Differential effects of condensed and hydrolyzable tannin on polyphenol oxidase activity of attine symbiotic fungus

The leaf-cutting antAtta cephalotes is a generalist herbivore of the neotropics and collects leaf material to cultivate a fungus. It appears that this fungus, a Basidiomycete, is responsible for the ability of the ants to utilize most of the available woody plant species. Tannins and other phenolics are ubiquitous secondary chemicals in woody plants, and Basidiomycete fungi produce enzymes, such as polyphenol oxidase, that are capable of polymerizing and inactivating the phenolics. This study evaluates the effects of a condensed and a hydrolyzable tannin on the activity of polyphenoi oxidase and the growth of the fungus. I hypothesized that low concentrations of tannin would not inhibit polyphenol oxidase activity but high concentrations would inhibit the enzyme. Consequently, I predicted that only high concentrations of tannin would inhibit fungal growth. Laboratory assays with the fungus indicated that hydrolyzable tannin (tannic acid) and condensed tannin (quebracho tannin) differ in the mechanism of inhibition. Tannic acid does not inhibit polyphenol oxidase activity but does inhibit fungal growth. Quebracho tannin, however, inhibits both polyphenol oxidase activity and fungal growth. As predicted, both tannic acid and quebracho tannin primarily inhibit the fungus at high concentrations.     

Factors affecting levels of some phenolic compounds,digestibility, and nitrogen content of the mature leaves ofBarteria fistulosa (Passifloraceae)

Levels of total phenolics, condensed tannins, acid detergent fiber, pepsin/cellulase digestibility, and nitrogen in mature leaves of 26 individuals of the ant-plant,Barteria fistulosa, have been determined. Analysis of the results in terms of the presence or absence of ants and the position of the branch from which the leaves were collected showed no relationship with concentrations of phenolics or fiber and only a weak relationship with digestibility and nitrogen. By contrast, light intensity strongly influenced levels of phenolics, notably condensed tannins, so that mature leaves of individuals growing in direct sunlight were less digestible and appeared to be of lower quality as food for herbivores than did mature leaves of individuals in shaded positions. Possible reasons for the variation in condensed tannin levels are discussed.     

Tannin Composition Affects the Oxidative Activities of Tree Leaves

We examined whether tannin composition plays an important role in explaining the oxidative activities of tree leaves of Acer saccharum (sugar maple) and Quercus rubra (red oak). Sugar maple leaves contained substantial amounts of ellagitannins, condensed tannins, and galloyl glucoses, whereas red oak leaves contained almost exclusively condensed tannins. Oxidative activities of the crude phenolics from both species, and the phenolic fractions from sugar maple, were measured with electron paramagnetic resonance (EPR) spectrometry and UV-visible spectrophotometry. The two assays produced similar results: (1) sugar maple phenolics produced larger semiquinone radical concentrations,and higher semiquinone decay rates and browning rates than did red oak phenolics;(2) ellagitannin levels were positively associated with the three measures of oxidative activity; and (3) condensed tannin and galloyl glucose levels were negatively associated with these measures. The negative relationship between condensed tannin levels and oxidative activity resulted from the antioxidant effects of condensed tannins on hydrolyzable tannins; several purified condensed tannins significantly decreased the concentrations of semiquinone radicals and browning rates of pedunculagin (an ellagitannin) and pentagalloyl glucose. As expected, whole-leaf extracts from sugar maple produced elevated levels of semiquinone radicals, but none were observed in red oak extracts when the two species were compared with an EPR time-course assay. We conclude that the oxidative activities of tree leaves may be affected by tannin composition, and that the prooxidant activity of ellagitannins may be decreased by co-occurring condensed tannins.     

