Network pharmacology- and molecular docking-based analyses of the antihypertensive mechanism of Ilex kudingcha.

Herein, network pharmacology was used to identify the active components in Ilex kudingcha and common hypertension-related targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted, and molecular docking was performed to verify molecular dynamic simulations. Six active components in Ilex kudingcha were identified; furthermore, 123 target genes common to hypertension were identified. Topological analysis revealed the strongly associated proteins, with RELA, AKT1, JUN, TP53, TNF, and MAPK1 being the predicted targets of the studied traditional Chinese medicine. In addition, GO enrichment analysis revealed significant enrichment of biological processes such as oxidative stress, epithelial cell proliferation, cellular response to chemical stress, response to xenobiotic stimulus, and wound healing. Furthermore, KEGG enrichment analysis revealed that the genes were particularly enriched in lipid and atherosclerosis, fluid shear stress and atherosclerosis, and other pathways. Molecular docking revealed that the key components in Ilex kudingcha exhibited good binding potential to the target genes RELA, AKT1, JUN, TP53, TNF, and IL-6. Our study results suggest that Ilex kudingcha plays a role in hypertension treatment by exerting hypolipidemic, anti-inflammatory, and antioxidant effects and inhibiting the transcription of atherosclerosis-related genes.

Ilex kudingcha C.J. Tseng Mitigates Phenotypic Characteristics of Human Autism Spectrum Disorders in a Drosophila Melanogaster Rugose Mutant.

Autism spectrum disorders (ASD) have heterogeneous etiologies involving dysfunction of central nervous systems, for which no effective pan-specific treatments are available. Ilex kudingcha (IK) C.J. Tseng is a nootropic botanical used in Asia for neuroprotection and improvement of cognition. This study establishes that a chemically characterized extract from IK (IKE) mitigates behavioral traits in the Drosophila melanogaster rugose mutant, whose traits resemble human ASD, and examines possible mechanisms. IKE treatment significantly ameliorated deficits in social interaction, short-term memory, and locomotor activity in Drosophila rugose, and significantly increased synaptic bouton number of size more than 2 µm2 in the neuromuscular junctions (NMJs) of Drosophila rugose. To clarify mechanism(s) of IKE action, methylphenidate (MPH), a dopamine transporter inhibitor, was included as a reference drug in the behavioral assays: MPH significantly improved social interaction and short-term memory deficit in Drosophila rugose; administration of the dopamine D1 receptor antagonist SCH23390 and dopamine D2 receptor antagonist sulpiride reversed the ameliorative effects of both MPH and IKE on the social interaction deficits of Drosophila rugose. To extend analysis of IKE treatment to the vertebrate central nervous system, ASD-associated gene expression in mouse hippocampus was studied by RNA-seq: IKE treatment altered the expression of genes coding phosphoinositide 3-kinases/protein kinase B (PI3K-Akt), proteins in glutamatergic, dopaminergic, serotonergic, and GABAergic synapses, cAMP response element-binding protein (CREB), and RNA transporter proteins. These results provide a foundation for further analysis of IKE as a candidate for treatment of some forms of ASD.

Modulation of gut microbiota by Ilex kudingcha improves dextran sulfate sodium-induced colitis.

Increasing evidence shows that the gut microbiota contributes to the occurrence and development of colitis. Kudingcha (KDC), made from the leaves of Ilex kudingcha, could mitigate inflammation, however, little is known about the relationship between modulatory effect on gut microbiota by KDC and improvement of colitis. In this study, the attenuating effects of KDC extract (KDCE) on dextran sulfate sodium (DSS)-induced colitis and gut microbiota in C57BL/6 mice were investigated. It was found that the supplementation of KDCE could alleviate typical symptoms of IBD including weight loss, colon shortening, intestinal barrier damage, and decreases in the colitis disease activity index and pro-inflammatory cytokines. Moreover, KDCE supplementation could reverse the alteration of gut microbiota in the colitic mice by increasing the abundances of potential beneficial bacteria, e.g. Odoribacter, Prevotella and Helicobacter, and decreasing the abundances of potential harmful bacteria, e.g. Parabacteroides, Bacteroides, Turicibacter, Parasutterella and Lachnospiraceae. The levels of short-chain fatty acids in feces, cecum contents and serum were also regulated by KDCE. Furthermore, the correlation analysis suggested that KDCE could attenuate DSS-induced colitis which might be related to the alteration of gut microbiota. Therefore, the modulation of gut microbiota by KDCE might be a potential strategy for improving inflammation-driven diseases.

