ANTI-BACTERIAL AND TOXICOLOGICAL ACTIVITY IN SILICO OF CINNAMOMUM CASSIA ESSENTIAL OIL

ANTI-BACTERIAL AND TOXICOLOGICAL ACTIVITY IN SILICO OF CINNAMOMUM CASSIA ESSENTIAL OIL

José Roberto Dantas de Andrade Santos ¹, Francisco Orlando Rafael Freitas ¹, Alanna Michely Batista de Morais ¹, Vanessa Passos Brustein ¹, Tiago Bezerra de Sá Sousa Nogueira ¹, Raquel Bezerra de Sá de Sousa Nogueira ¹, Milena Nunes Alves de Sousa ¹, Daysianne Pereira de Lira Uchoa ¹, Michelangela Suelleny de Caldas Nobre ¹, Luara de Sousa Monteiro Duarte ¹, Jorge Luiz Silva Araújo Filho ¹, Fabrício Kleber de Lucena ¹, Marcelo Alves Barreto ¹, Umberto Joubert de Morais Lima ¹, Geraldo Gonçalves de Almeida Filho ¹, Abrahão Alves de Oliveira Filho ³, Edeltrudes de Oliveira Lima ², Cássio Ilan Soares Medeiros^{*1, 2} and Hilzeth de Luna Freire Pessôa ⁴

Research Laboratory in Microbiology ¹, Integrated College of Patos, Patos, Brazil.

Mycology Laboratory, Department of Pharmaceutical Sciences ², Academic Unit of Biological Sciences ³, Department of Cell and Molecular Biology ⁴, Federal University of Paraíba, João Pessoa, Paraíba, Brazil.

ABSTRACT: The search for natural anti-microbials present in plants has been the target of intense scientific studies over the last years. The objective of this study was to assess the in-vitro antibacterial potential and in silico toxicology of the essential oil of Cinnamomum cassia (Chinese cinnamon) against E. coli and S. aureus strains. The essential oil was initially solubilized in tween 80 and dimethyl sulphoxide acid (DMSO). The antibacterial action was assessed by means of the minimum inhibitory concentration (MIC) determined from the microdilution in double-concentrated brain heart infusion broth (BHI) and the minimum bactericidal concentration (MBC) which was determined by the depletion in nutrient agar (NA) technique with aliquots of  10µL of the MIC, MIC × 2 and MIC × 4. The MIC and the MBC presented satisfactory and varied results; the E. coli LPM 4459, LPM 1243, LPM 4010 and LPM 4896 strains, had a MIC of 256 µg/mL and MBC of 512 µg/mL respectively. As for the E. coli LPM 2810 and S. aureus LPM 55 strains, they showed a MIC of 128 µg/mL and MBC of 256 µg/mL. In the in silico toxicological analysis, a toxic potential was observed only in the p-Cymene and 3-Carene fractions. However, in the p-Mentha-1,4(8)-dien fraction there was no evidence of toxicity. Therefore, the essential oil of Cinnamomum cassia has satisfactory anti-bacterial activity, in addition to having a low theoretical oral toxicity when compared to other compounds commonly used in the therapeutics of bacterial infections.

 Essential oil, Cinnamomum cassia, Antimicrobials, Toxicity

INTRODUCTION: The infectious diseases have become a global concern as the clinical efficiency of many existing antibiotics is being threatened by the rapid growth of multiresistant pathogens ¹.

There is a continuous need to discover new antimicrobial compounds with adequate chemical structures and a new mode of action against pathogenic agents ².

The natural products present many properties and versatilities for the development of new drugs, especially the anti-microbials, which may have a therapeutic potential for the treatment of infectious diseases ³. The essential oils (EOs) are suitable to be antimicrobials, both individually, and individually in their isolated compounds, including terpenes, terpenoids and aromatic compounds, in which have been shown antimicrobial activity against a wide range of pathogens with various spectrum of activities ⁴. The phytotherapeutic agents are the main sources of new medicines and may constitute an alternative to usual medicines ⁵.

The Chinese cinnamon is popularly used to treat inflammatory processes, pain, menstrual disorders, disorders of the blood circulation and fever ^{6 - 8}. Ethereal extracts of Cinnamomum cassia exhibit antioxidant activities and in traditional medicine are used for anti-ulcer effects ⁹. The anti-Candida and anti-dermatophytic effects of the EO of various species of Cinnamomum have been evidenced over the years ^{10, 11}.

