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. 2024 Sep 24;10(21):e38389. doi: 10.1016/j.heliyon.2024.e38389

Analysis of Eremostachys hyoscyamoides essential oil composition and assessing the antibacterial and antioxidant properties of the ethanol extract

Marjan Talebi a,b, Afsaneh Arefei Oskouie c, Arash Mahboubi d,e, Mohammad Khani a, Faraz Mojab b,f,⁎
PMCID: PMC11577149  PMID: 39568859

Abstract

The increasing issue of antibiotic resistance in bacterial infections has led to challenging and costly treatments. Free radicals significantly contribute to the progression of several diseases, such as cardiometabolic disorders, neurodegenerative conditions, and cancers. Antioxidants can help alleviate or prevent these health problems. This research aimed to assess the antibacterial and antioxidant effects of Eremostachys hyoscyamoides ethanol extract. Additionally, the chemical profile of the essential oil obtained from the aerial parts of E. hyoscyamoides was characterized through gas chromatography-mass spectrometry (GC-MS). The extract's antibacterial effect was tested against three Gram-positive bacteria (Micrococcus luteus, Staphylococcus epidermidis, and S. aureus), in addition to three Gram-negative bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli). The cup-plate method was employed to assess the antibacterial activity, which was subsequently followed by the determination of the minimum inhibitory concentration through the agar dilution method. To evaluate the antioxidant effects, the DPPH radical scavenging assay was used. The total phenolic content (TPC) and flavonoid content (TFC) of the extract were quantified using the Folin Ciocalteu and Aluminum Chloride methods as spectrophotometric-based techniques, respectively. The essential oil of E. hyoscyamoides was extracted via hydrodistillation and subjected to GC-MS analysis. The findings demonstrated that the ethanol extract of E. hyoscyamoides effectively inhibited the growth of the bacterial strains examined. The IC50 value measured in the DPPH test was 48.194 ± 0.61 μg/mL. The TPC was found to be 84.15 ± 2.5 mg GAE/g, while the TFC was determined to be 19.35 ± 1.3 mg RE/g. A total of 50 components, accounting for 93.6 % of the essential oil composition, were identified. High concentrations of elemol (6.8 %), 2,4-di-tert-butylphenol (10.7 %), and linalyl acetate (4.2 %) were putatively identified in the essential oil. In conclusion, the ethanol extract of E. hyoscyamoides exhibited hopeful potential as a natural source of antioxidants and antibacterial agents.

Keywords: Antibacterial effect, Antioxidant activity, Flavonoid, Eremostachys hyoscyamoides, Essential oil, GC-MS

1. Introduction

For millennia, natural compounds have been of noteworthy significance in the field of pharmaceutical research, particularly in the context of finding potential treatments for diseases such as cancer and infectious diseases [1,2].

The pathogenesis of numerous health conditions, such as heart and lung diseases, metabolic disorders, neurodegenerative conditions like Alzheimer's, and diverse types of cancer, have been linked to the detrimental effects caused by the presence of excessive bodily free radicals [3,4]. Oxidative stress has been implicated in mitochondrial dysfunction, DNA damage, aging, dysregulation of proteins, and lipids metabolism [[5], [6], [7]].

Due to the changes in human lifestyle particularly alterations in the dietary regimens, the prevalence of oxidative stress is increasing. Free radicals can be produced through various external sources, which include environmental pollution, exposure to toxic heavy metals and harmful substances, as well as exposure to various forms of radiation such as ultraviolet, gamma, and X-rays. Furthermore, they can also be generated through the action of intrinsic oxidizing agents [7]. So, the need for antioxidants that can effectively scavenge free radicals is of high interest. Medicinal plants and their specialized metabolites including phenolic compounds and flavonoids can be rich sources of antioxidants [8].

Infectious agents result in bodily dysfunctions and may cause morbidity and mortality worldwide. Owing to the growing microbial resistance to synthetic antibiotics and their potential adverse effects, the trend for investigating the antibacterial activities of natural products is a significant rationale [[9], [10], [11], [12], [13], [14]]. Medicinal plants have been traditionally used as therapeutic strategies to manage infectious diseases. They produce secondary metabolites, which function as components of their protective mechanisms against microorganisms [1].

