Abstract Background: Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia. In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. Methods: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. Results: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially β chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. Conclusion: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo.

Abstract The present study explored essential oil constituents of the medicinal plants used in traditional medicine and growing wild in the Western Ghats region of India, which is one of the 34 global biodiversity hotspots. The hydro distilled essential oils of Blumea lanceolaria (Roxb.) Druce. (Asteraceae), Heliotropium indicum L. (Boraginaceae) and Triumfetta rhomboidea Jacq. (Tiliaceae) were analyzed using gas chromatography equipped with a flame ionization detector (GC-FID) and gas chromatography coupled with mass spectroscopy (GC/MS). Thirty-six, twenty-seven, and fifty constituents were identified from the essential oils of B. lanceolaria, H. indicum, and T. rhomboidea, respectively comprising 97.3% (B. lanceolaria), 98.2% (H. indicum) and 97.6% (T. rhomboidea) of the total oil constituents. The major compounds of B. lanceolaria, H. indicum, and T. rhomboidea were identified as β-pinene (82.3%), methyl salicylate (54.3%), and β-caryophyllene (28.9%), respectively. The essential of B. lanceolaria was found to be rich in monoterpene hydrocarbons (90.4%), while H. indicum and T. rhomboidea oils were rich in phenyl derivative (72.8%) and sesquiterpene hydrocarbons (69.5%) type constituents, respectively. The common compounds viz., terpin-4-ol and eugenol were identified in the essential oils of B. lanceolaria, H. indicum, and T. rhomboidea.

Resumen El presente estudio exploró los componentes de los aceites esenciales de plantas medicinales utilizadas en la medicina tradicional y de plantas silvestres que crecen en la región occidental de Ghats, India, que es uno de los 34 puntos críticos de la biodiversidad mundial. Los aceites esenciales hidro destilados de Blumea lanceolaria (Roxb.) Druce. (Asteraceae), Heliotropium indicum L. (Boraginaceae) y Triumfetta rhomboidea Jacq. (Tiliaceae) se analizaron mediante cromatografía de gases equipada con un detector de ionización de flama (GC-FID) y por cromatografía de gases acoplada con espectrometría de masas (GC / MS). Se identificaron treinta y seis, veintisiete y cincuenta componentes de los aceites esenciales de B. lanceolaria, H. indicum y T. rhomboidea, respectivamente, que comprenden 97.3% (B. lanceolaria), 98.2% (H. indicum) y 97.6 % (T. rhomboidea) del total de los constituyentes del aceite esencial. Los principales compuestos de B. lanceolaria, H. indicum y T. rhomboidea se identificaron como β-pineno (82.3%), salicilato de metilo (54.3%) y β-cariofileno (28.9%), respectivamente. Se observó que el aceite esencial de B. lanceolaria era rico en hidrocarburos monoterpénicos (90.4%), mientras que los aceites esenciales de H. indicum y T. rhomboidea eran ricos en derivados de fenilo (72.8%) e hidrocarburos sesquiterpénicos (69.5%), respectivamente. Los compuestos comunes, a saber, terpin-4-ol y eugenol, se identificaron en los aceites esenciales de B. lanceolaria, H. indicum y T. rhomboidea.

Plectranthus es un género grande y extenso con una diversidad de usos etnobotánicos. En la medicina tradicional P.mollis se ha utilizado contra las mordeduras de serpiente, como estimulante respiratorio y vasoconstrictor, depresor cardiaco, cura para hemorragias, tratamiento del retraso mental y el reumatismo. Se informó que P. mollis presenta actividad relajante sobre los músculos lisos y esqueléticos, y tiene actividad promotora citotóxica y anti - tumoral, además puede ser utilizado en el tratamiento del cáncer. El objetivo del presente estudio fue identificar la composición química del aceite esencial de P. mollis para evaluar la eficacia antimicrobiana del aceite. El aceite esencial de las partes aéreas de las flores de P. mollis se obtuvo por hidro - destilación y se analizó por cromatografía de gases equipado con un detector de ionización de llama (GC-FID), y cromatografía de gases acoplada a espectrometría de masas (GC/MS). Se identificaron veintisiete compuestos, que comprenden el 98.6% de los constituyentes totales. El compuesto principal se identificó como fencona (32.3%), seguido de α-humuleno (17.3%), óxido de piperitenona (8.5 %), óxido de cis piperitona (6.0 %) y E-β-farneseno (5.9%). Se encontró que el aceite es rico en monoterpenos oxigenados de tipo constituyentes (52.0%), seguido de hidrocarburos de sesquiterpeno (40.2%), sesquiterpenos oxigenados (4.9%), e hidrocarburos monoterpenos (1.5 %). La actividad antimicrobiana del aceite esencial de P.mollis se ensayó frente a seis bacterias Gram-negativas y ocho Gram-positivas, y tres hongos, utilizando el método de dilución en tubo. El aceite fue activo contra las bacterias Gram-positivas y Gram-negativas y hongos ensayados, en un intervalo de concentración de 0.065±0.008 a 0.937±0.139mg/ml, 0.468±0.069 a 3.333±0.527mg/ml y 0.117±0.017 a 0.338±0.062mg/ml, respectivamente. El presente estudio reveló que los constituyentes del aceite de alguna manera fueron cualitativamente similares y cuantitativamente diferentes de los informes anteriores de diferentes partes del mundo. Se encontró que la actividad antimicrobiana del aceite esencial de P. mollis puede ser de utilidad en el tratamiento de diversas enfermedades infecciosas causadas por bacterias y hongos.

