Development of an efficient high-performance thin layer chromatography method for determination of jasmonic acid in leaf tissue of Stevia rebaudiana (Bertoni) Bertoni

Abstract Determination of endogenous levels of jasmonic acid (JA) is essential, as it plays a pivotal role in the physiological processes during a plant’s life cycle. A high performance thin layer chromatography (HPTLC) method was developed for the detection and quantification of JA in leaf extracts of medicinal plant, Stevia rebaudiana (Bertoni) Bertoni. The separation was achieved using the solvents ethyl acetate–benzene (1:1, v/v) as the mobile phase, followed by scanning and quantification at 295 nm. Densitometric analysis of leaf extract resulted in compact spots for JA at Rf = 0.45 ± 0.02. The linear regression analysis showed good relationship with r value. The recovery rate of JA indicated good reproducibility and repeatability of the assay. The statistical analysis proved the reproducibility of the method; therefore, it can be employed for routine quantification of JA in different tissue samples of S. rebaudiana and may also be extrapolated to other biological samples.


Introduction
Jasmonic acid (JA) and similar compounds (jasmonates) play a diverse role in plants. They mediate several developmental processes and also get activated as part of the plant's defence response (Pieterse et al. 2012). Beneficial micro-organisms have shown to modulate the JA signalling for establishment of long-term association with their host plant (Gimenez-Ibanez et al. 2016). Jasmonates are known to act as conserved elicitors of plant secondary metabolism (Zhao et al. 2005). Thus, elevated JA levels triggered by beneficial microorganisms can lead to concerted activation of the secondary metabolite pathways. Determination of JA is thus essential to study its physiological and biological activity during beneficial plant microbe interactions.
Stevia rebaudiana (Bertoni) Bertoni, a sweet herb is known to produce low-calorie sweetening glycosides, especially stevioside and rebaudioside-A (Brandle & Telmer 2007;Li et al. 2009). However, the licorice like aftertaste imparted due to the higher stevioside to rebaudioside-A ratio does pose a challenge. Different concentrations of JA have been found to play an important role in alteration of this ratio (Peynevandi et al. 2014). It has also been the prime aim of plant breeders to enhance commercial use of this plant. In view of these facts, it is imperative to develop an efficient method for quantification of JA in S. rebaudiana.
Endogenous levels of JA have been determined using enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and gas chromatography/mass spectrometry (GC/ MS), involving development of monoclonal antibodies and derivatisation, respectively. These methods are tedious and require expensive solvents. Thin layer chromatography (TLC) has been shown to separate JA in culture filtrate of a fungus, Lasiodiplodia theobromae (Dhandhukia & Thakkar 2008). However, there are no reports of JA quantification in S. rebaudiana as yet. Thus, in continuation with the previous study on interaction of beneficial micro-organisms; an endophytic fungus Piriformospora indica (Pi) and rhizobacteria Azotobacter chroococcum (Az) in association with S. rebaudiana (Kilam et al. 2015), the paper reports the development of an efficient HPTLC method for JA estimation.

Method development
An HPTLC method was developed for quantification of JA in the leaves of medicinal plant, S. rebaudiana. The developed method was found to be effective in separation of the desired compound on the TLC plate. The 3D patterns obtained from standard (JA) showed that the peaks obtained at R f 0.45 ± 0.02 were superimposable with the R f values of the sample extracts.

Method validation
The specificity of the method was ascertained by comparing the peak purity of the standard JA and test samples. In situ UV spectra between the standard (JA) and samples showed a good correlation. The peaks were super imposable, thus confirming the peak purity. The accuracy of the method was expressed as per cent recovery of JA in the samples, which ranged 96.50-104.9%, showing a good recovery rate. The percentage recovery values higher than 100% indicated that there was no interference of the excipients present in the formulation (Bhattacharyya et al. 2013). Intra-day and inter-day precisions (n = 3) for JA were found to be 0.07-0.25% and 0.03-0.17%, respectively. The linearity of the phytohormone JA was validated by linear regression equation and correlation coefficient. The calibration curve for JA, in the range of 40-280 μg/band, was found to be linear. Regression equation and r value were observed to be y = −161.098 + 22.809 * x and 0.998, respectively, showing a good linear regression for the developed method. The adherence of the system to Beer's law was validated by high correlation coefficient and SD for intercept value. LOD and LOQ values for JA were found to be 10 and 20 μg/band, respectively, indicating a good sensitivity for quantification of this compound. The robustness of the method was indicated by no significant changes in the R f values and band patterns. The percentage RSD was < 2% for slight changes to the mobile-phase composition, mobile-phase saturation and volume. The results obtained for all validation parameters used in the present study were found to be within the recommended limits.

HPTLC analysis of samples
The utility of the method was shown by applying this method for quantification of JA in control and treated (Pi, Az, Pi + Az) groups of medicinal plant, S. rebaudiana (Bertoni) Bertoni. All of the four evaluated samples were found to contain JA (Table 1). Thus, the developed method provides an economical and sensitive assay for detection and estimation of JA. It can be effectively used in routine estimation of JA in biological samples.

Conclusions
Plant interaction with beneficial microorganisms has shown JA to be a crucial component of plant processes; JA signalling pathway suppresses the early defence responses and activates the secondary metabolite biosynthesis. Thus, a rapid, sensitive, reliable and affordable HPTLC method has been developed for detection and quantification of JA in leaf tissue of S. rebaudiana. The method was suitably validated and used to analyse the leaf samples of plants treated with beneficial microorganisms. This simple method developed and reported in the present study, can be used as an analytical tool for detection of JA without derivatisation of samples. Further extrapolation of this work on other tissues/plants may prove it to be a versatile tool for estimation of JA in medicinal plants. the same lower case letters within each column are not significantly different at p ≤ 0.05 as determined by one-way aNoVa and duncan's multiple comparison test. Values are the treatment means ± sd, N = 3. Pi, P. indica-inoculated; az, A. chroococcum-inoculated; Pi + az, co-inoculated with P. indica and A. chroococcum.