Supercritical Fluid-extracted Citrus aurantium L. var. amara Engl. Essential Oil Nanoemulsion: Preparation, Characterization, and Its Sleep-promoting Effect.

To overcome the defects of Citrus aurantium L. var. amara Engl. essential oil (CAEO), such as high volatility and poor stability, supercritical fluid-extracted CAEO nanoemulsion (SFE-CAEO-NE) was prepared by the microemulsification method. Emulsifiers comprising Tween 80, polyoxyethylenated castor oil (EL-40), and 1,2-hexanediol, and an oil phase containing SFE-CAEO were used for microemulsification. We examined the physicochemical properties of SFE-CAEO-NE and steam distillation-extracted CAEO nanoemulsion (SDE-CAEO-NE), which were prepared using different concentrations of the emulsifiers. The mean particle size and zeta potential were 21.52 nm and -9.82 mV, respectively, for SFE-CAEO-NE, and 30.58 nm and -6.28 mV, respectively, for SDE-CAEO-NE, at an emulsifier concentration of 15% (w/w). SFE-CAEO-NE displayed better physicochemical properties compared with SDE-CAEO-NE. Moreover, its physicochemical properties were generally stable at different temperatures (-20-60℃), pH (3-8), and ionic strengths (0-400 mM). No obvious variations in particle size, zeta potential, and Ke were observed after storing this nanoemulsion for 30 days at 4℃, 25℃, and 40℃, suggesting that it had good stability. The sleep-promoting effect of SFE-CAEO-NE was evaluated using a mouse model of insomnia. The results of behavioral tests indicated that SFE-CAEO-NE ameliorated insomnia-like behavior. Moreover, SFE-CAEO- NE administration increased the serum concentrations of neurotransmitters such as 5-hydroxytryptamine and γ-aminobutyric acid, and decreased that of noradrenaline in mice. It also exerted a reparative effect on the function of damaged neurons. Overall, SFE-CAEO-NE displayed a good sleep-promoting effect.

Mechanic study based on untargeted metabolomics of Pi-pa-run-fei-tang on pepper combined with ammonia induced chronic cough model mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Pi-pa-run-fei-tang (PPRFT), a traditional Chinese medicine formula with long-standing history, demonstrated beneficial effect on chronic cough. However, the mechanism underlying efficacy unclear. In current research, we explored the impact and molecular mechanism of chronic cough mouse stimulating with capsaicin combined with ammonia. AIM OF THE STUDY: To investigate the metabolic modulating effects, and potential mechanisms underlying the therapeutic effect of PPRFT in chronic cough. MATERIALS AND METHODS: Chronic cough mouse models were created by stimulating mice by capsaicin combined with ammonia. Number of coughs and cough latency within 2 min were recorded. With lung tissue and serum samples collected for histopathology, metabolomics, RT-qPCR, immunohistochemistry, and WB analysis. Lymphocytes were isolated and flow cytometric assays were conducted to evaluate the differentiation between Th17 and Treg cell among CD4+ cells. RESULTS: Results indicated that PPRFT obviously reduced the number of coughs, prolonged cough latency, reduced inflammatory cell infiltration and lung tissues damage, and decreased the serum level of IL-6, IL-1ß, TNF-α, and IL-17 while increasing IL-10 levels. Notably, PPRFT suppressed Th17 cell divergence and promoted Treg cell divergence. Furthermore, serum metabolomic assays showed that 46 metabolites differed significantly between group, with 35 pathways involved. Moreover, mRNA levels of IL-6, NF-κB, IL-17, RORγT, JAK2, STAT3, PI3K and AKT in lung tissues remarkably reduced and mRNA levels of IL-10 and FOXP3 were elevated after PPRFT pretreatment. Additionally, PPRFT treatments decreased the protein levels of IL-6, NF-κB, IL-17, RORγT, p-JAK2, p-STAT3, p-PI3K, and p-AKT and increased the protein levels of IL-10 and FOXP3, but no significantly effects to the levels on JAK2, STAT3, PI3K, and AKT in the lungs. CONCLUSION: Conclusively, our result suggested the effect with PPRFT on chronic cough may be mediated through IL-6/JAK2/STAT3 and PI3K/AKT/NF-κB pathway, which regulate the differentiation between Th17 and Treg cell. This beneficial effect of PPRFT in capsaicin and ammonia-stimulated chronic cough mice indicates its potential application in treating chronic cough.

