbsp;plus, 0.5-10 &amp;micro;L</td><td>Eppendorf</td><td>3123000020</td><td></td></tr><tr><td>Eppendorf Research&amp;nbsp;plus, 0.5-5 mL</td><td>Eppendorf</td><td>3123000071</td><td></td></tr><tr><td>Eppendorf Research&amp;nbsp;plus, 100-1000 &amp;micro;L</td><td>Eppendorf</td><td>3123000063</td><td></td></tr><tr><td>Eppendorf Research&amp;nbsp;plus, 10-100 &amp;micro;L</td><td>Eppendorf</td><td>3123000047</td><td></td></tr><tr><td>Ethyl acetate, HPLC grade</td><td>Fisher Chemical</td><td>E195-4</td><td></td></tr><tr><td>Ferulic acid standard</td><td>Sigma Aldrich</td><td>128708-5G</td><td></td></tr><tr><td>Fluorescein</td><td>Fisher Scientific</td><td>AC119245000</td><td></td></tr><tr><td>Folin &amp;amp; Ciocalteu phenol reagent</td><td>Sigma Aldrich</td><td>F9252</td><td></td></tr><tr><td>Formic acid, 99%</td><td>Acros Organics, Janssen Pharmaceuticalaan 3a</td><td>27048-0010</td><td></td></tr><tr><td>Gallic acid standard</td><td>Sigma</td><td>G7384</td><td></td></tr><tr><td>High performance liquid chromatograph (HPLC), Waters 2695</td><td>Waters</td><td>960402</td><td></td></tr><tr><td>Methanol, HPLC grade</td><td>Fisher Chemical</td><td>A452-4</td><td></td></tr><tr><td>Micro pipet tips, 0.5-10 &amp;micro;L</td><td>Fisherbrand</td><td>21-197-2F</td><td></td></tr><tr><td>Microcentrifuge Sorvall Legend Micro 21 centrifuge</td><td>Thermo Scientific</td><td>75002435</td><td></td></tr><tr><td>Multichannel micropipette, Proline Plus, 30-300 &amp;micro;L</td><td>Sartorius</td><td>728240</td><td></td></tr><tr><td>Photodiode array detector, Waters 2996</td><td>Waters</td><td>720000350EN</td><td></td></tr><tr><td>Pipet tips, 1000 &amp;micro;L</td><td>VWR</td><td>83007-382</td><td></td></tr><tr><td>Pipet tips, 1-5 mL</td><td>VWR</td><td>82018-840</td><td></td></tr><tr><td>Potassium persulfate (K&lt;sub&gt;2&lt;/sub&gt;S&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;8&lt;/sub&gt;), &amp;ge;99.0%</td><td>Sigma Aldrich</td><td>216224-100G</td><td></td></tr><tr><td>Potassium phosphate dibasic anhydrous (K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>P288-500</td><td></td></tr><tr><td>Potassium phosphate monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>P285-500</td><td></td></tr><tr><td>PYREX 250 mL Short Neck Boiling Flask, Round Bottom</td><td>Corning</td><td>4321-250</td><td></td></tr><tr><td>Reversed phase C18 Analytical Column (100 x 3 mm) Accucore aQ</td><td>Thermo Scientific</td><td>17326-103030</td><td></td></tr><tr><td>Roto evaporator, IKA RV 10</td><td>IKA</td><td>&amp;nbsp;0010005185</td><td></td></tr><tr><td>Sodium carbonate (NaCO3) anhydrous</td><td>Fisher Chemical</td><td>S263-1</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Mallinckrodt AR&amp;reg;</td><td>7581</td><td></td></tr><tr><td>Sodium phosphate dibasic anhydrous (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>BP332-500</td><td></td></tr><tr><td>Sodium phosphate monobasic anhydrous (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher bioreagents</td><td>BP329-500</td><td></td></tr><tr><td>Standardization pipet tips 0-200&amp;micro;L</td><td>Fisherbrand</td><td>02-681-134</td><td></td></tr><tr><td>Syringe Driven Filter unit (0.22 &amp;micro;m)</td><td>&amp;nbsp;Millex&amp;reg;-GV</td><td>SLGVR04NL</td><td></td></tr><tr><td>Target micro-serts vial insert (400 &amp;micro;L)</td><td>Thermo Scientific</td><td>C4011-631</td><td></td></tr><tr><td>Ultrapure water (Direct Q-3 UV system with pump)</td><td>Millipore</td><td>ZRQSVP030</td><td></td></tr></tbody></table>

