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Effect of light wavelength on cell growth, content of phenolic compounds and antioxidant activity in cell suspension cultures of Thevetia peruviana
Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 87–91
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Effect of light wavelength on cell growth, content of phenolic compounds and antioxidant activity in cell suspension cultures of Thevetia peruviana J.P. Arias ⁎, K. Zapata, B. Rojano, M. Arias Escuela de Biociencias, Facultad de Ciencias, Universidad Nacional de Colombia, sede Medellín, Medellín, Antioquia, Colombia
a r t i c l e
i n f o
Article history: Received 24 April 2016 Accepted 10 August 2016 Available online 12 August 2016 Keywords: Plant cell suspension cultures Thevetia peruviana Phenolic Antioxidant activity Light-emitting diode (LED)
a b s t r a c t Thevetia peruviana (T. peruviana) has been considered as a potentially important plant for industrial and pharmacological application. Among the number of compounds which are produced by T. peruviana, antioxidants and polyphenols are of particular interest due to their benefits on human health. Cell suspension cultures of T. peruviana were established under different conditions: 1) constant illumination (24 h/day) at different light wavelengths (red, green, blue, yellow and white), 2) darkness and 3) control (12 h/12 h: day light/dark) to investigate their biomass, substrate uptake, polyphenols production and oxidizing activity. The results showed biomass concentrations between 17.1 g dry weight (DW)/l (green light) and 18.2 g DW/l (control) after 13 days. The cultures that grew under green light conditions consumed completely all substrates after 10 days, while other cultures required at least 13 days or more. The total phenolic content was between 7.21 and 9.46 mg gallic acid (GA)/g DW for all light conditions. In addition the ferric reducing antioxidant power and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid antioxidant activity ranged from 5.41–6.58 mg ascorbic acid (AA)/g DW and 82.93–110.39 μmol Trolox/g DW, respectively. Interestingly, the samples which grew under the darkness presented a higher phenolic content and antioxidant capacity when compared to the light conditions. All together, these results demonstrate the extraordinary effect of different lighting conditions on polyphenols production and antioxidant compounds by T. peruviana. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Thevetia peruviana (T. peruviana) is a common ornamental shrub that belongs to the Apocynaceae family. It has been considered as an important source of biologically active compounds which exhibit antitermite [1], antimicrobial [2,3], fungicide [4,5], anti HIV [6], anticancer [7] and antioxidant [8] activities. T. peruviana is particularly known for its production of cardiac glycosides such as thevetin, neriifolin and peruvoside, which are used as alternative drugs to digoxin during heart failure treatment [9]. Plants utilize light as an energy source for cell growth but also as an important external signal for a wide range of physiological responses such as production of phenolic compounds, terpenoids and alkaloids [10]. The effect of light on plant metabolism is completely speciedependent, therefore, it is necessary to design specific studies for each species of interest [11,12]. The light-emitting diode (LED) technology has widely been used in horticulture in order to improve energy efficiency, to promote plant growth and to increase the nutritional value ⁎ Corresponding author at: Laboratorio de Bioconversiones, Escuela de Biociencias, Facultad de Ciencias, Universidad Nacional de Colombia, sede Medellín, Carrera 59A # 63 – 20, Medellín, Antioquia, Colombia. E-mail address: [email protected] (J.P. Arias).
http://dx.doi.org/10.1016/j.jphotobiol.2016.08.014 1011-1344/© 2016 Elsevier B.V. All rights reserved.
of crops [13–15]. The biggest advantage of LED technology is its ability to produce light of a desired wavelength and hence allowing to study the effects of various wavelengths on the plant growth. Recent studies have reported several factors which affect cell growth and production of bioactive metabolites in cell suspension cultures of T. peruviana such as plant growth regulators, sucrose concentration and presence of elicitors [16–18]. However, to the best of our knowledge, there has not been published any work yet describing the effect of light on the behavior of cell suspension cultures of this species. Therefore, our study evaluates the impact of different light wavelengths (400–700 nm) on the cell growth, substrate uptake, phenolic production and antioxidant capacity in cell suspension cultures of T. peruviana. 2. Materials and Methods 2.1. Plant Material, Callus and Cell Suspension Cultures The protocol for obtaining callus and cell suspension cultures of T. peruviana from fruit pulp has already been described in detail [16]. Briefly, the fruits were disinfected using ethanol (70% v/v), sodium hypochlorite (10% w/v) and sterile distilled water. Next, fresh explants were extracted aseptically and sowed in solid Schenk and Hildebrandt
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(SH) medium supplemented with 30 g/l sucrose, 2 mg/1 of 2,4-D and 0.5 mg/l of kinetin. The cell suspension cultures were maintained at 25 °C and constant agitation (110 rpm). All cell suspension cultures used in the experiments were in exponential growth phase (6 days from the last subculture). 2.2. Effect of Light Wavelengths The study of various light wavelengths on the cell growth and the substrate uptake of the cell suspension cultures was performed using a custom lighting equipment. The equipment consisted of six compartments with individual LED lighting system located at the top and emitting specific light wavelengths: red (586–596 nm), green (525 nm), blue (465 nm), yellow (590 nm) and white. The walls of each compartment had mirror-like surfaces to optimize lighting. Three 500 ml Erlenmeyer flasks with 200 ml culture medium and an initial cell concentration of 2 g DW/l were placed in each compartment. Light intensity for all wavelengths was 52 μmol m−2 s−1. The cultures were subjected to constant light i.e. 24 h/day. Additionally, control and darkness conditions were performed with 12 h day light/12 h darkness, and 24 h of darkness, respectively.
