Background

Plants are essential in treating diseases with less or no side effects [1]. They contain chemical compounds with significant biological and pharmacological properties. Plants are a source of effective medications. There will continue to be crucial for screening novel lead compounds, as has been discovered over time. However, the demand for and interest in medicinal and aromatic plants for food, medicine and other applications is constantly rising. Plant secondary metabolites include many essential oils (EOs) [1]. However, despite their rich and complex composition, essential oils are used in cosmetics and perfumes. In order to produce beneficial uses in environmental health, agriculture, biology and pharmaceutics, it is vital to understand the chemistry and biological properties of essential oils and their unique constituents [1].

Essential oils constitute a small proportion of plants’ composition. They are composed of volatile compounds that typically have low molecular weight [2]. The volatile compounds are mainly terpenes with little traces of alcohols, heterocycles, aldehydes, ethers, ketones, amines, esters, phenols and amides [3]. Numerous other aromatic compounds range from aldehydes, alcohols and ketones, including fruity flavours like ((E)-nerolidol), floral (Linalool), citrus (Limonene), herbal (( +)-selinene), etc. can also be present. Additionally, non-terpenic compounds like cinnamaldehyde and safrole are produced during the biogenesis process of essential oils through the phenylpropanoids route [3, 4]. The antibacterial property of EOs and their components have been reported [5, 6] with notable characteristics—hydrophobicity. Hydrophobicity allows EOs to partition into the lipids of the cell membrane of micro-organisms, altering the structure and making it more porous [7], leading to the death of the organisms’ cells. Also, the constitutions of an EO play significant role in antioxidant activity. It is widely known that phenolic compounds and secondary metabolites with conjugated double bonds typically exhibit strong antioxidative effects [7].

Eremomastax speciosa (Hochst.) Cufod. (Acanthaceae) (Fig. 1) is widely distributed in the tropics of Africa [8]. It is a robust, polymorphous shrub that grows up to 2 m long and has a characteristic quadrangular stem and violets on the underside of leaves. It is known in Cameroon as pang nyemshe (red on one side) and in southern Nigeria (Ibibio) as Edem ididot (“golden seal” or “African blood tonic”) [8]. In addition to its application in treating female infertility and menstrual cramps, the plant has also been used in treating anaemia, dysentery, urinary tract infection, haemorrhoids and gastric ulcers [9,10,11]. The preliminary phytochemical analysis of aqueous leaf extract revealed the presence of flavonoids, tannins, alkaloids and saponins [12, 13]. The pharmacological relevance of the leaf includes promising fertility effect [14], antianemic, antimicrobial activities [15, 23] and antiulcer [16,17,18,19,20,21,22].

Fig. 1
figure 1

Picture of E. speciosa plant (Source: Picture captured at the point of collection on 15 April, 2022)

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However, no reports on the composition of the essential oil of E. speciosa exist in the literature. Given the various medicinal applications in ethno-medicine. Also, our continuing search for bioactive chemical compounds from Nigerian medicinal plants [29, 30], this paper now reports the results of the analysis of the essential oil, antioxidant and antimicrobial activities of the E. speciosa.

Methods

Plant collection and preparation

A fresh E. speciosa was harvested from Utit Uruan, Akwa Ibom, Nigeria. The fresh stem was identified and authenticated at the Forest Research Institute of Nigeria (FRIN), Oyo State, Nigeria, where a voucher specimen with the FRIN herbarium number FHI 113451 was deposited [30].

