Effect of Sodium Chloride on Flavone Biosynthesis and Antioxidant Activitiesin Hairy Root Culture of Scutellaria baicalensis

INTRODUCTION

Flavonoids play a vital role in environmental adaptations of plants, and are the most widely distributed secondary metabolites (Liu et al., 2021), with prominent applications in food and medicine.

Flavonoids have a C6-C3-C6 carbon skeleton structure characterized by at least two aromatic rings (labeled A and B) joined at the three carbons chain that has the potential to cyclize with ring A to form a heterocyclic ring containing oxygen ring C (Wang et al., 2018).

Flavonoids are further characterized as flavones, flavanols, flavanones, flavanols, isoflavones as well as leucoanthocyanidins and chalcones (Panche et al., 2016).

Flavones (from the Latin word flavus, “yellow”) are plant secondary metabolites found in plants and fungi and naturally exhibit yellow color and belong to one of the major groups of flavonoids that share a common 2-phenylchromen-4-one (2-phenyl-1-benzopyran-4-one) skeletal structure (Panche et al., 2016). Flavone is a prominent group of specialized metabolites that have essential functions during both plant signaling and defense strategy, however, many researchers find flavones are also valuable compounds in the human diet that can yield huge health-promoting benefits (Martens and Mithöfer, 2005; Jiang et al., 2016; Hostetler et al., 2017).

The root of S. baicalensis has been commonly used in Chinese herbal medicine and is rich in various bioactive flavones including major active compounds baicalin, baicalein, and wogonin (Kim et al., 2014a; Kim et al., 2014b; Liang et al., 2017).

Previous studies reported that wogonin exerts natural characteristics together with anti-cancer, restoring bonds in an inflammatory state, toward bacterial and viral infection (Huynh et al., 2020; Li et al., 2021; Wang et al., 2022).

Baicalein has conventionally been applied against a broad spectrum such as antioxidant, anti-inflammatory, anti-hepatotoxic, antiviral, and anticancer activities as well (Im et al., 2012; Sowndhararajan et al., 2017; Chagas et al., 2022; Chandrashekar and Pandi, 2022; Cui et al., 2022).

Soil salinity is another major abiotic stress that severely affects crop growth and yields around the world. That salinity induces osmotic pressure, ion toxicity, anabolic disorders, and oxidative stress (Munns and Tester, 2008; Arzani and Ashraf, 2016). Such factors may lead to physiological and biochemical abnormalities in plant tissues.

Low molecular weight non-enzymatic antioxidants are produced by plant cells and these phenolic compounds play a significant role in eliminating the ROS (Hodaei et al., 2018).

Due to their ability to protect biological systems from different kinds of oxidative stresses, phenolic compounds are important for redox homeostasis and could be a possible target in developing stress tolerance approaches in plants (Trchounian et al., 2016). So, the homeostasis of ROS and phytochemicals (such as polyphenols and flavonoids) also possibly influenced the plant adaptation to salt stress.

The objectives of this study were to determine the effect of salinity treatment with different concentrations of NaCl on flavone biosynthesis and their antioxidant activities in the hairy root culture of S. baicalensis.

MATERIALS AND METHODS

RESULTS

1. Phenotypic factors of different NaCl concentration treated S. baicalensis

The effect of different NaCl concentrations on the dry weight of S. baicalensis hairy roots highlights a gradual decline in biomass as salt concentration increases. At NaCl 0, 30, and 50 mM, the dry weight remains relatively high (–0.3 g/30 ㎖) and there is no statistical difference between the treatments.

This suggests that low to moderate salinity levels do not significantly impact root biomass production. However, at 100 mM NaCl, a marked decrease is observed, with dry weight significantly reduced.

At 200 and 300 mM NaCl, the dry weight declines further, reflecting no significant difference between them. This pattern demonstrates that high salinity levels adversely affect biomass accumulation, likely due to osmotic stress or ionic toxicity, suggesting a salt sensitivity threshold beyond 50 mM.

These findings provide insight into the tolerance limits of S. baicalensis roots under saline conditions, with potential implications for its cultivation in high-salinity environments (Fig. 1).

Fig. 1.

The dry weight of S. baicalensis hairy root culture was treated with different concentrations of NaCl.Values are means ± standard deviation of 3 replicates. nd; not detected. Means with each column followed by different alphabetical letters (a-c) indicating significant differences at 5% according to Duncan’s Multiple Range Test (DMRT, *p < 0.05).

