Introduction
Gastrointestinal infections caused by enteric pathogenic microorganisms remain a global threat to many lives and the medical community (Maroyi 2016). This in part has compounded the clinical and economic challenges faced by public health on a yearly basis (Moon et al. 2023). Notably, 19% of the mortality rate recorded out of the 10 million deaths among children of 0–5 years all over the world was because of pathogenic enteric infections. In many instances, gastroenteritis presents with diarrhoeal symptoms including pain, bloating or belly cramps, nausea or vomiting and sometimes bloody or mucus stools (Negrut et al. 2020). Diarrhoea continues to affect people of all age groups globally, with more than 68% occurrence among young children (Florez, Nino-Serna & Beltran-Arroyave 2020). Apart from the children, the elderly population of more than 60 years of age contributes about 40% of the 5% diarrhoeal episodes that occur in the general population (Fernández-Bañares et al. 2016). In 2017, the South African public health department reported more than 34 000 patients with diarrhoeal illnesses admitted to the hospital and one in every 14 South African needs to be treated from the pathogen causing diarrhoea (Potgieter et al. 2018). Studies have shown that the most isolated enteric bacterial pathogens associated with diarrhoea includes Bacillus cereus (Mbhele et al. 2021), Escherichia coli (Potgieter et al. 2023), Staphylococcus aureus (Galvao, Whitelaw & Reid 2015) and Salmonella choleraesuis now called Salmonella enterica (Kalule et al. 2019). In addition, other commensal gut bacteria including Enterococcus faecium, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterococcus faecalis are opportunistic microbes, capable of causing diarrhoeal sicknesses in people with weak immune system such as the young, the old and the immunocompromised (Al-Dahmoshi et al. 2019; Blanco et al. 2018; Sathe et al. 2023). Rehydration is the first line of treatment for diarrhoea (Florez et al. 2020). However, other medical options, including the use of antibiotics, probiotics and other medications that lessen electrolytes and water loss into the intestine, are increasingly prescribed to treat diarrhoea of bacteria origin (Ramatla et al. 2022; Schiller 2017). Phytochemicals are natural compounds that have been investigated and reported as having the ability to control the growth of many microorganisms through several mechanisms of action that cause alterations in bacterial metabolism including changes in intracellular pressure, disruption in cell wall synthesis and deoxyribonucleic acid production (Truong & Jeong 2022). Various classes of phytochemicals, such as flavonoids, tannins and saponins, have been reported to be responsible for the antidiarrhoeal properties of some medicinal plants (Czerkas et al. 2024; Tagousop et al. 2018; Truong & Jeong 2022; Yuan et al. 2025). Moreover, it has been established that a direct relationship exists between the phytochemical constituents of plants and their antioxidant properties on the one hand and between the antioxidant properties and antimicrobial properties of plants on the other hand (Mehmood et al. 2022). Oxidative stress has been identified as critical phenomenon in diarrhoeal gastrointestinal disorders caused by pathogenic microorganisms (Mousavi et al. 2020). It has been reported that during chronic inflammation of the gastrointestinal tract, there is activation of hydroxyl radicals. Hydroxyl radical is toxic to cells and impedes function of enteric smooth muscles that propel food and assist with reabsorption of water and nutrients along the digestive tract, thus increasing the chances of diarrhoea (Mousavi et al. 2020). Combretum imberbe, also known as leadwood, is a plant that belongs to the Combretaceae family (Peloewetse et al. 2008). It is found mainly in Africa, in regions ranging from Tanzania to KwaZulu-Natal in South Africa. The plant is a popular traditional medicine in southern Africa where it is used to treat gonorrhoea, malaria and bilharzia (Carr 1988; Silén et al. 2023). The Combretum genus is known to contain phytochemicals such as gallotannins, alkaloids, lignans, fatty acids, fatty alcohols, saponins and ellagitannins (Angeh et al. 2007). It was previously reported that the root, root bark, stem and leaves of C. imberbe contain amino acids, triterpenoids, terpenes, flavonoids, tannins and polyphenolics (Maroyi 2025; Silén et al. 2023). Decoctions, infusions macerations and tinctures of C. imberbe leaves have reportedly been utilised for treating many illnesses including diarrhoea and other bacterial infections in South Africa. Inhalation of burnt C. imberbe leaves was also considered a cure for coughs (Nyagumbo et al. 2024; Silén et al. 2023). However, there is still a need to further explore its potential antibacterial and antidiarrhoeal agent. Hence, the aim of the study was to determine the antibacterial and antidiarrhoeal activities of C. imberbe leaf crude extracts at various concentrations.
