Ilex Aquifolium Research Articles

Assessing Algerian Ilex aquifolium L.’s phenolic compounds, antioxidant and enzyme inhibitory properties to find a new natural remedy for Alzheimer’s, diabetes and skin diseases

This research explored the impact of methanolic extracts from the fruits and leaves of Ilex aquifolium L., a rare Algerian species, on several enzymes, focusing on tyrosinase, α-amylase, α-glucosidase, and cholinesterases, as well as their antioxidant potential in vitro assays, including phosphomolybdenum, DPPH, ABTS, CUPRAC, FRAP, and MCA. HPLC-DAD analysis identified chlorogenic acid, rosmarinic acid, hesperidin, and quercetin as predominant phenolic components. Both extracts exhibited notable antioxidant properties, up to 250.08 mg TE/g for fruits and 237.08 mg TE/g for leaf extracts. Enzyme inhibitory assays demonstrated significant inhibition against AChE and BChE with maximum activities of 2.34 mg GALAE/g in leaves and 4.32 mg GALAE/g in fruits. Additionally, considerable tyrosinase inhibition was observed in leaves at 39.69 mg KAE/g. These findings highlight I. aquifolium L. as a valuable source for investigating natural antioxidants and enzyme inhibitors, particularly for possible cosmetics and food additives applications.

Differential growth rate, water use efficiency and climate sensitivity between males and females of Ilex aquifolium in north-western Spain.

Dioecious plant species, i.e., those in which male and female functions are housed in different individuals, are particularly vulnerable to global environmental changes. For long-lived plant species, such as trees, long-term studies are imperative to understand how growth patterns and their sensitivity to climate variability differentially affect the sexes. Here, we explore long-term intersexual differences in wood traits, namely radial growth rates, water use efficiency quantified as stable carbon isotope abundance of wood cellulose, and their climate sensitivity in Ilex aquifolium trees growing in a natural population in NW Spain. We found that sex differences in secondary growth rates were variable over time, with males outperforming females in both radial growth rates and water use efficiency in recent decades. Summer water stress significantly reduced the growth of female trees in the following growing season, while the growth of male trees was primarily favoured by cloudy and rainy conditions the previous fall and winter combined with low cloud cover and warm conditions in summer. Sex-dependent lagged correlations between radial growth and water availability were found, with a strong association between tree growth and cumulative water availability in females at 30 months and in males at 10 months. Overall, our results point to greater vulnerability of female tress to increasing drought, which could lead to sex-ratio biases threatening population viability in the future.

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.

The genome sequence of the English holly, Ilex aquifolium L. (Aquifoliaceae).

We present a genome assembly from an individual Ilex aquifolium (the English holly; Eudicot; Magnoliopsida; Aquifoliales; Aquifoliaceae). The genome sequence is 800.0 megabases in span. Most of the assembly is scaffolded into 20 chromosomal pseudomolecules. The assembled mitochondrial and plastid genomes have lengths of 538.43 kilobases and 157.52 kilobases in length, respectively.

Twenty years of population dynamics in European beech-oak forest at their rear range margin anticipate changes in its structure and composition

There is an increasing interest in restoring degraded forests, which occupy half of the forest areas. Among the forms of restoration, passive restoration, which involves the elimination of degrading factors and the free evolution of natural dynamics by applying minimal or no management, is gaining attention. Natural dynamics is difficult to predict due to the influence of multiple interacting factors such as climatic and edaphic conditions, composition and abundance of species, and the successional character of these species. Here, we study the natural dynamics of a mixed forest located in central Spain, which maintained an open forest structure, due to intensive use, until grazing and cutting were banned in the 1960s. The most frequent woody species in this forest are Fagus sylvatica, Quercus petraea, Quercus pyrenaica, Ilex aquifolium, Sorbus aucuparia, Sorbus aria and Prunus avium, with contrasting shade and drought tolerance. These species are common in temperate European deciduous forest and are found here near their southern distribution limit, except for Q. pyrenaica. In order to analyze forest dynamics and composition, three inventories were carried out in 1994, 2005 and 2015. Our results show that, despite the Mediterranean influence, the natural dynamics of this forest has been mainly determined by different levels of shade tolerance. After the abandonment of grazing and cutting, Q. pyrenaica expanded rapidly due to its lower shade tolerance, whereas after canopy closure and forest densification, shade-tolerant species gained ground, particularly F. sylvatica, despite its lower drought and late-frost tolerance. If the current dynamics continue, F. sylvatica will overtake the rest of the species, which will be relegated to sites with shallow soils and steep slopes. Simultaneously, all the multi-centennial beech trees, which are undergoing a rapid mortality and decline process, will disappear.

