Antiviral and Cytotoxic Activities of Ilex aquifolium Silver Queen in the Context of Chemical Profiling of Two Ilex Species.

The leaves of Ilex paraguariensis (known as Yerba mate), used as a popular beverage, are a very well-recognized plant material with various biological activities, including analeptic (because of caffeine), anti-obesity (phenolics, saponins), antimicrobial, and antiviral (phenolics, saponins). Here, the chemical compositions of the leaves of two European Ilex species (× meserveae and aquifolium) with three varieties each were investigated. The terpenoid, saponin, and polyphenolic fractions were submitted for LC-MS or GC-MS analysis against a standard Mate leaf. In addition, the aroma profiles of all the species were analysed using HS-SPME-Arrow prior to GC-MS analysis. All fractions were subjected to antiviral and cytotoxic assays. We found 86 compounds in all accessions, with limonene, linalool, and p-cymene being predominant. There were minor similarities between the volatile compositions of the European and South American species. We found ursolic and oleanolic acid to be the main compounds in the terpenoid fraction. Mono-caffeoylquinic acids and di-caffeoylquinic acids were the main constituents of the polar fractions. About 180 compounds from the saponin group were tentatively identified, of which 9 and 3 were selected as distinctive markers for I. meserveae and I. aquifolium, respectively. Based on chemical screening, I. aquifolium Silver Queen was chosen as the source of terpenoid and saponin fractions and polyphenol extracts. The most substantial inhibition of cancer cell growth was observed with saponin in the case of the MCF7 (human breast cancer) cell line, while for LoVo and L929 cell lines (human colorectal cancer and reference mouse fibroblasts), it was slightly weaker. These results should be analysed further as a promising chemoprevention of colorectal and gastrointestinal cancers. Saponin and polyphenolic extracts exhibited similar activities against HSV-1 and HAdV-5, with 4-log reduction in virus titres. This study focuses our attention on a field of potential antiviral formulations derived from European holly.

Chemical components with biological activities in the roots of Ilex pubescens.

Two new triterpenoids, ilexsaponin U (1) and ilexsaponin V (2), and three new phenylpropanoids, pubescenoside S (3), pubescenoside T (38), and pubescenoside U (39), along with thirty-four existing compounds were isolated from the roots of Ilex pubescens. The elucidation of their structures involved comprehensive spectroscopic techniques, including IR, UV, HR-ESI-MS, and NMR experiments. The anti-inflammatory effects of almost all the compounds were evaluated in LPS-induced RAW264.7 cells. Among these, compounds 1, 4, 8, 11, 12, 26, 27, 29 and 33 exhibited varying degrees of inhibition of inflammatory factors. Notably, compounds 1, 4 and 8 significantly inhibited the mRNA levels of iNOS, IL-6, IL-1ß and TNFα, comparable to or exceeding the effect of the positive control (dexamethasone, DEX). We also evaluated the cardioprotective effects of these compounds in OGD/R-induced H9c2 cells. The results revealed that compounds 2, 3, 7, 8, 26, 35, 36 and 37 at 20 µM significantly increased cell viability by 24.9 ± 3.4%, 28.0 ± 0.3%, 37.6 ± 0.2%, 44.86 ± 0.5%, 9.47 ± 2.1%, 23.9 ± 0.4%, 39.5 ± 3.1% and 28.2 ± 0.1%, respectively. Some of them exhibited effects equal to or greater than that of the positive control (diazoxide, DZ) at 100 µM, showing a 21.9 ± 3.0% increase.

Integrated network pharmacology and metabolomics reveal vascular protective effects of Ilex pubescens on thromboangiitis obliterans.

