Ethnomedicinal Studies, Chemical Composition, and Antibacterial Activity of the Mammea americana L. Bark in the Municipality of Cértegui, Chocó, Colombia

Mammea americana L. is a plant with diverse medicinal uses in the municipality of Cértegui, Chocó, Colombia. This research characterized the ethnomedicinal, chemical, and antibacterial activities of the bark of Mammea americana. Through interviews and semistructured surveys with the community, its ethnomedicinal uses were determined. Compounds present in the bark extract were identified and quantified by gas chromatography-coupled to mass spectrometry (GC-MS), and a qualitative analysis was performed by preliminary phytochemistry. Antibacterial activity and minimum inhibitory concentration (MIC) were carried out by agar diffusion and dilution methods, respectively, using ethanolic and aqueous extracts. Ethnomedical data showed that the bark is used to treat 14 conditions, the most representative being gallstones, prostate inflammation, and malaria. Preliminary phytochemical analyses showed the existence of several secondary metabolites such as tannins, alkaloids, flavonoids, triterpenes and/or steroids, quinones, and saponins. A total of 29 compounds were identified; the most abundant were ethyl 5-oxo-4-(p-toluidine)-2,5-dihydro-3-furancarboxylate, phenol, 4,4′,4″-ethylidynetris, nerolidol, 19-hydroxy-13-epimanoyl oxide, α-elemene, and δ-cadinene. The results showed remarkable antibacterial activity of the ethanolic extract (20 mg/ml) against Staphylococcus aureus (22.6 mm) and Escherichia coli (19.6 mm) and of the crude water extract (20 mg/ml) against Staphylococcus aureus (18.5 mm) and Escherichia coli (12.4 mm). The strongest MIC was for the ethanolic extract with values of 0.357 and 0.897 mg/ml against S. aureus and E. coli strains, respectively, while in the aqueous extract, S. aureus (3.99 mg/ml) and E. coli (4.3 mg/ml) were recorded. It is assumed that the compounds identified in this study could be responsible for the antibacterial activity of the species, as well as the relationship of the identified compounds and metabolites with the ethnomedical uses given by the community, providing a scientific and traditional basis for its different traditional medical uses.


