Phenolic profile and effects of acetone fractions obtained from the inflorescences of Calluna vulgaris (L.) Hull on vaginal pathogenic and non-pathogenic bacteria.

The phenolic profile and the antibacterial activity against pathogenic commensal vaginal bacteria exhibited by different fractions of the acetone extract of heather were assessed. The acetone extract of Calluna vulgaris (L.) Hull was fractionated by silica gel column chromatography through an eluent system of increasing polarity, and 10 different fractions (Fr 1 to Fr 10) were obtained. The phenolic profile was analyzed by HPLC-DAD-ESI/MS. Type B (epi)catechin dimers, (-)-epicatechin and (+)-catechin, were the main phenolic compounds present in the fractions. The antibacterial activity was also analyzed against pathogenic bacteria and the effect on the beneficial microflora was also assessed. Some of the obtained fractions revealed the capacity to inhibit pathogenic microorganisms without affecting the beneficial microflora, especially Fr 7 and Fr 8. For instance, Neisseria gonorrhoeae was inhibited by both of the fractions, while Fr 7 was more active against Klebsiella pneumoniae and Morganella morganii, and Fr 8 was active against methicillin resistant Staphylococcus aureus (MRSA) and methicillin susceptible Staphylococcus aureus (MSSA), without affecting the Lactobacillus strains. This study corroborates the therapeutic use of this matrix in traditional medicine.

Screening of ionic liquids for extraction of flavonoids from heather.

Room temperature ionic liquids are novel solvents with the specific properties that makes them of interest for application for extraction for a wide range of compounds. In this work extraction efficiency of flavonoids from heather flowers using ionic liquids based on imidazolium cation were evaluated and compared with organic solvents. It was found that the anion of ionic liquid significantly influence the extraction yields. Flavonoid content as well as antioxidant activity based on radical scavenging on 1,1-diphenul-2-pirylhydrazyl radicals and cupric reducing antioxidant capacity increased in the order: [Bmim]PF6 < [Bmim]BF4 < [Bmim]Cl. The obtained extraction yield using [Bmim]Cl were higher than reported for 60% ethanol and ethyl acetate under similar conditions, thus, may be helpful for better utilization of heather flowers as the potential pharmaceutical and nutraceutical ingredients.

Calluna vulgaris (L.) Hull: chemical characterization, evaluation of its bioactive properties and effect on the vaginal microbiota.

The inflorescences of Calluna vulgaris were nutritionally and chemically characterized. Furthermore, different organic and aqueous extracts were prepared for the evaluation of their bioactive properties. From the obtained results, carbohydrates were the major compounds, followed by protein, lipid and ashes. It was possible to identify the sugars fructose and glucose, five organic acids, 26 individual fatty acids and the four tocopherol isoforms. Concerning the extract composition, 12 phenolic compounds were identified, with myricetin-3-O-glucoside and myricetin-O-rhamnoside predominating. Concerning the bioactive effects, the more polar extracts showed not only the highest amount in phenolic compounds, but also the strongest antioxidant and antibacterial activities. In contrast, for the anti-inflammatory and cytotoxic potential, the most effective extracts were the n-hexane and the ethyl acetate extracts, respectively. C. vulgaris presented a wide range of biological effects, highlighting their capacity to inhibit pathogenic bacteria without affecting beneficial microflora, corroborating their use in traditional medicine.

The phytochemical and bioactivity profiles of wild Calluna vulgaris L. flowers.

The nutritional composition of Calluna vulgaris flowers as well as the phytochemical profile, antioxidant (DPPH and FRAP assays), antimicrobial and cytotoxic (in human immortalized non-tumorigenic keratinocyte and fibroblasts) activities of aqueous, hydroalcoholic and ethanolic extracts were evaluated. A high content of fiber and carbohydrates (75%) and the prevalence of α-tocopherol as vitamer deserves attention. Linolenic (35%), linoleic (27%) and palmitic (21%) acids were the most abundant fatty acids. Qualitative and quantitative analysis by LC-MS and 1NMR indicated high levels of quercetin, kaempferol and myricetin derivatives as well as procyanidins. The hydro-alcoholic extract displayed the highest antioxidant activity and total phenolics (TPC) and flavonoid contents (TFC). No adverse effects on cells were observed until a concentration of 100 µg/mL and a good antimicrobial activity was reported against S. epidermidis and S. aureus with the hydro-alcoholic extract. The data obtained demonstrated that wild plants like heather, although not being a common nutritional reference, can be used in an alimentary base as a source of bioactive compounds, namely antioxidants.