Pi-Pa-Run-Fei-Tang alleviates lung injury by modulating IL-6/JAK2/STAT3/IL-17 and PI3K/AKT/NF-κB signaling pathway and balancing Th17 and Treg in murine model of OVA-induced asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Pi-Pa-Run-Fei-Tang (PPRFT) is an empirical TCM prescription for treating asthma. However, the underlying mechanisms of PPRFT in asthma treatment have yet to be elucidated. Recent advances have revealed that some natural components could ameliorate asthma injury by affecting host metabolism. Untargeted metabolomics can be used to better understand the biological mechanisms underlying asthma development and identify early biomarkers that can help advance treatment. AIM OF THE STUDY: The aim of this study was to verification the efficacy of PPRFT in the treatment of asthma and to preliminarily explore its mechanism. MATERIALS AND METHODS: A mouse asthma model was built by OVA induction. Inflammatory cell in BALF was counted. The level of IL-6, IL-1ß, and TNF-α in BALF were measured. The levels of IgE in the serum and EPO, NO, SOD, GSH-Px, and MDA in the lung tissue were measured. Furthermore, pathological damage to the lung tissues was detected to evaluate the protective effects of PPRFT. The serum metabolomic profiles of PPRFT in asthmatic mice were determined by GC-MS. The regulatory effects on mechanism pathways of PPRFT in asthmatic mice were explored via immunohistochemical staining and western blotting analysis. RESULTS: PPRFT displayed lung-protective effects through decreasing oxidative stress, airway inflammation, and lung tissue damage in OVA-induced mice, which was demonstrated by decreasing inflammatory cell levels, IL-6, IL-1ß, and TNF-α levels in BALF, and IgE levels in serum, decreasing EPO, NO, and MDA levels in lung tissue, elevating SOD and GSH-Px levels in lung tissue and lung histopathological changes. In addition, PPRFT could regulate the imbalance in Th17/Treg cell ratios, suppress RORγt, and increase the expression of IL-10 and Foxp3 in the lung. Moreover, PPRFT treatment led to decreased expression of IL-6, p-JAK2/Jak2, p-STAT3/STAT3, IL-17, NF-κB, p-AKT/AKT, and p-PI3K/PI3K. Serum metabolomics analysis revealed that 35 metabolites were significantly different among different groups. Pathway enrichment analysis indicated that 31 pathways were involved. Moreover, correlation analysis and metabolic pathway analysis identified three key metabolic pathways: galactose metabolism; tricarboxylic acid cycle; and glycine, serine, and threonine metabolism. CONCLUSION: This research indicated that PPRFT treatment not only attenuates the clinical symptoms of asthma but is also involved in regulating serum metabolism. The anti-asthmatic activity of PPRFT may be associated with the regulatory effects of IL-6/JAK2/STAT3/IL-17 and PI3K/AKT/NF-κB mechanistic pathways.

Crocetin confers neuroprotection and is anti-inflammatory in rats with induced glaucoma.

