Experiment-Based Optimization of LED Spectral Irradiance Ratios for Enhancing Biomass, Secondary Metabolite, and Essential Oil Yields and Compositions of Ocimum × africanum Lour. Under Controlled Environmental Conditions

1. Introduction

Lemon basil (Ocimum africanum Lour.) belonging to the Lamiaceae family is an aromatic annual herb widely distributed throughout tropical and subtropical regions of Asia, particularly in Vietnam and China. This plant is known by the synonym “Ocimum basilicum var. pilosum (Willd.) Benth.)” in Vietnamese literature. The species typically reaches 30–80 cm in height and is characterized by quadrangular stems, profuse branching, and the presence of fine pubescence. Leaves are arranged oppositely, ovate to broadly lanceolate in shape, with slightly serrated margins and light green laminae. The plant emits a distinctive fragrance attributed to its abundant essential oil glands and high essential oil content. Owing to its adaptability to warm, humid environments and tolerance to relatively high irradiance levels, lemon basil exhibits vigorous growth and rapid biomass accumulation under short cultivation cycles, making it an attractive crop for the commercial production of essential oils and bioactive phytochemicals [1]. Traditionally, its seeds are used in refreshing beverages, while the plant has been employed in folk medicine for treating snake bites and skin inflammation [2]. The leaves and flowers are rich in fragrant essential oils, making lemon basil an important subject of research for applications in the food, pharmaceutical, and cosmetic industries [3].

Essential oil accumulation occurs primarily in the leaves and flowers of lemon basil, with minimal content in the stem. During flowering, oil content can reach up to 1.25% in flowers and approximately 1.14% in leaves (dry weight). Previous studies showed that citral is the dominant compound, accounting for 39.73–40.64% of flower oil and 50.22–54.81% of leaf oil [3]. However, the chemical composition of lemon basil essential oil varies markedly with geographic origin and environmental conditions. Major constituents reported include linalool (0.6–46.1%), and methyl chavicol (3.7–84.0%), citral (16.6–33.6%) in lemon basil (syn. Ocimum × citriodorum) in the United States [4], linalool (29.68%), and (Z)-cinnamic acid methyl ester (21.49%) in China [1]. However, the RI values ​​of the constituent compounds of the essential oil in lemon basil were not published in these two literatures, leading to low reliability of these results. Other studies reported the chemotype "citral" of lemon basil, such as, a diverse combinations of neral (21.1–36.8%), geranial (15.6–33.4%), linalool, and methyl chavicol across different regions of Thailand [5], neral (15.6%), geranial (22.8%), β-caryophyllene (11.7%), and α-humulene (12.6%) in basil of Indonesia [6]. A recent investigation revealed substantial geographical variation in the chemical composition of lemon basil essential oil across different regions of China. Specifically, the contents of estragole (18.4%, 20.4%, and 21.4%), β-caryophyllene (13.3%, 7.9%, and 13.9%), and α-caryophyllene (11.1%, 7.8%, and 11.2%) were identified in essential oils obtained from Henan, Shandong, and Anhui provinces, respectively [7]. Besides geographical factor, other environmental conditions also affect the phytochemical characteristics of lemon basil, such as nutrient media supplemented with natural compound additives [8], the growth stages of a plant include the pre-flowering stage, the full flowering stage, and the post-flowering stage [9], and different organic fertilization conditions [10].

Lemon basil is widely utilized in the culinary, fragrance, and pharmaceutical industries, primarily through the extraction of its essential oil, particularly during the full-flowering stage [11]. In addition to its aromatic properties, lemon basil essential oil exhibits notable biological activities, particularly strong antifungal effects against plant-pathogenic fungi such as Fulvia fulva and Fusarium solani var. coeruleum, with EC₅₀ values below 20 ppm [1]. The antioxidant activity of lemon basil essential oil was shown to be stronger than that of the extract, and there are differences between parts of the plant. Specifically, the essential oil from the leaves has the strongest antioxidant activity, followed by the flowers, and the stem [12]. Essential oil from lemon basil in Indonesia was studied for its potential to inhibit bacterial growth on tofu and chicken fillets during storage and extend their shelf life up to respective 4 and 6 days [6,13]. In general, many species in the genus Ocimum have biological activities such as the ability to scavenge free radicals, reduce cellular oxidative stress, and support cardiovascular health. Studies on various Ocimum species showed that the total phenolic and total flavonoid content correlates strongly with the antioxidant activity of extracts and essential oils [14]. The phenolic compounds in lemon basil, in the form of rosmarinic and caffeic acids, are effective antioxidants, and it has also been shown that they can strongly inhibit some strains of harmful fungi [15,16]. As in all chlorophyll-containing green plants, chlorophyll in lemon basil plays a central role in capturing light energy for photosynthesis. Through this process, absorbed light energy is converted into chemical energy that drives carbon fixation and other metabolic activities [17]. Chlorophyll occurs in several forms, with chlorophyll a and chlorophyll b being the primary photosynthetic pigments. These pigments differ in their absorption characteristics and contribute complementary functions in light harvesting. Chlorophyll a predominantly absorbs R light around 665–680 nm, whereas chlorophyll b exhibits stronger absorption in the B region near 460 nm, thereby extending the spectral range available for photosynthesis [18]. In lemon basil, chlorophyll-related studies have primarily focused on chlorophyll fluorescence analysis as a non-destructive indicator of photosynthetic performance, plant growth, and stress responses under varying environmental conditions [19].

Under the increasing pressures of climate change, crop productivity and quality are becoming more susceptible to environmental fluctuations, thereby compromising the stability and sustainability of agricultural production systems. For medicinal plants, improving the consistency and quality of raw materials requires the optimization and standardization of cultivation, harvesting, and processing protocols. Environmental factors play a crucial role in regulating plant growth and secondary metabolite production, among which light is one of the most influential. In addition to serving as the primary energy source for photosynthesis, light acts as an essential environmental signal that regulates plant growth, development, and physiological processes through photoreceptor-mediated signaling networks [20,21,22]. Light quality has been shown to influence plant morphology, photosynthetic efficiency, organ development, and the biosynthesis of numerous primary and secondary metabolites, including proteins, carbohydrates, vitamins, phenolic compounds, flavonoids, and anthocyanins [20]. Among the light spectra, red (R) and blue (B) lights are considered the most effective in driving photosynthesis, as chlorophyll pigments predominantly absorb radiation within these regions. Nevertheless, other spectral components, including ultraviolet-A (UV-A), green (G), and far-red (Fr) light, also serve as important regulatory signals influencing plant physiology and development [23]. In particular, UV-A radiation has attracted considerable attention because of its ability to enhance photosynthetic performance and stimulate the biosynthesis of bioactive secondary metabolites, including phenolics and flavonoids [24]. For example, the supplementation of UV-A radiation to a red–white light spectrum (20% R:80% W) during the final three days before harvest significantly increased shoot fresh weight, plant height, and the number of leaves and branches in coriander [25]. Similarly, exposure to UV-A radiation (385 nm, 30 W m⁻²) for five days increased both biomass yield and phenolic content in kale (Brassica oleracea var. acephala) [26]. In lettuce (Lactuca sativa), UV-A supplementation (365 nm, 10–30 μmol·m⁻²·s⁻¹) for 13 days promoted fresh and dry biomass accumulation, leaf expansion, and antioxidant activity compared with plants grown without UV-A radiation [27]. Likewise, the combination of R light (215 μmol·m⁻²·s⁻¹) and UV-A light (35 μmol m⁻² s⁻¹) significantly enhanced flavonoid accumulation in tomato (Solanum lycopersicum) relative to R light alone [28]. Although G light has traditionally been considered less efficient for photosynthesis, its deeper penetration into leaf tissues facilitates improved carbon assimilation and can enhance crop productivity [29]. Beyond its role in light absorption, G light also regulates key physiological processes, including stomatal behavior, canopy architecture, and resource-use efficiency, thereby contributing to plant performance under controlled-environment conditions [30].

