Aloe barbadensis Miller leaf exudate is a potential treatment for bovine mastitis

Sampling and extraction of leaf exudate

A total of 30 plant samples were collected from 3-year-old Aloe vera (Aloe barbadensis) at random from a commercial grower (Naturama Sucos Integrais do Brasil Ltda®; Paulo Lopes, SC, Brazil) in March, June, September and December of 2015, and one leaf was taken from the mid-position of each plant. Thus, 30 leaves in total were collected for each month, representing each season. Leaves were cut at the base and maintained vertically for 3 h in a beaker to collect the LE at room temperature. Subsequently, the LE was lyophilized and stored at -20 °C. LEs of six plants were combined for a total of five repetitions for each season of the year.

Chemical profile of leaf exudate

Total phenolics. The total phenolic content was determined using the colorimetric method of Folin-Ciocalteau¹⁴ and an external standard curve of gallic acid (10–100 μg/mL) (y= 0.0197x / r²= 0.987). The results were expressed in µg of gallic acid equivalents (GAE)/mg of extract (µg of GAE/mg). All tests were performed in triplicate.

Aloin. The aloin content in LE was obtained on an UHPLC Thermo Scientific UltiMate 3000 RS Dual System (Thermo Fisher Scientific, San Jose, CA), using a Thermo Scientific C18 reverse-phase column (4.6 × 250 mm; 5 µm; 120Å (Acclaim^TM120, Thermo Scientific©) at 40°C, operating at 240, 260, 280, and 320 nm. The mobile phase was eluted at 1 mL/min flow rate, using a methanol/water (70/30) mixture²². The identification of aloin was based on a comparison of the chromatographic profile and retention time with the commercial standard (Sigma-Aldrich, St. Louis, MO, USA/ B6906). After the addition of the standard, samples were co-chromatographed to confirm identification of the compound. Aloin content was determined through an external standard curve of barbaloin (y= 302.73x / r²= 0.9822) and the result expressed in µg of aloin per mg of the sample (µg/mg).

Antimicrobial activity

Antimicrobial activity was evaluated using a broth microdilution method according to the Clinical and Laboratory Standards Institute Manual²³. We tested six different concentrations of LE (4000 to 125 µg/mL) against the standard strain of S. aureus ATCC 25923 (Collection of Reference Microorganisms on Health Surveillance, Fundação Oswaldo Cruz, Fiocruz, Brazil) and seven strains of mastitic milk isolates. Milk samples from cows were submitted to the California Mastitis Test (CMT). CMT-positive milk samples were plated on blood agar supplemented with 5% sterile ovine blood and incubated for 24–48 h at 37 °C. Gram-positive, catalase-positive, and rabbit plasma coagulase-positive samples were biochemically confirmed as Staphylococcus aureus²⁴. Each strain was considered one repetition of the experiment with five replicates/repetition. As such, we conducted eight repetitions for each LE sample.

The minimum inhibitory concentration (MIC) was determined through visual analysis of turbidity after 24 h of incubation on plates at 37 °C in addition to spectrophotometric reading at 600 nm to determine the percentage of inhibition of bacterial growth, using a previously described method²³.

Because LE is an exudate with a yellow color that easily oxidizes to a dark coloration¹⁴, we confirmed the MIC through a colorimetric method. We added 50 µL of resazurin dye (100 µg/mL, Sigma-Aldrich, St. Louis, MO, USA) to each well after reading the plates by spectrophotometer (600 nm). The plates were left to incubate at 37 °C for an additional 30 min²⁵.

Cytotoxicity of leaf exudate to MAC-T cells

Mammary epithelial cells of the MAC-T (Mammary Alveolar Cells-T) lineage were maintained in culture, as indicated by the supplier (Banco de Células do Rio de Janeiro, Brazil). Briefly, MAC-T cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) and supplemented with 100 U/mL of penicillin, 100 µg/mL of streptomycin, 20% (V/V) heat-inactivated fetal bovine serum (FBS, Gibco, CA, USA), 4 mmol L^-1-glutamine (Synth), 4.5 g/L glucose (Sigma-Aldrich, St. Louis, MO, USA), 1 mmol of sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), 1.5 g/L sodium bicarbonate (Vetec^TM Sigma-Aldrich, St. Louis, MO, USA), 5 μg/mL insulin (Sigma-Aldrich, St. Louis, MO, USA), and 1 μg/mL hydrocortisone (Sigma –Aldrich, St. Louis, MO, USA) at 37 °C and 5% CO₂ in a humidified incubator. We changed the medium every 48 h. After reaching confluence, the cells were treated with 0.25% trypsin with 1 mmol EDTA (Gibco, CA, USA) to prepare the cellular suspension (10⁵ cells/mL). The suspension was transferred a 96-well microplate (100 µL/well), followed by incubation (24 h) in culture conditions for adherence. Subsequently, varying concentrations (2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8 and 3.9 µg/mL) of LE from the summer samples were added for 24 h, and cytotoxicity was determined based on the MTT method (Sigma-Aldrich, St. Louis, MO, USA)²⁶. The formed formazan was dissolved with dimethyl sulfoxide (DMSO) to give a purple color with characteristic absorption at 540 nm. Intensity of purple color is directly proportional to the cell number, thus indicating cell viability. The experiments were performed in triplicate.