a generalized method for determining free soluble phenolic acid composition and antioxidant capacity of cereals and legumes

Phenolic acids are a class of organic compounds that bear both a phenolic group, and a carboxylic group. They are found in grains and concentrate in the bran of cereals or seed coat of legumes. They possess antioxidant properties that have generated much research interest in recent years, about their potential antioxidant protective health functions. This work presents a generalized method for the extraction of free soluble phenolic acids from whole grains and analysis of their antioxidant capacity. Five whole grain samples comprising two cereals (wheat and yellow corn) and three legumes (cowpea bean, kidney bean, and soybean), were used. The grains were milled into flour and their free soluble phenolic acids extracted using aqueous methanol. The compounds were then identified using a high-pressure liquid chromatograph (HPLC). The Folin-Ciocalteu method was used to determine their total phenolic content while their antioxidant capacities were determined using the DPPH radical scavenging capacity, Trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays. The phenolic acids identified included vanillic, caffeic, p-coumaric and ferulic acids. Vanillic acid was identified only in cowpea while caffeic acid was identified only in kidney bean. p-Coumaric acid was identified in yellow corn, cowpea, and soybean, while ferulic acid was identified in all the samples. Ferulic acid was the predominant phenolic acid identified. The total concentration of phenolic acids in the samples decreased in the following order: soybean &gt; cowpea bean &gt; yellow corn = kidney bean &gt; wheat. The total antioxidant capacity (sum of values of DPPH, TEAC and ORAC assays) decreased as follows: soybean &gt; kidney bean &gt; yellow corn = cowpea bean &gt; wheat. This study concluded that HPLC analysis as well as DPPH, TEAC, and ORAC assays provide useful information about the phenolic acid composition and antioxidant properties of whole grains.