BACKGROUND: Crocetin is a bioactive ingredient in saffron, derived from the Crocus sativus stigmas of the Iridaceae family. As a chemically carotenoid derivative, crocetin exhibites effects like anti-inflammatory, antioxidant, neuroprotective, etc. However, the protective effect of crocetin on glaucoma and its mechanism remains unclear. The current study assesed the neuroprotective and anti-inflammatory effects of crocetin on retinal neurons in glaucoma rats which were induced by 0.3% carbomer injection into the anterior chamber. METHODS AND RESULTS: The pathological structures on the retina and optic nerve were observed and examined by H&E staining and transmission electron microscopy. Immunohistochemical staining was used to detect the expression of TNF-α, IL-1ß, and IL-6 of the retina and the expression of a brain-derived neurotrophic factor (BDNF) in the primary visual cortex (PVC). Western blot was carried out to detect the expression of PI3K, Akt, and NF-κB in the retina. It was found that crocetin ameliorated the pathological changes of the retina and ON and reduced the number of apoptotic retinal ganglion cells. Immunohistochemical staining showed that crocetin could decrease the contents of TNF-α, IL-1ß, and IL-6 and increase the contents of BDNF. Western blot showed that crocetin was found to suppress the expression of PI3K, Akt, and NF-κB. CONCLUSION: The results obtained in this study have indicated that crocetin showes neuroprotective effects on retinal ganglion cells in glaucoma rats and inhibits retinal dysfunction. Meanwhile, crocetin exerted an anti-inflammatory effect to protect the retina by inhibiting the expression of the PI3K/Akt/NF-κB signaling pathway. This work provides substantial evidence that crocetin may be a potential drug for the treatment of glaucoma.

Saffron essential oil ameliorates CUMS-induced depression-like behavior in mice via the MAPK-CREB1-BDNF signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Saffron, the dried stigma of Crocus sativus L., has a long history of use in the treatment of depression in traditional Chinese medicine and Islamic medicine. The unique aroma of saffron, primarily derived from its volatile oil, has been widely used by folk to mitigate anxiety and depression via sniffing because the aroma of saffron has a pleasant and invigorating effect. AIM OF THE STUDY: This study aimed to investigate the antidepressant effect and the underlying mechanism of saffron essential oil (SEO) in mice exposed to chronic unpredictable mild stress (CUMS). MATERIALS AND METHODS: In this study, compounds of SEO were identified using gas chromatography-mass spectrometry analysis, while network pharmacology was used to predict potential active compounds, antidepressant targets, and related signaling pathways of SEO. The CUMS depression model was further used to explore the therapeutic effect and possible mechanism of SEO. During the modeling period, mice were regularly administered fluoxetine (3.6 mg/kg, i.g.) or diluted SEO (2%, 4%, and 6% SEO, inhalation). The antidepressant and neuroprotective effects of SEO were evaluated by behavior tests (the open field test, the sucrose preference test, the tail suspension test, and the forced swimming test), hematoxylin-eosin staining, and Nissl staining. The enzyme-linked immunosorbent assay kits were used to measure dopamine (DA), 5-serotonin (5-HT), brain-derived neurotrophic factor (BDNF), and γ-aminobutyric acid (GABA) levels in serum. The relative abundance of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B in the hippocampus was determined using western blot (WB). RESULTS: According to the network pharmacology analysis, seven active SEO compounds mediated 113 targets related to depression treatment, most of which were enriched in the 5-HT synapse, calcium signaling pathway, and cAMP signaling pathway. In vivo experiments indicated that fluoxetine and SEO improved depression-like behaviors in depressed mice. The levels of 5-HT, DA, BDNF, and GABA in serum increased significantly. Histopathological examinations revealed that fluoxetine and SEO ameliorated neuronal damage in the hippocampus. WB analysis showed that the relative expressions of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B were significantly higher in the fluoxetine and SEO groups than in the CUMS group. CONCLUSION: Overall, these findings suggest that SEO significantly alleviates the depressive symptoms in CUMS exposed mice and partially restores hippocampal neuronal damage. Meanwhile, the best efficacy was observed in 4% SEO. Furthermore, the antidepressant mechanism of SEO is primarily dependent on the regulation of the MAPK-CREB1-BDNF signaling pathway.

Effects of fulvic acid on growth performance, serum index, gut microbiota, and metabolites of Xianju yellow chicken.

