Effect of naringenin on the anti-inflammatory, vascularization, and osteogenesis differentiation of human periodontal ligament stem cells via the stromal cell-derived factor 1/C-X-C motif chemokine receptor 4 signaling axis stimulated by lipopolysaccharide / 华西口腔医学杂志

OBJECTIVES@#This study aimed to investigate how naringenin (Nar) affected the anti-inflammatory, vascula-rization, and osteogenesis differentiation of human periodontal ligament stem cells (hPDLSCs) stimulated by lipopolysaccharide (LPS) and to preliminarily explore the underlying mechanism.@*METHODS@#Cell-counting kit-8 (CCK8), cell scratch test, and Transwell assay were used to investigate the proliferation and migratory capabilities of hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red staining, lumen-formation assay, enzyme-linked immunosorbent assay, quantitative timed polymerase chain reaction, and Western blot were used to measure the expression of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), vascular endothlial growth factor (VEGF), basic fibroblast growth factor (bFGF), von Willebrand factor (vWF), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6.@*RESULTS@#We observed that 10 μmol/L Nar could attenuate the inflammatory response of hPDLSCs stimulated by 10 μg/mL LPS and promoted their proliferation, migration, and vascularization differentiation. Furthermore, 0.1 μmol/L Nar could effectively restore the osteogenic differentiation of inflammatory hPDLSCs. The effects of Nar's anti-inflammatory and promotion of osteogenic differentiation significantly decreased and inflammatory vascularization differentiation increased after adding AMD3100 (a specific CXCR4 inhibitor).@*CONCLUSIONS@#Nar demonstrated the ability to promote the anti-inflammatory, vascularization, and osteogenic effects of hPDLSCs stimulated by LPS, and the ability was associated with the stromal cell-derived factor/C-X-C motif chemokine receptor 4 signaling axis.

Inhibitory effects of naringenin on the activation of hepatic stellate cells through activating the apoptosis signal / 中国药房

OBJECTIVE To study the inhibitory effects and possible mechanism of naringenin on the activation of hepatic stellate cells. METHODS Using human hepatocytes LO2 as reference， based on drug intervention concentration screened by MTT assay， the effects of naringenin （Western blot assay and trypan blue staining test in 10， 20， 40 μmol/L， immunofluorescence assay in 40 μmol/L） on the expressions of liver fibrosis markers protein （collagen Ⅰ， α-SMA） and mRNA （α1-pro collagen Ⅰ， α-SMA） in human hepatic stellate cells LX2， and the expressions of cell apoptosis and apoptosis-related proteins （Bcl-2， Bax， cleaved caspase-3） were investigated. The apoptosis agents （Z-VAD-FMK， FMK）， ferroptosis pathway inhibitor ferrostatin-1， and programmed death pathway inhibitor necrostatin-1 were used to verify the mechanism of the above effects. RESULTS The naringenin could significantly down-regulate protein expressions of collagen Ⅰ （except for naringenin 10 μmol/L） and α-SMA， mRNA expressions of α1-pro collagen Ⅰ （except for naringenin 10 μmol/L） and α-SMA （P＜0.05）； it also induced LX2 cell apoptosis and increased its apoptotic ratio， down-regulated the protein expression of Bcl-2 while up-regulated the protein expressions of Bax （except for naringenin 10 μmol/L） and cleaved caspase-3 （except for naringenin 10 μmol/L）. FMK could reverse above effects of naringenin on LX2 cells （P＜0.05）. CONCLUSIONS Naringenin can inhibit the activation of hepatic stellate cells LX2 through activating the cell apoptosis signal， which plays ameliorative role in liver fibrosis.

