The antimicrobial activity of alcoholic extracts of leaves and bark from two varieties of ”Sangre de Drago” compared with the antimicrobial activity present in the latex of the same species

Botanical material

The vegetal material used from the two species of Sangre de Drago was collected in the localities of Talag and Canelos belonging to the provinces of Napo and Pastaza, respectively, and located at 1°, 3’, 57” south latitude; 77°, 54’, 26” west longitude; 40° west longitude; and 1°, 35’, 22” south latitude; 77°, 44’, 48” west longitude; 40° west longitude, respectively. The sample collection was permitted by the Ministerio del Medio Ambiente, Ecuador (MAE), (registration number: 2015-RO. Nro. 449 and 905-RO.- suplemento 553). Aerial parts (leaves and bark) and latex of the trees aged 3 to 4 years and in a flowering-fructification stage were used. The crude latex for the analysis was obtained from indigenous merchants in the areas. One of the two latex samples corresponding to the Canelos sector was extracted from the same tree from which the plant material was collected. The collecting folders for each species were prepared and sent to the National Herbarium of Ecuador (QCNE) for taxonomic identification. The folder regarding the material collected in the Province of Napo was identified as C. lechleri Muller Arg. and the one corresponding to the province of Pastaza as C. urucurana Baill. The aerial parts (leaves and bark) of the two species were dried at 49°C, in a JP SELECTA series 0385081 stove with circulating air for 3 days. The plant material was removed at least once a day for uniform drying until a constant weight was achieved. Next, the parts were crushed in a hammer mill until a moderately thick powder was obtained. The batches of the plant material were identified for the corresponding analyses with the letters “l” and “b” for leaves and bark, respectively, accompanied by the letters T and C to identify provenance from Talag and Canelos, respectively. The latex samples were refrigerated at 4°C for 2 weeks, during which time their analysis began from their acquisition. They were coded with the letters “T” and “C” and the letter “C” was accompanied with numbers “1” and “2” for the latex provided by the indigenous merchants and for the latex obtained from the same tree as the vegetal material was collected, respectively.

Physical-chemical evaluation

The extraction of the alcoholic extracts (20 % Tincture) of the leaves, bark and latex from the two Croton species was carried out by maceration using 70% alcohol as a menstruum, at room temperature, for 2 to 7 days, with shaking at least twice a day. A 20% tincture was obtained, from which the physical and chemical parameters were determined as indicated by the Ecuadorian Quality Control Standard for natural medicinal products. Under the same conditions, the physical-chemical constants of the crude latex of Sangre de Drago samples were determined. For the determination of the refractive index, a Clean-Prison series 003021 ABBE refractometer was used. pH determination was carried out in a Metrohm potentiometer, type 1.744.0010.

Chromatography analysis

The fractionation of the analysed alcoholic extracts was achieved by thin-layer chromatography, tapping 0.1 ml of the alcoholic extract by means of a glass capillary 1 cm from the bottom edge of 60GF254 silica gel plates. It was allowed to dry for a moment before each application and at the end of it. Then the plates were placed in saturated glass chambers for at least 30 minutes with steam from the components of each of the mobile phases used, which are listed below as ratios:

• Butanol-acetic acid-water (BAW): 4-1-5

• Chloroform-acetone-diethylamine: 7-3-1

• Toluene-ethyl acetate: 70–30

The following were used as developers: 254 nm and 366 nm ultraviolet light, Dragendorff reagent, ammonia fumes. Each chromatography experiment was repeated twice.

Microbiological quality

The microbiological quality of the alcoholic extracts of leaves and barks, as with crude latex, was verified as outlined by the World Health Organization (WHO)¹⁰. The tally of bacteria, fungi and yeast was read after 5 days of incubation as indicated by the WHO. Regarding the reading for the determination of pathogenic bacteria, this was performed at 24 and 48 h of incubation with two repeats. The results of bacterial growth were compared with that described by Porter and Kaplan¹¹ for each culture medium used, for the purpose of bacterial recognition.

Evaluation of antibacterial and antifungal Activity

The evaluation of the antimicrobial activity was carried out against six bacterial strains (Escherichia. coli, S. aureus, Pseudomonas aeruginosa, S. epidermidis, Bacillus subtilis and Streptococcus sp.) maintained in nutritious agar at 4°C according to Garcia and Uruburu¹². The antifungal activity was not assessed since growth problems were encountered for the strain Candida albicans ATCC10231. The method used was described in the “CYTED” Research Techniques Manual⁹. The extracts were solubilized using dimethylsulfoxide (DMSO) to their respective dilutions (5000, 2500, 1250, 1000, 500, 250 and 125 p.p.m.). Gentamicin sulfate (0.1 mg/0.1 ml) was used as a standard antibiotic and as a growth inhibition solution. As a negative control, Muller Hilton Agar and Muller Hilton Agar boxes with DMSO (0.1 ml) were prepared.

Physical and chemical constants

Table 1 shows the results of density, total solids, pH and refractive index of the alcoholic extracts of the bark, leaves and latex of the two Sangre de Drago varieties analyzed.

