Aerobic glycolysis in colon cancer is repressed by naringin via the HIF1Α pathway / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Metabolic reprogramming is a common phenomenon in cancer, with aerobic glycolysis being one of its important characteristics. Hypoxia-inducible factor-1α (HIF1Α) is thought to play an important role in aerobic glycolysis. Meanwhile, naringin is a natural flavanone glycoside derived from grapefruits and many other citrus fruits. In this work, we identified glycolytic genes related to HIF1Α by analyzing the colon cancer database. The analysis of extracellular acidification rate and cell function verified the regulatory effects of HIF1Α overexpression on glycolysis, and the proliferation and migration of colon cancer cells. Moreover, naringin was used as an inhibitor of colon cancer cells to illustrate its effect on HIF1Α function. The results showed that the HIF1Α and enolase 2 (ENO2) levels in colon cancer tissues were highly correlated, and their high expression indicated a poor prognosis for colon cancer patients. Mechanistically, HIF1Α directly binds to the DNA promoter region and upregulates the transcription of ENO2; ectopic expression of ENO2 increased aerobic glycolysis in colon cancer cells. Most importantly, we found that the appropriate concentration of naringin inhibited the transcriptional activity of HIF1Α, which in turn decreased aerobic glycolysis in colon cancer cells. Generally, naringin reduces glycolysis in colon cancer cells by reducing the transcriptional activity of HIF1Α and the proliferation and invasion of colon cancer cells. This study helps to elucidate the relationship between colon cancer progression and glucose metabolism, and demonstrates the efficacy of naringin in the treatment of colon cancer.

Naringin Inhibits Colorectal Carcinogenesis by Inhibiting Viability of Colorectal Cancer Cells / 中国结合医学杂志

OBJECTIVE@#To explore the therapeutic effect of naringin on colorectal cancer (CRC) and the related mechanism.@*METHODS@#Cell counting kit-8 (CCK-8) assay and annexin V-FITC/PI assay were used to detect the effect of naringin (50-400 µg/mL) on cell proliferation and apoptosis of CRC cells, respectively. The scratch wound assay and transwell migration assay were used to assess the effect of naringin on CRC cell migration. Four-week-old male nude mice were injected with HCT116 cells subcutaneously to establish the tumor xenograft model. Naringin was injected intraperitoneally at 50 mg/(kg·d), with solvent and 5-fluorouracil treatment as control. The width and length of the tumors were measured and recorded every 6 days, and tumor tissues were photographed and weighed on the last day of the 24-d observation period. Immunohistochemical staining for caspase-3, proliferating cell nuclear antigen and TUNEL assay were used to evaluate the effect of naringin on cell proliferation and apoptosis in tumor tissues. The body weight, food and water intake of mice were recorded, and the major organs in different treatment groups were weighed on the last day and stained with hematoxylin and eosin for histological analysis. Meanwhile, the routine blood indicators were recorded.@*RESULTS@#CCK-8 and annexin V-FITC/PI results confirmed that naringin (100, 200, and 400 µg/mL) could inhibit proliferation and promote apoptosis. The scratch wound assay and transwell migration assay results confirmed the inhibitory activity of naringin against CRC cells migration. In vivo results demonstrated the inhibitory effect of naringin on tumor growth with good bio-compatibility.@*CONCLUSION@#Naringin inhibited colorectal carcinogenesis by inhibiting viability of CRC cells.

Effect of naringenin on natural killer cell cytotoxicity against hepatocellular carcinoma cells in vitro and its mechanism / 临床肝胆病杂志

Objective To investigate the effect of naringenin on the killing rate of natural killer (NK) cells and related mechanism by amplification of human peripheral blood mononuclear cells into NK cells in vitro and co-culture with hepatocellular carcinoma (HCC) CLC5 cells at a ratio of 1∶ 1. Methods A lymphocyte separation medium was used to isolate human peripheral blood mononuclear cells, which were induced with recombinant human interleukin-2 in vitro to culture NK cells. CCK-8 assay was used to measure the proliferation of HCC cells after human HCC cells were treated with naringenin (0, 3.125, 6.25, 12.5, 25, and 50 μmol/L) for 0, 24, and 48 hours, and after human NK cells were treated with different concentrations of naringenin for 24 hours, CCK-8 assay was used to measure the proliferation of NK cells. CellTiter-LumiTM was used to measure the killing rate of NK cells after the NK-HCC cell co-culture system at the ratio of 1∶ 1 was treated with naringenin for 24 hours. Quantitative real-time PCR was used to measure the gene expression of the activating receptor NKG2D in NK cells and NKG2D ligands in HCC cells. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results After being induced and cultured by recombinant human interleukin-2, NK cells were amplified to 82.33%±0.70% of human peripheral blood mononuclear cells. After naringenin treatment for 24 hours, there was no significant difference in the proliferation rate of HCC CLC5 cells between all mass concentration groups (all P > 0.05), and in the 25 and 50 μmol/L mass concentration groups, naringenin significantly promoted the proliferation of NK cells (both P 0.05); it significantly upregulated the expression of the NKG2D ligands such as ULBP1 and ULBP3 in HCC cells (all P < 0.001). Conclusion Naringenin may increase the killing activity of NK cells by upregulating the expression of NKG2D ligands in HCC cells.

