Bioactive Compounds and the Antioxidant Activity of Selected Vegetable Microgreens: A Correlation Study

Abstract

In this study, the content of bioactive compounds and antioxidant activity was determined in five selected vegetable microgreens (daikon, i.e., Japanese radish; Chinese red radish; pea; beetroot; and onion). Total phenolics and chlorophylls were analyzed spectrophotometrically in all investigated microgreens. In addition, the content of betalains was determined spectrophotometrically in beetroot microgreens. HPLC analysis was used to identify and quantify individual phenolic compounds. The antioxidant activity of microgreens was determined by DPPH, ABTS and reducing power assays. The highest content of total phenolics, chlorophyll a and chlorophyll b was found in beetroot microgreens (639.85 mg GAE/100 g DW, 202.17 mg/100 g DW and 79.53 mg/100 g DW, respectively). In beetroot microgreens, the content of total betalains, betacyanins and betaxanthins was determined to be 57.27 mg/100 g DW, 43.58 mg BE/100 g DW and 13.68 mg VE/100 g DW, respectively. Among the investigated microgreens, beetroot microgreens showed the highest antioxidant activity, while pea microgreens exhibited the lowest antioxidant activity in all applied assays. The highest correlation was observed for the content of total phenolics and phenolic acids, as determined by HPLC analysis with antioxidant activity using all applied assays, indicating that these compounds were most important contributors to the antioxidant activity of the investigated vegetable microgreens.

1. Introduction

Microgreens are immature plant forms produced from the seeds of vegetables, cereals or herbs [1]. They are harvested without roots, after the development of the cotyledon leaves, or seed leaves, when the first true leaves have emerged [2,3]. Therefore, harvest takes place later than for sprouts and before the baby greens stage of plant growth, with variations among microgreen species, usually between 7 and 21 days after the germination process [1,2,3]. Microgreens can be easily produced by those with limited land area and throughout the year since young seedlings do not need any particular weather conditions. Due to their limited life cycle, to grow, microgreens do not require soil, fertilizers and pesticides and thus can be deemed organic foods [4].

After harvest, microgreens quickly deteriorate, and when stored at room temperature, they can be consumed safely within 1 to 2 days [1]. Therefore, on the market, microgreens are also available in the form of tablets, frozen juice, and powders [5]. In the form of powder, microgreens can be easily accessible for addition to many kinds of foods at daily level [1]. Nevertheless, microgreens are usually consumed raw to avoid the loss or degradation of nutrients during thermal food processing [4].

As very young plants, microgreens have a unique profile of phytochemicals that contribute to the characteristic flavor; hence, they are increasingly used in culinary preparations [3,4]. Recently, their popularity has been increasing due to consumers’ high interest in foods with a significant content of bioactive compounds [1]. According to one report, microgreens contain higher levels of micronutrients compared to mature plants of same species [2]. In addition, the bioavailability of nutrient compounds of microgreens is much higher than in mature plants, as at the initial stage of growth, epidermis development is minimal [4]. The phenolics content found in microgreens is a few times higher in comparison to mature leafy vegetables [4]. Therefore, the use of microgreens has increased, mostly because of their health benefits linked to the high content of bioactive compounds, such as phenolic compounds and pigments (chlorophylls, betalains, etc.) [5,6,7]. It is known that these compounds are beneficial against various diseases, primarily due to their antioxidative properties [5]. Accordingly, microgreens are an emerging class of fresh functional foods with high potential to improve the human diet [8].

This study was carried out to assess several vegetable microgreens, including their content of phenolics and pigments and their antioxidant activity, using different assays. In addition, correlation analysis was carried out to identify compounds contributing to the antioxidant potential in the investigated microgreens.

2. Materials and Methods

2.1. Chemicals and Instruments

Trolox, Folin–Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2ʹ-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), trichloroacetic acid and all standards (quercetin, rutin, epicatechin, catechin, kaempferol, gallic, protocatechuic, chlorogenic, ferulic, sinapic, p-coumaric, caffeic, p-hydroxybenzoic, syringic, rosmarinic acid, isoferulic, gentisic and vanillic acid) for HPLC analysis were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ferric chloride was obtained from J.T. Baker (Deventer, The Netherlands), and manganese (IV) oxide was from Alfa Aesar (Karlsruhe, Germany). All other chemicals and solvents used were of the highest analytical grade. For the preparation of distilled water, a DESA 0081 Water Still distiller purification system (POBEL, Madrid, Spain) was employed. For the spectrophotometric analysis, a Multiskan GO microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used.

2.2. Plant Material and Dried Microgreen Preparation

Fresh daikon (Japanese radish), Chinese red radish, pea, beetroot and onion microgreens were purchased; they are commonly available vegetable microgreens at the local market in Novi Sad, Serbia. Microgreen samples were harvested on the same day as purchase with sterilized scissors, cleaned carefully with tap water, and dried by weeping under air. The cleaned microgreens were frozen at −20 °C for 24 h and dried using a freeze-dryer (model Christ Alpha 2-4 LSC, Martin Christ, Osterode am Harz, Germany) at −40 °C under a pressure of 0.03 mbar for 24 h. The dried microgreens were ground, vacuumed in plastic bags and kept at −20 °C until further analysis. The dry matter content determined for daikon (Japanese radish), Chinese red radish, pea, beetroot and onion microgreens was 8.48%, 8.81%, 8.97%, 6.74% and 9.06%, respectively.

