Effects of nobiletin on gut microbiota and short-chain fatty acids in rats with simple obesity / 中国病理生理杂志

AIM:This study aims to investigate the impact of nobiletin(NOB)on the gut microbiota and short-chain fatty acids(SCFAs)in high-fat diet-induced obese rats.METHODS:A total of 22 SD rats were randomly di-vided into the control(CON)group and high-fat diet(HFD)group.The HFD induced obesity,upon successful modeling,the rats were further divided into the HFD and NOB group,each group consisting of 6 rats.The NOB group received nobi-letin solution(100 mg·kg-1·d-1)via gavage for 21 consecutive days.Body weight was measured bi-daily,and hematoxylin-eosin(HE)staining was employed to observe pathological changes in adipose tissue and liver.Serum lipid levels were as-sessed using an auto-biochemical analyzer.Analysis of the gut microbiota was performed using 16S ribosomal RNA(rRNA)sequencing,while high-performance liquid chromatography mass spectrometry(LC-MS)was used to determine SCFAs levels in rat feces.RESULTS:Compared with CON group,HFD-fed demonstrated a substantial increase in body weight(P<0.01),accompanied by an augmentation in adipocyte diameter and the presence of hepatic cell vacuolization,indicative of cellular steatosis and inflammation.Moreover,there was a notable elevation in TG and TC levels(P<0.05).At the phylum level,the HFD rats exhibited an altered composition,characterized by an increase in Firmicutes and a con-current decrease in Bacteroidetes.At the genus level,Bacteroides,Lactobacillus and Blautia experienced a significant de-crease,while Colidextribacter showed an increase.Notably,there was a substantial reduction in the expression of propion-ic acid and butyric acid.In comparison to the HFD group,rats administered with NOB demonstrated a marked decrease in body weight(P<0.05),a reduction in adipocyte diameter,and an amelioration in hepatic cell vacuolization and cellular steatosis.Furthermore,TG and TC levels exhibited a significant decrease(P<0.05).At the phylum level,Firmicutes de-creased,and Bacteroidetes increased.At the genus level,Bacteroides,Lactobacillus and Blautia exhibited a significant in-crease,while Colidextribacter displayed a decrease.Additionally,there was an up-regulation of propionic acid and butyric acid levels in the NOB group.CONCLUSION:Nobiletin,through its multifaceted actions,demonstrates a potential anti-obesity effect by effectively reducing body weight in obese rats.This includes the regulation of gut microbiota structure,modulation of SCFAs content,and enhancement of lipid metabolism.

Effect of Nobiletin on LPS-Induced Inflammatory Injury of Mesangium Cells by Regulating AMPK/NLRP3 Signaling Pathway / 中药新药与临床药理

Objective To investigate the effect of nobiletin(Nb)on lipopolysaccharide(LPS)-induced inflammatory injury of mesangium cells(HBZY-1)by regulating AMP-activated protein kinase(AMPK)/NOD-like receptor protein 3(NLRP3)signaling pathway.Methods HBZY-1 cells were separated into 5 groups:normal control(NC)group,LPS group(100 ng·mL-1 LPS),and Nb group(100 ng·mL-1 LPS+40 μmol·L-1 Nb),Rapamycin(Rap,AMPK/NLRP3 signaling pathway inhibitor)group[100 ng·mL-1 LPS+0.5 μmol·L-1 Rap],and Nb+Rap group(100 ng·mL-1 LPS+40 μmol·L-1 Nb+0.5 μmol·L-1 Rap).MTT was applied to detect the cytotoxicity and proliferation of HBZY-1 cells.ELISA was applied to detect the contents of interleukin(IL)-1β,IL-6,tumor necrosis factor-α(TNF-α),catalase(CAT),superoxide dismutase(SOD),and glutathione(GSH)in HBZY-1 cells.Flow cytometry was used to detect cell apoptosis.Western Blot was applied to detect the protein levels of AMPK/NLRP3 signaling pathway.Results Compared with the NC group,the levels of CAT,SOD,GSH,cell OD value,and the level of AMPK protein in the LPS group were significantly reduced(P＜0.05).The apoptosis rate,contents of IL-1β,IL-6,TNF-α,and the level of NLRP3 protein were significantly increased(P＜0.05).Compared with the LPS group,the levels of CAT,SOD,GSH,OD value,and the level of AMPK protein in the Nb group were significantly increased(P＜0.05).The apoptosis rate,contents of IL-1β,IL-6,TNF-α,and the level of NLRP3 protein were significantly decreased(P＜0.05),while the above indicators in the Rap group showed an opposite trend to the Nb group(P＜0.05).Compared with the Nb group,the above indicators in the Nb+Rap group also showed an opposite trend to the Nb group(P＜0.05).Conclusion Nb may alleviate LPS-induced inflammatory injury to MC cells by up-regulating the AMPK/NLRP3 signaling pathway.But down-regulation of the AMPK/NLRP3 signaling pathway may eliminate the improvement effect of Nb on LPS-induced inflammatory injury in MC cells.

