High Amounts Of Secondary Metabolites Research Articles

Evaluation of genetic stability using FRAPD markers as novel method along with antioxidant and anti-diabetic properties of micropropagated Salacia chinensis L.

Salacia chinensis L., a perennial medicinal plant, is well-known for its well-documented anti-diabetic properties. The daily growing demand in pharmaceutical industry is stimulating the conservation and wide-ranging production of the plant using plant tissue culture techniques (micropropagation). In the present study, the plants generated by direct micropropagation from nodal explants were assessed using fluorescently labeled RAPD (FRAPD) primers. Although standard RAPD primer bands in agarose gel showed genetic stability, using FRAPD analysis in genetic DNA sequencer as a novel strategy showed more accurate and reliable method has indicated by the evidence in 5% genetic variation. Antioxidant and anti-diabetic activities of micropropagated plants versus mother plant were examined using DPPH, FRAP, α-amylase, and α-glucosidase assays. The results showed that the micropropagated plants, which are able to produce higher amount of secondary metabolites than the mother plant, possess higher in vitro antioxidant and anti-diabetic properties.

PHYTOCHEMICAL SCREENING AND IN VITRO ANTIOXIDANT ACTIVITIES OF LEAF EXTRACTS OF ALTERNANTHERA BRASILIANA (L). KUNTZE AND ALTERNANTHERA BETTZICKIANA REGEL.

Objectives: The present study was carried out to determine the phytochemical constituents and in vitro antioxidant activities of leaf extracts of Alternanthera brasiliana (L). Kuntze (A. brasiliana) and Alternanthera bettzickiana regel (A. bettzickiana).Methods: Preliminary phytochemical screening was performed in petroleum ether, chloroform, acetone, ethanol, hydroethanolic and water extracts of leaves of A. brasiliana and A. bettzickiana. The level of alkaloids, flavonoids, total phenolic content, tannins, Vitamin C, Vitamin E, GSH, and total proteins were determined in hydroethanolic, ethanol, and water extracts. Antioxidant activity of the hydroethanolic leaf extracts of A. brasiliana and A. bettzickiana were determined by 2,2-diphenyl-1-picryl-hydrazyl-hydrate free radical scavenging assay, nitric oxide scavenging assay, superoxide anion scavenging assay, ferric reducing antioxidant power assay, total antioxidant capacity, and reducing power assay.Results: The phytochemical screening of six different extracts of A. brasiliana and A. bettzickiana revealed the presence of various phytonutrients. Quantitative analysis of secondary metabolites in ethanol, hydroethanolic and water extracts of leaves of A. brasiliana and A. bettzickiana showed the presence of high amount of secondary metabolites in the hydroethanolic extract. In vitro, antioxidant assay of two plant extracts revealed that A. bettzickiana was more potent than A. brasiliana in scavenging free radicals.Conclusion: The different extracts from A. brasiliana and A. bettzickiana and specifically the hydroethanolic extract of A. bettzickiana revealed several properties such as rich source of phytonutrients, higher free radical scavenging properties, and significant antioxidant capabilities. Therefore, the bioactive compound should be isolated in future studies and could be used as a safe and serve as a potential source of natural antioxidants.

Green synthesis of silver nanoparticles using Givotia moluccana leaf extract and evaluation of their antimicrobial activity

Present study reported a simple and green synthesis of silver nanoparticles (AgNPs) synthesized via rapid bio-reduction method. An aqueous extract of Givotia moluccana (G. moluccana) plant was serving as reducing and stabilizing agent. An aqueous extract was found to comprise significantly high amounts of secondary metabolites like phenolics, flavonoids, proteins and reducing sugars etc., which are responsible for reducing and capping agents. The synthesized AgNPs were characterized by UV–Visible spectroscopy, FTIR, XRD, FESEM and HRTEM with SAED pattern. Particle size distribution was measured by dynamic light scattering (DLS). Further, thermal analysis of nanoparticles were studied using TGA. Synthesized AgNPs exhibited antimicrobial activity against both gram positive and gram negative bacteria.

