- Email: [email protected]
Concentrations of perillaldehyde, limonene, and anthocyanin of Perilla plants as affected by light quality under controlled environments
Scientia Horticulturae 122 (2009) 134–137
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Short communication
Concentrations of perillaldehyde, limonene, and anthocyanin of Perilla plants as affected by light quality under controlled environments Tetsuro Nishimura a, Katsumi Ohyama b,*, Eiji Goto a, Nobuyuki Inagaki c a
Graduate School of Horticulture, Chiba University, Matsudo, Chiba 271-8510, Japan Centre for Environment, Health and Field Sciences, Chiba University, 6-2-1, Kashiwanoha, Kashiwa, Chiba 277-0882, Japan c Botanical Raw Materials Research Dept., Tsumura & Co., Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 October 2008 Received in revised form 5 March 2009 Accepted 9 March 2009
Perilla plants with red coloured leaves grown in red-enriched light treatments (i.e., red lamps alone (R), a mixture of blue and red lamps (BR), and a mixture of green and red lamps (GR)) had greater dry weight, bigger leaves, and more leaves than those grown in other treatments (i.e., blue lamps alone (B), a mixture of blue and green lamps (BG), and green lamps alone (G)). Although the concentrations of perillaldehyde and limonene in leaf tissues (mg g 1 leaf DW) were 1.6–1.9 and 1.5–1.9 times higher, respectively, in the G treatment than in red-enriched light treatments, the contents of perillaldehyde and limonene per plant (mg/plant) were 1.8 times higher in the BR treatment than those in the G treatment. The content of anthocyanin per plant was also 4.3 times higher in the BR treatment than that in the G treatment. Therefore, within the six different combinations of fluorescent lamps used in the present study, the BR treatment was a suitable light quality for producing plants with high contents of perillaldehyde, limonene, and anthocyanin under controlled environments. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Essential oil Fluorescent lamp Medicinal plant Perilla frutescens
1. Introduction Perilla frutescens (L.) Britt. belonging to the family Lamiaceae (Labiatae) is native to mountainous areas of China and India and is grown mainly in Asia (Yu, 1997). The Perilla plants were classified into seven chemotypes based on the main components of essential oil (Ito, 1970; Koezuka et al., 1986; Ito et al., 1999a,b, 2002), viz. perillaldehyde (PA-type), perilla-ketone (PK-type), elsholtziaketone (EK-type), citral (C-type), phenylpropanoid (PP-type), perillene (PL-type), and piperitenone (PT-type). Perilla frutescens with red coloured leaves is a PA-type plant species. The plants cultivated in Japan contain approximately 0.5% essential oil in fresh leaves, mainly consisting of 55% perillaldehyde and 20–30% limonene (Omer et al., 1998). Perillaldehyde is thought to have a depressive effect on the central nervous system (Sugaya et al., 1981) and antibacterial activity (Sato et al., 2006). Hence, leaves of the plants are used in traditional, Japanese herbal medicine (Kampo medicine). The quality of Perilla plants used for Kampo medicine is determined by the concentrations of essential oil components in leaves and
* Corresponding author. Tel.: +81 4 7137 8000; fax: +81 4 7137 8008. E-mail address: [email protected] (K. Ohyama). 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.03.010
the appearance such as redness of leaves attributed to anthocyanin (Nagao et al., 1974), so a method for increasing the concentrations of perillaldehyde, limonene and anthocyanin in the leaves while simultaneously promoting growth is essential for efficient production of the plants. Recently, Kampo medicine has become popular worldwide as an alternative therapy (e.g., Matsumoto et al., 1999; Kanda et al., 2005; Oka, 2006). As a result, the demand for the aerial parts of Perilla plants as a raw material of Kampo medicine has been increasing, and thus a stable supply of the plants to meet the projected increase in demand will be required in the near future. In the natural environment, there are a number of concerns associated with the use of medicinal plants, including biotic or abiotic contamination, adulteration of plant species and weeds, and quality variation of the medicinal plant products themselves (Zobayed et al., 2005). However, these problems can be solved by growing medicinal plants under controlled environments with artificial light. It was reported that red light increased the content of menthol, which is the main component of the essential oil in Mentha arvensis (Nishioka et al., 2008) and ultraviolet-B radiation suppressed anthocyanin production of Perilla plants (Nishimura et al., 2008) under controlled environments. It was also reported that light quality had effects on plant growth and development (Smith, 1982;
T. Nishimura et al. / Scientia Horticulturae 122 (2009) 134–137
McNellis and Deng, 1995; Goto, 2003). Light quality might alter the growth and concentrations of essential oil components and anthocyanin of red Perilla plants, but little information is available. The objective of our study was to evaluate the effects of light quality provided by coloured fluorescent lamps on the growth and concentrations of essential oil components and anthocyanin of Perilla plants in order to determine efficient production conditions under controlled environments.
135
differences in the reversible action of phytochrome among the plants grown in the six different treatments were small in this study. In the growth chamber, environmental conditions except for light quality were the same as those during the seedling production period. Since the plants were larger than the seedlings, the planting density was adjusted to 37 plants m 2. 2.3. Measurements Since the underground part of the plant is discarded in the commercial production of Perilla plants used for crude drug, in the present experiment only the aerial part of the plant was measured.