Roles of Ca2+ sensitive K+ channel, phosphodiesterase inhibitor, and ? adrenergic activity in Tridax procumbens leaf extract relaxation of trachea smooth muscle in male Wistar rats

Background: Although traditional use of Tridax procumbens aqueous leaf extract (TPALE) in the management of respiratory disorders is documented, validated scientific evidence is scarce. Aim and Objectives: Trachea smooth muscle (TSM) relaxant activity of TPALE ingestion was investigated in the presence or absence of key TSM relaxant agents. This was with the aim at elucidating relaxant activity of TPALE on TSM. Materials and Methods: Contractile activity of TSM excised from TPALE treated (100 mg/kg) and non-treated rats was assessed pre - and post-incubation in salbutamol (10?4 M), theophylline (10?4 M), caffeine (10?4 M), naringin (10?4 M), and naringenin (10?4 M) using organ chamber connected to a force isometric transducer (Model 7004; Ugo-Basile VArese, Italy). Results: TPALE treatment significantly inhibited contractile activity in TSM. TPALE treated rats showed significantly inhibited contractile activity of the TSM pre (45.6%) and post-incubation (35%) in theophylline when compared to control pre (90.6%) and post-incubation (60%). Incubation of TSM from control and TPALE treated rats in salbutamol, significantly inhibited contractile activity (33.2%) and (37.2%), respectively. After incubation in caffeine, TSM from TPALE treated rats showed significant inhibition in the contractile activity (30.7%) as TSM from control postincubation (38.4%). TSM of TPALE-treated group pre-incubation showed significant inhibition in contractile activity (41.8%) when compared to the TSM of TPALE-treated Group (59.3%) and control (64.5%) post-incubation in naringin. However, incubation of TSM of TPALE-treated rats in naringenin significantly inhibited contractile activity (40.4%) when compared to pre-incubation (45%) and control pre - and post-incubation, respectively (52% and 90%). Conclusion: Calcium-activated K+ channels, ?2 adrenergic stimulation, and antioxidant activity contribute to the mediation of relaxant activity by TPALE in TSM.

The effects of naringenin on pancreatic fibrosis in chronic pancreatitis mouse model and activation, proliferation and apoptosis of pancreatic stellate cells / 中华胰腺病杂志

Objective:To study the effects of naringenin on pancreatic fibrosis in the mouse model of chronic pancreatitis (CP) and its effects on the activation, proliferation and apoptosis of pancreatic stellate cells (PSCs).Methods:Eighteen C57BL/6 mice were randomly divided into control group, CP group and naringenin group, with 6 mice in each group. The CP mouse model was established by intraperitoneal injections of caerulein. Naringenin group was given naringenin (200 mg/kg/day) by gavage once a day from the first day of the fourth week of modeling process to the day before the killing; the control group and CP group were treated by gavage with an equivalent amount of drug solvent containing 0.5% sodium carboxymethyl cellulose (CMC-Na). Mice were killed 5 days after the last caerulein injection, and their pancreatic tissues were collected for hematoxylin-eosin staining and Sirius Red staining, pathological scoring and collagen sedimentation detection. Naringenin with different concentrations (0, 5, 10, 20, 50, 100, 150, 200 μmol/L) were used to intervene HPSC for 24 hours, and CCK-8 method was used to detect the cell activity. TGF-β1 recombinant protein (2 ng/ml) was used to induce PSCs for 1 hour (TGF-β1 stimulation group), and naringenin with low (50 μmol/L), middle (100 μmol/L) and high (150 μmol/L) concentration was used to intervene for 36 hours after TGF-β1 stimulation, respectively. Western Blotting was used to detect the expression of PSC activation related proteins FN and COL1A1, cell proliferation marker p21, anti-apoptotic protein Bcl-xL, pro-apoptotic protein Bax and Bid.Results:The pathological scores of pancreatic tissue [(7.33±1.15), (4.67±1.15)] and the percentage of collagen positive areas [(46±4), (28±2)%] in CP group and naringenin group were higher than those in the control group [0, (4±2)%]. However, these indexes in the naringenin group were lower than those in CP group, and the differences were all statistically significant (all P value <0.05). The relative expression of FN in control group, TGF-β1 stimulation group and low, medium and high naringenin group was 0.02, 0.76, 0.67, 0.34 and 0.07, respectively; the expression of COL1A1 in these groups was 0.51, 1.71, 1.34, 0.84 and 0.11. The expression of FN and COL1A1 in TGF-β1 stimulation group was significantly higher than that in control group, and the expression of FN and COL1A1 in low, medium and high naringenin group was significantly lower than that in TGF-β1 stimulation group, and the differences were all statistically significant (all P value <0.05). The expression of p21 in the above five groups was 0.87, 1.18, 1.27, 1.22 and 1.00. The expression of p21 in TGF-β1 stimulation group was higher than that in control group, and the expression of p21 in high naringenin group was obviously lower than that in TGF-β1 stimulation group, and the differences were all statistically significant (all P value <0.05). In addition, the expression of Bcl-xL in these groups was 2.09, 2.21, 2.38, 2.50 and 2.12; the expression of Bax was 0.98, 0.88, 0.98, 1.00 and 0.88; the expression of Bid was 1.15, 1.09, 1.14, 1.18 and 1.18. There was no statistically significant difference among these groups (all P value >0.05). Conclusions:Naringenin could significantly alleviate the inflammation, atrophy and fibrosis in the CP mouse model, and inhibit the activation and proliferation of PSCs. However, naringenin had no significant effect on the apoptosis of PSCs, indicating that naringenin may be potentially used to treat pancreatic fibrosis in CP.

