Papyriferic Acid Research Articles

Inhibition of Snowshoe Hare Succinate Dehydrogenase Activity as a Mechanism of Deterrence for Papyriferic Acid in Birch

The plant secondary metabolite papyriferic acid (PA) deters browsing by snowshoe hares (Lepus americanus) on the juvenile developmental stage of the Alaska paper birch (Betula neoalaskana). However, the physiological mechanism that reduces browsing remains unknown. We used pharmacological assays and molecular modeling to test the hypothesis that inhibition of succinate dehydrogenase (SDH) is a mode of action (MOA) of toxicity of PA in snowshoe hares. We tested this hypothesis by measuring the effect of PA on the activity of SDH in liver mitochondria isolated from wild hares. In addition, we used molecular modeling to determine the specific binding site of PA on SDH. We found that PA inhibits SDH from hares by an uncompetitive mechanism in a dose-dependent manner. Molecular modeling suggests that inhibition of SDH is a result of binding of PA at the ubiquinone binding sites in complex II. Our results provide a MOA for toxicity that may be responsible for the concentration-dependent anti-feedant effects of PA. We propose that snowshoe hares reduce the dose-dependent toxic consequences of PA by relying on efflux transporters and metabolizing enzymes that lower systemic exposure to dietary PA.

Papyriferic acid derivatives as reversal agents of multidrug resistance in cancer cells

Forty-one derivatives of papyriferic acid were prepared based on our previous finding that methyl papyriferate ( 3) showed potent reversing effect on cytotoxicity of colchicine against multidrug resistance (MDR) human cancer cells (KB-C2), and evaluated for their cytotoxicity and effect on reversing P-gp-mediated MDR against KB-C2 cells. 3- O-(Morpholino-β-oxopropanoyl)-12β-acetoxy-3α,25-dihydroxy-(20 S,24 R)-epoxydammarane ( 37) significantly increased the sensitivity of colchicine against KB-C2 cells by 185-fold at 5 μg/mL (7.4 μM), and the cytotoxicity of colchicine was recovered to nearly that of sensitive (KB) cells. The other several new amide derivatives also exhibited potent reversal activity comparable to or more potent than methyl papyriferate and verapamil.

Papyriferic Acid, An Antifeedant Triterpene From Birch Trees, Inhibits Succinate Dehydrogenase From Liver Mitochondria

Papyriferic acid (PA) is a triterpene that is secreted by glands on twigs of the juvenile ontogenetic phase of resin producing tree birches (e.g., Betula neoalaskana, B. pendula) and that deters browsing by mammals such as the snowshoe hare (Lepus americanus). We investigated the pharmacology of PA as a first step in understanding its antifeedant effect. After oral administration to rats, PA and several metabolites were found in feces but not urine, indicating that little was absorbed systemically. Metabolism involved various combinations of hydrolysis of its acetyl and malonyl ester groups, and hydroxylation of the terpene moiety. The presence of a malonyl group suggested a possible interaction with succinate dehydrogenase (SDH), a mitochondrial enzyme known to be competitively inhibited by malonic acid. The effect of PA on the oxidation of succinate by SDH was examined in mitochondrial preparations from livers of ox, rabbit, and rat. In all three species, PA was a potent inhibitor of SDH. Kinetic analysis indicated that, unlike malonate, PA acted by an uncompetitive mechanism, meaning that it binds to the enzyme-substrate complex. The hydrolysis product of PA, betulafolienetriol oxide, was inactive on SDH. Overall, the evidence suggests that PA acts as the intact molecule and interacts at a site other than the succinate binding site, possibly binding to the ubiquinone sites on complex II. Papyriferic acid was potent (K(iEIS) ranged from 25 to 45 microM in the three species) and selective, as malate dehydrogenase was unaffected. Although rigorous proof will require further experiments, we have a plausible mechanism for the antifeedant effect of PA: inhibition of SDH in gastrointestinal cells decreases mitochondrial energy production resulting in a noxious stimulus, 5-HT release, and sensations of nausea and discomfort. There is evidence that the co-evolution of birches and hares over a large and geographically-diverse area in Northern Europe and America has produced marked differences in the formation of PA by birches, and the tolerance of hares to dietary PA. The present findings on the metabolic fate and biochemical effects of PA provide a rational basis for investigating the mechanisms underlying differences among populations of hares in their tolerance of a PA-rich diet.

