This research focused on comparing the effects of Securinega virosa leaf extract and its partitioned fractions against methicillin-resistant Staphylococcus aureus (MRSA). The extraction process utilized 80% methanol, and the resulting crude extract underwent phytochemical screening using established protocols. Antimicrobial potency of the crude methanol extract and its aqueous and chloroform fractions was assessed using the agar well diffusion method. Additionally, their minimum inhibitory concentrations were determined. The phytochemical analysis revealed that the leaf extract of S. virosa contained various bioactive compounds, including alkaloids, flavonoids, saponins, steroids, tannins, terpenes, cardiac glycosides, anthraquinones, and reducing sugars. When tested for antibacterial activity, the crude extract at concentrations of 100, 50, 25, 12.5, and 6.25 mg/ml produced inhibition zones averaging 14.67 ± 0.82, 10.33 ± 0.82, 8.33 ± 0.41, 6.67 ± 0.41, and 4.33 ± 0.82 mm, respectively. For the partitioned fractions, the aqueous extract yielded inhibition zones of 10.50 ± 0.35, 6.67 ± 1.47, 4.67 ± 0.82, 1.67 ± 2.04, and 1.33 ± 1.63 mm across the same concentrations. The chloroform fraction resulted in inhibition zones of 10.33 ± 1.08, 6.67 ± 0.41, 4.33 ± 1.08, and 2.66 ± 1.78 mm, respectively.
Regarding the minimum inhibitory concentration, the crude extract displayed effectiveness starting at 25 mg/ml, while both the aqueous and chloroform fractions showed inhibitory activity at 50 mg/ml. Overall, all forms of S. virosa extracts tested (crude methanol, aqueous, and chloroform) demonstrated activity against MRSA, with the crude extract exhibiting the most pronounced antibacterial effect.
The global rise in multidrug-resistant bacterial infections remains a critical public health concern [1–3]. Pathogens of clinical importance, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci, and Escherichia coli strains producing extended-spectrum β-lactamase (ESBL), are spreading at an alarming rate [4, 5]. Consequently, the pursuit of novel therapeutic agents has become increasingly urgent. The persistence of bacterial drug resistance, recurrent infections in immunocompromised individuals, the emergence of new deadly viruses, and the surge in global fungal infections highlight the limitations of current medical interventions [6, 7].
For centuries, herbal remedies and plant-derived medicines have been integral to the prevention and treatment of various diseases [8]. In many African nations, traditional medicine serves as the primary approach for managing high-fever illnesses such as malaria in children, accounting for about 60% of first-line treatments [9]. The growing demand for herbal therapies is further driven by population expansion, with projections estimating over 7.5 billion people globally within the next 10–15 years. Much of this growth is expected in the southern hemisphere, where roughly 80% of the population still relies on herbal-based traditional medicine for basic health care [10]. These considerations provided a strong rationale for conducting this investigation into the potential effects of Securinega virosa extract and its partitioned fractions on MRSA.
Bacteria (in addition to causing infections) possess the ability to develop resistance to antimicrobial agents. This evolutionary adaptation reduces the long-term effectiveness of antibiotics. The improper or excessive use of these drugs has accelerated the emergence of resistance, leading to high mutation rates in bacterial strains and a rapid spread of resistant organisms [11]. With the increasing virulence and resistance of pathogens, the global burden of these infections has grown considerably. In the US and Europe alone, antimicrobial-resistant infections are estimated to cause around 50,000 deaths each year, with even greater numbers reported across other regions. If left unchecked, projections suggest drug-resistant infections may account for an additional 10 million deaths worldwide by 2050. In response, the World Health Organization has urged collaboration between governments and the pharmaceutical industry to enhance research investment in antibiotic development to meet urgent public health needs [12].
To address this pressing issue, researchers have turned to natural products, particularly plant-derived secondary metabolites, as promising candidates for new antimicrobial agents. These bioactive compounds offer an affordable and abundant source for combating resistant pathogens. In Nigeria, different parts of the S. virosa plant, including leaves, stem, and root bark, are traditionally used to treat various ailments [13, 14]. However, there remains limited scientific evidence regarding the antimicrobial potential of S. virosa leaf extract against MRSA. Therefore, this study was designed to assess the antibacterial properties of S. virosa extract and its partitioned fractions on methicillin-resistant Staphylococcus aureus.
The following equipment was used: a digital weighing balance (KERO BLG 300), a rotary evaporator, a refrigerator (Haier Thermocol®, Model: HRF-250E), and a hot air oven (Leader®, Model: GP/50/CLAD/250/HYD).
Leaves of S. virosa were collected from Esan Botanical Garden in Edo State. The plant material was identified and authenticated by Dr. O.E. Ikpefan, the Head of the Department of Pharmacognosy and Traditional Medicine. The harvested leaves were rinsed with clean water, chopped into small pieces, and left to air-dry. Once dried, they were ground into fine powder using an electric grinder. The resulting powdered material was weighed and stored in clean plastic containers in the laboratory at a temperature of 25 ± 2 °C until further use for extraction.
