A periodical of the Faculty of Natural and Applied Sciences, UMYU, Katsina
ISSN: 2955 – 1145 (print); 2955 – 1153 (online)
ORIGINAL RESEARCH ARTICLE
Sani Surajo1*, Abdussalam Auwal2, Abbas Baballe2, Zakariya Ali Muhammad2, Abdussalam Yunusa3, Dauda Jesse Michael4
1Department of Science Laboratory Technology, College of Science and Technology, Jigawa State Polytechnic, Dutse, P. M. B. 7040, Jigawa State, Nigeria
2Department of Biological Sciences, Faculty of Natural & Applied Science, Sule Lamido University Kafin Hausa, P. M. B. 048, Kafin Hausa, Jigawa State, Nigeria
3Department of Integrated Science, School of Secondary Science Education, Jigawa State College of Education, Gumel, P. M. B. 1002, Jigawa State, Nigeria
4Department of Integrated Science, Kaduna State College of Education, Gidan Waya, Kaduna , Nigeria
Corresponding Author: Sani Surajo 
surajosani@jigpoly.edu.ng
Multidrug-resistant bacterial strains represent a growing global public health concern, prompting increased interest in plant-derived antibacterial agents. Vernonia amygdalina is widely used in traditional medicine and has been reported to possess antimicrobial properties; however, the antibacterial potential of its volatile terpene-rich extracts has received limited investigation. Therefore, this study aimed to evaluate the antibacterial activity of a volatile terpene-rich crude extract from V. amygdalina leaves against selected enteric bacteria and to characterize its phytochemical constituents using gas chromatography–mass spectrometry (GC–MS). Dried powdered leaves of V. amygdalina were extracted by maceration using ethanol followed by aqueous reconstitution containing lead acetate solution and chloroform partitioning to obtain a volatile terpene-rich crude extract. Antibacterial activity of the extract was evaluated against clinical isolates of Salmonella typhi and Escherichia coli using agar well diffusion susceptibility tests, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays. Chemical profiling of the extract was performed by GC–MS analysis on an Agilent system equipped with an HP-5MS capillary column, using helium as the carrier gas. The extract exhibited concentration-dependent antibacterial activity against S. typhi, producing inhibition zones of 10.5 ± 0.7 mm, 6.5 ± 2.1 mm, and 5.5 ± 0.7 mm at 1000, 800, and 600 µg/ml, respectively, while E. coli showed no susceptibility. The MIC and MBC values against S. typhi were 800 µg/ml and 1000 µg/ml, respectively. GC–MS analysis identified 22 bioactive compounds, predominantly volatile terpenoids including farnesol, squalene, and lavandulol derivatives, that contributed to the observed antibacterial activity. These findings suggest that the volatile terpene-rich extract of V. amygdalina leaves possesses moderate antibacterial activity against clinical isolates of S. typhi but shows no activity against E. coli. This highlights the potential of these compounds as natural antibacterial agents with the possibility of optimization towards effective management of enteric bacterial infections.
Keywords: Terpene, GC-MS, V. amygdalina, Salmonella
Vernonia amygdalina Del. belongs to the family Asteraceae, and it is native to Africa. In Nigeria, it is commonly called bitter leaf but known locally in Yoruba as “Ewuro”, in Hausa as “Shuwaka”, in Igbo as “Onugbu” and in Efik as “Etidot” (Itah and Akinjogunla, 2024; Obi et al. 2024; Usman et al. 2025). The plant is commonly consumed in Nigeria as food or medicine due to its nutritional and health benefits. In traditional medicine, it is used to treat various ailments, including intestinal worms, bloating, malaria, urinary problems, menstrual pain, and skin infections, among others, either singly or synergistically with other plant and animal parts (Degu et al., 2024). Phytochemical classes such as flavonoids, terpenes, saponins, tannins, steroids, and alkaloids have been reported from the leaf section of the plant. Sesquiterpene lactones such as vanodalol, vernolide, epivernodalol, vernodalinol, flavonoids such as luteolin, cymaroside, phenolics such as quinic acid derivatives, among other compounds, have been isolated from the leaf section of the plant. Experimental studies showed that plant metabolites and isolated compounds possess anti-inflammatory, antidiabetic, antioxidant, anticancer, antiparasitic, and antidiabetic properties (Abere et al., 2025).