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.

Naringin regulates mitochondrial dynamics to protect against acetaminophen-induced hepatotoxicity by activating the AMPK/Nrf2 signaling pathway in vitro

Naringin (Nar) has been reported to exert potential hepatoprotective effects against acetaminophen (APAP)-induced injury. Mitochondrial dysfunction plays an important role in APAP-induced liver injury. However, the protective mechanism of Nar against mitochondrial damage has not been elucidated. Therefore, the aim of this study was to investigate the hepatoprotective effects of Nar against APAP and the possible mechanisms of actions. Primary rat hepatocytes and HepG2 cells were utilized to establish an in vitro model of APAP-induced hepatotoxicity. The effect of APAP and Nar on cell viability was evaluated by a CCK8 assay and detection of the concentrations of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase. The cellular concentrations of biomarkers of oxidative stress were measured by ELISA. The mRNA expression levels of APAP-related phase II enzymes were determined by real-time PCR. The protein levels of Nrf2, phospho (p)-AMPK/AMPK, and biomarkers of mitochondrial dynamics were determined by western blot analysis. The mitochondrial membrane potential (MMP) was measured by high-content analysis and confocal microscopy. JC-1 staining was performed to evaluate mitochondrial depolarization. Nar pretreatment notably prevented the marked APAP-induced hepatocyte injury, increases in oxidative stress marker expression, reductions in the expression of phase II enzymes, significant loss of MMP, mitochondrial depolarization, and mitochondrial fission in vitro. In conclusion, Nar alleviated APAP-induced hepatocyte and mitochondrial injury by activating the AMPK/Nrf2 pathway to reduce oxidative stress in vitro. Applying Nar for the treatment of APAP-induced liver injury might be promising.

Naringin attenuates oxidative stress, inflammation, apoptosis, and oxidative DNA damage in acrylamide-induced nephrotoxicity in rats

Objective: To explore the possible effects of naringin on acrylamide-induced nephrotoxicity in rats. Methods: Sprague-Dawley rats weighing 200-250 g were randomly divided into five groups. The control group was given intragastric (i.g.) saline (1 mL) for 10 d. The acrylamide group was given i.g. acrylamide in saline (38.27 mg/kg titrated to 1 mL) for 10 d. The treatment groups were administered with naringin in saline (50 and 100 mg/kg, respectively) for 10 d and given i.g. acrylamide (38.27 mg/kg) 1 h after naringin injection. The naringin group was given i.g. naringin (100 mg/kg) alone for 10 d. On day 11, intracardiac blood samples were obtained from the rats when they were under anesthesia, after which they were euthanized. Urea and creatinine concentrations of blood serum samples were analyzed with an autoanalyzer. Enzyme-linked immunosorbent assay was used to quantify malondialdehyde, superoxide dismutase, glutathione, glutathione peroxidase, catalase, tumor necrosis factor-β, nuclear factor-κB, interleukin (IL)-33, IL-6, IL-1β, cyclooxygenase-2, kidney injury molecule-1, mitogen-activated protein kinase-1, and caspase-3 in kidney tissues. Renal tissues were also evaluated by histopathological and immunohistochemical examinations for 8-OHdG and Bcl-2. Results: Naringin attenuated acrylamide-induced nephrotoxicity by significantly decreasing serum urea and creatinine levels. Naringin increased superoxide dismutase, glutathione, glutathione peroxidase, and catalase activities and decreased malondialdehyde levels in kidney tissues. In addition, naringin reduced the levels of inflammatory and apoptotic parameters in kidney tissues. The histopathological assay showed that acrylamide caused histopathological changes and DNA damage, which were ameliorated by naringin. Conclusions: Naringin attenuated inflammation, apoptosis, oxidative stress, and oxidative DNA damage in acrylamide-induced nephrotoxicity in rats.

Naringin alleviates early brain injury in rats with subarachnoid hemorrhage / 国际生物医学工程杂志

Objective:To investigate the effects of naringin on early brain injury in rats with subarachnoid hemorrhage and its possible mechanism of action.Methods:Rats were randomly divided into the sham operation group, the model group, and the naringin group. Each group had 8 rats. The SAH model was established by intravascular perforation, and then rats in the model group and the naringin group were administered 0.9% NaCl or naringin 40 mg/kg by intraperitoneal injection after 0.5 h. SAH score, neurological function score, cerebral edema, and blood-brain barrier permeability were detected. The level of NAD + and nflammatory factors were detected by ELISA. The expression of poly(ADP-ribose) polymerase-1 (PARP-1), apoptosis inducing factor (AIF), and protease-activated receptor (PAR) proteins was detected by Western Blot. The expression of PARP-1 mRNA was detected by quantitative real-time fluorescence PCR (qRT-PCR). Neuronal apoptosis was detected by an immunofluorescence assay. Results:Compared with the model group, naringin treatment improved neurological function ( P<0.01), reduced cerebral edema and Evans blue exudation (all P<0.01), increased the content of NAD + ( P<0.001), reduced IL-1β, IL-6 and TNF-α levels (all P<0.001), and reduced the expression of PARP-1/AIF pathway-related proteins in vivo (all P<0.001). In addition, naringin could inhibit neuronal apoptosis in early brain injury after SAH. Conclusions:Naringin can improve the early brain injury after SAH, which may be achieved by inhibiting the PARP-1/AIF pathway.

