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.
Chromatography, High Pressure Liquid/methods* ; Ilex/chemistry* ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Plant Leaves/chemistry* ; Triterpenes/analysis*

The chemical constituents from the leaves of Ilex guayusa were investigated. Sixteen triterpenoids were isolated from the 95% ethanol extract of dried leaves of I. guayusa by silica gel, Sephadex LH-20, and ODS column chromatographies and semi-prepa-rative HPLC. Those triterpenoids were identified by NMR, HR-MS, and literature analysis: 3β-hydroxy-11α,12α-epoxy-24-nor-urs-4(23)-ene-28,13β-olide(1), 3β-hydroxy-24-nor-4(23),12-oleanadien-28-methyl ester(2), oleanolic acid(3), 3β,28-dihydroxy-12-oleanene(4), 2α,3β-dihydroxy-11α,12α-epoxy-24-'nor-olean-4(23)-ene-28,13β-olide(5), ursolic acid(6), 3β,23-dihydroxy ursolic acid(7), 3β,28-dihydroxy-12-ursene(8), 3β-28-nor-urs-12-ene-3,17-diol(9), 3β-hydroxyurs-11-ene-28,13β-olide(10), 13β,28-epoxy-3β-hydroxy-11-ursene(11), 3β-hydroxy-28,28-dimethoxy-12-ursene(12), 3β-hydroxy-24-nor-urs-4(23),12-dien-28-oic acid(13), 3β-hydroxy-24-nor-urs-4(23),12-dien-28-methyl ester(14), 2α,3β-dihydroxy-11α,12α-epoxy-24-nor-urs-4(23)-ene-28,13β-olide(15) and 2α,3β-dihydroxy-11α,12α-epoxy-24-nor-urs-4(23),20(30)-dien-28,13β-olide(16). Compounds 1-2 were new compounds, and compounds 4-5, 7 and 9-16 were isolated from I. guayusa for the first time.
Drugs, Chinese Herbal ; Ilex guayusa ; Molecular Structure ; Oleanolic Acid ; Plant Leaves ; Triterpenes

This study intends to develop a high performance liquid chromatography-diode array detection(HPLC-DAD) method for simultaneous determination of chlorogenic acid, 2-hydroxymethyl-3-hydroxyl-1-butene-4-O-β-D-(6″-O-caffeoyl)-glucopyranoside, pubescenoside B, huazhongilexone-7-O-β-D-glucopyranoside, rutin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C in Ilex hainanensis. The HPLC conditions are as follows: Waters XBridge C_(18 )column(4.6 mm×250 mm, 5 μm), mobile phase of 0.5% formic acid in water(A)-acetonitrile(B), gradient elution(0-8 min, 5%-12% B; 8-18 min, 12%-18% B; 18-30 min, 18%-25% B; 30-40 min, 25%-30% B; 40-42 min, 30%-80% B; 42-45 min, 80% B) at the flow rate of 0.8 mL·min~(-1), detection wavelengths of 282, 324, and 360 nm, column temperature of 25 ℃, and injection volume of 5 μL. The content of the 8 phenols in 8 samples was 0.30-6.29, 0.29-3.27, 0.15-10.4, 0.51-5.85, 0.49-9.02, 0.51-4.68, 1.93-13.4, and 0.87-5.95 mg·g~(-1), respectively. Moreover, the content of phenols in the samples collected in October was higher than that of samples harvested in other months. The established method is accurate and sensitive for the determination of phenols in I. hainanensis, which is useful for the quality improvement of this herbal medicine.
Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; Ilex ; Phenols

Cardio-cerebral vascular disease induced by atherosclerosis is a serious cause of human health. The pathogenesis of AS is very complex,and the oxidized low-density lipoprotein( ox LDL) induced foam cells formation is considered to be the most important cytological change in AS. Based on the definition of " TCM chemical biology",we clarified the chemical composition of Ilex hainanensis,the effective substances of I. hainanensis on the activity of anti-AS were screened. Then we found that saponin BF523 had the good inhibitory effect on foam cell formation. In this research,we studied the BF523 as the research object to clarify the molecular target of the active compound of I. hainanensis by foam cell formation model. The results showed that BF523 significantly inhibited the oxidation of ox LDL-induced macrophage foaming and decreased the lipid content in macrophages. BF523 had inhibited the phagocytosis of ox LDL in macrophages by reducing the mRNA and protein levels of scavenger receptor CD36,thereby inhibiting the occurrence and development of AS. These findings not only clarified the mechanism of the inhibition of foam cell formation by saponin BF523,but also provided a useful exploration for the enrichment of the theory of " TCM chemical biology".
Atherosclerosis ; CD36 Antigens ; metabolism ; Cells, Cultured ; Foam Cells ; cytology ; drug effects ; Humans ; Ilex ; chemistry ; Lipoproteins, LDL ; adverse effects