Research methods and design
Plant selection and collection
Fresh C. imberbe Wawra leaves were collected from trees in Walter Sisulu National Botanical Garden located in Gauteng province, South Africa, in June 2023. The C. imberbe plant species was identified by the plant labels on the trees and authenticated by the National Botanical Garden Manager and Botanist. The leaves were transported to the Department of Life and Consumer Science of the University of South Africa.
Preparation of extracts
Combretum imberbe leaves were washed under running tap water and air-dried at room temperature for 1 week. The leaves were ground into fine powder using a kitchen coffee grinder (Eloff 1998). Ten grams of the coarse plant powder was dissolved separately in 100 mL of acetone, ethanol and distilled water at room temperature for 5 days under agitation (Ketpanyapong & Itharat 2016; Wang et al. 2011). A centrifuge was used at 10000 rpm for 10 min to spin and separate the supernatant from the pellets (Bharani & Namasivayam 2016). Millipore Whatman filter paper of size no.1 was inserted into a Buckner funnel to filter the samples, after which the residual solvents of the filtrate were allowed to evaporate at room temperature while the water extract was concentrated using a freeze dryer. The percentage yield ranged from 7.60 to 12.53%.
Phytochemical analysis
Phytochemical screening was conducted on the acetone extract, ethanol extract and water extract of C. imberbe (acetone extract of C. imberbe [ACI], ethanol extract of C. imberbe [ECI] and water extract of C. imberbe [WCI]) to determine the presence of phytochemicals. The presence of flavonoids, saponins, terpenoids and tannins was determined using a slightly modified method of Cyprian, Sewuese and Akacha (2019). The presence of anthocyanins was determined using the method of Hasan et al. (2018), with slight modifications.
Determination of total phenolic and total flavonoid content
The total phenolic compound content in the different plant extracts (acetone, ethanol and distilled water) from C. imberbe was determined using the Folin–Ciocalteu’s reagent as described by Jimoh, Afolayan and Lewu (2019) with a slight modification. The total flavonoid compound content of the plant crude extracts was measured using the aluminium chloride (AlCl3) colorimetric assay as described by Jimoh et al. (2019) with a slight modification. The total content of proanthocyanidin (condensed tannin) present in the dry plant extracts was determined using the catechin reagent method as described by Unuofin, Otunola and Afolayan (2018) with minor modifications.
<italic>In vitro</italic> antioxidant assays
The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity of the C. imberbe leaf extracts was determined using the method described by Mansour et al. (2022). ABTS (2,2′-azino-bis [3-ethylbenzothiazoline-6-sulphonic acid]) radical-scavenging activity of C. imberbe leaf extracts was determined by following the method described by Rao et al. (2023) with a few modifications.
Antimicrobial activity of <italic>Combretum imberbe</italic>
Microorganisms and growth media
The microorganisms that were used in the study were selected bacteria species of the American Type Culture Collection (ATCC) strain and were purchased from Thermofisher Scientific, Johannesburg, South Africa. The ATCC strains used for the in vitro antibacterial analysis include the gram-positive E. faecalis (ATCC29212), gram-positive S. aureus (ATCC43300), gram-positive B. cereus (ATCC10876), gram-positive E. faecium (ATCC700221), gram-negative E. coli (ATCC11775), gram-negative A. baumannii (ATCC19606), gram-negative P. aeruginosa (ATCC9027) and gram-negative S. choleraesuis (ATCC7001). These pathogenic bacteria were handled under Biosafety Level II (BSL-2) laboratory environment with biological safety cabinets (Jones & D’Orazio 2013; Vitko & Richardson 2013).
The starter culture of the eight previously mentioned bacterial strains were grown on nutrient agar aerobically overnight in a 37°C incubator. Cultures were then sub-cultivated on Mueller Hinton (MH) agar and re-suspended in MH broth medium (Sigma-Aldrich, Germany) using previous incubation conditions (Jones & D’Orazio 2013; Nuamsetti, Dechayuenyong & Tantipaibulvut 2012).