2.3. Extraction Procedure

For the determination of the content of phenolic compounds and chlorophylls and antioxidant activity, the extracts were obtained following the modified procedure described by Šeregelj et al. (2017) [9]. Each sample of dried microgreens was extracted using an acetone/ethanol mixture (50:50 v/v) with a solid-to-solvent ratio of 1:20 (w/v) at constant room temperature and under light protection for 30 min using an ultrasonic bath Sonic 12GT (Vims elektrik, Loznica, Serbia) at a frequency of 40 kHz, and for another 30 min at 400 rpm using a laboratory shaker (Unimax 1010, Heidolph Instruments GmbH, Kelheim, Germany). The obtained extract was separated, centrifuged for 5 min at 4000 rpm, and filtered (Whatman paper No. 1). This procedure was repeated with the residue obtained after extract separation and with same volume of solvents. The two obtained extracts were combined and stored in dark bottles at −20 °C for further analysis. For the determination of the content of betalains, a sample of dried beetroot microgreens was extracted by same procedure using ethanol/distilled water (50:50 v/v). Previously, for the extraction of betalains, a similar extraction procedure was applied by Vulić et al. (2012) [10].

2.4. Determination of Total Phenolic, Chlorophyll and Betalain Content

The total content of phenolics in sample extracts was determined spectrophotometrically using the Folin-Ciocalteu (FC) method [11], adapted for a 96 well microplate. Briefly, in a well of the microplate, the reaction mixture was prepared by mixing 15 μL of sample, 170 μL of distilled water, 12 μL of the Folin–Ciocalteu reagent (2 M), and 30 μL of 20% sodium carbonate. After 1 h of incubation at room temperature in dark conditions, the absorbance at 750 nm was measured. Distilled water was used as a blank. The results of total phenolics content were expressed as gallic acid equivalents (GAE) per 100 g of sample dry weight (DW). The content of chlorophyll a and chlorophyll b in microgreen extracts was analyzed spectrophotometrically using the method of Nagata and Yamashita (1992) [12], adapted to 96-well microplates. The absorbance of extract was measured at 663 and 645 nm. Total chlorophylls were calculated as the sum of chlorophyll a and chlorophyll b according to Andrejiová et al. (2017) [13]. Total chlorophylls, chlorophyll a and chlorophyll b were expressed as mg per 100 g of sample dry weight (DW). The content of betalains (i.e., betacyanins and betaxanthins) in the beetroot microgreen extract was measured according to von Elbe (2001) [14], adapted to 96-well microplates. In brief, the sample was diluted with 0.05 M phosphate buffer of pH 6.5 in a microplate well to a final volume of 250 µL. For the analysis of betacyanins and betaxanthins, absorbance was measured at 538 nm and 476 nm, while for the correction a wavelength of 600 nm was used. Phosphate buffer was used as a blank. Betacyanins were expressed as mg betanin equivalents (BE) per 100 g of sample dry weight (DW), while betaxanthins were expressed as mg vulgaxanthin-I equivalents (VE) per 100 g of sample dry weight (DW). Betalains content was calculated as the sum of betacyanins and betaxanthins. The results of betalains, betacyanins and betaxanthins content were expressed as mg per 100 g of sample dry weight (DW).

2.5. HPLC Analysis

The qualitative and quantitative analysis of phenolic compounds (flavonoids and phenolic acids) in microgreen extracts was performed using by HPLC in accordance with the method of Tumbas Šaponjac et al. (2019) [15]. Chromatograms were recorded at different wavelengths: for hydroxybenzoic acids and catechins at 280 nm, for hydroxycinnamic acids at 320 nm, and for other flavonoids at 360 nm. Separation was performed on a Luna 5 μm C-18 RP column of 250 × 4.6 mm (Phenomenex; Torrance, CA, USA), with a C18 guard column of 4 × 30 mm (Phenomenex; Torrance, CA, USA). Two mobile phases, A (acetonitrile) and B (1% formic acid), were used with the gradient profile in the following way: from 10% to 25% A (0–10 min), a linear rise up to 60% A (10–20 min), and a linear rise up to 70% A (20–30 min), followed by a reversal to the initial 10% A (30–40 min), with additional equilibration time (40–45 min). A flow rate of 1 mL/min was applied. The identification of phenolic compounds in samples was performed by matching the retention time and their spectral characteristics against those of the standards. For the quantification of phenolic compounds, the external standard method was used. Reference substances for the preparation of stock solutions were dissolved in 50% methanol. By diluting the stock solutions, solutions for calibration were created. Chromatogram peak areas were plotted versus known concentrations of standards, and equations were generated using linear regression. These equations were then used to determine the concentrations of phenolic compounds in the samples.

2.6. Determination of Antioxidant Activity

The antioxidant activity on DPPH radicals (DPPH assay) was estimated spectrophotometrically using a 96-well microplate reader, following the method described by Girones-Vilaplana et al. (2014) [16]. Briefly, in a microplate well, 200 μL of DPPH^• solution in methanol (0.89 mM) was mixed with 8 μL of the sample and left in the dark for 50 min at room temperature. Absorbance was measured at 515 nm, using methanol as a blank. Reducing power (RP) was performed with the method of Oyaizu (1986) [17] adapted for 96-well microplates. In short, a 120 μL sample or 120 μL water (blank), 120 μL sodium phosphate buffer (pH 6.6), and 120 μL of 1% potassium ferricyanide were mixed and incubated in a water bath at 50 °C for 20 min. After cooling, 120 μL of 10% trichloroacetic acid was added, and solutions were centrifuged at 3000 rpm for 10 min. In a microplate well, 80 μL of the supernatant was mixed with 80 μL of distilled water and 16 μL of 0.1% ferric chloride. Absorbances were measured immediately at 700 nm. The antioxidant activity on ABTS⁺ radicals (ABTS assay) was evaluated according to Tumbas Šaponjac et al. (2014) [18]. Briefly, in a microplate well 200 μL of ABTS^+• solution (prepared in 0.1 mol/L acetate buffer of pH 5.0 and activated by MnO₂) was mixed with 8 μL of the sample and left in the dark for 35 min at room temperature. Absorbance was measured at 414 nm, using water as a blank. For the determination of antioxidant activity using DPPH and RP tests, a calibration curve of Trolox was prepared in the concentration range from 0.0125 mg/mL to 0.4 mg/mL, while for the ABTS test, the concentration range was from 0.0125 mg/mL to 0.1 mg/mL. The results were expressed as mg Trolox equivalents (TE) per 100 g of sample dry weight (DW).

