Variation of Secondary Metabolite Contents and Activities against Bovine Diarrheal Pathogens among Zygophyllaceae Species in Benin and Implications for Conservation

Key words

substitution - Balanites aegyptiaca - Zygophyllaceae - ethnoveterinary uses - antibacterial activity - West Africa

Introduction

It is widely accepted that the selection and use of plants in traditional medicine is not a matter of chance but guided by reason-based processes, including observation and empirical experience of the past. Many scientists have wondered about how humans select and use plants, proposing different theories that are sufficiently detailed in Gaoué et al. [1]. These are as follows: the social dynamics and human traits, ecological biochemistry hypothesis, and the demographic traits of the plant. Among these major ethnobotanical theories that explain how and why local populations select plants for a wide range of uses, the role of the plant life-form and ecological biochemistry are the central determinants [1]. This article covers an important ethnobiological hypothesis of this theory known as the “optimal defense theory.” This hypothesis postulates that plant tissues unlikely to be attacked by herbivores or with low fitness value have low levels of quantitative defense but high production of qualitative defense compounds. However, tissues that are likely to be attacked or have a high fitness value should have high levels of quantitative defense and low production of qualitative compounds [1], [2]. Our hypothesis is linked to 2 main hypotheses: ecological apparency [3] and resource availability [4], which state that herbs and weeds, in particular, tend to be rich in bioactive secondary compounds. It is known that the secondary metabolites of plants are naturally synthesized: (i) for ecological functions such as allelopathy, where they inhibit the germination and growth of other plants, (ii) as attractants for insects and birds for pollination and seed dispersal, and (iii) for chemical defense against a wide range of insect pests, pathogens, and other herbivores [5], [6]. These numerous, diverse compounds, including terpenes, nitrogenous or alkaloid compounds, and phenolic compounds (flavonoids and tannins), are therapeutically important. The bioactive compounds most often developed in Angiosperms, especially in this regard, are phenolic compounds [7].

In the Republic of Benin, among the 74 botanical families reported to be used for their ethnoveterinary potential [8], 60 have been reported to possess gastrointestinal activities [9]. In this recent study, based on statistical analysis, the Zygophyllaceae was cited to be overexploited while Balanites aegyptiaca (L.) Delile is overrepresented in species used to treat diarrhea in cattle.

B. aegyptiaca is a xerophytic tree distributed in tropical and nontropical areas of North and West Africa and West Asia [10]. In Benin, it is distributed from the Guineo-Congolian zone in the coastal area (www.gbif.org) to the Sudanian zone with a zero record in the Sudano-Guinean zone [11]. In addition to its forage value for ruminants and wild ungulates [12], [13], the powder of B. aegyptiaca leaves and fruits is used to prepare a sauce for human consumption [14]. The socio-economic importance of this plant demonstrates that it is overexploited, and it runs the risks, in the medium term, of becoming rare and even extinct. Furthermore, regional trade is being developed around its fruits and seeds [14]. Fruit collection for various purposes (production of juice, oil, and other products) would negatively impact the natural regeneration and the demographic structure of natural populations. This cannot guarantee the sustainable use of this multipurpose tree. In this regard, investigations of plant species that could exhibit the same biological properties as B. aegyptiaca are needed to reduce the pressure on the species. According to Judd et al. [15] and Molares and Ladio [16], plants within the same family, with close evolutionary ties, are more likely to share similar secondary compounds with similar or equal medicinal properties.

