Biochemical Systematics and Ecology 27 (1999) 743}751
Nitro compounds in Astragalus species from Iran Hassan Ebrahimzadeh *, Vahid Niknam , Ali Asghar Maassoumi Department of Biology, Faculty of Sciences, University of Tehran, Tehran, Iran Herbarium of Botanical Garden, Research Institute of Forests and Rangelands, P.O. Box 13185-116, Tehran, Iran Received 8 September 1998; accepted 8 January 1999
Abstract Lea#ets of 460 specimens including 440 species of Astragalus from the Herbarium of the Research Institute of Forests and Rangelands, Tehran, Iran, were analysed for toxic aliphatic nitro compounds. The catabolites of nitro compounds found in species of Astragalus, 3-nitro-1propanol and 3-nitropropionic acid, are specially toxic to cattle and sheep. Nitro compounds in concentration of 4}25 mg NO /g of plant, were detected in 37 of 440 (8.4%) species. There is a chemotaxonomic relationship among nitro-bearing species of Astragalus and nitro compounds could be used to predict the presence or absence of these compounds in other taxa, relative concentrations and the probable toxicity of the plant to livestock. The occurrence of nitro-toxins in 16 species of Astragalus is reported for the "rst time. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Aliphatic nitro toxin; Poisonous plants; Astragalus; Leguminosae; Chemotaxonomy
1. Introduction The principal poisons associated with Astragalus are aliphatic nitro compounds. More than 450 species and varieties of Astragalus synthesize these compounds (Williams, 1981; Williams and Barneby, 1971). The most potent of these compounds is miserotoxin (1), a glycoside, "rst isolated from Astragalus miser var. oblongifolius (Rydb.) Cronq. (Stermitz et al., 1969), which is catabolized to its toxic component, 3-nitro-1-propanol (3-NPOH, 2), in the digestive tract of ruminants (Williams et al.,
*Corresponding author. E-mail:
[email protected]. 0305-1978/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S0 30 5 - 19 7 8( 9 9 )00 0 12 -5
744
H. Ebrahimzadeh et al./Biochemical Systematics and Ecology 27 (1999) 743}751
1970). Several species of Astragalus that synthesize miserotoxin have caused moderate to heavy losses of cattle and sheep on western rangelands of the United States (Cronin et al., 1981; Williams and James, 1975). Other species of Astragalus synthesize karakin, hiptagin, cibarian and other compounds that are catabolized to 3-nitropropionic acid (3-NPA, 3) in the digestive tract of ruminants (James et al., 1980; Williams and James, 1975; Williams et al., 1976). The oral toxic and lethal doses of 3-NPA for ruminants are considerably higher than doses of 3-NPOH (Williams, 1982). 3-NPOH and 3-NPA are respiratory toxins that inhibit mitochondrial enzymes essential to respiration (Anderson et al., 1998). Ruminal microbes can metabolize 3-NPOH and 3-NPA to their non-toxic amines, amino-propanol and b-alanine, respectively (Anderson et al., 1993). The rumen thus possesses a detoxi"cation potential that can be enhanced by selection for competent detoxifying microbes, and gradual adaptation of ruminal microbes to increasing toxic concentrations (Anderson et al., 1998). Previous investigations indicated that these compounds are remarkably stable, being preserved for up to 90}100 years in herbarium specimens of Astragalus (Williams and Parker, 1974). However, if one is to detect nitro compounds in herbarium specimens, the plants must have been properly dried to avoid bleaching and leaves must be green (Williams, 1981). Analysis of Astragalus for these compounds from herbarium specimens identify the species that are nitro bearing and therefore might be poisonous to livestock. Examination of the chemotaxonomic relationships among the nitro-bearing species suggests which related species might be nitro- bearing even if specimens are not available for analysis (Williams, 1981). Because Astragalus may be grazed by livestock, these species require toxicological investigation to determine whether nitro compounds are present and whether they occur in toxic concentrations. The interception of poisonous or weedy species before introduction or extensive development can save time and money in needless research, alleviate future losses of livestock and costly control measures. This paper identi"es several nitro-bearing species of Astragalus from Iran. The occurrence of nitro-toxins in 16 species is reported for the "rst time.