Effect of key enzymes ubiquitination sites on the biosynthesis of naringenin / 生物工程学报

Flavonoids have a variety of biological activities and have important applications in food, medicine, cosmetics, and many other fields. Naringenin is a platform chemical for the biosynthesis of many important flavonoids. Ubiquitination plays a pivotal role in the post-translational modification of proteins and participates in the regulation of cellular activities. Ubiquitinated proteins can be degraded by the ubiquitin-protease system, which is important for maintaining the physiological activities of cells, and may also exert a significant impact on the expression of exogenous proteins. In this study, a real-time in-situ detection system for ubiquitination modification has been established in Saccharomyces cerevisiae by using a fluorescence bimolecular complementation approach. The ubiquitination level of protein was characterized by fluorescence intensity. By using the approach, the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway have been obtained. The lysine residues of the relevant ubiquitination sites were mutated to arginine to reduce the ubiquitination level. The mutants of tyrosine ammonia-lyase (FjTAL) and chalcone synthase (SjCHS, SmCHS) showed decreased fluorescence, suggested that a decreased ubiquitination level. After fermentation verification, the S. cerevisiae expressing tyrosine ammonia-lyase FjTAL mutant FjTAL-K487R accumulated 74.2 mg/L p-coumaric acid at 72 h, which was 32.3% higher than that of the original FjTAL. The strains expressing chalcone synthase mutants showed no significant change in the titer of naringenin. The results showed that mutation of the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway could increase the titer of p-coumaric acid and have positive effect on naringenin biosynthesis.

Fermentación alcohólica por Saccharomyces cerevisiae y cuantificación de flavonoides del zumo de Citrus x clementina (naranja)

Objetivo: elaborar una bebida por fermentación alcohólica y la cuantificación de flavonoides del zumo de Citrus x clementina (naranja). Metodología: se utilizó el método de fermentación alcohólica por levadura de la variedad Saccharomyces cerevisiae, se fermento el jugo de naranja con una densidad de 1,050 glcm3 por 5 semanas y se cuantificó los flavonoides de la bebida alcohólica por el método de cromatografía HPLC. Resultados: después de las 5 semanas se analizó que la bebida por fermentación alcohólica tuvo un 11 % de alcohol y flavonoides de hesperidina 13,9 mgl100 ml y naringenina 6,3 mg/100 ml en su concentración.

SUMMARY Aim: to elaborate a drink by alcoholic fermentation and the quantification of flavonoids in Citrus x clementine (orange) juice. Methodology: the method of alcoholic fermentation by yeast of the Saccharomyces cerevisiae variety was used, the orange juice was fermented with a density of 1.050 glcm3 for 5 weeks and the flavonoids of the alcoholic beverage were quantified by the HPLC chromatography method. Results: after 5 weeks it was analyzed that the drink by alcoholic fermentation had 11 % alcohol and hesperidin flavonoids 13.9 mgl100 ml and 6.3 mg/100 ml naringenin in its concentration.

Objetivo: elaborar uma bebida por fermentação alcoólica e quantificação de flavonóides no suco Citrus x clementina (laranja). Metodologia: foi utilizado o método de fermentação alcoólica por levedura da variedade Saccharomyces cerevisiae, o suco de laranja foi fermentado com densidade de 1,050 glcm3 por 5 semanas e os flavonóides da bebida alcoólica foram quantificados pelo método de cromatografía HPLC. Resultados: após 5 semanas foi analisado que a bebida por fermentação alcoólica continha álcool a 11 % e flavonóides de hesperidina 13,9 mgl100 ml e 6,3 mg/100 ml naringenina em sua concentração.

