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Clastogenic effect of extracts obtained from Crotalaria retusa L. and Crotalaria mucronata Desv. on mouse bone marrow cells.

This work has evaluated the clastogenicity of six extracts (tea and aqueous extract of leaves, tea, aqueous and methanolic extracts of dried fruit, and tea of unripe fruit) obtained from Crotalaria retusa L. and three extracts (tea and methanolic extract of dried fruit, and tea of unripe fruit) obtained from Crotalaria mucronata Desv. The extracts were injected intraperitoneally into mice, and the animals were killed 24 h after treatment for preparation of bone marrow cells. The extracts obtained from fruits of Crotalaria retusa were found to cause a dose-dependent increase in the frequency of chromosomal aberrations in mice. On the other hand, no statistically significant increase in the frequency of aberrant cells was observed for the animals treated with leaf extracts obtained from Crotalaria retusa and with extracts from fruits of Crotalaria mucronata. The possibility that the pyrrolizidine alkaloid, monocrotaline, present in Crotalaria retusa exerts a clastogenic effect on mouse bone marrow cells is discussed. Our conclusion is based on studies using intraperitoneal treatments. Effects of oral exposure to extracts of Crotalaria retusa are unknown.

Animals↗

Genetic diversity of Crotalaria germplasm assessed through phylogenetic analysis of EST-SSR markers.

The genetic diversity of the genus Crotalaria is unknown even though many species in this genus are economically valuable. We report the first study in which polymorphic expressed sequence tag-simple sequence repeat (EST-SSR) markers derived from Medicago and soybean were used to assess the genetic diversity of the Crotalaria germplasm collection. This collection consisted of 26 accessions representing 4 morphologically characterized species. Phylogenetic analysis partitioned accessions into 4 main groups generally along species lines and revealed that 2 accessions were incorrectly identified as Crotalaria juncea and Crotalaria spectabilis instead of Crotalaria retusa. Morphological re-examination confirmed that these 2 accessions were misclassified during curation or conservation and were indeed C. retusa. Some amplicons from Crotalaria were sequenced and their sequences showed a high similarity (89% sequence identity) to Medicago truncatula from which the EST-SSR primers were designed; however, the SSRs were completely deleted in Crotalaria. Highly distinguishing markers or more sequences are required to further classify accessions within C. juncea.

Base Sequence↗

Hypercholeresis in turkeys following the ingestion of Crotalaria spectabilis seeds.

Young female turkeys received diets containing Crotalaria spectabilis seeds for 18 weeks. Mean values for total bile flow, biliary bile acid excretion, bile acid-dependent flow and bile acid-independent flow were significantly higher in crotalaria-fed turkeys than in controls. The hypercholeresis observed in crotalaria-fed birds involved both bile acid-dependent and bile acid-independent components of bile. Crotalaria-fed turkeys developed biliary hyperplasia. Liver weights were similar in the two groups. Since increases in 14C-erythritol clearances paralleled increases in total bile flow in crotalaria-fed turkeys (14C-erythritol bile to plasma ratios were similar in both groups), it was evident that the hypercholeresis in crotalaria-fed turkeys did not involve the ductal/ductular component of bile. Little or no ductal/ductular bile flow occurred in either group since total bile flow was equal to erythritol clearance and extrapolation to zero erythritol clearance yielded zero bile flow rates in both groups. Crotalaria-fed turkeys exhibited significantly higher biliary concentrations of chlorides (associated with lower sodium , potassium and bicarbonate concentrations) than did control birds. Validation of the use of 14C-erythritol clearance for the estimation of canalicular bile flow in turkeys was provided both by substantial decreases in bile to plasma 14C-erythritol concentration ratios following the injection of avian vaso-active intestinal peptide and the failure to demonstrate chromatographically the presence of radioactive metabolites of 14C-erythritol in plasma.

Animals↗

A comparative study on the hepatic toxicity and metabolism of Crotalaria assamica and Eupatorium species.