Our investigations were based on the 2 life-history strategies defined by MacArthur and Wilson [17], which are as follows: (i) r-strategist species are those species that produce many “cheap” offspring and live in unstable environments, and (ii) K-strategist are those species that produce few “expensive” offspring and live in stable environments. The objective was to contribute to the promotion and sustainable use of species of Zygophyllaceae in the treatment of diarrhea in cattle in Benin even if previously, no use of Guaiacum officinale, Tribulus terrestris, and Kallstroemia pubescens for ethnoveterinary uses had been reported in the literature. Specifically, 2 main hypotheses were tested. Since r-strategist species tend to invest in the quality of defense compounds and not in quantity [4], the first hypothesis stipulates that the r-strategist species (T. terrestris L. and K. pubescens [G.Don] Dandy) that belong to the Zygophyllaceae are less rich in phenolic compounds than the K-strategist species (B. aegyptiaca and G. officinale L.) of this botanical family. Additionally, due to this variability in diversity and secondary metabolites content, the second hypothesis predicts that the r-strategist species do not exhibit any antimicrobial activity and therefore cannot replace the K-strategist species. By rejecting the latter hypothesis, T. terrestris and K. pubescens could potentially substitute for B. aegyptiaca in treating bovine infectious diarrhea. The investigations were based on well-known antibacterial secondary compounds such as polyphenols (flavonoids and tannins).

Results

Twelve secondary metabolites (SMs) were identified among extracts of 4 studied species ([Table 1]). Out of these SMs, 11 (92%) were detected in r-strategist species; these included alkaloids, flavonoids, anthocyanins, leucoanthocyanins, anthraquinones, tannins, sterols, terpenes, quinones, coumarins, and saponosides ([Table 1]). According to the proportion test, this richness in SMs was significantly different (Z = 2.04; p = 0.041) compared to the diversity of SMs (7 SMs, 58%) in K-strategist species ([Table 1]). Thus, r-strategist species turned out to be richer in secondary metabolites than K-strategist species.

Table 1 Secondary metabolites composition of Balanites aegyptiaca (Bal), Guaiacum officinale (Guai), Tribulus terrestris (Trib), and Kallstroemia pubescens (Kal).

Secondary metabolites	K-strategist species		r-strategist species
	Bal	Guai	Trib	Kall
+: presence; −: absence
Alkaloids	+	+	–	+
Flavonoids	+	+	+	+
Anthocyanins	–	–	–	+
Leuco-anthocyanins	–	–	+	–
Anthraquinones	–	–	+	+
Reducing compounds	+	–	–	–
Sterols and terpenes	+	–	–	+
Catechic tannins	+	+	+	+
Gallic tannins	–	–	–	+
Sterols and terpenes	+	–	–	+
Mucilages	–	–	–	–
Quinones	+	–	+	–
Cyanogenic derivated	–	–	–	–
Coumarins	+	–	+	+
Saponosides	–	–	+	+

The results reported in [Table 2] revealed that K-strategist species were significantly richer in total phenolic content (TPC) than r-strategist species. The mean values of TPC for K-strategist species were 15.18 mg GAE/g extract and 41.84 mg GAE/g extract, respectively, for B. aegyptiaca and G. officinale ([Table 2]). In r-strategist species, the mean values of TPC were 4.82 mg GAE/g extract and 11.36 mg GAE/g extract, respectively, for T. terrestris and K. pubescens ([Table 2]).

Table 2 Composition and amount of phenolic compounds in aqueous extracts of 4 species of Zygophyllaceae.

	Species	TPC (mg GAE/g)	FC (mg QE/g)	TC (mg PE/mL)
TPC = total phenols, FC = flavonoids, and TC = tannins. Bal: Balanites aegyptiaca; Guai: Guaiacum officinale; Trib: Tribulus terrestris; Kall: Kallstroemia pubescens.
K-strategist	Bal	15.18 ± 0.43	57.11 ± 0.13	0.06 ± 0.01
	Guai	41.84 ± 0.85	55.14 ± 0.29	0.04 ± 0.01
r-strategist	Trib	4.82 ± 0.05	35.07 ± 0.05	0.05 ± 0.01
	Kall	11.36 ± 0.04	30.64 ± 0.35	0.04 ± 0.00

The trends were the same concerning flavonoids and tannins. K-strategist species showed higher yields ([Table 2]). The highest values were obtained for B. aegyptiaca with 57.11 mg QE/mg extract for flavonoids and 0.06 mg PE/mL extract for tannins. These facts suggested that K-strategist species produce more phenolic compounds than the r-strategist species.

