Effect of the application of elicitors on the amount of steviol glycosides in Stevia rebaudiana Bertoni. 1
Vázquez-Hernández Ma. Cristina. 1Alvarado-Mariana Aurora, 2Aguirre-Becerra Humberto. 3Soto-Zarazúa Genaro Martin. 2Feregrino-Pérez Ana Angélica. 1Guevara-Gonzalez Ramón G.
1 Cuerpo académico de Biosistemas. Facultad de Ingeniería. Campus Amazcala. Universidad Autónoma de Querétaro. Cuerpo académico de Bioingeniería. Facultad de Ingeniería. Campus Amazcala. Universidad Autónoma de Querétaro. 3 Cuerpo académico de Sistemas Embebidos y Aplicaciones. Facultad de Ingeniería. Campus Amazcala. Universidad Autónoma de Querétaro. * Corresponding author
[email protected], *
[email protected] 2
Abstract— Interest in Stevia rebaudiana Bertoni as a natural non-caloric sweetener has increased. To cope with biotic and abiotic environmental stresses, plants have developed a defense system that can be triggered by applying stress-simulating substances (elicitors or metabolic modifying factors) that may increase production of secondary metabolites of commercial interest such as steviol glycosides. Elicitors include salicylic acid, hydrogen peroxide, oligosaccharides, and chitosan, among others. The aim of this research was to analyze the amount of steviol glycosides in S. rebaudiana leaves due to a foliar application of hydrogen peroxide, chitosan and salicylic acid. The best result was obtained with treatment elicited with salicylic acid (0.1 mM) Keywords— Stevia; steviol glycosides; elicitors
I. INTRODUCTION The consumption of Stevia rebaudiana extract (Stevioside and Rebaudioside A) has increased in the food and beverage industry due to the nutritional and pharmaceutical importance it has received, in addition to its low-caloric sweetening power 100-300 times greater than saccharose [1], [2]. Several studies have proved its anti-inflammatory, anti-cariogenic, antihypertensive, anti-carcinogen, and blood glucose lowering properties [1], [2], [3]. A total of 8 diterpenoid esteviol glycosides known as Esteviol has been isolated from this extract, consisting of a white-yellow powder obtained after processing the dried leaves by physical-chemical methods [4]. Plants have developed defense mechanisms that give them an advantage for survival [5]. Elicitors are substances that may induce the production of secondary metabolites in plants (phenolic compounds, flavonoids, etc.) that create a similar effect of biotic and abiotic stress conditions [6]. S. rebaudiana synthesizes in its leaves, several specialized metabolites, including more than 30 steviol glycosides (SGs) in different concentrations [1], [2]. These compounds are responsible for
the high sweetening power and low caloric content. In this research, elicitors (Salicylic Acid, Hydrogen Peroxide and Chitosan) were applied to stevia plants grown under greenhouse conditions, and the effect on the content of steviol glycosides in leaves was determined [6], [7]. II. MATERIALS AND METHODS A. Biological material A total of 60 plants of Stevia rebaudiana Bertoni variety Morita II were used. Seedlings were purchased from Ing. Adriana Morales, owner of a greenhouse located in the city of Cuernavaca, Morelos, Mexico. The seedlings were grown from certified seed produced in Paraguay in 2008 and reproduced in Mexico with a growth of 3 months, a height of 10-12 cm and 10 to 15 true leaves. B. Cultivation conditions Crop consisted on 50 plants of Stevia rebaudiana var. Morita II (plus 10 plants for propagation by cutting), distributed in completely randomized blocks. At the beginning of the experiment, new plants were disinfected with a 2g/L copper sulfate solution, and an organic insecticide (Azanim 3% CE, brand BIOKRONE) applied at a concentration of 250 ml/100L of H2O to ensure innocuousness of cultivar and avoid generation of false-positive results [7]. Peat moss - perlite (50:50) substrate was used in order to have a good aeration and humidity in the roots. Plants were placed in pots of 1 L. Nutrition at the beginning of the crop was with a 50% Steiner solution, with an electrical conductivity of 1.5 and pH of 5.5 [3], [7].