Cinnamon is a dietary phytochemical which demonstrates anti-microbial properties and is particularly significant, in which the dietary chemical products are considered to be reliable and used routinely ¹². Thus, this work aimed to analyze the in-vitro antibacterial activity and in silico toxicology of the essential oil of Cinnamomum cassia against E. coli and S. aureus strains.

MATERIALS AND METHODS:

Phytoconstituent and Substances: The following substances used in this work were obtained commercially: essential oil (EO) of Cinnamomum cassia (purity ˃ 95%), dimethyl sulfoxide acid (DMSO) and tween 80 (0.02%) (all from Sigma-Aldrich, São Paulo, SP, Brazil). The tween 80 and the DMSO were solubilized in a proportion which did not exceed 0.5 % in the tests, subsequently was diluted in sterile distilled water with the EO of the cinnamon in order to obtain a doubly concentrated emulsion of 2048 μg/mL ^{13, 14}.

Bacterial Strains: The tests were carried out with bacterial strains: E. coli LPM 2810, LPM 4459, LPM 1243, LPM 4040, LPM 4896 (clinical isolates), E. coli ATCC 8859 and ATCC 2536 (standard strains), S. aureus (LPM 45 and 55) (clinical isolates) and two standard strains of S. aureus ATCC 6538 and ATCC 25213. All the samples belong to the collection of the Research in Microbiology Laboratory (LPM) of the Integrated Faculties of Patos (FIP). All the strains were maintained in NA at 4 °C. Cultures of 24 hrs incubated at 35 ± 2 °C were used in the test.

Inoculum: The suspensions were prepared from recent bacterial cultures streaked in NA and incubated at 35 ± 2 °C during 24hrs. After the incubation, approximately 4-5 colonies were transferred (with a sterile loop) to test tubes containing 5.0 mL of sterile saline solution (NaCl at 0.85%). The resulting suspensions were agitated during 15 seconds with the aid of a vortex mixer (Fanem Ltd., Guarulhos, SP, Brazil). The turbidity of the final inoculum was normalized using a barium sulfate suspension (tube of 0.5 in McFarland’s scale). The final concentration obtained was of 1-5 × 10⁸ colony forming units per milliliters (CFU/mL) ^{15, 16}.

Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC): The determination of the MIC of the product on the strains used in the biological tests was determined by the broth microdilution method as proposed by the National Committee for Clinical Laboratory Standards (NCCLS) ¹⁷ and Santurio et al., ¹⁸ A hundred microliters (100 µL) of the double concentrated BHI liquid medium were transferred to the cavities of a microdilution plate with 96 u-shaped wells (Alamar, Diadema, SP, Brazil). Subsequently, 100 µL of the emulsion of the double concentrated product were inoculated in the first horizontal row of the dish wells. Two-fold serial dilutions were carried out, and an aliquot of 100 µL was removed from the most concentrated well to the following one, producing concentrations of 1024-16 µg/mL.

Finally, 10µL of the bacterial suspensions were added to each well of the plate, in which each column represented a strain. In parallel, the controls were made for the bacterial viability and for the susceptibility to the standard antibiotic chloramphenicol (100 UI/mL). The plates were sealed and incubated at 35 ± 2 °C during 24hrs. After the appropriate incubation time, the presence (or absence) of microbial growth was visually observed. The formation of bunches of cells or “buttons” in the dish wells was considered. The MIC of the EO was defined as the lowest concentration of the product which produced a visible inhibition of growth of the bacteria.

The antimicrobial activity of the product was interpreted (considered active or not) according to the values of the MIC against the studied pathogens, where the proposed classification criteria are: strong/good activity (MIC < 100 µg/ mL), moderate activity (MIC 100-500 µg/mL), weak activity (MIC 500-1000 µg/mL) and inactive product/no anti-microbial effect (MIC > 1000 µg/mL) ^{19, 20}.

In order to determine the MBC, aliquots of 10µL of the MIC, MIC × 2 and MIC × 4 of the test product were subcultivated. The chloramphenicol (100UI/mL) was the bacterial growth control in Petri dishes containing NA. After 24 hrs of incubation at 35 ± 2 °C a reading was carried out to evaluate the MBC based on the controls. The MBC was defined as the lowest concentration of the product capable of inhibiting bacterial growth or allowing a growth inferior to three CFU, thus resulting in bactericide activity of 99.9 % ^{15, 21}.

The biological activity tests were carried out in duplicate and the results were expressed as the arithmetic average of the MIC and the MBC.