Fr radiation, which is only weakly absorbed by chlorophyll and is largely transmitted or reflected by leaves [17], has also been shown to substantially affect plant growth and productivity. Zhen & van Iersel [31] reported that the addition of Fr light consistently increased net photosynthesis in lettuce grown under R-B lighting by improving the efficiency of light utilization during the photosynthetic light reactions. Likewise, supplementing a R–B–W spectrum (70:20:10) with Fr radiation significantly increased plant height, internode length, leaf area, and fresh biomass in coriander [32]. In another study, the addition of 6% Fr light to a R–B spectrum increased total phenolic content and ascorbic acid concentration compared with R–B lighting alone [33]. Similar growth-promoting effects have been reported in ornamental crops, where the addition of 16–64 μmol m⁻² s⁻¹ Fr light to a R–B background increased shoot dry weight by 28–50% in geranium and snapdragon [34]. Furthermore, Zou et al. [35] observed increases of 49% in leaf area and 39% in biomass production in lettuce when 50 μmol m⁻² s⁻¹ Fr light was added to a R–B lighting regime. Collectively, these findings suggest that Fr supplementation is an effective strategy for enhancing light interception, photosynthetic performance, and biomass accumulation.

Increasing evidence suggests that light spectral composition can also markedly affect the synthesis of essential oils and other specialized metabolites in aromatic plants. For instance, McAusland et al. [36] demonstrated that light spectral quality not only influenced plant morphology and biomass but also substantially altered essential oil profiles in coriander (Coriandrum sativum). Plants grown under combined R:B (1:1) and R:B:G (35.8:37.8:26.4) spectra showed three- to four-fold higher concentrations of major essential oil compounds than those under monochromatic R or B light. The increased aromatic complexity observed under spectrally diverse lighting conditions was attributed to enhanced biosynthesis of secondary metabolites associated with plant defense, ecological interactions, and overall plant fitness.

For Ocimum x africanum, to date, some efforts have been devoted to understanding how light intensity and spectral ratios affect plant growth and development. Mat Daud et al. [37] evaluated the effects of four light intensities (50, 80, 120, and 150 µmol·m⁻²·s⁻¹) under a constant R-to-B ratio of 4:1 and observed significant variations in leaf, stem, and root biomass, as well as photosynthetic performance, in lemon basil. Net photosynthetic rate increased with increasing light intensity and peaked at 8.27 µmol CO₂ m⁻²·s⁻¹ under 150 µmol·m⁻²·s⁻¹. Likewise, the highest fresh and dry biomass were recorded under the 120 and 150 µmol·m⁻²·s⁻¹ treatments. These findings suggest that light intensity is a key determinant of photosynthetic efficiency and biomass production in lemon basil when spectral composition is held constant. Another research of Daud et al. [38] was to investigate the effects of R and B light spectral irradiance levels (at a ratio of 4:1, with two intensities of 80 and 160 µmol·m⁻²·s⁻¹), nutrient solution (electrical conductivity - EC), and their interaction on the plant growth, yield, and phytochemical contents. The authors reported that the application of combined R and B lighting and an intensity of 160 µmol·m⁻²·s⁻¹ in combination with a nutrient solution EC of 2.6 mS cm⁻¹ significantly enhanced plant growth, yield, phenolic, and flavonoid compound accumulation of lemon basil (syn. Ocimum citriodurum Vis.) cultivated under controlled environment conditions. Another study evaluated the influence of LED irradiance on biomass yield, antioxidant production, and antioxidant capacity in microgreens of five traditional Thai vegetable species, including lemon basil. Among the tested light intensities, a photosynthetic photon flux density (PPFD) of 330 µmol·m⁻²·s⁻¹ proved most effective in promoting dry matter accumulation, resulting in higher dry biomass, total phenolic and flavonoid content, and free radical scavenging than plants exposed to PPFD levels of 220 or 110 µmol·m⁻²·s⁻¹ and those grown under conventional fluorescent lighting (45 µmol·m⁻²·s⁻¹) [39]. On the other hand, Pitaloka et al. [40] evaluated the effect of three LED spectral treatments—100% B, 100% white (W), and a mixed spectrum consisting of 67% R, 20% B, and 13% W in combination with different conditions of planting medium ratio, and nutrients on the growth of lemon basil (syn. (Ocimum basilicum var. anisatum Benth.)). The authors reported that exposure to 100% W LED lighting combined with a nutrient solution concentration of 700 ppm resulted in optimal growth of lemon basil, as indicated by greater plant height, leaf area, fresh biomass, and chlorophyll a, chlorophyll b, and total chlorophyll contents. However, the lighting treatments differed not only in spectral composition but also in light intensity, making it difficult to isolate the effects of light quality. Specifically, the 100% W treatment provided the highest PPFD (95.92 µmol·m⁻²·s⁻¹), substantially exceeding that of the 100% B treatment (44.26 µmol·m⁻²·s⁻¹) and the mixed R:B:W treatment at the ratio of 67%:20%:13% (37.33 µmol·m⁻²·s⁻¹). Consequently, the superior growth observed under the 100% W treatment may have been influenced by differences in both spectral distribution and light intensity. A recent study evaluated the influence of daily light integral (DLI) and photoperiod on flowering responses in several Ocimum species, including lemon basil. Lemon basil exhibited the earliest flowering under high DLI conditions (22.2–23.3 mol·m⁻²·d⁻¹) and a 16-h photoperiod, indicating that both cumulative light exposure and day length are key environmental factors governing reproductive development in this species [41]. Another recent study investigated the influence of cultivation environments and lighting conditions on the growth and essential oil characteristics of lemon basil. Specifically, the authors compared plant height, essential oil content, and essential oil composition among plants grown under field condition, in a greenhouse without supplemental lighting, and in a city farming system illuminated with LEDs (blue:red:white:deep pink = 25:25:80:20) under a 14-h photoperiod. The results indicated that plant height was greatest in the greenhouse-grown plants and lowest in those cultivated under field condition. Furthermore, the chemical composition of the essential oil varied among the different cultivation environments, suggesting that growth conditions can substantially influence secondary metabolism in lemon basil. However, the study did not report retention index (RI) values for the identified essential oil constituents, which limits the reliability and reproducibility of compound identification and consequently reduces confidence in the reported compositional data [42].