Introduction
ere is a pressing need to develop new and innovative antimicrobial agents. Volatile secondary metabolites are one of the main antimicrobial compounds in plants [1] because the number of resistant microorganisms has increased and is becoming one of the greatest threats to global health affecting people of any age and anywhere in the world [2].
e WHO data show that 25% of deaths worldwide are caused by bacterial infections due to the difficulty to identify the microorganism for treatment [3].
Plants throughout the development of science have played a fundamental role in the search for compounds or extracts with medicinal characteristics. Herbal therapeutic solutions play an essential role in the primary healthcare of 80% of the world's population [4]. e plants produce secondary metabolites, which are an important source of drugs, especially in traditional medicine [5]. Secondary metabolites are responsible for multiple biological activities and may contribute to the treatment of diseases of bacterial origin such as gastroenteritis, diarrhea, urinary tract infections, sepsis, pneumonia, neonatal meningitis, and peritonitis [6][7][8].
Mammea americana L. (family Calophyllaceae) is a tree native to the West Indies and northern South America. It is cultivated in the Bahamas and, on a smaller scale, in Venezuela, Guyana, Suriname, French Guiana, Ecuador, and northern Brazil. In Colombia, it is found in humid forests, such as the Department of Chocó, and in temperate and warm zones, such as the Department of Cundinamarca. e Mammea americana L. tree can reach up to 25 m in height. Its trunk and branches are covered with a dark brown, rough bark. e fruit is a big flattened spherical dupa that can reach 10-25 cm in diameter wrapped in a thick brown skin with a leathery appearance [9]. It is commonly known as mamey, mamey apple, mamey de Santo, South American apricot, abricó-do-pará, abricó, Santo Domingo apricot, mamey amarillo, mamey de Cartagena, mata serrano, zapote mamey, and zapote de Santo Domingo [10,11].
is plant was included in Tramil, a comprehensive study of the medicinal plant resources of the Caribbean [12]. All its parts can be used for the treatment of various diseases, with differences in medicinal preparations depending on the country [13]. To these plants are attributed properties such as antioxidants, antidiabetic, antimalarial, fungal infections, eczema, skin diseases, treatment of periodontal diseases, indications as convulsive tonic, parasitic, against stomach pain, anthelmintic, and digestive diseases, and antibacterial [14][15][16][17][18][19][20]. Studies have shown the potential of Mammea americana bark to treat gastrointestinal disorders, including gastric ulcers in ethanolic, methanolic, and dichloromethane extracts [18]. Antimicrobial activity performed on crude ethanolic extract of endophytic fungi of M. americana showed that it has antimicrobial effects on S. aureus and E. coli [21]. Similarly, it has been reported that the ethanolic extract of the leaves and bark of this plant also possesses antibacterial activity against S. aureus [22], while its seeds were shown to have inhibitory activity for E. coli, Pseudomonas spp., and Salmonella ssp. [23].
Information on the phytochemicals present in M. americana; it was found that the ethanol extract of the plant contained alkaloids, coumarins, phenols, tannins, quinones, and flavonoids in leaves and bark [22] and terpenoids, phenolic compounds, and flavonoids in the pulp of their fruit [20,24,25]. e objective of this investigation is the first to determine the medicinal uses that the bark of these plants presents through a set of cultural and central knowledge of the community, for the different inhabitants of the municipality of Cértegui. Subsequently, we relate the medicinal uses with an analysis of the secondary metabolites identified by preliminary phytochemical analysis and gas chromatographycoupled mass spectrometer (GC/MS). Finally, the antibacterial activity of the extract from M. americana bark will be evaluated against four bacterial strains, two Grampositive and two Gram-negative. In this way, we intended to establish a relationship between the medicinal uses granted by the community, the antibacterial activity of the extract, and the identified secondary metabolites, which is important to scientifically justify traditional knowledge, giving added value to these plants.

Study Area.
is research was performed in the municipality of Cértegui, Department of Chocó, Colombia ( Figure 1). It has an area of 342 km 2 , located at 5°41′41″ north latitude and 76°39′40″ west longitude, with an altitude of 43 m above the sea level. e annual precipitation is 7000 mm; its average temperature is 28°C. It presents a topography characterized by low hills, according to the classification system reported by Rangel [26]. It is bordered to the north by the municipalities of Atrato and Lloró, to the south by the municipalities of Unión Panamericana and Tadó, to the east by the municipalities of Lloró and Bagadó, and to the west by the municipalities of Río Quito and Cantón del San Pablo [27].

Ethnomedical Phase and Data Analysis.
e ethnomedicinal information of the cortex of the species Mammea americana L. was obtained through interviews and semistructured surveys. e most relevant data taken into account by the interviewees were sex, age, and occupation to ensure that the medicinal use of the plant species came from the traditional knowledge of the community. e medicinal information of the plant was validated as in the studies reported in the literature. e data obtained were processed in Microsoft Excel, 2016. e plant material (bark) was pressed and transferred to the herbarium of the Universidad Tecnológica del Chocó Diego Luis Córdoba for taxonomic identification, presenting registration number 15142.