The protective effect of naringin on cardiac injury in diabetic rats via activating Maxi K / 中国药理学通报

Aim To investigate the protective effect of naringin ( NA) on diabetic cardiomyopathy by activating the large conduction Ca2+ activated K4 channels (Maxi K ).Methods SD rats were fed with high-fat diet combined with intraperitoneal injection of strepto- zotocin (STZ) to establish a diabetic rat model.Then the rats were randomly divided into model group ( DCM) , naringin group ( NA) and naringin + Maxi K-specific inhibitor group ( NA + PAX) , with 8 rats in each group.Hats in treatment group received administration for 12 weeks and blood glucose was monitored regularly during experiments.The changes of cardiac function, morphology and fibrosis were detected after the treatment.The changes of cx and (3 subunits of Maxi K in heart were detected.Results Cardiac ultrasound results showed that NA could partially restore the cardiac function of rats.However, the cardiac protec tive function of NA was significantly reduced in diabetic rats after Maxi K was specifically blocked.Fibrosis analysis showed that the expression of collagen and fi- bronectin in rats could be decreased after NA treatment, which could be partially reversed by PAX.Western blot results showed that the expression of Maxi K a and p-subunit decreased in DCM group, but there was no significant change after NA treatment.Conclusions NA has a cardioprotective effect on diabetic rats by promoting the opening of the Maxi K channel on the membrane surface rather than increasing its expression.

Naringin is a promising natural compound for therapy of iron-overload disorders

Abstract Naringin has been shown to exhibit satisfying iron chelation capacity. Considering the side effects of routinely-used iron chelator (desferrioxamine, DFO), we decided to evaluate the iron chelation potency of naringin to discover whether or not it can be a promising natural substitute for treatment of excessive iron-related diseases. 35 mice were classified into five groups of 7 and subjected to iron dextran administration to induce the iron-overload condition. Iron-overloaded mice were then treated with normal saline (as control), naringin or DFO Morphology changes, and iron deposition in liver tissues were studied using H&E and Perl's staining. The results revealed that naringin is more potent than DFO in removing excessive iron ions deposited in liver tissues, indicating that naringin is a promising natural compound for therapy of iron overload disorders

A new combination of naringin and trimetazidine protect kidney Mitochondria dysfunction induced by renal Ischemia / Reperfusion injury in rat

Abstract Ischemia/reperfusion (IR) injury leads to overproduction of Reactive Oxygen Species (ROS), and disrupts membrane potential that contributes to cell death. The aim of this study was to determine if naringin (NAR), trimetazidine (TMZ) or their combination, protect the kidney mitochondrial from IR injury. Forty rats were randomly allocated into five groups, harboring eight rats each: Sham, IR, NAR (100 mg/kg), TMZ (5 mg/kg) and NAR plus TMZ. Ischemia was induced by obstructing both renal pedicles for 45 min, followed by reperfusion for 4 hours. The mitochondria were isolated to examine the ROS, Malondialdehyde (MDA), Glutathione (GSH), mitochondrial membrane potential (MMP) and mitochondrial viability (MTT). Our findings indicated that IR injury resulted in excessive ROS production, increased MDA levels and decreased GSH, MMP and MMT levels. However, NAR, TMZ or their combination reversed these changes. Interestingly, a higher protection was noted with the combination of both, compared to each drug alone. We speculate that this combination demonstrates a promising process for controlling renal failure, especially with the poor clinical outcome, acquired with NAR alone. This study revealed that pretreatment their combination serves as a promising compound against oxidative stress, leading to suppression of mitochondrial stress pathway and elevation of GSH level.

Naringin and trimetazidine improve baroreflex sensitivity and nucleus tractus solitarius electrical activity in renal ischemia-reperfusion injury / Naringin and trimetazidine improve baroreflex sensitivity and nucleus tractus solitarius electrical activity in renal ischemia-reperfusion injury

