Effectiveness of silver nitrate application on plant growth and bioactive compounds in Agastache rugosa (Fisch. & C.A.Mey.) kuntze

Abstract

The objective of this study was to determine the optimal dose of silver nitrate (AgNO₃) for plant growth and to increase the main bioactive compounds in A. rugosa cultivated in a hydroponic system. The application of soaked diniconazole (120 μmol mol⁻¹) to all plants at 7 days after transplanting (DAT) for dwarfing plant height, optimizing cultivation space in the plant factory. Subsequently, plants were soaked with 50, 100, 200, and 400 μmol mol⁻¹ AgNO₃ for 10 min at 25 DAT and harvested at 39 DAT. The results indicated that 200 and 400 μmol mol⁻¹ treatments tended to severely decrease plant growth parameters compared to treatments with lower concentrations. The net photosynthetic rate was significantly reduced by the 200 and 400 μmol mol⁻¹ treatments compared to treatments with other concentrations. The 400 μmol mol⁻¹ treatment led to the lowest concentrations of chlorophyll a, chlorophyll a/b, total carotenoid, chlorophyll b, and the total chlorophyll. However, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was considerably increased in 50, 100, 200, and 400 μmol mol⁻¹ compared to that of the control plants. A higher rosmarinic acid (RA) concentration in the whole plant was noticed with the 400 μmol mol⁻¹ treatment compared with that of the untreated plants. The 100 μmol mol⁻¹ treatment exhibited the highest concentration and content of tilianin in the whole plant. Concentration of acacetin 1 significantly increased in the whole plant with 100 and 200 μmol mol⁻¹ treatments compared with that of the untreated plants. Concentrations of acacetin 2 and 3 in the whole plant were the highest with 100 and 200 μmol mol⁻¹ treatments, respectively. The results demonstrated that 100 μmol mol⁻¹ treatments can be used to increase bioactive compounds without severely limiting the plant growth and reducing chlorophyll concentrations of A. rugosa. Implementing this optimal dose can enable growers and researchers to cultivate A. rugosa more efficiently, enhancing bioactive compound content and overall plant performance, thus harnessing the potential health benefits of this valuable plant species.

1. Introduction

The Lamiaceae family (mint family) includes more than 7000 species distributed throughout subtropical, tropical, and temperate regions worldwide [1]. Agastache rugosa is a member of the mint family [2]. The market value of A. rugosa is primarily based on its essential oil content and has attracted attention because of its diverse application in pharmaceuticals, culinary, cosmetics, medicine, and food products [2]. This medicinal herb is known for its antioxidant [3], antimicrobial [4], anticancer [5], anti-inflammatory [6], analgesic [7], and cardiovascular properties [8], and is presumed to promote the human immune system response [9]. Plant polyphenols include simple phenols, hydroxybenzoic acids [10], hydroxycinnamic acids [11], and flavonoids such as flavones [12], flavanones [13], and flavonols [14,15]. Among these, the main bioactive compounds in A. rugosa are tilianin (an active flavonoid glycoside), acacetin (flavone), and rosmarinic acid (RA) (esters of caffeic acid and 3,4-dihydroxyphenyllactic acid) [16]. Acacetin is a flavone that is used to treat cancers, infections, inflammation, and metabolic disorders [17]. Tilianin has antidiabetic, cardioprotective, antihypertensive, neuroprotective, anti-atherogenic, antidepressant, anti-inflammatory, and antioxidant effects [18]. RA has many pharmacological uses, including antitumorigenic, antioxidative, anti-inflammatory, and anti-apoptotic [19]. The growing concerns regarding the bioactive quality and nutritional value of food sources, alongside the increasing economic and environmental considerations regarding sustainable crop management, underline the need for sustainable, profitable, and ecologically advantageous solutions to increase the quality of A. rugosa.

Environmental factors, including light, temperature, humidity, and carbon dioxide (CO₂), are controlled to allow year-round production and to increase plant productivity and quality [20]. Although A. rugosa can reach a height of 50–60 cm within 5–6 weeks, making it unsuitable for cultivation in the limited vertical space of a plant factory, we explored the use of diniconazole to reduce its height [21,22]. By controlling the plant height, our goal was to increase the number of cultivation layers in the plant factory, thereby maximizing space utilization and overall productivity. Therefore, this study utilized diniconazole to decrease the height of A. rugosa plants. Heavy metal exposure is an abiotic stress that alters primary metabolic processes and plant physiology, including respiration, photosynthesis, yield, plant growth, and secondary metabolites. The effects of silver nitrate (AgNO₃) on plants have been studied over the past few decades [[23], [24], [25]]. AgNO₃ has been widely applied in fields such as agriculture [23,26], medical science [27], and environmental management [28]. It is primarily used in agriculture to achieve a range of results, such as plant growth and development control, high-quality plants, and flowering control [[29], [30], [31]]. Although the potential of AgNO₃ is widely accepted, its use in agriculture to increase secondary metabolite levels in plants remains debated [23]. The influence of AgNO₃ on plant growth and secondary metabolites has been explored in crops such as Bacopa monnieri [32], Cucumis sativus L. [33], Brassica sp. [23], and pearl millet [34]. Brassica seedling growth decreases due to AgNO₃ accumulation, which can severely inhibit photosynthesis. AgNO₃ causes oxidative stress, as demonstrated by an increase in hydrogen peroxide and superoxide radicals [23]. The influence of toxic silver is correlated with the accumulation of oxidative damage and reactive oxygen species (ROS). In response, plants operate an antiradical enzymatic system to synthesize small antioxidant compounds [35]. Hydrogen peroxide content, total phenolic compounds, and lipid peroxidation in potatoes were considerably increased under AgNO₃ treatment compared with those in untreated explants [36]. Increases in catalase and peroxidase activities and total phenol content under AgNO₃ treatments were detected in the leaves, stems, and roots of Bacopa monnieri compared to control plants [32].