2.7. Statistical Analysis

All experiments were run in triplicate. The results are presented as mean value ± standard deviation (SD), N = 3. The Origin 7.0 SRO software package (OriginLab Corporation, Northampton, MA, USA, 1991–2002) and Microsoft Office Excel 2016 software (Microsoft, Redmond, WA, USA) were used for statistical analyses. Data were analyzed by one-way analysis of variance (ANOVA), and Tukey’s post hoc HSD test was used to test the mean differences among the samples (p < 0.05).

3. Results and Discussion

3.1. Bioactive Compounds in Microgreens

The content of bioactive compounds (phenolics and chlorophylls) in selected vegetable microgreens, determined spectrophotometrically, is presented in

Table 1. Content and ratio of bioactive compounds in microgreens.

Bioactive Compounds	Daikon (Japanese Radish)	Chinese Red Radish	Peas	Beetroot	Onion
Phenolics	462.28 ± 10.90 d	343.50 ± 10.78 c	308.11 ± 6.52 b	639.85 ± 17.56 e	268.41 ± 3.30 a
Chlorophyll a	46.26 ± 1.68 b	14.61 ± 0.49 a	46.42 ± 0.36 b	202.17 ± 4.99 d	124.92 ± 0.51 c
Chlorophyll b	17.21 ± 0.80 b	4.99 ± 0.11 a	17.81 ± 0.11 b	79.53 ± 1.91 d	52.14 ± 2.53 c
Chlorophyll a + b	63.47 ± 2.48 b	19.60 ± 0.59 a	64.23 ± 0.47 b	281.70 ± 6.90 d	177.06 ± 3.05 c
Chlorophyll a/b ratio	2.69 ± 0.03 c	2.93 ± 0.04 d	2.61 ± 0.00 bc	2.54 ± 0.00 b	2.40 ± 0.11 a

Data of bioactive compounds content are expressed in mg/100 g DW. Different letters (a–e) in rows indicate that there is significant difference at p ≤ 0.05, according to Tukey’s HSD test.

.

It is known that phenolic compounds exhibit antioxidant actions with proven benefits for human health due to their capacity to donate electrons to oxidants, scavenge free radicals, and chelate metal ions; they also indirectly exhibit antioxidant actions by either improving the activity of antioxidant enzymes or inhibiting pro-oxidant enzymes [19]. In our study, the total phenolics content in microgreens, expressed on the basis of dried weight (DW) and fresh weight (FW), decreased in the following order: beetroot (639.85 mg GAE/100 g DW; 43.10 mg GAE/100 g FW) > Japanese radish (462.28 mg GAE/100 g DW; 39.20 mg GAE/100 g FW) > Chinese red radish (343.50 mg GAE/100 g DW; 30.26 mg GAE/100 g FW) > peas (308.11 mg GAE/100 g DW; 27.64 mg GAE/100 g FW) > onion (268.41 mg GAE/100 g DW; 24.31 mg GAE/100 g FW). In the study of Acharya et al. (2021) [5], the phenolics level determined in fresh beetroot microgreens during growth was the lowest after 9 days of growth (678.01 mg GAE/100 g DW, i.e., 33.24 mg GAE/100 g FW) and highest after 15 days of growth (908.16 mg GAE/100 g DW, i.e., 42.84 mg GAE/100 g FW). The value determined after 9 days of growth was close to our result. Xiao et al. (2013) [20] determined the content of total phenolics to be approximately ten-times higher in lyophilized beet and China rose radish microgreens (303.0 mg/100 g FW and 465.5 mg/100 g FW, respectively) in relation to the content of phenolic compounds determined in radish and beetroot microgreens in our study (from 30.26 mg/100 g FW to 43.10 mg/100 g FW). Also, higher values of total phenolics in comparison to our results were determined in lyophilized radish cv. Aoush microgreens (135.74 mg GAE/100 g FW) [4], in lyophilized radish hydroponically grown microgreens (2111.19 mg GAE/100 g DW) [21], and in different varieties of fresh radish microgreens (from 132.78 mg GAE/100 g FW to 145.04 mg GAE/100 g FW) [19]. Ghoora et al. (2020) [6] determined a phenolics content of 61.8 mg GAE/100 g FW in lyophilized radish microgreens, which is closer to our results. Senevirathne et al. (2019) [22] reported a six-times higher total phenol content (1871 mg/100 g DW) in dried green-pea microgreens in comparison to the content found in our study. Truzzi et al. (2021) [23] found total phenolics of 641 μg GAE/g FW and 1073 μg GAE/g FW (i.e., 64.1 mg GAE/100 g FW and 107.3 mg GAE/100 g FW) in nitrogen frozen green-pea microgreens cultivated under different light conditions. Relatively comparable to our findings, Kowitcharoen et al. (2021) [19] reported a phenolics content of 38.14 mg GAE/100 g FW in fresh pea microgreens. Ghoora et al. (2020) [6] observed that the phenolics content of lyophilized onion microgreens (21.4 mg GAE/100 g FW) was among lowest of the investigated microgreens, which is in accordance with our observations. The total phenolics content in the microgreens of leafy vegetables ranged from 25.00 to 152.10 mg GAE/100 g FW [4]. In another study, the total phenolic content of ten culinary microgreens was in the range from 14.6 mg GAE/100 g FW to 73.6 mg GAE/100 g FW [6]. The total phenolic content in microgreens found in our study was in the range of previously reported results for various microgreens. The observed disagreement in the results determined for the same microgreen species can be attributed to several factors that affect the phenolic content, such as the conditions of plant growth, maturity at harvest time and even the method of sample preparation [6].