Nobiletin promotes fracture healing in osteoporosis rats by inhibiting STING/NF-κB signal pathway / 免疫学杂志

This study was performed to investigate the impact of nobiletin(NOB)on fracture healing in osteoporosis(OP)rats through the stimulator of interferon gene(STING)/nuclear transcription factor kappa B(NF-κB)signal pathway.A rat model of OP fracture was established by ovariectomy and right femoral shaft fracture intramedullary fixation;the rats after modeling were randomly grouped into model group,high dose(NOB-H,30 mg/kg NOB),medium dose(NOB-M,20 mg/kg NOB),low dose(NOB-L,10 mg/kg NOB)NOB group and NOB-H+ STING activator(DMXAA)group(30 mg/kg NOB+25 mg/kg DMXAA),and 18 rats experienced only ovaries expose were used as sham operation group.After the intervention,the fracture healing status of rats were measured;Micro-CT was used to detect the changes of bone trabecular microstructure in rats;commercial kits were used to detect the serum levels of bone metabolism related indicators(alkaline phosphatase(ALP),calcium,phosphorus)and inflammatory factors(tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β));HE was used to detect the morphological changes and trabecular area of femur,while Western blot was applied to detect the expression of STING/NF-κB pathway related proteins.Compared with the control group,the fracture line in the model group was clear,the trabecular structure was disordered and the gap was large,furthermore,the levels of TNF-α and IL-1β,the expression of STING and p-NF-κB p65/NF-κB p65 were significantly increased,the trabecular area,the levels of ALP,calcium,phosphorus,and bone mineral density(BMD),bone volume fraction(BV/TV),bone trabecular number(Tb.N)and bone trabecular thickness(Tb.Th)were significantly decreased(P<0.05);compared with the model group,the fracture line of NOB-L group,NOB-M group and NOB-H group gradually blurred,the trabecular structure arranged orderly,and the gap gradually decreased,and the trend of the index changes mentioned above were opposite to that of the model group(P<0.05).STING activators attenuated the promotion of fracture healing by NOB in OP rats and increased the inflammatory responses.In conclusion,NOB can reduce inflammatory reaction and promote fracture healing in OP rats,which may be related to the inhibition of STING/NF-κB signal pathway.

Predictive Analysis of Quality Markers of Jiangxi Aurantii Fructus Based on Fingerprint and Network Pharmacology / 中国实验方剂学杂志

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.

Simultaneous determination of seven constituents in Chenxiang Lubailu tablet by HPLC / 药学实践与服务

