Vegetal material. - Fresh B. megalandra leaves were collected at the Campus of the Universidad Central de Venezuela during May 2012, the plant was identified by Dr. S. Tillett and a specimen was deposited at the Herbarium “Manuel Ovalles” Faculty of Pharmacy of the Universidad Central de Venezuela under the number: MYF29251. The washed and dried leaves were cut into small pieces, 5 ml of distilled water were added per g, and the whole brought to the boil, cooled, then filtered and stored at -20º C until their use.

Animals and their treatment. - Sprague Dawley male rats of 200 ± 20 g were used, they were divided into 2 groups and kept in metabolic cages; the control group having free access to water and food and the experimental group receiving, during 7 consecutive days, the B. megalandra leaves extract, prepared as described above, in place of water, and received the same type of food as the control group. The urine of both groups of rats was collected, during the same 7 consecutive days, and stored at – 20º C until use.

Urine treatment. – Following Zhao et al. ⁽⁹⁾, the urines of both groups of animals were acidified with HCl (0.274 M) and 5 volumes of ice-cold acetone were added; later they were centrifuged at 1000 g for 15 min. at 4º C, the resulting precipitate was discarded and the supernatant after drying was stored at -20º C. An aliquot of the plant extract was treated with the same procedure.

The solid material obtained from the supernatant was fractionated using: chloroform, dichloromethane, acetone, ethanol, methanol and water respectively. For that, the solid material was resuspended in 100 ml of the solvent, sonicated (MSE, England) centrifuged at 2500 g for 15 min. at 4º C, the supernatant corresponding to each solvent fraction being dried and the precipitate treated with the next solvent using the same procedure. Each solvent fraction was dried using a rotatory evaporator Yamamoto (Japan) or a lyophilizer Virtis (USA). The solvent fractions were analyzed using TLC on silica gel 60 with fluorescence indicator UV₂₅₄, the mobile phase was dichloromethane-methanol (8:2; v/v), once the plates were dried, they were observed under a UV/Vis lamp or developed with ceric sulphate.

The acetone fractions of the urine of the experimental rats, showed a spot under TLC that was not present in the urine of control animals nor in the B. megalandra leaves extract (see results): in consequence we decided to fractionate it.

Treatment of the acetone fraction. – When the dry acetone fraction was resuspended in 15 ml of acetone a precipitate was formed which was discarded by centrifugation as above, due to the fact that the compound of interest was in the soluble fraction. When this fraction was dry some crystals were formed, extracted with hot acetone (45º C) and separated by filtration, again the compound of interest was in the soluble fraction which was dried at ambient temperature.

The last fraction of the above procedure was resuspended in a small volume of methanol, mixed with a small amount of silica gel, dried at room temperature and seeded on top of a dry silica gel chromatographic column and the elution was performed using dichloromethane-methanol (8:2; v/v) and 26 fractions of 2 ml were obtained. By TLC it was shown that the compound of interest was in fractions 17-23 which were combined, dried at room temperature, resuspended in dichloromethane, centrifuged as above and the precipitate washed with acetone in the same way. The precipitate of acetone was resuspended in methanol, centrifuged as above and the compound of interest was in the soluble fraction, which by concentration at room temperature yielded a yellow-brown solid. This solid was resuspended in 5 ml of methanol, centrifuged as above, the precipitate was discarded and the supernatant constituted the Fr_17-23, fraction enriched with the compound of interest and mixed with urea.

Spectroscopic methods. – The ¹H and ¹³C NMR of Fr_17-23 were carried out using a JEOL spectrometer, Eclipse 270 model with an application camp of 270 MHz for ¹H and 67.5 MHz for ¹³C and a spectrometer Bruker ¹H (500 MHz) and ¹³C (125 MHz).

Changes in glycaemia and glycosuria during the administration of the B. megalandra leaf extract. - To a control group of animals and other group of treated rats similar to those used in the previous experiment (b) food was withdrawn for 24 h., the urine was collected during that time and a sample of the tail blood was taken. In the urine and blood the glucose was measured by the glucose oxidase-peroxidase method ⁽¹⁰⁾.

g. All experiments were carried out following the ethical criteria for experimental animals of the Institute of Experimental Medicine of the Medicine Faculty of the Venezuela Central University.