Seriphidium herba-alba (Asso): A comprehensive study of essential oils, extracts, and their antimicrobial properties.

Seriphidium herba-alba (Asso), a plant celebrated for its therapeutic qualities, is widely used in traditional medicinal practices throughout the Middle East and North Africa. In a detailed study of Seriphidium herba-alba (Asso), essential oils and extracts were analyzed for their chemical composition and antimicrobial properties. The essential oil, characterized using mass spectrometry and retention index methods, revealed a complex blend of 52 compounds, with santolina alcohol, α-thujone, β-thujone, and chrysanthenone as major constituents. Extraction yields varied significantly, depending on the plant part and method used; notably, methanol soaking of aerial parts yielded the most extract at 17.75%. The antimicrobial analysis showed that the extracts had selective antibacterial activity, particularly against Staphylococcus aureus, and broad-spectrum antifungal activity against organisms such as Candida albicans and Aspergillus spp. The methanol-soaked extract demonstrated the strongest antimicrobial properties, indicating its potential as a natural antimicrobial source. This study not only underscores the therapeutic potential of Seriphidium herba-alba (Asso) in pharmaceutical applications but also sets a foundation for future research focused on isolating specific bioactive compounds and in vivo testing.

White wormwood, known scientifically as Seriphidium herba-alba (Asso), is a perennial shrub indigenous to North Africa and the Middle East. It is widely recognized for its use in traditional medicine. Numerous studies have explored its diverse pharmacological properties, particularly focusing on its antibacterial and antifungal effects [[1]]. The essential oil extracted from this plant is rich in various volatile compounds, believed to be the key contributors to its medicinal effectiveness [[2]] (S1 Table).

Among the critical bioactive components in Seriphidium herba-alba (Asso) are phenolic and saponin compounds, typically derived from its aerial parts. These components demonstrate notable antimicrobial activities. However, their yield and composition are subject to variation and are influenced by factors such as the specific plant parts used, and the extraction methods applied [[2]]. Notably, the crude phenolic extracts from this species have shown significant effects against bacteria and fungi [[4]], which can be attributed to their antioxidant properties and ability to disrupt microbes' cell membranes [[5]].

In response to the growing problem of antibiotic-resistant bacteria, research efforts have intensified to discover new plant antimicrobial agents [[7]]. Seriphidium herba-alba (Asso) extracts have been effective against gram-positive and gram-negative bacteria in various studies, highlighting their potential in combatting resistant bacterial strains, including Staphylococcus aureus and Pseudomonas aeruginosa [[8]].

The antifungal capabilities of Seriphidium herba-alba (Asso) are equally significant, particularly considering the limitations and side effects of many synthetic antifungal drugs. Research has shown that Artemisia extracts can inhibit fungi such as Candida albicans, which cause infections in immunocompromised individuals [[9]], and their efficacy against molds such as Penicillium and Aspergillus suggests their potential use as natural food preservatives [[7]].

However, despite extensive studies, there is still much to learn about the comprehensive antimicrobial properties of Seriphidium herba-alba (Asso). Many research efforts have concentrated on isolated compounds or specific extraction techniques, often without comparing different extracts and their bioactivities [[10]]. In addition, there is an ongoing need to link the chemical makeup of these extracts to their antimicrobial effectiveness for a clearer understanding of their action mechanisms.

The primary goals of this study include: analyzing the chemical composition of Seriphidium herba-alba (Asso)'s essential oils and extracts and identifying their major and minor components; comparing extraction yields from various plant parts using different methods; assessing the antibacterial efficacy of these extracts against a range of gram-positive and gram-negative bacteria; evaluating the antifungal effectiveness against common yeasts and molds; and examining the relationship between the chemical profiles of the extracts and their antimicrobial activity. This study deepens our understanding of Seriphidium herba-alba (Asso)'s pharmacological potential, emphasizing its traditional medicinal role and its possible modern therapeutic applications.

