Approach for elucidating the structure of phenolics in tomato

Abstract

Phenolics are key components for plant survival and have important medicinal uses for human beings. Nevertheless, the approach for identifying all four types of phenolics is still in need by now. In this study, we developed an approach to elucidate all these phenolics, including anthocyanins, flavanols, chlorogenic acid-related compounds and hydroxycinnamate derivatives in plants, taking purple tomato as material. We identified 58 phenolics and addressed their structures. Among them, there were 15 anthocyanins, 30 flavanols, 8 chlorogenic acid-related compounds and 5 hydroxycinnamate derivatives. In tomato, 29 out of 58 phenolics were not reported previously. By quantification, it was found that the total phenolics were more concentrated in the fruit peel, compared to plant leaves and the fruit flesh. Based on these phenolics, we proposed five possible pathways in flavonols, anthocyanins and hydroxycinnamate derivatives. These findings provide new insight into identification and metabolic variation of phenolics in plants.

Keywords: Elucidation; Identification; LC-MS; Phenolics; Separation.

Graphical abstract

Fig. 1

The samples were harvested from the tomato plants of the tomato cultivar Indigo Rose. At the red ripe fruits stage, the tomato fruits had the red and purple color on the fruit surface and were harvested from plant (a). The fruits (b) were cut (c) and the fruit peel was separated from the fruit flesh. The leaf from the plant was harvested and had the green and purple colors (d). The scale bar is 1 cm. The three sections of the work flow for the analysis of the phenolics (e). The three sections include the components separation, structure identification and metabolic pathway analysis. The components separation is composed of sampling, grinding of the samples, extraction of the components and the analysis of the components. The structure identification contains the ultraviolet-visible (UV–Vis) analysis, precursor ion and daughter ion identification for all the components. The metabolic pathway analysis includes the pathway classifications in anthocyanins, flavanols, chlorogenic acid-related compounds and hydroxycinnamate derivatives for all the components. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Liquid chromatograms were analyzed in positive mode by quadrupole time-of-flight mass spectrometry (a) and at 535 nm by photo-diode array (PDA) (b), as well as in negative mode by quadrupole time-of-flight mass spectrometry (c) and at 350 nm by PDA (d). The liquid chromatogram conditions were as follows: The mobile phase was composed of two elution solvents. Mobile phase A was acetonitrile supplemented with 3 % formic acid, and mobile phase B was water mixed with 5 % formic acid. The chromatographic run was at a constant flow rate of 0.15 ml/min. At the start, the proportion of mobile phase A was set at 2.5 % and maintained for 1 min. Subsequently, within a span of 2 min, the percentage of solvent A increased from 2.5 % to 12 %. This was followed by a more gradual change, where it rose from 12 % to 24 % over 17 min. Then, over a period of 13 min, it further advanced from 24 % to 35 %. Next, in 3 min, the proportion of solvent A changed from 35 % to 45 %. Finally, within 0.5 min, it went back to 2.5 % and was held at that level for an additional 0.5 min. The temperature of the column was maintained at 25 °C, while that of the FTN was kept at 20 °C. The wavelength range was set from 190 nm to 800 nm for PDA. Mass was set in positive ion mode for anthocyanin identification and in negative mode for other phenolics. The ESI source parameters were set as follows: The electrospray capillary voltage was set at 2.5 kV. The source temperature was maintained at 100 °C. Meanwhile, the desolvation temperature was set at 250 °C, to facilitate the formation of gas-phase ions. The desolvation gas flow rate was 600 L/h, and the cone gas flow was set at 50 L/h. The analyzer was operated in the sensitivity mode. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

The metabolic pathways were proposed for the 29 unreported components in tomato. Their contents in fruit peel, fruit flesh and leaf were also displayed beside the components in the metabolic pathway.

Fig. 4

The five possible metabolic pathways were proposed with detailed structure information in tomato. There were two possible metabolic pathways in flavanols, two possible metabolic pathways in anthocyanins and one possible metabolic pathway in hydroxycinnamate derivatives. The compound displaying a red color was the previously unreported one. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)