The effects of elevated CO2 and temperature on the resistance of winter‐dormant birch seedlings (Betula pendula) to hares and voles

AbstractThe effects of elevated CO2 and temperature on the resource allocation pattern and resistance against mammalian herbivores of silver birch (Betula pendula Roth) were studied. Birch seedlings were grown through two growing seasons in closed‐top chambers exposed to four different treatments: ambient CO2 and temperature, elevated atmospheric CO2 (700 ppm) and ambient temperature, elevated temperature (+3°C above ambient) and ambient CO2, and a combination of elevated CO2 and temperature. After winter hardening of the seedlings, the growth of the seedlings was measured and the concentration of secondary compounds such as phenolics and papyriferic acid determined. The top parts of the stem were fed to hares, and the basal parts of the same stems were offered to voles.Elevated CO2 increased the height and basal diameter of the shoots, shoot biomass and total biomass of the seedlings but did not have any effect on secondary chemistry. Elevated temperature increased the height and shoot biomass, but did not have a significant effect on the total biomass of the seedlings. Elevated temperature decreased the concentration of condensed tannins and their precursor, (+)‐catechin, in the top part of the stems, but only the concentration of (+)‐catechin in the basal part of the stems. There were no significant interactive effects between CO2 and temperature on phenolics in the stems, while the concentration of papyriferic acid showed significant interaction in the top part of the stems. This indicates high accumulation of papyriferic acid in ambient CO2 under increased temperature. Consequently, elevated temperature increased the resistance of birch against hares, but did not affect the resistance of the basal parts of the same birches to voles. Our results indicate that the predicted climatic change will not necessarily lead to increased browsing damage by the mountain hare and the field vole to silver birch.

Variation in birch bark secondary chemistry between and within clones: implications for herbivory by hares

We studied the variation in bark chemistry between and within 19 European white birch (Betula pendula) clones and its implications to resistance to the mountain hare (Lepus timidus). We used one‐year‐old clonal plantlets originating from randomly selected naturally regenerated parental trees. The same clones were used in both chemical analyses and in feeding experiments. The condensed tannins were analysed by an acid butanol assay, other phenolic components by HPLC‐DAD, and triterpenoid components by HPLC‐MS. The resistance to hare was tested in open‐field feeding experiment. The main phenolic compounds in birch bark were catechin derivatives, rhododendrin, platyphylloside, and condensed tannins, and the main triterpenoids were papyriferic acid and pendulic acid. Most of the variation in the concentrations of the studied compounds was found between clones for the studied phenolics and large variation for triterpenoid components were found both between clones and among plantlets within the same clone. Hares clearly selected among the studied clones. Our results suggest that birch bark chemistry play an important role in resistance to herbivory by hare. The total triterpenoids and total flavonoid‐aglycones showed significant negative correlation with hare feeding. It seems that a genetic basis for bark chemistry and birch resistance is strong. Such a high variation in secondary chemistry both between clones and within individual clones indicates that European white birch populations have a good resistance towards variable environmental conditions and varying pressures from herbivory.

Effects of long-term, elevated ultraviolet-B radiation on phytochemicals in the bark of silver birch (Betula pendula).

Long-term outdoor experiments were conducted to investigate the effects of elevated ultraviolet-B (UV-B, 280-320 nm) radiation on secondary metabolites (phenolics and terpenoids) and the main soluble sugars (sucrose, raffinose and glucose) in the bark of silver birch (Betula pendula Roth) saplings. Saplings were exposed to a constant 50% increase in erythemal UV irradiance (UV-B(CIE); based on the CIE (International Commission on Illumination) erythemal action spectrum) and a small increase in UV-A radiation (320-400 nm) for three growing seasons in an irradiation field in central Finland. Two control groups were used: saplings exposed to ambient radiation and saplings exposed to slightly increased UV-A radiation. Concentrations of sucrose, raffinose and glucose in bark were higher in UV-treated saplings than in saplings grown in ambient radiation, indicating that stem carbohydrate metabolism was changed by long-term elevated UV radiation. Saplings in the elevated UV-A + UV-B radiation treatment and the UV-A radiation control treatment had significantly increased concentrations of certain UV-absorbing phenolics, such as salidroside, 3,4'-dihydroxypropiophenone-3-glucoside, (+)-catechin and (-)-epicatechin compared with saplings in ambient radiation. In contrast, the radiation treatments had no effect on the non-UV-B-absorbing terpenoids, papyriferic acid and deacetylpapyriferic acid. We conclude that plant parts, in addition to leaves, accumulate specific phenolic UV-filters in response to UV radiation exposure.