As can be seen, there is no marked difference between the values of density, total solids and pH of the different extracts analysed. The organoleptic characteristics, on the other hand, differ clearly between the extracts of the two Croton species. In the same way, the capillary analysis shows a clear differentiation between the extracts and basically between the latex extracts (Figure 1). The presence of a fluorescent blue colour on the fringe of the capillary analysis image was characteristic in all the analysed extracts. When the wet paper strip was exposed to ammonia fumes, revealed with 366 nm UV light and the environment left to dry, a weaker blue color was observed on the paper strips with 366 nm UV light. According to the consulted literature¹³, these results are characteristic of flavonoid-anthocyanidin type metabolites.

Figure 1. Capillary analysis of alcoholic extracts of leaves, bark and latex of Croton lechleri and Croton urucurana.

Table 2 shows the values of the physical and chemical parameters of the crude latex of the two varieties of Sangre de Drago analysed. As can be seen, the organoleptic characteristics allow the two varieties to be clearly differentiated. In terms of density parameters, total solids and pH, the results vary for each species. The refractive index cannot be determined by the physical characteristics of the samples (colour and viscous consistency).

Chromatographic analysis

Figure 2 presents the results of the thin layer chromatography, RF of the spots and colours visualized with the developers: 366 nm UV light, 254 nm UV light, ammonia fumes, Drangendorf reagent, alcoholic extracts of leaves, bark and latex.

Figure 2. Chromatograms obtained for the alcoholic extract of the plant material and latex of the two Sangre de Drago species analysed in the following systems: 1) chloroform-acetone-diethylamine 70:20:10; 2) BAW 4:1:5 and 3) toluene ethylacetate 70:30.

As can be seen, the separation of the respective fractions occurs with greater clarity in the plates run in the mobile phases of BAW and 70–30 toluene-ethyl acetate. The different fractions obtained are observed as yellow at the point of application and faint orange to the naked eye during the course of the shift.

When revealed with 366 nm UV light, fluorescent orange spots are observed at different RF. When revealed with 254 nm UV light, faint grey spots are observed. The plates were passed through ammonia fumes and observed under 366nm UV light, where it was observed that the orange-coloured dots turned fluorescent blue. The phases were run in chloroform-acetone-diethylamine (7-2-1) mobile phase. Under natural light, no stain can be seen, but under the 366nm UV light, reddish spots are observed at a 0.48 RF and under 254 nm UV light, blue spots are observed. The RF of the calculated spots is closer to that reported in the literature for the alkaloid taspine (Theoretical RF of 0.50). When revealing the spots with Drangendorf’s reagent, a faint brown color was observed that disappeared after a few seconds. The presence of orange spots that turn blue in the presence of ammonia is characteristic of flavonoid-type phenolic compounds, and within these the anthocyanidins group, which are components present in Sangre de Drago¹⁴.

Microbiological control

The results obtained from the count of bacteria, fungi and yeasts of the alcoholic extracts of leaves, bark and crude latex in colony forming units/ml of sample are found in Table 3.

As can be observed, the values corresponding to the microbiological count of bacteria of the alcoholic extracts are within those established for crude drugs (see Table 4). The other two samples of latex did not present bacterial growth. The count of moulds and yeasts presents similar results to the bacterial count in the alcoholic extracts. The same cannot be said for the crude latex, because on the fifth day, the three analysed samples presented massive growth, which did not allow for the counting of the colony forming units. The presence of pathogenic batteries was negative for the alcoholic extracts and for the latex samples from “C1“ Talag and Canelos. This was not so for the “C2“ Canelos latex sample, which showed growth of pink colonies, characteristic of E. coli in red Violet Agar. The abundant bacterial growth with a metallic luster in Mac Conkey Agar of the planting in lines of characteristic colonies in Red Violet Agar confirmed the presence of E. coli. It was impossible to count the colony forming units, as they surpassed what was established by the WHO for this type of sample. All samples were negative for bacterial growth in Cetrimide Agar specific for P. aeruginosa and in Baird Parker Agar for S. aureus.

Antimicrobial activity

For the determination of the antibacterial activity by means of the method described in the CYTE Research Techniques Manual⁹, a battery of six bacteria was used (E. coli, S. aureus, P. aeruginosa, S. epidermidis, B. subtilis and Streptococcus sp.). The preliminary results of each of the bioassays performed are presented in Table 5, in which the moderate antimicrobial activity (±) for B. subtilis can be observed in the seven samples analysed (extracts and crude latex). For the remaining five bacteria, the antibacterial activity was negative (-) for the alcoholic extracts. The Sangre de Drago latex coded “T“ and “C1“ were active (+) for S. epidermidis at doses of 2500 p.p.m. and 5000 p.p.m. and the “C2“ latex is active (+) at a dose of 1250 p.p.m. For the four remaining bacteria, they did not exhibit antimicrobial activity. The negative activity (-) for E. coli does not agree with previous reports^15–17. Regarding B. subtilis, it could be said that it is similar to results from other authors’ studies. The literature does not present information relating to activity on S. epidermidis. As for fungal activity, this test was not performed due to growth problems that arose with the C. albicans strain.