Cultivar differentiation of Citri Reticulatae Pericarpium by a combination of hierarchical three-step filtering metabolomics analysis, DNA barcoding and electronic nose

Highlights

• CRP cultivars were differentiated by hierarchical filtering metabolomics analysis.

• 9 species-specific chemical markers of CRP were discovered.

• DNA barcoding analysis of CRP cultivars was investigated.

• Electronic nose was developed for discrimination of CRP cultivars.

Abstract

The traditional Chinese medicine Citri Reticulatae Pericarpium (CRP) was mainly originated from the dried pericarp of Citrus reticulata ‘Chachi’ (Crc), Citrus reticulata ‘Dahongpao’ (Crd), Citrus reticulata ‘Unshiu’ (Cru) and Citrus reticulata ‘Tangerina’ (Crt) in China. Since these four cultivars have great similarities in morphology, reliable methods to differentiate CRP cultivars have rarely been reported. To discriminate the differences of these CRP cultivars, herein an efficient and reliable method by combining metabolomics, DNA barcoding and electronic nose was first established. The hierarchical three-step filtering metabolomics analysis based on liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) indicated that 9 species-specific chemical markers including 6 flavanone glycosides and 3 polymethoxyflavones could be considered as marker metabolites for discrimination of the geoherb Crc from other cultivars. A total of 19 single nucleotide polymorphism (SNP) sites were found in nuclear internal transcribed spacer 2 (ITS2) of CRP, and three stable SNP sites (33, 128 and 174) in the ITS2 region can distinguish the four CRP cultivars. The electronic nose coupled with chemometrics could also be used to effectively distinguish Crc from other CRP cultivars. Therefore, our results indicated that the integrated method will be an effective strategy for discrimination of similar herbal medicines.

Introduction

Citri Reticulatae Pericarpium (CRP), the dried tangerine pericarp, has been broadly applied as a well-known traditional Chinese medicine (TCM), food and dietary supplement in China [1]. Modern pharmacological studies indicate that CRP has various bioactivities, such as effects on gastrointestinal and respiratory systems; antioxidant, anti-inflammatory, and anticancer activities; and the prevention of hyperlipidemia, obesity and type 2 diabetes [2,3]. CRP cultivars are widely distributed in different regions of China, of which Guangchenpi (Citrus reticulata ‘Chachi’, Crc), Dahongpao (Citrus reticulata ‘Dahongpao’, Crd), Wenzhou migan (Citrus reticulata ‘Unshiu’, Cru) and Fuju (Citrus reticulata ‘Tangerina’, Crt) are recorded in the Chinese Pharmacopeia [4]. These four cultivars share great similarities in morphology, chemical constituents, and genomic DNA sequences. Among the CRP cultivars, Crc is the geoherb produced in Xinhui, Guangdong province, and it is traditionally considered to have superior qualities and therapeutic effects [3]. In the Chinese Pharmacopoeia (2015 edition), hesperidin is used as the marker compound for quality control of CRP. However, as all of these four CRP cultivars contain abundant hesperidin, it is impossible to distinguish Crc from the other CRP cultivars by measuring hesperidin [2].

Plant secondary metabolites are an enormous group of small-molecule natural products with high structural diversity [5]. Metabolomics is a rapidly emerging field of postgenomic research that aims to comprehensively analyze the total population of metabolites in samples [6,7]. Recently, metabolomics analysis has been successfully applied to discriminate and differentiate plant phenotypes. For instance, Duan et al. [8] have applied metabolomics analysis to the discrimination of Oryza sativa L. Since CRP is derived from several closely related plant cultivars, little is known about the impact of cultivar variation on plant secondary metabolism. To discriminate different CRP cultivars, a comprehensive understanding of their secondary metabolome is of vital significance.

Methods for identifying species by amplifying short orthologous DNA sequences, known as ‘‘DNA barcoding’’, have been proposed for identification of plants, animals and fungi [9,10]. The barcodes could be located on nuclear rDNA (nrDNA), such as the internal transcribed spacer (ITS) and the internal transcribed spacer 2 (ITS2), or on chloroplast DNA (cpDNA), such as rbcL and the trnH-psbA intergenic spacer [5]. ITS2 is an ideal region, as it evolves comparably fast, contains highly conservative sites, and shows high levels of interspecific divergence, which allows classification of different specimens at the species level [2,9]. Chen et al. [11] suggested that integrated DNA barcodes could provide an efficient and reliable authentication system for herbal products, and the ITS2 region represented the most suitable region for identifying medicinal plant species.

Electronic noses, derived from numerous types of aroma-sensor technologies, are being used with increasing frequency because they allow the acquisition of real-time information about the chemical and physical nature and the quality of plants [12]. Electronic noses have demonstrated their versatility in the quality analysis of citrus fruit juice [13], the differentiation of Chinese robusta coffees [14] and the prediction of food additives [15]. Thus, flavor evaluation of CRP samples may be an effective approach to accurately discriminate these four CRP cultivars.

Integration of the genetic and metabolomic fingerprinting can provide a new approach to differentiate similar TCM materials. In the present study, an efficient and reliable method that combines metabolomics, DNA barcoding and electronic nose was first established to accurately distinguish the four CRP cultivars. A hierarchical three-step filtering metabolomics analysis based on liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) was employed to reveal differences in the secondary metabolomes of CRP cultivars. For molecular authentication of the CRP cultivars, the DNA barcoding of the ITS2 region was further utilized. A nondestructive analysis method based on electronic nose was also developed to distinguish the CRP cultivars by detecting volatile components.