Anthelmintic Effect of Leucaena leucocephala Extract and Its Active Compound, Mimosine, on Vital Behavioral Activities in Caenorhabditis elegans.

Helminth infections continue to be a neglected global threat in tropical regions, and there have been growing cases of anthelmintic resistance reported towards the existing anthelmintic drugs. Thus, the search for a novel anthelmintic agent has been increasing, especially those derived from plants. Leucaena leucocephala (LL) is a leguminous plant that is known to have several pharmacological activities, including anthelmintic activity. It is widely known to contain a toxic compound called mimosine, which we believed could be a potential lead candidate that could exert a potent anthelmintic effect. Hence, this study aimed to validate the presence of mimosine in LL extract and to investigate the anthelmintic effect of LL extract and mimosine on head thrashing, egg-laying, and pharyngeal pumping activities using the animal model Caenorhabditis elegans (C. elegans). Mimosine content in LL extract was confirmed through an HPLC analysis of spiking LL extract with different mimosine concentrations, whereby an increasing trend in peak heights was observed at a retention time of 0.9 min. LL extract and mimosine caused a significant dose-dependent increase in the percentage of worm mortality, which produced LC50s of 73 mg/mL and 6.39 mg/mL, respectively. Exposure of C. elegans to different concentrations of LL extract and mimosine significantly decreased the head thrashing, egg-laying, and mean pump amplitude of pharyngeal pumping activity. We speculated that these behavioral changes are due to the inhibitory effect of LL extract and mimosine on an L-type calcium channel called EGL-19. Our findings provide evidential support for the potential of LL extract and its active compound, mimosine, as novel anthelmintic candidates. However, the underlying mechanism of the anthelmintic action has yet to be elucidated.

Evaluation of nutraceutical properties of Leucaena leucocephala leaf pellets fed to goat kids infected with Haemonchus contortus.

BACKGROUND: Leucaena leucocephala, as a shrub legume rich in condensed tannins, is a candidate for the integrated control of gastrointestinal parasitic pathogen nematodes. Here, we investigated the anthelmintic potential of the nutraceutical L. leucocephala, transformed into pellets, against Haemonchus contortus. RESULTS: Creole goat kids were fed an iso-nitrogenous diet of Dichantium hay with alfalfa pellets or Leucaena pellets or an alfalfa-leucaena mixture in varying ratios. The artificial infection of kids with H. contortus led to infection levels comparable to those observed in the farm environment on the basis of egg excretion. The anthelmintic activity of Leucaena, compared to alfalfa, was demonstrated by its potential to reduce egg excretions (1524 vs. 3651 eggs/g) and the larval development of excreted eggs (3.5 vs. 24%). This anthelmintic potential was reported when the ratio of Leucaena incorporation in the diet was high (50% DM intake). The voluntary dry matter intake (79.3 vs. 77.0 g Large Weigth0.75), the total tract dry matter digestion (0.585 vs. 0.620), and the growth (57.1 vs. 71.3 g/d) of kids fed Leucaena compared to alfalfa indicate that Leucaena has a high feed value. The Leucaena, even at the highest intakes in the diets, has not shown any signs of poisoning in kids. CONCLUSIONS: Leucaena fulfilled the conditions to be a good nutraceutical, and pelleting is a good method for its use.

In Vitro Anthelmintic Evaluation of Gliricidia sepium, Leucaena leucocephala, and Pithecellobium dulce: Fingerprint Analysis of Extracts by UHPLC-Orbitrap Mass Spectrometry.

In the present work, the anthelmintic activity (AA) of ethanolic extracts obtained from Gliricidia sepium, Leucaena leucocephala, and Pithecellobium dulce was evaluated using the third-stage-larval (L3) exsheathment inhibition test (LEIT) and egg hatch test (EHT) on Haemonchus contortus. Extracts were tested at concentrations of 0.3, 0.6, 1.2, 2.5, 5.0, 10, 20, and 40 mg/mL. The larval exsheathment inhibition (LEI) results showed that G. sepium achieved the highest average inhibition of 91.2%, compared with 44.6% for P. dulce and 41.0% for L. leucocephala at a concentration of 40 mg/mL; the corresponding IC50 values were 22.4, 41.7, and 43.3 mg/mL, respectively. The rates of egg hatching inhibition (EHI) at a concentration of 5 mg/mL were 99.5% for G. sepium, 64.2% for P. dulce, and 54% for L. leucocephala; the corresponding IC50 values were 1.9 mg/mL for G. sepium, 3.9 mg/mL for P. dulce, and 4.3 mg/mL for L. leucocephala. The species extracts studied here were also analyzed by ultra-high performance liquid chromatography and Orbitrap high resolution mass spectrometry (UHPLC-Q/Orbitrap/MS/MS), resulting in the compounds' identification associated with AA. Glycosylated flavonoids and methoxyphenols were observed in all three species: fatty acids in G. sepium and P. dulce; phenylpropanoids, anthraquinone glycosides, amino acids and glycosylated phenolic acids in G. sepium; and flavonoids in L. leucocephala. Comparatively, G. sepium presented a greater diversity of compounds potentially active against the control of gastrointestinal nematodes, which was associated with the results obtained in the applied tests.