2.7. Antioxidant Capacity The Ferric Reducing Antioxidant Power (FRAP) values were determined following a previously described method [21] with some modifications. The protocol was based on the increase of absorbance due to the formation of 2,4,6-tripyridil-s-triazine (TPTZ)-Fe(II) in the presence of reducing agents. The FRAP reagent contained 2.5 ml of TPTZ (10 mM) in hydrochloric acid (40 mM), 25 ml of acetate buffer (0.3 mM pH 3.6) and 2.5 ml of iron(III) chloride (20 mM). A volume of 50 μl of extract was mixed with 950 μl FRAP reagent. The absorbance was measured at 590 nm. The FRAP values were expressed in mg of ascorbic acid (AA)/g DW, using AA as a standard in the calibration curve. The 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS) concentrations were established using a previously published protocol [22] with some modification. In order to evaluate the ability of the samples to trap the radical ABTS•+, 10 μl of extract was added to 990 μl of ABTS solution and after 30 min of reaction the absorbance at 732 nm wavelength was measured. Next, the absorbance was compared with the reference curve prepared with Trolox as a primary standard. The results were expressed in μmol of Trolox/g DW. 3. Statistical Analysis
2.3. Cell Growth Determination Cell growth was determined by vacuum filtration of culture aliquots (5 ml) for each treatment (wavelength) through a pre-dried (60 °C - 24 h) filter paper Whatman 595 (Sigma-Aldrich Bogotá, Colombia). Next, the retained sample was washed 3 times with distilled water and dried in a convection oven until it reached constant weight after 24 h at 60 °C (DW [g]).
All data are presented as the mean values ± Standard Deviation (SD) of at least two replicates. The data were analyzed by Fisher multiple comparison test (p = 0.05) using the Statgraphics Centurion XV version 15.2.05 software (Statpoint Technologies, Inc. Virginia USA). 4. Results and Discussion 4.1. Effect of Light Wavelength on Cell Growth and Substrate Uptake
2.4. Sugar Quantification To measure sucrose uptake a Shimadzu Prominence High Pressure Liquid Chromatography (HPLC) system with a pumping system LC20 CE coupled to a refractive index detector RID-10A and Solution LC software was used (Shimadzu Scientific Instruments, Inc. USA). Separation and quantification of sucrose was carried out using a reverse phase amino column RP-NH2 NUCLEODUR 100-5 (Macherey-Nagel Inc. PA. USA.) and mobile phase acetonitrile/water (79:21 v/v ratio), flow 2 ml/min, sample size 10 μl at 35 °C. 2.5. Metabolite Extraction
Cell suspension cultures of T. peruviana showed similar kinetic of cell growth in spite of different light wavelength conditions, natural photoperiod and darkness (Fig. 1). The cell suspension cultures did not show adaptation phase. The exponential phase lasted 10 days and was followed by a stationary phase (until the 16th day of cell growth) after which the cell's death started. The cell suspensions cultures were initiated with an inoculum concentration of 2.0 g DW/l, reaching maximum and minimum concentrations of biomass of 18.2 and 17.1 g DW/l, after 13 days of growth under control and green light conditions, respectively. Table 1 presents the specific cell growth rates (μ) and doubling times (td) for each lighting condition.
Biomass obtained after 23 days of incubation was freezed/dried (SYCLON-18 N) at −60 °C and 1 bar to avoid degradation of thermolabile compounds. The protocol of bioactive metabolite extraction has previously been described [19]. Briefly, 20 mg of biomass was homogenized with 2 ml of water-ethanol mixture (1:1 v:v) using Ultra-Turrax® homogenaizer T45 S5 (Janke & Kunkel GmbH & Co. KG IKA-WERK). Next, the extract was centrifuged at 4000 rpm for 10 min at room temperature and the supernatant was filtered on Whatman® qualitative filter paper grade 4 (Sigma-Aldrich Bogotá, Colombia) and stored at 4 °C until testing. 2.6. Total Phenolic Content The total phenolic content was determined using the adapted FolinCiocalteu method [20]. The extracts (50 ml) were mixed with 125 ml of Folin-Ciocalteu reagent and 400 ml of sodium carbonate solution (7.1% w/v) and the resulting solution was brought to a final volume of 1000 ml with distilled water. The mixture was stirred and stored at room temperature for 30 min in darkness. The absorbance was measured at 760 nm against a blank. Aqueous solutions of gallic acid (GA) were used for calibration and the results were expressed as mg GA/g DW.
Fig. 1. Cell growth curves of cell suspension cultures of T. peruviana under different lighting conditions. Results are presented as average ± SD (n = 2).
J.P. Arias et al. / Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 87–91
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Table 1 Kinetic parameters of cell suspension cultures of T. peruviana under different lighting conditions: maximum cell growth rate (μ [days−1]) and doubling time (td [days]). Lighting condition
μ [days−1]
td [days]
Control Darkness Yellow Red Blue White Green
0.2511 0.268 0.259 0.258 0.252 0.227 0.262
2.760 2.586 2.679 2.681 2.751 3.048 2.649
Fig. 2 presents the kinetics of sucrose uptake for each light wavelength. The cell suspension cultures subjected to green light and darkness showed a complete depletion of the substrate after 10 days of growth. In contrast, for the cell suspension cultures which grew under yellow, red, white light and in control conditions, sucrose concentration was close to 4.2 g/l at day 10. The highest sucrose concentration at day 10 was for the cell suspension cultures which grew under blue light condition 7.6 g/l. For all conditions at least 13 days were required to completely consume the sucrose. The time when the maximum concentration of cell growth is reached for specific light wavelength (Fig. 1) is consistent with the day of the carbon source depletion for each cell suspension culture (Fig. 2). These results indicate that sucrose was the only energy source for all conditions during the cell growth and the cell suspension cultures did not use the light energy from the diodes for the cell growth. Therefore, we were able to demonstrate that the cell suspension cultures of T. peruviana do not have the ability to utilize light as an energy source and hence present a strictly heterotrophic cell growth. 4.2. Effect of Light Wavelength on the Production of Polyphenols and Antioxidant Capacity The phenolic content, FRAP values and ABTS concentration of T. peruviana cell suspension cultures under different lighting conditions are presented in Figs. 3, 4 and 5, respectively. The results show that the production of polyphenols was affected by the type of light used during the cell growth (p b 0.05). The total phenolic content was between 7.21 and 7.91 mg GA/g DW for all light wavelengths, except for the darkness condition for which the phenolic content was 9.46 mg GA/g DW (Fig. 3). Interestingly, when comparing with the available literature, the production of polyphenols by cell suspension cultures of T. peruviana
Fig. 2. Sucrose uptake curves of cell suspension cultures of T. peruviana under different lighting conditions. Results are presented as average ± SD (n = 2).
