Network Pharmacology and Molecular Modeling Techniques in Unraveling the Underlying Mechanism of Citri Reticulatae Pericarpium aganist Type 2 Diabetic Osteoporosis.

Type 2 diabetic osteoporosis (T2DOP) is a common complication in diabetic patients that seriously affects their health and quality of life. The pathogenesis of T2DOP is complex, and there are no targeted governance means in modern medicine. Citri Reticulatae Pericarpium (CRP) is a traditional Chinese medicine that has a long history and has been used in the treatment of osteoporosis diseases. However, the molecular mechanism for the CRP treatment of T2DOP is not clear. Therefore, this study aimed to explore the underlying mechanisms of CRP for the treatment of T2DOP by using network pharmacology and molecular modeling techniques. By retrieving multiple databases, we obtained 5 bioactive compounds and 63 common targets of bioactive compounds with T2DOP, and identified AKT 1, TP 53, JUN, BCL 2, MAPK 1, NFKB 1, and ESR 1 as the core targets of their PPI network. Enrichment analysis revealed that these targets were mainly enriched in the estrogen signaling pathway, TNF signaling pathway, and AGE-RAGE signaling pathway in diabetics, which were mainly related to oxidative stress and hormonal regulation. Molecular docking and molecular dynamics simulations have shown the excellent binding effect of the bioactive compounds of CRP and the core targets. These findings reveal that CRP may ameliorate T2DOP through multiple multicomponent and multitarget pathways.

Citri Reticulatae Pericarpium Limits TLR-4-Triggered Inflammatory Response in Raw264.7 Macrophages by Activating RasGRP3.

Inflammation is an important immune response to pathogen invasion, but excessive inflammation leads to tissue injury and even cytokine storm. Therefore, proper response is needed depending on the intensity of the infection. Ras guanine nucleotide releasing protein 3 (RasGRP3) is a regulator of the TLR-mediated response. In low-intensity inflammation, it negatively regulates production of pro-inflammatory cytokines, especially IL-6. Citri Reticulatae Pericarpium, the peel of Citrus reticulata Blanco, is a major medicinal herb in Korean medicine. The present study aims to investigate whether the Citri Reticulatae Pericarpium extract (CRE) has immunomodulatory activity using the Raw264.7 macrophage. Also, we investigated the effect of CRE on RasGRP3 expression. In the present study, CRE reduced IL-6 production in the low-LPS environment (1 ng/mL) and did not in the high-LPS environment (100 ng/mL). The suppression of IL-6 production in the low-LPS environment (1 ng/mL) was abolished after the pretreatment of RasGRP3 siRNA. The reduced RasGRP3 protein content by 100 ng/mL LPS treatment was increased by CRE treatment. Additionally, nobiletin, a major component of CRE showed a suppressive effect on IL-6 production in the low-LPS environment (1 ng/mL). The present results suggest that CRE alleviates inflammatory response via activating RasGRP3 expression in low-intensity inflammation.

Reduction of Th2 inflammation and fibrosis in eosinophilic esophagitis in a murine model by citri reticulatae pericarpium.

ETHNOPHARMACOLOGICAL RELEVANCE: Inflammation and subepithelial fibrosis play major roles in the early pathology of eosinophilic esophagitis (EoE). However, there are currently no pharmacotherapeutic interventions that directly target eosinophilic esophagitis. Citri Reticulatae Pericarpium (CRP, known as Chen-Pi) is one of most frequently used qi-regulating drugs in Chinese medicine and nutrition. CRP is rich with flavonones and polymethoxy flavones, both of which exhibit superior anti-inflammatory, anti-allergic and anti-fibrosis effects. This study is to investigate intervention effect of CRP on EoE, to identify its active compounds and to explore its underlying mechanisms. METHODS: The CRP extract was obtained by liquid-liquid extraction with 70% ethanol, and its main components were identified by HPLC and TLC chromatography as hesperidin, nobiletin, tangeretin, and narirutin in turn. Furthermore, we evaluated its effect and underlying mechanisms in an PN (Peanut protein extract)-sensitized murine model of food allergy induced EoE. RESULTS: CRP treatment attenuated EoE model mice symptomatology, blocked hypothermia, reduced the production of PN-specific IgE and IgG1 and TH2 cytokines (interleukin (IL)-4 and IL-5), and increased the level of anti-inflammatory cytokines IL-10 and interferon (IFN)-γ. CRP treatment also significantly alleviated the pathological damage and reduced fibrosis in inflamed tissues like esophagus, lung, and intestine. These results were strongly associated with reducing the expression of p-p38 mitogen-activated protein kinase (MAPK), transforming growth factor beta1 (TGF-ß1) and p-Smad 3 proteins. CONCLUSION: CRP extract markedly inhibited TH2 immune response and attenuated subepithelial fibrosis with a dose-dependent manner through down-regulating MAPK/TGF-ß signaling pathway. It is suggested that CRP extract might serve as a potential therapy for food allergy-induced EoE like disease.

Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. herb pair suppresses breast cancer liver metastasis by targeting ECM1-mediated cholesterol biosynthesis pathway.

BACKGROUND: Liver metastasis is a frequent event in breast cancer that causes low survival rate and poor prognosis. Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. (CR), a traditional Chinese herb pair, is used for the treatment of breast cancer liver metastasis or cholesterol gallstone disease in clinics. PURPOSE: This study attempted to investigate the potential therapeutic target and mechanism of CR herb pair on breast cancer liver metastasis. METHODS: The anti-metastatic and cholesterol-lowering activities of CR extract were evaluated in triple-negative breast cancer (TNBC) cell lines and an experimental liver metastasis model. The role of extracellular matrix protein 1 (ECM1) in the cholesterol biosynthesis pathway was determined by the knockdown and overexpression of ECM1 gene of TNBC cells. Changes in the gene and protein expression levels of ECM1 and the cholesterol biosynthesis pathway after CR treatment were detected in vitro and in vivo by real-time PCR and Western blot. RESULTS: The invasive and metastatic potentials and hypercholesterol levels of TNBC cells were positively associated with ECM1 expression. ECM1 knockdown reduced tumor cholesterol levels via downregulating cholesterol biosynthesis genes, including ACAT2, HMGCS1, HMGCR, MVK, and MVD, whereas ECM1 overexpression elicited the opposite effects. CR herb pair exerts the potential therapeutic effects on TNBC liver metastasis, which is partially mediated by disrupting ECM1-activated cholesterol biosynthesis process in TNBC cells. CONCLUSION: This study reveals that ECM1 is a novel target for the activation of cholesterol biosynthesis to promote TNBC liver metastasis occurrence. CR herb pair, an ECM1 inhibitor, maybe be considered to serve as an adjuvant therapeutic drug for liver metastasis in clinical practice.

Progress of research on the role of active ingredients of Citri Reticulatae Pericarpium in liver injury.

