Luteoloside mitigates premature age-related macular degeneration by suppressing p53-p21-Rb1 axis: Insights from transcriptomic analysis, serum metabolomics and gut microbiota analysis.

Transcriptomics of dry age-related macular degeneration (AMD) patients with premature aging revealed the upregulated pathways involved in glycerolipid metabolism, tyrosine metabolism, and pentose and glucuronate interconversion. To investigate natural strategies for modulating these implicated pathways, we examined the impact and underlying mechanism of luteoloside on premature AMD using a stress-induced premature senescence (SIPS)-associated AMD animal model in middle-aged mice that mimicked the dysregulated pathways observed in dry AMD patients with premature aging. Luteoloside supplementation resulted in a significant reduction in serum levels of the pro-inflammatory cytokine IL-1ß and lipofuscin, along with increased serum activity of the antioxidant enzyme superoxide dismutase (SOD) and elevated levels of pigment epithelium-derived factor (PEDF), and preserved retinal thickness and structure in AMD mice. Furthermore, luteoloside supplementation effectively reversed the abnormal serum levels of metabolites, particularly by reducing harmful lysophosphatidylcholine (LysoPC) and increasing beneficial 4-guanidinobutanoic acid. In addition to its impact on metabolites, luteoloside modulated the composition of gut microbiota, promoting the enrichment of beneficial bacterial populations, including Lactobacillus, while reducing the abundance of harmful bacterial populations, including Bacteroides. Overall, our findings highlight the potential of luteoloside supplementation in regulating the dysregulated intestinal microbiota and metabolites in premature AMD, thereby reducing ocular levels of senescence-associated secretory phenotype (SASP) factors through the suppression of the p53-p21-retinoblastoma protein 1 (Rb1) axis.

Based on Virtual Screening and Simulation Exploring the Mechanism of Plant-Derived Compounds with PINK1 to Postherpetic Neuralgia.

Accumulating evidence strongly supports that PINK1 mutation can mediate mitochondrial autophagy dysfunction in dopaminergic neurons. This study was conducted to determine the role of PINK1 in the pathogenesis of postherpetic neuralgia (PHN) and find new targets for its treatment. A rigorous literature review was conducted to identify 2801 compounds from more than 200 plants in Asia. Virtual screening was used to shortlist the compounds into 20 groups based on their binding energies. MM/PBSA was used to further screen the compound dataset, and vitexin, luteoloside, and 2'-deoxyadenosine-5'-monophosphate were found to have a score of - 59.439, - 52.421, and - 47.544 kcal/mol, respectively. Pain behavioral quantification, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, western blotting, and transmission electron microscopy were used to confirm the effective mechanism. Vitexin had the most significant therapeutic effect on rats with PHN followed by luteoloside; 2'-deoxyadenosine-5'-monophosphate had no significant effect. Our findings suggested that vitexin could alleviate PHN by regulating mitochondrial autophagy through PINK1.

Luteoloside inhibits Aß1-42 fibrillogenesis, disintegrates preformed fibrils, and alleviates amyloid-induced cytotoxicity.

Abnormal aggregation and fibrillogenesis of amyloid-ß protein (Aß) can cause Alzheimer's disease (AD). Thus, the discovery of effective drugs that inhibit Aß fibrillogenesis in the brain is crucial for the treatment of AD. Luteoloside, as one of the polyphenolic compounds, is found to have a certain therapeutic effect on nervous system diseases. However, it remains unknown whether luteoloside is a potential drug for treating AD by modulating Aß aggregation pathway. In this study, we performed diverse biophysical and biochemical methods to explore the inhibition of luteoloside on Aß1-42 which is linked to AD. The results demonstrated that luteoloside efficiently prevented amyloid oligomerization and cross-ß-sheet formation, reduced the rate of amyloid growth and the length of amyloid fibrils in a dose-dependent manner. Moreover, luteoloside was able to influence aggregation and conformation of Aß1-42 during different fiber-forming phases, and it could disintegrate already preformed fibrils of Aß1-42 and convert them into nontoxic aggregates. Furthermore, luteoloside protected cells from amyloid-induced cytotoxicity and hemolysis, and attenuated the level of reactive oxygen species (ROS). The molecular docking study showed that luteoloside interacted with Aß1-42 mainly via Conventional Hydrogen Bond, Carbon Hydrogen Bond, Pi-Pi T-shaped, Pi-Alkyl and Pi-Anion, thereby possibly preventing it from forming the aggregates. These observations indicate that luteoloside, a natural anti-oxidant molecule, may be applicable as an effective inhibitor of Aß, and promote further exploration of the therapeutic strategy against AD.

