Biochemical Systematics and Ecology 31 (2003) 557–562 www.elsevier.com/locate/biochemsyseco
Toxic nitro compounds in Astragalus species Vahid Niknam a,*, Hassan Ebrahimzadeh a, Ali Asghar Maassoumi b a
b
University of Tehran, Faculty of Sciences, Department of Biology, Tehran, Iran Research Institute of Forests and Rangelands, Herbarium of Botanical Gardens, P.O. Box 1385-116, Tehran, Iran Received 4 August 2001; accepted 18 July 2002
Abstract Leaflets of 78 type herbarium specimens including 78 species of Astragalus species from Iran were analyzed for toxic aliphatic nitro compounds. The catabolites of nitro compounds, 3-nitropropanol and 3-nitropropionic acid are especially toxic to cattle and sheep. Nitro compounds were detected in six species of Astragalus and all the nitro-bearing species were herbaceous. The occurrence of nitro toxins in four species of Astragalus is reported for the first time. Nitro-toxins are more common among the simple haired Astragalus species. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Aliphatic nitro toxins; Astragalus; Simple haired; Bifurcate haired; Chemical defense; Fabaceae
1. Introduction Aliphatic nitro compounds are a group of toxins found in various milkvetches (Pass, 1994). More than 470 species and varieties of Astragalus are known to synthesize nitro compounds (Williams and Barneby, 1977a, 1977b; Williams, 1981; Ebrahimzadeh et al., 1999). Several species of Astragalus that synthesize miserotoxin (1), 3-nitro-1-propyl-β-D-glucopyranoside have caused moderate-to-heavy losses of cattle and sheep on western rangelands of the United States (Cronin et al., 1981) and Canada (Majak and Cheng, 1983). Two metabolic steps are involved in the
* Corresponding author. Tel.: +98-21-6112492; Fax: +98-21-6405141. E-mail address:
[email protected] (V. Niknam). 0305-1978/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0305-1978(02)00179-5
558
V. Niknam et al. / Biochemical Systematics and Ecology 31 (2003) 557–562
bioactivation of miserotoxin (Fig. 1). The first occurs in the rumen and the second probably occurs in the liver. By hydrolysis of the glycosidic bond 3-nitropropanol (2) is released. By enzymatic oxidation in the second step, 3-nitropropanol was converted to 3-nitropropionic acid (3) (Majak, 1991). Since 3-nitropropionic acid is degraded more rapidly than 3-nitropropanol by ruminal microbes, ruminants are less tolerant of 3-nitropropanol than 3-nitropropionic acid (Majak and Pass, 1989). Certain species of Coronilla, Indigofera, and Lotus also synthesize esters of 3-nitropropionic acid (Majak and Pass, 1989; Niknam et al., 1999). In North America, it is estimated that up to 5% of range cattle exposed to Astragalus miser var. serotinus in British Columbia are poisoned annually (Majak and Cheng, 1983). Acute clinical signs of toxicosis caused by nitro toxins include incoordination, distress, labored breathing, bluish skin or tongue, muscular weakness, and collapse. Death may occur within a few hours after ingestion of the toxin. In chronic poisoning, animals lose weight and develop respiratory distress, a poor hair coat, hind limb paralysis, and nasal discharge (Majak et al., 2001). Human poisoning caused by mildewed sugar cane has also been reported. There is no known cure for nitro toxin poisoning (Anderson et al., 1998). The genus Astragalus is generally considered the largest genus of vascular plants with an estimated 2500–3000 species (Podlech, 1986). According to the shape of leaf hairs, the genus Astragalus has been classified into two subgenera: simple haired subgenus of Astragalus and bifurcate haired subgenus of Cercidothrix. The Old World Astragalus species can be placed in an estimated 152 sections. Out of 152 sections in the genus Astragalus, 97 sections have simple hairs and 55 sections have bifurcate hairs (Maassoumi, 1998). Because Astragalus may be grazed by livestock or fed as fodder, these species require toxicological investigation. Moreover, according to the literature, analysis for nitro compounds can be used in correct identification and classification of nitrobearing species as well as to resolve synonymy (Williams, 1981; Williams, 1982).
Fig. 1. Two metabolic steps in bioactivation of miserotoxin. The first step occurs in the rumen and the second probably occurs in the liver. (1) miserotoxin; (2) 3-nitropropanol; (3) 3-nitropropionic acid; ADH: alcohol dehydrogenase (drawn based on Majak, 1991).