Eremostachys is a genus in the Lamiaceae family, comprising 60 species worldwide. Of these, 15 are found in Iran, with 5 being endemic to the region including E. adenantha, E. azerbayjanica, E. codonocalyx, E. hyoscyamoides, and E. pulvinaris. Previous research on the Eremostachys genus has reported the presence of alkaloids, coumarins, flavonoids, terpenes, iridoids, and phenylethanoids in the aerial parts. Additionally, phytochemical investigations have detected a variety of monoterpenes and sesquiterpenes in the essential oils of this genus [[15], [16], [17], [18]]. Numerous pharmacological indications have been reported in Eremostachys species including antioxidant, anti-parasitic [19,20], antibacterial, analgesic [21], antidepressant [22], and anti-inflammatory effects [15,23].

Eremostachys hyoscyamoides is a medicinal plant with an ethnobotanical background in the northeast province of Iran. E. hyoscyamoides has been traditionally used to treat infected wounds [15].

In this study we assessed the antibacterial and antioxidant properties of E. hyoscyamoides ethanol extract. Preliminary phytochemical investigations were performed to determine the TPC and TFC in the extract. Furthermore, the essential oil of E. hyoscyamoides was subjected to GC-MS analysis, which enabled the identification of the profile of volatile compounds. This is the first report of investigating the phytochemical and biological aspects of E. hyoscyamoides.

2. Materials and methods

2.1. Plant material and extraction

E. hyoscyamoides Boiss. & Buhse was collected in June 2023 from Mashhad, Khorasan Province, Iran. The plant was identified by Dr. F. Mojab, a qualified pharmacognosy professor. A voucher specimen (HPSRC-101) was deposited at the herbarium of the Pharmaceutical Sciences Research Center at Shahid Beheshti University of Medical Sciences. Aerial parts of E. hyoscyamoides were shade-dried and ground into fine powder. An aliquot of 150 g of powdered E. hyoscyamoides was macerated with ethanol (96 %) (3 × 450 mL) on a GFL 3017 orbital shaker (Burgwedel, Germany) for three days. The extracted solution was subjected to filtration utilizing Whatman filter paper and subsequently concentrated using a Heidolph rotary evaporator (Schwabach, Germany) at 45 °C and 70 rpm. The yield of extraction was 9 %. The dried extract was immediately stored at 4 °C in the dark to maintain its stability.

2.2. Bacterial strains

The antibacterial properties of the ethanol extract from E. hyoscyamoides were evaluated against six bacterial strains, including Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC 10031, Kocuria rhizophila ATCC 9341, Staphylococcus aureus ATCC 6538, S. epidermidis ATCC 12228, and Pseudomonas aeruginosa ATCC 9027. These microorganisms were acquired from the Iranian Research Organization for Science and Technology. The bacterial culture preparation involved the cultivation of the microorganisms on Merck Mueller Hinton Agar-MHA medium(Darmstadt, Germany) and maintaining them incubated at 37 °C for 24 h.

2.3. Antibacterial susceptibility test

The antibacterial properties of E. hyoscyamoides ethanol extract were assessed using the Cup-Plate method. In this procedure, 7 mm diameter wells were created in the MHA medium, that had been inoculated with a microbial suspension adjusted to a 0.5 McFarland standard (1 × 108 CFU/mL) in distilled water. The wells were then filled with 100 μL of the E. hyoscyamoides ethanol extract at varying concentrations (1000, 500, 250, 125, and 62.5 mg/mL), all dissolved in water. A well containing only the solvent was included as a negative control. The plates were subsequently incubated at 37 °C for 24 h. The experiment was conducted in triplicate, and the mean values of the results were reported [9,24].

2.4. Minimum inhibitory concentration (MIC)

The MIC was determined using the agar dilution technique following the recommendations set by the Clinical & Laboratory Standards Institute. Ethanol extract from E. hyoscyamoides was mixed with melted MHA to get homogeneous concentrations of 250, 125, 62.5, 31.25, 15.62, 7.81, 6.25, and 3.9 mg/mL. Bacterial suspensions, standardized to a 0.5 McFarland turbidity, were prepared and then diluted 10 times by using sterile normal saline. A volume of 2 μL from each diluted bacterial suspension was carefully applied to the surface of the agar mediums containing different concentrations of the E. hyoscyamoides ethanol extract. One plate served as a negative control, without the plant extract. All plates were then incubated at 37 °C for 24 h, and the MIC determinations were performed in triplicate for reliable results [24].