2. Materials and methods 2.1. Plant materials and growth conditions Seeds of Perilla (Perilla frutescens (L.) Britt. var. acuta Kudo f. purpurea Makino, cv. Sekiho) were sown in rock wool cubes. On day 21 after sowing, the seedlings were transplanted to individual pots (diameter: 9.0 cm; capacity: 330 ml), filled with a commercial soil mixture of vermiculite (50%) and peat moss (50%). The seedlings were grown in a growth chamber for 7 days at a photoperiod of 16 h d 1 provided by cool-white fluorescent lamps (FHF32-EX-N-H, Matsushita Electric Industrial Co. Ltd., Osaka, Japan). All seedlings were placed under a photosynthetic photon flux (PPF) of 200 mmol m 2 s 1 on the soil surface, an air temperature of 27/22 8C (photo-/dark periods), a relative humidity of 60/90% (photo-/dark periods), and a CO2 concentration of 1000 mmol mol 1. The planting density was 100 seedlings m 2. After germination, subirrigation was made once a day with a nutrient solution (Otsuka hydroponic composition, Otsuka Chemical Co. Ltd., Osaka, Japan). The nutrient solution, adjusted to electrical conductivity (EC) 1.2 dS/m and pH 6.0, contained 4.2 mmol l 1 NO3 , 1.3 mmol l 1 H2PO4 , 1.0 mmol l 1 Ca2+, 0.38 mmol l 1 Mg2+, 2.2 mmol l 1 K+, and 0.4 mmol l 1 NH4+. 2.2. Treatments The 28-day-old seedlings with six true leaves (dry weight: 38 mg, stem length: 1.6 cm, hereafter ‘‘plants’’) were subjected to six light quality treatments for 21 days. All plants were placed at the same PPF on the canopy surface of 200 mmol m 2 s 1 in different growth chambers. The lighting system of each treatment was provided by different coloured fluorescent lamps, including the following combinations of coloured fluorescent lamps: blue lamps (FLR40SEB/M, Matsushita Electric Works Ltd.) alone (B), green lamps (FLR40S-EG/M) alone (G), red lamps (FLR40S-ER/M) alone (R), a mixture of blue and green lamps (BG), a mixture of blue and red lamps (BR), or a mixture of green and red lamps (GR) (Table 1). Although the red/far-red photon flux ratio varied from 4.4 to 25.4 among the treatments in this study, the calculated values of phytochrome photoequilibrium using an equation proposed by Hanyu et al. (1996) was 0.8 in all treatments. Therefore, the
2.3.1. Growth On day 21, dry weights of the leaves and stems were measured with an electronic balance after drying in an oven (temperature 80 8C, 72 h), leaf area was measured with a leaf area meter (LI3100-C, LI-COR Inc., Lincoln, USA), and the number of true leaves of each plant was determined. 2.3.2. Perillaldehyde and limonene concentrations On day 21, the true leaf on the third or fourth node counted from the shoot apex of the main stem of the plants was sampled. Then, the analysis of perillaldehyde and limonene of the sampled leaves was conducted, as described previously (Nishimura et al., 2008). The concentrations of perillaldehyde and limonene in the sampled leaves were expressed as milligram per unit leaf dry weight (mg g 1 leaf DW). Contents of perillaldehyde and limonene per plant were estimated by multiplying these concentrations by the dry weight of all leaves per plant, and were expressed as milligram per plant (mg/plant). 2.3.3. Anthocyanin concentration Three leaf disks (2.4 cm2 total leaf disk area) were sampled from the true leaf on the third or fourth node counted from the shoot apex of the main stem of the plants. Analysis of anthocyanin of sampled leaves was conducted, as described previously (Nishimura et al., 2008). The concentrations of anthocyanin in the sampled leaves were expressed as milligram per unit leaf dry weight (mg g 1 leaf DW). Contents of anthocyanin per plant were estimated by multiplying these concentrations by the dry weight of all leaves per plant, and were expressed as milligram per plant (mg/plant). 2.4. Statistical analysis A completely randomized design with 9 seedlings per treatment and 2 replications was employed in the experiment. Six of these seedlings were used for the measurements of dry weight, leaf area, and number of true leaves, and all seedlings were used for the measurements of concentrations of perillaldehyde, limonene, and
Table 1 Spectral characteristics of each light treatment. 2
1 b
Treatment codea
Photon flux (mmol m 300–400 nm
400–500 nm
500–600 nm
600–700 nm
700–800 nm
B BG G BR GR R
1.1 1.9 2.5 1.0 1.6 1.0
153.0 95.8 29.4 68.4 20.0 14.2
36.3 88.0 148.9 37.6 79.8 39.1
10.7 16.2 21.7 94.0 100.2 146.7
2.5 1.9 0.9 11.6 11.3 17.5
s
)
a B, blue lamps alone; BG, a mixture of blue and green lamps; G, green lamps alone; BR, a mixture of blue and red lamps; GR, a mixture of green and red lamps; R, red lamps alone. b The photon fluxes of each wavelength band were measured using a portable spectroradiometer (LI-1800, LI-COR Inc., Lincoln, USA).
