Enhancing the Growth, Bioactive Compounds, and Antioxidant Activity of Kangkong (Ipomoea aquatica Forssk.) Microgreens Using Dielectric Barrier Discharge Plasma

Abstract

Enhancing the nutraceutical value of health-promoting foods is a strategy to mitigate non-communicable diseases (NCDs), which pose a global health threat. This study aimed to improve the growth, bioactive compound content, and antioxidant activity of kangkong (Ipomoea aquatica Forssk.) microgreens through the application of dielectric barrier discharge (DBD) plasma at different treatment durations. Seeds from two cultivars, Pugun 19 (PG) and Banhann (BH), were treated with DBD plasma for 5 to 20 min, compared to untreated seeds as the control. DBD plasma treatments had no significant effect on the dry weight of BH, whereas a 10 min treatment resulted in the highest dry weight in PG. Principal component analysis exhibited that treating PG seeds with 5 min of DBD plasma increased coumaric acid, total flavonoids, and DPPH and FRAP activities. Meanwhile, exposing BH seeds to 10 min DBD plasma treatment enhanced carotenoids content, as well as ABTS and antiglycation activities. Based on these results, the optimal time for DBD plasma treatment to improve the quality of kangkong microgreens was 5 min for PG and 10 min for BH. These findings indicate that DBD plasma treatment offers potential applications in sustainable agriculture and food biofortification.

1. Introduction

Rapid economic and societal changes have led to a convergence of behavioral risk factors, contributing significantly to the occurrence of non-communicable diseases (NCDs). These NCDs, including hypertension, elevated cholesterol, obesity, cancer, and diabetes, are significant global contributors to mortality and morbidity [1]. Addressing NCDs aligns with sustainable development goals and supports global economic and health security. Prioritizing personal well-being through a nutraceutical-rich diet of fruits and vegetables can prevent and reduce disease risks, as plants play a key role in this through their bioactive compounds [2]. The increasing focus on adopting healthier eating habits has ignited a worldwide passion for fresh, functional, and nutritious foods, especially microscale vegetables such as microgreens with superior phytochemical and nutrient density [3]. Microgreens are edible plants harvested at an early stage of growth, typically when the first and/or second true leaves or cotyledons have fully developed [4]. Recent studies have highlighted microgreens as an abundant source of functional components, with polyphenolic compounds and carotenoids being the primary active antioxidant compounds [3]. Considering these diverse properties, efforts are being made to find techniques to enhance the phytochemical content in microgreens, particularly in kangkong (Ipomoea aquatica). This could lead to their potential development as a future supplement food.

Kangkong is a highly nutritious leafy green, widely cultivated and consumed for its rich content of vitamins, minerals, fiber, protein, and carbohydrates [5]. The substance is characterized by high levels of phenolic compounds and flavonoids, as well as specific phenolic acids. It is associated with remarkable antioxidant activity and exhibits medicinal properties, such as inhibitory effects on α-glucosidase, antidiabetic effects, antiglycation properties, and anti-inflammatory capabilities [6,7]. To enhance bioactive compounds in microgreens, numerous methods exist, including the application of elicitors such as salicylic acid [8], methyl jasmonate, jasmonic acid [9], and hydrogen peroxide [10], as well as plasma treatment [11,12,13,14,15,16,17].

Plasma technology represents an innovative and eco-friendly approach, representing an emerging technology with diverse applications across various fields, such as semiconductor manufacturing, medicine, agriculture, and environmental decontamination [18,19,20]. Dielectric barrier discharge (DBD) plasma is a widely employed cold plasma technique. It is produced by gathering and releasing ions on a dielectric using alternating current (AC) electricity, characterized by its oscillating flow of electrons, which is employed in this process at a high voltage, usually under ambient air pressure or in alternative gaseous environments [21]. Plasma treatment releases crucial reactive oxygen species (ROS), including superoxide (O₂⁻), hydroxy radical (OH•), and hydrogen peroxide (H₂O₂). These components function as effective elicitors, stimulating processes such as seed germination, seedling growth, bioactive compound production, and metabolism [22]. Recent research has demonstrated the potential of non-thermal plasma, particularly DBD plasma, to enhance seed germination, seedling vigor, and the accumulation of bioactive compounds in various crops. DBD plasma treatments for 2 to 3 min improved radish seed germination and seedling growth [23]. In spinach, plasma exposure for 30 s to 3 min increased polyphenol and chlorophyll contents [11], while treatments of 120–240 s enhanced phenolic and flavonoid levels in sunflower sprouts [13]. Similarly, mustard green seedlings from seeds exposed to cold plasma for 5 min showed higher levels of phenolic, flavonoid, and antioxidant activity [14]. DBD plasma treatment of wheat seeds for 5 min resulted in increased root length and higher contents of chlorophyll, carotenoids, phenolics, and flavonoids, although no significant change in DPPH activity was observed [15]. In radish microgreens, a 2 min low-temperature argon plasma treatment increased chlorophyll a, b, phenolics, flavonoids, and antioxidant capacity (DPPH and FRAP), while extending the exposure to 3 min reduced these benefits [16]. In lettuce, NTP treatments at both low (2 L/min) and high (5 L/min) ionization levels enhanced chlorophyll and carotenoid content, although total phenolics and antioxidant capacity remained unchanged [17].

Incorporating insights from these studies, it is evident that the optimal plasma exposure time for enhancing seedling quality, bioactive compound accumulation, and antioxidant efficacy varies among plant species. In our previous study, DBD plasma treatment for 5 min was identified as the most effective condition for promoting seed germination in kangkong [24]. However, the effects of varying durations of DBD plasma treatment on the biochemical composition and antioxidant activity of kangkong microgreens have not yet been explored. We hypothesize that DBD plasma treatment enhances the growth and biochemical traits of kangkong by modulating physiological and antioxidative pathways. Therefore, the aim of this study was to investigate the effects of different durations of DBD plasma treatment on the growth, bioactive compound content, antioxidant activity, and antiglycation properties of kangkong microgreens.