Fig. 3. Total phenolic content in cell suspension cultures of T. peruviana under different lighting conditions. Significant differences (p b 0.05) among medias are expresses by different letters. Results are presented as average ± SD (n = 2).
is superior to the production of polyphenols by the plants cultivated in field, which exhibited a total phenolic content of 2.14 mg GA /g dry leaf [8]. The FRAP values were between 5.41 and 6.58 mg AA/g DW for all lighting conditions (Fig. 4), while the ABTS concentration was between 82.93 and 120.39 μmol Trolox/g DW (Fig. 5). It should be mentioned here that our study is the first so far, that has attempted to measure the antioxidant activity of cell suspension cultures of T. peruviana by using the FRAP and ABTS methods. Moreover, the FRAP and ABTS values have not been reported yet for other species of the Apocynaceae family. The phenolic content detected in our study was comparable with other important in vitro plant cultures. Ahmad et al. showed lower phenolic content in callus cultures of Stevia rebaudiana (0.1 mg GA/g DW) grown under different type of lights [23]. Giri et al. obtained phenolic content in cell suspension cultures of Habenaria edgeworthii between 5.28 and 14.70 mg GA/g DW [24]. Other studies have reported higher values of phenolic content and antioxidant activity. However, these high levels can be explain by using different extraction techniques which improve the recovery of phenolic compounds in 5 to 10 times [25,26]. The correlations between the matrices 1) Total Phenols and ABTS (for each type of light) and 2) Total Phenols and FRAP (for each type of light), were calculated from the Pearson Product-Moment Correlation Coefficient (PPMCC). The values of the PPMCC for the phenolic content versus ABTS and the phenolic content versus FRAP values were very close to one (Table 2). Such high values of PPMCCs indicate positive and perfect correlation between the two variables. Therefore, it is expected that when one variable increases (i.e. phenolic content), the other variable (ABTS or FRAP) increases in a proportional rate.
Fig. 4. Ferric Reducing Antioxidant Power (FRAP) of cell suspension cultures of T. peruviana under different lighting conditions. Different letters indicate significant differences between medias (p b 0.05). Results are presented as average ± SD (n = 2).
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with the current literature for other plant species. Nonetheless, further studies would be required to establish the enzymatic and molecular mechanisms involved in the changes in polyphenols production under light and darkness conditions. 5. Conclusions
Fig. 5. The 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS) capacity of cell suspension cultures of T. peruviana under different lighting conditions. Different letters indicate significant differences between medias (p b 0.05). Results are presented as average ± SD (n = 2).
Moreover, our results confirmed that the antioxidant activity of the samples was effectively conditioned by their phenolic content and not by the presence of reducing substances. Such reducing agents (i.e. citric acid and monosaccharide sugars) could lead to false positive findings. Importantly, cell suspension cultures which grew under dark condition exhibited the greatest antioxidant activity when compared to the cultures which grew under other lighting conditions (Fig. 4 and Fig. 5). These results indicate that the light has a deleterious effect on the production of phenolic compounds and the antioxidant activity of T. peruviana cultures. Some authors have reported similar findings in other plant species. Ghasemzadeh et al. [27] showed that flavonoid synthesis in the Halia bara species was improved if lower light intensities were used (310 μmol m−2 s−1) when compared to the cultures irradiated with higher light pulses (790 μmol m−2 s−1). Furthermore, the antioxidant activity determined by the 1,1-diphenyl-2 picrylhydrazyl (DPPH) methodology was greater for the leaves of Halia bentong and Halia bara species when the plants were subjected to lower intensity of light [28]. Liu et al. [10] have reported that under white and blue light conditions the production of shikonin (natural anti inflammatory phenol) was completely inhibited in the Onasma paniculatum cultures. The results were associated with the attenuation of expression of genes involved in the synthesis of phenolic metabolites. Moreover, Shohael et al. [29] have found a higher content of oxidants such as H2O2, malondialdehyde and lipoxygenase in E. senticosus somatic embryos treated with red light when compared to the embryos which developed without the light. These results were related to light sensitivity of the oxidative enzymes. Additionally, it has been shown that illuminated callus cultures of Catharanthus roseus accumulated less ajmalicine (an alkaloid with antihypertensive activity) than the cultures in darkness [30]. The authors explained this phenomenon by a possible conversion of the ajmalicine to serpentine due to enzymatic activation induced by visible light radiation. Finally, Yu et al. [31] stated that the hairy roots of Panax ginseng cultured in bioreactors showed lower biomass formation when the hairy roots were grown under different lighting conditions in contrast to the hairy roots incubated in darkness. Furthermore, the hairy roots under darkness produced higher amount of ginsenosides when compared to the irradiated samples. All together, our findings for T. peruviana cell suspension cultures are in agreement Table 2 The Pearson Product-Moment Correlation Coefficients (PPMCC) between phenolic content and antioxidant capacity of the T. peruviana cells suspension cultures under different lighting conditions. Phenolic content vs. Antioxidant activity