Resumo Fundamento: O núcleo do trato solitário (NTS) é uma área do cérebro que desempenha um papel fundamental na regulação renal e cardiovascular através dos impulsos dos barorreceptores. Objetivos: O objetivo deste estudo foi avaliar o efeito da Naringina (NAR) e trimetazidina (TMZ), isoladamente e combinadas, na atividade elétrica do NTS e na sensibilidade barorreflexa (SBR) na lesão de isquemia e reperfusão (I/R) renal. Métodos: Foram utilizados quarenta ratos machos Sprague-Dawley (200-250 g), alocados em 5 grupos com 8 ratos cada. Grupos: 1) Sham; 2) I/R; 3) TMZ 5 mg/kg; 4) NAR 100 mg/kg; e 5) TMZ5 + NAR100. A veia femoral esquerda foi canulada para infundir a solução salina ou droga e avaliar a SBR. A I/R foi induzida por oclusão dos pedículos renais por 45 min, seguida de reperfusão de 4 horas. O eletroencefalograma local do NTS foi registrado antes, durante a isquemia e durante a reperfusão. A fenilefrina foi injetada por via intravenosa para avaliar a SBR ao final do tempo de reperfusão. Os dados foram analisados por ANOVA de duas vias com medidas repetidas seguida pelo teste post hoc de Tukey. Um valor de p<0,05 foi considerado como significativo. Resultados: As ondas elétricas do NTS não se alteraram durante o tempo de isquemia, mas diminuíram significativamente durante todos os tempos de reperfusão. A atividade elétrica do NTS e a SBR foram reduzidas drasticamente em ratos com lesão I/R; no entanto, a administração de NAR e TMZ, isoladamente e combinadas, melhorou significativamente essas alterações em ratos com lesão I/R. Conclusões: Os resultados mostraram que a lesão de I/R leva à redução da atividade elétrica da SBR e do NTS, e pode haver uma ligação entre a I/R e a diminuição da SBR. Além disso, a NAR e a TMZ são agentes promissores para tratar complicações de I/R.

Abstract Background: Nucleus tractus solitarius (NTS) is a brain area that plays a key role in kidney and cardiovascular regulation via baroreceptors impulses. Objectives: The aim of this study was to evaluate the effect of naringin (NAR) and trimetazidine (TMZ) alone and their combination on NTS electrical activity and baroreceptor sensitivity (BRS) in renal ischemia- reperfusion (I/R) injury. Methods: Forty male Sprague-Dawley rats (200- 250 g) were allocated into 5 groups with 8 in each. 1) Sham; 2) I/R; 3) TMZ 5 mg/kg; 4) NAR 100 mg/kg; and 5) TMZ5+ NAR100. The left femoral vein was cannulated to infuse saline solution or drug and the BRS was evaluated. I/R was induced by occlusion of renal pedicles for 45 min, followed by 4 hours of reperfusion. The NTS local electroencephalogram (EEG) was recorded before, during ischemia and throughout the reperfusion. Phenylephrine was injected intravenously to evaluate BRS at the end of reperfusion time. The data were analyzed by two-way repeated measurement ANOVA followed by Tukey's post hoc test. A p-value <0.05 was considered significant. Results: NTS electrical waves did not change during ischemia time, while they significantly decreased during the entire reperfusion time. NTS electrical activity and BRS dramatically reduced in rats with I/R injury; however, administration of NAR, TMZ alone or their combination significantly improved these changes in rats with I/R injury. Conclusions: The results showed that I/R injury leads to reduced BRS and NTS electrical activity and there may be an association between I/R and decreased BRS. In addition, NAR and TMZ are promising agents to treat I/R complications.

Protective effect of naringin in rat model of renal ischemia reperfusion injury / Efecto protector de la naringina en modelo de rata con lesión por reperfusión de isquemia renal

ABSTRACT Objective: We aimed to research that naringin whether protects from renal ischemia/reperfusion induced renal damage in rats. Methods: Twenty-four Wistar albino female rats randomly were divided into three groups: 1) control group, in which the rats were only performed right nephrectomy; 2) a second group received right nephrectomy and left kidney ischemia (1 h) and reperfusion (24 h) group ischemia/reperfusion (I/R); 3) a third group received 50 mg/kg naringin orally once a day for two weeks before ischemia/reperfusion (I/R/N). Expression of cyclooxygenase-2 (COX-2), cytosolic phospholipase A2 (cPLA2), inducible nitric oxide synthase (iNOS), caspase-3, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated x protein (Bax), serum creatinine (Cr), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) were measured by using enzyme-linked immunosorbent assay (ELISA). Results: Naringin-treated rats that performed renal ischemia/reperfusion demonstrated significant decrease in Cr, IL-6 and TNF-α levels when compared to the only renal ischemia/reperfusion performed rats. While renal ischemia/reperfusion caused a decrease of bcl-2 (1.72 ± 0.20 pg/ml) levels, while an increase of COX-2 (11882 ± 642 pg/ml), cPLA2 (2448 ± 139 pg/ml), iNOS (4331 ± 438 IU/ml), cleaved caspase-3 (7.33 ± 0.76 ng/ml) and Bax (2.33 ± 0.44 ng/ml) levels. The treatment of naringin reversed these kidney effects (7.47 ± 60.35 pg/ml; 9299 ± 327 pg/ml; 2001 ± 78 pg/ml; 3112 ± 220 IU/ml; 3.38 ± 0.54 ng/ml; 2.33 ± 0.44 ng/ml, respectively) (p <0.05). Conclusion: This study showed that naringin treatment attenuated renal damage induced by ischemia/reperfusion in rats.

