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).

Chromosomal level genome of Ilex asprella and insight into antiviral triterpenoid pathway.

Ilex asprella is a widely used herbs in Traditional Chinese Medicine for treating viral infection and relieving inflammation. Due to the earlier fruiting period of I. asprella, it is the major food source for frugivores in summer. Despite its pharmacological and ecological importance, a reference genome for I. asprella is lacking. By using Illumina, stLFR and Omni-C sequencing data, we present the first chromosomal-level assembly for I. asprella. The genome assembly size is 804 Mbp, with Benchmarking Universal Single-Copy Orthologs (BUSCO) score 94.4% for eudicotyledon single copy genes. Transcriptomes of leaves, stems, flowers, premature fruits and roots were analyzed, providing 39,215 gene models. The complete set of genes involved in the triterpenoids production is disclosed for the first time. We have also found the oxidosqualene cyclases (OSCs), CYP716s and UDP-glycosyltransferases (UGTs), which are responsible for the modification of triterpenoid backbones, resulting in the high variety of triterpenoid saponins.

Polyphenols in Ilex latifolia Thunb. inhibit human lung cancer cell line A549 by regulation of the PI3K-Akt signaling pathway.

BACKGROUND: The leaves of the plant Ilex latifolia Thunb. can be made into Kuding tea, which is a drink rich in polyphenols. This study aimed to observe the effect of Ilex latifolia Thunb. polyphenols (ILTPs) on human lung cancer cell line A549 (A549 cells) by regulating the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. METHODS: In vitro cultured cells were treated with ILTPs; the proliferation of A549 cells and BEAS-2B human normal lung epithelial cells (Beas-2B cells) was observed using the 3-(4,5-dimethylazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the survival status of A549 cells was observed by fluorescence staining. The expression of A549 cells was observed by quantitative polymerase chain reaction (qPCR) assay and Western blot analysis, while the compound composition of ILTPs was detected using high-performance liquid chromatography (HPLC). RESULTS: The experimental results showed that the proliferation of Beas-2B cells was unaffected by treatment with 0-500 µg/mL of ILTPs, whereas the decreased proliferation of A549 cells was observed with the increasing concentrations of ILTPs. Additionally, ILTPs elevated the levels of lactate dehydrogenase (LDH) and reactive oxygen species (ROS) and promoted apoptosis in A549 cells. The results of qPCR experiments showed that ILTPs upregulated caspase-9 mRNA expression and downregulated phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), B-cell lymphoma-2 (Bcl-2), nuclear factor-κB (NF-κB), vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 alpha (HIF-1α), and cyclooxygenase-2 (COX-2) expression in A549 cells. The Western blot analysis results also showed that ILTPs could reduce the protein expression of PI3K and Akt. The HPLC results showed that the main compounds present in the ILTPs were rutin, kaempferol, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C. CONCLUSIONS: Thus, this study indicated that the polyphenols of I. latifolia act as a class of natural functional food materials that potently suppress cancer by exerting their inhibitory effects on A549 cell proliferation through five key polyphenolic compounds.

LC-Q-TOF/MS-oriented systemic metabolism study of pedunculoside with in vitro and in vivo biotransformation.

As a triterpene saponin, pedunculoside is one of the most abundant, representative and active components in plants of genus Ilex (Aquifoliaceae). Pedunculoside has been used to treat myocardial ischemia, ameliorate hyperlipidemia and prevent liver injury. In this paper, a systemic in vitro liver microsomes / S9 and intestinal bacteria incubation, and in vivo animal experiment were performed, using LC-Q-TOF/MS analysis and a three-step data processing protocol. As a result, Bifidobacterium adolescentis and Bifidobacterium breve were identified to potentially metabolize pedunculoside among the intestinal bacteria tested. A total of 11 metabolites were found and tentatively identified, with 6 in both microsomal and bacterial incubation systems, and 9 after rats orally administered with pedunculoside. The metabolites detected involving both phase I and phase II metabolism, mainly through deglycosylation (hydrolyzation), dehydrogenation, hydroxylation and conjugation, and some of them underwent more than one-step metabolic reactions. Most of the metabolites have not been reported before. In vitro, liver microsome and intestinal bacteria prefer to metabolize pedunculoside in totally different ways; while in vivo, intestinal tract is the most important site for the metabolism and excretion of pedunculoside, where both intestinal bacteria and the host metabolic enzymes participate in its metabolism and disposition. The importance of intestinal bacteria should be highlighted. This study would contribute to a better understanding of pedunculoside metabolism, which can provide scientific evidence for its pharmacodynamic mechanism research and prove its clinical application.