2.5. The DPPH radical scavenging assay

The DPPH radical scavenging assay was performed using a range of concentrations (6.25–800 μg/mL) of the ethanol extract from E. hyoscyamoides. These dilutions were transferred to 96-well plates and kept in the dark at 37 °C for 30 min. Following the addition of DPPH, the absorbance was read at 517 nm using a BioTek microplate reader (Winooski, Vermont, USA). Rutin and vitamin C were used as positive controls at concentration ranges of 1.563–6.25 and 0.781–6.25 μg/mL, respectively [25,26].

2.6. Total phenolic content

The analysis of the TPC of E. hyoscyamoides ethanol extract was performed using the Folin-Ciocalteu reagent. Absorbance was measured at 765 nm with a Multispec-1501 Shimadzu UV spectrophotometer (Kyoto, Kyoto, Japan). A calibration curve was established using gallic acid as the standard. All measurements of TPC were conducted in triplicate, and the TPC was reported as gallic acid equivalents (mg GAE per gram of sample) [27,28].

2.7. Total flavonoid content

The TFC was evaluated using the AlCl3 colorimetric technique. The measurement of absorbance was conducted at 420 nm. A calibration curve was constructed utilizing rutin as the standard, and the determination of TFC in E. hyoscyamoides ethanol extract was performed in triplicate. The quantification of these compounds was calculated based on the equation derived from the aforementioned line diagram. TFC was expressed as the amount of rutin equivalents in the dry extract matter, measured in milligrams per gram (mg RE/g) [29,30].

2.8. Extraction of the essential oil

The EO of E. hyoscyamoides was extracted through hydrodistillation employing a Clevenger-type apparatus for 4 h. Following extraction, the EO was desiccated utilizing anhydrous sodium sulfate and kept refrigerated at 4 °C until further analysis.

2.9. GC-MS analysis of the essential oil and identification of volatile compounds

The chemical composition of the nonderivatized EO obtained from E. hyoscyamoides was subjected to GC-MS analysis. The analysis was conducted on an Agilent 7000 Series MS instrument (Santa Clara, CA, USA) equipped with an HP-5ms column (30 m × 0.25 mm inner diameter, 0.25 μm film thickness), a triple quadrupole mass detector, and a computing system integrated with Wiley 7n.l and NIST version 2.0 libraries. The GC-MS procedure involved an initial temperature of 60 °C maintained for 5 min, followed by a gradual increase from 60 °C to 250 °C over a period of 30 min, and a final at 250 °C for 10 min, resulting in a total run time of 60 min. The injector temperature was set at 250 °C, with a 1 μl injection of the EO and a split ratio of 1:50. Helium served as the carrier gas, maintaining a flow rate of 1.1 mL/min. The mass spectrometer functioned under an ionization potential of 70 eV, an ionization current of 150 μA, an ion source temperature of 230 °C, a quadrupole temperature of 150 °C, and a mass range spanning from 25 to 1000 amu in full scan mode.

Volatile compound identification was achieved by matching the obtained EI-MS fragmentation patterns at 70 eV with those in the NIST MS Search algorithm (version 2.0) library. Retention indices (RI) were also used as supplementary identifiers, with the experimental values compared to literature reference indices within a tolerance of ±15 units. To calculate the RI for each respective peak, a 1 μL injection of an n-alkane mixture (ranging from C7 to C40; Sigma-Aldrich, St. Louis, Mo, USA) was performed under similar GC-MS conditions. The RI was obtained utilizing the formula.

  • (1)

    RI = 100n + 100(tx − tn)/(tn + 1 − tn)

In this formula, tx represents the Rt of the compound, while n and n + 1 denote the alkanes’ number of carbon atoms that elute just before and after the compound, respectively [31].

2.10. Statistical analysis

In all tables, data were shown as mean ± SD. The statistical analyses were implemented utilizing GraphPad Prism 9.1 (San Diego, CA, USA) and Microsoft Office Excel 2023. Each experiment was performed at least three times. IC50 values were derived from logarithmic regression curves and are conveyed with 95 % confidence intervals.

