Antimicrobial with time-kill kinetics, antioxidant, and anticancer properties of Rosmarinus officinalis L. oil extract based on its bioactive components

Abstract

There is a major clinical problem associated with antimicrobial resistance. Rosmarinus officinalis L. is an effective medicinal source. Its oil has been extracted and tested for its multiple therapeutic capabilities. The oil extract was found to be a broad-spectrum antimicrobial agent against Bacillus subtilis and Staphylococcus aureus as Gram-positive bacteria, Escherichia coli and Klebsiella pneumonia as Gram-negative bacteria, and Candida albicans as the most common pathogenic mold. The minimum inhibitory concentration of the oil extract was found to be 15.6 μg/mL against B. subtilis, S. aureus, and C. albicans, and 62.5 μg/mL and 125 μg/mL against E. coli and K. pneumonia, respectively. The bactericidal activity started at 150 and 180 min against Gram-positive and Gram-negative bacteria, respectively, with clear time-killing kinetics. The oil extract was able to scavenge DPPH free radicals with an IC₅₀ of 4.0 μg/mL. The oil extract was found to have high toxicity on the Caco2 cell line (colon tissue) at high dose 1000 μg/mL with IC₅₀ of 75.39 ± 0.56 μg/mL. The chemical composition of the oil extract was determined employing gas chromatography/mass spectrometry, in which 53 compounds were named at different surface area ratios, retention times,  and probability ratios.

Full Article

Antimicrobial with Time-kill Kinetics, Antioxidant, and Anticancer Properties of Rosmarinus officinalis L. Oil Extract Based on Its Bioactive Components

Emad Abada ,^a,b,* Abadi M. Mashlawi ,^a Abdelalim A. Gadallah ,^a,c Othman Hakami ,^d Mervat A. Ahmed El Alfy ,^e Abeer S. Meganid,^f and Reham M. Elbaz ^e,g,*

There is a major clinical problem associated with antimicrobial resistance. Rosmarinus officinalis L. is an effective medicinal source. Its oil has been extracted and tested for its multiple therapeutic capabilities. The oil extract was found to be a broad-spectrum antimicrobial agent against Bacillus subtilis and Staphylococcus aureus as Gram-positive bacteria, Escherichia coli and Klebsiella pneumonia as Gram-negative bacteria, and Candida albicans as the most common pathogenic mold. The minimum inhibitory concentration of the oil extract was found to be 15.6 μg/mL against B. subtilis, S. aureus, and C. albicans, and 62.5 μg/mL and 125 μg/mL against E. coli and K. pneumonia, respectively. The bactericidal activity started at 150 and 180 min against Gram-positive and Gram-negative bacteria, respectively, with clear time-killing kinetics. The oil extract was able to scavenge DPPH free radicals with an IC₅₀ of 4.0 μg/mL. The oil extract was found to have high toxicity on the Caco2 cell line (colon tissue) at high dose 1000 μg/mL with IC₅₀ of 75.39 ± 0.56 μg/mL. The chemical composition of the oil extract was determined employing gas chromatography/mass spectrometry, in which 53 compounds were named at different surface area ratios, retention times, and probability ratios.

DOI: 10.15376/biores.20.2.2728-2744

Keywords: Rosemary; Antimicrobial; Antioxidant; Cytotoxicity; GC-MS analysis

Contact information: a: Department of Biology, College of Science, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia; b: Environment and Nature Research Centre, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia; c: Zoology Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt; d: Department of Physical Sciences, Chemistry Division, College of Science, Jazan University, P.O. Box. 114, Jazan 45142, Kingdom of Saudi Arabia; e: Department of Biology, College of Science, University of Bisha, Bisha 61922, P.O. Box 551, Saudi Arabia; rmostafa@ub.edu.sa, maahmed@ub.edu.sa; f: College of Science and Humanities, Jubail, Imam Abdulrahman Bin Faisal University, Jubail, Saudi Arabia, asalmkand@iau.edu.sa; g: Botany and Microbiology Department, Faculty of Science, Helwan University, Cairo 11795, Egypt, reham_elbaz@science.helwan.edu.eg;

* Corresponding author: eabada@jazanu.edu.sa, reham_elbaz@science.helwan.edu.eg

INTRODUCTION

The medical field, especially the clinical one, has long been plagued by a major problem known as multidrug resistance, which is the ability of some microbes to resist different antimicrobial agents (Uddin et al. 2021; Alsolami et al. 2023; Saied et al. 2023).