Chemical diversity of several Betulaceae species: comparison of phenolics and terpenoids in northern birch stems

Phenolics and terpenoids characteristic for silver birch (Betula pendula Roth) were screened in current-growth twigs of seedlings (1-growing-season-old individuals) and current-growth twigs of saplings (3 to 5-year-old individuals) of several birch species. Seedlings were grown in a greenhouse for 1 growing season. Saplings were field cultivated or wild-stand individuals. Chemicals were methanol-extracted, purified and HPLC-diode array detector-, gas chromatograph-flame ionizaton detector- and gas chromatograph-mass selective-analyzed. Species-specific qualitative and quantitative variation of secondary chemicals in birch stems were considerable and dependent on the age of the individual. The chemical diversity and amount were lower in seedlings than in saplings. In saplings, (+)-catechin and its derivatives were found in nearly all species, while arylbutanoids (±rhododendrol glucosides), an arylheptanoid (platyphylloside) and dammaraneterpenoids (papyriferic acid, deacetylpapyriferic acid and pendulic acid) showed a more restricted distribution. The chemicals analyzed in birch stems are a useful tool for recognition of species.

Does ozone stress change the primary or secondary metabolites of birch (Betula pendula Roth.)?

SUMMARYIn field experiments three clones of two‐year‐old birch (Betula pendula Roth.) were exposed for one growing season to an ozone concentration 1–2 times higher than ambient. At the end of August, leaf and stem material was analyzed for a wide range of primary and secondary metabolites. Although most of these metabolites were not significantly affected by ozone exposure, ozone‐treated leaves contained larger concentrations of total sugars and reduced amounts of the phenolic glucoside, dehydrosalidroside. In the stems, greater amounts of catechin pentoside, hyperoside and papyriferic acid were found. The results indicated considerable inter‐ and intra‐clonal variation in the production of phytochemicals in both leaves and stems.

Isolation and structure determination of three triterpenes from bark resin of juvenile european white birch

3α- O-Malonyl-12β- O-acetyl-25-hydroxy-(20 S,24 R)-epoxydammarane (papyriferic acid) and two previously unknown dammarane triterpenes w

Triterpene constituents of the dwarf birch, Betula glandulosa

Juvenile stages of the dwarf birch, Betula glandulosa, from the Yukon, Canada, are rarely browsed by snowshoe hares ( Lepus Americanus). Chemical investigation of extracts of B. glandulosa which strongly deter browsing by snowshoe hares has led to the isolation and structure determination of the new metabolise, deacetoxypapyriferic acid. Darnmar-24-ene-12β- O-acetyl-20( S)-ol-3-one, a previously unknown natural product, was obtained from extracts which did not deter browsing. The known metabolise papyriferic acid was found to be a minor constituent of extracts of B. glandulosa.

Chemical determinants of resistance in winter-dormant seedlings of European white birch (Betula pendula) to browsing by the mountain hare

A sample of one-year-old seedlings of European white birch (Betula pendula) was analyzed to determine the content of sugars, phenolics and terpenoid compounds. Two vertical segments of each seedling were analyzed separately. The number of resin droplets, which correlates strongly and negatively with feeding by the mountain hare, was also counted on the bark of experimental seedlings. The variation in the palatability of birch seedlings to mountain hare was determined primarily by the most abundant terpenoid, papyriferic acid. On the other hand, the resistance to hare feeding at the seedling bases, which are frequently attacked by voles, appeared to be dependent on other components, apparently phenolic substances. Sugars did not affect the resistance of the tested seedlings. The variation among experimental seedlings was much greater for secondary substances, especially terpenoid compounds, than for sugars. It is suggested that this high variation in protective compounds may be an adaptive trait selected for by the feeding of generalist herbivores.

