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Comparison of the toxicities of Senecio jacobaea, Senecio vulgaris and Senecio glabellus in rats
Toxicology Letters, 15 (1983) 19-23 Elsevier Biomedical Press
19
COMPARISON OF TIiE TOXICITIES OF SENECIO JACORAEA, SENE~r~ VULGARIS AND ~ENE~IU GLARELLUS IN RATS (Senecio toxicity; pyrrolizidine
alkaloids; rats)
DOUGLAS E. GOEGER’, PETER R. CHEEKEar*, HOWARD S. RAMSDELLb, STEVEN S. NICHOLSON and DONALD R. BUHLERb ‘Department of Animal Science, and bDepartment of Agricultural Chemistry, Oregon State University Corvallii, OR 97331; ‘Louisiana Cooperative Extension Service, University Station, Baton Rouge, LA 70803 (U.S.A.) (Received April 2Oth, 1982) (Accepted July 8th, 1982)
SUMMARY The toxicity of Senecio jacobaea, S. vulgaris and S. glabeilus to rats was assessed in a feeding trial. The plants were of similar toxicity, with a plant dry matter intake of about 20% of initial body weight being a lethal dose. Gas chromatography-mass spectrometry analysis demonstrated the presence of seneciphylline, jacobine, jacozine and jacoline in S. jacobaea, senecionine and seneciphylline in S. vulgaris, and senecionine in S. gfabellus. An unidentified alkaloid was also found in al1 three plants.
There are more than 1200 species of Senecio found throughout the world. There are 50 species alone in North America of which 7 have been proven or suspected of being toxic [l]. S. vulgaris, also known as common groundsel, is an annual commonly found in gardens and cultivated areas. The PAS which it is known to contain are senecionine, seneciphylline, and retrorsine. These are found at concentrations of 0.21% of dried plant material [2]. S. jacobaea (tansy ragwort) contains senecionine, seneciphylline, jacobine jacoline, jaconine and jacozine at a total PA concentration of about 0.18% of the plant dry matter [2, 31. Although S. vulgaris does not cause as much economic loss as S. jmobaea, it has been implicated in the deaths of dairy cattle [4]. There has been little work reported on the toxicity of S. glubellus (S. *Author to whom correspondence
should be addressed.
Abbreviation: PA, pyrrolizidine alkaloid. 0378-4274/83/0000-0000/$03.00
0 Elsevier Biomedical Press
20
lo&a&s). Commonly known as bitterweed, it is found in wet soils of the southeastern United States north to Illinois and North Carolina [I]. The only PA that it is reported to contain is senecionine. This plant has been suspected of poisoning cattle in Florida (61. The purpose of the our present study is to compare the toxicity of S. ~~co~ue~, S. vulgaris and S. g~a~eliu~ by a feeding trial with rats. The alkaloid extracts were also compared by gas chromatography. MATERIALS AND METHODS
Three groups of 8 male Long Evans rats each were individually housed in hanging wire cages in temperature- and light-controlled rooms. Average individual weights were 153 g. Each group was fed ad lib. one of the Senecio plants at 10% of a cornsoy basal ration (Table I) for 18 days. They were then fed the OSU pelleted rat diet ad lib. for 51 days. Following this period they were again given the Senecio diets at the 5% level for 14 days followed by the OSU pelleted diet until the termination of the study at 121 days. Water was furnished ad lib. Feed consumption, weight gains, and mortalities were recorded. Dead animals were examined for gross and microscopic lesions. Isolation of PAS was done at room temperature using the Dowex-50 procedure [7]. This method allows a purer preparation to be obtained compared to the alternative Soxhlet method since many extractable compounds, other than bases, are not bound to the resin. Gas chromatographic analyses were obtained with 3% OV-17 on chromosorb WHP packed in a 10’ x 118 e Ni-200 column. The column oven was held at 250°C and a flame ionization detector was used. Gas chromatography-mass spectrometric analysis of the alkaloids was conducted, using a Varian CH-7 mass spectrometer equipped with a Systems Industries Model 150 data system. A lo-foot Ni-200 column packed with 3% OV-101 on Chromsorb-HP-W (SO/l00 mesh) was used with helium as the carrier gas and the column held at 235°C. The effluent was split betTABLE I PERCENT COMPOSITION
