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.

Qualitative and Quantitative Analyses of the Chemical Components of Peels from Different Pomelo Cultivars (Citrus grandis [L.] Osbeck) Based on Gas Chromatography-Mass Spectrometry, Ultraperformance Liquid Chromatography-Q-Exactive Orbitrap-MS, and High-Performance Liquid Chromatography-Photodiode Array Detection.

The volatile and nonvolatile phytochemicals in peels of 5 major pomelo cultivars (including Citrus grandis cv. Yuhuanyou, C. grandis cv. Liangpingyou, C. grandis cv. Guanximiyou, C. grandis cv. Duweiwendanyou, and C. grandis cv. Shatianyou) from 11 places in China were characterized. First, 194 volatile compounds in pomelo peels were identified by gas chromatography-mass spectrometry (GC-MS). Of these, 20 major volatile compounds were subjected to cluster analysis. The heatmap indicated that the volatile compounds in peels of C. grandis cv. Shatianyou and C. grandis cv. Liangpingyou were different from those in other varieties, while there was no difference among C. grandis cv. Guanximiyou, C. grandis cv. Yuhuanyou, and C. grandis cv. Duweiwendanyou from different origins. Second, 53 nonvolatile compounds were identified in pomelo peels by ultraperformance liquid chromatography-Q-exactive orbitrap tandem MS (UPLC-Q-exactive orbitrap-MS), of which 11 components were detected for the first time. Third, six major nonvolatile compounds were quantitatively analyzed with high-performance LC-photodiode array detection (HPLC-PDA). Combining the results of HPLC-PDA and the heatmap, 6 nonvolatile compounds in 12 batches of pomelo peel were well separated among varieties. Comprehensive analysis and identification of chemical components in pomelo peels are of great significance for their further development and utilization.

Comparative analysis of chemical constituents in Citri Exocarpium Rubrum, Citri Reticulatae Endocarpium Alba, and Citri Fructus Retinervus.

Citri Exocarpium Rubrum (CER), Citri Reticulatae Endocarpium Alba (CREA), and Citri Fructus Retinervus (CFR) are used as medicine and food, which derive from three different parts of the pericarp of Citrus reticulata Blanco through natural drying. To systematically investigate similarities and differences in phytochemicals about the three herbs, a series of analytic approaches were applied for the qualitative and quantitative analysis of chemical constituents in them. The results indicated a total of 48 volatile compounds were determined representing 99.92% of the total relative content of CER extracts, including 24 alkenes, 11 alcohols, 6 aldehydes, 2 ketones, and 2 phenols, while volatile compounds were not extracted from CREA and CFR. CER was abundant in volatile components that mainly existed in the oil gland. And a total of 32, 35, and 28 nonvolatile compounds were identified from CER, CREA, and CFR extracts, respectively. The total content of flavonoids and phenolic, and hesperidin in CFR was the highest, followed by CREA and CER. Conversely, CER was a rich source of polymethoxyflavones (PMFs), and the total polymethoxyflavone content (TPMFC), the content of nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF), tangeretin, and 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone (5-HPMF) in CREA and CFR were extremely low. Besides, CER and CREA had a higher concentration of synephrine than CFR. The phytochemicals of CER, CREA, and CFR were significantly different, which might provide chemical evidence for the comparative pharmacological activities' research and rational application of them.

Qualitative and quantitative analyses of chemical components of Citri Sarcodactylis Fructus from different origins based on UPLC-Q-Exactive Orbitrap-MS and GC-MS.

Ultra-high-performance liquid chromatography-Q-Exactive Orbitrap-mass spectrometry (MS) and gas chromatography (GC)-MS were performed for the qualitative and quantitative analyses of Citri Sarcodactylis Fructus (CSF) from different origins. The contents of eight major CSF components, namely 5,7-dimethoxycoumarin, scopoletin, hesperidin, tangeretin, nobiletin, limonin, nomilin, and stachydrine, were quantitatively analyzed. Clustering analysis and principal component analysis (PCA) were, respectively, performed to classify and compare the 10 CSF batches. One hundred and two volatile components were identified accordingly by comparing retention times, reference standards, parent peaks, fragment peaks, and findings from relevant literature. Moreover, high content of 5,7-dimethoxycoumarin and stachydrine was detected in all the CSFs, especially in CSF-Zhe. Therefore, the high content component coumarin "5,7-dimethoxycoumarin" was suggested to be quality analysis component rather than hesperidin. Additionally, characteristic compounds were found to distinguish different CSFs. This work was a comprehensive study about the components of various CSF. It distinguished the basic differences in the compositions of CSF from different origins. Eventually, it provided experimental and systematic bases for the quality control analysis of CSF, which has potential application in the further research.