BACKGROUND: Ilex pubescens Hook. et Arn (IP), traditionally known for its properties of promoting blood circulation, swelling and pain relief, heat clearing, and detoxification, has been used in the treatment of thromboangiitis obliterans (TAO). Despite its traditional applications, the specific mechanisms by which IP exerts its therapeutic effects on TAO remain unclear. AIM OF THE STUDY: This study aims to uncover the underlying mechanisms in the therapeutic effects of IP on TAO, employing network pharmacology and metabolomic approaches. METHODS: In this study, a rat TAO model was established by injecting sodium laurate through the femoral artery, followed by the oral administration of IP for 7 days. Plasma coagulation parameters were measured to assess the therapeutic effects of IP. The potential influence on the femoral artery and gastrocnemius muscle was histopathologically evaluated. Network pharmacology was employed to predict relevant targets and model pathways for TAO. Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) was used for the metabolic profile analysis of rat plasma. Immunohistochemistry (IHC) was used to verify the mechanisms by which IP promotes blood circulation in TAO. RESULTS: The study revealed that IP improved blood biochemical function in TAO and played a significant role in vascular protection and maintaining normal blood vessels and gastrocnemius morphologies. Network pharmacology showed that IP compounds play a therapeutic role in modulating lipids and atherosclerosis. Metabolomic analysis revealed that the pathways involved in sphingolipid metabolism and steroid biosynthesis were significantly disrupted. The joint analysis showed a strong correlation between lysophosphatidylcholine and IP components, including triterpenoid and iridoid components, which support the curative action of IP through the modulation of sphingolipid metabolism. Furthermore, decreased expression levels of SPHK1/S1PR1, TNF-α, IL-1ß, and IL-6 were observed in the IP-treated group, suggesting that IP exerts a protective effect on the vasculature primarily by regulating of the SPHK1/S1PR1 signaling pathway. CONCLUSION: In this study, we found that IP protects the vasculature against injury and treats TAO by regulating the steady-state disturbance of the sphingolipid pathway. These findings suggest that IP promotes vasculature by modulating sphingolipid metabolism and SPHK1/S1PR1 signaling pathway and reduce levels of inflammatory factors, offering new insights into its therapeutic potential.

UPLC-Q-TOF/MS based metabolite profiling and quality marker constituents screening of root, stem and rhizome extracts of Ilex asprella.

OBJECTIVE: The root of Ilex asprella (RIA) is a popular plant resource for treating inflammation-related diseases. The purpose of this study was to identify the secondary metabolites, to compare anti-inflammatory effects and to determine the quality marker components among root, stem and rhizome sections of IA. METHODS: Chemical fingerprints of stem, root and rhizome of IA was determined by high performance liquid chromatography (HPLC). A reliable method using ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was established for comprehensively determining the chemical constituents of the plants. Anti-inflammatory activities of IA and its ingredients were screened by in vivo mouse ear swelling and in vitro LPS-induced release of NO from RAW264.7 cells experiments. RESULTS: Root, stem and rhizome of IA have shown high similarity in chemical fingerprints. Totally 149 compounds were characterized in IA, including triterpenoids, triterpenoid saponins, phenolic acids and lignans. 44 of them were identified based on co-occurring Mass2Motifs, including 19 unreported ones, whilst 17 were tentatively confirmed by comparison with reference compounds. No significant anti-inflammatory activity difference among root, stem and rhizome parts of IA was found. Ilexsaponin B2, protocatechualdehyde, isochlorogenic acid B and quinic acid, were screened out as quality marker compounds in IA. CONCLUSION: A sensitive and rapid strategy was established to evaluate the differences on secondary metabolites of different parts of IA for the first time, and this study may contribute to the quality evaluation of medicinal herbs and provide theoretically data support for further analysis of different parts of IA.

Ilexchinene, a new seco-ursane triterpenoid from the leaves of Ilex chinensis with therapeutic effect on neuroinflammation by attenuating the MAPK/NF-κB signaling pathway.