BACKGROUND: Liver is a vital organ responsible for metabolizing and detoxifying both endogenous and exogenous substances in the body. However, it is susceptible to damage from chemical and natural toxins. The high incidence and mortality rates of liver disease and its associated complications impose a significant economic burden and survival pressure on patients and their families. Various liver diseases exist, including cholestasis, viral and non-viral hepatitis, fatty liver disease, drug-induced liver injury, alcoholic liver injury, and severe end-stage liver diseases such as cirrhosis, hepatocellular carcinoma (HCC), and cholangiocellular carcinoma (CCA). Recent research has shown that flavonoids found in Citri Reticulatae Pericarpium (CRP) have the potential to normalize blood glucose, cholesterol levels, and liver lipid levels. Additionally, these flavonoids exhibit anti-inflammatory properties, prevent oxidation and lipid peroxidation, and reduce liver toxicity, thereby preventing liver injury. Given these promising findings, it is essential to explore the potential of active components in CRP for developing new drugs to treat liver diseases. OBJECTIVE: Recent studies have revealed that flavonoids, including hesperidin (HD), hesperetin (HT), naringenin (NIN), nobiletin (NOB), naringin (NRG), tangerine (TN), and erodcyol (ED), are the primary bioactive components in CRP. These flavonoids exhibit various therapeutic effects on liver injury, including anti-oxidative stress, anti-cytotoxicity, anti-inflammatory, anti-fibrosis, and anti-tumor mechanisms. In this review, we have summarized the research progress on the hepatoprotective effects of HD, HT, NIN, NOB, NRG, TN, ED and limonene (LIM), highlighting their underlying molecular mechanisms. Despite their promising effects, the current clinical application of these active ingredients in CRP has some limitations. Therefore, further studies are needed to explore the full potential of these flavonoids and develop new therapeutic strategies for liver diseases. METHODS: For this review, we conducted a systematic search of three databases (ScienceNet, PubMed, and Science Direct) up to July 2022, using the search terms "CRP active ingredient," "liver injury," and "flavonoids." The search data followed the PRISMA standard. RESULTS: Our findings indicate that flavonoids found in CRP can effectively reduce drug-induced liver injury, alcoholic liver injury, and non-alcoholic liver injury. These therapeutic effects are mainly attributed to the ability of flavonoids to improve liver resistance to oxidative stress and inflammation while normalizing cholesterol and liver lipid levels by exhibiting anti-free radical and anti-lipid peroxidation properties. CONCLUSION: Our review provides new insights into the potential of active components in CRP for preventing and treating liver injury by regulating various molecular targets within different cell signaling pathways. This information can aid in the development of novel therapeutic strategies for liver disease.

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.

Quality control strategies of medicine food homology materials based on fingerprint profiling and chemometrics: Citri Reticulata Pericarpium as an example.

The study aimed to provide a reliable and feasible strategy for the comprehensive quality control of medicine food homology materials (MFHM). The high performance liquid chromatography (HPLC) fingerprints and Fourier transform mid-infrared (FT-MIR) quantized fingerprints were successfully developed to comprehensively evaluate overall quality of Citri Reticulata Pericarpium (CRP) by applying comprehensive quantified fingerprint method (CQFM). All samples were well distinguished and divided into 5 grades. In addition, through principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), the identification ability of HPLC fingerprints and FT-MIR fingerprints on CRP with different storage years was discussed. The results showed that HPLC fingerprints combined with PCA had good discrimination ability, and the PLS-DA model established by the preprocessed FT-MIR fingerprint data could accurately distinguish and predict the storage period of CRP. Finally, based on 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH•) scavenging assay, combined with bivariate correlation analysis, the fingerprint-activity relationship of offline antioxidant activity of CRP samples with the fingerprints peak were studied. In general, the comprehensive strategies provide a reliable and scientific reference scheme for the quality control of MFHM in the future.

Analysis of Suitable Processing Time of Rehmanniae Radix Praeparata Processed with Amomi Fructus and Citri Reticulatae Pericarpium Based on UPLC-Q-TOF-MS / 中国实验方剂学杂志

ObjectiveTo investigate the relative content changes of differential metabolites and reducing sugars during the processing process of Rehmanniae Radix Praeparata （RRP） processed with Amomi Fructus （AF） and Citri Reticulatae Pericarpium （CRP）， and to lay the foundation for revealing the processing principle of this characteristic variety. MethodThe samples of the 0-54 h processing process of RRP processed with AF and CRP were taken as the research object， and their secondary metabolites were detected by ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS）. The 0.1% formic acid aqueous solution （A）-acetonitrile （B） was used as the mobile phase for gradient elution （0-1 min， 1%-3%B； 1-10 min， 3%-9%B； 10-15 min， 9%-12%B； 15-22 min， 12%-18%B； 22-31 min， 18%-24%B； 31-35 min， 24%-100%B； 35-36 min， 100%-5%B； 36-40 min， 5%-1%B； 40-45 min， 1%B）， column temperature was 40 ℃， injection volume was 3 μL， flow rate was 0.3 mL·min-1. Electrospray ionization （ESI） was used to scan and collect MS data in the negative ion mode， the scanning range was m/z 50-1 250. Data analysis was carried out using PeakView 1.2 software， and the chemical composition of RRP processed with AF and CRP was identified by combining the literature information and chemical composition databases. The MS data were normalized by MarkerView 1.2， and then the multivariate statistical analysis was applied to screen the differential metabolites， and the changes of the relative contents of the differential metabolites with different processing times was analyzed， finally， correlation analysis was performed between the differential metabolites， the change of the reducing sugar content was combined to determine the most suitable processing time of RRP processed with AF and CRP. ResultA total of 121 compounds were identified from RRP processed with AF and CRP at different processing times， and 12 differential metabolites were screened out by multivariate statistical analysis， including catalpol， hesperidin， isoacteoside， acteoside， narirutin， echinacoside， isomartynoside， decaffeoylacteoside， 6-O-E-feruloylajugol， dihydroxy-7-O-neohesperidin， jionoside D， and rehmapicroside. With the prolongation of processing time， the relative contents of these 12 differential metabolites and reducing sugars changed slightly at 52-54 h. ConclusionUPLC-Q-TOF-MS can comprehensively and accurately identify the chemical constituents of RRP processed with AF and CRP at different processing times， and the suitable processing time of 52-54 h is determined according to the content changes of different metabolites and reducing sugars， which provides a basis for revealing the scientific connotation of the processing principle of this variety.