Objective To establish the method for the simultaneous determination of liquiritin, ammonium glycyrrhizinate, hesperidin, nobiletin;tangeretin;costunolide, dehydrocostuslactone in Chenxiang Lubailu tablet by HPLC. Methods ZORBAX Eclipse XDB-C18 chromatographic column (4.6 mm×250 mm, 5 μm) was used. The mobile phase was methanol-0.1% phosphoric acid solution. Gradient elution with flow rate of 1.0 ml/min was used. Column temperature was 35 ℃. Detection wavelength for liquiritin, ammonium, tangeretin, and costunolide was at 237 nm. Detection wavelength for glycyrrhizinate was at 283 nm. Detection wavelength for hesperidin and nobiletin was at 330 nm. Injection volume was 10 μl. 16 batches of samples were tested. Results The linear ranges for the detection of liquiritin, ammonium, glycyrrhizinate, hesperidin, nobiletin, tangeretin, and costunolide were 1.110 - 55.72 (r=0.9992), 22.15 - 1108 (r=0.9999), 6.140 - 307.2 (r=0.9995), 1.130 - 56.25 (r=0.9997), 0.3700 - 18.75 (r=0.9982), 0.5200 - 26.01 (r=0.9991), and 1.180 - 58.95 (r=0.9999) μg/ml respectively. The average recoveries were 98.71%, 98.12%, 98.44%, 98.22%, 99.17%, 99.18%, and 97.93%, and the RSDs were 0.16%, 0.67%, 0.57%, 0.62%, 0.48%, 0.56%, and 0.58% respectively. The contents of the seven components in 16 batches of samples were 0.1250 - 1.174, 2.354 - 7.426, 1.822 - 27.21, 0.0370 - 1.399, 0.0723 - 0.4433, 0.0140 - 0.1990, and 0.2207 - 1.407 mg/g respectively. Conclusion The method is accurate, reproducible and durable, which could be used to the quality control and evaluation of Chenxiang Lubailu tablet.

Nobiletin inhibits neonatal rat cardiomyocytes hypertrophy induced by high glucose / 中国临床药理学与治疗学

AIM: To investigate the effect of nobiletin (Nob) on cardiomyocyte hypertrophy induced by high glucose and its mechanism. METHODS: Neonatal rat cardiomyocytes (NRCMS) were stimulated with high glucose (HG) to establish cardiomyocyte hypertrophy and nobiletin was given. Cell viability was measured by MTT assay. C-myc and Nppa mRNA levels were detected by qRT-PCR. Cellular surface area was detected by immunofluorescence, and Nrf2 and HO-1 protein expressions were detected by Western blot. RESULTS: After stimulation with 33.3 mmol/L HG for 48 h, the survival rate of NRCMS was significantly decreased, C-myc, Nppa mRNA levels and cellular surface area were significantly increased, Nrf2 and HO-1 protein expression were significantly decreased (P<0.05). After Nob treatment, compared with HG group, cellular surface area, Nrf2 and HO-1 protein expression were significantly increased, C-myc and Nppa mRNA levels were significantly decreased. The above indexes were reversed by using Nrf2 inhibitor. CONCLUSION: Nob inhibits cardiomyocyte hypertrophy induced by high glucose, and its mechanism may be related to the activation of Nrf2/HO-1 signaling pathway.

Mechanism of Xiahuang Granules in treating opioid-induced constipation based on network pharmacology / 中草药

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.

HPLC fingerprints research on decoction pieces of Citri Reticulatae Pericarpium based on chemometrics methods combined with orthogonal partial least square discriminate analysis / 中草药

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.

Effects of Chinese herb ingredients with different properties on OAT4, URAT1 and serum uric acid level in acute hyperuricemia mice / 中草药

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.

Nobiletin attenuates palmic acid-induced lipidosis in hepatocytes and regulatory mechanism of lncLSTR / 中国病理生理杂志

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.

Simultaneous quantitative determination of nine active compounds from Aurantii Fructus by HPLC / 中草药

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.

Nobiletin attenuates neurotoxic mitochondrial calcium overload through K⁺ influx and ΔΨ(m) across mitochondrial inner membrane

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.

The inhibitory effects of nobiletin on P-glycoprotein and the study of molecular mechanism / 药学学报

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.

The research progress of anti-tumor effect of polymethoxyflavonoids / 中国药理学通报

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.

Simultaneous determination of six constituents in rukuaixiao granules by UHPLC-MS/MS / 中国药学杂志

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.

Anti-dementia Activity of Nobiletin, a Citrus Flavonoid: A Review of Animal Studies

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.

Composition of Flavonoids and Antioxidative Activity from Juice of Jeju Native Citrus Fruits during Maturation

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.

Citrus fiavonoids simultaneous HPLC analysis / 中成药

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.

Sensitizing effect of nobiletin on increased apoptosis in human cancer cells induced by chemotherapeutic agents / 中草药

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.

Immunological Mechanisms of Nobiletin,a Citrus Polymethoxy Flavonoid in Inhibiting Hepatocarcinoma Proliferation / 中药新药与临床药理

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.