PPMCC
Phenolic content vs. ABTS Phenolic content vs. FRAP
0.993 0.992
Although the cell suspension cultures of T. peruviana cultured under different light wavelengths did not present a significant difference in the cell growth, they showed significant differences in sucrose rate uptake. The changes in sucrose uptake together with the cell growth indicate that the growth of the cell suspensions cultures have a strictly heterotrophic behavior. Moreover, the presence of light has a deleterious effect on the production of phenols and antioxidant capacity on in vitro cell suspension cultures of T. peruviana. Accordingly, in darkness condition there is a protective effect on the production of phenols and the higher antioxidant activity. Finally, our study established that the cell suspension cultures of T. peruviana exhibit higher antioxidant capacity than the actual plants of the same specie as well as other farmed species in vitro. Acknowledgments The authors are grateful to the Facultad de Ciencias of Universidad Nacional de Colombia, Medellin, for the support to carry out this work. References [1] T.A. Tagbor, The anti-termite properties and basic phytochemicals of eight local plants and the chemical characterisation of Thevetia peruviana (pers) k. schum in Ghana, Ph.D thesis, Kwame Nkrumah University of Science and Technology, 2009. [2] M.M. Hassan, A.K. Saha, S.A. Khan, A. Islam, S.S.U. Ahmed, Studies on the antidiarrhoeal, antimicrobial and cytotoxic activities of ethanol-extracted leaves of yellow oleander (Thevetia peruviana), Open Vet. J. 1 (2011) 28–31. [3] P.G. Kareru, J.M. Keriko, G.M. Kenji, G.T. Thiong'o, A.N. Gachanja, H.N. Mukiira, Antimicrobial activities of skincare preparations from plant extracts, Afr. J. Tradit. Complement. Altern. Med. 7 (2010) 214–218. [4] Z. Ambang, J. Ngoh Dooh, G. Essono, N. Bekolo, G. Chewachong, C. Asseng, Effect of Thevetia peruviana seeds extract on in vitro growth of four strains of Phytophthora megakarya, Plant Omi. J. 3 (2010) 70–76. [5] L. Gata-Gonçalves, J.M.F. Nogueira, O. Matos, R.B. de Sousa, Photoactive extracts from Thevetia peruviana with antifungal properties against Cladosporium cucumerinum, J. Photochem. Photobiol. B Biol. 70 (2003) 51–54. [6] S. Tewtrakul, N. Nakamura, M. Hattori, T. Fujiwara, T. Supavita, Flavanone and flavonol glycosides from the leaves of Thevetia peruviana and their HIV-1 reverse transcriptase and HIV-1 integrase inhibitory activities, Chem. Pharm. Bull.(Tokyo) 50 (2002) 630–635. [7] R. Newman, P. Yang, A.D. Pawlus, K.I. Block, Cardiac glycosides as novel cancer therapeutic agents, Mol. Interv. 8 (2008) 36–49. [8] G. Zibbu, A. Batra, In vitro and in vivo determination of phenolic contents and antioxidant activity of desert plants of apocynaceae family, Asian J. Pharm. Clin. Res. 5 (2012) 75–83. [9] G. Zibbu, A. Batra, Thevetia peruviana (Pers.) Schum.: a plant with enormous therapeutic potential, J. Pharm. Res. 4 (2011) 4461–4464. [10] Z. Liu, J.L. Qi, L. Chen, M.S. Zhang, X.Q. Wang, Y.J. Pang, Y.H. Yang, Effect of light on gene expression and shikonin formation in cultured Onosma paniculatum cells, Plant Cell Tissue Organ Cult. 84 (2006) 39–46. [11] L. Chun-zhao, G. Chen, W. Yu-chun, O. Fan, Effect of light irradiation on hairy growth and artemisinin biosynthesis of Artemisia annua L, Process Biochem. 38 (2002) 581–585. [12] J. Zhao, W.H. Zhu, Q. Hu, Effects of light and plant growth regulators on the biosynthesis of vindoline and other indole alkaloids in Catharanthus roseus callus cultures, Plant Growth Regul. 33 (2001) 43–49. [13] M.C. Wu, C.Y. Hou, C.M. Jiang, Y.T. Wang, C.Y. Wang, H.H. Chen, H.M. Chang, A novel approach of LED light radiation improves the antioxidant activity of pea seedlings, Food Chem. 101 (2007) 1753–1758. [14] M.E.-B. Younis, M.N.A.-G. Hasaneen, H.M.M. Abdel-Aziz, An enhancing effect of visible light and UV radiation on phenolic compounds and various antioxidants in broad bean seedlings, Plant Signal. Behav. 5 (2010) 1197–1203. [15] G. Samuolienė, R. Sirtautas, A. Brazaitytė, P. Duchovskis, LED lighting and seasonality effects antioxidant properties of baby leaf lettuce, Food Chem. 134 (2012) 1494–1499. [16] M. Arias, M.J. Angarita, J.M. Restrepo, L.A. Caicedo, M. Perea, Elicitation with methyljasmonate stimulates peruvoside production in cell suspension cultures of Thevetia peruviana, In Vitro Cell Dev. Biol. Plant 46 (2009) 233–238. [17] P. Odhiambo, M. Makobe, H. Boag, A. Muigai, H. Kiesecker, In vitro regeneration of Thevetia peruviana Pers. K. Schum, Family Apocynaceae, Sci. Conf. Proc. (2010) 81–91.