T. Nishimura et al. / Scientia Horticulturae 122 (2009) 134–137
136
Table 2 Growth of Perilla plants on day 21 (49 days after sowing) grown under different light qualities. Treatment codea
Dry weight (g/plant) Leaf
B BG G BR GR R ANOVAc
0.98 1.07 0.62 1.74 1.57 1.66 **
Leaf area (cm2)
Number of true leaves
524.7 567.6 391.8 814.8 796.2 837.9 **
32.9 30.6 21.5 42.8 39.5 38.6 **
Stem bb b c a a a
0.15 0.14 0.07 0.28 0.26 0.30 **
b b c a a a
b b c a a a
c c d a ab b
a
For treatment codes and detailed descriptions, see Table 1. Each value represents the mean of 12 replicates. Means within a column followed by different letters are significantly different at P < 0.05 determined by the Tukey–Kramer test. c **Significance at P < 0.01. b
Table 3 Concentrations of perillaldehyde, limonene, and anthocyanin of red-leafed Perilla plants on day 21 (49 days after sowing) grown under different light qualities. Treatment codea B BG G BR GR R
Perillaldehyde concentration (mg/g leaf DW) 4.47 3.76 4.90 3.09 2.89 2.64
b
a ab a b b b
Limonene concentration (mg/g leaf DW)
Anthocyanin concentration (mg/g leaf DW)
0.53 0.50 0.58 0.38 0.33 0.31
2.41 2.39 2.00 3.07 3.25 3.42
ab abc a bcd cd d
b b b a a a
a
For treatment codes and detailed descriptions, see Table 1. Each value represents the mean of 18 replicates. Different letters in each column indicate significant differences between the treatments at P < 0.05, determined by the Tukey–Kramer test. b
anthocyanin. The data were quantified in terms of the means and standard errors obtained from replicate treatments, and were subjected to an analysis of variance (ANOVA). The means were compared among the treatments using the Tukey–Kramer test at a 5% level of significance. 3. Results and discussion The leaf and stem dry weights of the plants were 1.5–2.8 and 1.8–4.2 times greater, respectively, in the red-enriched light treatments (i.e., BR, GR, and R treatments) than those in the other treatments (B, BG, and G treatments) (Table 2). The number of true leaves and leaf area of the plants were 1.2–2.0 and 1.4–2.1 times greater, respectively, in the red-enriched light treatments than those in the other treatments. The greater leaf area and number of true leaves increase the amount of absorbed light (photons). Therefore, the net photosynthesis rates of the plants may increase in the red-enriched light treatments compared with those grown in other treatments. Hence, the growth of the plants in the redenriched light treatments was promoted compared with those in the other treatments. The results obtained in the present experiment were in agreement with those of previous studies on the growth of other plant species grown under red-enriched light (Warrington and Mitchell, 1976; Mortensen and Strømme, 1987; Inada and Yabumoto, 1989; Nishimura et al., 2006, 2007). The concentrations of perillaldehyde and limonene in leaf tissues of the Perilla plants were 1.6–1.9 and 1.5–1.9 times higher, respectively, in the G treatment than those in the red-enriched light treatments, but similar to those in the B and BG treatments (Table 3). Yoshida et al. (1969) reported that the concentrations of essential oil in leaf tissues of Perilla plants with green coloured leaves increased with increasing number of peltate glandular trichomes per unit leaf area. The peltate glandular trichomes, which develop from leaf epidermal cells, are the main sites for the biosynthesis and accumulation of essential oil (Yoshida et al., 1968; Nishizawa et al., 1992). Thus, we speculated that the number of peltate glandular trichomes per unit area might have been greater in the G treatment.
The anthocyanin concentration in leaf tissues of the Perilla plants was 1.3–1.7 times higher in the red-enriched light treatments than that in the other treatments. The main anthocyanin of the plants is a cyanidin-type compound called malonylshisonin (Saito and Yamazaki, 2002). The malonylshisonin and related compounds, which are accumulated in the epidermal cells in leaves and stems of the plants, are biosynthesized from primary photosynthate or carbohydrates. According to the overflow metabolism concept, when carbohydrate production exceeds the carbon demand associated with plant growth, the excess carbohydrates are channelled into biosyntheses of secondary metabolites (Matsuki, 1996). In this study, the increase in total carbon fixation due to the increase in total leaf area could cause not only the photosynthesis but also the secondary metabolites of the plant. Although the concentrations of perillaldehyde and limonene in leaf tissues were significantly higher in the G treatment than in red-enriched light treatments, the contents of perillaldehyde and limonene per plant were 1.8 times higher in the BR treatment than in the G treatment. The content of anthocyanin per plant was 4.3 times higher in the BR treatment than those in the G treatment. Hence, within the 6 different combinations of fluorescent lamps used in the present study, the BR treatment was a suitable light quality for producing plants with high contents of perillaldehyde, limonene, and anthocyanin. Acknowledgements The authors are grateful to Prof. Dr. Toyoki Kozai at the Centre for Environment, Health and Field Sciences, Chiba University for valuable discussions. We thank Mr. Hideo Yoshida, Ms. Sara Hassani Malayeri, and Ms. Naoko Nishioka for technical assistance. References Goto, E., 2003. Effects of light quality on growth of crop plants under artificial lighting. Environ. Control Biol. 41, 121–132.