There is a modern term called superbug microbes, which are resistant to different antibiotics at very high concentrations even above the approved minimum inhibitory concentrations (MICs), causing serious medical conditions. Numerous synthetic and semi-synthetic antibiotics are widely used worldwide to treat serious indications, but they have many side effects that emerge in some patients, especially if administered for a long time. Hence, the general medical trend resorts to using natural antibiotics extracted from microorganisms and plants, especially those with therapeutic properties called medicinal plants as studied by Almehayawi et al. (2024) and Selim et al. (2024). Although an extensive variety of therapeutic plants are known and widely exploited for this function as mentioned by Yahya et al. (2022) and Qanash et al. (2024), rosemary (Rosmarinus officinalis L.) still ranks first in use, because it is available, affordable, easy to grow, and has effective therapeutic properties (Belbachir et al. 2025).

Rosmarinus officinalis L. contains potent therapeutic essential oils (EOs) that include antibacterial agents with different mechanisms. Therefore, these EOs are widely employed in foodstuff preservation and pharmaceutical industry as an alternative medicine (Bakri et al. 2024). The R. officinalis extracts are rich in antibacterial volatile oils, including camphor, and α-pinene, besides phenols, including acids of rosmarinic and carnosic (Yeddes et al. 2021). R. officinalis is a perennial shrub and has many aromatic and medicinal components (Rafie and Soheila 2017). The leaves of R. officinalis are described as an ancient remedy, as they were consumed by the ancient Egyptians and Greeks to stop various diseases and were also used as preservatives and food flavorings (Couto et al. 2012). The R. officinalis extracts contain various antioxidant compounds, which play important roles, including preventing food decomposition and delaying diseases and aging. Antioxidants inhibit oxidation by preventing the initiation of oxidative chain reactions. The most common antioxidants rich in R. officinalis are phenols (flavonoids, tannins, hydroxycinnamate esters, and lignin), vitamin C, vitamin E, and glutathione, which limit the spread of reactive oxygen species (Khojasteh et al. 2020). The Lamiaceae family includes a wide range of medicinal plants, especially R. officinalis, which is rich in various therapeutic components, including powerful antioxidants, like caffeic acid, carnosic acid, and rosmarinic acid as polyphenolic compounds (Singh and Manna 2022).

The R. officinalis L. species is widely distributed throughout the world even in harsh environments, such as saline soils, so it has priority for use as a research material. Egypt is rich in R. officinalis, especially at the Mediterranean coast. Therefore, the Bedouin inhabitants of these coastal areas, who have inherited herbal medicine, use R. officinalis in many cases that require quick and effective treatment. Therefore, R. officinalis is widely used due to its antimicrobial, antioxidant, anti-inflammatory, antidiabetic, anticoagulant, anticancer, anti-obesity, dietary supplement, and hepatoprotective effects. Moreover, R. officinalis is used as an antispasmodic for renal colic and dysmenorrhea, a bronchodilator, a cough suppressant, an expectorant, a mucolytic, and a hair growth stimulant. Phenolic compounds have the upper hand in providing all of the above therapeutic properties to R. officinalis, and therefore, it is considered the dark horse in the treatment of many severe indications (Fernández-Ochoa et al. 2017; Nieto et al. 2018).

Rosmarinus officinalis has important medicinal uses, due to its ability to induce apoptosis of various cancer cell lines, including breast, colon, liver, and leukemia (Rafie et al. 2017). As reported in several studies, carnosic, carnosol, and ursolic acids showed strong toxicity against cancer cells even at advanced levels leading to complete cure (González-Vallinas et al. 2015; Chan et al. 2021). The anticancer activity of R. officinalis acts as a mediator in numerous mechanisms, comprising apoptosis, arrest of cell cycle, delay of angiogenesis, and alteration of signaling pathways required for cell division. Cancer cell apoptosis is induced by carnosic acid and carnosol, which activate caspase cascades, disrupt the mitochondrial membrane, and regulate the proteins design of pro- and anti-apoptotic. Other components present in R. officinalis extracts kill cancer cells by inhibiting cancer-promoting enzymes, including matrix metalloproteinase, which are involved in tumor invasion and metastasis (Bozin et al. 2007). As mentioned in the current introduction, there are many articles available on R. officinalis L. but the novelty in the present investigation focused on antimicrobial activity and studying the Time-kill Kinetics and its anticancer activity against cancer cells associated with the digestive system. Therefore our research paper aims to show the therapeutic potential of different components present in R. officinalis L. oil extract, as well as to recognize the chemical constitution of the ethanolic extract of the oil using the GC-MS technique.