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Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Effect of light wavelength on cell growth, content of phenolic compounds and antioxidant activity in cell suspension cultures of Thevetia peruviana J.P. Arias ⁎, K. Zapata, B. Rojano, M. Arias Escuela de Biociencias, Facultad de Ciencias, Universidad Nacional de Colombia, sede Medellín, Medellín, Antioquia, Colombia
a r t i c l e
i n f o
Article history: Received 24 April 2016 Accepted 10 August 2016 Available online 12 August 2016 Keywords: Plant cell suspension cultures Thevetia peruviana Phenolic Antioxidant activity Light-emitting diode (LED)
a b s t r a c t Thevetia peruviana (T. peruviana) has been considered as a potentially important plant for industrial and pharmacological application. Among the number of compounds which are produced by T. peruviana, antioxidants and polyphenols are of particular interest due to their benefits on human health. Cell suspension cultures of T. peruviana were established under different conditions: 1) constant illumination (24 h/day) at different light wavelengths (red, green, blue, yellow and white), 2) darkness and 3) control (12 h/12 h: day light/dark) to investigate their biomass, substrate uptake, polyphenols production and oxidizing activity. The results showed biomass concentrations between 17.1 g dry weight (DW)/l (green light) and 18.2 g DW/l (control) after 13 days. The cultures that grew under green light conditions consumed completely all substrates after 10 days, while other cultures required at least 13 days or more. The total phenolic content was between 7.21 and 9.46 mg gallic acid (GA)/g DW for all light conditions. In addition the ferric reducing antioxidant power and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid antioxidant activity ranged from 5.41–6.58 mg ascorbic acid (AA)/g DW and 82.93–110.39 μmol Trolox/g DW, respectively. Interestingly, the samples which grew under the darkness presented a higher phenolic content and antioxidant capacity when compared to the light conditions. All together, these results demonstrate the extraordinary effect of different lighting conditions on polyphenols production and antioxidant compounds by T. peruviana. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Thevetia peruviana (T. peruviana) is a common ornamental shrub that belongs to the Apocynaceae family. It has been considered as an important source of biologically active compounds which exhibit antitermite [1], antimicrobial [2,3], fungicide [4,5], anti HIV [6], anticancer [7] and antioxidant [8] activities. T. peruviana is particularly known for its production of cardiac glycosides such as thevetin, neriifolin and peruvoside, which are used as alternative drugs to digoxin during heart failure treatment [9]. Plants utilize light as an energy source for cell growth but also as an important external signal for a wide range of physiological responses such as production of phenolic compounds, terpenoids and alkaloids [10]. The effect of light on plant metabolism is completely speciedependent, therefore, it is necessary to design specific studies for each species of interest [11,12]. The light-emitting diode (LED) technology has widely been used in horticulture in order to improve energy efficiency, to promote plant growth and to increase the nutritional value ⁎ Corresponding author at: Laboratorio de Bioconversiones, Escuela de Biociencias, Facultad de Ciencias, Universidad Nacional de Colombia, sede Medellín, Carrera 59A # 63 – 20, Medellín, Antioquia, Colombia. E-mail address: [email protected] (J.P. Arias).
http://dx.doi.org/10.1016/j.jphotobiol.2016.08.014 1011-1344/© 2016 Elsevier B.V. All rights reserved.
of crops [13–15]. The biggest advantage of LED technology is its ability to produce light of a desired wavelength and hence allowing to study the effects of various wavelengths on the plant growth. Recent studies have reported several factors which affect cell growth and production of bioactive metabolites in cell suspension cultures of T. peruviana such as plant growth regulators, sucrose concentration and presence of elicitors [16–18]. However, to the best of our knowledge, there has not been published any work yet describing the effect of light on the behavior of cell suspension cultures of this species. Therefore, our study evaluates the impact of different light wavelengths (400–700 nm) on the cell growth, substrate uptake, phenolic production and antioxidant capacity in cell suspension cultures of T. peruviana. 2. Materials and Methods 2.1. Plant Material, Callus and Cell Suspension Cultures The protocol for obtaining callus and cell suspension cultures of T. peruviana from fruit pulp has already been described in detail [16]. Briefly, the fruits were disinfected using ethanol (70% v/v), sodium hypochlorite (10% w/v) and sterile distilled water. Next, fresh explants were extracted aseptically and sowed in solid Schenk and Hildebrandt
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(SH) medium supplemented with 30 g/l sucrose, 2 mg/1 of 2,4-D and 0.5 mg/l of kinetin. The cell suspension cultures were maintained at 25 °C and constant agitation (110 rpm). All cell suspension cultures used in the experiments were in exponential growth phase (6 days from the last subculture). 2.2. Effect of Light Wavelengths The study of various light wavelengths on the cell growth and the substrate uptake of the cell suspension cultures was performed using a custom lighting equipment. The equipment consisted of six compartments with individual LED lighting system located at the top and emitting specific light wavelengths: red (586–596 nm), green (525 nm), blue (465 nm), yellow (590 nm) and white. The walls of each compartment had mirror-like surfaces to optimize lighting. Three 500 ml Erlenmeyer flasks with 200 ml culture medium and an initial cell concentration of 2 g DW/l were placed in each compartment. Light intensity for all wavelengths was 52 μmol m−2 s−1. The cultures were subjected to constant light i.e. 24 h/day. Additionally, control and darkness conditions were performed with 12 h day light/12 h darkness, and 24 h of darkness, respectively.