Previously, it was reported that chlorophylls can exert preventive action against the oxidative damage of DNA and the peroxidation of lipids, both by reducing reactive oxygen species and DPPH radicals and through the chelation of metal ions, such as ferrous ion (Fe²⁺), which can participate in the formation of reactive oxygen species [24]. Despite chlorophylls being the most abundant pigments in nature, there is a scarcity of information about their action, which could be attributed to the difficulty of obtaining chlorophylls in a purified form and also their instability [25]. In addition, chlorophyll content is not only significant for the health benefits it provides; it also impacts the appearance of the microgreens, since the greenness of microgreens adds to their aesthetic appeal [6]. The highest content of chlorophyll a and chlorophyll b was recorded in beetroot (202.17 mg/100 g DW and 79.53 mg/100 g DW, respectively), while the lowest content was found in Chinese red radish microgreens (14.61 mg/100 g DW and 4.99 mg/100 g DW, respectively) (Table 1). Andrejiová et al. (2017) [13] determined the content of chlorophyll a in fresh microgreens cultivated with and without light. The results were as follows: radish (325.23 mg/kg FW and 465.91 mg/kg FW, respectively), garden pea (361.31 mg/kg FW and 578.09 mg/kg FW, respectively) and beetroot (291.87 mg/kg FW and 475.97 mg/kg FW, respectively). In the same study, the content of chlorophyll b was determined in fresh microgreens cultivated with and without light. The results were as follows: radish (139.06 mg/kg FW and 198.35 mg/kg FW, respectively), garden pea (123.00 mg/kg FW and 228.42 mg/kg FW, respectively) and beetroot (104.39 mg/kg FW and 178.64 mg/kg FW, respectively) [13]. Ghoora et al. (2020) [6] determined higher chlorophyll a and chlorophyll b contents in lyophilized radish (33.6 mg/100 g FW and 17.3 mg/100 g FW, respectively) in comparison to onion (16.9 mg/100 g FW and 12.6 mg/100 g FW, respectively) microgreens, which is in agreement with our observations. Our results were in the range of values determined by Acharya et al. (2021) [5], who observed in beetroot microgreens that during growth, the content of chlorophyll a was in the range from 90.10 mg/100 g DW to 362.71 mg/100 g DW, while chlorophyll b varied from 36.37 mg/100 g DW to 153.47 mg/100 g DW. In the study of Wojdyło et al. (2020) [1], the content of total chlorophylls in microgreens ranged from 195.6 to 638.5 µg/g FW and decreased in the following order: green peas (522.7 µg/g FW) > beetroot (258.7 µg/g FW) > radish (215.7 µg/g FW). Kyriacou et al. (2019) [8] reported somewhat higher levels of total chlorophylls (from 647.3 mg/kg FW to 1852.5 mg/kg FW) in various microgreens. Kowitcharoen et al. (2021) [19] determined the content of total chlorophyll as the sum of chlorophyll a and chlorophyll b in different varieties of fresh radish (which ranged from 36.61 mg/100 g FW to 59.21 mg/100 g FW) and in fresh pea microgreens (12.35 mg/100 g FW). In nitrogen-frozen pea microgreens cultivated under various light conditions, total chlorophyll content was approximately 300 μg/g FW and 1750 μg GAE/g FW (i.e., 30 mg/100 g FW and 175 mg/100 g FW), as reported by Truzzi et al. (2021) [23]. These previously reported contents of chlorophylls were mostly several times higher, with the exception of beetroot microgreens, than those obtained in our study. It should be noted that the conditions of cultivation (in particular, exposure to the sun or artificial light sources) and maturity also influenced the content of chlorophylls in microgreens [1,7]. The chlorophyll a/b ratio shows the conditions of microgreen cultivation [1]. A high chlorophyll a/b ratio indicates that microgreens were exposed to the sun or intense light, since the yellower color is caused by a higher content of chlorophyll b [1]. In earlier studies, the chlorophyll a/b ratio in microgreens ranged from 1.30 to 2.15, from 1.41 to 2.52, from 1.8 to 1.9, and from 2.85 to 4.20 [1,6,8,26]. The chlorophyll a/b ratios in our study were in agreement with previously reported results.

In addition to the content of phenolics and chlorophylls, in beetroot microgreens, the content of betalains, betacyanins and betaxanthins was determined (Figure 1). The content of betacyanins and betaxanthins was 43.58 ± 1.40 mg BE/100 g DW and 13.68 ± 0.58 mg VE/100 g DW, respectively, in beetroot microgreens; hence, betacyanins content was threefold higher (3.19) than betaxanthins. In the study of Acharya et al. (2021) [5], in fresh beetroot microgreens during growth, the content of betacyanins and betaxanthins was determined to be in a range from 511.15 mg/100 g DW to 841.85 mg/100 g DW, and from 248.48 mg/100 g DW to 422.99 mg/100 g DW, respectively, while the betacyanins/betaxanthin ratio gradually decreased with the days of growth of microgreens from 2.06 to 1.88. In comparison to the study of Acharya et al. (2021) [5], a lower content of betacyanins and betaxanthins in beetroot microgreens and a higher betacyanin/betaxanthin ratio was observed. Rocchetti et al. (2020) [27] found a total betalain content in red beet microgreens during ten days of storage at 4 °C in a range from 174.3 to 249.1 mg/100 g DW. These values were closer to the content of total betalains (57.27 ± 0.88 mg/100 g DW) found in our study in comparison to the values reported by Acharya et al. (2021) [5].

A HPLC analysis of vegetable microgreen extracts revealed their phenolic composition (

Table 2. Content of phenolic compounds in microgreens determined by HPLC analysis.