Statistical analysis

Data were expressed as the mean ± standard deviation (SD), of at least three independent experiments. We analyzed the data using analysis of variance with a Tukey adjustment (GraphPad Prism 5.0). We considered the effects statistically significant for P<0.05. The inhibitory concentrations capable of reducing cellular viability by 50% (IC₅₀) were calculated using a nonlinear regression of data obtained from the cellular viability tests with GraphPad Prism 5.0 software. The average accumulated precipitation (mm) in the study region was calculated using available data²⁷.

Leaf exudate chemical profile

The total phenolic and aloin content of LE from the aloe vera leaves varied based on the season during which the samples were collected. The highest levels were found in the samples taken during the summer and the lowest levels from the spring samples (P<0.05). The accumulation of total phenolics in the summer LE seems to be associated with the climatic conditions during the collection period (Figure 2a, b). Precipitation indices were lower during the summer months (January to March)²⁷ (Figure 2a), possibly causing hydric stress in the plants. In the spring, the lower total phenolic content of LE coincides with a period of greatest precipitation that year. Aloe is a plant comprised of 96% water; thus, its chemical composition is heavily influenced by precipitation levels²⁸, as well as other factors, such as the period of flowering²⁹.

Figure 2. Seasonal differences in rainfall, phenolic content and aloin content.

(A) Average accumulated precipitation (mm) in the study region (Paulo Lopes, SC, Brazil) during 2015; Source: INMET²⁶. (B) Total phenolic content (μg GAE/mg) of Aloe vera leaf LE from different seasons of the year (average of five repetitions ±SD) (P<0.05). (C) Average content (µg/mg) of aloin (average of three independent injections ± SD) in samples of LE collected from Aloe vera leaves during different seasons of the year. Data points with the same letter above them are not significantly different from each other (P<0.05 indicates a significant difference).

The husk of Aloe leaves has greater levels of total phenolics compared to the leaf interior and internal parenchyma. In the literature, these values range from 12.06 to 20.86 µg GAE/mg leaf, depending on the species¹⁴. Among the various phenolic compounds in the leaves of Aloe, anthraquinones are noteworthy, particularly aloin. Anthraquinones are free in phloem vessels directly below the leaf epidermis, and aloin, in particular, is distributed throughout the plant as part of its defense mechanism³⁰. In the present study, we found the highest levels of aloin in the summer samples and the lowest in the spring samples (Figure 2 b, c). These results are correlated with the total phenolic content found in the studied samples (Figure 2). Previous studies have also suggested the effect of seasonality on aloin content, and its synthesis is strongly influenced by precipitation levels. Dry periods have been correlated with greater content of aloin in the analysis of aloe vera leaves^15,31,32. However, other factors can influence aloin content of aloe vera leaves, including cultivation conditions, age, and plant health³³. For example, higher levels of barbaloin, isobarbaloin, and aloin in Aloe sp. plants were found during periods of the year with higher temperatures³².

Antimicrobial activity

Despite significant differences in the levels of total phenolics and aloin in the LE samples (Figure 2b, c), these levels did not influence antimicrobial activity against S. aureus. For all LE samples, the MIC was 1,000 µg/mL, as confirmed by resazurin oxidation. The concentrations below of 500 µg/mL were incapable of reducing bacterial growth to values greater than 70%. The effect of other concentrations between 500 and 1,000 µg/mL was not included in the study (Figure 3).

Figure 3. Percentage of inhibition (mean ± SD) of the standard strain of S. aureus ATCC (25923) and seven strains of mastitic milk isolates by different concentrations of LE samples taken during different seasons of 2015.