Variation of antibacterial activity according to the types of species

After extraction, for 100 g of powdered raw material per plant species, we had obtained 11.4; 10.3; 09.6, and 12.7 g of dry extract for B. aegyptiaca, G. officinale, K. pubescens, and T. terrestris, respectively. Regarding the origin (type of the species) of the extract, no bacterial activity was found for the following concentrations: 20 mg/mL, 40 mg/mL, 60 mg/mL, and 80 mg/mL. However, with a concentration equal to 100 mg/mL, 4 out of 5 strains were sensitive. Only Klebsiella oxytoca was not sensitive to the various extracts ([Table 3]). Extract from the r-strategist species K. pubescens held a wide action spectrum (active on 4 out of 5 strains) with an inhibition zone diameter ranging from 8 ± 1 mm on Klebsiella pneumoniae to 20.33 ± 1.52 mm on Salmonella spp. Furthermore, Escherichia coli ATCC 25 922 represents the most sensitive strain since it showed sensitivity to the 4 extracts ([Table 3]).

Table 3 Inhibition zone diameters (mm) of the 4 Zygophyllaceae species on the 5 bacterial strains.

	Inhibition zone diameters (mm)
Bacterial strains	K-strategist species		r-strategist species		Positive control (charge)
	B. aegyptiaca	G. officinale	T. terrestris	K. pubescens	Chloramphenicol (30 µg)
B. aegyptiaca: Balanites aegyptiaca; G. officinale: Guaiacum officinale; T. terrestris: Tribulus terrestris; K. pubescens: Kallstroemia pubescens
K. pneumoniae	–	–	–	8 ± 1	17.33 ± 0.04
K. oxytoca	–	–	–	–	19.33 ± 0.57
Salmonella spp.	–	–	15.66 ± 1.52	20.33 ± 1.52	18.67 ± 0.02
Salmonella choleraesuis	10.33 ± 1.52	16.66 ± 1.15	–	10.66 ± 1.15	17.33 ± 0.04
E. coli ATCC 25922	10.33 ± 0.00	8.33 ± 0.57	11 ± 0.00	14.66 ± 0.57	22 ± 1

Values of MIC and MBC are presented in [Table 4]. For the majority of the plant extracts tested, the MBC/MIC ratios were greater than or equal to 4, demonstrating a bacteriostatic power. On the other hand, for the extract of K. pubescens on Salmonella and E. coli strains, these ratios were respectively equal to 1 and 2, indicating its bactericidal power with a concentration of 100 mg/mL. Therefore, extracts of r-strategist species tend to exhibit higher antibacterial power than those from K-strategist species.

Table 4 Minimum inhibitory and bactericidal concentrations of 4 Zygophyllaceae species against the 5 bacterial strains used.

Extracts	Parameters (mg/mL)	Salmonella spp.	Salmonella choleraesuis	Klebsiella pneumoniae	Klebsiella oxytoca	Escherichia coli ATCC 25922
MIC = minimal inhibitory concentration; MBC = minimal bactericidal concentration; a. p. = antibacterial power of the aqueous extracts; a. p. with * = bactericidal power; a. p. without * = bacteriostatic power; B. aegyptiaca: Balanites aegyptiaca; G. officinale: Guaiacum officinale; T. terrestris; Tribulus terrestris; K. pubescens: Kallstroemia pubescens
B. aegyptiaca	MIC	–	6.25	–	–	6.25
	MBC	–	50	–	–	50
	a. p.	–	8	–	–	8
G. officinale	MIC	–	6.25	12.50	–	6.25
	MBC	–	50	> 50	–	50
	a. p.	–	8	–	–	8
K. pubescens	MIC	3.13	3.13	6.25	–	6.25
	MBC	3.13	3.13	25	–	12.50
	a. p.	1*	1*	4	–	2*
T. terrestris	MIC	6.25	–	–	–	6.25
	MBC	25	–	–	–	50
	a. p.	4	–	–	–	8
Chloramphenicol	MIC	0.195	0.781	0.391	0.391	0.098
	MBC	0.195	0.781	0.391	0.391	0.098
	a. p.	1*	1*	1*	1*	1*

Discussion

The phytochemical screening revealed that r-strategist species were richer in secondary metabolites than K-strategist species. Certain compounds present in r-strategist species, particularly anthocyanins, gallic tannins, and saponosides, appeared absent in the K-strategists.