Isolation of essential oil from E. speciosa stem

According to the British Pharmacopoeia guidelines described by Odeja et al. [30], the fresh, chopped E. speciosa stem (400 g) was placed in an all-glass Clevenger-type equipment hydrodistilled for 3 h. Essential oil collected was dry over anhydrous sodium tetraoxosulphate (vi), filtered, weighed (0.5 g) and stored in a refrigerator at 4 °C before analysis. The % yield of the essential oil was calculated using the formula:

$${\text{\% yield}} = { }\frac{{{\text{Weight of essential oil }}\left( {\text{g}} \right)}}{{{\text{Weight of the sample }}\left( {\text{g}} \right)}}{ } \times 100$$
(1)

Gas chromatography–mass spectrometry (GC–MS) analysis and identification of the essential oil constituents

About 1.5 mL of n-hexane was added to the GC vial and approximately 1.0 µL of the essential was added, centrifuged for about 5 min.

The constituents of the stem essential oil of E. speciosa were identified on an Agilent 7809A gas chromatograph hyphenated with an Agilent mass detector featuring a split/splitless injector interfaced to a mass selective detector operating at 70 eV. With a 1428 amu/sec scan rate, the ion source temperature was 200 °C with a mass spectral range of m/z 50–700. A 30 m long HP-5MS column with an internal diameter of 0.25 mm and a film thickness of 0.25 m was installed in the GC column. The oven temperature was adjusted as follows: 80 °C at first for 2 min, then 10 °C/min up to 240 °C/6 min. Helium was used as the carrier gas, with a 1 mL/min flow rate. Injection volume, linear velocity and pressure were adjusted at 1.0 μL, 362 cm/s and 56.2 kPa, respectively. The oven temperature was set at 60 °C, hold for 1 min to 180 °C for 3 min at 10 °C/min, then the final temperature was 280 °C for 2 min at 10 °C/min. Both injector and detector temperatures were fixed at 250 °C.

Based on their retention indices, which were calculated using homologous series of normal alkane and comparing the mass spectral fragmentation patterns (NIST data/base/Chemstation data system) with information previously published in the literature, the constituents of essential oil were identified [36].

Antioxidant activity

According to Brand-William et al. [33] and Odeja et al. [30], the free radical scavenging capacity of the essential oil and standards were determined by their ability to react with the radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH*). 3.94 mg of DPPH* was dissolved in 100 mL of methanol, producing a methanol-DPPH* solution (0.1 mM). The oil was dissolved in methanol and serially diluted to prepare five (5) concentrations of the oil (1000, 500, 250, 125 and 62.5 mg/mL). The oil was then combined with 2.0 mL of a methanol-DPPH* solution (0.1 mM). The mixture was agitated and incubated for 30 min at room temperature in the dark cupboard. A GS UV-12 UV–Vis spectrophotometer measured the absorbance at 517 nm. The identical approach was used in a control experiment, but no essential oil was used (DPPH* + methanol) and the absorbance was recorded as AC. The antioxidant capacities of ascorbic acid and butylated hydroxyanisole (BHA) were used as a baseline for comparison. Each test was conducted in triplicate and the essential oil’s capacity to free radical scavenging was determined using the formula to determine percentage inhibition (% I):

$$\% I = \frac{{ \left( {{\text{Ac}} - {\text{As}}} \right)}}{{\left( {{\text{Ac}}} \right)}} \times 100.$$
(2)

where Ac—Absorbance of Control, As—Absorbance of Sample.

50% inhibition concentration (IC50) of the sample was evaluated using GraphPadPrism 5.0.

Antimicrobial activity (broth dilution method)

The minimum inhibitory concentration (MIC) and the MBC of the essential and reference standards were assessed against ten (10) micro-organisms; six bacteria—Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhi and Klebsiella pneumoniae, four fungi—Candida albican, Aspergillus niger, Penicillium notatum.

Standardisation of inoculum

The micro-organisms were revived in tryptose sulphite-cycloserine agar (Oxoid) with d-cycloserine (Sigma) under anaerobic conditions at 36 °C for 24 h following British Standards Institute [37]. The inoculum's bacterial concentration was calibrated to be 0.5 on the McFarland turbidity scale or 108 CFU mL−1. To prepare a concentration of 107 CFU mL−1, an aliquot (1 mL) of this suspension was transferred to a sterile tube and the volume was increased to 10 mL using sodium chloride solution (0.8%, w/v). Using reinforced clostridial medium (RCM; Oxoid), 200 µL aliquots of this solution were divided into three test tubes. The contents were adjusted to 10 mL to produce working inoculums with final concentrations of 2.0 × 105 CFU mL−1 [38, 39].

