Effects of Leucaena leucocephala (lamk. ) shoot tips plus young leaf extract containing mimosine on reproductive system of male rats / Efectos de los brotes de Leucaena leucocephala (lamk. ) y del extracto de hojas jóvenes que contiene mimosina en el sistema reproductivo de ratas macho

This study aimed to determine the mimosine level and examine the male reproductive toxicity effects of Leucaena leucocephala (LL) shoot tips plus young leaf extract. Mimosine level in LL extract was determined by thin layer chromatography before administration in animals. Male rats were divided into control and LL (1,500 mg/KgBW) groups (n = 6). After 60 days of experiment, serum sex hormones, sperm quality, and testicular histopathology were assayed and observed. Malondialdehyde (MDA) level and expressions of steroidogenic acute regulatory (StAR) and phosphorylated proteins in testicular lysate were examined by western blotting. Results showed that mimosine levels in LL extract was 17.35 ± 1.12 % of dry weight. LL significantly decreased FSH & LH levels, sperm qualities, and seminiferous tubule diameter compared to the control (p<0.05). Seminiferous tubular atrophies, germ cell sloughing, and degenerations were observed in LL group. In addition, testicular MDA level and StAR protein expression were significantly decreased in LL group. LL extract could increase the expression of a 50 kDa phohorylated protein in testicular lysate. In conclusion, LL extract has mimosine and reproductive toxicity effects on males.

Este trabajo tuvo como objetivo determinar el nivel de mimosina y examinar los efectos de la toxicidad reproductiva de los brotes de Leucaena leucocephala (LL), más el extracto de hojas jóvenes, en ratas macho. El nivel de mimosina en el extracto de LL se determinó mediante cromatografía en capa fina antes de la administración en animales. Las ratas se dividieron en grupos de control y LL (1,500 mg / kgBW) (n = 6). Después de 60 días, se analizaron y observaron las hormonas sexuales séricas, la calidad de los espermatozoides y la histopatología testicular. A través de Western Blot se examinaron el nivel de malondialdehído (MDA), las expresiones de reguladores agudos esteroidogénicos (StAR) y las proteínas fosforiladas en el lisado testicular. Los resultados mostraron que los niveles de mimosina en el extracto de LL fueron 17.35 ± 1.12 % del peso seco. LL disminuyó significativamente los niveles de FSH y LH, la calidad de los espermatozoides y el diámetro de los túbulos seminíferos en comparación con el control (p <0,05). Se observaron atrofias en los túbulos seminíferos, desprendimiento de células germinales y degeneraciones en el grupo LL. Además, el nivel de MDA testicular y la expresión de la proteína StAR se redujeron significativamente en el grupo LL. El extracto de LL podría aumentar la expresión de la proteína fosforilada de 50 kDa en el lisado testicular. En conclusión, el extracto de LL tiene mimosina y efectos de toxicidad reproductiva en los hombres.

The co-occurrence of two pyridine alkaloids, mimosine and trigonelline, in Leucaena leucocephala.

Leucaena leucocephala is a nitrogen-fixing tropical leguminous tree that produces two pyridine alkaloids, i. e. mimosine [beta-(3-hydroxy-4-pyridon-1-yl)-L-alanine] and trigonelline (1-methylpyridinium-3-carboxylate). Mimosine has been detected in leaves, flowers, pods, seeds, and roots, and it is one of the principal non-protein amino acids that occurs in all organs. Asparagine was the most abundant amino acid in flowers. The mimosine content varied from 3.3 micromol/g fresh weight (FW) in developing flowers to 171 micromol/g FW in mature seeds. Trigonelline was also detected in leaves, flowers, pods, and seeds, but not roots. The trigonelline content was lower than that of mimosine in all organs. It varied from 0.12 micromol/g FW in developing seeds to 2.6 micromol/g FW in mature seeds. [2-14C]Nicotinic acid supplied to the developing seeds was incorporated into trigonelline but not mimosine. This indicates that the pyridine and dihydroxypyridine structures of these two alkaloids are derived from distinct precursors. The physiological functions of mimosine and trigonelline are discussed briefly.

Solid-phase synthesis of mimosine tetrapeptides and their inhibitory activities on neuraminidase and tyrosinase.

Neuraminidase is a rational target for influenza inhibition, and the search for neuraminidase inhibitors has been intensified. Mimosine, a nonprotein amino acid, was for the first time identified as a neuraminidase inhibitor with an IC(50) of 9.8 ± 0.2 µM. It was found that mimosine had slow, time-dependent competitive inhibition against the neuraminidase. Furthermore, a small library of mimosine tetrapeptides (M-A(1)-A(2)-A(3)) was synthesized by solid-phase synthesis and was assayed to evaluate their neuraminidase and tyrosinase inhibitory properties. Most of the tetrapeptides showed better activities than mimosine. Mimosine-FFY was the best compound, and it exhibited 50% neuraminidase inhibition at a low micromolar range of 1.8 ± 0.2 µM, whereas for tyrosinase inhibition, it had an IC(50) of 18.3 ± 0.5 µM. The kinetic studies showed that all of the synthesized peptides inhibited neuraminidase noncompetitively with K(i) values ranging from 1.9 -to 7.2 µM. These results suggest that mimosine could be used as a source of bioactive compounds and may have possibilities in the design of drugs as neuraminidase and tyrosinase inhibitors.

Selective determination of mimosine and its dihydroxypyridinyl derivative in plant systems.