EXPERIMENTAL

Oil Extraction from R. officinalis L. Plant

Fresh R. officinalis L. plant (100 g) was ground in a mortar to obtain a fine paste, then placed in a flask containing distilled water (1000 mL), followed by the addition of 200 mL of chloroform to obtain organic phase. The extraction time continued to 4 h. By means of a rotating evaporator (Buchi Rotavapor E-210, China), the solvent was removed at 25 °C. The concentrated extract was washed by 4 mL of ethyl alcohol and then transferred to an ampoule. Via a slight stream of 1 L/min nitrogen, the residual of ethanol was then evaporated and the extract was reconstituted in 2 mL of ethyl alcohol (Teixeira et al. 2007).

Dilution of Antimicrobial Agent

Dilution was completed utilizing the broth microdilution approach illustrated by Wiegand et al. (2008), where small volumes of broth medium were dispensed into sterile plastic microdilution trays, hence the name. Microdilution trays consist of 96 wells, and all wells were filled separately with broth (0.1 mL) using a fractionator in which a twofold medium of antimicrobial agent was volumetrically diluted into the broth.

Preparation and Storage of Antimicrobial Agent

The microbial inoculum was prepared by the direct suspension of colony technique. Sterile saline solution was inoculated with bacterial colonies grown on blood agar for 18 to 24 h, then incubated for 24 h. Turbid growth was adjusted to meet the 0.5 McFarland standard using colorimetry, where 1 to 2 × 10⁸ CFU/mL was obtained. Within 15 min, the bacterial suspension was diluted using sterile distilled water to obtain an inoculum of 2 to 8 × 10⁵ CFU/mL, and it was optimized at 5 × 10⁵ CFU/mL. Within 15 min, each well in the microdilution tray was inoculated with a bacterial inoculum (inoculum volume not exceeding 10% of the well volume). The inoculated microdilution trays were placed in plastic bags and sealed with plastic tape to avoid drying out and then incubated at 35 ± 2 °C for 16 to 20 h in an ambient air incubator at different distances and heights to avoid stacking to maintain the same incubation temperature for all bacterial cultures (Alghonaim et al. 2024).

Estimation of Minimal inhibitory Concentration (MIC)

A pure culture of the test bacterium was inoculated into trypsinized soy broth and incubated overnight. The bacterial suspension was diluted in trypsinized soy broth to obtain 1 × 10⁵ to 1 × 10⁶ CFU/mL. The antimicrobial agent stock was diluted to approximately 100 times the MIC limit. A stock solution of the extract containing 1000 μg/mL was diluted to obtain 500 µg/mL, and then diluted in a serial manner to obtain 1.95 μg/mL. Each dose was injected in the inoculated 96-well microtiter plate by cultured trypsinized soy broth with microorganisms. The negative control was twofold diluted (1000 – 1.95 µg/mL) free of microorganism inoculum. The positive control was the bacterial suspension grown in the same broth free of antimicrobial agent under the same conditions. The microtiter plates were incubated at 35 ± 2 °C for 16 to 20 h. The absorbance of the turbid growth was measured at 630 nm using a Bio-Tek 800 TS microplate reader. Bacterial growth was tested by colorimetry and compared in broth with and without antimicrobial agent, and the growth endpoint or MIC was precisely determined (Al-Rajhi et al. 2024).

Estimation of Minimal Bactericidal Concentration (MBC)

The MBC was determined based on the MIC dilution and the two most concentrated dilutions of the antimicrobial agent, which were plated and numbered to determine the viable CFU/mL. On Mueller Hinton agar plates enriched with 10% sheep blood, 100 mL of microbial culture was grown from each well that shown full inhibition of growth, from the final positive, and from the growth control to get the MBC. The plates underwent a 72-h microaerophilic incubation period at 35 °C. To ascertain the cidal or static impact of the examined extracts on microorganisms, the MBC/MIC ratios were computed (French 2006).