2. Materials and Methods

2.1. Setup for an Experiment, Initial Seed Sample, and Plasma Treatment

The dielectric barrier discharge (DBD) plasma device was set up following the methodology outlined by Ongrak et al. [24]. A schematic of the DBD apparatus is shown in Figure 1. The DBD plasma generator comprises (a) a high-voltage power supply (Trek 30/20A) operating at 5.5 kV and 4.0 kHz; (b) two electrodes—a bottom electrode (copper plate, 10 × 10 × 0.2 cm) and a top electrode (copper immersed in normal saline); and (c) a dielectric material, specifically a glass box. The distance between the bottom electrode and the glass box was fixed at 6 mm. The DBD generator operates under atmospheric pressure. The two kangkong cultivars, Pugun 19 (PG) and Banhann (BH), originated from seeds produced in the Sukhothai and Suphan Buri provinces of Thailand, respectively. PG seeds were obtained from Home Seeds Corporation Co. Ltd. located in Pathum Thani, Thailand, while a local seed supplier in Suphan Buri, Thailand, provided BH seeds. Three hundred seeds were evenly distributed in a single layer on a 10 × 10 cm copper plate within the generator. These seeds were treated with DBD plasma for 5, 10, 15, and 20 min. Untreated seeds were used as the control.

2.2. Growth of Microgreens

Seeds weighing 8 g were planted in a 10 × 15 × 7 cm plastic box, filled with about 4 cm of moist peat moss (REKYVA Peat Substrate Professional, Šiauliai, Lithuania). The peat moss was 100% milled peat with a black color and medium particle size. It had a pH of 5.47 ± 0.03 and an EC of 0.98 ± 0.01 mS/cm. Kangkong microgreens were grown at ambient temperature (30 °C) under fluorescent light with a 12/12 h light/dark photoperiod, providing a photosynthetically active radiation of 13.92 µmol/m²/s and 70% relative humidity. They were watered daily with 10 mL per plastic box. After ten days, the microgreens were harvested by cutting the stems above the peat moss. Shoot length was measured by sampling ten plants per replication, and the fresh weight was recorded for the entire box. The microgreens were kept at 70 °C for 72 h in a hot air oven to determine the dry weight. Fresh and dry weights were reported in kg/m² and g/m², respectively. The remaining microgreens were dipped in liquid nitrogen and then freeze-dried to obtain dehydrate microgreens using a freeze-dryer (Gold-sim FD5-9, Gold Sim Cellular Science LLC, Miami, FL, USA), and subsequently finely ground with a pestle and mortar to create a powder for extraction.

2.3. Chlorophyll and Carotenoids Contents

The contents of chlorophyll and carotenoids were assessed and adapted from Gunathilake and Ranaweera [25]. One gram of sample powder was mixed with 5 mL of 95% ethanol. The supernatant was measured at 470, 665, and 649 nm using a spectrophotometer (Shimadzu Europe UV-1208, Established Shimadzu Aerotech Manufacturing, Inc., Kyoto, Japan). The chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents were calculated based on formulas established by Lichtenthaler and Wellburn [26] and reported as µg/g dry weight.

2.4. Crude Extraction

One gram of sample powders was extracted in 75 mL of 70% ethanol and filtered through Whatman No.1 filter paper. The supernatant was evaporated using an Evaporator^® R-300 (Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd., Zhengzhou, China), then further dried with a freeze dryer to obtain the crude extract for the evaluation of bioactive compounds, as well as antioxidant and antiglycation activities [27].

2.5. Total Ascorbic Acid Content

The analysis of total ascorbic acid was evaluated using a method adapted from Kammapana et al. [28]. A 0.1 g quantity of crude extract was dissolved in 0.2 mL of 5% metaphosphoric acid. Then, 0.02% indophenol solution was added, and the mixture was incubated for 3 min. Subsequently, 2% thiourea and 2% 2,4-dinitrophenylhydrazine (DNP) were added. The mixture was incubated in a water bath at 50 °C for 70 min, followed by adding 85% sulfuric acid and kept at room temperature for 30 min. The supernatants were measured at 540 nm using the spectrophotometer. Total ascorbic acid was quantified utilizing the linear standard curve for ascorbic acid and reported as mg/g dry extract.

2.6. Total Phenolic Content

Total phenolic content was assessed using the Folin–Ciocalteu method [13]. The extract solution was mixed with a Folin reagent, 7.5% Na₂CO₃, and stored in darkness at room temperature. The reaction was estimated after 30 min at 765 nm with a 96-well microplate reader (BioTek-PowerWave XS, Marshall Scientific LLC, Hampton, NH, USA). Total phenolic content was reported as mg gallic acid equivalent (GAE)/g dry extract.

2.7. Total Flavonoid Content

The total flavonoid content was assessed by the aluminum chloride colorimetric method [13]. The extract solution was mixed with 5% NaNO₂ and left for 6 min, followed by the addition of 10% AlCl₃ and left for 6 min. Then, 1 M NaOH and distilled water were added. The reaction was estimated after 15 min at 510 nm by a 96-well microplate reader. The total flavonoid content was reported as mg quercetin equivalent (QE)/g dry extract.

2.8. Phenolic Profiling by HPLC

The extract solution was prepared in ethanol and filtered through a 0.45 µm syringe filter before injection into the HPLC system. The method used to analyze phenolic compounds was based on Kubola and Siriamornpun [29]. The reversed-phase HPLC system (Shimadzu Co., Ltd., Tokyo, Japan) consisted of Shimadzu LC-20AC pumps, an SPD-M20A diode array detector, and an Acclaim C18 column (GL Sciences Inc., Tokyo, Japan) with a particle size of 5 µm and dimensions of 250 × 4.6 mm². The mobile phase comprised acetonitrile as solvent A and purified water with orthophosphoric acid (pH 2.0) as solvent B, with a flow rate of 1 mL/min. Gradient elution was performed as follows: from 0 to 53 min, a linear gradient from 5% to 70% solvent A; from 52 to 60 min, a linear gradient from 70% back to 5% solvent A. The column temperature was set at 38 °C, the injection volume was 20 µL, and UV diode array detection was conducted at 280 nm for chlorogenic acid and coumaric acid, and at 360 nm for rutin, ferulic acid, and caffeic acid. Phenolic compounds were identified using an external standard method by comparing retention times and UV spectra with authentic standards.

2.9. Antioxidant Activity

2.9.1. DPPH Assay

The antioxidant activity was estimated using the DPPH radical scavenging assay [30]. The extract solution was mixed with the DPPH solution and stored in darkness for 30 min at room temperature. The reaction was recorded at 517 nm by a 96-well microplate reader. The percentage of inhibition was calculated using the formula: % inhibition = [(Abs_control − Abs_sample)/Abs_control] × 100.

2.9.2. FRAP Assay

The FRAP assay was performed based on the method described by Alrifai et al. [31]. The extract solution was mixed with the FRAP reagent. The reaction was measured after 8 min at 595 nm by a 96-well microplate reader. The ferric-reducing ability of the extracts was established from the linear standard curve ascorbic acid equation and reported as µmol ascorbic acid equivalent (AsAE)/mg dry extract.

