Chemical Composition of Essential Oil from Citrus reticulata Blanco cv. Chachiensis (Chachi) and Its Anti-Mosquito Activity against Pyrethroid-Resistant Aedes albopictus.

The overuse of synthetic insecticides has led to various negative consequences, including insecticide resistance, environmental pollution, and harm to public health. This may be ameliorated by using insecticides derived from botanical sources. The primary objective of this study was to evaluate the anti-mosquito activity of the essential oil (EO) of Citrus reticulata Blanco cv. Chachiensis (Chachi) (referred to as CRB) at immature, semi-mature, and mature stages. The chemical compositions of the CRB EO were analyzed using GC-MS. The main components were identified to be D-limonene and γ-terpinene. The contents of D-limonene at the immature, semi-mature, and mature stages were 62.35%, 76.72%, and 73.15%, respectively; the corresponding contents of γ-terpinene were 14.26%, 11.04%, and 11.27%, respectively. In addition, the corresponding contents of a characteristic component, methyl 2-aminobenzoate, were 4.95%, 1.93%, and 2.15%, respectively. CRB EO exhibited significant larvicidal activity against Aedes albopictus (Ae. albopictus, Diptera: Culicidae), with the 50% lethal doses being 65.32, 61.47, and 65.91 mg/L for immature, semi-mature, and mature CRB EO, respectively. CRB EO was able to inhibit acetylcholinesterase and three detoxification enzymes, significantly reduce the diversity of internal microbiota in mosquitoes, and decrease the relative abundance of core species within the microbiota. The present results may provide novel insights into the utilization of plant-derived essential oils in anti-mosquitoes.

Short-term steaming during processing impacts the quality of Citri Reticulatae 'Chachi' peel.

Citrus peel is a commonly used food-medicine material in the production of fast-moving consumer goods (FMCGs). For instance, Ganpu tea is manufactured by combining the peel of Citri Reticulatae 'Chachi' (PCRC) with Pu-erh tea. The alleviated irritation of PCRC through years of aging makes Citri reticulatae Pericarpium a traditional Chinese medicine. Herein, we introduced short-term steaming into the processing of PCRC to favor the quick removal of its irritation while retaining its food-medicine properties. Sensory evaluation and volatile component analysis showed that 60-s steaming reduced irritation of freshly prepared PCRC. Biological evaluations indicated no effects of steaming on the neuroprotective activity of PCRC. The process increased the contents of several bioactive ingredients, including hesperidin, nobiletin, tangeretin, and synephrine. In addition, physical indications of accelerating PCRC aging were observed. Taken together, our findings suggest that short-term steaming may offer a promising new possibility for enhancing the quality of citrus peel.

Flavonoids contribute most to discriminating aged Guang Chenpi (Citrus reticulata 'Chachi') by spectrum-effect relationship analysis between LC-Q-Orbitrap/MS fingerprint and ameliorating spleen deficiency activity.

To further explore the mechanism of "the longer storage time, the better bioactivity" of aged Guang Chenpi, the dry pericarp of Citrus reticulata 'Chachi' (CRC), a series of activity assessments were performed on spleen deficiency mice. The constituents in CRC with different storage years were analyzed by LC-Q-Orbitrap/MS. A total of 53 compounds were identified, and CRC stored for more than 5 years showed higher flavonoid content, especially that of polymethoxyflavones. Anti-spleen deficiency bioactivity analysis among various CRC with different storage years showed aged CRC (stored for more than 3 years) could significantly alleviate fatigue and depression behaviors much better, increase D-xylose and gastrin secretion, and upregulate the expression of the linking protein occludin in the colon walls. Results from 16S rDNA sequencing showed that aged CRC could downregulate the abundance of Enterococcus, Gemmata, Citrobacter, Escherichia_Shigella, and Klebsiella, which were significantly overrepresented in the model group. Bacteroides, Muribaculum, Alloprevotella, Paraprevotella, Alistipes, Eisenbergiella, and Colidextribacter were downregulated in the model group but enriched in the CRC groups. At last, the spectrum-effect relationship analysis indicated that flavonoids such as citrusin III, homoeriodictyol, hesperidin, nobiletin, and isosinensetin in aged CRC showed the highest correlation with better activity in ameliorating spleen deficiency by regulating gut microbiota. Flavonoids contribute most to discriminating aged CRC and could disclose the basis of "the longer storage time, the better bioactivity" of aged Guang Chenpi.