T. Nishimura et al. / Scientia Horticulturae 122 (2009) 134–137 Hanyu, H., Shoji, K., Ji, S., 1996. Evaluation of light quality variation through supplement of far-red light and the difference in the effects on growth of a pole-type and a bush-type kidney bean, Phaseolus vulgaris L. Environ. Control Biol. 34, 267–275 (in Japanese with English abstract). Inada, K., Yabumoto, Y., 1989. Effects of light quality, daylength and periodic temperature variation on the growth of lettuce and radish plants. Jpn. J. Crop. Sci. 58, 689–694. Ito, H., 1970. Studies on Folium perillae. VI. Constituent of essential oils and evaluation of Genus Perilla. Yakugaku zasshi 90, 883–892 (in Japanese with English abstract). Ito, M., Toyoda, M., Honda, G., 1999a. Chemical composition of the essential oil of Perilla frutescens. Nat. Med. 53, 32–36. Ito, M., Toyoda, M., Honda, G., 1999b. Essential oil composition of hybrids and amphidiploids of Japanese wild Perilla. Nat. Med. 53, 118–122. Ito, M., Toyoda, M., Kamakura, S., Honda, G., 2002. A new type of essential oil from Perilla frutescens from Thailand. J. Essent. Oil Res. 14, 416–419. Kanda, T., Yamakawa, J., Moriya, J., Saegusa, S., Kawaura, K., Yu, F., Kusaka, K., Takahashi, T., Itoh, T., 2005. Globalization of Kampo medicine. J. Kanazawa Med. Univ. 30, 297–301. Koezuka, Y., Honda, G., Tabata, M., 1986. Genetic control of the chemical composition of volatile oils in Perilla frutescens. Phytochemistry 25, 859–863. Matsuki, M., 1996. Regulation of plant phenolic synthesis: from biochemistry to ecology and evolution. Aust. J. Bot. 44, 613–634. Matsumoto, M., Inoue, K., Kajii, E., 1999. Integrating traditional medicine in Japan: the case of Kampo medicines. Comp. Ther. Med. 7, 254–255. McNellis, T.W., Deng, X.W., 1995. Light control of seedling morphogenetic pattern. Plant Cell 7, 1749–1761. Mortensen, L.M., Strømme, E., 1987. Effects of light quality on some greenhouse crops. Sci. Hortic. 33, 27–36. Nagao, Y., Komiya, T., Fujioka, S., Matsuoka, T., 1974. Studies on the quality of the Chinese drug ‘‘Soyo’’ and the cultivation of the original plant 1. J. Takeda Res. Lab. 33, 111–118 (in Japanese with English abstract). Nishimura, T., Zobayed, S.M.A., Kozai, T., Goto, E., 2006. Effect of light quality of blue and red fluorescent lamps on growth of St. John’s wort (Hypericum perforatum L.). J. SHITA 18, 225–229 (in Japanese with English abstract). Nishimura, T., Zobayed, S.M.A., Kozai, T., Goto, E., 2007. Medicinally important secondary metabolites and growth of Hypericum perforatum L. plants as affected by light quality and intensity. Environ. Control Biol. 45, 113–120.
137
Nishioka, N., Nishimura, T., Ohyama, K., Sumino, M., Malayeri, S.H., Goto, E., Inagaki, N., Morota, T., 2008. Light quality affected growth and contents of essential oil components of Japanese mint plants. Acta Hortic. 797, 431–436. Nishimura, T., Ohyama, K., Goto, E., Inagaki, N., Morota, T., 2008. Ultraviolet B radiation suppressed the growth and anthocyanin production of Perilla plants grown under controlled environments with artificial light. Acta Hortic. 797, 425–429. Nishizawa, A., Honda, G., Kobayashi, Y., Tabata, M., 1992. Genetic control of peltate glandular trichome formation in Perilla frutescens. Planta Med. 58, 188–191. Oka, T., 2006. The role of Kampo (Japanese traditional herbal) medicine in psychosomatic medicine practice in Japan. Intern. Congr. Ser. 1287, 304–308. Omer, E.A., Khattab, M.E., Ibrahim, M.E., 1998. First cultivation trial of Perilla frutescens L. in Egypt. Flavour Fragr. J. 13, 221–225. Saito, K., Yamazaki, M., 2002. Biochemistry and molecular biology of the late-stage of biosynthesis of anthocyanin: lessons from Perilla frutescens as a model plant. New Phytol. 155, 9–23. Sato, K., Krist, S., Buchbauer, G., 2006. Antimicrobial effect of trans-cinnamaldehyde, ( )-perillaldehyde, ( )-citronellal, citral, eugenol and carvacrol on airborne microbes. Biol. Pharm. Bull. 29, 2292–2294. Smith, H., 1982. Light quality, photoperception, and plant strategy. Ann. Rev. Plant Physiol. 33, 481–518. Sugaya, A., Tsuda, T., Obuchi, T., 1981. Pharmacological studies of Perillae herba. I. Neuropharmacological action of water extract and perillaldehyde. Yakugaku Zasshi 101, 642–648 (in Japanese with English abstract). Warrington, I.J., Mitchell, K.J., 1976. The influence of blue- and red-biased light spectra on the growth and development of plants. Agric. Meteorol. 16, 247– 262. Yoshida, T., Higashi, F., Ikawa, S., 1968. On the oil containing tissue, the essential oil contents and the chemical composition of essential oil in Perilla species. Nippon Sakumotsu Gakkai Kiji 37, 118–122 (in Japanese with English abstract). Yoshida, T., Morisada, S., Kameoka, K., 1969. On the distribution of oil gland and the accumulation of essential oil in Perilla species. Nippon Sakumotsu Gakkai Kiji 38, 333–337 (in Japanese with English abstract). Yu, H.-C., 1997. Introduction. In: Yu, H.-C., Kosuna, K., Haga, M. (Eds.), Perilla: the genus Perilla. Harwood Academic Publishers, Amsterdam, pp. 1–8. Zobayed, S.M.A., Afreen, F., Kozai, T., 2005. Necessity and production of medicinal plants under controlled environments. Environ. Control Biol. 43, 243–252.