Bacterial endosymbiont inhabiting Leucaena leucocephala leaves and their antioxidant and antidiabetic potential.

OBJECTIVES: Research on endosymbionts is emerging globally and is considered as a potential source of bioactive phytochemicals. The present study examines the antioxidant and antidiabetic of the endophytic crude extract isolated from Leucaena leucocephala leaves. METHODS: Endophytic bacteria were isolated from the leaves of L. leucocephala and 16S rRNA gene sequencing was used to establish their identity. The in vitro antioxidant effect of endophytic crude extract (LL) was evaluated using 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) free radical scavenging methods. The in vitro antidiabetic properties of LL were evaluated using α-amylase and α-glucosidase enzyme inhibition assay. RESULTS: The isolated endophytic bacteria were identified as Cronobacter sakazakii. LL displayed potent free radical scavenging effect against ABTS and DPPH radicals with an inhibitory concentration 50% (IC50) value of 17.49 ± 0.06 and 11.3 ± 0.1 µg/mL respectively. LL exhibited α-amylase and α-glucosidase inhibition with an IC50 value of 23.3 ± 0.08 and 23.4 ± 0.1 µg/mL respectively compared to the standard drug (acarbose). Both glucose loaded normoglycemic rats and STZ induced diabetic rats treated with LL (200 mg/kg) exhibited a considerable reduction in blood glucose levels p<0.01 after 8 h of treatment when compared to normal and diabetic control rats respectively. CONCLUSIONS: Thus, the study shows that LL has a wellspring of natural source of antioxidants, and antidiabetic agents and phytoconstituents present in endophytes could be the rich source for bioactive compounds.

Leucaena leucocephala extract has estrogenic and antiestrogenic actions on female rat reproduction.

Leucaena feed has been reported to cause disruptive effects on livestock reproduction, such as low calving percentages in cows, abortion in female goats and pigs, dead fetuses and fetal resorption in pregnant rats. In this study, the effects of Leucaena on different female reproductive variables were analyzed in two different reproductive conditions: gonadally intact and ovariectomized (OVX) female rats. Leucaena (LEU) was administered to females in both experimental conditions for 30 consecutive days. The effects of the legume extract were compared with those of Daidzein (DAI), a phytoestrogen, and of the female hormone estradiol (E2). In intact females, LEU disrupted the estrous cycle and female sexual behavior, decreased the number of follicles and corpora lutea, increased uterine and vaginal epithelium in proestrus and diestrus periods, increased uterine and vaginal relative weights during diestrus, and decreased serum progesterone during proestrus. All these effects were similar to those of DAI but lower than E2-induced effects. In OVX females, LEU decreased body weight, induced lordosis, stimulated vaginal epithelium cornification, increased vaginal weight, and augmented vaginal epithelium thickness. Again, these effects were similar to the effects of DAI and lower than the effects observed with E2. These results indicate that, in gonadally intact females, LEU can produce antiestrogenic effects in sexual behavior but estrogenic effects on uterine and vaginal weight and epithelia, without modifying serum levels of E2. In OVX females, in total absence of endogenous E2, LEU induced estrogenic effects on vaginal weight and epithelia, as well as on sexual behavior.