Analysis of Flavonoid Metabolites in Citrus reticulata 'Chachi' at Different Collection Stages Using UPLC-ESI-MS/MS.

Flavonoids are essential substances with antioxidant properties and high medicinal value. Citrus reticulata 'Chachi' peel (CRCP) is rich in flavonoids and has numerous health benefits. The different maturity periods of CRCP can affect the flavonoid contents and pharmacological effects. In this study, we successfully performed UPLC-ESI-MS/MS-based metabolic analysis to compare the metabolites of CRCP at different harvesting periods (Jul, Aug, Sep, Oct, Nov, and Dec) using a systematic approach. The results revealed the identification of a total of 168 flavonoid metabolites, including 61 flavones, 54 flavonols, 14 flavone C-glycosides, 14 dihydroflavones, 9 flavanones, 8 isoflavones, 3 flavanols, 3 dihydroflavonols, and 2 chalcones. Clustering analysis and PCA were used to separate the CRCP samples collected at different stages. Furthermore, from July to December, the relative contents of isoflavones, dihydroflavones, and dihydroflavonols gradually increased and flavanols gradually decreased over time. The relative content of flavonoid C-glycosides showed an increasing and then decreasing trend, reaching the highest value in August. This study contributes to a better understanding of flavonoid metabolites in CRCP at different harvesting stages and informs their potential future utilization.

Portable Microelectrochemical Sensors for Rapid and Sensitive Determination of Hesperidin in Citrus reticulate 'Chachi' Peel.

Portable and low-cost analytical devices are essential for rapid detection of bioactive substrates in agricultural products. This study presents the first highly integrated microelectrochemical sensor based on pencil graphite for rapid and sensitive detection of hesperidin in Citrus reticulate 'Chachi' peel. The surface morphology and characterization as well as the electrochemical property of pencil graphite was investigated and discussed. A high electrocatalytic efficiency of hesperidin has been found at used pencil graphite-based microelectrodes. Kinetic analysis was carried out to further understand the electrochemical process of hesperidin at a pencil graphite microelectrode. Consequently, a portable and highly-integrated microelectrochemical sensor exhibits a sensitivity of 0.7251 µA cm-2 µM-1 and a detection limit as low as 25 nM (S/N = 3), and high selectivity was fabricated. Proposed microelectrochemical sensors were applied to electrochemically determinate the hesperidin content in the extract of Citrus reticulata "chachi" peel. As a result, the concentration of hesperidin in the actual real sample detected electrochemically with the proposed portable and low-cost microelectrochemical sensors is highly consistent to that obtained with a common chromatographic method, thus indicating the good reliability and that it can be used in practical applications.

Comparison of the volatile organic compounds in Citrus reticulata 'Chachi' peel with different drying methods using E-nose, GC-IMS and HS-SPME-GC-MS.

Introduction: Citrus reticulata 'Chachi' peel (CRCP), which is named "Guangchenpi" in China, is a geographical indication product with unique flavor properties. CRCP has been used for centuries as a traditional genuine herb because of its excellent therapeutic effects. In addition, owing to its unique odor and high nutrition, it is widely used in various food preparations. Volatile organic compounds (VOCs) are regarded as an important quality marker for CRCP and are highly susceptible to effects in the drying process due to their thermal instability. Methods: In the current study, the main VOCs in CRCP were processed using different drying methods, including sun-drying, hot air drying, and vacuum-freeze drying. The VOCs were identified by the electronic nose (E-nose), gas chromatography-ion mobility spectrometry (GC-IMS), and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Results: The results showed that the CRCP dried by vacuum-freeze exhibited the highest VOCs contents and retained the richest compounds compared to those dried by other methods, which indicated that vacuum-freeze drying is the most suitable for CRCP production. Furthermore, the chemometrics analysis revealed that the primary differential metabolites of the samples generated using different drying methods were terpenes and esters. Discussion: Overall, our study would help better understand the VOCs present in CRCP with different drying methods. The outcomes of the current study would guide the drying and processing of CRCP, which is beneficial for large-scale storage and industrial production of CRCP.

The molecular mechanism for inhibiting the growth of nasopharyngeal carcinoma cells using polymethoxyflavonoids purified from pericarp of Citrus reticulata 'Chachi' via HSCCC.