Use of evaporative light scattering mass detection in high performance liquid chromatography of triterpenes in the bark resin of betula species

Plants often contain triterpenes that are difficult to analyse by high performance liquid chromatography (HPLC) due to lack of double bonds or other chromophores that can be readily detected spectrophotometrically. The present results show that light scattering mass detection (LS) is useful in such cases. We have been able to replace thin layer chromatography (TLC) with HPLC by using LS in evaluating triterpene contents in the bark resin of different birches (Betula pendula Roth., B. resinifera (Regel) Fern. & Raup., B. platyphylla Sucachev, B. ermanii Cham., B. papyrifera Marsh and B. pubescens Ehrh.). LS was five-fold more sensitive than UV for papyriferic acid, which was used as a reference compound, and HPLC/LS revealed new compounds and qualitative relations that were undetectable by UV or TLC. This paper describes detection of a new major triterpenoid from B. pendula, the age dependency of the triterpenoid content in B. pendula and B. papyrifera and gives an interspecies comparison of the triterpenoid content of the different species.

Response of winter chemical defense in Alaska paper birch and green alder to manipulation of plant carbon/nutrient balance.

Plant carbon/nutrient balance has been implicated as an important factor in plant defensive chemistry and palatability to herbivores. We tested this hypothesis by fertilizing juvenile growth form Alaska paper birch and green alder with N, P and N-plus-P in a balanced 2x2 factorial experiment. Additionally, we shaded unfertilized plants of both species. Fertilization with N and N-plus-P increased growth of Alaska paper birch, reduced the concentration of papyriferic acid in internodes and increased the palatability of birch twigs to snowshoe hares. Shading decreased birch growth, decreased the concentration of papyriferic acid in internodes and increased twig palatability. These results indicate that the defensive chemistry and palatability of winter-dormant juvenile Alaska paper birch are sensitive to soil fertility and shade. Conversely the defensive chemistry and palatability of green alder twigs to snowshoe hares were not significantly affected by soil fertility or shade. The greater sensitivity of Alaska paper birch defensive chemistry and palatability to snowshoe hares in comparison to green alder is in agreement with the hypothesis that early successional woody plants that are adapted to high resource availability are more plastic in their chemical responses to the physical environment than are species from less favorable environments.

Effects of Secondary Metabolites from Balsam Poplar and Paper Birch on Cellulose Digestion

