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.

Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry Identification of Metabolites in Winterberry Fruit Putatively Associated with Natural Disease Resistance to Diaporthe ilicicola.

Winterberry holly (Ilex verticillata) is an ornamental plant popularly used in landscape design and sold as cut branches for fall and winter seasonal decoration. Latent fruit rot of winterberry is an emerging disease caused by the fungus Diaporthe ilicicola, which can result in up to 100% crop loss. Diaporthe ilicicola infects open flowers in spring, but symptom onset does not occur until the end of the growing season when the fruit is fully mature. This study was conducted to identify compounds displaying significant variation in abundance during fruit maturation and that may be putatively associated with natural disease resistance observed when the fruit is immature. Winterberry 'Sparkleberry' fruits collected at four timepoints during the 2018 and 2019 seasons were extracted in methanol and analyzed using high resolution ultra-high performance liquid chromatography-tandem mass spectrometry. The results showed a distinct separation of metabolic profiles based on fruit phenological stage. The top 100 features that were differentially expressed between immature and mature fruit were selected from both electrospray ionization (ESI) (-) and ESI (+) datasets for annotation. Eleven compounds shown to decrease throughout the season included cinnamic acids, a triterpenoid, terpene lactones, stilbene glycosides, a cyanidin glycoside, and a furopyran. Nine compounds shown to accumulate throughout the season included chlorogenic acid derivatives, hydrolysable tannins, flavonoid glycosides, and a triterpene saponin. Future research will further confirm the exact identity of the compounds of interest and determine whether they are biologically active toward D. ilicicola or I. verticillata. The results could inform breeding programs, chemical management programs, and novel antifungal compound development pipelines.

Holly Ingraham.

Holly Ingraham examines biological sex differences in the brain and body across the lifespan from endocrine, metabolic, and neuroscientific perspectives. She highlights the significance of including females in experiments, the benefits of reading classic studies, and the importance of posing great questions.

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.

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.

Assembly and comparative analysis of the complete mitochondrial genome of Ilex metabaptista (Aquifoliaceae), a Chinese endemic species with a narrow distribution.

BACKGROUND: Ilex metabaptista is a woody tree species with strong waterlogging tolerance and is also admired as a landscape plant with high development prospects and scientific research value. Unfortunately, populations of this species have declined due to habitat loss. Thus, it is a great challenge for us to efficiently protect I. metabaptista resources from extinction. Molecular biology research can provide the scientific basis for the conservation of species. However, the study of I. metabaptista genetics is still in its infancy. To date, no mitochondrial genome (mitogenome) in the genus Ilex has been analysed in detail. RESULTS: The mitogenome of I. metabaptista was assembled based on the reads from Illumina and Nanopore sequencing platforms; it was a typical circular DNA molecule of 529,560 bp with a GC content of 45.61% and contained 67 genes, including 42 protein-coding genes, 22 tRNA genes, and 3 rRNA genes. Repeat sequence analysis and prediction of RNA editing sites revealed a total of 286 dispersed repeats, 140 simple repeats, 18 tandem repeats, and 543 RNA editing sites. Analysis of codon usage showed that codons ending in A/T were preferred. Gene migration was observed to occur between the mitogenome and chloroplast genome via the detection of homologous fragments. In addition, Ka/Ks analysis revealed that most of the protein-coding genes in the mitogenome had undergone negative selection, and only the ccmB gene had undergone potential positive selection in most asterids. Nucleotide polymorphism analysis revealed the variation in each gene, with atp9 being the most notable. Furthermore, comparative analysis showed that the GC contents were conserved, but the sizes and structure of mitogenomes varied greatly among asterids. Phylogenetic analysis based on the mitogenomes reflected the exact evolutionary and taxonomic status of I. metabaptista. CONCLUSION: In this study, we sequenced and annotated the mitogenome of I. metabaptista and compared it with the mitogenomes of other asterids, which provided essential background information for further understanding of the genetics of this plant and helped lay the foundation for future studies on molecular breeding of I. metabaptista.

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.

A first insight into the genomic background of Ilex pubescens (Aquifoliaceae) by flow cytometry and genome survey sequencing.

BACKGROUND: Ilex pubescens is an important traditional Chinese medicinal plant with many naturally occurring compounds and multiple pharmacological effects. However, the lack of reference genomic information has led to tardiness in molecular biology research and breeding programs of this plant. RESULTS: To obtain knowledge on the genomic information of I. pubescens, a genome survey was performed for the first time by next generation sequencing (NGS) together with genome size estimation using flow cytometry. The whole genome survey of I. pubescens generated 46.472 Gb of sequence data with approximately 82.2 × coverage. K-mer analysis indicated that I. pubescens has a small genome of approximately 553 Mb with 1.93% heterozygosity rate and 39.1% repeat rate. Meanwhile, the genome size was estimated to be 722 Mb using flow cytometry, which was possibly more precise for assessment of genome size than k-mer analysis. A total of 45.842 Gb clean reads were assembled into 808,938 scaffolds with a relatively short N50 of 760 bp. The average guanine and cytosine (GC) content was 37.52%. In total, 197,429 microsatellite motifs were detected with a frequency of 2.8 kb, among which mononucleotide motifs were the most abundant (up to 62.47% of the total microsatellite motifs), followed by dinucleotide and trinucleotide motifs. CONCLUSION: In summary, the genome of I. pubescens is small but complex with a high level of heterozygosity. Even though not successfully applied for estimation of genome size due to its complex genome, the survey sequences will help to design whole genome sequencing strategies and provide genetic information support for resource protection, genetic diversity analysis, genetic improvement and artificial breeding of I. pubescens.