2.7. Antioxidant Capacity The Ferric Reducing Antioxidant Power (FRAP) values were determined following a previously described method [21] with some modifications. The protocol was based on the increase of absorbance due to the formation of 2,4,6-tripyridil-s-triazine (TPTZ)-Fe(II) in the presence of reducing agents. The FRAP reagent contained 2.5 ml of TPTZ (10 mM) in hydrochloric acid (40 mM), 25 ml of acetate buffer (0.3 mM pH 3.6) and 2.5 ml of iron(III) chloride (20 mM). A volume of 50 μl of extract was mixed with 950 μl FRAP reagent. The absorbance was measured at 590 nm. The FRAP values were expressed in mg of ascorbic acid (AA)/g DW, using AA as a standard in the calibration curve. The 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS) concentrations were established using a previously published protocol [22] with some modification. In order to evaluate the ability of the samples to trap the radical ABTS•+, 10 μl of extract was added to 990 μl of ABTS solution and after 30 min of reaction the absorbance at 732 nm wavelength was measured. Next, the absorbance was compared with the reference curve prepared with Trolox as a primary standard. The results were expressed in μmol of Trolox/g DW. 3. Statistical Analysis
2.3. Cell Growth Determination Cell growth was determined by vacuum filtration of culture aliquots (5 ml) for each treatment (wavelength) through a pre-dried (60 °C - 24 h) filter paper Whatman 595 (Sigma-Aldrich Bogotá, Colombia). Next, the retained sample was washed 3 times with distilled water and dried in a convection oven until it reached constant weight after 24 h at 60 °C (DW [g]).
All data are presented as the mean values ± Standard Deviation (SD) of at least two replicates. The data were analyzed by Fisher multiple comparison test (p = 0.05) using the Statgraphics Centurion XV version 15.2.05 software (Statpoint Technologies, Inc. Virginia USA). 4. Results and Discussion 4.1. Effect of Light Wavelength on Cell Growth and Substrate Uptake
2.4. Sugar Quantification To measure sucrose uptake a Shimadzu Prominence High Pressure Liquid Chromatography (HPLC) system with a pumping system LC20 CE coupled to a refractive index detector RID-10A and Solution LC software was used (Shimadzu Scientific Instruments, Inc. USA). Separation and quantification of sucrose was carried out using a reverse phase amino column RP-NH2 NUCLEODUR 100-5 (Macherey-Nagel Inc. PA. USA.) and mobile phase acetonitrile/water (79:21 v/v ratio), flow 2 ml/min, sample size 10 μl at 35 °C. 2.5. Metabolite Extraction
Cell suspension cultures of T. peruviana showed similar kinetic of cell growth in spite of different light wavelength conditions, natural photoperiod and darkness (Fig. 1). The cell suspension cultures did not show adaptation phase. The exponential phase lasted 10 days and was followed by a stationary phase (until the 16th day of cell growth) after which the cell's death started. The cell suspensions cultures were initiated with an inoculum concentration of 2.0 g DW/l, reaching maximum and minimum concentrations of biomass of 18.2 and 17.1 g DW/l, after 13 days of growth under control and green light conditions, respectively. Table 1 presents the specific cell growth rates (μ) and doubling times (td) for each lighting condition.
Biomass obtained after 23 days of incubation was freezed/dried (SYCLON-18 N) at −60 °C and 1 bar to avoid degradation of thermolabile compounds. The protocol of bioactive metabolite extraction has previously been described [19]. Briefly, 20 mg of biomass was homogenized with 2 ml of water-ethanol mixture (1:1 v:v) using Ultra-Turrax® homogenaizer T45 S5 (Janke & Kunkel GmbH & Co. KG IKA-WERK). Next, the extract was centrifuged at 4000 rpm for 10 min at room temperature and the supernatant was filtered on Whatman® qualitative filter paper grade 4 (Sigma-Aldrich Bogotá, Colombia) and stored at 4 °C until testing. 2.6. Total Phenolic Content The total phenolic content was determined using the adapted FolinCiocalteu method [20]. The extracts (50 ml) were mixed with 125 ml of Folin-Ciocalteu reagent and 400 ml of sodium carbonate solution (7.1% w/v) and the resulting solution was brought to a final volume of 1000 ml with distilled water. The mixture was stirred and stored at room temperature for 30 min in darkness. The absorbance was measured at 760 nm against a blank. Aqueous solutions of gallic acid (GA) were used for calibration and the results were expressed as mg GA/g DW.
Fig. 1. Cell growth curves of cell suspension cultures of T. peruviana under different lighting conditions. Results are presented as average ± SD (n = 2).
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Table 1 Kinetic parameters of cell suspension cultures of T. peruviana under different lighting conditions: maximum cell growth rate (μ [days−1]) and doubling time (td [days]). Lighting condition
μ [days−1]
td [days]
Control Darkness Yellow Red Blue White Green
0.2511 0.268 0.259 0.258 0.252 0.227 0.262
2.760 2.586 2.679 2.681 2.751 3.048 2.649
Fig. 2 presents the kinetics of sucrose uptake for each light wavelength. The cell suspension cultures subjected to green light and darkness showed a complete depletion of the substrate after 10 days of growth. In contrast, for the cell suspension cultures which grew under yellow, red, white light and in control conditions, sucrose concentration was close to 4.2 g/l at day 10. The highest sucrose concentration at day 10 was for the cell suspension cultures which grew under blue light condition 7.6 g/l. For all conditions at least 13 days were required to completely consume the sucrose. The time when the maximum concentration of cell growth is reached for specific light wavelength (Fig. 1) is consistent with the day of the carbon source depletion for each cell suspension culture (Fig. 2). These results indicate that sucrose was the only energy source for all conditions during the cell growth and the cell suspension cultures did not use the light energy from the diodes for the cell growth. Therefore, we were able to demonstrate that the cell suspension cultures of T. peruviana do not have the ability to utilize light as an energy source and hence present a strictly heterotrophic cell growth. 4.2. Effect of Light Wavelength on the Production of Polyphenols and Antioxidant Capacity The phenolic content, FRAP values and ABTS concentration of T. peruviana cell suspension cultures under different lighting conditions are presented in Figs. 3, 4 and 5, respectively. The results show that the production of polyphenols was affected by the type of light used during the cell growth (p b 0.05). The total phenolic content was between 7.21 and 7.91 mg GA/g DW for all light wavelengths, except for the darkness condition for which the phenolic content was 9.46 mg GA/g DW (Fig. 3). Interestingly, when comparing with the available literature, the production of polyphenols by cell suspension cultures of T. peruviana
Fig. 2. Sucrose uptake curves of cell suspension cultures of T. peruviana under different lighting conditions. Results are presented as average ± SD (n = 2).
