1. Introduction

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Toxic plants can be devastating if they are grazed upon or unintentionally incorporated into feeds or as contaminants in grains for animals and humans respectively. The genus Crotalaria, which belongs to the family Fabaceae (Leguminosae), consists of about 700 species (Polhill, 1982; Lewis et al., 2005; Adema, 2006; Mabberley, 2008) distributed throughout the tropical and subtropical regions of the world, with highest concentration in Africa. Presently, the Crotalaria species are of health importance because some of them contain pyrrolizidine alkaloids (PAs), which were reported to be cytotoxic (Srinivaset al., 2014).Both the foliage and seeds of many Crotalaria plants contain PAs (Dare et al., 2013; Maia et al., 2013;Diaz et al., 2014), thus posing potential hazard to grazing livestock. Crotalaria poisoning can either be chronic or acute (Srinivasan and Liu, 2012;Merz and Schrenk, 2016).

The Crotalaria plant occurs in Nigeria (Mattocks and Nwude, 1988), and is found in damp sites along forest margins (Burkill, 1995).It is known by many names among various tribes in Nigeria and they include ‘fara bi rana’, ‘Akedimwo’, ‘Korupo’ and ‘Birijibei’ in Hausa, Igbo, Yoruba, and Fulani, respectively (Nuhu et al., 2009; Ibrahim et al., 2012).

In domestic animals, syndromes such as sluggishness, weakness, loss of appetite, wasting, ascites, jaundice, photosensitization and behavioural abnormalities had been observed with PAs poisoning, and were related to hepatic insufficiency (Keeler et al., 1978).Crotalaria lachnosema Stapf. was found to contain two PAs, dicrotaline and acetyl dicrotaline (Mattocks and Nwude, (1988). The folklore utilization of C. lachnosema plant in the management of liver diseases and de-worming in livestock by some communities in Zaria, Nigeria, who had no knowledge of the hazards it poses is on the increase (Nuhu et al., 2009). The plant combined with the powder of other species of Crotalaria is highly sought after for use in portion ingredients for love matters, acceptance; in written and washed Qur’anic verses in native medicine (Nuhu et al., 2009). In developed countries, attention had been focused on the potential hazards of low level PAs as contaminant of many food products mainly because it could lead to progressive and chronic diseases that may not present as overt hepatotoxicity (Molyneuxet al., 2011). Also, livestock poisoning, presenting primarily as liver damage had been reported worldwide following the consumption of plants containing PAs (Molyneux et al., 2011; Witabouna and Brahima, 2012).

Despite the likely dangers of grazing in C. lachnosema-infested lands and its folkloric medicinal use, information on the toxicological profile of this species are unavailable. The only report that Crotalaria lachnosema contain spyrrolizidine alkaloids in Nigeria were that of Mattock and Nwude (1988), previous studies (Okopido and Ogunbiyi, 1976; Nuhu et al., 2009; and Ibrahim et al., 2012) did not detect any alkaloids in the leaves of C. lachnosema Stapf. The need to document the phytochemistry and clinico-pathological changes associated with possible toxicity from the consumption of the plantin Nigeria is therefore imperative.

2. Methods And Materials

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2.1. Crotalaria Lachnosema, Collection And Identification
The fresh leaves and meristems of the plant species (C. lachnosema) Stapf. were collected from farmlands (Plate I) and along streams in Samaru area of Zaria. The plant was identified, confirmed and authenticated and given the voucher botanical number, 1885,at the Herbarium unit of the Department of Biological Science, Ahmadu Bello University, Zaria. The sample was air dried in the laboratory to a constant weight and then pulverized using a laboratory milling machine (5 mm sieve mesh). The powdered material was stored in a black polythene bagsat room temperature until used for extraction.

2.2. Plant Extraction
The extraction from the plant material was conducted according to the method described by Mattocks and Nwude (1988).

2.3. Phytochemical Determination
Identification of alkaloid and other phyto constituents in the plant extract was carried out according to the method described by Trease and Evans (1983) and Nash et al.(1992)

2.4. Preparation Of Test Solutions
The maximum convenient concentration (MCC) of the extract of C.lachnosema was estimated using the method described by Ibrahim et al., (1983) and Ibrahim (1984). The Stock solution of MCC was made and kept at 4°C until required for use.

2.5. Acute Toxicity Studies
Fifteen 10 to 14-week old male Wistar rats (161±6.0 g) were obtained from the animal house of the Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria and used in this study. They were fed commercial grower poultry feed, maize offal and groundnut cake at a ratio of 4: 2: 1, respectively. Water was provided ad libitum. Acute toxicity was conducted according to the method of Lorke (1983).

2.6. Clinical Observations
The rats were closely monitored during the period of exposure for signs of toxicity and death.