2. Content of Baicalin, baicalein, and wogonin in different NaCl concentration treated S. baicalensis

Fig. 2. represents the impact of varying NaCl concentrations on three flavonoids in S. baicalensis: baicalin, baicalein, and wogonin. In all cases, the content of these flavonoids demonstrates a dynamic response to increasing salinity levels. Baicalin content peaks significantly at 100 mM NaCl (around 13 ㎎/g DW), with this concentration statistically distinct from all others.

Fig. 2.

Flavonoid contents in S. baicalensis hairy root culture exposed to different NaCl concentrations.(A) Baicalin contents variation treated NaCl in S. baicalensis hairy roots, (B) baicalein contents variation treated NaCl in S. baicalensis hairy roots, and (C) wogonin contents variation treated NaCl in S. baicalensis hairy roots. Values means ± standard deviation of 3 replicates. nd: not detected. Means with each column followed by different alphabetical letters a-c indicating significant differences at at 5% according to Duncan’s Multiple Range Test (DMRT, *p < 0.05).

Lower concentrations (0, 30, and 50 mM) yield moderate baicalin levels (–5 ㎎/g DW), while higher concentrations (200 and 300 mM) show a sharp decline, suggesting potential damage to biosynthesis pathways under extreme salinity.

Similarly, baicalein content also peaks at 100 mM NaCl (–2.5 ㎎/g DW), showing that this concentration optimizes flavonoid accumulation, likely as a stress response mechanism. At 0 mM NaCl, moderate baicalein production occurs, followed by further reductions at 30 and 50 mM. Beyond 100 mM, levels drop sharply, with 200 mM yielding minimal amounts, and 300 mM showing undetectable content.

Wogonin follows a comparable trend, with a peak at 100 mM NaCl (–2.2 ㎎/g DW), while c ontent d eclines a s NaC l concentration increases from 0 to 50 mM. Notably, wogonin is undetectable at both 200 and 300 mM NaCl, highlighting its sensitivity to severe salinity stress (Fig. 2).

These findings suggest that 100 mM NaCl triggers a beneficial stress response, maximizing flavonoid production, whereas excessive salinity (200 mM - 300 mM) disrupts metabolic processes, diminishing flavonoid synthesis and accumulation. This trend emphasizes a critical threshold for optimal stress-induced flavonoid production in S. baicalensis.

3. Total phenol and total flavonoid content of different NaCl concentration treated S. baicalensis

TP content gradually increased with increasing concentration of NaCl. At the lowest concentrations (30 and 50 mM NaCl) and the highest concentrations (200 and 300 mM NaCl), the TP content was significantly lower than that of the control (0 mM NaCl). The highest TP content was achieved in the hairy root treated with 100 mM NaCl, whereas the lowest was obtained in the 300 mM NaCl treatment (Table 1). The TF content also showed a similar trend to that of the TP content. At the minimum NaCl concentration (30 and 50 mM NaCl) the TF content gradually increased, whereas at the highest NaCl concentrations (200 and 300 mM NaCl) the TF content was significantly decreased.

Table 1.

Total phenolic and total flavonoid content of S. baicalensis hairy root culture treated with different NaCl concentrations.

In both TP and TF content, the 100 mM NaCl showed significantly higher content than that of control and other NaCl-treated hairy root cultures. Hence, for the antioxidant analyses, we selected 100 mM NaCl as a treated culture, whereas 0 mM NaCl) were selected as a control .

4. Antioxidative activity of different NaCl concentration treated S. baicalensis

DPPH and ABTS radical scavenging activities of S. baicalensis extract under two NaCl treatment conditions (0 mM and 100 mM NaCl) across different extract concentrations (31.25 to 1,000 ㎎/ℓ).

Fig. 3a shows the DPPH radical scavenging activity, where ascorbic acid serves as a positive control with - 85% scavenging activity. The DPPH activity gradually increases with higher extract concentrations, with 100 mM NaCl-treated samples consistently showing higher scavenging activity than the 0 mM samples.

Fig. 3.

Antioxidant activities of S. baicalensis hairy root culture treated with 0 mM and 100 mM NaCl concentrations.(A) DPPH radical scavenging activity (%); (B) ABTS radical scavenging activity (%). Values are means ± standard deviation of 3 replicates. The x-axis represents the concentration of each extract, and the y-axis represents the sample’s radical scavenging activity. AA; ascorbic acid, ns; not significant. Asterisks indicate significant differences between the S. baicalensis hairy root culture treated with 0 mM and 100 mM NaCl concentrations, as determined by Sidak's multiple comparison test (*p < 0.05, **p < 0.01, and ***p < 0.001).