V. Niknam et al. / Biochemical Systematics and Ecology 31 (2003) 557–562
559
In this paper the distribution of nitro toxins among Astragalus species and sections and the possible involvement of aliphatic nitro compounds in the defensive strategy against herbivory and grazing is discussed. 2. Materials and methods Twenty-milligram samples of leaflets of 78 type specimens including 78 species of Astragalus from 22 taxonomic sections, collected from the Herbarium of Research Institute of Forests and Rangelands, Tehran, Iran, were analyzed qualitatively for toxic aliphatic nitro compounds according to the modified method of Williams and Barneby (1977a). The extracts of nitro-bearing species were centrifuged for 20 min at 10,000 rpm and the supernatants were used for spectrophotometric determination of aliphatic nitro content using the colorimetric method of Majak et al. (1982). Calibration was achieved with 3-nitropropionic acid (Sigma) solutions of 40–400 µg/ml. Beckman high-speed centrifuge (J2-21M) and Shimadzu UV-visible recording spectrophotometer (UV-160) with 10 mm matched quartz cells were used for centrifugation of the extracts and determination of the absorbance, respectively. 3. Results and discussion Nitro compounds were found in six species from five of 22 sections of Astragalus (Table 1). The occurrence of nitro toxins in A. ovalifoliolatus Maassoumi and Table 1 Nitro toxin content in nitro-bearing species of Astragalus from Iran. The only bifurcate haired species is highlighted in boldface. The results presented in this table were obtained from the analyses of type specimens of Astragalus Section
Species
Origin and Voucher Number
Date Collected
Nitro Content (mg/g/dw)
Alopecuroidei DC
Anthylloidei DC
Cystium Bunge
Glycyphyllos Bunge
Hemiphaca Bunge Hemiphaca Bunge
ovalifoliolatus Maassoumi and Ranjbar submitis subsp. Maassoumi, Tietz and Zarre mazandaranus Bunge var. trichocarpus Sirj. et Rech.f. botryophorus Maassoumi and Podlech minutistipulatus Podlech penetratus Maassoumi
Tehran: 64066
1985
19.13
Mazandaran: 55131 1986
31.00
Khorasan: 5065
1984
28.12
Hamadan: 59350
1987
32.10
Fars: 17661
1975
47.13
Chaharmahal: 573921985
1975
120.15
560
V. Niknam et al. / Biochemical Systematics and Ecology 31 (2003) 557–562
Ranjbar, A. botryophorus Maassoumi and Podlech, A.minutistipulatus Podlech and A. penetratus Maassoumi is reported for the first time. As mentioned elsewhere (Williams and Barneby, 1977a; Williams, 1981; Williams, 1982) the concentration of nitro toxins in Astragalus leaflets often indicated whether the toxic catabolite will be 3-nitropropanol or 3-nitropropionic acid. Miserotoxin, which is catabolized in the digestive tract of ruminants to 3-nitropropanol, is primarily found in Astragalus species that synthesize nitro toxins at concentrations up to 20 mg/g dry weight. Nitro toxins that are synthesized at concentrations more than 20 mg/g dry weight are catabolized to 3-nitropropionic acid in the digestive tract of ruminants. According to the present study 12.11% of the simple haired and 4.39% of bifurcate haired Astragalus species are nitro bearing. Of six nitro-bearing Astragalus species reported in this paper, only A. mazandaranus from section Cystium Bunge is bifurcate haired and the other five species are simple haired (Table 1). Thus, nitro-positive species are more common among simple haired Astragalus species. According to this study all the nitro-bearing species introduced in this paper (Table 1) and our previous research (Ebrahimzadeh et al., 1999) are herbaceous and not one of the shrubby, thorny, and tragacanthic Astragalus species contains nitro compounds. A plant grows in a highly competitive environment. It is continually threatened by other plants encroaching upon the space from which it draws its sustenance, by microorganisms, by insects, and by both large and small mammalian, avian, or reptilian herbivores. In order to survive, each plant must draw upon a complex of defenses, which may be physical, such as spines or leathery leaves, or chemical (Rosenthal and Janzen, 1979). The study of plant defense is unique in the field of chemical ecology in that no other area is so burdened with theory (Berenbaum, 1995). Ever since the landmark paper of Fraenkel (1959), scientists have fought to generate theories which adequately explain the strategies employed by plants to chemically defend themselves. The relative costs and types of defenses employed by plants were first assessed by Feeny (1976). His plant apparency theory has served as a primary paradigm for most research in this field. Feeny characterized plants as either “bound to be found” (apparent) or not predictably distributed (unapparent). Plants could be (un) apparent in space and/or time. Apparency theory predicts that apparent plants will be defended differently from unapparent ones. Apparent plants would be predicted to employ “quantitative” defenses. Quantitative defenses are dose-dependent compounds which are considered to be costly to produce. Feeding deterrents such as tannins, terpenes, and flavonoids are quantitative defenses. Conversely, qualitative defenses are the toxins which are lethal in small concentrations and are thus considered to be less costly to produce than quantitative defenses. In summary, analysis of Astragalus species for these compounds identify the species that are nitro bearing and therefore might be poisonous to livestock. Analysis for nitro compounds can be used to correctly identify and classify nitro-bearing species as well as resolve synonymy. Finally, it is likely that the presence of nitro toxins in
V. Niknam et al. / Biochemical Systematics and Ecology 31 (2003) 557–562
561
herbaceous and non-thorny Astragalus species could be a qualitative defense strategy against herbivory.