GC–MS is a widely used analytical technique for the identification and characterization of bioactive phytochemicals present in medicinal plants (Salisu et al. 2017; Zheng et al. 2018; Salisu and Shema 2019; Lokossou et al. 2022; Hamisu and Salisu 2025; Ibrahim et al. 2025; Musari et al. 2025; Said et al. 2025; Ado et al. 2026; Hassan et al. 2026). The technique enables the detection of volatile and semi-volatile compounds, such as terpenoids, fatty acids, alcohols, and other secondary metabolites, which may contribute to the biological activities of plant extracts (Al-Rubaye et al., 2017; Njoku et al., 2021). Several studies have employed GC–MS profiling to investigate the phytochemical composition of various medicinal plants and to relate the identified compounds to their antimicrobial properties. For instance, GC–MS analysis of several medicinal plant extracts revealed diverse bioactive constituents with significant antibacterial potential against pathogenic microorganisms (Nabi et al., 2022; Farooq et al., 2024; Endris et al., 2024). These findings highlight the importance of phytochemical profiling techniques such as GC–MS in the discovery of plant-derived compounds that may serve as potential alternatives to conventional antibiotics.
The increasing spread, persistence, and prevalence of multidrug-resistant bacterial strains have emerged as a significant global public health concern largely due to the overuse of antibiotics, which has resulted in increased resistance and subsequently treatment failures, which in turn have resulted in increased healthcare costs and higher mortality, particularly in developing countries (Itah & Akinjogunla, 2024). This has prompted an increased search for novel, potent, and safe alternative sources of antibiotics with a major focus on medicinal plants due to their relative safety. Studies have shown that medicinal plant metabolites affect their antibacterial potential via mechanisms such as disruption of bacterial membrane, inhibition of protein synthesis, and disruption of nucleic acid replication (Alozie et al., 2024). Volatile terpenes and terpenoids derived from medicinal plants have been reported to possess significant antimicrobial properties, primarily by altering membrane permeability, leading to the release of nucleic acids and proteins and a decrease in membrane potential (Masyita et al., 2022). In vitro studies have demonstrated that specific terpenes, such as thymol, carvacrol, and linalool, exhibit significant antibacterial activity against foodborne and pathogenic bacteria, primarily by disrupting microbial cell membranes and interfering with cellular metabolic processes (Di Matteo et al., 2024). Furthermore, recent reviews have highlighted the growing interest in plant-derived terpenes as potential agents to combat antimicrobial resistance, due to their diverse mechanisms of action and reduced likelihood of resistance development (Bardaji et al., 2025). Thus, highlighting the potential of terpene-based compounds as promising candidates against pathogenic bacteria and antimicrobial resistance. Previous studies on the phytochemical composition and antibacterial activities of V. amygdalina have largely focused on conventional extracts and pure isolated compounds (Olusola-Makinde et al., 2021; Tura et al., 2024; Degu et al., 2024). Limited attention has been given to the phytochemical composition and antibacterial activity of its volatile terpene-rich extracts. Therefore, this study aimed to evaluate the antibacterial activity of a volatile terpene-rich crude extract from V. amygdalina leaves against selected enteric bacteria and to characterize its phytochemical constituents using GC–MS platform.
The Vernonia amygdalina plant was collected from a nursery located in the Gwaram Local Government Area, Jigawa State, Nigeria. The plant material was identified by a taxonomist at the Herbarium unit of the Department of Plant Biology, Bayero University, Kano.
Fresh leaves were collected and rinsed thoroughly under running tap water to remove any excess silt. The leaves were shade-dried at room temperature for 14 days. Dried leaves were then pulverized into a powder using a mechanical grinder, and the powdered sample was was stored in an airtight container until extraction.
Extraction was carried out according to a previously described protocol (Watanabe et al., 2005) with modifications (Fig. 1). Briefly, 50 g of powdered plant material was extracted by cool maceration with 200ml ethanol (1:4 w/v) for 72 hours. Extraction was performed twice using fresh 200ml portions of ethanol. The crude ethanol extract was reconstituted with 200 mL of distilled water to obtain an aqueous extract, which was then treated with lead acetate solution to yield a precipitate. The solution was centrifuged at 3000 rpm for 3 min, and the supernatant was extracted with chloroform (150ml x2) to obtain a chloroform extract. The extract yield obtained was 0.10 g (0.2%). The dried extract was stored in an airtight container until further analysis.