At 1,000 ㎎/ℓL, both NaCl treatments exhibit their highest activities, with 100 mM NaCl achieving around 80% scavenging, at lower concentrations (31.25 to 125 ㎎/ℓ), there is little to no significant difference, except for a slight difference at 62.5 ㎎/ℓ, reflecting a threshold concentration for noticeable antioxidant activity (Fig. 3).

Figure 3b displays the ABTS radical scavenging activity, with a similar pattern as the DPPH assay. Ascorbic acid shows 65% scavenging activity, and extract activity increases with concentration. Again, the 100 mM NaCl-treated samples exhibit significantly higher ABTS activity at higher concentrations (500 and 1,000 ㎎/ℓ).

At lower concentrations (31.25 ㎎/ℓ – 125 ㎎/ℓ), no significant differences are observed between NaCl treatments. These results suggest that salt stress enhances the antioxidant capacity of S. baicalensis, possibly due to an adaptive response that boosts the production of antioxidant metabolites. The difference in scavenging activity between treatments demonstrates the importance of stress conditions in modulating the plant's bioactive properties, with potential implications for the use of S. baicalensis extracts in health-related applications (Fig. 3).

The IC₅₀ values (concentration required to reduce initial free radicals by 50%) for both DPPH and ABTS assays in S. baicalensis under two salinity treatments: 0 mM NaCl and 100 mM NaCl. Lower IC₅₀ values indicate stronger antioxidant activity, as less extract is needed to achieve 50% radical scavenging. For DPPH, the 100 mM NaCl treated sample shows a significantly lower IC₅₀ value (0.8 ± 0.01 ㎎/㎖) compared to the 0 mM NaCl sample (1.14 ± 0.02 ㎎/㎖), suggesting that salt stress enhances the antioxidant capacity for DPPH scavenging (Table 2).

Table 2.

IC50 value of DPPH and ABTS assays of S. baicalensis hairy root culture treated with 0 mM and 100 mM NaCl concentrations.

Similarly, for the ABTS assay, the 100 mM NaCl treatment also results in a lower IC₅₀ value (0.55 ± 0.01 ㎎/㎖) compared to 0 mM NaCl (0.73 ± 0.0 ㎎/㎖). The improvement in antioxidant efficiency under 100 mM NaCl implies that salinity induces stress responses that boost the production of antioxidant metabolites, potentially increasing the plant’s ability to neutralize free radicals (Table 2).

However, there is no significant difference was found in the 100 mM NaCl, which indicates that the difference in antioxidant capacity for the ABTS assay is not statistically significant, suggesting variability in how different types of radicals respond to salt-induced changes in the plant. These findings highlight the complex relationship between environmental stress and bioactive compound production in S. baicalensis.

The reducing power of S. baicalensis extracts treated with different NaCl concentrations (0 mM and 100 mM) across a range of extract concentrations (31.25 to 1,000 ㎎/ℓ). Ascorbic acid, used as the positive control, demonstrates the highest reducing power (–0.3), indicating strong electron-donating ability. For the extracts, the reducing power increases proportionally with concentration.

At low concentrations (31.25 ㎎/ℓ), both NaCl treatments exhibit similar and minimal reducing power, with no statistically significant difference (ns). However, at 62.5 and 125 ㎎/ℓ, the 100 mM NaCl-treated samples show significantly higher reducing power, suggesting enhanced antioxidant potential under moderate salt stress. This trend becomes more pronounced at higher concentrations: at 250 ㎎/ℓ, the difference becomes highly significant.

The overall results suggest that salt stress (100 mM NaCl) enhances the reducing power of S. baicalensis extracts, likely due to an increase in antioxidant compounds that act as electron donors to neutralize free radicals (Fig. 4). This improvement in reducing power indicates that salinity stress promotes the production of bioactive compounds with enhanced redox potential, making 100 mM NaCl-treated extracts more effective in antioxidant assays. These findings highlight the role of environmental stress in influencing the antioxidant properties of S. baicalensis, with potential implications for its use in therapeutic and health-related applications.

Fig. 4.