Fig. 3. Total phenolic content in cell suspension cultures of T. peruviana under different lighting conditions. Significant differences (p b 0.05) among medias are expresses by different letters. Results are presented as average ± SD (n = 2).
is superior to the production of polyphenols by the plants cultivated in field, which exhibited a total phenolic content of 2.14 mg GA /g dry leaf [8]. The FRAP values were between 5.41 and 6.58 mg AA/g DW for all lighting conditions (Fig. 4), while the ABTS concentration was between 82.93 and 120.39 μmol Trolox/g DW (Fig. 5). It should be mentioned here that our study is the first so far, that has attempted to measure the antioxidant activity of cell suspension cultures of T. peruviana by using the FRAP and ABTS methods. Moreover, the FRAP and ABTS values have not been reported yet for other species of the Apocynaceae family. The phenolic content detected in our study was comparable with other important in vitro plant cultures. Ahmad et al. showed lower phenolic content in callus cultures of Stevia rebaudiana (0.1 mg GA/g DW) grown under different type of lights [23]. Giri et al. obtained phenolic content in cell suspension cultures of Habenaria edgeworthii between 5.28 and 14.70 mg GA/g DW [24]. Other studies have reported higher values of phenolic content and antioxidant activity. However, these high levels can be explain by using different extraction techniques which improve the recovery of phenolic compounds in 5 to 10 times [25,26]. The correlations between the matrices 1) Total Phenols and ABTS (for each type of light) and 2) Total Phenols and FRAP (for each type of light), were calculated from the Pearson Product-Moment Correlation Coefficient (PPMCC). The values of the PPMCC for the phenolic content versus ABTS and the phenolic content versus FRAP values were very close to one (Table 2). Such high values of PPMCCs indicate positive and perfect correlation between the two variables. Therefore, it is expected that when one variable increases (i.e. phenolic content), the other variable (ABTS or FRAP) increases in a proportional rate.
Fig. 4. Ferric Reducing Antioxidant Power (FRAP) of cell suspension cultures of T. peruviana under different lighting conditions. Different letters indicate significant differences between medias (p b 0.05). Results are presented as average ± SD (n = 2).
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with the current literature for other plant species. Nonetheless, further studies would be required to establish the enzymatic and molecular mechanisms involved in the changes in polyphenols production under light and darkness conditions. 5. Conclusions
Fig. 5. The 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS) capacity of cell suspension cultures of T. peruviana under different lighting conditions. Different letters indicate significant differences between medias (p b 0.05). Results are presented as average ± SD (n = 2).
Moreover, our results confirmed that the antioxidant activity of the samples was effectively conditioned by their phenolic content and not by the presence of reducing substances. Such reducing agents (i.e. citric acid and monosaccharide sugars) could lead to false positive findings. Importantly, cell suspension cultures which grew under dark condition exhibited the greatest antioxidant activity when compared to the cultures which grew under other lighting conditions (Fig. 4 and Fig. 5). These results indicate that the light has a deleterious effect on the production of phenolic compounds and the antioxidant activity of T. peruviana cultures. Some authors have reported similar findings in other plant species. Ghasemzadeh et al. [27] showed that flavonoid synthesis in the Halia bara species was improved if lower light intensities were used (310 μmol m−2 s−1) when compared to the cultures irradiated with higher light pulses (790 μmol m−2 s−1). Furthermore, the antioxidant activity determined by the 1,1-diphenyl-2 picrylhydrazyl (DPPH) methodology was greater for the leaves of Halia bentong and Halia bara species when the plants were subjected to lower intensity of light [28]. Liu et al. [10] have reported that under white and blue light conditions the production of shikonin (natural anti inflammatory phenol) was completely inhibited in the Onasma paniculatum cultures. The results were associated with the attenuation of expression of genes involved in the synthesis of phenolic metabolites. Moreover, Shohael et al. [29] have found a higher content of oxidants such as H2O2, malondialdehyde and lipoxygenase in E. senticosus somatic embryos treated with red light when compared to the embryos which developed without the light. These results were related to light sensitivity of the oxidative enzymes. Additionally, it has been shown that illuminated callus cultures of Catharanthus roseus accumulated less ajmalicine (an alkaloid with antihypertensive activity) than the cultures in darkness [30]. The authors explained this phenomenon by a possible conversion of the ajmalicine to serpentine due to enzymatic activation induced by visible light radiation. Finally, Yu et al. [31] stated that the hairy roots of Panax ginseng cultured in bioreactors showed lower biomass formation when the hairy roots were grown under different lighting conditions in contrast to the hairy roots incubated in darkness. Furthermore, the hairy roots under darkness produced higher amount of ginsenosides when compared to the irradiated samples. All together, our findings for T. peruviana cell suspension cultures are in agreement Table 2 The Pearson Product-Moment Correlation Coefficients (PPMCC) between phenolic content and antioxidant capacity of the T. peruviana cells suspension cultures under different lighting conditions. Phenolic content vs. Antioxidant activity