Figure 1: Extraction flow chart of powdered Vernonia amygdalina leaf
The test organisms were identified as clinical isolates of Salmonella typhi and Escherichia coli obtained from the microbiology unit of the medical laboratory department of the federal medical center, Nguru, Yobe State. The bacterial isolates were confirmed using standard microbiological identification procedures, including Gram staining and biochemical tests, before use.
Nutrient agar (Titan Biotech LTD, Delhi, India) was used for the growth of the microorganisms. The media was prepared according to manufacturer’s instructions and sterilized in an autoclave at 121℃ for 15 min.
The agar well diffusion method was used to screen the extract as described by Sabo et al. (2019). Briefly, a standardized inoculum of test microorganisms was spread evenly over the surface of the medium. At the center of each inoculated medium was a well containing 0.2 ml of extract concentrations (1000, 800, and 600 μg/ml). The inoculated plates were then incubated at 37℃ for 24 h. Thereafter, the plates were observed for growth inhibition, and the diameters of the zones of inhibition were measured and recorded. The experiment was conducted using triplicate tests at each concentration.
MIC of the extracts was determined using the broth dilution method as described by Sani et al. (2016). Briefly, nutrient broth was prepared, sterilized (121 °C, 15 min), and dispensed into test tubes. The test microorganisms were inoculated into each tube containing different concentrations of the extract (1000, 800, and 600 μg/ml) and incubated at 37 ℃ for 24 h. The tubes were observed for turbidity (growth), and the lowest extract concentration in the broth that prevents turbidity indicates the MIC. The MIC assay was conducted in triplicate for each concentration.
Here, the MIC experiment was subcultured onto a freshly prepared medium plate and incubated at 37℃ for 24 hours. Each plate was observed for colony growth, and the MBC was determined as the lowest extract concentration at which no colonies grew (Sani et al., 2016). The MBC assay was conducted in triplicate for each concentration.
GC-MS analysis was carried out on Agilent 19091S-433UI system equipped with HP-5MS ultra inert capillary column (30 m × 0.25 mm × 0.25 μm). Helium was used as the carrier gas at a flow rate of 0.73 mL/min. The column oven temperature was held at 50°C to 325°C at 10°C/min for 5 min. An injection volume of 2 µL was used in split mode, with a total runtime of 40 min. The mass spectrometer was operated in electron ionization mode at 70 eV, with a m/z range of 100 –600. Identification of compounds was performed by comparing the obtained mass spectra with those in the National Institute of Standards and Technology (NIST 14) library database. Only compounds with a similarity index ≥80% were considered for identification.
Results were presented as mean ± standard deviation (SD) where applicable. Statistical analysis was performed using One-way analysis of variance (ANOVA) followed by Duncan Multiple Range Test (DMRT) for multiple comparisons. Differences among group means were considered statistically significant at p ˂ 0.05. All analyses were conducted using IBM SPSS Statistics version 29.
The volatile terpene-rich crude extract exhibited concentration-dependent inhibitory activity against S. typhi, with the highest inhibition observed at 1000 µg/ml, while E. coli showed resistance to the extract (Table 1), which suggest E. coli was highly resistant to both the extract and standard drug, a phenomenon most probably associated with the formation of biofilm by the pathogen. The resistance of E. coli to antibiotics is quite common and has been shown in previous studies (Ali et al., 2019; Onifade et al., 2024). Factors such as self-medication or inappropriate administration of antibiotics due to the lack of guidelines for selecting antibiotic drugs were most probably associated with this finding, given that the E. coli tested in this study was a clinical isolate (Onifade et al., 2024). Similar observations have been reported in previous studies, in which plant extracts exhibited lower activity against Gram-negative bacteria than against Gram-positive bacteria due to intrinsic resistance mechanisms (Nazzaro et al., 2013; Bassolé & Juliani, 2012). Furthermore, the resistance of E. coli in this study may also be due to the specific composition and concentration of the identified compounds, as well as possible absence or low abundance of highly active constituents required to exert inhibitory effects against Gram-negative organisms. This finding highlights the selective antibacterial activity of the extract and suggests that its efficacy may be more pronounced against susceptible bacterial strains such as S. typhi.