Preparation of the Extract.
e bark was washed and disinfected with a 2% NaClO solution. It was air-dried at room temperature for seven days. en, it was submitted to a temperature of 40°C, in an oven with circulating air (Memmert 854, Schwabach, Germany), always verifying humidity loss (three days approximately). e cortex was ground to a fine powder and then weighed. e crushed and weighed plant material was then subjected to cold maceration for three days in 96% ethanol. Successive concentrations of this extract were made at reduced pressure in a digital rotary evaporator (model R-124, vacuum controller V-800, Buchi) at 40°C to obtain an ethanolic extract, which was stored at 4°C until the phytochemical screening was performed. . e extraction of the volatile compounds of the vapor phase of the sample was performed through the technique of solid-phase microextraction (SPME), with monitoring in the vapor phase (head space), using a fused silica fiber coated with polydimethylsiloxane-divinyl benzene of 65 μm thickness (PDMSDVB-65 μm, Sigma) [30]. e chromatographic analysis was performed in a 6890 Series Plus Gas Chromatograph (Agilent Technologies, USA), coupled to a mass selective detector (Agilent Technologies MSD 5973, USA). e column used for the analysis was DB-5MS (5% phenyl poly (methylsilane), 30 m × 0.32 mm x 0.5 μm, Agilent); injection was performed with the SPME device. Identification of secondary metabolites was established based on their mass spectra (EI, 70 eV). e databases used were NIST98.l, NIST02.L, and NIST5a.L from the laboratory in question. ree biological replicates were used, and all analyses were done in duplicate.

Phytochemical
2.6. Antibacterial Activity Assessment. Antibacterial activity was evaluated using the agar diffusion method [31]. In this study, four bacterial strains were tested, two Gram-positive bacteria (Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 25923) and two Gram-negative bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 13076). e bacterial strains were first cultured in the Mueller-Hinton medium at 37°C for 24 hours before planting on nutrient agar. e inoculums of the bacteria used were stained on Mueller-Hinton agar plates using a sterile swab. e dilutions of the aqueous and ethanolic extracts used were 20, 10, 5, 2.5, and 0.5 mg/ml. e paper discs used were 5 mm, with the amount of extract in each disc 20 μl, the thickness of the agar 4 mm, and were placed on the surface of the agar, pressing gently on the surface of the agar with sterile forceps. Before incubating at 37°C, the plates were refrigerated at 4°C for three hours to allow the oil to diffuse. e positive control was streptomycin sulfate 10 μg/ml, and the negative control was TSA (trypticase soy agar). After the incubation period (24 hours), the diameters of the inhibition halos produced were measured. All tests were performed in triplicate, and the means were calculated as final results. To determine the sensitivity of the Mammea americana L. bark extract, it was classified by the diameter of the inhibition halos, following the methodology: not sensitive (−) for diameters below 8 mm, Advances in Pharmacological and Pharmaceutical Sciences sensitive (+) for diameters between 9 and 14 mm, very sensitive (++) for diameters ranging from 15 to 19 mm, and extremely sensitive (+++) for diameters above 20 mm [32].

Determination of Minimum Inhibitory Concentration (MIC) of Crude Extracts.
e MIC was performed on the ethanolic and aqueous extracts that showed bactericidal activity in the antibacterial activity evaluation test, i.e., the minimum concentrations of the extract at which no growth was observed in the solid medium. e minimum inhibitory concentration of the crude extracts of the bark of M. americana L. was determined by the agar dilution method. e culture media after preparation were sterilized by autoclaving. e sterilized culture media were allowed to cool to 50°C, and 20 ml of molten agar was added to test tubes containing 2.0 ml of different concentrations (aqueous extract, ethanol extract, and control). e concentrations used ranged 0.0020-5.0 mg/ml. Suspensions of the respective bacteria having density adjusted to McFarland 0.5 turbidity units (1.5 × 10 8 CFU/ml) were inoculated onto a series of agar plates using a standard loop. e dilution series was prepared using broth microdilution (1 : 100). Incubation was performed at 37°C for one day. e experiments were performed in duplicate and repeated three times (n � 3). e lowest concentration of the crude extract of M. americana was expressed as MIC in mg/ml [32].

Data Analyses.
e experimental data were expressed as the mean of three repetitions and their standard deviation. Data analysis was performed using GraphPad Prism 9 statistical program. Analysis of variance (ANOVA) was used with Tukey's multiple comparisons test, with a 95% confidence level. A p value of less than 0.05 was considered statistically significant.