However, to date, no studies have successfully examined the effects of AgNO₃ on plant growth or bioactive compound production in A. rugosa plants cultivated using the deep-flow technique in a plant factory. This study aimed to determine the optimal AgNO₃ dose to enhance bioactive compound accumulation without severely reducing the plant growth of A. rugosa and to suggest cultivation conditions for creating high-quality plants.

2. Materials and methods

2.1. Seedling growing conditions

A. rugosa seeds were obtained from Danong Co. Ltd (Seoul, Korea) and sown on a seed germination tray in an environment-controlled room. The seedling conditions were set up at 21 ± 3 °C with a relative humidity of 75 ± 10% and a photosynthetic photon flux density (PPFD) of 185 ± 15 μmol m⁻² s⁻¹ using a fluorescent lamp (TL5 14 W/865 Philips, Amsterdam, Netherlands) with 16 h of light and 8 h of darkness (16/8) photoperiod. The Hoagland solution with electrical conductivity (EC) 1.1 dS m⁻¹ and pH 6.0 was used for seedlings from 2 weeks after sowing.

2.2. Treatments

On day 28 after sowing, seedlings were moved to a deep flow technique system of dimensions 0.11 m × 1.2 m × 0.7 m; H × L × W placed in a plant factory. The cultivation conditions were controlled at an average temperature of 22.5 °C with a relative humidity of 65 ± 10%, 220 ± 10 μmol m⁻² s⁻¹ of PPFD with 14 h of light and 10 h of dark (14/10) photoperiod. The plants were cultivated for 39 d in Hoagland solution with a pH of approximately 6.5 and EC of 2.0 dS m⁻¹. All the plants were treated with diniconazole (120 μmol mol⁻¹) for 10 min by root soaking for dwarfing at 7 d after transplantation (DAT). Four AgNO₃ concentrations of 50, 100, 200, and 400 μmol mol⁻¹ (control treatment without AgNO₃) were treated by soaking the roots at 25 DAT for 10 min.

2.3. Plant growth parameter measurements

Thirty 9 d after transplantation, all samples were harvested. Leaf width, leaf number, stem length, leaf length, leaf area, shoot and root fresh weights/plant, and dry weights of shoots and roots per plant were recorded. Shoots and roots/plant were weighed to determine the fresh weights, then moved to an oven for 1 week at 70 °C, and the dry weights/plant were weighed. Leaves were detached from each plant to determine leaf area. A digital camera (Canon EOS 850D, Canon Inc., Tokyo 146–8501, Japan) and ImageJ software were used to determine leaf area (Fig. 1).

Fig. 1.

Representative A. rugosa leaf images of the different AgNO₃ treatments (0-control, 50 μmol mol⁻¹, 100 μmol mol⁻¹, 200 μmol mol⁻¹, and 400 μmol mol⁻¹) under study. Images are five plants per treatment (n = 5).

2.4. Photosynthetic activity

Photosynthetic characteristics, including the net photosynthetic rate (P_n; μmol CO₂ m⁻¹ s⁻¹), transpiration rate (T_r; mmol H₂O mol⁻¹), intercellular CO₂ concentration (C_i; μmol CO₂ mol⁻¹), and stomatal conductance (g_s; mol H₂O m⁻¹ s⁻¹), were determined using a portable photosynthesis system (LICOR 6400, Licor. Inc. Nebraska, NE, USA). Measurements were recorded in a leaf chamber with CO₂ concentration of 400 μmol mol⁻¹, PPFD of 1000 μmol m⁻² s⁻¹, leaf temperature of 25 °C, and an air flow rate of 500 cm³ s⁻¹.

2.5. Photosynthetic pigments and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity

Upon harvesting, the leaves, stems, and flowers of each A. rugosa plant replicate were promptly immersed in liquid nitrogen and stored in a deep freezer at −70 °C; subsequently, they were transferred to a dry freezer at −50 °C (TFD5503, IL Shinbiobase Co. Ltd, Gyeonggi-do, Korea) within 4 d. A porcelain mortar and pestle were employed to ground each sample, and the dry powder was filtered through mesh sieves. Twenty milligrams of dry powdered shoots was extracted with 2 mL of 90% MeOH. The obtained extract was centrifuged at 1308×g for 20 min. Chlorophyll (Chl) concentrations (Chl a and Chl b), total carotenoids (Car), and DPPH radical scavenging activity were analyzed using an Epoch microplate spectrophotometer (EPOCH-SN, Agilent Technologies, Inc., Santa Clara, CA 95051, USA). Following the method of Lichtenthaler, 1987 and Machado et al., 2020 [37,38], Chl a, Car, and Chl b were detected at 652.4, 470, and 665.2 nm, respectively. The DPPH radical scavenging assay was performed at 517 nm, according to Rahman et al., 2015 [39]. The Chl a, Chl b, Car, DPPH, Chl a and b ratio (Chl a/b), and total Chl a + b concentrations were calculated using the following formulas:

Chl a (mg^.g⁻¹) = (16.82 × A_665.2 − 9.28 × A_652.4)/10

Chl b (mg^.g⁻¹) = (36.92 × A_652.4 − 16.54 × A_665.2)/10

Total carotenoid (mg^.g⁻¹) = [(1000 × A₄₇₀ − 1.91 × Chl a −95.15 Chl b)/225]/10

DPPH (%) = (A_blank − A_sample)/A_blank × 100

Chl a and b ratio (Chl a/b) = Chl a/Chl b

Total Chl a + b (mg.g⁻¹) = Chl a + Chl b

where A is the absorbance at wavelength.