Molecular Docking and Molecular Dynamics Studies on Selective Synthesis of α-Amyrin and ß-Amyrin by Oxidosqualene Cyclases from Ilex Asprella.

Amyrins are the immediate precursors of many pharmaceutically important pentacyclic triterpenoids. Although various amyrin synthases have been identified, little is known about the relationship between protein structures and the constituent and content of the products. IaAS1 and IaAS2 identified from Ilex asprella in our previous work belong to multifunctional oxidosqualene cyclases and can produce α-amyrin and ß-amyrin at different ratios. More than 80% of total production of IaAS1 is α-amyrin; while IaAS2 mainly produces ß-amyrin with a yield of 95%. Here, we present a molecular modeling approach to explore the underlying mechanism for selective synthesis. The structures of IaAS1 and IaAS2 were constructed by homology modeling, and were evaluated by Ramachandran Plot and Verify 3D program. The enzyme-product conformations generated by molecular docking indicated that ASP484 residue plays an important role in the catalytic process; and TRP611 residue of IaAS2 had interaction with ß-amyrin through π-σ interaction. MM/GBSA binding free energy calculations and free energy decomposition after 50 ns molecular dynamics simulations were performed. The binding affinity between the main product and corresponding enzyme was higher than that of the by-product. Conserved amino acid residues such as TRP257; TYR259; PHE47; TRP534; TRP612; and TYR728 for IaAS1 (TRP257; TYR259; PHE473; TRP533; TRP611; and TYR727 for IaAS2) had strong interactions with both products. GLN450 and LYS372 had negative contribution to binding affinity between α-amyrin or ß-amyrin and IaAS1. LYS372 and ARG261 had strong repulsive effects for the binding of α-amyrin with IaAS2. The importance of Lys372 and TRP612 of IaAS1, and Lys372 and TRP611 of IaAS2, for synthesizing amyrins were confirmed by site-directed mutagenesis. The different patterns of residue-product interactions is the cause for the difference in the yields of two products.

LC-MS Metabolomics and Chemotaxonomy of Caffeine-Containing Holly ( Ilex) Species and Related Taxa in the Aquifoliaceae.

Ilex species have been consumed traditionally as medicinal teas worldwide. Though the presence of caffeine has been reported in several species, little is known about secondary-metabolite diversity within and among these taxa. Leaf samples of Ilex guayusa, Ilex paraguariensis, and Ilex vomitoria were analyzed by liquid chromatography-mass spectrometry and comparative metabolite profiling with Ilex cassine and other Ilex species to identify chemotaxonomic markers, delimit species, and provide an assessment of chemodiversity. Purine alkaloids were detected and quantified in I. guayusa, I. paraguariensis, and I. vomitoria. Reports of caffeine for I. cassine were not corroborated, suggesting that I. vomitoria was the traditional source of the Native North American tea yaupon. The tetramethyluric acid, theacrine, was detected for the first time in the genus Ilex as a low-level chemotaxonomic marker in I. vomitoria samples. Chemotaxonomy and metabolomics support a close relationship for caffeine-containing Ilex species.

Effects of TNT contaminated soil on vegetation at an explosive range by probing UPLC-qTOF MS profiling method.

Three tree species (Wild olive, Stinkwood and Cape Holy) and a shrub (Dovyalis caffra) were each potted in 20 L pots in order to evaluate the effect of 1,3,5-trinitrotoluene (TNT)-contaminated soil on vegetation. TNT contamination was established by dissolving flake TNT in acetone at 300 and 600 mg per kilogram soil concentrations. One pot for every species was left uncontaminated as control elements. A set of 16 samples, four contaminated, four uncontaminated aerial parts and their corresponding soils, were gathered. These were processed and subjected to a solid phase extraction method to isolate analytes of interest. A laboratory analytical method was applied using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-qTOF MS). For the UPLC-qTOF MS a gradient for the mobile phase was found which allowed the profiling and separation of metabolites in the aerial parts of the vegetation. This method allowed identification and quantification of major changes caused by TNT contaminated soil on vegetation. The Synapt High Definition Mass Spectrometer SYNAPT HDMS G1 was operated using the electrospray ionisation (ESI) technique in both positive and negative mode. A clear comparison of profiles was achieved and this has been demonstrated by the distinct newly-formed metabolites in the TNT contaminated vegetation understudy. The results have also shown that the chlorophyll region in the contaminated profile was also affected by the uptake of TNT degradation products. This has been observed in the contaminated profiles of Wild olive, Stinkwood and Cape Holly extracts indicating enhanced nutrient availability.

