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ObjectiveTo analyze and predict the potential quality markers （Q-Marker） in the Genuine medicinal materials Jiangxi Aurantii Fructus based on fingerprints and network pharmacology. MethodUltra-high performance liquid chromatography （UPLC） and gas chromatography-mass spectrometry （GC-MS） fingerprints were established for 18 batches of Jiangxi Aurantii Fructus ，combined with chemometric methods to screen out candidate Q-Marker components.Use network pharmacology to construct a "core component-target-pathway" network to predict the Q-Marker and core targets of Jiangxi Aurantii Fructus，and then verify the biological activity of Jiangxi Aurantii Fructus Q-Marker by molecular docking method. ResultThe 18 batches of Jiangxi Aurantii Fructus use UPLC，GC-MS fingerprints combined with chemometric analysis，a total of 9 Q-Marker candidate components were screened out.Through network pharmacological analysis，it is predicted that nobiletin，neohesperidin，meranzin，naringin and D-limonene are the Q-Marker of Jiangxi Aurantii Fructus，acting on the core targets transforming protein p21/H-Ras-1（HRAS），cellular tumor antigen p53 （TP53），mitogen-activated protein kinase 8 （MAPK8），transcription factor AP-1（JUN），glycogen synthase kinase-3 beta（GSK3B），tumor necrosis factor（TNF），cyclin-dependent kinase inhibitor 1（CDKN1A），cAMP-dependent protein kinase catalytic subunit alpha（PRKACA），cysteine aspartate-specific protease-9（Caspase-9），cyclic AMP-responsive element-binding protein 1（CREB1），exerting gastrointestinal motility and antidepressant，anti-inflammatory，anti-tumor，etc.； molecular docking shows that nobiletin，neohesperidin，meranzin，naringin and D-limonene and the selected 10 core targets have good binding ability，reflecting the better biological activity of the Q-Marker of Jiangxi Aurantii Fructus. ConclusionThe Q-Marker of Jiangxi Aurantii Fructus can be comprehensively predicted from the two aspects of volatile and non-volatile components，providing a reference for the quality control of Jiangxi Aurantii Fructus and the further study of its pharmacodynamic mechanism.
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PURPOSE: Diabetic nephropathy is one of the most important diabetic complications prompted by chronic hyperglycemia, characterized by glomerulosclerosis, tubular fibrosis, and it eventually causes kidney failure. Nobiletin is a polymethoxyflavone present in tangerine and other citrus peels, and has anti-cancer and anti-inflammatory effects. This study investigated the effects of nobiletin on glomerular fibrosis through inhibition of the transforming growth factor (TGF)-β1-Src-caveolin-1 pathway.METHODS: Human renal mesangial cells (HRMC) were incubated in media containing 33 mM glucose with or without 1–20 uM nobiletin for 3 day. The cellular expression levels of fibrogenic collagen IV, fibronectin, connective tissue growth factor (CTGF), TGF-β1, Src and caveolin-1 were all examined. In addition, TGF-β1, Src and caveolin-1 proteins were screened to reveal the relationship among TGF-β1-Src-caveolin-1 signaling in glomerular fibrosis.RESULTS: High glucose promoted the production of collagen IV, fibronectin and CTGF in HRMC, which was inhibited in a dose dependent manner by 1–20 uM nobiletin. The Western blot data showed that high glucose elevated the expression of TGF-β1, Src, caveolin-1 and Rho GTPase. When nobiletin was treated to the HRMC exposed to high glucose, the expression of TGF-β1-Src-caveolin-1 was dampened. Finally, TGF-β1-Src-caveolin-1 signaling pathway was activated in high glucose-exposed HRMC, and such activation was encumbered by nobiletin.CONCLUSION: These result demonstrated that nobiletin blunted high glucose-induced extracellular matrix accumulation via inhibition of the TGF-β1-Src-caveolin-1 related intracellular signaling pathway. Nobiletin may be a potent renoprotective agent to counteract diabetes-associated glomerular fibrosis that leads to kidney failure.
