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. 2019 Jul 29;9(40):23373-23381.
doi: 10.1039/c9ra03740c. eCollection 2019 Jul 23.

Chemical and genetic discrimination of commercial Guangchenpi (Citrus reticulata 'Chachi') by using UPLC-QTOF-MS/MS based metabolomics and DNA barcoding approaches

Peng Wang  1 , Jing Zhang  1 , Yating Zhang  1 , He Su  1 , Xiaohui Qiu  2 , Lu Gong  1 , Juan Huang  1 , Junqi Bai  1 , Zhihai Huang  2 , Wen Xu  1
Affiliations

Chemical and genetic discrimination of commercial Guangchenpi (Citrus reticulata 'Chachi') by using UPLC-QTOF-MS/MS based metabolomics and DNA barcoding approaches

Peng Wang et al. RSC Adv. .

Abstract

CRP (Citri Reticulatae Pericarpium), a famous traditional Chinese medicine, has also been extensively used in foods and condiments in dietary practice for centuries. According to the Chinese Pharmacopeia (2015 edition) it contains two subtypes, Guangchenpi (GCP) and Chenpi (CP). GCP exclusively originates from the pericarp of Citrus reticulata 'Chachi' cultivar and it's generally believed that GCP has superior qualities compared with the other main cultivars (CP). In the present study, an integrated approach combining LC-QTOF MS-based untargeted metabolomics analysis and DNA barcoding molecular identification was conducted to study the genetic diversity and chemical differences between GCP and CP. A validated UPLC-QTOF MS metabolomics method was established to identify markers by using PCA and OPLS-DA models. 34 identified metabolites could be used as chemical markers to distinguish effectively between the two subtypes. Among them polymethoxyflavones (PMF) such as hexamethoxyflavone (nobiletin and natsudaidain), pentamethoxyflavone (tangeretin and sinensetin), and tetramethoxyflavone are the most influential markers. Support vector machines were employed to classify all the samples and these markers showed good prediction accuracy (100%). The results of DNA barcoding showed that the secondary structure of the ITS2 sequences were significantly different among GCP and other three cultivars. The study indicated the integrated method could be a powerful and reliable analytical tool for differentiating GCP from CP.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. The typical base peak chromatograms (BPC) of GCP and CP samples in positive and negative modes. (A) BPC of GCP in positive MS mode; (B) BPC of CP in positive MS mode; (C) BPC of GCP in negative MS mode; and (D) BPC of CP in negative MS mode.
Fig. 2
Fig. 2. (A) PCA score plot in positive mode (R2X = 0.873, R2Q = 0.723). (B) OPLS-DA score plot in positive mode with statistical parameters (R2X = 0.656, R2Y = 0.989, Q2 = 0.974). (C) PCA score plot in negative mode (R2X = 0.888, R2Q = 0.741). (D) OPLS-DA score plot in negative mode with statistical parameters (R2X = 0.549, R2Y = 0.994, Q2 = 0.982). Blue triangles represent GCP samples, green triangle represent CP samples and pentacles represent QC samples.
Fig. 3
Fig. 3. Differences of the 34 chemical markers between GCP and CP.
Fig. 4
Fig. 4. The MS fragmentation pathways of diosmin.
Fig. 5
Fig. 5. The characteristics of the dominant haplotype sequence of four species (the variation sites were framed).
Fig. 6
Fig. 6. The secondary structures of: (A) Citrus reticulata ‘Chachi’; (B) Citrus reticulata ‘Dahongpao’; (C) Citrus reticulata ‘Unshiu’; (D) Citrus reticulata ‘Tangerina’.
Fig. 7
Fig. 7. The psbA-trnH sequences of the four Species.
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