Efectividad de hongos micorrizo-arbusculares nativos en suelos con diferentes usos agropecuarios / Native arbuscular mycorrhizal fungi effectiveness in soils with different agricultural uses

RESUMEN Objetivo. Determinar la efectividad de hongos micorrizo-arbusculares (HMA) de suelos con diferentes usos agropecuarios, en el valle medio del río Sinú, Colombia. Materiales y métodos. Bajo condiciones de invernadero se realizó el experimento, donde se utilizó como planta indicadora Leucaena leucocephala, la cual se sembró en materos con suelo de un Andisol (horizonte B) usado como sustrato. Este sustrato, se inoculó separadamente con muestras de siete suelos (horizonte Ap), como fuente de HMA, provenientes del Centro de Investigación Turipaná de Agrosavia. Como controles se incluyeron inoculación con HMA Glomus fasciculatum y uno no-inoculado El efecto de la inoculación micorrizal se evaluó mediante el monitoreo del contenido de P foliar en las plantas de L. leucocephala, en función del tiempo hasta 64 días cuando se determinó la masa seca de su parte aérea y la colonización micorrizal. Resultados. El crecimiento de L. leucocephala y el contenido de P foliar fue significativamente superior cuando el suelo se inoculó con G. fasciculatum, en comparación a aquel observado con los demás tratamientos. De igual forma, la colonización micorrizal fue muy alta en las raíces de L. leucocephala que crecieron en el suelo inoculado con G. fasciculatum e inferior en los otros tratamientos. Conclusiones. Los resultados indican que los HMA nativos de suelos con diferentes usos, provenientes del C.I Turipaná de Agrosavia, exhibieron bajo potencial para desarrollar simbiosis micorrizal, lo que limitó el crecimiento y la concentración de P foliar en la planta hospedera.

ABSTRACT Objective. To determine the effectiveness of mycorrhizal-arbuscular fungi (AMF) of soils with different agricultural uses, in the middle valley of the Sinú river, Colombia. Materials and methods. The experiment was carried out under greenhouse conditions, where Leucaena leucocephala was used as the indicator plant, which was planted in masons with an Andisol soil (horizon B) used as a substrate. This substrate was inoculated separately with samples from seven soils (Ap horizon), as a source of AMF, from the Turipaná Research Center in Agrosavia. As controls we included inoculation with HMA Glomus fasciculatum and one non-inoculated one. The effect of mycorrhizal inoculation was evaluated by monitoring the foliar P content in L. leucocephala plants, as a function of time to 64 days, when the dry mass of its aerial part and mycorrhizal colonization was determined. Results. The growth of L. leucocephala and the foliar P content was significantly higher when the soil was inoculated with G. fasciculatum, in comparison to that observed with the other treatments. Likewise, mycorrhizal colonization was very high in the roots of L. leucocephala that grew in the soil inoculated with G. fasciculatum and lower in the other treatments. Conclusions. The results indicate that the native AMF of soils with different uses, from Turipaná, exhibited low potential to develop mycorrhizal symbiosis, which limited the growth and concentration of leaf P in the host plant.

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.

Flavonoids, a Potential New Insight of Leucaena leucocephala Foliage in Ruminant Health.

We investigated the constituents of Leucaena leucocephala foliage collected from Guangdong province in China and isolated 17 diverse flavonoids (1-17), including flavones (5-9, 11, and 12), flavonols (1, 10, and 16), flavanone 4, flavanonol 15, and flavonol glycosides (2, 3, 13, 14, and 17). Flavonoids quercetin (1), quercetin-3- O-α-rhamnopyranoside (2), and myricetin-3- O-α-rhamnopyranoside (17) were the major flavonoids components in L. leucocephala leaves, at a total concentration of about 2.5% of dry matter. pHRE-Luc inductive activity to mimic the activation of erythropoietin (EPO) gene, anti-inflammatory, antidiabetic, and antioxidant activities of isolated flavonoids (1-17) were evaluated. Flavonoids 7, 10, and 13 could strongly induce the transcriptional activity of pHRE-Luc, which indicated their potential to induce the expression of EPO. Flavonoids 7, 10, 13, and 17 displayed strong anti-inflammatory activity, relatively equal to the positive control dexamethasone. Flavonoids 1, 2, 3, 11, 12, 16, and 17 showed stronger antioxidant activities of DPPH radical scavenging capacity than ascorbic acid. Flavonoids 1, 2, and 10 showed weak cellular antioxidant activities against tert-butyl hydroperoxide (tBHP) induced ROS formation. Flavonoid rhamnoside 2 and arabinoside 3 undergone deglycosylation to the aglycone quercetin under anaerobic incubation with cattle rumen microorganisms. Furthermore, the potential health benefits for ruminant of flavonoids, which was rich in L. leucocephala foliage, was also discussed.