RESUMEN Objetivo: Nuestro objetivo fue investigar si la naringina protege del daño en los riñones provocado por isquemia-reperfusión renal en ratas. Material y métodos: De forma aleatoria, dividimos 24 ratas albinas Wistar hembras en tres grupos: 1) grupo control, en el que solo se les realizó a las ratas una nefrectomía derecha; 2) un segundo grupo isquemia-reperfusión, con nefrectomía derecha e isquemia de riñón izquierdo (1 h) y reperfusión (24 h); 3) un tercer grupo al que se le administró 50 mg/kg de naringina por vía oral una vez al día durante dos semanas antes de la isquemia-reperfusión. Por medio de un ensayo inmunoabsorbente ligado a enzimas (ELISA), se midieron las siguientes expresiones: ciclooxigenasa-2 (COX-2), fosfolipasa citosólica A2 (cPLA2), óxido nítrico sintetasa inducible (ONSi), caspasa-3, linfoma de células B2 (Bcl-2), proteína X asociada a Bcl-2 (Bax), creatinina sérica (Cr), factor de necrosis tumoral alfa (FNT-α) e interleucina 6 (IL-6). Resultados: Las ratas tratadas con naringina por isquemia-reperfusión renal mostraron un descenso significativo en los niveles de Cr, IL-6 y FNT-α en comparación con las ratas a las que se les indujo isquemia-reperfusión renal pero que no se les suministró naringina. La isquemia-reperfusión renal provocó un descenso de los niveles de Bcl-2 (1,72 ± 0,20 pg/ml) y un ascenso en los niveles de COX-2 (11882 ± 642 pg/ml), cPLA2 (2448 ± 139 pg/ml), ONSi (4331 ± 438 UI/ml), caspasa-3 escindida (7,33 ± 0,76 ng/ml) y Bax (2,33 ± 0.,44 ng/ml). El tratamiento con naringina diminuyó estos efectos en el riñón (7,47 ± 60,35 pg/ml; 9299 ± 327 pg/ml; 2001 ± 78 pg/ml; 3112 ± 220 UI/ml; 3.38 ± 0.54 ng/ml; 2.33 ± 0,44 ng/ml, respectivamente) (p <0,05). Conclusión: En este estudio se demostró que el tratamiento con naringina atenuó el daño renal producido por isquemia-reperfusión en ratas.

Protective effects of naringin phospholipid complex on oxidative injury in ARPE-19 cells associated with activation of Nrf2 pathway / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To investigate the protective effects of naringin(Nar)phospholipid complex(NPC)on oxidative injury in retinal pigment epithelium cells(ARPE-19 cells)induced by tert-butyl hydroperoxide(t-BHP)and elucidate the underlying mechanism.<p>METHODS:The NPC was prepared by solvent method. Experimental cells are divided into seven groups: control group \〖cultured with dimethylsulfoxide(DMSO)\〗, model group(intervention with 200μmol/L t-BHP), nuclear factor erythroid 2-related factor 2(Nrf2)-siRNA group(cell transfection for Nrf2 gene), naringin group(add 200μmol/L t-BHP after pretreatment with 200μmol/L naringin medium), NPC group(add 200μmol/L t-BHP after pretreatment with 200μmol/L NPC medium), Nrf2-siRNA+ naringin group(after 200μmol/L naringin pretreatment, Nrf2 gene interference, then add 200μmol/L t-BHP)and Nrf2-siRNA+ NPC group(after 200μmol/L NPC pretreatment, Nrf2 gene interference, then add 200μmol/L t-BHP). The intracellular levels of superoxide dismutase(SOD), malondialdehyde(MDA)and total antioxidant capacity(T-AOC)were detected, intracellular level of reactive oxygen species(ROS)was detected by DCFH-DA staining method. The mRNA and protein expressions of HO-1, NQO-1, GCL and Nrf2 were detected by real-time PCR and western blot, respectively. <p>RESULTS:NPC more significantly increased the levels of SOD and T-AOC, reduced the contents of ROS and MDA than naringin in t-BHP-treated ARPE-19 cells. After naringin and NPC pre-protected ARPE-19 cells, the relative expression and protein expression of Nrf2, HO-1, NQO-1 and GCL mRNA were higher than those of the model group and Nrf2-siRNA group. There were statistically significant differences in the relative expression of 4 genes and the expression levels of 4 proteins in the naringin group and the NPC group, the Nrf2-siRNA+naringin group and the Nrf2-siRNA+NPC group. The expression of Nrf2, HO-1 and NQO-1 protein in the Nrf2-siRNA+naringin group was not significantly different than that in the Nrf2-siRNA group. Compared with the Nrf2-siRNA group, the expression of 4 proteins in the Nrf2-siRNA+NPC group was statistically significant, and the effect of NPC was significantly stronger than that of naringin.<p>CONCLUSION: After naringin forms a phospholipid complex, it can significantly increase the antioxidant capacity in cells and reduce the oxidation level. It up-regulates the expression of Nrf2 and its downstream antioxidant enzymes and phase Ⅱ detoxification enzymes by activating the Nrf2/ARE antioxidative stress pathway to better protect ARPE-19 cells from oxidative damage.