The presence of flavonoids, alkaloids, tannins, sterols, and saponosides in all extracts, seems to agree with the findings of Nayeem et al. [18], Pendyala et al. [19], Nigam [20], Abdulhamid and Sani [21], who respectively investigated T. terrestris, G. officinale, K. pubescens, and B. aegyptiaca. Furthermore, Sharma et al. [22] did not detect the presence of flavonoids or tannins in T. terrestris extracts in India. This inconsistency may be attributed to the variation of environmental conditions that may affect the biosynthesis of secondary metabolites in the plant.

The quantification of phenolic compounds showed that K-strategist species are significantly richer than r-strategist species in total polyphenols, total flavonoids, and tannins, confirming our first hypothesis. These results support the optimal defense theory, which is directly linked to the ecological apparency. Indeed, the primary prediction of this hypothesis is that species with a short lifespan (non-apparent) face lower herbivore pressure and are more likely pressured and invest in more quantitative defense [3]. Quantitative defense uses digestibility reducers such as tannins and lignins, which are immobile, are difficult for herbivores to adapt to, and have low turnover rates. Qualitative defense uses highly active molecules such as alkaloids and glycosides in low concentrations and with low metabolic cost investments [3], [23], [24].

However, the present study focused only on the quantitative defense compounds. As pointed out by Almeida et al. [25], it would be interesting to investigate qualitative compounds (alkaloids, quinones) to fully validate this theory in the Zygophyllaceae species. In their study, Almeida et al. [25] found that K-strategist plants tended to have high levels of both quantitative and qualitative defense compounds.

In addition, phenolic compounds are synthesized in plants not only for biological requirements and the need to control evolutionary defense [26] but also for tolerance to environmental stresses [27]. Considering their respective habits, the K-strategists, as they are trees, are more sensitive to stress than the r-strategists. As a result, the increasing concentration of phenolic compounds in K-strategist species is a response to the mechanisms of tolerance to the various abiotic and biotic stresses. These mechanisms reduce the impact of stress on the plant due to factors such as drought, extreme temperatures, salinity, and cold [28]. Studies have shown that the high content of these compounds in plants can also be associated with their protective role against UV-B light [29].

All bacterial strains tested showed a relative susceptibility to different plant extracts, except K. oxytoca. Extracts of K-strategist plants were active on 2 bacteria while those of r-strategist plants exhibited effects on 4 bacteria. Thus, 83% of r-strategist species exhibited better antibacterial activity than K-strategist species, with K. pubescens the most effective. These results were completely different from our predictions; thus, the second hypothesis was rejected. According to Albuquerque et al. [30], from an ethnobotanical perspective, the incidence of chemical defense implies that r-strategist plants (short-lived, herbaceous) are more likely to be used in traditional medicine than K-strategist plants (perennial, woody, dominant plants). Therefore, herbaceous species K. pubescens could be considered potential surrogate plants for the overexploited B. aegyptiaca in treating bovine infectious diarrhea.

Otherwise, our results contrast with the findings of Almeida et al. [31], who showed that K-strategist plants in Caatinga (Northeast Brazil) tended to have higher antimicrobial activity than r-strategist plants, which often display qualitative defense compounds but with lower antimicrobial activity. For these authors, plants with a short life cycle in an arid zone tend to require a greater investment in growth to complete their life cycle than in the production of alkaloids, for example. Their results conformed with the resource availability hypothesis. Otherwise, the fact that r-strategist species showed better antibacterial activity in the present study could be due to the synergistic effect of all metabolites present in their extracts. Qualitative compounds such as alkaloids, terpenes, or quinones also possess antimicrobial properties [32].