Exploring the pharmacological mechanisms and key active ingredients of total flavonoids from Lamiophlomis rotata (Benth.) Kudo against rheumatoid arthritis based on multi-technology integrated network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Lamiophlomis rotata (Benth.) Kudo (LR, Lamiaceae) is a traditional Tibetan medicinal material in China. Tibetan medicine classic and research report suggested that LR could be used to cure rheumatoid arthritis (RA). However, the anti-RA active ingredients and pharmacological mechanisms of LR have not been elucidated. AIM OF THE STUDY: To explore the mechanisms and key active ingredients of total flavonoids from LR (TFLR) against RA. MATERIALS AND METHODS: First, the mechanisms of TFLR against RA were investigated on collagen-induced arthritis (CIA) rat model by analyzing paw appearance, paw swelling, arthritis score, spleen index, thymus index, inflammatory cytokine (TNF-α, IL-1ß, IL-6 and IL-17) levels in serum, histopathology of ankle joint and synovium from knee joint (hematoxylin-eosin, safranin O-fast green and DAB-TUNEL staining), and apoptosis-related protein (PI3K, Akt1, p-Akt, Bad, p-Bad, Bcl-xL and Bcl-2) levels in the synovium of ankle joints (Western blot). Then, the crucially active ingredients of TFLR against RA were explored by network pharmacology, ingredient analysis, in vitro metabolism and TNF-α-induced human RA synovial fibroblast MH7A proliferation assays. Network pharmacology was applied to predict the key active ingredients of TFLR against RA. The ingredient analysis and in vitro metabolism of TFLR were performed on HPLC, and MH7A proliferation assay were applied to evaluate the predicted results of network pharmacology. RESULTS: TFLR shown excellently anti-RA effect by reducing paw swelling, arthritis score, spleen index, thymus index and inflammatory cytokine (IL-1ß, IL-6 and IL-17) levels, and improving the histopathological changes of ankle joint and synovium from knee joint in CIA rats. Results of Western blot indicated that TFLR reversed the changes of PI3K, p-Akt, p-Bad, Bcl-xL and Bcl-2 levels in the ankle joint synovium of CIA rats. Results of network pharmacology exhibited that luteolin was identified as the pivotal active ingredient of TFLR against RA. The ingredient analysis of TFLR indicated that the main ingredient in TFLR was luteoloside. The in vitro metabolism study of TFLR suggested that luteoloside could be converted to luteolin in artificial gastric juice and intestinal juice. Results of MH7A proliferation assay showed that there was no significant difference between TFLR and equal luteoloside on the viability of MH7A cells, indicating that luteoloside was the key active ingredient of TFLR against RA. Additionally, the luteolin (same mol as luteoloside) showed better inhibitory effect on the viability of MH7A cells than luteoloside. CONCLUSION: TFLR showed anti-RA effect, and the mechanism was related to promoting synovial cell apoptosis mediated by PI3K/Akt/Bad pathway. Meanwhile, this work indicated that luteoloside was the key active ingredient of TFLR against RA. This work lays a foundation for providing TFLR product with clear mechanism and stable quality to treat RA.

Luteoloside Induces G0/G1 Phase Arrest of Neuroblastoma Cells by Targeting p38 MAPK.

Luteoloside has shown anti-inflammatory, antiviral, and antitumor properties. However, the effect and mechanism of luteoloside on neuroblastoma cells remain unknown. The proliferation of human neuroblastoma cells (SH-SY5Y and SK-N-AS) treated with different concentrations of luteoloside (0, 12.5, 25, and 50 µM) was detected by the MTT assay and colony formation assay. Cell apoptosis and cell cycle were examined by Hoechst staining and flow cytometry. A subcutaneous tumorigenesis model was established in nude mice to evaluate the effect of luteoloside on tumor growth in vivo. Bioinformatics, molecular docking techniques, and cellular thermal shift assays were utilized to predict the potential targets of luteoloside in neuroblastoma. The p38 MAPK inhibitor SB203580 was used to confirm the role of p38 MAPK. Luteoloside inhibited the proliferation of neuroblastoma cells in vitro and in vivo. Luteoloside slightly induced cellular G0/G1 phase arrest and reduced the expression levels of G0/G1 phase-related genes and the proteins cyclin D1, CDK4, and C-myc, which are downregulated by p38 MAPK pathways. Meanwhile, p38 was identified as the target of luteoloside, and inhibition of p38 MAPK reversed the inhibitory effect of luteoloside on neuroblastoma cells. Luteoloside is a potential anticancer drug for treating neuroblastoma by activating p38 MAPK.