BACKGROUND: Neuroinflammation is a vital factor participating in the whole pathogenetic process of diverse neurodegenerative disorders, but accessible clinical drugs are still insufficient due to their inefficacy and side effects. Triterpenoids are reported to possess potential anti-neuroinflammatory activities, and the leaves of Ilex chinensis are a commonly used herbal medicine containing many ursane-type and oleanane-type triterpenoids. However, the novel triterpenoids from I. chinensis and their underlying mechanisms are still elusive. PURPOSE: To isolate novel seco-ursane triterpenoids with anti-neuroinflammatory effects from the leaves of I. chinensis and reveal their underlying mechanisms. STUDY DESIGN AND METHODS: The novel compound was purified by column chromatography and identified by comprehensive spectroscopic experiments. The LPS-induced BV-2 cell model and LPS-induced acute murine brain inflammation model were used to assess the anti-neuroinflammatory effect of the structure and further understand its underlying mechanisms by cell viability, ELISA, Western blot analysis, qRT‒PCR analysis, behavior analysis, H&E staining, and immunofluorescence staining experiments. RESULTS: Ilexchinene is a novel ursane-type triterpenoid with a rare 18,19-seco-ring skeleton that was first isolated and identified from I. chinensis. Ilexchinene evidently reduced the overexpression of inflammatory substances in vitro. A mechanistic study suggested that ilexchinene could decrease NF-κB activation to prevent the formation of the NLRP3 inflammasome in the early neuroinflammatory response; in addition, it could prevent the phosphorylation of ERK and JNK. In vivo, ilexchinene remarkably improved LPS-induced mouse behavioral deficits and diminished the number of overactivated microglial cells. Furthermore, ilexchinene evidently diminished the overexpression of inflammatory substances in mouse brains. A mechanistic study confirmed that ilexchinene markedly suppressed the MAPK/NF-κB pathway to relieve the neuroinflammatory response. CONCLUSION: We identified a novel 18,19-seco-ursane triterpenoid from the leaves of I. chinensis and revealed its underlying mechanism of neuroinflammation for the first time. These findings suggest that ilexchinene might possess promising therapeutic effects in neuroinflammation.

Pharmacokinetic and tissue distribution study of six saponins in the rat after oral administration of Ilex pubescens extract using a validated simultaneous UPLC-qTOF-MS/MS assay.

Ilex pubescens Hook. et Arn is a medicinal plant of the Ilex family that is mainly used for the treatment of cardiovascular diseases. Its main medicinal ingredients are total triterpenoid saponins (IPTS). However, the pharmacokinetics and tissue distribution of the main multi-triterpenoid saponins are lacking. This is the first report that demonstrates a sensitive ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-qTOF-MS/MS) method for the quantification of ilexgenin A (C1), ilexsaponin A1 (C2), ilexsaponin B1 (C3), ilexsaponin B2 (C4), ilexsaponin B3 (DC1) and ilexoside O (DC2) in rat plasma and various tissues of the heart, liver, spleen, lungs, kidney, brain, stomach, duodenum, jejunum, ileum, colon and thoracic aorta. The chromatographic separation was carried out on an Acquity HSS T3 UPLC column (2.1 × 100 mm, 1.8 µm, Waters, USA) with a mobile phase consisting of 0.1% (v/v) formic acid (A) and acetonitrile containing 0.1% (v/v) formic acid (B) at a flow rate of 0.25 mL/min. The MS/MS detection was performed by electrospray ionization (ESI) using selected ion monitoring (SIM) in negative scan mode. The developed quantification method showed good linearity over the concentration range of 10-2000 ng/mL for plasma and 25-5000 ng/mL for tissue homogenates with R2 ≥ 0.990. Lower limits of quantification (LLOQ) was 10 ng/mL in plasma and 25 ng/mL in tissue homogenates. The intra- and inter-day precision were less than 10.39%, and the accuracy was between - 1.03% and 9.13%. The extract recoveries, dilution integrity and matrix effect were well within satisfactory limits. Using the validated method, the pharmacokinetic parameters, including half-life, AUC, Cmax, CL, and MRT, of six triterpenoid saponins in rats after oral administration were provided by establishing their plasma concentration-time curves, while their absolute quantification in various tissues after oral administration was also determined at first, which provides a scientific basis for their clinical application.

Identification and characterization of the chemical constituents in the roots of Ilex asprella by high performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry.