Antimicrobial Activity Determination

Antimicrobial effect was operated by the agar well manner versus Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Klebsiella pneumonia ATCC 13883, Candida albicans ATCC 10221, and Aspergillus brasiliensis ATCC 16888. Nutrient agar/potato dextrose agar was operated for bacteria/fungi inoculation with the best inoculum size for the test microorganisms, then transferred into plates, which are called seeded plates. The agar surface was cut using a sterile corkborer to make wells (6 mm in diameter). Each well was filled with 100 µL of oil extract compared to those loaded with 100 µL of standard antibiotic (1.0 mg/mL). Agar plates were incubated at 37 ℃ for 24 h and at 25 ℃ for 72 h with bacteria and fungi, respectively. The inhibition zones that appeared around the wells were used to indicate antimicrobial activity, and vice versa (Qanash et al. 2022).

Time-kill Kinetics Test

This test was used to identify the type of antimicrobial activity (bacteriostatic or bactericidal) of the oil extract of R. officinalis L. against Gram-positive and Gram-negative bacteria over time. In bactericidal activity, more than 99.9% was killed, which is equivalent to 3 log₁₀ times the colony forming units (CFU) of the bacterial sample in a given time. Nutrient broth was inoculated with a test bacterial strain and incubated at 37 ℃ for 24 h. Bacterial suspension was supplemented with the oil extract at MIC. Nutrient broth without inoculations was considered as a positive control, while nutrient broth inoculated with a reference bacterial strain was considered as a negative control. The CFU/mL log was set at zero, 30, 60, 120, 150, and 180 min (Chinedu et al. 2024).

Evaluation of Antioxidant Activity

Antioxidant action was evaluated by measuring DPPH (Abdelghany et al. 2019). Three milliliters of test material at varying concentrations (1.95, 3.9, 7.8, 15.62, 31.25, 62.5, 125, 250, 500, and 1000 μg/mL) were combined with 1.0 milliliter of the DPPH solution (0.1 mM) and ethanol. After giving the mixture a good shake, it was allowed to sit at room temperature for half an hour. A Milton Roy UV-VIS spectrophotometer (Nicolet evolution 100, Cambridge, MA, USA) was applied to notice absorbance (517 nm). The investigation was carried out employing ascorbic acid as a standard. The log dose-prevention curve was applied to decide the IC₅₀. Higher free radical potential was indicated by means of the reaction mixture’s low absorbance. The DPPH scavenging effect percentage was calculated using Eq. 1:

(1)

Determination of Cytotoxicity of Test Sample

Wells containing tissue culture medium in the plate were inoculated with 1 × 10⁵ CFU/mL and well-kept at 37 °C up to one day. The growth medium was harvested from the wells after the appearance of a monolayer, and then it was washed twice through water. Double dilution was performed in RPMI medium twice in the presence of 2% serum. Each dilution fraction (0.1 mL) was analyzed in all wells except 3 wells (control). Then the cytotoxicity was tested to detect abnormalities compared to controls (Qanash et al. 2023). The wells were supplemented with 20 µL of MTT solution and incubated in a under shaking at 37 °C and 150 rpm for 5 min, then the period of incubation completed at 37 °C for 4 h and at 5% CO₂ The medium was disposed of, and the plate was dried by sterile tissue to eliminate impurities. The reaction product was supplemented with 200 µL of DMSO and incubated under shaking at 37 °C and 150 rpm for 5 min, and then at 560 nm, the absorbance was deliberate.

GC-MS Analysis

The chemical alignment of the sample was determined operating a Trace GC1310-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) through a TG–5MS direct capillary column (film thickness 30 m × 0.25 mm × 0.25 μm). The column oven temperature was held at 35 °C and then extended 3 °C/min to 200 °C for 3 min and then increased to the final temperature of 280 °C by 3 °C/min and held for 10 min. The temperature of the injector and MS transfer line was maintained at 250 and 260 °C, respectively. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The solvent delay was 3 min and 1 μL diluted samples were automatically injected using the Autosampler AS1300 coupled to the GC in split mode. The EI mass spectra were collected at an ionization voltage of 70 eV over the range of 40 to 1000 m/z in full scan mode. The ion source temperature was set at 200 °C. The components were identified by comparing their retention times and mass spectra with those in the WILEY 09 and NIST 11 mass spectra databases (Al-Rajhi and Abdelghany 2023).