Phytochemical composition and biological screening of two Lophophytum species / Composición fitoquímica y cribado biológico de dos especies de Lophophytum

Lophophytum species are holoparasites that grow on tree roots. The objectives of the work were to explore the chemical composition of the tubers of two Lophophytum species and to analyze the antioxidant, anti-inflammatory and antilithiatic activity of their extracts using in vitro methods. The chemical composition was determined by histochemical, phytochemical and TLC tests. In addition, the profile of phenolic compounds was determined by HPLC-MS. The presence of secondary metabolites of recognized activity was demonstrated. The results of the HPLC-MS/MS allowed the tentative identification of catechin, luteolin and glycosides of eriodictyol, naringenin and luteolin in the extract of Lophophytum leandriand eriodictyol, naringenin, luteolin and their glycosylated derivatives in Lophophytum mirabile. The extracts showed promising antioxidant (DPPH, ABTS and ß-carotene-linoleic acid), anti-inflammatory (inhibition of 5-LOX) and anti-urolytic (by bioautographic TLC) activity. It is noteworthy that these are the first results of the phytochemical composition and biological activity of L. mirabile. However, in vivo studies are required to corroborate these activities.

Las especies de Lophophytumson holoparásitas que crecen en raíces de árboles. Los objetivos del trabajo fueron explorar la composición química del túber de dos especies de Lophophytum y analizar la actividad antioxidante, antiinflamatoria y antilitiásica de sus extractos usando métodos in vitro. La composición química se determinó mediante pruebas histoquímicas, fitoquímicas y por TLC. Además, se determinó el perfil de compuestos fenólicos por HPLC-MS/MS. Se demostró presencia de metabolitos secundarios de reconocida actividad. Los resultados del HPLC-MS/MS permitieron identificar tentativamente catequina, luteolina y glucósidos de eriodictiol, naringenina y luteolina en el extracto de Lophophytum leandriy eriodictiol, naringenina, luteolina y sus derivados glicosilados en Lophophytum mirabile. Los extractos mostraron prometedora actividad antioxidante (DPPH, ABTS y ß-caroteno-ácido linoleico), antiinflammatoria (inhibición de la 5-LOX) y antiurolitiásica (por TLC bioautográfica). Es de destacar que estos son los primeros resultados de composición fitoquímica y actividad biológica de L. mirabile. Sin embargo, se requieren estudios in vivo para corroborar dichas actividades.

Improved performance of naringenin herbosomes over naringenin in streptozotocin-induced diabetic rats: In vitro and in vivo evaluation

Objective: To prepare naringenin herbosome and evaluate its antidiabetic activity. Methods: Herbosomes were prepared by the solvent evaporation method. In vitro parameters like particle size, polydispersity index, zeta potential, and entrapment efficiency were estimated and in vitro diffusion study was performed. The in vivo studies were also performed in streptozotocin-induced diabetic male Sprague Dawley rats to evaluate blood glucose, total cholesterol, triglyceride, blood urea nitrogen, total protein, albumin level, aspartate aminotransferase, and alanine aminotransferase levels. Results: The optimized herbosome batch showed a particle size of 564.4 nm, a polydispersity index of 0.412, and zeta potential of-39.3 mV. The percentage entrapment of this formulation was 84.04%, with complete drug release within 8 h. Treatment of diabetic rats with naringenin herbosomes for 28 d significantly reduced the elevated level of plasma glucose as compared to plain naringenin. In biochemical parameters, the treatment showed a significant decrease in total cholesterol, triglyceride, and blood urea nitrogen; while elevated levels of aspartate aminotransferase and alanine aminotransferase were returned to normal. Pure naringenin and herbosome formulation at high dose increased the total protein whereas albumin level significantly increased in naringenin herbosomes at the highest dose but not in the pure naringenin treatment group. Conclusions: Naringenin herbosomes could improve the metabolic profile of diabetic rats, indicating enhanced antidiabetic activity of herbosome formulation.