Despite growing evidence highlighting the pivotal role of light quality in regulating plant growth, physiology, and secondary metabolism, studies investigating the effects of LED lighting on lemon basil have largely focused on variations in light intensity rather than spectral composition. Consequently, there remains a significant lack of information regarding the effects of different multispectral light ratios applied at a constant PPFD on plant growth and the biosynthesis of secondary metabolites. In particular, the influence of LED spectral composition on the yield and chemical profile of lemon basil essential oil remains poorly understood. To address this knowledge gap, the present study evaluated the effects of multispectral LED lighting with varying proportions of UV-A, B, G, R, and Fr lights on the growth, secondary metabolite accumulation, and essential oil production of lemon basil cultivated in controlled environment growth chambers. By maintaining a similar PPFD across treatments, the effects of spectral quality could be assessed independently of light intensity. Five lighting formulations were designed as follows:

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B:G:R:Fr = 17.14:29.8:47.4:5.66 (F1),

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UV-A:B:R:Fr = 6.60:45.15:29.23:19.02 (F2),

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UV-A:B:R:Fr = 3.30:47.37:29.24:20.09 (F3),

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B:G:R:Fr = 13.85:43.50:39.30:3.35 (F4), and

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F5–consisting of natural sunlight transmitted through the greenhouse at approximately 25% of ambient solar irradiance, which served as the control.

Treatment F1 was developed using a white LED background supplemented with R light (660 nm), whereas F2 and F3 were composed of monochromatic LEDs (UV-A (360 nm), B (460 nm), R (660 nm), and Fr (730 nm)). Treatment F4 was based on a broad-spectrum white LED source. The selected spectral combinations were based on previous findings indicating that R and B light maximize photosynthetic performance, UV-A promotes secondary metabolite biosynthesis, Fr light enhances photosynthetic efficiency and biomass production, and G light improves canopy light penetration and overall light-use efficiency. The spectral distributions of the five used lighting treatments are illustrated in the Figure 6 of the subchapter 4 in this paper.

Accordingly, the present study aimed to evaluate the effects of different LED spectral compositions on the growth, physiological performance, and phytochemical characteristics of lemon basil cultivated under controlled-environment conditions. Specifically, plant height, biomass production, photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoids), anthocyanin content, total phenolic content (TPC), total flavonoid content (TFC), essential oil yield, and essential oil composition were investigated. By systematically modifying the spectral distribution of LED lighting, this study sought to identify optimal light combinations that simultaneously maximize biomass productivity and the accumulation of high-value bioactive compounds. To the best of our knowledge, this is the first study to comprehensively assess the effects of multispectral LED lighting on growth, physiological traits, phytochemical accumulation, and essential oil composition in lemon basil. The findings are expected to provide new insights into light-mediated metabolic regulation and support the development of sustainable, precision-lighting strategies for the commercial production of high-quality aromatic and medicinal plants.

2. Results

2.1. The Effect of LED Light Conditions on Biomass and Essential oil Yield of Ocimum x Africanum

The different light spectra had distinct effects on growth, metabolite, and essential oil biosynthesis in the aerial parts of Ocimum x africanum Lour. (lemon basil). The plant height increased progressively throughout the four-week cultivation period under all five lighting treatments (F1–F5), although significant differences among treatments became apparent from week 1 onward. At the beginning of the experiment (week 0), plant height was similar among treatments, averaging approximately 14.11 cm. After one week, plants grown under F2 (UV-A:B:R:Fr = 6.60:45.15:29.23:19.02) and F3 (UV-A:B:R:Fr = 3.30:47.37:29.24:20.09) exhibited significantly greater heights (32.65–33.27 cm) than those under F1, F4, and F5 (24.63–25.58 cm). This trend became more pronounced during subsequent weeks. At week 2, plants exposed to F2 and F3 reached 51.45 and 49.18 cm, respectively, whereas those under F5 showed the lowest height (31.97 cm). By week 3, F2 produced the tallest plants (65.07 cm), followed closely by F3 (62.46 cm), while F5 remained significantly shorter (33.28 cm). At the end of the experiment (week 4), the maximum plant height was recorded under F2 (76.62 cm), which was statistically comparable to F3 (72.45 cm) but significantly higher (p< 0.01) than F1 (51.18 cm), F4 (47.84 cm), and F5 (35.11 cm). Overall, the F2 and F3 lighting conditions promoted superior stem elongation and plant height, whereas F5 resulted in the least plant height throughout the cultivation period (Figure 1).

Light conditions markedly affected biomass production and essential oil accumulation in lemon basil. Among the five treatments, F1 (B:G:R:Fr = 17.14:29.8:47.4:5.66) produced the highest fresh shoot yield (8.30 ± 0.77 ton ha⁻¹) and dry shoot yield (1.44 ± 0.14 ton ha⁻¹), which were significantly (p < 0.01) greater than those obtained under the other treatments. In contrast, plants grown under F5 exhibited the lowest biomass, with fresh and dry yields of only 1.61 ± 0.20 and 0.14 ± 0.02 ton ha⁻¹, respectively. Water content varied significantly among treatments and ranged from 82.14 ± 0.09% in F1 to 91.27 ± 0.12% in F5. Treatments F2 and F3 showed relatively high water contents (88.05–88.59%), whereas F4 exhibited an intermediate value (84.08%). The essential oil content of the shoots was strongly influenced by the lighting regime. The highest oil content was recorded in F4 (B:G:R:Fr = 13.85:43.50:39.30:3.35) (0.83 ± 0.002% w/w, dry basis), followed closely by F5 (0.82 ± 0.000%), whereas F2 and F3 produced significantly lower values (0.66 ± 0.001%). Although F4 exhibited the highest essential oil concentration, the greatest essential oil yield per hectare was obtained in F1 (10.89 ± 1.04 L ha⁻¹) due to its superior biomass production. Essential oil yield decreased progressively in F4 (8.58 ± 1.04 L ha⁻¹), F2 (5.59 ± 0.65 L ha⁻¹), and F3 (4.47 ± 0.91 L ha⁻¹), while F5 produced the lowest yield (1.16 ± 0.14 L ha⁻¹) (Table 1).

These results indicate that light quality not only affected plant growth and biomass accumulation but also influenced essential oil biosynthesis. Consequently, the overall essential oil productivity of lemon basil depended on the combined effects of biomass production and oil concentration, with F1 providing the most favorable balance between these two factors.