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.

Understanding Environmental and Physiological Factors Affecting the Biology of Diaporthe ilicicola, the Fungus Causing Latent Fruit Rot in Winterberry.

Fruit rot in winterberry is associated with a complex of fungal pathogens. Among them, Diaporthe ilicicola plays a unique role by infecting flowers at bloom, resulting in symptom development in mature fruit. This research aimed to identify at what stage of maturation Ilex fruit can develop disease symptoms and correlate changes in fruit physiology (sugar and phenolic content) and environment (temperature and light intensity) with disease incidence. Correlation data informed in vitro studies testing the ability of putative factors to alter growth of D. ilicicola and select opportunistic fungi within the fruit rot complex: Alternaria alternata, Colletotrichum fioriniae, and Epicoccum nigrum. Results indicated that Ilex fruit do not develop symptoms until 81 to 108 days after inoculation. Temperature and fruit phenolic content were negatively correlated with disease incidence, while fruit sugar concentration and light intensity were positively correlated. In vitro assays revealed that sugar concentration had no effect on the growth of D. ilicicola, but increased light intensity increased hyphal growth and pycnidium formation. Additionally, phenolics extracted from fruit inhibited spore germination in A. alternata, induced secondary conidiation in C. fioriniae, and late season phenolic extracts increased hyphal melanization and pycnidial formation in D. ilicicola. Finally, drops in field temperatures, when replicated in vitro, resulted in a decrease in hyphal growth and spore germination for all fungi. These results suggest that changes in Ilex fruit phenolics during maturation and the increased exposure to light following defoliation may play a role in symptom development by altering D. ilicicola growth within the fruit.

Bioassay guided isolation of caffeoylquinic acids from the leaves of Ilex pubescens Hook. et Arn. and investigation of their anti-influenza mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Ilex pubescens Hook. et Arn. (Maodongqing, MDQ) is a common herbal tea ingredient in Southern China for heat clearance and anti-inflammation. Our preliminary screening showed that 50% ethanol extract of its leaves has anti-influenza virus activity. In this report, we proceed to identify the active components and clarify the related anti-influenza mechanisms. AIM: We aim to isolate and identify the anti-influenza virus phytochemicals from the extract of the MDQ leaves, and study their anti-influenza virus mechanism. MATERIAL AND METHODS: Plaque reduction assay was used to test the anti-influenza virus activity of fractions and compounds. Neuraminidase inhibitory assay was used to confirm the target protein. Molecular docking and reverse genetics were used to confirm the acting site of caffeoylquinic acids (CQAs) on viral neuraminidase. RESULTS: Eight CQAs, 3,5-di-O-caffeoylquinic acid methyl ester (Me 3,5-DCQA), 3,4-di-O-caffeoylquinic acid methyl ester (Me 3,4-DCQA), 3,4,5-tri-O-caffeoylquinic acid methyl ester (Me 3,4,5-TCQA), 3,4,5-tri-O-caffeoylquinic acid (3,4,5-TCQA), 4,5-di-O-caffeoylquinic acid (4,5-DCQA), 3,5-di-O-caffeoylquinic acid (3,5-DCQA), 3,4-di-O-caffeoylquinic acid (3,4-DCQA), and 3,5-di-O-caffeoyl-epi-quinic acid (3,5-epi-DCQA) were identified from the MDQ leaves, in which Me 3,5-DCQA, 3,4,5-TCQA and 3,5-epi-DCQA were isolated for the first time. All these eight compounds were found to inhibit neuraminidase (NA) of influenza A virus. The results of molecular docking and reverse genetics indicated that 3,4,5-TCQA interacted with Tyr100, Gln412 and Arg419 of influenza NA, and a novel NA binding groove was found. CONCLUSION: Eight CQAs isolated from the leaves of MDQ were found to inhibit influenza A virus. 3,4,5-TCQA was found to interact with Tyr100, Gln412 and Arg419 of influenza NA. This study provided scientific evidence on the use of MDQ for treating influenza virus infection, and laid the foundation for the development of CQA derivatives as potential antiviral agents.

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.