Plectranthus is a large and widespread genus with a diversity of ethnobotanical uses. In traditional medicine P. mollis has been used against snakebites, respiratory stimulant and vasoconstrictor, cardiac depressant, cure for haemorrahage, treatment of mental retardation and rheumatism. P. mollis is reported to exhibit relaxant activity on smooth and skeletal muscles, and has cytotoxic and anti-tumour promoting activity, and can be used in the treatment of cancer. The aim of the present study was to identify chemical composition of the essential oil of P. mollis and to evaluate antimicrobial efficacy of the oil. The essential oil of the flowering aerial parts of P. mollis as obtained by hydro-distillation and analyzed by gas chromatography equipped with a flame ionization detector (GC-FID) and gas chromatography coupled with mass spectrometry (GC/MS). Twentyseven compounds were identified, which comprised 98.6% of the total constituents. The main compound was identified as fenchone (32.3%), followed by α-humulene (17.3%), piperitenone oxide (8.5%), cis-piperitone oxide (6.0%) and E-β-farnesene (5.9%). The oil was found rich in oxygenated monoterpenes type constituents (52.0%), followed by sesquiterpene hydrocarbons (40.2%), oxygenated sesquiterpenes (4.9%), and monoterpene hydrocarbons (1.5%). Antimicrobial activity of the essential oil of P. mollis was tested against six Gram-positive and eight Gram negative bacteria, and three fungi, by using the tube dilution method. The oil was active against the tested Gram-positive and Gram-negative bacteria, and fungi at a concentration range of 0.065±0.008-0.937±0.139mg/mL, 0.468±0.069-3.333±0.527 mg/mL and 0.117±0.0170.338±0.062mg/mL respectively. The present study revealed that the oil constituents somehow were qualitatively similar and quantitatively different than earlier reports from different parts of the world. The essential oil of P. mollis has found to be antimicrobial activity which can be usefulness in the treatment of various infectious diseases caused by bacteria and fungi.

O presente estudo relata o isolamento e caracterização de bactérias moderadamente halofilicas e alcalífilicas de um habitat salino no oeste da Índia. Oito cepas diferentes de bactérias foram isoladas empregando técnicas de enriquecimento em NaCl a 20% (p/v) e pH 10. As cepas apresentaram diversidade em relação à coloração de Gram e à morfologia das colônias e células. As cepas foram capazes de multiplicar e produzir protease alcalina em uma ampla faixa de concentração de NaCl (5 a 20%) e pH (8 a 10). Nenhuma das cepas foi capaz de se multiplicar em pH 7, e uma não se multiplicou nem em pH 8.0. Proteases naturais e parcialmente purificadas da cepa S5 foram submetidas à caracterização com relação ao pH, estabilidade salina, e estrutura protéica. Atividade e estabilidade ótimas da protease foram obtidas com 10% de sal e pH 9-9,5. A cinética de denaturação da protease de S5, juntamente com uma protease de referencia, foi avaliada com uréia 8M seguida de renaturação. A protease alcalina de S5 foi renaturada a 32% da atividade original. Apesar de provenientes do mesmo local, as oito cepas mostraram grande diversidade em relação à exigência de sal para multiplicação e secreção enzimática. Enquanto o efeito do pH na multiplicação foi menos marcante, o efeito na produção de protease foi significativamente afetada. A cepa S5 produziu uma quantidade substancial de protease alcalina e halotolerante. A atividade e estabilidade da protease alcalina em uma faixa mais ampla de pH e sal tornam essa enzima uma importante candidata para diversas aplicações industriais.

The present study deals with the isolation and characterization of the moderately halophilic-alkaliphilic bacteria from a saline habitat in western India. Eight different bacterial strains were isolated using enrichment techniques at 20% (w/v) NaCl and pH 10. The isolates exhibited diversity towards gram's reaction, colony and cell morphology. They were able to grow and produce alkaline protease over a broad range of NaCl, 5-20% (w/v) and pH, 8-10. None of the isolates could grow at pH 7, and one could not grow even at pH 8. Crude and partially purified proteases from strain S5 were subjected to characterization with reference to pH, salt stability and protein folding. Optimum protease activity and stability was recorded at 10% salt and pH 9-9.5. Denaturation kinetics of S5 alkaline protease along with a reference protease was studied at 8M urea followed by renaturation. The S5 alkaline protease could be partially renatured up to 32% of the original activity. Despite of the fact that all the 8 isolates were from the same site, they displayed significant diversity with respect to their salt requirement for growth and enzyme secretion. While the effect of pH was less demarcated on growth, the protease production was significantly affected. Isolate S5 produced substantial amount of halotolerant and alkaline protease. The activity and stability of the alkaline protease in a broader range of pH and salt would definitely make this enzyme an important candidate for various industrial applications.