Artificial color light sources and precursor feeding enhance plumbagin production of the carnivorous plants Drosera burmannii and Drosera indica

Journal of Photochemistry & Photobiology, B: Biology 199 (2019) 111628

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Artificial color light sources and precursor feeding enhance plumbagin production of the carnivorous plants Drosera burmannii and Drosera indica

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Panitch Boonsnongcheepa,b, Worapol Sae-fooa, Kanpawee Banpakoata, Suwaphat Channaronga, Sukanda Chitsaithana, Pornpimon Uafuaa, Wattika Puthaa, Kanchanok Kerdsiria, ⁎ Waraporn Putaluna,b, a

Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand Research Group for Pharmaceutical Activities of Natural Products using Pharmaceutical Biotechnology (PANPB), National Research University, Khon Kaen University, Khon Kaen 40002, Thailand

b

ARTICLE INFO

ABSTRACT

Keywords: Carnivorous plant Plant tissue culture Naphthoquinone LEDs Secondary metabolite Drosera burmannii Drosera indica Plumbagin

Plumbagin is the main pharmacologically active compound of carnivorous plants in the genera Drosera. It possesses various pharmacological activities, including anticancer and antimalarial activities, and is used in traditional medicine. In this study, we reported a sustainable production system of plumbagin by adding sodium acetate and L-alanine as precursors to in vitro cultures of Drosera burmannii Vahl and Drosera indica L. In addition, plumbagin production was reported in the cultures subjected to different color LED lights. The highest plumbagin level (aerial part 14.625 ± 1.007 mg·g−1 DW and root part 1.806 ± 0.258 mg·g−1 DW) was observed in D. indica cultured under blue LED light for 14 days, and further culturing did not increase plumbagin production. In addition, plumbagin enhancement by precursor feeding (9.850 ± 0.250 mg·g−1 DW, 1.2-fold) was observed in the aerial part of D. indica treated with 50 mg·L−1 sodium acetate for 3 days. Comparing both plants, up to 700-fold higher plumbagin was observed in D. indica than in D. burmannii. Moreover, in both plants, the aerial part accumulated higher plumbagin (up to 10-fold) than the roots. This is the first report on the effect of artificial LED lights on the plumbagin level of Dorsera plants. The culturing of D. indica under blue LED light showed enhanced plumbagin levels and suggests a fast and simple system for the in vitro production of plumbagin.

1. Introduction Plants in the genus Drosera are carnivorous plants that are distributed in many regions of the world [1]. These plants have been used in traditional medicine, of which naphthoquinone and flavonoid compounds are responsible for many pharmacological activities [2,3]. The main active compound of Drosera spp. is the naphthoquinone plumbagin [4,5]. The range of biological and pharmacological activities of plumbagin has been reported [6]. In particular, anti-cancer and antimalarial activities have been studied in both in vitro and in vivo models [7–9]. Plant tissue culture and related techniques have been used for the sustainable production of plant secondary metabolites for decades [10]. The controllable environment of in vitro culture, e.g., light quality, media composition, and temperature, is preferable and beneficial over field cultivation [10,11]. In vitro production of plumbagin was reported in root, hairy root, and cell cultures of several species of Plumbago [12–16] and in vitro cultures of Drosera [4,17–20]. Some of these studies ⁎

also employed various techniques to enhance the production of plumbagin, i.e., elicitation and precursor feeding. Drosera burmannii Vahl and Drosera indica L. are Thai medicinal plants that produce plumbagin. The plant’ habitats are limited to wetlands, and therefore, cultivation in the field is difficult [21]. Hence, plant in vitro culture is an effective alternative. Previously, in vitro culturing of both plants showed increased levels of plumbagin production using elicitation with methyl jasmonate and yeast extract [17,18]. However, there are few studies on other factors that may enhance the plumbagin production in D. burmannii and D. indica cultures. Precursor feeding is a technique that has been used to increase the amount of desirable secondary metabolites in plants [22,23]. Plumbagin is biosynthesized through the acetate and mevalonate pathways [5,24,25]. Acetate was previously proposed as a building block for plumbagin biosynthesis in higher plants [24]. Later, L-alanine was also reported to be a precursor of plumbagin [26]. Moreover, L-alanine was reported to enhance plumbagin production in Plumbago indica root cultures [12].