Table 1: Shows zones of inhibition by extracts on bacterial pathogens
| Extract concentrations (μg/ml) | Zones of inhibition (mm) | |
|---|---|---|
| S. typhi | E. coli | |
| 1000 | 10.5 ± 0.7a | 0.0 ± 0.0 |
| 800 | 6.5 ± 2.1b | 0.0 ± 0.0 |
| 600 | 5.5 ± 0.7b | 0.0 ± 0.0 |
| 5 (Ciprofloxacin) | 12.5 ± 0.7a | 0.0 ± 0.0 |
Values are expressed as mean ± SD of independent experiments. Means with different superscript letters within the same column are significantly different (p < 0.05) using one-way ANOVA followed by DMRT.
Fig. 2. Bar chart showing antibacterial activity of volatile terpene-rich crude extract of V. amygdalina against S. typhi and E. coli. Values represent mean inhibition zone ± SD of triplicate experiments.
The MIC and MBC values of the volatile terpene-rich crude extract against S. typhi are presented in Table 2. The extract exhibited inhibitory activity, with MIC and MBC values of 800 µg/mL and 1000 µg/mL, respectively (Figure 2). The higher MBC value relative to the MIC suggests that the extract was bacteriostatic at lower concentrations and bactericidal at higher concentrations against S. typhi. These findings indicate that the extract possesses measurable antibacterial activity against S. typhi.
Table 2: Shows the MIC and MBC of the extract against S. typhi at different concentrations
| Test organism | Extract concentrations | MIC | MBC |
|---|---|---|---|
| S. typhi | 1000 | - | * |
| 800 | * | + | |
| 600 | + | ++ |
- = clear (no growth), * = MBC, + = moderate colony growth, ++ = heavy colony growth
The retention times, library identification, compound classes, molecular formulas, and molecular weights of the phytochemical constituents identified in the volatile terpene-rich extract of V. amygdalina are presented in Figure 3 and Table 3. A total of 22 bioactive compounds were identified through GC–MS analysis. The chromatogram (Figure 3) revealed several prominent peaks, indicating the presence of both major and minor constituents within the extract.
Notable compounds corresponding to prominent peaks in the chromatogram include oleic acid, squalene, farnesol (2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl-), lavandulol derivative, and aspidocarpine, occurring at retention times of 38.27, 37.63, 37.48, 35.41, and 34.28 (Table 3). The phytochemical compounds identified by GC–MS were further grouped into chemical classes, as shown in Table 4. Terpenoids constituted the largest proportion of compounds detected, followed by fatty acids and their derivatives, while other minor classes included organosilicon compounds, ketones, alcohols, and alkaloids. The distribution of these compound classes is illustrated in Figure 4.
Terpenoids are widely reported to possess significant antimicrobial, antifungal, and anti-inflammatory properties, largely due to their ability to disrupt microbial cell membranes and interfere with essential metabolic processes (Bakkali et al., 2008; Guimarães et al., 2019). Among the identified compounds, farnesol, an acyclic sesquiterpene alcohol, has been reported to exhibit antibacterial activity against a range of Gram-positive and Gram-negative bacteria. Its mechanism of action involves altering membrane permeability and inhibiting biofilm formation, thereby reducing bacterial survival and virulence (Jabra-Rizk et al., 2006; Gomes et al., 2018).
Similarly, lavandulol, a monoterpene alcohol commonly found in essential oils, has been associated with antimicrobial activity through disruption of microbial membrane integrity and inhibition of key enzymatic systems (Burt, 2004). The presence of such compounds may contribute to the inhibitory activity observed in this study against S. typhi.
In addition, the triterpene squalene was identified in the extract. Although primarily known for its antioxidant and pharmacological properties, squalene may also contribute to antimicrobial activity through membrane interactions and enhancement of the bioactivity of other phytochemicals (Reddy & Couvreur, 2009). The combined presence of these compounds suggests possible synergistic interactions that may enhance the extract's overall antibacterial efficacy.
The antibacterial activity observed in this study may therefore be attributed to the collective action of multiple phytoconstituents, particularly terpenoids and fatty acid derivatives. It has been reported that complex mixtures of phytochemicals often exhibit greater biological activity than isolated compounds due to synergistic effects (Bassolé & Juliani, 2012).
However, the presence of these bioactive compounds supports the antibacterial activity of the volatile terpene-rich extract of V. amygdalina and highlights its potential as a source of natural antimicrobial agents.