2.2. The Effect of LED Light Conditions on Essential oil Composition of Ocimum × Africanum

The chemical composition of the essential oils extracted from lemon basil varied moderately among the five lighting treatments, with a total of 12–15 compounds identified, representing 97.8–99.5% of the total oil. In all treatments, the oils were dominated by oxygenated monoterpenes, accounting for 70.6–91.4% of the total composition. The principal constituents were geranial (trans-citral) and neral, which together constituted the citral chemotype characteristic of lemon basil. Geranial was the most abundant compound in all samples, ranging from 41.1% in F5 to 49.9% in F3, while neral varied between 27.5% (F5) and 37.3% (F2). Consequently, the combined citral content (geranial + neral) exceeded 80% in treatments F1–F4 but decreased to approximately 68.6% under F5. Minor oxygenated monoterpenes, including linalool (0.8–2.3%), terpinen-4-ol (1.2–2.3%), nerol (trace–1.1%), and geraniol (trace–0.9%), were detected in all or most treatments. In contrast, sesquiterpene hydrocarbons increased markedly under F5, reaching 27.1%, compared with only 7.7–11.7% in F1–F4. This increase was mainly associated with elevated levels of (E)-β-caryophyllene (9.9%), germacrene D (6.1%), and (E)-α-bisabolene (5.3%), all of which were considerably higher than those observed under the other lighting conditions. Treatment F4 also showed a moderate enrichment of sesquiterpenes, with (E)-β-caryophyllene and germacrene D reaching 4.5% and 3.1%, respectively. Monoterpene hydrocarbons and benzene derivatives were detected only in trace amounts (<0.2%) in all treatments. Overall, the results indicate that while all lighting regimes maintained the citral-rich chemotype of lemon basil, treatment F5 induced a noticeable shift from oxygenated monoterpenes toward sesquiterpene hydrocarbons, thereby altering the relative proportions of secondary metabolites in the essential oil (Table 2).

The yields of the three main constituents, namely neral (Ne), geranial (Ge), and (E)-β-caryophyllene (Ca), in lemon basil essential oils varied significantly under five different lighting conditions. Among all treatments, F1 produced the highest yields of neral and geranial, reaching approximately 4.0 and 5.4 L ha⁻¹, respectively. F4 ranked second, with yields of about 2.9 L ha⁻¹ for neral and 4.0 L ha⁻¹ for geranial. In contrast, F5 resulted in the lowest yields for all constituents. The yield of (E)-β-caryophyllene was much lower than those of neral and geranial across all treatments, with the highest value observed under F4 (around 0.4 L ha⁻¹). Statistical analysis indicated significant differences among lighting treatments, as shown by different letters above the bars (p < 0.01) (Figure 2).

2.3. The Effect of LED Light Conditions on Pigments of Ocimum × Africanum

Chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Caro) are the major photosynthetic pigments involved in light harvesting and energy conversion in plants. The concentrations of these pigments in fresh leaves of lemon basil were significantly affected by different LED lighting conditions. Among the five treatments, F2 produced the highest contents of chlorophyll a and chlorophyll b, reaching 7.41 and 4.73 mg/100 g fresh leaves, respectively. These values were significantly greater (p < 0.01) than those observed under other treatments. F4 also supported substantial chlorophyll accumulation, whereas F1 and F3 exhibited intermediate levels. In contrast, F5 resulted in the lowest levels of both chlorophyll pigments. Carotenoid content followed a different trend, with the highest value recorded under F1 (3.74 mg/100 g fresh leaves), follwed by F2, F3, and F4 , while F5 again exhibited the lowest concentration. Overall, chlorophyll a levels were more abundant than chlorophyll b and carotenoid across all treatments (Figure 3).

Anthocyanins are water-soluble flavonoid pigments responsible for the red, purple, and blue coloration observed in many plant tissues. Besides their role in pigmentation, anthocyanins function as powerful antioxidants and contribute to plant protection against various environmental stresses, including excessive light and oxidative damage. Their biosynthesis is highly responsive to external factors, particularly light quality and intensity, making anthocyanin accumulation an important indicator of plant physiological adaptation to different lighting conditions. The anthocyanin concentration in fresh leaves of lemon basil was significantly influenced by different lighting conditions. The highest anthocyanin content was recorded under F4 (16.50 mg/100 g fresh leaves), followed closely by F1 (16.07 mg/100 g fresh leaves), and these two treatments were not significantly different (p > 0.05). Intermediate values were observed in F3 and F2, with concentrations of 13.78 and 12.00 mg/100 g fresh leaves, respectively. In contrast, F5 resulted in the lowest anthocyanin content, reaching only 7.87 mg/100 g fresh leaves (Figure 4).

2.4. The Effect of LED Light Conditions on Total Phenolic and Total Flavonoid of Ocimum × Africanum

Total phenolic content (TPC) and total flavonoid content (TFC) are important indicators of the antioxidant potential and phytochemical quality of plants. Phenolic compounds and flavonoids contribute significantly to plant defense mechanisms by scavenging reactive oxygen species and protecting tissues against environmental stresses. In addition to their physiological roles in plants, these secondary metabolites are associated with numerous health-promoting properties, including antioxidant, anti-inflammatory, antimicrobial, and anticancer activities. The total phenolic content (TPC) and total flavonoid content (TFC) in fresh leaves of lemon basil were significantly affected by different LED lighting conditions. The highest TPC values were observed under F2, F3, and F4, ranging from 25.14 to 25.78 mg/g fresh leaves, and these treatments were not significantly different from each other. In contrast, F5 showed the lowest TPC value (19.01 mg/g fresh leaves). For TFC, F4 produced the highest concentration (14.17 mg/g fresh leaves), followed by F2 (11.07 mg/g fresh leaves). The lowest TFC content was recorded under F3 (7.42 mg/g fresh leaves). Overall, F4 appeared to be the most favorable lighting condition for enhancing both phenolic and flavonoid accumulation (Figure 5).

3. Discussion

The relative increase in plant height of Ocimum × africanum varied markedly among the five lighting treatments throughout the cultivation period. All plants exhibited continuous growth over time; however, the magnitude of height increment differed considerably depending on the light condition. After one week, plants grown under F2 and F3 showed the greatest increases in height, reaching 133.31% and 128.00% above their initial values, respectively, whereas F4 and F5 exhibited the lowest increases (76.43% and 80.56%). This trend persisted during the subsequent weeks. By week 2, the height increase under F2 had reached 260.80%, followed by F3 (243.44%), while F5 showed the smallest increase (127.71%). At week 3, plants under F2 and F3 recorded height increments of 356.31% and 336.17%, respectively, which were substantially higher than those observed under F1 (238.85%), F4 (198.78%), and F5 (137.04%). At the end of the experiment (week 4), F2 produced the highest relative increase in plant height (437.31%), followed by F3 (405.94%). In contrast, plants exposed to F5 exhibited the lowest increase, reaching only 150.07% above the initial height. Overall, the F2 and F3 lighting regimes were the most effective in promoting stem elongation and plant growth, whereas F5 had the least stimulatory effect on height development. However, the height of lemon basil plants in the present study is higher than previously published values ​​for many varieties with different species accessions [4].