Biophenols and antioxidant activity in wild and cultivated heather.

Over the last decade, there has been an increasing interest to study the antioxidants from natural sources which can be applied to replace the synthetic compounds commonly used in food or as cosmetic ingredients. Calluna vulgaris (L.) Hull (heather), a plant grown in most parts of Europe and Northern America, has been used in ethnopharmacology. The content of selected biophenols and the antioxidant capacity of the extracts of wild heather collected from natural environmental localities of central Poland were assessed in this study and compared with cultivated plant. Chlorogenic acid was the major biophenolic compound present in the extracts, followed by a high amount of catechins. The reducing power of the extracts evaluated by Folin-Ciocalteau assay was in the range of 75.7-89.1 mg GA/g dry flowers. The cultivated plant extract showed the largest radical scavenging on 1,1-diphenyl-2-pirylhydrazyl (DPPH) radicals.

Disruption of trophic interactions involving the heather beetle by atmospheric nitrogen deposition.

Elevated nitrogen (N) deposition impacts the structure and functioning of heathland ecosystems across Europe. Calluna plants under high N-inputs are very sensitive to secondary stress factors, including defoliation attacks by the heather beetle. These attacks result in serious damage or death of Calluna, its rapid replacement by grasses, and the subsequent loss of heathland. We know very little about the mechanisms that control the populations and trigger outbreaks of the heather beetle, impeding proper management measures to mitigate the damage. We investigated the effects of N deposition on the relationships between the heather beetle, its host plant, and two arthropod predators at building (rejuvenated through fire) and mature heathlands. The study combines field manipulation experiments simulating a range of N deposition rates (0, 1, 2, 5 g N m-2 year-1 for 2 years, and 5.6 g N m-2 year-1 for 10 years), and food-choice laboratory experiments testing the preferences of adults and larvae of the heather beetle for N-treated Calluna plants, and the preferences of predators for larvae grown on plants with different N-content. The larvae of the heather beetle achieved the highest abundances after the long-term (10-year) addition of N at mature Calluna plots in the field. Contrary to the adults, the larvae foraged preferentially on the most N-rich Calluna shoots under laboratory conditions. Predators showed no aggregative numerical responses to the accumulation of heather beetle larvae at high N-input experimental plots. During the feeding trials, predators consumed a small number of larvae, both in total and per individual, and systematically avoided eating the larvae reared on high-N Calluna shoots. Our study showed that the most severe defoliation damage by the heather beetle is inflicted at the larval stage under prolonged availability of high-N inputs, and that arthropod predators might not act as effective regulators of the beetle's populations.

Combination of herbivore removal and nitrogen deposition increases upland carbon storage.

Ecosystem carbon (C) accrual and storage can be enhanced by removing large herbivores as well as by the fertilizing effect of atmospheric nitrogen (N) deposition. These drivers are unlikely to operate independently, yet their combined effect on aboveground and belowground C storage remains largely unexplored. We sampled inside and outside 19 upland grazing exclosures, established for up to 80 years, across an N deposition gradient (5-24 kg N ha(-1) yr(-1) ) and found that herbivore removal increased aboveground plant C stocks, particularly in moss, shrubs and litter. Soil C storage increased with atmospheric N deposition, and this was moderated by the presence or absence of herbivores. In exclosures receiving above 11 kg N ha(-1) year(-1) , herbivore removal resulted in increased soil C stocks. This effect was typically greater for exclosures dominated by dwarf shrubs (Calluna vulgaris) than by grasses (Molinia caerulea). The same pattern was observed for ecosystem C storage. We used our data to predict C storage for a scenario of removing all large herbivores from UK heathlands. Predictions were made considering herbivore removal only (ignoring N deposition) and the combined effects of herbivore removal and current N deposition rates. Predictions including N deposition resulted in a smaller increase in UK heathland C storage than predictions using herbivore removal only. This finding was driven by the fact that the majority of UK heathlands receive low N deposition rates at which herbivore removal has little effect on C storage. Our findings demonstrate the crucial link between herbivory by large mammals and atmospheric N deposition, and this interaction needs to be considered in models of biogeochemical cycling.

Supercritical fluid extraction of heather (Calluna vulgaris) and evaluation of anti-hepatitis C virus activity of the extracts.

