1.   Introduction

Vitex agnus-castus L. (Lamiaceae), commonly known as chasteberry or monk’s pepper, is a small flowering deciduous tree or shrub, that typically grows to an average of 1.5 m to 2 m tall with leaves around 7.6–10 cm in diameter, and is native to southern Europe and Central Asia, mainly the Mediterranean region [1]. The ethnopharmacology and phytochemistry of V. agnus-castus have been reviewed [2–7]; the plant has been used to treat various female conditions such as menstrual disorders, premenstrual dysphoric disorder, corpus luteum deficiency, and menopausal problems [1,8]. The important constituents of V. agnus‑castus essential oil are 1,8‑cineole, sabinene, α‑pinene, (E)‑β‑farnesene, (E)‑β‑caryophyllene, and α‑terpinyl acetate [4, 9]. As part of our ongoing interest in essential oils of aromatic and medicinal plants of Nigeria, this study is aimed to investigate the chemical characterization, enantiomeric distribution, and antibacterial efficacy of the essential oil of V. agnus-castus growing in north-central Nigeria.

2.   Materials and methods

2.1. Plant sample collection and identification

The fresh plant of Vitex agnus-castus was collected in July 2023 along new Jos Road, Zaria (11^o 5′ 0.2544″N, 7^o 42′ 48.726″ 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 ABU0841. The fresh aerial parts, leaves, and seeds of the plant were air-dried in the shade for seven days and then pulverized using an electric blender before extraction.

2.2.   Hydrodistillation of the essential oil

Each of the air-dried plant samples (500 g) was placed in a 5-L flask, and distilled water was added to cover the sample. Hydrodistillation was carried out for four hours in an all-glass Clevenger apparatus in accordance with the British Pharmacopoeia. The distillate was extracted with n-hexane, transferred to a pre-weighed amber sample vial, and dried with anhydrous sodium sulfate to remove any remaining water.  The essential oil yields ranged from 1.2 to 4.5% (v/w) with a yellowish coloration. The oils were refrigerated at 4 °C until ready for analysis.

2.3. Gas chromatographic– mass spectral analysis

The essential oils were analyzed by GC-MS as reported previously [10]: Shimadzu GCMS-QP2010 Ultra (Shimadzu Scientific Instruments, Columbia, MD, USA), electron impact (EI) mode (electron energy = 70 eV), scan range = 40-100 atomic mass units, scan rate = 3.0 scan/s,  ZB-5 fused silica capillary GC column (30 m ´ 0.25 mm ´ 0.25 μm film); He carrier gas, column head pressure = 553 kPa, flow rate =1.3 mL/min; injector temperature = 250 °C, ion source temperature = 200 °C; GC oven temperature program, 50 °C initial temperature, increased to 260 °C at 2 °C/min. A 5% w/v solution of each essential oil in CH₂Cl₂ was prepared and 0.1 μL was injected, 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 [11–14].

2.4. Chiral gas chromatographic– mass spectral analysis

Chiral GC-MS of the essential oils of Vitex agnus-cactus carried out as previously reported [10]: Shimadzu GCMS-QP2010S (Shimadzu Scientific Instruments, Columbia, MD, USA), EI mode (electron energy = 70 eV), scan range = 40–400 amu, scan rate = 3.0 scans/s, Restek B-Dex 325 capillary column (Restek Corp., Bellefonte, PA, USA) (30 m × 0.25 mm ID × 0.25 μm film). The oven temperature program, 50 °C initial temperature, increased to 120 °C at 1.5 °C/min, increased to 200 °C at 2 °C/min, kept at 200 °C for 5 min; He carrier gas, flow rate = 1.8 mL/min. Essential oil samples were diluted to 3% w/v with CH₂Cl₂, and a 0.1 μL was injected, split mode = 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 screening

The A. agnus-castus leaf essential oil was screened for antibacterial activity using the microbroth dilution assay as previously reported [10]: 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); 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 serially diluted in a 96-well microdilution plate (essential oil concentrations = 2500, 1250, 625, 312.5, 156.3, 78.1, 39.1, and 19.5 μg/mL); bacteria were added to each well at concentrations of 1.5 × 10⁸ CFU/mL; plates incubated at 37 °C for 24 h; minimum inhibitory concentration (MIC) was determined as the lowest concentration with no turbidity; positive antibiotic control = streptomycin (Sigma-Aldrich, St. Louis, MO), negative control = DMSO (50 μL DMSO diluted in 50 μL broth medium, serially diluted. 1,8-Cineole and α-terpineol (Sigma-Aldrich, St. Louis, MO) were individually screened for activity.

2.6. Hierarchical cluster analysis

Hierarchical cluster analysis (HCA) analysis was carried out on the V. agnus-castus essential oils using XLSTAT v. 2018.1.1.62926 (Addinsoft, Paris, France). The HCA was performed using the concentrations of the 12 most abundant components (1,8-cineole, sabinene, α-pinene, (E)-β-farnesene, (E)-β-caryophyllene, α-terpinyl acetate, terpinen-4-ol, α-terpineol, bicyclogermacrene, caryophyllene oxide, limonene, and τ-cadinol) from this current work as well as those previously reported compositions from the literature [15–40]. Dissimilarity was used to determine clusters, considering Euclidean distance, and Ward’s method was used to define agglomeration.

