Use of Eucalyptus Wood Vinegar as Antiseptic in Goats

Abstract

The use of wood vinegar Eucalyptus urograndis is used with antiparasitic, antibacterial, antifungal, but its action in combination with glycerin or matodextrin has not been demonstrated. In this way, we will inform this chapter this question, as well as the cytotoxicity in cells of the mammary gland. It was checked in the laboratory and in animals. It has been verified that the action with glycerin is better than the use with maltodextrin and that there is no cytoxicity in the mammary gland of lactating animals.

1. Introduction

The term pyroligneous comes from “pyrolysis”, a process that uses energy biomass for the production of the wood vinegar. It needs to go through a process of combustion. This process is called pyrolysis, a term for thermal decomposition of materials containing carbon in the absence of oxygen, in this process coal is obtained greenhouse gases with a negative environmental impact. This impact can be decreased if the oven is adapted to collect a condensable fraction, also known wood vinegar the remaining part is called non-condensable gases (CNG) [1]. This process is carried out with several plants, as Eucalyptus. This originates in Australia. Its first plantings were in Europe, Asia and Africa in the early eighteenth century, arriving in Brazil a century later [2]. The genus Eucalyptus sp. is the most planted in the whole country. The plant has rapid growth, high density and productivity, its use includes, fence constructions, pulp extraction for paper production, bioproducts, animal feed [3]. Several organic compounds are described are acetic acid, formic acid, propionic acid, methanol, maltol, ether, methyl alcohol, alcohols, acetaldehydes, acetone, ketone, phenols, guayacol, furan derivatives, and pyran, esters, cresol, derived from carbohydrates and nitrogen compounds [4]. Research on the use of pyroligneous acid (PA) against gram-positive, gram-negative and yeasts was confirmed in vitro [5]. Another study analyzed the effect in vivo of wood vinegar as a cutaneous antiseptic in ruminants [6] and there were found a lower bacterial count. Other studies have been observed as insecticides [3, 7].

2. Methodogy

2.1 Production of Eucalyptus wood vinegar

Wood samples from Eucalyptus urophylla x Eucalyptus grandis hybrid (clone I144) were collected from 8-year-old plantations in the experimental area of the Agricultural Sciences Unit, Universidade Federal do Rio Grande do Norte (05° 51′30″S and 35° 21′ 14″ W), municipality of Macaíba, Rio Grande do Norte State, Brazil. One hundred 3.0 cm-thickness wood disks were collected by following the procedures established by [8] and divided into four wedges each. Samples were oven-dried (Sterilifer, model SX cr/80, São Paulo, Brazil).at 103 + 2°C for 48 hours until reaching absolute dryness.

For the carbonization runs, the dry samples of each material were placed, separately, in a steel container inside a laboratory muffle equipped with a condensation apparatus to collect the total pyrolysis liquids. The condensation device was water-cooled aiming to maintain its temperature around 25–30°C, providing conditions for the condensation of vapors from the carbonization bed. For each type of woody material, 15 carbonization runs were carried out with about 500 g of plant material each. After each was concluded, the liquid products were mixed to make at the end of the experiment one composite sample of vinegar from each woody material. The carbonization process was carried out from the ambient temperature until reaching 450°C, with a heating rate of 0.7°C min 1, totalizing 8 hours. Composite samples of eucalyptus and bamboo vinegar were distilled under a 20-mmHg vacuum until the range of 100–103°C to remove tar and heavy oils from them. After the distillation, the resulting products were stored in amber bottles, previously sanitized with boiling water, and stored under refrigeration at 6°C for further procedures and experiments.

2.2 Cytotoxicity

Efficient protocols that ensure the verification of the cytotoxic potential of components of natural origin are essential for the proper use of substances for medicinal and/or therapeutic purpose. In general, the culture of animal cells allows obtaining information with greater productivity and speed [9]. In this sense, cytotoxicity can be conceptualized as the ability of a substance to inhibit the proliferative activity of cells, or cause damage to cells, resulting in cell death [10]. Since different substances, whether of endogenous or exogenous origin, can reduce cell proliferation, drugs developed in human and/or veterinary medicine must be previously tested.