Polymethoxyflavonoids (PMFs), the main bioactive compounds naturally occurring in the pericarp of Citrus reticulata 'Chachi' (CRCP), possess significant antitumor action. However, the action of PMFs in nasopharyngeal carcinoma (NPC) is currently unknown. The present research study was conducted to investigate the inhibitory mechanisms of PMFs from CRCP on NPC growth in vivo and in vitro. In our research, we used high-speed counter-current chromatography (HSCCC) to separate four PMFs (nobiletin (NOB), 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF), tangeretin (TGN), and 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone (5-HPMF)) from CRCP. CCK-8 assay was used to preliminarily screen cell viability following exposure to the four PMFs. Colony formation, Hoechst-33258 staining, transwell, and wound scratch assays were performed to assess the anti-proliferation, invasion, migration, and apoptosis-inducing effects of HMF on NPC cells. NPC tumors in xenograft tumor transplantation experiments were also established to explore the effect of HMF (100 and 150 mg/kg/day) on NPC. The histopathological changes in the treated rats were observed by H&E staining and Ki-67 detection by immunohistochemical techniques. The expressions of P70S6K, p-P70S6K, S6, p-S6, COX-2, p53, and p-p53 were measured by Western blot. The four PMFs were obtained with high purity (>95.0%). The results of the preliminary screening by CCK-8 assay suggested that HMF had the strongest inhibitory effect on NPC cell growth. The results of the colony formation, Hoechst-33258 staining, transwell, and wound scratch assays indicated that HMF had significant anti-proliferation, invasion, migration, and apoptosis-inducing ability in NPC cells. Moreover, HMF suppressed NPC tumor growth in xenograft tumor transplantation experiments. Further investigation suggested that HMF regulated NPC cells proliferation, apoptosis, migration, and invasion by activating AMPK-dependent signaling pathways. In conclusion, HMF-induced AMPK activation inhibited NPC cell growth, invasion, and metastatic potency by downregulating the activation of the mTOR signaling pathway and COX-2 protein levels, as well as enhancing the p53 phosphorylation level. Our study provides a crucial experimental basis for the clinical treatment of NPC, as well as the development and utilization of PMFs from CRCP.

Soil conditions and the plant microbiome boost the accumulation of monoterpenes in the fruit of Citrus reticulata 'Chachi'.

BACKGROUND: The medicinal material quality of Citrus reticulata 'Chachi' differs depending on the bioactive components influenced by the planting area. Environmental factors, such as soil nutrients, the plant-associated microbiome and climatic conditions, play important roles in the accumulation of bioactive components in citrus. However, how these environmental factors mediate the production of bioactive components of medicinal plants remains understudied. RESULTS: Here, a multi-omics approach was used to clarify the role of environmental factors such as soil nutrients and the root-associated microbiome on the accumulation of monoterpenes in the peel of C. reticulata 'Chachi' procured from core (geo-authentic product region) and non-core (non-geo-authentic product region) geographical regions. The soil environment (high salinity, Mg, Mn and K) enhanced the monoterpene content by promoting the expression of salt stress-responsive genes and terpene backbone synthase in the host plants from the core region. The microbial effects on the monoterpene accumulation of citrus from the core region were further verified by synthetic community (SynCom) experiments. Rhizosphere microorganisms activated terpene synthesis and promoted monoterpene accumulation through interactions with the host immune system. Endophyte microorganisms derived from soil with the potential for terpene synthesis might enhance monoterpene accumulation in citrus by providing precursors of monoterpenes. CONCLUSIONS: Overall, this study demonstrated that both soil properties and the soil microbiome impacted monoterpene production in citrus peel, thus providing an essential basis for increasing fruit quality via reasonable fertilization and precision microbiota management. Video Abstract.

Comparative study on comprehensive quality of Xinhui chenpi by two main plant propagation techniques.