2.6. Analyzation of acacetin, tilianin, three acacetin glycosides, and rosmarinic acid concentrations and contents

The roots, stems, leaves, and flowers of A. rugosa from each replicate were promptly immersed in liquid nitrogen and stored in a deep freezer at −70 °C; subsequently, they were transferred to a dry freezer at −50 °C (TFD5503, IL Shinbiobase Co. Ltd, Gyeonggi-do, Korea) within 4 d. Subsequently, a porcelain mortar and pestle were used to grind each sample, and the dry powder was filtered through a mesh sieve. A total of 200 mg dry powder of flowers, leaves, roots, and stems was dissolved in 10 mL of methanol and sonicated for 30 min to determine the concentrations of rosmarinic acid (RA), tilianin, acacetin, and three acacetin glycosides: acacetin 7-O-(2″-O-acetyl)β-d-glucopyranoside (acacetin 1), acacetin 7-O-(6″-O-malonyl)β-d-glucopyranoside (acacetin 2), and acacetin 7-O-(2″-O-acetyl-6″-O-malonyl)β-d-glucopyranoside (acacetin 3). The mixed extract was centrifuged at 1358×g for 20 min. One mL of the extract solution was passed through a 0.45 μm filter before being analyzed by high-performance liquid chromatography (1260 Infinity, Agilent Technologies, Santa Clara, CA, USA). The mobile phase was composed of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). The gradient program was as follows: 0–5 min: 20% B, 5–10 min: 50% B, 10–20 min: 50% B, and 20–22 min: 100% B. The injection volume and flow rate were 10 μL and 0.8 mL min⁻¹, respectively. High-performance liquid chromatography (HPLC) analysis was carried out at a temperature of 30 °C using a C18 column (250 mm × 4.6 mm, 5 μm; RS tech, Daejeon, Korea). The HPLC chromatogram was monitored at 330 nm according to An et al., 2018 and Hong et al., 2021 [40,41]. Standard compounds from Sigma−Aldrich (St. Louis, MO, USA) were used to generate the calibration curves. RA, tilianin, and acacetin were detected at 11.655 min, 12.542 min, and 19.659 min, respectively. Acacetins 1, 2, and 3 were detected at 13.131, 14.485, and 15.351 min, respectively [40,41]. The concentrations of RA, tilianin, acacetin, and three acacetin glycosides (bioactive compounds) in the root, leaf, stem, and flower were expressed as per unit dry weight (DW) (mg·g⁻¹ DW). The concentration of each bioactive compound in the entire plant (mg·g⁻¹ DW) was determined by summing up the product of the amount of each compound present in every plant part and the percentage of dry weight for each plant organ relative to the total dry weight of the entire plant, as indicated in equation (1).

BCx in the entire plant (mg^.g⁻¹ DW) = Σ (amount of BCx in each part × % dry weight of each plant organ per dry weight of the entire plant). (1)

BC:Concentration of bioactive compound

x: Tilianin, Acacetin a, Acacetin b, Acacetin c, or RA or Acacetin.

The BC content of the entire plant (mg/plant DW) was expressed as the BC concentration (mg^.g⁻¹ plant DW) multiplied by the entire plant DW (g). For each replicate per AgNO₃ treatment, BC concentrations were analyzed using three measurements (n = 3).

2.7. Statistical analysis

For each treatment, growth parameters were collected from five plants (n = 5) and photosynthetic characteristics, photosynthetic pigments, DPPH radical scavenging activity, and bioactive compounds were determined from three plants (n = 3). The experiment was conducted using a completely randomized design and comprised of two replicates. Statistical data were analyzed using SPSS version 20.0 software (SPSS 20, IBM Corp., Armonk, N.Y., USA). The results of the experiment were consolidated and examined using a one-way ANOVA. Tukey's multiple range test was used to compare the means of the treatment groups, and significant differences were assessed at the 5% level.

The BC concentrations and contents in the plant organs were visualized using a heat map. Rows and columns were grouped using the GraphPad Prism distance and average linkage methods for heatmap construction. The color scale in the heatmap represents numeric differences, with red indicating the largest values, black representing medium values, and green indicating the smallest values for each parameter.

3. Results

3.1. Plant growth parameters

The results evidenced that plant growth significantly declined with increasing concentrations of AgNO₃ (Table 1 and Fig. 2). All the measured growth characteristics, that is, the leaf length, leaf area, stem length, shoot and root fresh weights/plant, and root length were significantly decreased at 200 and 400 μmol mol⁻¹ of AgNO₃ compared to those of the untreated plants. Additionally, the application of 400 μmol mol⁻¹ of AgNO₃ resulted in a significant decrease in the number of leaves, shoot and root dry weights/plant, and flower and stem dry weights/plant, when compared to those of the untreated plants (Table 1 and Fig. 2). The lowest values of plant growth parameters were found with 400 μmol mol⁻¹ of AgNO₃ treatment. The stem lengths exhibited a significant reduction of 48.75% compared to those of the untreated plants, as a result of the 400 μmol mol⁻¹ AgNO₃. The number of leaves and leaf area under 400 μmol mol⁻¹ AgNO₃ were 20.50% and 46.76%, respectively, being significantly lower than those of the control plants. 400 μmol mol⁻¹ treatment significantly decreased the shoot fresh weight and root fresh weight by 51.37% and 45.28%, respectively, compared with those of the untreated plants. The shoot and root dry weights/plant, and flowers and stem dry weights/plant were significantly reduced in the 400 μmol mol⁻¹ treatment compared to those of the control plants. These results evidenced that 400 μmol mol⁻¹ of AgNO₃ strongly reduced the plant growth characteristics of A. rugosa.

Table 1.

The growth characteristics of A. rugosa at different AgNO₃ treatments (0-control, 50 μmol mol⁻¹, 100 μmol mol⁻¹, 200 μmol mol⁻¹, and 400 μmol mol⁻¹), subjected to root soaking.