Phenolic Compounds	Daikon (Japanese Radish)	Chinese Red Radish	Peas	Beetroot	Onion
Gallic acid	7.3 ± 0.3 a	7.4 ± 0.2 a	8.4 ± 0.2 a	47.4 ± 2.0 c	30.0 ± 0.1 b
Protocatechuic acid	0.6 ± 0.1 a	0.6 ± 0.1 a	1.1 ± 0.1 a	84.2 ± 3.8 c	52.8 ± 2.2 b
Epicatechin	7.9 ± 0.3 a	10.5 ± 0.4 b	36.5 ± 1.7 c	nd	nd
Catechin	0.7 ± 0.1 a	2.5 ± 0.1 b	3.0 ± 0.1 c	nd	nd
Chlorogenic acid	nd	nd	nd	nd	41.0 ± 1.8 a
Ferulic acid	41.6 ± 2.0 d	33.9 ± 1.4 c	2.3 ± 0.1 a	62.7 ± 2.0 e	6.6 ± 0.1 b
Sinapic acid	3.6 ± 0.1 ab	4.7 ± 0.1 ab	1.2 ± 0.1 a	70.1 ± 2.9 c	6.1 ± 0.3 b
p-Coumaric acid	42.6 ± 2.0 d	28.0 ± 1.3 b	nd	34.5 ± 1.3 c	17.7 ± 0.6 a
Caffeic acid	25.6 ± 1.1 d	14.4 ± 0.6 b	38.6 ± 1.7 e	21.8 ± 0.7 c	1.1 ± 0.2 a
p-Hydroxybenzoic acid	9.7 ± 0.3 a	13.9 ± 0.5 b	46.4 ± 2.1 e	34.0 ± 1.1 c	39.2 ± 1.6 d
Syringic acid	5.2 ± 0.2 a	7.1 ± 0.2 b	nd	nd	nd
Epicatechin galat	16.6 ± 0.7 a	nd	nd	nd	nd
Rosmarinic acid	33.6 ± 1.5 b	18.6 ± 0.8 a	nd	121.0 ± 5.1 c	nd
Isoferulic acid	5.0 ± 0.1 c	2.3 ± 0.1 a	nd	3.8 ± 0.1 b	nd
Gentisic acid	nd	12.5 ± 0.5 a	nd	97.4 ± 4.1 c	25.0 ± 2.0 b
Vanillic acid	19.9 ± 0.7 b	8.8 ± 0.4 a	nd	nd	32.2 ± 1.1 c
Kaempferol	nd	nd	2.9 ± 0.1 a	nd	nd
Quercetin	5.8 ± 0.1 a	9.8 ± 0.3 c	7.5 ± 0.3 b	nd	nd
Rutin	32.9 ± 1.3 c	15.1 ± 0.6 b	9.1 ± 0.4 a	nd	nd
Hydroxybenzoic acids	42.7 ± 1.6 a	50.3 ± 1.9 a	55.9 ± 2.4 a	263.0 ± 11 c	179.2 ± 7.0 b
Hydroxycinnamic acids	152.0 ± 6.8 d	101.9 ± 4.3 c	42.1 ± 1.9 a	313.9 ± 12.1 e	72.5 ± 3.0 b
Phenolic acids	194.7 ± 8.4 c	152.2 ± 6.2 b	98.0 ± 4.3 a	577.0 ± 23.1 e	251.7 ± 10.0 d
Flavonoids	63.9 ± 2.5 b	37.9 ± 1.4 a	59.0 ± 2.6 b	nd	nd
Total phenolics	258.4 ± 10.9 b	190.2 ± 7.6 a	156.9 ± 6.9 a	577.0 ± 23.1 c	251.7 ± 10.0 b

Data are expressed in mg/100 g DW; nd means not detected. Different letters (a–e) in rows indicate that there is significant difference at p ≤ 0.05, according to Tukey’s HSD test.

).

The polyphenol profiles of the different microgreen species varied considerably. The highest content of phenolics was observed in beetroot microgreens (577 mg/100 g DW), whereas the lowest phenolic content was found in pea microgreens (156.9 mg/100 g DW). Also, the differences were noted in relation to the major phenolic components. Phenolic acids were the most abundant class of phenolic compounds in all investigated microgreens. This can be expected since phenolic acid is among the phenolics that are primary compounds synthesized via the phenylpropanoid metabolic pathway during plant ontogenesis [1]. Our results calculated on the basis of the fresh weight of total phenolics in daikon (Japanese radish) (21.91 mg/100 g FW), Chinese red radish (16.76 mg/100 g FW), pea (14.07 mg/100 g FW) and beetroot (38.88 mg/100 g FW), as determined by HPLC, were in accordance with the content determined by Wojdyło et al. (2020) for radish (24.3 mg/100 g FW) and beetroot (40.7 mg/100 g FW) and lower than in pea (108.5 mg/100 g FW) microgreens [1]. In a recent study of 13 microgreen species, the total phenolic content determined by HPLC analysis varied from 691 mg/kg DW (2.8 mg/100 g FW) to 5920 mg/kg DW (35.4 mg/100 g FW) [8]. In our study, the sum of phenolic acids determined by HPLC in daikon (Japanese radish) (16.51 mg/100 g FW), Chinese red radish (13.41 mg/100 g FW), pea (9.76 mg/100 g FW) and beetroot (38.88 mg/100 g FW) was higher than the content of phenolic acids determined by Wojdyło et al. (2020) in radish (1.5 mg/100 g FW) and beetroot (16.6 mg/100 g FW) but lower than in pea (41.9 mg/100 g FW) microgreens [1]. Variations in the sum of flavonoids in our study ranged from 0 to 63.9 mg/100 g DW (i.e., 0 to 5.42 mg/100 g FW) were consistent with the total flavonoids determined photometrically in various microgreen varieties (1.1 to 6.5 mg/100 g FW) [6]. In our study, all samples contained phenolic acids, such as ferulic, sinapic, protocatechuic, gallic, caffeic and p-hydroxybenzoic acid. In daikon (Japanese radish) and Chinese red radish, the highest content of coumaric and ferulic acids was found, which were also identified previously in radish microgreens [8]. In onion microgreens, the highest content of protocatechuic acid was determined. A high content of this phenolic acid was determined in onion samples [28,29]. Among the identified phenolic compounds, the highest content of rosmarinic acid was found in beetroot microgreens, and the highest content of p-hydroxybenzoic acid was found in pea microgreens. These acids were previously determined in a higher amount in beetroot leaves and pea hydrolyzed hull, cotyledon and seed samples [30,31,32]. Higher values of phenolics determined spectrophotometrically, in comparison to the content of phenolics determined by HPLC, could be caused by substances (in particular ascorbic acid, sugars, aromatic amines, etc.) present in extracts, which may interfere in determination and result in higher total phenolics being determined by the Folin–Ciocalteu method [33].