Tolerance of acridids to ingested condensed tannin

Four species of Acridoidea were fed on wheat leaves with and without the condensed tannin, quebracho. In no case was it deleterious to survival and growth at levels below about 10% dry weight on the food. Similarly, consumption, digestibility, and utilization of food were unaffected at up to 10% dry weight. At higher concentrations, however, the consumption and the efficiency of conversion of digestion were reduced, although digestibility was little affected. The possible mechanisms for such tolerance are discussed, and contrast made with insects which are very sensitive to ingested condensed tannin.     

Loss of Tannins and Other Phenolics from Willow Leaf Litter

A litter bag experiment was conducted to determine the fate of condensed tannins during the initial phase of willow leaf decomposition. Chemical methods and HPLC were used to identify and quantify the phenolics in aqueous acetone extracts of the leaves during the first eight weeks after leaf fall. Extractable phenolics and tannins were lost rapidly from the leaves and had half lives of about 2.4 weeks. Lower molecular weight tannins were lost more rapidly than higher molecular weight tannins, suggesting that the primary route for loss of tannins is leaching. There was no evidence for decomposition of the tannin during the leaf decay process. After leaching, the tannin apparently sorbs tightly to the soil.     

Effects of large mammalian herbivores and ant symbionts on condensed tannins of Acacia drepanolobium in Kenya

Condensed tannins have been considered to be important inducible defenses against mammalian herbivory. We tested for differences in condensed tannin defenses in Acacia drepanolobium in Kenya over two years among different large mammalian herbivore treatments [total exclusion, antelope only, and megaherbivore (elephants and giraffes) + antelope] and with four different ant symbiont species on the trees. We predicted that (1) condensed tannin concentrations would be lowest in the mammal treatment with the lowest level of herbivory (total exclusion), (2) trees occupied by mutualist ants that protect the trees most aggressively would have lower levels of tannins, and (3) if chemical defense production is costly, there would be a trade-off between tannin concentrations, growth, and mechanical defenses. Mean tannin concentrations increased from total exclusion treatments to wildlife-only treatments to megaherbivore + antelope treatments. In 1997, condensed tannin concentrations were significantly lower in trees occupied by the ant Crematogaster nigriceps, the only ant species that actively removed axillary buds. Contrary to our prediction, trees occupied by ant species that protect the trees more aggressively against mammalian herbivores did not have lower overall levels of condensed tannins. There was no consistent evidence of a trade-off between tannin concentrations and growth rate, but there was a positive correlation between mean thorn length and mean tannin concentrations across species of ant inhabitants and across herbivore treatments in 1997. Contrary to our expectation, trees had higher tannin concentrations in the upper parts of the canopy where there is little herbivory by mammals.     

Methods and pitfalls of extracting condensed tannins and other phenolics from plants: Insights from investigations onEucalyptus leaves

Optimal conditions for extraction of tannins and other phenolics from tree foliage and their subsequent storage rarely have been investigated. We investigated methods of drying leaves, optimal solvents, and the effects of light and temperature on the extractability and stability of condensed tannins (proanthocyanidins) and total phenolics from leaves ofEucalyptus trees. Aqueous acetone was a better solvent than aqueous methanol for condensed tannins and total phenolics, but condensed tannins were less stable in aqueous acetone than aqueous methanol. Stability of condensed tannins also was decreased substantially by room temperature versus 4°C and by exposure to indirect sunlight, although the assay for total phenolics was unaffected. For quantitative estimation of condensed tannins, extraction with 50% acetone was better than methods of direct analysis of leaf tissue. The highest estimates of total condensed tannins were obtained by exhaustive extraction with 50% acetone followed by direct analysis of the residue. Lyophilization of fresh leaf increased yield of condensed tannin (although usually by less than 10%). Lyophilization and subsequent storage of extracts had little effect on assays for condensed tannins or total phenolics.     