Luteoloside Prevents Sevoflurane-induced Cognitive Dysfunction in Aged Rats via Maintaining Mitochondrial Function and Dynamics in Hippocampal Neurons.

Postoperative cognitive dysfunction (POCD) is characterized by impaired cognitive function, such as decreased learning and memory after anesthesia and surgery. This study aimed to explore the effect of luteoloside, a flavonoid extracted from natural herbs, on sevoflurane-induced cognitive dysfunction. Aged Sprague-Dawley male rats (20 months old) were treated with luteoloside for 7 days prior to sevoflurane exposure. After evaluation using an open field, novel object recognition, and Y-maze tests, it was determined that luteoloside effectively prevented sevoflurane-induced cognitive dysfunction. Sevoflurane exposure led to hippocampal neuron apoptosis in vivo (n = 6) and in vitro (n = 3), while this injury was prevented by luteoloside in a dose-dependent manner. Mechanistically, luteoloside maintained mitochondrial function and dynamics, as evidenced by the restored adenosine triphosphate (ATP) production and mitochondrial membrane potential as well as the upregulated levels of mitochondrial fission (optic atrophy protein 1 (Opa1) and mitofusin1 (Mfn1)) and downregulated mitochondrial fusion (mitochondrial fission 1 (Fisl) and dynamin-related protein 1 (Drp1)) factors. Notably, silencing Opa1 blocked the protective effect of luteoloside on hippocampal neurons and mitochondrial function. In summary, luteoloside prevented sevoflurane-induced cognitive dysfunction in aged rats, which may be achieved by regulating mitochondrial dynamics. Our study reveals the potential of luteoloside in preventing POCD in aged patients.

Luteoloside pretreatment attenuates anoxia-induced damage in cardiomyocytes by regulating autophagy mediated by 14-3-3η and the AMPKα-mTOR/ULK1 pathway.

The relation between ischemia and heart failure is well demonstrated, and several studies suggested that realizing the physiological role of autophagy will be of great importance. Luteoloside (Lut) is one of the main components of Lonicera japonica flos and exhibits antioxidant, anti-inflammatory, and cardioprotective properties. To determine if Lut pretreatment enhanced autophagy by 14-3-3η expression and the AMPKα-mTOR/ULK1 pathway and protected the neonatal rat cardiomyocytes (NRCMs) against anoxia damage, NRCMs were treated using 20 µM Lut for 36 h, and the anoxia damage model was established using NRCMs. The indexes reflecting the condition of NRCMs, oxidative stress level, and mitochondrial function were evaluated. In addition, the expression and phosphorylation of 14-3-3η and AMPKα/mTOR/ULK1, and autophagy markers (LC3II, P62) and the abundance of autophagy lysosomes were detected. Results revealed that Lut pretreatment alleviated anoxia- induced damage in NRCMs, that is, Lut pretreatment could increase cell viability, decrease LDH activity and apoptosis, suppressed ROS generation and oxidative stress, restored intracellular ATP levels, stabilized MMP levels, and inhibited mPTP opening. Furthermore, Lut pretreatment could enhance autophagy via upregulating 14-3-3η, LC3II expression and increasing p-AMPKα/AMPKα and p-ULK1/ULK1 level, whereas P62 expression and p-mTOR/mTOR level decreased; the fluorescence intensity of autolysosomes also increased. However, in the NRCMs treated with pAD/14-3-3η RNAi or incubated with 3-MA (an autophagy inhibitor), the abovementioned effects of Lut pretreatment were reduced. Taken together, Lut pretreatment could enhance autophagy by upregulating 14-3-3η expression to influence the AMPKα-mTOR/ ULK1 pathway against anoxia-induced damage in NRCMs.

The hypolipidemic mechanism of chrysanthemum flavonoids and its main components, luteolin and luteoloside, based on the gene expression profile.

