Antibacterial and antioxidant activities of plants consumed by western lowland gorilla (Gorilla gorilla gorilla) in Gabon

Zoopharmacognosy is the study of the self-medication behaviors of non-human animals that use plant, animal or soil items as remedies. Recent studies have shown that some of the plants employed by animals may also be used for the same therapeutic purposes in humans. The aim of this study was to determine the antioxidant and antibacterial activity of Ceiba pentandra, Myrianthus arboreus, Ficus subspecies (ssp.) and Milicia excelsa bark crude extracts (BCE), plants consumed by western lowland gorillas (Gorilla gorilla gorilla) in Moukalaba-Doudou National Park (MDNP) and used in traditional medicine, and then to characterize their phytochemical compounds. DPPH (2,2-Diphenyl-1-Picrylhydrazyl), phosphomolybdenum complex and β-carotene bleaching methods were used to assess antioxidant activity. Antimicrobial susceptibility testing was performed using the diffusion method, while minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were assessed using the microdilution method. The highest level of total phenolics was found in Myrianthus arboreus aqueous extract [385.83 ± 3.99 mg [gallic acid equivalent (GAE)/g]. Total flavonoid (134.46 ± 3.39) mg quercetin equivalent (QE)/100 g of extract] were highest in Milicia excelsa, tannin [(272.44 ± 3.39) mg tannic acid equivalent (TAE)/100 g of extract] in Myrianthus arboreus and proanthocyanidin [(404.33 ± 3.39) mg apple procyanidins equivalent (APE)/100 g of extract] in Ceiba pentandra. Ficus ssp. (IC50 1.34 ±3.36 μg/mL; AAI 18.57 ± 0.203) ethanolic BCE and Milicia excelsa (IC50 2.07 ± 3.37 μg/mL; AAI 12.03 ± 0.711) showed the strongest antioxidant activity. Myrianthus arboreus ethanolic BCE (73.25 ± 5.29) and Milicia excelsa aqueous BCE (38.67 ± 0.27) showed the strongest percentage of total antioxidant capacity (TAC). Ceiba pentandra ethanolic BCE (152.06 ± 19.11 mg AAE/g) and Ficus ssp aqueous BCE (124.33 ± 39.05 mg AAE/g) showed strongest relative antioxidant activity (RAA). The plant BCE showed antimicrobial activity against multidrug resistant (MDR) E. coli (DECs) isolates, with MICs varying from 1.56 to 50 mg/mL and inhibition diameters ranging from 7.34 ± 0.57 to 13.67 ± 0.57mm. Several families of compounds were found, including total phenolic compounds, flavonoids, tannins and proanthocyanidins were found in the plant BCEs. The plant BCEs showed antioxidant activities with free radical scavenging and antimicrobial activities against 10 MDR E. coli (DECs) isolates, and could be a promising novel source for new drug discovery.