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Short communication
Concentrations of perillaldehyde, limonene, and anthocyanin of Perilla plants as affected by light quality under controlled environments Tetsuro Nishimura a, Katsumi Ohyama b,*, Eiji Goto a, Nobuyuki Inagaki c a
Graduate School of Horticulture, Chiba University, Matsudo, Chiba 271-8510, Japan Centre for Environment, Health and Field Sciences, Chiba University, 6-2-1, Kashiwanoha, Kashiwa, Chiba 277-0882, Japan c Botanical Raw Materials Research Dept., Tsumura & Co., Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 October 2008 Received in revised form 5 March 2009 Accepted 9 March 2009
Perilla plants with red coloured leaves grown in red-enriched light treatments (i.e., red lamps alone (R), a mixture of blue and red lamps (BR), and a mixture of green and red lamps (GR)) had greater dry weight, bigger leaves, and more leaves than those grown in other treatments (i.e., blue lamps alone (B), a mixture of blue and green lamps (BG), and green lamps alone (G)). Although the concentrations of perillaldehyde and limonene in leaf tissues (mg g 1 leaf DW) were 1.6–1.9 and 1.5–1.9 times higher, respectively, in the G treatment than in red-enriched light treatments, the contents of perillaldehyde and limonene per plant (mg/plant) were 1.8 times higher in the BR treatment than those in the G treatment. The content of anthocyanin per plant was also 4.3 times higher in the BR treatment than that in the G treatment. Therefore, within the six different combinations of fluorescent lamps used in the present study, the BR treatment was a suitable light quality for producing plants with high contents of perillaldehyde, limonene, and anthocyanin under controlled environments. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Essential oil Fluorescent lamp Medicinal plant Perilla frutescens
1. Introduction Perilla frutescens (L.) Britt. belonging to the family Lamiaceae (Labiatae) is native to mountainous areas of China and India and is grown mainly in Asia (Yu, 1997). The Perilla plants were classified into seven chemotypes based on the main components of essential oil (Ito, 1970; Koezuka et al., 1986; Ito et al., 1999a,b, 2002), viz. perillaldehyde (PA-type), perilla-ketone (PK-type), elsholtziaketone (EK-type), citral (C-type), phenylpropanoid (PP-type), perillene (PL-type), and piperitenone (PT-type). Perilla frutescens with red coloured leaves is a PA-type plant species. The plants cultivated in Japan contain approximately 0.5% essential oil in fresh leaves, mainly consisting of 55% perillaldehyde and 20–30% limonene (Omer et al., 1998). Perillaldehyde is thought to have a depressive effect on the central nervous system (Sugaya et al., 1981) and antibacterial activity (Sato et al., 2006). Hence, leaves of the plants are used in traditional, Japanese herbal medicine (Kampo medicine). The quality of Perilla plants used for Kampo medicine is determined by the concentrations of essential oil components in leaves and
* Corresponding author. Tel.: +81 4 7137 8000; fax: +81 4 7137 8008. E-mail address: [email protected] (K. Ohyama). 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.03.010
the appearance such as redness of leaves attributed to anthocyanin (Nagao et al., 1974), so a method for increasing the concentrations of perillaldehyde, limonene and anthocyanin in the leaves while simultaneously promoting growth is essential for efficient production of the plants. Recently, Kampo medicine has become popular worldwide as an alternative therapy (e.g., Matsumoto et al., 1999; Kanda et al., 2005; Oka, 2006). As a result, the demand for the aerial parts of Perilla plants as a raw material of Kampo medicine has been increasing, and thus a stable supply of the plants to meet the projected increase in demand will be required in the near future. In the natural environment, there are a number of concerns associated with the use of medicinal plants, including biotic or abiotic contamination, adulteration of plant species and weeds, and quality variation of the medicinal plant products themselves (Zobayed et al., 2005). However, these problems can be solved by growing medicinal plants under controlled environments with artificial light. It was reported that red light increased the content of menthol, which is the main component of the essential oil in Mentha arvensis (Nishioka et al., 2008) and ultraviolet-B radiation suppressed anthocyanin production of Perilla plants (Nishimura et al., 2008) under controlled environments. It was also reported that light quality had effects on plant growth and development (Smith, 1982;
T. Nishimura et al. / Scientia Horticulturae 122 (2009) 134–137
McNellis and Deng, 1995; Goto, 2003). Light quality might alter the growth and concentrations of essential oil components and anthocyanin of red Perilla plants, but little information is available. The objective of our study was to evaluate the effects of light quality provided by coloured fluorescent lamps on the growth and concentrations of essential oil components and anthocyanin of Perilla plants in order to determine efficient production conditions under controlled environments.
135
differences in the reversible action of phytochrome among the plants grown in the six different treatments were small in this study. In the growth chamber, environmental conditions except for light quality were the same as those during the seedling production period. Since the plants were larger than the seedlings, the planting density was adjusted to 37 plants m 2. 2.3. Measurements Since the underground part of the plant is discarded in the commercial production of Perilla plants used for crude drug, in the present experiment only the aerial part of the plant was measured.
