---
title: "Effects of steaming, microwaving and boiling on phenolics and antioxidant capacity of dark green l"
id: "plos-one-11-impact-of-traditional-cooking-methods-on-total-phenolics-flavonoids-and"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-11-impact-of-traditional-cooking-methods-on-total-phenolics-flavonoids-and"
content_type: "clinical_feed_article"
specialty: "General"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0358478"
published_at: "2026-09-17T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Effects of steaming, microwaving and boiling on phenolics and antioxidant capacity of dark green l
## Provenance & Clinical Metadata
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- **Specialty:** [General](https://medichelpline.com/clinical-feed/general.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0358478)
- **Published At:** 2026-09-17T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- This study compared four common domestic cooking methods—**steaming**, **microwaving**, **low-water boiling** and **high-water boiling**—for their effects on phenolic composition and antioxidant capacity in five dark green leafy vegetables: sorrel, stinging nettle, spinach, Swiss chard and purslane. - Total phenolics, phenolic acids, flavonoids and tannins were quantified spectrophotometrically; antioxidant capacity was measured by **DPPH**, **ABTS** and **CUPRAC** assays to capture different reaction mechanisms. - Cooking effects were species- and method-dependent. Total phenolic content fell significantly in sorrel (p = 0.005), while other vegetables showed either stable or slightly increased total phenolics after some treatments. - Phenolic acids rose substantially in stinging nettle and purslane, most notably after **low-water boiling** (+279.3% and +275.0%) and **steaming** (+153.2% and +159.5%). - Flavonoid concentrations tended to decline with **high-water boiling** but were relatively stable in purslane across treatments. - Tannin-related phenolics decreased in nettle and sorrel during boiling; conversely, purslane showed increases in tannin-associated compounds under several cooking methods. - Antioxidant capacity generally decreased after high-water boiling; **steaming** and **microwaving** tended to preserve or even enhance certain activities (DPPH and CUPRAC) in purslane. - Strong positive correlations were found between total phenolic content and antioxidant assay results (correlation coefficients r = 0.853–0.956), indicating that phenolic levels largely explained antioxidant capacity in these samples. - Overall, the authors conclude that **steaming** and **microwaving** are the most effective household techniques for retaining phenolic compounds and antioxidant activity in the tested leafy vegetables, whereas **high-water boiling** produced the largest losses.
## Clinical Analysis & Structured Key Points
Impact of traditional cooking methods on total phenolics, flavonoids, and antioxidant capacities of dark green leafy vegetables | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Reader Comments Figures Figures Abstract This study evaluated the effects of four domestic cooking methods—steaming, microwaving, low-water boiling and high-water boiling—on the phenolic composition and antioxidant capacities of five dark green leafy vegetables (sorrel, stinging nettle, spinach, Swiss chard and purslane). Total phenolics, phenolic acids, flavonoids and tannins were quantified spectrophotometrically, while antioxidant capacities were assessed by DPPH, ABTS and CUPRAC assays. Cooking induced species- and method-dependent changes in phenolic profiles. Total phenolic content significantly decreased in sorrel (p = 0.005), whereas other vegetables showed stable or slightly increased values. Phenolic acids increased markedly in stinging nettle and purslane, particularly after low-water boiling (+279.3% and +275.0%) and steaming (+153.2% and +159.5%). Flavonoid levels declined mainly under high-water boiling but remained stable in purslane. Tannin-associated phenolic compounds decreased in nettle and sorrel during boiling, whereas purslane exhibited increases across several treatments. Antioxidant capacities generally decreased with high-water boiling, while steaming and microwaving preserved or enhanced DPPH and CUPRAC activity in purslane. Strong correlations were observed between total phenolic content and antioxidant assays (r = 0.853–0.956). Overall, steaming