LED Light Treatments Enhance the Synthesis of Bioactive Compounds in Salvia lavandulifolia Vahl

TL;DR

This study shows that using specific LED light colors can boost the production of health-boosting compounds in a Mediterranean plant called Salvia lavandulifolia.

Contribution

The study demonstrates that LED light spectra can be tailored to enhance bioactive compound synthesis in S. lavandulifolia ecotypes.

Findings

Red LED light increased photosynthetic pigments and non-enzymatic antioxidants in ecotype 1.

White/blue light improved catalase and antioxidant activity in ecotype 2 without affecting pigments.

Both ecotypes showed increased α-tocopherol and hydroxycinnamic acid derivatives with no oxidative damage.

Abstract

Salvia lavandulifolia Vahl., a species native to the Western Mediterranean, is valued for its bioactive compounds and beneficial biological properties. Commonly propagated in greenhouses, it may benefit from exposure to tailored light-emitting diode (LED) light to enhance antioxidant defense and metabolite production. This study examined the effects of various spectra on two S. lavandulifolia ecotypes from southeastern Spain. Plants were propagated in vitro and grown for 30 days under white, red, blue, red/blue (70:30), white/blue, or white/red LED light, under a 16/8 h light/dark photoperiod (light intensity of 115 µmol m−2 s−1). Photosynthetic pigments, enzymatic antioxidants (superoxide dismutase and catalase), non-enzymatic antioxidants (tocopherols and polyphenols), antioxidant capacity (FRAP and DPPH•), and lipid peroxidation (MDA) were assessed. In ecotype 1, red LED light significantly increased the content of photosynthetic pigments and non-enzymatic antioxidants while reducing enzymatic antioxidant activity. In contrast, ecotype 2 showed higher catalase and non-enzymatic antioxidant activity under white/blue light, without changes in pigment content. In both ecotypes, these treatments increased α-tocopherol and hydroxycinnamic acid derivative content, strengthening antioxidant defenses without inducing oxidative damage. Overall, the results highlight the need to customize LED light spectra for each ecotype, as genetic background may significantly influence plant responses.

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1. Introduction

The Salvia genus, which includes over 1000 species [1], is known for its diverse phytochemical profile and is utilized globally in traditional medicine [2], culinary applications [3], and cosmetics [4]. A notable member of this genus is Salvia lavandulifolia Vahl., commonly known as Spanish sage, which is native to the Iberian Peninsula and found from the southeastern region of Morocco to the Mediterranean regions of France [5]. This aromatic plant has a long history of use in both cooking and traditional medicine [6]. It has been employed for enhancing cognitive performance [7], and diabetes management [8], as well as food preservation [9], and as an additive in animal feed [10]. Its effectiveness is based on strong antioxidant, antimicrobial, and neuroprotective properties, primarily linked to its essential oil [11] and polyphenolic content [8].

In this regard, polyphenols play an important role in plant defense and human health [12]. Their synthesis is associated with plant stress tolerance and growth, functioning as key secondary metabolites with diverse protective and regulatory effects [13,14]. These factors motivate producers to enhance the synthesis of these components in plants. To this end, researchers have proposed several strategies, including treatments with elicitors such as methyl jasmonate [15], genetic or biotechnological approaches [16], and modification of light quantity and quality [17].

Light plays a crucial role as an environmental factor in regulating the synthesis of plant secondary metabolites. It affects the production and accumulation of these compounds through various regulatory mechanisms [17]. In relation to this, several studies have provided valuable insights into how different light-emitting diode (LED) spectra influence plant physiology, secondary metabolite production, and stress resilience, particularly in the genus Salvia. Research on Salvia miltiorrhiza [18], S. bulleyana [19], S. viridis [20], and S. atropatana [15] has shown that mixed red and blue LED light, especially with a dominance of red, generally promotes shoot regeneration, biomass accumulation, and overall plant growth compared to monochromatic light or fluorescent control conditions. These results are further supported by molecular data, including the light-induced upregulation of key phenylpropanoid pathway genes in S. miltiorrhiza [18]. Overall, the available evidence indicates that tailored LED lighting represents a powerful and versatile tool for improving growth performance and maximizing the yield of phenolic compounds, including rosmarinic acid, in Salvia species.