Triterpenoids from the leaves of Ilex chinensis.

Eleven previously undescribed compounds including two triterpenes, ilexchinenin A and ilexchinenin B, and nine triterpenoid saponins, ilexchinenosides A-I, along with twelve known triterpenoids were isolated from the leaves of Ilex chinensis Sims (Aquifoliaceae). Their structures were elucidated by spectroscopic analysis and comparison with known compounds. Furthermore, eight compounds exhibited significant inhibitory effects on NO production of lipopolysaccharide (LPS)-induced murine macrophages, while nine compounds exhibited potent hepatoprotective activity against N-acetyl-p-aminophenol (APAP)-induced HepG2 cell damage in in vitro assays.

One-pot biosynthesis of silver nanoparticles using Iboza Riparia and Ilex Mitis for cytotoxicity on human embryonic kidney cells.

Plant extracts continue gaining significant prominence in green synthesis of silver nanoparticles (AgNPs), due to their potential applications in nano-medicine and material engineering. This work reports on green synthesis of silver nanoparticles (AgNPs) from aqueous extracts of Iboza Riparia leaf and Ilex Mitis root bark with diterpenes (DTPs) and saponins (SPNs) as major components. After TEM, DLS, TGA/DSC, ATR, XRD and UV-Vis characterization, the relevant cytotoxicity studies were conducted with the MTT assay on human embryonic kidney cells (HEK293T) followed by antioxidant activity with ABTS. Overall, the AgNPs-DTPs (156nm) were found to be less toxic with 49.7% cell viability, while AgNPs-SPNs (50nm) and AgNPs-PVA (44nm) had cell viability of 40.8 and 28.0% respectively at 400µM. Based on the cytotoxicity and antioxidant activity, it is fair to report that these plant extracts have potential reducing and capping agents as they retain chemical properties on the surface of the nanoparticles.

Neuroprotective effect of total flavonoids from Ilex pubescens against focal cerebral ischemia/reperfusion injury in rats.

Ilex pubescens is commonly used in traditional Chinese medicine to treat cardiovascular and cerebrovascular diseases, such as coronary artery disease and stroke. However, the underlying mechanisms remain to be fully elucidated. The aim of the present study was to investigate the effects of Ilex pubescens total flavonoids (IPTF) on neuroprotection and the potential mechanisms in a rat model of focal cerebral ischemia/reperfusion (I/R) injury. Rats were pretreated with intragastric administration of IPTF at doses of 200 and 100 mg/kg for 5 days; middle cerebral artery occlusion surgery was then performed to induce cerebral I/R injury. Neurological deficits were determined using the 5­point neurological function score evaluation system, brain infarct sizes were determined by 2,3,5­triphenyltetrazolium chloride staining and alterations in brain histology were determined by hematoxylin and eosin staining. The neurological deficit score, the infarcted area and the brain tissue pathological injury were significantly reduced when the rats were pretreated with IPTF. In addition, inflammatory mediators and neurotrophic factors in the brain were investigated. IPTF pretreatment decreased the activities of total nitric oxide synthase (TNOS), induced NOS (iNOS) and constitutive NOS (cNOS), and the levels of nitric oxide (NO), interleukin­1ß (IL­1ß) and tumor necrosis factor­α (TNF­α), however, it increased the levels of IL­10 in brain tissues. Furthermore, pretreatment with IPTF also increased the protein expressions of brain­derived neurotrophic factor, glial cell­derived neurotrophic factor and vascular endothelial growth factor, when compared with the model group. In conclusion, the results of the present study demonstrated that IPTF has a neuroprotective effect against focal cerebral I/R injury in rats. The mechanism may be associated with the decreased production of certain proinflammatory cytokines including NO, IL­1ß, TNF­α, TNOS, iNOS and cNOS, the increased production of the anti­inflammatory cytokine IL­10 and the increased secretion of neurotrophic factors.