Elevated CO2 does not improve seedling performance in a naturally regenerated oak woodland exposed to biotic stressors

IntroductionInsect herbivores and biotrophic pathogens are major stressors influencing natural regeneration in woodlands. Information on the effect of elevated CO2 (eCO2) on plant-insect-pathogen interactions under natural conditions is lacking.MethodsWe studied the effects of eCO2 on leaf-out phenology, as well as on levels of insect herbivory and powdery mildew (PM), i.e., reduction of leaf photosynthetic material. We then assessed the combined impacts of these biotic stressors and eCO2 on seedling photosynthesis and growth. A total of 92 naturally recruited and 114 potted seedlings of 5 temperate tree species (Quercus robur, Acer pseudoplatanus, Corylus avellana, Crataegus monogyna, and Ilex aquifolium) within a mature oak woodland were studied.ResultsWe found that eCO2 advanced leaf-out phenology and was a significant explanatory variable for growth and physiological performance in potted seedlings. Potted oak seedlings experienced 11-fold higher tissue loss from insect herbivory than natural seedlings. The earliest leaf-flushing species, hawthorn, and the evergreen holly were resistant to insect attack and were not affected by PM. Oak was defoliated most but showed the highest regeneration capacity. Hazel was more resistant to PM infection than oak and sycamore. Despite being highly infected by PM, sycamore was less affected than oak. The more vigorous sycamore and oak seedlings suffered more severe PM disease.ConclusionNo evidence emerged that eCO2 enhances natural regeneration under biotic stress for any of the species studied.

Leaf Stomatal Characteristics of Some Species in Bush Form Used in Kastamonu Urban Landscape

Nowadays, the rapidly increasing population has brought many problems such as building concrete structures everywhere, air pollution, and traffic congestion. It is known that parks and gardens, which are an important element for the urban landscape, not only balance the human-nature relationship in cities, but also improve urban living conditions. Parks and gardens that utilize urban areas in a functional and aesthetic way to meet the recreational needs of people, to recover the lost nature and to create healthy living spaces are extremely important. In this study, it was aimed to determine the leaf stomatal characteristics of Buxus microphylla Siebold & Zucc., Prunus laurocerasus L, Ligustrum delavayanum Har., Berberis thunbergii DC., Ilex aquifolium L., Euonymus japonicus Wall. var. aureomarginatus Rehder, Cotoneaster lacteus W.W.Sm., Mahonia aquifolium (Pursh) Nutt., Pyracantha coccinea M. Roem. species, one of the species used in the park, garden and central landscaping studies in Kastamonu province. With micromorphological investigations, stomatal characteristics were determined by SEM on the species used in the study. With respect to stomatal density in the unit area on species, it was determined that Pyracantha coccinea species had the maximum stomatal density (81) while Cotoneaster lacteus species had the minimum stomatal density (10). Furthermore, it was determined that Prunus laurocerasus (34.82 µm) had mean the longest stomatal length and Mahoni aquifolium (7.183 µm) had the mean shortest stomatal length.

