Development of antimicrobial soaps using essential oil of Schinus terebinthifolius and

A cosmetic product consists of natural or synthetic substances, the purpose of which is to wash, scent, correct and protect the various parts of the human body. Human skin has pores that connect to the oil glands, which produce sebum, an oily liquid that carries dead cells through the hair follicles to the surface of the epidermis, causing the appearance of skin lesions and the growth of microorganisms. In the present work, two antimicrobial soaps were formulated in solid and liquid form using essential oil of Schinus terebinthifolius and Piper nigrun, analysed and compared with other antimicrobial soaps available on the market. In addition, the microbiological safety of the products developed was tested, according to ANVISA criteria. All products developed in this work have microbiological safety, but the best inhibitory results regarding the growth of the microorganisms Staphylococcus aureus and Escherichia coli were obtained with the soaps in liquid form.

Herbal Cosmetics are products formulated using one or more herbal ingredients to provide cosmetic benefits and are known as Cosmeceuticals. The demand of these products is increasing due to their lack of side effects. Cosmeceuticals are cosmetic products intended to improve the health and beauty of the skin by providing a specific result (Joshi & Pawar, 2015). In June 2006, the Decree No. 5.813 (Brasil, 2006) was signed, approving the National Policy on Medicinal Plants and Herbal Medicines, whose main objective is to guarantee to society safe access and correct handling of both herbal medicines and medicinal plants. The measure also aims to promote the sustainable use of biodiversity and the development of the sector's industry (Brasil, 2006). Among the various sources of natural products, Schinus terebinthifolius (commonly known as pink pepper) is a species belonging to the Anacardiaceae family, also known as aroeirada-praia, aroeira, aroeira-Vermelha, pink pepper, cambuí, among other names (Lorenzii & Matos, 2008 ). It is native to South America and can be found in Europe, Asia and other regions of America, with increasing pharmacological use being considered by popular medicine as astringent, antidiarrheal, anti-inflammatory, depurative, diuretic and febrifugal (Paiva & Aloufa, 2009) . The aroeira fruits have from 5.50 to 8.41% of essential oil, which has a predominant chemical composition of monoterpenes, the most abundant being δ-3-carene, limonene, α-felandrene, α-pinene, mircene and o-cimene, followed by the trans-caryophylene sesquiterpenes, -murulene. However, variations in the composition of essential oils can occur depending on the time of hydrodistillation of the fruits for oil extraction, the geographic region in which the fruits were collected and also on their maturation time (Oliveira Junior et al., 2013).
Piper nigrun (commonly known as black pepper), on the other hand, is a species of shrub creeper, belonging to the Piperaceae family, popularly known as black pepper or black pepper, originating in the tropical regions of India (Bomtempo, 2007;Ee, Lim, Rahmani, Shaari. & Bong, 2010;Carnevalli & Araújo, 2015). Black pepper is a plant rich in retinol (vitamin A), ascorbic acid (vitamin C), iron and potassium, among other compounds. In addition, this spice has a wide variety of metabolites that are distributed in different classes of compounds, such as: amides, alkaloids and propenylphenalins, which have important Development, v. 9, n. 11, e69491110328, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10328 5 biological actions, especially anti-inflammatory and antioxidant (Bomtempo, 2007;Ee, Lim, Rahmani, Shaari. & Bong, 2010), analgesic, healing, circulatory, as well as combating the signs of aging caused by free radicals when consumed in appropriate amounts (Carnevalli & Araújo, 2015) Thus, this study aimed to evaluate the antimicrobial activity of the essential oils of S. terebinthifolius and P. nigrum and use them in the development of bar and liquid soaps, assessing their antiseptic activity in the prepared formulations, as well as their stability , comparing its antibacterial prospecting with other antimicrobial soaps available on the market, in solid and liquid conditions, such as Dettol, Protex, Lifebuoy, Biocrema and Asepxia. In addition, the microbiological safety of the products was also assessed.