3.2. Antioxidant Activity of Microgreens

Since antioxidants can act by various mechanisms (such as the chelation of transition metal ion catalysts, reducing capacity and free radical scavenging, peroxide decomposition, preventing continuous hydrogen abstraction, etc.) to exert their antioxidant properties, it is important to use more than one test, i.e., methodologies with distinct mechanisms, to evaluate the antioxidant activity of plant extracts with complex mixtures of different antioxidant compounds [25,34,35]. In our study, ABTS, DPPH and RP assays were used for the assessment of the antioxidant activity of microgreens. In both the ABTS and DPPH assays, the absorption was measured as a function of time, and the color decrease in the respective radical (DPPH or ABTS⁺) was detected in the presence of antioxidants in the samples [36]. The decrease in absorbance (in percent) was then used to calculate the decrease in the initial radical concentration (in percent) [36]. In the RP assay, the presence of antioxidants (i.e., reductants) in the samples causes the reduction of the Fe³⁺/ferricyanide complex to the ferrous form [37]. Therefore, the amount of Fe²⁺, monitored by measuring the formation of Perl’ Prussian blue at 700 nm is dependent on the reducing power of antioxidant samples [37]. The antioxidant activity of microgreens obtained in DPPH, ABTS and RP assays is summarized in

Table 3. Antioxidant activity (AA) and specific antioxidant activity (SAA) of selected vegetable microgreens.

AA and SAA	Daikon (Japanese Radish)	Chinese Red Radish	Peas	Beetroot	Onion
AA by DPPH	194.24 ± 4.95 c	135.55 ± 5.98 b	87.46 ± 1.28 a	764.48 ± 4.18 e	208.12 ± 5.70 d
AA by RP	196.88 ± 6.04 bc	150.06 ± 2.11 b	75.66 ± 1.99 a	1199.04 ± 42.35 d	247.30 ± 5.12 c
AA by ABTS	82.37 ± 3.23 c	47.24 ± 0.30 b	30.82 ± 1.33 a	242.36 ± 0.83 e	116.64 ± 1.31 d
SAA by DPPH	0.42 ± 0.01 b	0.39 ± 0.02 b	0.28 ± 0.00 a	1.19 ± 0.01 d	0.78 ± 0.02 c
SAA by RP	0.43 ± 0.01 b	0.44 ± 0.01 b	0.25 ± 0.01 a	1.87 ± 0.07 d	0.92 ± 0.02 c
SAA by ABTS	0.18 ± 0.01 c	0.14 ± 0.00 b	0.10 ± 0.00 a	0.38 ± 0.00 d	0.43 ± 0.00 e

Antioxidant activity (AA) is presented in mg TE/100 g DW of microgreens. Specific antioxidant activity (SAA) is presented in mg TE/mg of phenolics expressed as gallic acid equivalent. Different letters (a–e) in rows indicate that there is significant difference at p ≤ 0.05, according to Tukey’s HSD test.

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Among the investigated microgreens, beetroot microgreens showed the highest antioxidant activity, while pea microgreens exhibited the lowest antioxidant activity in all applied assays. In the study of Wojdyło et al. (2020) [1], the antioxidant activity determined using an ABTS assay of lyophilized radish, green pea and beetroot microgreens produced results of 0.6 mmol TE/100 g FW, 0.7 mmol TE/100 g FW and 0.2 mmol/100 g FW, respectively, and in FRAP assay, the results were 0.1 mmol TE/100 g FW, 0.1 mmol TE/100 g FW and 0.2 mmol TE/100 g FW, respectively. The antioxidant activity determined by an ABTS assay of daikon (Japanese radish) (82.37 mg TE/100 g DW, i.e., 0.028 mmol TE/100 g FW), Chinese red radish (47.24 mg TE/100 g DW, i.e., 0.017 mmol TE/100 g FW), pea (30.82 mg TE/100 g DW, i.e., 0.011 mmol TE/100 g FW) and beetroot (242.36 mg TE/100 g DW, i.e., 0.065 mmol TE/100 g FW) microgreens evaluated in our study was lower in comparison to results of Wojdyło et al. (2020) [1]. The antioxidant activity determined by an RP assay of daikon (Japanese radish) (196.88 mg TE/100 g DW, i.e., 0.067 mmol/100 g FW), Chinese red radish (150.06 mg TE/100 g DW, i.e., 0.053 mmol/100 g FW) and pea (75.66 mg TE/100 g DW, i.e., 0.027 mmol/100 g FW) microgreens evaluated in our study was also lower, while the AA determined by an RP assay of beetroot (1199.04 mg TE/100 g DW, i.e., 0.323 mmol/100 g FW) microgreens was higher in relation to the antioxidant activity found in the FRAP assay reported by Wojdyło et al. (2020) [1]. In comparison to the antioxidant activity in FRAP, DPPH and ABTS assays of lyophilized radish cv. Aoush (17.77 µmol TE/g FW, 2.07 µmol TE/g FW and 4.97 µmol TE/g FW, respectively) estimated by Yadav et al. (2019) [4], the radish genotypes investigated in our study had lower antioxidant activity in the RP assay (0.67 µmol TE/g FW and 0.53 µmol TE/g FW), DPPH assay (0.66 µmol TE/g FW and 0.48 µmol TE/g FW) and ABTS assay (0.28 µmol TE/g FW and 0.17 µmol TE/g FW). The antioxidant activity in the ABTS assay of daikon (Japanese radish) (82.37 mg/100 g DW, i.e., 329.10 µmol TE/100 g DW) and Chinese red radish (47.24 mg/100 g DW, i.e., 188.74 µmol TE/100 g DW) microgreens determined in our study, although lower, was closer to the antioxidant activity of radish (488.65 µmol TE/100 g DW) reported by de la Fuente et al. (2019) [21]. In the study of Xiao et al. (2019) [38], microgreens of ruby radish were found to have a remarkably high radical scavenging capacity using the DPPH test (806.3 μmol TE/100 g FW), while radish China rose (304.6 μmol TE/100 g FW), radish daikon (279.8 μmol TE/100 g FW), and radish red (259.7 μmol TE/100 g FW) microgreens exhibited lower radical scavenging activity. The antioxidant activity in a DPPH assay of onion microgreens (208.12 mg TE/100 g DW, i.e., 0.75 µmol TE/g FW) was within the range from 0.60 to 4.31 µmol TE/g FW, while the antioxidant activity of onion microgreens using an RP assay (247.30 mg TE/100 g DW, i.e., 0.90 µmol TE/g FW) and an ABTS assay (116.64 mg TE/100 g DW, i.e., 0.42 µmol TE/g FW) was below the range of AA (from 2.15 to 17.77 µmol TE/g FW for RP assay and from 4.25 to 13.14 µmol TE/g FW for ABTS assay) determined for different leafy microgreens by Yadav et al. (2019) [4]. Previously, in the study of Ghoora et al. (2020), the antioxidant activity of onion microgreens found using DPPH and ABTS assays was lower in a group of ten culinary microgreens [6]. In our study, it also can be observed that a higher antioxidant activity of microgreens was estimated by DPPH and RP tests, in comparison to the ABTS assay. Previously, higher antioxidant capacity was also detected by the DPPH method in comparison to the ABTS method [39]. This could be explained by various reaction mechanisms, since different kinds or contents of antioxidants in extracts lead to different results in the applied tests. Currently, for the action of DPPH radicals, an electron transfer (ET) approach with a hydrogen atom transfer (HAT) mechanism as the minor reaction pathway is established [40]. Also, an ABTS radical cation reaction through ET and the HAT mechanism is equally applicable, while for the method based on the reduction of ferric to ferrous ions, ET is the proposed mechanism [40].