AgNO3w (μmol⋅mol−1)	Leaf length (cm)	Leaf width (cm)	Number of leaves (leaves)	Leaf area (cm2)	Stem length (cm)	SFWP (g/plant)	RFWP (g/plant)	Root length (cm)	SDWP (g/plant)	RDWP (g/plant)	LDWP (g/plant)	FDWP (g/plant)	Stem DWP (g/plant)
0	6.26a	5.70	56.60 ab	532.67a	20.80a	17.21a	11.24a	77.08a	2.91a	0.87a	1.67	0.65a	0.61 ab
50	6.04 ab	5.64	64.80a	574.76a	18.92 ab	16.65a	11.97a	62.50b	2.62 ab	0.86a	1.40	0.58a	0.63a
100	5.90 ab	5.60	54.40abc	473.34 ab	18.70 ab	13.88 ab	11.37a	61.03b	2.58 ab	0.78 ab	1.50	0.63a	0.46BCE
200	5.64b	5.40	51.80bc	390.67bc	17.62b	11.26bc	7.03b	56.06b	2.58 ab	0.69 ab	1.48	0.53a	0.57 ab
400	5.54b	5.24	45.00c	283.58c	10.66c	8.37c	6.15b	54.96b	1.96b	0.60b	1.25	0.37b	0.34c
Significancez	**	NS	***	***	***	***	***	***	**	**	NS	***	***
Ly	***	***	***	***	***	***	***	***	***	**	*	**	***
Qx	***	**	**	***	***	***	***	***	***	**	NS	***	**

^WAgNO₃; NS: not significant (p > 0.05); ^zsignificant at * p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001; ^yL: linear; ^xQ: quadratic in regression analysis. Values are the means of five samples (n = 5). Different letters are significant differences among treatments at p ≤ 0.05, according to Tukey's test. SFWP: Shoot fresh weight/plant; RFWP: Root fresh weight/plant; SDWP: Shoot dry weight/plant; RDWP: Root dry weight/plant; LDWP: Leaf dry weight/plant; FDWP: Flower dry weight/plant, and stem DWP: Stem dry weight/plant.

Fig. 2.

Images of A. rugosa plants growth at different AgNO₃ treatments (0-control, 50 μmol mol⁻¹, 100 μmol mol⁻¹, 200 μmol mol⁻¹, and 400 μmol mol⁻¹), subjected to root soaking.

3.2. Photosynthetic activity

The photosynthetic parameters of A. rugosa under different concentrations of AgNO₃ stress and the control are illustrated in Fig. 3. The net photosynthetic rate (P_n) and stomatal conductance (g_s) evidenced a decreasing tendency with increased AgNO₃ concentrations. When the AgNO₃ concentration was raised to 400 μmol mol⁻¹, the P_n and g_s were reduced to the lowest value of 4.71 μmol CO₂ m⁻² s⁻¹ and 0.06 mol H₂O m⁻² s⁻¹, respectively (Fig. 3A and B). The intercellular CO₂ concentration (C_i) was not significantly different between the AgNO₃ treatments and control (Fig. 3C). The transpiration rate (T_r) decreased by 35.65% in the 400 μmol mol⁻¹ AgNO₃ treated group compared to that of the untreated plants (Fig. 3D). These results indicate that high AgNO₃ concentrations strongly influenced the photosynthetic system of A. rugosa.

Fig. 3.

Net photosynthetic rate (P_n) (A), stomatal conductance (g_s) (B), intercellular CO₂ concentration (C_i) (C), and transpiration rate (T_r) (D) of A. rugosa at different AgNO₃ treatments (0-control, 50 μmol mol⁻¹, 100 μmol mol⁻¹, 200 μmol mol⁻¹, and 400 μmol mol⁻¹). Each value indicates the mean ± SE of three samples (n = 3). Different letters represent significant differences at p ≤ 0.05, as assessed by ANOVA according to Tukey's multiple range test.

3.3. Photosynthetic pigments and DPPH radical scavenging activity

The chlorophyll a, total carotenoid, chlorophyll a/b, total chlorophyll, and chlorophyll b contents of A. rugosa were not significantly different among the control and 0, 50, 100, and 200 μmol mol⁻¹ treatments. However, chlorophyll a, total carotenoid, chlorophyll a/b, total chlorophyll, and chlorophyll b were the lowest in the 400 μmol mol⁻¹ treatment (Fig. 4A, B, 4C, 4D, and 4E). The DPPH radical scavenging activity was significantly higher in the 50, 100, 200, and 400 μmol mol⁻¹ treatments by 20.94%, 19.04%, 16.95%, and 13.33%, respectively, compared to that in the control plants (Fig. 4F). These results demonstrated that the lowest values of chlorophyll and total carotenoid were in the 400 μmol mol⁻¹ treatment. However, all AgNO₃ treatments increased the DPPH radical-scavenging activity.

Fig. 4.

Chlorophyll a (A), chlorophyll b (B), chlorophyll a/b ratio (C), total chlorophyll (D), total carotenoid (E), and DPPH (F) of A. rugosa under different AgNO₃ treatments (0-control, 50 μmol mol⁻¹, 100 μmol mol⁻¹, 200 μmol mol⁻¹, and 400 μmol mol⁻¹). Each value indicates the mean ± SE of three samples (n = 3). Different letters represent significant differences at p ≤ 0.05, as assessed by ANOVA according to Tukey's multiple range test.