Chemical and Biological Assays to Evaluate Bacterial Inhibition by Tannins

The interactions among purified tannins from three different plants with five strains of ruminal bacteria were investigated. Tannins were purified from crude quebracho (Schinopsis balansae spp.), desmodium (Desmodium ovalifolium), and myrtle (Mirtus communis). The ruminal bacteria studied were Streptococcus bovis JB1, Ruminococcus albus 8, Fibrobacter succinogenes S85, Prevotella ruminicola H₁4, and a recently isolated tannin-tolerant bacterium. Bacterial binding to tannins, rate of tannin binding, and the inhibitory concentrations for each bacterium to each tannin were evaluated. The effects of tannins on glucose utilization and fermentation product formation also were studied. Tannin binding was rapid with little additional binding occurring 10 min after tannin addition. There was variation among bacteria in the amount of tannins bound and in the amount of tannin required to inhibit growth. Condensed tannins from myrtle and desmodium had the highest binding capacity and were most inhibitory to microbes. Absorbance readings from the total phenolics assay were highest for myrtle and lowest for quebracho. Similarly, readings for condensed tannins were highest for desmodium, which also had the largest polymer size. Myrtle and quebracho gave the largest precipitate rings in the radial diffusion assay, a biological assay that measures precipitation of bovine serum albumin by tannins. The relative absorbance values obtained from the total phenolics assay were the most useful predictor of tannin inhibition of microbial growth. The acid–butanol assay and the radial diffusion assay using bovine serum albumin as the precipitable protein were much less useful predictors of tannin biological activity against the bacteria studied.     

Effects of Acacia condensed tannins on urinary parameters,body mass,and diet choice of an Acacia specialist rodent,Thallomys nigricauda

The aim of this study was to investigate the dietary and physiological effects of condensed tannin ingestion on foregut fermenters, using Thallomys nigricauda, a folivorous rodent, as a model. We initially investigated the variability in physiological parameters, such as daily body mass (DM_b), daily feed intake, daily fecal energy loss (FE), daily energy intake (DEI), daily urine pH, and daily urinary ammonia and urea concentrations, in response to different diets with low condensed tannin levels. This experiment was conducted to identify which physiological variables showed the least variation in the absence of tannin. In a second experiment, we investigated the response of the same dietary and physiological parameters to the effects of high dietary condensed tannin ingestion in T. nigricauda. We hypothesized that DM_b, daily feed intake, FE, and DEI of T. nigricauda would be adversely affected by high dietary tannin content. We predicted that detoxification activity by T. nigricauda would increase at higher tannin levels. Ingestion of tannins affected the nutritional status of T. nigricauda, as shown by a decrease in body mass at high tannin levels. We also found that fewer ammonium ions were excreted in the urine by T. nigricauda, as would be expected if this were a means of regulating metabolic acidosis. The urine produced was more alkaline. This result indicates that T. nigricauda is not metabolizing these allelochemicals. Urea production was initially reduced, indicating conservation of bicarbonate ions that will neutralize blood acidity if there is detoxification. A diet choice experiment showed that tree rats avoid high tannin diets, even to the extent that they lose body mass on an alternative diet. This last-mentioned result is noteworthy because previous studies of the effects of tannins on herbivorous mammals have shown that there is physiological control rather than behavioral avoidance of the negative effects of tannin ingestion.     

Chestnut bark tannin assays and growth of chestnut blight fungus on extracted tannin

Tannins extracted from the green bark of each of two Chinese, Japanese, and American chestnut trees were assayed in a protein-binding test. Four levels of tannins were added to a buffered, minimal growth medium, and a standard, virulent strain of the chestnut blight fungus was grown. There were only slight differences in protein binding between the extracts from different species. Fungal growth was better with tannin than without, but there was no difference between species extracts in their ability to improve fungal growth rate. There was also no inhibition of blight fungus growth by any of the tree tannins, so tannin toxicity is not the reason for Asian chestnut tree resistance.     