The differences in plant height observed among the five lighting treatments can be attributed primarily to variations in spectral composition and daily light supply. Treatments F2 and F3 consistently produced the tallest plants, with final height increases of 437.31% and 405.94%, respectively. These two treatments were characterized by a high proportion of B light (45.15–47.37%) combined with relatively high Fr radiation (19.02–20.09%) under identical irradiance (220 ± 10 µmol·m⁻²·s⁻¹) and photoperiod (16 h d⁻¹). Previous studies have demonstrated that Fr radiation plays an important role in regulating phytochrome-mediated shade-avoidance responses, promoting stem elongation and increasing plant height [34,43]. In addition, B light is essential for maintaining photosynthetic efficiency and supporting biomass accumulation through cryptochrome-mediated signaling pathways [44]. Although F1 provided the highest proportion of R light (47.4%), its lower Fr fraction (5.66%) resulted in only moderate stem elongation compared with F2 and F3. R light is highly efficient for photosynthesis because chlorophyll absorbs strongly in this spectral region; however, plant morphology is also strongly influenced by the R-to-Fr balance rather than R light alone [17]. Consequently, the limited Fr component in F1 may have reduced the stimulation of elongation growth. Plants grown under F4 exhibited lower height increments despite receiving the same PPFD and photoperiod as F1–F3. This treatment contained a relatively high proportion of G light (43.5%) and only a small fraction of Fr light (3.35%). Although G light can penetrate deeper into the canopy and contribute to photosynthesis, they are generally less effective than R and B light in regulating plant architecture and promoting shoot elongation [30]. The results obtained for the F1-F4 treatment in the present study are in agreement with previous findings demonstrating that a reduced R-to-Fr ratio stimulates stem elongation and enhances plant height, while an elevated R/Fr ratio restricts elongation growth, resulting in a more compact growth habit [45]. The lowest height increase was observed under F5, which reached only 150.07% above the initial height at week 4. Unlike the other treatments, F5 provided only a low DLI (2.35 mol·m⁻²·d⁻¹) because the amount of sunlight and sky light transmitted through the greenhouse roof was only 25%. Light quantity is a major determinant of photosynthetic carbon assimilation and biomass production, and insufficient daily light exposure can severely limit plant growth [46]. Therefore, despite containing appreciable Fr radiation (17.26%), the low overall light input in F5 restricted plant development. Overall, these results indicate that plant height in lemon basil is influenced by the interaction between spectral quality and light quantity. A lighting regime enriched with B and Fr lights under adequate irradiance and photoperiod conditions appears to be particularly effective in promoting stem elongation and vegetative height growth.

The marked differences in biomass production, water content, essential oil concentration, and essential oil yield among the five lighting treatments suggest that both light quality and daily light supply strongly influenced the physiological performance of lemon basil. Treatment F1 produced the highest fresh and dry biomass, whereas F5 resulted in the lowest productivity. F1 contained a balanced spectrum composed mainly of R light (47.4%), moderate G light (29.8%), and a smaller proportion of B light (17.14%), under a PPFD of 220 ± 10 µmol·m⁻²·s⁻¹ with a 16-h photoperiod. R light is recognized as the most efficient light for photosynthesis because chlorophyll pigments absorb strongly in the R region, thereby enhancing carbon fixation and biomass accumulation [17]. In addition, G light penetrates deeper into leaf tissues and lower canopy layers, contributing to whole-canopy photosynthesis [30]. The adequate daily light integral (12.67 mol·m⁻²·d⁻¹) in F1 therefore promoted dry matter accumulation and resulted in the greatest fresh and dry yields. In contrast, F5 received only a low supplemental light dose (2.35 mol·m⁻²·d⁻¹), which limited photosynthetic carbon assimilation and consequently reduced biomass production. Similar observations were reported by Poorter et al. [46], who demonstrated that insufficient light availability is a major limiting factor for plant growth.

The water content of shoots was lowest in F1 (82.14%) and highest in F5 (91.27%). Because water content is inversely related to dry matter accumulation, plants producing greater amounts of structural carbohydrates generally exhibit lower moisture percentages. The higher water contents observed in F2, F3, and especially F5 indicate reduced accumulation of dry biomass relative to water uptake. Similar relationships between dry matter accumulation and tissue water content have been described in aromatic herbs grown under different light environments [47]. Interestingly, the highest essential oil concentration was observed in F4 (0.83%) and F5 (0.82%), despite their lower biomass compared with F1. The F4 was characterized by the highest proportion of G light (43.5%) and a moderate R component, whereas F5 contained substantial Fr radiation together with UV-A and infrared (IR) light. Environmental stresses and suboptimal growth conditions frequently stimulate the biosynthesis of secondary metabolites, including terpenoids and essential oils, as part of plant defense mechanisms [48]. Several studies on Ocimum species showed that moderate stress or reduced growth rates may increase essential oil concentration even when biomass decreases [47,49]. Therefore, the elevated oil content in F4 and F5 may reflect a shift in carbon allocation from primary growth toward secondary metabolism. Nevertheless, essential oil yield per hectare depended on both oil concentration and biomass production. Although F4 exhibited the highest essential oil content, F1 generated the greatest essential oil yield (10.89 L ha⁻¹) because of its superior dry biomass production. Similar results have been reported in basil cultivation, where essential oil yield is determined primarily by the interaction between biomass accumulation and oil concentration rather than by oil content alone [50]. In contrast, the extremely low biomass in F5 limited total oil production, resulting in the lowest essential oil yield (1.16 L ha⁻¹) despite its relatively high oil concentration. Overall, these results indicate that different light spectra affect primary and secondary metabolism differently in lemon basil. While balanced R-dominant lighting with adequate daily light integral favored biomass accumulation and maximized essential oil yield (F1), treatments associated with lower growth rates tended to enhance essential oil concentration. Thus, optimization of lighting strategies should consider the desired production objective, whether maximizing biomass, increasing essential oil concentration, or achieving the highest total oil yield.

The essential oil composition of lemon basil obtained in the present study was generally consistent with previous reports describing this species as a citral chemotype, characterized by the predominance of geranial and neral. Across all lighting treatments, geranial (41.1–49.9%) and neral (27.5–37.3%) were identified as the major constituents, together accounting for approximately 68.6–85.8% of the total oil composition. Similar findings were reported by Pisutthanan and Pisutthanan [5], who analyzed lemon basil populations collected from different regions of Thailand and found that neral (21.1–36.8%) and geranial (15.6–33.4%) were the principal components, confirming the existence of a citral-rich chemotype in this species. Likewise, Padalia et al. [51] reported total citral contents ranging from 55.0 to 75.5% in lemon basil essential oils and identified linalool, nerol, geraniol, and β-caryophyllene as important minor constituents.

Compared with previous studies, the present samples, particularly those from treatments F1–F4, exhibited relatively higher proportions of citral, indicating that these LED conditions effectively maintained the characteristic aroma profile of lemon basil. Minor oxygenated monoterpenes such as linalool (0.8–2.3%), terpinen-4-ol (1.2–2.3%), nerol, and geraniol were also detected, although their concentrations were lower than those reported in some earlier investigations. For example, Pisutthanan and Pisutthanan [5] observed linalool contents ranging from approximately 5 to 9% in certain populations, whereas the present study recorded values below 2.5%. In addition, methyl chavicol (estragole), which is frequently encountered in several Ocimum chemotypes, was detected only in trace amounts (<0.1%), indicating that the plants investigated here clearly belonged to the citral type rather than the estragole or linalool chemotypes commonly reported in basil species [52]. A remarkable difference was observed under treatment F5, where the proportion of oxygenated monoterpenes decreased to 70.6%, accompanied by a substantial increase in sesquiterpene hydrocarbons (27.1%). This shift was mainly associated with elevated levels of (E)-β-caryophyllene (9.9%), germacrene D (6.1%), and (E)-α-bisabolene (5.3%). Similar variations have been reported in previous studies, suggesting that environmental factors such as geographical origin, harvesting season, developmental stage, and cultivation conditions can significantly affect terpenoid biosynthesis in Ocimum species. Padalia et al. [51] demonstrated that harvest time and post-harvest processing markedly influenced the relative abundance of monoterpenes and sesquiterpenes in lemon basil. Likewise, Chang et al. [49] reported that different irradiance levels altered volatile oil composition in basil plants, indicating that light conditions can modulate secondary metabolism. Overall, despite the differences induced by the LED treatments, all samples retained the characteristic citral-rich profile of lemon basil. However, treatment F5 promoted a noticeable metabolic shift toward sesquiterpene accumulation, suggesting that light quality may influence the partitioning of carbon flux between monoterpene and sesquiterpene biosynthetic pathways. These findings highlight the potential of adjusting light environments to tailor the chemical profile of lemon basil essential oil according to specific industrial or pharmacological applications.