Ethnomedical Phase.
Traditional applications of the plant species by community residents in medicine show that the bark is used to treat diseases related to gallstones and kidney stones, prostate, and malaria, among others; it is also used as a laxative. Table 1 presents the ethnomedicinal information and demographic characteristics of the people surveyed, which were 110, including men and women, with women being the ones who know the most about the plant. e age of the participants varied between 21 and 80 years (mean age 50.5 ± 17.4 years), with the highest range being between 41-50 years and 51-60 years. Regarding the occupation or economic activity they performed, most of them stated that they are dedicated to mining and in the second instance to the exploitation of wood.

Phytochemical Screening.
e presence of secondary metabolites was performed according to the individual qualitative rather test for each group. Table 2 provides the results of the phytochemical screening performed on the ethanolic extract of the bark of M. americana L., where a high diversity of secondary metabolites is observed in this species, such as tannins, alkaloids, flavonoids, triterpenes and/or steroids, quinones, saponins, and sapogenins. It can be said that these bioactive components could be responsible for the bioactivity demonstrated in the ethanol extract.
A compound by itself or in combination with others likely contributes to the bioactivity present in the extract.

Antibacterial Activity Assessment.
e antibacterial activity of M. americana L. against bacterial strains was qualitatively evaluated through the diameter of the inhibition zone. e antibacterial activity was analyzed in ethanolic extract, and then, it was proceeded to evaluate its general bioactivity in aqueous extract (crude water), in order to simulate the traditional preparation used by the community of the municipality of Cértegui. e antibacterial activity of M. americana L. against the bacterial strains tested was qualitatively evaluated through the diameter of the inhibition zone. e results indicate that the ethanolic extract from the bark of the plant exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli strains but in a concentration-dependent manner. e positive control, streptomycin control, was optimal in this test. e highest inhibition zone diameters were recorded for the S. aureus strain (22.6 mm), showing the strong activity of the plant bark which has extremely sensitive activity against this strain. e concentrations evaluated in this strain presented diameter ranges between sensitive and extremely sensitive; on the other hand, the E. coli strain inhibition diameter ranged from nonsensitive to very sensitive as the extract concentration decreased, registering a value of 6.4 mm for the minimum concentration used. e results of this activity are presented in Table 4.

Minimum Inhibitory Concentration (MIC) of the Crude Extracts.
e effectiveness of ethanolic and aqueous extracts of M. americana was evaluated using the minimum inhibitory concentration (MIC) in those extracts that showed significant antibacterial activities, i.e., those extracts that showed halos greater than 8.0 mm in the zone of inhibition. Bacteria were added to diluted ethanolic and aqueous extracts at concentrations ranging from 0.0503 mg/ ml to 0.191 mg/ml (Table 5). e strongest MIC was for the ethanolic extract with values of 0.0503 and 0.074 mg/ml against S. aureus and E. coli strains, respectively. e results also showed that the most significant MIC was against S. aureus.

Ethnomedical Information.
In general, plant species are used in ethnomedical practices for the treatment of different conditions; however, a large part of the world's plant population remains unexplored [33]. M. americana is used within traditional medicine in the municipality of Certeguí, Department of Chocó, Colombia, for the treatment of different diseases. Within the population surveyed in this study, there was a predominance of women over men to collect information on the plant; this is because the work activities    Table 1; however, the results allowed us to affirm that the people surveyed acquired knowledge about this plant by the teaching of their parents or grandparents. According to Manya et al. [37], ethnomedicinal practice can be performed by men or women and is mainly practiced by adults and older people. On the other hand, [38] express that the family context is common in herbalists or ethnomedical practitioners, recognizing this knowledge as a family legacy, where the family is considered a social unit that subsists in an everyday life in which customs, norms, and practical knowledge such as traditional medicine are transmitted. However, some also state that their knowledge comes from years of experience and from their natural space where they have related with coworkers, capturing the knowledge of others who use medicinal plants [39].
Similar studies have been conducted in Chocó where plants such as Jacaranda caucana Pittier (Gualanday) (family Bignoniaceae) are used as infusion and/or plaster by the communities of Tutunendo and Villa Conto for rheumatoid arthritis pain, Columnea cruenta Morley (chicken's blood) (family Gesneriaceae) is used in the form of infusion by the community of Novita (Region of San Juan) for the relief in renal affections, and Besleria barclayi L.E. Skog (Sparkling) (family Gesneriaceae) is used by the community of Pacurita (Quibdó) as a disinfectant agent, for menstrual ailments and problems of sexual impotence [40,41].
Since ancient times, humans have been using traditional medicine, either through herbal preparations, to treat, manage, and cure various ailments. erefore, this type of medicine provides a solid foundation for the discovery of new agents against health-threatening pathogens [42].