Decomposition and nutrient release from pruning residues of two indigenous agroforestry species during the wet and dry seasons

The decomposition of leaves from Cordia africana Lam. and Albizia gummifera G. F.Gmel was investigated during the wet and dry seasons at Wondo Genet (Ethiopia). Litterbags of leaves were buried in soils under farmland and shaded-coffee agroforestry systems. Residual matter was recovered after 4, 8, 12, and 16 weeks and analysed for nitrogen (N), phosphorus (P), potassium (K), cellulose, lignin, soluble polyphenol and condensed tannin content. Mass-loss and release of N, polyphenols and condensed tannins were greater from Albizia leaves than from Cordia leaves, suggesting that a high polyphenol content does not necessarily retard decomposition. The rates of mass loss and release of the majority of leaf constituents were considerably faster during the wet season than during the dry season. Lignin decomposition, however, proceeded more rapidly during the dry season, and no significant seasonal differences were observed for polyphenol decomposition. The decomposition kinetics of most leaf components during the wet season were best described by a single-exponential model, but a quadratic model provided the best fit during the dry season. Initial leaf chemistry and season were important decomposition factors, while land-use effects were negligible. However, land-use effects showed distinct seasonal differences, with leaf litter decomposing more rapidly in soil under shaded-coffee than under farmland management, especially during the wet season. This study also demonstrated that polyphenol content does not show the predictive effects it has been attributed to have and that other constituents, such as condensed tannins, would be better suited for this purpose.     

Patterns and sources of leaf tannin variation in yellow birch (Betula allegheniensis) and sugar maple (Acer saccharum)

Leaves from forest-grown sugar maple (Acer saccharum Marsh) and yellow birch (Betula allegheniensis Britt.) trees were analyzed for four tannin measures (hydrolyzable and condensed tannins, total phenolics, and protein binding) at three times during the growing season. Fifteen-year-old half-sib sugar maples from four provenances, representing the geographical extremes of the sugar maple range and growing in a common garden, were examined for the same traits. We found no significant geographic or seed source component to variation in three of the four tannin measures. We found significant seasonal changes in both birch and maple leaf tannins. Withincanopy leaf tannin variation tended to obscure differences between trees in maple, but in birches between-tree differences in leaf tannin content were more readily found. We also found a significant negative correlation between leaf protein binding capacity and leaf wet weight.     

Inheritance patterns of phenolics in F1, F2, and back-cross hybrids of willows: implications for herbivore responses to hybrid plants

The aim of this study was to determine the inheritance pattern of phenolic secondary compounds in pure and hybrid willows and its consequences for plant resistance to leaf-feeding insects. F1, F2, and back-cross hybrids along with pure species were produced by hand pollination of pure, naturally-growing Salix caprea (L., Salicaceae) and S. repens (L.) plants. Leaf concentrations of condensed tannins and seven different phenolic glucosides were determined by using butanol-HCl and HPLC analyses. Insect herbivore leaf damage was measured on the same leaves as used for chemical analyses. We found hybrids to be approximately intermediate between the parental species: S. caprea with high levels of condensed tannins and no phenolic glucosides, and S. repens with low levels of condensed tannins and high levels of phenolic glucosides. We also found a negative correlation between concentrations of condensed tannins and phenolic glucosides, suggesting a trade-off in production of these two substances. F2 hybrids and the hybrid back-crossed to S. caprea were significantly more damaged by insect herbivores than the parental species and the F1 hybrid, indicating reduced resistance and possibly a selective disadvantage for these hybrid categories.     