Clonal Diversity and Fine-Scale Genetic Structure of a Keystone Species: Ilex aquifolium

Resprouting species, such as English holly (Ilex aquifolium L.), can be severely affected by long-lasting forest management. In the present study, the clonal and fine-scale spatial genetic structure (FSGS) of English holly are evaluated in a sub-Mediterranean mixed forest of Central Spain, where this species was subjected to different and intense human interventions for centuries, including coppicing in dense structures and in open woody pastures. Using seven microsatellite loci, we assessed the contribution of clonal and sexual reproduction to the diversity of holly in both areas and the fine-scale genetic structure was evaluated. For the dense coppiced area, the results showed high clonal diversity derived mainly from stem resprouting. Stems were separated an average of 3 m and up to 10 m, and dense aggregations of stems sometimes included various genotypes. The results also showed recent seed recruitment leading to small unique and isolated genotypes. Furthermore, FSGS was detected at close distances as being stronger in the coppiced area than in the open woody pasture area, especially for individuals of greater diameter at breast height. The results obtained suggest that current management strategies are not negatively affecting the genetic diversity of this species.

Assessing Phenotypic Variability in Some Eastern European Insular Populations of the Climatic Relict Ilex aquifolium L.

Through its natural or cultivated insular population distribution, Ilex aquifolium L. is a paramount species which is exceptionally suitable for studying phenotypic variability and plasticity through the assessment of morphological, physiological, biochemical and genomic features with respect to acclimation and/or adaptation efficiency. The current study is focused on four insular populations of Ilex aquifolium from Eastern Europe (i.e., in Romania, Hungary, Serbia and Bulgaria), and presents an initial evaluation of phenotypic variability in order to conclude our research on phylogenetic relationships and phytochemical profiles, including several descriptive and quantitative morphological traits. Taken together, the data from different methods in this paper indicate that the Bulgarian and Romanian populations can be distinguished from each other and from Serbian and Hungarian populations, while the latter show a higher level of resemblance with regards to their quantitative morphological traits. It is likely that these morphological traits are determined through some quantitative trait loci implicated in stress responses generated by light, temperature, soil water, soil fertility and salinity conditions that will need to be analysed in terms of their physiological, genomic and metabolomics traits in future studies.

Biochemical and Molecular Investigation of the Effect of Saponins and Terpenoids Derived from Leaves of Ilex aquifolium on Lipid Metabolism of Obese Zucker Rats.

Ilex paraguariensis, the holly tree, is a plant with recognized biological properties, whose aqueous infusions are known as “Yerba mate”, that regulate lipid metabolism, reduce obesity, and improve brain stimulation. In the present study, the effect of standardized saponin and terpenoid fractions of a European taxon, Ilex aquifolium, on blood biochemical parameters in a rat model of metabolic disorder, (fa/fa) Zucker, are presented. The profiles of the volatile fractions of two species and six European varieties of Ilex were investigated. After selecting the best variety, the saponin and terpenoid fractions were isolated and standardized, and animals were fed 10 mg kg−1 b.w. for 8 weeks. A statistically significant decrease in liver adiposity was observed, confirmed by histology and quantitative identification (gas chromatography–mass spectrometry analyses of hepatic lipids. RT-qPCR analysis of gene expression in the aorta revealed that the administration of the terpenoid fraction downregulated LOX-1, suggesting a reduction in atherosclerotic stimuli. In addition, a statistically significant reduction (p < 0.05) in PPARγ for the saponin fraction was observed in the liver. The expression of the ACAT-1 gene in the liver, responsible for the formation of cholesterol esters, increased significantly in the group receiving the terpenoid fraction compared to the control, which was also confirmed by the analysis of individual blood biochemical parameters. The opposite effect was observed for saponins. Taking the above into account, it is shown for the first time that Ilex aquifolium can be a source of compounds that positively influence lipid metabolism.