Soyisoflavone diversity in wild soybeans (Glycine soja Sieb. & Zucc.) from the main centres of diversity

Wild soybeans are naturally found in China, and the Korean Peninsula, Japanese archipelago and Russian Far East, which are considered their centres of diversity. The presence of higher amounts of secondary metabolites in wild soybeans makes them the ideal choice to increase soyisoflavone content in cultivated soybeans through modern breeding and biotechnological techniques. Such a superiority calls for soyisoflavone profiling of germplasm resources. The soyisoflavone diversity of 298 wild soybean accessions collected from main centres of diversity, i.e. China, South Korea, Japan, and the Russian Far East were subjected to high-performance liquid chromatography. The profiles of six soyisoflavone components from whole seeds were quantified. The analysis of variance, principle component analysis, and Pearson's correlation analysis clearly revealed that the 298 wild soybean accessions studied had low variation in terms of the six soyisoflavone components, i.e. daidzin, glycitin, genistin, malonyl-daidzin, malonyl glycitin, and malonyl genistin. The accessions with higher soyisoflavone contents are good candidates for future breeding strategies.

QUALITATIVE AND QUANTITATIVE ANALYSIS OF PHYTOCHEMICALS AND IN VITRO ANTIOXIDANT ASSAYS IN THE TUBER OF SOLENA AMPLEXICAULIS (LAM.) GANDHI. (CUCURBITACEAE)

Most of the traditional medicinal plants in India are not scientifically validated. Scientific evaluation of medicinal property along with traditional knowledge is essential to obtain effective drugs for commercial purpose. Solena amplexicaulisbelongs to the family, Cucurbitaceae, a traditional medicinal plant species ofTamil Nadu, India is being prescribed to cure various diseases. In this study, the objective was to investigate the qualitative and quantitative determinations of certain phytochemicals and in vitro antioxidant capabilities of various alcoholic and aqueous tuber extracts of S. amplexicaulisby adapting standard procedures. In all the assays methanolic tuber extract registered significantly high amount of secondary metabolites and also it effectively scavenge the free radicals in a concentration dependent manner than the other extracts. These results were compared with synthetic (BHA and BHT) as well as natural antioxidants (rutin and quercetin). The outcome of the study revealed most valuable information and also supports the continued sustainable use of this species in traditional systems of medicine.

STUDY OF ANTIMICROBIAL ACTIVITY OF THE FOLKLORE MEDICINAL PLANT, ACALYPHA FRUTICOSA FORSSK.

The purpose of the study is to examine the antimicrobial efficacy of root extracts of the folklore medicinal plant species, Acalypha fruticosa Forssk by using three alcoholic solvents viz; petroleum ether, ethyl acetate and methanol were tested against ten human pathogenic bacteria viz., Pseudomonas aeruginosa, P. stutzeri, Escherichia coli, Micrococcus sp., Lactobacillus sp., Servatia sp., Moraxetta sp., Bacillus subtilis, B. thuriengensis, and Klebsiella pneumoniae and ten human pathogenic fungi viz., Aspergillus niger, A. flavus, A.baumannii, Fusarium oxysporum, F. solani, Mucor rouxii, Alternaria alternata, Candida albicans, Cladosporium sp. and Rhizopus sp. for assessing the antimicrobial properties by adapting disc diffusion method. The results of the study revealed that all extracts showed varied degree of antimicrobial activity against the tested pathogens. However, the methanol extracts exhibited higher inhibition zone (21.83 mm) against the bacterium, Bacillus subtilis and ethyl acetate extracts showed higher inhibiotion zone (24.83 mm) against the fungus, Rhizopus sp. Results concluded that this spices contain high amount of secondary metabolites due to these metabolites they have high antimicrobial activity and it can be used as good bio- preservater and it can also use for medicinal purpose.

Enhancement of anthraquinone production and release by combination of culture medium selection and methyl jasmonate elicitation in hairy root cultures of Rubia tinctorum