Acknowledgements The authors acknowledge the financial support of the Faculty of Sciences, University of Tehran. The authors wish to express their deep appreciation to Dr. M. Coburn Williams, retired scientist of Poisonous Plant Research Laboratory, USDA, Logan, UT 84321, for his research articles and helpful communications. The first author also wishes to thank Dr. F. Ghahremani-Nejad for his help and excellent suggestions.
References Anderson, R.C., Majak, W., Rasmussen, M.A., Allison, M.J., 1998. Detoxification potential of a new species of ruminal bacteria that metabolize nitrate and naturally occuring nitrotoxins. In: Garland, T., Bar, A.C. (Eds.), Toxic Plants and Other Natural Toxicants. CAB International, Wallingford, Oxon, UK, pp. 154–158. Berenbaum, M.R., 1995. The Chemistry of Defense: Theory and Practice. Proc. Nat. Acad. Sci. USA 92, 2–8. Cronin, E.H., Williams, M.C., Olsen, J.D., 1981. Toxicity and control of Kelsey milkvetch. J. Range Manag. 34, 181–183. Ebrahimzadeh, H., Niknam, V., Maassoumi, A.A., 1999. Nitro compounds in Astragalus species from Iran. Biochem. Syst. Ecol. 27, 743–751. Feeny, P., 1976. Plant apparency and chemical defense. In: Wallace, J.W., Nansel, R.L. (Eds.), Biological Interactions Between Plants and Insects. Recent Advances in Phytochemistry, Vol. 10. Plenum Press, NY, pp. 1–40. Fraenkel, G.S., 1959. The raison d’etre of secondary plant substances. Science 129, 1466–1470. Maassoumi, A.A., 1998. Astragalus in the Old World. Research Institute of Forests and Rangelands, Tehran. Majak, W., 1991. Metabolism and absorption of toxic glycosides by ruminants. J. Range Manag. 45, 67–71. Majak, W., Cheng, K.J., 1983. Recent studies on ruminal metabolism of-nitropropanol in cattle. Toxicon. (Supplement) 3, 265–268. Majak, W., Cheng, K.J., Hall, W.J., 1982. The effect of cattle diet on the metabolism of 3-nitro-propanol by ruminal microorganisms. Can. J. Anim. Sci. 62, 855–860. Majak, W., Hall, J.W., McAllister, T.A., 2001. Practical measures for reducing risk of Alfalfa bloat in cattle. J. Range Manag. 54, 490–493. Majak, W., Pass, M.A., 1989. Aliphatic nitro compounds. In: Cheeke, P.R. (Ed.), Toxicants of Plant Origin, Vol. II: Glycosides. CRC Press, Boca Raton, FL, pp. 143–159. Niknam, V., Ebrahimzadeh, H., Maassoumi, A.A., 1999. Analysis of the species of Coronilla, Indigofera and Lotus from Iran for toxic nitro compounds. Research and Reconstruction 40, 65–67. Pass, M.A., 1994. Toxicity of plant-derived aliphatic nitrotoxins. In: Colegate, S.M., Dorling, P.R. (Eds.), Plant-associated Toxins: Agricultural, Phytochemical and Ecological Aspects. CAB International, Wallingford, Oxon, UK, pp. 541–545. Podlech, D., 1986. Taxonomic and phytogeographical problems in Astragalus of the Old World and southwest Asia. Proc. Roy. Soc. Edinburgh 89, 37–43. Rosenthal, G.A., Janzen, D.H., 1979. Herbivores. Their interaction with secondary plant metabolites. Academic Press, NY. Williams, M.C., 1981. Nitro compounds in foreign species of Astragalus. Weed Sci. 29, 261–269.
562
V. Niknam et al. / Biochemical Systematics and Ecology 31 (2003) 557–562
Williams, M.C., 1982. 3-Nitropropionic acid and 3-nitro-1-propanol in species of Astragalus. Can. J. Bot. 60, 1956–1963. Williams, M.C., Barneby, R.C., 1977a. The occurrence of nitro-toxins in North American Astragalus (Fabaceae). Brittonia 29, 310–326. Williams, M.C., Barneby, R.C., 1977b. The occurrence of nitro-toxins in Old World and South American Astragalus (Fabaceae). Brittonia 29, 327–331.
