Anthocyanin and spermidine derivative hexoses coordinately increase in the ripening fruit of Lycium ruthenicum
Introduction
Solanaceae species Lycium ruthenicum (Lr) is a halophyte mainly distributed in northwestern China. Lr is also known as “black goji” because its fruit is abundant in anthocyanin. Lr fruit has been extensively utilized as a medicinal herb because of its health-promoting properties conferred by its abundant bioactive components such as anthocyanin, flavonoids, hydroxycinnamic acid derivatives, and other phenolic compounds (Kosar, Altintas, Kirimer, & Baser, 2003).
Thirty-seven anthocyanins have been reported in Lr fruits (Wang, Li et al., 2018), among which petunidin glycosides are dominant components (Chen et al., 2013, Zheng et al., 2011). Studies have shown that anthocyanins from Lr fruits have numerous activities, such as reducing inflammation and accelerating fat decomposition (Yin & Wu, 2017), neuroprotection of PC12 cells (Tang et al., 2017), and maintaining intestinal health (Yan et al., 2018). The structure of Lr anthocyanins show multiple modifications such as glycosylation and acylation, which enhance the stability of compounds (Sadilova et al., 2006, Tang and Giusti, 2018) during human or animal consumption.
Other flavonoids (Zhang, Chen, Zhao, & Xi, 2016), diverse polysaccharides (Peng et al., 2012, Lv et al., 2013), and alkaloids (Wu et al., 2016, Nzeuwa et al., 2017) have also been identified in Lr fruits. Particularly, 24 spermidine alkaloids were reported in Lr fruits. Most contain caffeoyl or dihydrocaffeoyl moieties and some have glycosylation (named as spermidine derivative hexoses in this paper) (Wang H. et al. 2018). Different types of spermidine alkaloids have been identified in other plants such as Arabidopsis seed (Luo et al., 2009) and lulo (Solanum quitoense Lam.) fruit (Forero, Masatani, Fujimoto, Cou-Barrera, Peterson, & Osorio, 2016). These molecules may defend against pathogens in plants (Muroi et al., 2009) and inhibit HIV-1 protease according to in vitro assays (Ma, Nakamura, & Hattori, 2001).
Glycosylation of the anthocyanin and spermidine derivative hexoses mentioned above is catalyzed by glycosyltransferase. This enzyme involved in plant secondary metabolism transfers a sugar moiety from an active donor such as a UDP-sugar to various metabolites to increase their stability during transportation or storage (Jones & Vogt, 2001). In the biosynthesis of anthocyanin, glycosylation performed by glucosyltransferase at the 3-O-position is the first step in increasing anthocyanin stability and is a prerequisite for further modifications (Springob, Nakajima, Yamazaki, & Saito, 2003).
Metabolite profiling is useful for investigating compound accumulation and fruit development. For instance, in tomato, metabolites profile and gene regulatory network analyses revealed new candidate genes controlling fruit composition and development (Mounet et al., 2009); Lombardo et al. (2011) determined the peach metabolic programs by comparing the metabolite profiles during the early development and postharvest ripening stages, and then explored the relationships between these profiles with some regulatory genes. Studies of metabolite profiles in Lr have been limited to tracing fruits from different geographical origins (Wang, Yan et al., 2018) or comparing different genotypes/species (Wang et al., 2018, Zhang et al., 2016). No studies have examined Lr fruit metabolites during development and explored the detailed mechanisms.
In this study, we performed ultra-high-performance liquid chromatography (UHPLC) with tandem mass spectrometry (MS) to provide full MS and MS2 data (MS/MS) for metabolites analysis during 4 developmental stages of Lr fruit. The results showed that anthocyanin and spermidine derivative hexoses increased synergistically during fruit ripening, and that glycosyltransferase HG27071 used both anthocyanidin and spermidine derivative as substrates to produce glycosides. The detailed metabolic dynamics in L. ruthenicum ripening fruits were also evaluated.
Section snippets
Chemicals
Acetonitrile and formic acid used in UHPLC-MS/MS were LC/MS-grade. Both were purchased from Thermo Fisher Scientific (Waltham, MA, USA). HPLC-grade acetonitrile and formic acid were purchased from New Material Tech Co. (Guangzhou, China) and Aladdin (Shanghai, China), respectively. Water was generated from a Milli-Q system (Millipore, Billerica, MA, USA). Petunidin and petunidin-3-O-glucoside standard were purchased from Yuanye company (Shanghai, China). Dihydrocaffeoyl-caffeoyl spermidine was
Tentative compound identification of L. ruthenicum fruits
A total of 490 compounds with valid MS data were obtained after pre-treatment by Compound Discoverer 2.1. The formula, molecular weight, retention time (RT), and peak area of each compound in the 4 developmental stages are shown in Supplementary Table 1. Among them, 406 compounds had MS2 data. Combined local/online database (mzCloud, mzVault, MassLists, and ChemSpider) searching using the software with compounds previously described in the literature identified 49 compounds (Supplementary Table
Conclusion
Lr fruit is health-promoting functional food that has been widely used in China, particularly by Tibetans, for thousands of years because of its high levels of bioactive compounds. Different types of metabolites that showed various activities were found in the fruit. We profiled the metabolites during the dynamic process of fruit development and ripening. Five categories of compounds were identified using UHPLC-MS/MS, and 9 compounds, mainly including anthocyanin and spermidine derivative
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by grants from National Key R&D Program of China [2018YFD1000607]; National Natural Science Foundation of China [31770334, 31470391]; Youth Innovation Promotion Association CAS [2015286]; Science and Technology Program of Guangzhou [201904010167]; Ningxia Agricultural Comprehensive Development Science and Technology Project [NTKJ2018-07]; and Chinese Academy of Sciences [XDA13020604].
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