Aloe barbadensis Miller leaf exudate is a potential treatment for bovine mastitis [version 1; peer review: 1 approved, 1 not approved]

Background: Aloe barbadensis Miller is a well-known phytotherapeutic, and parts of its leaves are used for a wide range of medicinal purposes. This study seeks to assess the in vitro antimicrobial and cytotoxic effects of leaf exudate (LE) from A. barbadensis leaves against Staphylococcus aureus and MAC-T bovine mammary epithelial cells. Methods: Seasonal LE samples were collected, and the effect on total phenolic and aloin contents was determined. Antimicrobial activity of LE was evaluated using the broth microdilution method, and toxicity to MAC-T cells was determined by MTT assay. Results: Samples collected during different seasons of the year showed a seasonal effect on the chemical profile of LE (P<0.05). However, despite these chemical variations, we found no differences in antimicrobial activity against S. aureus. For all studied samples, the minimum inhibitory concentration (MIC) was 1,000 µg/ml. Furthermore, we found an elevated cytotoxic effect of LE on MAC-T cells with a significant reduction in cellular viability at 7.8 µg/ml (P<0.05) and an IC50 of 91.89 µg/ml. Conclusions: Despite the antimicrobial effects of LE, the high toxicity for MAC-T cells suggests that it is unsuitable for intramammary use, but does have potential as a topical antimicrobial.


Introduction
Bovine mastitis, which is characterized by inflammation of the mammary gland, is the most frequent infection found in dairy herds worldwide 1 .The treatment recommended for mastitis is the administration of intramammary antimicrobials.However, control of infections caused by Staphylococcus aureus, the principal etiological agent of bovine mastitis 2 , is very difficult.In addition to inactivating several antimicrobials, this microorganism can also survive in the intracellular environment after phagocytosis.As a consequence, the cure rate of mastitis caused by S. aureus is low, with a high incidence of recurrence 3 .As such, interest in the search for methods of control and prevention has increased, including the identification of new antimicrobials [4][5][6][7] .
In vivo methods are still commonly used to study bovine mastitis, but in vitro testing has been recommended 8 .Based on in vitro models, studies have produced a wide range of results, from identifying the prevalence of etiological agents of mastitis to evaluating the direct effects of products on the susceptibility of studied microorganisms 9,10 .Among these, in vitro tests on antimicrobials are some of the most widely used 8 .In vitro studies with bovine mammary gland explants or mammary epithelial cells (MEC) are commonly used to assess the different functions of mammary glands, such as the response to initial infection 11,12 .Recently, primary cultures of mammary explants and MECs were also suggested as adequate models in the search for new therapeutic agents 5,13 .In the case of mastitis, such in vitro methods can help evaluate the toxicity of antimicrobials, enabling the determination of safe doses and minimizing the potential risks during in vivo validation.
Aloe vera (Aloe barbadensis Miller) is a plant widely used and recognized for its antimicrobial, anti-inflammatory, woundhealing, antitumor, and antioxidant pharmacological properties 14 .Yet, until now, few studies have reported on its potential as a treatment for bovine mastitis.Most research on the pharmaceutical potential of Aloe vera has studied the mucilaginous gel, commonly known as aloe vera gel, that is rich in complex carbohydrates, particularly acemannans 15,16 .However, along with the gel, a yellow exudate with a strong odor and bitter taste, known as leaf exudate (LE), can also be extracted from the leaves 17 .Its release occurs as soon as the leaves are cut and it can be found within the phloem vessels 18,19 .Despite being composed of large amounts of 1,8-dihydroxyanthraquinone derivatives and their glycosides, the industry that uses aloe vera gel as a raw material considers LE a residue.Among the anthraquinones found in LE, the major compounds are a mixture of two readily oxidizable diastereomers, aloin A and aloin B, which are sometimes undesirable because of their toxic and cathartic potential.However, these compounds may be of therapeutic interest in the control of antimicrobial and tumor cell proliferation 20,21 .
Thus, the current study seeks to investigate the potential of LE from Aloe vera leaves in the control of bovine mastitis through in vitro models that evaluate the antimicrobial effects against S. aureus and cytotoxicity to MAC-T cells.

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-Ciocalteau 14 and an external standard curve of gallic acid (10-100 μg/ml) (y= 0.0197x / r 2 = 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 x 250 mm; 5 μm; 120Å (Acclaim TM 120, 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, v/v) mixture 22 .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 / r 2 = 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 Manual 23 .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 24-48h at 37°C.Gram-positive, catalase-positive, and rabbit plasma coagulase-positive samples were biochemically confirmed as Staphylococcus aureus 24 .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 hours of incubation on plates at 37°C in addition to spectrophotometric reading at growth, using a previously described method 23 .
Because LE is an exudate with a yellow color that easily oxidizes to a dark coloration 14 , 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 minutes 25 .