A total of 1.2 kg of the powdered S. virosa leaf sample was subjected to extraction using the cold maceration technique. The extraction and subsequent phytochemical screening followed a slightly modified version of the method described in Dhivya and Manimegalai [15].
Nutrient broth was prepared according to the manufacturer’s instructions and distributed into small bottles, which were then sterilized using an autoclave. After sterilization, the bottles were allowed to cool before inoculating them with methicillin-resistant Staphylococcus aureus (MRSA) obtained from the Pharmaceutical Microbiology laboratory. The inoculated broth was incubated at 37°C for 24 hours using a sterile wire loop.
Mueller Hinton agar was prepared according to the manufacturer’s guidelines and sterilized via autoclaving. MRSA strains were evenly spread across the agar plates. A 10 mg sample of cefuroxime underwent serial tenfold and twofold dilutions to obtain a 5 µg concentration. Sterile paper discs were then soaked with the 5 µg cefuroxime solution and placed on the surface of each inoculated agar plate. These plates were incubated at 37 °C for 24 hours. After the incubation period, inhibition zones were recorded based on CLSI standards [16].
Mueller Hinton agar was prepared and sterilized as described earlier, poured into petri dishes, and allowed to solidify. After solidification, bacterial cultures were spread on the agar surface. Serial twofold dilutions of the crude methanol extract of S. virosa were made to obtain concentrations of 100, 50, 25, 12.5, and 6.25 mg. Using a sterile 6 mm cork borer, five wells were created on each plate and labeled according to the concentration. Each well was filled with the corresponding volume of methanol extract. The plates were left for diffusion and then incubated at 37 °C for 24 hours. Following incubation, zones of inhibition corresponding to each concentration were measured and recorded [17].
The antibacterial activity of the crude extract and its various partitioned fractions was assessed and reported as the Minimum Inhibitory Concentration (MIC). Serial two-fold dilutions of the extract and fractions were prepared at five different concentrations: 100, 50, 25, 12.5, and 6.25 mg/mL. Five Petri dishes were labeled corresponding to these concentrations for both the crude extract and the partitioned fractions. Each dish was divided into sections, with MRSA organisms inoculated onto these divisions. The appropriate concentrations of the crude and partitioned extracts were then added to their designated plates. Sterile Mueller-Hinton agar was poured into each plate and gently swirled to ensure proper mixing before allowing the agar to solidify. The inoculated plates were incubated at 37 °C for 24 hours. The lowest concentration at which bacterial growth was inhibited was recorded as the MIC for both the crude extract and its partitioned phases.
From 1.5 kg of powdered S. virosa leaves, a total extract yield of 156 g was obtained, corresponding to approximately 10.4%. The partitioned extract yielded 80 g, from which the aqueous fraction accounted for 42 g (percentage yield) and the chloroform fraction 23 g (approximately 2.21%).
Phytochemical analysis of the leaf extract and its fractions of S. virosa is summarized in Table 1.
Table 1. Phytochemical screening of S. virosa leaves extract and fractions
Phytochemical groups | Extract | Fractions | |
Aqueous | Chloroform | ||
Alkaloids | ++ | - | - |
Flavonoids | ++ | + | - |
Saponin | + | ++ | - |
Steroids | + | - | ++ |
Tannins | +++ | + | - |
Cardiac glycosides | ++ | + | + |
Terpenes | ++ | + | +++ |
Anthraquinones | +++ | ++ | ++ |
Key: +++: appreciable amount; ++: moderate amount; +: minute amounts; -: not detected.
The formation of clear zones around the wells demonstrated inhibition of bacterial growth. The size of these inhibition zones was recorded in millimeters (mm) using a meter rule, as detailed in Table 2.
Table 2. Zone of inhibition (mm) of the extract and fractions against MRSA
Extract | Conc. mg/ml | 1 (mm) | 2 (mm) | 3 (mm) | Mean ± SD (mm) |
Crude | 100 | 14 | 16 | 14 | 14.67 ± 0.82 |
| 50 | 9 | 11 | 11 | 10.33 ± 0.82 |
| 25 | 8 | 9 | 8 | 8.33 ± 0.41 |
| 12.5 | 6 | 7 | 7 | 6.67 ± 0.41 |
| 6.25 | 3 | 5 | 5 | 4.33 ± 0.82 |
Aqueous | 100 | 11 | 10.5 | 10 | 10.50 ± 0.35 |
| 50 | 9 | 6 | 5 | 6.67 ± 1.47 |
| 25 | 6 | 4 | 4 | 4.67 ± 0.82 |
| 12.5 | 5 | - | - | 1.67 ± 2.04 |
| 6.25 | 4 | - | - | 1.33 ± 1.63 |
Chloroform | 100 | 9 | 12 | 10 | 10.33 ± 1.08 |
| 50 | 7 | 6 | 7 | 6.67 ± 0.41 |
| 25 | 3 | 4 | 6 | 4.33 ± 1.08 |
| 12.5 | 3 | - | 5 | 2.66 ± 1.78 |
| 6.25 | - | - | - | - |
Control (cefuroxime) | - |
|
|
| - |
KEY: no inhibition (-)
Antimicrobial evaluation expressed as minimum inhibitory concentration (MIC) for crude extract and partitioned fractions of the leaf of S. virosa (Table 3).