Corresponding author at: Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail address: [email protected] (W. Putalun).

https://doi.org/10.1016/j.jphotobiol.2019.111628 Received 7 June 2019; Received in revised form 22 July 2019; Accepted 11 September 2019 Available online 12 September 2019 1011-1344/ © 2019 Elsevier B.V. All rights reserved.

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Light is one of the most important factors affecting the growth and secondary metabolite production of plants [27]. In plant in vitro cultures, white-fluorescent light was conventionally used as a light source. However, in recent years, the use of artificial light emitting diodes (LEDs) has been increasingly studied. LEDs provide several benefits over fluorescent lamps, including durability, a relatively cool emitting surface, and a specific wavelength [27–29]. Plant in vitro cultures are grown under artificial lighting conditions; therefore, light sources could be modified and controlled in various aspects. Artificial LEDs have been used to enhance the production of plant metabolites [27,30–32]. In particular, blue (450–500 nm) and red (610–760 nm) LEDs are commonly used for improved secondary metabolite production in various plants [27,29]. However, the effect of light on the biosynthesis pathway for most plant secondary metabolites is still unclear [10,22,27]. Some studies reported the upregulation of gene expression in plants towards secondary metabolism after exposure to red and blue lights [33,34]. However, there is a lack of information about the effect on secondary metabolite production of D. burmannii and D. indica. The appropriate conditions for precursor feeding and artificial lighting are not universal and need to be optimized for each plant [11,23]. In this study, we tested the effect of two precursors, sodium acetate and L-alanine, along with the effect of artificial LED light sources on the production of plumbagin in D. burmannii and D. indica in vitro cultures. Optimum conditions for the in vitro production of plumbagin are discussed.

the addition of precursors. Sodium acetate and L-alanine were individually dissolved in deionized water and sterilized by filtration. The solution of sodium acetate was added to the culture at concentrations of 10, 50, and 100 mg·L−1, while the L-alanine solution was added at concentrations of 0.1, 1.0, and 5.0 mM. The cultures were harvested at 3, 6, and 9 days after the addition of precursors. 2.5. Extraction of Plant Samples and Analysis of Plumbagin Contents

2. Materials and Methods

Harvested plant samples were separated into aerial and root parts. They were then dried at 50 °C for 48 h and ground into powder. Thirty milligrams of sample was extracted with 500 μL of methanol 4 times in an ultrasonic bath. The extracts were combined and evaporated to dryness before resuspension in 1 mL of methanol. Plumbagin contents of D. burmannii and D. indica samples were analyzed using high-performance liquid chromatography (HPLC) using a previously described method with modifications [36]. Samples were analyzed using a Spectra-Physics® Spectra system HPLC with a P4000 pump and a UV 2000 detector. The mobile phase was an isocratic system of methanol and 1% aqueous acetic acid (55:45) operated at a flowrate of 1 mL·min−1. The stationary phase was a Merck LiChrospher® 100 RP-18 (125 × 4 mm, 5 μm) column, and plumbagin was detected at 254 nm. The regression equation for quantification (y = 35,584× + 12,670, where y is the area under the curve and x is the concentration) was obtained from the plotted calibration curve of standard plumbagin in the concentration range of 6.25 to 100 μg·mL−1.

2.1. Chemicals

2.6. Statistical Analysis

Plumbagin was obtained from Wako Pure Chemical Industries (Osaka, Japan), while sodium acetate and L-alanine were purchased from Ajax Finechem (Auckland, New Zealand). All other chemicals were of analytical grade.

Each experiment was performed in three biological replicates, and HPLC analysis was performed in three technical replicates. The data were expressed as the mean ± SD. The difference between samples was analyzed using a one-way analysis of variance (ANOVA) and Tukey's test at the P-value < .05 in SPSS version 16.0 (SPSS Inc., Chicago, IL). The graphs were created using Microsoft Excel 2010.

2.2. Plant Materials Drosera burmannii Vahl and Drosera indica L. seeds were obtained from Ubon Ratchathani, Thailand. The seeds were surface sterilized with a 2% sodium hypochlorite solution for 20 min followed by immersion in 70% ethanol for 1 min and were then washed with sterile distilled water. Seeds were germinated into whole plants on a halfstrength Murashige and Skoog solid medium (MS) [35] containing 3% sucrose and solidified using 2.5 g·L−1 gellan gum but without plant growth regulator. Both plants were grown under 16/8 h light/dark conditions at 25 ± 2 °C. The plants were subcultured onto the same media every 3 weeks. They were used in every experiment as the whole plantlets.