Statistical Analysis

The findings were demonstrated as mean ± standard deviation (SD) and mean ± standard error (SE), which were computed with SPSS v.25 and Microsoft Excel 365. One-way investigation of variance (ANOVA) was applied to assess quantitative data with a standard distribution across various treatments at a 0.05 possibility level.

RESULTS and DISCUSSION

The oil extract of R. officinalis L. was tested for its antimicrobial ability versus fungi and versus bacteria belonging to both Gram-positive and negative (Table 1 and Fig. 1). The oil extract activity against microorganisms was compared with 1.0 mg/mL of gentamycin and fluconazole as standard antibacterial and antifungal antibiotics, respectively. The oil extract showed broad-spectrum antimicrobial action, affecting B. subtilis, S. aureus, E. coli, K. pneumonia, and C. albicans but not A. brasiliensis. Gram-positive bacteria were most affected by the oil extract, followed by C. albicans and bacteria of Gram-negative. Naturally, the oil extract showed less activity against bacteria development of Gram-negative, due to their different line of defense and resistance mechanisms to antimicrobial agents. The oil extract showed inhibition regions of 28 ± 0.1 and 31 ± 0.1 mm compared to gentamycin activity of 27 ± 0.2 and 26 ± 0.2 mm for B. subtilis and S. aureus, correspondingly. Therefore, the oil extract was more effective than gentamycin, and S. aureus was more sensitive to the activity of the oil extract than B. subtilis. The oil extract showed inhibition zones of 23 ± 0.1 mm and 20 ± 0.2 mm compared to gentamycin activity of 25 ± 0.1 mm and 20 ± 0.1 mm for E. coli and K. pneumonia, respectively. Therefore, the oil extract was less effective than gentamycin, and E. coli was more susceptible to the activity of the oil extract than K. pneumonia. The oil extract revealed an inhibition area of 30 ± 0.1 mm compared to fluconazole activity of 31 ± 0.1 mm for C. albicans. Therefore, the oil extract was less effective than fluconazole. The oil extract did not show antifungal activity against A. brasiliensis compared to fluconazole activity of 25 ± 0.2 mm. This may be because of cell wall structure where it is different from the wall structure of bacteria. Yeddes et al. (2022) informed that rosemary essential oil showed bactericidal activity against bacteria belonging to Gram-negative, including Campylobacter jejuni, Salmonella enterica, Pseudomonas aeruginosa, Enterobacter aerogenes, and Escherichia coli, besides Gram-positive, including Bacillus subtilis, Staphylococcus aureus, and Enterococcus faecalis. Bosnić et al. (2006) informed that rosemary (Bosnian origin) essential oil exhibited prevention action for growth of E. coli and P. aeruginosa as Gram-negative bacteria, as the essential oil was found to contain high levels of 1,8-cineole (Kifer et al. 2016), camphor (Zainudin and Mohd 2015), and α-pinene (Ngân et al. 2019).

Table 1. Antimicrobial Activity of Volatile Oil Extract of R. officinalis L. Against Pathogens

NA: No activity

The oil extract showed different MICs against different pathogens (Table 2). The oil extract showed MIC of 15.6 µg/mL against B. subtilis, S. aureus, and C. albicans. The oil extract showed MICs of 62.5 and 125 µg/mL toward E. coli and K. pneumonia, respectively. The oil extract showed different MBCs against different pathogens (Table 3). The extract of oil showed MBC of 31.2 µg/mL toward B. subtilis and S. aureus. The extract of oil showed MBCs of 125 µg/mL and 500 µg/mL against E. coli and K. pneumonia, respectively. The oil extract showed MBC of 62.5 µg/mL versus C. albicans. Aseer et al. (2021) registered that the leaves extract via ethanol of rosemary presented potent activity against growth of clinical isolates, including S. aureus, Enterococcus sp., Streptococcus pyogenes, Salmonella sp., Shigella sp., Klebsiella pneumoniae, P. aeruginosa, E. coli, Proteus sp., and Campylobacter sp. Furthermore, the MIC ​​of the ethanolic extract of rosemary at dose from 4 × 10³ μg/mL to 32 × 10³ μg/mL with MBC ​​ at dose from 8 × 10³ μg/mL to 32 × 10³ μg/mL. The MIC and MBC were shown toward S. aureus, while their top quantities were shown against E. coli.