Previous studies using lipid extracts of heather (Calluna vulgaris) leaves showed the presence of high concentrations of ursolic and oleanolic acid. These two compounds have been reported to present antiviral activity against hepatitis C virus (HCV). In this work, the supercritical fluid extraction of heather was studied with the aim of assessing a potential anti-HCV activity of the extracts owing to their triterpenic acid content. Supercritical extraction assays were carried out exploring the pressure range of 20-50 MPa, temperatures of 40-70°C and 0-15% of ethanol cosolvent. The content of oleanolic and ursolic acid in the extracts were determined, and different samples were screened for cellular cytotoxicity and virus inhibition using a HCV cell culture infection system. Antiviral activity was observed in most extracts. In general, superior anti-HCV activity was observed for higher contents of oleanolic and ursolic acids in the extracts.

In vitro activity of heather [Calluna vulgaris (L.) Hull] extracts on selected urinary tract pathogens.

Calluna vulgaris L. Hull (Ericaceae) has been used for treatment of urinary tract infections in traditional medicine. In this study we analyzed in vitro antibacterial activity of the plant extracts on different strains of Escherichia coli, Enterococcus faecalis and Proteus vulgaris, as well as the concentrations of total phenols and flavonoids in the extracts. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The concentrations of total phenols were examined by using Folin-Ciocalteu reagent and ranged between 67.55 to 142.46 mg GAE/g. The concentrations of flavonoids in extracts were determined using spectrophotometric method with aluminum chloride and the values ranged from 42.11 to 63.68 mg RUE/g. The aqueous extract of C. vulgaris showed a significant antibacterial activity. The values of MIC were in the range from 2.5 mg/ml to 20 mg/ml for this extract. Proteus vulgaris strains were found to be the most sensitive. The results obtained suggest that all tested extracts of C. vulgaris inhibit the growth of human pathogens, especially the aqueous extract.

Simulated climate change impact on summer dissolved organic carbon release from peat and surface vegetation: implications for drinking water treatment.

Uncertainty regarding changes in dissolved organic carbon (DOC) quantity and quality has created interest in managing peatlands for their ecosystem services such as drinking water provision. The evidence base for such interventions is, however, sometimes contradictory. We performed a laboratory climate manipulation using a factorial design on two dominant peatland vegetation types (Calluna vulgaris and Sphagnum Spp.) and a peat soil collected from a drinking water catchment in Exmoor National Park, UK. Temperature and rainfall were set to represent baseline and future conditions under the UKCP09 2080s high emissions scenario for July and August. DOC leachate then underwent standard water treatment of coagulation/flocculation before chlorination. C. vulgaris leached more DOC than Sphagnum Spp. (7.17 versus 3.00 mg g(-1)) with higher specific ultraviolet (SUVA) values and a greater sensitivity to climate, leaching more DOC under simulated future conditions. The peat soil leached less DOC (0.37 mg g(-1)) than the vegetation and was less sensitive to climate. Differences in coagulation removal efficiency between the DOC sources appears to be driven by relative solubilisation of protein-like DOC, observed through the fluorescence peak C/T. Post-coagulation only differences between vegetation types were detected for the regulated disinfection by-products (DBPs), suggesting climate change influence at this scale can be removed via coagulation. Our results suggest current biodiversity restoration programmes to encourage Sphagnum Spp. will result in lower DOC concentrations and SUVA values, particularly with warmer and drier summers.

Anthelmintic effect of heather in goats experimentally infected with Trichostrongylus colubriformis.