Minimal inhibitory concentrations

Anaerobic conditions were used for all microbiological experiments. MIC analyses were conducted in 96-well microplates following the Clinical and Laboratory Standards Institute [39] suggested protocols. To prepare a stock solution containing 40 mg of oil per mL, essential oil (200 mg) was dissolved in 40 µL of dimethyl sulphoxide before the volume was increased to 5 mL with sterile RCM that contained 1% Tween 80. Essential oil stock was serially diluted twice with RCM to produce final concentrations ranging from 20 to 0.625 µg/mL. The diluted samples (100 µL) were added to the microplate wells and thoroughly mixed using a micropipette. The negative controls consisted of sterile RCM alone in combination with dimethyl sulphoxide (DMSO). At the same time, gentamycin (for bacteria at 10 µg/mL) and tioconazole (for fungi at 0.07 µg/mL) were used as a positive control. The control wells had sterile RCM, but no inoculum, so aseptic conditions could be determined. The inoculated microplates were incubated in anaerobic conditions at 36 °C for 48 h. The bacterial growth was verified by adding 10 µL of a sterile 0.5% aqueous solution triphenyltetrazolium chloride (TTC, Sigma-Aldrich) and incubating at 36 °C for 30 min [40, 41]. Pink/red 1,3,5-triphenyl formazan was produced from yellow TTC by the live bacteria (TPF). Each assay was performed in triplicate.

Minimum bactericidal concentrations and Minimum fungicidal concentrations

According to the Ministério da Agricultura, Pecuária eAbastecimento's recommendations as described by Radaelli et al. [40], MBCs and MFCs were evaluated by inoculating the test mixtures from the wells showing no microbial growth onto the surface of sterile Shahidi-Ferguson Perfringens agar medium. The plates were subjected to ocular inspection after being incubated anaerobically for 24 h in an oven at 36 °C. The essential oil sample had bactericidal and fungicidal activities if there was no microbial growth on the medium, which suggested that the oil sample had bacteriostatic and fungistatic activities.

Results

Phytoconstituents of stem essential oil of E. speciosa

The hydro-distillation of stem essential oil of E. speciosa gave colourless fluid (0.13% yield) with a characteristic herbal-like scent. GC/MS qualitative and quantitative data on the stem essential oil of E. speciosa are shown in Fig. 2 and summarised in Table 1. Forty-three (43) compounds were identified, representing 62.87% of the oil constituents. Non-terpene derivatives were the main constituents identified in the stem essential oil (32.67%; Table 2) followed by monoterpene hydrocarbons (9.72%) and sesquiterpene hydrocarbons (9.59%). Moderate quantifications were observed in oxygenated monoterpenes (5.03%). Minor quantification of the essential oil was oxygenated sesquiterpenes (1.03%), diterpenes (0.8%) and triterpene (4%). The most abundant compound was (14β)-p (17.58%). Other significant compounds identified in the essential oil include n-decane (2.3%), norbornane (2.2%), (−)-α-Copaene (1.5%), 10-epizonarene (1.5%), thymol (1.25) and (−)-α-phellandrene (1.12%).

Fig. 2
figure 2

GC-TIC Chromatogram of essential oil of E. speciosa stem

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Table 1 Essential Oil Composition of Eremomastax speciosa stem
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Table 2 Class of compounds identified in stem essential oil of Eremomastax speciosa
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Antioxidant activity

The results of the scavenging ability to stem essential oil of E. speciosa on DPPH* assay and IC50 values are summarized in Table 3. The percentage inhibition of the essential oil was concentration-dependent, as shown in Fig. 3.