The significant differences in photosynthetic pigment accumulation observed under the five lighting treatments can be explained by the spectral composition of each LED combination and their effects on chlorophyll biosynthesis and photomorphogenic responses. Among the treatments, F2 (6.6% UV-A, 45.15% B, 29.23% R, and 19.02% Fr light) produced the highest chlorophyll a and chlorophyll b contents. The significantly higher chlorophyll content observed under F2 compared with F3 may be attributed to the synergistic interaction between UV-A and B radiation. Although both treatments contained a high proportion of B light, F2 provided twice the UV-A fraction of F3 (6.60% vs. 3.30%). UV-A and B lights are perceived primarily by cryptochromes and phototropins, which regulate chloroplast development, chlorophyll biosynthesis, and the expression of photosynthesis-related genes. Enhanced activation of these photoreceptors under F2 may have promoted chloroplast biogenesis and the accumulation of photosynthetic pigments, resulting in higher chlorophyll concentrations. Previous studies have demonstrated that B light enhances chlorophyll accumulation and photosynthetic capacity in many aromatic and medicinal plants [44,53]. In addtition, UV-A supplementation can increase chlorophyll content and photosynthetic capacity in horticultural crops by stimulating the development of the photosynthetic apparatus and enhancing light-harvesting efficiency [54]. Furthermore, the combined action of UV-A and B light was reported to exert a stronger regulatory effect on photomorphogenesis and chloroplast differentiation than B light alone through cryptochrome-mediated signaling pathways [55]. The presence of a moderate proportion of Fr radiation in F2 may also have improved photosystem balance and light-harvesting efficiency through the Emerson enhancement effect [56], contributing to the superior chlorophyll levels observed. F4, characterized by a high proportion of G light (43.5%) and moderate R light (39.3%), also maintained relatively high chlorophyll concentrations. G light can penetrate deeper into leaf tissues and lower canopy layers, thereby complementing B and R light and improving overall photosynthetic performance [30]. In contrast, F5 received only natural sunlight and sky light with a much lower daily light integral (2.35 mol m⁻² d⁻¹), resulting in the lowest chlorophyll a and b contents. Insufficient light availability generally suppresses chlorophyll biosynthesis and reduces leaf photosynthetic activity [17]. Overall, the chlorophyll a and chlorophyll b contents in lemon basil of the present study are lower than previously published values, possibly due to the older age of the plants [40]. Unlike chlorophylls, carotenoid accumulation was highest under F1, which contained the greatest proportion of red light (47.4%) together with moderate B light and G light. Carotenoids function not only as accessory pigments in photosynthesis but also as photoprotective compounds that dissipate excess excitation energy and scavenge reactive oxygen species [57]. The relatively high R light fraction in F1 may have increased photosynthetic electron transport and induced greater demand for photoprotection, thereby stimulating carotenoid biosynthesis. Conversely, the lowest carotenoid content under F2 suggests that the enhanced chlorophyll production under B‒enriched light reduced the requirement for carotenoid-mediated photoprotection. Overall, chlorophyll pigments were consistently more abundant than carotenoids in all treatments, reflecting their central role in light harvesting and carbon assimilation in lemon basil. The results confirm that the accumulation of chlorophylls and carotenoids in lemon basil is highly responsive to LED spectral composition and light intensity.

The anthocyanin content of lemon basil leaves was significantly affected by the different LED lighting conditions, indicating that light quality strongly regulates flavonoid biosynthesis. The highest anthocyanin concentrations were observed under F4 (16.50 mg/100 g FW) and F1 (16.07 mg/100 g FW), whereas F5 produced the lowest value (7.87 mg/100 g FW). The elevated anthocyanin accumulation under F4 may be associated with its high proportion of G light (43.5%) combined with R light (39.3%), which could induce moderate photooxidative stress and stimulate the phenylpropanoid pathway. Anthocyanins are known to function as photoprotective pigments by absorbing excess radiation and scavenging reactive oxygen species generated under stressful light environments [58,59]. In contrast, the lower anthocyanin content observed under F2, despite exhibiting the highest chlorophyll a and chlorophyll b concentrations, suggests that B‒enriched conditions favored photosynthetic pigment synthesis and efficient light utilization rather than the accumulation of protective secondary pigments. Similar inverse relationships between chlorophyll and anthocyanin accumulation have been reported in several species, where improved photosynthetic performance reduces the need for photoprotective anthocyanins [60]. Interestingly, F1 simultaneously promoted high carotenoid and anthocyanin contents, indicating enhanced photoprotection through both carotenoid-dependent quenching mechanisms and anthocyanin-mediated antioxidant activity. Carotenoids and anthocyanins often act synergistically to protect the photosynthetic apparatus against oxidative damage caused by excessive light [57]. Conversely, the low anthocyanin content under F5 corresponded with the lowest chlorophyll and carotenoid concentrations, which is likely attributable to the reduced daily light integral and limited photosynthetic activity under this treatment. Overall, the present results suggest that the accumulation of anthocyanins in lemon basil is closely linked to the balance between photosynthetic efficiency and photoprotective requirements imposed by different light environments.