Discovery of the key active compounds in Citri Reticulatae Pericarpium (Citrus reticulata "Chachi") and their therapeutic potential for the treatment of COVID-19 based on comparative metabolomics and network pharmacology.

Edible herbal medicines contain macro- and micronutrients and active metabolites that can take part in biochemical processes to help achieve or maintain a state of well-being. Citri Reticulatae Pericarpium (CRP) is an edible and medicinal herb used as a component of the traditional Chinese medicine (TCM) approach to treating COVID-19 in China. However, the material basis and related mechanistic research regarding this herb for the treatment of COVID-19 are still unclear. First, a wide-targeted UPLC-ESI-MS/MS-based comparative metabolomics analysis was conducted to screen for the active metabolites of CRP. Second, network pharmacology was used to uncover the initial linkages among these metabolites, their possible targets, and COVID-19. Each metabolite was then further studied via molecular docking with the identified potential SARS-CoV-2 targets 3CL hydrolase, host cell target angiotensin-converting enzyme II, spike protein, and RNA-dependent RNA polymerase. Finally, the most potential small molecule compound was verified by in vitro and in vivo experiments, and the mechanism of its treatment of COVID-19 was further explored. In total, 399 metabolites were identified and nine upregulated differential metabolites were screened out as potential key active metabolites, among which isorhamnetin have anti-inflammatory activity in vitro validation assays. In addition, the molecular docking results also showed that isorhamnetin had a good binding ability with the key targets of COVID-19. Furthermore, in vivo results showed that isorhamnetin could significantly reduced the lung pathological injury and inflammatory injury by regulating ATK1, EGFR, MAPK8, and MAPK14 to involve in TNF signaling pathway, PI3K-Akt signalling pathway, and T cell receptor signaling pathway. Our results indicated that isorhamnetin, as screened from CRP, may have great potential for use in the treatment of patients with COVID-19. This study has also demonstrated that comparative metabolomics combined with network pharmacology strategy could be used as an effective approach for discovering potential compounds in herbal medicines that are effective against COVID-19.

Comparison of Geqingpi and Sihuaqingpi based on ultra-high-performance liquid chromatography-tandem mass spectrometry combined with multivariate statistics, network pharmacology analysis, and molecular docking.