Seriphidium herba-alba (Asso) specimens were collected from the Botanical Garden of Hashemite University, Zarqa, Jordan (S1 Fig). Prof. A. El-Oqlah performed taxonomic verification at the Biological Sciences Department, Yarmouk University, Irbid, Jordan. A representative sample was archived at the Medicinal Chemistry and Pharmacognosy Department, Jordan University of Science and Technology.

In this study, the tested bacterial strains encompassed gram-positive and gram-negative strains. Specifically, Staphylococcus aureus (ATCC 25923) represented gram-positive bacteria, whereas the gram-negative group included Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 700603), and Proteus mirabilis (ATCC 12453). In addition, the pathogenic yeast Candida albicans (ATCC 10231) and various pathogenic molds such as Penicillium, Fusarium, Aspergillus, and Rhizopus species were examined. These microbial strains were obtained from the microbiology laboratory at Al-Bashir Hospital in Amman, Jordan.

The plant was air-dried to a constant weight in a shaded area and then pulverized through a 0.25 mm sieve using a Wiley mill. The resulting powder was stored in airtight glass containers at 5°C until extraction.

Aerial parts and flowers (20 g) were subjected to Soxhlet extraction using petroleum ether for phenolic compounds, methanol for saponins, n-hexane for lipids and terpenoids, ethyl acetate for more polar substances, and ethanol for polar compounds. Each solvent extract was concentrated at 40–50°C under vacuum, and the dry extracts were weighed and stored at -20°C (S4 Fig).

Plant parts (50 g) were macerated in ethanol and methanol and separately stored for three weeks. Homogenization was concentrated at 40–50°C under reduced pressure. The residues were weighed and stored at -20°C.

Twenty grams of powder of the aerial part was extracted with 200 mL petroleum ether for 12 h at 45°C, then filtered with cheesecloth, and the residue was collected. The residue was then extracted with 100 mL of absolute methanol for 6 h at 45°C. The methanolic extract was filtered and then concentrated to a small volume by evaporation under vacuum at 45°C. Fifty milliliters of ether were added to 50 mL of 10% sodium hydroxide and extracted using a separatory funnel. The aqueous layer was removed. Concentrated HCl was added to the remaining aqueous layer for acidification. Butanol (3X20 mL) was used to extract the aqueous layer using a separatory funnel. The same aqueous layer was extracted with petroleum ether (3x20 mL). Finally, the ether extract was dried using an evaporator. The butanol extract was added to the flask and evaporated using an evaporator [[12]]. The phenolic residue was weighed and stored in a freezer until further examination (S2 Fig).

Detection was performed using a drop of 5% (w/v) FeCl2 solution in distilled water added to the plant extract. Green was observed, confirming the detection of phenol. Detection can also be performed using an ethanolic KOH solution, which gives a yellow to phenolic compounds [[12]].

Twenty grams of dried powder was shaken vigorously for 15 min in a 500-mL round-bottom flask (screwcap) with chloroform (3x70 mL) to exclude fatty substances. The extract was left separate, and the supernatant was completely decanted (filtration). Then, 80% methanol (200 mL) was added to the flask and boiled for 24 h using the Soxhlet method. The procedure involved filtering the extract into a 500 mL beaker using cheesecloth or filter paper immediately after extraction. Following this, the filtrate was subjected to vacuum evaporation at 45°C. The remaining residue was then dissolved in 100 mL of distilled water. Subsequently, the water-soluble portion of the residue was extracted using butanol and applied in three separate 50 mL quantities. The sterilized filtrate was concentrated under a vacuum at 40–45°C until a dry residue was obtained. The residue was weighed and stored in a freezer at -21°C until further examination [[12]] (S3 Fig).

Saponin compounds were detected by adding a drop of chlorosulfonic acid to the extract. A reddish-brown color was observed, confirming saponin production [[12]].

Two methods were used to obtain water extraction: The first method was according to An et al. [[13]]. Water extraction was performed by pouring distilled water (50 mL) into 5 g of dried powder. The mixture was left to stand for 15–20 min. The mixture was then filtered through a layer of cheesecloth, and the resulting filtrate was centrifuged at 6000 rpm for 15 min. The supernatant was removed and filtered through a Millipore microfilter (0.2 μm). The sterilized filtrate was concentrated under a vacuum at 40–45°C until a dry residue was obtained. The residue was weighed and stored in a freezer at -21°C until further examination.