and microwaving were identified as the most effective techniques for retaining phenolic compounds and antioxidant capacity, whereas high-water boiling caused the greatest losses. These findings highlight the importance of selecting appropriate cooking methods to preserve the nutritional quality of leafy vegetables. Citation: Yalçın Çeliktaş B, Garipağaoğlu M, Karaman Ersoy Ş (2026) Impact of traditional cooking methods on total phenolics, flavonoids, and antioxidant capacities of dark green leafy vegetables. PLoS One 21(9): e0358478. https://doi.org/10.1371/journal.pone.0358478 Editor: Marcello Iriti, University of Milan, ITALY Received: March 17, 2026; Accepted: September 1, 2026; Published: September 17, 2026 Copyright: © 2026 Yalçın Çeliktaş et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Introduction Green leafy vegetables are widely recognized as “protective foods” due to their richness in vitamins, minerals, dietary fibre and phenolic compounds [ 1 ]. Their abundant bioactive constituents—including polyphenols, carotenoids and glucosinolates—have been associated with improved insulin sensitivity, reduced cardiovascular risk, antihypertensive effects and protection against several cancer types [ 2 , 3 ]. Current dietary guidelines recommend at least five servings (400 g/day) of fruits and vegetables, with two servings specifically derived from green leafy varieties [ 4 ]. Dark green leafy vegetables, in particular, exhibit strong biological activity owing to their diverse phytochemical profiles, including phenolic compounds, flavonoids, carotenoids, vitamins, minerals and essential fatty acids [ 5 ]. These profiles vary across species, contributing to different health-promoting properties. For instance, nettle (Urtica dioica) contains high levels of phenolics and flavonoids- predominantly chlorogenic acid, caffeic acid derivatives, and rutin- that underpin its antioxidant activity [ 6 ], while spinach (Spinacia oleracea) provides lutein, β-carotene and phenolic acids — principally p -coumaric acid derivatives — alongside a unique flavonoid fraction dominated by patuletin and spinacetin glycosides-supporting immune and metabolic functions [ 7 ]. Swiss chard (Beta vulgaris subsp. vulgaris) demonstrates benefits for metabolic syndrome due to its betalain and phenolic content including syringic acid and vitexin-derived apigenin flavonoids- [ 8 ], and purslane (Portulaca oleracea) —rich in α-linolenic acid and vitamins E and C, caffeic acid, quercetin, kaempferol, and luteolin—exhibits anti-inflammatory and hypolipidemic effects [ 9 ]. Sorrel (Rumex acetosa) also shows high antioxidant potential attributable to its flavonoid and phenolic acid composition — particularly quercetin derivatives, chlorogenic acid, caffeic acid, and rutin — [ 10 ]. However, the levels of these bioactive compounds are highly influenced not only by species and genotype but also by cultivation conditions, post-harvest handling and particularly home cooking practices [ 11 ]. In everyday nutrition, most vegetables are consumed cooked, with boiling, steaming and microwaving being the most common household methods [ 12 ]. These thermal treatments can markedly influence the physical attributes, sensory quality and bioactive compounds of vegetables [ 13 ]. Depending on the species, raw material quality and applied method, heating may produce both beneficial and adverse outcomes [ 14 ]. Nutrient losses, tissue softening and colour changes often occur during cooking, yet bioavailability of certain bioactive components may increase and anti-nutritional factors may be reduced. Such changes are largely attributable to the disruption of the vegetable matrix, through which low molecular weight compounds released during heating can interact with nutrients, either reducing or enhancing their accessibility [ 15 ]. Although previous studies have examined the influence of individual cooking methods on total phenolic content and antioxidant activity, the findings remain inconsistent across species and experimental conditions [ 16 – 18 ]. Recent comprehensive reviews also highlight substantial variability in reported outcomes and emphasize the lack of a standardized methodology, making it difficult to identify the most beneficial cooking technique [ 19 ]. Moreover, limited research has simultaneously quantified key phenolic subclasses—phenolic acids, flavonoids and tannins—and linked them to multiple antioxidant