Methodology
The work was a laboratory research on a quali/quantitative basis (Pereira et al., 2018).
Obtaining essential oils: S. terebinthifolius essential oil was purchased from the company Destilaria Bauru, while P. nigrum essential oil was purchased from Ferquima Indústria e Comércio Ltda.
Obtaining commercial soaps: The soaps described in their packaging as antibacterial, in common use, used comparatively in this work, were purchased at cosmetic stores in the city of Uberaba-MG, but are widely marketed in the country. These were used in comparative tests of effectiveness as antiseptics. The bar soaps used were Asepxia, Biocrema, Dettol, Lifebuoy and Protex, while the brands of liquid soap used were Dettol, Lifebuoy and Protex.
Evaluation of antimicrobial activity of essential oils: The suspensions of S. aureus (ATCC 29213) and E. coli (ATCC 35218) were diluted 1:100 based on the amount of Muller-Hinton Agar medium placed in the Petri dishes (CLSI, 2018). Sterile 5 mm diameter filter paper discs impregnated with the essential oils of S. terebinthifolius and P. nigrum were placed in 9.2 cm diameter Petri dishes. Then the plates were incubated at 37°C for 24 hours. The evaluation of the antimicrobial activity of the formulated soaps was carried out in triplicate.

Preparation of the formulation of the antiseptic bar soap:
The formulation of the bar soap was based on a basic composition with the presence of surfactants that provide detergency, as Research, Society and Development, v. 9, n. 11, e69491110328, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10328 6 well as quantity and quality of foam, viscosity agents, overfat, preservatives and ion scavengers following the formula in Table 1. Three soap formulations were prepared in solid form: adding essential oil of S. terebinthifolius (SSPR), adding essential oil of P. nigrum (SSPP) and adding both essential oils (SSPP/PR).

Preparation of the formulation of an antiseptic liquid soap:
For the formulation of liquid soaps, a soap base was used whose components are sodium lauryl ether sulfate, amide, water, sodium chloride, amphoteric acid, EDTA and citric acid. Distilled water and essential oils were added slowly, to avoid previous foam formation. Three soap formulations were prepared in liquid form: adding essential oil of S. terebinthifolius (SLPR), adding essential oil of P.
Organoleptic parameters analysed: Samples of commercial and formulated soaps were analysed for colour, odour and homogeneity, according to ANVISA (2012).
Physical-Chemical Analysis: for pH analysis, formulations were evaluated on a pH meter; to analyses the viscosity of the formulated liquid soaps, a Brooksfield Viscometer was used; to assess the density of the formulated liquid soaps, a clean, dry and calibrated pycnometer with a capacity of 100 mL was obtained. The density analysis was performed according to the guidelines of the Brazilian Pharmacopoeia (Farmacopéia Brasileira, 1988). All tests were done in triplicate. Research, Society and Development, v. 9, n. 11, e69491110328, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10328 Evaluation of the stability of the formulations: Based on the combinations made, the organoleptic and physical-chemical parameters were evaluated using the centrifugation test, in which the liquid soaps were subjected to centrifugation at 3,000 rpm for 30 minutes and the formation or not of sediments, phase separation and formation of agglomerates; Foam analysis, in which the foam index was determined using 100 mL beaker, with 5 mL of sample, addition of distilled water to the 50 mL mark, followed by stirring for 10 seconds, the foam formed was measured in cm, with the aid of a ruler, at times 0, 5, 10 and 15 minutes. All tests were done in triplicate.

Evaluation of the antimicrobial activity of commercial soaps:
The suspensions of S.
aureus and E. coli were diluted 1: 100 based on the amount of Muller-Hinton Agar medium placed in Petri dishes (CLSI, 2018). Sterile 5 mm diameter filter paper discs impregnated with the samples of the soaps, once diluted in 5mL of distilled water, were placed in 9.2 cm diameter Petri dishes. Then the plates were incubated at 37°C for 24 hours for the bacteria.
The evaluation of the antimicrobial activity of essential oils was carried out in triplicate.

Evaluation of antiseptic properties of formulated soaps:
The antiseptic activity of formulations containing bioactive essential oils was verified by measuring the zone of inhibition of bacterial growth and compared with each other and with the antibacterial activity of commercially available soaps. The methodology used for this evaluation was the same used for the evaluation of the antiseptic properties of commercial soaps. All tests were done in triplicate.