H. Ebrahimzadeh et al./Biochemical Systematics and Ecology 27 (1999) 743}751
745
2. Materials and methods Twenty milligrams samples of leaves of 460 specimens including 440 species of Astragalus from 48 sections, collected from the herbarium of the Research Institute of Forests and Rangelands, Tehran, Iran, were analysed for toxic aliphatic nitro compounds. The names of species listed in this paper, are those found on the herbarium sheets, and the nomenclature and section classi"cation used herein is based on Maassoumi (1998). The quantitative test for aliphatic nitro-toxins was developed by Cooke (1955) and modi"ed by Williams and Parker (1974) for qualitative determination of aliphatic nitro compounds in 10 mg plant samples. Ten milligrams of lea#ets was placed into each of the two test tubes and macerated to a "ne powder with a stirring rod. One millilitre of 1 N HCl was added to each test tube, and the solutions allowed to stand with frequent stirring for 2 h. One millilitre of 20% KOH was added to each tube, and the test tube kept at room temperature for another 2 h. One millilitre of glacial acetic acid, followed immediately by 1 ml of Griess-Ilosvay reagent was then added to one test tube. Two millilitre of glacial acetic acid was added to the second tube which was the control blank. Colour was allowed to develop for 3 min. Solutions that contained nitro-toxins turned pink to red within a few seconds. The intensity of the red colour was determined visually. Nitro content was rated on a scale of 1}5. Ratings and their approximate equivalent in mg NO /g plant were: 1"4}8; 2"9}13; 3"14}19; 4"20}25; 5"over 25. Shimadzu UV-visible recording spectrophotometer (UV-160) with 10 mm matched quartz cells was used for recording the absorption spectrum between 400}800 nm, after the coloured reaction mixture was "ltered through No. 1 "lter paper. Maximum colour is developed 3 min after adding of the last reagent (Williams and Barneby, 1977). In our analyses the absorption spectra were recorded after 15 min.
3. Results and discussion Nitro compounds were found in 37 species from 20 of 48 taxonomic sections of Astragalus, based on a scale of 1}4 (Table 1).These compounds were not detected in sections Astragalus, Asciocalyx Bunge, Caraganella Bunge, Cercidophaca Maassoumi and Podlech, Cystodes Bunge, Eremophysa Bunge, Erioceras Bunge, Grammocalyx Bunge, Halicacabus Bunge, Herpocaulos Bunge, Hololeuce Bunge, Hymenostegis Bunge, Hypoglottidei DC., ¸aguropsis Bunge, ¸axi-ori Agerger-Kirchhof, ¸eucocercis Bunge, Macrophyllium Boiss, Macrosemium Bunge, Microphysa Bunge, Onobrychioidei DC., Ornithopodium Bunge, Paracystium Gontsch, Plagiophaca Maassoumi and Podlech, Platyglottis Bunge, Poterion Bunge, Rhacophorus Bunge, Stereothrix Bunge and Xiphidium Bunge. The numerical ranking assigned each nitro-containing species re#ects only the approximate concentration of nitro-toxin and though providing a basis for predictability, does not necessarily re#ect absolute toxicity to ruminants. The concentration of
remotifolius Boiss and Hausskn megalotropis C.A.Mey kirindicus Boiss meridionalis Bunge ammodendroides Bornm laricus Boiss & Hohen corrugatus Betrol corrugatus Betrol murinus Boiss raswendicus Hausskn and Bornm remoti-orus Boiss ssp. melanograma (Boiss) Zarre remoti-orus Boiss ssp. melena remoti-orus Boiss ssp. remoti-orus submitis Boiss ssp. submitis -orulentus(Boiss and Hausskn)Podlech hamosus L. argyrostachys Boiss vanillae Boiss angusti-orus ssp. angusti-orus Boiss apricus Bunge maymanensis Podlech masandaranus Bunge daenensis Boiss zerdanus Boiss campylotrichus Bunge robustus Bunge deickianus Bornm elegans Bunge macrostachys DC.