The oral LD50 of Crotalaria assamica, which contains mainly monocrotaline, was found to be 154 mg/kg in mice. Neither liver necrosis nor morbidity was demonstrated with Eupatorium extract at a dose level of 144 mg/kg, which was equivalent to the LD20 of Crotalaria. Pretreatment with phenobarbitone enhanced the toxicity of both plant extracts in mice. In in vitro studies, "metabolic pyrrole" was formed by incubating Eupatorium japonicum extracts with liver microsomes. The rate of "pyrrole" formation was similar to that of Crotalaria extract and pure monocrotaline alkaloid, but was much slower than retrorsine. The rate of N-oxide formation was, in descending order, retrorsine, Eupatorium japonicum and Crotalaria/monocrotaline. It is concluded that the alkaloid in Eupatorium species is metabolized to "pyrrole" and an N-oxide metabolite in the liver, but hepatotoxicity is much lower when compared with that caused by Crotalaria.

Alkaloids↗

Development of Crotalaria pulmonary hypertension: hemodynamic and structural study.

In awake rats with indwelling catheters, the development of pulmonary hypertension after feeding Crotalaria spectabilis seeds is followed. Hypoxemia is excluded as a factor. Other hemodynamic changes are found before hypertension. After 7 days, pulmonary artery pressure (Ppa) is normal, 17.17 +/- 0.30 (SE) mmHg and from 14 days significantly increased (P < 0.01). Oxygen consumption (Vo2) is significantly increased by day 7 (control 22.72 +/- 2.13 ml . min-1 . kg-1; Crotalaria 42.47 +/- 2.95; P < 0.001). and cardiac index (CI) is significantly above normal after 7, 14, and 21 days (control 350 +/- 31 ml . min-1 . kg-1; 7 days Crotalaria 476 +/- 28; P < 0.02); pulmonary vascular resistance (PVR) increases to six times normal at day 33 (control 0.033 +/- 0.003 U/kg; 33 days Crotalaria 0.194 +/- 0.020; P < 0.001). The pulmonary arteries of these same rats were studied by quantitative morphometric techniques. The first change, muscle in smaller and more peripheral arteries than normal, is detected when Vo2 and CI are increased. Increased medial thickness of arteries < 200 micrometer diameter follows with Ppa rises. Even later, the larger arteries increase their media as the number of peripheral arteries falls and right ventricular hypertrophy becomes apparent, and hypertension and increased PVR are well established.

Animals↗

Hemodynamic and metabolic effects of exercise in Crotalaria-induced pulmonary hypertension in rats.

The hemodynamic and metabolic effects of exercise were measured in Crotalaria-induced pulmonary hypertension in rats. The Crotalaria group had increased preexercise heart rate, mean pulmonary arterial pressure (PAP), arteriovenous O2 content difference, right ventricular work index (RVWI), and total pulmonary vascular resistance index (TPVRI) and decreased mean systemic blood pressure (BP), arterial O2 content (CaO2), venous O2 content (CvO2), cardiac index (CI), stroke volume index (SVI), and left ventricular work index (LVWI). The Crotalaria group during exercise had increased PAP, RVWI, TPVRI, and total systemic vascular resistance index and decreased BP, O2 consumption, CaO2, CvO2, CI, SVI, LVWI, O2 pulse index, and exercise duration. It is hypothesized that abnormal right ventricular function was a primary factor in the reduced exercise tolerance of the Crotalaria group.

Adenosine Triphosphatases↗

Influence of selenium on aflatoxin B1 or crotalaria toxicity in turkey poults.

This research compared the toxic effects of aflatoxin B1 and monocrotaline, the active principle of Crotalaria spectabilis, and the additive effect between aflatoxin B1 and monocrotaline in turkey poults. It was of interest whether selenium fed at dosage levels of 0.1, 5, or 10 micrograms/g of feed would protect against the toxic effect of aflatoxin and/or monocrotaline, and whether the toxicants would result in detectable residues in poult tissues. A total of 180 healthy 1-day-old male turkey poults was assigned at random to 12 treatment groups (15 birds/group). Body and liver weight losses, and low serum concentrations in total protein (TP), albumin (A), alpha-globulin (alpha G), and beta-globulin (beta G), as well as high values in gamma-globulin (gamma G), were produced in the groups fed crotalaria. Pathologic changes were induced by monocrotaline with no protection afforded by the added selenium. Low values in TP, A, alpha G, and beta G and in body and liver weights were observed in groups given the combination of aflatoxin plus crotalaria. Gross lesions were associated with an additive toxic effect and a lack of protective effect of selenium against this combination. However, higher values in TP, A, alpha G, and beta G, and liver weights in groups fed aflatoxin B1 plus selenium indicated that selenium had a protective effect against aflatoxin toxicity. Residues of aflatoxin B1 and aflatoxin M1 were found in the kidneys of poults fed aflatoxin B1; also, dehydroretronecine (the metabolite of monocrotaline) was detected in livers of poults fed Crotalaria spectabilis seeds.