3.4. Acacetin, tilianin, and rosmarinic acid concentrations and contents

The concentrations of RA, tilianin, and acacetin differed in diverse organs of A. rugosa. The highest concentrations of RA were found in the roots, whereas the highest concentrations of tilianin and acacetin were found in the flowers. The difference in bioactive compound concentrations in each part of A. rugosa may be attributed to the movement of bioactive compounds from the root to the shoot. Stems and flowers of A. rugosa treated with 400 μmol mol⁻¹ AgNO₃ accumulated highest amounts of RA (Fig. 5). Roots treated with 50 μmol mol⁻¹ AgNO₃ exhibited the highest levels of RA concentration. Tilianin concentration was the highest in the treatment group with 100 μmol mol⁻¹ AgNO₃. The application of AgNO₃ did not influence the tilianin concentration in the stems, roots, or leaves compared with that in the untreated plants. Nevertheless, AgNO₃ treatment reduced acacetin concentrations in flowers compared to that in untreated plants (Fig. 5).

Fig. 5.

Heatmap analysis of rosmarinic acid (RA), tilianin (Ti), and acacetin (Aca) concentrations (mg g⁻¹ DW) in flowers (F), stems (S), leaves (L), and roots (R) of A. rugosa grown at different AgNO₃ treatments (0-control, 50 μmol mol⁻¹, 100 μmol mol⁻¹, 200 μmol mol⁻¹, and 400 μmol mol⁻¹). The color scale in the heatmap represents numeric differences, with red indicating the largest values, black representing medium values, and green indicating the smallest values for each parameter, respectively. AgNO₃ treatments are grouped in the rows; RA, Ti, and Aca concentrations in each plant organ are grouped in the columns.

Due to the reduction in dry weight, higher concentrations of AgNO₃ (400 μmol mol⁻¹) resulted in a tendency of decreased RA content in the stems, leaves, and roots in comparison to untreated plants. The lowest tilianin content in flowers was observed under the concentration of 400 μmol mol⁻¹. The tilianin content under 100 μmol mol⁻¹ in leaves was higher than those of the other treatments (Fig. 6). AgNO₃ treatment reduced acacetin content in flowers and leaves compared to control plants (Fig. 6).

Fig. 6.

Heatmap analysis of rosmarinic acid (RA), tilianin (Ti), and acacetin (Aca) contents (mg/plant organs DW) in flowers (F), stems (S), leaves (L), and roots (R) of A. rugosa grown at different AgNO₃ treatments (0-control, 50 μmol mol⁻¹, 100 μmol mol⁻¹, 200 μmol mol⁻¹, and 400 μmol mol⁻¹). The color scale in the heatmap represents numeric differences, with red indicating the largest values, black representing medium values, and green indicating the smallest values for each parameter, respectively. AgNO₃ treatments are grouped in the rows; RA, Ti, and Aca concentrations in each plant organ are grouped in the columns.

Plants exposed to 400 μmol mol⁻¹ had significantly higher RA concentrations (1.10 times) in the entire plant than the untreated plants (Fig. 7A). Treatment with 100 μmol mol⁻¹ resulted in significantly higher tilianin concentrations (1.26 times) in the entire plant than that in the untreated plants (Fig. 7B). However, AgNO₃ treatment restrained acacetin production in the entire plant compared to that in the untreated plants (Fig. 7C). Furthermore, the RA content was reduced by increasing the dosage of AgNO₃ because of the decrease in the dry weight of the entire plant (Fig. 7D). Especially, tilianin contents were greatly increased by 17.88% in the presence of 100 μmol mol⁻¹, compared to that in the control plants (Fig. 7E). In contrast, increasing the dosage of AgNO₃ led to a progressive reduction in acacetin content due to the low dry weight of the whole plant. Further details reveal that the application of treatment with a concentration of 400 μmol mol⁻¹ resulted in the maximal reduction of acacetin contents accumulated in the whole plant, exhibiting an impressive reduction of 77.37% compared to that of the untreated plants (Fig. 7F).

Fig. 7.

Rosmarinic acid (RA) concentration (A) and content (D), tilianin concentration (B) and content (E), acacetin concentration (C) and content (F) in whole plant of A. rugosa at different AgNO₃ treatments (0-control, 50 μmol mol⁻¹, 100 μmol mol⁻¹, 200 μmol mol⁻¹, and 400 μmol mol⁻¹). Each value indicates the mean ± SE of three samples (n = 3). Different letters represent significant differences at p ≤ 0.05, as assessed by ANOVA according to Tukey's multiple range test.

3.5. Acacetin 7-O-(2″-O-acetyl)β-d-glucopyranoside (acacetin 1), acacetin 7-O-(6″-O-malonyl)β-d-glucopyranoside (acacetin 2), and acacetin 7-O-(2″-O-acetyl-6″-malonyl)β-d-glucopyranoside (acacetin 3) concentrations and contents

Concentration of acacetin 1 in the leaves with 100 μmol mol⁻¹ was highest value (Fig. 8). Application of AgNO₃ at 50, 200 and 400 μmol mol⁻¹ indicated higher values of acacetin 2 concentration in the flowers compared with that in the untreated plants. Concentration of acacetin 2 in the stems and leaves under treatment with AgNO₃ was increased compared with that in the untreated plants. The concentration of acacetin 3 in the flowers was higher under 50 and 200 μmol mol⁻¹ among the AgNO₃ treatments and the control group, whereas in the stems, under treatment with AgNO₃, it was increased compared with that in the untreated plants. The most notable concentrations of acacetin 3 were discovered in the leaves under 200 μmol mol⁻¹, demonstrating the highest values (Fig. 8).

Fig. 8.