Table 2 shows the phenolic acids that were identified in the cereal and legume grains. Based on available authentic standards, four phenolic acids were identified in the samples and they are: vanillic, caffeic, p-coumaric, and ferulic acids. Vanillic acid is a hydroxybenzoic acid while the other three are hydroxycinnamic acids. Vanillic acid was identified only in Blackeye cowpea bean while caffeic acid was identified only in kidney bean. p-Coumaric acid was identified in yellow corn, cowpea bean, and soybean. Its concentration ranged between 7.57 and 12.48 &#181;g/g flour on dry weight basis, with yellow corn having the lowest value while soybean had the highest value. Ferulic acid was the only phenolic acid that was identified in all the samples. Its concentration ranged between 5.69 and 41.76 &#181;g/g flour on dry weight basis. Among the samples, ferulic acid concentration was highest in soybean followed by cowpea bean while wheat, yellow corn, and kidney bean had comparable values (Table 2). When all the phenolic acid concentrations were summed up for each sample, their values decreased in the following order: soybean &#62; cowpea bean &#62; yellow corn = kidney bean &#62; wheat.
Table 3 showed the total phenolic content (TPC) and antioxidant capacity of the cereal and legume grains. The antioxidant capacity comprised DPPH radical scavenging capacity, TEAC, and ORAC. The summation of these three values therefore gave the total antioxidant capacity of the samples. TPC was measured both in ferulic acid equivalent and gallic acid equivalent for the purpose of comparison. TPC ranged between 1.16 and 2.78 mg FAE/g flour and 0.63 to 1.48 mg GAE/g flour, on dry weight basis. Irrespective of the equivalent unit, TPC of the samples decreased in the following order: soybean &#62; wheat = yellow corn = kidney bean &#62; cowpea bean.
DPPH radical scavenging capacity of the samples ranged between 4.48 and 14.87 &#181;mol TE/g flour on dry weight basis. Soybean had the highest DPPH scavenging capacity, followed by kidney bean, while yellow corn and cowpea bean had comparable values. Wheat had the lowest ability to scavenge the DPPH radical. The Trolox equivalent antioxidant capacity (TEAC) of the samples ranged between 12.82 and 57.24 &#181;mol TE/g flour on dry weight basis. Again, Soybean had the highest TEAC value, followed by kidney bean, and then wheat, while yellow corn and cowpea bean had comparably low TEAC values. Oxygen radical absorbance capacity of the samples were between 0.35 and 1.67 &#181;mol TE/g flour on dry weight basis, with the cereal grains having the lowest value while soybean had the highest value. When all the antioxidant capacities were summed up, their values in the samples decreased in the following order: soybean &#62; kidney bean &#62; yellow corn = cowpea bean &#62; wheat.
There was a strong positive correlation between the two TPC values, and the values for TEAC, ORAC and total antioxidant capacity (TAC) (Table 4). However, there was weak correlation between the TPC values and DPPH.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62467/62467fig01.jpg" /> 
Figure 1: Hydroxybenzoic acids identified in cereals and legumes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62467/62467fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62467/62467fig02.jpg" /> 
Figure 2: Hydroxycinnamic acids identified in cereals and legumes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62467/62467fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Preparation of Trolox concentrations for DPPH and ABTS standard curves. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62467/Table 1 Preparation of Trolox concentrations.docx" target="_blank">Please click here to download this Table.</a>
Table 2: Phenolic acid content of some whole grain cereals and legumes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62467/Table 2 Phenolic acid content of some whole grains.docx" target="_blank">Please click here to download this Table.</a>
Table 3: Total phenolic content (TPC) and antioxidant capacity of some whole grain cereals and legumes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62467/Table 3 TPC and antioxidant capacity of some whole grains.docx" target="_blank">Please click here to download this Table.</a>
Table 4: Pearson correlation matrix for total phenolic content and antioxidant capacities of some cereal and legume grains. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62467/Table 4 Correlation matrix of TPC and antioxidant capacity.docx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes at JoVE.com

A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes

Phenolic acids are important phytochemicals that are present in whole grains. They possess bioactive properties such as antioxidant protective functions. This work aimed at reporting on a generalized method for the HPLC identification, total phenolic content estimation, and determination of the antioxidant capacity of phenolic acids in cereals and legumes.

Phenolic acids are a class of organic compounds that bear both a phenolic group, and a carboxylic group. They are found in grains and concentrate in the ...

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6.7K Views.  University of Manitoba. Een gegeneraliseerde methode voor het bepalen van de vrij oplosbare fenolzuursamenstelling en antioxidantcapaciteit van granen en peulvruchten. Volle granen, waaronder granen en peulvruchten, vormen een aanzienlijk deel van de menselijke voeding. Hun voedingsrelevantie voor de mens is al lang erkend.Meer recent zijn echter hun antioxiderende beschermende gezondheidsvoordelen gemeld. Fenolzuren in de buitenste graanlagen van granen en de zaadlaag van peulvruchten dragen bij aan de antio...

Video: A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes

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The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to inorganic arsenic through diet, information about distribution of arsenic species in various food types must be generated. A method, previously validated in a collaborative trial, has been applied to determine inorganic arsenic in a wide variety of food matrices, covering grains, mushrooms and food of marine origin (31 samples in total). The method is based on detection by flow injection-hydride generation-atomic absorption spectrometry of the iAs selectively extracted into chloroform after digestion of the proteins with concentrated HCl. The method is characterized by a limit of quantification of 10 &#181;g/kg dry weight, which allowed quantification of inorganic arsenic in a large amount of food matrices. Information is provided about performance scores given to results obtained with this method and which were reported by different laboratories in several proficiency tests. The percentage of satisfactory results obtained with the discussed method is higher than that of the results obtained with other analytical approaches.