2.9.3. ABTS Assay

Decolorization of ABTS^•+ was determined using the method described by Puccinelli et al. [32]. ABTS reagent was prepared by dissolving ABTS^•+ in distilled water and combining it with potassium persulfate dissolved in distilled water. After being stored in the darkness for 12–16 h at room temperature, the mixture was diluted to achieve an absorbance ranging from 0.68 to 0.72 at 734 nm. The extract solution was mixed with ABTS reaction and incubated for 6 min at room temperature. The reaction was determined at 734 nm by a 96-well microplate reader. The percentage of inhibition was calculated using the formula: % inhibition = [(Abs_control − Abs_sample)/Abs_control] × 100.

2.10. Antiglycation Activity

Antiglycation activity was determined using the glucose assay method adapted from Rahbar et al. [33] and Wu and Yen [34]. The bovine serum albumin (BSA) control solution comprised BSA (50 mg/mL) and D-glucose (0.8 M) in a phosphate buffer (1.5 M, pH 7.4). The crude extract and positive controls (aminoguanidine and rutin, both at 1 mg/mL) were dissolved in phosphate buffer (1.5 M, pH 7.4) in the test solution and sterilized with 0.3 g/L NaN₃. Both solutions underwent incubation at 37 °C for 7 days. A fluorescence spectrophotometer (Thermo Scientific Varioskan LUX, Thermo Fisher Scientific, Waltham, MA, USA) was used to assess the fluorescence intensity with excitation at 330 nm and emission at 410 nm. The estimation of advanced glycation end-product inhibition was conducted as follows: % inhibition = 1 − [(fluorescence of the solution with inhibitors)/(fluorescence of the solution without inhibitors)] × 100.

2.11. Experimental Design and Statistical Analysis

The experimental design followed a 2 × 5 factorial in a completely randomized design. Factor A consisted of two kangkong cultivars, PG and BH, while factor B included five DBD plasma treatment durations: 0, 5, 10, 15, and 20 min. Three replications were conducted, with one plastic box representing each replication. Growth, bioactive compounds, and antioxidant analyses were performed in triplicate. Statistical assumptions, such as normality and homogeneity, were validated using the Shapiro–Wilk test and Levene’s test, respectively. When the normality assumption was not met, the Kruskal–Wallis test was employed. Results were considered statistically significant at p < 0.05. Pairwise comparisons were conducted using Dunn’s test. Data analysis was performed using the Statistix program (ver. 10.0, Analytical Software, Tallahassee, FL, USA). For data that satisfied the normality assumption, analysis was performed using IBM SPSS Statistics ver. 21.0 (IBM Corp., Armonk, NY, USA). Analysis of variance (ANOVA) was conducted, and mean separation was carried out using Tukey’s Honestly Significant Difference (HSD) test at a significance level of p < 0.05. Principal component analysis (PCA) was performed using JMP statistical software (ver. Free trial, JMP^®, SAS Institute Inc., Cary, NC, USA). Effect size for each principal component (PC) was calculated based on the proportion of variance explained (PVE), using the following formula, PVE for PC_i = ${\lambda }_i/\sum _{j=1}^n{\lambda }_j$, where λ_i is the eigenvalue associated with the i-th PC, $\sum _{j=1}^n{\lambda }_j$ is the sum of all the eigenvalues from all PCs and n is the total number of components. Bootstrap methods were used to generate 95% confidence intervals (CIs) for the variance explained by PC1 and PC2 using MATLAB R2024b (trial version, MathWorks, Natick, MA, USA). Pearson correlation coefficients between 19 variables and PC1 or PC2 were computed using the corr function in MATLAB R2024b to assess the strength and direction of the relationships.

3. Results

3.1. Growth of Microgreens

The interaction between cultivar and DBD plasma treatment was found in the shoot length and dry weight of kangkong microgreens (

Table 1. Shoot length (SL), fresh weight (FW), and dry weight (DW) of kangkong microgreens cv. PG and BH derived from seeds treated with DBD plasma for different durations.

Cultivar (A)	DBD Plasma Treatment (min; B)	SL (cm)	FW (kg/m2)	DW (g/m2)
PG		12.37 ± 0.53 b 1	2.99 ± 0.59 b 2	169.45 ± 15.18 a 1
BH		12.81 ± 0.41 a	3.25 ± 0.27 a	160.74 ± 6.81 b
	Untreated	12.04 ± 0.54 c	2.97 ± 0.25	160.23 ± 5.40 ab
	5	13.00 ± 0.18 a	3.11 ± 0.96	167.78 ± 13.86 a
	10	13.02 ± 0.37 a	3.25 ± 0.09	165.96 ± 13.06 ab
	15	12.37 ± 0.32 b	3.23 ± 0.94	165.49 ± 9.36 ab
	20	12.27 ± 0.11 bc	3.15 ± 0.37	155.21 ± 5.00 b
PG	Untreated	11.62 ± 0.18 f	2.89 ± 0.23	161.06 ± 7.31 b
	5	13.12 ± 0.13 ab	3.18 ± 3.28	177.10 ± 6.17 ab
	10	12.66 ± 0.11 b–e	3.30 ± 3.38	192.03 ± 2.22 a
	15	12.15 ± 0.23 ef	3.19 ± 3.29	174.01 ± 5.45 ab
	20	12.32 ± 0.11 c–e	2.99 ± 0.08	151.85 ± 3.44 b
BH	Untreated	12.68 ±0.13 b–e	3.03 ± 0.25	159.94 ± 1.72 b
	5	12.83 ± 0.04 bc	3.07 ± 0.16	155.22 ± 9.90 b
	10	13.38 ± 0.04 a	3.25 ± 0.02	168.00 ± 1.10 b
	15	12.69 ± 0.04 b–d	3.26 ± 0.22	163.14 ± 5.61 b
	20	12.21 ± 0.06 de	3.33 ± 0.08	162.63 ± 0.82 b
	A	**	*	**
	B	**	ns	**
	A × B	**	ns	*

¹ Means ± SD within each column sharing different letters are significantly different at p < 0.05 by Tukey’s test. ² Medians ± interquartile range (IQR) within column sharing different letters are significantly different at p < 0.05 by Dunn’s test. ‘ns’ denotes non-significance, ‘**’ indicates significance at p < 0.01, and ‘*’ signifies significance at p < 0.05.

