**1. Introduction**

*Fritillaria,* a bulbiferous and perennial monocot plant genus, belongs to the family Liliaceae. The genus consists of about 130 species distributed in the temperate regions of Central Asia and the Mediterranean region [1]. Though some *Fritillaria* species are grown as ornamental plants, several *Fritillaria* species possess valuable medicinal properties. *Fritillaria* bulbs composed of fleshy, farinaceous scales constitute essential plant parts and have been used to relieve cough for centuries [2].

In different *Fritillaria* species, a majority of bioactive compounds (86%) identified so far (~130) consist of isosteroidal alkaloid skeletons [3]. Alkaloids in *Fritillaria* bulbs are the main bioactive

compounds responsible for relief from coughs [3,4]. However, the quantities and types of alkaloids vary depending on species [1,3–5].

In a recent study, it was found that peimine, an alkaloid from *Fritillaria,* blocked the Nav1.7 ion channel and inhibited the Kv1.3 ion channel in HEK 293 cell lines, indicating that the compound has a role in relieving pain and possesses anti-inflammatory properties [6]. More recently, Liu and co-workers investigated the potential effect and mechanism of six isosteroidal alkaloids on oxidative stress. The findings showed that *F. cirrhosa* D. Don bulbs might play a protective role in cellular oxidative stress by activating the Nrf2-mediated antioxidant pathway [7].

China is the center of diversity of the *Fritillaria* genus. *F. cirrhosa* D. Don (FC) is an important traditional Chinese medicine commonly known as "Chuanbeimu" (川貝母), and is one of the most exploited species. Due to scarcity, the price of wild *F. cirrhosa* D. Don bulbs escalated almost nine times from \$60 to \$560 USD between 2002–2017 [8]. Due to the excessive collection of FC bulbs from natural habitats, the species is now under protection [9]. Therefore, alternative propagation methods of FC bulbs and the production of critical isosteroidal alkaloids by tissue culture techniques must be optimized.

The culture of plant tissues and organs is an important bio-technique to produce secondary plant metabolites under controlled environmental conditions in a laboratory. Under culture conditions, callus (an undifferentiated mass of cells) can easily be induced, practically from any living plant part. Induced callus can be cultured and multiplied in vitro on a defined nutrient medium with or without plant growth regulators. There are several advantages of using callus cultures as a source of valuable secondary metabolites, including (i) ease of induction and multiplication; (ii) production of bioactive compounds throughout the year independent of season; (iii) whole plants do not need to be cultivated, especially rare and endangered species; (iv) amenability of scaling by bioreactors for mass production, etc. More recently, we have reported on the micropropagation of bulblets and the production of isosteroidal alkaloids in tissue culture-derived materials of *F. cirrhosa* D. Don [10]. Several recent studies have demonstrated that the light quality not only affects morphogenetic responses in plants but has significant effects on their physiological processes, including metabolic pathways and the production of secondary metabolites [11,12].

A recent review listed several studies on the effects of light quality on the production of secondary metabolites in different plant species [11]. In the present study, four isosteroidal alkaloids (peimisine, sipeimine, peiminine, and peimine) were analyzed considering their therapeutic effects. Peiminine (Pm) is one of the major isosteroidal alkaloids in *Fritillaria* that is reported to have extensive pharmacological activities, including anti-inflammatory [6], anti-cancer [13], and antioxidant [14] capabilities. In another report, sipeimine and peiminine from bulbs of *F. wabuensis* inhibited pro-inflammatory mediators in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophage cells [15]. More recently, it was demonstrated that peimine relieved inflammatory effects in IL-1β-induced chondrocytes, indicating that peimine might be a potential therapeutic agent for osteoarthritis [16]. Antitussive, expectorant, and anti-inflammatory effects of several alkaloids, including sipeimine, chuanbeinone, peiminine, and peimine isolated from Bulbus *Fritillaria cirrhosa* were demonstrated in mice by using a phytochemical method [17]. A most recent study confirmed the anti-cancer effects of sipeimine obtained from bulbs of *F. cirrhosa* against non-small cell lung cancer (NSCLC) both in vivo and in vitro [18]. This anti-cancer property of sipeimine is largely due to anti-inflammation action affected by NF-κB inhibition, making it a potential drug candidate for treating cancer at early stages [18]. Recently, Yin and co-workers have reported several therapeutic properties of peimine, including anti-cancer, anti-inflammatory, antitussive, expectorant, and sedative [19]. A more recent study has also demonstrated cough relief by peimine by affecting the systemic network of proteins and pathways [20].