The roots of Ilex asprella (Rla) are a well-known traditional Chinese medicine for the treatment of viral and bacterial infectious diseases, such as influenza, tonsillitis, sphagitis, and trachitis. However, due to the complexity of the chemical constituents in Rla, few investigations have acquired a comprehensive understanding of material basis. High-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS/MS) was used for the identification of chemical constituents from the extract of Rla in negative ion mode. Their chemical structures were tentatively elucidated based on exact formulas, fragmentation patterns and literature data. A total of 32 compounds were discovered and tentatively characterised in Rla, including 4 phenolic glycosides and 28 triterpenoid glycosides. 10 compounds have not been previously reported in Rla and 8 of them have not been previously reported in the literature. The chemical composition of Rla was identified and summarised, providing a basis for further study on Rla.

Five glycosylated phenolic derivatives from the bark of Ilex rotunda Thunb. and their anti-inflammatory activities.

Five new glycosylated phenolic derivatives, rotundosides A-E (1-5), and three known glycosides (6-8) were isolated from the 95% alcohol extract of the bark of Ilex rotunda. Their structures were elucidated by extensive spectroscopic analysis and comparison with the literature data. All new compounds possessed a [5-O-(E)-caffeoyl]-ß-D-apiofuranosyl-(1→6)-ß-D-glucopyranosyl group. The anti-inflammatory properties of all isolated compounds were evaluated using a modified nitric oxide (NO) production in lipopolysaccharide (LPS)-induced leukemia cells in mouse macrophage (RAW264.7) method. Compound 8, dracunculifoside H, showed significant anti-inflammatory activity in vitro.

Triterpenoid saponins of Ilex pubescens against TNF-α induced inflammation and apoptosis in human umbilical vein endothelial cells via autophagy pathway.

OBJECTIVES: Triterpenoid saponins of Ilex pubescens (IPTS), the main active components of Ilex pubescens, has a therapeutic effect on atherosclerosis (AS). The ingredients in IPTS that could be intracellularly transported by human umbilical vein endothelial cells (HUVECs) may play an essential role in AS. This study attempted to explore its mechanism from the perspectives of HUVECs' inflammation, apoptosis, and autophagy. METHODS: By using a tumour necrosis factor-α (TNF-α)-induced HUVECs injury model, cell viability and the expression of intercellular adhesion molecule 1 (ICAM1), matrix metalloproteinase 9 (MMP9), cleave-caspase-3 and cleave-caspase-9, in combination with the results of flow cytometry, JC-1 and Hoechst 33258 staining were investigated to evaluate the anti-inflammatory and anti-apoptotic impact effects of IPTS on HUVECs. Afterwards, the expression of microtubule-associated proteins light chain 3II (LC3II) and sequestosome 1 (p62) was determined to test the effect of IPTS on autophagy. Finally, by adding an autophagy inhibitor 3-methyladenine (3-MA), we investigated whether IPTS exerts anti-inflammatory and anti-apoptotic effects through the autophagy pathway. KEY FINDINGS: We firstly demonstrated that pretreatment with IPTS could increase the cell viability, maintain the cell morphology and reduce TNF-α-induced inflammation and apoptosis of HUVECs. Moreover, IPTS pretreatment was proved to raise the expression of LC3II /LC3I while decreasing the expression of p62, which indicated that IPTS could activate HUVECs' autophagy. IPTS has been shown for the first time to exert anti-inflammatory and anti-apoptotic effects through autophagy and thereby resisting TNF-α-induced inflammatory injury of HUVECs. CONCLUSIONS: This study preliminarily confirmed that IPTS ameliorated HUVECs' inflammation and apoptosis by increasing autophagy.

[Determination of absolute configuration of a new triterpenic acid in leaves of Ilex hainanensis].

This study aimed to explore the triterpenic acid components in leaves of Ilex hainanensis. Alkaline water extraction, macroporous resin adsorption, and high performance liquid chromatography were used to separate and purify the triterpenic acid components in leaves of I. hainanensis. The physical and chemical property analysis, MS, NMR spectroscopy, and literature comparison were performed to identify the structures, and a new triterpene acid compound was discovered:(3S, 4R, 5R, 8R, 9R, 10R, 14S, 17S, 18S, 19R)-3,19-dihydroxyursa-12,20(30)-diene-24,28-dioic-acid, and named ilexhainanin F. In addition, according to its structural characteristics, the ~(19)F-NMR Mosher method was further employed to study its absolute configuration. By comparison of the ~(19)F-NMR chemical shifts of Mosher esters, it was determined that the absolute configuration of the 3-position chiral center of the compound was the S configuration.

