Comparison of the Antibacterial Activity of the Ethanol Extract vs Hydroalcoholic Extract of the Leaves of Mangifera indica L. (Mango) in Different Concentrations: An In Vitro Study

INTRODUCTION

Bacterial resistance is a public health problem that involves the world’s population. This is because bacteria that cause infectious diseases become resistant to antibiotics, so science has been in a position to take measures such as the development of new antibacterial that can cope with these multidrug-resistant bacteria. On the other hand, traditional medicine and phytotherapy has been gaining increasing strength since a wide variety of plants show that they have biological properties for the different conditions that man presents, so there is a great empirical source of active ingredients, it is convenient to clarify those properties.^1–4

Currently, bacterial resistance against antibiotics has been increasing, resulting in a worldwide concern since mild infections cannot be controlled. This caused by the inappropriate and excessive use of drugs in addition to deficiencies in the prevention and control of infections. Since it is not possible to use first-line antibiotics, it is necessary to use more expensive medicines, which increases the costs for the family and society.^5–8

In health sciences, for example, the use of antibiotics is justified in the presence of odontogenic infections and as prophylaxis in at-risk patients. Among stomatological infections, bacteremia and osteomyelitis are common as a result of invasive dental procedures whose multifactorial origin includes a very varied microflora, finding opportunistic microorganisms such as S. aureus, which is related to the onset of these diseases.^7,9,10

In recent years, research shows various medicinal plants of stomatological interest, including Mangifera indica L., to which antibacterial properties are attributed due to its active substance that is mangiferin. As the phenolic compound is one of the most abundant chemical components in this plant, it is considered that it gives it the ability to inhibit or block the growth of certain microorganisms. Some bacteria that cause oral diseases such as S. mutans, E. faecalis, C. albicans, S. aureus and E. coli among others that have proven to be susceptible to Mangifera indica L.^11–15 In addition, according to the literature, the leaves of this plant have certain antibacterial properties due to the presence of saponins, steroids, alkaloids, anthracenocides, coumarins, flavonones, among other chemical compounds that are considered responsible for providing antimicrobial properties to this natural resource.^4,11

Therefore, the main objective of this research was to compare the in vitro antibacterial effect of two types of extracts (ethanol and hydroalcoholic) based on the Mangifera indica L. (mango) leaf of Northern Peru against strains of Staphylococcus aureus ATCC 6538™ so that it can provide scientific evidence to the profile of this plant and incorporate it as a possible source of antibacterial agent.

MATERIALS AND METHODS

The study was experimental in vitro, the execution of the present investigation was carried out in the Pharmaceutical Production Center (CENPROFARMA), in the Microbiology Laboratory of the Faculty of Pharmacy and Biochemistry of the National University of San Marcos (UNMSM). The sample was obtained using the means comparison formula with the Stata® 12.0 software with a 95% confidence level, with a statistical power of 0.80 from the results of the pilot test, determining a sample size of n = 48 discs embedded with the experimental substances in each of the six groups which was made up of n = 8 discs.

1. Group I: 50% MEE facing S. aureus
2. Group II: 100% MEE facing S. aureus
3. Group III: 50% MHE facing S. aureus
4. Group IV: 100% MHE facing S. aureus
5. Group V: chlorhexidine 0.12% vs S. aureus as a positive control
6. Group VI: alcohol 96% facing S. aureus as a negative control

RESULTS

When evaluating the antibacterial activity of the Mangifera indica L. (mango), it was shown that between 50% and 100% MEE concentrations they presented an inhibitory halo at 21.3 ± 0.5 and 24.1 ± 0.8 mm, respectively. In addition, with respect to the 50% and 100% MHE an activity of 24.6 ± 0.5 and 33.5 mm was found, respectively. Finally, in relation to the hydroalcoholic extracts with the positive (chlorhexidine 0.12%) and negative (alcohol 96%) control group, an antimicrobial activity of 25.3 ± 1.5 and 6.0 ± 0.0 mm was found. Finally, all groups presented normal distribution with a p > 0.05 (Table 1 and Fig. 3).

Table 1 shows that when performing the inferential statistics between the antibacterial activities of the 50% and 100% MEE, differences were found to be significantly significant with a p %3C; 0.001. Likewise, when comparing the MHE at 50% and 100%, it was also evident that there were statistically significant differences with a p < 0.001. Finally, when comparing the ethanol and hydroalcoholic extracts with the control group (chlorhexidine 0.12%), statistically significant differences were found (p < 0.001). In relation to the comparison of the negative control group (alcohol 96%) and positive control group (chlorhexidine 0.12%), there were also statistically significant differences between these groups. The latter being the one that had the greatest antimicrobial effect p < 0.001.