The effects of heather (composed primarily of Calluna vulgaris with a smaller content of Erica umbellata and Erica cinerea) consumption on the establishment of incoming infective larvae (experiment 1, preventive treatment) and an adult worm population (experiment 2, curative treatment) were investigated in Cashmere goats experimentally infected with Trichostrongylus colubriformis. In experiment 1, 12 castrated male goats were divided into two groups: heather-supplemented vs. non-supplemented animals. After 2 weeks of adaptation to the diet, all goats were experimentally infected per os with 6,000 T. colubriformis third-stage larvae. Three weeks post-infection, the goats were slaughtered, and worm counts as well as female worm fecundity and development were determined. Heather consumption was associated with a close to significant (P=0.092) reduction (mean 14 %) in larvae establishment. No effect on fecundity was observed, but the length of female worms in supplemented goats was greater (P<0.001). In experiment 2, 15 non-lactating does were experimentally infected with 6,000 T. colubriformis third-stage larvae. At 6 weeks post-infection, three groups were established: control, heather-supplemented and heather-supplemented with polyethylene glycol. Individual faecal nematode egg output was measured twice weekly to assess gastrointestinal nematode egg excretion. The goats were slaughtered 5 weeks after heather administration (11 weeks post-infection), and worm counts as well as female worm fecundity and development were subsequently determined. Heather administration was associated with a significant (P<0.001) decrease (between 47 and 66 % compared with control group) in egg excretion from 45 to 76 days post-infection. Although worm counts and female fecundity were lower in supplemented goats, no significant differences were observed. Overall, the results showed a reduction in T. colubriformis larvae establishment and a decrease in nematode egg excretion when heather was administered in experimentally infected goats. The heather plus polyethylene glycol treatment reduced nematode egg excretion levels at the same proportion as heather, thereby suggesting that the threshold of tannins required for an anthelmintic effect is most likely quite low.

Nitrogen deposition reduces plant diversity and alters ecosystem functioning: field-scale evidence from a nationwide survey of UK heathlands.

Findings from nitrogen (N) manipulation studies have provided strong evidence of the detrimental impacts of elevated N deposition on the structure and functioning of heathland ecosystems. Few studies, however, have sought to establish whether experimentally observed responses are also apparent under natural, field conditions. This paper presents the findings of a nationwide field-scale evaluation of British heathlands, across broad geographical, climatic and pollution gradients. Fifty two heathlands were selected across an N deposition gradient of 5.9 to 32.4 kg ha(-1) yr(-1). The diversity and abundance of higher and lower plants and a suite of biogeochemical measures were evaluated in relation to climate and N deposition indices. Plant species richness declined with increasing temperature and N deposition, and the abundance of nitrophilous species increased with increasing N. Relationships were broadly similar between upland and lowland sites, with the biggest reductions in species number associated with increasing N inputs at the low end of the deposition range. Both oxidised and reduced forms of N were associated with species declines, although reduced N appears to be a stronger driver of species loss at the functional group level. Plant and soil biochemical indices were related to temperature, rainfall and N deposition. Litter C:N ratios and enzyme (phenol-oxidase and phosphomonoesterase) activities had the strongest relationships with site N inputs and appear to represent reliable field indicators of N deposition. This study provides strong, field-scale evidence of links between N deposition--in both oxidised and reduced forms--and widespread changes in the composition, diversity and functioning of British heathlands. The similarity of relationships between upland and lowland environments, across broad spatial and climatic gradients, highlights the ubiquity of relationships with N, and suggests that N deposition is contributing to biodiversity loss and changes in ecosystem functioning across European heathlands.

Antimicrobial activity of different Finnish monofloral honeys against human pathogenic bacteria.

The antimicrobial activity and phenolic compounds of five Finnish honey products against important human pathogens Streptococcus pneumoniae, S. pyogenes, Staphylococcus aureus, and methicillin-resistant S. aureus were analyzed. Microbroth dilution method and HPLC-DAD were used in antimicrobial testing and phenolic compound determination, respectively. Significant antimicrobial activity (p < 0.01) against all the tested pathogens was found from willow herb (Epilobium angustifolium), heather (Calluna vulgaris), and buckwheat (Fagopyrum esculentum) honeys. This is the first report on antimicrobial activity of Finnish monofloral honeys against streptococcal and staphylococcal bacteria. To our knowledge this is also the first report on the antimicrobial effect of honey against S. pneumoniae.

Triterpenoid profile of flower and leaf cuticular waxes of heather Calluna vulgaris.

Analysis of the main triterpenoid profile of chloroform-soluble cuticular waxes of heather flowers and leaves by GC-MS revealed the following composition: five triterpene acids - betulinic, oleanolic, ursolic, 3-oxo-olean-12-en-28-oic and 3-oxo-ursan-12-en-28-oic; eight monohydroxyalcohols - α-amyrin, ß-amyrin, cycloartanol, 24-methylenecycloartanol, friedelinol, germanicol, lupeol and taraxasterol; three dihydroxyalcohols - betulin, erythrodiol and uvaol; two aldehydes - oleanolic and ursolic; four ketones - α-amyrenone, 4-epi-friedelin, friedelin and taraxerone and seven steroids - campesterol, cholesterol, sitostanol, sitosterol, stigmasterol, stigmasta-3,5-dien-7-one and stigmastane-3,6-dione. Triterpenoids accounted for 20% and 65% by mass of flower and leaf waxes, respectively, which suggest that heather leaves represent a very promising source of these compounds. Ursolic acid was the principal triterpenoid in the cuticular wax of both organs, whereas among the neutral triterpenes, friedelin and uvaol were the most abundant in flowers and leaves, respectively. This report provides the first thorough overview of the triterpenoid composition of cuticular waxes of heather.