3. Results

3.1. Results of antibacterial activities of E. hyoscyamoides ethanol extract by well diffusion method

Table 1 illustrates the inhibition zone diameters produced by the ethanol extract of E. hyoscyamoides. The negative control, distilled water, demonstrated no inhibitory impact on the examined bacterial strains. The obtained results from Table 1 indicated that the ethanol extract had antimicrobial activity against the selected microorganisms with a diameter zone of inhibition that ranged from 11 to 24 mm. Among the bacteria tested, S. epidermidis was the most sensitive to the E. hyoscyamoides ethanol extract, represented by the larger inhibition zone.

Table 1.

The mean diameter of the growth inhibition diameter (mm) of E. hyoscyamoides ethanol extract by well diffusion assay. ∗ATCC (American Type Culture Collection), diameter well (7 mm), mean ± SD value of three independent experiments (n = 3).

No. Bacteria Gram ATCC∗ Extract concentration (mg/ml) Mean (mm) ± SD
1 Staphylococcus epidermidis + 12228 1000 24 ± 0.5
500 18 ± 0.4
250 17 ± 1
125 13 ± 0.5
62.5 11
2 Staphylococcus aureus + 6538 1000 16 ± 1
500 15 ± 0.2
250 13.5 ± 0.5
125 11
62.5 –
3 Pseudomonas aeruginosa _ 9027 1000 16 ± 0.1
500 14 ± 0.8
250 13 ± 0.5
125 –
62.5 –
4 Escherichia coli _ 8739 1000 20 ± 0.4
500 16 ± 1
250 14
125 12.5 ± 1
62.5 –
5 Klebsiella pneumonia _ 10031 1000 14 ± 0.6
500 13 ± 0.4
250 12 ± 0.5
125 –
62.5 –
6 Kocuria rhizophila + 9341 1000 14 ± 1
500 12.5 ± 0.5
250 11 ± 0.5
125 –
62.5 –
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3.2. Results of minimum inhibitory concentration (MIC) of E. hyoscyamoides ethanol extract in agar dilution method

Table 2 represents the results of an agar dilution test conducted on several types of bacteria. The purpose of this test was to determine the MIC of an extract against these bacteria. The extract effectively inhibited Gram-positive species (S. epidermidis and S. aureus) at higher concentrations (15.62 mg/mL and 31.25 mg/mL, respectively). However, it inhibited Gram-negative species (P. aeruginosa, K. pneumoniae, and E. coli) at lower concentrations (≤125 mg/mL). Additionally, K. rhizophila exhibited intermediate susceptibility, inhibited at 62.5 mg/mL.

Table 2.

Results of agar dilution test on studied bacteria, (n = 3). ✓: growth of bacterial colonies.

Extract concentration (mg/mL) 250 125 62.5 31.25 15.62 7.81 3.9
Bacteria
S. epidermidis × × × × ✓ ✓ ✓
S. aureus × × × ✓ ✓ ✓ ✓
P. aeruginosa × ✓ ✓ ✓ ✓ ✓ ✓
K. pneumoniae × ✓ ✓ ✓ ✓ ✓ ✓
K.rhizophila × × ✓ ✓ ✓ ✓ ✓
E. coli × × ✓ ✓ ✓ ✓ ✓
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As shown in Table 3, the MIC values of E. hyoscyamoides ethanol extract on tested microorganisms are expressed. The MIC values obtained in this investigation ranged from 15.62 to 125 mg/mL. E. hyoscyamoides ethanol extract exhibited the lowest MIC (15.62 mg/mL) against S. epidermidis. This suggested that S. epidermidis displayed the highest level of sensitivity among the six microorganisms examined in this study. K. pneumoniae and P. aeruginosa demonstrated the greatest resistance (MIC of 125) to E. hyoscyamoides ethanol extract.

Table 3.

MIC (mg/ml) of E. hyoscyamoides ethanol extract (n = 3).