3. Results and Discussion 3.1. Effect of Light Source On Plumbagin Accumulation D. burmannii and D. indica plantlets cultured under different LED light sources are shown in Fig. 1. Plumbagin levels in the aerial part of the plants were approximately 10-fold higher than those in the root part. At 14 days, plumbagin levels in D. burmannii cultured under different lights were ranged from 0.453 ± 0.013 to 0.521 ± 0.048 mg g−1 DW in the aerial part and 0 to 0.062 ± 0.006 mg g−1 DW in root part. However, plumbagin levels in the D. burmannii aerial part showed no difference under different lighting conditions (Fig. 2). In the root part, significantly different plumbagin levels were only observed in the white light culture (Fig. 2). The highest level of plumbagin (0.521 ± 0.048 mg g−1 DW) in D. burmannii was observed in the aerial part cultured under white LED light. However, at 28 days, plumbagin was completely depleted in every group. Similar to D. burmannii plumbagin levels in the aerial part of D. indica were approximately 9-fold higher than those of theroot part (Fig. 3). At 14 days, the levels of plumbagin in the aerial part of D. indica were 6.119 ± 0.193 to 14.625 ± 1.007 mg g−1 DW, and the levels in the roots were 0.295 ± 0.030 to 1.896 ± 0.258 mg g−1 DW. After 14 days of culture, the aerial part of D. indica cultured under white, blue, and red LED light showed no significant difference in plumbagin level. However, the plants cultured under dark conditions accumulated less plumbagin than the other groups. In the root part, blue and red LED light showed higher levels of plumbagin. The highest

2.3. Effect of Artificial LED Treatments Two grams each of D. burmannii and D. indica plantlets was inoculated into 125 mL flasks containing 30 mL of MS media without the solidifying agent. The cultures were placed on a rotary shaker operated at 80 rpm. They were placed under white (400–700 nm), blue (425–500 nm), and red (600–700 nm) LEDs (NVC lighting technology corporation; Guangdong, China) with 16/8 h light/dark or complete dark conditions. The distance between LEDs and plants was 30 cm, and the LEDs irradiated at 61.5 W·m2. After 14 and 28 days, the plants were collected for extraction and chemical analysis. 2.4. Precursor Preparation and Treatment The plants were inoculated into liquid MS media in a similar manner as in the LED experiment and were grown for 21 days prior to 2

Journal of Photochemistry & Photobiology, B: Biology 199 (2019) 111628

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Plumbagin (mg·g-1DW)

Fig. 1. In vitro cultures of D. burmannii (A, B, C, and D) and D. indica (E, F, G, and H) under different light sources at 14 days. A and E: white light, B and F: blue light, C and G: red light, D and H: dark; bar = 1 cm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 0.70

Aerial

0.60

a

a

level of plumbagin was observed in the plants cultured under blue light at 14 days (aerial part: 14.625 ± 1.007 mg g−1 DW, root part: 1.806 ± 0.258 mg g−1 DW). This is the treatment that accumulated the highest plumbagin in this report. At 28 days, although the plants cultured under white and blue light treatments showed significantly higher levels of plumbagin than those cultured under red light and dark conditions (Fig. 3), the level of plumbagin decreased in every treatment. The levels at day 28 ranged from 5.950 ± 0.518 to 9.240 ± 0.578 mg g−1 DW in the aerial part and 0.458 ± 0.051 to 0.854 ± 0.098 mg g−1 DW in the root part. Compared with the plant growth in the normal culture room under fluorescent lighting (Table 1), the highest levels of plumbagin cultured under LED were 2.8-fold higher in D. burmannii and 1.9-fold higher in D. indica. Culturing under blue LEDs showed the highest plumbagin level (Fig. 3), which coincided with other studies. In Scutellaria lateriflora shoot cultures, the levels of flavonoids, phenolics, and phenylpropanoid glycosides were stimulated to the highest level when cultured under blue lights [32]. In another study, blue light treatment helped promote the growth and accumulation of total flavonoids and total polyphenols of the medicinal plant Anoectochilus roxburghii [37]. Blue LED light was effective in promoting the production of phenolic compounds, as reported in several studies [38–42]. Red light positively affected the level of phytochemicals, including monoterpenes of Achillea millefolium [43], cucurbitacins of Aquilaria agallocha [44], anthocyanins of Perilla frutescens [45], and eleutherosides of Eleutherococcus senticosus [46].