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% (IC 50 ) 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 data 27 .

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 1a, b).Precipitation indices were lower during the summer months (January to March) 27 (Figure 1a), 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 levels 28 , as well as other factors, such as the period of flowering 29 .
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 species 14 .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 mechanism 30 .In the present study, we found the highest levels of aloin in the summer samples and the lowest in the spring samples (Figure 1 b, c).These results are correlated with the total phenolic content found in the studied samples (Figure 1).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 health 33 .For example, higher levels of barbaloin, isobarbaloin, and aloin in Aloe sp.plants were found during periods of the year with higher temperatures 32 .
Antimicrobial activity Despite significant differences in the levels of total phenolics and aloin in the LE samples (Figure 1b, 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 lowest tested concentration of 500 μg/ml was incapable of reducing bacterial growth to values greater than 80%.The effect of other concentrations between 500 and 1,000 μg/ml was not included in the study (Figure 2).
The effectiveness of LE from aloe vera leaves as an antimicrobial agent has been demonstrated for a wide variety of Grampositive 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][37][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 others 39 ; 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 contrast 36 , 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 Gibbons 41 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 3).At higher concentrations, such as 500 μg/ml, the reduction in the percentage of viable cells was greater than 80%.The IC 50 was 91.89 μg/ml.It is worth noting that the MIC of S. aureus growth was 1,000 μg/ml (Figure 2), a concentration that had a strong effect on the viability of mammary epithelial cells (Figure 3).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.
In an in vivo situation, the administration of a toxic product to bovine mammary glands can result in the development of inflammation 42 , which is more severe than that caused by the infection of pathogens 42 .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 lineage 45 is an established model that has been frequently used in the investigation of mammary glandular functions and mediators of inflammatory processes 46 .
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 emodin 47 .These anthraquinones induce the apoptosis of cells caused by a reduction in the proportion of cells in the mitotic phase 48 .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 effect 49 .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 recommended.In this case, its potential as a disinfectant should be investigated.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 temperature 50 .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.

Conclusion
Although seasonality interferes with the chemical composition of aloe vera LE, the seasonal samples we evaluated did not differ in relation to antimicrobial activity with a MIC of 1,000 μg/ml found for all samples.At this concentration, aloe vera LE shows strong toxic effects on bovine mammary epithelial cells of the MAC-T lineage.Despite the demonstrated antimicrobial activity of aloe vera LE, we suggest caution in recommending its intramammary use to treat bovine mastitis; instead, the topical use of this product on an external area, such as the udder, may be both efficacious and safe.
However, the study they performed is incomplete.I do not understand, why the investigators did not try the Aloin that they purified to determine the inhibition of bacterial growth and the degree of toxicity on mammary cells with this compound as they did with the total leaf exudate?The leaf exudate has too many compounds.Not only phenols, the exudate may have amino acids, anthraquinones, chlorophyll, and other pigments, etc.At least testing the Aloin then you could know if this compound has an antibacterial effect by itself and/or, causes the toxicity of mammary epithelial cells.If the results are positive for Aloin as antimicrobial without causing toxicity they will know that Aloin can be used for the mastitis treatment.If not, they will know that the leaf exudate has to be free of Aloin to treat the mastitis.
The literature of the manuscript is missing important new and relevant literature on the uses of Aloe barbadensis Miller as a prebiotic and inhibitor of colon cancer development.
In my opinion the need to perform the analyses considering the aloin.In my opinion, the manuscript should be approved after the authors complete these analyses.

Are sufficient details of methods and analysis provided to allow replication by others?
© 2018 Diaz-Muñoz G.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Gaspar Diaz-Muñoz
Department of Chemistry, Federal University of Minas Gerais, Belo Horizonte, Brazil I read with interest the manuscript entitled: "Aloe barbadensis Miller leaf exudate is a potential treatment for bovine mastitis".The manuscript is very interesting.In general, I found clearly written and only a few typos and errors were found as listed in the attached file that already were corrected.
I did not understand why in the discussion of antimicrobial activity, the authors cited only the results of concentrations between 500 and 1000 ug/mL.In the methodology cited, the use of six concentrations between 500 and 4000 μg/mL.I think the authors should consider the possibility of Aloe exudate being toxic to epithelial cells as it showed toxicity to the MAC T cells.I suggest making a cytotoxicity test for this cell before suggesting this exudate as a disinfectant.
Overall, I believe that this otherwise very good manuscript could benefit of a minor revision before indexing in F1000Reasearch.I have attached a commented copy of the manuscript for the authors to consider in the revision.

If applicable, is the statistical analysis and its interpretation appropriate? Not applicable
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 1 .
Figure 1.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: INMET 26 .(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<0.05indicates a significant difference).

Figure 2 .
Figure 2. 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.

Figure 3 .
Figure 3. 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<0.05indicates a significant difference).