Naringenin promotes cell autophagy to improve high-fat-diet-induced atherosclerosis in ApoE-/- mice

Naringenin (NAR) is a major flavanone in citrus fruits that has multiple pharmacological attributes such as anticancer and antiatherogenic. This study aims to investigate the mechanism of NAR in high-fat-diet (HFD)-induced atherosclerosis (AS) in apolipoprotein E-knockout (ApoE-/-) mice. A HFD-induced AS ApoE-/- mouse model was established. The mice were treated with HFD, different doses of NAR and simvastatin (Simv). After drug treatment, the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), superoxide dismutase (SOD), and alanine aminotransferase (ALT) were determined. The expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was detected using qRT-PCR and enzyme-linked immunosorbent assay. The plaque area of the aorta of AS mice was determined using oil red O staining. Western blot analysis was applied to measure the levels of autophagy-related proteins [protein 1 light chain 3B (LC3B), beclin 1, and p62]. The TC, TG, LDL-C, TNF-α, ALT, and MDA levels were significantly increased while the HDL-C, SOD, and GSH-Px levels were decreased in the HFD-induced AS ApoE-/- mice. NAR treatment reversed the expression of the above indicators in mice. After they were treated with different doses of NAR, the LC3B and beclin 1 levels were improved while the p62 protein level was decreased. This study suggested that NAR could promote cell autophagy to improve HFD-induced AS in ApoE-/- mice.

Naringenin protects ischemia-reperfusion-induced acute kidney injury by nuclear factor κB / 中华肾脏病杂志

Objective:To explore the effect and involved mechanism of naringenin on acute kidney injury (AKI) induced by ischemia-reperfusion (IR).Methods:The IR-AKI rat model was constructed using the classic bilateral renal pedicle clamping method, then renal function and pathological change were assessed, as well as inflammation-associated genes were detected by quantitative real-time PCR. The hub genes were selected through differential gene analysis and protein-protein interaction network analysis, and their transcription factors were predicted, which constructed a protein library together. The proteins binding to naringenin were selected by reverse molecular docking analysis and further their binding patterns were predicted to explore the mechanism of naringenin. Finally, the results of bioinformatics were verified by experimental methods.Results:Compared with the AKI group, the kidney pathology of the rats in the naringenin pretreatment group was significantly improved, and the renal tubular injury score was reduced ( P<0.01); meanwhile the serum creatinine level and the mRNA expression of the kidney injury molecule 1 (KIM-1) were significantly decreased (both P<0.05). Compared to sham group, IR-AKI increased the level of nuclear factor κB (NF-κB), tumor necrosis factor-α and interleukin-1β (all P<0.05), which reversed by naringenin indicated that naringenin inhibited inflammation in IR-AKI. Differential gene analysis was performed on the GSE98622 data set, and 359 differential genes were obtained. In reverse molecular docking, the proteins with smallest binding energy including NFKBIA, BCL3, NFKB2 and RELA were considered to be related to the preventive effect of naringenin, which were mainly enriched in NF-κB-related inflammation pathways. Domain functional analysis of NF-κB-related genes showed that naringenin could stably bind to its key domain. According to quantitative real-time PCR results, naringenin increased BCL3 level after AKI ( P<0.05), and further decreased the expression level of RELA and NFKB2 (both P<0.05). Conclusion:Naringenin protects IR-AKI by alleviating inflammation, and its mechanism is related to increasing BCL3 and thereby inhibiting the NF-κB pathway.

Chemical constituents from Pogonatum inflexum / 中草药

Objective: To investigate the chemical constituents from Pogonatum inflexum. Methods: The constituents were separated by column chromatographic methods of silica gel, Sephadex LH-20 column chromatography and liquid phase preparation, and the structure of the compounds was identified by comparing the physicochemical properties of the compounds with the spectral data. Results: A total of 16 compounds were obtained from P. inflexum, which were identified as tricin (1), irisflorentin (2), 3,5,4'-trihydroxy-7,3'-dimethoxyflavone (3), 3,5,3'-trihydroxy-7,4'-dimethoxyflavanone (4), 5,2'-dihydroxy-6,7-methylenedioxy- flavanone (5), 5,2',3'-trihydroxy-6,7-methylenedioxyflavanone (6), apigenin (7), kaempferol (8), kaempferide (9), naringenin (10), quercetin (11), baicalein (12), luteolin (13), protocatechuic aldehyde (14), 4-hydroxy-3-methoxy-benzaldehyde (15), and 2-hydroxy-5-(2-hydroxy-4- methoxybenzyl)-4-methoxybenzaldehyde (16). Conclusion: All compounds are isolated from the genus Pogonatum for the first time, among them, dihydroflavones may be the characteristic components of this genus.