The accumulation of total phenolic compounds and flavonoids in lemon basil was markedly influenced by the spectral composition of the LED treatments, reflecting the close relationship between light quality, photosynthetic performance, and secondary metabolism. Treatments F2, F3, and F4 produced the highest TPC values (25.14–25.78 mg/g FW), whereas F5 resulted in the lowest phenolic concentration. Similarly, F4 exhibited the greatest TFC (14.17 mg g⁻¹ FW), indicating that this lighting regime was particularly effective in stimulating flavonoid biosynthesis. Light is a major environmental factor regulating the phenylpropanoid pathway through the activation of enzymes such as phenylalanine ammonia-lyase (PAL), which plays a central role in the synthesis of phenolic compounds and flavonoids [17,59]. The relatively high proportions of B light in F2 and F3 and the high G light component in F4 may have enhanced the expression of genes involved in secondary metabolism, thereby promoting phenolic accumulation. Similar responses have been reported in basil and other medicinal plants, where blue-enriched LED spectra significantly increased phenolic and flavonoid contents [23,47]. Interestingly, the patterns of TPC and TFC showed clear relationships with the pigment data obtained in the present study. F2, which exhibited the highest chlorophyll a and chlorophyll b contents, also maintained high TPC values, suggesting that improved photosynthetic capacity provided sufficient carbon skeletons and reducing power for secondary metabolite biosynthesis. Enhanced photosynthesis has been shown to increase the availability of assimilates required for phenolic production [17]. In contrast, F4, which accumulated relatively high chlorophyll levels and showed the highest anthocyanin concentration, also produced the highest flavonoid content. Since anthocyanins belong to the flavonoid family, the simultaneous increase in anthocyanins and TFC under F4 indicates activation of the phenylpropanoid pathway and enhanced antioxidant defense mechanisms [58]. Moreover, F1, which exhibited the highest carotenoid concentration, showed only intermediate TPC and TFC values, suggesting that photoprotection under this treatment relied more heavily on carotenoid-mediated quenching rather than flavonoid accumulation. Carotenoids and phenolic compounds are known to complement each other in protecting plants against oxidative stress [57]. Conversely, F5 consistently produced the lowest chlorophyll, carotenoid, anthocyanin, and TPC values, which can be attributed to the low daily light integral and reduced photosynthetic activity under this treatment. Limited light availability decreases carbon assimilation and suppresses the biosynthesis of both photosynthetic pigments and secondary metabolites [46]. However, TOC and TFC values ​​in lemon basil under all light treatments of the present study were higher than previously reported [38]. Overall, the present findings indicate that pigment accumulation and phenolic metabolism in lemon basil are closely interconnected, and that LED spectral composition can modulate both primary and secondary metabolism. Among the tested treatments, F4 provided the most favorable balance between pigment synthesis and antioxidant metabolite accumulation, thereby improving the phytochemical quality of the crop.

Overall, the present study demonstrated that light environment plays a crucial role in modulating both primary and secondary metabolic processes in lemon basil (Ocimum × africanum). Differences in LED spectral composition significantly affected plant growth and productivity, as reflected by variations in plant height, biomass production, essential oil yield, and essential oil composition. In addition, light quality strongly influenced the accumulation of photosynthetic pigments, phenolic compounds, and flavonoids, underscoring the potential of targeted spectral management as an effective approach for enhancing crop productivity and improving the phytochemical value of lemon basil.

4. Materials and Methods

4.1. Plant Materials and Lighting Conditions

The experiment was conducted in growth chambers in the laboratory of the Technical University Darmstadt, Germany. The seeds of Ocimum x africanum (lemon basil), cultivar RADO 189, purchased from Rang Dong seed Ltd. company in Ho Chi Minh city, Vietnam, Lemon basil seeds were sown on 14 October 2025. After a 7-day germination period, seedlings were transplanted into small pots (110 mL volume; 7 cm diameter; 5.2 cm height). Sixteen days later, the plants were repotted into larger containers (750 mL volume; 12 cm diameter; 9.5 cm height). After an additional 5 days, the potted plants were transferred to five growth chambers with different lighting conditions for subsequent investigations. The techniques for planting, caring, fertilizing, and harvesting lemon basil plants were carried out according to the previous document [3]. Throughout the experiment, the ambient temperature was maintained at 22 ± 1 °C and the relative humidity at 68%. The lighting treatments were applied for 4 weeks, from 11 November to 09 December 2025. In four chambers from F1 to F4, a 16 h/8 h light/dark photoperiod was used. The PPFD was set to 220 ± 10 µmol m⁻² s⁻¹ and measured at canopy height using an MQ-650 ePAR meter with an underwater sensor (Apogee Instruments, Inc., Logan, Utah, USA). The fifth chamber was a greenhouse receiving only natural global light (sunlight and sky light), with a transmission rate of 25%. Distinct spectral treatments were characterized using a CSS-45 Spectroradiometer of the company Gigahertz-Optik GmbH, Türkenfeld, Germany (Table 3).

To compare the spectral ratios between the five treatments, their common point is that they all contain R, B, and Fr spectra. The difference is that treatments F1 and F4 contain the highest amount of R and G, lowest amount of Fr, and no UV-A compared to the other three treatments. Treatments F2 and F3 have no G light, but contain the highest amount of B, relative high amount of Fr, and a small amount of UV-A. Treatment F5 contains the highest amount of Fr, a small amount of UV-A and IR (Figure 6).

Figure 6. The relative spectra with different proportions of radiation regions.

Figure 6. The relative spectra with different proportions of radiation regions.

The LED lamps with a length of 1.2 m were installed above the growth chamber at an approximate distance of above 20 cm from the plant canopy to ensure uniform light distribution and spectral blending. The lamps were centrally positioned and aligned longitudinally along the growth chamber. Each treatment was conducted in one separated chamber and included 10 individual plants. The aerial parts of lemon basil were harvested at the end of the cultivation period, when most of the plants are full blooming, for subsequent analysis and evaluation. Each experimental treatment consisted of 10 plants grown under identical conditions (Figure 7). For physiological measurements, 10 plants from each treatment were measured, and the mean value per treatment was used for statistical analysis. The values of plant physiological parameters were calculated per ha of growing, on a basis of density of 20 × 20 cm. For pigment and secondary metabolite analyses (sections 4.4-4.6), leaves from two plants within each treatment were combined to constitute one biological replicate. Sampling was standardized by selecting leaves from the fourth branch/node below the apical meristem. A total of 25 leaf samples (five biological replicates per treatment) were collected from lemon basil plants subjected to five different treatments. Immediately after harvest, the samples were wrapped in aluminum foil, flash-frozen in liquid nitrogen, and ground prior to subsequent biochemical analyses. Absorbance values ​​were determined photometrically at appropriate wavelengths using a FoodALYT photometer (Omnilab˗Laborzentrum GmbH and Co. KG, Bremen, Germany).

4.2. Essential oil Isolation

Each lemon basil sample, consisting of 72–401 g of aerial biomass, was shredded and subjected to hydrodistillation for 2.5 hours using a Clevenger-type apparatus [61]. The obtained essential oil was then separated and stored at –5 °C for subsequent analysis. For essential oil extraction, plant material from 10 plants within each treatment was divided into 2 portions (except for the sample from F5, which had only one part due to its small weight), and the extraction was performed separately for each portion. Subsequently, the essential oil from each treatment was combined and analyzed in triplicate to determine its chemical composition.

4.3. Essential oil GC-MS Analysis

GC–MS analysis was performed on a Thermo Scientific TRACE™ 1310 gas chromatograph coupled with an ISQ™ 7000 single quadrupole mass spectrometer (Thermo Scientific, Austin, TX, USA) operating in electron ionization (EI) mode at 70 eV. Separation was performed on an HP-5MS fused silica capillary column (60 m × 0.25 mm i.d., 0.25 μm film thickness). Helium was used as the carrier gas at a flow rate of 1.0 mL/min. The injector was set at 250 °C, with a 1 μL injection volume in split mode (1:100). The oven program started at 60 °C and was increased to 260 °C at 4 °C/min. Detector temperatures were maintained at 280 °C. For MS analysis, conditions included an interface temperature of 280 °C, electron ionization (EI) at 70 eV, a scan rate of 4.0 scans/s, and a mass range of 35–450 Da. Constituents were identified by comparing their relative retention indices (determined by co-injection with a homologous series of n-alkanes, C7–C30) and mass spectral fragmentation patterns with reference libraries (NIST2020, Wiley12, HPCH1607). Data was processed using Freestyle 1.8 and MassFinder 4.0. Relative concentrations were calculated from TIC peak areas without standardization. [62,63,64]. Additionally, the samples were analysed with the same GC setup decribed above, but coupled with a Orbitrap Exploris (Thermo Scientific) operating in electron ionization (EI) mode at 70 eV in order to proof fragments with their exact masses.