Introduction
Natural medicines from plants have been used to enhance human and veterinary health since time immemorial, as revealed in ancient tales, scriptures and other historical literature [1].This practice is experiencing a resurgence [1,2].The World Health Organization (WHO) estimates that approximately 80% of the world's population uses medicinal plants (MPs) for their health and care needs [3].Recent studies have focused on the potential to develop antioxidant and antimicrobial drugs from plants [4][5][6].These antioxidants, for example, reduce the incidence of many metabolic diseases [7].Antioxidants also reduce the incidence of chronic inflammation by reinforcing immunity [8], which would ultimately contribute to the efficacy of antimicrobial therapy.Antimicrobial agents are also used as antibiotics to control infections in the human body, but can cause many side effects, especially by increasing reactive oxygen species (ROS) [9].ROS are very dangerous to human health and well-being and can contribute to the development of cancer [10]; further, they are aggravating factors for the emergence of various other metabolic diseases [11].Finally, the use of these antimicrobial agents as antibiotic drugs can lead to resistance selection pressure in microorganisms such as bacteria [12].
Antimicrobial resistance is considered by the WHO to be one of the world's three greatest threats to human health because of the extensive spread of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) [13].Infectious diseases caused by multidrug-resistant (MDR) bacteria affect millions of people worldwide [14].Furthermore, many zoonoses caused by pathogenic microorganisms such as enterobacteria (e.g.; MDR Escherichia coli), have been a human public health problem for decades [15].E. coli is a Gram-negative bacteria and gut commensal in animals, including non-human primates (NHPs) [16].The close phylogenetic relationship between humans and other hominids, combined with a rapidly expanding human-animal interface, enables pathogen transmission across species, leading to morbidity and mortality in great ape populations throughout the world [17,18].Several studies have shown that wildlife [19,20], including wild primates such as western lowland gorillas (Gorilla gorilla gorilla), could transmit this kind of pathogens to humans [21,22], or that humans could transmit the pathogens to wildlife [23,24].This situation has great potential for the discovery of new antimicrobial agents [25,26].Many recent studies show that natural products from medicinal plants or plants consumed by animals continue to play a role in drug discovery and development [27,28].
Zoopharmacognosy is the study of non-human animals self-medicating, using plants, animals and natural compounds, such as soil, as a preventative or direct medicinal cure to regain health in their natural habitat [2,29].Most great apes, including western lowland gorillas (Gorilla gorilla gorilla) have a predominantly frugivorous diet [30,31].However, bark is the main fallback food for gorillas [32].These feeding practices appear to be beneficial to the wellbeing and health of these animals but also to those of humans [33].The use of great ape pharmacopoeia or zoopharmacognosy is a very promising strategy for management of human diseases because of the phylogenetic proximity of humans and great apes [34].Several studies have shown that plants from the diet of great apes, including western lowland gorilla, are also used as MPs by healers in traditional African medicine [35,36].
In view of the physiological (pathological, infectious) state of the gorillas in the PNMD, linked to the presence of potentially pathogenic enterobacteria, multi-resistant to antimicrobials used in human therapy, including MDR enterobacteria such as MDR E. coli (DECs) obtained in a previous study, how did these animals manage to host and control these microorganisms?In this study, we hypothesized that "the immunity-enhancing consumption of certain plant items (such as bark) by gorillas could be responsible for their ability to host and control these infectious microorganisms without developing serious disease".
Gabon, with its exceptional biodiversity, constitutes a vast reservoir of unexplored potential active biomolecules [37].This study aims to evaluate the chemical composition (secondary metabolites families), antioxidant and antimicrobial activities of four plant species consumed by gorillas living in MDNP to control microbial infections within their communities and, used as traditional MPs by healers in Gabon.Ceiba pentandra [38,39], Myrianthus arboreus [40,41], Ficus ssp [42] and Milicia excelsa [43,44] are the four plants selected for this study on the basis of ethnobotanical and ethnopharmacological surveys carried out among local populations.The ethnopharmacological activities of these four plants in traditional medicine have already been reported in recent literature.

Study area and field research authorization
Sampling of the bark of four plants consumed by western lowland gorillas was carried out under field research authorization N˚003/20/DG/JBLD/N˚306, from August 1 to 11, 2022 in MDNP, during the daily monitoring of gorillas by observing plants items they consumed in their natural environment during this period, using the non-invasive method as previously described [45].Research Institute for Tropical Ecology (IRET) provided all the necessary authorizations to carry out this study on the MDNP site.No special permits were required, due to the scientific and technical agreements established between the Centre National de Recherches Scientifiques et Techniques (CENAREST) and the Centre Interdisciplinaire de Recherches Me ´dicales de Franceville (CIRMF).

Data and sample collection
A survey was carried out among 27 inhabitants of the village of Doussala, including a number of traditional healers and herbalists, both men and women, known to the local peoples, using a questionnaire, as previously described by Obiang et al. [46].Fresh bark plant samples collected were identified by a team of botanists leading by Prof. Brama Ibrahim from the Department of Biology, Faculty of Sciences of the University of Science and Technology of Masuku (USTM) in Franceville (Gabon).Voucher specimens of Ceiba pentandra (BRLU/4618), Myrianthus arboreus (BRLU-LBV/8324), Ficus ssp (USTM/#443) and Milicia excelsa (USTM/739) has been deposited at the herbarium of the same institute.The choice of the bark of the selected plants was made based on local traditional medicinal use and the fact that gorillas also consumed them.

Therapeutic indications.
Descriptive statistical methods were used to analyze the ethnopharmacological survey data and various quantitative indices, including use value (UV) and relative frequency of citation (RFC).Data were reported in proportions and percentages [46].Use Value (UV) and Relative Frequency of Citation (RFC) were calculated according to the following formula: UV ¼ U=n and RFC ¼ FC=Nð0 < RFC < 1Þ:

Treatment of plant material
2.3.1.Extraction.From 100 g of bark powder of each selected plant, an extraction by maceration under agitation was carried out for 72 h with 2 L of each solvent (ethanol 99.8% and water).After filtration of the two (ethanolic and aqueous) maceration, using Whatman N˚1 filter paper, the aqueous extract was directly lyophilized, while the ethanolic extract was concentrated and dried in an oven.