Anticomplement triterpenoids from the roots of Ilex asprella.

Five new (1-5) and twenty-eight known (6-33) triterpenoids were isolated from the roots of Ilex asprella. The structures of the new compounds were elucidated by the detailed spectral analysis. The ursane and oleanane triterpenoids were found to show anticomplement activity with some structure-activity relationships. Several triterpenoids (1-3, 6-7) exhibited potent anticomplement activity with the CH50 and AP50 values of 0.058-0.131mg/mL and 0.080-0.444mg/mL, respectively. It was found that caffeoyl group could enhance activity remarkably, followed by coumaroyl and feruloyl group. The 28-carboxyl group was also important to anticomplement activity for the triterpenoids. However, the triterpenoids with lactone ring (4, 9-14) exhibited weak activity and triterpenoid glycosides (5, 23-33) showed no inhibition. The targets of several bioactive triterpenoids in complement activation cascade were identified as well.

Hydrolysis of Dicaffeoylquinic Acids from Ilex kudingcha Happens in the Colon by Intestinal Microbiota.

Monocaffeoylquinic acids (mono-CQAs) can be hydrolyzed or metabolized by pancreatin, intestinal brush border esterase, and microbiota in the colon. Data about the conversion of dicaffeoylquinic acids (diCQAs) in digestion are scarce. The diCQA-rich fraction including 3,4-, 3,5-, and 4,5-diCQAs was prepared from Ilex kudingcha, and the conversion in simulated gastricintestine was investigated. Artificial saliva, gastric and pancreatic fluids, Caco-2 monolayer cells, and anaerobic fermentation model were utilized to mimic digestions of the oral cavity, stomach, small intestine, and colon in vitro. The results revealed that diCQAs remained intact in simulated saliva, gastric, and pancreatic fluids and within Caco-2 cells. In anaerobic fermentation with human fecal slurry, diCQAs were hydrolyzed to mono-CQAs and caffeic acid, which were further metabolized to caffeic acid and dihydrocaffeic acid, respectively. The hydrolysis of diCQAs depended on the chemical structures, carbohydrates in the culture medium, and microbial compositions. Our research demonstrated that hydrolysis of diCQAs happened in the colon by intestinal microbiota.

Metabolic classification of South American Ilex species by NMR-based metabolomics.

The genus Ilex to which mate (Ilex paraguariensis) belongs, consists of more than 500 species. A wide range of metabolites including saponins and phenylpropanoids has been reported from Ilex species. However, despite the previous works on the Ilex metabolites, the metabolic similarities between species which can be used for chemotaxonomy of the species are not clear yet. In this study, nuclear magnetic resonance (NMR) spectroscopy-based metabolomics was applied to the classification of 11 South American Ilex species, namely, Ilex argentina, Ilex brasiliensis, Ilex brevicuspis, Ilex dumosa var. dumosa, I. dumosa var. guaranina, Ilex integerrima, Ilex microdonta, I. paraguariensis var. paraguariensis, Ilex pseudobuxus, Ilex taubertiana, and Ilex theezans. (1)H NMR combined with principal component analysis (PCA), partial least square-discriminant analysis (PLS-DA) and hierarchical cluster analysis (HCA) showed a clear separation between species and resulted in four groups based on metabolomic similarities. The signal congestion of (1)H NMR spectra was overcome by the implementation of two-dimensional (2D)-J-resolved and heteronuclear single quantum coherence (HSQC). From the results obtained by 1D- and 2D-NMR-based metabolomics it was concluded that species included in group A (I. paraguariensis) were metabolically characterized by a higher amount of xanthines, and phenolics including phenylpropanoids and flavonoids; group B (I. dumosa var. dumosa and I. dumosa var. guaranina) with oleanane type saponins; group C (I. brasiliensis, I. integerrima, I. pseudobuxus and I. theezans) with arbutin and dicaffeoylquinic acids, and group D (I. argentina, I. brevicuspis, I. microdonta and I. taubertiana) with the highest level of ursane-type saponins. Clear metabolomic discrimination of Ilex species and varieties in this study makes the chemotaxonomic classification of Ilex species possible.

Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering.

Details of the recently elucidated biosynthetic pathways of caffeine and related purine alkaloids are reviewed. The main caffeine biosynthetic pathway is a sequence consisting of xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine. Genes encoding N-methyltransferases involved in three of these four reactions have been isolated and the molecular structure of N-methyltransferases investigated. Pathways for the catabolism of caffeine have also been studied, although there are currently no reports of enzymatic and genetic studies having been successfully carried out. Metabolism of purine alkaloids in species including Camellia, Coffea, Theobroma and Ilex plants is summarised, and evidence for the involvement of caffeine in chemical defense and allelopathy is discussed. Finally, information is presented on metabolic engineering that has produced coffee seedlings with reduced caffeine content, and transgenic caffeine-producing tobacco plants with enhanced disease resistance.

Seasonal variations in water relations in current-year leaves of evergreen trees with delayed greening.

We investigated seasonal patterns of water relations in current-year leaves of three evergreen broad-leaved trees (Ilex pedunculosa Miq., Ligustrum japonicum Thunb., and Eurya japonica Thunb.) with delayed greening in a warm-temperate forest in Japan. We used the pressure-volume method to: (1) assess the extent to which seasonal variation in leaf water relations is attributable to leaf development processes in delayed greening leaves versus seasonal variation in environmental variables; and (2) investigate variation in leaf water relations during the transition from the sapling to the adult tree stage. Leaf mass per unit leaf area was generally lowest just after completion of leaf expansion in May (late spring), and increased gradually throughout the year. Osmotic potential at full turgor (Psi(o) (ft)) and leaf water potential at the turgor loss point (Psi(w) (tlp)) were highest in May, and lowest in midwinter in all species. In response to decreasing air temperature, Psi(o) (ft) dropped at the rate of 0.037 MPa degrees C(-1). Dry-mass-based water content of leaves and the symplastic water fraction of total leaf water content gradually decreased throughout the year in all species. These results indicate that reductions in the symplastic water fraction during leaf development contributed to the passive concentration of solutes in cells and the resulting drop in winter Psi(o) (ft). The ratio of solutes to water volume increased in winter in current-year leaves of L. japonicum and E. japonica, indicating that osmotic adjustment (active accumulation of solutes) also contributed to the drop in winter in Psi(o) (ft). Bulk modulus of elasticity in cell walls fluctuated seasonally, but no general trend was found across species. Over the growing season, Psi(o) (ft) and Psi(w) (tlp) were lower in adult trees than in saplings especially in the case of I. pedunculosa, suggesting that adult-tree leaves are more drought and cold tolerant than sapling leaves. The ontogenetic increase in the stress resistance of I. pedunculosa may be related to its characteristic life form because I. pedunculosa grows taller than the other species studied.

Saturation to improve pollutant retention in a rain garden.

Rain gardens have been recommended as a best management practice to treat stormwater runoff. Replicate rain gardens were constructed in Haddam, CT, to treat roof runoff. The objective of this study was to assess whether the creation of a saturated zone in a rain garden improved retention of pollutants. The gardens were sized to store 2.54 cm (1 in) of runoff. Results show high retention of flow; only 0.8% overflowed. Overall, concentrations of nitrite+ nitrate-N, ammonia-N, and total-N (TN) in roof runoff were reduced significantly by the rain gardens. Total-P concentrations were significantly increased by both rain gardens. ANCOVA results show significant reductions in TN (18%) due to saturation. Redox potential also decreased in the saturated garden. Rain garden mulch was found to be a sink for metals, nitrogen, and phosphorus, but rain garden soils were a source for these pollutants. The design used for these rain gardens was effective for flow retention, but did not reduce concentrations of all pollutants even when modified. These findings suggest that high flow and pollutant retention could be achieved with the 2.54 cm design method, but the use of an underdrain could reduce overall pollutant retention.

Classification of Ilex species based on metabolomic fingerprinting using nuclear magnetic resonance and multivariate data analysis.