Specific antioxidant activity (SAA) is usually used as a parameter to estimate the effectiveness of a mixture of phenolics in exerting their antioxidant properties [41]. In our study, this parameter was calculated using the total phenolic values obtained by the Folin–Ciocalteu method, as in the study of Vizzotto et al. (2014) [42]. A higher value of SAA indicates that phenolic compounds have a higher capacity to stabilize free radicals or act as antioxidants [41]. Phenolic compounds in beetroot microgreen extracts demonstrated the highest SAA in DPPH and RP assays, while the phenolics in the onion microgreen extracts showed the highest SAA in the ABTS assay (Table 3). Previously, it was observed that specific antioxidant activity tended to be similar for some samples (e.g., between plum varieties in the range from 0.3 to 1.4 mg Trolox/mg phenolics) and in a wider range of values in other samples (e.g., between peach varieties in the range of 0.25 to 2.2 mg Trolox/mg phenolics) [42,43]. Our values of specific antioxidant activity were mostly within the range of previously reported results. The estimation of specific antioxidant activity can be used for the selection of genotypes, with the aim of increasing the consumption of plant foods rich in phenolics with high antioxidant capacity [41].

3.3. Correlation between Contents of Bioactive Compounds and Antioxidant Activities of Microgreens

Previous studies reported that antioxidant activity can be related to the content of phenolics and other bioactive compounds [1]. Pearson’s correlation coefficient analysis was used to test the relationship between the bioactive compounds and antioxidant activity in microgreen extracts. The results of the correlation analyses are shown in

Table 4. Pearson correlation coefficient analysis of phenolic and chlorophyll contents with antioxidant activities by DPPH, RP and ABTS assays of selected vegetable microgreens.

Pearson Correlation Coefficient (r)	DPPH	RP	ABTS
Total phenolics by FC method	0.88 *	0.87	0.79
Chlorophyll a	0.89 *	0.89 *	0.95 *
Chlorophyll b	0.87	0.86	0.94 *
Chlorophyll a + b	0.88 *	0.88 *	0.95 *
Gallic acid	0.89 *	0.89 *	0.95 *
Protocatechuic acid	0.86	0.86	0.94 *
Epicatechin	−0.55	−0.52	−0.68
Catechin	−0.63	−0.60	−0.79
Chlorogenic acid	−0.14	−0.15	0.08
Ferulic acid	0.77	0.75	0.68
Sinapic acid	0.99 **	1.00 **	0.94 *
Coumaric acid	0.44	0.41	0.45
Caffeic acid	−0.07	−0.05	−0.25
p-Hydroxybenzoic acid	0.12	0.15	0.15
Syringic acid	−0.38	−0.39	−0.44
Epikatehin galat	−0.17	−0.21	−0.14
Rosmarinic acid	0.96 **	0.96 **	0.88 *
Isoferulic acid	0.45	0.42	0.38
Gentisic acid	0.98 **	0.98 **	0.96 *
Vanillic acid	−0.34	−0.37	−0.11
Kaempferol	−0.39	−0.36	−0.49
Quercetin	−0.67	−0.66	−0.82
Rutin	−0.44	−0.47	−0.49
Hydroxybenzoic acids	0.86	0.87	0.93 *
Hydroxycinnamic acids	0.95 *	0.94 *	0.89 *
Phenolic acids	0.99 **	0.99 **	0.99 **
Flavonoids	−0.64	−0.65	−0.75
Total phenolics by HPLC	1.00 **	0.99 **	0.98 **

* Significance at p < 0.05; ** Significance at p < 0.01.