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Humans , Blotting, Western , Caveolin 1 , Citrus , Collagen , Connective Tissue Growth Factor , Diabetes Complications , Diabetic Nephropathies , Extracellular Matrix , Fibronectins , Fibrosis , Glucose , GTP Phosphohydrolases , Hyperglycemia , Mesangial Cells , Renal Insufficiency , Transforming Growth FactorsABSTRACT
Objective: To explore the potential mechanism of Xiahuang Granules in the treatment of opioid-induced constipation. Methods: Various medicinal ingredients and targets information of Xiahuang Granules were found in TCMSP database. In Genecards database, "opiod constipation", "opioid-induced bowel dysfunction" and "opioid-induced constipation" were used as keywords to search for targets related to opioid-induced constipation, and the active targets mapping of Xiahuang Granules were selected as the research targets. The common targets were imported into the STRING database to build the targets interaction network diagram, and Cytoscape 3.3.0 software was used for visual processing to screen out the core targets. The OmicsBean analysis platform and STRING database were used to conduct GO function enrichment and KEGG pathway enrichment analysis on the targets. Results: A total of 55 chemical constituents, 158 candidate target genes, 86 common targets after mapping Venny, 49 corresponding chemical components, 12 core targets and 19 main chemical components of Xiahuang Granules were obtained by screening. GO functional enrichment analysis showed 4 150 biological process items, involving chemical stimulus cell reactions, chemical reactions, biological quality control and other processes; A total of 302 cell composition items, involving voxel projection, extracellular space, and whole membrane processes; A total of 459 molecules function items, involving processes such as protein binding, molecular transduction activity, and enzyme binding were obtained. KEGG enrichment analysis revealed 149 signaling pathways related to the effect of Xiahuang Granules, involving the AGE-RAGE signaling pathway in diabetic complications and tumor necrosis factor signaling pathway, etc. The network of "medicinal herb-component-target-pathway" of Xiahuang Granules was established. Conclusion: The main chemical components of Xiahuang Granules including naringenin, nobiletin, aloe emodin, rhein may regulate endocrine resistance and tumor necrosis factor signaling pathways by acting on key proteins such as TNF, MAPK3, IL-6, VEGFA, and PTGS2, thus play a role in laxative, antispasmodic, and promoting gastrointestinal motility, which provides theoretical basis for Xiahuang Granules to treat opioid-induced constipation and is consistent with the preliminary verification results of Xiahuang granules.
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Objective To study the effects of Chinese herb ingredients with different properties on transporters (URAT1 and OAT4) involved in renal urate reabsorption and serum uric acid level in acute hyperuricemia mice. Methods The OAT4, URAT1- overexpressed monoclonal cell line (MDCK-hOAT4, HEK293-hURAT1) was constructed. The inhibition effect and the half maximal inhibitory concentration (IC50) of different ingredients to transport activity of OAT4 and URAT1 mediating 14C-uric acid were determined. The effects of protocatechuic, liquiritigenin and isoliquiritigenin on serum uric acid levels in acute hyperuricemia mice were studied by the acute hyperuricemia mice induced by potassium oxonate and xanthine. Results The results indicated that nobiletin,liquiritigenin, isoliquiritigenin, licochalcone A with bitter flavor showed strong inhibition to OAT4. The IC50 of nobiletin, liquiritigenin, isoliquiritigenin, and licochalcone A on OAT4 were 0.556 μmol/L, 18.40 μmol/L, 6.831 μmol/L, and 6.825 μmol/L, respectively. Protocatechuic acid and liquiritigenin showed strong inhibition to URAT1 with IC50 of 7.709 μmol/L and 14.54 μmol/L, respectively. Liquiritigenin can significantly reduce the level of serum uric acid of acute hyperuricemia mice, increase the excretion of uric acid, and reduce the level of serum creatinine and blood urea nitrogen. Conclusion Nobiletin, liquiritigenin, isoliquiritigenin and licochalcone A can inhibit the transport activity of OAT4, while protocatechuic acid and liquiritigenin can inhibit the transport activity of URAT1. Liquiritigenin can significantly reduce the level of serum uric acid in acute hyperuricemia mice and protect kidney, the mechanism of which may be associated with the decreasing reabsorption of uric acid by inhibiting the activity of URAT1 and OAT4.