The oil extract showed antibacterial activity with different time-kill kinetics over time ranging from zero to 180 min (Table 3). In Gram-positive bacteria, bactericidal activity was found to start at 150 min, while in other groups of bacteria (Gram-negative) it was found to start at 180 min. The oil extract revealed superior antibacterial potential toward Gram-positive if compared to Gram-negative group. At zero time, the bacterial counts were found to be 112 × 10⁵ ± 2.0 CFU/mL, 55 × 10⁵ ± 1.0 CFU/mL, 17 × 10⁵ ± 2.0 CFU/mL, and 21 × 10⁵ ± 2.0 CFU/mL with B. subtilis, S. aureus, E. coli, and K. pneumonia, respectively. The count of bacteria gradually decreased over time until they were completely absent at 150 min for B. subtilis and S. aureus, and at 180 min for E. coli and K. pneumonia.

Fig. 1. Antimicrobial activity of oil extract of R. officinalis L. (O) and standard (Gentamycin / Fluconazole) (S), and DMSO (D) against pathogens

Table 2. Determination of MIC and MBC (µg/mL) of Volatile Oil Extract of R. officinalis L. Against Pathogens

+; The growth is present, -; The growth is absent (no growth)

Chinedu et al. (2024) reported that time-kill kinetics of ethanolic extract of Allium sativum at quantities of 0.5, 1.0, and 2 mg/mL were measured to repress S. aureus and P. aeruginosa. The ethanolic extract showed bacteriostatic activity at 0.25 mg/mL, while it showed bactericidal activity at 0.5 and 1 mg/mL versus S. aureus and P. aeruginosa after 24 and 12 h, correspondingly. A significant decline (p ≤ 0.05) in the count of viable P. aeruginosa was observed over multiple time periods at 1.0 mg/mL at 0.09 log10 to 1.20 log10 after 10 h, and at 0.5 mg/mL at 0.02 log10 to 0.52 log10 after 12 h. The reduction in viable P. aeruginosa ranged from ≥ 18.35% to ≤ 99.9% at 1.0 mg/mL, and ≥ 5.30% to ≤ 99.9% at 0.5 mg/mL between 2 h and 24 h. A significant decrease (p ≤ 0.05) in the count of viable S. aureus was observed over multiple time periods at 1.0 mg/mL at 0.09 log10 to 0.97 log10 after 10 h, and at 0.5 mg/mL at 0.03 log10 to 0.47 log10 after 12 h. The reduction in viable S. aureus ranged from ≥ 18.44% to ≤ 99.9% at 1.0 mg/mL, and ≥ 6.0% to ≤ 99.9% at 0.5 mg/mL between 2 h and 24 h.

Table 3. Time-kill Kinetics Assay of Antibacterial Activity of Volatile Oil Extract of R. officinalis L.

Antioxidant activity was determined on the basis of prevention of lipid peroxidation, so scavenging DPPH free radicals. Standard ascorbic acid and oil extract of R. officinalis L. were prepared at different concentrations, which were experienced for their capability to scavenge DPPH by calculating its absorbance at 517 nm. The oil extract of R. officinalis L. presented antioxidant action by scavenging DPPH across IC₅₀ of 4.0 μg/mL contrasted to the IC₅₀ of ascorbic acid (standard) of 3.13 μg/mL (Table 4). Thus, there is a slight difference between the antioxidant activities of standard ascorbic acid and the oil extract of R. officinalis L., with the former showing a higher ability to scavenge DPPH. The DPPH scavenging rate started at its peak at 1000 μg/mL with standard ascorbic acid and R. officinalis L. oil extract, then gradually decreased with lower concentrations until it reached its lowest level at 1.95 μg/mL. Ahmad (2024) registered that the antioxidant potential of rosemary leaf extracts was tested in accordance with different components, including total contents of phenolic (TPC), flavonoid (TFC), and Tannin (TTC). Where the ethanolic extract showed maximum TPC (72.3 GAE mg/g) and TFC (26.8 RE mg/g), while the aqueous extract reflected TTA (20.2 GAE mg/g), and the methanolic extract reflected scavenging of free radicals NO (86.7 RE mg/g) and DPPH (138 GAE mg/g). Also, the aqueous extract reflected antioxidant activity in ABTS (125 TE mg/g), and ferric reducing power (144.5 AScE mg/g) compared to FRAP (130.5 AScE mg/g) and total antioxidant activity (179 GAE mg/g) utilizing the ethanolic extract of rosemary leaves. The outcomes of the paper of Ahmad (2024) exhibited that the highest coefficient of determination showing the link among the phytocomponents content and the antioxidant approaches used was among TPC, FRAP, and TFC.

