1.   Introduction

The genus Acanthospermum belongs to the family Asteraceae (Compositae), which is known to be the largest family of flowering plants comprising over 1600 genera and 25,000 species widely distributed across the world [1]. Acanthospermum originates from the two Greek words akanthos (spiny) and sperma referring to seed. The specific epithet hispidum is derived from the Latin word hispidus meaning rough, hairy, or bristly [2]. A. hispidum is popularly known as bristly starbur or goat head. it is an annual herb characterized by dichotomous (Y-shaped) branching with hairy stems. The plant has elliptic and obovate leaves that range from 1.5 to 7.0 cm long, though some may be as long as 11.5 cm. It produces yellow flowers. The fruits of the plant are spiny and flattened, and are shaped like triangles that range from 5 cm to 10 cm in length [3]. It is native to tropical America, but has been introduced to Europe, Africa, India, and Asia [4] and is locally called kashin-yaawoo (Hausa), dagunro (Yoruba) and yaawoo (“Ron” tribe) in Nigeria. In Kandlemullu it is commonly referred to as kannada [4]. A. hispidum has been traditionally used in ethnomedicine to treat various ailments, including headaches, abdominal pains, convulsions, coughs, eruptive fevers, snake bites, scabies, asthma, bronchitis, dysentery, and fevers, and as an expectorant [5, 6].

Sesquiterpene lactones, including melampolides, germacranolides, and guaianolides have been isolated and characterized from A. hispidum [7, 8]. A. hispidum has been reported to exhibit various pharmacological activities such as molluscicidal [5], antibacterial [5], anthelmintic [9], antitumor [10], and antitrypanosomal activity [4]. Ethnopharmacological activities of two major sesquiterpene lactones isolated from A. hispidum, have been reported to show effective antiparasitic activities against Trypanosoma brucei [11] and serve as a promising anticancer, antimicrobial and antioxidative agent [12]. This study is therefore aimed at investigating the chemical composition, enantiomeric distribution, and bactericidal activities of the essential oil of A. hispidumfrom Nigeria.

2.   Materials and methods

2.1. Plant material and identification

The fresh plant of A. hispidum was collected in June 2023 from a local farm in Hayin Liman, Sabon Gari village (11°06′60.00″ N, 7°43′59.99″ E), located in Kaduna South Local government area of Kaduna State, Nigeria. The plant was authenticated by Mr. Namadi Sunusi of the Botany Department, Ahmadu Bello University, Zaria, with voucher number ABU07053. The fresh leaves of the plant were air-dried in the shade for 7 days and then pulverized using an electric blender before extraction.

2.2. Isolation of the essential oils

The air-dried leaves (500 g) were introduced into a 5-L flask and distilled water was added until it covered the sample. Hydrodistillation was carried out for four hours in an all-glass Clevenger apparatus according to the British pharmacopeia. The distillate was extracted with n-hexane, transferred to a pre-weighed amber sample bottle and dried using anhydrous sodium sulfate to eliminate traces of water. The oils were kept under refrigeration (4 °C) until ready for analysis.

2.3. Gas chromatographic–mass spectral analysis

The essential oil was analyzed by Gas Chromatography – Mass Spectrometry (GC-MS) using Shimadzu GCMS-QP2010 Ultra operated in the electron impact (EI) mode (electron energy = 70 eV), scan range = 40-100 atomic mass units, scan rate = 3.0 scan/s. The GC column was a ZB-5 fused silica capillary column (30 m length ´ 0.25 mm inner diameter with a 5% phenyl polydimethylsiloxane stationary phase and a film thickness of 0.25 μm. The carrier gas was helium with a column head pressure of 553 kPa and a flow rate of 1.37 mL/min. The injector temperature was 250 °C and the ion source temperature was 200 °C. The GC oven temperature was programmed for 50 °C initial temperature, then increased at the rate of 2 °C/min to 260 °C. A 5% w/v solution of the sample in CH₂Cl₂ was prepared and 0.1 μL was injected with a splitting mode (30:1). Identification of the volatile oil constituents was achieved based on their retention indices and by comparison of their mass spectral fragmentation pattern with those reported in databases [13–16]. The quantification of the constituents of the essential oil was done using an external standard method using calibration curves generated by running a GC analysis of representative standard compounds for each class [17].

