Effect of salicylic acid and silver nitrate on rutin production by Hyptis marrubioides cultured in vitro/Efeito do acido salicilico e nitrato de prata na producao de rutina por Hyptis marrubioides cultivada in vitro.
Hyptis marrubioides, which is native to the Brazilian Cerrado, is known as field-mint and has been used to treat gastrointestinal infections, skin infections, pains, and cramps in folk medicine; its essential oil composition has been extensively studied. (MCNEIL et al., 2011).
Several in vitro propagation strategies have been employed to increase the productivity of natural compounds that have great social and economic value (MARASCHIN et al., 2002). One of these strategies has been to use elicitors (chemical, physical, or biological agents) to alter metabolic routes, which affects the resulting molecules qualitatively and quantitatively (DJILIANOV et al., 2005; GIRI and ZAHEER 2016; RAMIREZ-ESTRADA et al., 2016).
This study has focused on enhancing rutin production in H. marrubioides cultured in vitro. To this end, two kinds of exogenous elicitors salicylic acid (SA) and silver nitrate (SN) were used at 30 [micro]M or 60 [micro]M in two different experiments.
H. marrubioides seeds were obtained at the experimental field of the Laboratory of Plant Tissue Culture of Instituto Federal de Educacao, Ciencia e Tecnologia Goiano, Campus Rio Verde. A voucher specimen (HRV71) was deposited at the Herbarium of the Institute (Herbarium HRV).
The seeds were surface-sterilized with two systemic fungicides--0.2% Bendazol (carbendazim) and 0.2% Alterno (tebuconazole)--for 1 h, subsequently treated with 1% sodium hypochlorite for 30min, and rinsed thrice with sterile distilled water. Next, the seeds were cultured in MS medium (MURASHIGE & SKOOG, 1962) supplemented with 30g [L.sup.-1] sucrose and 3.5g [L.sup.-1] agar; the pH was adjusted to 5.8 before autoclaving. The cultures were maintained in a growth room at an average temperature of 23 [+ or -] 1[degrees]C for 30 days, under a 16-hour photoperiod. Two treatments were conducted, and two kinds of exogenous elicitors, salicylic acid (SA) or silver nitrate (SN), at 30 [micro]M or 60 [micro]M, were employed. For procedure I, the plants were subcultured in glass flasks containing 50mL of MS medium for 10 days and then placed in a new flask containing MS medium and the desired elicitor for 20 days. The control plants were placed in fresh MS medium without elicitor. For procedure II, the plants were subcultured in glass flasks containing 50mL of MS medium. On day 20, the desired elicitor was added to the cultures at the desired concentration and was allowed to remain in contact with the medium for three or six days. Four flasks with five explants were used for each treatment, which amounted to 20 flasks and 100 plants in procedure I including the control group. For procedure II, a total of 40 flasks and 200 plants were used including the control. In both experiments, the plants were maintained in a growth room at 23 [+ or -] 1[degrees]C, under a 16-hour photoperiod. After treatment with the desired elicitor, the plants were harvested. Next, the shoot length, expanded leaf number, and fresh mass were measured, and the biomass was dried at 35[degrees]C until constant weight to evaluate the dry mass. The dry H. marrubioides plants were extracted with 4mL of HPLC grade methanol by ultrasonic-assisted extraction (30min), filtered through 0.2-[micro]m PTFE filter, and used for HPLC analysis. These procedures were performed in triplicate. The HPLC-DAD analysis for rutin quantitation was carried out on a Shimadzu Prominence LC-20AD binary system equipped with a DGU-20A5 degasser, an SPD-20A series diode array detector, a CBM-20A communication bus module, an SIL-20A HT autosampler, and a CTO-20A column oven (Shimadzu).The stationary phase was a Gemini ODS column (250 x 4.6mm, 5 [micro]m; Phenomenex) equipped with a pre-column; the mobile phase was C[H.sub.3]OH/[H.sub.2]O/HOAc (5:94.9:0.1, v/v/v) delivered in a linear gradient until 100% C[H.sub.3]OH was reached within 30 min, followed by 10-min elution with 100% C[H.sub.3]OH. A total of 20min was allowed for the system to return to the initial conditions. The flow rate was 1.0mL [min.sup.-1]; the injection volume was set at 20 [micro]L; and UV detection was set at 254nm and 40[degrees]C. A standard curve was plotted for different concentrations (0.063mg m[L.sup.-1] to 0.500mg m[L.sup.-1]) of authentic rutin in methanol (HPLC grade, J. T. Baker); each point was measured in triplicate. Rutin was quantified on the basis of the peak area as compared to the rutin calibration curve. The obtained regression equation was y = 4.0 x [10.sup.7]x-69442 with a correlation coefficient ([R.sup.2]) of 0.9998. The external standard rutin was acquired from the standard bank of the Natural Products Group of Universidade de Franca. Statistical analysis was accomplished with the Sisvar 5.3 software (FERREIRA, 2011). The experiment was conducted for each treatment in a completely randomized design with four replications. The averages were compared by the Scott-Knott test at 5% probability.