Table 3 Absorbance reading of stem essential oil of Eremomastax speciosa and the reference standards
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Fig. 3
figure 3

Percentage inhibition of stem essential oil of E. speciosa and reference standards

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Antimicrobial activity of stem essential oil of E. speciosa

Experimental data obtained from the in vitro antimicrobial activity of E. speciosa stem essential oil are summarised in Table 4. The essential oil of E. speciosa stems exhibited potent antimicrobial activities with 10–28 mm inhibitory zones against bacterial strains and 10–20 mm against fungal strains compared to the controls (Gentamicin: 40–38 mm and Tioconazole: 20–28 mm). The MBCs between 4.0 and 6.5 µg/mL against bacterial strains and MFCs between 3.5 and 5 µg/mL against fungal strains when compared to the controls (Gentamicin: 3.0 µg/mL and Tioconazole: 3.5 µg/mL). It was also found that the strains of fungi (C. albican, A. niger, P. notatum and R. spp.) were more susceptible to essential oil than the strains from bacteria (S. aureus, E. coli, B. subtilis, P. aeruguinosa, S. typhi and K. pneumoniae). Initial screening tests prove that, in the 100 µg/mL concentration, the oil had the highest fungistatic and bacteriostatic effects (Table 4). A 100% growth inhibition of the tested fungi (R. spp.) and almost 71% for the rest fungi, while about 68% growth inhibition was obtained in the bacteria strains. The EO shows strong growth inhibition against all the tested micro-organisms at 100 µg/mL. Additionally, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) values were determined for the oil of E. speciosa stem against the pathogenic fungi tested (Table 5). The tests were conducted in the 100–3.125 µg/mL concentration range. The minimum inhibitory concentration (MIC) values of the fungi species in the tested oils were in the range of 3.5–5.0 mg/ml and the minimum fungicidal concentrations (MFC) were in the range of 100–> 3.0 µg/mL (Table 5). The highest activity of these oils was observed against Rhizopus spp. The MIC and MFC values of E. speciosa oil for individual strains were C. albican, A. niger and P. notatum (MIC = MFC = 5.0 µg/mL). EO was bacteriostatic against E. coli, B. subtilis and S. aureus, with bactericidal concentrations re-evaluated at MIC 4, 4.5 and 5 µg/mL, respectively.

Table 4 Inhibitory zone of the stem essential oil of E. speciosa and reference standards
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Table 5 Minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and minimal fungicidal concentration of stem essential oil of Eremomastax speciosa and reference standard
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Data analysis

All results were conveyed as mean ± standard deviation. Analysis of variance was used to determine any significant difference between groups using the statistical analysis software package GraphPad 5.0. Values with p < 0.05 were regarded as significant. All graphs/charts were drawn with GraphPad 5.0.

Discussion

The phytoconstituents of essential oil of E. speciosa stem identified (14β)-Pregnane (17.58%) as the major component of the stem EO. (14β)-pregnane is a parent hydrocarbon for two series of steroids stemming from 5α-pregnane to 5β-pregnane. The C21 steroid has been reported in the urine of pregnant women. It exhibited anaesthetic, hypnotic and sedative effects [24]. (14β)-pregnane has been reported as the major constituent identified in the EO of Allium rotundum flower and the significant bacteria strains inhibition was attributed to (14β)-pregnane per cent composition [25]. Decane has been reported as the major component of Hypericum perfortum EO and possesses strong insecticidal activity [26]. Norbornane, a bicyclo-[2.2.1]heptane used as a pharmaceutical intermediate [27], (−)-α-Copaene-exhibited cytotoxic, antioxidant and antigenotoxic activities. Also, 10-epizonarene was identified in the essential oil of Mentha piperita [28]. Thymol was identified as a major component of Asparagus flagellaris essential oils of leaf and root [29, 30]. 1-octen-3-ol (11.9%)—a natural product derived from linoleic acid during oxidative breakdown [31] and also known as mushroom alcohol, has been confirmed as an antimicrobial [32].