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The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to ...

Environment

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties (Lactuca sativa L.) and Crop Wild Relatives (Lactuca spp.)

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant capacity. Lettuce is one of the most popular vegetables among consumers worldwide. An accurate protocol to measure vitamin C content in lettuce and other related species is crucial. We describe here a method using the ultra-high-performance liquid chromatography-ultraviolet (UPLC-UV) technique, in which sample preparation, vitamin extraction and chromatography conditions were optimized.
Samples were collected to represent the entire plant, frozen at -80 &#176;C and lyophilized to prevent undesirable oxidation and make their manipulation easier. The extraction of vitamin C was carried out in acidic media, which also contributed to its stability. As vitamin C can be present in two different interconvertible forms, ascorbic acid (AA) and dehydroascorbic acid (DHAA), both compounds should be measured for accurate quantification. The DHAA was quantified indirectly after its reduction to AA because AA shows a higher absorptivity than DHAA in the UV range of the spectrum. From the same extract, two measurements were carried out, one before and one after that reduction reaction. In the first case, we were quantifying the AA content, and in the second one, we quantified the sum of AA and DHAA (TAA: total ascorbic acid) in the form of AA. Then, DHAA quantity was indirectly obtained by subtracting AA coming from the first measurement from TAA. They were determined by UPLC-UV, using a commercial AA standard to build a calibration curve and optimizing the chromatographic procedure, to obtain AA peaks that were completely resolved in a short time. This protocol could be easily extrapolated to any other plant material with slight or no changes. Its accuracy revealed statistically significant differences otherwise unperceived. Other strengths and limitations are discussed more in depth in the manuscript.

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties Lactuca sativa L. and Crop Wild Relatives Lactuca spp.

We present a fast and reliable method to quantify vitamin C in Lactuca spp. using UPLC-UV, potentially transferable to other plants. The key steps are the sample preparation and vitamin C extraction under stable conditions, the reduction of dehydroascorbic acid to ascorbic acid and the optimization of the chromatographic procedure.

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant ...

Biochemistry

Semi-Targeted Ultra-High-Performance Chromatography Coupled to Mass Spectrometry Analysis of Phenolic Metabolites in Plasma of Elderly Adults

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss of muscle mass). Plasma samples were taken at the beginning of the intervention and after 30 days of consuming the functional meals. A semi-targeted ultra-high-performance chromatography coupled with tandem mass (UPLC-MS/MS) analysis was carried out to identify phenolic compounds and their metabolites. Plasma proteins were precipitated with ethanol and the samples were concentrated and resuspended in the mobile phase (1:1 acetonitrile: water) before injection into the UPLC-MS/MS instrument. Separation was carried out with a C18 reverse-phase column, and compounds were identified using their experimental mass, isotopic distribution, and fragment pattern. Compounds of interest were compared to those of data banks and the internal semi-targeted library. Preliminary results showed that the major metabolites identified after the intervention were phenylacetic acid, glycitin, 3-hydroxyphenylvaleric acid, and gomisin M2.

The goal of this protocol is to detect phenolic metabolites in plasma using a semi-targeted chromatography-mass spectrometry method.

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss ...

Lignin Micro-/Submicron Particle Synthesis for Bioflavonoid Release

Green Synthesis, Characterization, Encapsulation, and Measurement of the Release Potential of Novel Alkali Lignin Micro-/Submicron Particles

The applicability of biopolymer micro-/nano- technology in human, veterinary medicine, pharmaceutical, and food technology is rapidly growing due to the great potential of biopolymer-based particles as effective carrier systems. The use of lignin as a basic heteropolymer biomatrix for the design of innovative micro-/submicron formulations allows the achievement of increased biocompatibility and offers various active functional groups presenting opportunities for customization of the physicochemical properties and bioactivities of the formulations for diverse applications. The aim of the present study was to develop a simple and ecofriendly methodology for the synthesis of lignin particles with micro- and submicron size; to evaluate their physicochemical, spectral, and structural characteristics; and to examine their capacity for encapsulation of biologically active molecules and potential for in vitro release of bioflavonoids in simulated gastrointestinal media. The presented methodologies apply cheap and green solvents; easy, straightforward, quick, and sensitive processes requiring little equipment, non-toxic substances, and simple methods for their characterization, the determination of encapsulation capacity towards the poorly water-soluble bioactive compounds morin and quercetin, and the in vitro release potential of the lignin matrices.