). DBD plasma treatments for 5 to 20 min significantly increased shoot length in PG by 4.56% to 12.91%, while treatments for 5 to 10 min statistically enhanced it in BH by 1.18% to 5.52% over the control. The 10 min DBD plasma treatment increased the dry weight of PG by 19.23% compared to the control, whereas the 20 min treatment resulted in a significant reduction. No significant differences in dry weight were observed for BH across DBD plasma treatments. There was no interaction between cultivar and DBD plasma treatment on fresh weight (Table 1). The fresh weight of BH microgreens was higher than that of PG, and there was no significant difference among the DBD plasma treatments.

3.2. Chlorophyll and Carotenoids Contents

There was no interaction between cultivar and DBD plasma treatment in the chlorophyll and carotenoids contents of kangkong microgreens (

Table 2. Chlorophyll a (ChA), chlorophyll b (ChB), total chlorophyll (TCh), and carotenoids (CAR) contents of kangkong microgreens cv. PG and BH derived from seeds treated with DBD plasma for different durations.

Cultivar (A)	DBD Plasma Treatment (min; B)	ChA	ChB	TCh	CAR
		(µg/g Dry Weight)
PG		19.93 ± 0.98	31.98 ± 3.53	51.70 ± 2.63	27.13 ± 1.54
BH		19.55 ± 0.91	32.48 ± 4.07	52.03 ± 3.18	26.96 ± 2.15
	untreated	20.76 ± 0.36 a 1	27.32 ± 2.36 b	48.04 ± 2.00 b	24.41 ± 1.35 b
	5	19.85 ± 0.94 ab	31.47 ± 3.80 ab	51.32 ± 2.85 ab	27.13 ± 1.53 ab
	10	19.25 ± 1.19 b	35.59 ± 2.56 a	54.38 ± 1.91 a	29.57 ± 1.37 a
	15	19.30 ± 0.76 ab	33.67 ± 2.97 a	52.96 ± 2.22 a	27.57 ± 1.60 a
	20	19.53 ± 0.36 ab	32.93 ± 1.62 a	52.47 ± 1.28 a	27.34 ± 0.84 a
	A	ns	ns	ns	ns
	B	*	**	**	*
	A × B	ns	ns	ns	ns

¹ Means ± SD within each column sharing different letters are significantly different at p < 0.05 by Tukey’s test. ‘ns’ denotes non-significance, ‘**’ indicates significance at p < 0.01, and ‘*’ signifies significance at p < 0.05.

). No significant differences in chlorophyll a, chlorophyll b, total chlorophyll, or carotenoid levels were observed between the two cultivars. However, the chlorophyll and carotenoid contents varied significantly depending on the DBD treatment duration. The 10 min DBD plasma treatment showed a significantly lower chlorophyll a content compared to the control. DBD plasma treatments for 10 to 20 min increased chlorophyll b by 20.53% to 30.27%, total chlorophyll by 9.22% to 13.19%, and carotenoids levels by 12.00% to 21.13% compared to the control.

3.3. Bioactive Compounds

3.3.1. Total Ascorbic Acid Content

The interaction between the cultivar and DBD plasma treatment significantly affected the content of ascorbic acid (

Table 3. Total ascorbic acid (AsA) content, total phenolic content (TPC), and total flavonoid content (TFC) of kangkong microgreens cv. PG and BH derived from seeds treated with DBD plasma for different durations.

Cultivar (A)	DBD Plasma Treatment (min; B)	AsA (mg/g Dry Extract)	TPC (mg GAE/g Dry Extract)	TFC (mg QE/g Dry Extract)
PG		285.67 ± 13.33 b 1	22.71 ± 2.69 a 2	238.40 ± 22.00 a 1
BH		312.33 ± 11.33 a	21.04 ± 2.45 b	202.40 ± 30.00 b
	Untreated	274.33 ± 15.50 b	18.36 ± 0.84 c	181.40 ± 32.00 b
	5	304.34 ± 15.99 a	25.90 ± 1.16 a	211.40 ± 64.50 ab
	10	303.34 ± 31.50 a	21.99 ± 1.05 b	219.40 ± 33.50 ab
	15	304.00 ± 39.33 ab	21.31 ± 1.27 b	249.40 ± 24.00 a
	20	299.34 ± 22.00 ab	21.82 ± 1.70 b	220.40 ± 39.00 ab
PG	Untreated	270.33 ± 15.33 b	18.48 ± 1.16	208.40 ± 32.00 ab
	5	297.67 ± 5.34 ab	26.41 ± 1.48	242.00 ± 14.00 ab
	10	289.00 ± 3.33 ab	22.88 ± 0.18	238.40 ± 12.00 ab
	15	279.00 ± 14.00 ab	22.30 ± 0.48	236.40 ± 28.00 ab
	20	285.67 ± 6.00 ab	23.46 ± 0.25	242.40 ± 18.00 ab
BH	Untreated	274.33 ± 14.00 ab	18.24 ± 0.14	178.40 ± 0.00 b
	5	312.33 ± 1.33 ab	25.39 ± 0.00	180.40 ± 4.00 b
	10	319.67 ± 2.00 a	21.09 ± 0.76	206.40 ± 8.00 ab
	15	318.33 ± 3.33 ab	20.32 ± 1.02	256.40 ± 6.00 a
	20	307.37 ± 0.67 ab	20.17 ± 0.54	208.40 ± 6.00 ab
	A	**	**	**
	B	**	**	**
	A × B	**	ns	**

¹ Medians ± IQR within each column sharing different letters are significantly different at p < 0.05 by Dunn’s test. ² Means ± SD within column sharing different letters are significantly different at p < 0.05 by Tukey’s test. ‘ns’ denotes non-significance, ‘**’ indicates significance at p < 0.01.

). However, there was no significant difference in the total ascorbic acid content of PG and BH across the DBD plasma treatments.

3.3.2. Total Phenolic Content

There was no interaction between the cultivar and DBD plasma treatment in terms of total phenolic content (Table 3). The total phenolic content of the PG was higher than that of the BH. The 5 min DBD plasma treatment resulted in the highest increase in total phenolic content, with a 41.07% increase over the control. DBD plasma treatments for 10 to 20 min enhanced the content by 16.07% to 19.77% compared to the control.

3.3.3. Total Flavonoid Content

The interaction between the cultivar and DBD plasma treatment had a significant impact on the total flavonoid content (Table 3). There was no significant difference in the total flavonoid content of PG across the DBD plasma treatments. However, a 15 min treatment significantly increased the content in BH by 43.73% compared to the control.

3.3.4. Phenolic Profile

The interaction between cultivar and DBD plasma treatment significantly affected the contents of caffeic acid and ferulic acid. However, no significant interaction was detected in the contents of chlorogenic acid, coumaric acid, and rutin (

Table 4. Chlorogenic acid (CHL), coumaric acid (COU), caffeic acid (CAF), ferulic acid (FER), and rutin (RUT) contents of kangkong microgreens cv. PG and BH derived from seeds treated with DBD plasma for different durations.