On the other hand, the optimal light spectrum and intensity for plants vary based on the species and tissue type, requiring careful experimental optimization for each specific application [21]. For example, red and blue light are the most effective for photosynthesis: red light promotes biomass growth, while blue light enhances morphology, stomatal function, and pigment synthesis [22]; however, using only red or blue light can lead to abnormal growth. In line with this, studies confirm that red-blue LED combinations are more effective than broad-spectrum lighting by boosting crop yields alongside essential oil content, phenolics, and antioxidant reserves in multiple species [23]. Previous research in our laboratories on two ecotypes of Thymus mastichina (Spanish marjoram), each adapted to distinct environmental conditions and representing different essential oil chemotypes, showed that varying LED light spectra differentially affects the production of photosynthetic pigments and the antioxidant defense system.

The impact is influenced not only by the wavelength of light used but also by the plant’s chemotype [24]. The study concluded that exposure of the eucalyptol chemotype to red light or the linalool chemotype to a combination of red and blue light (ratio of 7:3) enhances the plants’ antioxidant system, increasing their antioxidant capacity and reducing lipid peroxidation.

Despite substantial progress in understanding light-mediated metabolic regulation in several Salvia species, the effects of LED light spectra on secondary metabolite biosynthesis in Salvia lavandulifolia remain largely unexplored. Based on the hypothesis that customized light treatments can improve plant health and optimize agricultural practices, this study aimed to evaluate whether modifying light spectra enhances physiological performance and the synthesis of bioactive compounds in two ecotypes of S. lavandulifolia (Spanish sage). These adjustments in light quality were expected to influence key physiological responses and strengthen the plants’ antioxidant defense mechanisms, and the results address a gap in the literature concerning the application of LED light optimization for this species.

2. Results

Table 1 presents the ANOVA results showing how the six LED light treatments affected the two S. lavandulifolia ecotypes individually and their combined interactions.

2.1. Photosynthetic Pigments (Chlorophyll a, Chlorophyll b, Total Chlorophylls, and Carotenoids)

The ANOVA results presented in Table 1 indicate that ecotype significantly influenced the levels of photosynthetic pigments, with the exception of Chl b. Conversely, analyzing the data by treatment type, Chl b levels were found to be affected by the different LED spectra. Additionally, no ecotype-treatment interaction was seen in the levels of Chl b or carotenoids (Car).

Nonetheless, analyzing the effects of the light treatments by ecotype, significant differences were observed between the various LED spectra (Table 2). Plants from ecotype 1 exposed to red LED (R) light exhibited the highest levels of Chl T, as well as elevated levels of Chl b, while the lowest levels of photosynthetic pigments were found in untreated plants (the control condition, C). Moreover, the production of Car was the highest under the combination of red and blue LED (R + B) treatment.

In contrast, the results for ecotype 2 indicate that exposure to blue LED (B) light promoted the synthesis of Chl a and b. Conversely, exposure to R light resulted in a degradation or lower synthesis of Chl T. Similarly, exposure to R + B light, as well as R light alone, led to a reduction in Car content in this ecotype.

Regarding the ratio of Chl a to Chl b, the response to different LED light spectra varies significantly by ecotype (Table 1). In both ecotypes, however, the lowest ratios were observed with monochromatic R light (Table 2).

2.2. Non-Enzymatic Antioxidants

The ANOVA results reported in Table 1 indicate statistically significant differences in α-tocopherol (α-T) and plastochromanol-8 (PC-8) content between ecotypes; however, there was no clear trend by treatment type because the response varied with ecotype.

2.2.1. α-Tocopherol and Plastochromanol-8

As shown in Figure 1, ecotype 1 produced lower levels of both tocochromanols than ecotype 2. However, the combination of light sources used had a different impact on each ecotype. Specifically, monochromatic B light significantly increased the levels of α-T in ecotype 1, yielding the highest levels among all treatments. In contrast, ecotype 2 achieved the highest values with treatments that involved supplementing white light with either blue LED (C + B) or red LED (C + R) light.

On the other hand, the lowest α-T values in ecotype 1 were obtained under the control condition and C + B light. In contrast, levels in ecotype 2 were negatively affected by exposure to monochromatic R light. Notably, under this treatment, the α-T levels in ecotype 1—which are generally lower—reached values comparable to those in ecotype 2.

In the case of PC-8, B light resulted in the highest concentration in ecotype 1. For ecotype 2, R + B light produced the highest content of this tocochromanol. Conversely, the treatments that negatively affected PC-8 production were C and C + R light for ecotypes 1 and 2, respectively.

2.2.2. Phenolic Profile

High-performance liquid chromatography (HPLC) diode array detection analysis enabled the identification and quantification of 10 major polyphenolic compounds in Salvia lavandulifolia (Figure 2 and Table 3).