The objective of the present study is to investigate the effects of different LED lights on growth and development in embryogenic calli and the contents of four isosteroidal alkaloids (peimisine, sipeimine, peiminine, and peimine) in in vitro cultures of *F. cirrhosa* D. Don. Findings in the study may be of help to produce certain alkaloids under laboratory conditions irrespective of the season and thus avoid having to collect *F. cirrhosa* D. Don bulbs from the wild.

#### **2. Material and Methods 2. Material and Methods**

#### *2.1. Callus Multiplication 2.1. Callus Multiplication*

Callus obtained in our previous experiments, as reported earlier [10], was further multiplied in the liquid medium. Callus was cultured in 125 mL Erlenmeyer flasks, each containing 20 mL basal salts and vitamins of Murashige and Skoog [21] medium (MSBM) supplemented with 2,4-D (0.5 mg/L) and 2% sucrose (Sigma). The culture flasks were placed on an orbital shaker (Model SK-302A, Sun Kaun Instruments Co., Taichung, Taiwan) set at 100 rpm and incubated at 25 ± 1 ◦C in the dark. Callus obtained in our previous experiments, as reported earlier [10], was further multiplied in the liquid medium. Callus was cultured in 125 mL Erlenmeyer flasks, each containing 20 mL basal salts and vitamins of Murashige and Skoog [21] medium (MSBM) supplemented with 2,4-D (0.5 mg/L) and 2% sucrose (Sigma). The culture flasks were placed on an orbital shaker (Model SK-302A, Sun Kaun Instruments Co., Taichung, Taiwan) set at 100 rpm and incubated at 25 ± 1 °C in the dark.

*Plants* **2020**, *9*, x FOR PEER REVIEW 3 of 15

#### *2.2. Influence of Di*ff*erent Light Spectra on Morphogenesis in Embryogenic Calli and Contents of Isosteroidal Alkaloids 2.2. Influence of Different Light Spectra on Morphogenesis in Embryogenic Calli and Contents of Isosteroidal Alkaloids*