The metabolomic analysis of 11 Ilex species, I. argentina, I. brasiliensis, I. brevicuspis, I. dumosavar. dumosa, I. dumosa var. guaranina, I. integerrima, I. microdonta, I. paraguariensis var. paraguariensis, I. pseudobuxus, I. taubertiana, and I. theezans, was carried out by NMR spectroscopy and multivariate data analysis. The analysis using principal component analysis and classification of the (1)H NMR spectra showed a clear discrimination of those samples based on the metabolites present in the organic and aqueous fractions. The major metabolites that contribute to the discrimination are arbutin, caffeine, phenylpropanoids, and theobromine. Among those metabolites, arbutin, which has not been reported yet as a constituent of Ilex species, was found to be a biomarker for I. argentina,I. brasiliensis, I. brevicuspis, I. integerrima, I. microdonta, I. pseudobuxus, I. taubertiana, and I. theezans. This reliable method based on the determination of a large number of metabolites makes the chemotaxonomical analysis of Ilex species possible.

Sensitivity analyses of woody species exposed to air pollution based on ecophysiological measurements.

GOAL, SCOPE AND BACKGROUND: Air pollution has been of a major problem in the Pearl River Delta of south China, particularly during the last two decades. Emissions of air pollutants from industries have already led to damages in natural communities and environments in a wide range of the Delta area. Leaf parameters such as chlorophyll fluorescence, leaf area (LA), dry weight (DW) and leaf mass per area (LMA) had once been used as specific indexes of environmental stress. This study aims to determine in situ if the daily variation of chlorophyll fluorescence and other ecophysiological parameters in five seedlings of three woody species, Ilex rotunda, Ficus microcarpa and Machilus chinensis, could be used alone or in combination with other measurements for sensitivity indexes to make diagnoses under air pollution stress and, hence, to choose the correct tree species for urban afforestation in the Delta area. METHODS: Five seedlings of each species were transplanted in pot containers after their acclimation under shadowing conditions. Chlorophyll fluorescence measurements were made in situ by a portable fluorometer (OS-30, Opti-sciences, U.S.A). Ten random samples of leaves were picked from each species for LA measurements by area-meter (CI-203, CID, Inc., U.S.A). DW was determined after the leaf samples were dried to a constant weight at 65 degrees C. LMA was calculated as the ratio of DW/LA. Leaf N content was analyzed according to the Kjeldhal method, and the extraction of pigments was carried out according Lin et al. RESULTS AND DISCUSSION: The daily mean Fv/Fm (Fv is the variable fluorescence and Fm is the maximum fluorescence) analysis showed that Ilex rotunda and Ficus microcarpa were more highly resistant to pollution stress, followed by Machilus chinensis, implying that the efficiency of photosystem II in I. rotunda was less affected by air pollutants than the other two species. Little difference in daily change of Fv/Fm in I. rotunda between the polluted and the clean site was also observed. However, a relatively large variation of Fv/Fm appeared in the other two species, particularly in M. chinensis, suggesting that they were more sensitive to air pollutants than I. rotunda. The mean LA was reduced for all species growing at the polluted site. The mean LMA for all species exceeded the sclerophylly threshold given by Cowling and Campbell and increased for those under pollution stress, which could be explained as one of the acclimation strategies for plants to air pollution stress. Little difference in leaf chlorophyll content was observed in F. microcarpa and M. chinensis, while remarkable differences were found in I. rotunda growing at the polluted and the clean site. Content of leaf carotenoids was largely reduced in I. rotunda growing at the polluted site, but increased in F. microcarpa and M. chinensis, compared with plants growing at the clean site. Plants growing at the clean site had a lower leaf N content than those growing at the polluted site. In addition, species with a higher resistance to pollution stress showed less difference in leaf N content than those sensitive species. CONCLUSION: Based on Fv/Fm measurements of the three woody species, I. rotunda showed the highest resistance to air pollutants from ceramic industries, followed by F. microcarpa. M. chinensis was the most sensitive species to air pollution, had lowest capacities to cope with the air pollution stress, which was consistent with visual injury symptoms observed in the crown profiles of plants at the polluted site. Fv/Fm, LAM, LA, leaf pigments and N content could be used alone or in combination to diagnose the extent of the physiological injury. The ratio of Fv/Fm, however, was the best and most effective parameter. RECOMMENDATION AND OUTLOOK: Tree species which have higher air-pollutant resistance, as diagnosed by such ecophysiological parameters, should be considered first and planted widely for urban afforestation or forest regeneration in areas where the forest was seriously degraded or forest health was markedly effected by the same kind of air pollutants.