RSC Advances View Article Online PAPER View Journal | View Issue Chemical and genetic discrimination of commercial Guangchenpi (Citrus reticulata ‘Chachi’) by using Cite this: RSC Adv.,2019,9, 23373 UPLC-QTOF-MS/MS based metabolomics and DNA barcoding approaches† Peng Wang,‡a Jing Zhang,‡a Yating Zhang,a He Su,a Xiaohui Qiu,b Lu Gong,a Juan Huang,a Junqi Bai,a Zhihai Huang*b and Wen Xu *a 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 Creative Commons Attribution 3.0 Unported Licence. 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 tetramethoxyflavonearethemost Received 18th May 2019 influential markers. Support vector machines were employed to classify all the samples and these markers Accepted 2nd July 2019 showed good prediction accuracy (100%). The results of DNA barcoding showed that the secondary DOI: 10.1039/c9ra03740c structure of the ITS2 sequences were significantly different among GCP and other three cultivars. The study This article is licensed under a rsc.li/rsc-advances indicated the integrated method could be a powerful and reliable analytical tool for differentiating GCP from CP. CRP is divided into two subtypes, Guangchenpi (GCP) and Open Access Article. Published on 29 July 2019. Downloaded 10/2/2021 9:01:06 PM. 1. Introduction Chenpi (CP), as stated unambiguously in the current version of Citri Reticulate Pericarpium (CRP), the dried matured tangerine Chinese Pharmacopeia.7 Generally, GCP is dened as pericarp of pericarp, is one of the most well-known Chinese medicinal a specic cultivar of Citrus reticulata,namelyCitrus reticulata materials which has long been extensively used for health ‘Chachi’, while other main cultivars refer to CP, including Citrus purposes. According to traditional Chinese Medicine theory, CRP reticulata ‘Dahongpao’, Citrus reticulata ‘Unshiu’, Citrus reticulata has been used as a Qi-regulating herb to treat nausea, vomiting, ‘Tangerina’, and so on. As a rule, Citrus reticulata ‘Chachi’, indigestion, anepithymia, diarrhea, coughs, expectoration, and cultivated and harvested in a limited area, Xinhui county of so on.1 Besides a medical purpose, CRP has been frequently used Guangdong Province in China, is regarded as the genuine source in foods, drinks, snacks, health-care products and condiments in of GCP,8 while CP is widely distributed cultivated within China. In China and Southeast Asia.2 In recent years, many researchers spite of moderate differences in morphological traits between CP have focused on the pharmacological actions of CRP, such as and GCP, their commercial products are frequently confused as benecial effects on the gastrointestinal and respiratory systems, some morphological traits have been lost aer processing. There antioxidant, anti-inammatory, and anti-cancer activities, as well is no systemic method to distinguish GCP and CP, however, the as protective effects on the liver and nervous system.3–6 current method relies on visual and morphological evaluation by articial experience. According to recent studies, chemical composition of GCP and CP oen vary among varieties, which aThe Second Clinical College of Guangzhou University of Chinese Medicine, could affect their quality and pharmacological effects.9,10 Thus, it Guangzhou, China. E-mail: [email protected] ff bGuangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese is imperative to investigate their genetic and chemical di erences Medicine Syndrome, Guangdong Provincial Hospital of Traditional Chinese for discrimination and quality control of these similar subtypes. Medicine, Guangzhou, China. E-mail: [email protected] DNA barcoding methods have been used to overcome the † Electronic supplementary information (ESI) available. See DOI: limitations of morphological identication in plant discrimi- 10.1039/c9ra03740c nation based on the DNA sequences, such as the ITS region of ‡ These authors have contributed equally to this work. This journal is © The Royal Society of Chemistry 2019 RSC Adv.,2019,9, 23373–23381 | 23373 View Article Online RSC Advances Paper nrDNA, and chloroplast DNA (cpDNA) rbcL, psbA-trnH, and  Taq PCR Mix was obtained from Aidlab Biotechnologies Co., Ltd matK.11,12 Previous studies have demonstrated the ability of (Beijing, China). LC-MS grade formic acid was obtained from DNA barcoding