The second method was performed according to Al-Charchafchi et al. [[14]]. As mentioned, 50 mL of boiling distilled water was poured onto 5 g of dried powder. The process involved boiling the mixture for 10 min. After boiling, it was filtered through a layer of cheesecloth. The filtrate obtained from this step was centrifuged at 6000 revolutions per minute for 15 min. After centrifugation, the supernatant was carefully separated and passed through a Millipore microfilter with a pore size of 0.2 μm for further purification. The sterilized filtrate was concentrated under a vacuum at 40–45°C until a dry residue was obtained. The residue was weighed and stored in a freezer at -21°C until further examination.

GC analyses were performed using an HP-5890 Series II instrument equipped with HPWAX and HP-5 capillary columns (30 m × 0.25 mm, 0.25 μm film thickness) and set to the following conditions: temperature program of 60°C for 10 min, followed by an increase of 5°C /min to 220°C; injector and detector temperatures at 250°C; carrier gas nitrogen (2 mL/min); detector dual FID; split ratio 1:30; and injection of standards of 0.5 μL). The chemicals were identified for both columns by comparing their retention times with those of pure, authentic samples and using their linear retention indices (LRI) relative to the series of n-hydrocarbons. The relative proportions of each component, expressed as percentages, were determined by FID peak area normalization (mean of three replicates). GC/EIMS analyses were performed using a Varian CP-3800 gas chromatograph equipped with a DB-5 capillary column (30 m × 0.25 mm; coating thickness, 0.25 μm) and a Varian Saturn 2000 ion trap mass detector. Analysis conditions included: injector and transfer line temperatures at 220 and 240°C, respectively; the oven temperature programmed from 60°C to 240°C at 3°C /min; carrier gas helium at 1 mL/min; injection of 0.2 μL (10% hexane solution); split ratio 1:30. The identification of the components was based on comparing the retention times with those of authentic samples, comparing their linear retention indices relative to the series of n-hydrocarbons, and by computer matching against commercial (NIST 98 and ADAMS 95) and home-made library mass spectra built from pure substances and components of known essential oils [[15]].

The bacterial inoculum was prepared from a nutrient broth culture incubated for 24 h at 37°C. The suspension density was adjusted to approximately 104 colony-forming units per mL by comparing its turbidity with a McFarland 0.5 BaSO4 standard. The standard was prepared by adding 0.5 mL of 0.048 M BaCl2 to 99.5 mL of 0.36 N H2SO4. Aliquots of 4 to 6 mL were dispensed into screwcap tubes and stored in the dark at room temperature. The bacterial suspension and McFarland tube were positioned side by side to adjust the turbidity and viewed against a backed background. The suspension was supplemented or diluted as required.

The antimicrobial activity of various plant extracts such as n-hexane, ethyl acetate, and ethanol extracts, as well as phenols, saponin, ethanol-soaked, methanol-soaked, and water extracts extracted from various parts of Seriphidium herba-alba (Asso), were analyzed separately using the disk agar diffusion method [[55]] and evaluated against the microorganisms mentioned above.

The disks were impregnated with the various plant extracts to be tested, allowed to dry, and then placed on the plates within an hour of pouring. Before disk placement, the plate surface was inoculated with a swab dipped in the standardized bacterial suspension. The surface of the plates was wiped in three directions to ensure an even and complete distribution of the inoculum throughout the plate. Discs were placed on plates and incubated at 37°C for 24 h [[17]].

The diameter of the inhibition zones was measured in mm. Each test was conducted in triplicate for this experiment to ensure reliability and accuracy. The average of these three replicates was then calculated to obtain a more accurate and representative result. Standard antibiotics such as vancomycin 30 μL for S. aureus, streptomycin 10 μL for E. coli, K. pneumonia, and P. mirabilis, carbenicillin 100 μL for P. aeruginosa, and amphotericin B 9.6 μg for fungi (yeast and molds) were used as a positive control and phosphate-buffered saline was used as a negative control.