Microorganism MIC (mg/mL)
Staphylococcus epidermidis 15.62
Staphylococcus aureus 31.25
Pseudomonas aeruginosa 125
Klebsiella pneumonia 125
Kocuria rhizophila 62.5
Escherichia coli 62.5
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3.3. Results of DPPH radical scavenging assay

As shown in Fig. 1, E. hyoscyamoides ethanol extract exhibited noticeable concentration-dependent anti-radical activity. The ethanol extract of E. hyoscyamoides exhibited notable antioxidant activity, with an IC50 value of 48.19 ± 0.61 μg/mL. The standards including rutin and vitamin C showed antioxidant activity, with IC50 values of 9.26 ± 0.25 and 2.29 ± 0.22 μg/mL, respectively.

Fig. 1.

Fig. 1

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IC50 of extract, rutin, and vitamin C.

3.4. Results of TPC and TFC in E. hyoscyamoides ethanol extract

TPC was calculated as 84.15 ± 2.5 mg GAE/g which was equal to 21.03 % TPC/extract according to the standard calibration curve (y = 0.005x + 0.044, r2 = 0.99).

TFC was calculated as 19.35 ± 1.3 mg RE/g which was equal to 4.83 % TFC/extract based on the standard calibration curve (y = 0.0142x + 0.0472, r2 = 0.99).

3.5. Results of GC-MS analysis of E. hyoscyamoides essential oil

Table 4 presents the detailed results of a GC-MS analysis of the EO extracted from E. hyoscyamoides. This analysis was conducted to elucidate the chemical composition of the EO, which is crucial for understanding its biological activities and potential applications. According to the comprehensive GC-MS analysis, a total of 93.6 % of the EO's chemical composition was successfully identified, encompassing a diverse array of 50 compounds. Several terpenes were present, including limonene, terpinolene, α-terpineol, linalyl acetate, neryl acetate, geranyl acetate, (E)-caryophyllene, humulene, germacrene D, bicyclogermacrene, β-bisabolene, nerolidol. Compounds like methyl palmitate, linolenic acid methyl ester, oleic acid, and n-octadecyl ethanoate suggested the presence of fatty acids and their esters. A series of n-alkanes were identified, ranging from n-decane to n-nonacosane.

Table 4.

Chemical compounds identified in E. hyoscyamoides essential oil by GC-MS analysis.

No. Compound Rt Area (%) Exp. RI L. RI
1 n-Decane 5.24 0.1 997 1000
2 Limonene 5.91 0.1 1027 1027
3 Terpinolene 7.50 1.0 1099 1098
4 α-Terpineol 9.82 1.5 1195 1200
5 Linalyl acetate 11.41 4.2 1255 1257
6 Neryl acetate 14.20 0.8 1362 1362
7 Geranyl acetate 14.68 2.3 1381 1381
8 (E)-Caryophyllene 15.64 2.4 1420 1420
9 Humulene 16.49 0.5 1454 1455
10 Germacrene D 17.16 3.7 1482 1482
11 Bicyclogermacrene 17.53 0.5 1497 1495
12 β-Bisabolene 17.78 0.1 1507 1509
13 2,4-Di-tert-butylphenol 17.90 10.7 1512 1512
14 Elemol 18.78 6.8 1550 1549
15 Nerolidol 19.57 1.2 1584 1583
16 n-Hexadecane 19.83 1.0 1595 1600
17 10-epi-γ-Eudesmol 20.70 0.2 1634 1632
18 (E)-4-Hexadecen-6-yne 21.13 0.1 1653 MS
19 α-Eudesmol 21.20 0.1 1656 1653
20 3-Methylhexadecane 21.52 0.4 1671 1673
21 n-Heptadecane 22.04 0.7 1694 1700
22 n-Octadecane 24.18 9.3 1799 1800
23 3-cyclohexyl- dodecane 24.35 0.9 1807 MS
24 Hexahydrofarnesyl acetone 25.13 2.9 1846 1846
25 Farnesyl acetone 25.65 0.2 1871 1881
26 n-Nonadecane 26.21 0.4 1899 1900
27 Farnesyl acetone 26.64 0.1 1921 1919
28 Methyl palmitate 26.75 0.1 1927 1927
29 2-Methylnonadecane 27.21 0.9 1951 1966
30 Dibutyl phthalate 27.52 2.7 1967 1967
31 2,6,10,14-Tetramethylheptadecane 27.60 0.7 1971 MS
32 n-Heptadecanol 28.03 0.1 1993 1986
33 n-Eicosane 28.15 10.5 1999 2000
34 5E-Eicosene 28.40 3.3 2013 MS
35 n-Heneicosane 30.00 0.4 2101 2100
36 Linolenic acid, methyl ester 30.04 0.5 2103 2101
37 Oleic acid 30.13 0.4 2108 2110
38 Phytol 30.37 0.7 2121 2119
39 5-Methylheneicosane 30.92 0.5 2151 2151
40 8-Heptylpentadecane 31.29 0.4 2172 MS
41 n-Docosane 31.78 6.7 2198 2200
42 n-Octadecyl ethanoate 32.00 0.3 2211 2211
43 n-Heptadecylcyclohexane 32.10 1.7 2217 MS
44 n-Tricosane 33.49 3.4 2298 MS
45 n-Tetracosane 35.13 2.3 2398 2400
46 n-Pantacosane 36.71 0.5 – 2500
47 n-Hexacosane 37.96 0.8 – 2600
48 n-Heptacosane 39.99 1.4 – 2700
49 n-Octacosane 42.11 0.7 – 2800
50 n-Nonacosane 44.84 2.4 – 2900
Total Identified 93.6 %
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The major compounds present in the EO were 2,4-di-tert-butylphenol (10.7 %), n-eicosane (10.5 %), elemol (6.8 %), n-docosane (6.7 %), and Linalyl acetate (4.2 %). 2,4-di-tert-butylphenol, an alkylphenol, is accomplished by scavenging free radicals and combating oxidative damage as well as the possession of antifungal effects [32,33]. Elemol is a sesquiterpene alcohol acknowledged as a fragrance ingredient and used for insecticidal and anti-termite properties [34,35]. Linalyl acetate, the acetate ester of the monoterpene alcohol linalool has admired sedative and anxiolytic properties [36]. Additionally, n-docosane is a long-chain saturated hydrocarbon.