Photochemoprotective effect of Calluna vulgaris extract on skin exposed to multiple doses of ultraviolet B in SKH-1 hairless mice.

UVB is a major cause of nonmelanoma skin cancer in humans. Photochemoprevention represents an important strategy in protecting the skin against the detrimental effects of ultraviolet B (UVB). We investigated the activity of Calluna vulgaris (Cv) delivered via a hydrogel on 3 main pathways (oxidative stress, inflammation, DNA damage) on skin exposed to multiple doses of UVB in SKH-1 mice. Fifty female mice were divided randomly into 5 groups: control, vehicle, UVB irradiated, Cv + UVB irradiated, and Cv + vehicle + UVB irradiated. The extract was applied topically on the skin in a dose of 4 mg polyphenols/cm2 30 minutes before each UVB (240 mJ/cm2) exposure over 10 consecutive days. Malondialdehyde, reduced glutathione, tumor necrosis factor-α, interleukin-6, cyclobutane pyrimidine dimer (CPD) levels, sunburn cell formation and epidermal thickness, and the number of epidermal cell layers in skin were evaluated 24 hours after the last treatment. UVB increased cytokine levels (P < 0.001), formation of CPDs (P < 0.001) and sunburn cells (P < 0.001), and the epidermal thickness and number of epidermal cell layers (P < 0.001) compared with the control group. The topical application of Cv protected the skin against inflammation and DNA damage, as shown by a decreased number of CPDs (P < 0.001) and sunburn cells (P < 0.001). The administration of Cv via hydrogel may be a viable method for chemoprevention..

Photoprotective effect of Calluna vulgaris extract against UVB-induced phototoxicity in human immortalized keratinocytes.

There is an increasing interest in the use of natural antioxidants as photoprotective agents against skin damages produced by ultraviolet radiation. The aim of our study was to investigate the protective effect of a Calluna vulgaris extract in human keratinocytes (HaCaT) exposed to ultraviolet B (UVB) radiation. HaCaT cells were treated with C. vulgaris extract 30 minutes prior to irradiation with UVB. The protective effect was evaluated by assessing cell viability using tetrasolium salt (MTT) assay; the generation of lipid peroxides was evaluated using malondialdehide assay (MDA); and DNA damage was evaluated using the comet assay and the quantification by ELISA of specific DNA photolesions [i.e., cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs)]. After irradiation with cytotoxic doses of UVB (300 and 500 mJ/cm(2)), HaCaT cells pretreated with C. vulgaris extract (50 µg GAE/ml) showed significantly increased viability compared to control cells exposed to UVB only. Irradiation alone increased MDA levels in a dose-dependent fashion. Pretreatment with 12 µg GAE/ml extract lowered MDA levels both at 100 mJ/cm(2) (ρ<0.01) and 300 mJ/cm(2) (ρ<0.001). Treatment with C. vulgaris extract before exposure to UVB also reduced DNA damage: Lesion scores in a comet assay were significantly reduced at UVB doses of 50 mJ/cm2 (ρ<0.01) and 100 mJ/cm(2) (ρ<0.05), while CPDs and 6-4PPs (via ELISA) were significantly lower after irradiation with 100 mJ/cm(2) in the protected cells (ρ<0.05 for CPDs and ρ<0.001 for 6-4PPs). These results recommend the use of the C. vulgaris extract as photoprotective agent, in combination with sunscreens and/or other natural products with similar or complementary properties.

Photoprotective effects of two natural products on ultraviolet B-induced oxidative stress and apoptosis in SKH-1 mouse skin.