In this study, different crude extracts of Seriphidium herba-alba (Asso) were used to determine the minimum inhibitory concentration (MIC) against various microorganisms. To prepare each extract for testing, 100 mg of the extract was first dissolved in 1 mL of dimethyl sulfoxide (DMSO, supplied by Sigma). A series of twofold dilutions was then created. This was accomplished by adding 1 mL of the dissolved extract to test tubes containing an equal volume (1 mL) of phosphate-buffered saline (PBS) with a pH of 6.8. Bacterial cell suspensions were prepared and adjusted to give a final inoculum concentration of approximately 1 x 106 cells per mL (Spectronic Instruments, USA). The extract buffer tubes were inoculated with 0.5 mL of the prepared inoculum and incubated at 37°C. The tubes were read after 24 h, and 0.1 mL of each test tube was spread on the surface of a nutrient agar plate and incubated at 37°C for 24 h to count CFU/mL.

The values behind the means, standard deviations, and other measures reported in the manuscript were calculated using appropriate statistical methods, including analysis of variance (ANOVA), where applicable. All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation. Data analysis was conducted using the statistical software SPSS.

Table 1 provides a detailed chemical profile of the essential oils of Seriphidium herba-alba (Asso) and a complex series of 52 identified compounds. These were predominantly identified using mass spectrometry and retention index methods, with some verified pure reference compounds. The oil was characterized by several main components, including santolina alcohol, α-thujone, β-thujone, chrysanthenone, cis-chrysanthenyl acetate, 1,8-cineole, camphor, and limonene, which were present in significant amounts ranging from 6.34% to 18.12%. These key components were instrumental in defining the oil's aromatic and potential therapeutic profiles. The listed compounds exhibited various polarities and volatilities, as indicated by their linear retention indices on the apolar and polar columns. While most components were in lower concentrations, some compounds were reported without reported percentages, suggesting trace amounts or unquantified presence. Overall, the essence of Seriphidium herba-alba (Asso) is a rich complex of terpenes, alcohols, and ketones, reflecting the typical heterogeneity of essential oils used in various applications from the medical to the perfume industry.

Graph

Table 1 Chemical composition of Seriphidium herba-alba (Asso) essential oil.

1 a Linear retention index (apolar column).

• 2 b Linear retention index (polar column).
• 3 c trace amounts < 0.1.
• 4 e identification: MS = mass spectrometry, RI = retention index, ST = pure reference compound.

Table 2 shows the extraction yields from different parts of the Seriphidium herba-alba (Asso) plant, with all initial samples weighing 20 g. The extraction methods examined resulted in different extract weights, which were categorized according to the type of extract and the type of plant used.

Graph

Table 2 Various extracts of Seriphidium herba-alba (Asso). The weight of the plant parts was 20 g.

For crude phenolic extracts, aerial parts with flowers gave a variable weight percentage between 4.40% and 13.80%, with an average of 9.02%. The median weight percentage for these extracts was 8.10%, with the most common value being 5.15%. In contrast, immature flowers alone gave a significantly higher yield of crude phenolic extract of 13.8%, whereas mature flowers gave a yield of 8.1%.

Crude saponin extracts were also derived from different plant parts. The combined aerial parts with flowers provided a mean extract weight of 5.89%, with a range of 4.95%–7.32% and a median of 5.66%. However, the aerial parts without flowers yielded 5.14%, immature flowers yielded 7.32%, and mature flowers yielded the lowest yield at 4.95%.

Other extraction methods were used on aerial parts with flowers, resulting in consistent yields across single measurements. The ethyl acetate and ethanol extracts had uniform weights of 9.15% and 7.67%, respectively. Soaking the aerial parts in methanol gave the highest extract weight at 17.75%, whereas ethanol soaking gave 15.53%. The two different water extraction methods each yielded 1%.

Notably, some extracts have detailed statistical data, such as crude saponin from aerial parts with flowers, indicating multiple measurements and variations in the extraction process. However, for several extracts, only single values were provided without additional statistical data, suggesting that either a single measurement was taken, or the variability needed to be documented.