3.   Results and discussion

3.1. Essential oil compositions

The essential oils of the aerial parts, leaves, and seeds of V. agnus-castus were analyzed by GC-MS (Table 1). The major components in the aerial parts essential oil were 1,8-cineole (26.4%), terpinen-4-ol (8.9%), α-terpineol (7.4%), sabinene (5.8%), and (E)-β-farnesene (5.5%). The leaf essential oil showed 1,8-cineole (31.6%), sabinene (9.4%), α-pinene (8.1%), terpinen-4-ol (7.5%), and α-terpineol (5.7%) as major compounds. The essential oil from the seeds of V. agnus-castus were rich in 1,8-cineole (20.6%), (E)-β-farnesene (8.1%), α-terpinyl acetate (7.7%), sabinene (7.3%), τ-cadinol (6.9%), manoyl oxide (6.3%), and α-pinene (5.5%).

Table 1. Chemical compositions (percent) of essential oils of Vitex agnus-castus from North-Central Nigeria.

There have been numerous previous examinations of essential oils of V. agnus-castus [15–40]. In order to place the present study into perspective, a hierarchical cluster analysis (HCA) was carried out based on the major essential oil components (Fig. 1). The HCA shows three well-defined clusters for the leaf and aerial parts essential oils of V. agnus-castus: (1) a 1,8-cineole/sabinene/(E)-β-caryophyllene cluster, which includes the samples from this work; (2) an α-pinene cluster, which includes samples from Iran with little or no 1,8-cineole; and (3) a 1,8-cineole/sabinene/α-pinene cluster.

Figure 1. Dendrogram obtained by hierarchical cluster analysis of 44 essential oil samples (aerial parts or leaves) of Vitex agnus-castus. Cetin [19], Varcin [39], Habbab [23], Senatore [36], Neves [31], Stojkovic [37], Khairi [26], Goncalves [17], Hamid [24], Marongiu [30], Kustrak [29], Duymus [20], Ulukanli [38], Al Saka [16], Inal [25], Ouali [33], Rezaei [34], Khalilzadeh [27], Farokhzad [21], Novak [32], Zoghbi [40], Abou-Zied [15], Khedri [28], Ricarte [35], Galletti [22], Bakr [18].

3.2. Enantiomeric Distribution

The V. agnus-castus essential oils were subjected to enantioselective GC-MS in order to determine the enantiomeric distribution of chiral monoterpenoid components (Table 2). The (+)-enantiomers were the major stereoisomers for α-pinene, α-phellandrene, and β-phellandrene, while the (–)-enantiomers predominated for α-thujene, sabinene, limonene, terpinen-4-ol, and α-terpineol. As far as we are aware, there have been no previous chiral gas chromatographic examinations of V. agnus-castus or any Vitex essential oils.

Table 2. Enantiomeric distribution (percent of each enantiomer) of chiral monoterpenoids in Vitex agnus-castus essential oils. 

RI_db = Retention index from our in-house database based on commercially available compounds available from Sigma-Aldrich and augmented with our own data. RI_calc = Calculated retention index based on a series of n-alkanes on a Restek B-Dex 325 capillary column. n.o. = not observed.

3.3. Antibacterial Activity

The V. agnus-castus leaf essential oil was screened for antibacterial activity against a panel of Gram-positive (Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis) and Gram-negative (Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhi) organisms (Table 3). The essential oil showed strong antibacterial activity (MIC = 312.5 μg/mL) against S. aureus and E. coli and moderate activity (MIC = 625 μg/mL) against S. faecalis and P. aeruginosa [42, 43]. Of the major components, 1,8-cineole and α-terpineol showed antibacterial activity against S. aureus, E. coli, P. aeruginosa, and S. typhi (Table 3). Previous work has shown sabinene to be relatively inactive as an antibacterial [44, 45]. However, α-pinene has shown antibacterial activity against B. subtilis, S. aureus, E. coli, P. vulgaris, P. aeruginosa, and S. typhi [46, 47]; and terpinen-4-ol has shown activity against B. subtilis, E. coli, P. vulgaris, P. aeruginosa, and S. aureus [44,48]. Thus, the antibacterial activity of V. agnus-castus leaf essential oil can be attributed to the major components 1,8-cineole, α-pinene, terpinen-4-ol, and α-terpineol. Consistent with the antibacterial observations in this report, there have been previous reports on the antibacterial activities of V. agnus-castus essential oils, including activities against B. subtilis [18], Streptococcus mutans [17], P. aeruginosa [23], Bacillus cereus [28], and Salmonella enterica serovar Typhimurium [37].

Table 3. Antibacterial activity of Vitex agnus-castus leaf essential oil, 1,8-cineole, and α-terpineol. 

4.   Conclusions

The essential oil compositions and antibacterial activities of V. agnus-castus from north-central Nigeria are comparable to compositions (largely dominated by oxygenated monoterpenoids and monoterpene hydrocarbons) and antibacterial activities (good antibacterial activity against S. aureus and E. coli) from other geographical locations. This is the first report on the enantiomeric distributions of chiral monoterpenoids in V. agnus-castus essential oils, however, this adds to our knowledge on this important medicinal plant. Additional research is needed on the enantioselective GC-MS of other Vitex essential oils to identify any trends in enantiomeric distributions.

Authors’ contributions

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