Aflatoxins↗

Purification and characterization of a lectin from Crotalaria paulina seeds.

A lectin was purified from Crotalaria paulina seeds by ion-exchange and FPLC molecular exclusion chromatography. CrpL had an apparent molecular mass of 30 kDa, as determined by SDS-PAGE under non-reducing and reducing conditions. CrpL effectively agglutinated human and cow erythrocytes, and this activity was not affected by 20 mM EDTA, showing no dependence of divalent cations. Hemagglutination was inhibited by N-acetyl-D-galactosamine, D-galactose and was also inhibited by glycoproteins, fetuin and asialofetuin. The N-terminal amino acid sequence of CrpL was identical to those of other lectins from the genus Crotalaria, and amino acid composition showed high amounts of Asx and Glx, and was rich in Gly, Ala and Ser, as also reported for lectins from other Crotalaria species. CrpL inhibited the growth of Xanthomonas axonopodis pv. phaseoli and Xanthomonas axonopodis pv. passiflorae, suggesting a role of this lectin in the defense of seeds against bacterial infections.

Amino Acid Sequence↗

Molecular characterisation and evolutionary relationships of a potyvirus infecting Crotalaria in Brazil. Brief report.

The 3' terminal genomic region of a potyvirus causing mosaic disease in several Crotalaria species has been cloned and sequenced. Comparisons of the nucleotide and deduced amino acid (aa) sequences of the cloned cDNA with those from other potyviruses show that the Crotalaria-infecting virus (designated Crotalaria mosaic virus; CrMV) is closely related to Cowpea aphid-borne mosaic virus (CABMV). Maximum identity (95.4%) at the coat protein (CP) aa level was observed between CrMV and a Brazilian strain of CABMV. Phylogenetic analyses derived from the sequence alignments of the CP and 3' untranslated region confirmed the identification of CrMV as a strain of CABMV and the name CABMV-Cr is suggested.

3' Untranslated Regions↗

Lectins from seeds of Crotalaria pallida (smooth rattlebox).

A lectin from the seeds of Crotalaria pallida (CPL), with an apparent molecular mass of 30 kDa, determined by SDS-polyacrylamide gel electrophoresis, showed human type A and B erythrocytes agglutination activity, which is inhibited by raffinose and galactose. The lectin requirement for divalent cation was demonstrated with EDTA/EGTA blocking hemagglutination activity. Although the N-terminal amino acid sequence of CPL is identical to another lectin from Crotalaria striata, which is taxonomically synonymous to Crotalaria pallida, these lectins differ in amino acid composition and hemagglutination properties.

Amino Acid Sequence↗

[ Nodulation of certain legumes of the genus Crotalaria by the new species Methylobacterium].

We studied a collection of 126 rhizobial isolates from eight species of Crotalaria (C. comosa, C. glaucoides, C. goreensis, C. hyssopifolia, C. lathyroides, C. perrottetii, C. podocarpa, and C. retusa) growing in Senegal. Nodulation and nitrogen-fixation tests on nine Crotalaria species revealed two specificity groups within the genus Crotalaria. Group I consists of plants solely nodulated by very specific fast-growing strains. Group II plants are nodulated by slow-growing strains similar to promiscuous Bradyrhizobium spp. strains already reported to nodulate many tropical legumes. SDS-PAGE studies showed that slow-growing strains grouped with Bradyrhizobium while fast-growing strains constituted a homogeneous group distinct from all known rhizobia. Amplified ribosomal DNA restriction analysis (ARDRA) of 10 representative strains of this group using four restriction enzymes showed a single pattern for each enzyme confirming the high homogeneity of group I. The 16S rDNA sequence analysis revealed that this specific group belonged to the genus Methylobacterium, thus constituting a new branch of nodulating bacteria.