Notwithstanding, the EO from E. speciosa stem contains several constituents with distinct antioxidant properties. Since the radical molecule is stable, the DPPH* test is a reliable, simple and affordable way to assess the antioxidants' capacity to scavenge free radicals [33, 34]. The assay's process is based on how the antioxidant agent reduces the radical, either by donating an electron or by reducing DPPH to its reduced form, DPPH-H, a stable diamagnetic molecule, changing its colour from purple to yellow [35]. It was observed that the stem EO of E. speciosa exhibited significant antioxidant activity at the tested concentrations (Fig. 2 and Table 2) comparable to the reference standard vitamin C and BHA. The order of activity based on the IC50 is as follows: Vitamin C (0.8728 mg/mL) ≤ BHA (0.8729 mg/mL) < ESEO (0.7296 mg/mL). Statistical analysis of the data subjected to a one-way analysis of variance (ANOVA) showed no significant difference between the stem essential oil and the reference standard at p < 0.05. The considerable antioxidant activity observed in this study can be attributed to the synergetic action of oxygenated monoterpenes and oxygenated sesquiterpenes coupled with conjugated double bond [7] constituents identified in E. speciosa EO either by hydrogen atom transfer mechanism or by the sequential proton loss-electron-transfer [36].

The emerging multidrug resistance of bacterial strains is critically lessening the potentially effective antimicrobials used in health care. One reason might have been the long-term consequence of the uncontrolled use and misuse of antimicrobial agents and products. Natural products like EOs can be potential alternatives to fight multidrug-resistant species. The tested essential oil showed vital to moderate effectiveness against the tested bacteria and fungi strains. The antimicrobial of E. speciosa stem essential oil was compared with the earlier report of the ethanolic, n-hexane and aqueous leaf extracts of E. speciosa. Okokon et al. [15] reported that all the extracts showed moderate to higher activity against the tested organism, the same trend was observed in the present study. Following evaluation of the MBC, EO has shown practically equal bactericidal and bacteriostatic effects against both gram-positive and -negative bacteria. It has been purported that EO can be used as an effective antiseptic against several micro-organism species, including S. aureus, E. coli, B. subtilis, P. aeruguinosa, S. typhi and K. pneumoniae. The significant antimicrobial activity exhibited in this study might be due to the chemical composition of the EO, such as (14β)-pregnane [25] and the synergetic effect of the identified compounds in the EO.

Conclusion

The E. speciosa stem essential oil comprises important constituents such as (14β)-pregnane, thymol, daucene, panaginsene, (−)-α-Copaene, α-longipinene, β-cis-guaiene, among others. Although the constituents identified were quantified in small amounts possessed different pharmaceutical properties including generic antimicrobial and free radical scavenging activities. The free radical activity of the E. speciosa oil against DPPH* radical was significantly higher than those of the reference standard (vitamin C and BHA), demonstrating activity even at a lower concentration. The inhibitory power of E. speciosa oil against tested micro-organisms was also exhibited at lower concentrations when compared to the reference standard for different strains of gram-positive and -negative bacteria and fungi. The EO was bacteriostatic against E. coli, B. subtilis and S. aureus and fungistatic against all the tested fungi strains, but R. spp. Inhibitory power was comparable to the Tioconazole. Thus, this plant is a rich source of secondary metabolites of medicinal importance and the results of this study provide some scientific basis for the utilisation of the plant in ethno-medicine for menstrual cramps and treat female infertility, anaemia, dysentery and urinary tract infection. Also, this is the first study on the E. speciosa stem essential oil. The present study shows that E. speciosa EO possesses the significant antioxidant and antimicrobial capacity and can readily be used as a natural preservative to minimise or prevent product losses from oxidative processes and for infectious diseases.