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A high correlation (p < 0.01) was observed between total phenolic content using HPLC and antioxidant activity determined by DPPH, ABTS and RP assays (r = 1, r = 0.98, and r = 0.99, respectively). Additionally, a high correlation was found between antioxidant activity determined by DPPH and RP assays (r = 1; p < 0.01). Although many studies have demonstrated a correlation between total phenolics content and antioxidant activities in microgreens in general, research looking at the correlation of individual phenolics (in particular phenolic acids) and antioxidant activities in microgreens was limited. According to Table 4, the correlation between total phenolic acids, as determined by HPLC analysis, and antioxidant activity in all assays, as well as sinapic, rosmarinic and gentisic acids and antioxidant activity, as evaluated by DPPH and RP assays of microgreens, was highly significant (r ≥ 0.96; p < 0.01), indicating that phenolic acid highly contributes to antioxidant activity, while there was no significant correlation between flavonoids determined by HPLC analysis and antioxidant activity. A correlation at a significant level was found for the content of total chlorophylls and chlorophyll a with antioxidant activity in all assays, as well as for the content of chlorophyll b with antioxidant activity in the ABTS assay (with an r in the range from 0.88 to 0.95; p < 0.05). Previously, in several studies, a good correlation was found between the total phenolics and antioxidant properties of microgreens [44]. In barley and wheat microgreens, total phenolics were highly correlated (>0.91) to antioxidant activities, determined by both DPPH and FRAP assays, while low correlations of total CHLs were reported with antioxidant activities, as determined during cultivation in the dark [44]. The absence of a significant correlation for the most-abundant flavonoid glycosides of thirteen microgreen species with antioxidant activity was observed by Kyriacou et al. (2019) [8]. This can be supported by the previous finding that antioxidant activity evaluation depends on the capacity of the individual phenolic compounds present in a sample and the type of assay used in the investigation [8,45]. In addition, glycosylation leads to lower antioxidant activity for flavonoids, since the loss of two hydroxyl groups significantly reduces the activity [8,46]. In the study of Xiao et al. (2019) [38], a strong correlation was found between total phenolic content and radical scavenging capacity, as determined by DPPH assay (r = 0.896 **, significant at the 0.01 level (two-tailed)). Wojdyło et al. (2020) [1] reported that antioxidant capacity, evaluated by ABTS and FRAP assays, was correlated with the content of phenolics (r² = 0.682 and 0.538), in particular flavan-3-ols (r² = 0.742 and 0.649), while the content of polymeric procyanidins was correlated with the results of the ABTS assay (r² = 0.758). According to Yadav et al. (2019) [4], the total phenolic and flavonoid contents of leafy vegetables (in both microgreen and mature stages) were highly positively (p value < 0.0001) correlated with antioxidant activity, as determined by FRAP (r = 0.95 and 0.88), CUPRAC (r = 0.95 and 0.90), and DPPH (r = 0.82 and 0.86) assays, while an absence of correlation was found with antioxidant activity, as found by ABTS assay (r = 0.40 and 0.44). Significant correlations between the total phenolic content of selected culinary microgreens and antioxidant activity, as found by DPPH (r = 0.730; p ≤ 0.05) and FRAP (r = 0.768; p ≤ 0.01) assays, and between the total flavonoid content and antioxidant activity, as found by DPPH (r = 0.719; p ≤ 0.05), FRAP (r = 0.808; p ≤ 0.01) and ABTS (r = 0.828; p ≤ 0.01) assays, were reported in the study of Ghoora et al. (2020) [6]. Other bioactive compounds, which are not identified and quantified in the microgreen samples, should not be ignored, as they may contribute to the antioxidant activity of microgreens [1].

4. Conclusions

The presented results indicated that the content of bioactive compounds (phenolics and chlorophylls) and antioxidant activity differed in the studied vegetable microgreens (daikon, i.e., Japanese radish; Chinese red radish; pea; beetroot; and onion). The highest content of total phenolics (639.85 mg GAE/100 g DW), total chlorophylls (281.70 mg/100 g DW), chlorophyll a (202.17 mg/100 g DW) and chlorophyll b (79.53 mg/100 g DW), as determined spectrophotometrically, was found in beetroot microgreens. According to HPLC analysis, the highest content of total phenolics (i.e., phenolic acids) (577.0 mg/100 g DW) was also observed in beetroot microgreens. In addition, in beetroot microgreens, a significant content of betalains (57.27 mg/100 g DW), betacyanins (43.58 mg BE/100 g DW) and betaxanthins (13.68 mg VE/100 g DW) was found. As expected, according to the determined contents of antioxidant compounds (phenolics, chlorophylls and betalains), among the studied vegetable microgreens, beetroot microgreens showed the highest antioxidant activity in all applied assays. A significant correlation was observed for the content of total chlorophylls and chlorophyll a with antioxidant activity in all assays, as well as for the content of chlorophyll b with antioxidant activity in the ABTS assay. A highly significant correlation was found for the content of total phenolics and phenolic acids, determined by HPLC analysis, and antioxidant activity by all assays, as well as for the content of sinapic, rosmarinic and gentisic acids and antioxidant activity, as evaluated by DPPH and RP assays, indicating that these compounds were the most important contributors to the antioxidant activity of the investigated vegetable microgreens. The obtained information of phenolic profiles, overall pigment compositional constitution, and antioxidant activity of the studied vegetable microgreens can be used in contribution to the rapidly growing microgreen industry, as the relative abundance of bioactive compounds across species and its implications for their functional quality may support future species selection. Accordingly, this study provides an assessment of the variation encountered across five vegetable microgreens species. In future research, it would be beneficial to evaluate bioavailability and stability during the storage of bioactive compounds in lyophilized microgreens, in order to choose a suitable delivery system of bioactive compounds in powder form to be used as functional ingredient in the formulation of innovative food products. Certainly, the investigated vegetable microgreens are abundant in bioactive compounds, which can provide protection against different chronic and metabolic diseases. Due to this, the inclusion of microgreens in the daily diet offers the possibility of improving human health.