Adiaspastus Bunge Alopecuroidei DC. Alopecuroidei DC. Alopecuroidei DC. Ammodendron Bunge Annulares DC. Annulares DC. Annulares DC. Anthylloidei DC. Anthylloidei DC. Anthylloidei DC.
Anthylloidei DC. Anthylloidei DC. Anthylloidei DC. Brachycalyx Bunge Bucerates DC. Campylanthus Bunge Chronopus Bunge Caprini DC. Caprini DC. Caprini DC. Cystium Bunge Hemiphaca Bunge Hemiphaca Bunge Heterodontus Bunge Incani DC. Malacothrix Bunge Malacothrix Bunge Malacothrix Bunge
Species
Section
Table 1 Nitro bearing species of Astragalus from Iran
Arak: 48332 Fars: 24 Karaj:15561 Esfahan: 46052 Khuzestan: 62464 Fars: 45769 Tehran: 11606 Chaharmahal: 62081 Azarbayejan: 57036 Khorasan: 55854 Mazandaran: 33037 B Yazd:18019 Esfahan: 46167 Khorasan: 50864 Azarbayejan: 30445 Hamadan: 59381 Azarbayejan: 56977 Azarbayejan: 30497
Kordestan: 16883 Azarbayejan: 34519 Esfahan: 2937 Bandar-Abbas Gilan: 18298 Lar: 15409 Gorgan: 47572 Khorasan: 50246 Esfahan: 46094 Arak: 47960 Arak: 644002
Origin and Voucher Number
1984 4.5.1987 23.6.1973 14.7.1983 14.3.1986 31.5.1983 20.5.1974 12.6.1987 27.6.1986 28.4.1986 23.6.1979 23.6.1975 16.7.1983 30.5.1984 30.6.1978 13.5.1987 25.6.1986 30.6.1978
11.5.1975 20.6.1980 11.6.1983 5.5.1985 13.7.1975 18.7.1972 8.5.1984 22.5.1984 15.7.1983 14.6.1984 9.7.1985
Date collected
2 2 1 1 2 1 1 1 1 1 2 4 2 1 1 1 1 1
1 1 1 1 1 3 2 1 2 1 2
Nitro* concentration
746 H. Ebrahimzadeh et al./Biochemical Systematics and Ecology 27 (1999) 743}751
crispocarpus Nabelek oxyglottis Stev. and M.B. oxyglottis Stev. var. psiloglottis oxyglottis Stev. and M.B. var. psiloglottis oxyglottis Stev. and M.B. var. oxyglottis vicarius Lipsky schmalhausenii Bunge biovulatus Bunge coronilla Bunge kerkukiensis Bornm ispahanicus Boiss ispahanicus Boiss ispahanicus Boiss siliquosus ssp. siliquosus emend-Podlech siliquosus ssp. stramineus (Boiss and Kotschy) Podlech magistratus Maassoumi, Ghahreman and Moza!arian odoratus Lam. Azarbayejan: 57015
Tehran: 35244
Lorestan: 26529
Zanjan: 64771 Semnan: 28838 Azarbayejan: 19265 Qum-Esfahan: 15156 Rudshur: 9920 Gorgan: 47570 Qazvin: 55146 Baluchestan: 22826 Tehran: 19506 Fars: 17753 Arak: 47981 Khorasan: 50371 Tehran: 11583 Tehran: 59230
26.6.1986
9.6.1981
2.7.1977
24.5.1987 4.5.1978 24.4.1976 22.7.1972 8.5.1968 8.5.1984 3.6.1986 25.4.1977 7.5.1976 1.6.1975 14.6.1984 23.5.1984 1974 4.5.1986
* The approximate concentration (mg NO /g dry weight) represented by the scores are: 1"4}8; 2"9}13; 3"14}19; 4"20}25.