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Objective To establish an HPLC method for the fingerprints analysis of decoction pieces of tangerine peel, so as to provide reference for the quality control of it. Methods Fingerprints of 17 batches of decoction pieces of tangerine peel were established by HPLC and evaluated by cluster analysis (CA), principal component analysis (PCA) and orthogonal partial least square discriminate analysis (OPLS-DA). Results The method of fingerprint of decoction pieces of tangerine peel was established, the similarities were greater than 0.9. There were 25 common peaks in the HPLC fingerprint, of which variable importance projection (VIP) of 14 peaks were greater than 1. Compared with the spectrogram of reference substances, peak 13 was narirutin, peak 14 was hesperidin, peak 23 was nobiletin, peak 24 was 3,5,6,7,8,3’,4’-heptamethoxy flavone, and peak 25 was hesperetin. Conclusion This simple and reliable method can be used for the identification and quality control of decoction pieces of Citri Reticulatae Pericarpium.
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Nobiletin is a kind of polymethoxyflavonoid with many pharmacological effects, such as antiinflammatory and antioxidation activities. This study was carried out to investigate the inhibitory effects of nobiletin on P-glycoprotein (P-gp) in vitro and in vivo. The molecular mechanism for structure-inhibition relationships of nobiletin with P-gp was investigated. Nobiletin exhibited significant inhibition (IC50=2.21 μmol·L-1) on P-gp in MDR1-MDCKⅡ cells. In the cell toxicity test, the paraquat-treated cell viability was decreased with nobiletin by inhibiting P-gp activity. In the rats PK study, the AUC0-t of digoxin was increased 2.02 folds while the Cmax of digoxin was increased 3.29 folds, when nobiletin was used in the pretreatment of SD rats. Molecular docking analysis elucidated that the formation of Pi-Pi bonds with Phe974 was the key factor for P-gp inhibition. The research findings provide important guideline for prediction of potential interaction between nobiletin and P-gp.
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Objective: To establish an HPLC method for simultaneous determination of 7-hydroxycoumarin, narirutin, naringin, hesperidin, neohesperidin, nobiletin, 3,5,6,7,8,(3,4)-heptamethoxyflavone, tangeretin, and auraptene from Aurantii Fructus, determinate and compare the content of various chemical components from Aurantii Fructus with different perimeters. Methods: The HPLC system consisted of a diamon C18 (250 mm × 4.6 mm, 5 μm), the mobile phase was methanol (A)-water (adjusted pH 3.0 with phosphorous acid). The gradient elution conditions: 0-25 min, 30%-50% A; 25-35 min, 50%-70% A; 35-40 min, 70%-75% A; 40-55 min, 75%-100% A. The flow rate was 1.0 mL/min and the column temperature was 30 ℃. The detection wavelength was 320 nm and the injection volume was 20 μL. Results: The calibration curves of 7-hydroxycoumarin, narirutin, naringin, hesperidin, neohesperidin, 3,5,6,7,8,(3,4)-heptamethoxyflavone, nobiletin, tangeretin, and auraptene had good linear relationship in the ranges of 0.002 18-0.07 μg (r = 0.999 8), 0.021 8-0.70 μg (r = 0.999 9), 0.242 5-7.76 μg (r = 0.999 8), 0.024 38-0.78 μg (r = 0.999 4), 0.523 76-16.76 μg (r = 0.999 3), 0.003 13-0.10 μg (r = 0.999 3), 0.004 13-0.132 μg (r = 0.999 6), 0.002 75-0.088 μg (r = 0.999 6), and 0.000 93-0.03 μg (r = 0.999 3); And the average recoveries (n = 6) of the nine components were 98.50%, 98.80%, 99.51%, 98.43%, 99.64%, 99.21%, 100.03%, 98.75%, and 101.11%, respectively. Conclusion: This method can be applied to determinating the components from Aurantii Fructus, including 7-hydroxycoumarin and 3,5,6,7,8,(3,4)-heptamethoxyflavone, etc.