assays within a unified experimental design. Evidence is particularly scarce for dark green leafy vegetables commonly consumed in Türkiye, such as nettle, spinach, sorrel, Swiss chard and purslane, especially under standardized domestic cooking conditions. To address these gaps, this study assessed the effects of four traditional cooking methods—steaming, microwaving, low-water boiling and high-water boiling—on total phenolic content, phenolic acids, flavonoids, tannins and antioxidant capacities (DPPH, ABTS, CUPRAC) in these vegetables. The three antioxidant assays were selected because they operate through distinct reaction mechanisms: DPPH and ABTS measure hydrogen atom transfer (HAT) and single electron transfer (SET)-based radical scavenging activity, while CUPRAC assesses electron transfer capacity via cupric ion reduction. Their combined use provides a more comprehensive and mechanistically diverse evaluation of antioxidant capacity than any single assay alone [ 20 ]. By integrating detailed phenolic characterisation with multiple antioxidant assays and correlation analysis, the study offers method- and species-specific insights that advance current understanding and inform practical dietary and culinary recommendations for optimising the nutritional quality of cooked leafy vegetables. Materials and methods Chemicals All chemicals were of analytical grade and purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, USA). The reagents included Folin-Ciocalteu reagent, sodium carbonate (Na 2 CO 3 ), sodium hydroxide (NaOH), sodium potassium tartrate (NaKC 4 H 4 O 6 ), copper(II) sulfate pentahydrate (CuSO 4 ·5H 2 O), gallic acid, ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt], potassium persulfate (K 2 S 2 O 8 ), DPPH [1,1-diphenyl-2-picrylhydrazyl], copper(II) chloride (CuCl 2 ), neocuproine (Nc), ammonium acetate (NH 4 Ac), aluminum chloride (AlCl 3 ), quercetin, sodium acetate (CH 3 COONa), sodium molybdate (Na 2 MoO4), sodium nitrite (NaNO2), hydrochloric acid (HCl) and Trolox [6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid], and caffeic acid. Instruments Analyses were performed using a UV–Vis spectrophotometer (PG Instruments T80 + , UK), incubator (Elektromag Lab., M6040 BP, Türkiye), analytical balance (Weightlab Instruments, WSA-224, Türkiye), centrifuge (Elektromag, M4812M, Türkiye), shaking water bath (Nükleon Laboratuvar, TT107–30 L, Türkiye), and vortex mixer (Dizgi Analitik, Türkiye). Micropipettes (10–100 µL and 100–1000 µL, Ertick, Türkiye) were used for precise liquid handling. Laboratory glassware (beakers, tubes, glass pipettes; ISOLAB, Germany) was employed in all assays. For sample preparation, a microwave oven (Arçelik MD 574, Türkiye), hand blender (Sinbo SHB 3107, Türkiye), and stainless-steel cooking pot (Karaca Galaxy Series, Türkiye) were utilized. Plant material and sample preparation Fresh samples of five dark green leafy vegetables-Nettle (Urtica dioica), Spinach (Spinacia oleracea), Sorrel (Rumex acetosa), Swiss chard (Beta vulgaris subsp. cicla), and Purslane (Portulaca oleracea) —were purchased from local farmers’ markets in Istanbul, Türkiye. Approximately 3 kg of each vegetable was procured. Immediately after purchase, all samples were cleaned, washed under running tap water, and gently dried with paper towels. To prevent nutrient loss, no storage was applied; samples were processed directly. Vegetables were chopped into uniform pieces and weighed into 100 g portions using an analytical balance to ensure consistency across experimental replicates. Each vegetable was divided into three independent sample portions, which were subjected separately to each cooking treatment, representing biological replicates. Subsequently, for each biological replicate, all chemical and antioxidant measurements were carried out in analytical triplicate to ensure measurement precision. The overall experimental workflow is illustrated in Fig 1 . Download: PNG larger image TIFF original image Fig 1. Graphical overview of the experimental study design. https://doi.org/10.1371/journal.pone.0358478.g001 Cooking procedures Cooking conditions were standardized according to previously published protocols with minor modifications [ 21 – 23 ]. The two boiling conditions were selected to reflect contrasting domestic practices commonly reported in the literature: low-water boiling (50 mL per 100 g) simulates minimal-water cooking as described by Turkmen et