Anthraquinones (AQs) are secondary metabolites widely distributed in nature. Interesting applications of plant extracts containing AQs include the treatment of Hepatitis C and cancer. Plant in vitro culture is an alternative for producing plant-derived pharmaceuticals in controlled conditions and with low environmental impact. Moreover, it allows the application of different strategies for enhancing secondary metabolite production. Hairy root cultures, obtained after Agrobacterium rhizogenes infection, are able to produce high amounts of secondary metabolites at high growth rates. In this work, growth kinetics of hairy root cultures of Rubia tinctorum and AQ production were evaluated in two different culture media, Gamborg B5 with half of the saline strength (B51/2) and Lloyd & Mc Cown’s Woody Plant Medium (WPM). Although WPM allowed higher biomass production (58.6% higher) than B51/2, specific AQ production was higher in B51/2 (between 1.2 and 2.1 fold increases from day 21 to the end of the experiment). Moreover, AQ release to the culture medium was observed in B51/2 (∼10% of total AQs). The different performance of hairy roots in these culture media may be due to a limiting nutrient (other than carbon source) in B51/2. Elicitation in B51/2 with methyl jasmonate (100μM) resulted in a massive accumulation of intracellular (between 1.5 and 2.4-fold increases) and also extracellular AQs (up to 8.1 fold-increase compared with control at 4days post-elicitation), which could ease AQ purification. These results prove the usefulness of combining different approaches to enhance secondary metabolite accumulation in plant in vitro cultures, in order to develop an optimized productive process.

Variation in bioactive contents and anatomical characteristics of different fennel ( Foeniculum vulgare Mill.) populations as affected by self-pollination

The production of self pollinated plant genotypes could be critical for improving medicinal plants. Pollination in Apiaceae family can also affect secondary metabolites. In this study, 23 fennel populations were used to assess the effect of self pollination on essential oil yield, antioxidant activity (based on three model system), total phenolic (TPC) and flavonoid content (TFC). First, some plant inflorescences were divided in two parts. Then the half was bagged and the second half was permitted for out crossing. The self and outcross pollinated seeds were sown in a randomized complete block design (RCBD) in three replicates. Results revealed that inbreeding led to increase in the secretary ducts number (9.36%) as well as essential oil yield (25.61%) in all fennel populations. Essential oil yield ranged from 2.4% to 6.4% in seeds produced via out crossing, while it varied from 3.5% to 6.5% in self pollinated ones. Furthermore, self pollination increased TPC (21.66%), TFC (49.40%) and antioxidant activity (6.23%). Among the populations derived from self pollinated seeds Tabriz showed the highest TFC (8.4 mgQUEg -1 DW) and antioxidant activity (IC 50 =83.1μg/ml), whereas Semirom possessed the highest TPC (150 mgTAEg -1 DW), respectively. In overall, self pollination can produce lead to the populations with higher amount of secondary metabolites.

STUDY OF VARIOUS FIVE EXTRACTS OF TRACHYSPERMUM AMMI SEEDS ON SELECTED FOUR GRAM-NEGATIVE COOKED FOOD SPOILAGE BACTERIAL STRAINS BY DISK DIFFUSION METHOD

Bacterial foodborne diseases are caused by consumption of foods contaminated with bacteria or their toxins. This study evaluated antibacterial properties of Trachyspermum ammi Acetone, ethanol, methanol, cold water and hard water extracts of spice (Ajowan) seeds against four gram negative strains of pathogenic foodborne bacteria, E. coli O157:H7 ATCC 43888, ATCC 25922, ATCC 8739 and ATCC 43895 that cause infection and intoxication. E. coli ATCC 43888 was observed to be highly susceptible to all extracts of ajowan with absolute zones of inhibition in the range of 16mm – 23mm in diameter. This study demonstrated that spice extracts have antimicrobial activity against food-borne bacterial species and should be considered as potential antibacterial agents for addition to ready meals. The spice contain high amount of secondary metabolites due to these metabolites they have high antimicrobial activity and it can be used as good bio- preserver and it can also use for medicinal purpose.

Biochemical Profile of Leaf, Silk and Grain Samples of Eight Maize Landraces (Z ea mays L.) Cultivated in Two Low-Input Agricultural Systems