In this study, the following four groups of mice with hyperlipidemia were involved: the model control group (MC), the Chrysanthemum flavonoids group (CF), the luteolin group, and the luteoloside group. The whole gene expression profile was detected in the liver tissues of each group. Differential genes significantly enriched in the biological process of gene ontology (GO) items and Kyoto Encyclopedia of Genes and Genomes (KEGG) were selected, and 4 differential genes related to lipid metabolism were selected for further real-time quantitative PCR verification. Compared with the MC, 41 differential genes such as Sqle, Gck, and Idi1 were screened in the CF intervention group; 68 differential genes such as Acsl3, Cyp7a1, and Lpin1 were screened in the luteolin intervention group (CF); and 51 differential genes such as Acaca, Cyp7a1, and Lpin1 were screened in the luteoloside group. The mechanism of CF to improve hyperlipidemia is very complex, mainly involving biological processes such as cholesterol and fatty acid metabolism and glycolysis, luteolin mainly involves the synthesis and transport of cholesterol, and luteoloside mainly involves fatty acid metabolism. The functional pathways of CF may not be completely the same as luteolin and luteoloside, and further study is needed on the mechanism of action of other components.

Study on the interaction mechanism between luteoloside and xanthine oxidase by multi-spectroscopic and molecular docking methods.

Gout is an inflammatory joint disease caused by urate crystal deposition, which is associated with hyperuricemia. Gout will take place when the uric acid accumulates. Xanthine oxidase (XO) is a crucial enzyme in the formation of uric acid. Inhibiting XO is one of the means to ameliorate gout. Luteoloside is a kind of natural flavonoid, which has an excellent prospect for relieving gout. But there are few reports on the interaction mechanism between luteoloside and XO currently. In this study, the interaction mechanism between luteoloside and XO was explored using spectroscopy and molecular docking. The fluorescence spectroscopy results indicated that luteoloside could make the intrinsic fluorescence of XO quenched, and the binding constant between luteoloside and XO was (1.85 ± 0.22) × 103 L mol-1 at 298 K. The synchronous fluorescence spectroscopy results showed that the absorption peaks of Tyr and Trp shifted blue, and the hydrophobicity of the microenvironment increased. Moreover, CD spectra showed that α-helix of XO decreased, ß-sheet and ß-turn increased after adding luteoloside. The results of molecular docking analysis showed that XO could combine with luteoloside through hydrogen bonds and hydrophobic force. The results indicated that luteoloside could remarkably interact with XO. Insights into the interaction mechanism provide a necessary basis for the search for low-toxic natural products as targets of XO. HIGHLIGHTS: Luteoloside and xanthine oxidase was a strong binding mode and had only one binding site. Luteoloside could cause α-helix reduced, ß-sheet and ß-turn increased, and change the secondary structure of XO. The binding between luteoloside and xanthine oxidase was a spontaneous process. The main binding force was hydrophobic force between luteoloside and xanthine oxidase.

Luteoloside protects the vascular endothelium against iron overload injury via the ROS/ADMA/DDAH II/eNOS/NO pathway.

Iron overload injury is considered to be a part of blood stasis syndrome of arthralgia in traditional Chinese medicine. Its primary therapies include clearing heat and detoxification, activating blood circulation, and removing blood stasis. Lonicera japonica flos (LJF) has long been known as an excellent antipyretic and antidote. Luteoloside (Lut) is one of the main components of LJF and exhibits antioxidant, anti-inflammatory, and cytoprotective properties. However, the protection of Lut against iron overload injury and its underlying mechanisms remain unclear. Therefore, HUVECs were exposed to 50 µmol·L-1 iron dextran for 48 h to establish an iron overload damage model and the effects of Lut were assessed. Our results showed that 20 µmol·L-1 Lut not only increased cell viability and weakened LDH activity, but also significantly up-regulated DDAHⅡ expression and activity, increased p-eNOS/eNOS ratio and NO content, and reduced ADMA content in HUVECs exposed to iron overload. Furthermore, Lut significantly attenuated intracellular/mitochondrial ROS generation, improved SOD, CAT, and GSH-Px activities, reduced MDA content, maintained MMP, inhibited mPTP opening, prevented cyt c from mitochondria released into cytoplasm, reduced cleaved-caspase3 expression, and ultimately decreased cell apoptosis induced by iron overload. The effects of Lut were similar to those of L-arginine (an ADMA competitive substrate), cyclosporin A (a mPTP blocker agent), and edaravone (a free radical scavenger) as positive controls. However, addition of pAD/DDAH II-shRNA adenovirus reversed the above beneficial effects of Lut. In conclusion, Lut can protect HUVECs against iron overload injury via the ROS/ADMA/DDAH II/eNOS/NO pathway. The mitochondria are the target organelles of Lut's protective effects.