Heatmap analysis of acacetin 7-O-(2″-O-acetyl)β-d-glucopyranoside (Aca 1), acacetin 7-O-(6″-O-malonyl)β-d-glucopyranoside (Aca 2), and acacetin 7-O-(2″-O-acetyl-6″-malonyl)β-d-glucopyranoside (Aca 3) concentrations (mg g⁻¹ DW) in flowers (F), stems (S), leaves (L), and roots (R) of A. rugosa grown at different AgNO₃ treatments (0-control, 50 μmol mol⁻¹, 100 μmol mol⁻¹, 200 μmol mol⁻¹, and 400 μmol mol⁻¹). The color scale in the heatmap represents numeric differences, with red indicating the largest values, black representing medium values, and green indicating the smallest values for each parameter, respectively. AgNO₃ treatments are grouped in the rows; RA, Ti, and Aca concentrations in each plant organ are grouped in the columns.

Application of 400 μmol mol⁻¹ AgNO₃ reduced acacetin 1 contents in the flowers (Fig. 9). Treatment with 50 and 200 μmol mol⁻¹ AgNO₃ resulted in higher acacetin 1 content in the stems compared to that in the untreated plants (Fig. 9). The acacetin 1 content in the leaves was decreased in response to treatments with 50 and 400 μmol mol⁻¹ AgNO₃, when compared to that in the untreated plants. The application of 400 μmol mol⁻¹ AgNO₃ treatments decreased the acacetin 2 contents in flowers when compared to that in the untreated plants. The treatments with 100 and 200 μmol mol⁻¹ AgNO₃ led to elevated acacetin 2 contents in leaves, surpassing those observed in the control plants. In contrast, the application of 400 μmol mol⁻¹ AgNO₃ resulted in the lowest acacetin 3 content in flowers compared to that in the other treatments. The application of 50 and 200 μmol mol⁻¹ AgNO₃ treatments enhanced the acacetin 3 content in stems compared to that in untreated plants. Among the various treatments, the acacetin 3 content in leaves reached its highest value under the application of 200 μmol mol⁻¹ (Fig. 9).

Fig. 9.

Heatmap analysis of acacetin 7-O-(2″-O-acetyl)β-d-glucopyranoside (Aca 1), acacetin 7-O-(6″-O-malonyl)β-d-glucopyranoside (Aca 2), and acacetin 7-O-(2″-O-acetyl-6″-malonyl)β-d-glucopyranoside (Aca 3) contents (mg/plant organs DW) in flowers (F), stems (S), leaves (L), and roots (R) of A. rugosa grown at different AgNO₃ treatments (0-control, 50 μmol mol⁻¹, 100 μmol mol⁻¹, 200 μmol mol⁻¹, and 400 μmol mol⁻¹). The color scale in the heatmap represents numeric differences, with red indicating the largest values, black representing medium values, and green indicating the smallest values for each parameter, respectively. AgNO₃ treatments are grouped in the rows; RA, Ti, and Aca concentrations in each plant organ are grouped in the columns.

A slight increase in acacetin 1 concentration was recorded in the 100 and 200 μmol mol⁻¹ treatments (8.99% and 5.31%, respectively) compared to that in the untreated plants (Fig. 10A). The plants treated with 100 μmol mol⁻¹ exhibited significantly increased acacetin 2 concentration by 12.78%, compared to that in the untreated plants (Fig. 10B). The application of 200 μmol mol⁻¹ resulted in a significant increase of 11.90% in the concentration of acacetin 3 compared to that in the untreated plants (Fig. 10C). These results demonstrated that AgNO₃ positively affects the accumulation of bioactive compounds in A. rugosa. In contrast, 50 and 400 μmol mol⁻¹ treatments significantly decreased the acacetin 1 content by 15.47% and 44.15%, respectively, compared with that in the control, from the reduction in the dry weight of the entire plant (Fig. 10D). However, a significant reduction in acacetin 2 and acacetin 3 contents was only recorded under the 400 μmol mol⁻¹ treatment. The mean acacetin 2 and 3 contents decreased by 27.87% and 40.25% in the 400 μmol mol⁻¹ treatment, respectively, compared to that in the untreated plants (Fig. 10E and F).

Fig. 10.

Acacetin 7-O-(2″-O-acetyl)β-d-glucopyranoside (Acacetin 1) (A and D), Acacetin 7-O-(6″-O-malonyl)β-d-glucopyranoside (Acacetin 2) (B and E), and Acacetin 7-O-(2″-O-acetyl-6″-malonyl)β-d-glucopyranoside (Acacetin 3) (C and F), concentrations and contents in whole plant of A. rugosa at different AgNO₃ treatments (0-control, 50 μmol mol⁻¹, 100 μmol mol⁻¹, 200 μmol mol⁻¹, and 400 μmol mol⁻¹). Each value indicates the mean ± SE of three samples (n = 3). Different letters represent significant differences at p ≤ 0.05, as assessed by ANOVA according to Tukey's multiple range test.