Anti-inflammatory effects of dicaffeoylquinic acids from Ilex kudingcha on lipopolysaccharide-treated RAW264.7 macrophages and potential mechanisms.

Increasing evidence has shown that dicaffeoylquinic acids (DiCQAs) have anti-inflammatory activity. However, the underlying molecular mechanisms of the anti-inflammatory effects of DiCQAs are still unclear. In the present study, the anti-inflammatory effects of DiCQAs from the leaves of Ilex kudingcha and the potential molecular mechanisms on LPS-induced inflammatory responses in RAW264.7 macrophage cells were investigated. The results showed that pretreatment with DiCQAs could suppress the production of NO, PGE2 and also pro-inflammatory cytokines TNF-α, IL-1ß and IL-6, and the mRNA expression of two major inflammatory mediators of COX-2 and iNOS. The phosphorylated IκBα, ERK, JNK and p38 proteins in LPS-treated cells were significantly increased, which could be reversed by pretreatment with DiCQAs in a concentration-dependent manner. Taken together, the results suggest that DiCQAs from I. kudingcha have potent anti-inflammatory effects on LPS-induced inflammatory responses by inhibiting the NF-κB and MAPKs pathways and may be a prophylactic for inflammation.

Multi-Component Analysis of Ilex Kudingcha C. J. Tseng by a Single Marker Quantification Method and Chemometric Discrimination of HPLC Fingerprints.

The quantitative analysis of multiple components with a single marker (QAMS) method was firstly established for simultaneous determination of 18 active components in Ilex kudingcha C. J. Tseng by HPLC. Using rutin, isochlorogenic acid A and kudinoside A as internal refererence substances (IRS), compatibility results showed that the relative correction factors (RCFs) of all compounds showed good reproducibility under different chromatographic conditions. On the basis of previous studies, the accuracy of the QAMS method was systematically evaluated by investigating the influences of curve intercept, analytes and IRS concentration. The results showed that the concentration (especially at low level) of analytes and curve intercept were the major influencing parameters for the LRG-QAMS method (LRG = linear regression), whereas the influence of IRS concentration seemed more apparent in terms of the AVG-QAMS method (AVG = average). The two approaches were complementary with each other. In addition, hierarchical clustering analysis (HCA), principal components analysis (PCA) and similarity analysis (SA) were performed to differentiate and classify the samples based on the contents of 18 marker compounds. The results of the different chemometric analyses were completely consistent with each other, and could be supported by the quantification results.

Kudinoside-D, a triterpenoid saponin derived from Ilex kudingcha suppresses adipogenesis through modulation of the AMPK pathway in 3T3-L1 adipocytes.