Quantitative phytochemical analysis
2.5.1.Total phenol content.To determine the total phenol content, the Folin-Ciocalteu method was used [49].Absorbance was measured at 735 nm.Phenolic compounds were expressed as mg gallic acid equivalents (GAE) /dry weight of extract.
2.5.2.Total flavonoid content.Aluminum trichloride method was used to determine the flavonoid content and absorbance was measured at 435 nm.Flavonoid content was expressed in quercetin equivalent (QE) [50].
2.5.3.Tannin content.Tannin content was determined using the method described previously by Sima-Obiang et al. [51].Absorbance was measured at 525 nm and tannic acid was used as a standard.Tannin contents were expressed in mg of tannic acid equivalent (TAE)/g of dry extract.
2.5.4.Proanthocyanidin content.Proanthocyanidins were determined using the HCl-Butanol method [52].Absorbance was read at 550 nm and apple procyanidin was applied as standard.Proanthocyanidin levels were expressed in apple procyanidins equivalent (APE)/g of dry extract.[53], based on the DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical test, was used to determine the Antioxidant Activity Index (AAI).Briefly, DPPH solution was prepared by dissolving 10 mg of DPPH powder in 200 mL methanol ([DPPH] = 0.05 mg/mL).400 μL of each of the eight BCE (at 1mg/mL concentration) were added to 1.6 mL of methanol to obtain a stock solution.Cascade dilutions to 1/2 were made from the stock solution into test tubes containing 1 mL of methanol beforehand.Then, 1 mL of DPPH was added to each of the tubes.Absorbencies were measured at 517 nm after 30 min incubation at room temperature in the dark against a blank.Ascorbic acid (vitamin C) was used as reference.The ability to scavenge DPPH radical (RSA) was calculated by the following equation: % RSAðRelative Scavenging ActivityÞ ¼ ½ðA control À A sample Þ=A control � � 100:

Bioactive properties of bark crude extracts of selected species consumed by western lowland gorilla
A = Absorbance at 517 nm.The IC 50 (concentration providing 50% inhibition) of BCE and standards was determinate using regression curves in the linear range of concentrations.The AAI was then calculated as follows: AAIðAntioxidant Activity IndexÞ ¼ ½DPPH� fðmg:mL À 1 Þ=IC50ðmg:mL À 1 Þ: ½DPPH� f is the final concentration of DPPH: 2.6.1.2.Phosphomolybdenum complex method for total antioxidant capacity.Spectrophotometric evaluation of total antioxidant activity was carried out through the formation of a phosphomolybdenum complex.The assay was based on the reduction of Mo (VI) to Mo (V) and subsequent formation of a green phosphate/Mo (V) complex in acid pH [54,55].A total volume of 0.3 mL of each bark crude extract (at 5mg/mL concentration) dissolved in methanol was added to 3 mL of reagent solution (0.6 mol/L H 2 SO 4 , 28 mmol/L Na 3 PO 4 and 4 mmol/L ammonium molybdate).The mixtures were incubated at 95˚C for 90 min the cooled to room temperature.The absorbance was measured at 695 nm.The total antioxidant activity of each plant BCE was expressed as the number of equivalence of ascorbic acid (mg AAE/g).
2.6.1.3.β-Carotene bleaching assay.The β-carotene-linoleic acid model system is prepared by dissolving 0.5 mg β-carotene in 1 mL chloroform followed by the addition of 40 μL linoleic acid and 500 μL Tween-20.A rotary evaporator was used to completely evaporate the chloroform added into the system.To the resulting solution, 100 mL of oxygenated distilled water is added followed by vigorous shaking.500 μL of BCE (at 1mg/mL concentration) or antioxidant solution of the reference (Ascorbic acid; 1mg/mL concentration) was added to 2.5 mL of the previous emulsion.The absorbance was measured at 490 nm before and after heat treatment with regular time intervals for 48 h.The measurement of the absorbance continued until the color of β-carotene disappears [56].
2.6.2.2.Antimicrobial activities.To test the antimicrobial activity of BCEs and the antimicrobial susceptibility, antibiograms were performed using the Kirby-Bauer disk diffusion method, according to the CLSI (Clinical and Laboratory Standards Institute) protocols [59].The agar was suspended in distilled water, heated to complete dissolution autoclaved at 121˚C, and poured into Petri dishes.Whatman paper discs (6 mm) were inoculated with suspensions (10 8 CFU/mL) and dispersed on the surface of Mueller-Hinton agar plates (bioMe ´rieux, France) [60].Then, the discs were impregnated with 20 μL of each bark crude plant extracts.All plates were incubated for 24 h at 37˚C.The diameters of the inhibition zones were determined after incubation.Antimicrobial activity was estimated after incubation at 37˚C for 24 h by measuring the zone of inhibition against the tested microorganisms.Antimicrobials tested were amoxicillin/clavulanic acid (AMC, 30 μg) and gentamycin (GEN, 10 μg).Breakpoints provided by the CLSI were used for the designation of isolates as resistant (R), intermediately susceptible (IS) or susceptible (S).For subsequent data analysis, the isolates with an I result were grouped with the isolates that gave an R result and defined as resistant.Multidrugresistant isolates were identified based on the definition of MDR as bacteria that are resistant to three or more classes of antimicrobial agents [61,62].
2.6.2.3.Minimum Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) assays.In microplate wells (96 wells), 10 μL of each dilution of plant bark crude extracts varying from 30 mg/mL to 0.014 mg/mL were combined with Mueller Hinton broth (bioMe ´rieux, France) (170 μL) and bacterial inoculums (20 μL) and set to a final microbial concentration of 5 × 10 5 CFU/mL according to NCCLS standards methods, with some modifications [63].The ethanol content in each well was less than 3.5% in an overall amount of 200 μL.For the negative control, the same percentage of ethanol was used.The MIC is the lowest concentration that does not emit a red color after 2 h of incubation.To assess MBC, a portion of each well where the concentrations are > or = (MIC) was sub-cultured on Muller-Hinton agar (MHA) (bioMe ´rieux, France) and incubated for 24 h at 37˚C.The MBC is described as the extract concentration at which 99.9% of the inoculated bacteria were destroyed [64].