Analysis of the chemical components of pomelo peels (Citrus grandis [L.] Osbeck) from different cultivars by using supercritical CO2 fluid extraction and ultra-high-performance liquid chromatography-tandem mass spectrometry.

Five pomelo cultivars (i.e., Citrus grandis cv. Shatianyou, Citrus grandis cv. Guanximiyou, Citrus grandis cv. Yuhuanyou, Citrus grandis cv. Duweiwendanyou and Citrus grandis cv. Liangpingyou) from different origins in China were selected to analyze their components by using supercritical CO2 fluid extraction coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry. A total of 45 compounds were identified in the supercritical CO2 fluid extracts of the pomelo peels from the five cultivars. These compounds included eight flavonoids, 18 coumarins, four organic acids, three aldehydes, and 12 other compounds, which were identified using the obtained MS data and by comparison with commercial standards, orbitrap Chinese Traditional Medicine Library, and previous literature. Twenty-five of the identified compounds were detected for the first time in the pomelo peel extracts. Results suggested that the pomelo peels of C. grandis cv. Shatianyou contained the most natural chemical compositions. The pooled result may offer scientific evidence for further development and utilization of pomelo peels and a route for screening appropriate varieties for various demands.

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.

Intraspecific DNA Barcoding and Variation Analysis for Citri Reticulatae Pericarpium of Citrus reticulata "Chachi".

Citri Reticulatae Pericarpium, the desiccative mature peel of Citrus reticulata Blanco or its cultivated varieties, is a national geographical indicated product that has the concomitant function of both medicine and foodstuff. The primary source of Citri Reticulatae Pericarpium is Citrus reticulata "Chachi," called "Guang chenpi," while it differs in variety, propagation, grafting rootstock, and tree age, and the hereditary stability of its biological information between intraspecific plants is worthy of our attention. Homologous analysis result of 4 DNA barcodings in the ribosome or the chloroplast showed that the homology of them (ITS2, rbcl, matK, and psbA-trnH) of 22 samples was 100.00%, 99.97%, 99.99%, and 99.81%, respectively, which indicated that 4 DNA barcodes maintained a high degree of genetic stability in Citrus reticulata "Chachi." Also, ITS2 was considered to identify Citrus reticulata "Chachi" from other varieties because it presented not only low variability within a certain taxon but also a high level of interspecies variability. Simultaneously, variant site detection of Citrus reticulata "Chachi" was analyzed by comparing with the reference Citrus reticulata genome, and 2652697 SNP sites and 533906 InDel sites were detected from whole-genome resequencing data of 22 samples, providing the data resources and theoretical foundation for the future study about the relevant molecular makers of "Guang chenpi."

Identification of Components in Citri Sarcodactylis Fructus from Different Origins via UPLC-Q-Exactive Orbitrap/MS.

To systematically analyze the chemical constituents of Citri Sarcodactylis Fructus (CSF) from different origins, an efficient approach based on ultraperformance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap/MS) detection for the discrimination of chemical components from of 15 batches of CSF from four main origins was used in this research. Through parent peaks, fragment peaks, fragmentation characteristics, and comparative analysis with the literature and reference standards, a total of 77 components from the methanol extracts including 18 coumarins, 24 flavonoids, seven organic acids, three limonoids, and 25 other compounds were detected and identified. Among them, 15 components have not been reported previously in the CSF. Notably, the stachydrine peak initially showed a higher content in the total ion current chromatogram. Overall, CSF produced in the Zhejiang province contained a richer variety of chemical compositions. These observations provided a theoretical basis for the further quality assessment and application of CSF.