Cultivar (A)	DBD Plasma Treatment (min; B)	CHL	COU	CAF	FER	RUT
		(mg/g Dry Extract)
PG		0.44 ± 0.06	0.19 ± 0.04 a	0.14 ± 0.03 b	0.79 ± 0.19 b	0.25 ± 0.03
BH		0.48 ± 0.05	0.16 ± 0.03 b	0.17 ± 0.04 a	1.09 ± 0.30 a	0.24 ± 0.03
	Untreated	0.38 ± 0.05 b 1	0.11 ± 0.01 b	0.11 ± 0.01 b	0.57 ± 0.11 b	0.24 ± 0.02
	5	0.48 ± 0.02 a	0.19 ± 0.02 a	0.16 ± 0.01 a	1.04 ± 0.38 a	0.28 ± 0.03
	10	0.46 ± 0.02 a	0.18 ± 0.02 a	0.18 ± 0.02 a	1.14 ± 0.10 a	0.24 ± 0.02
	15	0.46 ± 0.04 a	0.19 ± 0.01 a	0.16 ± 0.04 a	1.04 ± 0.11 a	0.25 ± 0.03
	20	0.52 ± 0.02 a	0.20 ± 0.04 a	0.16 ± 0.04 a	0.97 ± 0.19 a	0.23 ± 0.01
PG	Untreated	0.35 ± 0.00	0.11 ± 0.00	0.10 ± 0.01 c	0.46 ± 0.01 e	0.22 ± 0.01
	5	0.47 ± 0.06	0.20 ± 0.02	0.16 ± 0.01 a–c	0.74 ± 0.01 de	0.29 ± 0.03
	10	0.46 ± 0.00	0.20 ± 0.01	0.18 ± 0.03 ab	1.08 ± 0. 06 bc	0.25 ± 0.01
	15	0.44 ± 0.01	0.20 ± 0.01	0.13 ± 0.02 bc	0.93 ± 0.01 b–d	0.27 ± 0.02
	20	0.51 ± 0.01	0.23 ± 0.02	0.13 ± 0.00 bc	0.78 ± 0. 01 cd	0.24 ± 0.02
BH	Untreated	0.43 ± 0.04	0.10 ± 0.01	0.11 ± 0.01 c	0.65 ± 0.09 de	0.25 ± 0.02
	5	0.49 ± 0.04	0.18 ± 0.02	0.15 ± 0.01 a–c	1.48 ± 0.17 a	0.28 ±0.01
	10	0.47 ± 0.03	0.16 ± 0.00	0.18 ± 0.00 ab	1.20 ± 0.10 ab	0.23 ± 0.02
	15	0.50 ± 0.04	0.18 ± 0.01	0.19 ± 0.03 ab	1.15 ± 0.02 b	0.23 ± 0.02
	20	0.52 ± 0.02	0.16 ± 0.01	0.21 ± 0.01 a	1.16 ± 0.02 b	0.23 ± 0.01
	A	ns	**	**	**	ns
	B	**	**	**	**	ns
	A × B	ns	ns	**	**	ns

¹ Means ± SD within each column sharing different letters are significantly different at p < 0.05 by Tukey’s test. ‘ns’ denotes non-significance, ‘**’ indicates significance at p < 0.01.

).

The 10 min DBD plasma treatment significantly increased caffeic acid levels in PG by 80.00%, while 10 to 20 min DBD treatments enhanced the levels in BH by 63.64% to 90.91% compared to the control (Table 4). DBD plasma treatments for 10 to 20 min significantly boosted ferulic acid content in PG by 69.56% to 134.78%, whereas 5 to 20 min DBD treatments statistically increased the content in BH by 76.92% to 127.69%, compared to the control (Table 4).

The contents of chlorogenic acid and rutin did not differ between the two cultivars. However, PG exhibited a higher content of coumaric acid than the BH. Regarding the DBD plasma treatment duration, treatments for 5 to 20 min resulted in a significant increase in chlorogenic acid content (21.05% to 36.84%) and coumaric acid content (63.64% to 81.82%) compared to the control. On the other hand, rutin content showed no significant differences among the DBD plasma treatments (Table 4).

3.4. Antioxidant Activity

The interaction between cultivar and DBD plasma treatment was evident in the DPPH and ABTS assays (

Table 5. Antioxidant activity using DPPH, ABTS, and FRAB assays, and antiglycation activities (AG) of kangkong microgreens cv. PG and BH derived from seeds treated with DBD plasma for different durations.

Cultivar (A)	DBD Plasma Treatment (min; B)	DPPH (% Inhibition)	ABTS (% Inhibition)	FRAP (µmol AsAE/ mg Dry Extract)	AG (% Inhibition)
PG		56.06 ± 6.38 a 1	35.60 ± 2.38 1	23.43 ± 1.62 a 1	58.49 ± 3.66 2
BH		43.98 ± 6.76 b	36.73 ± 5.56	21.51 ± 1.74 b	60.70 ± 4.24
	Untreated	40.71 ± 5.80 c	30.16 ± 2.27 c	19.76 ± 1.15 b	52.12 ± 6.35 b
	5	50.65 ± 8.46 ab	35.10 ± 1.34 b	22.38 ± 1.70 a	59.53 ± 2.56 ab
	10	47.19 ± 8.09 b	28.78 ± 4.53 a	23.71 ± 1.29 a	57.44 ± 3.29 ab
	15	53.91 ± 2.30 a	38.59 ± 1.86 a	23.23 ± 1.51 a	61.33 ± 1.03 a
	20	55.64 ± 9.61 a	37.96 ± 1.34 ab	22.98 ± 1.26 a	61.67 ± 1.70 a
PG	Untreated	45.87 ± 4.30 c–e	31.95 ± 0.80 cd	20.57 ± 0.72	56.77 ± m ab
	5	57.45 ± 2.22 ab	35.07 ± 1.34 bc	24.02 ± 0.57	58.33 ± m ab
	10	54.54 ± 1.50 a–c	35.37 ± 0.57 bc	24.99 ± 0.19	57.27 ± 0.18 ab
	15	54.19 ± 3.84 bc	38.49 ± 1.68 ab	23.54 ± 0.40	61.65 ± m ab
	20	64.84 ± 4.74 a	37.12 ± 0.53 bc	24.04 ± 0.89	60.91 ± 1.81 ab
BH	Untreated	35.56 ± 3.18 e	27.46 ± 0.65 d	18.53 ± 0.23	50.59 ± 1.87 b
	5	40.45 ± 1.12 de	35.14 ± 1.90 bc	20.73 ± 0.08	60.27 ± 2.51 ab
	10	39.83 ±5.64 de	43.89 ± 3.87 a	22.44 ± 0.26	60.51 ± 3.28 ab
	15	53.63 ± 0.95 bc	38.75 ± 3.39 ab	22.92 ± 2.05	61.33 ± 0.61 ab
	20	46.44 ± 0.61 cd	39.21 ± 1.77 ab	21.92 ± 0.39	62.08 ± 1.16 a
	A	**	ns	**	ns
	B	**	**	**	**
	A x B	**	**	ns	**