Xinhui chenpi (XHCP), the sun-dried peel of the mandarin orange, Citrus reticulata "Chachi," is the most famous crude drug, as well as a traditional seasoning in Chinese cooking. The main cultivation methods of XHCP are cutting and grafting, but it is generally considered that the quality of XHCP after cutting is superior to that obtained from plants propagated by graftings, which had a negative impact on the marketing of the finished product. In our study, a total of 25 samples of XHCP obtained from plants cultivated by either traditional methods (i.e., from cuttings) or by grafting were collected to compare the contents of four types of metabolites (essential oils, flavonoids, synephrine, and total polysaccharides) as well as antioxidant activity. The results revealed that the quality of XHCP did not decline after cutting, and marked individual differences between XHCP samples, even when prepared from plants grown in the same way. In general, grafting had no significant effect on the most essential oils components, total polysaccharides, synephrine, total flavonoids, total polymethoxylated flavones, hesperidin, nobiletin, tangeretin content, and antioxidant activity. Nevertheless, five volatile compounds can be used as potential chemical markers (p < 0.05) to distinguish between cutting XHCP and grafted XHCP, while four volatile compounds showed high content in grafted XHCP. Our study is expected to provide a theoretical basis for XHCP breeding and cultivation, and thereby further standardize the market of XHCP.

Mass Spectrometry Rearrangement Ions and Metabolic Pathway-Based Discovery of Indole Derivatives during the Aging Process in Citrus reticulata 'Chachi'.

The rapid analysis and characterization of compounds using mass spectrometry (MS) may overlook trace compounds. Although targeted analysis methods can significantly improve detection sensitivity, it is hard to discover novel scaffold compounds in the trace. This study developed a strategy for discovering trace compounds in the aging process of traditional Chinese medicine based on MS fragmentation and known metabolic pathways. Specifically, we found that the characteristic component of C. reticulata 'Chachi', methyl N-methyl anthranilate (MMA), fragmented in electrospray ionization coupled with collision-induced dissociation (CID) to produce the rearrangement ion 3-hydroxyindole, which was proven to exist in trace amounts in C. reticulata 'Chachi' based on comparison with the reference substance using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Combining the known metabolic pathways of 3-hydroxyindole and the possible methylation reactions that may occur during aging, a total of 10 possible indole derivatives were untargeted predicted. These compounds were confirmed to originate from MMA using purchased or synthesized reference substances, all of which were detected in C. reticulata 'Chachi' through LC-MS/MS, achieving trace compound analysis from untargeted to targeted. These results may contribute to explaining the aging mechanism of C. reticulata 'Chachi', and the strategy of using the CID-induced special rearrangement ion-binding metabolic pathway has potential application value for discovering trace compounds.

Content assay and metabolic study of flavonoids from Citrus reticulata 'Chachi' / 药学学报

In this study, a high-performance liquid chromatography method was established to simultaneously determine three flavonoids including hesperidin (HES), nobiletin (NOB) and tangeretin (TAN) in 10 batches of Citrus reticulata 'Chachi' planted and collected in Xinhui District, Jiangmen City, Guangdong Province. Moreover, we studied the metabolism and transformation of three flavonoids in liver and intestinal flora in vitro, and sequenced 16S rRNA of bacteria flora samples after incubation. The RP-HPLC system consisted of Alltima C18 column (250 mm × 4.6 mm, 5 μm) and a mobile phase of water (A) - methanol (B). The column temperature was 25 ℃ and the detection wavelength was both 283 nm and 330 nm while the flow rate was 1.0 mL·min-1. The results showed that the retention time of HES, NOB and TAN ranged from 12.313 min to 34.271 min. The content of HES, NOB and TAN in 10 batches of Citrus reticulata 'Chachi' was 26.81-39.80 mg·g-1, 4.06-7.90 mg·g-1 and 1.81-3.93 mg·g-1, respectively. There were differences in the content of flavonoids in different batches and growing areas. The three flavonoids were metabolized in various degrees after incubation of rat and human liver S9, cytosol, microsomes or intestinal flora in vitro, especially HES. The results of 16S rRNA showed that the main flavonoids of Citrus reticulata 'Chachi' could regulate lipid metabolism by regulating intestinal flora related to energy metabolism. This study established a rapid, simple, reproducible and stable quantitative analysis method for detecting the main flavonoids in Citrus reticulata 'Chachi' which evaluated the content of flavonoids from Citrus reticulata 'Chachi' in different growing areas and different storage periods. The intestinal bacteria can metabolize and transform the flavonoids of Citrus reticulata 'Chachi' to varying degrees, which provides a valuable scientific basis for the subsequent study on the material basis of the efficacy of Citrus reticulata 'Chachi' from the perspective of metabolism. Animal experiments were approved by the Medical Ethics Committee of Guangdong Jiangmen Chinese Medicine College (No. 20190419).

Transcriptome and Metabolome Analyses Provide Insights into the Flavonoid Accumulation in Peels of Citrus reticulata 'Chachi'.