The leaves of Ilex Kudingcha, locally named "Kudingcha" in China, has been traditionally applied for treating obesity. Studies have demonstrated that the ethanol extract of Ilex kudingcha have anti-adipogenic effects. However, the constituent which was responsible for its anti-obesity and its underlying molecular mechanism has not yet been elucidated. This research explored the anti-obesity effect of kudinoside-D which was a main natural component of triterpenoid saponin from the ethanol extract of Ilex kudingcha, on lipid accumulation and the potential mechanism of action of adipogenesis in 3T3-L1 adipocytes. The adipocytes were treated with various concentrations of kudinoside D (0 to 40µM) during differentiation. The image-based Oil Red O staining analyses revealed that KD-D, dose dependently reduced cytoplasmic lipid droplet in 3T3-L1 adipocytes with the IC50 is 59.49µM. Meanwhile, major adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein-α (C/EBPα) and sterol regulatory element-binding protein 1c (SREBP-1c) were significantly repressed as well as their target genes. The phosphorylation of AMP-activated protein kinase (AMPK) and its downstream target phosphorylated-acetyl CoA carboxylase (ACC) expression were also increased. In addition, the inhibitory effects of KD-D on the expressions of PPARγ and C/EBPα were weakened when cells were cotreated with AMPK inhibitor Compound C. These results indicated KD-D exerts anti-adipogenic effects through modulation of adipogenic transcription factors via AMPK signaling pathway. And the current findings demonstrated that KD-D was a potential therapeutic candidate for alleviating obesity and hyperlipidemia.

Modulating Effects of Dicaffeoylquinic Acids from Ilex kudingcha on Intestinal Microecology in Vitro.

Dietary polyphenols have been considered as novel prebiotics, and polyphenols could exert their functions through modulating intestinal microbiota. The diverse bioactivities of kudingcha could derive from its phenolic compounds, but the effects of dicaffeoylquinic acids (diCQAs) from Ilex kudingcha on intestinal microbiota have not been investigated. In the present study, high-throughput sequencing and anaerobic fermentation in vitro were utilized to investigate the microecology-modulating function of I. kudingcha diCQAs. As a result, diCQAs raised the diversity and exhibited a more considerable impact than a carbon source on the microbial profile. DiCQAs increased the relative abundances of Alistipes, Bacteroides, Bifidobacterium, Butyricimonas, Clostridium sensu stricto, Escherichia/Shigella, Parasutterella, Romboutsia, Oscillibacter, Veillonella, Phascolarctobacterium, Lachnospiracea incertae sedis, Gemmiger, Streptococcus, and Haemophilus and decreased the relative abundances of Ruminococcus, Anaerostipes, Dialister, Megasphaera, Megamonas, and Prevotella. DiCQAs also affected the generation of short-chain fatty acids through microbiota. The contents of acetic and lactic acids were raised, while the production of propionic and butyric acids was reduced. Conclusively, diCQAs from I. kudingcha had significant modulating effects on intestinal microbiota in vitro, which might be the fundamental of diCQAs exerting their bioactivities.

Simultaneous Qualitation and Quantitation of Chlorogenic Acids in Kuding Tea Using Ultra-High-Performance Liquid Chromatography-Diode Array Detection Coupled with Linear Ion Trap-Orbitrap Mass Spectrometer.

Kuding tea, the leaves of Ilex Kudingcha C.J. Tseng, has been applied for treating obesity, hypertension, cardiovascular disease, hyperlipidemia, and so on. The chlorogenic acids (CGAs) in Kuding tea have shown excellent antioxidative, antiobesity, anti-atherosclerotic and anticancer activities. Nevertheless, the chemical profiles of CGAs in Kuding tea have not been comprehensively studied yet, which hinders further quality control. In the present study, a sensitive ultra-high-performance liquid chromatography-diode array detection coupled with a linear ion trap-Orbitrap (UHPLC-DAD-LTQ-Orbitrap) method was established to screen and identify CGAs in Kuding tea. Six CGA standards were first analyzed in negative ion mode with a CID-MS/MS experiment and then the diagnostic product ions (DPIs) were summarized. According to the retention behavior in the RP-ODS column, accurate mass measurement, DPIs and relevant bibliography data, a total of 68 CGA candidates attributed to 12 categories were unambiguously or preliminarily screened and characterized within 18 min of chromatographic time. This was the first systematic report on the distribution of CGAs in Kuding tea. Meanwhile, the contents of 6 major CGAs in Kuding tea were also determined by the UHPLC-DAD method. All the results indicated that the established analytical method could be employed as an effective technique for the comprehensive and systematic characterization of CGAs and quality control of the botanic extracts or Chinese medicinal formulas that contain various CGAs.