Lessons from the past showed that the continuous use of a species could lead to its overexploitation and ultimately to its extinction if it is not managed judiciously. According to Cunningham [33], the most vulnerable species are the most popular, which grow and reproduce slowly, require a specific habitat, and whose distribution is limited. Despite numerous measures introduced in developing countries, biodiversity loss is still rising at an alarming scale and rate [34]. Therefore, the sustainable use of wild biological resources should be an effective conservation alternative. Besides conventional strategies such as in situ and ex situ conservation, potential plant surrogates should be a better option for alleviating pressure on overexploited species. This practice will reduce the pressures on threatened medicinal plants and maintain their natural populations through in situ conservation strategy.

Many studies showed that the substitution of critical plant parts (root, barks, etc.) by others is a good measure of conservation of medicinal plants [35], [36]. However, in the context of overexploitation, the substitution of the whole plant is more interesting because even leaf overharvesting can compromise the healthy growth and development of the plant [37]. According to the same authors, the substitution can result from improved processes or research in available and less expensive products that give the same or even better results.

The present study has shown the possible substitutions for the B. aegyptiaca tree by herbaceous species of the same family. Further research such as clinical studies should be envisaged to validate the efficacy of these potential alternative species and test the effects on noninfectious diarrhea in cattle. In addition, it has also highlighted the antibacterial properties of G. officinale and K. pubescens [20], [21], [38].

Materials and Methods

Plant material collection and extraction

Randomly selected samples of the leafy stems from wild-growing plants of 2 categories were collected: 1) r-strategists (T. terrestris and K. pubescens) and 2) K-strategists (B. aegyptiaca and G. officinale). The material collection was mainly done on March 8, 2019, in the morning at Abomey-Calavi and Grand-Popo in the same climatic zone (Guineo-Congolese zone in the south of Benin). However, samples of B. aegyptiaca were collected in the same conditions by 2 co-authors, Donald Djidohokpin and Abraham Favi, in the Sudanian zone (Malanville) since our previous studies showed that the extracts of leafy stems of B. aegyptiaca from the Guineo-Congolese zone did not exhibit any antimicrobial activity. Botanist Professor Hounnankpon Yedomonhan from the University of Abomey-Calavi (Benin) identified the plants. The voucher specimen were registered under numbers YH 628/HNB, YH 629/HNB, YH 630/HNB, YH 631/HNB (respectively for B. aegyptiaca, G. officinale, K. pubescens, and T. terrestris), and they were deposited at the National Herbarium of Benin.

All of the collected parts were washed with distilled water and dried under air conditioning at a temperature of 25 ± 1 °C for 2 wk. Then, they were ground to homogenous powder for aqueous extraction so that the experiments complied with traditional prescriptions. Thus, 4 aqueous extracts were prepared.

For antimicrobial tests, a total of 4 bacterial strains were provided by the Food Microbiology section of the National Laboratory of Public Health, Ministry of Public Health, Benin. These were isolated from cattle feces samples: Salmonella spp., Samonella choleraesuis, K. pneumoniae, and K. oxytoca and 1 reference strain: E. coli ATCC 25 922. Salmonella spp. and some pathogenic E. coli strains are generally responsible for diarrhea in cattle. In addition, the presence of Klebsiella strains in the feces samples of diarrheic cattle may be explained by co-infection due to immune weakness of the diseased cattle or by external contamination of the feces by Klebsiella strains on the farm [39].

Preparation of the extracts

We macerated 100 g of the powder in 500 mL of distilled water under an agitator for 48 h at ambient temperature. The homogenate was filtered once using absorbent cotton and once with Whatman N°1 filter paper. The obtained filtrate was then dried at 50 °C in an incubator and served as the aqueous extract.