Our observations on the growth stimulatory nature of mimosine, (beta-(3-hydroxy-4-pyridon-1-yl)-L-alanine), the toxic non-protein plant amino acid, in some model experimental systems, warranted sensitive and selective routine estimations. For the determination of both mimosine and DHP, an indirect spectrophotometric method was developed based on their individual reaction with known excess of DZSAM and by estimating the remaining DZSAM with N-(1-naphthyl)ethylene-diamine (NEDA). The resultant decrease in the secondary coupled product was measured at 540 nm. On equimolar basis, DHP had 40% of the reactivity of mimosine while interference from other relevant compounds was 15-35%. The determination of mimosine and DHP in tissue samples under different physiological conditions was effected after paper chromatographic separation of mimosine and DHP with distinctly differing Rf, from other compounds. The indirect method is superior in terms of absolute selectivity, sensitivity and ease of applicability with linear decreases in absorbance, proportional to increasing concentrations of mimosine from 0.1 to 0.75 microM or DHP from 0.2 to 1.5 microM and with recoveries of 99.2 to 100.5%.

Nutritional and economic benefits of Leucaena and Gliricidia as feed supplements for small ruminants in humid West Africa.

Considering leguminous trees Leucaena and Gliricidia as good sources of quality food, on-station and on-farm studies were conducted in the humid zone of West Africa to establish animal responses to levels, times and forms of browse supplementation, to develop alternative feeding strategies for utilising limited feed supply and to assess the economic benefits of feed supplements as against the use of tree foliage as mulch for crop production. Results indicate that at any level of supplement, sheep grew twice as fast as goats. The main benefits of supplementation came through increased growth and survival. Form and level of supplementation had significant effect on intake. Economic analyses showed that crop response to mulching was the principal competing determinant of whether the use of tree foliage as feed supplement was economic.

Determination of mimosine and 3,4-dihydroxypyridine in milk and plasma of goats.

A simple method for determination of mimosine and 3,4-dihydroxypyridine (3,4-DHP) in plasma and milk was developed. Milk and plasma, with tyrosine as internal standard, were deproteinized using 9% trichloracetic acid and extracted with diethyl ether. Metabolites were separated by isocratic high-performance liquid chromatography, with 0.02 M orthophosphoric acid (pH 2.5) at 0.5 ml/min and a Hypersil ODS microbore column. Mimosine, 3,4-DHP and tyrosine were detected at 275 nm. The recovery of the mimosine added to the plasma samples was 101.6 +/- 2.3% and 103.3 +/- 1.0% for milk samples. 3,4-DHP recovery for plasma samples was 101.2 +/- 0.9% and for milk samples 100.8 +/- 1.4%. The reproducibility of the method was evaluated by analyzing six plasma samples and six goat milk samples. The analyses yielded relative standard deviations of 2.65 and 2.82%, respectively.

Technical note: tissue residues of mimosine and 2,3-dihydroxypyridine after intravenous infusion in goats.

Sixteen growing Alpine wethers (average BW 35 +/- 2 kg) were assigned to one of four treatments to evaluate tissue retention of the leucaena toxins mimosine (MIM) and 2,3-dihydroxypyridine (2,3-DHP). Treatments were infused i.v. for 2 d and were 1) saline control, 2) MIM (200 mg.kg BW-.75.d-1), 3) 2,3-DHP (200 mg.kg BW-.75.d-1), or 4) MIM (100 mg.kg BW-.75.d-1) + 2,3-DHP (100 mg.kg BW-.75.d-1). Immediately after the infusion, the goats were slaughtered and tissue concentrations of MIM and 2,3-DHP were determined via HPLC. No detectable levels of either toxin were found in spleen, heart, lung, or muscle; however, appreciable amounts of MIM and 2,3-DHP were found in plasma, kidney, and liver samples. Kidney MIM content was greater (P < .01) than that of liver, although liver tended to retain slightly more 2,3-DHP (P > .05). Infusion of MIM resulted in a plasma MIM content of 39 to 54 mumol/L and reduced (P < .01) plasma PHE and LEU. Infusion of 2,3-DHP resulted in a plasma 2,3-DHP content of 9.4 mumol/L and increased plasma THR, ARG, VAL, PHE, ILE, LEU, and LYS concentrations (P < .10). Humans consuming offals from ruminants consuming large amounts of the leguminous forage leucaena may be exposed to appreciable quantities of MIM and 2,3-DHP.

Spectrophotometric determination of mimosine and 3-hydroxy-4-(1H)-pyridone--the toxic principles of Leucaena leucocephala.

A sensitive and selective spectrophotometric method for the estimation of the toxic factors mimosine and 3-hydroxy-4-(1H)-pyridone (DHP) has been developed based on the intense yellow-colored azodye formed with p-nitroaniline which showed a sharp absorption maximum at 400 nm. The method was optimized based on relative sensitivity of the reaction with various aromatic primary amino compounds and under different conditions of pH. Interference from a variety of structurally related compounds and phenols was tested and found insensitive to this method. The molar extinction coefficient at 400 nm for the azodye formed with mimosine was 5.31 x 10(4) M-1 cm-1 and that for DHP was 1.699 x 10(4) M-1 cm-1. The applicability of the method was tested using different plant extracts and recovery was found to be at 100 +/- 0.3%. The method is suitable for accurate estimation of both mimosine and DHP after paper chromatographic separation of extracts of different biological samples.