Tropical legume supplementation influences microbial protein synthesis and rumen ecology.

Four rumen-fistulated male swamp buffaloes, 5-year-old with initiated live weight at 360 ± 12 kg, were randomly assigned according to a 4 × 4 Latin square design to investigate the effect of feeding high level of dried Leucaena leaf (DLL) on feed intake, fermentation efficiency and microbial protein synthesis. The dietary treatments were the feeding levels of DLL at 0, 2, 4 and 6 kg/head/day. All buffaloes were supplemented with concentrate mixtures at 0.1% of body weight, and rice straw was fed ad libitum with the availability of water and mineral block at all time. The results revealed that the total feed intake and nutrient digestibility were significantly improved with the increasing levels of DLL feeding, and the highest was in the buffaloes consuming DLL at 6 kg/head/day. Feeding high levels of DLL did not affect on ruminal pH and temperature, while ammonia nitrogen, blood urea nitrogen and volatile fatty acid concentration were significantly enhanced. Moreover, methane production was dramatically reduced by increasing levels of DLL feeding. Total direct counts of the micro-organism population were increased with the increasing levels of DLL feeding. According to the application of quantitative PCR to quantity cellulolytic bacteria (16S rRNA) targets, it was found that the population of total bacteria and Fibrobactor succinogenes was affected by treatments, while Ruminococcus flavefaciens and methanogen population were significantly decreased as buffaloes were fed with DLL. The nitrogen balance and microbial nitrogen supply were remarkably improved with the increasing levels of DLL feeding. Based on this study, it could be concluded that high levels of DLL feeding at 6 kg/head/day could enhance feed intake, nutrient digestibility, rumen fermentation efficiency and microbial protein synthesis in swamp buffaloes fed on rice straw without any adverse effect.

Antioxidant capacity and acute testicular toxicity of Leucaena leucocephala aqueous shoot tips plus young leaves extracts / Capacidad antioxidante y toxicidad testicular aguda de brotes acuosos con extracto de hojas nuevas de Leucaena leucocephala

This study aimed to determine the antioxidant capacity of the Leucaena leucocephala aqueous shoot tips plus young leaves (LL-spl) extracts among three different fractions (LL-spl 10, 20, and 40 min) and to examine its acute toxicity on male reproductive parameters. The amount of the total phenolics in LL-spl extract was determined using a Folin-Ciocalteu reagent method and its antioxidant capacity was analyzed using 1, 1-diphenyl l-2-picrylhydrazyl radical scavenging and ferric reducing antioxidant powder methods. The LL-spl extract fraction with highest antioxidant capacity was used in animal treating. Male rats were divided into three groups (n= 5); control and groups treated with LL-spl 400 and 600 mg/Kg body weight for consecutive 40 days. The results showed that the LL-spl 40 min fraction possessed the highest antioxidant capacity. In addition, the LL-spl 400 and 600 groups showed no differences in weights of body, testis and epididymis, serum testosterone levels, and expression of testicular steroidogenic acute regulatory (StAR) protein. Significantly, LL-spl extract reduced the weight of seminal vesicle, sperm concentration, and seminiferous diameters compared with control. Moreover, LL-spl extract had adverse effect on testicular histology in inducing of seminiferous atrophy and degeneration including dilated blood vessels in interstitial tissue. It was concluded that although LL-spl extract possessing antioxidant capacity, in short term consumptions, it could be toxic to some male reproductive organs especially damaging testicular tissues.

El objetivo fue determinar la capacidad antioxidante del extracto de brotes acuosos con hojas nuevas de Leucaena leucocephala (LL-spl) en tres fracciones diferentes (LL-SPL 10, 20 y 40 min), además de examinar su toxicidad aguda sobre los parámetros reproductivos masculinos. Se determinó la cantidad de los fenoles totales en el extracto de LL-spl utilizando un método reactivo de Folin-Ciocalteu. La capacidad antioxidante se analizó por medio de 1-difenil-2-picrilhidracilo y/o métodos de reducción férrica de la capacidad antioxidante. La fracción de extracto LL-spl con mayor capacidad antioxidante fue utilizada en el tratamiento de los animales. Ratas macho fueron divididas en tres grupos (n= 5): el control y los grupos tratados con LL-spl 400 y 600 mg/kg peso corporal por 40 días consecutivos. El resultado mostró que la fracción LL-spl 40 min poseía la mayor capacidad antioxidante. Además, los grupos 400 y 600 LL-spl no mostraron diferencias según el peso corporal, testículos y epidídimo, niveles de testosterona y la expresión de proteínas testiculares. El extracto de LL-spl redujo de manera significativa el peso de la vesícula seminal, la concentración de espermatozoides y los diámetros de los túbulos seminíferos en comparación con el control. Por otra parte, el extracto de LL-spl tuvo un efecto adverso sobre la histología testicular por la inducción de atrofia y degeneración de los túbulos seminíferos, incluyendo a vasos sanguíneos dilatados en el tejido intersticial. Si bien el extracto LL-spl posee una capacidad antioxidante, ésta podría ser tóxica en el consumo a corto plazo para algunos órganos reproductores masculinos y especialmente dañino para los tejidos testiculares.