Citri Reticulatae Pericarpium Viride is used in traditional Chinese medicine as Geqingpi and Sihuaqingpi varieties. We used the ultra-high-performance liquid chromatography-quadrupole-Exactive Orbitrap-mass spectrometry method and high-performance liquid chromatography-triple quadrupole-tandem mass spectrometry to analyze the chemical compounds in these varieties. Principal components analysis and orthogonal partial least squares discriminant analysis were used to analyze the quantitative results. Network pharmacology and molecular docking technology were used to forecast Citri Reticulatae Pericarpium Viride treatment mechanisms in irritable bowel syndrome. We identified 44 main compounds in Citri Reticulatae Pericarpium Viride. Compared to Sihuaqingpi, Geqingpi had higher narirutin, didymin, naringenin, and hesperetin, and lower hesperidin, isosinensetin, nobiletin, 3,5,6,7,8,3',4'-hexamethoxyflavone, tangeretin. Tangeretin, nobiletin, narirutin, didymin, and isosinensetin were the main compounds distinguishing Geqingpi from Sihuaqingpi. We found that the MAPK signaling pathway, which is closely related to irritable bowel syndrome, was an important target pathway. TP53, HRAS, MAPK1, AKT1, and EGFR were important targets in this pathway. Eriodictyol-7-O-rutinoside, narirutin, limonin, and hesperidin showed a good binding ability to the five targets. Orientin, unique to Sihuaqingpi, bound well to TP53, MAPK1, AKT1, and EGFR, while rhoifolin bound well to TP53, HRAS, MAPK1, AKT1, and EGFR. Hesperetin, unique to Geqingpi, bound well to TP53, HRAS, and MAPK1, while naringenin bound well to HRAS. Hesperidin and didymin bound well to TP53, MAPK1, AKT1, and EGFR.

[Spleen deficiency and phlegm dampness syndrome model in rats treated by Citri Reticulatae Pericarpium based on metabolomics].

The present study analyzed the effect of Citri Reticulatae Pericarpium on endogenous metabolites in spleen deficiency and phlegm dampness syndrome by metabolomics, and explored the underlying mechanism of Citri Reticulatae Pericarpium in the treatment of spleen deficiency and phlegm dampness syndrome.The model of spleen deficiency and phlegm dampness syndrome was induced in rats by the multi-factor modeling method.The intervention effects of Citri Reticulatae Pericarpium on rats with spleen deficiency and phlegm dampness syndrome were preliminarily evaluated by observing the pathological changes of rat liver tissues and measuring the plasma content of pathological and biochemical indexes such as triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C), and high-density lipoprotein cholesterol(HDL-C).Immunohistochemistry was used to detect the expression of AQP2 in the kidney, AQP3 in the colon, and AQP5 in the submandibular gland, and the effect of Citri Reticulatae Pericarpium on aquaporin expression in rats with spleen deficiency and phlegm dampness syndrome was evaluated.Furthermore, UHPLC-ESI-MS/MS was used to analyze the metabolic profiles of rat plasma samples.Multiple methods, such as principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were used for pattern recognition.Differential metabolites were screened out by t-test and variable importance in projection(VIP), followed by pathway analysis based on MetaboAnalyst 5.0.As revealed by experimental results, Citri Reticulatae Pericarpium could improve the pathological changes of liver tissues, increase the levels of HDL-C in the plasma, reduce the levels of TC, TG, and LDL-C, and enhance the expression of AQP2 in the kidney, AQP3 in the colon, and AQP5 in the submandibular gland of rats with spleen deficiency and phlegm dampness syndrome.In addition, 87 differential metabolites of spleen deficiency and phlegm dampness syndrome were screened out by UHPLC-ESI-MS/MS(the levels of 39 metabolites increased significantly and the levels of 48 metabolites decreased significantly), with the representatives of glycine, L-isoleucine, N-acetyl-L-tyrosine, xanthine, hypoxanthine, and trigonelline.The differential metabolites were mainly enriched in the pathways of steroid hormone biosynthesis, linoleic acid metabolism, and purine metabolism.This study distinguished and revealed the characteristic metabolic pattern of spleen deficiency and phlegm dampness syndrome by metabolomics.The preliminary construction of the OPLS-DA model provides an objective basis for the differentiation of spleen deficiency and phlegm dampness syndrome in traditional Chinese medi-cine(TCM), as well as ideas and methods for exploring the biological basis of TCM syndrome from the molecular level and the overall level.

Mechanism of Citri Reticulatae Pericarpium as an Anticancer Agent from the Perspective of Flavonoids: A Review.