Data analysis
All tests were performed as triplicate and the results are showed present as the mean.Excel software for Microsoft was used to analyze data.Analysis of variance (ANOVA) followed by Student's tests were used to test for significant differences between means.Differences were considered statistically significant at p < 0.05.Adobe Illustrator software was used to plot the histograms.The Pearson correlation was determined between the antimicrobial and antioxidant activity of plant BCE and total phenolic, flavonoid, proanthocyanidin and tannin content.To visualize the correlation data, a heatmap of three colors: red (r = ─1), white (r = 0) and blue (r = -1) analysis was plotted with the packages Gplot using R 4.0.2.

Inclusivity in global research
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Ethnobotanical and ethnopharmacological survey
Western lowland gorilla living in MDNP consumed 27 plants [30].Local people use different parts of these plants (bark, root, fruit and leaves) in medicinal preparation (maceration, decoction, lotion, pomade and infusion) (Table 1).The cross-referenced results of ethnobotanical and ethnopharmacological surveys on the traditional use of plants by traditional healers in their pharmacopoeia to treat various human illnesses enabled us to select the four plants consumed by western lowland gorillas living in MDNP for this study.This information was recovered from autochthone Vungu people, living in Doussala village in MDNP.
Table 2 shows the results of ethnobotanical and ethnopharmacological surveys about traditional uses of most cited Ceiba pentandra L., Myrianthus arboreus P. Beauv., Ficus ssp.and Milicia excelsa Welw C.C. berg.bark by traditional healers in their pharmacopoeia to treat various human diseases, plants consumed by western lowland gorillas living in MDNP.These

Extraction yields
Extraction yields varied according to the solvents used.Water proved more efficient with higher yields than ethanol.

Antioxidant potential of bark crude extracts of selected species consumed by western lowland gorilla
The results of antioxidant potential of the four plant bark crude extracts are shown in Figs 1 and 2.