Root

a

0.50

a

0.40 0.30 0.20

a

b

b

White

Blue

Red

0.10 0.00

Dark

White

Blue

14 days

Red

Dark

28 days

Light sources

Fig. 2. Plumbagin accumulation in D. burmannii culture grown under artificial lighting conditions for 14 and 28 days. The letters indicate a significant difference between treatment groups within a plant part at P-value < .05. 18.00

Aerial

Plumbagin (mg·g-1DW)

14.00

Root

a

16.00

a

a

12.00

a

10.00

a b

8.00

b

b

6.00

4.00

a 2.00

a

b

c

a

b

b

b

Blue

Red

Dark

Table 1 Plumbagin levels in D. burmannii and D. indica under normal culture conditions and florescent lighting.

0.00

White

Blue

Red

Dark

White

14 days

28 days

Plumbagin (mg g-1 DW)

Light sources

Fig. 3. Plumbagin accumulation in D. indica culture grown under artificial lighting conditions for 14 and 28 days. The letters indicate a significant difference between treatment groups within a plant part at P-value < .05.

D. burmannii D. indica

3

Aerial

Root

0.184 ± 0.020 7.718 ± 0.553

0.012 ± 0.001 0.131 ± 0.029

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Fig. 4. In vitro cultures of D. burmannii (A, B, and C) and D. indica (D, E, and F) after the addition of precursors at 9 days. A and D: control, B and E: 100 mg·L−1 sodium acetate, C and F: 5.0 mM L-alanine bar = 1 cm.

The exact mechanisms behind light and secondary metabolite production are still unclear. However, Li et al. [34] reported that blue light positively regulated genes involved in grape plant growth, while culturing under red and green light resulted in gene expression similar to a shade-avoidance syndrome response. In addition, OuYang et al. [33] reported that blue light upregulated genes involved in secondary metabolism and that red light affected the regulation of gibberellic acid in Norway spruce. In the case of plumbagin, therefore, the gene involved in the biosynthesis of the acetate-mevalonate pathway leading to the production of plumbagin [24] may be upregulated after exposure to blue and red LED light.

plumbagin in the D. burmannii aerial part were approximately 10-fold higher than those in the root part (Fig. 5A and B). The levels of plumbagin ranged from 0.161 ± 0.008 to 0.288 ± 0.025 mg g−1 DW in the aerial part and from 0.010 ± 0.001 to 0.030 ± 0.002 mg g−1 DW in the root part. Three days after the addition of precursors, the levels of plumbagin in both the aerial and root parts of D. burmannii were increased when compared with the control group of the same plant parts (Fig. 5A). In addition, significant differences were observed in the aerial part that was treated with 10 mg·L−1 sodium acetate and in the root part treated with 50 and 100 mg·L−1 sodium acetate (Fig. 5A). However, higher concentrations of 50 and 100 mg·L−1 sodium acetate did not enhance the production of plumbagin in aerial parts. In contrast, L-alanine showed no effect on the plumbagin level of the D. burmannii aerial part at 3 days after addition, but significant enhancement was observed in the root part of plants treated with 1.0 and 5.0 mM L-alanine. The

3.2. Effects of Sodium Acetate And L-alanine On Plumbagin Accumulation After addition of precursor for 9 days, there was no different in the appearance of D. burmanii and D. indica (Fig. 4). Overall, the levels of Control

Act 10

Act 50

Act 100

Control

A

Ala 0.5

Ala 1.0

Ala 5.0

B

0.35

0.35

* 0.30

Plumbagin (mg·g-1DW)

Plumbagin (mg·g-1DW)

0.30 0.25 0.20 0.15 0.10 0.05

0.25 0.20 0.15 0.10 0.05

* *

0.00

*

*

0.00

Aerial

Root Day 3

Aerial

Root Day 6

Aerial

Aerial

Root

Root Day 3

Day 9

Aerial

Root Day 6

Aerial

Root Day 9

Fig. 5. Plumbagin accumulation in D. burmannii cultures after feeding with different concentrations of precursors for 3, 6, and 9 days. A) Sodium acetate; control: no precursor, Act 10, 50, and 100: sodium acetate 10, 50, and 100 mg L−1. B) L-alanine; control: no precursor, Ala 0.5, 1.0 and 5.0: L-alanine 0.5, 1.0, and 5.0 mM. *indicates a significant difference compared to the control group at the P-value < .05. 4