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AIM:To observe the effects of nobiletin on palmic acid (PA)-induced lipidosis in hepatocytes and to discuss the regulatory mechanism of lncLSTR. METHODS:AML12 cells were cultured in vitro. The control group, PA group (0.2 mmol/L) and protection group (exposure to nobiletin at 1 mg/L, 5 mg/L, 15 mg/L or 50 mg/L for 2 h, fol-lowed by treatment with 0.2 mmol/L PA) were established according to the experimental requirements. The lipid accumula-tion was morphologically observed by Oil red O staining in the cells. The qPCR was applied to detect mRNA expression, and the protein expression was determined by and Western blot. RESULTS:PA treatment (0.2 mmol/L) induced lipidosis, while 50 mg/L nobiletin pretreatment suppressed the lipidosis. Compared with control group, the mRNA expression of Apoc2 in PA group was significantly down-regulated (P<0.05), but increased by nobiletin pretreatment compared with PA group (P<0.05). The protein expression of Apoc2 in PA group was significantly down-regulated (P<0.05), but increased by nobiletin pretreatment (P<0.05). The expression of lncLSTR in PA group was significantly increased (P<0.05) and that was inhibited by nobiletin pretreatment (P<0.05). A negative correlation between Apoc2 protein and lncLSTR expression in PA group (R2=0.717 9, P<0.01) was observed. A negative correlation between Apoc2 protein and lncLSTR expression in protection group (R2=0.525 3, P<0.05) was also found. CONCLUSION:Nobiletin has anti-lipidosis effect on hepa-tocytes. The mechanism is partially related to the inhibition of up-regulation of lncLSTR, thus down-regulating Apoc2 expres-sion.
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Mitochondrial calcium overload is a crucial event in determining the fate of neuronal cell survival and death, implicated in pathogenesis of neurodegenerative diseases. One of the driving forces of calcium influx into mitochondria is mitochondria membrane potential (ΔΨ(m)). Therefore, pharmacological manipulation of ΔΨ(m) can be a promising strategy to prevent neuronal cell death against brain insults. Based on these issues, we investigated here whether nobiletin, a Citrus polymethoxylated flavone, prevents neurotoxic neuronal calcium overload and cell death via regulating basal ΔΨ(m) against neuronal insult in primary cortical neurons and pure brain mitochondria isolated from rat cortices. Results demonstrated that nobiletin treatment significantly increased cell viability against glutamate toxicity (100 µM, 20 min) in primary cortical neurons. Real-time imaging-based fluorometry data reveal that nobiletin evokes partial mitochondrial depolarization in these neurons. Nobiletin markedly attenuated mitochondrial calcium overload and reactive oxygen species (ROS) generation in glutamate (100 µM)-stimulated cortical neurons and isolated pure mitochondria exposed to high concentration of Ca²⁺ (5 µM). Nobiletin-induced partial mitochondrial depolarization in intact neurons was confirmed in isolated brain mitochondria using a fluorescence microplate reader. Nobiletin effects on basal ΔΨ(m) were completely abolished in K⁺-free medium on pure isolated mitochondria. Taken together, results demonstrate that K⁺ influx into mitochondria is critically involved in partial mitochondrial depolarization-related neuroprotective effect of nobiletin. Nobiletin-induced mitochondrial K⁺ influx is probably mediated, at least in part, by activation of mitochondrial K⁺ channels. However, further detailed studies should be conducted to determine exact molecular targets of nobiletin in mitochondria.