al. [ 21 ] and Şengül et al. [ 22 ], while high-water boiling (250 mL per 100 g) represents conventional excess-water boiling widely used in household settings [ 23 ]. These conditions were deliberately chosen to represent the full range of water-contact cooking, enabling assessment of the role of water volume as an independent variable in phenolic retention. Low-water boiling: 100 g of vegetable samples were added to 50 mL of boiling distilled water in a stainless-steel pot, covered, and simmered for 5 min. High-water boiling: 100 g of samples were cooked in 250 mL boiling distilled water for 5 min. Steaming: 100 g of samples were placed in a metal strainer over 300 mL boiling distilled water, covered, and steamed for 5 min. Microwave cooking: 100 g of vegetables with 15 mL distilled water were placed in a porcelain dish (25 cm diameter, 5 cm depth) and microwaved at 600 W for 3 min. Cooking times of 3–5 min were selected to reflect typical household culinary practices reported in the literature for leafy vegetables [ 21 , 22 ], and to ensure visual and textural doneness without excessive heat exposure. These durations are consistent with times previously applied in comparative cooking studies on similar plant matrices [ 23 ]. It is acknowledged that optimal cooking time may vary among species; however, a standardised duration was applied across all vegetables to enable direct comparison of cooking method effects independent of time variables. After cooking, samples were cooled to room temperature, homogenized with a hand blender for 30 s and processed for further analysis. Dry matter determination To eliminate the influence of variable moisture content among samples, all analytical data were expressed on a dry weight (DW) basis. Following each cooking treatment, homogenized samples were placed in pre-weighed aluminum dishes and dried in an incubator at 70 °C until a constant weight was achieved (approximately 48 h). Dry matter content was then calculated from the difference between the initial and final sample weights. Dry matter content was calculated using the following equation 1 : (1) where W 1 is the fresh weight of the homogenized sample (g) and W 2 is the weight of the sample after drying to constant mass (g). The dried samples were subsequently used for extraction and further analyses; therefore, the results reflect the combined effects of cooking and subsequent drying. Preparation of extracts The extraction procedure was adapted from Türkmen et al. [ 21 ] with minor modifications. Approximately 2 g of dried and homogenized raw or cooked vegetable samples were extracted with 10 mL of 70% methanol in a shaking water bath at 25 °C for 2 h. Extracts were centrifuged (5000 rpm, 15 min), and the supernatants were collected. The residues were re-extracted under the same conditions, and pooled supernatants were filtered through Whatman No. 1 filter paper. The clarified extracts were stored at −18 °C until analysis. Determination of total phenolic content Total phenolic content (TPC) was determined using the Folin–Ciocalteu method described by Singleton et al. [ 24 ], implemented via a modified protocol incorporating copper-alkaline reagent solutions (Lowry A, B, and C). This modification is based on the principle originally described by Lowry et al. [ 25 ]. In this approach, an alkaline copper pre-treatment is applied prior to the addition of the Folin–Ciocalteu reagent. Although this modification may yield values that differ quantitatively from those obtained using the conventional Folin–Ciocalteu procedure [ 26 ], all samples and calibration standards were processed under identical analytical conditions, ensuring internal consistency for comparative purposes. Three solutions were prepared: Lowry A solution: 2% (w/v) Na 2 CO 3 dissolved in 0.1 M NaOH. Lowry B solution: 1% (w/v) NaKC 4 H 4 O 6 and 0.5% (w/v) CuSO 4 . Lowry C solution: prepared by mixing 50 mL Lowry A with 1 mL Lowry B. Briefly, 0.1 mL of sample extract, 1.9 mL distilled water, and 2.5 mL Lowry C solution were added to test tubes and incubated at room temperature for 10 min. Subsequently, 0.25 mL of Folin-Ciocalteu reagent diluted 1:3 with distilled water was added. After 30 min incubation, absorbance was measured at 750 nm using the UV-Vis spectrophotometer against a blank. Gallic acid standards (6.00 × 10 − 5 –3.00 × 10 − 4 mol/L) were subjected to the same analytical procedure, including the Lowry C