To identify the metabolites primarily responsible for the chemical variance in our dataset, a principal component analysis (PCA) was performed (Figure 2). The first two components explained 75% of the total variance. While the PCA did not reveal discrete clusters based on light treatments or ecotypes, it was instrumental for variable identification.

The first component accounted for approximately 58% of the total variance and was correlated positively with salvianolic acid B and luteolin-7-glucoside, and negatively with rosmarinic acid. The second component explained 17% of the variance, showing positive correlations with salvianolic acid K and salvianic acid. Hence, these compounds can be identified as the most sensitive indicators of changes in the plant’s secondary metabolism in response to spectral quality.

The major phenolic compounds quantified in both ecotypes are rosmarinic acid, followed by salvianolic acids K and B, as can be seen in Table 3. Notably, ecotype 2 shows a higher synthesis of polyphenolic components than ecotype 1.

In ecotype 1, the synthesis of hydroxycinnamic acid derivatives (4-caffeoyl-quinic acid, 5-caffeoylquinic acid, caffeic acid, and salvianolic acid B) seemed to increase after exposure to C + R light. Meanwhile, we observed a negative effect of pure B or R light on the concentration of these components. In contrast, the presence of other hydroxycinnamic acid derivatives, including rosmarinic acid, salvianolic acid K, the di-hydroxyphenyl lactic acid (salvianic acid), and the flavones (luteolin-7-glucuronide and luteolin-7-glucoside), increased after R + B or B light treatments. In ecotype 2, under C + B light, increases were observed in the concentrations of hydroxycinnamic acid derivatives 4-caffeoyl-quinic acid and 5-caffeoyl-quinic acid, and also in salvianolic acid B, while B and R light exposure negatively influenced all these components. On the other hand, rosmarinic acid and salvianolic K were favored by exposure to C + R treatment, and flavone concentrations increased under R + B or B spectra.

2.3. Enzymatic Antioxidants

To assess the enzymatic antioxidant activity, two enzymes were measured: superoxide dismutase (SOD) and catalase (CAT).

2.3.1. Superoxide Dismutase Activity

In relation to SOD (Figure 3), ecotype 2 exhibited greater enzymatic activity than ecotype 1. Notably, while the response to specific spectra varied by ecotype, activity levels were generally lower with all treatments than under control conditions.

In particular, SOD activity in ecotype 1 decreased significantly under both B and R light supplementation (C + B and C + R, respectively), while in ecotype 2, it was most strongly disrupted by monochromatic B light treatment.

2.3.2. Catalase Activity

In relation to the CAT activity, enzyme levels were found to be similar in the two ecotypes, but their responses to light treatment varied (Figure 4). Specifically, CAT activity in ecotype 1 was highest under the control condition, decreasing significantly with all treatments and most markedly with monochromatic B light.

In contrast, in ecotype 2, enzyme activity increased significantly when plants were grown under white light supplemented with either blue or red (C + B or C + R), while it decreased when the treatment used was a combination of monochromatic blue and monochromatic red light (R + B).

2.4. Antioxidant Activity and Lipid Peroxidation

Antioxidant status of Spanish sage under six different LED light treatments was measured using DPPH^•^ radical scavenging activity and ferric reducing antioxidant power (FRAP), while lipid peroxidation was evaluated based on malondialdehyde (MDA) production.

2.4.1. DPPH• and FRAP Activity

To assess antioxidant activity, two different methods were employed: the DPPH^•^ and FRAP assays (Figure 5). As anticipated, the results varied depending on the nature of the method used.

Nonetheless, as shown in Figure 5, both methods indicated that antioxidant activity was higher in ecotype 2 than in ecotype 1, and the response to LED treatments was ecotype dependent. Specifically, both methods showed the highest activity with R + B light in ecotype 1 and with C + B in ecotype 2. Conversely, ecotype 1 displayed the lowest activity under the control condition, while ecotype 2 demonstrated significantly diminished activity in response to B light treatment.

Pearson correlation analysis revealed that antioxidant activity, as measured by both DPPH^•^ and FRAP assays, was strongly and significantly related to the production of rosmarinic acid (r = 0.92 and r = 0.91, respectively) and that of luteolin-7-glucuronide (r = 0.74 and r = 0.85, respectively). The correlation also showed some variation across antioxidant activity methods. In particular, antioxidant activity was correlated with salvianic acid levels only when considering DPPH^•^ results (r = 0.72), and with salvianolic B acid levels considering FRAP results (r = 0.83).