Author Spotlight: Development and Characterization of Eco-Friendly Lignin-Based Microparticles for Enhanced Delivery of Bioflavonoids

We describe novel, simple methodologies of synthesis and characterization of biocompatible lignin micro- and submicron particles. These formulations provide a facile approach for the utilization of the heteropolymer, as well as an alternative for the rational design of multifunctional carrier matrices with potential applicability in biomedicine, pharmaceutical technology, and the food industry.

The applicability of biopolymer micro-/nano- technology in human, veterinary medicine, pharmaceutical, and food technology is rapidly growing due to the ...

Polyol-Based Microwave-Assisted Extraction for Phenolic and Antioxidant Yields

Microwave-Assisted Extraction of Phenolic Compounds and Antioxidants for Cosmetic Applications Using Polyol-Based Technology

The utilization of polyols as green solvents for extracting bioactive compounds from plant materials has gained attention due to their safety and inert behavior with plant bioactive chemicals. This study explores the sustainable extraction of phenolic compounds and natural antioxidants from coffee silverskin using the microwave-assisted extraction (MAE) method with polyol-based solvents: glycerin, propylene glycol (PG), butylene glycol (BG), methylpropanediol (MPD), isopentyldiol (IPD), pentylene glycol, 1,2-hexanediol, and hexylene glycol (HG). A comparative analysis was conducted on conventional and non-conventional solvent extractions, focusing on their impact on the bioactive compounds of MAE, encompassing parameters such as total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activities like the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay (DPPH), the 2,2&#8242;-azino-bis(-3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging assay (ABTS), and the ferric reducing antioxidant power assay (FRAP). The highest values were observed for TPC with aqueous-1,2-hexanediol extraction (52.0 &#177; 3.0 mg GAE/g sample), TFC with aqueous-1,2-hexanediol extraction (20.0 &#177; 1.7 mg QE/g sample), DPPH with aqueous-HG&#160;extraction (13.6 &#177; 0.3 mg TE/g sample), ABTS with aqueous-pentylene glycol extraction (8.2 &#177; 0.1 mg TE/g sample), and FRAP with aqueous-HG extraction (21.1 &#177; 1.3 mg Fe (II) E/g sample). This research aims to advance eco-friendly extraction technology through natural plant components, promoting sustainability by minimizing hazardous chemical use while reducing time and energy consumption, with potential applications in cosmetics.

Author Spotlight: Eco-Friendly Extraction of Bioactive Compounds Using Polyol-Based Microwave-Assisted Techniques

This protocol details the utilization of a polyol-based microwave-assisted extraction method for extracting phenolic compounds and natural antioxidants, representing a practical and environmentally sustainable approach to the development of ready-to-use extracts.

The utilization of polyols as green solvents for extracting bioactive compounds from plant materials has gained attention due to their safety and inert ...

Evaluation of Wild Peanut Species for Resistance to Aflatoxin Accumulation

Non-destructive SPE-UPLC-based Quantification of Aflatoxins and Stilbenoid Phytoalexins in Single Peanut (Arachis spp.) Seeds