The Extract of Ilex cornuta Bark Promotes Bone Healing by Activating Adenosine A2A Receptor.

Introduction: Bone fracture is a common reason causing human disability. The delay union and nonunion rates are approximately 5-10% despite patients receiving active treatment. Currently, there is a limited number of drugs directly accelerating bone healing, especially direct extracts from plants. Moreover, the pharmacological effects of Ilex cornuta bark are still unknown. This study aimed to explore the effects and mechanisms of Ilex cornuta bark in bone healing. Methods and Results: First, the promoting effects of Ilex cornuta bark on bone healing were verified by the mice femur fracture model as Ilex cornuta bark increased the callus formation and enhanced the biomechanical stability during the bone healing process. Second, the target gene of Ilex cornuta bark in bone healing identified by bioinformatics analysis and immunofluorescence validation was ADORA2A. Third, 410 main compound compositions of Ilex cornuta bark were explored by a non-target metabolomic analysis, where 190 of them were neg ion mode, and 220 were pos ion mode. Molecular docking was used to predict the regulatory effect of the compounds on adora2a (adenosine A2A receptor), and ursonic acid had the lowest binding energy with adora2a. Finally, nfkb1 was the transcription factor (TF) of adora2a, and ursonic acid also had the lowest binding energy by bioinformatic analysis and molecular docking. Conclusion: Overall, Ilex cornuta bark water extract was a new plant extract on promoting bone healing; in addition, the mechanism of it might be activating adora2a though Nfkb1.

3,4-Dicaffeoylquinic Acid from the Medicinal Plant Ilex kaushue Disrupts the Interaction Between the Five-Fold Axis of Enterovirus A-71 and the Heparan Sulfate Receptor.

While infections by enterovirus A71 (EV-A71) are generally self-limiting, they can occasionally lead to serious neurological complications and death. No licensed therapies against EV-A71 currently exist. Using anti-virus-induced cytopathic effect assays, 3,4-dicaffeoylquinic acid (3,4-DCQA) from Ilex kaushue extracts was found to exert significant anti-EV-A71 activity, with a broad inhibitory spectrum against different EV-A71 genotypes. Time-of-drug-addition assays revealed that 3,4-DCQA affects the initial phase (entry step) of EV-A71 infection by directly targeting viral particles and disrupting viral attachment to host cells. Using resistant virus selection experiments, we found that 3,4-DCQA targets the glutamic acid residue at position 98 (E98) and the proline residue at position 246 (P246) in the 5-fold axis located within the VP1 structural protein. Recombinant viruses harboring the two mutations were resistant to 3,4-DCQA-elicited inhibition of virus attachment and penetration into human rhabdomyosarcoma (RD) cells. Finally, we showed that 3,4-DCQA specifically inhibited the attachment of EV-A71 to the host receptor heparan sulfate (HS), but not to the scavenger receptor class B member 2 (SCARB2) and P-selectin glycoprotein ligand-1 (PSGL1). Molecular docking analysis confirmed that 3,4-DCQA targets the 5-fold axis to form a stable structure with the E98 and P246 residues through noncovalent and van der Waals interactions. The targeting of E98 and P246 by 3,4-DCQA was found to be specific; accordingly, HS binding of viruses carrying the K242A or K244A mutations in the 5-fold axis was successfully inhibited by 3,4-DCQA.The clinical utility of 3,4-DCQA in the prevention or treatment of EV-A71 infections warrants further scrutiny. IMPORTANCE The canyon region and the 5-fold axis of the EV-A71 viral particle located within the VP1 protein mediate the interaction of the virus with host surface receptors. The three most extensively investigated cellular receptors for EV-A71 include SCARB2, PSGL1, and cell surface heparan sulfate. In the current study, a RD cell-based anti-cytopathic effect assay was used to investigate the potential broad spectrum inhibitory activity of 3,4-DCQA against different EV-A71 strains. Mechanistically, we demonstrate that 3,4-DCQA disrupts the interaction between the 5-fold axis of EV-A71 and its heparan sulfate receptor; however, no effect was seen on the SCARB2 or PSGL1 receptors. Taken together, our findings show that this natural product may pave the way to novel anti-EV-A71 therapeutic strategies.