2.4.2. Malondialdehyde Production

The lipid peroxidation results shown in Figure 6 indicate that ecotype 1 produces more MDA than ecotype 2. However, the extent of lipid peroxidation in each ecotype was influenced by the light treatments. Specifically, ecotype 1 experienced more lipid peroxidation when exposed to C + B light than under the control condition. It is also noteworthy that neither C + R nor R light treatments led to significant increases in MDA levels.

In the case of ecotype 2, only the treatments using monochromatic B or R light spectra resulted in increased levels of MDA compared to the control condition, with B light leading to the highest level of oxidative stress. That is, none of the other treatments was associated with any increase in MDA levels compared to the control.

3. Discussion

Light is a major factor influencing the biosynthesis of specialized metabolites in plants. It triggers a cascade of responses in photoreceptors and influences the expression of genes and corresponding biosynthetic pathways [25]. In this context, LED lighting, especially when customized with specific blue and red spectra, is an effective tool for enhancing chlorophyll production and increasing the accumulation of valuable secondary metabolites in plants [26]. The effects of light treatment are, however, likely to differ based on the plant species and the specific lighting conditions used [21]. Additionally, our research indicates that responses may vary even within the same species, depending on the ecotype.

When interpreting the physiological and biochemical responses of Salvia lavandulifolia, it is important to consider the light dosage used, namely, 115 µmol m^−2^ s^−1^. According to the biphasic response model, plant secondary metabolism can be stimulated by moderate light levels or inhibited by excessive high-intensity stress. In the case of the Salvia genus, the photosynthetic photon flux density applied can be categorized as ‘moderate’ and non-stressful. For example, Kozłowska et al. [27] defined 130 µmol m^−2^ s^−1^ as a moderate intensity for Salvia species in controlled environments, as this level promotes metabolic acclimation without causing photoinhibition. Additionally, as a Mediterranean species that has adapted to high solar radiation, S. lavandulifolia has a high threshold for light-induced damage [28].

According to our results, the synthesis of chlorophylls in ecotype 1 is enhanced by exposure to various light treatments, with the most significant effects observed under R light. On the other hand, for ecotype 2, exposure to R light reduces the content of both Chl a and b. Instead, B light exposure significantly increases the levels of both forms of chlorophyll in this ecotype. These contrasting responses highlight the need to adjust LED light spectra to the specific needs of individual ecotypes within each plant species, especially when no prior selection or breeding programs have been implemented.

Both forms of chlorophyll are essential for plant photosynthesis. Chl a primarily drives photochemical reactions, while Chl b expands the range of light that can be absorbed [29]. A decrease in chlorophyll content after the application of R light may be linked to the impact of this light spectrum on the efficiency of photosystem II (PSII). Longer wavelengths primarily excite photosystem I (PSI), and excessive excitation of PSI can limit the efficiency of PSII [30,31].

In contrast, exposure to a combination of 660 nm red light with white LEDs has significantly increased chlorophyll content in certain species, such as lettuce. This enhancement improves the electron transport efficiency in PSII and leads to a greater accumulation of photosynthetic products [32].

Similarly, the effect of B light can vary depending on the species, ecotype, and dosage. For example, exposure to B light increases chlorophyll content in potato plants [33], whereas it can induce stress in saffron plants, resulting in photoinhibition and reduced chlorophyll levels [34].

The balance of Chl a and b in chloroplasts is essential for a plant’s adaptation to varying light conditions, its photosynthetic efficiency, and its ability to protect itself from excess light. This balance allows the light-harvesting complex to adjust to specific conditions [35]. Exposure to R light decreases the Chl a/b ratio in both ecotypes. A lower chlorophyll ratio suggests that the plant is modifying its light-harvesting pigments to enhance photosynthetic efficiency.

In ecotype 1, the reduction in the chlorophyll ratio is related to the observed increase in Chl b content, possibly indicating adaptation to low-light energy environments. This effect can be reduced by increasing the dosage of B light, as seen with the R + B treatment, which enhances the proportion of chlorophyll in this ecotype. In ecotype 2, the decrease in the chlorophyll ratio is linked to reductions in the concentrations of both Chl a and b. This may result from exposure to R light, as noted by Su et al. [31], since excessive excitation of PSI can impair the efficiency of PSII.

Carotenoids play an auxiliary role in plant photosynthesis by transferring absorbed radiation energy to chlorophylls and protecting against photooxidative damage [36]. Therefore, the relationship between chlorophylls and carotenoids is crucial for photosynthetic efficiency in plants [37].