Luteoloside protects the vascular endothelium against iron overload injury via the ROS/ADMA/DDAH II/eNOS/NO pathway / 中国天然药物

Iron overload injury is considered to be a part of blood stasis syndrome of arthralgia in traditional Chinese medicine. Its primary therapies include clearing heat and detoxification, activating blood circulation, and removing blood stasis. Lonicera japonica flos (LJF) has long been known as an excellent antipyretic and antidote. Luteoloside (Lut) is one of the main components of LJF and exhibits antioxidant, anti-inflammatory, and cytoprotective properties. However, the protection of Lut against iron overload injury and its underlying mechanisms remain unclear. Therefore, HUVECs were exposed to 50 μmol·L-1 iron dextran for 48 h to establish an iron overload damage model and the effects of Lut were assessed. Our results showed that 20 μmol·L-1 Lut not only increased cell viability and weakened LDH activity, but also significantly up-regulated DDAHⅡ expression and activity, increased p-eNOS/eNOS ratio and NO content, and reduced ADMA content in HUVECs exposed to iron overload. Furthermore, Lut significantly attenuated intracellular/mitochondrial ROS generation, improved SOD, CAT, and GSH-Px activities, reduced MDA content, maintained MMP, inhibited mPTP opening, prevented cyt c from mitochondria released into cytoplasm, reduced cleaved-caspase3 expression, and ultimately decreased cell apoptosis induced by iron overload. The effects of Lut were similar to those of L-arginine (an ADMA competitive substrate), cyclosporin A (a mPTP blocker agent), and edaravone (a free radical scavenger) as positive controls. However, addition of pAD/DDAH II-shRNA adenovirus reversed the above beneficial effects of Lut. In conclusion, Lut can protect HUVECs against iron overload injury via the ROS/ADMA/DDAH II/eNOS/NO pathway. The mitochondria are the target organelles of Lut's protective effects.

Hypolipidemic Effects and Preliminary Mechanism of Chrysanthemum Flavonoids, Its Main Components Luteolin and Luteoloside in Hyperlipidemia Rats.

This study aimed to investigate the key constituents and preliminary mechanism for the hypolipidemic activity of chrysanthemum flavonoids. Hyperlipidemia (HPL) rats were divided into five groups: the model control group (MC); Chrysanthemum flavone intervention group (CF); luteolin intervention group; luteoloside intervention group and simvastatin intervention group. The body weight, organ coefficient, serum lipids, antioxidant activity, and lipid metabolism enzymes were detected. Hematoxylin and eosin (H&E) staining was used to observe the liver and adipose tissue. Chrysanthemum flavonoids, luteolin, and luteoloside can reduce the weight and levels of total cholesterol (TC), triglycerides (TG), and LDL-C, and increase the level of HDL-C in the blood and reduce liver steatosis. Indicators of liver function (AST, ALT, and ALP) improved. The antioxidant activity (GSH-Px, CAT, SOD) and enzymes associated with lipid catabolism (FAßO, CYP7A1, and HL) increased, while lipid peroxidation products (MDA) and enzymes associated with lipid synthesis (FAS, HMG-CoA, and DGAT) decreased. Chrysanthemum flavonoids had a better effect on the antioxidant level and lipid metabolism-related enzyme activity. There was no significant difference in the effects of the chrysanthemum flavonoids, luteolin, and Luteoloside on improving blood lipids and hepatic steatosis-mechanisms that may be related to antioxidant levels and regulating enzymes involved in the metabolism of fatty acids, cholesterol, and triglycerides in the liver. However, chrysanthemum flavonoids had a stronger antioxidant and lipid metabolism regulation ability, and the long-term effects may be better.

Active components from Lagotis brachystachya maintain uric acid homeostasis by inhibiting renal TLR4-NLRP3 signaling in hyperuricemic mice.

Lagotis brachystachya Maxim is a herb widely used in traditional Tibetan medicine. Our previous study indicated that total extracts from Lagotis brachystachya could lower uric acid levels. This study aimed to further elucidate the active components (luteolin, luteoloside and apigenin) isolated from Lagotis brachystachya and the underlying mechanism in vitro and in vivo. The results showed that treatment with luteolin and luteoloside reversed the reduction of organic anion transporter 1 (OAT1) levels, while apigenin attenuated the elevation of urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) levels in uric acid-treated HK-2 cells, which was consistent with the finding in the kidneys of potassium oxonate (PO)-induced mice. On the other hand, hepatic xanthine oxidase activity was inhibited by the components. In addition, all of these active components improved the morphology of the kidney in hyperuricemic mice. Moreover, molecular docking showed that luteolin, luteoloside and apigenin could bind Toll-like receptor 4 (TLR4) and NLR family pyrin domain containing 3 (NLRP3). Congruently, western blot analysis showed that the components inhibited TLR4/myeloid differentiation primary response 88 (MyD88)/NLRP3 signaling. In conclusion, these results indicated that luteolin, luteoloside and apigenin could attenuate hyperuricemia by decreasing the production and increasing the excretion of uric acid, which were mediated by inhibiting inflammatory signaling pathways.