The quality of Chinese medicinal materials depends on the content of bioactive components, which are affected by the environmental factors of different planting regions. In this research, integrated analysis of the transcriptome and metabolome of C. reticulata 'Chachi' was performed in two regions, and three orchards were included in the analysis. In total, only 192 compounds were found in fresh peels, and among 18 differentially accumulated flavonoid metabolites, 15 flavonoids were enriched in peels from the Xinhui planting region. In total, 1228 genes were up-regulated in peels from Xinhui, including the CHS and GST genes, which are involved in the salt stress response. Overall, based on the correlation analysis of flavonoid content and gene expression in peels of C. reticulata 'Chachi', we concluded that the authenticity of the GCRP from Xinhui may be closely related to the higher content of naringin and narirutin, and the increase in the content of these may be due to the highly saline environment of the Xinhui region.

Identification of Crucial Polymethoxyflavones Tangeretin and 3,5,6,7,8,3',4'-Heptamethoxyflavone and Evaluation of Their Contribution to Anticancer Effects of Pericarpium Citri Reticulatae 'Chachi' during Storage.

Pericarpium Citri Reticulatae 'Chachi' (PCR-C), rich in polymethoxyflavones (PMFs), has potential anticancer bioactivity and its quality will be improved during storage. However, the main factors influencing the PCR-C quality during its storage remain unclear. In this study, multivariate analysis was performed to investigate free and bound PMFs of PCR-C during storage. The anticancer effects of purified PCR-C flavonoid extracts (PCR-CF) and the important PMFs were evaluated using A549 cells. The results showed that PCR-C samples exhibited remarkable differences in free PMFs during storage, which fell into three clusters: Cluster 1 included fresh (fresh peel) and PCR-C01 (year 1); Cluster 2 consisted of PCR-C03 (year 3) and PCR-C05 (year 5); and PCR-C10 (year 10) was Cluster 3. 3,5,6,7,8,3',4'-heptamethoxyflavone, tangeretin, and isosinensetin were identified as the most important PMFs distinguishing the various types of PCR-C according to its storage periods. Moreover, PCR-CF inhibited A549 cell proliferation and induced cell cycle arrest at G2/M phase, cell apoptosis, and ROS accumulation, and all anticancer indices had an upward tendency during storage. Additionally, tangeretin and 3,5,6,7,8,3',4'-heptamethoxyflavone exhibited anticancer effects on A549 cells, whereas isosinensetin displayed no anticancer effect, indicating that tangeretin and 3,5,6,7,8,3',4'-heptamethoxyflavone jointly contributed to anticancer activity of PCR-C during storage. PCR-CF and the most important PMFs killed cancer cells (A549 cells) but had no cytotoxicity to normal lung fibroblast cells (MRC-5 cells). Overall, the high quality of long-term stored PCR-C might be due to the anticancer effects of tangeretin and 3,5,6,7,8,3',4'-heptamethoxyflavone.

Aged Pericarpium Citri Reticulatae 'Chachi' Attenuates Oxidative Damage Induced by tert-Butyl Hydroperoxide (t-BHP) in HepG2 Cells.

This study investigated the protective effects of aged Pericarpium Citri Reticulatae 'Chachi' (PCR-C) on tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in HepG2 cells. According to HPLC analysis, PCR-C aged 10 years (PCR-C10) had the highest flavonoids content, especially polymethoxyflavones (PMFs), compared with the fresh peel of Citrus reticulata cv. 'Chachiensis' and PCR-C aged 1, 3, and 5 years. Then, flavonoids-rich PCR-C samples and non-flavonoids-rich PCR-C samples (NF) were prepared by extracting and purifying PCR-C of different aging periods, for further cell experiments. Pretreatment with flavonoids-rich PCR-C samples (particularly PCR-C10) considerably reversed t-BHP-induced oxidative damage in HepG2 cells by improving cell viability, increasing SOD activity and GSH levels and reducing the overproduction of ROS and MDA. Correlation analysis further indicated that the accumulation of PMFs, mainly 5,6,7,4'-tetramethoxyflavone and nobiletin, was the main reason that PCR-C10 maintained the redox balance in HepG2 cells. These findings provided direct evidence for the cellular antioxidant activity of aged PCR-C and a guide for PCR-C's classification, authentication and rational use.

Dynamic variation of the phytochemical and volatile compounds in the pericarp of Citrus reticulata ''Chachi'' (Rutaceae) during 2 years of storage.