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C. Elicitation Stevia plants were elicited with hydrogen peroxide (2 mM, 20 mM, and 200 mM), salicylic acid (0.1 mM, 1 mM, 6.7 mM), chitosan (10 μg/ml, 670 μg/ml, and 1000 μg/ml), and distilled water as control. Weekly applications for a period of four weeks by means of foliar sprinkling were made. Samples were collected 24 h after the last application [7]. D. HPLC analysis For extraction, purification and quantification of stevia extracts, the methodology proposed by [4] was used. S. rebaudiana freeze-dried leaves (1 g) were mixed with 10ml of a mobile phase (Acetonitrile: Water 80:20) during 40 min. Rebaudioside A was identified and quantified by the highpressure liquid chromatography method (HPLC, Hewlett Packard, 1100 model), in which 20 μL were taken and were injected into the chromatography equipment. The used column was Zorbax Carbohydrate with a flow of 0.1 ml/min [4]. E. Statistical analysis Experimental design for the evaluation of DNA methylation levels was a completely randomized block with three replicates. An ANOVA test was performed for the statistical significance analysis (P=0.05, and 0.01) using GraphPad PRISM 6 software, Windows 10 version. Dunett’s multiple comparison tests were used at a significance level of 5% and 0.01% (p < 0.05) [6]. III. RESULTS AND DISCUSSION
Fig. 1. Quantification of Rebaudioside A in freeze-dried leaves. T1: AS 0.1 mM, T2: AS 6.7 mM, T3: QN 10 mg/ml, T4: QN 1000 mg/ml T5: H2O2 2 mM, T6: H2O2 200 mM.
Phenological changes were observed due to the application of elicitors (Figure 2). Treatments 1 and 6 had a significant difference on number of leaves compared against control. These results indicate that foliar application of salicylic acid at a concentration of 0.1mM and hydrogen peroxide at 200mM induces an increase in the number of leaves, which is favorable to producers. In addition, the ratio of steviol glycosides (Rebaudioside A) is also increased when these elicitors are applied.
Results show significant differences in the content of Rebaudioside A. Figure 1 shows significant differences in the effect of elicitation compared against the control treatment. Treatment 1 and Treatment 2 show an approximate increase of 20 to 25% production of Reabudioside A. Treatment with Chitosan at a concentration of 10 mg/ml decreased the content of this steviol glycoside by 25%, in addition to number of leaves decreased by 45% compared to control (Figure 2). Since these results are unfavorable for the purpose of this research, it is suggested to avoid this elicitor. It has been reported that the elicitation of stevia (with peroxide of hydrogen, salicylic acid, among others) has a positive effect on the production of secondary metabolites [1]. This agrees with results obtained in this research since the effect of via foliar application of elicitors in low concentrations to stevia plants induces the production of Rebaudioside A, a molecule of great economic importance in the food and beverage industry as a substitute for sugar.
Fig. 2. Number of leaves in plants of S. rebaudiana Bertoni var. Morita II. T1: AS 0.1 mM, T2: AS 6.7 mM, T3:QN 10 mg/ml,. T4: QN 1000 mg/ml, T5: H2O2 2 mM, T6: H2O2 200 mM).
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IV. CONCLUSIONS The content of Rebaudioside A in stevia leaves is significantly increased with the use of elicitors. The best result in the content of steviol glycosides and number of leaves per plant was treatment with salicylic acid (0.1 mM). This methodology can be used to improve yield of steviol glycosides in plants of S. rebaudiana Bertoni var. Morita II. V. ACKNOWLEDGMENT Our most sincere thanks to DGEST for the financing of this project, to CONACYT for the scholarship granted, and to Dr. Mario González Chavira INIFAP campus Bajio.
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