According to the HPLC-DAD analysis, procedure I, during which the seedlings were cultured in MS medium for 10 days followed by culture in MS medium containing the desired elicitor for 20 days, led to 1.28 times higher rutin content in the H. marrubioides methanolic extracts in the presence of SN (30 [micro]M) as compared to the untreated culture (Table 1). In contrast, for procedure II, during which the seedlings were cultured in MS for 20 days followed by addition of the desired elicitor for three or six days, SA (30 [micro]M) enhanced rutin production by 16.56-fold as compared to the control, whereas SN (30 [micro]M) increased the rutin content by 1.17-fold (Table 1). Our results agreed with the data obtained by PEREZ et al., (2014) in their study of the effect of chemical elicitors on peppermint (Mentha piperita) plants. According to the latter authors, plants treated with SA at 0.5 and 1mM had increased rutin production as compared to non-treated plants (PEREZ et al., 2014). In the same way, bean sprouts (Phaseolus vulgaris L.) treated with SA presented increased rutin content (41-fold) as compared to controls (MENDOZA-SANCHEZ et al., 2016). HOU et al. (2015) developed an in vitro regeneration system using the buckwheat species Fagopyrum esculentum and Fagopyrum tataricum. The authors detected the highest rutin content (5.01mg g [FW.sup.-1]) in regenerated plantlets in F. tataricum treated with SA for 24h. In contrast, rutin did not accumulate significantly in F. esculentum (HOU et al., 2015).
Elicitation with silver nitrate (SN) also provided a positive response in the case of Sussurea medusa suspension cultures with increasing the concentration of the flavones jaceosidin and hispidulin. The maximumjaceosidin and hispidulin yields were 49.90 and 4.95mg [L.sup.-1] after treatment with SN at 0.01mmol [L.sup.-1] on the inoculation day, respectively (ZHAO et al., 2005).
Parameters such as concentration, selectivity, length of exposure to the elicitor, culture age, and nutrient composition can affect the elicitation process (NAMDEO 2007).
Rutin production stimulation might be associated with defense responses promoted by selected elicitors. Indeed, some authors consider that SA is a phytohormone that participates in plant defense reactions, to induce an acquired systemic response, whereas SN can inhibit ethylene biosynthesis. (CURTIS et al., 2004; AL-KHAYRI & AL-BAHRANY, 2001; YELDA et al., 2005).
Compared to the controls, the variables elicitor type, elicitor concentration, and length of exposure to the elicitor did not affect the H. marrubioides growth parameters shoot length, leaf number, or average fresh and dry weight during procedures I and II (Table 2 and Table 3).
In contrast, (BRANDAO, 2014) described decreased growth of Alternanthere tenella colla plants treated with AS concentrations of up to 400 [micro]M. However, explant inoculation followed a different procedure from the protocol used in our study: (BRANDAO, 2014) inoculated the explant directly in the medium containing the elicitor, whereas we inoculated the explants in fresh medium and only transferred them to the medium containing the elicitor 10 days later. Therefore, in our case, any possible toxic elicitor effect on the plant may have been reduced or avoided because the seedling was already established when it came into contact with SA or SN (BRANDAO et al., 2014).