Phytochemical Screening.
e medicinal properties of this plant are associated with the biological activities of one or a group of its compounds. Characterization of the active compounds present in the ethanolic extract of the bark was performed by qualitative phytochemical screening. Some of the secondary metabolic chemicals have matched the report of Rodriguez et al. [22], alkaloids, tannins, quinones, and flavonoids. In this investigation work, the presence of alkaloids, phenols, and tannins, quinones, and flavonoids in the extract of its leaves was also reported. Similarly, the studies of [18,20,23] indicated that flavonoids, alkaloids, saponins, and tannins were present in the fruit of this plant. e presence of alkaloids, flavonoids, saponins, and quinones in the seed has also been determined [23]. In general, phytochemical investigations of various parts of M. americana have revealed the presence of flavonoids, terpene compounds, and volatile compounds [43]. Phytochemical studies in Mammea species have reported the presence of triterpenes and pentacyclic steroids [44]. Flavonoids, saponins, and alkaloids are reported to be present in the aqueous and ethanol extracts of M. suriga root bark [45].
Community surveys and interviews conducted in this research could attribute analgesic, anti-inflammatory,    Advances in Pharmacological and Pharmaceutical Sciences antimalarial, antibacterial, antianemic, anthelmintic, or laxative properties. However, it is necessary to determine other biological activities in future studies to scientifically validate the community's knowledge. In agreement with the above, other studies have shown that this plant has antibacterial, antiulcerogenic, and antimalarial activities in the leaves and bark [14,18,22,52], antioxidant activity in the fruit pulp [24], and antitumor activity in the oil of its seeds [53].

Advances in Pharmacological and Pharmaceutical Sciences
Although phytochemical analysis provides important qualitative data, it is not a reliable tool to establish a relationship between ethnomedical use and secondary metabolites present in the plant. From this point of view, GC/MS was used, which provides more precise information on the constituents of the plant bark.