OF THE BASAL RAT DIET
Ingredient
Percent
Senecio Ground corn Soybean meal Sucrose Corn oi1 Mineral mixa Vitamin mixb
10 48.5 30.0 5.0 3.0 3.0 0.5
aJones and Foster 19) bCheeke et al. [lo]
21
ween a flame ionization detector and the separator inlet. An ionization potential of 70 eV was utilized. Samples were injected as methanol solutions. RESULTS AND DISCUSSION
The S. glubellus-containing diet was extremely unpalatable, as assessed by the poor growth (Table II) and visual observation of the animals. Therefore, after 18 days, the rats were taken off all the Senecio-containing diets to prevent these animals from dying of starvation, and were fed a pelleted diet for 51 days, after which they were again fed the Senecio-containing diets. All 8 rats of the S. vulgaris group died before the termination of the study at 121 days (Table II). This might be expected since it contains the three most toxic alkaloids of the seven contained in the three plants. The LDs~ in the rat of these alkaloids are: retrorsine 38 mg/kg, seneciphylline 77 mg/kg and senecionine 85 mg/kg [5]. S. glabellus, which contains the comparatively toxic senecionine, killed 7 of the 8 rats. The LDx-, of the crude alkaloid extract from S. jacobaea is approx. 140 mg/kg for the rat [8]. Thus the toxicity of the S. jacobaea would be expected to be less than for the other two plants in view of the LDso of the alkaloids in which they contain. This is also evident from the fewer deaths from this plant than the others. Dead animals showed typical hepatotoxic lesions associated with PA intoxication. The gas chromatograms of the PAS isolated from the three plants are shown in Fig. 1. Jaconine of S. jacobaea and retronecine of S. vulgaris were not evident from the chromatograms. It appears that there may be another alkaloid (F) in S. glubellus in addition to senecionine which it has been previously reported to contain. However, mass spectrometric analysis of this peak showed it to have a molecular and fragmentation pattern identical to that of senecionine (Fig. 2). The difference in gas chromatographic retention time could be a reflection of two different diastereomeric forms. For example, a geometric isomer of senecionine, integerrimine, differing only in the configuration around the double bond of the macrocyclic ring, has been found in other Senecio species [5]. This trial has shown that S. glabellus contains at least one PA, and that it is of
TABLE II TOXICITY OF DIETARY XJACOBAEA, Plant
S. jacobaea S. glabelhs S. vulgaris aMeans*SEM;
S. GLABELLUS
AND S. VULGARIS
TO THE RAT
Rats dead at 121 days
Average total gain (g) for first 18 days
Average survival time (days)’
Average intake (g)
Senecio intake as % of initial body weight
S/8
29.9
32 + 1
7:8
0.8
65 f
11
32.1 + 2.2 30.7 * 3.0
21 20
818
32.5
78 f
9
35.1 + 2.4
23
Senecioa
survival time calculated for animals that died during the 121 days test period.
22
_
C
S globelius --
S.jocoboea
I Y
4 A
0
B F
i Fig. 1. Gas chromatograms
of Senecio alkaloid preparations measured by a flame ionization detector.
Fig. 2. M~s-s~ctromet~c glabellus.
analysis of alkaloid peaks of A (sen~ion~ne) and F (unidenti~ed)
of S.
23
similar hepatotoxicity to rats as S. jacobaeaand S. vulgar&.Since both of the latter are important livestock-poisoning plants, the significance of S. glabehs as a plant poisonous to livestock should be further assessed. It has previously been implicated in the poisoning of cattle in Florida [6].