Journal of Photochemistry & Photobiology, B: Biology 199 (2019) 111628

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Control

Act 10

Act 50

Act 100

Control

A

*

Ala 1.0

B

Ala 5.0

*

10.00 Plumbagin (mg·g-1DW)

10.00 Plumbagin (mg·g-1DW)

Ala 0.5

12.00

12.00

8.00 6.00 4.00

*

2.00

**

**

8.00 6.00 4.00 2.00

0.00

0.00

Aerial

Root Day 3

Aerial

Root Day 6

Aerial

Aerial

Root

Root Day 3

Day 9

Aerial

Root Day 6

Aerial

Root Day 9

Fig. 6. Plumbagin accumulation in D. indica culture after feeding with different concentrations of precursors for 3, 6, and 9 days. A) Sodium acetate; control: no precursor, Act 10, 50, and 100: sodium acetate 10, 50, and 100 mg L−1. B) L-alanine; control: no precursor, Ala 0.5, 1.0 and 5.0: L-alanine 0.5, 1.0, and 5.0 mM. *indicates a significant difference compared to the control group at the P-value < .05.

highest level of plumbagin in D. burmannii cultures (0.288 ± 0.025 mg g−1 DW) was observed in the aerial part treated with the 10 mg L−1 sodium acetate solution for 3 days. Nonetheless, plumbagin completely disappeared from the samples after treatment with both precursors for 6 and 9 days, suggesting an optimum treatment time of 3 days. Similar to D. burmannii, the aerial part of D. indica accumulated approximately 5-fold more plumbagin than the root part (Fig. 6A and B). However, the levels of plumbagin in D. indica were 30-fold higher than those in D. burmannii. The values ranged from 7.051 ± 0.144 to 9.850 ± 0.250 mg g−1 DW in the aerial part of D. burmannii and 0.407 ± 0.010 to 1.784 ± 0.058 mg g−1 DW in the root part. In addition, plumbagin was detected in D. indica cultures at 3, 6, and 9 days. Significant enhancement of plumbagin levels was only detected in the aerial part treated with 50 mg·L−1 sodium acetate and 5 mM Lalanine at day 3 (Fig. 6). In addition, enhanced plumbagin accumulations were observed in the roots of D. indica on days 3 and 9 of the sodium acetate-treated groups, although the levels were much lower than those in the aerial part (Fig. 6A). The highest level (9.850 ± 0.250 mg g−1 DW) was observed in the aerial part of D. indica treated with 50 mg L−1 sodium acetate for 3 days (Fig. 6A). Prolonged culture for 6 and 9 days showed a decline in plumbagin level. Therefore, the optimum time for harvesting after precursor treatment was 3 days. The harvesting time after precursor treatment could have an impact on plant secondary metabolite production [23], as shown in a previous study on P. indica [12]. Our results also confirmed that the optimum time for harvesting after precursor treatments in Drosera cultures is critical to achieve maximum plumbagin levels. The results from our study indicate the ability to induce a higher accumulation of plumbagin by the addition of both precursors, although at different concentrations. This finding is supported by previous studies showing that both acetate and L-alanine are precursors for the production of plumbagin [24–26]. Moreover, L-alanine was reported to enhance the plumbagin level of P. indica root culture [12]. In every experiment, the results indicate that plumbagin levels differ between the aerial and root parts. In addition, when the level of both aerial and root parts was combined, the highest level of total plumbagin was observed in D. indica cultured under blue LED light (aerial and root parts combined, 16.431 mg·g−1 DW; Fig. 3). This level is 1.8-fold higher than the yeast extract-elicited cultures of D. burmannii seen in previous reports for both D. indica [17] and D. burmannii [18]. Compared with other plants, the levels are higher than in Plumbago zeylanica callus elicited with yeast extract [14] and Plumbago rosea cell suspension cultures [13] but are comparable with those in P. indica hairy root cultures [16] and P. indica root cultures fed with L-alanine [12]. On the other hand, D. burmannii plumbagin levels in our study were up to 700-