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Animals , Rats , Brain , Calcium , Cell Death , Cell Survival , Citrus , Fluorescence , Fluorometry , Glutamic Acid , Membrane Potential, Mitochondrial , Membrane Potentials , Membranes , Mitochondria , Neurodegenerative Diseases , Neurons , Neuroprotective Agents , Reactive Oxygen SpeciesABSTRACT
Tumor is a disease with high mortality rate,which seriously threatens the human life and health.To study the antitumor effect,sensitization and anti-drug-resistant action of effective ingredients extracted from traditional Chinese medicine or natural medicine is a hotspot.Polymethoxyflavone is a class of flavonoids with strong anti-tumor activity found in recent years.In this paper,the research progress on anti-tumor action and its mechanisms of polymethoxyflavonoids is reviewed.
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OBJECTIVE: To establish a UHPLC-MS/MS method for simultaneous determination of tangertin, hesperetin, 5-demethylnobiletin, nobiletin, vitexin, and narirutin in Rukuaixiao granules. METHODS: The analysis method was performed on a Waters ACQUITY UPLC® BEH C18 eolumn (2.1 mm×50 mm, 1.7 μm) with gradient elution of acetonitrile (containing 0.1% formic acid)-0.1% formic acid at a flow rate of 0.6 mL·min-1, and the column temperature was set at 50℃. Electrospray ionization (ESI) source with positive mode and the detector with multiple reaction monitoring (MRM) mode were adopted to determine the analytes by mass spectormeter. RESULTS: Satisfactory linearities were obtained for the six constituents in the investigated ranges (r>0.9958): The lower limit of quantitation (LOQ) was 0.80, 1.10, 1.30, 0.66, 2.00, 7.10 and 0.70 ng·mL-1, respectively. All the recoveries of samples were between 95.78%-101.35% with RSDs less than 2.1%. CONCLUSION: The method is simple, rapid, sensitive, and highly reproducible. It can be applied to the quality control of Rukuaixiao granules.
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Alzheimer's disease (AD), the most common form of dementia among the elderly, is characterized by the progressive decline of cognitive function and has a detrimental impact worldwide. Despite intensive laboratory and clinical research over the last three decades, pharmacological options for the prevention and effective long-term treatment of AD are not currently available. Consequently, successful therapeutic and preventive treatments for AD are needed. When researching materials from natural resources having anti-dementia drug activity, we identified nobiletin, a polymethoxylated flavone from the peel of Citrus depressa. Nobiletin exhibited memory-improving effects in various animal models of dementia and exerted a wide range of beneficial effects against pathological features of AD including amyloid-beta (Abeta) pathology, tau hyperphosphorylation, oxidative stress, cholinergic neurodegeneration and dysfunction of synaptic plasticity-related signaling, suggesting this natural compound could become a novel drug for the treatment and prevention of AD.
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Aged , Animals , Humans , Alzheimer Disease , Citrus , Natural Resources , Dementia , Memory , Models, Animal , Oxidative Stress , PathologyABSTRACT
This study aims to evaluate the changes of flavonoid contents and antioxidants activity of Jeju native citrus fruits juice according to the harvest date. Flavonoids such as quercatagetin, narirutin, hesperidin and neohesperidin were contained most plentifully in the juice of Jigak (Citrus aur- antium) by 573.73 mg/100 mL, Sadoogam (C. pseudogulgul) by 393.99 mg /100 mL, Soyooja by 29.63 mg/100 mL and Jigak (C. aurantium) by 201.23 mg/100 mL in the late August, respectively. The highest contents of nob-iletin, sinensetin and tangeretin among polymethoxyflavones were found in the juice of Hongkyool (C. tachibana) by 7.39 mg/100 mL, 2.24 mg/100 mL, 0.63 mg/100 mL in the late August, respectively. 3,5,6,7,8,3',4'- Heptamet- hoxyflavone recorded the highest amount in Punkyool (C. tangerina) by 0.27 mg/100 mL in the late August, but the other polymethoxyflavones including 3',4',7,8-tetramethoxyflavone, 3',4'-dimethoxyflavone, 4'-methoxyflavone, 5,6,7,3',4',5'-hexamethoxyflavone, scutellarein tetramethylether were observed only trace amount in all the citrus fruits. Flavonoid contents in the citrus fruit juices were the highest during early maturation and decreased rapidly while ripening. Total polyphenol contents were the highest in the late August and decreased with ripening. However from the late December, the contents were increased again. Antioxidant activities of the fruits were evaluated as electron donating ability and were the lowest in the late September and increased with the fruit ripening. These results suggest that quercetagetin among all the flavonoids was most plentiful in Jigak and Dangyooja (C. grandis), so that the fruits could be used for industrial material of flavonoids and antioxidant agents.