PPMCC
Phenolic content vs. ABTS Phenolic content vs. FRAP
0.993 0.992
Although the cell suspension cultures of T. peruviana cultured under different light wavelengths did not present a significant difference in the cell growth, they showed significant differences in sucrose rate uptake. The changes in sucrose uptake together with the cell growth indicate that the growth of the cell suspensions cultures have a strictly heterotrophic behavior. Moreover, the presence of light has a deleterious effect on the production of phenols and antioxidant capacity on in vitro cell suspension cultures of T. peruviana. Accordingly, in darkness condition there is a protective effect on the production of phenols and the higher antioxidant activity. Finally, our study established that the cell suspension cultures of T. peruviana exhibit higher antioxidant capacity than the actual plants of the same specie as well as other farmed species in vitro. Acknowledgments The authors are grateful to the Facultad de Ciencias of Universidad Nacional de Colombia, Medellin, for the support to carry out this work. References [1] T.A. Tagbor, The anti-termite properties and basic phytochemicals of eight local plants and the chemical characterisation of Thevetia peruviana (pers) k. schum in Ghana, Ph.D thesis, Kwame Nkrumah University of Science and Technology, 2009. [2] M.M. Hassan, A.K. Saha, S.A. Khan, A. Islam, S.S.U. Ahmed, Studies on the antidiarrhoeal, antimicrobial and cytotoxic activities of ethanol-extracted leaves of yellow oleander (Thevetia peruviana), Open Vet. J. 1 (2011) 28–31. [3] P.G. Kareru, J.M. Keriko, G.M. Kenji, G.T. Thiong'o, A.N. Gachanja, H.N. Mukiira, Antimicrobial activities of skincare preparations from plant extracts, Afr. J. Tradit. Complement. Altern. Med. 7 (2010) 214–218. [4] Z. Ambang, J. Ngoh Dooh, G. Essono, N. Bekolo, G. Chewachong, C. Asseng, Effect of Thevetia peruviana seeds extract on in vitro growth of four strains of Phytophthora megakarya, Plant Omi. J. 3 (2010) 70–76. [5] L. Gata-Gonçalves, J.M.F. Nogueira, O. Matos, R.B. de Sousa, Photoactive extracts from Thevetia peruviana with antifungal properties against Cladosporium cucumerinum, J. Photochem. Photobiol. B Biol. 70 (2003) 51–54. [6] S. Tewtrakul, N. Nakamura, M. Hattori, T. Fujiwara, T. Supavita, Flavanone and flavonol glycosides from the leaves of Thevetia peruviana and their HIV-1 reverse transcriptase and HIV-1 integrase inhibitory activities, Chem. Pharm. Bull.(Tokyo) 50 (2002) 630–635. [7] R. Newman, P. Yang, A.D. Pawlus, K.I. Block, Cardiac glycosides as novel cancer therapeutic agents, Mol. Interv. 8 (2008) 36–49. [8] G. Zibbu, A. Batra, In vitro and in vivo determination of phenolic contents and antioxidant activity of desert plants of apocynaceae family, Asian J. Pharm. Clin. Res. 5 (2012) 75–83. [9] G. Zibbu, A. Batra, Thevetia peruviana (Pers.) Schum.: a plant with enormous therapeutic potential, J. Pharm. Res. 4 (2011) 4461–4464. [10] Z. Liu, J.L. Qi, L. Chen, M.S. Zhang, X.Q. Wang, Y.J. Pang, Y.H. Yang, Effect of light on gene expression and shikonin formation in cultured Onosma paniculatum cells, Plant Cell Tissue Organ Cult. 84 (2006) 39–46. [11] L. Chun-zhao, G. Chen, W. Yu-chun, O. Fan, Effect of light irradiation on hairy growth and artemisinin biosynthesis of Artemisia annua L, Process Biochem. 38 (2002) 581–585. [12] J. Zhao, W.H. Zhu, Q. Hu, Effects of light and plant growth regulators on the biosynthesis of vindoline and other indole alkaloids in Catharanthus roseus callus cultures, Plant Growth Regul. 33 (2001) 43–49. [13] M.C. Wu, C.Y. Hou, C.M. Jiang, Y.T. Wang, C.Y. Wang, H.H. Chen, H.M. Chang, A novel approach of LED light radiation improves the antioxidant activity of pea seedlings, Food Chem. 101 (2007) 1753–1758. [14] M.E.-B. Younis, M.N.A.-G. Hasaneen, H.M.M. Abdel-Aziz, An enhancing effect of visible light and UV radiation on phenolic compounds and various antioxidants in broad bean seedlings, Plant Signal. Behav. 5 (2010) 1197–1203. [15] G. Samuolienė, R. Sirtautas, A. Brazaitytė, P. Duchovskis, LED lighting and seasonality effects antioxidant properties of baby leaf lettuce, Food Chem. 134 (2012) 1494–1499. [16] M. Arias, M.J. Angarita, J.M. Restrepo, L.A. Caicedo, M. Perea, Elicitation with methyljasmonate stimulates peruvoside production in cell suspension cultures of Thevetia peruviana, In Vitro Cell Dev. Biol. Plant 46 (2009) 233–238. [17] P. Odhiambo, M. Makobe, H. Boag, A. Muigai, H. Kiesecker, In vitro regeneration of Thevetia peruviana Pers. K. Schum, Family Apocynaceae, Sci. Conf. Proc. (2010) 81–91.
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