2. Materials and methods 2.1. Plant materials and growth conditions Seeds of Perilla (Perilla frutescens (L.) Britt. var. acuta Kudo f. purpurea Makino, cv. Sekiho) were sown in rock wool cubes. On day 21 after sowing, the seedlings were transplanted to individual pots (diameter: 9.0 cm; capacity: 330 ml), filled with a commercial soil mixture of vermiculite (50%) and peat moss (50%). The seedlings were grown in a growth chamber for 7 days at a photoperiod of 16 h d 1 provided by cool-white fluorescent lamps (FHF32-EX-N-H, Matsushita Electric Industrial Co. Ltd., Osaka, Japan). All seedlings were placed under a photosynthetic photon flux (PPF) of 200 mmol m 2 s 1 on the soil surface, an air temperature of 27/22 8C (photo-/dark periods), a relative humidity of 60/90% (photo-/dark periods), and a CO2 concentration of 1000 mmol mol 1. The planting density was 100 seedlings m 2. After germination, subirrigation was made once a day with a nutrient solution (Otsuka hydroponic composition, Otsuka Chemical Co. Ltd., Osaka, Japan). The nutrient solution, adjusted to electrical conductivity (EC) 1.2 dS/m and pH 6.0, contained 4.2 mmol l 1 NO3 , 1.3 mmol l 1 H2PO4 , 1.0 mmol l 1 Ca2+, 0.38 mmol l 1 Mg2+, 2.2 mmol l 1 K+, and 0.4 mmol l 1 NH4+. 2.2. Treatments The 28-day-old seedlings with six true leaves (dry weight: 38 mg, stem length: 1.6 cm, hereafter ‘‘plants’’) were subjected to six light quality treatments for 21 days. All plants were placed at the same PPF on the canopy surface of 200 mmol m 2 s 1 in different growth chambers. The lighting system of each treatment was provided by different coloured fluorescent lamps, including the following combinations of coloured fluorescent lamps: blue lamps (FLR40SEB/M, Matsushita Electric Works Ltd.) alone (B), green lamps (FLR40S-EG/M) alone (G), red lamps (FLR40S-ER/M) alone (R), a mixture of blue and green lamps (BG), a mixture of blue and red lamps (BR), or a mixture of green and red lamps (GR) (Table 1). Although the red/far-red photon flux ratio varied from 4.4 to 25.4 among the treatments in this study, the calculated values of phytochrome photoequilibrium using an equation proposed by Hanyu et al. (1996) was 0.8 in all treatments. Therefore, the
2.3.1. Growth On day 21, dry weights of the leaves and stems were measured with an electronic balance after drying in an oven (temperature 80 8C, 72 h), leaf area was measured with a leaf area meter (LI3100-C, LI-COR Inc., Lincoln, USA), and the number of true leaves of each plant was determined. 2.3.2. Perillaldehyde and limonene concentrations On day 21, the true leaf on the third or fourth node counted from the shoot apex of the main stem of the plants was sampled. Then, the analysis of perillaldehyde and limonene of the sampled leaves was conducted, as described previously (Nishimura et al., 2008). The concentrations of perillaldehyde and limonene in the sampled leaves were expressed as milligram per unit leaf dry weight (mg g 1 leaf DW). Contents of perillaldehyde and limonene per plant were estimated by multiplying these concentrations by the dry weight of all leaves per plant, and were expressed as milligram per plant (mg/plant). 2.3.3. Anthocyanin concentration Three leaf disks (2.4 cm2 total leaf disk area) were sampled from the true leaf on the third or fourth node counted from the shoot apex of the main stem of the plants. Analysis of anthocyanin of sampled leaves was conducted, as described previously (Nishimura et al., 2008). The concentrations of anthocyanin in the sampled leaves were expressed as milligram per unit leaf dry weight (mg g 1 leaf DW). Contents of anthocyanin per plant were estimated by multiplying these concentrations by the dry weight of all leaves per plant, and were expressed as milligram per plant (mg/plant). 2.4. Statistical analysis A completely randomized design with 9 seedlings per treatment and 2 replications was employed in the experiment. Six of these seedlings were used for the measurements of dry weight, leaf area, and number of true leaves, and all seedlings were used for the measurements of concentrations of perillaldehyde, limonene, and
Table 1 Spectral characteristics of each light treatment. 2
1 b
Treatment codea
Photon flux (mmol m 300–400 nm
400–500 nm
500–600 nm
600–700 nm
700–800 nm
B BG G BR GR R
1.1 1.9 2.5 1.0 1.6 1.0
153.0 95.8 29.4 68.4 20.0 14.2
36.3 88.0 148.9 37.6 79.8 39.1
10.7 16.2 21.7 94.0 100.2 146.7
2.5 1.9 0.9 11.6 11.3 17.5
s
)
a B, blue lamps alone; BG, a mixture of blue and green lamps; G, green lamps alone; BR, a mixture of blue and red lamps; GR, a mixture of green and red lamps; R, red lamps alone. b The photon fluxes of each wavelength band were measured using a portable spectroradiometer (LI-1800, LI-COR Inc., Lincoln, USA).