to identify Citrus plants.13 The internal tran- Thermo Fisher Scientic Company China Branch (Shanghai, scribed spacer 2 (ITS2) which is part of the eukaryotic nuclear China). Analytical grade methanol was obtained from Guangzhou rDNA cistron and lies between the 5.8S and 28S rDNA, as a novel Chemical Reagent (Guangzhou, China). Deionized water (18 MU) DNA barcode for identifying medicinal plant species.14 was puried by a Mill-Q ultrapure water system (Millipore, USA). Compared with other sequences, the advantage of ITS2 is the Reference compounds nobiletin, tangeretin, 3,5,6,7,8,30,40-hepta- standard sequence that can be obtained through the website methoxyavone, rutin, eriocitrin and hesperetin were obtained annotation (http://its2.bioapps.biozentrum.uni-wuerzburg.de/ from Shanghai Yuanye Co, Ltd. (Shanghai, China). )15 and predicted secondary structure of ITS2 from the data- base helps us to learn more about the genetic information of species for identication. A previous study has shown that there 2.3 LC-MS analysis are differences in the secondary structures of ITS2 sequence 2.3.1 Sample preparation for LC-MS analysis. Each CRP between Citrus reticulata ‘Chachi’ and other two cultivars.16 sample was powdered by a mill, and passed through 60 mesh Recently developed metabolomics analysis method is regar- sieve. 0.2 g of each powdered sample was accurately weighed ded as a powerful technology that could systematically and and extracted with 15 mL methanol in an ultrasonic bath (40 comprehensively investigate the metabolic proles in different kHz, 250 W) for 30 min. Aer cooling at room temperature, the samples, which enable us to reveal the differences of chemical extracted solution was added with methanol to the original À features among the closely related species of traditional weight, and followed by centrifugation at 5000 r min 1 for Chinese Medicines.9,10 Previously studies have successfully 10 min. Then the supernatant was ltered through a 0.22 mm applied GC-MS and/or LC-MS based metabolomics analysis for membrane prior to use. discriminating the Citrus genus products such as the Citrus The quality control (QC) sample was used for validating the 9 17 18 Creative Commons Attribution 3.0 Unported Licence. peel, fruit, and/or fruit juice. However, basing on a synthetic reproducibility and stability of the UPLC-QTOF-MS/MS method- strategy combing metabolomics analysis and DNA barcoding ology. It was made up by 20 mL of each CRP sample (prepared as methods could be a more practical solution to make integrated described previously) and the mixed solution was vortexed for evaluation on their cultivar differentiation. 1min,andthenltered through a 0.22 mmmembranepriortouse. In present study, 51 batches of commercial CRP samples, 2.3.2 LC-MS conditions. The LC analysis was carried out on including four cultivars originated from Hunan, Jiangxi, Zhe- an ACQUITY™ UPLC™ H-CLASS instrument (Waters Corp., jiang, Hubei, Sichuan and Guangdong province in China were Milford, MA, USA) equipped with an automatic degasser, collected. We aim to clarify the genetic and chemical diversities a quaternary pump, a column oven and an autosampler. Sepa- between GCP and CP by combining deoxyribonucleic acid ration was performed on a Waters ACQUITY™ UPLC™ HSS T3 This article is licensed under a (DNA) barcoding method using mainly sequences of ITS2 and column (100  2.1 mm, 1.7 mm) at 30 C. The mobile phase psbA-trnH, with a metabolomics approach using ultra- consisted of solvent A (water containing 0.1% formic acid) and performance liquid chromatography coupled with quadrupole solvent B (acetonitrile), and the elution program was optimized Open Access Article. Published on 29 July 2019. Downloaded 10/2/2021 9:01:06 PM. time-of-ight mass spectrometry (UPLC-QTOF-MS/MS). as follows: 0–3.5 min, 5–15% B; 3.5–6 min, 15–25% B; 6–15 min, 25–40% B; 15–18 min, 40–55% B; 18–20 min, 55–85% B; 20– À 22 min, 85–95% B. The ow rate was set to 0.2 mL min 1, with 2. Experimental an injection volume set to 2 mL. 2.1 Plant materials The MS analysis was performed on a TripleTOF™ 5600+ In present study, a total of 51 batches of CRP samples were mass spectrometer (AB SCIEX, Foster City, CA, USA) equipped ™ purchased from herbal medicine market in southern and eastern with a DuoSpray