¹ Means ± SD within each column sharing different letters are significantly different at p < 0.05 by Tukey’s test. ² Medians ± IQR within column sharing different letters are significantly different at p < 0.05 by Dunn’s test. ‘m’ denotes missing data, ‘ns’ denotes non-significance, ‘**’ indicates significance at p < 0.01. For antiglycation activity, aminoguanidine (74.01 ± 0.94% inhibition) and rutin (80.82 ± 0.24% inhibition) were used as positive controls. Values are expressed as mean ± SD.

). For the DPPH assay, DBD plasma treatments for 5 and 20 min significantly increased antioxidant activity in PG by 25.24% and 41.36%, respectively, whereas treatments for 15 and 20 min improved antioxidant activity in BH by 50.81% and 30.60%, respectively, over the control (Table 5). Regarding the ABTS assay, the 15 min DBD plasma treatment significantly boosted antioxidant activity in PG by 20.46%, compared to the control. DBD plasma treatments for 5 to 20 min statistically enhanced antioxidant activity in BH by 27.97% to 59.83% over the control (Table 5).

In the FRAP assay, no significant interaction was observed between the cultivar and DBD plasma treatment (Table 5). PG exhibited a higher ferric-reducing ability compared to BH. DBD plasma treatments for 5 to 20 min significantly increased this activity by 13.26% to 19.99% compared to the control.

3.5. Antiglycation Activity

An interaction between cultivar and DBD plasma treatment affected the antiglycation activity (Table 5). There was no significant difference in the antiglycation activity of PG across the DBD plasma treatments. However, a 20 min treatment significantly increased the activity in BH by 22.72% compared to the control.

3.6. Principal Component Analysis

Principal component analysis (PCA) was conducted on 19 dependent variables. The variables were obtained from two cultivars of kangkong microgreens grown from untreated seeds and seeds treated with DBD plasma for 5, 10, 15, and 20 min. The first two principal components accounted for 67.0% of the total variation, as shown in Figure 2. The variance explained by PC1 was 49.2% (95% CI: 16.03, 45.11), indicating a large contribution to the total variance. For PC2, the variance explained was 17.8% (95% CI: −28.05, 28.89), suggesting a moderate contribution with some uncertainty, as indicated by the confidence interval range. The effect size for PC1, as indicated by the proportion of variance explained, was 49.2%, suggesting a substantial effect in explaining the variability of the data. In contrast, the effect size for PC2 was 17.8%, which suggested a more limited contribution to the total variance. According to the Pearson correlation coefficients (

Table 6. Pearson correlation coefficients between original variables and principal components of kangkong microgreens cv. PG and BH derived from seeds treated with DBD plasma for different durations.

Variable	PC1 (49.2%)	PC2 (17.8%)	Variable	PC1 (49.2%)	PC2 (17.8%)
Shoot length	0.11	0.89	chlorogenic acid	0.46	−0.66
Fresh weight	0.73	−0.14	coumaric acid	0.46	0.02
Dry weight	0.40	0.18	caffeic acid	0.61	−0.65
Chlorophyll a	0.02	0.54	ferulic acid	0.63	0.19
Chlorophyll b	−0.73	−0.19	rutin	0.71	0.45
Total chlorophyll	0.82	0.10	DPPH	−0.06	−0.45
Carotenoids	0.83	0.09	ABTS	0.84	0.09
Total ascorbic acid	0.82	0.04	FRAP	0.53	−0.60
Total phenolic content	0.65	0.52	antiglycation	0.78	−0.11
Total flavonoid content	0.41	−0.48

Correlation coefficients were interpreted as follows: values > 0.70 were considered strong, 0.40–0.69 as moderate, and <0.40 as weak.

), ABTS (0.84), carotenoids (0.83), total ascorbic acid (0.82), total chlorophyll (0.82), antiglycation activity (0.78), fresh weight (0.73), and rutin (0.71) exhibited strong positive correlations with PC1, whereas chlorophyll b (−0.73) demonstrated a strong negative correlation. These findings suggest that these variables substantially contribute to the variation accounted for by the first principal component. In contrast, shoot length (0.89) displayed a strong positive correlation with PC2, while chlorogenic acid (−0.66), caffeic acid (−0.65), and FRAP (−0.60) exhibited moderate negative correlations. This indicates that the second principal component captures a distinct and independent source of variation relative to PC1.

The loading plot graph (Figure 2) demonstrated that PC1 had a positive correlation with carotenoids, chlorophyll b, total chlorophyll, ABTS, and antiglycation activity, but a negative correlation with chlorophyll a. PC2 showed a positive association with DPPH, FRAP, coumaric acid, and total flavonoids. Based on the PCA score plot, BH microgreens derived from seeds treated with 10 min of DBD plasma were positively clustered in PC1, showing associations with high levels of carotenoids, chlorophyll b, total chlorophyll, ABTS, and antiglycation activity. PG microgreens from seeds exposed to 5 min of DBD plasma showed positive clustering in PC2, indicating associations with high levels of coumaric acid, total flavonoids, DPPH, and FRAP activities. Conversely, microgreens from untreated seeds of both cultivars loaded negatively in PC1, presenting associations with low levels of bioactive compounds and antioxidant and antiglycation activities.

4. Discussion

High-quality seeds, characterized by attributes such as a high germination rate and uniformity in seedling establishment, are typically essential for successful microgreens production. DBD plasma treatment has been reported to increase seed germination and the subsequent seedling growth in various plant species such as radish [23], spinach [11], sunflower [35], black gram [36], rice [37], and wheat [15]. In the current study, the use of DBD plasma to produce high-quality kangkong microgreens was evaluated. Kangkong microgreens are commonly consumed fresh in salads. They are not only rich in nutrients but also contain high levels of bioactive compounds, especially those generated from seeds treated with DBD plasma. As a result, consuming kangkong microgreens grown from DBD plasma-treated seeds may offer health benefits and potentially help prevent or reduce the risk of NCDs.