DISCUSSION

Biofilm is a layer of homologous extracellular polysaccharides produced by some strains of S. aureus. This extracellular network serves as an aid for the adhesion of the bacterial community to different surfaces. It is also involved in the persistence and colonization of this bacterium in probes, prostheses and catheters. This biofilm can prolong the time of colonization and infection, as well as the dispersion to different parts of the human body.^4,5,7 Infections develop from the deepening of this bacterium by a solution of tissue continuity due to trauma, skin or surgical lesions. These infections generate purulent material forming abscesses and can cause minor infections of the skin and soft tissues and invasive infections such as: infections of the central nervous system, respiratory, gastrointestinal and urinary tract, bacteraemia and osteomyelitis.^16–20

In the present investigation the antibacterial activity of different mango leaf extracts on S. aureus strain ATCC 6538 was evaluated, the ethanolic and hydroalcoholic extracts were used at 50% and 100% dilutions on bacterial cultures, using the diffusion method from Kirby–Bauer wells and it was found that in effect the four dilutions have extremely sensitive activity, there are significant differences between them and in comparison with the positive control (chlorhexidine 0.12%). The results obtained in this research agree with similar studies carried out by other authors.

For example, in the study conducted by Madduluri et al.²¹ the antibacterial activity of the leaves of five native plants was determined, including Mangifera indica L., comparing the effects of methanol and ethanol extracts and using the diffusion agar disk method in five different strains, including S. aureus. It was found that the five strains showed sensitivity to the extracts of the five plants being one of the most susceptible S. aureus with halos of 11.5 mm for the ethanolic extract and 15 mm for the methanolic extract of Mangifera indica L. concluding an antibacterial activity positive of the mango leaves coinciding with the present study. However, smaller halos were obtained here and a concentration of 10 mg/mL was used with native leaves of India while in the present study concentrations of 50% and 100% were used with native leaves of Peru. Similarly, Diso et al.²² evaluated the antibacterial activity of leaves and bark of mango tree native to Nigeria with aqueous extracts and chloroform using the diffusion method on methicillin-resistant S. aureus and verified that the extracts possess antimicrobial activity since they formed inhibition halos between 11 mm and 17 mm around the wells at 120 mg/mL which classifies this activity as very sensitive coinciding with the present work since that a positive antibacterial activity was found, although concentrations of 50% and 100% were used in the present study and the inhibition halos were between 21.38 mm and 33.5 mm, these results suggest a sensitivity of these microorganisms to the evaluated plant. And further Diso et al. used a strain resistant to methicillin. Another study that found similar results was that described by Anand et al.²³ who made an ethanolic extract of walnut and mango leaves at 20 mg/mL in India and applied it on oral bacterial strains including S. aureus with the diffusion method, which showed inhibition halos with an average of 14.67 mm that according to the Duraffourd scale, the bacterium is very sensitive to the antibacterial applied, thus verifying the effectiveness of the extract, possibly being attributed to the presence of bioactive compounds. This is consistent with the present study since it also used ethanol as a solvent although finding halos larger than 21.38 mm and 24.13 mm at 50% and 100%, respectively being interpreted as highly sensitive. All these results are consistent with those described in the present study since we also found an antibacterial activity of the mango based ethanol and hydroalcoholic extract.

Within the main limitations of this research, we have found that when performing two types of extracts and several concentrations, the demand for supplies and reagents was extremely considerable, so this effect could not be proven against other oral germs. In addition, the low availability of strains pure bacterial, limited this study. On the other hand, another limitation was the need to travel to the North of Peru to get this natural resource and transfer it to the capital (Lima), so time and logistics resources had to be invested to guarantee the correct transport of this plant to the microbiology laboratory.

The importance of this study is that it was verified that the leaves of the mango tree are found in large quantities in Northern Peru, and these have the ability to act as an antibacterial agent against a strain found in greater percentage in certain diseases such as osteomyelitis and other bacteremia. In addition, it had a methodological justification because it identified pharmacological effects and contributed scientific evidence to the profile of the mango plant on the S. aureus ATCC 6538, so that it motivates the elaboration of subsequent investigations. Finally, this plant can be used as a possible alternative source for the production of new antibacterial agents of natural origin and accessible to the population.

CONCLUSION

It was determined that the ethanolic and hydroalcoholic extracts of the 50% and 100% mango leaves formed halos of inhibition at 24 hours against the strains of S. aureus ATCC 6538. In addition, when comparing the extracts, the 100% hydroalcoholic evidenced a halo of greater inhibition with statistically significant difference compared to the other concentrations of the extracts evaluated.

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