Antimicrobial activity of bark crude extracts of selected species consumed by western lowland gorilla
The ethanolic BCE tested showed more significant activity against MDR E. coli (DECs) than the aqueous BCE.Of the four plants tested, all extracts were active against at least one MDR E. coli (DECs) isolate (Table 4 and Fig 4).Ceiba pentandra aqueous BCE showed remarkable activity against all the tested MDR E. coli (DECs) isolates with inhibition zones ranging from 8.34 ± 0.57 mm to 13.67 ± 0.57 mm (Table 4).Ceiba pentandra ethanolic BCE also showed reasonable activity against all the tested MDR E. coli (DECs) isolates with inhibition zones ranging from 8.34 ± 0.57 mm to 11.67 ± 0.57 mm.Ficus ssp aqueous and ethanolic BCE showed activity against MDR E. coli (DECs) isolates with inhibition zones ranging from 8.34 ± 0.57 mm to 10.4 ± 0.57 mm and 7.67 ± 1.54 mm to 10.67 ± 0.57 mm respectively.Myrianthus arboreus aqueous BCE showed activity only against two MDR E. coli (DECs) isolates with inhibition zones ranging from 7.34 ± 0.57 mm to 8.34 ± 0.57 mm.Myrianthus arboreus ethanolic BCE showed activity against six tested MDR E. coli (DECs) isolates with inhibition zones ranging from 8.67 ± 0.57 mm to 10.67 ± 0.57 mm.Milicia excelsa Welw C.C berg.aqueous BCE were the least active.Ethanol (98%) and water were used as control and did not have any effect on all studied MDR E. coli (DECs) isolates.
MIC values were between 1.56 mg/mL and 50 mg/mL, and MBC values were between 3.12 mg/mL and >50 mg/mL (Table 5).

Correlations analysis
There was a positive correlation between the overall total phenolic, flavonoid, proanthocyanidin and tannin content and antimicrobial activity and a negative correlation with antioxidant activity assessed by DPPH IC 50 , PM TAC and BCB RAA (Figs 5 and 6).

Discussion
The use of MPs for their pharmacological properties and biological activities is a practice that is increasingly reported across the world [65].The WHO estimates that more than 25% of prescription drugs derive from MPs [66].Research on and development of evidence-based phytomedicines is a priority for Africa, including Gabon [67].Zoopharmacognosy is an original approach that could achieve these objectives [68].Since the introduction of zoopharmacognosy as a scientific discipline, many drugs that are now in use have been found by studying animal self-medication behaviors [2].Great apes use plants to heal themselves and thus control their parasitemia, viremia and bacteremia [69].Moreover, traditional healers use plants items usually consumed by great apes for their pharmacological properties and biological activities [70].However, these traditional uses of plants are not based on Western science and analysis [71].
The results in this study showed a negative correlation between extraction yield and solvent.The percentage yield of the different solvents obtained for the four plant bark extracts studied can be compared to those reported in the literature.For Ceiba pentandra, studies report a yield of 0.34% of the ethanolic extract [72], and 9% for the aqueous bark extract [73].For Myrianthus arboreus, the yield of the ethanolic extract was 9.87% [74], and 0.05% for leaf aqueous extract [75].For Milicia excelsa the yield .65% for stem bark ethanolic extract [76].There are no reports on the yield of Ficus ssp.The differences observed between the results of this study and those in the literature could be explained by the many factors taken into account during the extraction process [77].
Preliminary phytochemical screening revealed the presence of several classes of secondary metabolites in the bark extracts of the four plants consumed by gorillas living in MDNP.These analyses suggest that the four plants bark investigated are good sources of natural products, secondary metabolites endowed pharmacological properties and biological activities.Similar results have been obtained in other studies of the phytochemical content and antimicrobial activities of C. pentandra extracts [78,79].For Myrianthus arboreus, studies in Cameroon [80] and Ghana [81] showed comparable results to those obtained here.One study showed only the presence of phenolic compounds and reducing sugars [82].Another detected phenolic compounds and flavonoids [83].Ficus spp are used as medicine to reduce the risk of cancer and heart disease [84].Comparable results to those obtained here have been reported for Ficus species in Tunisia [85], Turkey [86] and Italy [87].For Milicia excelsa BCE, a study in Nigeria, showed the presence of tannins, alkaloids, flavonoids and saponins in Nigeria [88].Flavonoids and phenolic compounds were also found in Nigeria [89].The secondary metabolites found in the BCE of the four plants studied possess pharmacological properties and are endowed with biological activities, suggesting that they can be used in traditional medicine as MP by healers and in pharmaceutical products [90,91].