T. Nishimura et al. / Scientia Horticulturae 122 (2009) 134–137
136
Table 2 Growth of Perilla plants on day 21 (49 days after sowing) grown under different light qualities. Treatment codea
Dry weight (g/plant) Leaf
B BG G BR GR R ANOVAc
0.98 1.07 0.62 1.74 1.57 1.66 **
Leaf area (cm2)
Number of true leaves
524.7 567.6 391.8 814.8 796.2 837.9 **
32.9 30.6 21.5 42.8 39.5 38.6 **
Stem bb b c a a a
0.15 0.14 0.07 0.28 0.26 0.30 **
b b c a a a
b b c a a a
c c d a ab b
a
For treatment codes and detailed descriptions, see Table 1. Each value represents the mean of 12 replicates. Means within a column followed by different letters are significantly different at P < 0.05 determined by the Tukey–Kramer test. c **Significance at P < 0.01. b
Table 3 Concentrations of perillaldehyde, limonene, and anthocyanin of red-leafed Perilla plants on day 21 (49 days after sowing) grown under different light qualities. Treatment codea B BG G BR GR R
Perillaldehyde concentration (mg/g leaf DW) 4.47 3.76 4.90 3.09 2.89 2.64
b
a ab a b b b
Limonene concentration (mg/g leaf DW)
Anthocyanin concentration (mg/g leaf DW)
0.53 0.50 0.58 0.38 0.33 0.31
2.41 2.39 2.00 3.07 3.25 3.42
ab abc a bcd cd d
b b b a a a
a
For treatment codes and detailed descriptions, see Table 1. Each value represents the mean of 18 replicates. Different letters in each column indicate significant differences between the treatments at P < 0.05, determined by the Tukey–Kramer test. b
anthocyanin. The data were quantified in terms of the means and standard errors obtained from replicate treatments, and were subjected to an analysis of variance (ANOVA). The means were compared among the treatments using the Tukey–Kramer test at a 5% level of significance. 3. Results and discussion The leaf and stem dry weights of the plants were 1.5–2.8 and 1.8–4.2 times greater, respectively, in the red-enriched light treatments (i.e., BR, GR, and R treatments) than those in the other treatments (B, BG, and G treatments) (Table 2). The number of true leaves and leaf area of the plants were 1.2–2.0 and 1.4–2.1 times greater, respectively, in the red-enriched light treatments than those in the other treatments. The greater leaf area and number of true leaves increase the amount of absorbed light (photons). Therefore, the net photosynthesis rates of the plants may increase in the red-enriched light treatments compared with those grown in other treatments. Hence, the growth of the plants in the redenriched light treatments was promoted compared with those in the other treatments. The results obtained in the present experiment were in agreement with those of previous studies on the growth of other plant species grown under red-enriched light (Warrington and Mitchell, 1976; Mortensen and Strømme, 1987; Inada and Yabumoto, 1989; Nishimura et al., 2006, 2007). The concentrations of perillaldehyde and limonene in leaf tissues of the Perilla plants were 1.6–1.9 and 1.5–1.9 times higher, respectively, in the G treatment than those in the red-enriched light treatments, but similar to those in the B and BG treatments (Table 3). Yoshida et al. (1969) reported that the concentrations of essential oil in leaf tissues of Perilla plants with green coloured leaves increased with increasing number of peltate glandular trichomes per unit leaf area. The peltate glandular trichomes, which develop from leaf epidermal cells, are the main sites for the biosynthesis and accumulation of essential oil (Yoshida et al., 1968; Nishizawa et al., 1992). Thus, we speculated that the number of peltate glandular trichomes per unit area might have been greater in the G treatment.
The anthocyanin concentration in leaf tissues of the Perilla plants was 1.3–1.7 times higher in the red-enriched light treatments than that in the other treatments. The main anthocyanin of the plants is a cyanidin-type compound called malonylshisonin (Saito and Yamazaki, 2002). The malonylshisonin and related compounds, which are accumulated in the epidermal cells in leaves and stems of the plants, are biosynthesized from primary photosynthate or carbohydrates. According to the overflow metabolism concept, when carbohydrate production exceeds the carbon demand associated with plant growth, the excess carbohydrates are channelled into biosyntheses of secondary metabolites (Matsuki, 1996). In this study, the increase in total carbon fixation due to the increase in total leaf area could cause not only the photosynthesis but also the secondary metabolites of the plant. Although the concentrations of perillaldehyde and limonene in leaf tissues were significantly higher in the G treatment than in red-enriched light treatments, the contents of perillaldehyde and limonene per plant were 1.8 times higher in the BR treatment than in the G treatment. The content of anthocyanin per plant was 4.3 times higher in the BR treatment than those in the G treatment. Hence, within the 6 different combinations of fluorescent lamps used in the present study, the BR treatment was a suitable light quality for producing plants with high contents of perillaldehyde, limonene, and anthocyanin. Acknowledgements The authors are grateful to Prof. Dr. Toyoki Kozai at the Centre for Environment, Health and Field Sciences, Chiba University for valuable discussions. We thank Mr. Hideo Yoshida, Ms. Sara Hassani Malayeri, and Ms. Naoko Nishioka for technical assistance. References Goto, E., 2003. Effects of light quality on growth of crop plants under artificial lighting. Environ. Control Biol. 41, 121–132.