2.7. Pathological Examination
Immediately following the death of the MLECL-exposed rats, postmortem examination was carried out and lesions observed were recorded. Specimens from the heart, kidneys, liver, lungs, and spleen were collected and fixed in 10% buffered formalin and later processed according to standard histological techniques (Luna, 1960). The prepared histopathological slides were examined using light microscope. The lesions were recorded and photographed using a digital camera (SAMSUNG S760 Auto, 18.9mm wide-angle lens, 7.2mega pixels, CHINA) at 400 magnification.

3. Results

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3.1. Test Plant Preparation, Extraction And Yield
The weight of the ground leaves of Crotalaria lachnosema was 800 g, which were from a freshly collected samples that weighed 2269 g. The crude methanolic soxhlet extract obtained from the ground leaves was 118.48 g, thus, giving a percentage yield of 14.81%.

3.2. Phytochemical Qualitative Determination
The phytochemical qualitative analysis revealed that the crude methanolic leaf extract of C. lachnosema contained alkaloids (Mayer’s and Dragendorff’s tests), carbohydrates (Molisch’s test), cardiac glycosides (Kella-killiani test), flavonoids (Sodium hydroxide test), saponins (Frothing test), steroids and triterpenes (Lieberman-Burchards test), and tannins (Ferric chloride test). The pyrrolizidine alkaloid obtained was 360 mg (0.09 %) from 400 g of ground dried leaves of Crotalaria lachnosema.

3.3. Acute Toxicity
In phase I of the study, no death was observed following exposure of the rats to the extract at 10mg/kg, 100mg/kg, and 1000mg/kg body weight. In the phase II, all the dose levels of the extract, 1600mg, 2900 mg and 5000 mg/kg body weight, caused death of the 3 rats within the first 24-hour observation period. A repeat of the phase II at dose levels of 1000mg, 1200mg, and 1400mg/kg was done, only the 1400mg/kg dose level caused death of the rat. Thus, an LD50of 1300 mg/kg was arrived at for MLECL.

3.4. Clinical Signs Of Acute Toxicity
The clinical signs observed in the rats in this acute phase of the study were oral irritation, nose bleeding, restlessness, arched-back, hyperpnoea, depression/weakness, hind limbs dragging, bloat (Plate II) and death.

3.5. Gross Pathological Findings From Acute Toxicity Of MLECL
The gross post-mortem lesions observed in the dead rats during this study were slight congestion of the liver, kidneys and red hepatization of the lungs

3.6. Histopathological Findings In The MLECL-Exposed Rats
Theacute exposure of the rats to MLECL resulted in severe congestion of the kidneys (Plate III) and lungs (Plate V) of the rats. There were noobservable histological lesions in the liver, heart and spleen of the control (Unexposed) rats.

4. Discussion

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In the present study, the oral LD50 of 1300 mg/kg obtained indicate that the methanolic leaf extract of C. lachnosema Stapf. was slightly toxic to rodents based on the reported classifications of the severity of the oral LD50 (Matsumura and Mizushima, 1975; Corbett et al., 1984; OECD, 2008). The classification of the LD50 in this study notwithstanding, the finding that the kidneys and the lungs were also severely congested suggests that the leaves of Crotalaria lachnosema could pose potential hazards to animals grazing in lands infested with this plant or endanger humans who make folkloric use of it in the management of different health ailments.

The phytochemical qualitative analysis revealed that the crude methanolic leaf extract of C. lachnosema contained alkaloids (Mayer’s and Dragendorff’s tests), carbohydrates (Molisch’s test), cardiac glycosides (Kella-killiani test), flavonoids (Sodium hydroxide test), saponins (Frothing test), steroids and triterpenes (Lieberman-Burchards test), and tannins (Ferric chloride test). The crystalline alkaloid obtained in this study was quantified at 360 mg (0.09 %) from 400 g dried ground leaves of Crotalaria lachnosema using the method described by Mattocks and Nwude, 1988. Phytotoxicological studies have always been considered vital prior to plant usage as foods, cosmetics and medicine indiscriminately without recourse to potential side effects, which could vary from mild to moderate or severe and life threatening (WHO, 2000).

5. Conclusion

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In conclusion, the present toxicity studies have provided insight on the toxicological status of the leaves of the plant,C. lachnosema Stapf. found in Nigeria. For the first time we have been able to show that; the oral LD50 of 1300 mg/kg obtained indicate that the methanolic leaf extract of C. lachnosema Stapf. was slightly toxic to rodents. The clinical signs observed in the acute phase of the study were oral irritation, nose bleeding, restlessness, arched-back, hyperpnoea, depression/weakness, hind limbs dragging, bloat and death, as well as congested kidneys and lungs. The phytochemical qualitative analysis revealed that the crystalline alkaloid obtained in this study was quantified at 360 mg (0.09 %) from 400 g dried ground leaves of Crotalaria lachnosema using the method described by Mattocks and Nwude, 1988.

Recommendations

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Herbal medicinal products containing toxic pyrrolizidine alkaloids (even in very low amounts) should be regarded as very hazardous. Extensive and intensive studies on the toxicity profiles of plants containing pyrrolizidine alkaloids in Nigeria and elsewhere in the world should be sustained.