The effectiveness of LE from aloe vera leaves as an antimicrobial agent has been demonstrated for a wide variety of Gram-positive and Gram-negative bacteria, including S. aureus and others^34,35. In the literature, the MIC of aloe vera extracts against S. aureus varies. Previous studies have shown lower (195 µg/mL), similar (1560 µg/mL), and higher (5,000 µg/mL) MIC values compared to those in the present study^36–38. This variation may be related to diverse factors, such as the Aloe species studied, the part of the aloe leaf used in the tests, and the type of extraction and resuspension vehicle used. In the current study, the LE samples were collected directly from the cut leaf without any type of posterior extraction of the compounds of interest. Some solvents are capable of extracting certain compounds that may possess greater antimicrobial activity than others³⁹; however, resuspension in water may be the easiest way to use aloe vera leaf subproducts, making it accessible, even to the producer.

An interesting aspect to consider in the present study is that the concentration of total phenolics and aloin in the LE samples does not seem to affect antimicrobial activity. By contrast³⁶, the antimicrobial and anti-inflammatory activity of aloe vera LE has been associated with the concentration of phenolic and aloin compounds, suggesting that older leaves have higher levels of these compounds, and as such, have greater biological activity and defense against microorganisms and herbivores.

For Fabry et al.⁴⁰, the potentially useful activity defined for crude plant extracts with organic solvents is considered good when MIC values are <8000 µg/mL, while Gibbons⁴¹ suggests that phytochemical isolates must have MIC values <1,000 µg/mL. As such, the antimicrobial action of the LE samples in the present study can be considered good, even though neither extraction nor isolation of the principal components took place.

Cytotoxicity of leaf exudate

The LE showed high toxicity to MAC-T lineage cells, causing significant reduction in cellular viability at concentrations greater than 7.8 μg/mL (Figure 4). At higher concentrations, such as 500 μg/mL, the reduction in the percentage of viable cells was greater than 80%. The IC₅₀ was 91.89 μg/mL. It is worth noting that the MIC of S. aureus growth was 1,000 μg/mL (Figure 3), a concentration that had a strong effect on the viability of mammary epithelial cells (Figure 4). This result is significant because it suggests that caution must be exercised when considering the intramammary use of LE in order to avoid inflammation, owing to the death of epithelial cells. However, future studies are needed with aloin purified to determine the inhibition of bacterial growth and the degree of toxicity on mammary cells.

Figure 4. Percent of cellular viability (mean ± SD) of the MAC-T lineage after exposure to different concentrations of LE samples in the summer of 2015.

Data shown are an average of three independent experiments. Data points with the same letter above them are not significantly different from each other (P<0.05 indicates a significant difference).

In an in vivo situation, the administration of a toxic product to bovine mammary glands can result in the development of inflammation⁴², which is more severe than that caused by the infection of pathogens⁴². In these cases, the attempt to combat inflammation leads to the formation of connective tissue at the affected site, which can diminish the alveolar area responsible for the synthesis of milk and, consequently, reduce milk production. In more severe cases, the loss of complete mammary glandular function, or even death of the animal, can occur^43,44.

The MAC-T cellular lineage⁴⁵ is an established model that has been frequently used in the investigation of mammary glandular functions and mediators of inflammatory processes⁴⁶. Nonetheless, studies reporting on the effects of Aloe sp. extract, or fractions on this type of cell, are scarce. The toxic effects of aloe vera LE on other types of cells are discussed in the literature and have been associated with the presence of aloin and aloe emodin⁴⁷. These anthraquinones induce the apoptosis of cells caused by a reduction in the proportion of cells in the mitotic phase⁴⁸. Another hypothesis is that disruptions to the cell cycle and cellular differentiation, stimulation of the immune system, and antioxidant activity also have an anti-proliferative effect⁴⁹.

While the results found for MAC-T cells show that aloe vera LE has a high toxic potential for bovine mammary glands, the topical use of this product on the external area of the udder, for example pre- and post-dipping, or on instruments used during the management of milking, can be suggested. In this case, its potential as a disinfectant should be investigated, and cytotoxicity tests on epithelial cells will be performed.

Furthermore, it is important to highlight that the compounds present in the Aloe vera LE liquid oxidize easily in the presence of light, oxygen, and at room temperature⁵⁰. As such, very high concentrations are required in order to achieve antimicrobial efficacy against S. aureus, concentrations that would be toxic to mammary epithelial cells. Thus, we suggest the standardization of a methodology that can preserve and conserve these oxidative compounds, such as nanoencapsulation, which can maintain the desired antimicrobial activity and diminish the toxic effects.