4. Discussion

E. hyoscyamoides is an endemic plant that has been ethnobotanically used in the wound healing process. This study was the first evaluation of the analysis of E. hyoscyamoides chemical composition. We also assessed the antibacterial and antioxidant effects of E. hyoscyamoides ethanol extract for the first time.

The application of synthetic preservatives for prevention and antibiotics to combat infectious diseases can yield a variety of undesirable consequences such as allergic reactions, hypersensitivity, and a weakened immune system. Hence, there exists a growing necessity for developing new, natural alternatives that can serve as preservatives or antimicrobial agents [37,38].

By considering the antibacterial activities of the ethanol extract, it is inferred that among the Gram-positive bacteria strains, E. hyoscyamoides ethanol extract showed the most effectiveness on S. epidermidis with mean growth inhibition diameter of 24 and 18 mm at 1000 and 500 mg/mL concentrations, respectively and MIC value equaled to 15.62 mg/mL. In other Gram-positive bacteria strains, acceptable antibacterial effects were observed as well. Among the Gram-negative bacteria strains, E. hyoscyamoides ethanol extract displayed the most effectiveness on E. coli with mean growth inhibition diameters of 20 and 16 mm at 1000 and 500 mg/mL concentrations, respectively and MIC values equaled 62.5 mg/mL. In two Gram-negative strains including P. aeruginosa and K. pneumoniae, no growth inhibition zone was observed at 125 mg/mL consternation and the lower ones. According to the results of the study, the largest inhibition zone in the six tested bacteria strains was observed at 1000 mg/mL concentration, which could confirm that the antibacterial activities of E. hyoscyamoides ethanol extract was dose-dependent.

Modarressi et al. discovered that iridoid glycosides including phloyoside I, phlomiol, and pulchelloside I isolated from the rhizomes of E. laciniata exhibited low to moderate levels of antibacterial impacts with MIC values ranging from 0.05 to 0.50 mg/mL. Among these phytochemicals, pulchelloside I was the most effective, showing activity against Bacillus cereus, Proteus mirabilis, penicillin-resistant E. coli, and S. aureus with a MIC of 0.05 mg/mL [39]. Vahedi et al. reported that the methanol extract of E. labiosiformis had antibacterial effects against phytopathogens such as Xanthomonas campestris, Pseudomonas viridiflava, and Rathayibacter rathayi [40]. Hariri et al. found that E. binalodensis methanolic extract prevented S. mutans biofilm formation at 4.096 mg/mL concentration [41]. The E. hyoscyamoides ethanol extract showed promising, species-specific antibacterial activity, warranting further investigation into active compound isolation, in vivo efficacy evaluation, and structure-activity relationships investigation to enhance specificity and potency. These antibacterial potentials were in accordance with findings of previous studies conducted on other species of Eremostachys.

