2.3.3. Blue and Red LED in Combination

Several studies have reported the use of blue and red LEDs in combination on one or more plants simultaneously. Many reports suggested that the use of different LEDs in combination can enhance the production of bioactive compounds to several folds. Thus, their combination can be employed in eliciting various cultures that have gained importance pharmacologically [95,97,98]. Since ancient times, root extracts of *Rehmannia glutinosa* Libosch (Chinese foxglove) have been used to treat many disorders such as anemia and hypertension [96]. The therapeutic use of *R. glutinosa* roots is well known due to the presence of bioactive compounds such as catalpol, aucubin, and rehmaglutin [96,110]. In a study, plant development as well as phytochemicals with both defensive and prospective medicinal characteristics, such as phenolics and flavonoids, were influenced by spectral characteristics in both leaf and root tissues of *R. glutinosa*. On irradiation with blue LED, leaf extracts were found to have the highest total phenol concentration (35 ± 0.05 µg GAE/mg) as compared to root extracts. On the other hand, red LED exposure also increased the overall flavonoid content of the leaf extract by 33.6% and the root extracts by 61.7%. As a result, blue and red LEDs may be the most enticing light sources for *R. glutinosa* proliferation in in vitro conditions [95].

Bioactive compounds like phenolics and flavonoids are of significant importance in therapeutic plants because they can act as free radical scavengers [111]. The effects of red and blue LEDs on the accumulation of phenolic and flavonoids in *Ocimum basilicum* callus cultures were investigated in a recent study. Rosmarinic acid and eugenol were considerably enhanced under blue light (2.46 and 2.25 times greater than control). While under red light, the highest amounts of cyanidin (0.1216 mg/g DW) and peonidin (0.127 mg/g DW) were detected. Chicoric acid accumulation was about 4.52 times higher than callus grown under the control of continuous white light (81.40 mg/g DW) [97]. This suggests that employing LEDs to accelerate the production of physiologically active chemicals in vitro could be beneficial.

Fruits, nuts, medicinal herbs, and vegetables all contain phenylpropanoid, a type of secondary metabolite that is involved in reducing the risk of diabetes and heart disease

through inhibiting carcinogenesis [112,113]. Plants are protected from bacteria by the antimicrobial activities of some phenylpropanoids during their interactions [114]. The generation of phenylpropanoid metabolites was examined using LEDs as an elicitor in wheat sprouts, and qRT-PCR and HPLC results revealed that white light (380 nm) was the best wavelength for epicatechin biosynthesis in wheat sprouts. Blue light (470 nm) was involved in the increased accumulation of gallic acid and quercetin, whereas red light (660 nm) increased the aggregation of ferulic acid on the 8th day and p-coumaric acid on the 12th day [98]. Similarly, flavonoids and anthocyanins, which also belong to a class of phenylpropanoids, were also enhanced in buckwheat, *Fagopyrum* sp. under red + blue (combination), and blue LED, respectively [99]. Likewise, phenolics and flavonoids were enhanced in seeds of *Ocimum basilicum* under red–blue (2R:1B) LED. In addition, the highest flavonoid content of 16.79 mmol/g FW was also achieved for protocorm-like bodies pretreated with white LED for more than three cultures cycles under blue–red (1:1) LED [101]. Thus, red and blue LED ratios can be altered to generate improved growth and phenolic content in both red and green basil microgreens as a practical technique for generating superior quality foods [100]. Furthermore, in the callus culture of *Cnidium officinale*, mixed (red–blue) LED illumination enhanced the synthesis of phenolics and flavonoids [10]. In another study, in vitro generated *Eclipta alba* callus culture was subjected to multispectral lighting under regulated aseptic conditions. Results showed that the red light enhanced the production of phenolics (57.8 mg/g) and flavonoids (11.1 mg/g). whereas, on exposure to blue light, the production of four major compounds coumarin (1.26 mg/g), eclalbatin (5.00 mg/g), wedelolactone (32.54 mg/g), and demethylwedelolactone (23.67 mg/g), as well as two minor compounds β-amyrin (0.38 mg/g) and luteolin (0.39 mg/g) were increased [102].