Quantification of Identified Compounds by GC/MS.
e compounds identified within their chemical structure present carbon chains or rings, which make them not very soluble in water, giving them lipophilic characteristics, and they could be identified by GC-MS.
In our study, the alkaloids were the most abundant compounds. Alkaloids are medically known as stimulants, local anesthetics, antibacterials, antihypertensives, and analgesics [54], relating it to the different ethnomedicinal uses of our research (menstrual cramps, toothache, abdominal pain, and pain produced by gallstones and kidney stones). Interestingly, among the compounds identified, the alkaloid ethyl 5-oxo-4-(p-toluidino)-2,5-dihydro-3-furancarboxylate was the main compound in the bark of the plant, which is strange, since there is no information about this compound. However, to establish a relationship between the biological activities of the compound and the ethnomedicinal information, the alkaloids of the p-toluidine ring, such as p-toluidine 4-(3-aminopropyl) morpholino and p-toluidine methylamino, possible antimalarials, were taken as reference [55].
Phenolic compounds have been implicated as important antioxidants in natural products, since they possess an ideal structural pattern for free radical stabilization [63]. Among phenolic compounds, phenol 4,4′,4″-ethylidynetris was identified, which reports antiestrogenic activity in vivo [64]. Estrogens are considered one of the main risk factors for the development of breast and uterine cancer [65].
Terpenoids were the group with the highest number of compounds identified (17) ( Table 3). Terpenes are known for their antimicrobial or bactericidal, analgesic, and anti-inflammatory activities [48,50,66]. Nerolidol is a compound with antimalarial, antiparasitic, and antileishmanial activities [67][68][69], which could be related to the diseases reported by the community in this study, such as malaria and intestinal parasites. Other compounds identified were α-elemene and δ-cadinene, which have anticancer activity in both in vitro and in vivo models [70] and antimicrobial, antifungal, and larvicidal activities against malaria, dengue, and filariasis mosquitoes [71][72][73], respectively. Squalene has activity against high cholesterol, is an antioxidant, and inhibits the development of several tumors [74,75]. Although most of the compounds identified within the terpenes group present relative amounts lower than 1%, they could contribute to different activities considering they can act in synergy with other compounds. e labdane-type diterpene compound, 19-hydroxy-13epimanoyl oxide, was identified with a relative amount of 4.28%. is compound has been revealed to have antimicrobial and antifungal activities in plants such as Cistus creticus L. (Cistaceae) [76]; the essential oil from the leaves of this plant has, among its main constituents, 13-epimanoyl oxide, showing strong microbial activity against strains of S. aureus and Bacillus subtilis [77], matching those reported for the antibacterial activity of M. americana, compared to the strong activity exhibited by S. aureus. Moreover, this activity coincides with the data provided by the studied community for the treatment of intestinal parasites.
Finally, groups such as fatty acids and phenylpropanoids only present one compound and in amounts less than 1%; ethyl oleate has been reported to have significant larvicidal, repellent, ovicidal, and adulticidal properties against Tetranychus cinnabarinus [78]. e results of this study are important for future research, as most of the identified compounds do not report their biological activities. Although most of the identified compounds are related to antimalarial activity, it is necessary to perform this activity in a way that it verifies the medicinal use of this plant for treatment, since compounds that are in concentrations above 4%, such as nerolidol and ethyl 5-oxo-4-(p-toluidino)-2,5-dihydro-3-furancarboxylate, are assumed to be the main contributors to this activity. e results of the compounds identified by GC/MS provided information that allowed relating them to ethnomedicinal use and biological activities. Ethnomedicinal uses such as pain caused by gallstones and kidney stones, toothache, abdominal pain, menstrual cramps, and prostate Advances in Pharmacological and Pharmaceutical Sciences inflammation were related to analgesic and anti-inflammatory activities, malaria to antimalarial activity, intestinal parasites to antiparasitic, anthelmintic, antibacterial, and/or antimicrobial activity, and ovarian cysts to antioxidant, anticancer, and antitumor activities. It is necessary to isolate the compounds and test their activity individually and/or in combination to see the correlative effect between them.

