Ann E. Hagerman

High molecular weight plant polyphenolics (tannins) as biological antioxidants

Representative condensed and hydrolyzable tannins and related simple phenolics were evaluated as biological antioxidants using cyclic voltammetry, the metmyoglobin assay, and the deoxyribose assay. The redox potentials of the tannins were similar to those of structurally related simple phenolics. However, the tannins were 15-30 times more effective at quenching peroxyl radicals than simple phenolics or Trolox. One of the tannins, polygalloyl glucose, reacted an order of magnitude more quickly with hydroxyl radical than mannitol. These results suggest that tannins, which are found in many plant-based foods and beverages, are potentially very important biological antioxidants.

Role of Tannins in Defending Plants Against Ruminants: Reduction in Protein Availability

We tested the hypothesis that tannins defend plants against large herbivores by decreasing protein availability. Digestion trials were conducted with mule deer (Odo- coileus hemionus) and results from previous trials with white-tailed deer (O. virginianus), moose (Alces alces), caribou/reindeer (Rangifer tarandus), and elk (Cervus elaphus) were summarized to evaluate dietary factors affecting protein availability. The digestibility of plant protein in feeds with minimal tannins, such as grasses and agriculturally produced legumes and grains, was a highly predictable function of the total protein content and the amount of nondigestible, fiber-bound protein. Digestible protein in plants containing sig? nificant tannins was lower than predicted from regressions for low-tannin feeds. The re? duction in digestible protein was proportional to the protein-precipitating capacity of the plant tannins. Deciduous browse stems collected in winter had very low levels of protein- precipitating tannins and only a slightly lower protein availability than predicted. Tannins are not important in the defense of most deciduous tree and shrub stems consumed by these herbivores. Tannins in flowers and forb, tree, and shrub leaves markedly reduced protein availability. Tannins must be considered in understanding the defensive strategies of leaves and flowers. Voluntary intake of the high-phenolic forages was significantly re? duced below ingestion rates for grasses, legumes, and pelleted diets. It is hypothesized that soluble phenolics that do not inhibit digestion but are absorbed and reduce intake through their toxicity are more important in defending some plant parts against ruminants than are digestion-reducing tannins.

RADIAL DIFFUSION METHOD FOR DETERMINING TANNIN IN PLANT EXTRACTS

Tannin in plant extracts can be determined by reacting the tannin with a protein and quantitating the precipitated complex. In the new assay described here, a tannin-containing solution is placed in a well in a protein-containing agar slab. As the tannin diffuses into the gel and complexes with protein, a visible ring of precipitation develops. The area of the ring is proportional to the amount of tannin in the extract. The detection limit of the method is 0.025 mg tannic acid or condensed tannin and the precision is 6% (relative standard deviation). Tests with extracts of a variety of plants show that the new method gives results comparable to other precipitation methods and that the new method is superior for samples of unusual composition, such as aspen buds. The method has several advantages over other methods for determining tannin: The new method is very simple and requires neither complex reagents nor instruments. Components of the plant extract such as non-tannin phenolics or water-insoluble compounds do not interfere with the method. The assay is not subject to interference from the organic and aqueous solutions which are commonly used to extract tannin from plants.

Role of tannins in defending plants against ruminants: reduction in dry matter digestion

Polyphenolic allelochemicals, such as tannins, are widely thought to reduce the digestibility of plants consumed by herbivores by binding to digestive enzymes and dietary proteins. While the apparent digestibility of protein and, therefore, cell solubles is reduced in mule deer (Odocoileus hemionus) and white-tailed deer (O. virginianus) consuming tanniferous forages, digestion of the plant cell wall is not reduced beyond that predicted from its content of lignin, cutin, and silica. The lack of a tannin effect on cell wall digestion in deer is in contrast to studies with domestic sheep and numerous in vitro studies. Herbivores adapted to consume tanniferous forages may defend against such allelochemicals by producing salivary proteins that bind tannins in a highly specific manner. These tannin-salivary protein complexes would reduce apparent digestibilities of protein and cell solubles and, if completely effective, would not reduce cell wall digestion. The occurrence of such proteins in ruminants is reported here for the first time. The saliva composition of mule deer (a mixed feeder that commonly consumes browse) and domestic cattle and sheep (predominant grazers) are compared, and the higher potential of the deer saliva to neutralize tannins is related to their feeding habits. Salivary proteins that preferentially bind tannins may minimize fecal nitrogen losses by maximizing the efficiency of tannin-binding per unit of protein and may reduce the absorption of hydrolyzable tannins and the potential for tannin toxicity.