The efficiency and yield of the extraction process depend on both the plant part being processed and the extraction method used. The table provides an overview of how different techniques and plant parts contribute to the yield of Seriphidium herba-alba (Asso) extracts.

Table 3 evaluates the antibacterial efficacy of various extracts from Seriphidium herba-alba (Asso) using agar diffusion and minimum inhibitory concentration (MIC) methods against a spectrum of bacteria. The findings reveal that the plant extracts exhibit a range of activities against both gram-positive and gram-negative bacteria, with varying levels of effectiveness.

Graph

Table 3 Antibacterial activity of various extracts of Seriphidium herba-alba (Asso) by agar diffusion and MIC methods.

• 5 DDM, disc diffusion method; MIC, minimal inhibition concentration
• 6 *Inhibition zone in mm ± SE (S.E., Standard error).

Several extracts demonstrated antibacterial action against the gram-positive bacterium S. aureus, indicated by inhibition zones measuring 11 and 20 mm. In particular, extracts 2, 4, 5, 6, 7, 8, and 9 showed inhibition, with the most potent being extract 10. The MIC values complemented these findings, with some extracts displaying substantial bacteriostatic properties at concentrations as low as 3.125 μg/mL. Notably, extracts 1, 3, 12, and 13 did not inhibit S. aureus, suggesting they lack the necessary components or concentrations to affect this bacterium.

Regarding gram-negative bacteria, the extracts generally exhibited less activity. E. coli was resistant to most extracts, except for extract 10, which inhibited growth with an inhibition zone of 13 mm and a MIC of 3.125 μg/mL. P. aeruginosa was only susceptible to extract 1, which displayed a moderate inhibition zone and MIC indicative of moderate effectiveness. For P. mirabilis, extracts 10 and 11 were effective, with extract 10 showing a particularly significant inhibition zone and a corresponding MIC value. K. pneumoniae was similarly affected by extracts 10 and 11, with notable inhibition zones and MIC values, although the MIC for extract 10 against this bacterium was relatively high at 50 μg/mL.

The positive control, which likely represents a standard antibiotic, consistently inhibited all tested bacterial strains, affirming the validity of the assay. Conversely, the negative control exhibited no antibacterial activity, as expected for a control. Including standard error values with the inhibition zones suggests that the experiments were conducted multiple times to determine reliability, reflecting the variability often inherent in biological testing.

Overall, the results from Table 3 demonstrate that Seriphidium herba-alba (Asso) extracts have selective antibacterial properties, with certain extracts, particularly extract 10, showing broad-spectrum effectiveness. The variation in activity across different extracts highlights the importance of extract type and specific bacterial strain when considering the potential therapeutic applications of these plant extracts.

Table 4 provides a detailed analysis of the antifungal activities of Seriphidium herba-alba (Asso) extracts, using both agar diffusion method and minimum inhibitory concentration (MIC) tests against various fungal species, including yeasts and molds.

Graph

Table 4 Antifungal activity of various extracts of Seriphidium herba-alba (Asso) by agar diffusion and MIC.

• 7 DDM: Disc diffusion method; MIC: Minimal inhibition concentration
• 8 *Inhibition zone in mm ± SE (S.E., Standard error).

For the yeast Candida albicans, multiple extracts showed varying degrees of efficacy. Extract 6 was the most potent, with a 23-mm inhibition zone and an MIC value of 3.125 μg/mL. Extract 2 also demonstrated significant activity (20-mm zone, MIC of 25 μg/mL). Extracts 4, 12, and 13, however, showed no activity against this yeast.

Regarding molds, the results were more varied. In the case of Penicillium spp., only extract 10 showed substantial inhibitory action (23 mm zone, MIC of 3.125 μg/mL), while other extracts, including extracts 1, 2, 3, 6, 7, 12, and 13, were ineffective.

For Fusarium spp., extract 9 was particularly effective (21 mm zone, MIC of 3.125 μg/mL). This pattern of effectiveness was also observed against Aspergillus spp., with extract 9 again demonstrating high activity (18 mm zone, MIC of 3.125 μg/mL).