A series of in vitro assays can be proposed to evaluate the toxicity of substances in cells [11, 12]. The most widely used assays involve the use of microscopy and spectrophotometry, relating assessments on cellular morphology, viability and metabolism, which mirror the proliferative activity of cells. In cytotoxicity assays of the eucalyptus wood vinegar in cultured goat cells [6], the analyzes have been those related to morphological evaluation by brightfield microscopy, viability by the cell membrane integrity assay using trypan blue and metabolic activity by the -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For all analyses, the conditions of isolation and in vitro culture of the cells are fundamental for the accuracy of the results.

2.2.1 Step by step in the elaboration of a cytotoxicity assay in goat cells

In general, an in vitro cytotoxicity protocol involves the stages of isolation and in vitro characterization of the cells to be evaluated, and applications of cell analysis methodologies [13]. In cytotoxicity assays of the eucalyptus wood vinegar in cultured goat cells, initially, goat mammary gland skin tissues are aseptically collected and transported to the laboratory in Dulbecco’s modified minimal essential medium (DMEM) plus 10% fetal bovine serum (FBS), and 2% antibiotic-antimycotic solution at 4°C for up to 1 h. In the laboratory, these tissues are fragmented into 9.0 mm³ (3 × 3 × 1 mm), washed and cultured in pretri dishes containing culture medium [DMEM, 10% FBS and 2 of 2% antibiotic-antimycotic solution] at 38.5°C and 5% CO₂ until cell detachment. This cell detachment occurs in up to 7 days, where the first cell subculture is carried out. After three subcultures, cells are cryopreserved or evaluated for cytotoxicity assays.

For the in vitro cytotoxicity assays, cells are evaluated before and after incubation with the eucalyptus wood vinegar, which has been for a period of 10 min [6]. Thus, in morphological analyses, cells are evaluated using an inverted microscope and the characteristics of cytoplasms with elongations, evident nucleus are evaluated. For cell viability, the trypan blue assay consists of an analysis to evaluate the integrity of the membrane. Briefly, after incubation with 0.4% trypan blue, cells in blue (broken membrane) and colorless (membrane intact) are counted in a hematocytometer and the viability rate calculated [14, 15].

Finally, the metabolic assay is performed using the MTT reagent and consists of a colorimetric assay for assessing cell metabolic activity [16]. It is based on the conversion of MTT into formazan crystals, which is associated with mitochondrial and cytoplasmic cellular functioning. Briefly, the assay is based on the metabolic reduction of MTT by dehydrogenases linked to NADH and NADPH that cleaves the tetrazolium salt to formazan crystals by metabolically active cells, reflecting in the number of viable cells present. This by-product has a dark color, which can be measured in a spectrophotometer. The absorbance result is calculated based on metabolism, because the darker the cell residue, the more viable cells metabolically produced it and the lower the cytotoxicity to metabolism promoted by the substance tested.

All in vitro cytotoxicity assays have advantages and limitations and the associated use of several tests guarantees safe results (Table 1). Cell health can be checked by a variety of methods [17] and the choice of a good cytotoxicity assay depends on the specific issues of each substance to be tested, relating cell type conditions and in vitro culture conditions [10].

In vitro analyzes	Advantages	Limitations
Morphological evaluation	Easy execution when compared to other in vitro analyses No need for pretreatment on cells	Subjective analysis method Use of inverted analysis microscopy
Viability by the cell membrane integrity assay	Low cost method Easy execution when compared to metabolic activity	Cells can be dead without necessarily having their membrane broken Cells with small pores may have dye penetration and not be unviable
Metabolic activity	Assay widely used to assess proliferative activity Accurate assessment of proliferative activity	Use of reagents to assess metabolic activity Need for spectrophotometer within the wavelength.

Table 1.

Advantages and limitations of the main analyzes used in in vitro toxicity tests.