Effects of Naringin on neuronal apoptosis in mice with memory consolidation disorder / 国际中医中药杂志

Objective:To observe the effect of Naringin on neuronal apoptosis in mice with memory consolidation disorderinduced by sodium nitrite.Methods:Fifty mice were randomly divided into blank group, model group, standardized protocol group, high-dose Naringin group and low-dose Naringin group, with 10 mice in each group. The standardized protocol group was given Donepezil 1 mg/kg, the Naringin high and low dose groups were gavaged with Naringin solution 100 and 50 mg/(kg·d), blank group and model group were gavaged with equal volume of distilled water once a day for 21 days. The model was established on the 22nd day. The blank group was intraperitoneally injected with normal saline, and the other groups were intraperitoneally injected with 100 mg/(kg·d) sodium nitrite solution for 7 days. The cognitive ability of mice in each group was evaluated by platform jumping test, and the hippocampal synaptic structure was observed by electron microscope. The contents of acetylcholine (ACh), SOD, MDA and NO in hippocampus and the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) was detected by ELISA. The expression of N-methyl-D-aspartate receptor (NMDAR), glutamine receptor 2 (GluR), calcium/calmodulin dependent protease Ⅱ (CaMK Ⅱ), Caspase-3, Bcl-2 and Bad proteins in hippocampus of model mice were detected by Western blot.Results:The number and morphology of hippocampal neurons were normal, nucleus, mitochondria, rough endoplasmic reticulum and synaptic membrane of hippocampal neurons in high-dose Naringin group were clear. Compared with the model group, the latency of mice in the high-dose Naringin group was prolonged and the number of errors was reduced ( P<0.01). The levels of MDA and NO in hippocampus of mice in the high-dose Naringin group significantly decreased ( P<0.01), and the activity of SOD significantly increased ( P<0.01). The content of ACh (23.682 ± 2.835 μg/mg prot vs. 14.939 ± 2.901 μg/mg prot), ChAT (163.302 ± 21.278 U/g vs. 89.612 ± 11.497 U/g) increased, AChE (0.367 ± 0.015 U/mg prot vs. 0.471 ± 0.014 U/mg prot) activity decreased ( P<0.01); The expression of Bad (0.441 ± 0.010 vs. 0.633 ± 0.010), Caspase-3 (0.425 ± 0.036 vs. 0.537 ± 0.024) significantly decreased, and the expression of Bcl-2 (0.890 ± 0.014 vs. 0.727 ± 0.009) significantly increased ( P<0.01); The expression of CAMKⅡ (1.043 ± 0.037 vs. 1.475 ± 0.043) significantly decreased ( P<0.01), and the expression of NMDAR1 (0.407 ± 0.037 vs. 0.345 ± 0.012), GluR2 (1.125 ± 0.033 vs. 0.664 ± 0.023) significantly increased ( P<0.01). Conclusion:Naringin could play the role of protecing the neuron and improving the cognition of mice with memory consolidation disorder by regulating the balance of ACh and glutamate system and reducing neuronal apoptosis and antioxidant stress.

Simultaneous Determination of 5 Components in the Classical Formula Huaihua San by HPLC Wavelength Switching Method / 中国药房

OBJECTIVE：To develop a method for simultaneous determination of 5 components in classical formula Huaihua san，including rutin ，naringin，neohesperidin，quercetin and pulegone. METHODS ：HPLC wavelength switching method was adopted. The determination was performed on Cosmosil C 18 column with mobile phase consisted of acetonitrile- 0.05％ phosphoric acid solution （gradient elution ）at the flow rate of 1.0 mL/min. The detection wavelengths were set at 257 nm for rutin ，283 nm for naringin and neohesperidin ，254 nm for quercetin ，252 nm for pulegone ，respectively. The column temperature was set at 30 ℃， and sample size was 10 μL. RESULTS：The linear range was 21.7-2 170 μg/mL for rutin，46-4 600 μg/mL for naringin，22.3- 2 230 μg/mL for neohesperidin，0.96-96 μg/mL for quercetin，2.7-270 μg/mL for pulegone（all r＞0.999），respectively. RSDs of precision，stability（24 h）and reproducibility tests were all lower than 2％（n＝6）. Average recoveries were 100.70％，99.31％， 101.10％，100.03％ and 99.63％（all RSD ＜2％，n＝9）. Among 3 batches of Huaihua san samples ，the contents of above 5 components were 20.055-22.615，25.557-27.806，11.428-13.250，0.350-0.478，2.372-4.011 mg/g，respectively. CONCLUSIONS ： Established method is simple ，accurate and reproducible ，and could be used for the simultaneous determination of 5 components in Huaihua san.