The activity against Rhizopus spp. was noteworthy in extracts 5, 8, 9, 10, and 11. Extracts 9 and 10 showed the largest zones of inhibition (24 mm), although their MIC values varied, indicating differences in their efficacy.

The positive control showed consistent inhibition across all fungi, confirming the assay's effectiveness. The negative control, with no inhibition, validated the absence of inherent antifungal properties in the testing medium. The standard error values associated with the inhibition zones indicate that the measurements were repeated for precision.

Overall, the results demonstrate that Seriphidium herba-alba (Asso) extracts, particularly extracts 9 and 10, have potential as broad-spectrum antifungal agents. However, the efficacy of these extracts varies depending on the fungal species, highlighting the need for further research to isolate and understand the active compounds responsible for these effects.

The present study explores the field of ethnopharmacology and phytochemistry, focusing on Seriphidium herba-alba (Asso), a medicinal plant. This study aligns with the growing interest in natural products for their potential therapeutic applications, particularly antimicrobial agents. It delves into the extraction, chemical analysis, and evaluation of antimicrobial properties of various extracts from Seriphidium herba-alba (Asso), a plant well-known in traditional medicine.

This study also contributes to new insights into the chemical composition and bioactivity of Seriphidium herba-alba (Asso), particularly regarding its essential oils and extracts. The study stands out in its comprehensive approach, examining a wide range of extracts and their efficacy against bacterial and fungal pathogens. It introduces new data on the efficacy of different extraction methods, the specific bioactivity of various extracts, and their minimum inhibitory concentrations against a spectrum of microorganisms. This includes common pathogenic bacteria and fungi, offering a broad perspective on the plant's antimicrobial potential.

The chemical composition of the essential oil (Table 1) reveals a rich tapestry of volatile compounds, with α-thujone, β-thujone, and santolina alcohol as the predominant constituents. These findings align with those of Ouguirti et al. [[19]] and Houti et al. [[20]], who reported similar profiles in their studies of Artemisia species, emphasizing the consistency in essential oil composition across different geographical locations.

The extraction yields (Table 2) varied significantly depending on the part of the plant and the extraction method, which is in accordance with the results obtained by Brendler et al. [[21]] and corroborates the findings of Adam et al. [[22]] who noted that the extraction method profoundly affects the yield and composition of plant extracts.

The antibacterial activities (Table 3) against both gram-positive and gram-negative bacteria were noteworthy, particularly for extracts 9 (ethanol) and 10 (soaked in methanol), which showed broad-spectrum activity. These results are consistent with those of Liu et al. [[23]] and Bisht et al. [[24]], who observed substantial activity of Artemisia extracts against S. aureus and P. aeruginosa. However, our results suggest higher efficacy than the minimal inhibition concentrations reported by Ahameethunisa et al. [[25]].

Antifungal activities (Table 4) against various fungi, including C. albicans and Aspergillus spp. showcase some extracts with significant inhibitory properties. This is particularly interesting compared with the moderate activity reported by Mehani et al. [[26]], suggesting that the specific strains of Seriphidium herba-alba (Asso) used in this study may possess unique or more potent antifungal compounds.

The potent antibacterial activity of crude phenolic extracts from immature flowers against K. pneumoniae aligns with previous studies that have documented the antibacterial properties of phenolic compounds [[27]]. The stronger antifungal efficacy against C. albicans is particularly noteworthy because it adds to the evidence supporting the use of phenolics as antifungal agents [[29]]. The low MIC observed for mature flower extracts against S. aureus and C. albicans is consistent with the known bioactive profiles of mature plant parts, as reported in other studies [[31]–[33]].

Our results regarding crude saponin extracts are consistent with the literature that acknowledges their antimicrobial properties [[34]]. The findings that extracts from both the aerial parts with and without flowers possess significant antibacterial effects against S. aureus corroborate previous research [[36]], while the efficacy against C. albicans agrees with recent studies highlighting the potential of saponins in antifungal therapy [[38]].