Natural compounds may exhibit antioxidant properties by offering protection against free radicals, which can induce modifications in the composition of DNA, signaling pathways, and cellular membranes [42]. Phenolic compounds represent a group of phytochemicals derived from plants, characterized by the presence of an aromatic ring connected to hydroxyl groups. These compounds are recognized with numerous advantages for human health, including cardiovascular diseases and neurodegenerative disorders [43].

E. hyoscyamoides ethanol extract showed remarkable antioxidant activity with IC50 equaled to 48.19 ± 0.61 μg/mL. TPC was calculated as 84.15 ± 2.5 mg GAE/g and TFC was measured as 19.35 ± 1.3 mg RE/g. It is inferred that the promising antioxidant potential of E. hyoscyamoides ethanol extract might be attributed to the presence of phenolic and flavonoid compounds. Asnaashari et al. reported that the methanol extract of E. azerbaijanica showed significant scavenging activity [44]. Bajalan and coworkers revealed that E. laciniata methanol extract showed DPPH radical scavenging activity with IC50 equaled to 54.64 μg/mL [45].

This study reported a detailed profile of the EO of E. hyoscyamoides for the first time. The different components (n = 50) were identified in E. hyoscyamoides EO. Based on the findings derived from the GC-MS analysis, a significant portion of the chemical composition of the EO, amounting to 93.6 %, was successfully identified. This composition consisted of a total of 50 compounds. Among these compounds, the primary ones identified in E. hyoscyamoides essential oil were 2,4-Di-tert-butylphenol (10.67 %), n-Eicosane (10.54 %), Elemol (6.82 %), n-Docosane (6.75 %), and Linalyl acetate (4.20 %). According to the literature review, there has been chemical variability reported in the EOs composition of various species from the Eremostachys genus and also in different plant parts of each species of varied geographical regions and at different flowering stages [[16], [17], [18],[46], [47], [48], [49]].

5. Conclusion

This research was done in order to identify the chemical composition of E. hyoscyamoides EO and study the antioxidant and antimicrobial effectiveness of its ethanol extract. The GC-MS analysis of the E. hyoscyamoides EO yielded the identification of 50 compounds. The high content of 2,4-Di-tert-butylphenol, n-Eicosane, Elemol, n-Docosane, and Linalyl acetate were detected. With the TPC and TFC in high levels, E. hyoscyamoides ethanol extract exhibited high antioxidant activities during the DPPH assay. Furthermore, the antibacterial effects of the ethanol extract from E. hyoscyamoides were observed against S. aureus, S. epidermidis, M. luteus, E. coli, P. aeruginosa, and K. pneumoniae. Our findings offer a valuable source of information regarding the EO and ethanol extract derived from E. hyoscyamoides.

Funding

This work was financially supported by the Research Council of Shahid Beheshti University of Medical Sciences (Grant no. 43008632).

Compliance with ethical standards:

All experiments were conducted according to the ethical guidelines of SBMU Pharmacy School, Tehran, Iran (IR.SBMU.PHARMACY.REC.1402.276).

Data availability statement

All data are within the manuscript.

CRediT authorship contribution statement

Marjan Talebi: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Investigation, Formal analysis, Data curation. Afsaneh Arefei Oskouie: Writing – review & editing, Validation, Software, Investigation, Formal analysis. Arash Mahboubi: Writing – review & editing, Validation, Software, Investigation. Mohammad Khani: Visualization, Validation, Resources, Methodology, Investigation. Faraz Mojab: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This article is part of Mohammad Khani's Pharm.D. thesis project. The authors gratefully acknowledge the financial support provided by Shahid Beheshti University of Medical Sciences in Tehran, Iran, which made this research possible. Before submission, all authors have thoroughly reviewed the manuscript and approved the final version.

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