Reducing power assay of S. baicalensis hairy root culture treated with 0 mM and 100 mM NaCl concentrations.Values are means ± standard deviation of 3 replicates. The x-axis represents the concentration of each extract, and the y-axis represents the sample’s reducing power. AA; ascorbic acid, ns; not significant. Asterisks indicate significant differences between the S. baicalensis hairy root culture treated with 0 mM and 100 mM NaCl concentrations, as determined by Sidak's multiple comparison test (*p < 0.05; **p < 0.01, and ***p < 0.001).

The effect of varying concentrations of NaCl on biomass production. As the NaCl concentration increases, there is a clear decline in biomass, suggesting that higher salinity levels negatively a ffect growth. At 0 mM NaC l, the b iomass is a t its highest, with a mean value of 0.33 ± 0.01 g/30 mL. However, as the concentration rises to 30 mM and 50 mM, the biomass decreases to 0.32 ± 0.02 g/30㎖ and 0.27 ± 0.03 g/30 ㎖, respectively. This trend continues more sharply at higher concentrations, with biomass values reducing to 0.24 ± 0.01 g/30㎖ at 100 mM, 0.12 ± 0.01 g/30㎖ at 200 mM, and reaching the lowest value of 0.09 ± 0.01 g/30㎖ at 300 mM NaCl.

These findings indicate a dose-dependent inhibition of biomass production with increasing salinity, likely due to the osmotic stress and ionic toxicity imposed by elevated NaCl concentrations (Table 2).

The impact of different NaCl concentrations on the flavonoid content, specifically focusing on baicalin, baicalein, and wogonin, measured in ㎎/g dry weight (DW). Baicalin content initially increases with mild salinity, reaching a peak of 11.39 ± 1.53 ㎎/g DW at 100 mM NaCl, indicating that moderate salt stress may enhance its accumulation.

However, at higher concentrations (200 mM and 300 mM), baicalin levels sharply decline to 1.22 ± 0.75 ㎎/g DW and 0.39 ± 0 ㎎/g DW, respectively, suggesting a detrimental effect of excessive salinity. Similarly, baicalein content peaks at 2.35 ± 0.05 ㎎/g DW at 100 mM NaCl but is drastically reduced at higher concentrations, with no detectable levels at 300 mM. Wogonin follows a comparable pattern, with a peak of 2.26 ± 0.07 ㎎/g DW at 100 mM, followed by a complete absence at 200 mM and 300 mM NaCl.

These results indicate that moderate salinity may stimulate flavonoid production, while excessive NaCl concentrations inhibit their accumulation, likely due to stress-induced metabolic limitations.

The antioxidant activity was measured through DPPH, ABTS, and reducing power assays, at varying concentrations of extracts under two NaCl treatments: 0 mM and 100 mM. In the DPPH assay, antioxidant activity improves with increasing extract concentration, with NaCl 100 mM consistently showing higher activity across all concentrations, reaching 60.08 ± 2.56 ㎎/ℓ at 1,000 ㎎/ℓ compared to 44.69 ± 6.27 ㎎/ℓ for NaCl 0 mM.

A similar trend is observed in the ABTS assay, where NaCl 100 mM treatment enhances antioxidant potential, peaking at 80.74 ± 2.89 ㎎/ℓ at 1,000 ㎎/ℓ, in contrast to 67.18 ± 3.83 ㎎/ℓ for NaCl 0 mM.

The reducing power assay also shows a slight improvement with NaCl 100 mM, where the activity increases from 0.07 ± 0.0 to 0.27 ± 0.0 at the highest concentration, compared to 0.21 ± 0.0 for NaCl 0 mM.

These results suggest that exposure to moderate salinity (100 mM NaCl) enhances the antioxidant properties of the extracts, likely due to an upregulation of stress-responsive metabolites, improving free radical scavenging and reducing power capabilities across the assays.

DISCUSSION

Salinity stress had an observable effect on the biomass of hairy roots, as shown by the decrease in root biomass with increasing NaCl concentration. At the early concentrations (0, 30, and 50 mM), biomass was only slightly diminished (0.3 g/30 ㎖) indicating that panicoids can tolerate low levels of salinity without serious growth impairment (Wang et al., 2018).

At 100 mM NaCl, biomass was significantly lower than in control conditions suggesting that the process of growth suppression associated with stress has begun. The drastic reduction in plant height at 200 and 300 mM NaCl reflects the high sensitivity of L. sativa germination to elevated salinity, presumably due to osmotic stress and ionic toxicity which hinder water uptake and disturb cellular homeostasis (Fang et al., 2023; Solov’eva et al., 2023).