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.

Identification of flavonoids 3-hydroxylase from Silybum marianum (L.) Gaertn and its application in enhanced production of taxifolin / 生物工程学报

(2S)-taxifolin is an important flavonoid that has anti-inflammatory and anti-oxidation effects. It is widely used in pharmaceutical and nutraceutical industries. Flavone 3-hydroxylase (F3H) can catalyze the synthesis of (2S)-taxifolin and other 3-hydroxylated flavonoids from (2S)-eriodictyol. Due to the low catalytic efficiency of F3H, the titer of many 3-hydroxyflavones, such as taxifolin, synthesized by microbial method is relatively low. In this study, a SmF3H was identified from the transcriptome of Silybum marianum (L.) Gaertn. The results of fermentation showed that SmF3H can catalyze the flavone 3-hydroxylation reaction, and its catalytic efficiency was significantly higher than that of commonly used SlF3H from Solanum lycopersicum. Six promoters with different transcription strength were selected to optimize the synthesis pathway from the flavonoid precursor (2S)-naringenin to (2S)-taxifolin. The results showed that the highest titer of (2S)-taxifolin (695.90 mg/L in shake flask) could be obtained when the P(GAL7) promoter was used to control the expression of SmF3H. The titer of (2S)-taxifolin was further improved to 3.54 g/L in a 5-L fermenter, which is the highest titer according to current available literatures.

Effect of Naringenin on Oxidative Stress and Tau Protein Phosphorylation of Aβ25-35-induced PC12 Cell Injury / 中国实验方剂学杂志

Objective:To investigate the effects of naringenin on oxidative stress and Tau protein phosphorylation of adrenal pheochromocytoma（PC12） cells injured by β-amyloid（Aβ）25-35 and its relationship with estrogen receptor（ER） and phosphatidylinositol -3 kinase/protein kinase B（PI3K/Akt） signaling pathway. Method:The PC12 cells were intervened with Aβ25-35 to prepare the injury model. The experiment was divided into blank group， model group， naringenin（400，40，4，0.4，0.04，4×10-3，4×10-4，4×10-5 μmol·L-1）group， positive drugs estradiol（E2）（1 nmol·L-1）+Aβ25-35 group， naringenin（0.4，0.04，4×10-3，4×10-4，4×10-5 μmol·L-1）+Aβ25-35 group， E2+Aβ25-35+ER antagonist（ICI182780）（1 μmol·L-1） group， naringenin+Aβ25-35+ICI182780 group， E2+Aβ25-35+PI3K blocker（LY294002）（50 μmol·L-1） group， naringenin+Aβ25-35+LY294002 group. Methye thiazolye telrazlium（MTT）method was used to detect the cell proliferation index， 2'，7'-Dichlorodi -hydrofluorescein diacetate（DCFH-DA） was used as a fluorescent probe to detect the content of reactive osygen species（ROS）， the content of malondialdehyde（MDA） and the activity of superoxide dismutase（SOD） were measured by thiobarbituric acid（TBA） and oxidase methods， Western blot was used to detect the expression of phosphorylated Tau protein/total Tau protein（p-Tau/t-Tau）. Result:According to the results of MTT experiment， 0.4 μmol·L-1 was selected as the best effective concentration of naringenin， compared with the blank group， the cell proliferation index of model group decreased significantly （P<0.01）， compared with model group， the cell proliferation index of naringenin+Aβ25-35 group increased significantly （P<0.01）. In addition， compared with blank group， the content of ROS， MDA and the expression of p-Tau/t-Tau in the model group increased significantly （P<0.01）， and the activity of SOD decreased significantly （P<0.01）， compared with model group， the content of ROS， MDA and the expression of p-Tau/t-Tau in naringenin+Aβ25-35 group decreased significantly （P<0.01）， and the activity of SOD increased significantly （P<0.01）， compared with naringenin+Aβ25-35 group， the addition of ICI182780 and LY294002 significantly reversed the role of naringenin in the above indicators （P<0.01）. The effect of naringenin was similar to that of E2. Conclusion:Naringenin can improve the cell proliferation index and protect PC12 cells from Aβ25-35 injury， which may be achieved by activating ER and PI3K/Akt signaling pathway to reduce ROS， MDA content， p-Tau/t-Tau expression and promote SOD activity.