Aflatoxins are highly carcinogenic secondary metabolites of some fungal species, particularly Aspergillus flavus. Aflatoxins often contaminate economically important agricultural commodities, including peanuts, posing a high risk to human and animal health. Due to the narrow genetic base, peanut cultivars demonstrate limited resistance to fungal pathogens. Therefore, numerous wild peanut species with tolerance to Aspergillus have received substantial consideration by scientists as sources of disease resistance.
Exploring plant germplasm for resistance to aflatoxins is difficult since aflatoxin accumulation does not follow a normal distribution, which dictates the need for the analyses of thousands of single peanut seeds. Sufficiently hydrated peanut (Arachis spp.) seeds, when infected by Aspergillus species, are capable of producing biologically active stilbenes&#160;(stilbenoids) that are considered defensive phytoalexins. Peanut stilbenes inhibit fungal development and aflatoxin production. Therefore, it is crucial to analyze the same seeds for peanut stilbenoids to explain the nature of seed resistance/susceptibility to the Aspergillus invasion. None of the published methods offer single-seed analyses for aflatoxins and/or stilbene phytoalexins.
We attempted to fulfill the demand for such a method that is environment-friendly, uses inexpensive consumables, and is sensitive and selective. In addition, the method is non-destructive since it uses only half of the seed and leaves the other half containing the embryonic axis intact. Such a technique allows germination and growth of the peanut plant to full maturity from the same seed used for the aflatoxin and stilbenoid analysis. The integrated part of this method, the manual challenging of the seeds with Aspergillus, is a limiting step that requires more time and labor compared to other steps in the method. The method has been used for the exploration of wild Arachis germplasm to identify species resistant to Aspergillus and to determine and characterize novel sources of genetic resistance to this fungal pathogen.

Author Spotlight: Quantification of Aflatoxins and Phytoalexins in Peanut Seeds to Identify Genetic Resistance Against Aspergillus

We demonstrate a medium-throughput method for quantification of aflatoxins and stilbenoid phytoalexins in single peanut seeds using ultra performance liquid chromatography. This method was specifically developed for the analyses of wild Arachis species challenged by the aflatoxigenic Aspergillus species.

Aflatoxins are highly carcinogenic secondary metabolites of some fungal species, particularly Aspergillus flavus. Aflatoxins often contaminate ...

Harnessing Gracilaria gracilis: Bioactive Potential for Sustainable Innovation

Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach

The interest in seaweeds as an abundant feedstock to obtain valuable and multitarget bioactive ingredients is continuously growing. In this work, we explore the potential of Gracilaria gracilis, an edible red seaweed cultivated worldwide for its commercial interest as a source of agar and other ingredients for cosmetic, pharmacological, food, and feed applications.
G. gracilis growth conditions were optimized through vegetative propagation and sporulation while manipulating the physicochemical conditions to achieve a large biomass stock. Green extraction methodologies with ethanol and water were performed over the seaweed biomass. The bioactive potential of extracts was assessed through a set of in vitro assays concerning their cytotoxicity, antioxidant, and antimicrobial properties. Additionally, dried seaweed biomass was incorporated into pasta formulations to increase food&#39;s nutritional value. Pigments extracted from G. gracilis have also been incorporated into yogurt as a natural colorant, and their stability was evaluated. Both products were submitted to the appreciation of a semi-trained sensorial panel aiming to achieve the best final formulation before reaching the market.
Results support the versatility of G. gracilis whether it is applied as a whole biomass, extracts and/or pigments. Through implementing several optimized protocols, this work allows the development of products with the potential to profit the food, cosmetic, and aquaculture markets, promoting environmental sustainability and a blue circular economy.
Moreover, and in line with a biorefinery approach, the residual seaweed biomass will be used as biostimulant for plant growth or converted to carbon materials to be used in water purification of the in-house aquaculture systems of MARE-Polytechnic of Leiria, Portugal.

Author Spotlight: Exploring Seaweed's Bioactive Compounds for Sustainable Innovations in Industries 

Here, we describe several protocols aiming at an integrated valorization of Gracilaria gracilis: wild species harvesting, in-house growth, and extraction of bioactive ingredients. The extracts&#39; antioxidant, antimicrobial, and cytotoxic effects are evaluated, along with the nutritional and stability assessment of food enriched with whole seaweed biomass and pigments.

The interest in seaweeds as an abundant feedstock to obtain valuable and multitarget bioactive ingredients is continuously growing. In this work, we ...

A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes

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