These results correspond with the studies on other medicinal plants (e.g. S. baicalensis and S. pycnoclada) which reported that the high salinity reduced root biomass and inhibited metabolic activities in roots (Lee et al., 2013).

The S. baicalensis hairy roots showed a non-linear responsiveness in flavonoid biosynthesis to concentration of NaCl. The yield peak of baicalin, baicalein, and wogonin was at 100 mM NaCl treatment, indicating that medium salinity could activate a positive stress physiology that promotes the accumulation of flavonoids. At 100 mM NaCl, the content of baicalin increased to 11.39 ± 1.53 ㎎/g DW, and then dropped sharply at 200 mM and further declined at 300 mM NaCl suggesting that high salinity induces disturbances in metabolic pathways critical for flavonoids (Kim et al., 2012).

This trend corroborates with findings showing that stress conditions like salinity can enhance the production of secondary metabolites through cellular metabolic changes (Wang et al., 2018), but too high an extent of stress inhibits biosynthetic processes.

The significantly more potent antioxidant capacity of S. baicalensis extracts subjected to salt stress is confirmed by DPPH and ABTS assays.

DPPH scavenging activity at all extract concentrations showed higher antioxidant activity in the NaCl-treated sample (100 mM) than in the untreated samples, where 80% was DPPH scavenging activity at a concentration of extract 1,000 ㎎/ℓ.

In ABTS this pattern is similarly observed, with the NaCl 100 mM samples leading to an abolition of scavenging activity (80.74 ㎎/ℓ), significantly higher than for NaCl 0 mM. This indicates that NaCl stress activates a response, which causes the plant to be primed to synthesize antioxidant metabolites useful in defending against oxidative stress (Schenk and Hildebrandt, 1972). Nevertheless, the IC₅₀ values indicated decreased antioxidant activity at 300 mM NaCl, which is an indication that high salinity stress can harm metabolic efficiency (Kim et al., 2012; Park et al., 2011).

The reducing power assay further demonstrated that the electron-donating ability of S. baicalensis extracts improves under moderate salt stress. At higher extract concentrations (250 ㎎/ℓ - 1,000 ㎎/ℓ), NaCl-treated samples (100 mM) showed significantly higher reducing power compared to untreated samples, suggesting enhanced production of bioactive compounds with redox potential. This increase in reducing power corresponds to the plant’s ability to synthesize flavonoids such as baicalin, baicalein, and wogonin, which possess strong antioxidant properties.

The observed trend is consistent with findings that salinity induces the accumulation of stress-responsive metabolites in hairy root cultures, enhancing their pharmacological properties (Hostetler et al., 2017; Park et al., 2021).

Studies on other strains of S. baicalensis and related species highlight similar trends in response to environmental stress. For example, S. pycnoclada showed higher flavonoid accumulation under optimized stress conditions, although the species lacked some biosynthetic enzymes present in S. baicalensis (Park et al., 2011; Solov’eva et al., 2023). This suggests that environmental factors play a crucial role in modulating secondary metabolism in these medicinal plants. Additionally, genetic modifications through metabolic engineering have been proposed as a strategy to further enhance flavonoid production under stress conditions (Park et al., 2021).

The findings offer vital information on the interaction of salinity conditions and metabolic behaviors in S. baicalensis. Flavonoid synthesizing and antioxidant activity are maximized by moderate salt stress (100 mM NaCl) revealing a crucial limit for adaptive responses to stress Hey, but high salinity (200 mM - 300 mM) hinders these processes and highlights the need for environmental management during cultivation.

Our results have valuable implications for the pharmaceutical industry because metabolic changes induced by stress on S. baicalensis may be further exploited to promote its therapeutic potential. Future studies should investigate more genetic and environmental interactions to maximize flavonoid production of this medicinal plant and develop sustainable cultivation approaches (Liu et al., 2021).

In conclusion, S. baicalensis responds dynamically to varying NaCl concentrations, with 100 mM NaCl enhancing flavonoid production (baicalin, baicalein, wogonin) and antioxidant activity through improved radical scavenging and reducing power. However, higher salinity (200–300 mM) impairs biomass and flavonoid synthesis due to osmotic stress and ionic toxicity. Moderate salinity thus offers a strategic approach to boost the plant’s pharmacological potential, while excessive levels hinder its benefits, highlighting the need for optimized growing conditions.

Acknowledgments

This research was supported by the Bio and Medical Tehcnology Development Program of the National Reserach Foundation(NRF) and funded by the Korean Government (MSIT, No. 2022M3E5E6018649).

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