The present study was conducted to investigate anti-nociceptive and anti-inflammatory effects of the leaves of Ilex latifolia Thunb (I. latifolia) in in vivo and in vitro. Writhing responses induced by acetic acid and formalin- and thermal stimuli (tail flick and hot plate tests)-induced pain responses for nociception were evaluated in mice. I. latifolia (50 – 200 mg/kg, p.o.) and ibuprofen (100 mg/kg, p.o.), a positive non-steroidal anti-inflammatory drug (NSAID), inhibited the acetic acid-induced writhing response and the second phase response (peripheral inflammatory response) in the formalin test, but did not protect against thermal nociception and the first phase response (central response) in the formalin test. These results show that I. latifolia has a significant anti-nociceptive effect that appears to be peripheral, but not central. Additionally, I. latifolia (50 and 100 µg/mL) and 3,5-di-caffeoyl quinic acid methyl ester (5 µM) isolated from I. latifolia as an active compound significantly inhibited LPS-induced NO production and mRNA expression of the pro-inflammatory mediators, iNOS and COX-2, and the pro-inflammatory cytokines, IL-6 and IL-1β, in RAW 264.7 macrophages. These results suggest that I. latifolia can produce antinociceptive effects peripherally, but not centrally, via anti-inflammatory activity and supports a possible use of I. latifolia to treat pain and inflammation.
Acetic Acid ; Animals ; Cyclooxygenase 2 ; Cytokines ; Ibuprofen ; Ilex ; In Vitro Techniques ; Inflammation ; Interleukin-6 ; Macrophages ; Mice ; Nitric Oxide ; Nociception ; Pain Measurement ; Quinic Acid ; RNA, Messenger

Phytochemical investigation on Ilex asprella stems by using various chromatographic techniques led to the isolation of 18 phenolic constituents. Based on spectroscopic data analyses and/or comparison of the spectroscopic data with those in literature, these constituents were identified, including two lignans (1, 2), five phenylpropanes (3-7), six chlorogenic analogues (8-13), and five benzoic analogues (14-18). Among them, compounds 3-7, 9, 11, 13, 14, 17, and 18 were isolated from genus Ilex for the first time, and 2, 8, 10, 15, and 16 were isolated from this species for the first time. The in vitro anti-inflammatory assay results showed that compounds 8, 9, 11, 13, and 15 possessed moderate inhibition on the NO production in RAW264.7 cells with IC₅₀ values of 51.1-85.8 μmol·L⁻¹. The present study brought preliminary reference for the clarification of therapeutic ingredients of I. asprella with anti-inflammatory efficacy and its quality evaluation.
Animals ; Anti-Inflammatory Agents ; isolation & purification ; pharmacology ; Ilex ; chemistry ; Mice ; Nitric Oxide ; metabolism ; Phenols ; chemistry ; Phytochemicals ; isolation & purification ; pharmacology ; Plant Stems ; chemistry ; RAW 264.7 Cells

Previous studies have indicated that the Ilex genus exhibits antioxidant, neuroprotective, hepatoprotective, and anti-inflammatory activities. However, the pharmacologic action and mechanisms of Ilex cornuta against cardiac diseases have not yet been explored. The present study was designed to investigate the antioxidant and cardioprotective effects of Ilex cornuta root with in vitro and in vivo models. The anti-oxidative effects of the extract of Ilex cornuta root (ICR) were measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging and MTT assays as well as immunoassay. Furthermore, a rat model of myocardial ischemia was established to investigate the cardioprotective effect of ICR in vivo. Eight compounds were isolated and identified from ICR and exhibited DPPH free-radical scavenging activities. They also could increase cell viability and inhibit morphological changes induced by HO or NaSO in H9c2 cardiomyocytes, followed by increasing the SOD activities and decreasing the MDA and ROS levels. In addition, it could suppress the apoptosis of cardiomyocytes. In the rat model of myocardial ischemia, ICR decreased myocardial infarct size and suppressed the activities of LDH and CK. Furthermore, ICR attenuated histopathological alterations of heart tissues and the MDA levels, while increasing SOD activities in serum. In conclusion, these results suggest that ICR has cardioprotective activity and could be developed as a new food supplement for the prevention of ischemic heart disease.
Animals ; Antioxidants ; metabolism ; pharmacology ; therapeutic use ; Apoptosis ; Cardiovascular Agents ; pharmacology ; therapeutic use ; Cell Survival ; drug effects ; Hydrogen Peroxide ; metabolism ; Ilex ; Malondialdehyde ; metabolism ; Myocardial Infarction ; Myocardial Ischemia ; drug therapy ; metabolism ; pathology ; Myocardium ; cytology ; pathology ; Myocytes, Cardiac ; drug effects ; Oxidative Stress ; drug effects ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Plant Roots ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism

In the present study, three new triterpenoids, 23-hydroxyurs-12, 18-dien-28-oic acid 3β-O-α-L-arabinopyranoside (1), 23-hydroxyurs-12, 18-dien-28-oic acid 3β-O-β-D-glucuronopyranoside-6-O-methyl ester (2), and urs-12, 18-dien-28-oic acid 3β-O-β-D-glucuronopyranoside-6-O-methyl ester (3), and a known triterpenoid, 3β-hydroxy-urs-2, 18-dien-28-oic acid (4, randialic acid B), were isolated from the aerial parts of Ilex cornuta. Their structures were identified by the spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, and 1D and 2D NMR) and chemical reactions. Compound 4 showed significant cell-protective effects against HO-induced H9c2 cardiomyocyte injury. Compounds 1-4 did not show any significant DPPH radical scavenging activity.
Animals ; Biphenyl Compounds ; metabolism ; Cardiovascular Agents ; chemistry ; isolation & purification ; pharmacology ; Hydrogen Peroxide ; metabolism ; Ilex ; chemistry ; Molecular Structure ; Myocardium ; cytology ; pathology ; Myocytes, Cardiac ; drug effects ; Picrates ; metabolism ; Plant Components, Aerial ; chemistry ; Plant Extracts ; chemistry ; pharmacology ; Rats ; Triterpenes ; chemistry ; isolation & purification ; pharmacology

BACKGROUND: The extract and hemiterpene glycosides of Ilex Rotunda Thunb exert antioxidant and anti-inflammatory effects. The effect of rotundarpene on apoptosis in neuronal cells caused by the 1-methyl-4-phenylpyridinium (MPP⁺) has not been reported previously. METHODS: Using differentiated PC12 cells and human neuroblastoma SH-SY5Y cells, we investigated the effect of rotundarpene on MPP⁺-caused apoptosis in relation to the cell death process. RESULTS: MPP⁺-induced cell death was identified using the MTT and neutral red uptake tests. Apoptosis was induced by eliciting decreases in the cytosolic levels of Bid and Bcl-2 proteins, increases in the cytosolic levels of Bax and p53, disruption of the mitochondrial transmembrane potential, and the release of cytochrome c and the activation of caspase-8, -9, and -3 in differentiated PC12 cells and SH-SY5Y cells. Treatment with rotundarpene reduced the MPP⁺-induced changes in the levels of apoptosis-regulated proteins, formation of reactive oxygen species, depletion and oxidation of glutathione, and cell death in both PC12 and SH-SY5Y cells. CONCLUSIONS: Rotundarpene may reduce MPP⁺-induced apoptosis in neuronal cells by suppressing the activation of the mitochondria-mediated pathway and the caspase-8 and Bid pathways. Rotundarpene appears to act by inhibiting the production of reactive oxygen species and by the depletion and oxidation of glutathione.
1-Methyl-4-phenylpyridinium ; Animals ; Apoptosis ; Caspase 8 ; Cell Death* ; Cytochromes c ; Cytosol ; Glutathione ; Glycosides ; Humans ; Ilex ; Membrane Potentials ; Neuroblastoma ; Neurons ; Neutral Red ; PC12 Cells ; Reactive Oxygen Species

This study was carried out to evaluate the anti-inflammatory and free radical scavenging activities of flavans from flex centrochinensis S. Y. Hu in vitro and their structure-activity relationship. LPS-stimulated RAW 264.7 macrophage was used as inflammatory model. MTT assay for cell availability, Griess reaction for nitric oxide (NO) production, the content of TNF-alpha, IL-1beta, IL-6 and PGE, were detected with ELISA kits; DPPH, superoxide anion and hydroxyl free radicals scavenging activities were also investigated. According to the result, all flavans tested exhibited anti-inflammatory effect in different levels. Among them, compounds 1, 3, 4 and 6 showed potent anti-inflammatory effect through the inhibition of NO, TNF-alpha, IL-lp and IL-6, of which 1 was the most effective inhibitor, however, 2 and 5 were relatively weak or inactive. The order of free radical scavenging activities was similar to that of anti-inflammatory activities. Therefore, these results suggest that 3, 4 and 6, especially of 1, were,in part responsible for the anti-inflammatory and free radical scavenging activity of Ilex centrochinensis. Hydroxyl group at 4'-position of B-ring plays an important role in the anti-inflammatory and free radical scavenging capacities.
Animals ; Anti-Inflammatory Agents, Non-Steroidal ; chemistry ; pharmacology ; Cell Line ; Cyclooxygenase 2 ; immunology ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Flavanones ; chemistry ; pharmacology ; Free Radical Scavengers ; chemistry ; pharmacology ; Ilex ; chemistry ; Interleukin-6 ; immunology ; Macrophages ; drug effects ; immunology ; Mice ; Nitric Oxide ; immunology ; Tumor Necrosis Factor-alpha ; immunology