Other methodologies can be cited to evaluate the cytotoxicity of extracts, such as apoptosis assays by flow cytometry, and comet assays. In the first assay, cells are labeled with fluorescent probes, such as annexin and propidium iodide, and evaluated by flow cytometry [15]. In the second assay, cultured cells are treated with the alkaline comet method, and evaluated by fluorescence microscopy [18].

2.3 Action in vivo

Research on the action of the wood vinegar associated with matodextrin and glycerin in vivo was carried out on the dairy goat property in the Independência Mossoró settlement/RN. The animals were randomly selected, separated into stalls, and daily subjected to antisepsis for 21 consecutive days. The experiment was authorized by opinion CEUA 02/2021. Twenty animals were divided into four distinct groups (A), (B) (C) and (D). Group A belongs to animals that received daily application of a conventional antiseptic (2% iodine tincture) - this being the positive control group. In the second group B was the animals that were treated daily with sterile distilled water, this being the negative control group. Test group C was submitted to treatment with the eucalyptus wood vinegar with maltodextrin and without glycerin and D was group with wood vinegar associated with glycerin.

The teat of each animal was immersed in the mentioned solutions, daily, once a day, for a period of 28 days. Four samples were collected with an interval of 7 days, being 0, 7, 14 21 and 28 days with sterile swab on the lateral part of the teat in an area of 1 cm².

After applying the product on the surface, 10 minutes were timed, referring to the time of action. After the product’s time of action, a sterile swab was used to pass over the entire surface of the teat, this swab was then refrigerated at 8°C for further processing and serial dilution.

From the collected swab, it was processed; a serial dilution of the swab collected from the surface of the mammary gland roof was made, and an aliquot of each dilution of 10⁻¹, 10⁻² and 10⁻³ was plated. Then, 1 ml of each dilution was distributed in Petri dishes containing Agar Plate Count (PCA) and subsequently taken to the bacteriological oven for 24 hours at a temperature ranging from 37 + − 0.5°C, to promote growth and development bacterian. After the time in the greenhouse, they were removed and counted. The numbers of viable colony-forming units, obeying the lower limit of 30 and the upper limit of 300 colonies [19].

After the isolation of the bacteria found in the negative control group, these were cultivated in BHI broth for 24 hours at 37° C until the log phase for approximately 24 to 48 hours, adjusted by the Macfarland scale. The microorganisms were identified through cytology and biochemical tests [20].

3. Results and discussion

3.1 Main results of the eucalyptus wood vinegar in goat cells

In cells isolated from the mammary gland of adult goats [6], the in vitro toxicity of 20% of the eucalyptus wood vinegar was evaluated from a compilation of three assays [morphology, viability and metabolic activity]. Comparisons were made with a group without the presence of any antiseptic substance and another group of cells incubated with 2% iodine solution, a commercial antiseptic currently used in many productive sectors. All these assays correspond to what we conceptualize as cell viability, through proliferative analysis. Thus, in goat cells, the eucalyptus wood vinegar did not affect cell morphology, being evidenced in all groups of cells with healthy morphology, with evident cytoplasm and nucleus and with evident cytoplasmic prolongations.

Interestingly, the viability assessed by the membrane integrity assay showed no difference between the groups, with viability values ranging from 57.6 to 88.6%. Nevertheless, when the metabolic activity was evaluated, both antiseptics (iodine solution and eucalyptus wood vinegar) caused a reduction of this parameter with values ranging from 31.8 to 47.6%, while cells not incubated with any substance presented values of 100%. Probably, the chemical composition of eucalyptus wood vinegar may be responsible for this reduction in metabolic rate; although it was similar to the values found in the iodine solution [6]. Moreover, cytotoxicity assays do not have the same principle [15], that is, while morphology assesses the surface conditions of cells, and trypan blue assesses cell membrane integrity, the MTT assay assesses metabolic activity associated with mitochondrial function, justifying thus differential responses of the eucalyptus wood vinegar on the cells.

3.2 Action in vivo of Eucalyptus wood vinegar associated a maltodextrin and glycerin

Lack of hygiene may result in loss of quality of milk matter, as well as its derivatives, leading to financial losses due to non-marketing of contaminated milk. Hygiene practices during and after milking considerably control cases of mastitis in the herd, contributing to standardized milk production with superior quality. Currently, the chemical products most used as antiseptics for the teats of dairy cows, before and after milking, are products based on 0.5% iodine and chlorhexidine [21].