Reduning injection and its effective constituent luteoloside protect against sepsis partly via inhibition of HMGB1/TLR4/NF-κB/MAPKs signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Reduning injection (RDN), a popular traditional Chinese medicine, formulated by three herbs (i.e., Artemisia carvifolia Buch.-Ham. ex Roxb., Lonicera japonica Thunb., and Gardenia jasminoides J. Ellis), has been widely used to treat upper respiratory infectious diseases in China. AIM OF THE STUDY: To investigate the protective effect of RDN on both lipopolysaccharides (LPS)- and cecal ligation and puncture (CLP)-induced septic mice. To identify the potentially effective constituent, and to determine its protective effect and underlying mechanism in vivo and in vitro. MATERIALS AND METHODS: Male C57BL/6 mice were used to establish septic model by tail intravenous injection of 4 mg/kg LPS or CLP surgery. After modeling, mice were administered by tail intravenous injection of RDN in the dose of 16 or 8 mL/kg/day. The mortality, histopathology, plasma levels of inflammatory cytokines were evaluated respectively. In addition, we screened the potentially effective substances of RDN against sepsis by detecting the nitric oxide (NO) production in LPS-stimulated Raw 264.7 cells and verified the effect of luteoloside in CLP-induced septic mice subsequently. Finally, the underlying mechanisms of RDN and luteoloside were investigated in the inflammatory model in vitro. RESULTS: Administration of RDN significantly reduced the mortality and increased the survival rate in both LPS- and CLP-induced septic mice. Meanwhile, RDN reduced the release of inflammatory cytokines accompanied by alleviating the organs damage of lung, liver, and kidney in CLP-induced septic mice. Moreover, several components from Gardenia jasminoides J. Ellis extract (ZZ) or Lonicera japonica Thunb and Artemisia carvifolia Buch.-Ham. ex Roxb extract (JQ) as well as the constituents of luteoloside, quercetin, and caffeic acid were screened out to have obvious anti-inflammatory activity, which may be the potentially effective substances of RDN against sepsis. We further verified the protective role of luteoloside in CLP-induced septic mice. In addition, RDN and luteoloside significantly inhibited both the secretion and translocation of mobility group box (HMGB)1, and HMGB1-mediated activation of TLR4/NF-κB/MAPKs signaling pathways. CONCLUSION: RDN and its effective constituent luteoloside exhibited a significant protective effect against sepsis, which were potential candidate drugs for treatment of sepsis. The mechanism of antisepsis partly was related to inhibition of HMGB1/TLR4/NF-κB/MAPKs signaling pathways. The results provide an evidence base for the follow-up clinical application of RDN in treatment of sepsis.

One step assay of the four plant ingredients in Yangxue Anshen syrup by HPLC method / 药学实践杂志

Objective To establish a HPLC method for simultaneous determination of quercitrin, luteoloside, rutin and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in Yangxue Anshen syrup. Methods Waters symmetry C18 column (250 mm×4.6 mm, 5 μm) was used with 0.1% acetic acid (A) and methanol (B) as the mobile phase. Gradient elution was performed at a flow rate of 1.0 ml/min, 0-15 min, 95%-90%A; 15-35 min, 90%-70%A; 35-55 min, 70%-60%A; 55-85 min, 60%-50%A; 85-95 min, 10%A. The detection wavelengths were 256 nm and 320 nm. Column temperature was 30 ℃ and the injection volume was 10 μl. Results Quercitrin, luteoloside, rutin and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside showed good linear relationship within the range of 10-300, 5.0-150.0, 5.0-150.0, 20.0-600.0 µg/ml(r≥0.9989), respectively. The average recovery was (96.75±1.41)%, (99.61±1.01)%, (97.18±1.96)% and(99.12±0.97)% (n=6), respectively. Conclusion The established method is simple, accurate and stable, which can be used for the simultaneous determination of 4 components in Yangxue Anshen syrup.