This work was supported by grants from the National Institutes of Health No. ES-00210 and CA-22524, and project 156 of the Oregon Agriculture Experiment Station. The manuscript was issued as Oregon Agricultural Expe~ment Station Technical Paper No. 6305.
I J.M. Kingsbury, Poisonous Plants of the United States and Canada, Prentice-Hall, Englewood Cliffs, NJ, 1964. 2 G.W. Buckmaster, P.R. Cbeeke and L.R. Shull, Pyrrolizidine alkaloid poisoning in rats: Protective effects of dietary cysteine, J. Anim. Sci., 43 (1976) 464. 3 J.O. Dickinson, M.P. Cooke, R.R. Kind and P.A. Mohamed, Milk transfer of pyrrolizidine alkaloids in cattle, J. Am. Vet. Med. Assoc., 169 (1976) 1192. 4 M.D, Fowler, Pyrrolizidine alkaloid poisoning in calves, J. Am. Vet. Med. Assoc., 152 (I968) 1131. 5 L.B. Bull, C.C.J. Culvenor and A.T. Dick, The Pyrrolizidine Alkaloids, Wiley, New York, 1968. 6 J.F. Morton, Ornamental plants with poisonous properties, Proc. Florida State Hort. Sot., 71(1958) 372. 7 A.R. Mattocks, Extraction of heat-labile alkaloids from plants, Nature, 191 (1961) 1281. 8 L.R. Shuil, G.W. Buckmaster and P.R. Cheeke, Factors influencing pyrrolizidine (Senecio) alkaloid metabolism: species, liver sulfhydryls, and rumen fermentation, J. Anim. Sci., 43 (1976) 1247. 9 J.H. Jones and C. Foster, A salt mixture for use with basal diets either high or low in phosphorus, J. Nutr., 24 (1942) 245. IO P.R. Cheeke, J.H. Kinzeff and M.W. Pedersen, Inffuenee of saponins on alfalfa utilization by rats, rabbits and swine, J. Anim. Sci., 46 (1977) 476.
19
COMPARISON OF TIiE TOXICITIES OF SENECIO JACORAEA, SENE~r~ VULGARIS AND ~ENE~IU GLARELLUS IN RATS (Senecio toxicity; pyrrolizidine
alkaloids; rats)
DOUGLAS E. GOEGER’, PETER R. CHEEKEar*, HOWARD S. RAMSDELLb, STEVEN S. NICHOLSON and DONALD R. BUHLERb ‘Department of Animal Science, and bDepartment of Agricultural Chemistry, Oregon State University Corvallii, OR 97331; ‘Louisiana Cooperative Extension Service, University Station, Baton Rouge, LA 70803 (U.S.A.) (Received April 2Oth, 1982) (Accepted July 8th, 1982)
SUMMARY The toxicity of Senecio jacobaea, S. vulgaris and S. glabeilus to rats was assessed in a feeding trial. The plants were of similar toxicity, with a plant dry matter intake of about 20% of initial body weight being a lethal dose. Gas chromatography-mass spectrometry analysis demonstrated the presence of seneciphylline, jacobine, jacozine and jacoline in S. jacobaea, senecionine and seneciphylline in S. vulgaris, and senecionine in S. gfabellus. An unidentified alkaloid was also found in al1 three plants.
There are more than 1200 species of Senecio found throughout the world. There are 50 species alone in North America of which 7 have been proven or suspected of being toxic [l]. S. vulgaris, also known as common groundsel, is an annual commonly found in gardens and cultivated areas. The PAS which it is known to contain are senecionine, seneciphylline, and retrorsine. These are found at concentrations of 0.21% of dried plant material [2]. S. jacobaea (tansy ragwort) contains senecionine, seneciphylline, jacobine jacoline, jaconine and jacozine at a total PA concentration of about 0.18% of the plant dry matter [2, 31. Although S. vulgaris does not cause as much economic loss as S. jmobaea, it has been implicated in the deaths of dairy cattle [4]. There has been little work reported on the toxicity of S. glubellus (S. *Author to whom correspondence
should be addressed.