;liginosi Gray
¹richolobus Bunge
¹heiochrus Bunge
Oxyglottis Bunge Oxyglottis Bunge Oxyglottis Bunge Oxyglottis Bunge Oxyglottis Bunge Oxyglottis Bunge Oxyglottis Bunge Sesamei DC. Sesamei DC. Sesamei DC. ¹heiochrus Bunge ¹heiochrus Bunge ¹heiochrus Bunge ¹heiochrus Bunge
2
1
3
3 1 2 2 2 4 4 4 2 1 2 2 2 2
H. Ebrahimzadeh et al./Biochemical Systematics and Ecology 27 (1999) 743}751 747
748
H. Ebrahimzadeh et al./Biochemical Systematics and Ecology 27 (1999) 743}751
nitro-toxin in Astragalus lea#ets often indicated whether the toxic catabolite will be 3-NPOH or 3-NPA. This taxonomic character provides an estimate of the toxicity of the species to ruminants. Miserotoxin, which is catabolized in the digestive tract of ruminants to 3-NPOH, is primarily found in Astragalus species that synthesize nitro-toxins at levels up to 3. Nitro-toxins that are synthesized at levels 4 and 5 are catabolized to 3-NPA in the digestive tracts of ruminants (Williams and Barneby, 1977). We did not "nd species with a score of 5. The nitro-toxin concentration listed in Table 1 should be considered a minimum concentration. Age may have lowered the nitro-toxin level in some species and may have lowered the nitro-toxin concentration below a detectable level in others (Williams, 1981). To minimise this possibility we collected our samples from the most recently acquired specimens that had leaves of good green colour. Williams (1981) has analysed nitro compounds in many species of Astragalus, including some species of this genus from Iran, collected from di!erent herbaria of Europe, and reported 221 nitro-bearing species of Astragalus, 190 of them from the Old World. This paper identi"es 37 nitro-bearing species of Astragalus from Iran. Of the 37 positive-testing species, 21 had previously been reported as nitro-bearing (Williams, 1981). In this paper, the occurrence of nitro-toxins in 16 of 37 species is reported for the "rst time. These species are A. remotifolius Boiss and Hausskn, A. ammodendroides Bornm, A. corrugatus Betrol., A. -orulentus (Boiss and Hausskn) Podlech, A. angusti-orus ssp. angusti-orus Boiss, A. apricus Bunge, A. maymanensis Podlech, A. masandaranus Bunge, A. daenensis Boiss, A. campylotrichus Bunge, A. robustus Bunge, A. deickianus Bornm, A. elegans Bunge, A. macrostachys DC., A. kerkukiensis Bornm and A. magistratus Maassoumi, Ghahreman and Moza!arian. We report nitro compounds in A. kirindicus Boiss, A. hamosus L., A. crispocarpus Nabelek, A. oxyglottis Stev. and M.B., A. vicarius Lipsky, A. schmalhausenii Bunge, A. biovulatus Bunge and A. odoratus Lam., for the "rst time, for specimens collected from Iran. Williams (1981) has reported these compounds in A. oxyglottis Stev., collected from the former USSR, with score of 2. In our analyses, A. oxyglottis Stev. and M.B. ranked 1, and A. oxyglottis Stev. var. psiloglottis, A. oxyglottis Stev. and M.B. var. psiloglottis, and A. oxyglottis Stev. and M.B. var. oxyglottis ranked 2. A. remoti-orus Boiss, here ranked 2 in three di!erent subspecies (Table 1), was ranked 1 in a previous study (Williams, 1981). In the species of Astragalus examined, the capacity to synthesize nitro compounds was species characteristic. Nitro compounds were always detected in the same species, or subspecies or varieties of these species (Table 1). The correct identi"cation of the species is a major problem. The genus Astragalus is complex and is under constant revision. Species may be renamed, transfered to di!erent sections, reduced to a subspecies or variety, or several species may be combined into one. Nitro-bearing sections as well as species may be chemotaxonomically related. Analysis for nitro compounds can be used to correctly identify and classify nitro-bearing species as well as resolve synonymy. The presence of nitro compounds tends to restrict a species to a limited number of sections. Some sections
H. Ebrahimzadeh et al./Biochemical Systematics and Ecology 27 (1999) 743}751
749
are nitro-bearing and some others are nitro-free. Thus nitro-bearing species within otherwise nitro-free sections should be reexamined for proper identi"cation and section classi"cation. All nitro-bearing species within taxonomic sections thus far examined synthesize nitro compounds that hydrolyze to 3-NPOH or 3-NPA, but not mixtures of the two.