Antibacterial Activity Assessment.
e traditional approach of preparing the extract using water was used, which produced less antibacterial activity than the preparation with the ethanol extract. is could be due to the polarity of the solvents used (water and ethanol), where the solubility of various plant compounds in solvents of different polarities is involved. It is established that the polarity of the solvent affects the qualitative and quantitative compositions of bioactive compounds enriched in various solvents [79], which is reflected in this study, since ethanol is an extractant more powerful than water, thus justifying the high inhibition zones of the ethanol extract. e bark extract of M. americana L. showed the capacity to inhibit the bacterial growth of Staphylococcus aureus and Escherichia coli, with S. aureus having a greater activity in each of the concentrations used, coinciding with other studies conducted on the plant, where the extracts were more active against Gram-positive bacteria than Gramnegative bacteria [21,22]. e behavior in the inhibition zone for the ethanolic and aqueous extracts was similar, since in both extracts the diameter of the inhibition halos decreased as the concentrations of the extracts decreased.
As the ethanolic extract produced higher inhibition activity than the raw water extract in the worked concentrations, a significant difference was shown against the worked concentrations in each of the extracts, which were the same for each of the bacterial strains, in that sense, as for the S. aureus strain, evaluating the concentrations of the ethanolic extract vs. the aqueous extract (ANOVA: 20 mg/ ml, mean diff. 4.067, p � 0.0018; 10 mg/ml, mean diff.: 3.433, p < 0.0001; 5 mg/ml, mean diff.: 2.967, p � 0.0117), and E. coli (ANOVA: 20 mg/ml, mean diff.: 7.200, p < 0.0001; 10 mg/ml, mean diff.: 6.667, p � 0.0021; 5 mg/ml, mean diff.: 5.067, p � 0.0154).
Although the concentrations of the extracts used were in the range of 10 2 times more than the antibiotic used as a positive control (streptomycin), the extracts showed remarkable antibacterial activity, as shown by their inhibition zones, highlighting the value of 22.6 ± 0.1 for the case of S. aureus and 19.6 ± 0.17 for the case of E. coli. e above could be due to the active components of the extracts, which present only a fraction of them, deducing that, if the components are isolated and purified, they could show greater antibacterial activity, represented through their inhibition zone. It is expressed that the concentration of the active components in the extracts could be much lower than the standard antibiotics [72]. In the strains of B. subtilis and P. aeruginosa, there was no antibacterial activity for any of the plant fractions, which could be related to the low permeability of the cell wall to antibacterial agents [22].
Similarly, Mosquera et al. [21] reported that endophyte fungal extracts of the leaves and seeds of this plant inhibited the growth of Staphylococcus aureus and Escherichia coli strains by producing inhibition zone diameters ranging 12-22 mm and 12-23 mm, respectively. Similarly, in the work of Rodriguez et al. [22], the cortex was only active against the S. aureus strain, with inhibition halos of 17 mm with a concentration of 780 mg/ml. e antimicrobial activity of the ethanolic extract of M. americana showed higher inhibition zones against Grampositive bacteria, specifically against the bacterial strain S. aureus, with inhibition zones (22.6 mm) very close to those demonstrated by the positive control, standard drug (streptomycin: 28.4 mm). Dissanayake et al. [33] indicated that the root bark of P. indica has Plumbagin as an active compound, revealing a strong antimicrobial activity in the methanolic extract against Gram-positive bacteria S. aureus, with an inhibition zone around 24 mm, a value equivalent or similar to that obtained in the assay with the standard drug chloramphenicol, with an inhibition zone of 28.0 mm. Gram-positive bacteria are more susceptible to antibacterial activity. is resistance of Gram-negative bacteria could be due to the low permeability of the cell wall to bacterial agents, high genetic capacity to express a wide repertoire of resistance mechanisms, by mutations in chromosomal genes that regulate the expression of resistance genes and by the acquisition of foreign resistance genes via plasmids, transposons, and bacteriophages [80].
In addition to the above, previous findings of another species of the Mammea genus, Mammea suriga, exhibited strong antibacterial activity in aqueous and ethanolic extracts showing zones of inhibition between 32 and 33 mm against S. aureus; on the other hand, a zone of inhibition between 20 and 25 mm was presented against B. subtilis; as for the bacterial strain E. coli, a zone of moderate inhibition was registered in all the extracts in which they were evaluated [45].
e results show that this antibacterial activity could be related to the secondary metabolites found in this study, such as tannins, alkaloids, flavonoids, terpenes, and quinones [7,47,48,81,82]. e antibacterial activity could be justified by the presence of the compounds identified in this study. Compounds, such as nerolidol, δ-cadinene, caryophyllene, calarene, caryophylladienol II, epizonarene, and c-gurjunene, present this activity against S. aureus and E. coli in several groups of plants, which may be due to the synergistic effect of the components found [49,[83][84][85][86][87][88].