fold lower than the D. indica plumbagin levels, and the levels were lower than those reported in a previous study [18]. This might be the result of the variation in the long-term subculture plants and the difference in plumbagin quantification methods. With regard to time, plumbagin production was higher in the earlier culture period and declined in the later phase (Figs. 2, 3, 5 and 6), which may be due to conversion to other derivative forms [5]. However, the shorter culture time of this system could reduce the production cost. In addition, this is the first report on the effects of artificial lighting on the production of plumbagin in plant in vitro culture. 4. Conclusion In this study, we reported an alternative method for the enhanced production of plumbagin using artificial LED light, particularly blue LED light, which represented a fast and simple plumbagin production system. In addition, two precursors, acetate and L-alanine, can be used for enhancing plumbagin production of D. burmannii and D. indica when harvested within 3 days after addition. This provided another application for further study. Acknowledgment This work was supported by funding from The Thailand Research Fund (IRN61W0005) and financial support from a scholarship under the Postdoctoral Training Program from the Research Affairs and Graduate School, Khon Kaen University, Thailand (Grant no. 58439-2). References [1] F. Rivadavia, K. Kondo, M. Kato, M. Hasebe, Phylogeny of the sundews, Drosera (Droseraceae), based on chloroplast rbcL and nuclear 18S ribosomal DNA sequences, Am. J. Bot. 90 (2003) 123–130. [2] S.P. Devi, S. Kumaria, S.R. Rao, P. Tandon, Carnivorous plants as a source of potent bioactive compound: naphthoquinones, Trop. Plant Biol. 9 (2016) 267–279. [3] P.A. Egan, F. van der Kooy, Phytochemistry of the carnivorous sundew genus Drosera (droseraceae) – future perspectives and ethnopharmacological relevance, Chem. Biodivers. 10 (2013) 1774–1790. [4] R. Caniato, R. Filippini, E.M. Cappelletti, Naphthoquinone contents of cultivated Drosera species Drosera binata, D. binata var. dichotoma, and D. capensis, Int. J. Crude. Drug. Res. 27 (1989) 129–136. [5] P. Babula, V. Adam, L. Havel, R. Kizek, Noteworthy secondary metabolites naphthoquinones - their occurrence, pharmacological properties and analysis, Curr. Pharm. Anal. 5 (2009) 47–68. [6] S. Padhye, P. Dandawate, M. Yusufi, A. Ahmad, F.H. Sarkar, Perspectives on medicinal properties of plumbagin and its analogs, Med. Res. Rev. 32 (2012) 1131–1158. [7] Y. Liu, Y. Cai, C. He, M. Chen, H. Li, Anticancer properties and pharmaceutical applications of plumbagin: a review, American J.Chinese. Med. 45 (2017) 423–441. [8] W. Sumsakul, T. Plengsuriyakarn, W. Chaijaroenkul, V. Viyanant, J. Karbwang, K. Na-Bangchang, Antimalarial activity of plumbagin in vitro and in animal models,

