Quality assessment and Q-markers discovery in Citri Sarcodactylis Fructus by integrating serum pharmacochemistry and network pharmacology.

INTRODUCTION: Citri Sarcodactylis Fructus (CSF), a common fruit and traditional Chinese medicine (TCM), has been hindered in its further development and research owing to the lack of comprehensive and specific quality evaluation standards. OBJECTIVE: This study aimed to establish clear TCM quality standards related to the therapeutic mechanisms of CSF and to provide a basis for subsequent research and development. METHODS: Ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-orbitrap HRMS) technology was used to comprehensively identify CSF components and explore their absorbance levels in rat serum. Network pharmacology research methods were employed to investigate the potential mechanisms of action of the identified components in the treatment of major clinical diseases. Subsequently, a combination of HPLC chromatographic fingerprinting for qualitative analysis and multi-index content determination was used to evaluate the detectability of the identified quality markers (Q-markers). RESULTS: Twenty-six prototype components were tentatively characterized in rat serum. Network pharmacology analysis showed six effective components, namely 7-hydroxycoumarin, isoscopoletin, diosmin, hesperidin, 5,7-dimethoxycoumarin, and bergapten, which played important roles in the treatment of chronic gastritis, functional dyspepsia, peptic ulcer, and depression and were preliminarily identified as Q-markers. The results of content determination in 15 batches of CSF indicated significant differences in the content of medicinal materials from different origins. However, compared with the preliminarily determined Q-markers, all six components could be measured and were determined as Q-markers of CSF. CONCLUSION: The chemical Q-markers obtained in this study could be used for effective quality control of CSF.

Identification of in vivo metabolites of Citri Sarcodactylis Fructus by UHPLC-Q/Orbitrap HRMS.

INTRODUCTION: Citri Sarcodactylis Fructus has the effects of relieving cough, removing phlegm, and reducing asthma, but little is known about the metabolic and distribution of its chemical constituents in vivo. Therefore, it is necessary to study the metabolism of Citri Sarcodactylis Fructus in vivo. OBJECTIVE: We aimed to (1) analyze the distribution of prototype compounds and metabolites of the chemical constituents of Citri Sarcodactylis Fructus in rat and (2) infer the metabolites and metabolic pathways of the chemical constituents. MATERIALS AND METHODS: A C18 column (3 × 100 mm, 2.6 µm) was used. The mobile phase was water containing 0.1% formic acid (eluent A) and acetonitrile containing 0.1% formic acid (eluent B) at a discharge rate of 0.3 mL/min. Mass spectra of biological samples were collected in electrospray ionization (ESI) positive ion mode in the m/z 100-1500 scan range. The obtained biological samples were then subjected to chemical analysis, including plasma, urine, feces, and heart, liver, spleen, lungs, kidneys, stomach, and small intestine tissues. Prototype compounds and metabolites were identified. RESULTS: In all, 40 prototype compounds and 78 metabolites, including 26 phase I metabolites and 52 phase II metabolites, were identified using UHPLC-Q/Orbitrap HRMS. Eight possible metabolic pathways (reduction, hydrolysis, dehydration, methylation, hydroxylation, sulfation, glucuronidation, and demethylation) were proposed. The prototype compounds were predominantly distributed in lung tissues. The metabolites were mainly distributed in plasma and kidney tissues. CONCLUSION: We systematically investigated the metabolites of Citri Sarcodactylis Fructus in vivo. We suggest metabolic pathways that might be relevant for further metabolic studies and screening of active ingredients of Citrus Sarcodactylis Fructus in vivo.

Analysis of the characteristic compounds of Citri Sarcodactylis Fructus from different geographical origins.