Abbreviation: PA, pyrrolizidine alkaloid. 0378-4274/83/0000-0000/$03.00
0 Elsevier Biomedical Press
20
lo&a&s). Commonly known as bitterweed, it is found in wet soils of the southeastern United States north to Illinois and North Carolina [I]. The only PA that it is reported to contain is senecionine. This plant has been suspected of poisoning cattle in Florida (61. The purpose of the our present study is to compare the toxicity of S. ~~co~ue~, S. vulgaris and S. g~a~eliu~ by a feeding trial with rats. The alkaloid extracts were also compared by gas chromatography. MATERIALS AND METHODS
Three groups of 8 male Long Evans rats each were individually housed in hanging wire cages in temperature- and light-controlled rooms. Average individual weights were 153 g. Each group was fed ad lib. one of the Senecio plants at 10% of a cornsoy basal ration (Table I) for 18 days. They were then fed the OSU pelleted rat diet ad lib. for 51 days. Following this period they were again given the Senecio diets at the 5% level for 14 days followed by the OSU pelleted diet until the termination of the study at 121 days. Water was furnished ad lib. Feed consumption, weight gains, and mortalities were recorded. Dead animals were examined for gross and microscopic lesions. Isolation of PAS was done at room temperature using the Dowex-50 procedure [7]. This method allows a purer preparation to be obtained compared to the alternative Soxhlet method since many extractable compounds, other than bases, are not bound to the resin. Gas chromatographic analyses were obtained with 3% OV-17 on chromosorb WHP packed in a 10’ x 118 e Ni-200 column. The column oven was held at 250°C and a flame ionization detector was used. Gas chromatography-mass spectrometric analysis of the alkaloids was conducted, using a Varian CH-7 mass spectrometer equipped with a Systems Industries Model 150 data system. A lo-foot Ni-200 column packed with 3% OV-101 on Chromsorb-HP-W (SO/l00 mesh) was used with helium as the carrier gas and the column held at 235°C. The effluent was split betTABLE I PERCENT COMPOSITION
OF THE BASAL RAT DIET
Ingredient
Percent
Senecio Ground corn Soybean meal Sucrose Corn oi1 Mineral mixa Vitamin mixb
10 48.5 30.0 5.0 3.0 3.0 0.5
aJones and Foster 19) bCheeke et al. [lo]
21
ween a flame ionization detector and the separator inlet. An ionization potential of 70 eV was utilized. Samples were injected as methanol solutions. RESULTS AND DISCUSSION
The S. glubellus-containing diet was extremely unpalatable, as assessed by the poor growth (Table II) and visual observation of the animals. Therefore, after 18 days, the rats were taken off all the Senecio-containing diets to prevent these animals from dying of starvation, and were fed a pelleted diet for 51 days, after which they were again fed the Senecio-containing diets. All 8 rats of the S. vulgaris group died before the termination of the study at 121 days (Table II). This might be expected since it contains the three most toxic alkaloids of the seven contained in the three plants. The LDs~ in the rat of these alkaloids are: retrorsine 38 mg/kg, seneciphylline 77 mg/kg and senecionine 85 mg/kg [5]. S. glabellus, which contains the comparatively toxic senecionine, killed 7 of the 8 rats. The LDx-, of the crude alkaloid extract from S. jacobaea is approx. 