Our findings concerning the effects of different LED light spectra on carotenoid content in two ecotypes studied show that exposure to R + B or R light improved the content of these pigments in one case (ecotype 1), but had a negative effect in the other (ecotype 2). For ecotype 2, B light seems to increment the synthesis of chlorophylls, but maintain the accumulation of carotenoids. Consequently, the variations in Chl and Car responses to B or R light among different ecotypes can be attributed to differences in photoreceptor sensitivity as well as gene regulation and metabolic pathways [38,39]. These ecotypes (as described in the Materials and Methods section) were collected from various locations in southeastern Spain, where environmental conditions differ. This variation may explain the differences in adaptation to various light conditions (intensities and wavelengths).

Light can cause distress (and even lasting damage) at extreme intensities or unsuitable spectra, or eustress that enhances antioxidant systems and secondary metabolites without loss of yield [40]. To cope with stressful conditions, plants have natural defense mechanisms that include both non-enzymatic and enzymatic antioxidant systems. Enzymes function as isoforms that target chloroplasts, mitochondria, peroxisomes, the apoplast, and the cytosol, allowing for localized control of reactive oxygen species (ROS) where they are produced [41]. Additionally, other metabolites (carotenoids, tocopherols, and polyphenols) support ROS scavenging and protect membranes and photosystems [14].

In this respect, a recent study by Jené and Munné-Boch [42] revealed an association between PSII activity, chlorophyll content, and tocopherols (vitamin E) in parasitic plants. They described the antioxidant and photoprotective role of vitamin E in plants with reduced photosynthetic capacity. In Spanish sage, different light spectra had distinct effects on α-T and PC-8 levels in both ecotypes. As reported by Stange and Flores [43], tocopherols easily respond to light-driven manipulations, as their metabolic pathways are related to photosynthetic pigments [44]. In our experiments, exposure to B LED light resulted in a significant increase in the content of these tocochromanols in ecotype 1; however, the positive correlation between the synthesis of photosynthetic pigments and tocochromanols was more evident after exposure to R light. In ecotype 2, α-tocopherol levels decreased following exposure to R light, coinciding with the degradation of both chlorophyll forms (a and b). The B light treatment did not affect the vitamin E content of this sage ecotype, but Chl a and b content increased. Therefore, it can be stated that exposure to R light has a more significant impact on the synthesis of photosynthetic pigments and tocochromanols than exposure to other light spectra, with a positive correlation between these two parameters.

Similar behavior was observed in the quantitative analysis of the polyphenolic profile; as with tocochromanols, these components are directly related to the plant’s antioxidant defense system. Spanish sage also showed an ecotype-dependent response to different LED light spectra in terms of the concentration of these phenolic components. In ecotype 1, the application of R + B (70:30) LED light spectra generally promoted the most beneficial effects, as indicated by the number of components whose concentrations increased or remained unchanged following exposure to this spectrum. Under this light spectrum, the concentrations of salvianic acid, rosmarinic acid, and the flavonoids luteolin-7-glucuronide and luteolin-7-glucoside significantly increased compared to the control treatment. In contrast, for ecotype 2, supplementing white light with red (C + R) or blue light (C + B) seemed to increase the content of hydroxycinnamic acids and hydroxyphenyl lactic acid (salvianic acid).

These results are important for maintaining a more active antioxidant defense against photo-oxidation. It is known that the quality of LED light influences the expression of phenylpropanoid and flavonoid biosynthetic genes, resulting in polyphenol accumulation in aromatic and medicinal plants [45]. According to Zhang et al. [18], in Salvia miltiorrhiza, the combination of blue and red light upregulates the expression of several key enzyme-encoding genes (SmPAL1 and Sm4CL1) in the rosmarinic acid pathway. However, the ecotype-dependent responses in terms of polyphenolic component synthesis to the LED light spectra applied in our experiments highlight the importance of selecting the appropriate light spectrum for achieving the desired phenylpropanoid and flavonoid profiles. Additionally, in relation to the enzymatic antioxidant defense system, SOD and CAT activities generally decreased following exposure to the LED light spectra tested. SOD is consistently the first-line ROS detox enzyme, converting O_2_^−^• to H_2_O_2_, which is then removed by CAT, ascorbate peroxidase (APX), and peroxidase [46]. When antioxidant enzymatic activity decreases under a specific light spectrum or intensity, ROS accumulate, and oxidative damage usually increases, unless other antioxidants compensate for these changes [47]. Previous research shows that light, especially from red and blue LED sources, interacts with photoreceptors such as phytochromes and cryptochromes. This interaction activates transcription factors like elongated hypocotyl 5 and the myeloblastosis family, which in turn upregulate the expression of genes responsible for antioxidant enzymes (such as SOD, CAT, and APX) as well as those involved in phenylpropanoid biosynthesis (including phenylalanine ammonia-lyase and chalcone synthase) [48].