Toxic and allergenic plant species in primary school yards of Zagreb’s Lower Town district

Abstract The green infrastructure of Zagreb’s Lower Town is made up of row-planted trees and green spaces within blocks of buildings. All schools are surrounded by green spaces that have a positive impact on the environment. Although most green spaces contribute to improving the quality of life, they also contain toxic and allergenic species which are potentially hazardous, particularly to children. This paper aims to make an inventory of plant species in the environment of primary schools in Zagreb’s Lower Town district, and to determine the presence of toxic and allergenic species. The field research was conducted from March to the end of May 2021 at 7 primary school locations. 13 either annual or biennial plant families were catalogued. In total, 34 species of herbaceous perennials and geophytes, and 36 species of trees from 17 plant families were listed. A total of 38 species of shrubs and climbers were found. Also, a total of 35 poisonous and 28 allergenic plant taxa are planted in the green spaces surrounding primary schools. The most common poisonous species found are Hedera helix and Taxus baccata, and Ilex aquifolium, a highly poisonous species, was also found. No moderately poisonous species have been found at all. As far as allergenic species are concerned, school environments are dominated by those that produce low and moderate levels of pollen concentration in the air. Species that produce high levels of pollen concentration (e.g., Betula pendula, Robinia pseudoacacia, etc.) are the least represented ones, while species that produce a very high concentration of pollen have not been recorded in any of the localities.

Sex and tree rings: Females neither grow less nor are less water-use efficient than males in four dioecious tree species

Females of woody dioecious species usually expend more resources on reproduction than males. Therefore, it is expected that females incur greater costs of reproduction than males, and, as a result, trade-offs between reproduction and growth should emerge. The aim of this study is to test those hypotheses by analyzing the differences between genders regarding radial growth (basal area increment) and wood carbon isotope composition (δ13C), a proxy of water-use efficiency. We compared these two variables in males and females of four dioecious tree species inhabiting drought-prone Mediterranean sites in Spain (Pistacia terebinthus, Ilex aquifolium, Juniperus thurifera and Ailanthus altissima). We analyzed the influence of sex on the radial growth patterns throughout the tree life considering the growth stage of individuals, the differences in the response of genders to climate variables (air temperature, precipitation and drought severity), and the δ13C during a severe drought period. One site was studied for each species and 21–33 trees per species were sampled in each site. No differences in growth were found between genders for any of the four species throughout their life span. No significant interactions between gender and precipitation were found, although A. altissima males were more responsive to summer (June-July) temperature. No differences in δ13C were found between genders excepting for P. terebinthus, indicating that the males of this species show a less efficient water use during drought events than the females. These results do not support the broad assumption that females of woody dioecious plants show lower growth and are less water-use efficient than males or that they respond differently to precipitation variability, except for P. terebinthus during drought events. Further analyses could be performed in other dioecious species inhabiting seasonally dry regions to confirm or reject our conclusions.

Building a Barrier: The Influence of Different Wax Fractions on the Water Transpiration Barrier of Leaf Cuticles.

Waxes are critical in limiting non-stomatal water loss in higher terrestrial plants by making up the limiting barrier for water diffusion across cuticles. Using a differential extraction protocol, we investigated the influence of various wax fractions on the cuticular transpiration barrier. Triterpenoids (TRPs) and very long-chain aliphatics (VLCAs) were selectively extracted from isolated adaxial leaf cuticles using methanol (MeOH) followed by chloroform (TCM). The water permeabilities of the native and the solvent-treated cuticles were measured gravimetrically. Seven plant species (Camellia sinensis, Ficus elastica, Hedera helix, Ilex aquifolium, Nerium oleander, Vinca minor, and Zamioculcas zamiifolia) with highly varying wax compositions ranging from nearly pure VLCA- to TRP-dominated waxes were selected. After TRP removal with MeOH, water permeability did not or only slightly increase. The subsequent VLCA extraction with TCM led to increases in cuticular water permeabilities by up to two orders of magnitude. These effects were consistent across all species investigated, providing direct evidence that the cuticular transpiration barrier is mainly composed of VLCA. In contrast, TRPs play no or only a minor role in controlling water loss.

Biological Potential and Chemical Profile of European Varieties of Ilex.