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[29] S. Dutta Gupta, B. Jatothu, Fundamentals and applications of light-emitting diodes (LEDs) in in vitro plant growth and morphogenesis, Plant. Biotechnol. Rep. 7 (2013) 211–220. [30] K. Kitazaki, A. Fukushima, R. Nakabayashi, Y. Okazaki, M. Kobayashi, T. Mori, T. Nishizawa, S. Reyes-Chin-Wo, R.W. Michelmore, K. Saito, K. Shoji, M. Kusano, Metabolic reprogramming in leaf lettuce grown under different light quality and intensity conditions using narrow-band LEDs, Sci. Rep. 8 (2018) 7914. [31] F. Bantis, T. Ouzounis, K. Radoglou, Artificial LED lighting enhances growth characteristics and total phenolic content of Ocimum basilicum, but variably affects transplant success, Sci. Hortic. 198 (2016) 277–283. [32] B. Kawka, I. Kwiecień, H. Ekiert, Influence of culture medium composition and light conditions on the accumulation of bioactive compounds in shoot cultures of Scutellaria lateriflora L. (american skullcap) grown in vitro, Appl. Biochem. Biotechnol. 183 (2017) 1414–1425. [33] F. OuYang, J.-F. Mao, J. Wang, S. Zhang, Y. Li, Transcriptome analysis reveals that red and blue light regulate growth and phytohormone metabolism in Norway spruce [Picea abies (L.) karst.], PLoS One 10 (2015) e0127896. [34] C.-X. Li, Z.-G. Xu, R.-Q. Dong, S.-X. Chang, L.-Z. Wang, M. Khalil-Ur-Rehman, J.M. Tao, An RNA-seq analysis of grape plantlets grown in vitro reveals different responses to blue, green, red led light, and white fluorescent light, Front. Plant Sci. 8 (2017). [35] T. Murashige, F. Skoog, A revised medium for rapid growth and bio assays with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473–497. [36] S. Sakamoto, W. Putalun, R. Tsuchihashi, S. Morimoto, J. Kinjo, H. Tanaka, Development of an enzyme-linked immunosorbent assay (ELISA) using highlyspecific monoclonal antibodies against plumbagin, Anal. Chim. Acta 607 (2008) 100–105. [37] W. Wang, M. Su, H. Li, B. Zeng, Q. Chang, Z. Lai, Effects of supplemental lighting with different light qualities on growth and secondary metabolite content of Anoectochilus roxburghii, PeerJ 6 (2018) e5274. [38] S. Dutta Gupta, A. Karmakar, Machine vision based evaluation of impact of light emitting diodes (LEDs) on shoot regeneration and the effect of spectral quality on phenolic content and antioxidant capacity in Swertia chirata, J. Photochem. Photobiol. B Biol. 174 (2017) 162–172. [39] B.R. Jeong, I. Sivanesan, Direct adventitious shoot regeneration, in vitro flowering, fruiting, secondary metabolite content and antioxidant activity of Scrophularia takesimensis Nakai, Plant Cell, Tissue and Organ Culture (PCTOC) 123 (2015) 607–618. [40] E.S. Jung, S. Lee, S.-H. Lim, S.-H. Ha, K.-H. Liu, C.H. Lee, Metabolite profiling of the short-term responses of rice leaves (Oryza sativa cv. Ilmi) cultivated under different LED lights and its correlations with antioxidant activities, Plant Sci. 210 (2013) 61–69. [41] A. Manivannan, P. Soundararajan, N. Halimah, C.H. Ko, B.R. Jeong, Blue LED light enhances growth, phytochemical contents, and antioxidant enzyme activities of Rehmannia glutinosa cultured in vitro, Hortic. Environ. Biotechnol. 56 (2015) 105–113. [42] S.T. Silva, S.K.V. Bertolucci, S.H.B. da Cunha, L.E.S. Lazzarini, M.C. Tavares, J.E.B.P. Pinto, Effect of light and natural ventilation systems on the growth parameters and carvacrol content in the in vitro cultures of Plectranthus amboinicus (Lour.) Spreng, Plant Cell, Tissue and Organ Culture (PCTOC) 129 (2017) 501–510. [43] I.C.A. Alvarenga, F.V. Pacheco, S.T. Silva, S.K.V. Bertolucci, J.E.B.P. Pinto, In vitro culture of Achillea millefolium L.: quality and intensity of light on growth and production of volatiles, Plant Cell, Tissue and Organ Culture (PCTOC) 122 (2015) 299–308. [44] T.C.-Y. Kuo, C.-H. Chen, S.-H. Chen, I.H. Lu, M.-J. Chu, L.-C. Huang, C.-Y. Lin, C.Y. Chen, H.-F. Lo, S.-T. Jeng, L.-F.O. Chen, The effect of red light and far-red light conditions on secondary metabolism in agarwood, BMC Plant Biol. 15 (2015) 139. [45] Y.G. Park, H.J. Oh, B.R. Jeong, Growth and anthocyanin concentration of Perilla frutescens var. acuta kudo as affected by light source and DIF under controlled environment, Hortic. Environ. Biotechnol. 54 (2013) 103–108. [46] A.M. Shohael, M.B. Ali, K.W. Yu, E.J. Hahn, R. Islam, K.Y. Paek, Effect of light on oxidative stress, secondary metabolites and induction of antioxidant enzymes in Eleutherococcus senticosus somatic embryos in bioreactor, Process Biochem. 41 (2006) 1179–1185.