The flavonoid constitunts of Leucaena leucocephala. Growing in Egypt, and their biological activity.

BACKGROUND: Leucaena leucocephala is native to Southern Mexico and Northern Central America, but is now naturalized throughout the tropics. The phyto-chemical data of L. leucocephala revealed the presence of terpenes, flavonoids, coumarins and sterols. Various parts of L. leucocephala have been reported to have medicinal properties. MATERIALS AND METHODS: Flavonoids were isolated from the aerial parts of L. leucocephala. Antioxidant activity of the extracts and the isolated compounds was evaluated using (DPPH), as well as their cytotoxic activity using a single tumor [Ehrlish ascites carcinoma cells]. RESULTS: The flavonoidal constituents isolated from chloroform, ethyl acetate and n-butanol fractions of the aqueous alcoholic extract of aerial parts of Leucaena leucocephala were identified as Caffeic acid, Isorhamnetin, Chrysoeriol, Isorhamnetin 3-O-galactoside, Kaempferol-3-O-rubinoside, Quercetin-3-O-rhamnoside and Luteolin-7-glucoside. Chemical structures of the isolated compounds were identified by TLC, PC and spectral techniques (UV, (1)H-NMR and MS). The ethyl acetate fraction and the isolated flavonoidal compounds showed high antioxidant activity compared to Trolox (standard antioxidant compound). The different fractions and isolated compounds of Leucaena leucocephala exhibited no cytotoxic activity against Ehrlich-ascitis carcinoma cell line at the tested concentrations. CONCLUSION: This is the first record of the flavonoids in the aerial parts of Leucaena leucocephala (L.) except Quercetin-3-O-rhamnoside.

Immunostimulatory effect of artificial feed supplemented with indigenous plants on Clarias gariepinus against Aeromonas hydrophila.

The antibacterial activity of methanol extracts of Ficus benghalensis (prop-root) and Leucaena leucocephala (pod seed) was evaluated by measurement of zone of inhibition against pathogenic bacteria, Escherichia coli and Aeromonas hydrophila. Control artificial feed and artificial feed supplemented with 5% powder of F. benghalensis and L. leucocephala were prepared. Juvenile Clarias gariepinus were divided into four groups, acclimatized to laboratory conditions and fed with respective feeds for 20 days prior to the experiment. Immunomodulatory response of supplementary feed was studied by challenging the fish intraperitoneally at weekly intervals, with A. hydrophila. One set of fish, not challenged with A. hydrophila was used as a negative control, to analyze any detrimental effect of supplementary feed, while positive control, comprised of challenged fish fed with non-supplemented feed. Other two groups of fish were challenged with A. hydrophila and fed with respective supplementary feeds. Blood was collected on weekly intervals for four weeks and serum samples were analyzed to evaluate the damage of fish by A. hydrophila through liver function tests. The increase in the levels of Serum glutamic oxaloacetic transaminase (SGOT) and Serum Glutamic pyruvate transaminase (SGPT) in positive control group indicated the damage of liver & kidney. However the levels did not change significantly in fish fed with supplementary feeds when compared to negative control group. Nitric oxide, SOD, ALP and lipid peroxidase indicated lower stress levels in these fish compared to positive control. Fish fed with supplementary feed showed increased lysozyme activity and phagocytic index indicating an increase in non-specific immune response. The immunoglobulin levels of in serum were analyzed by homologous sandwich ELISA, which showed higher antibody production in fish fed with supplementary feed. The current study suggests conclusively, immunostimulatory role of F. benghalensis (prop-roots) and L. leucocephala (pod seed) in C. gariepinus when supplemented in artificial feed.