The efficacy of the ethyl acetate extract against S. aureus and its moderate effect against C. albicans add to the compound's profile as a selective antimicrobial agent [[39]]. In contrast, the ethanol extracts exhibited lower activity, which may be attributed to the solvent's polarity affecting the solubility of active compounds [[41]].

Notably, the methanolic extracts demonstrated broad-spectrum activity. This agrees with the literature suggesting that methanol may be more effective at extracting antimicrobial compounds than other solvents [[42]]. However, the reduced efficacy against K. pneumonia raises questions about the specific interactions between the extract's constituents and bacterial cells [[44]].

The ethanol-extracted extracts' consistent MIC of 50 μg/mL indicates lower efficacy, possibly due to the solvent's extraction efficiency or the microorganisms' intrinsic resistance [[45]]. This highlights the importance of solvent choice in the extraction process, a theme well-established in phytochemical research [[46]–[48]].

Our study's suggestion that the extraction method substantially impacts the antimicrobial potency of Seriphidium herba-alba (Asso) extracts is a significant contribution to the field. Methanolic extract yields the most potent extracts, which could guide future research into optimizing extraction techniques for medicinal plants [[49]].

The variability in efficacy demonstrates the complexity of plant-microbe interactions and suggests that extracts can be tailored to specific microbial targets [[51]]. This tailoring could lead to developing new antimicrobial agents that are more effective against resistant strains, a critical need given the rising concern over antibiotic resistance [[53]].

It is important to note the variability in the literature regarding the antimicrobial properties of Seriphidium herba-alba (Asso). While Mehani et al. [[26]] reported lower antimicrobial activity, our study aligns more closely with the potent bioactivities observed by El-Shatoury et al. [[55]]. Such discrepancies could be attributed to variations in plant chemotypes, extraction methodologies, or microbial strains used in the assays, as discussed by Atef et al. [[31]] and Barashkova et al. [[56]].

The results are significant in the context of increasing antibiotic resistance and the ongoing search for novel antimicrobial compounds. By highlighting the antimicrobial properties of Seriphidium herba-alba (Asso) extracts, the study adds valuable information to the field of natural product research and antimicrobial therapy. It underscores the importance of traditional medicinal plants as potential sources of new and effective antimicrobial agents. Additionally, the research provides a framework for future studies aiming to isolate and characterize specific bioactive compounds from Seriphidium herba-alba (Asso), further advancing our understanding of its therapeutic potential. The study's findings could have implications for developing new, naturally derived antimicrobial drugs, which are crucial in the face of the global challenge of antibiotic resistance.

This study demonstrated that Seriphidium herba-alba (Asso) possesses various chemical constituents, particularly within its essential oil. It has shown variable yet significant antibacterial and antifungal activities across different extracts. The results suggest that both the chemical composition and extraction technique are crucial in determining the efficacy of the extracts against various microorganisms. Although this study provides valuable insights into the potential use of Seriphidium herba-alba (Asso) as a natural source of antimicrobial agents, the findings are constrained by limitations such as the lack of in vivo testing, potential seasonal and geographical variation in plant chemistry, and the need for further toxicological assessment. Future investigations are warranted to explore the full therapeutic potential and safety of these extracts and the mechanisms underlying their bioactivity.

S1 Fig

Seriphidium herba-alba (Asso) composite, the Botanical Garden of Hashemite University, Zarqa, Jordan.

(DOCX)

S2 Fig

Extraction of phenolic compounds.

(DOCX)

S3 Fig

Extraction of saponin fraction.

(DOCX)

S4 Fig

n-hexane extract (R1), ethyl acetate extract (R2), and ethanol extract (R3).

(DOCX)

S1 Table

Uses of Seriphidium herba-alba (Asso) composite.

(DOCX)

Tripathi Vijay Academic Editor

31 Jan 2024

PONE-D-23-41380Artemisia herba alba: A Comprehensive Study of Essential Oils, Extracts, and Their Antimicrobial PropertiesPLOS ONE

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Reviewer #1: 1. Please check the accepted plant name. According to the WFO Plant List (http://wfoplantlist.org) the accepted plant name is Seriphidium herba-alba (Asso) Y.R.Ling.