Citri Reticulatae Pericarpium (CRP), also known as "chenpi", is the most common qi-regulating drug in traditional Chinese medicine. It is often used to treat cough and indigestion, but in recent years, it has been found to have multi-faceted anti-cancer effects. This article reviews the pharmacology of CRP and the mechanism of the action of flavonoids, the key components of CRP, against cancers including breast cancer, lung cancer, prostate cancer, hepatic carcinoma, gastric cancer, colorectal cancer, esophageal cancer, cervical cancer, bladder cancer and other cancers with a high diagnosis rate. Finally, the specific roles of CRP in important phenotypes such as cell proliferation, apoptosis, autophagy and migration-invasion in cancer were analyzed, and the possible prospects and deficiencies of CRP as an anticancer agent were evaluated.

Citri Reticulatae Pericarpium (Chenpi): A multi-efficacy pericarp in treating cardiovascular diseases.

Citri Reticulatae Pericarpium (CRP) has been utilized as a versatile medicinal herb with wide cardiovascular benefits in Asian nations for centuries. Accumulating evidence suggests that CRP and its components are effective in preventing cardiovascular diseases (CVDs) such as atherosclerosis, myocardial infarction, myocardial ischemia, arrhythmia, cardiac hypertrophy, heart failure, and hypertension. Studies show that the two most bioactive components of CRP are flavonoids and volatile oils. The cardiovascular protective effects of CRP have attracted considerable research interest due to its hypolipidemic, antiplatelet activity, antioxidant and anti-inflammatory effects. Hereby, we provide a rigorous and up-to-date overview of the cardiovascular protective properties and the potential molecular targets of CRP, and finally highlight the pharmacokinetics and the therapeutic potential of the main pharmacologically active components of CRP to treat CVDs.

Bioinformatics-Guided Identification of Ethyl Acetate Extract of Citri Reticulatae Pericarpium as a Functional Food Ingredient with Anti-Inflammatory Potential.

Citri Reticulatae Pericarpium (CRP) is one of the most commonly used food supplements and folk medicines worldwide, and possesses cardiovascular, digestive, and respiratory protective effects partially through its antioxidant and anti-inflammatory functions. The unique aromatic flavor and mild side effects make CRP a promising candidate for the development of anti-inflammatory functional food. However, recent studies show that the crude alcoholic extract and some isolated compounds of CRP show compromised anti-inflammatory activity, which became the main factor hindering its further development. To identify the bioactive compounds with anti-inflammatory potential, and improve the anti-inflammatory effects of the extract, a bioinformatics-guided extraction protocol was employed in this study. The potential bioactive candidates were identified by combing network pharmacology analysis, molecular docking, principal components analysis, k-means clustering, and in vitro testing of reference compounds. Our results demonstrated that 66 compounds in CRP could be grouped into four clusters according to their docking score profile against 24 receptors, while the cluster containing flavonoids and phenols might possess a more promising anti-inflammatory function. In addition, in vitro anti-inflammatory tests of the seven reference compounds demonstrated that hesperitin, naringenin, and gardenin B, which were grouped into a cluster containing flavonoids and phenols, significantly decreased LPS-induced NO, TNF-α, and IL-6 production of macrophages. While the compounds outside of that cluster, such as neohesperidin, naringin, hesperidin, and sinensetin showed little effect on alleviating LPS-induced NO and proinflammatory cytokine production. Based on the chemical properties of selected compounds, ethyl acetate (EtOAc) was selected as the solvent for extraction, because of its promising solubility of flavonoids and phenols. Furthermore, the ethanol alcoholic extract was used as a reference. The chemical profiling of EtOAc and crude alcoholic extract by HPLC/MS/MS also demonstrated the decreased abundance of flavonoid glycosides in EtOAc extract but increased abundance of phenols, phenolic acid, and aglycones. In accordance with the prediction, the EtOAc extract of CRP, but not the crude alcoholic extract, significantly decreased the NO, IL-6, and TNF-α production. Taken together, the results suggested selective extraction of phenols and flavonoids rich extract was able to increase the anti-inflammatory potential of CRP partially because of the synergistic effects between flavonoids, phenols, and enriched polymethoxyflavones. Our study might pave the road for the development of ethyl acetate extract of CRP as a novel functional food with anti-inflammatory function.