Highlights We analyzed total carotenoids, anthocyanins and phenolic acids in grains, leaves and silks of maize landraces. Higher amounts of secondary metabolites were found in leaves and silks. High performance liquid chromatography revealed the xanthophylls lutein and zeaxanthin as the major carotenoids in grains. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of crude extracts showed a higher richness of anthocyanin in leaf samples than in silks. Abstract This research aimed to determine the biochemical profile of leaf, silk and grain samples of eight maize landraces (Zea mays L.) cultivated in southern Brazil. To accomplish this, reverse-phase high performance liquid chromatography-UV-visible (RP-HPLC-UV-vis), UV-vis spectrophotometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and chemometrics were used to examine carotenoids and their isomers, anthocyanin and phenolic acids. Leaf tissue samples showed higher amounts of carotenoids (838.6 μg/g – Língua de Papagaio variety), anthocyanins (405.8 μg/g – Palha Roxa variety) and phenolics (655.0 μg/g – Roxo variety), followed by maize silks and grains. RP-HPLC-UV-vis analysis of grain extracts revealed xanthophylls lutein and zeaxanthin as the major compounds. The anthocyanin profile by MALDI-TOF MS identified pelargonidin, cyanidin, peonidin, malvidin and glucoside derivatives in leaf extracts, and silk samples were shown to contain cyanidin, peonidin and 7-methoxy-apigeninidin glucoside. These findings indicate that the discarded material, i.e., leaves and silks, can be prospected as bioactive biomasses useful to industrial sectors. Practical Applications The present study indicates that leaves and silks of maize landraces, typically discarded in the normal process of agricultural production, can be prospected in food systems as potential sources of antioxidant compounds, such as carotenoids, anthocyanins and phenolics. Moreover, acquisition of these raw materials for development of new products in the pharmaceutical, food and cosmetics industries can be accomplished with no or negligible cost. Thus, the preservation of this valuable germplasm is to be encouraged, as it will increase the possibility of economic return to small farmers.

Quantification of phytochemicals and in vitro antioxidant potential of various solvent extracts of certain species of Acanthaceae

Background: Scientific evaluation of traditional knowledge is essential to obtain effective drugs for commercial purposes. The present study is guide to the invention of new antioxidant sources from the Acanthaceae members. Aim: Quantification of certain secondary metabolites and in vitro antioxidant activities of alcoholic and aqueous leaf extracts of Nilgirianthus heyneanus and Peristrophe bicalyculata. Materials and Methods: Both the leaf extracts were tested for various phytochemical constituents such as phenols, tannins, condensed tannins, flavonoids, vitamin C and saponin contents. The extracts were further screened for different antioxidant assays such as reducing power, DPPH▪, nitric oxide, metal chelating, ABTS▪+ scavenging activity, lipid peroxidation preventive property and anti‑haemolytic activities. Results: Acetone fractions of Nilgirianthus heyneanus leaf registered significantly high amount of secondary metabolites and also it effectively scavenged the free radicals in a concentration dependent manner than Peristrophe bicalyculata leaf extracts. Results were compared with standard antioxidants such as rutin, quercetin, BHA and BHT. Conclusions: This study further confirmed the traditional use of N. heyneanus and P. bicalyculata as a natural source of antioxidants and prevents free radical‑induced life‑threatening diseases. Key words: In vitro antioxidant activity, Nilgirianthus heyneanus, Peristrophe bicalyculata, quantitative estimations

Enhanced Salt Stress Tolerance in Transgenic Potato Plants Expressing IbMYB1, a Sweet Potato Transcription Factor

IbMYB1, a transcription factor (TF) for R2R3-type MYB TFs, is a key regulator of anthocyanin biosynthesis during storage of sweet potatoes. Anthocyanins provide important antioxidants of nutritional value to humans, and also protect plants from oxidative stress. This study aimed to increase transgenic potatoes' (Solanum tuberosum cv. LongShu No.3) tolerance to environmental stress and enhance their nutritional value. Transgenic potato plants expressing IbMYB1 genes under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter (referred to as SM plants) were successfully generated through Agrobacterium-mediated transformation. Two representative transgenic SM5 and SM12 lines were evaluated for enhanced tolerance to salinity, UV-B rays, and drought conditions. Following treatment of 100 mM NaCl, seedlings of SM5 and SM12 lines showed less root damage and more shoot growth than control lines expressing only an empty vector. Transgenic potato plants in pots treated with 400 mM NaCl showed high amounts of secondary metabolites, including phenols, anthocyanins, and flavonoids, compared with control plants. After treatment of 400 mM NaCl, transgenic potato plants also showed high DDPH radical scavenging activity and high PS II photochemical efficiency compared with the control line. Furthermore, following treatment of NaCl, UV-B, and drought stress, the expression levels of IbMYB1 and several structural genes in the flavonoid biosynthesis such as CHS, DFR, and ANS in transgenic plants were found to be correlated with plant phenotype. The results suggest that enhanced IbMYB1 expression affects secondary metabolism, which leads to improved tolerance ability in transgenic potatoes.