Luteoloside Exerts Analgesic Effect in a Complete Freund's Adjuvant-Induced Inflammatory Model via Inhibiting Interleukin-1ß Expression and Macrophage/Microglia Activation.

BACKGROUND: Flavonoid monomers are proved to have an anti-inflammatory effect and may also be promising for chronic pain treatment. In the present study, the analgesic effect and the relevant mechanisms of luteoloside, one of the flavonoid monomers, were investigated. METHODS: The analgesic effect of luteoloside was first evaluated in complete Freud's adjuvant induced inflammatory model by von Frey test and Hargreaves test in both male and female mice. The interleukin-1ß levels in plantar tissue, serum, dorsal root ganglion, and the dorsal horn of the spinal cord were determined by enzyme-linked immunosorbent assay or immunofluorescence. The activation of macrophage/microglia was tested by Iba-1 staining. RESULTS: Our data showed that luteoloside exhibited both acute and chronic analgesic phenotypes. Every single dose of luteoloside solution reached the peak transient analgesic effect 2 h after administration and lasted less than 6 h. About 14 consecutive days administration (one dose per day) later, luteoloside showed a sustained analgesic effect which lasted more than 24 h. Celecoxib 20 mg/kg combined with luteoloside 40 mg/kg achieved a similar analgesic effect as celecoxib 40 mg/kg alone. Luteoloside inhibited interleukin-1ß expression in plantar tissue, dorsal root ganglion, the dorsal horn of spinal cord, and serum, after 14 days of continuous administration. Furthermore, our results also showed that the activation of macrophage/microglia in dorsal root ganglions were significantly inhibited 2 h after each single dose in daily luteoloside administration and recovered to a higher level 6 h later. These findings might be involved in the mechanisms of the acute analgesic effect of luteoloside. CONCLUSION: Luteoloside presents an analgesic effect via anti-inflammatory and other mechanisms such as inhibiting the activation of macrophage/microglia.

Simultaneous determination of thirteen components in Ainsliaea fragrans by UHPLC-MS/MS / 中草药

Objective: To establish an UHPLC-MS/MS method for rapid and simultaneous determination of the content of 13 components in Ainsliaea fragrans. Methods: The UHPLC-MS/MS method was performed on UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) with acetonitrile-water (containing 0.1% formic acid and 5 mmol ammonium formate) as mobile phase for gradient elution. Flow rate was 0.25 mL/min and column temperature was 40 ℃. A triple quadrupole mass spectrometer equipped with electrospray ionization source (ESI) was applied in negative ion mode with the following parameters: ion spray, -4 500 V, ion source temperature, 500 ℃, curtain gas, 344.5 kPa, nubulizer (GS1), 344.5 kPa, heater gas (GS2), 206.7 kPa, and multiple reaction monitoring (MRM) was performed for quantitative analysis of these compounds. Results: Under the optimized MS/MS condition, the linearity ranges of protocatechuic acid, caffeic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, luteolin, luteoloside and apigenin were 1.099-274.800 ng/mL, 1.006-100.600 ng/mL, 1.080-1 080.000 ng/mL, 83.12-8 312.00 ng/mL, 19.92-996.00 ng/mL, 1.076-269.000 ng/mL, 5.555-555.500 ng/mL, 4.420-4 420.000 ng/mL, 44.76-17 904.00 ng/mL, 48.00-9 600.00 ng/mL, 2.108-210.800 ng/mL, 4.136-413.600 ng/mL, 1.070-107.000 ng/mL (r ≥ 0.9958), respectively, with good linearity. The average recovery rate of thirteen compounds in the samples were in the range of 96.54%-99.75%, and the RSD range was from 0.48% to 0.96%. The RSD values of precision, stability and repeatability test were all less than 3.90%. Conclusion: The method had good repeatability, high specificity, stability and controllability, and could be used for quality control of A. fragrans and its preparations.

Identification and evaluation of Lonicera japonica flos introduced to the Hailuogou area based on ITS sequences and active compounds.