Plants of the genus Ilex are widespread throughout the world, with its best-known representative being Ilex paraguraiensis from South America. The European species Ilex aquifolium shows similarities in its terpenoid, sugar and phenolic acid profiles. Using aqueous extracts of Ilex aquifolium as a supplement in Wistar rats showed that, despite the lack of caffeine, it had strong hypocholesterolemic effects. In addition, a reduction in oxidative lipid degradation and a decrease in hepatic steatosis in histopathological studies were observed. The results of this study suggest that extracts from the European species Ilex aquifolium may have potential as an alternative treatment for hyperlipidemia.

Efficacy of Different Concentrations of NAA on Selected Ornamental Woody Shrubs Cuttings

Ornamental woody shrubs are used in landscape design worldwide. Their propagation can be made generatively and vegetatively. Vegetative propagation methods are mostly used by nurseries, as such methods are quick and the newly propagated plants inherit the genetics of the mother plant. However, rooting in some woody plants is slow and, unfortunately, sometimes produces only a small number of rooted cuttings. In this study, shoot cuttings from six selected ornamental woody shrubs were subjected to different concentrations of rooting stimulators (0.5 (NAA5) and 0.8 (NAA8) % concentrations of 1-Naphthylacetic acid; cuttings without treatment were considered as control) and propagated in two different periods (spring and summer). Our results show that significant changes were obtained in the plants under the different treatments. Most of the plants showed a positive response to both treatments, expect for Cotinus coggygria ‘Royal Purple’, which, compared to control, registered decreases in all the tested parameters under NAA5 treatment. Ilex aquifolium was the species that showed increments in all the parameters when NAA treatments were applied. In conclusion, our research suggests that NAA increases rooting in ornamental woody shrubs, although in some cases rooting could be a species-dependent process.

Water Sorption and Desorption of Isolated Cuticles From Three Woody Species With Focus on Ilex aquifolium.

The cuticle is a lipid-rich layer that protects aerial plant organs against multiple stress factors such as dehydration. In this study, cuticle composition and structure in relation to water loss are examined in a broad ecophysiological context, taking into consideration leaf age and side from Ilex aquifolium (holly) in comparison with Eucalyptus globulus (eucalypt) and Prunus laurocerasus (cherry laurel). Enzymatically isolated cuticular membranes from holly leaves were studied under three treatment conditions: natural (no chemical treatment), after dewaxing, and after methanolysis, and the rate of water loss was assessed. Structural and chemical changes were evaluated using different microscopy techniques and by Fourier transform infrared (FTIR) spectroscopy. The potential mechanisms of solute absorption by holly leaves were additionally evaluated, also testing if its prickly leaf margin may facilitate uptake. The results indicate that the treatment conditions led to structural changes, and that chemical composition was hardly affected because of the occurrence of cutan. Structural changes led to more hydrophilic adaxial surfaces, which retained more water and were more efficient than natural cuticles, while changes were not significant for abaxial surfaces. Across natural cuticles, age was a significant factor for eucalypt but not for holly. Young eucalypt cuticles were the group that absorbed more water and had the lowest water loss rate. When comparing older leaf cuticles of the three species, cherry laurel was found to absorb more water, which was, however, lost more slowly, compared with the other species. Evidence was gained that holly leaves can absorb foliar-applied solutes (traced after calcium chloride application) through the adaxial and abaxial surfaces, the adaxial mid veins, and to a lower extent, the spines. In conclusion, for the species examined, the results show variations in leaf cuticle composition and structure in relation to leaf ontogeny, and water sorption and desorption capacity.

Tree phyllospheres are a habitat for diverse populations of CO-oxidizing bacteria.