These results provide a theoretical basis for the use of the abiotic elicitors SA and SN to favor H. marrubiodes growth in vitro and to obtain increased rutin content in the plant, considering that previous studies on the use of chemical elicitors in in vitro cultures of H. marrubioides are non-existent.
This study was financed in part by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP). The authors thank Fundacao de Amparo a Pesquisa do Estado de Goias (FAPEG) for a fellowship to RCNP.
DECLARATION OF CONFLICTING INTERESTS
The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
All authors contributed equally for the conception and writing of the manuscript. All authors critically revised the manuscript and approved of the final version.
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Rita Cassia Nascimento Pedroso (1)(iD) Nubia Angelica de Avila Branquinho (1)(iD) Alessandra Cristina Boffino de Almeida Monteiro Hara (1)(iD) Fabiano Guimaraes Silva (1)(iD) Luis Claudio Kellner Filho (2)(iD) Mareio Luis Andrade Silva (2)(iD) Wilson Roberto Cunha (2)(iD) Patricia Mendonca Pauletti (2)(iD) Ana Helena Januario (2) * (iD)
(1) Instituto Federal de Educacao, Ciencia e Tecnologia Goiano, Rio Verde, GO, Brasil.
(2) Nucleo de Pesquisa em Ciencias Exatas e Tecnologicas, Universidade de Franca (UNIFRAN), Av. Dr. Armando Salles Oliveira, 201, 14404-600, Franca, SP, Brasil. E-mail:firstname.lastname@example.org. * Corresponding author.
Received 04.03.18 Approved 12.17.18 Returned by the author 01.18.19 CR-2018-0278.R1
Table 1--Effect of abiotic elicitor type and concentration on rutin production (mg [g.sup.-1] of dry weight) in Hyptis marrubioides seedlings. Evaluation time (days) Treatments 3 Rutin teor Salicylic acid 0.3812 [+ or -] 0.017 (1) A (2) 30 [micro]M Salicylic acid 0.2392 [+ or -] 0.007 E 60 [micro]M Silver nitrate 0.3483 [+ or -] 0.014 B 30 [micro]M Silver nitrate 0.2742 [+ or -] 0.003 D 60 [micro]M Control 0.2958 [+ or -] 0.006 C CV(%) 3.66 Evaluation time (days) Treatments 6 20 Rutin teor Salicylic acid 4.8049 [+ or -] 0.321 A 0.1041 [+ or -] 0.001 D 30 [micro]M Salicylic acid 0.2902 [+ or -] 0.009 B 0.1051 [+ or -] 0.002 D 60 [micro]M Silver nitrate 0.2785 [+ or -] 0.045 B 0.2317 [+ or -] 0.013 A 30 [micro]M Silver nitrate 0.2207 [+ or -] 0.009 B 0.1250 [+ or -] 0.007 D 60 [micro]M Control 0.2902 [+ or -] 0.0034 B 0.1808 [+ or -] 0.003 C CV(%) 12.3 4.92 (1) Mean [+ or -] standard error. (2) Means followed by the same uppercase letter in the column do not differ significantly according to the Scott-Knott test at 5% probability. CV: Coefficient of Variation. Table 2--Effect of abiotic elicitor type and concentration on H. marrubioides seedling shoot length and leaf number. Evaluation time (days) Treatments 3 6 Average shoot length (cm) Salicylic 6.620 [+ or -] 0.364 (1) 7.500 [+ or -] 0.896Aa acid 30 [micro]M Aa (2) Salicylic 6.780 [+ or -] 0.510Aa 6.475 [+ or -] 0.501Aa acid 60 [micro]M Silver nitrate 6.845 [+ or -] 0.339Aa 7.290 [+ or -] 0.547Aa 30 [micro]M Silver nitrate 5.235 [+ or -] 0.304Bb 7.655 [+ or -] 0.519Aa 60 [micro]M Control 7.390 [+ or -] 0.694Aa 7.405 [+ or -] 0.351Aa CV (%) 14.54 14.54 Evaluation time (days) Treatments 20 3 Average shoot Average leaf number length (cm) Salicylic 4.200 [+ or -] 0.287A 14.300 [+ or -] 1.526Aa acid 30 [micro]M Salicylic 4.085 [+ or -] 0.389A 14.700 [+ or -] 2.068Aa acid 60 [micro]M Silver nitrate 4.812 [+ or -] 0.546A 15.500 [+ or -] 1.567Aa 30 [micro]M Silver nitrate 4.800 [+ or -] 0.386A 12.350 [+ or -] 1.008Aa 60 [micro]M Control 5.412 [+ or -] 0.481A 14.400 [+ or -] 1.071Aa CV (%) 25.68 18.62 Evaluation time (days) Treatments 6 20 Average leaf number Salicylic 15.600 [+ or -] 1.665 Aa 13.950 [+ or -] 0.395A acid 30 [micro]M Salicylic 15.100 [+ or -] 1.034Aa 11.771 [+ or -] 1.079A acid 60 [micro]M Silver nitrate 16.800 [+ or -] 1.564Aa 11.550 [+ or -] 0.715A 30 [micro]M Silver nitrate 16.700 [+ or -] 1.382Aa 12.400 [+ or -] 0.534A 60 [micro]M Control 14.450 [+ or -] 0.865Aa 12.775 [+ or -] 0.479A CV (%) 18.62 14.71 (1) Mean [+ or -] standard error. (2) Means followed by the same uppercase letter in the column do not differ significantly according to the Scott-Knott test at 5% probability. CV: Coefficient of Variation Table 3--Effect of abiotic elicitor type and concentration on H. marrubioides seedling fresh and dry weight. Evaluation time (days) Treatments 3 6 Fresh weight (g) Salicylic acid 0.289 [+ or -] 0.039 0.364 [+ or -] 0.071Aa 30 [micro]M (1) Aa (2) Salicylic acid 0.312 [+ or -] 0.025Aa 0.434 [+ or -] 0.131Aa 60 [micro]M Silver nitrate 0.356 [+ or -] 0.064Aa 0.336 [+ or -] 0.033Aa 30 [micro]M Silver nitrate 0.284 [+ or -] 0.036Aa 0.362 [+ or -] 0.051Aa 60 [micro]M Control 0.273 [+ or -] 0.036 Ab 0.504 [+ or -] 0.055 Aa CV(%) 34.39 34.39 Evaluation time (days) Treatments 20 3 Fresh weight (g) Dry weight (g) Salicylic acid 0.192 [+ or -] 0.013A 0.039 [+ or -] 0.005Aa 30 [micro]M Salicylic acid 0.263 [+ or -] 0.025 A 0.039 [+ or -] 0.004Aa 60 [micro]M Silver nitrate 0.256 [+ or -] 0.039A 0.044 [+ or -] 0.005Aa 30 [micro]M Silver nitrate 0.241 [+ or -] 0.019A 0.035 [+ or -] 0.003Aa 60 [micro]M Control 0.251 [+ or -] 0.024A 0.044 [+ or -] 0.004Aa CV(%) 30.26 19.28 Evaluation time (days) Treatments 6 20 Dry weight (g) Salicylic acid 0.042 [+ or -] 0.002Aa 0.023 [+ or -] 0.001 A 30 [micro]M Salicylic acid 0.044 [+ or -] 0.006Aa 0.026 [+ or -] 0.003A 60 [micro]M Silver nitrate 0.038 [+ or -] 0.002Aa 0.027 [+ or -] 0.003 A 30 [micro]M Silver nitrate 0.042 [+ or -] 0.004Aa 0.030 [+ or -] 0.002A 60 [micro]M Control 0.051 [+ or -] 0.003Aa 0.026 [+ or -] 0.002A CV(%) 19.28 26.34 (1) Mean [+ or -] standard error. (2) Means followed by the same uppercase letter in the column do not differ significantly according to the Scott-Knott test at 5% probability. CV: Coefficient of Variation.
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|Author:||Pedroso, Rita Cassia Nascimento; de Avila Branquinho, Nubia Angelica; de Almeida Monteiro Hara, Ales|
|Date:||Feb 1, 2019|
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