Alexiteric activity of Costus pulverulentus C. Presl., Desmodium adscendens (Sw.) DC., Begonia glabra Aubl. and Equisetum bogotense on the poison of Bothrops asper (equis)

Obtaining plant samples

The plant samples were collected in areas of northwestern Pichincha (Table 1) and species were identified at the National Herbarium of Quito (Quito, Ecuador).

The parts used for the plant species, according to the traditional use of the inhabitants of the area, to be tested are as follows:

Preparation of extracts

Plant samples were dried in an oven at 37 degree C for 48 h, and then were ground to a particle size of 5 mm from which extracts were prepared by maceration with solvents of increasing polarity (n-hexane < alcohol < water). Hexane extract: 25 g of dried plant material was macerated with 350 mL of n-hexane at room temperature for 48 h. Then it was filtered and concentrated to dryness in rotary evaporator at 40 degree C and stored in an amber vial. The dry residue of this extraction was used to obtain the following alcoholic extract. Alcoholic extract: The dry residue from the previous extraction was macerated with 350 mL of absolute ethyl alcohol at room temperature for 48 h. Then it was filtered and concentrated to dryness in rotary evaporator at 40 degree C and stored in an amber vial. The dry residue of this extraction was used to obtain the following aqueous extract. Aqueous extract: The dry residue from the previous extraction was macerated with 350 mL of distilled water at room temperature for 48 h. Then it was filtered and concentrated to dryness in a water bath at 40 degree C and stored in an amber vial.

Obtaining the poison of B. asper

The sample of poison was obtained by manual milking of specimens in captivity (Figure 1), carried out by the staff of the Herpetological Foundation "Gustavo Orcés" (Vivarium of Quito, Quito, Ecuador). These specimens corresponded to three "pools" of B. asper venom from of the Ecuadorian coast, according to the specifications in Table 2.

Figure 1. Manual milking and samples of Bothrops asper specimens.

The pools of venom were stored in a vacuum desiccator protected from light and refrigerated at 4°C until the tests were carried out in the laboratories of the Life Sciences Area of the Universidad Politécnica Salesina, Campus Giron- Quito.

Determination of the Minimum Hemolytic Dose (MHD)

The technique described in 7 was followed. Briefly, on a Petri dish containing blood-phosphatidylcholine agar that was prepared by mixing 1% agar, 0.1 mm CaCl2, 0.33% egg yolk and 1.2% washed erythrocytes⁸, 15 µL of each dilution of venom, in physiological saline (4.76%, 2.44%, 1.23%, 0.62% v/v), was sown. The petri dishes were incubated for 20 h at 37°C. The halo of hemolysis was measured in mm. The lowest dose of venom that generated a 10 mm halo was taken as MHD.

Determination of the indirect antihemolytic activity of the extracts

The technique described in 8 was followed. Briefly, plant extract (1.6µg/mL) was pre-incubated with the MHD of the ophidian venom (ratios of 1: 7; 1:10; 1:20; poison: extract) for 30 minutes at 37°C. µL. 15 µL of this mixture was plated on a 6 mm-diameter petri plate containing blood-agar-phosphatidylcholine and incubated for 20 h at 37°C. The test was carried out in triplicate for each extract and for each concentration.

Statistical analysis

Analysis of variance was performed and after verifying significant differences, the Tukey test was applied using the Infostat/L program, version 11.1.2014, with a significance of 95%.

Minimum hemolytic dose (MHD)

Table 3 shows the values obtained for the minimum dose hemolytic (MIDH) poison of B. asper from different areas of the Ecuadorian coastal region. It is shown that pool V2, from Esmeraldas an Manabí province, presents greater hemolytic activity compared with V1 and V3, from the Northwest of the Pichincha and Bolivar provinces, respectively. These two pools have the same activity; however, must consider that the three poisons are mixtures obtained from specimen captive males and females, adults and young from each of the above locations.

Example photographs of the MHD can be seen in Supplementary File 1.

Indirect antihemolytic activity

Table 4 shows the results, assigning the sign (+), for the treatment that reduced the diameter of hemolysis produced by the MHD of each pool by 100%. The alcoholic extract of E. bogotense has very good inhibitory activity of the hemolytic effect produced by the venom of B. asper, which has also been shown in previous research on Bothrops atrox (Yarlequé amp; et al., 2012). However, in the case of B. glabra, its aqueous extract as well as its alcoholic extract is also effective. This shows that this plant is the most effective species of all tested.

The alcoholic extracts of E. bogotense and D. adscendens present antihemolytic activity in all treatments and for the three pools of venom, which shows they are the most active species against the hemolytic effect of Bothrops asper venom. Neither the aqueous nor the hexane extract of these two species demonstrate this activity.