4. Discussion

4.1. Plant growth parameters

Any abiotic stress reduces growth [42], and causes various physiological changes in plants, including the creation of osmotic stress [43], ROS, oxidative stress [44], reduction of photosynthetic activity [45], and macro-and micronutrients imbalance, including [46]. It also triggers biochemical changes in enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) [47], and non-enzymatic antioxidants such as phenols [48], hydroxybenzoic acids [10], flavanols [12], beta-carotene [49], flavanones [50], xanthophylls [51], hydroxycinnamic acids [52], flavonols [53], flavones [54], ascorbic acids [55], antiradical capacity [56], and carotenoids [57]. This study was conducted to determine the optimal AgNO₃ concentration for diniconazole base in A. rugosa plants. Diniconazole decreased almost all plant growth parameters of A. rugosa, except for root length, compared with those in the control plants [22]. Diniconazole, a growth regulator belonging to the triazole family, restricts elongation of stems [58]. Diniconazole, an N-containing heterocyclic compound, inhibits cytochrome of oxidative demethylation mediated by cytochrome P450. This is closely associated with the conversion of kaurene to kaurenoic acid, which plays a role in the biosynthesis of gibberellin [59]. By inducing stem elongation, diniconazole curbs gibberellin activity within the plant cells, resulting in reduced stem length [60]. Diniconazole is a useful tool for decreasing the height of A. rugosa plants, allowing for the expansion of cultivation layers in plant factories [22]. Exposure to high AgNO₃ concentrations significantly reduced plant growth characteristics in A. rugosa, which may be directly related to decreased photosynthetic performance. A significant decrease in stem length, leaf area, leaf length, the fresh weights of shoot and root, and root length under 200 and 400 μmol mol⁻¹ treatments was observed compared to those in the control treatment (Table 1, Figs. 1, and Fig. 2). A similar result was found by Krishnaraj et al., 2012 [32] who reported that using AgNO₃ did not lead to intense toxic effects on morphology, as observed on scanning electron microscopy. Plant growth is restricted at high AgNO₃ concentrations (Krishnaraj et al., 2012). The fresh weight, shoot and root lengths, and photosynthetic characteristics of Brassica sp. under AgNO₃ treatment were lower than those of the untreated plants. Moreover, AgNO₃ accumulation in the roots was greater than that in other organs of Brassica sp. plants [23]. Tobacco root growth was significantly promoted at lower silver nanoparticle (AgNP) concentrations of 25 and 50 μM and significantly decreased after exposure to higher concentrations of 75, 100, and 150 μM [61]. Seedling growth in Brassica napus L. was restricted by AgNO₃ [62]. The growth characteristics of live plants are reduced by high AgNO₃ concentrations because of the increased absorption of silver [63]. Corn root elongation is negatively affected by AgNP toxicity [64]. The association between silver ions (Ag⁺) and sunflower plant roots distorts the epidermal structure and changes its anatomical properties. Therefore, root hairs are damaged [65]. The high silver accumulation in plants associated with the root and the translocation element ([Ag]shoot/[Ag]root) was relatively low, and a high dose of Ag (40 mg L⁻¹) reduced leaf growth of wetland plants up to 55% [66]. In this study, plant growth of A. rugosa was reduced by higher AgNO₃ concentrations (200 and 400 μmol mol⁻¹), which may be due to the absorption of AgNO₃ through the roots, changes in the cell and membrane structures, defensive mechanisms, high AgNO₃ accumulation in plants, and AgNO₃ toxicity.

4.2. Photosynthetic parameters, photosynthetic pigment, and DPPH radical scavenging activity

The net photosynthetic rate was decreased with higher AgNO₃ exposure (200 and 400 μmol mol⁻¹) (Fig. 3). The results of this study are consistent with those of previous studies, which indicate that high concentrations of AgNPs and AgNO₃ inhibit photosynthetic parameters in several plant species, such as Chlamydomonas reinhardtii [67], aquatic plants [68], Brassica sp. [23], pearl millet [34], and tobacco seedlings [61]. Photosynthetic parameters of this study were lower under higher AgNO₃ concentrations (200 and 400 μmol mol⁻¹) than those in the control plants because of the toxicity influence of AgNO₃. A similar study conducted by Khan et al., 2019 [34] found that photosynthetic parameters were lower in AgNP-treated plants than those in untreated plants. A reduction in photosynthesis and plant growth may be the result of the destructive effects of ROS on the photosynthetic machinery and might be related to oxidative stress [33]. AgNO₃ caused a significant decrease in photosystem II activity, which finally reduced the photosynthetic parameter values and decreased the plant chlorophyll content [34]. This decrease in photosynthesis under high AgNO₃ concentrations may be attributed to ROS-mediated damage to biomolecules related to the photosynthetic system, which is predominantly related to the membranes of the energy transaction system.

Photosynthetic ability can be explored by determining the chlorophyll parameters under AgNO₃ stress and normal conditions [23]. The results verified that under 400 μmol mol⁻¹ exposure, the chlorophyll a, total carotenoid, chlorophyll a/b, total chlorophyll, and chlorophyll b contents of A. rugosa were reduced (Fig. 4). The substantial reduction in chlorophyll and total carotenoid content indicated modifications in the function and structure of the photosynthetic process. This may be related to decreased biomass accumulation in A. rugosa. Heavy metals can alter physiological processes, particularly by reducing the synthesis of photosynthetic pigments [69,70]. This may indicate that Ag⁺ caused a water imbalance. Therefore, plant photosynthesis is restricted to the highest concentration of AgNO₃. The decrease in chlorophyll content under high AgNO₃ concentrations may be ascribed to ROS-mediated damage to the biomolecules connected to the photosynthetic system, which is predominantly related to the membranes of the energy transaction system [71]. AgNO₃ treatment increased DPPH free radical scavenging activity (Fig. 4F). ROS are generated because AgNO₃ stress damages macromolecules and ultimately cellular structure in plants [72]. Abiotic stress causes oxidative damage in a considerable proportion of plants. However, free radical scavenging and antioxidant system capacities are also controlled [73]. AgNPs and AgNO₃ significantly increased the DPPH free radical scavenging activity of Echium amoenum [74]. In line with these results, AgNPs demonstrated to enhance DPPH radical scavenging activity in Erythrina suberosa [75]. The data of this study also evidenced that AgNO₃ increased the DPPH scavenging activity (Fig. 4F).