Zn/Pb-tolerant lichens with higher content of secondary metabolites produce less phytochelatins than specimens living in unpolluted habitats

Many lichens can cope with heavy-metal stress, however, the mechanisms of lichen tolerance are still not fully understood. Some lichen secondary metabolites (depsides and depsidones), produced in lichens by the fungal symbiont and accumulated on the outer surface of its hyphae, are supposed to play an important role in the extracellular immoblilization of heavy metals. Lichen photobionts (algal partners in the symbiosis), although surrounded by the mycobiont hyphae, may also accumulate high amounts of trace metals. This can lead to physiological disruptions and morphological damage in algal cells and hence affect the lichen physiological status. We hypothesized that lichen species/specimens living in heavily polluted sites and showing HM tolerance possess a higher content of secondary metabolites than those living in unpolluted sites. Hence, their photobionts can be better protected from the excess of metal ions and need to produce less metal-complexing phytochelatins (PCn) to combat metal toxicity. Specimens of Hypocenomyce scalaris, Cladonia furcata and Lepraria spp. sampled from Zn/Pb-polluted and control sites were compared for the accumulation of Zn/Pb and secondary metabolites, as well as for their production of phytochelatins and glutathione in response to experimental Zn or Pb exposure. Generally, the lichen specimens sampled from the HM-polluted site contained higher amounts of Zn and Pb as well as lichen substances (different depsides and depsidones) than those from the control site. A strong positive correlation was found between the accumulation of secondary metabolites and Zn/Pb accumulation ( R 2 = 0.98 and 0.63, respectively). For the first time, production of phytochelatins (PC 2–3) in response to Zn and Pb (50–200 μM) exposure was found in H. scalaris, L. elobata, L. incana and C. furcata. In both species of Lepraria also cysteine, a substrate for GSH and PCs synthesis was detected. The lichens from the polluted site produced under the same exposure conditions, or in response to higher metal concentrations, lower amounts of PCn than those sampled from the control site. It strongly suggests that less Zn and Pb ions reached the photobiont cells of the lichens containing higher amounts of secondary metabolites (lecanoric, fumarprotocetraric, stictic, constictic acids, antranorin). The results obtained support the putative role of some metabolites in heavy-metal tolerance of the lichens inhabiting metal-polluted habitats.

Molecular genetic analysis of Asparagus racemosus L. accessions using RAPD markers

Ten different accessions of Asparagus racemosus L. originated from Himachal Pradesh and Tamil Nadu were assessed for the genetic variation at molecular level. The genomic DNA was isolated using Stange et al. (1998) protocol with modifications to remove the secondary metabolites as Asparagus racemosus contains high amounts of secondary metabolites that interfere with DNA extraction. Twenty random decamer primers, selected from fifty initially screened, were used to assess variation. All selected primers produced polymorphic amplification products, however, the extent of polymorphism varied with each primer. The similarity coefficient and UPGMA clustering method were employed to construct the phylogenetic tree. The value of similarity coefficient ranged from 0.44 to 1.00. Four accessions (ASP-5, ASP-2, ASP-3, ASP-4) belonging to Mandi, Himachal Pradesh were genetically the most similar ones as validated by their similarity index. All ten accessions were grouped into two major clusters. Accessions from Himachal Pradesh were grouped in a separate subcluster. The second cluster consisted of three accessions of Tamil Nadu. In general, the genetic variability between the accessions of H.P. was less than the accessions of Tamil Nadu. The results warn prevailing of high homogeneity in A. racemosus accessions deployed for cultivation.

Coisolation of genomic and organelle DNAs from 15 genera and 31 species of plants