BMC Complement. Altern. Med. 14 (2014) 15. [9] P. Abedinpour, V.T. Baron, A. Chrastina, G. Rondeau, J. Pelayo, J. Welsh, P. Borgström, Plumbagin improves the efficacy of androgen deprivation therapy in prostate cancer: a pre-clinical study, Prostate 77 (2017) 1550–1562. [10] F. Bourgaud, A. Gravot, S. Milesi, E. Gontier, Production of plant secondary metabolites: a historical perspective, Plant Sci. 161 (2001) 839–851. [11] I. Smetanska, Production of secondary metabolites using plant cell cultures, in: U. Stahl, U.E.B. Donalies, E. Nevoigt (Eds.), Food Biotechnol, Springer Berlin Heidelberg, Berlin, Heidelberg, 2008, pp. 187–228. [12] A. Jaisi, P. Panichayupakaranant, Enhanced plumbagin production in Plumbago indica root cultures by ʟ-alanine feeding and in situ adsorption, Plant Cell Tissue Organ Cult. 129 (2017) 53–60. [13] P.K. Silja, G.P. Gisha, K. Satheeshkumar, Enhanced plumbagin accumulation in embryogenic cell suspension cultures of Plumbago rosea L. following elicitation, Plant Cell Tissue Organ Cult. 119 (2014) 469–477. [14] U. Sharma, V. Agrawal, In vitro shoot regeneration and enhanced synthesis of plumbagin in root callus of Plumbago zeylanica L.—an important medicinal herb, In Vitro Cell. Dev. Biol. Plant 54 (2018) 423–435. [15] P. Nayak, M. Sharma, S.N. Behera, M. Thirunavoukkarasu, P.K. Chand, High-performance liquid chromatographic quantification of plumbagin from transformed rhizoclones of Plumbago zeylanica L.: inter-clonal variation in biomass growth and plumbagin production, Appl. Biochem. Biotechnol. 175 (2015) 1745–1770. [16] M. Gangopadhyay, S. Dewanjee, S. Bhattacharya, Enhanced plumbagin production in elicited Plumbago indica hairy root cultures, J. Biosci. Bioeng. 111 (2011) 706–710. [17] J. Thaweesak, S. Seiichi, T. Hiroyuki, P. Waraporn, Elicitation effect on production of plumbagin in in vitro culture of Drosera indica L, J.Med.Plants. Res. 5 (2011) 4949–4953. [18] W. Putalun, O. Udomsin, G. Yusakul, T. Juengwatanatrakul, S. Sakamoto, H. Tanaka, Enhanced plumbagin production from in vitro cultures of Drosera burmanii using elicitation, Biotechnol. Lett. 32 (2010) 721–724. [19] I.J. Crouch, J.F. Finnie, J. van Staden, Studies on the isolation of plumbagin from in vitro and in vivo grown Drosera species, Plant Cell Tissue Organ Cult. 21 (1990) 79–82. [20] A. Krolicka, A. Szpitter, E. Gilgenast, G. Romanik, M. Kaminski, E. Lojkowska, Stimulation of antibacterial naphthoquinones and flavonoids accumulation in carnivorous plants grown in vitro by addition of elicitors, Enzym. Microb. Technol. 42 (2008) 216–221. [21] K. Jayaram, M. Prasad, Drosera indica L. and D. burmanii Vahl., medicinally important insectivorous plants in Andhra Pradesh-regional threats and conservation, Curr. Sci. 91 (2006) 943–946. [22] T. Isah, S. Umar, A. Mujib, M.P. Sharma, P.E. Rajasekharan, N. Zafar, A. Frukh, Secondary metabolism of pharmaceuticals in the plant in vitro cultures: strategies, approaches, and limitations to achieving higher yield, Plant Cell Tissue Organ Cult. 132 (2018) 239–265. [23] H.N. Murthy, E.-J. Lee, K.-Y. Paek, Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation, Plant Cell Tissue Organ Cult. 118 (2014) 1–16. [24] R. Durand, M.H. Zenk, Biosynthesis of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) via the acetate pathway in higher plants, Tetrahedron Lett. 12 (1971) 3009–3012. [25] K. Springob, S. Samappito, A. Jindaprasert, J. Schmidt, J.E. Page, W. De-Eknamkul, T.M. Kutchan, A polyketide synthase of Plumbago indica that catalyzes the formation of hexaketide pyrones, FEBS J. 274 (2007) 406–417. [26] H. Rischer, A. Hamm, G. Bringmann, Nepenthes insignis uses a C2-portion of the carbon skeleton of l-alanine acquired via its carnivorous organs, to build up the allelochemical plumbagin, Phytochemistry 59 (2002) 603–609. [27] D.S. Batista, S.H.S. Felipe, T.D. Silva, K.M. de Castro, T.C. Mamedes-Rodrigues, N.A. Miranda, A.M. Ríos-Ríos, D.V. Faria, E.A. Fortini, K. Chagas, G. Torres-Silva, A. Xavier, A.D. Arencibia, W.C. Otoni, Light quality in plant tissue culture: does it matter? In Vitro Cell. Dev. Biol. Plant 54 (2018) 195–215. [28] J.J. Bello-Bello, J.A. Perez-Sato, C.A. Cruz-Cruz, E. Martinez-Estrada, Light-emitting diodes: Progress in plant micropropagation, in: E. Jacob-Lopes (Ed.), Chlorophyll, InTech, 2017, pp. 93–103.

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