Determination of minimum inhibitory concentration
The broth microdilution method was performed to determine the minimum inhibitory concentration (MIC) for the crude extracts against the test microorganisms using 96-well plates with some modifications (Eloff 1998). The MIC represents the lowest concentration of antibacterial treatment that maintains a blue colour after 24 h based on metabolic activity, an indication of microbial inhibition (Tabit et al. 2016).
Statistical analysis
All data were expressed as mean ± s.d. (standard deviation) of three replicates and were statistically analysed using Student t-test of the GraphPad Prism’s software. Values were considered significant at p ≤ 0.05. The MINITAB 17 statistical package software was used to calculate Fischer’s least significant difference (LSD) mean separation. The data obtained from the cytotoxicity result were analysed using graph pad prism 8.0.1 software.
Ethical considerations
Ethical clearance of this study was obtained from the Ethics Committee of College of Agriculture and Environmental Sciences (CAES), University of South Africa, with Health Research Ethics Committee (REC) clearance number 2019/CAES_HREC/186.
Discussion
Medicinal plants have remained invaluable as a source of therapeutic products because they contain various biologically active compounds that act in synergy and interact simultaneously with several biological targets. Studies have established their potential in the management of several ailments, including diarrhoea (Ansari et al. 2025). This study therefore evaluated the antioxidant and antidiarrhoeal effects of acetone, ethanol and water extracts of C. imberbe.
Extraction is an important first step in the investigation of medicinal plants as it is essential to bring out the desired biologically active components from the plant material for additional characterisation processes (Sasidharan et al. 2011). In this study, the extraction yield of C. imberbe extracts increased with the polarity of the solvent, with the water extract having the highest yield, while the acetone extract had the lowest yield. This might be because of the electronegative functional groups on most of the phytochemicals that made them more compatible with water (Abbas et al. 2021). This result conforms with earlier studies in which polar solvents yielded a higher yield of extracts when compared with non-polar solvents (Gonelimali et al. 2018; Nawaz et al. 2020).
Studies have shown that identification of phytochemical constituents of medicinal plants is essential for predicting their pharmacological activities; therefore, phytochemicals are considered probable leads for the discovery of new drugs (Adil et al. 2024). In this study, flavonoids, tannins and saponins were detected in the acetone, ethanol and water extracts of C. imberbe. However, terpenoids and anthocyanins were not detected. In addition, quantitative evaluation of phytochemicals in the extracts showed that the acetone extract had the highest total phenolic, total flavonoids and proanthocyanidin contents, while the ethanol extract contained the lowest contents of total phenolic and total flavonoids.
The results of quantitative evaluation of phytochemicals in the three extracts utilised in this study are in line with the study of Nawaz et al. (2020) where a less polar extract yielded higher phenolic and flavonoid contents. Plant-derived bioactive compounds were also reportedly present at higher quantities in acetone extracts when compared with extracts of other solvents (Rao et al. 2023). This is instructive as a previous study ascribed the ability of medicinal plants to treat microbial infections of the alimentary canal to the presence of phytochemicals, including flavonoids, tannins and saponins (Belapurkar, Goyal & Tiwari-Barua 2014). In addition, flavonoids and tannins were reportedly responsible for antidiarrhoeal activity of medicinal plants (Palombo 2006). It is therefore plausible that the antidiarrhoeal activity of C. imberbe extracts evaluated in this study could be because of the presence of the classes of phytochemicals that were detected in them. Findings from this study align with previous studies that reported a positive correlation of total flavonoid and total phenolic contents with antioxidant capacity of plant extracts, which in turn has a positive correlation with the antibacterial properties of such extracts (Mehmood et al. 2022).
The presence of phytochemicals has been identified as a vital contributor to the antioxidant capacity of medicinal plants (Khan et al. 2022). In addition, total phenolic content, total flavonoid content and DPPH and ABTS inhibitory assays are routinely utilised as a means of determining the antioxidant activity of plant extracts (Irshad, Jawad & Mushtaq 2025; Rayan et al. 2020; Wołosiak et al. 2022). Results obtained from this study expressed as IC50 values showed that all the three extracts of C. imberbe possess significant DPPH and ABTS radical-scavenging activities. The results suggest that the water extract is the most effective in scavenging DPPH radicals, while the acetone extract has the best ABTS radical scavenging activity.