A large number of medicinal and aromatic plant species naturally grown in the Mediterranean basin of Turkey contain and produce secondary metabolites such as alkaloids, Xavanoids, phenols, polysaccharides, terpenes, and quinones that are used in the food, pharmaceutical, cosmetic, and pesticide industries. Many medicinal and aromatic plant species are undergoing domestication and cultivar development. Unfortunately some of these plant species are among the most endangered species which need to be protected to ensure their sustainable use in this region. However, medicinal and aromatic plants under study contain exceptionally high amounts of polysaccharides, polyphenols, and other secondary metabolites. Our preliminary studies showed that most medicinal and aromatic plant species were recalcitrant to several DNA extraction protocols tested including one commercial DNA extraction kit (Qiagen, Valencia, CA, USA). Although the kit used produced high-quality DNA, the amount of DNA was very low for many samples and it could not produce DNA in some samples. Also several other protocols described for plant DNA isolation failed to produce good quality and quantity of DNA or failed in PCR or restriction enzyme digestions from some of the Origanum, Salvia, Thymus, Sideritis, Saturaja, Calaminta, Rosemarunus, and Nerium spp., indicating that these plants contain exceptionally high amounts of secondary metabolites that interfere with DNA isolation [1–3]. These situations necessitate the development of protocols for isolating DNA from diVerent plant species. To address this problem we developed

Isolating high-quality RNA from mangroves without liquid nitrogen

Mangroves form unique communities in tropical coastal regions and tidal lowlands. Isolating RNA from mangrove leaves is difficult because of high amounts of secondary metabolites and polysaccharides. Conventional extraction methods produce poor-quality mangrove RNA. We present a simple, fast, and convenient protocol for isolating RNA from 5 mangrove species:Aegiceras corniculatum, Bruguiera gymnorrhiza, Ceriops tagal, Kandelia candel, andSonneratia apetala. Isolating RNA from other mangrove species is also possible. Obtained RNA was of high quality and used in an RT-PCR reaction that amplified 0.6 kb of theA. corniculatum CPI-1 gene.

DNA isolation from fresh, dry plant samples with highly acidic tissue extracts

Current DNA isolation methods are limited in their ability to obtain quality and/or quantity DNA from plants, such asEmblica officinalis, Terminalia belerica, andTerminalia chebula, which have low pH and high amounts of secondary metabolites in tissue extracts. Our modified DNA isolation method yields good-quality, high-molecular-weight DNA that is free of contaminants and colored pigments and is suitable for PCR amplification. This method is also useful for isolating DNA from dry powders.

Isolation of functional RNA from cactus fruit

Isolating RNA from cactus fruit is notoriously difficult because the fruit contains high amounts of secondary metabolites and polysaccharides. These form insoluble complexes with nucleic acids during extraction and can inhibit enzyme action. Our procedure allows for the extraction of RNA from finely ground tissue. The RNA we isolated was of high quality and undegraded, as gauged by spectrophotometry and electrophoresis in agarose gels. Quality was further assessed through use of the RNA in RT-PCR and northern blot analysis, indicating that it could be used to construct cDNA libraries. Using this modified protocol, 90μg of RNA was routinely obtained from 1 g of dried cactus fruit. Isolating RNA from other polysaccharide-rich fruits was also possible.

DNA isolation methods for medicinal and aromatic plants

Several protocols described for plant DNA isolation fail to produce good quality DNA from medicinal herbs and aromatic plants. These plants contain exceptionally high amounts of secondary metabolites that interfere with DNA isolation. To address this problem, we developed 2 DNA isolation methods for sundew and tarragon that produce DNA suitable for molecular biological applications. One of the methods also is applicable for milfoil and Siberian ginseng.

Niche separation in two species of hare. Metabolic costs of plant phenolics

The distributions of mountain hare (Lepus timidus) and European hare (L. europaeus) overlap in central Sweden, but they occupy separate food niches in winter. In Scandinavia, the European hare is mainly a grazer while mountain hare is a predominant browser. Browse contain high amounts of secondary metabolites, such as phenols, compared to grass. This raises the question if the two hare species differ in their metabolic tolerance of plant phenols and that these differences influence their food choice. Phenolic excretion in urine increased significantly with phenolic intake in both species. Excretion of glucuronic acid conjugates, one of the major pathways of elimination of phenols in both hare species, is positively correlated to phenolic intake and excretion. However, the extent of excretion of phenolics by this route was different in the two species of hare. European hares excreted substantially more glucuronic acid per amount of phenolics than mountain hare. The phenols were metabolized to a larger extent in the mountain hare, indicating a higher detoxification capacity. From these results it is likely that European hare have a higher cost for the detoxification of plant phenols compared to mountain hare. This cost and negative effect on sodium balance when feeding on browse may prevent exploitation of forest habitats by European hares, whereas mountain hare are better able to do so.