Myrianthus arboreus
3.12 6.25 3.12 6.25 3.12 6.25 3.12 6.25 Milicia excelsa Ethanol (98%) Traditional African healers make use of MPs to treat microbial diseases without western scientific basis [92,93].The results for total phenolic, flavonoid and tannin content obtained here in Ceiba pentandra are consistent with those reported in recent studies [38,73].For Myrianthus arboreus, total phenolic and flavonoid content [94], proanthocyanidins content [95] and tannins content [81] have been found as in this study.Recent work on the genus Ficus has also revealed the presence of total phenolic compounds and total flavonoids [96], and total tannins [97].Similar results to those obtained here have been reported for excelsa extracts [98].Therefore, the use of these four plant species in traditional medicine could be attributed to the high content of phenolic [99], flavonoids (flavones and flavonones) [100], tannins [101] and proanthocyanidins [102] compounds which are known for their antimicrobial, antioxidant, anticancer, antidiarrheal, anti-inflammatory, antimalarial, anti-cytotoxic and antispasmodic activities.The phenolic, flavonoid tannin and proanthocyanidin compounds found in this study may justify their bioactive effects for diarrheal diseases, those related to oxidative stress caused by ROS and diseases related to MDR E. coli (DECs).
Several studies have been conducted on MPs and their extracts with the aim of determining their antioxidant capacity, total phenolic content and characterizing the respective phenolic components [103].Most mentioned plant secondary metabolites associated with antioxidant activity are alkaloids, sulfur compounds, terpene/terpenoids, essential oil, carotene/carotenoid, polyphenol/phenol, flavonoid, tannin, and coumarin [104].However, variations exist, even among plant species belonging to the same genus.This could be attributed to differences in the extraction method during sample preparation [105], differences in harvest time [106], differences in the variety of the analyzed sample [107], as well as differences in the climatic and soil conditions and their origins [108].Antioxidant compounds from MPs or nutritional plants display numerous beneficial effects such as a remarkable scavenger ability against various radical species [109].As the latter explain their action through different mechanisms, usually, various tests should be explored to fully estimate the antioxidant capacity of bark crude extracts for example [110].Extraction of plant by solvent is a commonly used method to obtain antioxidants.However, no single solvent can extract all the secondary metabolites with antioxidant activities from plant because of its variation in solubility and polarity [111,112].In the present study, solvents with different polarities including water and ethanol and were used as solvents to extract antioxidants from Ceiba pentandra L., Myrianthus arboreus P. Beauv., Ficus ssp and Milicia excelsa Welw C.C berg.bark.The most natural antioxidants are multifunctional.Therefore, a reliable antioxidant evaluation protocol requires different antioxidant activity assessments to take into account various mechanisms of antioxidant action [113].
In this study, DPPH assay showed that the radical scavenging activity of the BCEs is dosedependent.All plant BCE studied here have DPPH radical scavenging activities.Ficus ssp ethanolic BCE showed a DPPH scavenging activity (IC 50 ) (1.31 ± 0.20 mg/mL) higher than Ficus brevibracteata leaves with a IC 50 value of 1.30 μg/ml (85.18 ± 0.22% of DPPH scavenging) [114], and lower than Ficus variegata stem bark ethanolic extract with 91% of DPPH scavenging [115].Studies show a high relative scavenging activity (RSA) (about 50% of DPPH scavenging and 73.7 ± 8.4% of DPPH scavenging) [80,116,117] and Myrianthus arboreus aqueous extract has antioxidant activities [75].In addition, antioxidant activities results obtained in the present study for Myrianthus arboreus are close to those observed in a previous study, which obtained a IC 50 value of 13.3 μg/mL for the ethanolic extract [95].For Ceiba pentandra ethanolic BCE, we observed similar antioxidant activities to those in a previous study, where the DPPH radical scavenging activity of ethanolic Ceiba pentandra leaves extract increased in dose dependent manner ranging from 10-50 μg/ml [118].For Ceiba pentandra aqueous BCE, studies have obtained comparable results from aqueous extracts of stem bark [119] which induced also a concentration-dependent radical scavenging activity on DPPH, trunk [120] and leaf bark extracts [121].Milicia excelsa showed radical scavenging activity on DPPH [122,123].Previous studies highlighted total antioxidant activities using phosphomolybdenum total antioxidant activity assay of Ficus nota [124], Ficus carica which possessed lower reducing ability with EC 50 value of 39 μg/ml [125], Ficus sycomorus with EC 50 value of 25 μg/ml [125] and Ficus Benghalensis methanolic extract with a IC 50 value for phosphor-molybdenum of 31.84 ± 0.12 μg/ml [126].A recent study showed Myrianthus arboreus aqueous root bark extract (40.3 ± 3.9 mg TE/g) and ethanolic extract (161.1 ± 11.9 mg TE/g) total antioxidant activities using phosphomolybdenum total antioxidant activity assay [95].Sinha et al. reported Bombax ceiba (Ceiba pentandra) flowers phenolic extract total antioxidant activities using phosphomolybdenum complex assay [127].There have been no reports on the effects of Milicia excelsa BCE using this assay.The β-carotene bleaching activities of all plant BCE depended on concentration.The antioxidant activity was observed in order of Ceiba pentandra.>Myrianthus arboreus> Ficus ssp> Milicia excelsa.There are no reports in the literature on the antioxidant activities of the BCEs of the four plants we studied using β-carotene bleaching assay.Plant antioxidants are a natural reservoir of bioactive compounds and play important roles in plant acclimation and adaptation to environmental challenges, and are also beneficial to human health [104].Recent work on pollen extracts of three plants from the Palestinian pharmacopoeia reported antioxidant activities of flavonoids compounds such as flavones and flavonols in these extracts [48].The antioxidant capacities of these all secondary compounds support the human body's battle against diseases by absorbing free radicals and chelating metal ions that could catalyze the production of ROS, which facilitates lipid peroxidation [128].Antioxidants break radical chain reactions, preventing oxidative stress-related damage [129].In brief, antioxidants can act according to two major mechanisms, either by transfer of hydrogen atom or by electron transfer [130].