Preserving leaves for tannin and phenolic glycoside analyses: A comparison of methods using three willow taxa

Members of the Salicaceae often produce phenolic glycosides and condensed tannins. There is much debate on the best method for the preservation of leaf material prior to chemical analysis. Published results indicate freeze-drying, a method commonly used for tannin analysis, may be inappropriate for phenolic glycosides, unless done in a manner to prevent thawing during the drying process. Another commonly employed method, air-drying, is appropriate for phenolic glycosides but inappropriate for condensed tannins. I present evidence using willow leaves that demonstrates that: (1) leaves freeze-dried in external flasks without temperature control contain lower concentrations of phenolic glycosides (salicortin and 2-cinnamoylsalicortin), (2) air-dried leaves have reduced concentrations of condensed tannins, while (3) vacuum-dried fresh leaves have high concentrations of both phenolic glycosides and condensed tannins. Freeze-drying caused salicortin and 2-cinnamoyl salicortin concentrations to drop by 20 mg/g and 4 mg/g, respectively. Salicin, a product of salicortin and 2-cinnamoyl salicortin degradation, is absent in vacuum-dried leaves, present in air-dried leaves and very high in freeze-dried leaves. Thus, the presence of salicin in this system is an artifact of the preservation technique. Condensed tannin concentrations dropped nearly 20 mg/g when leaves were air-dried. Thus, vacuum-drying fresh leaves allows researchers to quantify phenolic glycosides and condensed tannins from the same leaf material.     

Do defoliation and subsequent phytochemical responses reduce future herbivory on oak trees?

Perennial plants are thought to respond to partial or complete defoliation by producing new foliage that is less susceptible to herbivores because of induction of allelochemicals. Here, I tested this hypothesis by manually removing primary foliage from branches ofQuercus emoryi (Fagaceae) at two different times in the season and monitoring changes in protein and tannin levels and the amount of herbivory relative to control branches. New, secondary leaves had 2.5 × greater hydrolyzable tannin content than mature foliage of control branches. Condensed tannins, which constitute a relatively low fraction of leaf mass, were lower, while protein content was temporarily greater, in new secondary leaves relative to mature leaves. Despite large increases in hydrolyzable tannins, herbivory levels were greater on refoliated branches than on control branches. New foliage is susceptible to herbivory regardless of when it is produced in the season, possibly because lower toughness and higher water content override any induced or developmentally related changes in allelochemistry. My results do not support the hypothesis that postherbivore changes in phytochemistry protect perennial plants from future herbivory, at least within a growing season.     

Inhibition of Cyanogenesis by tannins

During isolation of two biosynthetic types of cyanogenic glycosides fromCarica papaya, weak cyanide tests were obtained from initial fractions. Upon final purification, strongly positive cyanide tests were obtained. Pretreatment of extracts to remove polyphenolics alleviated inhibition of cyanogenesis, which led us to suspect that tannins were inhibitory agents. Qualitative and quantitative measures of inhibition were made using standard cyanogenic glycosides and polyphenolics. Cyanogenesis was inhibited quantitatively when condensed tannins (quebracho, wattle, and chestnut), or hydrolyzable tannin (tannic acid) were added. When tannins were precipitated from the reaction mixture, cyanide tests proceeded optimally. These results stress the need to interpret negative cyanide tests with care and indicate possible ecological synergisms between plant defensive chemicals.     

Phenolics in black oak bark and leaves

Catechin, quercitrin, robinin, quercetin 3-methyl ether, scopoletin, cholorogenic acid, several leucoanthocyanins, and condensed and hydrolyzable tannins were identified in bark and leaves ofQuercus velutina Lamarck. The concentrations of most phenolics in leaves increased as the growing season progressed, whereas those of most phenolics in bark remained essentially unchanged. Qualitative differences in bark and leaf phenolics among different trees were negligible.     