Evaluation of the antiseptic properties of antibiotic discs:
The antiseptic activity of antibiotic discs (Bio-Rad, was performed for some antibiotics commonly used for comparison purposes. The methodology used for this evaluation was the same used for the evaluation of the antiseptic properties of antibiotics). commercial soaps All tests were performed in triplicate.
Evaluation of the microbiological safety of the formulated soaps: for the evaluation of the microbiological safety of the formulated soaps, the research of Escherichia coli was carried out, as well as, of Staphylococcus and Pseudomonas, according to ANVISA (2012). For E.
coli research, 1 g of sample was aseptically transferred to 9 mL of saline. Then, 1 ml of the solution was transferred to the Petri dish containing Eosin Methyl Blue agar (EMB) and the Research, Society and Development, v. 9, n. 11, e69491110328, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10328 8 dish was incubated at 36 ± 1ºC for 24 hours. After this period, it was observed whether there was a growth of colonies, as well as their characteristics (if present, they are black metallic colonies). When there is growth of suspicious colonies, the biochemical series for the identification of E. coli must be followed. For the research of Staphylococcus and Pseudomonas, 1 g of sample was aseptically transferred to 9 mL of soy-casein broth and this broth was incubated at 36 ± 1 ºC for 24 to 48 hours. Then, it was sown in Petri dishes containing Vogel Johnson agar for Staphylococcus research, and in cetrimide agar plates for Pseudomonas research. The plates were incubated at 36 ± 1 ºC for 24 hours. After this period, it was observed whether there was growth and the characteristics of the colonies. For Sthalylococcus, the presence of yellowish colonies or the appearance of specific characteristics must be confirmed with the catalase and coagulase tests. For Pseudomonas, on the other hand, the presence of blue-green colonies must be confirmed by the microorganism by the biochemical series.

Results and Discussion
Antimicrobial activity of essential oils used: The antibacterial activity of essential oils was evaluated. Figure 1 shows the inhibitory zones presented by the essential oils used in this study.
The inhibition halos are symmetrical, proportional, with significant and similar sizes around 18 mm. In addition to the compounds that make up essential oils that have antimicrobial activity, it is noted that their volatility and odour can influence the diameter of the inhibitory zones.
Organoleptic Analysis: The organoleptic characteristics of the commercialized soaps and the formulations developed in this work were evaluated. Colour, odour, and general appearance were evaluated, and the brands evaluated were: Asepxia, Biocrema, Dettol, Lifebuoy and Protex. In all evaluated samples, the properties were shown to be adequate, as shown in Tables 2 and 3.   The organoleptic parameters observed were within the standards and comparable among all soaps, which showed color, odor and characteristic aspects, differing according to each brand or formulation. None of the samples showed visual alteration, nor was any solid matter found in any formulation. All brands had an odor compatible with the product and its composition. Table 4 shows the results obtained for pH, viscosity and density, relative to soaps in liquid form. Among the physical-chemical characteristics, pH is the fundamental analysis, since the pH of the skin, especially the skin of the face, where there is a higher concentration of infections due to the presence of numerous microorganisms, is a more neutral pH and requires the formulation of cosmetic product has a pH compatible to the region thus helping to maintain your health and protect the skin region. The soap that has both essential oils (SLPP/PR) showed a pH equal to six, closer to the skin's pH and close to the pH values of the tested commercial soaps. This is important because, with a pH close to neutral, the cells of the epidermis are not attacked by the product.