Investigation of Chemical Constituents and Sources of Zhishi Xiebai Guizhi Decoction by LC-MS/MS / 中国药学杂志

OBJECTIVE: To study the chemical constituents of Zhishi Xiebai Guizhi decoction and its single agent by high performance liquid chromatography-mass spectrometry to provide a scientific basis for the quality evaluation and control. METHODS: Agilent XDB-C18(4.6 mm×250 mm, 5 μm) column was used for HPLC analysis with acetonitrile-water as mobile phase by gradient elution. Using liquid chromatography-mass spectrometry technology to collect data in positive and negative ion mode, combined with literature reports, the main chemical components of Zhishi Xiebai Guizhi decoction were analyzed and identified. RESULTS: A total of 19 compounds were identified from Zhishi Xiebai Guizhi decoction. The main components are naringin, neohesperidin, etc. The main component types include alkaloids, steroidal saponins, glycosides, flavonoids, etc. CONCLUSION: This study comprehensively clarified the chemical composition of Zhishi Xiebai Guizhi decoction, and it has important reference value for the identification and quality evaluation of Zhishi Xiebai Guizhi decoction.

Discussion and prediction of application prospects of Citri Grandis Exocarpium on COVID-19 based on literature analysis and molecular docking / 中草药

Objective: The present study aims to explore the application prospects of Citri Grandis Exocarpium for protecting against novel coronavirus pneumonia (COVID-19). Methods: The pharmacological effects, including expectorant, anti-acute lung injury, anti-inflammatory, anti-pulmonary fibrosis, relieving cough, anti-oxidation, anti-liver injury, anti-kidney injury effects and etc, of both Citri Grandis Exocarpium and its main chemical constituents were analyzed through literature review. The constituents of Citri Grandis Exocarpium were collected by using traditional Chinese medicine systems pharmacology (TCMSP) database and literature searching. The molecular docking study was performed to evaluate the binding ability between the chemical constituents and angiotensin converting enzyme 2 (ACE2), 3C-like main protease (Mpro), papain-like protease (PLP), and dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) to predict its potential activity in inhibiting the infection and replication of SARS-CoV-2 virus. Results: The literature review indicated that Citri Grandis Exocarpium possesses expectorant, anti-acute lung injury, anti-inflammatory, anti-pulmonary fibrosis, relieving cough, anti-oxidation, anti-liver injury, anti-kidney injury effects, and so on. Molecular docking results indicated that naringin, neohesperdin, rhoifolin, poncirin, and sitogluside were the main active flavonoids due to showing strong interactions with ACE2, MPro, PLP and DC-SIGN with potential activity in inhibiting the infection and replication of SARS-CoV-2 virus. Conclusion: Citri Grandis Exocarpium may probably delay the progression of COVID-19 through a variety of pharmacological activities and the inhibition of the infection and replication of SARS-CoV-2 due to targeting ACE2, MPro, PLP, and DC-SIGN, reminding that Citri Grandis Exocarpium may possess a potential capacity to protect against COVID-19.

Simultaneous Determination of the Contents of 12 Flavonoids in Quzhiqiao from Different Collection Places by HPLC / 中国药房

OBJECTIVE：To establish a method for the simultan eous determination of the contents of 12 flavonoids in Quzhiqiao. METHODS ：HPLC method was adopted. The determination was performed on Agilent Extend C 18 column with mobile phase consisted of 0.1％ formic acid-acetonitrile （gradient elution ）at the flow rate of 1.0 mL/min. The column temperature was set at 35 ℃. The detection wavelength was set at 330 nm，and sample size was 10 μL. The contents of 12 components（such as eriocitrin，narirutin，naringin，naringenin，hesperidin，neohesperidin，hesperide hydrate ，luteolin，hesperide，nobiletin，hesperetin and hesperidolactone ）in 10 batches of Quzhiqiao from different collection places were determined. RESULTS ：The linear range of eriocitrin，narirutin，naringin，naringenin，hesperidin，neohesperidin，hesperide hydrate ，luteolin，hesperide，nobiletin，hesperetin and hesperidolactone were 1.65-16.51，4.50-45.02，35.41-354.12，4.11-41.12，2.29-22.86，34.96-349.56，1.42-14.15，1.50-15.04， 1.83-18.28，1.51-15.08，1.61-16.12，1.28-12.84 μg/mL，respectively（all r＞0.999 7）. The detection limits were 0.165 1，0.450 2， 3.541 2，0.411 2，0.228 6，3.495 6，0.141 5，0.150 4，0.182 8，0.150 8，0.161 2，0.128 4 μg/mL，respectively. The limits of quantitation were 0.547 8，1.487 4，11.663 3，1.360 3，0.758 3，11.594 9，0.466 3，0.497 1，0.601 2，0.499 9，0.532 3，0.424 6 μg/mL，respectively. RSDs of precision （n＝6），reproducibility（n＝6）and stability （24 h，n＝7）tests were all lower than 3％. The average recoveries were 99.50％，99.61％，98.18％，98.85％，98.48％，98.50％，98.25％，99.91％，103.13％，98.82％， 98.44％ ， 100.29％ （RSD＝1.49％ -2.38％ ， n＝6）. The contents of the above 12 components in 10 batches of samples from different collection places were 1.995 5-2.648 8，4.317 7- 5.005 1，33.215 5-34.054 6，3.140 4-3.471 5，3.221 2-3.748 8， 42.746 6-44.026 6，0.202 7-0.239 4，0.191 2-0.208 8，0.080 3- 0.097 9，0.291 9-0.307 1，0.119 9-0.149 1，0.082 7-0.089 8 mg/g. CONC LUSIONS：The method is accurate ，reliable，simple and efficient，which can be used to simultaneous determination of the contents of 12 flavonoids in Quzhiqiao ，and to provide reference for the establishment of quality control standards of Quzhiqiao.