2.4. Chiral gas chromatographic–mass spectral analysis

Chiral GC-MS of the essential oil of A. hispidum was carried out using a Shimadzu GC-MS QP2010S (Shimadzu Scientific Instruments, Columbia, MD, USA) operated in the EI mode (electron energy = 70 eV) with a scan range of 40–400 amu and scan rate of 3.0 scans/s. The GC was equipped with a Restek B-Dex 325 capillary column (Restek Corp, Bellefonte, PA, USA) (30 m × 0.25 mm ID × 0.25 μm film). The oven temperature was programmed as follows: Start at 50 °C, temperature increased to 120 °C at a rate of 1.5 °C/min, then increased to 200 °C at 2 °C/min, and kept at 200 °C for 5 min. Helium was the carrier gas with a flow rate of 1.8 mL/min. The sample was diluted to 3% w/v with CH₂Cl₂, and a 0.1 μL sample was injected in a split mode at a split ratio of 1:45. The terpenoid enantiomers were identified by comparison of retention indices with authentic samples obtained from Sigma-Aldrich (Milwaukee, WI, USA). Relative enantiomer percentages were determined based on peak areas.

2.5. Antibacterial activity

The essential oils were screened for antimicrobial activity against the bacteria Staphylococcus aureus (ATCC No. 25923), Bacillus subtilis (ATCC No.6633), Streptococcus faecalis (ATCC No.9790), Salmonella typhi (ATCC No. 6539), Proteus vulgaris (ATCC No. 6380), Escherichia coli (ATCC No.25922), and Pseudomonas aeruginosa (ATCC No. 27853) using the micro-broth dilution technique as previously described [18, 19]. All bacteria were cultured using tryptic soy agar (Sigma-Aldrich, St. Louis, MO). A 1% stock solution of the essential oil in DMSO (50 μL) and 50 μL of cation–adjusted Mueller Hinton broth (CAMHB) (Sigma-Aldrich, St. Louis, MO) was added to the top wells of a 96-well microdilution plate. The essential oil solution was diluted serially in CAMHB (1:1) to obtain concentrations of 2500, 1250, 625, 312.5, 156.3, 78.1, 39.1, and 19.5 μg/mL. All microbes were harvested from a fresh culture, and then added to each well at a concentration of approximately 1.5 × 10⁸ CFU/mL The 96-well microdilution plates were incubated at 37°C. The minimum inhibitory concentration (MIC) was determined as the lowest concentration with no turbidity. The positive antibiotic control used was streptomycin obtained from (Sigma-Aldrich, St. Louis, MO) while DMSO was used as the negative control (50 μL DMSO diluted in 50 μL broth medium, and then serially diluted as above). (E)-β-Caryophyllene, caryophyllene oxide, and α-bisabolol (Sigma-Aldrich, St. Louis, MO) were individually screened for activity.

3.   Results and discussion

3.1. Essential oil composition

Hydrodistillation of A. hispidum air-dried leaves yielded a pale-yellow essential oil, 0.46% (v/w) yield. The chemical constituents of the essential oil were identified using gas chromatography-mass spectrometry. The GC-MS analysis revealed a total of 70 chemical components presenting 99.2% of the total oil composition (Table 1). The essential oil was dominated by monoterpenes (9.5%) and sesquiterpenes (87.6%). The predominant sesquiterpenes were (E)-β-caryophyllene (21.8%), α-bisabolol (20.7%), bicyclogermacrene (7.9%), caryophyllene oxide (6.6%), germacrene D (6.1%), α-humulene (5.9%), trans-β-elemene (3.6%), α-copaene (2.1%) and δ-cadinene (1.8%). The major monoterpenes wereα-pinene (3.0%), 1,8-cineole (1.8%), thymylmethyether (1.4%), sabinene (1.1%), and limonene (0.6%).

Table 1. Chemical composition of the essential oil of Acanthospermum hispidum from north-central Nigeria.

RT = Retention time in minutes. RI_calc = Retention index determined using a homologous series of n-alkanes on a

ZB-5ms column. RI_db = Reference retention index from the databases. tr = trace (< 0.05%).

Previous reports on the essential oil of A. hispidum obtained from Nigeria [20], Congo [21], and Argentina [5] revealed similar major constituents in varying quantities with some different constituents as shown in Table 2. Carvacryl methyl ether was identified in the previous study [21] but was absent in this study. Differences in the essential oil composition observed in the current and previous research studies may be attributed to certain factors, which include climatic, altitude, geographical, and environmental factors (temperature, day length, light, fertilizers) [22, 23]. The essential oil of A. hispidum obtained from Nigeria in this present study also showed similarity in the essential oil constituents obtained from Congo [21] and Argentina [5], which were dominated by sesquiterpenes and represent the uniqueness of the class of chemical constituents present in this essential oil.