Physical-Chemical Analysis:
Research, Society and Development, v. 9, n. 11, e69491110328, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10328  A small variation in viscosity values was observed in Table 4. It is noted that the soap with higher viscosity, flows through the packaging with more difficulty and slowly, while the soaps that presented lower viscosity, flowed through the packaging. This fact is linked to waste due to accelerated flow. Regarding viscosity, all formulated liquid soaps present values like or higher than those of the analysed brands, showing better options than the soaps found in the national market.
The Relative density is the amount of mass per volume of a substance. The liquid soaps formulated in this work showed the highest density values among the analysed samples.
Dettol soap had the lowest density (1,003 g/mL), while the soap containing the mixture of essential oils (SL PP/PR), the highest density among the samples analysed (1,099 g/mL).
However, the relative densities of liquid soaps did not differ from ideality, and were similar, remaining within the range of 0.9 -1.1 g/mL.
Evaluation of the stability of the formulations: After the centrifugation test, none of the samples showed any change, such as agglomerates, phase separation, or precipitation. All Development, v. 9, n. 11, e69491110328, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10328 12 samples remained stable during the test, so the results were satisfactory for all samples analysed, commercial or formulated in this work. Table 5 shows the foam indices during fifteen minutes of analysis.  In general, S. aureus was more sensitive to the soaps tested. However, the Assepxia soap showed a maximum inhibition zone of 24.33mm in diameter. Despite presenting smaller inhibition zones, the other brands also showed good inhibition results for the growth of S.
aureus, such as Lifebuoy -21.33mm, Dettol -17.33mm, Protex -12.67mm and Biocrema -11.67mm. For E. coli, the inhibition zones were not as significant as for S. aureus. The Assepxia brand soap showed the best result with a 20.16mm of inhibition zone, followed by Protex soaps with 18.33mm, Dettol with 13.67mm, Biocrema with 10mm and Lifebuoy with 9.17mm. Evaluation of antiseptic properties of bar formulations: to evaluate the antibacterial activity of formulated bar soaps, the same methodology used to evaluate the antimicrobial activity of commercially available antibacterial bar soaps was used. Figure 3 shows the values of the zones of inhibition (in mm) for bar soaps.   the areas presented by commercially acquired liquid soaps, since the isolated essential oils showed antimicrobial activity considerable. Figure 6 shows the Petri dish used in the bioassay of antimicrobial activity of the liquid formulation containing black pepper essential oil (SLPP) against S. aureus.  The E. coli bacteria, proved to be more resistant to the effects of formulated liquid soaps, as well as to the effects of bar soaps. These showed satisfactory zones of inhibition, however, with smaller diameters, when compared to the Protex Liquid brand against the same microorganism. Figure 8 shows the diameters of the zones of inhibition for formulations containing only the essential oil of black pepper (A), only the essential oil of pink pepper (B) and the formulation with both essential oils (C), with diameters approximately 32.67 mm, 30 mm and 29.33 mm, respectively.  Table 6.
The bar soaps formulated in this work had an antibacterial action comparable to commercial bar soaps with better antibacterial action, such as Asepxia and Lifebuoy. The antibacterial action against S. aureus of the black pepper bar soap had an antibacterial action comparable to the antibiotic Vancomycin. The Asepxia soap had an action comparable to the antibiotic Chloramphenicol. When the antibacterial action against E. coli is compared, the antibacterial action of the pink pepper bar soap, the bar soap containing both essential oils and the Asepxia soap had an action comparable to the antibiotic Cefaclor.
Comparing the liquid soaps, the liquid soap containing both essential oils and the Protex soap had the best results, having no antibiotics, of those tested, with comparable action. All formulated liquid soaps and Lifebuoy liquid soap had antibacterial action comparable to the antibiotics ceftriaxone and ciprofloxacin. Thus, it is evident that the soaps in pink pepper bar and with both essential oils, as well as all formulated liquid soaps are proving to be new, promising and innovative options for the segment of antimicrobial soaps.
Evaluation of the microbiological safety of formulated soaps: according to the microbiological safety assessment of the products studied, there was in E. coli, just as there was in Staphylococcus and Pseudomonas growth. In this way, it is possible to conclude that the essential oils used are effective antimicrobial additives, as none of the products developed showed growth of pathogenic microorganisms in the research carried out. Therefore, the products developed in this work are in accordance with RDC 07/2015 (ANVISA, 2015) which deals with the microbiological control of cosmetic products.
Following Lima et al. (2020), any cosmetic products are subject to microbial contamination. However, the lack of hygiene in the development and the low stability of the constituents of the formulation contribute to the contamination of the products and cause visible changes.

Conclusions
The present work brought the development of liquid and bar formulations of antimicrobial soaps, using the essential oils of black pepper, pink pepper and both, in order to prevent skin infections aggravated by microorganisms, emphasize the importance of their use and thus guide through the data the importance of phytocosmetics. The products developed in this work are in accordance with Resolution 07/2015, which deals with the microbiological control of cosmetic products. Thus, it is concluded that the soaps in pink pepper bar and with both essential oils, as well as all formulated liquid soaps are new, promising and innovative products for the segment of antimicrobial soaps.
In future studies other essential oils will be used to test the same properties.