Fulvic acid (FA) is a mixture of polyphenolic acid compounds extracted from humus, peat, lignite, and aquatic environments; it is used in traditional medicine to treat digestive tract diseases. The purpose of the present study was to investigate the effect of FA on growth performance, inflammation, intestinal microbiota, and metabolites in Xianju yellow chicken. The 240 Xianju yellow chickens (age, 524 days) included were randomly categorized into 4 treatments with 6 replicates per treatment and 10 birds per replicate. Birds received a basal diet or a diet supplemented with 500, 1,000, or 1,500 mg/kg of FA, for a period of 42 days. Dietary supplementation of FA improved average daily gain (ADG) and feed conversion ratio (FCR) (P > 0.05). Compared with the control group, the serum level of TNF-α in birds supplemented with FA was significantly decreased (P < 0.05), and that of IL-2 was significantly increased after administration of 1,500 mg/kg FA (P < 0.05). Analysis of gut microbiota indicated that FA reduced the relative abundance of genus Mucispirillum, Anaerofustis, and Campylobacter, but enriched genus Lachnoclostridium, Subdoligranulum, Sphaerochaeta, Oscillibacter, and Catenibacillus among others. Untargeted metabolomic analyses revealed that FA increased 7-sulfocholic acid, but reduced the levels of Taurochenodeoxycholate-7-sulfate, LysoPC 20:4 (8Z, 11Z, 14Z, 17Z), LysoPC 18:2, Phosphocholine and other 13 metabolites in the cecum. The results demonstrated that FA may potentially have a significant positive effect on the growth performance and immune function of Xianju yellow chicken through the modulation of the gut microbiota.

Exploring the Protective Effects and Mechanism of Crocetin From Saffron Against NAFLD by Network Pharmacology and Experimental Validation.

Background: Non-alcoholic fatty liver disease (NAFLD) is a burgeoning health problem but no drug has been approved for its treatment. Animal experiments and clinical trials have demonstrated the beneficial of saffron on NAFLD. However, the bioactive ingredients and therapeutic targets of saffron on NAFLD are unclear. Purpose: This study aimed to identify the bioactive ingredients of saffron responsible for its effects on NAFLD and explore its therapy targets through network pharmacology combined with experimental tests. Methods: Various network databases were searched to identify bioactive ingredients of saffron and identify NAFLD-related targets. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were conducted to enrich functions and molecular pathways of common targets and the STRING database was used to establish a protein-protein interaction network (PPI). The effect of crocetin (CCT) on NAFLD was evaluated in a mouse model of NAFLD by measuring the biomarkers of lipid, liver and renal function, oxidative stress, and inflammation. Liver histopathology was performed to evaluate liver injury. Nuclear factor erythroid-related factor (Nrf2) and hemeoxygenase-1 (HO-1) were examined to elucidate underlying mechanism for the protective effect of saffron against NAFLD. Results: A total of nine bioactive ingredients of saffron, including CCT, with 206 common targets showed therapeutic effects on NAFLD. Oxidative stress and diabetes related signaling pathways were identified as the critical signaling pathways mediating the therapeutic effects of the active bioactive ingredients on NAFLD. Treatment with CCT significantly reduced the activities of aspartate aminotransferase (AST), alanine transaminase (ALT), and the levels of total cholesterol (TC), triglyceride (TG), malondialdehyde (MDA), blood urea nitrogen (BUN), creatinine (CR), and uric acid (UA). CCT significantly increased the activities of superoxide dismutase (SOD), and catalase (CAT). Histological analysis showed that CCT suppressed high-fat diet (HFD) induced fat accumulation, steatohepatitis, and renal dysfunctions. Results of ELISA assay showed that CCT decreased the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and increased the expression of HO-1 and Nrf2. Conclusion: This study shows that CCT is a potential bioactive ingredient of saffron that treats NAFLD. Its mechanism of action involves suppressing of oxidative stress, mitigating inflammation, and upregulating Nrf2 and HO-1 expression.