Preparation and Characterization of Naringenin Nicotinamide Co-crystal / 中国药学杂志

OBJECTIVE: To prepare a novel naringenin co-crystal to improve the poor water solubility of the compound. METHODS: Solvent volatilization method was used to prepare naringenin nicotinamide co-crystal, with ethyl acetate as solvent. It is characterized by the technology of infrared spectroscopy (IR), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), scanning electron microscopy (SEM) and other techniques. RESULTS: By measuring the equilibrium solubility of different pH solutions, it was found that the naringenin nicotinamide co-crystal has a significantly improved equilibrium solubility compared with naringenin. CONCLUSION: This method can improve the solubility of naringenin, which is simple and easy, and lays the foundation for further study of naringenin co-crystal.

Studies of flavonoids from Glycyrrhizae Radix et Rhizoma / 中草药

Objective: To study the chemical constituents of flavonoids from Glycyrrhizae Radix et Rhizoma. Methods: The compounds were isolated and purified by column chromatography over HP-20 macroporous resin, silica gel, Sephadex LH-20, and preparative RP-HPLC. Their structures were elucidated by physicochemical properties and spectral analyses. Results: Ten flavonoids were isolated and identified as 4’,6,7-trihydroxy-2’-methoxyl-chalcone (1), 3’,4’,5,7-tetrahydroxy-8-(3-hydroxy-3- methylbutyl)-isoflavone (2), isoliquiritigenin (3), isoliquiritin (4), echinatin (5), orobol (6), ononin (7), 2(S)-3’,5’,7-trihydroxy- flavanone (8), 2(S)-naringenin-4’-O-β-D-glucopyranoside (9), and 4’,7-dihydroxyflavone (10). Conclusion: Compounds 1 and 2 are new compounds named isolicochalcone B and licoisoflavone G, while compound 9 is isolated from the genus for the first time.

Content Comparison of Mannose,Glucose and Narinhenin in Dendrobium officinale and Dendrobium huoshanense / 中国实验方剂学杂志

Objective: To optimize the pre-column derivation high performance liquid chromatography (HPLC) content determination method of D-mannose and D-glucose as well as the content determination method of narinhenin in Dendrobium officinale and D. huoshanense, and compare the contents of D-mannose,D-glucose and narinhenin between D. officinale and D. huoshanense. Method: A pre-column derivation HPLC method modified by Chinese Pharmacopoeia(Ch.P) 2015 was used to simultaneously determine the contents of D-mannose and D-glucose,with acetonitrile-0.02 mol·L-1 ammonium acetate solution as mobile phase for gradient elution. Kromasil 100-5 C18 was performed with the wavelength set at 250 nm,and the flow rate was 1 mL·min-1;column temperature was 30℃. HPLC content determination of narinhenin was performed on Kromasil 100-5 C18 with the acetonitrile-methanol-0.4% phosphoric acid solution as mobile phase for gradient elution,and the wavelength was set at 290 nm; the flow rate was 0.8 mL·min-1,and column temperature was 40℃. Result: D-mannose and D-glucose showed a good linear relationship within the range of 0.15-3.0 μg and 0.075-2.25 μg (r=0.999 9); and their average recoveries were 99.01% (RSD 2.1%) and 101.69% (RSD 2.0%) respectively. In addition, the other methodological researches such as repeatability and durability all met the requirements. The contents of D-mannose(Cm),D-glucose(Cg) and sum of them (Cm+Cg) were 12.75%-36.40%,2.93%-18.39% and 19.23%-54.58% in 43 batch of D. officinale. Almost all of the results except very few samples reached the D-mannose standard in Ch.P 2015, and the total content of D-mannose and D-glucose was also up to the total polysccharide standard in Ch.P. The correlation between content and origin was not significant. The contents of D-mannose(Cm),D-glucose(Cg) and sum of them (Cm+Cg) were 14.33%-29.47%,6.64%-15.20%,and 25.73%-44.37% in 12 batch of D. huoshanense. These contents and ratio of peak areas of D-mannose to D-glucose (Am/Ag) were within the scope of D. officinale's; in addition, their average contents were basically the same with those in D. officinale (about 33%).Next,naringenin showed a good linear relationship within the range of 0.020 8-0.832 0 μg (r=0.999 9),and its average recovery was 101.96% (RSD 1.8%). The content of naringenin was 0.053 2-0.122 4 mg·g-1 (average value of 0.081 0 mg·g-1) in 11 batch of D. officinale, slightly higher than 0.040 3-0.090 0 mg ·g-1 (average value of 0.068 3 mg ·g-1) in 7 batch of D. huoshanense. All of these results of narinfenin did not reach the content lower limit in Ch.P. Conclusion: The method used to determinate the content of D-mannose and D-glucose is reproducible, and their sum content is possible to substitute the total polysccaride determination (with higher errors) in D. officinale; monosaccharide content determination can be used for quantitative quality control of D. huoshanense. However, it could not distinguish D. officinale and D. huoshanense by determining the contents of polysccharide,D-glucose,D-mannose and narinhenin, and shall be combined with other specificity methods for further identification.