The optimal parameters for cultivating kangkong microgreens have not yet been clearly established, although growing conditions can greatly impact plant growth and biochemical composition. To minimize environmental variation, we followed the International Seed Testing Association (ISTA) guidelines, maintaining a germination temperature of 30 °C. Other factors—including photoperiod, relative humidity, watering regime, and growth substrate—were kept consistent throughout the experiment. As such, the primary experimental variables were the kangkong cultivar and the duration of DBD plasma treatment. Our results indicated that both cultivar type and treatment duration influenced the growth and bioactive properties of the microgreens. The PG and BH cultivars of kangkong belong to the narrow-leaved segment. Both cultivars are commercially available in Thailand and are supplied by seed companies that typically adhere to genetic purity standards as part of their seed quality certification processes. Their seeds were sourced from different stocks and produced in different locations. These differences may result in variations between the two cultivars. Notably, BH showed no significant change in dry weight across treatments, unlike PG (Table 1). In the case of PG, a 10 min DBD plasma treatment resulted in a significant increase in dry weight, whereas a 20 min treatment led to a substantial reduction. The results of this study were consistent with observations in sunflowers, indicating an increase in seedling growth following exposure to DBD plasma for 5 to 15 s. However, a duration of 30 s led to a decline in growth [35]. For black gram seeds, exposure to DBD plasma for 120 s resulted in the highest seedling growth, while it decreased under 180 s of treatment [36]. Prolonged plasma exposure was associated with oxidative stress, which may have negatively affected biomass accumulation. This assumption is further supported by the elevated levels of malondialdehyde detected in seeds treated with DBD plasma for 20 min, indicating increased lipid peroxidation as a result of oxidative damage [24]. Chlorophyll, an essential pigment, plays a crucial role in photosynthesis, which is vital for plant growth as it provides the primary energy source. The amount and presence of chlorophyll significantly influence photosynthetic capacity, directly affecting plant growth [38]. In the current study, treating seeds with DBD plasma for 5 to 20 min increased chlorophyll b and total chlorophyll; however, this effect was not observed in chlorophyll a. The high chlorophyll b content detected in this study might be attributed to the microgreens receiving fluorescent light during cultivation, as plants in low-light environments typically exhibit a higher chlorophyll b-to-chlorophyll a ratio, which enables them to capture and utilize limited light more effectively [39]. An increase in chlorophyll content not only enhances photosynthetic efficiency and promotes plant growth but also improves the plant’s nutritional value and stress tolerance [38,40]. Chlorophyll is often associated with health-promoting compounds, such as carotenoids and antioxidants, which collectively contribute to the nutritional quality of plants. Carotenoids, including beta-carotene, serve as precursors to vitamin A and, along with chlorophyll, play a crucial role in protecting cells from oxidative stress. Furthermore, elevated chlorophyll levels are indicative of improved stress resilience, as they help mitigate the effects of oxidative damage [40].

In addition, DBD plasma treatment induces seedling growth by initiating biochemical pathways during germination. It is well established that plant growth and development are regulated by phytohormones, particularly auxins and cytokinins [41,42]. The generation of ROS during plasma exposure induces oxidative stress, which, at optimal levels, can activate the shikimate pathway. This activation increases the synthesis of tryptophan, a key precursor in the biosynthesis of indole-3-acetic acid (IAA) [42]. Additionally, Stolárik et al. [43] reported that pea seeds exposed to low-temperature plasma for 2 min exhibited enhanced biosynthesis of both auxins and cytokinins, which contributed to improved germination and early seedling growth. The activation of these biochemical pathways also triggers the release of other hormones, such as gibberellins, as well as hydrolytic enzymes involved in seed germination [11], thereby enabling more efficient utilization of nutrients stored within the seeds. Furthermore, the generation of ROS through plasma treatment promoted plant growth by producing intracellular chemical signals, ultimately enhancing growth [44]. These data suggested that DBD plasma treatment improved the growth of kangkong microgreens through synergistic mechanisms within the plant involving various pathways.

In the case of nutraceutical foods, microgreens are distinguished for their health-promoting secondary metabolites [3]. Most bioactive compounds primarily originate from plants, including phenolic compounds, flavonoids, and ascorbic acid [45]. Upon consumption, these compounds undergo metabolic processes in the body, manifesting synergistic effects that include antioxidant, anti-inflammatory, and antiglycation properties, ultimately contributing to overall health [46,47]. A high concentration of bioactive compounds in kangkong microgreens could result from DBD plasma treatment. The contents of total flavonoid, caffeic acid, and ferulic acid varied depending on the cultivars and DBD plasma duration. However, the duration of DBD plasma treatment did not affect the levels of carotenoids, chlorogenic acid, or coumaric acid, as no significant differences were observed between treatments lasting 5 to 20 min (Table 2 and Table 4). Based on the PCA (Figure 2), the BH microgreens grown from seeds treated with DBD plasma for 10 min had high levels of carotenoids. Meanwhile, the PG microgreens grown from seeds treated with DBD plasma for 5 min had high contents of coumaric acid and total flavonoid. These results indicated that a short duration of DBD plasma treatment, specifically 5 and 10 min, could be optimal for inducing the accumulation of bioactive compounds in kangkong microgreens, depending on cultivars. DBD plasma treatment typically stimulates the production of bioactive compounds through the ROS system [48]. ROS produced during plasma treatment accelerated the accumulation of bioactive compounds by activating the shikimate pathway, which served as a source of phenylalanine, a precursor of the phenylpropanoid pathway [49]. The signal triggered by ROS-activated phenylpropanoid genes, resulting in the upregulation of phenylalanine ammonialyase (PAL), cinnamic acid 4-hydroxylase (C4H), and coumarate 3-hydroxylase (C3H) enzymes in the phenylpropanoid pathway [50]. This enzymatic activation led to the synthesis of phenolic and flavonoid compounds within plants through the flavonoid branch pathway [51]. In our previous study [24], a significant increase in H₂O₂ content and catalase (CAT) activity in kangkong seeds under DBD plasma treatment was observed. H₂O₂ and CAT are associated with the plant’s oxidative stress response [51,52], suggesting that the plasma treatment acts as a mild abiotic stressor. In response to this stress, plants may upregulate the biosynthesis of secondary metabolites, including phenolics and flavonoids, as part of their antioxidant defense system. Enhancement of bioactive compounds by DBD plasma has been reported in various plant species. When sunflower seeds cv. Arfael underwent DBD plasma for 120 and 240 s, resulting in seedlings exhibiting a notable increase in phenolic content by 7.07% and 7.96%, respectively [13]. Iranbakhsh et al. [53] also observed an 82.3% increase in the phenolic content of Capsicum annuum seedlings derived from seeds treated with a 120 s DBD plasma treatment. A 5 min DBD plasma treatment applied to wheat seeds led to elevated levels of chlorophyll, carotenoids, phenolic compounds, and flavonoids [15]. Similarly, treating seeds with DBD plasma for 20 min improved the chlorophyll, carotenoids, and total flavonoid levels in basil seedlings [12]. These suggested that the efficacy of DBD plasma treatment in enhancing bioactive compounds depended on the duration of treatment and the specific plant species.