In ecotype 1, lower activity of both enzymes studied (SOD and CAT) was observed after applying different LED light spectra compared to the control treatment. However, only the clones exposed to R light did not show more oxidative damage, with MDA levels being similar to those in the control group. Under R light, there was an increase in the content of photosynthetic pigments. Although the highest levels of bioactive components were not reached with this light treatment, there was an increase in tocochromanols and polyphenolic compounds—namely, salvianic acid, rosmarinic acid, salvianolic acid K, and the flavonoids luteolin-7-glucoside and luteolin-7-glucuronide. These increases were associated with higher antioxidant capacities than under control conditions. Therefore, for this ecotype, exposure to R light seemed to strengthen the non-enzymatic antioxidant defense system, leading to higher levels of bioactive components with enhanced antioxidant capacities in their extracts, without causing oxidative damage compared to the control group.

In ecotype 2, lower SOD activity was observed for all LED light treatments compared to the control conditions. However, in this ecotype, higher CAT activity was observed after exposure to C + R and C + B light spectra. Additionally, exposing these clones to white light supplemented with blue light did not alter the content of photosynthetic pigments, but did increase the levels of α-tocopherol, hydroxycinnamic acid derivatives, and hydroxyphenyl lactic acid. This suggests a higher efficacy of antioxidant defense in this ecotype, with antioxidant capacities being higher, while no oxidative damage was detected.

The reduction in oxidative damage observed under specific LED light spectra in both ecotypes indicates a light-mediated upregulation of antioxidant defense mechanisms, likely involving enhanced ROS scavenging and improved redox homeostasis, thereby increasing tolerance to environmental stress [14].

4. Materials and Methods

4.1. Plant Material In Vitro Growth and LED Light Treatment

Treatment

The experimental material consisted of two Salvia ecotypes collected from two different locations in Spain: Moratalla (38°12′52″ N, 2°5′48″ O, 760 m above sea level) in the province of Murcia, referred to as ecotype 1, and Letur (38°14′27″ N, 2°10′12″ W, 1185 m above sea level) in the province of Albacete, referred to as ecotype 2. This material was pre-selected based on the chemotypes of their essential oils identified in our previous research, specifically, 45% camphor in ecotype 1 and 48% 1,8-cineole for ecotype 2. These chemotypic differences likely reflect genetic variability arising from adaptation to distinct environmental conditions. Clonal lines were established following the micropropagation system described by Cáceres-Cevallos et al. [49]. Plants were propagated in vitro, and after acclimatization, grown in a controlled chamber for 2 months. The controlled chamber was kept at 24 ± 2 °C, with a relative humidity of 70% to 75%, and a 16/8-h light/dark cycle. For the next 30 days, 60 clones of each ecotype were exposed to one of the following six LED light treatments.

The experimental design, light treatments, and sampling strategy were based on a protocol previously optimized for Thymus mastichina under similar LED conditions [24] and were adapted here for the S. lavandulifolia ecotypes.

Plants were grown in a growth chamber equipped with programmable multi-spectral LED panels (HE-LIABIS GH; NTE, Murcia, Spain), which allowed for independent control of several narrow light bands. Six different light regimes were tested: a broad-spectrum white light (control), white light supplemented with additional blue (C + B) or red (C + R) photons, a red/blue mixture with a 70:30 ratio (R + B), and monochromatic blue (B) or red (R) light. The incident photon flux at canopy height was set to 115 μmol m^−2^ s^−1^ and was verified using a spectral PAR meter (PG200N, UPRtek, Miaoli County, Taiwan). Chamber temperature was maintained at 24 ± 1 °C with 75 ± 5% relative humidity, following a light/dark cycle of 16 h of light and 8 h of darkness.

After the 30-day light treatment period, half of the plants from each combination were randomly selected from an initial sample of 60. They were immediately frozen in liquid nitrogen, lyophilized, and then stored at −80 °C until biochemical analyses were conducted.

4.2. Photosynthetic Pigments Extraction and Quantification

Chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were determined according to Szekely-Varga et al. [50] with minor modifications. Briefly, 100 mg of freeze-dried leaf tissue was homogenized in absolute methanol, and clarified extracts were read spectrophotometrically at 665.2 for Chl a, 652.4 for Chl b, and 470 nm for Car (UV-2401 PC, Shimadzu, Japan). Pigment concentrations were calculated using Lichtenthaler and Buschmann’s equations and normalized to the dry weight of the samples [51].