INTRODUCTION: Citri Sarcodactylis Fructus (CSF) is widely used as a food ingredient and a traditional Chinese medicine. In China, CSF is cultivated in many places, including Sichuan, Guangdong, Zhejiang, and Fujian provinces. The types and chemical contents of CSF from different origins may vary greatly due to the difference in climate and environmental conditions. Therefore, comparing the chemical composition of CSF from various places is vital. OBJECTIVE: To rapidly select potential characteristic compounds for differentiating CSF from different origins. MATERIAL AND METHODS: Thirty-one batches of CSF samples from different regions were analysed using ultra-performance liquid chromatography with hybrid quadrupole-orbitrap high-resolution mass spectrometry. Thereafter, chemometric methods, including principal component analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA), were employed to find differential metabolites among the CSF samples from various origins. RESULTS: PCA revealed 77.9% of the total variance and divided all CSF samples into three categories corresponding to their origins. OPLS-DA displayed better discrimination of CSF from different sources, with R2 X, R2 Y, and Q2 of 0.801, 0.985, and 0.849, respectively. Finally, 203 differential metabolites were obtained from CSF from different origins using the variable importance in projection of the OPLS-DA model, 30 of which were identified, and five coumarin compounds were selected as marker compounds discriminating CSF from different origins. CONCLUSION: This work provides a practical strategy for classifying CSF from different origins and offers a research foundation for the quality control of CSF.

[Herbalogical study on original plant and medicinal and edible values of Citri Sarcodactylis Fructus].

Citri Sarcodactylis Fructus is a both medicinal and edible species specified by the China Ministry of Health, with a long history in China. According to the ancient monographs about materia medica, it was found that the records of the Citri Sarcodactylis Fructus on the original plants were confused. This paper reviewed the ancient monographs about materia medica, and made a summarization and textual research on the name, origin, habitat, processing methods, medicinal properties and clinicacy efficacy of Citri Sarcodactylis Fructus based the comprehensive analysis on modern literatures and authoritative books of Chinese herbal medicine. The results indicated that there were many bynames of Citri Sarcodactylis Fructus. Before the Yuan Dynasty, there was a mixed use of Citri Sarcodactylis Fructus and Citri Fructus, which were not distinguished from each other in terms of nature and taste until the Yuan dynasty. Citri Sarcodactylis Fructus was a varietas of Citri Fructus. The main shape of the original plant of Citri Sarcodactylis Fructus is "like a human hand with fingers" as recorded in ancient monographs about materia medica. The main places of origin of Citri Sarcodactylis Fructus were Guangdong, Zhejiang, Fujian, Sichuan, which were relatively stable. There were fewer records about medicinal proces-sing methods of Citri Sarcodactylis Fructus. Only steaming and baking methods were found in ancient monographs about materia medica, and the steaming method could reduce the irritability of Citri Sarcodactylis Fructus. The processing of therapeutic dietary of Citri Sarcodactylis Fructus was widely used in folk, which was represented by Chaozhou Laoxianghuang, a traditional succade made of Citri Sarcodactylis Fructus. According to the 2015 edition of Chinese Pharmacopoeia, Citri Sarcodactylis Fructus had effects in soothing liver and regulating gas, relieving pain in the stomach, eliminating dampness and resolving phlegm, which was basically consistent with the descriptions in ancient monographs about materia medica. This paper defined the original plant of Citri Sarcodactylis Fructus, and sorted out and summarized the processing methods, nature and taste of Citri Sarcodactylis Fructus, so as to provide data support for the standardization of the processing technology and the development and utilization of Citri Sarcodactylis Fructus.

UHPLC-Q/Orbitrap HRMS combined with spectrum-effect relationship and network pharmacology to discovery the gastrointestinal motility-promoting material basis in Citri Sarcodactylis Fructus.