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Antioxidants , Apigenin , Chromones , Citrus , Disaccharides , Electrons , Flavanones , Flavones , Flavonoids , Fruit , HesperidinABSTRACT
AIM: To develop a rapid HPLC method for quality control of traditional Chinese medicinal ingredients consisted of citrus flavonoids, naringin, hesperidin, neohesperidin, sinensetin and nobiletin. METHODS:Gradient elution with non-salt mobile phase ( methanol and water only) HPLC method on a Kromasil column ( 100-5C18-250A, 4.6 mm ×250 mm, 5 μm, C18 reverse phase) with peaks identification through DAD full UV wavelength scan. UV 284 nm and 332 nm profiles were observed. RESULTS: Satisfactory resolution, linearity, 95%～ 102% of recovery and 1.88 ～ 2.93% of repeatability were obtained for those five citrus flavonoids. Content of 6 Citrus aurantium L. based TCM ingredients were analyzed and identified. CONCLUSION: Rapid HPLC test method on citrus flavonoids was developed and can be in LC-MS identification.
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Objective To investigate whether nobiletin could enhance chemotherapeutic drug-induced cell proliferation inhibition and apoptosis in breast carcinoma cells MDA-MB-231 in vitro.Methods MDA-MB-231 cells were exposed to nobiletin and different chemotherapeutic drugs at different concentration.The cell proliferation inhibitory rate was observed by MTT,apoptosis was detected by DNA Ladder,flow cytometry (FCM),and ELISA,and an NF-?B electrophoretic mobility shift was conducted by EMSA and ELISA.Results The combination of nobiletin (20 ?mol) with Taxol (1 nmol) or Doxorubicin (50 ng/mL) resulted in a significant inhibitory rate of cell growth and more apoptosis in MDA-MB-231 cells of 75.1% and 82.6% compared with either of them alone (40% and 45%),respectively.DNA Ladder and FCM assay showed that nobiletin could promote the cell apoptosis induced by chemotherapeutic drug.EMSA and ELISA assay showed that nobiletin could inhibit NF-?B activity in nuclear and cytoplasm of MDA-MB-231 cells.Conclusion The current results suggest that nobiletin could enhance the inhibition of chemotherapeutic drug on cell proliferation and cell apoptosis of breast carcinoma cells MDA-MB-231,the mechanism may be related to down-regulate the NF-?B activity in vitro.
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Objective To investigate the effect of nobiletin,a citrus polymethoxy flavonoid,on hepatocarcinoma Heps in mice and to explore its immunological mechanism.Methods Mice tumor models were made by transplantation of Heps hepatocarcinoma cell strain.The tumor mass was weighed to calculate the tumor-inhibitory rates.The whole cellular immunity was observed by delayed type hypersensitivity(DTH).MTT assay was used to detect the proliferation of T and B lymphocyte,and the activities of cytotoxic T lymphocytes(CTL) and natural killing cells(NKC) were measured by lactate dehydrogenase(LDH) releasing method.Results Nobiletin could markedly inhibit the growth of hepatocarcinoma Heps(solid tumor).The decreased DTH in hepatocaricinoma mice was enhanced significantly,and T and B lymphocyte proliferation were increased by nobiletin.Meanwhile,the killing activities of CTL and NKC were promoted obviously.Conclusion Improvement of the immune function may be a vital route of nobiletin in inhibiting the proliferation of hepatocarcinoma Heps in mice.