Minimum Inhibitory Concentration (MIC) of the Crude
Extracts. MIC is considered a fundamental parameter for testing the sensitivity of a bacterium to an antibacterial [89]. e minimum inhibitory concentration performed in this study showed that aqueous and ethanolic extracts of M. americana exhibited strong activity against S. aureus and E. coli strains. e MIC of the extracts ranged from 0.0503 to 0.191 mg/ml. Among the two bacterial strains tested, S. aureus proved to be more sensitive, with a MIC of 0.0503 mg/ml for the ethanolic extract. According to Avellana et al., a bacterial strain is very sensitive when the substance tested has a MIC below 12.5 mg/ml, medium sensitivity between 12.5 and 50 mg/ml, and low sensitivity for values between 50 and 100 mg/ml [90]. In this order of ideas, the results obtained in this study indicate a high bacterial sensitivity in the extracts evaluated, since the MIC values were below 12.5 mg/ml, thus determining the antibacterial potential of the species. However, Kuete states that the activity of crude extracts is significant if MIC values are below 100 μg/ml, moderate when 100 < MIC < 625 μg/ml, or low when MIC > 625 μg/ml [91], which would indicate that in this study, the bacterial activity would be between significant and moderate, in the ethanol and aqueous extracts, respectively. Few articles have reported the antibacterial potential of M. americana. Mosquera et al. demonstrated that the endophytic fungi found in the leaves and seeds of this species possess bactericidal characteristics against S. aureus and E. coli, with MIC ranging from 1.0 μl/ml to 2.0 μl/ml [21]. Bactericidal activity is reported in fruit peel extracts with hexane, ethyl acetate, and acetone and seed extracts in methanol against S. aureus, with MIC of 8, 16, 4, and 2 μg/ ml, respectively [52,92,93].
Among studies conducted in other Mammea species (M. africana), the extract of this plant and its active component Mammea A/AA did not inhibit the growth of E. coli but did inhibit the growth of other bacteria such as Streptococcus pneumonia, Clostridium difficile, and Campylobacter jejuni, with MIC ≤ 2 μg/ml [94]. e presence of lipopolysaccharides in the outer membrane of Gram-negative bacteria prevents the presence of hydrophobic antimicrobial agents, so they are less susceptible to extracts or essential oils containing hydrophobic antimicrobial compounds [95].

Conclusions
is research presents different ethnomedical uses of the bark of M. americana L. in the municipality of Cértegui. Diseases or symptoms such as gallstones, prostate inflammation and pain, malaria, and intestinal parasites are the most representative.
e phytochemical analysis carried out confirmed the existence of different secondary metabolites in its ethanolic extract, such as tannins, alkaloids, flavonoids, triterpenes and/or steroids, quinones, saponins, and sapogenins. In addition, 29 compounds were identified and quantified. e information given by the phytochemical analysis followed by its identification and quantification served to associate these results with the medicinal uses given by the community under study; thus, the analgesic and anti-inflammatory activities were related to diseases such as pain caused by kidney stones, gallstones, toothache, menstrual cramps, inflammation and pain in the prostate, antimalarial activity of malaria, and antitumor activity of ovarian cysts.
e antibacterial activity carried out on the extract of the bark of Mammea americana showed a significant effect against the bacterial strains Staphylococcus aureus and Escherichia coli. e presence of identified compounds in this plant, such as nerolidol, δ-cadinene, caryophyllene, calarene, caryophylladienol II, epizonarene, and c-gurjunene, could scientifically justify this activity; however, work is required such as the isolation and purification of the compounds, in such a way that it is possible to decipher the mechanism of action.
Further studies are needed to validate the industrial applications of M. americana L. for medicinal use. It is recommended to carry out more research studies on the pharmacological properties of this species in vivo, which allows validating the different activities it presents according to the medicinal reports given by the community, which allows associating the disease with antimalarial, analgesic, anti-inflammatory, and antimicrobial activities.
Data Availability e data used to support the findings of this study are included within the supplementary information file.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Lina Mosquera-Chaverra performed the experiments, data/ evidence collection, and wrote the initial draft. Manuel Salas-Moreno provided reagents, materials, instrumentation, and computing resources for mass spectrometry analysis and did the critical review, commentaries, and revision for the pre-publication stage. Jos'e Marrugo-Negrete did the critical review, commentaries, and revision for the pre-publication stage and formulated the evolution of overarching research goals.