To investigate the effects of LED lights on the morphogenesis of embryogenic calli and the contents of isosteroidal alkaloids, embryogenic calli from liquid cultures were taken out and kept for 1 min on sterilized filter paper in a laminar flow before inoculation to glass bottles. Callus (3.0 g) was cultured in glass bottles (650 mL capacity), each containing 100 mL of MSBM medium with 2% sucrose and 0.4% gellan gum powder (GPP), a gelling agent (PhytoTechnology Laboratories®, USA). The pH of the medium was adjusted to 5.7 ± 0.1 before the addition of GPP and autoclaving at 1.05 kg/cm for 15 min. To facilitate LED light exposure to cultures, each bottle was closed with a piece of transparent, autoclavable plastic sheet. These bottles were incubated in a specially designed plant growth chamber equipped with eight different LED lights (Nano Bio Light Technology Co., Ltd., Taiwan). The chamber had two tiers, and each tier had four partitions (Figure 1a). Culture bottles were kept in these eight sections and exposed to different light spectra by eight specially designed LED lids (CW-5000K, WW-2700K, 8R1B, 7R1G1B, 3R3B3IR, 6R, 6B, and 6IR) (Figure 1b). LED lids CW-5000K and WW-2700K represented cool (C) and warm (W) white (W) light, while 5000K and 2700K represented color temperature, respectively. As reported in a previous study from our laboratory [22], six of these LED lids emitted single or combinations of four different light spectra with wavelengths such as blue (450 nm), green (525 nm), red (660 nm), and far-red (730 nm). The symbols in each LED lid code and the spectral distribution (quantum ratio) in eight LED lids were as follows: blue (B), green (G), red (R), infrared (IR), CW-5000K (28:43:29:0), WW-2700K (8:46:46:0), 8R1B (16:0:84:0), 7R1G1B (17:9:74:0), 3R3B3IR (57:0:43:37), 9R (0:0:100:0), 9B (100:0:0:0), and 9IR (0:0:0:100). In this work, the number (9, 7, 3, 1) in each LED lid code represents the number of LED chips in a particular lid. The light intensity of each LED lid was as follows: CW-5000K\* (57 µmol m−<sup>2</sup> s −1 ); WW-2700K (56 µmol m−<sup>2</sup> s −1 ); 7R1G1B (56 µmol m−<sup>2</sup> s −1 ); 8R1B (57 µmol m−<sup>2</sup> s −1 ); 9B (57 µmol m−<sup>2</sup> s −1 ); 9R (56); 9IR (10 µmol m−<sup>2</sup> s −1 ); 3R3B3IR (56 µmol m−<sup>2</sup> s −1 ). To investigate the effects of LED lights on the morphogenesis of embryogenic calli and the contents of isosteroidal alkaloids, embryogenic calli from liquid cultures were taken out and kept for 1 min on sterilized filter paper in a laminar flow before inoculation to glass bottles. Callus (3.0 g) was cultured in glass bottles (650 mL capacity), each containing 100 mL of MSBM medium with 2% sucrose and 0.4% gellan gum powder (GPP), a gelling agent (PhytoTechnology Laboratories®, USA). The pH of the medium was adjusted to 5.7 ± 0.1 before the addition of GPP and autoclaving at 1.05 kg/cm for 15 min. To facilitate LED light exposure to cultures, each bottle was closed with a piece of transparent, autoclavable plastic sheet. These bottles were incubated in a specially designed plant growth chamber equipped with eight different LED lights (Nano Bio Light Technology Co., Ltd., Taiwan). The chamber had two tiers, and each tier had four partitions (Figure 1a). Culture bottles were kept in these eight sections and exposed to different light spectra by eight specially designed LED lids (CW-5000K, WW-2700K, 8R1B, 7R1G1B, 3R3B3IR, 6R, 6B, and 6IR) (Figure 1b). LED lids CW-5000K and WW-2700K represented cool (C) and warm (W) white (W) light, while 5000K and 2700K represented color temperature, respectively. As reported in a previous study from our laboratory [22], six of these LED lids emitted single or combinations of four different light spectra with wavelengths such as blue (450 nm), green (525 nm), red (660 nm), and far-red (730 nm). The symbols in each LED lid code and the spectral distribution (quantum ratio) in eight LED lids were as follows: blue (B), green (G), red (R), infrared (IR), CW-5000K (28:43:29:0), WW-2700K (8:46:46:0), 8R1B (16:0:84:0), 7R1G1B (17:9:74:0), 3R3B3IR (57:0:43:37), 9R (0:0:100:0), 9B (100:0:0:0), and 9IR (0:0:0:100). In this work, the number (9, 7, 3, 1) in each LED lid code represents the number of LED chips in a particular lid. The light intensity of each LED lid was as follows: CW-5000K\* (57 μmol m−2 s−1); WW-2700K (56 μmol m−2 s−1); 7R1G1B (56 μmol m−2 s−1); 8R1B (57 μmol m−2 s−1); 9B (57 μmol m−2 s−1); 9R (56); 9IR (10 μmol m−2 s−1); 3R3B3IR (56 μmol m−2 s−1).

**Figure 1.** (**a**) Plant growth chamber with LED lights. Bar = 8.3 cm; (**b**) Eight different LED lights: (**a**) CW-5000 K, (**b**) WW-2700 K, (**c**) 7R1G1B, (**d**) 8R1B, (**e**) 9B, (**f**) 9R, (**g**) 9IR, (**h**) 3R3B3IR. Bar = 2 cm. Red (R): 660 nm; green (G): 525 nm; blue (B): 450 nm; infrared (IR): 730 nm. **Figure 1.** (**a**) Plant growth chamber with LED lights. Bar = 8.3 cm; (**b**) Eight different LED lights: (**a**) CW-5000 K, (**b**) WW-2700 K, (**c**) 7R1G1B, (**d**) 8R1B, (**e**) 9B, (**f**) 9R, (**g**) 9IR, (**h**) 3R3B3IR. Bar = 2 cm. Red (R): 660 nm; green (G): 525 nm; blue (B): 450 nm; infrared (IR): 730 nm.

The LED growth chamber was set on a 16 h light and 8 h dark cycle, kept in a culture room set at 25 ± 2 ◦C, and fully covered by a thick, dark cloth to cut off outside light. Morphological features of embryogenic callus under each light condition were recorded after three months of incubation. In addition, cultures under different LED lights were analyzed by LC-MS/MS for contents of peimisine, sipeimine, peiminine, and peimine.