Generation of reactive oxygen species after exhaustive aerobic and isometric exercise

UNLABELLED Many studies have implicated elevated oxygen consumption (VO2) associated with aerobic exercise as contributing to oxidative stress. Only a few studies have investigated nonaerobic exercise and its relation to pro-oxidant and antioxidant activities. PURPOSE The purpose of this study was to compare biomarkers of oxidative stress: lipid peroxidation, protein oxidation, and total antioxidants in blood after exhaustive aerobic (AE) and nonaerobic isometric exercise (IE). METHODS Blood samples were collected from 12 subjects who performed a maximum AE and IE test and were analyzed for thiobarbituric acid (TBARS), carbonyls, lipid hydroperoxides (LH), and oxygen radical absorbance capacity (ORAC). RESULTS VO2 increased 14-fold with AE compared with 2-fold with IE. Protein carbonyls increased 67% (P < 0.05) pre- to immediately and 1 h post-AE, and 12% pre- to immediately post-IE and returned to baseline 1 h post-IE. TBARS did not increase significantly with either treatment. LH increased 36% above rest during IE compared with 24% during AE (P < 0.05). ORAC increased 25% (P < 0.05) pre- to post-AE, compared with 9% (P < 0.05) pre- to post-IE. CONCLUSION There was evidence of oxidative stress after both exhaustive aerobic and isometric exercise. Lipid hydroperoxides, protein carbonyls, and total antioxidants increased after both IE and AE. Due to the different metabolic demands of aerobic and isometric exercise, we can rule out a mass action effect of VO2 as the sole mechanism for exercise-induced oxidative stress.

Choosing appropriate methods and standards for assaying tannin

Tannins are chemically diverse polyphenolics that have multiple biological activities. Attempts to establish the ecological significance of tannins have been hindered by the complexities of tannin analysis. A multitude of analytical procedures for tannins has been described, but it is difficult for the nonspecialist to select appropriate methods. We have classified the most common procedures for determining tannin as either chemical assays, appropriate for determining the amount and the chemical nature of the tannin in a sample, or as protein-binding assays, suitable for determining the potential biological activity of the tannin in a sample. We have recommended procedures that are particularly reliable and straightforward for general use. We have also considered the problems encountered in selecting appropriate standards for tannin analysis and have recommended standards that are readily available.

Variation in Mammalian Physiological Responses to a Condensed Tannin and Its Ecological Implications

Mule deer ( Odocoileus hemionus ), domestic sheep ( Ovis aries ), and American black bears ( Ursus americanus ) were fed quebracho ( Schinopsis sp.) tannin to determine the contribution of salivary proteins to nitrogen- and fiber-digestive efficiencies and tannin metabolism. These values were compared to previously published values for laboratory rats ( Rattus rattus ) and prairie voles ( Microtus ochrogaster ). Mule deer, black bears, and laboratory rats consuming this condensed tannin produced tannin-binding salivary proteins that reduced fecal-nitrogen losses per unit of ingested tannin and reduced tannin metabolism relative to domestic sheep and prairie voles. Digestibility of the plant fiber was reduced significantly by tannins in domestic sheep, but not in mule deer. Although virtually all ingested tannin (98.3 ± 5.0%) was recovered in feces of mule deer and black bears, ca. 25% was not recovered in feces of domestic sheep and presumably was metabolized. The defensive role of tannins as digestion inhibitors or toxins is dependent upon the molecular characteristics of the tannin interacting with the physiological capability of the animal. Results from one plant-mammal interaction cannot be used to interpret others without an understanding of the characteristics of the tannins and the physiology, ecology, and evolution of the animal.

Tannin-binding proteins in saliva of deer and their absence in saliva of sheep and cattle.

A method has been developed for detecting tannin-binding proteins in the saliva of herbivores. The method is simple and requires only small quantities of crude saliva. The saliva of deer, a browsing ruminant, has been compared to that of domestic sheep and cow, which are grazing ruminants. The browser, which normally ingests dietary tannin, produces tannin-binding proteins, while the grazers do not produce such proteins. The tannin-binding protein from deer saliva is a small glycoprotein containing large amounts of proline, glycine, and glutamate/glutamine. The protein is not closely related to the proline-rich salivary proteins found in rats and other nonruminant mammals.

Phenylalanine ammonia-lyase and hydroxycinnamate: CoA ligase in maize mesocotyls inoculated with Helminthosporium maydis or Helminthosporium carbonum☆

Abstract Phenylalanine ammonia-lyase (E.C. 4.3.1.5) (PAL) and hydroxycinnamate:CoA ligase (E.C. 6.2.1.12) activities were measured in extracts from maize mesocotyls resistant and susceptible to Helminthosporium maydis and resistant to H. carbonum . CoA ligase activity increased in response to infection with H. maydis in both the resistant and susceptible cultivars. Activity began to increase between 6 and 9 h after inoculation and in the resistant cultivar continued to increase throughout a 48-h period. In susceptible cultivars activity ceased to increase at approximately 12 h after inoculation. The results demonstrate that the increase in CoA ligase activity is detectable as early as the onset of penetration by the fungus. No significant change in PAL activity was observed in either resistant or susceptible combinations with H. maydis , suggesting that PAL and CoA ligase are not coordinately regulated in interactions involving this fungus. Neither enzyme was found to change as a result of inoculation of any cultivar with H. carbonum .

Extraction of tannin from fresh and preserved leaves

The extractability of tannin from fresh, lyophilized, and dried leaves collected at various times in the growing season was determined using the radial diffusion assay for protein-precipitating phenolics. The amount of tannin extracted depended on the method of leaf preservation and on the maturity of the leaf. Early in the season, more tannin was extracted from lyophilized leaves than from fresh leaves, but late in the season more tannin was extracted from fresh leaves. At all times, more tannin was extracted with aqueous acetone than with aqueous or acidic methanol.