ETHNOPHARMACOLOGICAL RELEVANCE: The prevalence of gastrointestinal motility disorders (GMD) is increasing and is characterized by long-term recurrence. Citri Sarcodactylis Fructus (CSF), as a traditional Chinese medicine (TCM) known in "regulating qi and harmonizing the stomach", has therapeutic effects on GMD. However, the material basis of its efficacy is not clear. AIM OF THE STUDY: The aim of this study was to evaluate the gastrointestinal motility-promoting activity of CSF extracts and to screen their active ingredients and to perform a preliminary validation. METHODS: The chemical composition spectrum of different extracts of CSF were established by ultra high-performance liquid chromatography coupled with quadrupole orbitrap high-resolution mass spectrometry (UHPLC-Q/Orbitrap HRMS). The gastrointestinal motility-promoting activities of CSF were investigated by determining the intestinal propulsion rate, gastric emptying rate, acetylcholinesterase activity, and motilin content in L-arginine-induced GMD mice. Spectrum-effect relationship and network pharmacology analysis were used for the screening of potential active ingredients. A zebrafish gastrointestinal motility model traced with Nile Red was established to validate the active ingredients. Molecular docking prediction was used to explore the mechanism of action of the active ingredient. Finally, Western blotting and TUNEL staining were performed to validate the molecular docking predictions. RESULTS: In total, 42 shared components were identified. The main active fraction of CSF to promote gastrointestinal motility was 70% ethanol elution fraction. Eleven potential active ingredients were screened by grey correlation analysis, orthogonal partial least squares analysis, and "active ingredient-target" network. Six compounds were confirmed as the pharmacodynamic substances of CSF by zebrafish gastrointestinal motility model, namely, quercetin, kaempferol, isorhamnetin, diosmetin, hesperetin, and 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone. Molecular docking predictions and Western blotting assays indicated that CSF may act on AKT and MMP9 targets to exert gastrointestinal motility-promoting activity. CONCLUSION: This study provided a foundation for elucidating the gastrointestinal motility-promoting activity of CSF and its material basis by integrating spectrum-effect relationship and network pharmacology. It also provided a theoretical basis for quality control of CSF and a new idea for the discovery and validation of pharmacodynamic substances in TCM.

Discovery of antitussive material basis and mechanisms in Citri Sarcodactylis Fructus by coupling UHPLC-Q/Orbitrap HRMS combined spectrum-effect relationship and metabolomics analyses.

Citri Sarcodactylis Fructus (CSF) is widely used as food raw material and traditional Chinese medicine. Fingerprints of different fractions of CSF were established for spectrum-effect relationship analysis, and the main compounds were identified by UHPLC Quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q/Orbitrap HRMS). The antitussive effect was evaluated using a classical mouse model of cough induced by ammonia water. One-way ANOVA was used to determine differences in efficacy. The potential active compounds were screened by spectrum-effect relationship with grey relational degree analysis (GRA), Pearson bivariate correlation analysis (Pearson's), and partial least squares analysis (PLS) analyses. Differential metabolites associated with cough in serum were screened and identified using orthogonal partial least squares-discriminant analysis, HMDB database, and UHPLC-Q/Orbitrap HRMS. Metabolic pathway analysis was performed using MetaboAnalyst 5.0. Results indicate that 70 % ethanol elution fraction (70 % EF) is the major active fraction, and 8 components were identified to possess antitussive effects. Metabolomic analysis showed that 19 metabolites are potential biomarkers related to cough, and 70 % EF can remarkable restore 13 of them to normal levels (P < 0.05). These biomarkers are mainly involved in glycerophospholipid metabolism and sphingolipid metabolism. This study aims to reveal the main pharmacodynamic active sites and potential active ingredients of CSF's antitussive effect. In addition, metabolomics was used to preliminarily elucidate the in-vivo regulatory mechanism of the antitussive effect of the 70 % EF of CSF.

Enhancing effects of 60Co irradiation on the extraction and activities of phenolic components in edible Citri Sarcodactylis Fructus.

In this study, the impact of 60Co-γ ray irradiation treatment on the content of active chemicals and their functions in Citri Sarcodactylis Fructus (CSF) was assessed. Scanning electron microscopy, Fourier transform infrared spectroscopy, and γ-ray diffraction revealed physical structure changes in CSF powder. According to the findings, the content of total flavonoids in the ethanol extract of CSF increased by 9.5%-21.62%, 7-hydroxycoumarin, hesperidin, 5,7-dimethoxycoumarin, and 5-methoxypsoralen increased by 5.31%-51.8%, 10.07%-99.81%, 6.6%-62.29%, and 3.03%-300%, respectively, when the irradiation dosage was raised, and the antibacterial, anti-inflammatory, antioxidant, and anticancer properties were all raised considerably. These results imply that the principal components and activity changes are proportional to the irradiation dosage. At present, the findings of this study serve as a reference for the use of irradiation technology in assisting extraction and enhancing the effects of functional foods.

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.