[Research advances of chemical constituents and analytical methods of Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium].

Citri Reticulatae Pericarpium Viride(CRPV) and Citri Reticulatae Pericarpium(CRP) are two commonly used Chinese medicinal materials. They have the same origin while are harvested in different seasons and have different clinical effects. They contain similar chemical components, like flavonoids, terpenes, volatile oils, and alkaloids. Although it has been demonstrated that differential components exist between them, there is still a lack of systematic comparison. Many studies have reported the chemical composition and quality evaluation of CRPV and CRP, including the characterization of flavonoids, alkaloids, and volatile oils via thin-layer chromatography, high-performance liquid chromatography, gas chromatography, infrared spectroscopy, and ultraviolet spectroscopy. A few studies have explored the differences between CRPV and CRP. In this paper, we systematically summarized the reported chemical composition, analytical methods, and quality evaluation of CRPV and CRP in recent ten years, aiming to facilitate the research on the pharmacodynamic material basis, quality evaluation, and standard improvement of CRPV and CRP.

Optimization of mechanically assisted coamorphous dispersion extraction of hydrophobic compounds from plant tea (Citri Reticulatae Pericarpium) using water.

This study aimed to establish a novel mechanically assisted coamorphous dispersion extraction (MADE) method for the extraction of hydrophobic compounds (hesperidin, nobiletin and tangeretin) from Citri Reticulatae Pericarpium using water. The surface morphology, particle size distributions, phase states and functional groups of the coground product surface were characterized by Scanning Electron Microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. The parameters affecting the efficiency of extraction method were optimized by single-factor experiments and response surface methodology. The method showed good linear relationships in the range of 1-500 µg/mL with correlation coefficients (R2) ≥ 0.9990, low limits of detection ranging from 3.0 to 28.3 ng/mL, and acceptable recoveries ranging from 87.0 to 91.0%. Therefore, the proposed MADE method is a promising, efficient and organic solvent-free method for the extraction of hydrophobic compounds from plant tea.

Analyses of Physical and Chemical Compositions of Different Medicinal Specifications of CRPV by Use of Multiple Instrumental Techniques Combined with Multivariate Statistical Analysis.

Citri Reticulatae Pericarpium Viride (CRPV) is the processed product of Citrus reticulata Blanco. We systematically analyzed two CRPV types, Geqingpi (GQP) and Sihuaqingpi (SHQP), based on powder color, microscopic characteristics, and chemical composition. In addition, we characterized their constituents via ultra-high-performance liquid chromatography with hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS). Both showed significant differences in their powder color and microscopic characteristics. Fourier-transform infrared (FT-IR) spectroscopic analysis results showed that the C=O peak absorption of carboxylic acids and their carbonyl esters in SHQP was higher than that of GQP, while the C-OH and C-H plane bending peaks of polysaccharides were lower than those of GQP. We analyzed these data via similarity analysis, PCA, and OPLS-DA. GQP and SHQP had large distinct differences. Based on the mass measurements for molecular and characteristic fragment ions, we identified 44 main constituents from CRPV, including different flavonoid glycosides and flavonoid aglycones in SHQP and GQP, respectively. We found luteolin-6-C-glucoside, orientin, rhoifolin, and pilloin solely in SHQP, and naringenin and hesperetin only in GQP. The peak area measurements showed GQP having a higher flavonoid glycoside (narirutin, hesperidin, etc.) content, whereas SHQP had a higher polymethoxyflavone (nobiletin, tangeretin, etc.) content. Since we holistically analyzed two CRPV types, the results can not only support future pharmacological research, but also provide a scientific basis for formulating more reasonable CRPV quality standards and guide its clinical potential as a precision medicine.

[Contributions of flavonoids from citri reticulatae pericarpium to gastric hormones, CD3~+ and TFF3 mRNA expression in rats with spleen deficiency intervened by Liujunzi Decoction].

