High speed centrifugal countercurrent chromatography (HSCCC) was successfully applied to preparative isolation of the main polar phenolics from the Peruvian Caricaceae Vasconcellea quercifolia A. St.-Hil. High-performance liquid chromatography with diode array (HPLC-DAD) and electrospray ionisation mass spectrometric detection (ESI-MS) was used to carry out a comprehensive characterisation of phenolic compounds from fruits and leaves of this plant. The main phenolics were the quercetin diglycoside rutin and the triglycoside manghaslin. Nine minor flavonoids were tentatively identified as kaempferol, quercetin, isorhamnetin and methoxymyricetin glycosides. HSCCC combined with other chromatographic methods allowed the isolation of the main phenolics from the plant. HPLC-DAD-MS analysis shows that the main phenolic compounds in V. quercifolia are similar to those described for V. pubescens but differs in the identity of the minor constituents.

Bothrops bilineatus is a tree snake whose bite causes many accidents in peruvian jungle. Using whole venom, we made an exploration of its coagulant activity using citrated plasma and commercial fibrinogen, as well as different chromogenic substrates. In this way, we assayed coagulation time on human citrated plasma and bovine fibrinogen 5 mg/ml in Tris-HCl buffer 0,05 M pH 7,4, and also amidolitic and esterasic activities on BApNA, TAME y BAEE, respectively. On the other hand, this venom was fractionated in a size exclusion chromatography using Sephadex G-100 in ammonium acetate buffer 0,05 M pH 6,0, measuring coagulant and amidolitic activities on each fraction. Furthermore, we obtained electrophoretic profiles using SDS-PAGEgels.As a result of this study,we found B. bilineatus venom has activity on plasma (0,59 U/mg) and fibrinogen (0,17 U/mg), being these values 60% lower in compare with the most coagulant venom studied in Peru ( ). Also, fractions obtained with coagulant activity were associated with amidolitic and esterasic activities. Chromatographic profiles showed three main peaks eluted with Ve/Vo of 1,1, 1,5 and 2,4, respectively, while electropherograms obtained, at least 10 protein bands between 15 and 70 kDa.

Bothrops bilineatus es una serpiente arboricola causante de numerosos accidentes humanos en la amazonia peruana. A partir del veneno completo se ha realizado una exploracion de la actividad coagulante empleando plasma citratado y fibrinogeno comercial, asi como diversos sustratos cromogenicos. Para ello, se han realizado ensayos de tiempo de coagulacion con plasma humano citratado y fibrinogeno bovino 5 mg/ml en buffer Tris-HCl 0,05 M pH 7,4, midiendose tambien la actividad amidolitica sobre BApNA y esterasica sobre TAME y BAEE. Adicionalmente, el veneno fue fraccionado en una columna de filtracion molecular de Sephadex G-100 con buffer acetato de amonio 0,05 M pH 6,0, analizando la actividad enzimatica en cada una de las fracciones. Ademas, se ha obtenido el perfil electroforetico mediante analisis de PAGE-SDS. Como resultado de este estudio se ha determinado que el veneno total de posee una actividad de 0,59 U/mg sobre plasma y 0,17 U/mg sobre fibrinogeno, siendo estos valores 60% menores en comparacion con el veneno ofidico mas coagulante estudiado en el Peru (Lachesis muta ). Asi tambien, las fracciones con actividad coagulante estan asociadas a la actividad amidolitica y esterasica. El perfil cromatografico obtenido corresponde a tres picos proteicos eluidos con valores de Ve/Vo de 1,1, 1,5 y 2,4, mientras que los electroferogramas obtenidos muestran al menos 10 bandas proteicas entre los 15 y 70 kDa.

We have determined some biochemical properties of a thrombin-like enzyme (TLE) isolated from Bothrops atroxperuvian snake venom "jergon". In this concern, TLE was purified until homogeneity using three chromatographical steps: on Sephadex G-75, CM-Sephadex C-50 and Agarose-PAB. Furthermore, molecular weight was determinate by PAGE-SDS and associated carbohydrates by hydrolysis and analysis of hexoses, hexosamines and sialic acid. Then, fibrinocoagulant, amidolytic and sterasic activities were measured on bovine fibrinogen, BApNAand BAEE, respectively.As a result of these analyses, we determined that this enzyme represents 1,7% of total venom and was 25,5-fold purified with a 43,3% yield,using BApNA as substrate. This enzyme had 29,6 kDa, where 14,2% was associated carbohydrates. The TLE of B. atrox produced coagulation of bovine fibrinogen and had enzymatic activity on BAEE and BApNA. In conclusion, we have purified and characterized the main component of B. atrox venom related to its coagulant activity.

Se ha determinado algunas propiedades bioquímicas de la enzima similar a trombina (EST) de la serpiente peruana "jergón". Para este fin, la enzima fue purificada utilizando tres pasos cromatrográficos: Sephadex G-75, CM-Sephadex C-50 y Agarosa-PAB. Asimismo, se determino el peso molecular por PAGE-SDS y el porcentaje de carbohidratos asociados mediante hidrolisis y análisis de hexosas, hexosaminas y acido siálico. Se ensayaron las actividades fibrinocoagulante, amidolìtica y esterásica, sobre fibrinógeno bovino, BApNA y BAEE, respectivamente. Como resultado del análisis se determino que la enzima constituye el 1,7% del veneno completo, siendo purificada 25,5 veces y con un rendimiento del 43,3% utilizando BApNA como sustrato. La enzima presenta un peso molecular de 29,6 kDa, del cual el 14,2% lo constituyen los carbohidratos asociados. La EST de B. atrox produjo coagulación del fibrinógeno bovino y presento actividad sobre BAEE y BApNA. De esta manera se ha logrado purificar y caracterizar al principal componente del veneno de B. atrox relacionado con su actividad coagulante.

Micrurus spixii venom was studied after fractionation by Sephadex G-100 SF gel filtration chromatography. Several enzymatic activities and biological effects were investigated in whole venom and fractions. The venom was resolved in four peaks in a range of about 73.2-10.7 kDa molecular weight. Alkaline phosphatase and acetylcholinesterase activities were found in peak I, and procoagulant activity was seen in peak II. Phospholipase A2, hemorrhagic, and proteolytic activities were detected in peak III. A second procoagulant factor and proteinase were present in peak IV. Thrombin-like enzyme and direct hemolytic activities were not found in any assayed samples.