Fig. 1. Absorption spectrum of coloured solution of nitro compounds of a specimen with a score of 3, using the reagent.
Fig. 2. Absorption spectrum of coloured solution of nitro compounds of a specimen with a score of 4, using the reagent.
750
H. Ebrahimzadeh et al./Biochemical Systematics and Ecology 27 (1999) 743}751
From these chemotaxonomic data for Astragalus we can predict: (a) the presence or absence of nitro compounds in individual species or within taxonomic sections; (b) whether the compounds yield 3-NPOH or 3-NPA upon hydrolysis; (c) the relative concentration of the nitro compounds; and, (d) the probable toxicity of the plant to livestock. These data provide valuable information on nitro synthesis in most of the world's species of Astragalus from which to determine their suitability for introduction and developmental research (Williams, 1985). Spectrum measurement of the coloured solutions, showed j at 527.5 nm. All the
coloured solutions showed the same j . Two of the spectra are given in Figs. 1 and 2.
Obtained j in our analyses is in agreement with the results of Cooke (1955) and
Williams and Norris (1969) using pure 3-nitropropionic acid.
Acknowledgements Financial support for this work was provided by a grant-in-aid for Ph.D. research projects from the Faculty of Sciences, University of Tehran. The authors wish to express their deep appreciation to Dr. M. Coburn Williams, Poisonous Plant Research Laboratory, USDA, Logan, UT 84321, for his research articles and helpful communications. The authors also thank Research Institute of Forests and Rangelands for permitting us to remove leaf samples from herbarium specimens.
References Anderson, R. C., Rasmussen, M. A., Allison, M. J., 1993. Metabolism of the plant toxins, nitropropionic acid and nitropropanol by ruminal microorganisms. Appl. Environ. Microbiol. 59, 3056}3061. Anderson, R. C., Majak, W., Rasmussen, M. A., Allison, M. J., 1998. Detoxi"cation 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, pp. 154}158. Cooke, A. R., 1955. The toxic constituent in Indigofera endecaphylla. Arch. Biochem. Biophys. 55, 114}120. Cronin, E. H., Williams, M. C., Olsen, J. D., 1981. Toxicity and control of Kelsey milkvetch. J. Range Manag. 34, 181}183. James, L. F., Hartley, W. J., Williams, M. C., Van Kampen, K. R., 1980. Field and experimental studies in cattle and sheep poisoned by nitro-bearing Astragalus or their toxins. Am. J. Vet. Res. 41, 377}382. Maassoumi, A.A., 1998. Astragalus in the Old World. Research Institute of Forests and Rangelands, Tehran. Stermitz, F. R., Norris, F. A., Williams, M. C. 1969. Miserotoxin, a new naturally occuring nitro compounds. J. Am. Chem. Soc. 91, 4599}4600. Williams, M. C., 1981. Nitro compounds in foreign species of Astragalus. Weed Sci. 29, 261}269. 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., 1977. The occurrence of nitro-toxins in North American Astragalus (Fabaceae). Brittonia 29, 310}326. Williams, M. C., James, L. F., 1975. Toxicity of nitro-containing Astragalus to sheep and chicks. J. Range Manag. 28, 260}263.
H. Ebrahimzadeh et al./Biochemical Systematics and Ecology 27 (1999) 743}751
751
Williams, M. C., James, L. F., Bleak, A. T., 1976. Toxicity of introduced nitro-containing Astragalus to sheep, cattle, and chicks. J. Range Manag. 29, 30}33. Williams, M. C., Norris, F. A., 1969. Distribution of miserotoxin in varieties of Astragalus miser Dougl.ex.Hook. Weed Sci. 17, 236}238. Williams, M. C., Norris, F. A., Kampen, K. R., 1970. Metabolism of miserotoxin to 3-nitro-1-propanol in bovine and ovine ruminal #uids. Am. J. Vet. Res. 31, 259}262. Williams, M. C., Parker, R., 1974. Distribution of organic nitrites in Astragalus. Weed Sci. 22, 259}262.