However, in this study, regarding the three methods used (DPPH radical capacity and Phosphomolybdenum complex for total antioxidant capacity and β-Carotene bleaching assay), ethanolic extracts of Ceiba pentandra L., Ficus ssp and Milicia excelsa Welw C.C berg.excepted for Myrianthus arboreus P. Beauv., presented relatively higher antioxidant activity than the aqueous ones.In a practical way, plant extracts antioxidant activity depends on several factors, for example: the concentration of the extracts, the method of evaluation, the sensitivity of the antioxidants to the temperature of the test, and the water-or fat-soluble nature of the antioxidant [131,132].
Regarding antimicrobial activity, previously published reports report that the inhibitory activity of MPs extracts against Gram-positive and Gram-negative bacteria has been widely highlighted in the literature [133], and that MPs extracts with MIC values less than 100 mg/mL can be considered to have very good antimicrobial activity [134,135].In addition, the antimicrobial activity of these MPs extracts could be different toward various kinds of bacteria and different kinds of extract [136,137].This can be explained by the highest resistance of Gramnegative bacteria due to the complexity of their cell wall, containing a double membrane as opposed to the unique glycoprotein/teichoic acid membrane of Gram-positive bacteria [138].In light of these indications, the antimicrobial activity of the bark crude extracts from the four plants studied and consumed by western lowland gorillas, against MDR E. coli (DECs) isolates, varied with different extraction solvents.
Results of the antimicrobial activities from the four plant BCEs investigated against MDR E coli (DECs), found in this study are comparable to those reported in the literature.Ceiba pentandra ethanolic extract showed antimicrobial activities against E. coli (an organism frequently implicated in gastroenteritis and pelvic inflammation) with a zone of inhibition value of 10.55 ± 1.45 mm and showed a MIC value of 12.5 mg/ml against E. coli [72].A previous study also showed that Ceiba pentandra ethanolic extract was highly active against E. coli and that the antimicrobial activity of leaf extract increased as the concentration of ethanolic extract of Ceiba pentandra leaves increased [118].Parulekar et al. [78] reported moderate antimicrobial activities from aqueous extract and strong antimicrobial activities ethanolic extract against E. coli, which increased as the concentration of the extracts increased; and Njokuocha et al. [79] also reported antimicrobial activities of Ceiba pentandra extracts.Ceiba pentandra aqueous extract has shown antimicrobial activities against Gram-negative bacteria, including E. coli [78,79].A recent study showed that the mean zone of inhibition of Myrianthus arboreus was zero for E. coli ATCC 25922 with aqueous and ethanolic extracts.However, for clinical MDR isolates, the Myrianthus arboreus inhibition zone diameter value was 0.4-2.2mm for ethanolic extracts, showing low antimicrobial activities [82].Ethanolic extracts of taxa belonging to the genus Ficus have been studied for their antimicrobial potential [139].Biologically, Milicia excelsa Welw C.C berg.extract antimicrobial activities against enterobacteria have been demonstrated [44].The flavonoid neocyclomorusin isolated from Milicia excelsa extract exhibited antimicrobial activity against K. pneumoniae ATCC11296 and E. cloacae BM47, with MIC values of 4 μg/mL each [43].Padayachee et al. reported zero antimicrobial activity of Milicia excelsa extract against E. coli [140].
Many of these bioactive compounds are believed to have been used by plants and their parts, during their evolution, to protect against bacteria and are responsible for antimicrobial activity [141].A possible mechanism for this phytochemical activity may be either through inhibiting the growth of microbes, inducing cellular membrane perturbations, interference with certain microbial metabolic processes, or modulation of signal transduction or gene expression pathways.However, these mechanisms may all occur at the same time as a result of the synergistic effect between the compounds [142].The roles of secondary metabolites are relatively straightforward; for instance, they participate in general protective roles (antioxidant, free radical scavenging, UV light absorbing, and antiproliferative agents) and protect the plant from herbivorous animals (grazing) including different pathogenic microorganisms such as bacteria, fungi, and viruses.They also manage interplant relationships, acting as allelopathic defenders of the plant's growing space against competitor plants [143].
Several phytochemicals have already been identified using GC-MS or HPLC-MS for Ceiba pentandra [118,120], for Myrianthus arboreus [41,95] and for Milicia excelsa [43].These bioactive components are known to exhibit medicinal property [144].In addition, in the current study, a positive correlation was observed between total phenolic, flavonoid, proanthocyanidin and tannin content with antimicrobial and antioxidant activity.The antioxidant activity of an extract or compound is often associated with their redox proprieties, which allow them to act as reducing agents [145].Several other studies have also reported positive correlations between plant extracts secondary metabolites such as phenolic [146], flavonoid [147], proanthocyanidin [148], tannin [149] and antimicrobial activity.
Biodiversity contributes significantly towards human livelihood and development and thus plays a predominant role in the well-being of the global population [150].Valorization of plants with medicinal value is a world challenge that meets the objectives of biodiversity conservation [104,151].The latter could involve the study of phytochemistry, pharmacological properties, antioxidant and antimicrobial activities of plants consumed by non-human animals, including great apes such as western lowland gorilla [2,152].Additionally, new drug discovery from natural sources involve a multifaceted approach combining botanical, phytochemical, biological, and molecular techniques [153,154].These bioprospecting practices have implications for medicine, environment, economy, public health, and culture [155].Unfortunately, the potential benefits of plant-based medicines have led to unscientific exploitation of natural resources, a phenomenon that is being observed globally.The decline in biodiversity and loss due to species extinction is largely the result of the rise in the global population, rapid and sometimes unplanned industrialization, indiscriminate deforestation, overexploitation of natural resources, illegal trade, pollution, and finally global climate change [155,156].Therefore, it is of utmost importance that biodiversity is preserved, to provide future structural diversity and compounds for the sustainable development of human civilization [153,157].This becomes even more important for low and middle-income nations, where well-planned bioprospecting coupled with non-destructive commercialization could help in the conservation of biodiversity, ultimately benefiting humankind in the long run [153,157].