pre-treatment, and were used to construct the calibration curve (y = 6100x + 0.0158, R 2 = 0.9984). Results were calculated from the calibration curve and expressed as µmol gallic acid equivalents per gram dry weight (µmol GAE/g DW) according to Equation 2 . (2) Determination of total flavonoid content Total flavonoid content (TFC) was determined according to the spectrophotometric method developed by Chang et al. [ 27 ], with minor modifications. In brief, 0.5 mL of vegetable extract was mixed with 1.5 mL methanol, 0.1 mL of 10% aluminum chloride (AlCl 3 ) solution, 0.1 mL of 1 M sodium acetate (CH 3 COONa), and 2.8 mL distilled water. The mixture was thoroughly vortexed and incubated at room temperature for 30 min. Following incubation, the absorbance was measured at 427 nm. Calibration was performed with quercetin standards (5.00 × 10 − 6 -3.00 × 10 − 5 mol/L) (y = 21600x + 0.0056, R² = 0.998). Results were expressed as µmol QE/g DW (QE: Quercetin equivalent) with equation 3 . (3) Determination of total phenolic acid content (Arnow Assay) Total phenolic acids (TPA) were determined using the Arnow assay as described by Gawlik-Dziki et al. [ 28 ]. It should be noted that the Arnow assay selectively detects ortho-dihydroxy phenolic compounds — primarily caffeic acid, chlorogenic acid, and related catechol-type derivatives — through their reaction with molybdate-nitrite reagent under alkaline conditions. Phenolic acids lacking the ortho-dihydroxy (catechol) structural motif, such as gallic acid, ferulic acid, and p-coumaric acid, do not yield a chromogenic response in this assay. Therefore, the values reported here represent ortho-dihydroxy phenolic acid fractions rather than total phenolic acids in the broadest sense, and should be interpreted accordingly. Reaction mixtures contained 0.5 mL extract, 2.5 mL distilled water, 0.5 mL 0.5 M HCl, 0.5 mL Arnow reagent (10% (w/v) sodium molybdate and 10% (w/v) sodium nitrite), 0.5 mL 10% NaNO 2 , and 0.5 mL 1 M NaOH, adjusted to 5.0 mL with distilled water. Absorbance was measured at 490 nm. Caffeic acid standards (2.0 × 10 − 5 -2.4 × 10 − 4 mol/L) were used for calibration (y = 3642x + 0.0451, R² = 0.9985). Results were expressed as µmol CAE/g DW (CAE: caffeic acid equivalent) by using the following equation 4 . (4) Determination of tannin-associated phenolic compounds Effects on tannin-associated phenolic compounds (TAPC) were determined following Zargar et al. [ 29 ] using the Folin–Ciocalteu method. A 0.1 mL extract was diluted with 7.5 mL distilled water, then mixed with 0.5 mL Folin reagent and 1.0 mL 35% Na 2 CO 3 . After vortexing, dilution to 10 mL and incubation for 30 min, absorbance was measured at 725 nm. Gallic acid standards (5.00 × 10 − 6 –3.00 × 10 − 5 mol/L) were used for calibration (y = 291497x – 0.0158, R 2 = 0.9998). Results were expressed as µmol GAE/g DW according to equation 5 . It should be noted that the Folin–Ciocalteu reagent reacts non-specifically with a broad range of reducing compounds, including non-tannin phenolics, ascorbic acid, reducing sugars, and amino acids [ 20 ]. In the absence of a prior tannin-specific isolation step – such as PVPP adsorption, protein precipitation, or condensed tannin-specific methods (e.g., vanillin–HCl or butanol–HCl assays) –the values reported here do not selectively quantify tannins; they are therefore designated as tannin-associated phenolic compounds (TAPC) and should be interpreted as operational Folin-reactive estimates within a comparative analytical framework. Furthermore, it is acknowledged that the Folin–Ciocalteu, AlCl 3 (flavonoid), and Arnow (phenolic acid) assays employed in this study exhibit overlapping reactivity toward certain phenolic compound classes; consequently, the four parameters reported– TPC, TPA, TFC, and TAPC – represent operationally defined spectrophotometric fractions rather than strictly isolated biochemical categories. This limitation is inherent to colorimetric phenolic analysis and has been widely recognized in the literature [ 20 ]. Chromatographic approaches such as HPLC-DAD or LC-MS/MS would be required for definitive subgroup separation and are recommended for future studies. (5) ABTS radical scavenging assay ABTS radical scavenging activity was evaluated according to Re et al. [ 30 ]. A 7 mM ABTS solution was prepared by dissolving 0.1920 g ABTS in distilled water and mixing with 0.0331 g K
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