New benzoic acid and caffeoyl derivatives with anti-inflammatory activities isolated from leaves of Ilex kaushue.

A new benzoic acid, 3-[2-(2-hydroxyphenyl)acetoxy]benzoic acid (1), and two new caffeoyl derivatives, methyl (3E,5Z)-di-O-caffeoylquinate (2) and dhurrin 6'-O-caffeate (3), along with 20 known compounds were isolated from the leaves of Ilex kaushue collected in Vietnam. Their structures were elucidated on the basis of 1 D and 2 D NMR spectroscopy, and high-resolution MS spectrometry. The absolute configuration of 2 and 3 was unambiguously established by comparison of experimental and calculated ECD spectra and/or chemical reactivity. In addition, new compounds were evaluated for inhibitory effects of their tumor necrosis factor-α (TNF-α) production and cell cytotoxicity on lipopolysaccharide-induced RAW264 macrophage cells. All of those moderately suppressed TNF-α production in ratios of approximately 50% or higher at 25-100 µM, without cell cytotoxicity.

Uncovering the molecular mechanisms of Ilex pubescens against myocardial ischemia-reperfusion injury using network pharmacology analysis and experimental pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Ilex pubescens (I. pubescens), has been widely used to treat cardiovascular disease (CVD) in South China. Several studies have revealed aspect of its phytochemistry and pharmacological activities in cardiovascular diseases, but its active compounds and mechanisms of action are still unclear. The aim of this study was to search for the active compounds and the pharmacological mechanisms of I. pubescens for myocardial ischemia-reperfusion injury (MI/RI) by an integrative pharmacology-based investigation. MATERIALS AND METHODS: The main targets of compounds in I. pubescens were predicted using the TargetNet webserver (http://targetnet.scbdd.com). The network between compounds and predicted targets related to MI/RI and compounds was constructed. Functional enrichment analysis was performed to investigate the specific functions and pathways involved in the candidate I. pubescens targets acting on MI/RI, which were further validated by in vitro and in vivo experiments. RESULTS: A total of 191 targets were predicted for 64 chemical compounds in I. pubescens. Following Venn's analysis, we found that 38 candidate targets of I. pubescens were associated with protective effects against MI/RI. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that these targets were related to estrogen signaling pathway. Importantly, the cardioprotective effects of I. pubescens and its active compounds were evaluated and the regulatory effects on key targets of heat shock protein 90 alpha family class A member 1 (HSP90AA1) and Estrogen receptor 1 (ESRα) in estrogen signaling pathway were validated in vitro and in vivo. CONCLUSION: Our discoveries revealed that I. pubescens ameliorated MI/RI by regulating HSP90AA1 and ESRα in estrogen signaling pathway.

Four New Triterpenoid Saponins from the Root of Ilex centrochinensis.

One new siaresinolic acid saponin (1) and three new rotundic acid saponins (2-4) were isolated from the roots of Ilex centrochinensis. Their structures were confirmed by detailed analysis of standard spectroscopic data (IR, MS, 1D and 2D NMR). Compounds 1-4 exhibited anti-inflammatory activity by inhibiting nitric oxide production in a lipopolysaccharide-induced RAW264.7 cell inflammatory model. However, they showed no significant lipid-lowering activity against the production of triglycerides in the lipid-accumulation model of HepG2 cells induced by oleic acid.

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.

New triterpenoid saponins from the leaves of Ilex chinensis and their hepatoprotective activity.

Seven new triterpenoid saponins, including five ursane-type saponins, ilexchinenosides R-V (1-5), and two oleanane-type saponins, ilexchinenosides W-X (6-7), with four known triterpenoid saponins (8-11) were isolated from the leaves of Ilex chinensis. Their structures were elucidated by comprehensive spectroscopic 1D and 2D NMR and HR-ESI-MS data. Their sugar moieties were determined by HPLC analysis compared with standards after hydrolysis and derivatization. Compounds 1, 2, 4, 9 and 10 exhibited potential hepatoprotective activity against N-acetyl-p-aminophenol (APAP)-induced HepG2 cell injury in vitro.