Minimum inhibitory concentration is defined as the minimum concentration of an antimicrobial agent that inhibits the observable growth of the tested microorganism (Zouine et al. 2024). Antibacterial activity of plant extracts and pure compounds increase with low MIC values (Sharma et al. 2017). Usually, plant extracts that have MIC values less than 8 mg/mL are regarded as active. Extracts with MIC values below 100 µg/mL are considered as having significant activity, extracts with MIC values between 100 and 625 µg/mL are considered moderately active, while those above 625 µg/mL are regarded as having low activity (Zouine et al. 2024).
In this study, acetone and ethanol extracts of C. imberbe showed significant antibacterial activity against most of the diarrhoea-causing bacterial strains tested in this study except against B. cereus, E. coli and S. choleraesuis where they both showed moderate activity. Water extract of C. imberbe largely showed low antibacterial activity against almost all the diarrhoea-causing bacterial strains used in this study, except against E. faecium and A. baumannii, where it showed moderate antibacterial activity. These results suggests that acetone and ethanol extracts of C. imberbe might contain potent antibacterial compounds that accounted for the observed low MIC values. The acetone extract of C. imberbe used in our study was significantly more effective against S. aureus, E. coli and P. aeruginosa than the one used in an earlier study (Eloff 1999). Results obtained from this study correspond to those of Donkor et al. (2025) who reported that ethanol leaf extract of Combretum adenogonium (a member of the Combretaceae family) had a higher antibacterial effect than the aqueous extract against both Gram-positive and Gram-negative bacteria used in their study (Donkor et al. 2025). Our findings support the local utilisation of C. imberbe against diarrhoea as tinctures, but not as decoctions, infusions and macerations (Magwenzi, Nyakunu & Mukanganyama 2014).
The better antibacterial activity of the acetone extracts in this study agrees with findings from previous studies that reported that non-polar extracts demonstrated better antibacterial effects when compared with polar extracts and that water does not usually extract antimicrobial compounds from several plants (Junior et al. 2009; Kotzé, Eloff & Houghton 2002). The observed antibacterial activity of the ethanol extract of C. imberbe may be because ethanol, although largely polar, could have extracted some antibacterial compounds that are not extractable by water, an absolute polar solvent. This observation is in accordance with Ibrahim and Kebede (2020) who reported that methanol extracts of medicinal plants had better antibacterial activities than aqueous extracts.
Previous studies have reported the antibacterial properties flavonoids, saponins and tannins (Czerkas et al. 2024; Tagousop et al. 2018; Yuan et al. 2025). Flavonoids have been reported to exert their bactericidal effects via several mechanisms including inhibition of energy metabolism, inhibition of cytoplasmic membrane function and inhibition of nucleic acid synthesis (Sharma et al. 2017; Cushnie & Lamb 2005). Moreover, the ability of saponins and tannins to cause cell lysis that disrupts the membrane permeability of cells and eventually lead to cell death has been regarded as one of its mechanisms of antibacterial effects (Kaczmarek 2020; Maestrini et al. 2020). We propose that the potential antidiarrhoeal effects of the extracts might be via the bactericidal effects of the class of phytochemicals identified in them.
According to the American National Cancer Institute (NCI), plant crude extracts are considered cytotoxic if they have IC50 values < 20 µg/mL or 10 µM after incubation for 48 h or 72 h (Canga et al. 2022). In vitro cytotoxicity tests provide a means for quick identification of the potential toxicity of a test substance, including plant extracts and biologically active compounds (Bácskay et al. 2018). Cellular toxicity studies are crucial for determining the essential minimal to no toxicity status of pharmaceutical products (Çelik 2018). This study showed that HEK 293 cells are most susceptible to WCI extract, which has the lowest IC50 value, compared to ECI and ACI extracts. Nevertheless, the high IC50 values (> 20µg/mL) obtained suggested that all three extracts were non-toxic and relatively safe for use and hence less likely to cause harm. Therefore, the results demonstrated the biocompatibility of C. imberbe extracts towards healthy normal cells, which would be beneficial for many biological applications.
Based on their considerable antibacterial activity and non-toxicity in HEK 293 cells, the acetone and ethanol extracts of C. imberbe leaves had comparable antibacterial and safety profiles and were the most effective among the three extracts.