• 2. The species name has to be written as herba-alba.
• 3. The units have to be given uniformly (ml, mL)
• 4. There are certain typographical and grammatical errors that has to be corrected.
• 5. There are sentences that has to be restructured, which has been marked.
• 6. I feel it will be more appropriate if the results are validated statistically using a suitable test (eg. t test).
• 7. In a few places, it is mentioned as the extract soaked in methanol, does it refer to the methanolic extract, if so, please change the sentence structure accordingly.
• 8. In the results section line 292, a subset of extracts is mentioned, but only one extract is discussed. Check.
• 9. In the results section line 295, several extracts are mentioned, but only one extract detail is given. Check.
• 10. References has to be given in the proper journal format and uniformly.
• 11. A clear picture of the habit and habitat of the plant should be provided.

Reviewer #2: The manuscript is a good piece of work in the concerned area, but few things need to be addressed as under

1. The abstract looks like that of a review article, please provide some data so as to make it ore impactful.

• 2. In materials method, the pathogens require accession no., please provide.
• 3. Check for syntax and typographical errors.
• 4. Add few nice references, like https://doi.org/10.3390/coatings11040484, https://doi.org/10.3390/coatings10080761 etc.

*** 

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Reviewer #1: No

Reviewer #2: No

***

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Attachment

Submitted filename: PONE-D-23-41380_reviewer (Recovered).pdf

25 Feb 2024

Dear Reviewer #1,

I hope this letter finds you well. I sincerely appreciate the time and effort you have dedicated to reviewing my manuscript, "Seriphidium herba-alba (Asso): A Comprehensive Study of Essential Oils, Extracts, and Their Antimicrobial Properties" submitted to PLOS ONE Journal. Your insightful comments and suggestions have significantly contributed to improving the manuscript. In response to your valuable feedback, I have made the necessary revisions as outlined below:

Once again, I would like to express my gratitude for your thorough review and constructive feedback. I believe these revisions have strengthened the quality and clarity of the manuscript. Please do not hesitate to contact me if further clarification or modifications are needed.

Thank you for your consideration.

Sincerely.

Hazem Aqel

Dear Reviewer #2,

I hope this message finds you well. I would like to express my gratitude for taking the time to review my manuscript titled "Seriphidium herba-alba (Asso): A Comprehensive Study of Essential Oils, Extracts, and Their Antimicrobial Properties" submitted to PLOS ONE Journal. Your constructive feedback has been invaluable in refining the quality of the manuscript. Below, I outline the actions taken in response to your insightful comments:

Once again, I sincerely appreciate your thoughtful review and constructive feedback. Please let me know if there are any further revisions or clarifications required.

Thank you for your time and consideration.

Regards.

Hazem Aqel

Attachment

Submitted filename: Rebuttal Letter.docx

Tripathi Vijay Academic Editor

2 Apr 2024

Seriphidium herba-alba (Asso): A Comprehensive Study of Essential Oils, Extracts, and Their Antimicrobial Properties

PONE-D-23-41380R1

Dear Dr. Aqel,

We're pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you'll receive an e-mail detailing the required amendments. When these have been addressed, you'll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at https://www.editorialmanager.com/pone/ and clicking the 'Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they'll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Vijay Tripathi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the "Comments to the Author" section, enter your conflict of interest statement in the "Confidential to Editor" section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

***

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

***

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

***

4. Have the authors made all data underlying the findings in their manuscript fully available?

The http://www.plosone.org/static/policies.action#sharing requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

***

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

***

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Almost most of the suggestions given by the referees has been incorporated. I feel the manuscript can be accepted for publication in the journal.

Reviewer #2: I recommend the editor to accept the manuscript for publication in the present form from my side as all the suggestions have been incorporated by the authors.

***

7. PLOS authors have the option to publish the peer review history of their article (https://journals.plos.org/plosone/s/editorial-and-peer-review-process#loc-peer-review-history). If published, this will include your full peer review and any attached files.

If you choose "no", your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our https://www.plos.org/privacy-policy.

Reviewer #1: No

Reviewer #2: No

***