In recent years, research focused on the use of medicinal plants has achieved excellent results by the scientific community in alternative and complementary treatment in veterinary medicine, both through the use of fresh vegetables or plant extracts and the use of herbal medicines administered in a complementary way or as inputs [22]. Thus, the objective of the present work is to prove the antiseptic action of the wood vinegar of eucalyptus sp. at 1% and also associated with maltodextrin or glycerin in Saanen goats with dairy ability as an alternative to the use of industrial antiseptics.

3.2.1 Bacteria count according to the action of wood vinegar

The data obtained in this research reveal that the treatment using vinegar wood associate a glycerin after iodine 2%. (Table 2). The results regarding vinegar wood associated a glycerin were better than the data that reveal the use of vinegar wood associated with maltodextrin. The best results regarding glycerin are justified due to the better fixation performed by this compound [23]. The glycerin potentiates the action wood vinegar of Eucalyptus and states that the presence of phenolic compounds that exist mainly as simple phenols, such as phenol, cresols and 1,2-benzenediol [6].

Groups	Day 0	Day 7	Day 14	Day 21	Day 28
Vinegar wood associated a glycerin	2.58	3.50	3.91	3.61	3.70
Vinegar wood associated a maltodextrin	5.39	4.86	2.59	2.40	5.34
Iodine 2%	2.53	3.42	2.59	2.40	3.19
Water Distilled	2.26	4.41	5.22	4.73	4.71

Table 2.

Number of bacteria (log 10) from the teats of animals that received natural and conventional antiseptics.

When is using the bacteria count according to the action of associated with maltodextrin had no antibacterial effect on the post dipping of goat teats. It is s suggested that maltodextrin has provided nutritional conditions for bacteria. This statement can justificated where studies show that there are important proteins that bind to maltodextrin and are ideal for the colonization of pathogenic bacteria’s [24]. Noting that the number of bacteria was higher than the negative control.

The antimicrobial effects of these phenolic compounds are mainly due to the chemical structure of these phenols, which allows them to act as proton exchangers that can lower the pH gradient across the cytoplasmic membrane, causing microbial cell death [25], it was not observed when wood vinegar associated maltodextrin.

The increase in the number of bacteria when maltodextrin is used may be associated with the absence of active principles such as guaiacol [5]. This is derivatives along with other phenols and furfural in the bacteria count according to the action of may explain the antibacterial and antifungal activities.

3.3 Influence of wood vinegar on goat milk

Does the wood vinegar used in post-dipping of dairy goats as a preventive measure for mastitis cause some interference in milk? This study in particular opens an interesting perspective for its use in processes involving the production of food intended for human food. As a measure of food safety, all food produced must present nutritional quality, which involves the microbiological quality that needs to be in accordance with current legislation, in this case, normative instruction No. 37 of October 31, 2000 [26].

According to this legislation, there are some practices, care and mandatory processes to minimize possible health-related risks. These risks may be of chemical, physical and biological origin. For goat’s milk, one of the main risks presented is organic, since food because it is one of the most complete, has physical-chemical properties conducive to the development of deteriorating and pathogenic microbial. The parameters that can be evaluated and are related to the degree of contamination in milk are: protein, lactose, pH, fat content and conductivity [27].

The fat percentage, non fat solid content, protein content, lactose content, conductivity and pH in the analyzed samples were the parameters are in accordance with the legislation for 28 days when utilized E. urograndis, except electrical conductivity [6]. This parameter were above the levels established by the legislation, and this represented a possible contamination of the milk that facilitated conductivity. This is supported by the observed correlation obtained between the increase colony-forming units of PA of E. urograndis and this physical parameter [28] and the discussion need be investigated.

4. Conclusions

The vinegar wood of E. urograndis associated a glycerin is better than the use of vinegar wood associated with maltodextrin and does not present cytoxicity to mammary gland cells. Research should be conducted for better knowledge of the rational.