T. Nishimura et al. / Scientia Horticulturae 122 (2009) 134–137 Hanyu, H., Shoji, K., Ji, S., 1996. Evaluation of light quality variation through supplement of far-red light and the difference in the effects on growth of a pole-type and a bush-type kidney bean, Phaseolus vulgaris L. Environ. Control Biol. 34, 267–275 (in Japanese with English abstract). Inada, K., Yabumoto, Y., 1989. Effects of light quality, daylength and periodic temperature variation on the growth of lettuce and radish plants. Jpn. J. Crop. Sci. 58, 689–694. Ito, H., 1970. Studies on Folium perillae. VI. Constituent of essential oils and evaluation of Genus Perilla. Yakugaku zasshi 90, 883–892 (in Japanese with English abstract). Ito, M., Toyoda, M., Honda, G., 1999a. Chemical composition of the essential oil of Perilla frutescens. Nat. Med. 53, 32–36. Ito, M., Toyoda, M., Honda, G., 1999b. Essential oil composition of hybrids and amphidiploids of Japanese wild Perilla. Nat. Med. 53, 118–122. Ito, M., Toyoda, M., Kamakura, S., Honda, G., 2002. A new type of essential oil from Perilla frutescens from Thailand. J. Essent. Oil Res. 14, 416–419. Kanda, T., Yamakawa, J., Moriya, J., Saegusa, S., Kawaura, K., Yu, F., Kusaka, K., Takahashi, T., Itoh, T., 2005. Globalization of Kampo medicine. J. Kanazawa Med. Univ. 30, 297–301. Koezuka, Y., Honda, G., Tabata, M., 1986. Genetic control of the chemical composition of volatile oils in Perilla frutescens. Phytochemistry 25, 859–863. Matsuki, M., 1996. Regulation of plant phenolic synthesis: from biochemistry to ecology and evolution. Aust. J. Bot. 44, 613–634. Matsumoto, M., Inoue, K., Kajii, E., 1999. Integrating traditional medicine in Japan: the case of Kampo medicines. Comp. Ther. Med. 7, 254–255. McNellis, T.W., Deng, X.W., 1995. Light control of seedling morphogenetic pattern. Plant Cell 7, 1749–1761. Mortensen, L.M., Strømme, E., 1987. Effects of light quality on some greenhouse crops. Sci. Hortic. 33, 27–36. Nagao, Y., Komiya, T., Fujioka, S., Matsuoka, T., 1974. Studies on the quality of the Chinese drug ‘‘Soyo’’ and the cultivation of the original plant 1. J. Takeda Res. Lab. 33, 111–118 (in Japanese with English abstract). Nishimura, T., Zobayed, S.M.A., Kozai, T., Goto, E., 2006. Effect of light quality of blue and red fluorescent lamps on growth of St. John’s wort (Hypericum perforatum L.). J. SHITA 18, 225–229 (in Japanese with English abstract). Nishimura, T., Zobayed, S.M.A., Kozai, T., Goto, E., 2007. Medicinally important secondary metabolites and growth of Hypericum perforatum L. plants as affected by light quality and intensity. Environ. Control Biol. 45, 113–120.
137
Nishioka, N., Nishimura, T., Ohyama, K., Sumino, M., Malayeri, S.H., Goto, E., Inagaki, N., Morota, T., 2008. Light quality affected growth and contents of essential oil components of Japanese mint plants. Acta Hortic. 797, 431–436. Nishimura, T., Ohyama, K., Goto, E., Inagaki, N., Morota, T., 2008. Ultraviolet B radiation suppressed the growth and anthocyanin production of Perilla plants grown under controlled environments with artificial light. Acta Hortic. 797, 425–429. Nishizawa, A., Honda, G., Kobayashi, Y., Tabata, M., 1992. Genetic control of peltate glandular trichome formation in Perilla frutescens. Planta Med. 58, 188–191. Oka, T., 2006. The role of Kampo (Japanese traditional herbal) medicine in psychosomatic medicine practice in Japan. Intern. Congr. Ser. 1287, 304–308. Omer, E.A., Khattab, M.E., Ibrahim, M.E., 1998. First cultivation trial of Perilla frutescens L. in Egypt. Flavour Fragr. J. 13, 221–225. Saito, K., Yamazaki, M., 2002. Biochemistry and molecular biology of the late-stage of biosynthesis of anthocyanin: lessons from Perilla frutescens as a model plant. New Phytol. 155, 9–23. Sato, K., Krist, S., Buchbauer, G., 2006. Antimicrobial effect of trans-cinnamaldehyde, ( )-perillaldehyde, ( )-citronellal, citral, eugenol and carvacrol on airborne microbes. Biol. Pharm. Bull. 29, 2292–2294. Smith, H., 1982. Light quality, photoperception, and plant strategy. Ann. Rev. Plant Physiol. 33, 481–518. Sugaya, A., Tsuda, T., Obuchi, T., 1981. Pharmacological studies of Perillae herba. I. Neuropharmacological action of water extract and perillaldehyde. Yakugaku Zasshi 101, 642–648 (in Japanese with English abstract). Warrington, I.J., Mitchell, K.J., 1976. The influence of blue- and red-biased light spectra on the growth and development of plants. Agric. Meteorol. 16, 247– 262. Yoshida, T., Higashi, F., Ikawa, S., 1968. On the oil containing tissue, the essential oil contents and the chemical composition of essential oil in Perilla species. Nippon Sakumotsu Gakkai Kiji 37, 118–122 (in Japanese with English abstract). Yoshida, T., Morisada, S., Kameoka, K., 1969. On the distribution of oil gland and the accumulation of essential oil in Perilla species. Nippon Sakumotsu Gakkai Kiji 38, 333–337 (in Japanese with English abstract). Yu, H.-C., 1997. Introduction. In: Yu, H.-C., Kosuna, K., Haga, M. (Eds.), Perilla: the genus Perilla. Harwood Academic Publishers, Amsterdam, pp. 1–8. Zobayed, S.M.A., Afreen, F., Kozai, T., 2005. Necessity and production of medicinal plants under controlled environments. Environ. Control Biol. 43, 243–252.