Integrated HS-GC-IMS and UPLC-Q-Orbitrap HRMS-based metabolomics revealed the characteristics and differential volatile and nonvolatile metabolites of different citrus peels

Abstract

Citrus is an important genus in the Rutaceae family, and citrus peels can be used in both food and herbal medicine. However, the bulk of citrus peels are discarded as waste by the fruit processing industry, causing environmental pollution. This study aimed to provide guidelines for the rational and effective use of citrus peels by elucidating the volatile and nonvolatile metabolites within them using metabolomics based on headspace-gas chromatography-ion mobility spectrometry and ultra-high-performance liquid chromatography-Q-Orbitrap high-resolution mass spectrometry. In addition, the antioxidant activities of the citrus peels were evaluated using DPPH radical scavenging, ABTS radical scavenging, and ferric reducing antioxidant power. In total, 103 volatile and 53 nonvolatile metabolites were identified and characterized. Alcohols, aldehydes, and terpenes constituted 87.36% of the volatile metabolites, while flavonoids and carboxylic acids accounted for 85.46% of the nonvolatile metabolites. Furthermore, (Z)-2-penten-1-ol, L-pipecolinic acid, and limonin were identified as characteristic components of Citrus reticulata Blanco cv. Ponkan (PK), C. reticulata 'Unshiu' (CLU), and C. reticulata 'Wo Gan' (WG), respectively. Principal component analysis and partial least squares discriminant analysis indicated that C. reticulata Blanco 'Chun Jian' (CJ), PK, CLU, and C. reticulata 'Dahongpao' (DHP) were clustered together. DHP is a traditional Chinese medicine documented in the Chinese Pharmacopoeia, suggesting that the chemical compositions of CJ, PK, and CLU may also have medicinal values similar to those of DHP. Moreover, DHP, PK, C. reticulata 'Ai Yuan 38'(AY38), CJ, C. reticulata 'Gan Ping'(GP), and C. reticulata 'Qing Jian'(QJ) displayed better antioxidant activities, recommending their use as additives in cosmetics and food. Correlation analysis suggested that some polyphenols including tangeritin, nobiletin, skullcapflavone II, genistein, caffeic acid, and isokaempferide were potential antioxidant compounds in citrus peel. The results of this study deepen our understanding of the differences in metabolites and antioxidant activities of different citrus peel varieties and ultimately provide guidance for the full and rational use of citrus peels.

Keywords: Antioxidant activity; Citrus peel; HS-GC-IMS; LC-Qtrap-MS; Volatile and nonvolatile metabolites.

Graphical abstract

Fig. 1

Different varieties of citrus peels. (A) C. reticulata ‘Dahongpao’; (B) C. reticulata ‘Unshiu’; (C) C. reticulata Blanco cv. Ponkan; (D) C. reticulata ‘Ai Yuan 38'; (E) C. reticulata ‘Chun Jian’; (F) C. reticulata ‘Wo Gan’; (G) C. reticulata ‘Bu Zhi Huo’; (H) C. reticulata ‘Gan Ping’; (I) C. reticulata ‘Qing Jian’; (J) C. reticulata ‘Mingri Jian’; (K) C. sinensis Osb. var. brasiliensis Tanaka; (L) C. sinensis ‘blood orange'. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Volatile metabolites in different citrus varieties. (A) Three-dimensional topographic plots; (B) Two-dimensional topographic plots; (C) Circular graph of relative content of various volatile metabolites; (D) Volatile metabolites category distribution sun diagram.

Fig. 3

Average content of volatile metabolites in citrus peels. (A) Alcohols; (B) Aldehydes; (C) Esters; (D) Ketones; (E) Terpenes; (F) Sulfur compounds and others.

Fig. 4

The fingerprints of volatile metabolites in different varieties of citrus peel. (A) Alcohols; (B) aldehydes; (C) esters; (D) ketones; (E) terpenes; (F) sulfur compounds and others; (G) unidentified compounds.

Fig. 5

Total ion current diagram, secondary mass spectrum, and classification of typical nonvolatile metabolites in citrus peels. (A) Total ion current diagram; (B) Second-order mass spectrometry of hesperidin compounds; (C) Second-order mass spectrometry of hesperetin metabolites; (D) Circular graph of the relative contents of nonvolatile metabolites.

Fig. 6

Average content of nonvolatile metabolites in citrus peels. (A) Carboxylic acids and derivatives; (B) cinnamic acids and derivatives; (C) coumarins and derivatives; (D) other compounds; (E) flavonoids; (F) flavonoid glycosides; (G) O-methoxylated flavonoids.

Fig. 7

Correlation heat map between different varieties of citrus peels and nonvolatile metabolites. Red indicates a strong correlation, blue indicates a weak correlation, and the redder the color, the greater the correlation coefficient (r ≥ 0.9 and P < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

PCA and PLS-DA based on UPLC–Q-Orbitrap HRMS and HS-GC–IMS data. (A) two-dimensional PCA plot; (B) 3D PCA plot; (C) PLS-DA score plot; (D) variable importance in projection scores; and (E) cross-validation results.

Fig. 9

Correlation between antioxidant activities and phenolic compound of the different varieties of citrus peels. (A) DPPH radical scavenging activity; (B) ABTS radical scavenging activity; (C) Ferric reducing antioxidant power; and (D) Correlation heat map. The error bars indicate the standard deviation. Statistical analysis was performed using analysis of variance plus post-hoc Ducan's test, and * (P < 0.05) indicates a significant difference from DHP.