Figure 3: Total ion chromatogram showing retention time and abundance of compounds
Table 3: Phytoconstituents identified in volatile terpene-rich crude extract from V. amygdalina leaf by GC-MS
| Compounds | Retention time (min) | Library/ID | Compound class | Molecular formular | Molecular weight (g/mol) |
|---|---|---|---|---|---|
| 1 | 5.99 | Octanoic acid, ethyl ester | Fatty acid acyl | C10H20O2 | 172.26 |
| 2 | 8.91 | Cyclohexasiloxane, dodecamethyl | Organosilicon | C12H36O6Si6 | 444.92 |
| 3 | 13.72 | dihydroactinidiolide | Benzofuran (lactone) | C11H16O2 | 180.24 |
| 4 | 17.54 | 3-Hydroxy-5,6-epoxy-I (2)-ionone | acyclic ketones | C13H20O3 | 224.3 |
| 5 | 18.54 | Oplopanone | Sesquiterpenoid | C15H26O2 | 238.37 |
| 6 | 21.10 | cis-Z-α-Bisabolene epoxide | Sesquiterpenoid | C15H24O | 220.35 |
| 7 | 23.86 | Palmitic acid ethyl ester | Fatty acid | C18H36O2 | 284.5 |
| 8 | 24.12 | Longifolenaldehyde | Sesquiterpenoid | C15H24O | 220.35 |
| 9 | 24.18 | 2(1H)-Naphthalenone | Sesquiterpenoid | C10H8O | 144.17 |
| 10 | 27.10 | Elaidic acid ethyl ester | Fatty acid ester | C20H38O2 | 310.5 |
| 11 | 27.60 | Ethyl stearate | Fatty acid | C20H40O2 | 312.12 |
| 12 | 30.64 | Oleamide | Fatty amide | C18H35NO | 281.5 |
| 13 | 34.28 | Aspidocarpine | Alkaloid | C22H30N2O3 | 370.5 |
| 14 | 37.63 | Squalene | Triterpene | C30H50 | 410.7 |
| 15 | 38.27 | Oleic Acid | Fatty acid | C18H34O2 | 282.5 |
| 16 | 15.33 | Octanoic acid, ethyl ester | Fatty acid ester | C10H20O2 | 172.26 |
| 17 | 18.41 | cis-p-mentha-1(7),8-dien-2-ol | Sesquiterpenoid | C10H16O | 152.23 |
| 18 | 26.40 | 3-Isopropoxy-1,1,1,7,7,7-hexamethyl-3,5,5-tris(trimethylsiloxy)tetrasiloxane | Organosilicon | C18H52O7Si7 | 577.2 |
| 19 | 29.32 | Cyclopentadecanone, 4-methyl- | Ketone | C16H30O | 238.41 |
| 20 | 32.72 | Ethanol, 2-(octadecyloxy)- | Alcohol | C20H42O2 | 314.5 |
| 21 | 35.41 | Trifluoroacetyl-lavandulol (S-Lavandulol) | Monoterpene alcohol | C10H18O | 154.25 |
| 22 | 37.48 | 2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl- (Farnesol) | Sesquiterpene alcohol | C15H26O | 222.37 |
Table 4: classification of GC-MS compounds by chemical class
| Chemical class | Number of compounds | Percentage (%) |
|---|---|---|
| Terpenoids | 8 | 36.4 |
| Fatty acids & derivatives | 7 | 31.8 |
| Organosilicon compound | 2 | 9.1 |
| Ketones | 2 | 9.1 |
| Alcohols | 1 | 4.5 |
| Alkaloids | 1 | 4.5 |
| Benzofuran/Lactone | 1 | 4.5 |
Figure 4: Bar chart showing compound classes identified in the volatile terpene-rich extract of V. amygdalina leaves by GC–MS analysis.
The present study demonstrated that the volatile terpene-rich extract of V. amygdalina exhibited antibacterial activity against S. typhi, with MIC and MBC values of 800 µg/ml and 1000 µg/ml, respectively. GC–MS analysis revealed the presence of 22 phytochemicals, predominantly terpenoids such as squalene, farnesol, bisabolene, and lavandulol, which may contribute to the observed antibacterial activity. These findings highlight the potential of the volatile terpene extract of V. amygdalina as a natural source of antibacterial agents. Further studies to isolate specific bioactive compounds and evaluate their mechanisms of action are recommended to better understand their therapeutic potential.
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