Phytochemical screening

The 4 extracts were screened for major bioactive compounds using standard qualitative methods described by Koudoro et al. [40]. These include tannins, saponosides, flavonoids, alkaloids, sterols, terpenes, glycosides, and anthraquinones. The screening tests were conducted in duplicates, following the recommendations of the Laboratory of Research in Applied Chemistry of the Polytechnique School of Abomey-Calavi (University of Abomey-Calavi).

Test for alkaloids

Five ml of each extract were evaporated to dryness and the alcohol residue was taken in 5 mL of 2% hydrochloric acid, saturated with sodium chloride, and filtered. To 2 – 3 ml filtrate, we added a few drops of Dragendroffʼs reagent, forming an orange-brown precipitate.

Test for glycosides

We added 1 mL of benzene and 0.5 mL of dilute ammonia solution to the extracts. A reddish color was formed in the presence of glycosides.

Test for carbohydrates

We added 10 mL of Fehling solution (copper sulfate in alkaline condition) to the concentrated extracts and heated it on a steam bath. Brick-red precipitates indicated the presence of carbohydrates.

Test for sterols and terpenes

We dissolved 2 mg of dry extract in acetic anhydride, heated to boiling, and cooled. Then, 1 mL of concentrated sulphuric acid was added along the sides of the test tube.

Test for phenols

We added 0.5 mL of FeCl₃ (w/v) solution in 2 mL of test solution. The formation of an intense color indicates the presence of phenols.

Test for anthocyanins

We added 2.5 mL of the extracts to 1 mL of 20% HCl. A pink-red color is accentuated, and then the addition of 5 mL of NH₄OH turns the solution to purplish-blue.

Test for tannins

To 1 – 2 mL of the extract, we added a few drops of 5% w/v FeCl₃ solution. A green color indicated the presence of gallic tannins, while brown color indicated the presence of catechic tannins.

Test for flavonoids

Adding 3 drops of isoamyl alcohol to the extract intensified a pink-orange or violet color.

The free compounds or combined quinones have been put in evidence according to the Borntraeger reaction. The saponosides have been identified by the calculation of the moss indication.

Determination of total phenolic content

The Folin-Ciocalteu method was used to determine the total phenolic content (TPC), as reported by Singleton and Rossi [41]. Each solution was filtered (100 µL) and then mixed with 750 µL of 1/10 Folin-Ciocalteu reagent for 5 min, before adding 750 µL of sodium carbonate. After incubation in the dark for 90 min, the absorbance at 725 nm was evaluated using a spectrophotometer. Gallic acid was used to generate the calibration curve. The TPC was expressed in mg gallic acid equivalents (GAE) per gram of extract.

Flavonoid content was estimated using the aluminum trichloride method described by Bahorun et al. [42]. Concisely, 100 µL of sample, 0.4 mL H₂O and 0.03 mL NaNO₂ (5%) were incubated for 5 min at 25 °C, then 0.02 mL AlCl₃ (10%) was added. After 5 min, the reaction mixture was alkalinized with 0.5 mL of 1 M NaOH and 0.25 mL H₂O. Finally, the absorbance was recorded at 510 nm. Results were expressed in mg quercetin equivalents (QE) per g of extract.

The condensed tannins content was determined using the method modified by Heimler et al. [43]. We mixed 500 µL of each extract with 3 mL of methanolic solution of vanillin (4%) and 1.5 mL of concentrated hydrochloric acid. The mixture was incubated at room temperature for 15 min, and the absorbance was determined at 500 nm. A calibration curve of pyrogallol was prepared, and the results were expressed as mg pyrogallol equivalents (PE) per mL of extract.

Evaluation of the antibacterial activity of extracts

The aqueous extracts were reconstituted in distilled water at a concentration of 100 mg/mL. The prepared solutions were sterilized by filtration using filter-syringes on 0.22 µm Millipore membrane. The sterility of the stock solutions was verified by culturing aliquots of each solution on Mueller Hinton and incubated at 37 °C for 24 to 48 h.