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4.3. Non-Enzymatic Antioxidant and Lipid Peroxidation Analysis

4.3.1. α-Tocopherol and Plastochromanol-8 Extraction

Lipophilic antioxidants (α-T and PC-8) were extracted from freeze-dried leaf tissue using a hexane/ethyl acetate mixture using a modified version of the method described by Quílez et al. [52]. Approximately 100 mg of finely ground material was mixed with 1.5 mL of cold solvent (6:4, v/v) and gently agitated for 4 h at 15 °C in darkness under an inert atmosphere. The extracts were then treated with anhydrous sodium sulfate to remove residual water and centrifuged (2061× g, 10 min, 15 °C). The organic phase was filtered through 0.45 µm nylon membranes and evaporated at 35 °C under reduced pressure using a concentrator. The resulting residues were re-dissolved in ethyl acetate and stored at −80 °C until chromatographic analysis.

4.3.2. HPLC with Fluorescence and Diode Array Detection Qualitative Analyses

Chromatographic analyses were conducted using an HPLC 1200 system comprising a G1311A binary pump, G1315A photodiode array UV-Vis detector, and G1321A fluorescence detector (Agilent, Waldbronn, Germany), configured for excitation at 290 nm and emission at 330 nm. Aliquots of 15 µL were injected onto a reversed-phase ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 µm particle size, Agilent Technologies, Santa Clara, CA, USA) fitted with a matching guard column (4.6 mm × 125 mm, 5 µm), operating at 25 °C and 1.0 mL min^−1^ flow rate.

For α-T separation, the mobile phase consisted of methanol (A) and tert-butyl methyl ether (B), eluted with the following gradient: initial 1% B, rising to 2% B at 3 min, 3% B at 6 min, 4% B at 9 min, 5% B at 12 min, 11% B at 25 min, 25% B at 28 min, returning to 1% B at 30 min and held until 35 min. PC-8 separation followed the method of Gruszka and Kruk with modifications: 15% B at 0 min, 45% B at 3 min, 55% B at 5 min, 65% B at 8 min, then back to 15% B at 10 min until 12 min [53]. Limits of detection and quantification for both compounds were determined as described by Cáceres-Cevallos et al. [54].

4.3.3. Lipid Peroxidation Analysis

Malondialdehyde (MDA), a primary marker of lipid peroxidation, was quantified by reversed-phase HPLC according to the method of Cáceres et al. [55], with minor modifications. Detection was performed using a fluorescence detector set at excitation/emission wavelengths of 515/553 nm. Sample volumes of 20 µL were injected at a 0.8 mL min^−1^ flow rate onto the analytical column, maintained at 35 °C.

The mobile phase comprised 0.05% formic acid in water (solvent A) and methanol (solvent B). Elution followed this gradient profile: 95% A at 0 min; 75% A at 5 min; 5% A at 10 min (held until 15 min); rapid return to 95% A at 15.1 min and maintained until 20 min. MDA concentrations were determined by linear regression using calibration curves prepared from 1,1,3,3-tetraethoxypropane standards.

4.4. Enzymatic Antioxidant Activity Analysis

SOD and CAT were extracted from lyophilized leaf tissue following a modified protocol from Zhao et al. [56]. Samples containing 0.2 g material were ground in 2 mL ice-cold extraction buffer (50 mM potassium phosphate, pH 7.0, 0.5 mM EDTA, 1 mM ascorbic acid). Homogenates were filtered through 0.44 µm membranes and centrifuged (12,000× g, 10 min, 4 °C) to obtain clear supernatants, which were stored at −20 °C pending spectrophotometric analysis using equipment described in Section 4.2.

4.4.1. Superoxide Dismutase Activity Assay

SOD activity was quantified with a commercial assay kit (CS0009, Sigma-Aldrich, Milan, Italy) following manufacturer instructions, monitoring absorbance at 450 nm. Enzyme activity was expressed as SOD units per mg dry weight.

4.4.2. Catalase Activity Assay

CAT activity was determined using a commercial kit (EIACATC, Invitrogen™, Carlsbad, CA, USA) with readings taken at 560 nm. Results were reported as CAT units per g dry weight.

4.5. Polyphenolic Profile and Antioxidant Activity

Phenolic compounds were extracted from 100 mg of lyophilized material following the procedure of Cáceres et al. [54]. Extraction was performed in a Soxhlet apparatus (B-811, Büchi, Flawil, Switzerland) using 50 mL of methanol for 2 h under a nitrogen atmosphere. Extracts were concentrated at 40 °C under vacuum (Syncore Polyvap R-96; Büchi), and the residue was reconstituted in 5 mL of pure methanol. Final extracts were transferred to vials and stored at −80 °C prior to HPLC analysis.