Carbon monoxide (CO) is both a ubiquitous atmospheric trace gas and an air pollutant. While aerobic CO-degrading microorganisms in soils and oceans are estimated to remove ~370 Tg of CO per year, the presence of CO-degrading microorganisms in above-ground habitats, such as the phyllosphere, and their potential role in CO cycling remains unknown. CO-degradation by leaf washes of two common British trees, Ilex aquifolium and Crataegus monogyna, demonstrated CO uptake in all samples investigated. Based on the analyses of taxonomic and functional genes, diverse communities of candidate CO-oxidizing taxa were identified, including members of Rhizobiales and Burkholderiales which were abundant in the phyllosphere at the time of sampling. Based on predicted genomes of phyllosphere community members, an estimated 21% of phyllosphere bacteria contained CoxL, the large subunit of CO-dehydrogenase. In support of this, data mining of publicly available phyllosphere metagenomes for genes encoding CO-dehydrogenase subunits demonstrated that, on average, 25% of phyllosphere bacteria contained CO-dehydrogenase gene homologues. A CO-oxidizing Phyllobacteriaceae strain was also isolated from phyllosphere samples which contains genes encoding both CO-dehydrogenase as well as a ribulose-1,5-bisphosphate carboxylase-oxygenase. These results suggest that the phyllosphere supports diverse and potentially abundant CO-oxidizing bacteria, which are a potential sink for atmospheric CO.

Vegetative Propagation and ISSR-Based Genetic Identification of Genotypes of Ilex aquifolium ‘Agrifoglio Commune’

The market demand for interesting varieties and cultivars of Ilex aquifolium necessitates the exploration and sustainable exploitation of plant individuals thriving in nature without human care. In this work, an effort was made to develop a simple and reliable protocol for vegetative propagation of I. aquifolium plants, with desirable-for-market characteristics, grown in a mountain area of Halkidiki, Greece, and at the same time to proceed with their genetic identification using molecular markers. From these plants, new plants were vegetatively produced which afterward were used as stock plants for providing the needed shoot cuttings for the experiments of rooting and leaves for their genetic analysis. Factors studied in formulating a propagation protocol included the season of cutting collection and the application of 0.2% 1-naphthaleneacetic acid (NAA), as well as the type of shoot cuttings (terminal, subterminal) and the application of auxin. It was found that application of NAA was crucial for rooting response and number of roots formed, whereas the season effect was not significant on rooting. Terminal cuttings treated with 0.2% NAA exhibited the highest rooting percentage (100%) and formed abundant roots (25.7) compared to subterminal ones. All rooted cuttings, after being potted and transferred to acclimatization greenhouse, were successfully hardened. In the spring of the next year, the produced plants blossomed abundantly and formed fruits (bright red berries) presenting their characteristic ornamental appearance that was maintained until Christmas. For the identification procedure, the genotypic profile of the stock plants was also investigated by inter-simple sequence repeat (ISSR) genetic analysis, revealing that they were genetically the same both among themselves and when compared with a certified I. aquifolium ‘Agrifoglio Commune’ individual, but they differed genetically from I. aquifolium ‘Argentea Marginata’ and I. aquifolium ‘Hellas’.

Ilex aquifolium (Aquifoliaceae) and the relics of Tertiary forest vegetation with Colchic affinity in Sicily (C-Mediterranean)

Ilex aquifolium (Aquifoliaceae) and the relics of Tertiary forest vegetation with Colchic affinity in Sicily (C-Mediterranean)

Noteworthy new floristic records from Greece

This contribution to the Greek flora includes distribution extensions and taxonomic notes for some noteworthy plant taxa. New records for certain phytogeographical regions of Greece are presented for: Arum cylindraceum, Convolvulus pentapetaloides. Cruciata taurica, Galanthus reginae-olgae subsp. vernalis, Ilex aquifolium, Linum decumbens, Lythrum thymifolia, Sedum praesidis, Silene congesta and Trifolium diffusum. Allium karistanum, Helichrysum amorginum, Limonium antipaxorum and L. cephalonicum have been recorded for the first time on some islands. New populations of Allium maniaticum and A. ritsii, previously known only from their loci classici, were discovered in the Peloponnese. Valeriana alliariifolia, Silene auriculata subsp. auriculata and Symphytum creticum were rediscovered in central Evvia, confirming long-lost historical records from previous centuries. The presence of V. alliariifolia in Greece and Europe is confirmed for the first time since the 19th century.