The present study established the spectrum-effect relationship model of flavonoids in Citri Reticulatae Pericarpium(CRP) from 15 batches of Liujunzi Decoction and statistically analyzed the correlation between chemical peaks and efficacy to identify the main effective components. HPLC fingerprints of flavonoids in CRP from 15 batches of Liujunzi Decoction were established. HPLC analysis was carried out on the Venusil XBP C_(18)(L) column(4.6 mm×250 mm, 5 µm) at 30 ℃ with acetonitrile-water(containing 0.1% formic acid) as mobile phase for gradient elution, a flow rate of 1.0 mL·min~(-1), and detection wavelength of 300 nm to obtain chemical fingerprints. Additionally, the effects of flavonoids from CRP in 15 batches of Liujunzi Decoction on the content of GAS, MTL, and VIP, TFF3 mRNA expression, and percentage of CD3~+ T-cells of model rats with spleen deficiency were determined. The spectrum-effect relationship model was established by gray correlation analysis. The results showed that the main characteristic peaks with great contribution to the regulation of gastrointestinal tract were peak 16(vicenin-2), peak 63(sinensetin), peak 64(isosinensetin), peak 65(nobiletin), peak 67(3,5,6,7,8,3',4'-heptemthoxyflavone), peak 68(tangeretin), and peak 69(5-desmethylnobiletin). Therefore, there was a linear correlation between flavonoids from CRP in Liujunzi Decoction and the efficacy, and the medicinal effect was achieved by multi-component action. This study is expected to provide a new idea for exploring the material basis of the effect, i.e., regulating qi prior to replenishing qi, of CRP in Liujunzi Decoction.

Citri Reticulatae Pericarpium alleviates postmyocardial infarction heart failure by upregulating PPARγ expression.

Heart failure after myocardial infarction (MI) is the leading cause of death worldwide. Citri Reticulatae Pericarpium (CRP) is a traditional Chinese herbal medicine that has been used in the clinic for centuries. In this study, we aimed to investigate the roles of CRP in cardiac remodelling and heart failure after MI, as well as the molecular mechanisms involved. Male C57BL/6 mice aged 8 weeks were subjected to coronary artery ligation to mimic the clinical situation in vivo. Echocardiography was used to assess the systolic function of the mouse heart. Masson trichrome staining and Wheat germ agglutinin (WGA) staining were utilised to determine the fibrotic area and cross-sectional area of the mouse heart, respectively. Cardiomyocytes and fibroblasts were isolated from neonatal rats aged 0-3 days in vitro using enzyme digestion. TUNEL staining and EdU staining were performed to evaluate apoptosis and proliferation, respectively. Gene expression changes were analysed by qRT-PCR, and protein expression changes were assessed by Western blotting. Our findings revealed that CRP attenuated cardiac hypertrophy, fibrosis and apoptosis and alleviated heart failure after MI in vivo. Furthermore, CRP mitigated cardiomyocyte apoptosis and fibroblast proliferation and differentiation into myofibroblasts. In addition, the PPARγ inhibitor T0070907 completely abolished the abovementioned beneficial effects of CRP, and the PPARγ activator rosiglitazone failed to further ameliorate cardiac apoptosis and fibrosis in vitro. CRP alleviates cardiac hypertrophy, fibrosis, and apoptosis and can ameliorate heart failure after MI via activation of PPARγ.

A Traditional Chinese Herb Citri Reticulatae Pericarpium Induced Melanonychia.

The common causes for melanonychia include melanoma, repetitive trauma, underlying systemic diseases, onychomycosis, pseudomonas infection and drugs. Drug-induced melanonychia usually affects numerous nails and appears as light brown to black pigmentation on the deck or nail bed with longitudinal, transverse or diffuse distribution. In previous cases, a number of chemotherapeutic agents such as azathioprine, bleomycin sulfate, cyclophosphamide, hydroxyurea and methotrexate were usually linked to melanonychia. Citri reticulatae pericarpium (CRP) is a traditional Chinese herb which is widely used in many foods and health care products in China. Up till now, there were no adverse reactions of CRP reported throughout the literature. Herein, we firstly reported a case of melanonychia in a 67-year-old man caused by CRP for external use.