Plasma treatment has been reported to enhance antioxidant capacity in plants through both enzymatic and non-enzymatic mechanisms. Previous research suggests that plasma can stimulate key protective enzymes such as CAT, superoxide dismutase (SOD), and peroxidase (POD), alongside increasing the synthesis of secondary metabolites like phenolics and flavonoids [54]. In the current study, the kangkong microgreens obtained from seeds treated with DBD plasma for 5 to 20 min demonstrated a marked enhancement in their radical-scavenging ability (Table 5). The significant increase in total antioxidant activity measured by DPPH, ABTS, and FRAP implies an enhanced redox-regulating capacity in response to DBD plasma exposure. The interplay between enzymatic and non-enzymatic antioxidant systems under plasma treatment remains an important avenue for future research. PCA and Pearson correlation analysis (Table 6) revealed that antioxidant-related parameters, such as ABTS (r = 0.84), carotenoids (r = 0.83), total ascorbic acid (r = 0.82), and total chlorophyll (r = 0.82), were strongly associated with PC1. These findings suggest that these compounds substantially contribute to the overall antioxidant capacity observed in kangkong microgreens, highlighting their potential role in enhancing the plant’s antioxidant profile. The results in this study were consistent with those of mustard green and rat-tailed radish microgreens, which were grown from seeds treated with cold plasma for 5 min. According to Saengha et al. [14] and Luang-In et al. [55], these microgreens have high amounts of isothiocyanates and total phenolics, leading to significantly increased antioxidant activities, especially in DPPH and FRAP assays. Meanwhile, Park et al. [56] discovered that germinated brown rice, which came from seeds treated with a corona discharge plasma jet for 10 min, had a higher total phenolic content and increased antioxidant activity as evaluated by the DPPH and ABTS assays, compared to the control. Thus, DBD plasma treatment demonstrated the potential to sustain the plant’s antioxidant activity system, enhancing its resistance to oxidative damage caused by ROS. In addition to the parameters evaluated in this study, the post-harvest stability of antioxidants was not assessed. However, considering the importance of antioxidant retention during storage, future research should explore how antioxidant levels in kangkong microgreens change over time under various storage conditions.

Kangkong exhibits significant potential as a valuable food supplement for preventing and managing diabetes [57]. In the present study, seeds exposed to DBD plasma for 5 to 20 min showed an improvement in the antiglycation activity of microgreens (Table 5). However, when compared to the standard positive controls, aminoguanidine (74.01 ± 0.94%) and rutin (80.82 ± 0.24%), the antiglycation activity of the kangkong microgreens derived from DBD plasma-treated seeds was relatively lower. This may be attributed to the fact that kangkong is a common vegetable rather than a medicinal plant with high levels of potent antiglycation compounds. In the context of PCA and Pearson correlation analysis (Table 6), antiglycation activity exhibited a strong positive correlation with PC1 (r = 0.78), which also showed strong associations with antioxidant-related compounds, including carotenoids (r = 0.83), total ascorbic acid (r = 0.82), and total chlorophyll (r = 0.82). These associations suggest a potential mechanistic link between antioxidant capacity and glycation inhibition. Additionally, ABTS radical scavenging activity was strongly correlated with PC1 (r = 0.84), further supporting the role of antioxidant compounds in contributing to antiglycation effects in kangkong microgreens. Antioxidants play a crucial role in protecting against free radicals generated from glycation, and they have been proposed as potential therapeutic agents [58]. A more effective approach to treating diabetes involves utilizing compounds with antioxidant and antiglycation properties rather than addressing each factor individually [59]. In addition, plasma treatment generates ROS that enhance the activity of antioxidant enzymes in plants, which inhibit glycation. This increased enzymatic activity disrupts the interaction between sugars and proteins, reducing glycation and ultimately improving the plant’s antiglycation activity [60]. The results in this study were consistent with Ephedra fragilis, where the presence of total phenolic and flavonoid contents contributed to the improvement of antiglycation efficiency [61]. Consequently, kangkong microgreens grown from DBD plasma-treated seeds can be used for the prevention and regulation of diabetes.

DBD plasma treatment has emerged as a promising technology for enhancing the growth, bioactive compound contents, and antioxidant and antiglycation activities in kangkong microgreens. The impact of DBD plasma treatment varied depending on the cultivar and treatment duration, suggesting that future studies should investigate the optimal parameters for different cultivars and growth conditions. This technique shows promise for enhancing the bioactive and health-promoting properties of kangkong microgreens, potentially making them a nutritionally beneficial food for consumers. In addition, the DBD system shows potential for commercial application, particularly with the integration of a conveyor system to facilitate the continuous movement of seeds into and out of the plasma treatment zone. However, scalability in commercial settings may face challenges such as the need for consistent energy output, uniform treatment distribution, and the handling of large volumes of seeds. These factors must be addressed to ensure efficient and cost-effective large-scale implementation. For food safety, plasma is a non-thermal, eco-friendly technology that does not leave toxic residues when applied under appropriate conditions. However, long-term toxicity and potential byproducts still require further investigation to fully ensure consumer safety [62,63].

5. Conclusions

Our study demonstrates that DBD plasma treatment significantly enhances the growth, bioactive compound content, and antioxidant and antiglycation activities of kangkong microgreens. Given its potential to improve microgreen quality without chemical treatments, DBD plasma emerges as a promising technology for enhancing the nutritional and functional properties of microgreens. It offers considerable potential for both small- and large-scale agricultural applications, particularly in the context of food biofortification and promoting health benefits.

Future research should focus on optimizing DBD plasma treatment parameters for different cultivars and verifying its effects on microgreens and leafy greens across various plant species, including different segments of kangkong, such as the broad-leaf type. Investigations into sensory attributes, safety, and economic feasibility are also needed to support commercial application. Furthermore, field-based trials and metabolomic or molecular studies, including gene expression analysis, would help elucidate the underlying mechanisms involved in plasma-induced enhancement of bioactive compounds.