In silico Analysis (Osiris): The prediction process of the biological effects executed by the Osiris software is based on a pre-computerized set of molecular fragments which give origin to the toxicity alerts, in the case of being found in the currently designed molecular structure. The toxicity predictions of the  Osiris result in molecular groups capable of producing effects of mutagenicity, tumorigenicity, irritability, possible damages to the reproductive system, cLogP, druglikeness and drug-score of the molecules ²².

Lipinski's rule of five ²³ evaluates the similarity of drugs. It determines if a chemical compound with validated pharmacological or biological activity has properties which make it a probable active drug orally in human beings. This rule describes the molecular properties which are important for the pharmacokinetics of a chemical compound in the human body including its absorption, distribution, metabolism and excretion (ADME).

The established rule for the majority of "drug-like" molecules has  cLogP ≤ 5, molecular weight ≤ 500 Da, number of hydrogen acceptors  ≤10 (nALH ≤ 10) and the number of hydrogen donors ≤ 5 (nDLH ≤ 5). Molecules which violate one of these parameters may have serious problems with bioavailability ^{23, 24}.

RESULTS: The results of the antibacterial activity of the EO of Cinnamomum cassia were found from the MIC and the MBC determined by broth microdilution. The values of the MIC for S. aureus LPM 45 was 32 µg/mL and the MBC 128 µg/mL. The strains of E. coli LPM 4459, LPM 1243, LPM 4040 and LPM 4896, had MIC of 256 µg/mL and MBC 512 µg/mL. The strains S. aureus LPM 55 and S. aureus ATCC 25213 had MIC 128 and 256 µg/mL, MBC 256 and 1024 µg/mL respectively. The strains E. coli ATCC 2536, ATCC 8859 and LPM 2810 had MIC of 256, 256 and 128 µg/mL, as well as a MBC of 1024, 1024 and 256 µg/mL respectively (Table 1 and 2).

Besides the values of the MIC and the MBC, in silico toxicological analysis of the components of the EO of cinnamon was also carried out, as well as its pharmacological properties enabling an evaluation of the parameters mutagenicity, tumorigenicity, irritability and the possible damage to the repro-ductive system caused by these compounds in comparison with standard antibiotics Table 3.

TABLE 1: MIC VALUES (μg/mL) OF CINNAMON ESSENTIAL OIL AGAINST BACTERIAL STRAINS

(+) inhibition (-) no inhibition

TABLE 2: MBC VALUES (μg/mL) OF CINNAMON ESSENTIAL OIL AGAINST BACTERIAL STRAINS

(+) inhibition (-) no inhibition

TABLE 3: OSIRIS CALCULATIONS OF TOXICITY RISKS AND DRUG-SCORE OF COMPOUNDS p-CYMENE, P-MENTHA-1,4(8)-DIEN AND 3- CARENE MONOTERPENES COMPARED TO THE STANDARD ANTIBIOTICS DRUGS

DISCUSSION: The essential oils obtained have been showing bactericidal potential over the years. In which, Cinnamomum species have already had reported bactericidal potential from plant species ²⁵. Naveed et al., ² showed in researches related to antibacterial activity of EOs that the Cinnamomun verum obtained excellent activity against the bacterial strains in study, including Salmonella typhi G7 and Pseudomonas fluorescens.

The antimicrobial activity of the EO of Cinnamomun cassia was investigated against 10 bacterial strains, in which showed activity against all the tested strains, which include seven strains of E. coli and three of S. aureus. The low MIC and MBC values especially against strains of  S. aureus LPM 45, LPM 55 and E. coli LPM 2810 (Table 1 and 2), confirm the excellent bactericidal potential of the Ciannamomum cassia. These results are quite similar to the findings of Unlu et al., ²⁶ in which the Cinnamomum zeylanicum inhibited the growth of gram-positive and gram-negative bacteria thereby demonstrating to be an excellent antimicrobial agent.

The S. aureus bacteria is one of the main causes of human infections worldwide, the severity of these infections vary a lot from minor skin infections up to fatal necrotizing pneumonia ²⁷. In addition, these bacteria have an excellent capacity of acquiring resistance to antibiotics ²⁸. The S. aureus is one of the most prevalent clinical pathogens isolated from hospital settings, and recently has become generalized in community settings ²⁹.

The bacteria E. coli occurs naturally in the human intestine, however, certain strains which can cause infections, are becoming resistant to antibiotics. These bacteria are responsible for urinary tract infections (UTIs) and are also associated to blood stream infections ³⁰. The identification of the dissemination of nosocomial infections and to interrupt the development of outbreaks caused by E. coli is becoming a demanding challenge due to the rapid global growth and constant and increasing influx of these bacteria from the community to the hospital environment ³¹.