Table 3. Minimum inhibitory concentration for the extract and fractions of S. virosa against MRSA
Concentration (mg/ml) | 100 | 50 | 25 | 12.5 | 6.25 |
Aqueous | - | - | + | ++ | ++ |
Crude | - | - | - | + | ++ |
Chloroform | - | - | + | ++ | ++ |
Key: Growth = +, ++, and no growth = -
The outcomes of the qualitative phytochemical assessment confirmed the existence of various secondary metabolites, including alkaloids, saponins, terpenes, tannins, cardiac glycosides, flavonoids, steroids, reducing sugars, and anthraquinones. These results support the findings of Chouhan et al. [18], who previously identified similar secondary metabolites in the methanol extract of S. virosa. In a related investigation, Adrien et al. [19] examined both the phytochemical profile and antifungal effects of different solvent extracts of S. virosa, likewise reporting the presence of these bioactive compounds.
Although multiple studies have focused on the sedative effects [20] and psychopharmacological potential of S. virosa [14], the current research centers on evaluating the antimicrobial properties of the methanol crude extract and its partitioned fractions from the leaves. Findings from this investigation reveal that S. virosa leaves demonstrate inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA), with the crude extract showing the most prominent antimicrobial effect among all tested samples, as indicated by the largest zones of inhibition.
Specifically, the aqueous extract showed antimicrobial activity only at concentrations of 12.5 mg/mL and 6.25 mg/mL, while the chloroform extract lacked activity at the lowest tested dose of 6.25 mg/mL. In contrast, the crude extract demonstrated inhibition across all concentrations evaluated. The most significant inhibition zone was recorded at 100 mg/mL concentration in all three extracts—crude, aqueous, and chloroform—measuring 14.67 ± 0.82 mm, 10.50 ± 0.35 mm, and 10.33 ± 0.82 mm, respectively.
This antimicrobial effect appears to be concentration-dependent. Furthermore, the chloroform fraction showed no efficacy at reduced concentrations. This observation diverges from the findings of Maria et al. [21], who reported strong antibacterial action of chloroform extract from S. virosa, with an inhibition zone diameter of 13 mm. However, the results from Amenu et al. [22] are consistent with the present findings, where the ethanol extract of S. virosa leaves exhibited substantial antibacterial activity against various test pathogens.
Cefuroxime, used as a standard control at 5 µg, failed to show any inhibitory effect against MRSA, indicating that the S. virosa extracts were more effective in this context. This also lends support to the ethnopharmacological use of the plant in treating infectious conditions. The minimum inhibitory concentration (MIC) values for the crude, aqueous, and chloroform extracts were 25 mg/mL, 50 mg/mL, and 50 mg/mL, respectively. The crude extract, having the lowest MIC, showed superior efficacy, suggesting it is the most potent of the tested samples.
Overall, the data from this study confirm that S. virosa possesses notable antimicrobial activity against infectious agents such as MRSA and that its effectiveness varies based on the concentration and type of extract used.
The aqueous, chloroform, and crude methanol extracts of S. virosa demonstrated antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) across multiple concentration levels. Among these, the crude extract exhibited the most potent effect, likely due to its synergistic compounds that remained intact before fractionation. These findings provide scientific validation for the traditional application of this plant in managing bacterial infections.
In view of these results, medicinal plants like S. virosa should be considered as accessible alternatives for treating bacterial diseases in resource-limited regions. Nonetheless, further studies are necessary to isolate and characterize the bioactive compound(s) chiefly responsible for its antimicrobial action. Additionally, comprehensive evaluations of the plant’s safety profile and toxicity are crucial.
The authors gratefully acknowledge the invaluable assistance of Mr. Micheal Oghenejobo, Chief Laboratory Technologist in the Department of Pharmaceutical Microbiology and Biotechnology, along with the dedicated personnel of the Department of Pharmacognosy and Traditional Medicine.
None
This research was financially supported by the Department of Pharmaceutical Microbiology and Biotechnology and the Department of Pharmacognosy and Traditional Medicine.
Approval for this research was granted by the Research and Ethics Committee, Faculty of Science, Delta State University, Abraka, Nigeria, during its session on November 4th, 2020.
Open Access The author(s) retain copyright. This article is licensed under the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. It may be shared and adapted for non-commercial purposes with appropriate attribution, an indication of changes, and distribution of adaptations under the same license. Third-party material may be subject to separate terms identified in its credit line. View the license at https://creativecommons.org/licenses/by-nc-sa/4.0/.
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