Table 2. Major components (%) of Acanthospermum hispidum leaf essential oils from different geographical locations.

The enantiomeric distribution of terpenoid components of A. hispidium is summarized in Table 3. The chiral GC-MS analysis of A. hispidum essential oil showed the presence of five pairs of enantiomeric monoterpenoids and six enantiomerically pure chiral sesquiterpenoids (α-copaene, trans-β-elemene, (E)-β-caryophyllene, germacrene D, β-bisabolene and δ-cadinene). The ratio of the enantiomer presence in the essential oil gives information which could be related to the biosynthesis of the plant essential oil components and the biological activity of the essential oil [24, 25]. The enantiomeric excess (ee, %) was determined for α-pinene (85.7%), sabinene (70.6%), β-pinene (4.8%), limonene (65.4%) and linalool (82.7%). β-Pinene showed almost a racemic mixture with 52.38% (+) and 47.08% (–).

Table 3. The enantiomeric distributions of chiral terpenoid constituents of Acanthospermum hispidum essential oil from north-central Nigeria.

 RT = Retention time in minutes. RI_calc = Retention index determined using a homologous series of n-alkanes on a Restek B-Dex 325 capillary column. ED = Enantiomeric distribution. ee = enantiomeric excess.RI_db = Retention index from our in-house database. nd = compound not detected.

3.2. Antibacterial activity

The essential oil of A. hispidumwas screened for antibacterial activity using the micro-dilution method against selected pathogenic bacteria, namely Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Streptococcus faecalis ATCC 9790, Salmonella typhi ATCC 6539, Proteus vulgaris ATCC 6380, Escherichiacoli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 (Table 4). The antimicrobial assay revealed a wide spectrum of activity with minimum inhibitory concentrations (MICs) ranging from 125 to1250 µg/mL. Antimicrobial activities of plants have been defined to exhibit good anti-microbial activity with MIC < 100 μg/mL, moderate activity (MIC 100-500 μg/mL), weak activity (MIC 500-1000 μg/mL), and considered inactive(MIC > 1000 μg/mL) [26]. Notably, the essential oil of A. hispidum showed good antimicrobial activity against all selected pathogenic bacteria with Salmonella typhi and Proteus vulgaris moderately susceptible (MIC = 625 μg/mL) and strong susceptibility against Staphylococcus aureus (MIC = 125 μg/mL). The antimicrobial activity displayed by the essential oil of A. hispidum may be attributed to the presence of identified (E)-β-caryophyllene and α-bisabolol constituents. (–)-α-Bisabolol, a monocyclic sesquiterpene alcohol has been reported to have promising activities as anti-inflammatory, anti-irritant, and antibacterial [27, 28]. Synergistic effects likely play a role in the activities [29, 30].

 Table 4. Antibacterial activities (MIC, μg/mL) of Achanthospermum hispidum leaf essential oil from north-central Nigeria.

4.   Conclusions

In conclusion, chiral GC-MS is an efficient method of analysis of reporting enantiomeric distribution of chiral terpenoids present in the essential oil. The results revealed the importance of enantiomers of sesquiterpenoid compounds as antibacterial agents. The major compounds observed in the present study, (E)-β-caryophyllene (21.8%) and α-bisabolol (20.7%), complement the previous investigations of A. hispidum essential oil. Future studies should be considered for the prospect of A. hispidum as an antimicrobial agent. That is, the essential oil may be considered for formulation as an antibacterial in the pharmaceutical or cosmetics industries.

Authors’ contributions

Conceptualization, M.S.O; Methodology, D.S.R.O., M.S.O., P.S., and W.N.S.; Software, P.S.; Validation, L.A.O., W.N.S., Formal Analysis, A.P., and W.N.S.; Investigation, D.S.R.O., M.S.O., P.S., A.P., and W.N.S.; Resources, D.S.R.O., M.S.O., P.S. and W.N.S.; Data Curation, W.N.S.; Writing – Original Draft Preparation, D.S.R.O. and M.S.O; Writing – Review & Editing, M.S.O. and W.N.S.; Project Administration, D.S.R.O. and M.S.O.

Acknowledgements

This work was carried out as part of the activities of the Aromatic Plant Research Center (APRC, https://aromaticplant.org/). We are grateful to Dr. P.A. Akinduti for the antibacterial screening.

Funding

This research received no specific grant from any funding agency.

Availability of data and materials

All data will be made available on request according to the journal policy.

Conflicts of interest

The authors declare no conflict of interest.

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