The variation of the susceptibility of the tested microorganisms may be attributed to their intrinsic properties which are related to the cell surface permeability the molecules which constitute the EO in study ³². In the present study, these variations are well established; the EO and its fractions demonstrated variable activities in strains of a same species. Notoriety well represented between the standard strain S. aureus ATCC 25213 (MIC 256 µg/mL) and clinical strains S. aureus LPM 55 (128 µg/mL) and S. aureus LPM 45 (MIC 32 µg/mL), bearing in mind that the later has proved to be more susceptible to the compound.

In contrast, the MBC of the compound followed the methodology of Hafidh et al., ³³ considering that a phytoconstituent presents bactericidal property when the coefficient between the MBC/MIC is between 1 and 2, and effect which is capable of causing the eventual death of the microorganism. Thus, the Cinnamomun cassia presents bactericidal effect, proving to be effective against the majority of the strain present in this study.

The term “in silico toxicity” refers to computational experiments, mathematical calculations or scien-tific analysis of data of chemical substances by means of computational tools which analyze them and make the prediction of a possible toxicological activity ³⁴. The software Osiris generates alerts about the mutagenicity, tumorigenicity, irritability and damage to the reproductive system. The predictions are registered by means of a color code which indicates the degree of the effects. With the color red indicating risks of undesirable effects, yellow, moderate risk and green, absence of risk ³⁵.With respect to the in silico analysis, the toxicological effects produced by constituents of the Cinnamomun cassia were based on the principles of Morales et al., ²⁰ and Ursu ³⁶. However, the determination of the pharmacokinetic profile of the phytoconstituent follows the methodology proposed by Lipinski et al., ²⁴ so that the compound has to have at least three of the four proposed requirements (nDLH ≤ 5, nALH ≤10, DA ≤ 500 Da and cLogP ≤ 5). The major fractions of the EO, which consist of p-Cymene, p-Mentha-1,4(8)-dien and 3-Carene, were analyzed. The results obtained demonstrate that the phytoconstituents present low levels of toxicity when compared to other phytochemicals and to conventional antibiotics, presenting Drug-likeness values (-2.50, -3.01 and -3.35) respectively and Drug-Score (0.22, 0.44, 0.16) respectively. Of the monoterpenes which constitute the essential oil, the only one which demonstrated to have an evident toxic potential was the p-Cymene, revealing itself as a tumorigenic and irritating agent. The 3-Carene showed to have a slight tumorigenic and irritating potential and the p-Mentha-1,4(8)-dien, however, did not reveal any toxic potential. By contrast, the chloramphenicol, broad-spectrum antibiotic frequently used to treat severe bacterial infections, proved to be strongly toxic with irritating, tumorigenic characteristics and possible damages to the reproductive system, as well as mutagenic effects.

CONCLUSION: In conclusion, the EO of Cinnamomum cassia has satisfactory antibacterial activity. Once again confirming the phytoconstituents as an efficient alternative in medicinal therapeutics. In this study, the Cinnamomum cassia proved to be effective against E. coli and S. aureus strains. The in silico analysis also proved to be interesting, seen as all the fractions of the EO respected the criteria proposed by Linpinski et al., ²⁴ in relation to the pharmacokinetics, with exceptions only for the toxic potential of two of the molecules.  Therefore, this essential oil and its major components are promising bactericides. However, subsequent studies are necessary for a more detailed investigation of the in-vitro and in-vivo pharmacological and toxicological properties of these molecules.

ACKNOWLEDGEMENT: The authors greatly acknowledge the research laboratory in micro biology of Integrated College of Patos, for providing the bacterial cultures for the present investigation.

CONFLICT OF INTEREST: The authors declare that there is no conflict of interests regarding the publication of this paper.

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    How to cite this article:

    Santos JRDA, Freitas FOR, Morais AMB, Brustein VP, Nogueira TBSS, Nogueira RBSS, Sousa MNA, Uchoa DPL, Nobre MSC, Duarte LSM, Filho JLSA, Lucena FK, Barreto MA, Lima UJM, Filho GGA, Filho AAO, Lima EO, Medeiros CIS and Pessôa HLF: Anti-bacterial and toxicological activity in silico of Cinnamomum cassia essential oil. Int J Pharm Sci Res 2018; 9(6): 2281-86. doi: 10.13040/IJPSR. 0975-8232.9(6).2281-86.

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