Triterpenoids from the genus Ilex attenuate free fatty acid-induced lipid accumulation in HepG2 cells by regulating lipid metabolism disorder and the AMPK signalling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Various traditional Chinese medicines from the genus Ilex (Aquifoliaceae) have been reported to have excellent hypolipidaemic effects. Although triterpenoids have been found to be the main active components, the underlying mechanisms have not been clarified. AIM OF THE STUDY: This study aimed to investigate the lipid-lowering effect, structure-activity relationship and action mechanism of triterpenoids from the genus Ilex. MATERIALS AND METHODS: FFA was used to induce HepG2 cells to establish a classical lipid-lowering activity screening model for the activities of 31 triterpenoids, and the contents of intracellular lipids, TC, and TG were measured. Furthermore, the structure-activity relationship was discussed. Mechanistically, UPLC-Q/TOF-MS-based metabolomics and lipidomics studies were performed, and metabolic pathways were analysed to investigate the lipid-lowering mechanism. Moreover, western blotting was performed to analyse the expression of key proteins of lipid metabolism and predict the targets of action. RESULTS: Thirteen triterpenoids significantly reduced intracellular lipid accumulation and decreased the levels of TG and TC. Among them, rotundic acid (RA) showed stronger lipid-lowering activity than the simvastatin-positive group, and structure-activity relationship analysis indicated that the hydroxyl groups at C-3 and C-19, hydroxymethyl groups at C-23, and carboxyl groups at C-28 may be the key groups for biological activity. Twenty-two metabolites in the metabolomics study and 19 metabolites in the lipidomics study were identified. The identified biomarkers were primarily glycerophosphocholine, LysoPCs, PCs, TAGs, LysoPEs, LysoPIs and sphingolipids, which are involved in glycerophospholipid and sphingolipid metabolism. Moreover, western blotting analysis showed that the expression of SREBP-1 and HMGCR decreased, while AMPK and ACC phosphorylation and the expression of CPT1A and CYP7A1 increased in the RA-treated group. CONCLUSION: The results suggested that triterpenoids from the genus Ilex showed significant lipid-lowering effects and that RA may be a novel hypolipidaemic drug candidate. Moreover, the underlying mechanism indicated that RA showed a lipid-lowering effect by regulating glycerophospholipid and sphingolipid metabolism and activating the AMPK pathway.

Anti-inflammatory activity of caffeic acid derivatives from Ilex rotunda.

Ilex rotunda Thunb. has been used in traditional medicine for treating rheumatoid arthritis, relieving pain and indigestion. In the present study, we isolated three new caffeic acid benzyl ester (CABE) analogs (1-3) along with eight known compounds (4-11) from the extract of I. rotunda. The absolute configuration of α-hydoxycarboxylic acid in 1 was assigned with the phenylglycine methyl ester (PGME) method. We further investigated their anti-inflammatory activities in lipopolysaccharide (LPS)-induced macrophages (RAW 264.7) cells. Among them, compounds 2-4, 7, 8, 10, and 11 suppressed the production of nitric oxide (NO), pro-inflammatory mediators. It was additionally confirmed that the anti-inflammatory effect of active compound 2 was through significant suppression of cytokines, including interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α, and IL-8 in LPS-stimulated RAW 264.7 cells and colon epithelial (HT-29) cells. Western blot analysis revealed that compound 2 decreased the LPS-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and phosphorylated extracellular regulated kinase (pERK)1/2. The following molecular docking simulations showed the significant interactions of compound 2 with the iNOS protein. These results suggested that the compound 2 can be used as potential candidate for treating inflammatory diseases such as inflammatory bowel disease (IBD).

Morpho-physiological and demographic responses of three threatened Ilex species to changing climate aligned with species distribution models in future climate scenarios.

The success of a species in future climate change scenarios depends on its morphological, physiological, and demographic adaptive responses to changing climate. The existence of threatened species against climate adversaries is constrained due to their small population size, narrow genetic base, and narrow niche breadth. We examined if ecological niche model (ENM)-based distribution predictions of species align with their morpho-physiological and demographic responses to future climate change scenarios. We studied three threatened Ilex species, viz., Ilex khasiana Purkay., I. venulosa Hook. f., and I. embelioides Hook. F, with restricted distribution in Indo-Burma biodiversity hotspot. Demographic analysis of the natural populations of each species in Meghalaya, India revealed an upright pyramid suggesting a stable population under the present climate scenario. I. khasiana was confined to higher elevations only while I. venulosa and I. embelioides had wider altitudinal distribution ranges. The bio-climatic niche of I. khasiana was narrow, while the other two species had relatively broader niches. The ENM-predicted potential distribution areas under the current (2022) and future (2050) climatic scenarios (General Circulation Models (GCMs): IPSL-CM5A-LR and NIMR-HADGEM2-AO) revealed that the distribution of highly suitable areas for the most climate-sensitive I. khasiana got drastically reduced. In I. venulosa and I. embelioides, there was an increase in highly suitable areas under the future scenarios. The eco-physiological studies showed marked variation among the species, sites, and treatments (p < 0.05), indicating the differential responses of the three species to varied climate scenarios, but followed a similar trend in species performance aligning with the model predictions.

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.