An Improved Synthesis of Water-Soluble Dual Fluorescence Emission Carbon Dots from Holly Leaves for Accurate Detection of Mercury Ions in Living Cells.

BACKGROUND: Carbon dots (CDs) emitting near-infrared fluorescence were recently synthesized from green leaves. However, the Hg2+ detection of CDs was limited because of the insufficient water solubility, low fluorescence and poor stability. METHODS: Dual fluorescence emission water-soluble CD (Dual-CD) was prepared through a solvothermal method from holly leaves and low toxic PEI1.8k. PEG was further grafted onto the surface to improve the water solubility and stability. RESULTS: The Dual-CD solution can emit 487 nm and 676 nm fluorescence under single excitation and exhibit high quantum yield of 16.8%. The fluorescence at 678 nm decreased remarkably while the emission at 470 nm was slightly affected by the addition of Hg2+. The ratiometric Hg2+ detection had a wide linear range of 0-100 µM and low detection limit of 14.0 nM. In A549 cells, there was a good linear relation between F487/F676 and the concentration of Hg2+ in the range of 0-60 µM; the detection limit was 477 nM. Furthermore, Dual-CD showed visual fluorescence change under Hg2+. CONCLUSION: Dual-CD has ratiometric responsiveness to Hg2+ and can be applied for quantitative Hg2+ detection in living cells.

Chemical Composition, Bioactivity and Safety Aspects of Kuding Tea-From Beverage to Herbal Extract.

Kuding tea (KT) is a bitter-tasting herbal tea that has been commonly used in traditional Chinese medicine (TCM). The large-leaved Ku-Ding-Cha (Aquifoliaceae) is composed of its representative species Ilex latifolia Thunb and Ilex kudingcha C.J. Tseng. Because of its potential lipid-lowering, body weight-reducing and blood-glucose-lowering properties, KT has increasingly been recognised for its importance over the past several decades. KT is no longer used only as a beverage, and various extraction methods have been applied to obtain aqueous and ethanolic KT extracts (KTE) or their fractions, which could potentially be used as dietary supplements. The major bioactive components of KT are triterpene saponins and polyphenols, but the composition of KT differs substantially between and among the different KT species. This in turn might affect the physiological effects of KT. KT exhibits antiobesity properties, possibly partly by affecting the intestinal microbiota. In addition, KT may mediate putative antioxidative, anti-inflammatory and anticancer activities. However, there is evidence that high KTE supplementation can adversely affect liver metabolism. The physiological relevance of KT in humans remains rather unclear since the potential health benefits of KT and its constituents reviewed here are mainly derived on the basis of in vitro and animal studies.

A heteropolysaccharide purified from leaves of Ilex latifolia displaying immunomodulatory activity in vitro and in vivo.

A novel polysaccharide (ILP50-2) was extracted, isolated and purified from the leaves of Ilex latifolia Thunb. Its structure was characterized as a repeating unit consisting of α-L-Araf-(1→, →3)-α-L-Araf-(1→, →5)-α-L-Araf-(1→, →3,5)-α-L-Araf-(1→, →2)-α-L-Rhap-(1→, →2,4)-α-L-Rhap-(1→, ß-D-Galp-(1→, →4)-ß-D-Galp-(1→, →4)-ß-D-Glcp-(1→, →6)-α-D-Manp-(1→, and →3,6)-α-D-Galp-(1→. The absolute molecular weight of ILP50-2 was 1.49 × 105 g/mol, which adapted a compact coil conformation in 0.1 M NaCl solution with Rz of 25.4 nm. Furthermore, ILP50-2 exhibited immunoregulatory activity, mainly through enhancing the phagocytosis ability of macrophages and prompting the release of nitric oxide (NO) and cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß). Simultaneously, ILP50-2 was found to significantly increase the release of ROS and NO in zebrafish embryos, showing immunoregulatory effects in vivo.