Ethnopharmacology surveyStudy area

The study was carried out from January 5 to March 15, 2019, in two regions of Cameroon. The Western region included two divisions namely Koung-Khi (Bandjoun) and Mifi (Bafoussam II subdivision), while the Centre region included the Mfoundi (Yaoundé I, Yaoundé VI, and Yaoundé VII subdivision), Mefou-et-Afamba (Awaé), and Lékié (Monatélé). The whole study area is located between 5°30′ N and 10°30′ E, 3°51′ to 3°53′ N and 11°25′ to 11°27′ E (Fig. 1).

Fig. 1

Location of the study sites

With a population of more than 1720 047 and a density of 123.8 inhabitants per Km2, the West region covers an area of 14 000 km2, making it one of the most densely populated regions in Cameroon. The relief of the West region is mountainous (plateaus and plains), with the tropical Sudanese's climate composed of two main seasons, including the dry (December–March) and the rainy season (March-November). Through the year, the temperatures vary between 15 and 30 °C on average, with a solid daily variation (Tchetgna 2012). On the other hand, the centre region with more than 1.299.369 inhabitants extends over 69.0000 km2. With the sub-equatorial climate and four seasons including a large dry (mid-November to late March), a short rainy (April to mid-June), a short dry (mid-June to mid-August) and a long rainy (mid-August to mid-November) (Nematchoua et al. 2014), it is an agroecological region of humid forest with a bimodal rainfall varying between 1500 and 2500 mm/year, a relative humidity of 70–90% and the temperature averaging 25 °C through the year (Moustapha et al. 2021). These localities were selected preliminarily based on small-scale rabbit’s farmers using medicinal plants to treat diarrhoea and related symptoms. Subsequently, the identification of plants with associated recipes was achieved through face-to-face interaction with farmers.

Data collection

The authorisation for the survey was obtained from the regional delegation of the ministry of livestock, fisheries and animal industries from West and Centre regions of Cameroon (supplementary materials 3). Before the start of the ethnobotanical data collection, we explained the goal of our study to informants. We requested their consent for capitalising their knowledge in a published document to avoid knowledge erosion through time. Contacted via the office of their respective regional delegations of livestock management, the forty-seven rabbit farmers, both men and women (20–50 plus years) from both regions who give consent to participate in the study, were interviewed using the snowball sampling technique according to a semi-structured questionnaire (supplementary material 4) which was previously prepared in the French language (Martin 1995). Using both an individual and focus group discussion, breeders were surveyed for their general profile, including their breeding experience, the mortality rate often recorded and the principal causes, the primary diseases affecting rabbits and the treatment and prevention methods employed. Moreover, the information on each medicinal plant, including the vernacular name, medical use, plant parts, mode of preparation, route of administration, the solvent used, the dosage, the length of treatment and the degree of satisfaction relative to each treatment was also surveyed. Each informant was interviewed at least twice to verify the relevance and confirm the information collected.

With the oral consent and aid from each local breeder, fresh plant specimens were collected, dried and stored in the laboratory of the Department of Biochemistry of the Faculty of Science of the University of Yaoundé 1. They were later identified at the National Herbarium of Cameroon, Yaoundé, where their full scientific names and voucher number were obtained. Further literature investigations were also conducted relatively to their therapeutic and/or pharmacological effects and their phytochemical composition if any.

Preparation of crude extracts

The fresh plant materials were washed separately with fresh tap water to remove dirt and other contaminants, shade-dried for three weeks until constant weight was recorded, and ground to fine powders. 100 g of powder was transferred in different Erlenmeyer flasks and macerated under shaking for 72 h in 500 mL of cold distilled water to prepare macerated extracts, heated for 5–10 min to prepare infusion extracts and boiling in distilled water for 30 min to prepare decocted extracts. The aqueous, infusion and decoction filtrates were sieved using hydrophilic cotton and filtered using Whatman filter paper No1. The organic filtrates were dried at 25 °C under constant ventilation to obtain crude extracts. The extracts were preserved in sterile bottles and conserved at 4 °C for further experiments.

In vitro antimicrobial assayMicrobial species and culture media

Three bacteria, including Clostridium perfringens WAL-14572 HM-310, obtained from BEI Resources (BEI Resources, Rockville, MD 20, 852), Enteropathogenic Escherichia coli (EPEC) and Salmonella enterica obtained from the Institute of Agricultural Research for Development (IRAD) of Wakwa, Ngaoundéré, Cameroon were used. The microorganisms were maintained on an agar slope at 4 °C and sub-cultured for 24 h before each experiment.

Preliminary screening and MIC determination

The forty-five extracts were screened at 5 mg/mL for their ability to inhibit bacteria species according to the following procedure: ninety-two microliters (92 µL) of Muller Hinton broth (MHB) were aseptically introduced into the wells of a 96-well micro-plate. Eight microliters (8 µL) of each extract, initially prepared at 125 mg/mL, were added in wells followed by 100 μL of standardised bacterial suspension (106 CFU/mL) to obtain final volumes of 200 μL. The tests were performed simultaneously for negative control (MHB + pathogens) and sterility control (MHB alone). Oxytetracycline 20% was used as the positive control and tested at 0.5 mg/mL. The plates were incubated at 37 °C for 24 h. The test was performed in duplicate and repeated thrice and the selected active extracts were further submitted to the dose–response study for MIC determination.