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.

UPLC-Q-Exactive Orbitrap MS Analysis for Identification of Lipophilic Components in Citri Sarcodactylis Fructus from Different Origins in China Using Supercritical CO2 Fluid Extraction Method.

To thoroughly evaluate the quality of Citri Sarcodactylis Fructus (CSF) and acquire knowledge of the lipophilic components of CSF from different origins, a simple and efficient approach based on supercritical fluid extraction (SFE) combined with ultraperformance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap/MS) detection for the discrimination of components from CSF was set up for the first time in this work. Eight batches of CSF samples from five main producing areas were extracted by SFE under optimized conditions, and then SFE extracts were dissected via UPLC-Q-Exactive Orbitrap/MS. The results indicated that 39 lipophilic compounds were successfully separated and unambiguously or tentatively identified, where 4 coumarins, 6 polymethoxyflavones, 3 phthalides, 6 terpenes, and 4 phenolics were not reported formerly. It was illustrated that CSF may be abundant in polymethoxyflavones, as in coumarins. Moreover, there were significant differences in the components of CSF from different origins. Especially, coumarin, dehydrocostus lactone, atractylenolide II, and atractylenolide I were exclusively found in CSF from the Guangdong province; isopsoralen was almost exclusively found in CSF from the Guangxi province; and ferulic acid was exclusively found in CSF from the Zhejiang province. These observations indicated that SFE joint with UPLC-Q-Exactive Orbitrap/MS owing to the potential of characterizing the lipophilic components could be used to promote quality assessment and chemotaxonomic investigation in phytology sciences of CSF.

[Conversion Characteristics of Combustible Particles from Coal-fired Flue Gas in WFGD and WESP].

Monitoring of condensable particulate matter (CPM) emitted from stationary pollution sources has often been neglected. To reduce the emission of CPM, it is necessary to study its transformation rules in flue-gas cleaning devices. The results show that the wet flue gas desulfurization (WFGD) and wet electrostatic precipitator (WESP) have a good synergistic effect on the removal of CPM. The concentration of CPM in flue gas is higher than that of filterable particulate matter, but the concentration of total particulate matter (sum of the condensable particulate matter and filterable particulate matter) reaches ultra-low emission requirements. The organic mass concentration of CPM in the WFGD inlet and outlet is larger than that of inorganic components, which is equalized during the treatment of WESP. Based on measurements of the components and concentrations of water-soluble ions in the inorganic part of the CPM, PM0.3 can be joined during the CPM trapping process and an acid mist is generated during the condensable particulate matter formation. The acid mist is strengthened through the treatment of WFGD and WESP. The experiment results also show that SO42- is the main component of water-soluble ions in the inorganic part of CPM.

[Efficacy of integrative medicine for the treatment of rheumatoid arthritis and its effect on glucocorticoid receptor expression].

OBJECTIVE: To investigate the efficacy and mechanism of Fengshikang (FSK) in treating rheumatoid arthritis (RA). METHODS: Thirty-eight patients with RA of damp-heat block syndrome type in Chinese medicine (CM), were randomized into two groups. They all received glucocorticoid therapy for 1 month, but FSK was administered to the 20 patients in the CM group in addition, while to the 18 in the WM group, MTX was given. Besides, a normal control group with 20 healthy subjects was set up. Blood levels of RA related indices were measured and compared. RESULTS: After treatment, the levels of rheumatoid factor (RF) and C-reactive protein (CRP) were significantly reduced in both groups (P<0.05), but the reduction was more significant in the CM group (P<0.05). No significant change was found in the level of ESR after treatment and showed no significant difference between groups (P>0.05). Both hGRalpha mRNA and hGRbeta mRNA expressions were significantly higher in the patients than in the normal controls (P<0.01), they were lowered after treatment in the CM group (P<0.01), but significant reduction could only be found in level of hGRbeta mRNA in the control group (P<0.01). CONCLUSION: FSK combined with glucocorticoid can effectively control the inflammatory reaction, decrease the level of hGRalpha and hGRbeta expressions and enhance the clinical efficacy in treating RA.