Lonicera japonica flos (LJF), the dried flower buds of L. japonica Thunb., have been used in traditional Chinese herbal medicine for thousands of years. Recent studies have reported that LJF has many medicinal properties because of its antioxidative, hypoglycemic, hypolipidemic, anti-allergic, anti-inflammatory, and antibacterial effects. LJF is widely used in China in foods and healthcare products, and is contained in more than 30% of current traditional Chinese medicine prescriptions. Because of this, many Chinese villages cultivate LJF instead of traditional crops due to its high commercial value in the herbal medicine market. Since 2005, the flower buds of L. japonica are the only original LJF parts considered according to the Chinese Pharmacopoeia of the People's Republic of China. However, for historical and commercial reasons, some closely related species of Lonicera Linn. continue to be mislabeled and used as LJF. Currently, there are hundreds of commercial varieties of LJF on the market and it is difficult to choose fine LJF varieties to cultivate. In this study, a total of 21 varieties labeled as LJF on the market were planted in the Hailuogou area. In order to choose the optimum variety, internal transcribed spacer (ITS) sequence alignment analysis was used to test whether the 21 varieties were genuine LJF or not. Cluster analysis of active components based on the content of chlorogenic acid and luteoloside in flower buds, stems and leaves was used to evaluate the quality of the varieties. Results demonstrated that four of the varieties were L. macranthoides Hand.-Mazz., while the other 17 varieties were L. japonica, and genuine LJF. The ITS sequence analysis was proven to be highly effective in identifying LJF and Lonicerae flos. Among the 17 L. japonica varieties, the amounts of chlorogenic acid and luteoloside in flower buds, stems and leaves were significantly different. Based on the cluster analysis method, the variety H11 was observed to have the highest level of active components, and is therefore recommended for large-scale planting in the Hailuogou area.

Luteoloside Inhibits IL-1ß-Induced Apoptosis and Catabolism in Nucleus Pulposus Cells and Ameliorates Intervertebral Disk Degeneration.

Intervertebral disk degeneration (IDD) is the major cause of low back pain (LBP), which affects 80% of the world's population. Interleukin 1 beta (IL-1ß) is a major inflammatory factor that accelerates disk degeneration, and IL-1ß levels increase in degenerative disks. It has recently been reported that luteoloside-a type of flavonoid glycoside-has anti-inflammatory properties. In the present study, we investigated the protective potential of luteoloside in IDD. We found that luteoloside maintains cell morphology and inhibits apoptosis (indicated by the reduced expression of cleaved caspase 3) in IL-1ß-treated nucleus pulposus (NP) cells. It also suppresses inflammatory mediators-nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS)-in IL-1ß-treated NP cells. Furthermore, we found increased collagen II and aggrecan expression and reduced MMP13 and ADAMTS5 expression in luteoloside-treated NP cells in the presence of IL-1ß. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is involved in apoptosis, inflammation, and extracellular matrix (ECM) homeostasis. Mechanistic studies revealed that the NF-κB signaling pathway is inhibited by luteoloside, and Nrf2 is involved in the regulation of luteoloside in NF-κB signaling because Nrf2 knockdown reduced the suppressive effect of luteoloside on NF-κB signaling. We also established a puncture-induced rat IDD model and demonstrated that the persistent intraperitoneal injection of luteoloside ameliorates the progression of IDD. In conclusion, we demonstrated that luteoloside activates the Nrf2/HO-1 signaling axis and is a potential therapeutic medicine for IDD.

Chemical Constituents of Salix babylonica L. and Their Antibacterial Activity Against Gram-Positive and Gram-Negative Animal Bacteria.

The principle of animal wellbeing, which states that animals should be free from pain, injury, and disease, is difficult to maintain, because microorganisms are most frequently found to be resistant or multi-resistant to drugs. The secondary metabolites of plants are an alternative for the treatment of these microorganisms. The aim of this work was to determine the antibacterial effect of Salix babylonica L. hydroalcoholic extract (SBHE) against Escherichia coli, Staphylococcus aureus and Listeria monocytogenes, and identify the compounds associated with the activity. The SBHE showed activity against the three strains, and was subjected to a bipartition, obtaining aqueous fraction (ASB) with moderate activity and organic fraction (ACSB) with good activity against the three strains. The chromatographic separation of ACSB, allowed us to obtain ten fractions (F1AC to F10AC), and only three showed activity (F7AC, F8AC and F10AC). In F7AC, five compounds were identified preliminary by GC-MS, in F8AC and F10AC were identified luteolin (1) and luteolin 7-O-glucoside (2) by HPLC, respectively. The best antibacterial activity was obtained with F7AC (Listeria monocytogenes; MIC: 0.78 mg/mL, MBC: 0.78 mg/mL) and F8AC (Staphylococcus aureus; MIC: 0.39 mg/mL; MBC: 0.78 mg/mL). The results indicated that the compounds obtained from SBHE can be used as an alternative treatment against these microorganisms and, by this mechanism, contribute to animal and human health.