Table 4. In vitro Assay of Antioxidant Activity of Oil Extract and Ascorbic Acid

SD; Standard deviation, SE; Standard error

The oil extract was tested for its effect (toxicity/viability) on the Caco2 cell line (colon tissue) compared to a normal cell line that appeared free of abnormal signs. Different concentrations of rosemary extract were experienced for their influence on the cell line (Table 5). The oil extract presented the highest toxicity of 97.6% and the lowest viability of 2.41% at 1000 μg/mL. The cytotoxicity decreased and the viability increased with the slope trend of rosemary extract until the lowest cytotoxicity of 1.62% and the highest viability of 98.4% were reached at 31.2 μg/mL. Different concentrations of the oil extract showed cytotoxic and viability effect on colon tissues with IC₅₀ of 75.39 ± 0.56 (Table 6). Additionally, morphological changes were observed on treated cancer cells to rosemary oil extract with clear apoptosis as cleared in Fig. 2, particularly at high concentrations. Eleni et al. (2022) reported that R. officinalis L. is extensively worked as a potent pharmaceutical plant due to its high cytotoxicity on different cell lines, including cancer cell lines of rhabdomyosarcoma and glioblastoma. The methanolic extract showed potent cytotoxicity based on time and dose on the two cell lines. The treated cell lines showed the lowest IC₅₀ values at 72 h corresponding to 0.249 mg/mL for cell line of rhabdomyosarcoma and 0.577 mg/mL for cell line of glioblastoma.

Table 5. Cytotoxicity Effect of Rosemary on Caco2 Cell Line

SD; standard deviation, SE; standard error

Fig. 2. (A) Caco2 cell line in colon tissues as a positive control; rosemary extract doses including (B) 31.25 µg/ mL, (C) 62.5 µg/ mL, (D) 125 µg/ mL, (E) 250 µg/ mL, (F) 500 µg/ mL, and (G) 1000 µg/mL

The oil extract was investigated using GC-MS and was noticed to contain 53 compounds at different retention times (RTs), surface area ratios, and probability ratios (Table 6), as shown in Fig. 3. The current findings showed that the RT increased towards peak 1 to peak 60, due to the increased association of the compound with the column as a stationary phase. Thus, the compound at peak 1 had the weakest association with the column, and then the association gradually increased until it reached the strongest state with the compound at peak 61. However, the boiling point and solubility of the compound in the stage of liquid, as well as the column temperature, affect the RT. Therefore, if the boiling point of the compound rises above the column temperature, the RT increases, and if the solubility of the compound in the liquid stage increases, the RT increases. Mehdi et al. (2011) reported that the ingredients of the essential oil of Iranian rosemary were identified through GC-MS analysis corresponding to 68 components. The analysis revealed that the most significant and influential components were 1,8-cineole (23.5%), α-pinene (21.7%), verbenone (7.57%), camphor (7.21%), and eucalyptol (4.49%).

Table 6. GC-MS Examination of Rosemary Extract

*RT; Retention time, **Prob; Probability, ***MW; Molecular weight

Fig. 3. Chromatogram from GC-MS analysis of rosemary extract

CONCLUSIONS

1. The present oil extract of was analyzed via GC-MS technique and found to be rich with numerous bioactive compounds, from which 5-amino-2-(P-methoxymethyl)-2- methyl-2H-triazolo triazine and (+)-sesamin were detected with the highest area 9.45 % and 9.39, respectively.
2. Oil extract of Rosmarinus officinalis L. possesses excellent antimicrobial (C. albicans, B. subtilis, S. aureus, E. coli, and K. pneumonia) and antioxidant (IC₅₀ 4.0 µg/mL) activities.
3. Anticancer activity of R. officinalis L. extract was reported with IC₅₀ of 75.39 ± 0.56 µg/mL against the Caco2 cell line.

ACKNOWLEDGMENT

“The authors gratefully acknowledge the funding of the Deanship of Graduate Studies and Scientific Research, Jazan University, Saudia Arabia, through project number: (RG24-S035)”.

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Article submitted: November 28, 2024; Peer review completed: February 5, 2025; Revisions received and accepted: February 9, 2025; Published: February 18, 2025.

DOI: 10.15376/biores.20.2.2728-2744