Rapid Herbivore-Induced Changes in Mountain Birch Phenolics and Nutritive Compounds and Their Effects on Performance of the Major Defoliator, Epirrita autumnata

Insect damage changes plant physiology and chemistry, and such changes may influence the performance of herbivores. We introduced larvae of the autumnal moth (Epirrita autumnataBorkh.) on individual branches of its main host plant, mountain birch (Betula pubescens ssp. czerepanovii (Orlova) Hämet-Ahti) to examine rapid-induced plant responses, which may affect subsequent larval development. We measured systemic responses to herbivory by analyzing chemistry, photosynthesis, and leaf growth, as well as effects on larval growth and feeding, in undamaged branches of damaged and control trees. Larvae reared on leaves from intact branches of the herbivore-damaged trees grew faster than those reared on leaves of control trees, indicating systemic-induced susceptibility. Herbivore damage did not lead to systemic changes in levels of primary nutrients or phenolic compounds. The analyses of photosynthetic activity and individual hydrolyzable tannins revealed a reversal of leaf physiology-herbivore defense patterns. On control trees, consumption by E. autumnata larvae was positively correlated with photosynthetic activity; on damaged trees, this correlation was reversed, with consumption being negatively correlated with photosynthetic activity. A similar pattern was found in the relationship between monogalloylglucose, the most abundant hydrolyzable tannin of mountain birch, and leaf consumption. Among the control trees, consumption was positively correlated with concentrations of monogalloylglucose, whereas among herbivore-damaged trees, this correlation was reversed and became negative. Our results suggest that herbivore performance is related to both concentrations of phenolic compounds and photosynthetic activity in leaves. This linkage between herbivore performance, leaf chemistry, and physiology was sensitive to induced plant responses caused by slight herbivore damage.     

Evaluation of Methods for Measuring Polyphenols in Conifer Foliage

There is a resurgence of interest in the quantification of polyphenols in plant tissues because of their presumed ecological importance in plant–litter–soil and plant–animal interactions. The influence of sample preparation, extracting solvent, foliage quality, and assay method was investigated for the quantification of total phenols and condensed tannins in conifer foliage. Our results suggest that it is not possible to recommend a single optimal protocol for quantification of total phenol and condensed tannin fractions from plant materials. In general, the use of aqueous acetone (50–70% v/v) with freeze-dried materials gave the highest recovery. The Folin-Ciocalteau method for total phenols and the butanol–HCl hydrolysis method for condensed tannins appear superior to other common assays tested. There were large differences (1.4–2.2 times) in the reactivity of purified condensed tannins among species, indicating the importance of an appropriate standard for polyphenol quantification. A solid-state ¹³C NMR method with an improved "interrupted decoupling" pulse sequence yielded the highest concentrations for condensed tannins. Assuming that ¹³C NMR provides an accurate measure of total condensed tannin, the other extraction/assay methods used in this study recovered 50–86% of the condensed tannin fraction. The recovery rate is correlated with the nitrogen content of the foliage, which suggests that the formation of protein–tannin complexes may limit the extractability of condensed tannins. While ¹³C NMR condensed tannin values may give the best value for total condensed tannin concentrations, the water-soluble fraction may have the greatest physiological and/or ecological significance.     

Synthesis,characterization, and thermal behaviors of tannin stearates prepared from quebracho and pine bark extracts

Reported is the preparation of various condensed tannin stearates and the associated chemical characterization and thermal properties of these products. Stearate esters of condensed tannins from both quebracho and pine bark extracts were prepared in generally high, isolated yields from reaction with stearic acid chloride. Tannin esterification was confirmed by both Fourier transform infrared and nuclear magnetic resonance (NMR), and the average degree of stearate substitution was calculated from ¹H‐NMR analysis. Product degree of substitution (DS) was observed to proportionately increase with higher stearic acid chloride ratio with maximum DS values of 4.0 and 5.1 achieved for quebracho and pine tannins, respectively. Thermal analysis revealed that tannin stearate products have increased thermal stability with a degradation onset at significantly higher temperature for those samples possessing relatively greater DS. Analysis using differential scanning calorimetry revealed isolated products to consist of multiple components which exhibit interesting melt behaviors, a likely result from their work up on isolation. However, multiple melt features of the individual components in products were lost on heating to give single, broad melt endotherms due to coalescence of sample components. Furthermore, tannin stearate samples with high DS show relatively greater endothermic melting at higher temperature than those samples with lower DS. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010