Preparation of bacterial suspensions and sensitivity test

A pure colony of each bacteria strain 24 h of age and from Mueller Hinton was emulsified in 5 mL of physiological water and adjusted to McFarland 0.5 standard. Each inoculum was cultured onto Mueller-Hinton II agar plates. Using the tip of the sterile pastor pipette, we dug wells of 6 mm of diameter into the agar, and 50 µL of each extract was transferred to the wells. A well containing sterile distilled water served as the negative control. A chloramphenicol disc loaded at 30 µg was used for the positive control. The Petri dishes were kept at ambient temperature 1 h for a prediffusion of the substances before being incubated at 37 °C for 18 h [44], [45]. Meanwhile, swabs of each inoculum were cultured onto MH II plates, and reference antibiotic disks were used as positive controls. The tests were repeated 3 times, and the antibacterial activity of the extracts was determined by measuring the inhibition zone diameters around each well ([Table 5]).

Table 5 Standard values used to interpret the results of the sensitivity tests of the plant extracts [44].

Inhibition zone diameter (Δ)	Degree of sensitivity of the germ	Symbol
Δ < 7 mm	Resistant	–
7 mm ≤ Δ < 8 mm	Susceptible	+
8 mm ≤ Δ < 9 mm	Fairly susceptible	++
Δ ≥ 9 mm	Very susceptible	+++

Determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)

This was performed using the 96 well plate method described by Agbankpé et al. [44], where we placed 100 µL of Mueller-Hinton broth (MHB) in each well of the microplate (1 to 10 wells). We placed 100 µL of the stock solution of each extractʼs stock solution in the first well. After homogenization by suction-discharge using a micropipette, 200 µL of extract solution at 50 mg/mL was obtained, and 100 µL of this new solution was taken and mixed with the MHB contained in the second well. This series of dilution was carried out from well to well until the 10th, of which 100 µL was discarded to have concentrations of 50 mg/mL; 25 mg/mL; 12.5 mg/mL; 6.25 mg/mL; 3.125 mg/mL; 1.562 mg/mL; 0.781 mg/mL; 0.391 mg/mL; 0.195 mg/mL and 0.098 mg/mL. Finally, 100 µL of the bacterial suspension was added to each well.

The 11th and 12th wells represented the positive and the negative controls. These respectively consisted of 100 µL of MHB + 100 µL of bacterial suspension and 100 µL of MHB + 100 µL of the stock solutions of the extracts that were being tested. Chloramphenicol was used as a control antibiotic to determine concentrations (positive test). Chloramphenicol concentrations also ranged from 0.098 to 50 mg/mL. The microplates were covered with parafilm paper and incubated for 24 h at 37 °C. The MIC was determined by the addition of tetrazolium. All wells of the MIC at higher concentrations were cultured on MH agar and incubated at 37 °C for 24 h to determine the MBC.

The MBC is the smallest concentration of extract at which any bacteria colony can be observed. Each extractʼs antibiotic power (ap) was thereafter calculated with the formula MBC/MIC.

Statistical analysis

Analyses were performed using R (R.3.0.5) and Minitab 14 Release. All analyses were performed in triplicate, if not otherwise stated. Results were reported as mean ± standard error of the mean of at least 3 independent experiments. The statistical significance of the data was evaluated by 1-way ANOVA followed by a Pearsonʼs test. A p-value < 0.05 was considered significant.

Contributorsʼ Statement

Data collection: J. M.-A. S. Ouachinou, J. Agbankpé, Y. A. Koudoro, G. A. Favi, D. Djidohokpin. Design of the study: J. M.-A. S. Ouachinou, G. H. Dassou. Statistical analysis: G. H. Dassou, G. A. Favi. Analysis and interpretation of the data: J. Agbankpé, Y. A. Koudoro, P. Agbangnan. Drafting the manuscript: J. M.-A. S. Ouachinou, G. H. Dassou, A. C. Adomou, N. Hidalgo Triana. Critical revision of the manuscript: N. Hidalgo Triana, A. C. Adomou, G. H. Dassou.

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