The minimum inhibitory concentration (MIC) of selected extracts was determined according to the M07-A9 Clinical Laboratory Standards Institute (CLSI) microdilution method using 96-wells microtitre plates. Briefly, 4 µL of extracts and reference drug (Oxytetracycline 20%) from stock solutions were introduced in the well, followed by the addition of 96 µL of bacteria inoculum standardised at 106 CFU/mL. A blank column was included for sterility control, while bacterial strains in culture medium without any inhibiting substance were negative control. The concentrations of extracts ranged from 0.625 to 5 mg/mL, and that of Oxytetracycline 20% ranged from 0.1 × 10–6 to 0.50 mg/mL. After 24 h of incubation at 37 °C, the turbidity was observed as an indication of growth. MIC was defined as the lowest concentration inhibiting the visible growth of microorganisms. All tests were performed in duplicate and repeated thrice.

Antibacterial activity of combined extracts against Enteropathogenic Escherichia coli

Three extracts from the two plants were combined to give three interspecies combinations, with no combination between extracts from the same plant. Moreover, the combination between extracts and Oxytetracycline 20% were also evaluated. The checkerboard microdilution assay was used to determine fractional inhibitory concentration indexes (FICIs) of combination of extracts against Enteropathogenic E. coli. The checkerboard broth microdilution method based on CLSI recommendations consisted of diluting extracts/compound in the two directions of a 96-wells microplate with the mixed concentrations in wells ranging from 1/64 × MIC to 2 × MIC. The bacterial inoculum was adjusted to give 106 CFU/mL an ultimate concentration in a final volume of 100 µL. Therefore, the plates were incubated at 37 °C for 24 h, and each test was performed in duplicate and repeated thrice. The changes in the Fractional Inhibitory Concentration Indexes (FICIs) were calculated using the following formula, and the type of interaction was determined according to previously described criteria.

FICI(X1/X2) = MICX1 in combination/MICX1 alone + MICX2 in combination/MICX2 alone.

Based on these criteria, a combination was considered to be synergistic when the FICI was ≤ 0.5, additive when it was > 0.5 to ≤ 1, indifferent when it was > 1.0 to ≤ 4.0. Antagonism was obtained with FICI > 4.0 (van Vuuren and Viljoen 2011).

Phytochemical screening

The presence of major phytochemical classes of compounds such as anthraquinones, flavonoids, glycosides, phenols, saponins, steroids, tannins, and terpenoids was qualitatively evaluated in the plant extracts using standard preliminary phytochemical analysis as previously described (Harborne 1998).

Test for anthraquinones: About 1.0 mg of each extract was reacted with 2.0 mL benzene, shaken properly, and filtered through Whatman’s no. 1 filter paper. Then, the filtrates were allowed to react with 2.5 mL of 10% ammonia solution and shaken properly. The presence of pink, red, or violet colour in ammonia solution in the lower phase indicates a positive result.

Test for glycosides: About 1.25 mg of each extract was allowed to react with 0.5 mL chloroform and mixed carefully. About 0.5 mL of concentrated sulfuric acid was then carefully added to form a lower layer. The reddish-brown colour at the interface indicates the presence of a steroidal ring, the glycone portion of glycosides.

Test for flavonoids: About 7.5 mg of each dry extract was dissolved in 0.5 mL of ethanol, concentrated HCl, and magnesium turnings. A yellowish coloration indicates the presence of flavonoids.

Test for phenols: The crude extract of the plant material was treated with 3 to 4 drops of ferric chloride solution, or dissolving 5.0 mg of dry extract in 0.5 mL of 1% ferric chloride solution. The formation of bluish-black colour indicates the presence of phenolic compounds.

Test for saponins: About 2.5 mg of the plant extract was dissolved with 5.0 mL water and shaken properly in a test tube. Samples showing froth were warmed. Persistent foam formation indicates the presence of saponin.

Tests for steroids: In a 10 mL glass tube, a mixture of 1.0 mL of extract, 1.0 mL of chloroform and 1.0 mL of acetic anhydride was prepared. Then 2 drops of concentrated H2SO4 was added from the side of test tube. The development of red, then blue and finally green colour was indicative of the presence of steroids.

Test for tannins: About 2.5 mg of each plant extract was boiled in 5.0 mL of water in a test tube and then filtered through Whatman’s No. 1 filter paper. Two to three drops of 0.1% ferric chloride added and read for brownish green or a blue-black precipitate indicating a positive result.

Test for terpenoids: About 0.5 mL of the chloroform extract of the dried extracts was evaporated to dryness on a water bath and heated with 3.0 mL of concentrated sulfuric acid for 10 min on a water bath. The gray colour indicates the presence of terpenoids.

Statistical analyses

Microsoft Excel 2016 software for Windows was used to calculate the number of recurrences and percentage of the plant use, knowledge on the use of medicinal plants. The sex and age of participant compared using khi2 and fishers tests. The results were presented as the Mean ± SD of the replicated values for each independent experiment.