4.5.1. HPLC with Diode Array Detection Analyses

The polyphenolic profile was determined using the HPLC system detailed in Section 4.3.2, according to Jordan et al. [57] with minor adjustments. The mobile phase consisted of 0.05% formic acid in water (solvent B) and acetonitrile (solvent A). Separation was achieved with the following gradient elution: initial 95% B, decreasing to 85% B at 10 min, 75% B at 30 min, 70% B at 35 min, 45% B at 50 min, 10% B at 55 min, 0% B at 57 min (held for 10 min), then returned to initial conditions. The column was operated at 1.0 mL min^−1^ with detection at 280, 306, and 330 nm.

Quantification relied on triplicate linear regression curves prepared from serial dilutions of authentic standards (six concentration levels). Results were expressed as mg g^−1^ dry weight. Standards of salvianic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, caffeic acid, luteolin-7-glucuronide, luteolin-7-glucoside and rosmarinic acid were purchased from Sigma-Aldrich (Milan, Italy); rosmarinyl glucoside, salvianolic acid K and salvianolic acid B were obtained from Cymit Quimica S.L. (Pamplona, Navarra, Spain).

4.5.2. Antioxidant Capacity (FRAP and DPPH•)

The reducing capacity and radical scavenging ability of extracts were evaluated using the FRAP and DPPH^•^-assays, respectively.

Ferric-Reducing Antioxidant Power (FRAP) Assay

The FRAP assay was determined following Szabó et al. [58] with modifications. The FRAP reagent was freshly prepared by combining 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s-triazine in 40 mM HCl, and 20 mM FeCl_3_·6H_2_O (10:1:1 v/v/v). Aliquots of 40 µL extract were mixed with 1.2 mL FRAP reagent and incubated in darkness at 37 °C for 30 min. Absorbance was recorded at 593 nm using a spectrophotometer. Results were quantified against FeSO_4_ standards and expressed as µmol Fe^2+^ equivalents g^−1^ dry weight.

DPPH• Assay

Radical scavenging activity was measured according to Farhat et al. [59]. Reaction mixtures containing 500 µL extract and 1 mL 0.1 mM DPPH- solution in methanol were incubated in the dark at room temperature for 20 min. Absorbance was read at 517 nm against blanks prepared with 500 µL extract and 1 mL methanol. Antioxidant capacity was calculated from Trolox calibration curves and reported as µmol Trolox equivalents g^−1^ dry weight.

4.6. Statistical Analysis

Data are reported as mean ± standard deviation (n = number of replicates). Homogeneity of variances was first verified using Levene’s test. Significant differences among treatments were identified using one- and two-way ANOVA followed by Tukey’s multiple-comparison test (p < 0.05). Pearson correlation coefficients were calculated to explore relationships between measured variables. Multivariate patterns were examined using principal component analysis (PCA) of the correlation matrix to identify key variables driving sample discrimination. All analyses were performed using STATGRAPHICS Centurion XVI.I and the GraphPad Prism 8.0 software.

5. Conclusions

The study assessed the effects of different LED light spectra on two ecotypes of Salvia lavandulifolia Vahl. on the plants’ antioxidant defense system. The results indicated that no specific spectral range can be generally recommended for optimizing the antioxidant defense system in this species.

In ecotype 1, exposure to various light spectra decreased the activity of antioxidant enzymes, namely, SOD and CAT. However, exposure to a combination of red and blue light (70:30) had a more positive effect on the non-enzymatic antioxidant system. This resulted in higher concentrations of bioactive compounds and increased antioxidant capacity, but it did not reduce the oxidative damage associated with this specific spectral range. Conversely, exposure to pure red light modestly increased the concentration of bioactive components and enhanced antioxidant activity without causing detectable oxidative damage. We conclude that exposure to red light may be a useful treatment for strengthening the antioxidant defense system in this ecotype without compromising yield due to oxidative damage.

For ecotype 2, exposure to white light supplemented with blue (C + B) resulted in improvements in both the enzymatic and non-enzymatic antioxidant systems of these clones. This treatment increased CAT activity, maintained levels of photosynthetic pigments, and enhanced the content of tocochromanols and hydroxycinnamic acid derivatives, leading to a greater antioxidant capacity and no observed oxidative damage.

These results confirm the need to adjust the LED light spectral range for individual ecotypes rather than just species, since genetics may play a crucial role in the response to light treatment, and Spanish sage plants from different sites are genetically distinct.