140 mg/kg for the rat [8]. Thus the toxicity of the S. jacobaea would be expected to be less than for the other two plants in view of the LDso of the alkaloids in which they contain. This is also evident from the fewer deaths from this plant than the others. Dead animals showed typical hepatotoxic lesions associated with PA intoxication. The gas chromatograms of the PAS isolated from the three plants are shown in Fig. 1. Jaconine of S. jacobaea and retronecine of S. vulgaris were not evident from the chromatograms. It appears that there may be another alkaloid (F) in S. glubellus in addition to senecionine which it has been previously reported to contain. However, mass spectrometric analysis of this peak showed it to have a molecular and fragmentation pattern identical to that of senecionine (Fig. 2). The difference in gas chromatographic retention time could be a reflection of two different diastereomeric forms. For example, a geometric isomer of senecionine, integerrimine, differing only in the configuration around the double bond of the macrocyclic ring, has been found in other Senecio species [5]. This trial has shown that S. glabellus contains at least one PA, and that it is of
TABLE II TOXICITY OF DIETARY XJACOBAEA, Plant
S. jacobaea S. glabelhs S. vulgaris aMeans*SEM;
S. GLABELLUS
AND S. VULGARIS
TO THE RAT
Rats dead at 121 days
Average total gain (g) for first 18 days
Average survival time (days)’
Average intake (g)
Senecio intake as % of initial body weight
S/8
29.9
32 + 1
7:8
0.8
65 f
11
32.1 + 2.2 30.7 * 3.0
21 20
818
32.5
78 f
9
35.1 + 2.4
23
Senecioa
survival time calculated for animals that died during the 121 days test period.
22
_
C
S globelius --
S.jocoboea
I Y
4 A
0
B F
i Fig. 1. Gas chromatograms
of Senecio alkaloid preparations measured by a flame ionization detector.
Fig. 2. M~s-s~ctromet~c glabellus.
analysis of alkaloid peaks of A (sen~ion~ne) and F (unidenti~ed)
of S.
23
similar hepatotoxicity to rats as S. jacobaeaand S. vulgar&.Since both of the latter are important livestock-poisoning plants, the significance of S. glabehs as a plant poisonous to livestock should be further assessed. It has previously been implicated in the poisoning of cattle in Florida [6].
This work was supported by grants from the National Institutes of Health No. ES-00210 and CA-22524, and project 156 of the Oregon Agriculture Experiment Station. The manuscript was issued as Oregon Agricultural Expe~ment Station Technical Paper No. 6305.
I J.M. Kingsbury, Poisonous Plants of the United States and Canada, Prentice-Hall, Englewood Cliffs, NJ, 1964. 2 G.W. Buckmaster, P.R. Cbeeke and L.R. Shull, Pyrrolizidine alkaloid poisoning in rats: Protective effects of dietary cysteine, J. Anim. Sci., 43 (1976) 464. 3 J.O. Dickinson, M.P. Cooke, R.R. Kind and P.A. Mohamed, Milk transfer of pyrrolizidine alkaloids in cattle, J. Am. Vet. Med. Assoc., 169 (1976) 1192. 4 M.D, Fowler, Pyrrolizidine alkaloid poisoning in calves, J. Am. Vet. Med. Assoc., 152 (I968) 1131. 5 L.B. Bull, C.C.J. Culvenor and A.T. Dick, The Pyrrolizidine Alkaloids, Wiley, New York, 1968. 6 J.F. Morton, Ornamental plants with poisonous properties, Proc. Florida State Hort. Sot., 71(1958) 372. 7 A.R. Mattocks, Extraction of heat-labile alkaloids from plants, Nature, 191 (1961) 1281. 8 L.R. Shuil, G.W. Buckmaster and P.R. Cheeke, Factors influencing pyrrolizidine (Senecio) alkaloid metabolism: species, liver sulfhydryls, and rumen fermentation, J. Anim. Sci., 43 (1976) 1247. 9 J.H. Jones and C. Foster, A salt mixture for use with basal diets either high or low in phosphorus, J. Nutr., 24 (1942) 245. IO P.R. Cheeke, J.H. Kinzeff and M.W. Pedersen, Inffuenee of saponins on alfalfa utilization by rats, rabbits and swine, J. Anim. Sci., 46 (1977) 476.