Bradyrhizobium↗

Development of pulmonary arterial changes in rats fed Crotalaria spectabilis.

The early changes in pulmonary artery structure and the timing and development of right ventricular hypertrophy were examined in rats after ingestion of Crotalaria spectabilis; the arteries were distended with a Micropaque--gelatin mixture. The earliest change, detected at Day 3, is the appearance of new muscle in normally nonmuscular arteries. Increased medial wall thickness of the normally muscular arteries is apparent from Day 7, reaching significance in the smaller arteries from Day 10 and in the larger arteries from Day 14. A reduction in number of small, filled peripheral arteries is apparent at Day 14 and is significant at Day 21 as is the increase in the ratio of alveoli to arteries per unit area of section. Right ventricular hypertrophy is detected and significant at Day 21 and, like all the arterial changes, continues to increase to Day 35. Hypoxia produces similar changes to those described above, although without the inflammation seen after Crotalaria ingestion, but the time table is different. New muscle appears in both models at the same time, but the other changes appear more slowly after ingestion of Crotalaria spectabilis.

Animals↗

Toxic effects of Crotalaria saltiana in mice.

Dry Crotalaria saltiana shoots were fed to strain ASL mice at 50, 10 and 1% of the diet. Feeding of Crotalaria shoots at 50 and 10% produced toxicity and death after 7 to 29 days. The signs of Crotalaria poisoning were inappetence, dullness, dyspnea and recumbency. The main lesions were necrosis, portal fibroplasia and hemorrhage in the liver, pulmonary congestion and emphysema, focal catarrhal enteritis, and degeneration of the cells of the renal tubules.

Animals↗

Crotalaria-induced pulmonary hypertension. Uptake of 3H-thymidine by the cells of the pulmonary circulation and alveolar walls.

Feeding with Crotalaria spectabilis seeds induces structural changes in the pulmonary arterial circulation characteristic of pulmonary hypertension: increased medial and adventitial thickness, the appearance of muscle in smaller arteries than normal, and reduction in the number of peripheral arteries. By autoradiographic techniques, after injection of 3H-thymidine into rats fed Crotalaria for 3, 7, 14, 21, 28, or 35 days, the contribution of hyperplasia to these changes has been assessed at two levels of the pulmonary artery--the hilum and the periphery. In the hilar pulmonary artery, a biphasic increase in labeling index (LI) is seen in each cell type. After 3 days of feeding, the medial smooth muscle cells show a slight but significant increase (1.5 times the control value), and, after 7 days, so do the adventitial fibroblasts (3 x) and the endothelial cells (EC) (2 x). After 14 days LI for all three cell types is again at control values, but after 21 days (wall thickness is no increased) each cell type shows at least a fivefold increase; by 35 days all are again near control levels. In the intra-acinar region, by 14 days, "newly" muscularized arteries are identified and increase in number and proportion up to 35 days; 3H-thymidine uptake is not evident in this cell type until 35 days have passed. The ECs of these arteries, however, show a striking increase in LI after 14 days as do those of the alveolar capillaries. The ECs of the intra-acinar veins show a biphasic response being increased after 7, 28, and 35 days. The present study has shown that Crotalaria ingestion induces hyperplasia and hypertrophy of pulmonary arterial cells at pre- and intra-acinar levels. The early increase in LI probably represents a response to the original cell injury, the later changes, a response to continuing damage or, in part, adaptation to the pulmonary hypertension now present.

Animals↗

Flavonoids of Crotalaria sessiliflora.

Phytochemical investigation of the whole plants of Crotalaria sessiliflora L. led to the isolation of four flavonoids. The structures of these compounds were identified as 2',4',5,7-tetrahydroxyisoflavone (1), 2',4',7-trihydroxyisoflavone (2), 4',7-dihydroxyflavone (3), and isovitexin (4) using spectroscopic analysis. Among these, compounds 2, and 3 have not been reported from Crotalaria species, whereas compounds 1, and 4 were reported from this plant for the first time.

Crotalaria↗