Enantioselective biotransformation of sterically hindered amine substrates by the fungus Stemphylium lycopersici.

AIMS: To screen 20 micro-organisms for ω-transaminase (ω-TA) activity by the kinetic resolution of rac-1-phenylethylamine, followed by testing rac-amines of pharmaceutical interest with bulky substituents and to conduct the asymmetric synthesis of a chiral amine. METHODS AND RESULTS: Stemphylium lycopersici was selected as the best biocatalyst. By the central composite rotatable design (CCRD), it was found that, at lower pH (5·5 and 6·5), the lyophilized micro-organism biocatalysed the kinetic resolution of rac-1-phenylethylamine with 99% enantiomeric excess (e.e.) ((R)-enantiomer) with acetophenone conversions ranged from 41 to 45%. Interestingly, the lyophilized crude enzymatic extract lead to better results at pH from 7·0 to 9·0, with conversions up to 47% and about 99% e.e. We also attested that as much as higher is the pyruvate (amino acceptor) concentration, higher is the acetophenone conversion, corroborating the presence of ω-TA-type enzymes. Among different sterically hindered racemic amines tested, rac-1,2,3,4-tetrahydro-1-naphthylamine and rac-phenylbutylamine were satisfactorily kinetically resolved in up to 91% e.e. (R). The results for the asymmetric synthesis showed excellent conversion (>85%) for the S-1-phenylethylamine, indicating (S)-stereopreference. CONCLUSION: Stemphylium lycopersici showed to be an important tool for broader substrate scope transaminases and a relevant player on the development of new biocatalysts with ability in asymmetric synthesis reactions. SIGNIFICANCE AND IMPACT OF THE STUDY: Here in, we contribute to the improvement of the biocatalytic toolbox for chiral amines synthesis. Interestingly, we have found that the crude enzymatic extract of the endophytic fungus S. lycopersici could accept bulky substrates with reasonable activity, compared to the wild-type transaminase already published over literature, and with high enantioselectivity.

Kalanchoe pinnata inhibits mast cell activation and prevents allergic airway disease.

Aqueous extract of Kalanchoe pinnata (Kp) have been found effective in models to reduce acute anaphylactic reactions. In the present study, we investigate the effect of Kp and the flavonoid quercetin (QE) and quercitrin (QI) on mast cell activation in vitro and in a model of allergic airway disease in vivo. Treatment with Kp and QE in vitro inhibited degranulation and cytokine production of bone marrow-derived mast cells following IgE/FcɛRI crosslinking, whereas treatment with QI had no effect. Similarly, in vivo treatment with Kp and QE decreased development of airway hyperresponsiveness, airway inflammation, goblet cell metaplasia and production of IL-5, IL-13 and TNF. In contrast, treatment with QI had no effect on these parameters. These findings demonstrate that treatment with Kp or QE is effective in treatment of allergic airway disease, providing new insights to the immunomodulatory functions of this plant.

Effectiveness of the immunomodulatory extract of Kalanchoe pinnata against murine visceral leishmaniasis.

Previously, we described the protective action of the immunomodulatory extract of Kalanchoe pinnata (Kp) in murine and human cutaneous leishmaniasis. In the present study, we investigated the effectiveness of Kp against visceral leishmaniasis, using the BALB/c mouse model of infection with Leishmania chagasi. Mice receiving oral daily doses of Kp (400 mg/kg) for 30 days displayed significantly reduced hepatic and splenic parasite burden, when compared with untreated animals. Protectiveness was accompanied by a reduction in parasite-specific IgG serum levels, and impaired capacity of spleen cells to produce IL-4, but not IFN-gamma and nitric oxide upon antigen recall in vitro. The reference drug Pentostam (72 mg/kg) given by the intra-peritoneal route on alternate days produced an anti-leishmanial effect similar to oral Kp. Our findings show that the oral efficacy of Kp, seen previously in murine cutaneous leishmaniasis, extends also to visceral leishmaniasis caused by L. chagasi, a difficult to treat and lethal disease of man.

Immunomodulatory pretreatment with Kalanchoe pinnata extract and its quercitrin flavonoid effectively protects mice against fatal anaphylactic shock.

Previously, we reported the immunosuppressive action of the aqueous extract of Kalanchoe pinnata (Kp) in mice. In the present study, we report on the protective effect of Kp in fatal anaphylactic shock, likewise a Th2-driven immunopathology, and the identification of its active component. Mice daily treated with oral Kp during hypersensitization with ovalbumin were all protected against death when challenged with the allergen, as compared with the 100% mortality in the untreated group. A single intraperitoneal dose 3 h prior to challenge was partially effective. Oral protection was accompanied by a reduced production of OVA-specific IgE antibodies, reduced eosinophilia, and impaired production of the IL-5, IL-10 and TNF-alpha cytokines. In vitro, Kp prevented antigen-induced mast cell degranulation and histamine release. Oral treatment with the quercitrin flavonoid isolated from Kp prevented fatal anaphylaxis in 75% of the animals. These findings indicate that oral treatment with Kp effectively downmodulates pro-anaphylactic inducing immune responses. Protection achieved with quercitrin, although not maximal, suggests that this flavonoid is a critical component of Kp extract against this extreme allergic reaction.

High selective leishmanicidal activity of 3-hydroxy-2-methylene-3-(4-bromophenyl)propanenitrile and analogous compounds.

Sixteen not new aromatic compounds were prepared by one-pot reaction i.e. through Baylis-Hillman reaction and were the first time evaluated against promastigote Leishmania amazonensis and infected mammalian cells. Most of the compounds were selectively more active against amastigotes than the reference drug sodium stibogluconate (Pentostam, IC(50)=44.7 microM). We found that 3-hydroxy-2-methylene-3-(4-bromophenyl) propanenitrile (13) was the most active (IC(50)=12.5 microM) and safer compound (0.0 (0.9); % macrophage LDH release), being the lead compound.