Conclusion
The results of this study, which examined the antioxidant and antimicrobial activity of Ceiba pentandra, Myrianthus arboreus, Ficus ssp and Milicia excelsa BCEs, plants are consumed by western lowland gorilla living in MDNP and used in traditional medicine by Gabonese healers, revealed some important facts.Indeed, all plant BCEs studied showed antioxidant and antimicrobial activities.The asymptomatic nature of theses gorillas with regard MDR E. coli (DECs) could be explained by their consumption of the bark of the four plants tested.The scientific results obtained during pharmacological analyses could justify the use of these plants in the traditional pharmacopoeia against various human diseases.The BCEs of the four plants studied could be promising sources for new bioactive molecules discovery in the pharmaceutical, cosmetics and food industries.One of the potential challenges of this study was to address the issue of potential alternative solutions to the problem of antimicrobial resistance, using a zoopharmacognosy approach.
These results show that the BCE of these plants could be used as an effective treatment for diseases caused by free radicals and diseases caused by antimicrobial-resistant bacterial strains.Then, all this founding could comfort the self-medication hypothesis of non-human animals, including great apes.The results of our study suggest that all plant BCEs studied could potentially be candidate improved traditional medicines (ITMs) in the application of new therapeutic protocols against infectious diseases of bacterial origin.
In addition, the identification of all plant BCEs studied bioactive compounds, using HPLC-MS or a LC-MS/MS and molecular network approach, would add quality to the results obtained in our study.This approach is a valuable tool for revealing the metabolomes of plants extracts, groups secondary metabolites into molecular families based on their spectral

Table 1 .
(Continued) Use Value indicates the relative importance of uses of plant species.RFC: Relative Frequency of Citation indicates the local importance of each species.