Inhibitory effects of metabolites from balsam poplar (Pops Unpublished data, P.B. Reichardt) and the relative amounts present in juvenile and mature plants and plant parts are being quantified. In addition, bioassays are being conducted to evaluate deterrent properties of different resin fractions isolated (Bryant and Kuropat 1980, Bryant 1981, Bryant et al. 1983, Unpublished data, P.B. Reichardt). Bryant and Kuropat (1980) speculated that current annual growth (CAG) twigs ofjuvenile Alaska paper birch are less digestible than CAG twigs from mature-growth-form plants due to their higher resin content, which may be toxic to rumen microbes. Previous studies have demonstrated antibacterial effects or inhibition of digestion by rumen microbes exposed to various terpenoids. Nagy et al. (1964) found that essential oils in sagebrush (Artemisia tridentata) inhibited bacterial growth and decreased gas and volatile fatty acid production in rumen of mule deer (Odocoileus hemionus). Oh et al. (1967) and Longhurst et al. (1968) found that oxygenated monoterpenes present in Douglas fir (Psuedotsuga menziesii) needles strongly inhibited Columbian black-tailed deer (0.h.’ columbianus) rumen microbial activity. Schwartz et al. (1980) reported that volatile oils present in various junipers (Juniperus spp.) reduced cellulose digestion in vitro by up to 40%. In this paper, we present evidence that benzyl alcohol, cineok papyrifcric acid, and a steam distillate fraction from juvenile Alaska paper birch depress in vitro fermentation of cellulose SUSpended in rumen fluid from wapiti (Cervus elaphus nelsoni). Material and Methods Four resin components, 2 each from balsam poplar and paper Authorsare research assistant, Department of Biological Sciences, MichiganTechnological University, Houghton 49931; research assistants, Department of Animal Sciences, University of Alberta, Edmonton T6G 2P5. The authors thank the following persons for their support and assistance with various aspects of this study: J. Bryant, P. Reichardt, R. Hudson, L. Jebson, D. Renecker, J. Aalhus, D. Summers, and T. Fenton. Financial support for this study was prowded by Michigan Technological University and the University of Alberta. Manuscript accepted October 15, 1984. 370 birch, were chosen for testing because of their ability to deter browsing by snowshoe hare (Lepus americanus) (Unpublished data, J.P. Bryant). Papyriferic acid, a triterpene carboxylic acid, and a steam distillate fraction, composed primarily of sesquiterpenes, from juvenile Alaska paper birch were provided by Dr. P.B. Reichardt (Department of Chemistry, University of Alaska, Fairbanks). Papyriferic acid was isolated from diethyl ether extract of CAG twigs from winterdormant juvenile plants by column chromatography. The steam distillable fraction of juvenile paper birch was isolated by diethyl ether extraction of the steam distillate (Personal communication, P. Reichardt). In addition, cineole (an isoprenoid) and benzyl alcohol, 2 compounds present in balsam poplar bud resin, were obtained commercially (Sigma Corp., St. Louis, MO.). Sample Preparation Concentrations of resin fractions used for in vitro trials were based on the amount of each resin component available and on the amount estimated to be present in CAG twigs from both mature and juvenile forms of paper birch and balsam poplar (Unpublished data, P.B. Reichardt). Because of the limited availability of papyriferic acid and paper birch steam distillate, treatment concentrations for these compounds were 5 and 20 mg per gram of substrate. Cineole and benzyl alcohol were tested at concentrations of 5, 10, 20,40, and 100 mg per gram of substrate. All treatment concentrations were prepared on a dry weight basis. Commercial purified cellulose (“Alpha floe”, Lee Chemicals Ltd., Toronto, Ont.) was used as a substrate. Resin components were dissolved in excess acetone, placed into a 150-ml round-bottom flask with the cellulose substrate, and swirled until the material was saturated. The flask was then placed on a vacuum rotodistiller with a water bath temperature of 50°C for approximately 10 minutes, or until the acetone had evaporated. The treated cellulose was removed from the flask, placed into metal dishes, and oven-dried at 4O“C for 48 hours. Following drying, samples were mixed in a micro mill and stored in sealed glass containers. In Vitro Techniques The in vitro techniques used to measure digestion of dry matter were developed by Tilley and Terry (1963) and modified by Morgantini and Hudson (in press). Rumen inoculum was obtained from a fistulated wapiti steer which was maintained on pelleted aspen concentrate, alfalfa hay, freshly cut browse, and fresh grass available ad libitum in a l-ha pasture. Triplicate samples were tested at each concentration level. Acetone controls were also tested for antimicrobial effects. Differences were tested by one-way analysis of variance and Duncan’s new multiple range test (KO.05).

Defense of winter-dormant Alaska paper birch against snowshoe hares.

Mature growth-phase internodes of Alaska paper birch (Betula resinifera) are preferred by the snowshoe hare (Lepus americanus) over juvenile growth-phase internodes due to the low food value of the latter. While the mature over juvenile preferencec cannot be explained by the levels of inorganic nutrients or gross chemical fractions (resins or phenols), it can be explained by the striking differences in secondary metabolites of the two growth phases. The principle compound which renders the juvenile phase internodes unpalatable is papyriferic acid, a triterpene which is a demonstrated feeding deterrent to snowshoe hares and which is present in juvenile internodes at concentrations 25 times greater than those in mature internodes.

ChemInform Abstract: PAPYRIFERIC ACID: A TRITERPENOID FROM ALASKAN PAPER BIRCH

Chemischer InformationsdienstVolume 13, Issue 19 Natural Products ChemInform Abstract: PAPYRIFERIC ACID: A TRITERPENOID FROM ALASKAN PAPER BIRCH P. B. REICHARDT, P. B. REICHARDTSearch for more papers by this author P. B. REICHARDT, P. B. REICHARDTSearch for more papers by this author First published: May 11, 1982 https://doi.org/10.1002/chin.198219348Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume13, Issue19May 11, 1982 RelatedInformation