The pericarp of Citrus reticulata "Chachi" (CRCP) is used as nutritional food and traditional medicine in China, usually harvested at three periods, namely, immature (CRCP-G1), semi-mature (CRCP-G2), and fully mature (CRCP-G3). Traditionally, if the CRCP is stored for a longer period, then the quality will be better. In this study, the dynamic variation of phytochemical and volatile compounds was profiled in the same batches of CRCP during 2 years of storage. Results illustrated that most of the phytochemical compounds showed a decreasing trend during storage, that is, total flavonoids, total phenolic acids, hesperidin, 3,5,6,7,8,3',4'-heptamethoxyflavone, 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone, synephrine, and limonin. The ferulic acid increased significantly, whereas no significant changes were observed in the total polymethoxyflavones, nobiletin, and tangeretin after 2 years of storage. In addition, we found that the extraction yield of volatile oil decreased significantly in CRCP-G1 during storage, and the herb odors were enhanced with the increase of phenols and esters. No significant difference in the extraction yield of volatile oil of CRCP-G2 and CRCP-G3 was found after 2 years of storage, but the citrus-like notes were increased with the promoted generation of alkenes. In particular, the multivariate statistical analysis indicated that 7 volatiles showed a higher level after 1 year of storage, whereas 11 volatiles decreased and 4 volatiles increased after 2 years of storage, respectively. This study could show the early aging mechanism of CRCP harvested at different periods and provide a scientific guidance in the storage of CRCP. PRACTICAL APPLICATION: This study indicated a comprehensive method for rapid analysis of phytochemical and volatile compounds in pericarp of Citrus reticulata ''Chachi'' (Rutaceae) (CRCP) harvested at different periods during 2 years of storage. The results obtained from this study would be valuable for revealing the early aging mechanism and sustainable storage of CRCP.

Rapid Discrimination of Citrus reticulata 'Chachi' by Electrospray Ionization-Ion Mobility-High-Resolution Mass Spectrometry.

A common idea is that some dishonest businessmen often disguise Citrus reticulata Blanco varieties as Citrus reticulata 'Chachi', which places consumers at risk of economic losses. In this work, we combined high-resolution ion mobility (U-shaped mobility analyzer) with high-resolution mass spectrometry to rapidly distinguish Citrus reticulata 'Chachi' from other Citrus species. The samples were analyzed directly through simple extraction and the analytes were separated in one second. It only took about 1 min to perform a cycle of sample analysis and data acquisition. The results showed that polymethoxylated flavones and their isomers were separated easily by the ion mobility analyzer and preliminarily identified according to the accurate mass. Moreover, the collision cross-section values of all analytes, which could be used as auxiliary parameters to characterize and identify the compounds in the samples, were measured. Twenty-four samples were grouped as two clusters by multivariate analysis, which meant that Citrus reticulata 'Chachi' could be effectively differentiated. It was confirmed that the developed method had the potential to rapidly separate polymethoxylated flavones and distinguish between Citrus reticulata 'Chachi' and other Citrus reticulata Blanco varieties.

Evaluation of dynamic changes in the bioactive components in Citri Reticulatae Pericarpium (Citrus reticulata 'Chachi') under different harvesting and drying conditions.

BACKGROUND: The Citrus reticulata 'Chachi' pericarp (CRCP) is one cultivar of Citri Reticulatae Pericarpium (Chenpi), which is widely applied in medicine and food. To determine the potential value of CRCP harvested at different stages and subjected to different drying processes, the dynamic changes in the bioactive components were profiled and evaluated in this study. RESULTS: The contents of all non-volatile components, i.e. synephrine, limonin, phenolic acids and flavonoids, decreased with delayed harvest time. The volatiles thujene, α-pinene, ß-pinene, d-citronellol, d-citronellal, decanal, linalool, geraniol, l-cis-carveol, terpinen-4-ol, α-terpineol, carvacrol, perillaldehyde, methyl 2-(methylamino)benzoate and d-limonene were considered the characteristic components for distinguishing CRCP harvested at different stages. Phenolic acids, synephrine and limonin were stable at different drying temperatures; however, high-temperature drying at 60 °C induced a significant transformation in the flavonoids (especially polymethoxyflavones) and volatile substances in CRCP. CONCLUSIONS: The results suggested that most of the bioactive components declined with the growth of Citrus reticulata 'Chachi'. And it is believed that the fresh peel should be naturally sun-dried or dried at low temperature (30 or 45 °C) rather than at high temperature (60 °C) to prevent excessive loss of nutrients. © 2020 Society of Chemical Industry.