Solar ultraviolet radiation (UV) is the major cause of nonmelanoma skin cancer in humans. Photochemoprevention with natural products represents a simple but very effective strategy for the management of cutaneous neoplasia. We studied the photoprotective activity of Calluna vulgaris and red grape seed (Vitis vinifera L, Burgund Mare variety [BM]) extracts in vivo in an SKH-1 hairless mice skin model. Fifty 8-week-old female SKH-1 hairless mice were randomly divided into 5 groups (n = 10 each): controls, UVB-irradiated, C. vulgaris plus UVB-irradiated, BM plus UVB-irradiated, and epigallocatechin gallate (EGCG) plus UVB-irradiated. A dose of 4 mg/mouse per cm² of skin area for both extracts was topically applied to the mice 30 minutes before a single-dose (240 mJ/cm²) UVB exposure. EGCG dissolved in phosphate-buffered saline (pH 6.6; 0.067 M) was administered at 2 mg/mouse per cm². Glutathione peroxidase and catalase activities, reduced glutathione (GSH), malondialdehyde, nitric oxide, and caspase 3 activity were determined in skin homogenates 24 hours after irradiation. A single dose of UVB increased GSH levels and glutathione peroxidase activity in the exposed skin. C. vulgaris and BM pretreatment significantly decreased GSH formation and glutathione peroxidase activity (P < .001) and inhibited UVB-induced lipid peroxidation (P < .0001) and nitric oxide production (C. vulgaris: P < .06). Topical treatments with C. vulgaris and particularly BM extracts (P < .002) significantly reduced caspase 3 activity, indicating that the cells were protected against apoptosis. These results suggest that C. vulgaris and BM extracts might be chemopreventive candidates for reducing UV-induced risk for skin cancer.

MAO-A inhibitory activity of quercetin from Calluna vulgaris (L.) Hull.

AIM OF THE STUDY: This study investigated MAO-A inhibitory activity of methanol extract of Calluna vulgaris (L.) Hull., which traditionally has been used as a nerve calming remedy. MATERIALS AND METHODS: A methanolic extract of Calluna vulgaris was partitioned against heptane, ethyl acetate and water. The three fractions were tested in a photometric peroxidase linked MAO-A bioassay. The ethyl acetate phase showed the highest MAO-A inhibitory activity. Quercetin was isolated by VLC through bioassay-guided fractionation and purified by re-crystallisation. The structure was elucidated by LC-MS and (1)H NMR. RESULTS: The IC(50)-value for MAO-A inhibition by quercetin was 18+/-0.2 microM in an assay where the IC(50)-value for MAO-A inhibition by clorgylin was 0.2+/-0.02 microM. CONCLUSION: The content of quercetin in Calluna vulgaris might explain the reported nerve calming effect of the plant.

Influence of altitudinal variation on the content of phenolic compounds in wild populations of Calluna vulgaris, Sambucus nigra, and Vaccinium myrtillus.

This study deals with the effect of altitudinal variation on the content of phenolic compounds in three traditional herbal plants, which are also consumed as food in Central Europe. Herbs of Calluna vulgaris (L.) HULL, flowers and fruits of Sambucus nigra L., and berries of Vaccinium myrtillus L. collected in the Naturpark Solktaler (Austria) were extracted using accelerated solvent extraction (ASE). Identification and quantification of the constituents in the polar extracts (methanol 80%, v/v) were achieved by means of RP-HPLC-PDA and/or LC-PDA-MS analysis with external standards. 3,5- O-Dicaffeoylquinic acid was identified in flowers of S. nigra for the first time. Rising concentrations of flavonoids and especially flavonol-3- O-glycosides with adjacent hydroxyl groups in ring B in C. vulgaris and S. nigra with increasing altitude were observed. Anthocyanins from the berries of both S. nigra and V. myrtillus occurred in decreasing amounts with rising altitude. C. vulgaris showed the best radical scavenging capacity based on the DPPH assay.

Interactive effects of nitrogen deposition and fire on plant and soil chemistry in an alpine heathland.

The response of alpine heathland vegetation and soil chemistry to N additions of 0, 10, 20 and 50 kg N ha(-1) year(-1) in combination with simulated accidental fire (+/-) was monitored over a 5-year period. N addition caused rapid and significant increases in plant tissue N content and N:P and N:K of Calluna vulgaris, suggesting increasing phosphorus and potassium limitation of growth. Soil C:N declined significantly with N addition, indicating N saturation and increasing likelihood of N leakage. Fire further decreased soil C:N and reduced potential for sequestration of additional N. This study shows that alpine heathlands, which occupy the headwaters of many rivers, have limited potential to retain deposited N and may rapidly become N saturated, leaking N into downstream communities and surface waters.