Study on quality regionalization of Lycii Fructus / 中国中药杂志

Lycii Fructus is a commonly used Chinese herbal medicine. Due to limited wild resources,Lycii Fructus is mainly derived from the cultivation of Lycium barbarum when it circulate in the market. Related studies have shown that there are some differences in the quality of Lycii Fructus from different regions. In August 2017,our research team went to the county areas of Ningxia,Gansu,Qinghai,Xinjiang and Inner Mongolia,which included 13 counties and planted L. barbarum. The county areas obtained 71 batches of samples. According to the content of four chemical components of polysaccharides,protein,vitamins and naringin in 71 batches,the quality of zoning was studied by SPSS and Arc GIS. The results showed that there were significant differences in the contents of polysaccharides,protein,vitamins and naringin in the sampling area. Among them,the vitamin content in Inner Mongolia was relatively high. The content of polysaccharide in Xingjiang was relatively high,the protein content of Ningxia was relatively high. The content of naringin in Inner Mongolia was relatively high. The quality of Lycii Fructus from different producing areas requires a comprehensive evaluation of multiple indicators. Through the analysis of the four chemical components and the eco-environmental factors,the chemical composition with high percentage of percentage content is affected by climatic factors,and the chemical composition with low proportion is greatly affected by soil factors. The literatures such as " Chinese Flora" are mostly recorded that L. barbarum are planted in the northwest and south central areas. There is no record of artificial planting L. barbarum in the northeast region. Through this study,it is found that Liaoning and Jilin province also have a small amount of L. barbarum cultivated. The result shows that in the middle temperature zone and warm temperature zone there are also the cultivation of L. barbarum.

Evaluation of Anti-inflammatory and Regenerative Efficiency of Naringin and Naringenin in Degenerated Human Nucleus Pulposus Cells: Biological and Molecular Modeling Studies

STUDY DESIGN: Development of an in vitro model for assessing the anti-inflammatory efficacies of naringin (Nar) and naringenin (NG).PURPOSE: To evaluate the efficacy of natural flavonoids as therapeutic drugs against anti-inflammatory processes in the nucleus pulposus (NP) cells using in-vitro and in-silico methods.OVERVIEW OF LITERATURE: Intervertebral disc (IVD) disease is a common cause of low back pain. Chronic inflammation and degeneration play a significant role in its etiopathology. Thus, a better understanding of anti-inflammatory agents and their role in IVD degeneration and pro-inflammatory cytokines expression is necessary for pain management and regeneration in IVD.METHODS: We performed primary cell culture of NP cells; immunocytochemistry; gene expression studies of cytokines, metalloproteases, extracellular proteins, and apoptotic markers using quantitative polymerase chain reaction and reverse transcription-polymerase chain reaction (RT-PCR); cytotoxicity assay (MTT); and molecular docking studies using AutoDock 4.2 software (Molecular Graphics Laboratory, La Jolla, CA, USA) to confirm the binding mode of proteins and synthesized complexes. We calculated the mean±standard deviation values and performed analysis of variance and t-test using SPSS ver. 17.0 (SPSS, Inc., Chicago, IL, USA).RESULTS: Molecular docking showed that both Nar and NG bind to the selected genes of interest. Semi-quantitative RT-PCR analysis reveals differential gene expression of collagen (COL)9A1, COL9A2, COL9A3, COL11A2, COMT (catechol-O-methyltransferase), and THBS2 (thrombospondin 2); up regulation of ACAN (aggrecan), COL1A1, COL11A1, interleukin (IL)6, IL10, IL18R1, IL18RAP, metalloprotease (MMP)2, MMP3, MMP9, ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5), IGF1R (insulin-like growth factor type 1 receptor), SPARC (secreted protein acidic and cysteine rich), PARK2 (parkin), VDR (vitamin D receptor), and BCL2 (B-cell lymphoma 2); down regulation of IL1A, CASP3 (caspase 3), and nine genes with predetermined concentrations of Nar and NG.CONCLUSIONS: The present study evaluated the anti-inflammatory and regenerative efficiencies of Nar and NG in degenerated human NP cells. Altered gene expressions of cytokines, metalloproteases, extracellular proteins, apoptotic genes were dose responsive. The molecular docking (in silico) studies showed effective binding of these native ligands (Nar and NG) with genes identified as potent inhibitors of inflammation. Thus, these natural flavonoids could serve as anti-inflammatory agents in the treatment of low back pain and sciatica.