Comprehensive Comparison on the Chemical Profile of Guang Chen Pi at Different Ripeness Stages Using Untargeted and Pseudotargeted Metabolomics.

The peel of Citrus reticulata 'Chachi' (GCP), which is highly valued in China for its health-promoting effects, is usually collected at different development stages to be processed into various functional foods. In the present work, a rapid method based on ultra-high-performance liquid chromatography Q Exactive Orbitrap mass spectrometry-incorporated untargeted and pseudotargeted metabolomics analysis was developed to investigate the chemical variations in GCP at different ripeness stages. Samples that originated from an individual tree were collected at immature, near mature, and mature stages. A total of 112 compounds were identified or tentatively identified, and flavonoids malonyl glycosides and polymethoxyfolavones glycosides were reported for the first time. Untargeted metabolomics analysis indicated the distinct chemical profiles and significant changes during ripeness stages. Then, a validated pseudotargeted metabolomics method based on parallel reaction monitoring was further applied with a wide coverage of targeted compounds. The GCP samples were found differing in the content variations of flavonoid aglycones, flavonoid O-/C-glycosides, polymethoxyfolavones, limonoids, alkaloids, and phenolic acid, which are important for phenotypic variations at different development stages. The present study is expected to provide new insight on comprehensive utilization of citrus peels at different ripeness stages.

A novel fluorescence sensing strategy based on nanoparticles combined with spectral splicing and chemometrics for the recognition of Citrus reticulata 'Chachi' and its storage year.

BACKGROUND: The fluorescence sensing method has been increasingly applied in food quality control because it is fast and sensitive. However, its application in quality evaluation is challenging. Using Citri Reticulatae Pericarpium (CRP; dried mandarin orange peel) as an example, we developed a simple and low-cost fluorescence sensing strategy based on nanoparticles combined with spectral splicing and chemometrics for quality evaluation. This method can recognize Citrus reticulata 'Chachi' (CRC) from other CRP cultivars and further identify the storage year. RESULTS: Nanogold particles and cadmium telluride quantum dots were selected as nanosensors and mixed with aqueous extracts of CRP separately to produce fluorescence quenching spectra. Then, a simple spectral splicing procedure was applied to obtain spliced spectra comprising different combinations of the self-fluorescence and fluorescence quenching spectra of CRP samples. With the aid of partial least-squares discriminant analysis, the new strategy achieved recognition rates of 100% in distinguishing CRC samples from other CRP samples, as well as recognition rates of 100% for the training set and 98.04% for the prediction set in the discrimination of the storage year of CRC. The recognition mechanism is dominated by interactions between the nanoparticles and the fluorescent components in the CRP samples, but other components also have concurrent effects. CONCLUSIONS: This novel fluorescence sensing strategy not only provides a new tool for the quality evaluation of CRC but also has good prospects for the authentication and traceability of other foods and herbs. Crucially, the developed method is convenient, simple and effective. © 2020 Society of Chemical Industry.

Rapid discrimination of Citrus reticulata 'Chachi' by headspace-gas chromatography-ion mobility spectrometry fingerprints combined with principal component analysis.

Dried pericarps of Citrus reticulata 'Chachi', named Guangchenpi (GCP), have been used as both food and medicine in China for centuries. Due to its special flavour and taste, the price of GCP is much higher than those of dried pericarps from other Citrus reticulata Blanco varieties (collectively called "Chenpi", CP). Therefore, some CP have been disguised has GCP by dishonest businessmen to obtain a higher profit. In this study, a simple, rapid, effective and economic analytical method based on headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) was established to discriminate GCP and CP by their volatile organic compounds (VOCs). Principal component analysis (PCA) was performed to extract meaningful attributes from VOCs based on the GC-IMS chromatograms and to give the analysis results. Twelve, 12 and 14 markers were identified and established as the characteristic fingerprints of GCP analysis of 45 batches of samples via SE-54, OV-1701 and Inter Cap WAX gas chromatographic columns, respectively. The PCA results indicated that GCP and CP could be effectively discriminated. This study confirmed the potential of HS-GC-IMS combined with PCA as a reliable analytical screening technique to discriminate between GCP and CP.