4.4. Chlorophylls and Carotenoid Analysis

The contents of chlorophyll a, chlorophyll b, and carotenoid were determined using a spectrophotometric method based on the characteristic light absorption properties of photosynthetic pigments in organic solvents. Following extraction with 90% methanol, the pigments were dissolved in the extract and exhibited specific absorption maxima at characteristic wavelengths. Chlorophyll a showed maximum absorption at approximately 665 nm, chlorophyll b at around 652 nm, whereas carotenoids absorbed predominantly at 470 nm. These photosynthetic pigments were extracted using 90% methanol, and 100 µL of the resulting extract was used for spectrophotometric analysis. Absorbance values were recorded photometrically at 665, 652, and 470 nm [65]. The contents of pigments were calculated according to the equations:

Chlorophyll a (mg/L) = 16,82 * A(665) – 9,28 * A(652)

(1)

Chlorophyll b (mg/L) = 36,92 * A(652) – 16,54 * A(665)

(2)

Carotenoid (mg/L) = ([1000 * A470] – [1,91 x Chlorophyll a] – [95,15 * Chlorophyll b])/225

(3)

4.5. Anthocyanin Analysis

Monomeric anthocyanins exhibit reversible structural transformations in response to pH changes, resulting in distinct color variations at pH 1.0 and pH 4.5. Under highly acidic conditions (pH 1.0), anthocyanins predominantly exist in the colored flavylium (oxonium) cation form, whereas at pH 4.5 they are mainly converted into the colorless hemiketal form. Consequently, the difference in absorbance measured at 520 nm between these two pH conditions is directly proportional to the anthocyanin concentration. Anthocyanin content was expressed as cyanidin-3-glucoside equivalents using a molecular weight of 449.2 g mol⁻¹ and a molar extinction coefficient of 26,900 L mol⁻¹ cm⁻¹.

For the pH differential assay, two buffer solutions were prepared: 0.025 M potassium chloride buffer adjusted to pH 1.0 with HCl, and 0.4 M sodium acetate buffer adjusted to pH 4.5 with acetic acid. The pH values were verified using a pH meter (Hanna Instruments, Woonsocket, RI, USA). Equal proportions of extract and buffer were used, with 40 µL of sample extract mixed with 160 µL of the corresponding buffer solution in microplates. After an incubation period of 20–50 min, absorbance readings were recorded photometrically at 520 and 700 nm. Total monomeric anthocyanin content was determined according to the pH differential method described by Lee et al. [66], and calculated using the following equations:

A (absorbance) = (A₅₂₀ – A₇₀₀)_pH1 – (A₅₂₀ – A₇₀₀)_pH4,5

(4)

Anthocyanin content (mg/L) = (A * MW * DF * 1000)/(Ɛ × 1)

(5)

where:

MW= molecular weight of cyanidin-3-O-glucoside (449,2 g/mol)

DF = Dilution factor (40:160 µl, adjustable if you don't see the pink color change)

Ɛ = molar extinction coefficient of cyanidin-3-O-glucoside (26900 L/cm*mol)

l = path length of cuvette (typically = 1 cm)

Total anthocyanin content was calculated in the sample as mg per g of fresh weight (FW)

4.6. Total Phenolic and Total Flavonoid Concentration Analysis

To evaluate the effects of different light regimes on phenolic and flavonoid production, rapid spectrophotometric assays based on the chemical properties of these secondary metabolites were employed. Phenolic compounds are aromatic molecules containing free hydroxyl groups that become deprotonated under alkaline conditions, generating phenolate ions with reducing capacity. Upon addition of the Folin–Ciocalteu reagent, which contains phosphomolybdic and phosphotungstic acids, these phenolate ions reduce the metal complexes present in the reagent, leading to the formation of blue-colored molybdenum complexes. The intensity of the resulting blue coloration is proportional to the concentration of reducing phenolic compounds in the sample and was quantified photometrically at 735 nm. Gallic acid was used to make the standard curve at the concentrations of 250, 200, 150, 100 mg/L.

Similarly, the determination of flavonoids relies on the reactivity of their hydroxyl groups. In the presence of aluminum chloride, these groups form stable chelate complexes with aluminum ions. Subsequent addition of sodium hydroxide promotes deprotonation of the hydroxyl groups, thereby enhancing complex formation and producing an intense red coloration. The absorbance of this colored complex was then be measured photometrically at 510 nm to estimate flavonoid content. Catechin was used to make the standard curve at the concentrations of 250, 200, 150, 100 mg/L.

Based on these principles, total phenolic and flavonoid contents were determined following the methods described by Waterhouse [67], and Zhishen et al. [68], Dou et al. [69], respectively, with appropriate modifications to the reagent and sample volumes.

4.7. Statistical Analysis

The effects of the lighting treatments on lemon basil were analyzed using a completely randomized design with one-way analysis of variance (ANOVA). Significant treatment effects were further evaluated by comparing means using the least significant difference (LSD) test at p ≤ 0.05. Statistical analyses were performed using IRRISTAT version 5.0 (International Rice Research Institute, Philippines).

5. Conclusions

Overall, the results demonstrated that lighting conditions exerted profound influences on both the primary and secondary metabolism of lemon basil (Ocimum × africanum). Variations in LED spectral composition significantly affected plant growth and productivity, including plant height, biomass accumulation, essential oil yield, and essential oil composition, as well as the biosynthesis of photosynthetic pigments, total phenolics, and total flavonoids. A lighting regime characterized by a high proportion of red light, moderate levels of blue and green light, and a low far-red component promoted biomass production and carotenoid accumulation. In contrast, treatments enriched in blue and red wavelengths, combined with moderate far-red radiation and elevated UV-A levels, favored stem elongation and enhanced chlorophyll a and chlorophyll b contents. Meanwhile, a spectral combination containing high proportions of red and green light, moderate blue light, and limited far-red radiation proved most effective in stimulating the accumulation of essential oils, anthocyanins, phenolic compounds, and flavonoids. These findings provide valuable insights into the physiological and biochemical responses of lemon basil to different light spectra and contribute to the growing understanding of light-mediated regulation of plant metabolism. Furthermore, the results establish a scientific foundation for the development of precision lighting strategies aimed at maximizing crop productivity and improving phytochemical quality for applications in agriculture, pharmaceutical industries, and cosmetic products. Based on the knowledge from this study, further LED spectral compositions and regimes will be optimized and experimentally validated for lemon basil and other similar species in order to increase the plant growth and biosynthesis performance.