Quantitative effect of the expression level of key genes in naringenin synthesis on the accumulation level of target products / 生物工程学报

Naringenin is a natural flavonoid compound with anti-inflammatory, anti-oxidation, anti-viral, anti-atherosclerosis and other pharmacological activities. It is also an important precursor of other flavonoid synthesis and with great value of application. At present, the production of flavonoids such as naringenin by microbial methods has a low yield due to imbalance of metabolic pathways, which greatly limits its industrial application. In this study, a naringenin-producing strain of Saccharomyces cerevisiae Y-01 was used in the research object. The expression levels of 4-coumaric acid: CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI) were controlled by promoter and copy numbers to investigate the quantitative effect of key enzyme expression level on the accumulation level of target products. The results showed that the correlation between naringenin production and 4CL or CHI expression was not significant while there was a positive correlation with the expression level of CHS. Strain Y-04 with high yield of naringenin was obtained by regulating the expression level of chs gene, and the yield was increased by 4.1-folds compared with the original strain Y-01. This study indicated that CHS is a key regulatory target of naringenin synthesis. Rational regulation of CHS expression can significantly promote the accumulation of naringenin. The related results provide an important theoretical reference for the use of metabolic engineering to strengthen microbial synthesis of important flavonoids such as naringenin.

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.

Naringenin ameliorates renal impairment by increasing 20-HETE in diabetic mice / 中国药理学通报

Aim: To investigate the ameliorative effects of naringenin on renal injury and its roles in 20-HETE in diabetic mice. Methods: A diabetic model was established by long-term high-energy feeding combined with streptozotocin. The structural and functional changes of kidney were evaluated by renal pathological examination and blood urea nitrogen(BUN), serum creatinine(SCr), urinary albumin levels. The expression of CYP4A protein was detected by Western blot. The level of 20-HETE was measured by ELISA kit. Results: After four weeks of high-energy feeding, streptozotocin (40 mg. kg-1. d-1, IP) was given for five days. Then after seven days, fasting blood glucose of mice exceeded 11.1 mmol L-1, which suggested the establishment of diabetic model. After four weeks of diabetic onset, the levels of BUN, SCr, urinary albumin increased significantly(P <0. 01); kidney index(renal weight/body weight) and glomerular volume were raised(P <0. 01); increased collagen fiber and thickened basement membrane were observed in diabetic mice. Meanwhile, the expression of CYP4A protein in the kidney and the content of 20-HETE in serum decreased in model group (P < 0. 01). With naringenin supplementation(25 or 75 mg · kg-1 · d-1) for four weeks, both the structure and function of kidney were improved in diabetic mice, with the up-regulation of CYP4A protein expression and the increase of 20- HETE level(P < 0. 05). Conclusion: Naringenin can ameliorate renal injury in diabetic mice, which may be related to activating CYP4A and increasing 20-HETE.