The influence of ripening stage and region on the chemical compounds in mulberry fruits (Morus atropurpurea Roxb.) based on UPLC-QTOF-MS
Graphical abstract
Introduction
The mulberry (Morus atropurpurea Roxb.) is widely cultivated in China, especially in the south for its leaves and fruits. Mulberry fruits (MFs) have historically been used in traditional Chinese medicine to reduce the risk of human cancer, liver disease, obesity, diabetes, and cardiovascular disease (Huang et al., 2013, Song et al., 2009). Modern research has revealed that the biological actions of MFs are largely due to their nutrients and bioactive components, including phytochemicals, such as polyphenols (Haminiuk, Maciel, Plata-Oviedo, & Peralta, 2012).
As the functional components of MFs have gradually been reported, consumer demand for these fruits has increased, and MFs are usually eaten fresh or processed as jam, jelly, marmalade, etc. (Vijayan et al., 2014). However, fresh, fully ripened MFs are rarely commercialized because of their susceptibility to spoilage and instability in commonly used storage conditions (Tchabo, Ma, Engmann, & Zhang, 2015). Additionally, the development of MFs is usually completed within one month, and it is not uncommon to find the green, red (ripening stage) or the over-mature MFs on sale. For the processing industry, a majority of the unripened MFs are wasted during quality control. Thus, it is necessary to identify the chemical ingredients and marker compounds whose contents vary dramatically during the mulberry development stages, which enables processing companies to maximize the use of rejected fruits and also provides basic knowledge to optimize harvests (Gibson, Rupasinghe, Forney, & Eaton, 2013).
However, current literature is mainly focused on the analysis of one or several classes of compounds, such as polyphenols and polysaccharides, instead of a comprehensive identification of the chemical components in MFs (Bae and Suh, 2007, Jin et al., 2015). MFs at their maximum ripeness have been broadly studied, but there are only a few reports associated with unripe MFs (Lou et al., 2012, Mahmood et al., 2012). Liquid chromatography-mass spectrometry (LC-MS) has been used for structure elucidation and characterization of low molecular mass organic molecules in these reports; however, the LC-MS-based workflow can be time-consuming and requires human intervention at almost every step (Zhang, Li, et al., 2016). With the advantages of high resolution, high sensitivity, and accurate mass measurement, ultra performance liquid chromatography (UPLC) combined with time-of-flight (TOF) mass spectrometry (MS) has become an alternate choice for unbiased compound screening and has been used to analyze natural fruits, vegetables and herbal medicines (Stark et al., 2015, Zhang et al., 2016 & Zhang, Zhang, et al., 2016). In addition, Progenesis QI (Nonlinear Dynamics, Newcastle, UK), a small molecule discovery analysis software, provides an informatics platform to reliably identify compounds even without reference standards, which alleviates workload (Zhang, Yang, et al., 2016). By performing alignment, peak-picking, and mining of metabolomics data, this new software platform can be used to identify important molecular alterations among sample groups (Arapitsas, Langridge, Mattivi, & Astarita, 2016). Several studies have used this platform for both in vivo and in vitro experiments to identify and quantify metabolites in their multisamples (Geenen et al., 2013, Hua et al., 2016, Jing et al., 2017)
Therefore, our first aim was to quickly identify the chemical constituents in the Dashi cultivar of MFs at different ripening stages from two regions in China utilizing UPLC-QTOF-MS coupled with Progenesis QI data analysis software. Then, we used principal component analysis (PCA) to build a predicting model using orthogonal partial least squares discriminant analysis (OPLS-DA) to screen for potential marker compounds that would differentiate samples based on ripening stage and cultivation region.
Section snippets
MF samples
Two regions (Xinjiang and Jiangsu) were selected as sampling sites due to their large production of MFs. Their meteorological conditions during collection are summarized in Table 1. MFs without injury and apparent contamination were collected according to color uniformity. In total, 9 different samples from the Jiangsu region (4 ripening stages) and the Xinjiang region (5 ripening stages) were picked randomly from twenty to forty trees at each site. Each sample weighed approximately 1–3 kg. All
Metabolic profiling analysis
Once the data in negative and positive mode were separately processed on the Progenesis QI platform, all the matching components were listed for further verification. Fig. 1 displays the negative and positive ion base peak intensity (BPI) chromatographic profiles of MFs (Dashi) at Stage 5 (ST5) from the Xinjiang region. Other comparisons of the chromatographic profiles of unripe and ripened MFs from various cultivars and regions are presented in Appendix Supplementary Fig. S2 and Fig. S3.
Conclusions
In this work, we identified individual chemical compounds from nine different groups (phenolic acids, flavonols, hydroxycoumarins, dihydroflavonols, anthocyanins, organic acids, amino acids, carbohydrates, and vitamins) in MFs from two different regions during ripening based on UPLC-QTOF-MS data. We also evaluated the data for potential markers that differentiate MFs at different ripening stages and from different cultivation regions using multivariate statistical analysis models (the
Acknowledgement
We would like to thank the National Natural Science Foundation of China for financial support (No. 31571840).
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