4.3. Acacetin, tilianin, rosmarinic acid, and three acacetin glycoside concentrations and contents

The concentration of RA in the entire plant of A. rugosa was higher than that of tilianin and acacetin (Fig. 7). A similar finding was reported by Tuan et al., 2012 [76], who observed that the concentration of RA in A. rugosa was higher than that of tilianin and acacetin. Furthermore, RA is an important phenolic compound, with its primary concentration observed in the hairy roots of A. rugosa [77]. Our findings are consistent with this observation as we detected the highest concentration of RA in the roots of A. rugosa compared to that in other plant organs (Fig. 5). Similarly, the roots of A. rugosa exhibited the highest concentration of RA compared to that in other organs [78]. This distribution pattern can be attributed to the movement of bioactive compounds from the roots to other parts of the plant, influencing their concentrations in different organs. The effects of AgNO₃ treatment on the accumulation of these bioactive compounds were also investigated. The stems and flowers treated with 400 μmol mol⁻¹ AgNO₃ exhibited the highest accumulation of RA. This suggests that the application of AgNO₃ at this concentration enhanced RA production in these plant parts. In the case of tilianin, AgNO₃ treatment with 100 μmol mol⁻¹ increase its concentration in the flower compared to that in the other plants. This indicates that AgNO₃ application may have a positive effect on tilianin accumulation in flowers. Conversely, AgNO₃ treatment led to a reduction in acacetin concentration in flowers. This study provides valuable insights into the differential accumulation of RA, tilianin, and acacetin in different organs of A. rugosa and the influence of AgNO₃ treatment on their concentrations. These findings highlight the potential use of AgNO₃ to enhance the production of bioactive compounds in specific plant parts, which may have implications for their use in various applications.

AgNO₃ causes considerable phytotoxicity in plants, which can be observed by analyzing various physiological, physical, structural, and biochemical traits [23]. AgNO₃ has been reported to be effective in promoting RA in A. rugosa [79]. Similarly, the RA concentrations of A. rugosa were significantly higher with exposure to high AgNO₃ concentrations (400 μmol mol⁻¹) (Fig. 7A). Among the different treatments, the highest value of tilianin concentration and content was achieved at 100 μmol mol⁻¹ (Fig. 7B and E). The results depicted in Fig. 10A demonstrate that the concentrations of acacetin 1 were notably higher in the plants treated with 100 and 200 μmol mol⁻¹ compared to those in the untreated plants. Fig. 10B reveals a significant elevation in the concentration of acacetin 2 in the plants treated with 100 μmol mol⁻¹ when compared to that in the untreated plants. Additionally, Fig. 10C displays that the application of 200 μmol mol⁻¹ resulted in the highest concentration of acacetin 3 among treatments. The individual phenolic compounds, flavonoid content, and total phenolic content in Cucumis anguria were increased by the AgNPs [80]. Malondialdehyde, hydrogen peroxide, glucosinolates, and phenolic compounds were significantly enhanced by AgNPs [81]. Superoxide dismutase, peroxidase, guaiacol peroxidase, total phenolics, glutathione reductase activity, ascorbate peroxidase, total flavonoid content, proline content, and catalase activity in pearl millet were increased by AgNO₃ [34]. Ag ⁺ released from AgNO₃ are severely toxic to plants [82]. Ag⁺ stress stimulates ROS production and promotes oxidative damage. Therefore, plants have created an antioxidant defense system to deal with oxidative stress in cells [68]. Ag ⁺ enhance ROS production and restrict enzymatic antioxidants. AgNO₃ stress in plants leads to increased ROS production, which affects plant metabolism by destroying defense mechanisms and reducing antioxidant activities [83]. ROS are produced in cells because of Ag⁺ toxicity [84]. AgNO₃ damages cell membranes and interrupts DNA replication and ATP production [82]. The subsequent generation of oxidative stress and ROS production results in different toxic effects and may affect gene expression and DNA degradation [85]. Total proline and phenolic contents in Solanum tuberosum were considerably increased by AgNO₃ and AgNP treatments compared to those in the untreated plants [36]. The accumulation of indole and phenolic compounds, including hydroxycamalexin malonyl-hexoside, camalexin, and hydroxycamalexin O-hexoside in Arabidopsis thaliana was increased by Ag⁺ and silver nanoparticle treatments [86]. In line with previous results, in this study RA, tilianin, and concentration of acacetin 1, 2, and 3 in A. rugosa significantly increased at various AgNO₃ concentrations.

5. Conclusion

These results demonstrate that AgNO₃ improved the bioactive compounds in A. rugosa under hydroponic culture conditions in a plant factory. The treatments with 200 and 400 μmol mol⁻¹ AgNO₃ led to a significant decrease in plant growth parameters compared to those of other treatments. However, 400 μmol mol⁻¹ exhibited an elevated concentration of RA in the whole plant, while the highest tilianin concentration and content were observed in the 100 μmol mol⁻¹ treatment. Concentration of acacetin 1 in the whole plant significantly increased under 100 and 200 μmol mol⁻¹ treatments compared to that in the untreated plants, while concentrations of acacetin 2 and 3 were the highest in the 100 and 200 μmol mol⁻¹ treatments, respectively. This study suggests that the 100 μmol mol⁻¹ treatment of AgNO₃ can be utilized to enhance the bioactive compound content in A. rugosa without severely impeding plant growth or significantly reducing chlorophyll concentrations. This optimal dose may provide a practical and effective approach for growers and researchers to cultivate A. rugosa and harness its health benefits. Given that we soaked selected concentrations of AgNO₃ for 10 min in this study, further studies are required to explore the optimal soaking duration for AgNO₃ from an application perspective.

Author contribution statement

Vu Phong Lam and Jongseok Park: Conceived and designed the experiments; Performed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Lee Beomseon: Performed the experiments; Wrote the paper.

Vu Ky Anh: Conceived and designed the experiments; Performed the experiments; Contributed reagents, materials, analysis tools or data.

Dao Nhan Loi: Conceived and designed the experiments; Performed the experiments; Contributed reagents, materials, analysis tools or data.

Sunwoo Kim and Lee Kwang-ya: Analysis tools or data; Wrote the paper.

Data availability statement

Data included in article/supp. material/referenced in article.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is/are the supplementary data to this article: