Demonstrating the antioxidant, antibacterial, and anticancer properties of the Achillea fragrantissima shrub growing in Riyadh, Saudi Arabia

Demonstrating the antioxidant, antibacterial, and anticancer properties of the Achillea fragrantissima shrub growing in Riyadh, Saudi Arabia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Demonstrating the antioxidant, antibacterial, and anticancer properties of the Achillea fragrantissima shrub growing in Riyadh, Saudi Arabia Fatimah Al-Otibi, Mohamed Taha Yassin, Tarad Abalkhail, Buthainah Khalid Alsughair, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6874992/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Achillea fragrantissima is a known medicinal plant in traditional Saudi culture. The current study aimed to evaluate the chemical composition of A. fragrantissima and its biological activity as a possible antioxidant, antibacterial, and anticancer agent. The phenolic content of A. fragrantissima was studied using Fourier transform infrared spectroscopy (FTIR) and gas chromatography/mass spectrometry (GC/MS). The antibacterial activity was investigated using the well-diffusion method. The anticancer was investigated against A594 lung cancer cells by MTT, annexin V/Propidium iodide assay, Cell cycle analysis, and Western blotting. The chemical analysis showed that A. fragrantissima includes volatile oils, fatty acids, and other aromatic chemicals. A. fragrantissima ethanolic extract (AFEE) had a 38% scavenging of free radicals at 12 µg/ml and 48% at 100 µg/ml ( p < 0.001). Using a 40% dose, the antibacterial test revealed substantial efficacy against all species, particularly S. aureus . The cytotoxic effect showed dose-dependent growth inhibition of A594 cells with an IC 50 of 14.01 µg/ml. At 20 µg/ml, AFEE caused 33.27% of cellular apoptosis. At 40 µg/ml, G2/M phase arrest increased to 36.77% ( p < 0.001). Mechanistically, AFEE reduced the anti-apoptotic protein Bcl-2 by 56.14% and increased caspase-3 by 66.27% while boosting Cyclin B levels by 74.35%. These findings emphasize AFEE's potential as an antioxidant, antibacterial, and anticancer agent. Biological sciences/Cancer Biological sciences/Microbiology Biological sciences/Plant sciences Achillea fragrantissima FTIR GC/MS antibacterial properties anticancer agent Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Saudi Arabia's flora is diverse, with herbs accounting for 70%, shrubs for 27%, and trees for only 3% [ 1 ]. Compounds obtained from numerous herbal treatments exhibited radical scavenging and antibacterial properties. They can be considered the human body's defense against pathogens and cellular oxidation. Because of their antibacterial, antiviral, anticancer, and antioxidant properties, these components are useful raw materials for producing many types of herbal medicine [ 2 , 3 ]. As a result of the extracted herbal active components, various studies have focused on developing antibacterial, antiviral, and anticancer medications with fewer side effects [ 4 , 5 ]. Achillea is a genus of more than 130 flowering and perennial plants native to Europe and temperate Asia. These plants are recognized by their hairy, scented leaves and flat clusters of little flowers at the stem's tip. Because these blooms appear in various colors, several varieties make appealing garden plants [ 6 ]. Achillea fragrantissima is a medicinal and aromatic shrub widely planted in Saudi Arabia. It has been stated to have medical applications and is utilized in traditional cultures and folkloric medicine among Saudis [ 7 ]. A. fragrantissima has long been used to treat fever and chronic illnesses such as arthritis and diabetes, with anti-inflammatory, antioxidant, and antiproliferative effects on cancer cells [ 8 ]. A. fragrantissima is a small, strongly scented, white-woolly shrub that grows to a height of 30–60 cm. This yarrow is native to North Africa and the Middle East (including Egypt, Jordan, Palestine, Syria, Lebanon, Iraq, and Saudi Arabia). It flourishes in deserts and semi-deserts [ 9 ]. Its ability to grow in arid environments underlines its potential application in sustainable agriculture, ecological restoration, and even extraterrestrial settlement, where resource-efficient medicinal plant production is critical. Currently, phrenological strategies using A. fragrantissima pure extracts or active metabolites to overcome the resistance of more dangerous microbes are widespread. The use of A. fragrantissima extracts in ethanol reduces metal ions to the base metal in a relatively short amount of time, at room temperature and pressure, and on a large scale [ 10 ]. A. fragrantissima has received attention for its powerful anticancer and antioxidant effects. This plant, which is high in bioactive substances including flavonoids, phenolics, and terpenoids, has strong free radical-scavenging activity, which helps protect cells from oxidative damage, a major cause in cancer development. Extracts of A. fragrantissima have been proven in studies to induce apoptosis, decrease cancer cell growth, and restrict migration in a variety of cancer models, indicating their potential as a natural therapy [ 11 , 12 ]. Its antioxidant qualities enhance its anticancer benefits by lowering oxidative stress and regulating critical signaling pathways involved in tumor growth. A. fragrantissima's anti-inflammatory and neuroprotective qualities make it a promising candidate for rehabilitation and disability research, in addition to cancer research. Many chronic illnesses that cause disability, including arthritis, neurological disorders, and muscle deficits, have been related to oxidative stress and inflammation [ 13 ]. A. fragrantissima 's traditional use in pain reduction and inflammatory control suggests that it has the potential to be used in the development of novel treatments in rehabilitation. For example, bioactive chemicals from this plant may aid in tissue regeneration, reduce muscle and joint inflammation, and promote neurological recovery after injuries or neurodegenerative disorders such as Parkinson's and Alzheimer's [ 14 ]. A. fragrantissima's pharmacological capabilities and resilience in severe desert climates make it not only a prospective option for cancer prevention and therapy, but also a possible important species for future bioregenerative life support systems. Research into its bioactive chemicals and medical applications is consistent with the larger goals of inhabitation research, which emphasizes the importance of sustainable, self-sufficient ecosystems in maintaining human health in remote or severe locations. So, the study seeks to provide scientific justification for the traditional use of A. fragrantissima , growing in Saudi Arabia, by examining its chemical composition and biological activity, as well as to investigate its potential as an antioxidant, antibacterial, and anticancer agent represents its rationale. Results Functional groups analysis of AFEE The phytochemical properties of A. fragrantissima ethanolic extract (AFEE) were analyzed using FTIR and GC/MS techniques. FTIR spectroscopy identified the presence of seven distinct functional Groups. A strong, broad peak at 3276 cm − 1 was identified as the O-H stretching group, which indicated the presence of a carboxyl alcohol. Another broad peak was detected at 2985 cm − 1 , which was translated as the N-H Stretching group, indicating an Amine salt, while a strong peak at 1733 cm − 1 was more likely a C = O Stretching Aldehyde. Finally, two medium peaks at 1591 cm − 1 and 1594 cm − 1 were likely to be N-H Bending Amine, while the two medium peaks at 1249 cm − 1 and 1026 cm − 1 were identified as C-N Stretching Amine groups (Fig. 1 ). Phenolic compounds analysis of AFEE GC/MS analysis revealed a variety of bioactive compounds in AFEE, including the fatty acid esters of cis-3-Hexenyl caproate (23.82%) and Ethyl (E)-3,4,4-trimethylpent-2-enoate (11.65%). Other major constituents included the di-alkyl ether E-1-Methoxy-4-hexene (11.67%), the long-chain fatty acids of Palmitic acid (10.06%), cis-vaccenic acid (8.67%), and Stearic acid (6%). Minor terpenoid constituents were reported and included R-Citronellene (0.4%), Norbornane (0.28%), Caryophyllene oxide (0.28%), and Norpatchoulenol (0.62%) (Table 1 ). Table 1 Phenolic constituents of AFEE. Phenolic compound Formula Molecular weight Area (Ab*s) Peak area (%) Classification 2,5-dimethyl-2,3,4-Hexatriene C 8 H 12 108.094 117263 0.88 Branched unsaturated hydrocarbons (1S,2S)-Cyclohexane-1,2-diyldimethanol C 8 H 16 O 2 144.115 33936 0.26 Primary alcohols 5,9-dimethyl-4,8-Decadien-3-ol C 12 H 22 O 182.167 16781 0.13 Monoterpenoids Cyclohexylacetone C 9 H 16 O 140.12 40075 0.3 Ketones 2-Dodecanone C 12 H 24 O 184.183 29956 0.23 Ketones 2-Pentyn-1-ol C 5 H 8 O 84.058 30495 0.23 Primary alcohols 2-Methylthiazoline C 4 H 7 NS 101.03 45219 0.34 Thiazoles Caproic acid C 6 H 12 O 2 116.084 35006 0.26 Fatty acids m-Cymene C 10 H 14 134.11 53866 0.41 Cumenes +-α-Thujone C 10 H 16 O 152.23 55361 0.42 Monoterpenoids (1S)-1-methylcyclohex-2-en-1-ol C 7 H 12 O 112.089 1106850 8.34 Tertiary alcohols 3-Methyltetrahydrofuran C 6 H 10 O 98.073 34770 0.26 Tetrahydrofurans N-Formylpiperidine C 6 H 12 N 2 S 113.084 60428 0.46 Piperidines 3,5-Dihydroxy-6-methyl-2H-pyran-4(3H)-one C 6 H 8 O 4 144.042 242731 1.83 Dihydropyranones R-Citronellene C 10 H 18 138.141 53685 0.4 Monoterpenoids cis-3-Hexenyl caproate C 12 H 22 O 2 198.162 3163564 23.82 Fatty acid esters E-1-Methoxy-4-hexene C 7 H 14 O 114.104 1549339 11.67 Dialkyl ethers (E)-3-Undecen-5-yne C 11 H 18 150.141 145947 1.1 Enynes Ethyl (E)-3,4,4-trimethylpent-2-enoate C 10 H 18 O 2 170.131 1547363 11.65 Fatty acid esters 1-(1-methylene-2-propenyl)-cyclopentanol C 9 H 14 O 138.104 33680 0.25 Cyclopentanols 4,4-Dimethyl-2-pentyne C 7 H 12 96.094 343739 2.59 Alkynes Furfuryl amine C 5 H 7 NO 97.053 34095 0.26 Aralkylamines 3,5-Octadien-2-one C 8 H 12 O 124.089 457042 3.44 Unsaturated ketones 3-hydroxy-2(2-oxopropyl)-2-cyclohexene-1-one C 9 H 12 O 2 152.084 105798 0.8 Ketones Norbornane C 10 H 18 138.141 37368 0.28 Monoterpenoids Ascaridole C 10 H 16 O 2 168.115 54515 0.41 1,2-dioxanes Caryophyllene oxide C 15 H 24 O 220.183 37679 0.28 Sesquiterpenoids 8-Methoxy- [ 1 , 2 , 4 ] triazolo[4,3-a] pyridine-3-thiol C 6 H 5 N 3 S 181.031 101449 0.76 Triazolopyridines D-glycero-D-gulo-Heptonic acid, delta-lactone C 7 H 12 O 7 208.058 50611 0.38 Monosaccharides Palmitic acid C 16 H 32 O 2 256.24 1336396 10.06 Fatty acids cis-Vaccenic acid C 18 H 34 O 2 282.256 1150818 8.67 Fatty acids Stearic acid C 18 H 36 O 2 284.272 796602 6 Fatty acids 15-hydroxy-pentadecanoic acid C 15 H 30 O 3 258.219 45385 0.34 Fatty acids Norpatchoulenol C 14 H 22 190.172 82694 0.62 Tricyclic terpenoid 2-Palmitoylglycerol C 19 H 38 O 4 330.277 247888 1.87 Monoacylglycerols The antibacterial activities of AFEE The antimicrobial activity of AFEE was evaluated using three different doses against various bacterial species, including S. aureus, P. aeruginosa, B. subtilis , and E. coli. The inhibition zone diameters were compared to erythromycin as a positive control. AFEE significantly inhibited the growth of the gram-positive species S. aureus and B. subtilis ( p < 0.001). For E. coli , only the highest dose of AFEE (40 µg/ml) was effective ( p < 0.001). Conversely, P. aeruginosa displayed the greatest resistance (Table 2 ). Table 2 Antibacterial effects of AFEE (diameter of zone of inhibition (mm)). Species DMSO Cephalexin 10 µg/ml 20 µg/ml 40 µg/ml MIC E. coli Mean ± SD 0 18 ± 0 0 0 11.7 ± 0.6 > 40 µg/ml Median (Min-Max) 0 18 (18–18) 0 0 12 (11–12) % Inhibition 0% 20.45% 0% 0% 13.26% P -value 1 < 0.001* 1 1 < 0.001* B. subtilis Mean ± SD 0 25.8 ± 0.8 0 9.8 ± 0.6 13.2 ± 0.3 15 µg/ml Median (Min-Max) 0 26 (25-26.5) 0 10 (9–10) 13 (13-13.5) % Inhibition 0% 29.36% 0% 10.98% 14.96% P -value 1 < 0.001* 1 1 40 µg/ml Median (Min-Max) 0 20 (19–20) 0 0 0 % Inhibition 0% 22.35% 0% 0% 0% P -value 1 < 0.001* 1 1 1 S. aureus Mean ± SD 0 26.8 ± 0.8 0 13.2 ± 0.3 14.8 ± 0.8 15 µg/ml Median (Min-Max) 0 27 (26-27.5) 0 13 (13-13.5) 15 (14-15.5) % Inhibition 0% 30.49% 0% 14.96% 16.86% P - value 1 < 0.001* 1 1 < 0.001* *Significant at p < 0.05. MIC: Minimum Inhibition Concentration. AFEE is an antioxidant agent The Scavenging activity assay results revealed that showed significant free-radical scavenging of almost 38% by 12 µg/ml of AFEE and reached more than 48% at 100 µg/ml, in comparison to that of ascorbic acid ( p < 0.001) (Fig. 2 ). AFEE increased the cytotoxicity of A594 cells The MTT assay showed potential cytotoxicity activity that affected the growth and proliferation of A594 cells. The lowest significant inhibitory concentration was 12.5 µg/ml, which induced about 35% inhibition, whereas about 73% inhibition was obtained by 200 µg/ml ( p < 0.001). The calculated IC 50 was 14.0.1 µg/ml (Fig. 3 ). AFEE-triggered apoptosis of A594 cells As shown in Fig. 4 , AFEE increased the cellular apoptosis of A594 cells at 20 µg/ml to 33.27% compared to the control (1.44%). Most cells (22.85%) underwent early apoptosis at a 20 µg/ml dose, while only 10.42% were processed to late apoptosis or complete cell death. At the highest concentration of 40 µg/ml, it was noticed that the total apoptotic effect seemed to decrease; however, as noticed in Fig. 4 A, the number of dots was lower than those in the control, 10 µg/ml, and 20 µg/ml. This is because most cells shrieked and shifted to the debris area near the zero point, located outside the gated area of the FSC and SSC dot blot. AFEE induced G2/M phase arrest and increased cellular death In comparison to the untreated control cells, it was noticed that the G2/M phase levels increased significantly from 19.18–27.97% ( p < 0.05), 31.17% ( p < 0.001), and 36.77% ( p < 0.001) in the cells treated with 10, 20, and 40 µg/ml of AFEE, respectively (Fig. 5 ). On the other hand, a dramatic decrease in the G0/G1 phase levels was observed. Furthermore, the number of cells arrested at the Sub G stage increased significantly ( p < 0.001), which indicated increased cellular death. Effect of AFEE on the cellular proteins integrated in the apoptotic and cell cycle pathways To confirm the apoptotic effect of AFEE in A594 cells, the expression of the apoptotic suppressor (Bcl-2) and apoptotic activator (caspase 3) was in a dose-dependent manner (Fig. 6 ). Bcl-2 levels decreased by 56.14%, while the expression levels of Caspase 3 increased by 66.27% at the dose of 40 µg/ml, compared to the control ( p < 0.001). The levels of Cyclin B increased up to 74.35% at the highest dose of AFEE, compared to the control, which might explain how AFEE impairs Cyclin B, causes its accumulation, and induces a cellular arrest at the G2/M phase ( p < 0.001). Discussion The current study investigated the phytochemical properties of A. fragrantissima by FTIR and GC/MS analysis to evaluate the common functional groups and phenolic constituents, respectively. The FTIR spectrum confirmed that AFEE was rich in five groups of amine salts (N-H bending, N-H stretching, and C-N stretching), a strong peak of aldehyde (C = O stretching), and a strong peak of carboxylic/alcoholic group (O-stretching) at 3276 cm − 1 , but no alkenes. Several studies on different plants found comparable chemical makeup of an Asteraceae family member. FTIR examination confirmed the presence of alkyl halides, alkanes, alkenes, aldehydes, and amide groups in recent research on the aqueous extract of the leaves of Matricaria chamonbmilla [ 15 ]. Another study confirmed the existence of aromatic compounds containing hydroxyl and carbonyl groups by FTIR examination of Centaurea cyanus flower extract with additional acetyl, C = C, and C = O functional groups [ 16 ]. In another investigation, FTIR spectroscopy was used to determine the existence of the C-H stretch, C = O stretch, O-H stretch, and C-O stretch functional groups in the essential oils of Arnica montana, Echinacea purpurea, Calendula officinalis, Tagetes erecta , and Chamomilla recutita [ 17 ]. The chemical composition of AFEE was phytochemically examined by GC/MS analysis. The GC/MS analysis revealed the presence of multiple phenolic biomolecules, including phenolics, fatty acids, ketones, terpenoids, and antioxidants. The GC/MS results showed that the most represented compounds in the A. fragrantissima extract were cis-3-Hexenyl caproate (23.82%), E-1-Methoxy-4-hexene (11.67%), Ethyl (E)-3,4,4-trimethylpent-2-enoate (11.65%), Palmitic acid (10.06%), cis-Vaccenic acid (8.67%), 1-methylcyclohex-2-en-1-ol (8.34%), and Octadecanoic acid (6%). Similar findings were reported by Qader et al. (2018), in which the phytochemical constituents of the essential oils of A. fragrantissima leaves were reported as m-cymene (0.8%) and α-Thujone (1.15%) [ 18 ]. In the study conducted by Alsohaili and Al-Fawwaz (2014), the GC/MS analysis of the A. frangantissima essential oil revealed that the major constituents were Artemisia ketone (20%), α-Thujone (12%), Carvacrol (13%), p-Cymene (2%), and β-Sesquiphellandrene (15%), which is quite similar to the current findings [ 19 ]. Another study showed that the major fractions of the essential oil of A. fragrantissima were (1S, 4S, 5R)-1-isopropyl-4-methylbicyclo [3.1.0] hexan-3-one (cis-thujone) and 3, 3, 6-trimethyl-1, 5-heptadien-4-one (artemisia ketone), whereas for Achillea biebersteinii , cis-ascaridole and P-cymene were the most represented [20]. Furthermore, for the oil of Achillea santolina , fragranyl acetate and 1,6-dimethyl-1,5-cyclooctadiene represented the highest concentrations, while chamazulene and b-pinene were more common in Achillea millefolium [20]. A previous study analyzing the constituents of the essential oil of different members of the Asteraceae family reported that ascaridole (43.22%), iso-ascaridole (37.17%), and 1,8-cineole (45.2%) were detected in A. biebersteinii [ 21 ], chamazulene (52.60%) in Achillea kellalensis [ 22 ], chrysanthenone (38.8%), trans-carveol (27.5%), neoiso-dihydrocarveol acetate (25.2%), filifolone (19.7%), apinene (11.8%), trans-piperitol (11.7%), (E)-caryophyllene (11.2%), (E)-nerolidol (10.8%), and lavandulyl acetate (26.19%) in Achillea wilhelmsii [23]. All these reports and studies confirm the current findings about the robust phytochemical composition of different extracts of A. fragrantissima. AFEE showed substantial antioxidant activity, with 38% activity at 12 µg/ml and over 48% at 100 µg/ml, comparable to ascorbic acid. These findings are consistent with earlier research on other Achillea species, emphasizing their potent antioxidant effects due to high phenolic and flavonoid content. For example, a study revealed that Achillea atrata L. and Achillea millefolium L. are rich in luteolin, apigenin, centaureidin, and nevadensin, which are known as their effective 2,2-diphenyl-picryl hydrazyl radical scavengers [ 24 ]. This shows that different species in the Achillea genus have a similar bioactive profile, which contributes to their strong antioxidant effects. Comparable research from Saudi Arabia highlights the high antioxidant activity of natural medicinal herbs. Research of essential oils extracted from A. fragrantissima grown in Saudi Arabia and Egypt discovered substantial radical-scavenging activity, with IC 50 values of 30.94 and 28.72 mg/L, respectively [ 25 ]. This highlights the importance of A. fragrantissima as a prospective natural source of antioxidants. In the study conducted by Elsharkawy et al. (2020), the antioxidant activity of A. fragmentisma growing in different regions of Saudi Arabia significantly differed, where those from the Arar and Tabuk regions exhibited their scavenging activities at IC 50 s of 0.21 ± 0.01 and 1.12 ± 0.03 mg/ml, respectively [ 8 ]. These commonalities show that plants in this family share bioactive chemicals that provide variable antioxidant effects, which might be affected by weather parameters, and that supports their usage in traditional medicine. The observed antioxidant potential of AFEE can be attributable to its high phenolic and flavonoid content, as corroborated by prior phytochemical research on A. fragrantissima . The inclusion of volatile oils, fatty acids, and aromatic components boosts its free radical scavenging activity. Given the growing interest in plant-based antioxidants for pharmaceutical and nutraceutical uses, A. fragrantissima's high activity supports its promise as a natural alternative to synthetic antioxidants. In the current study, three doses of AFEE were used to assess their antibacterial activities on the growth of different species ( S. aureus, P. aeruginosa, B. subtilis , and E. coli ). The inhibition zone diameter was measured and compared to the positive control of the antibiotic (Cephalaxin). A significant inhibition of the two gram-positive species ( S. aureus and B. subtilis ). Furthermore, the growth of E. coli was affected only by the highest dose of AFEE (40 mg/ml). P. aeruginosa was the most resistant species to the bactericidal effects of AFEE. Following these findings, a recent study found that A. fragrantissima essential oil has antibacterial action against Bacillus cereus, E. coli, S. aureus, Aspergillus niger, Penicillium sp., and Rhizopus sp. in tomato media [ 21 ]. A previous study discovered that aqueous and hydroethanolic extracts of A. fragrantissima aerial parts were ineffective against E. coli, MRSA, Streptococcus pneumoniae, Enterococcus faecalis, Shigella sonnei, P. aeruginosa, Candida albicans, Candida glabrata , and Salmonella typhimurium , while Klebsiella pneumonia , Bacillus cereus , and S. aureus were more sensitive [ 26 ]. Other species in the Asteraceae family demonstrated antibacterial activity. The antimicrobial activity of the essential oils of the Achillea biebersteinii Afan. from Turkey was effective against Salmonella enterica serovar typhimirium, S. saureus subsp., Yersinia pseudotuberculosis, Bacillus cereus, Enterobacter aerogenes, B. subtilis and Proteus vulgaris [27]. The gram-positive bacteria B. subtilis, B. cereus, S. aureus , and Staphylococcus epidermidis were more susceptible to the essential oil of A. millefolium than the gram-negative bacteria S. typhimurium, Salmonella enteritidis, Salmonella agona , and E. coli [ 28 ]. A. fragrantissima extracts' antimicrobial action could be attributed to their diverse chemical compositions of fatty acids, terpenoids, and ketones, besides, the sesquiterpene of β-Caryophyllene, which showed antibacterial, antifungal, and antioxidant effects against many gram-positive bacteria [ 29 ]. Palmitic and octadecanoic acids (stearic acid), which account for 10% and 6% of the current GC/MS study, respectively, have been shown to exhibit strong antibacterial effects against P. aeruginosa and S. aureus [ 30 ]. Furthermore, caproic acid has a strong antibacterial impact against Campylobacter jejuni and Campylobacter coli [31]. All these studies, in addition to the current findings, suggest the susceptibility of A. fragrantissima as a possible antibacterial agent. AFEE inhibited A594 cell proliferation in a dose-dependent manner, with an IC 50 of 14.01 µg/mL. These findings are consistent with prior research on A. fragrantissima and other Achillea species, which have shown strong anticancer activity because of their high bioactive content. Previous studies reported the cytotoxic effect of A. fragrantissima against breast cancer cells of MCF-7 [ 11 ], pancreatic cancer cells of MiaPaCa-2, prostate cancer cells of PC-3, and lung cancer cells of A594 [ 7 ]. Furthermore, the essential oil extract of A. fragrantissima demonstrated cytotoxic effects on breast (MCF7) and colorectal cancer (HCT-116) cells, with IC 50 values ranging from 0.51–0.91 µg/ml [ 32 ]. Similarly, Achillea membranacea extracts revealed high cytotoxic action against A2780 (ovarian cancer) and HT29 (colon cancer) cells with an IC 50 of around 12.99 and 14.02 µg/ml, respectively [ 33 ]. These findings indicate that distinct Achillea species share bioactive chemicals responsible for their anticancer properties, such as flavonoids, sesquiterpenes, and phenolics. Comparable research from Saudi Arabia supports the medicinal herbs' high cytotoxic action against cancer cells. The observed cytotoxicity in A594 cells could be ascribed to the high concentration of flavonoids, terpenoids, and phenolic chemicals in AFEE, which have previously been shown to have anticancer effects. These findings indicate that Saudi Arabian medicinal plants, particularly those in the Asteraceae family, have potent anticancer characteristics that could be further investigated for therapeutic purposes. AFEE's potential to induce apoptosis was validated by flow cytometry. Treatment with 20 µg/ml increased overall apoptosis to 33.27% compared to 1.44% in the control. Early apoptosis made up 22.85% of the total, with 10.42% proceeding to late apoptosis or full cell death. The highest dose of 40 µg/ml reduced total apoptotic % due to cell debris outside the gated area of the dot plot. This behavior is frequently seen in cells experiencing late-stage apoptosis or necrosis, in which cells shrink, lose integrity, and form apoptotic bodies [34]. To confirm the processes driving AFEE-induced apoptosis and cell cycle arrest, protein expression analysis was performed. AFEE treatment decreased Bcl-2 expression by 56.14% at 40 µg/ml compared to the control, indicating a dose-dependent effect. In contrast, caspase-3 expression increased by 66.27%, indicating apoptosis. Similar results were found for the aqueous extract of A. fragrantissima against breast cancer (MCF7) and HepG2 cells by targeting apoptotic network indicators such as Caspase-3, Caspase-8, Caspase-9, Bcl-2, BAX, and Bcl-xl [ 35 ]. On the other hand, other species from the Asteraceae family showed similar behavior. The aqueous extract of Achillea thracica seeds induced the cytotoxicity of HT1080 fibrosarcoma cells at an IC 50 of 9.85 g/l [36]. The hydroethanolic extract of Achillea millefolium induced apoptosis in NCI-H460 (lung cancer) and HCT-15 (colorectal cancer), caused an increase in p53 and p21, and reduced XIAP expression levels [ 37 ]. The essential oils of Achillea millefolium revealed similar findings on the Panc-1 and MCF-7 cancer cell lines [ 38 ]. Extracts from other species, including Achillea membranacea and Achillea Wilhelmsii C. Koch , were also apoptosis inducers against A2780 (ovarian cancer) and HeLa (cervical cancer) cells by altering the LIN28B and p53 genes expression [12, 34]. These findings demonstrate that AFEE exerts its anticancer effects by regulating critical apoptotic regulatory proteins, highlighting its potential as a natural cancer treatment. Cell cycle studies demonstrated that AFEE administration caused considerable G2/M phase arrest in A594 cells. Cells in the G2/M phase rose from 19.18% in untreated control cells to 27.97%, 31.17%, and 36.77% at 10, 20, and 40 µg/ml, respectively. This increase was followed by a significant decrease in the G0/G1 phase population and an increase in sub-G, indicating a high amount of cellular death. Cyclin B levels increased by 74.35% at 40 µg/ml, indicating that AFEE impairs mitotic progression and causes G2/M phase arrest. In comparable studies, A. fragrantissima caused cycle arrest through G2-phase cell cycle arrest, which caused the elongation of the human chronic myeloid leukemia (K562) cells [31]. Also, the 48-hour exposure to the secondary metabolite, rupicoline from Achillea grandifolia , induced a G2/M cell cycle arrest of U87MG and T98G Glioblastoma cells [ 39 ]. The observed G2/M arrest indicates that AFEE may interfere with cell division by targeting critical mitotic progression regulators, resulting in reduced cancer cell viability. This study offers a complete phytochemical characterization of A. fragrantissima , revealing its rich composition of phenolics, fatty acids, ketones, and terpenoids. It focuses on the plant's powerful antioxidant, antibacterial, and anticancer capabilities, as well as its substantial cytotoxic effects on A594 lung cancer cells. Unlike prior studies that focused on A. fragrantissima from other countries, this study looks particularly at specimens produced in Saudi Arabia, providing region-specific insights into its bioactive potential. The findings show that A. fragrantissima from Saudi Arabia has high antioxidant and antibacterial activity, implying that the country's particular climatic conditions may influence its chemical composition. These findings lend credence to the plant's prospective pharmacological and nutraceutical applications, notably as a natural alternative to synthetic antioxidants and antibacterial agents. The chemical composition, antioxidant, antibacterial, and anticancer properties of the ethanolic extract from the aerial parts of Achillea fragrantissima have important implications for disability research, particularly in the treatment of chronic illnesses, infections, and cancer-related disabilities. Chronic illnesses, including diabetes, arthritis, and cancer, are frequently connected with oxidative stress, which can worsen impairment by destroying cells and tissues. The study emphasizes AFEE's powerful antioxidant action, which may help buffer oxidative stress and improve the quality of life for those with disabilities caused by these illnesses. Furthermore, infections, particularly those produced by antibiotic-resistant bacteria, can result in serious consequences and disability. The study reveals that AFEE has substantial antibacterial action against both gram-positive and gram-negative bacteria, including S. aureus and E. coli. This shows that AFEE may be a natural alternative to infection control, lowering the likelihood of disability caused by severe or recurring infections. On the other hand, cancer and its therapies, such as chemotherapy and radiation, frequently cause physical disabilities owing to tissue damage, discomfort, and functional impairments. The study found that AFEE causes apoptosis and cell cycle arrest in A594 cells, showing its potential as a natural anticancer agent. By targeting cancer cells, AFEE may lessen the need for chemotherapy treatments, which frequently result in impairment, thereby improving patient outcomes. Conclusions The findings emphasized A. fragrantissima 's potential antibacterial efficacy against harmful bacteria, hinting that its active metabolites may be a hidden source of powerful bioactive compounds. This work successfully characterized the complex phytochemical composition of A. fragrantissima from Saudi Arabia, indicating a high phenolic content and powerful bioactive properties. The plant extract exhibits substantial antioxidative, antibacterial, and anticancer activities, indicating its potential utility in both traditional and modern medicine. Its strong cytotoxic activity and induction of apoptosis in lung cancer cells highlight its potential as a natural anticancer medication, prompting further investigation into its therapeutic applications. Given these findings, A. fragrantissima from Saudi Arabia could be researched further for pharmaceutical development, particularly for natural antibacterial and anticancer formulations. Future research should analyze the phytochemical content of different regions and conduct in vivo studies to assess the clinical potential. Materials and Methods Plant Material and Extract Preparation A. fragrantissima aerial parts were obtained in March 2023 from the Rafha region of the Kingdom of Saudi Arabia, located at 29°38′19″N 43°30′5″E. The collection and handling of the plant material were according to the International Union for Conservation of Nature (IUCN) Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. The plant was deposited in the herbarium of the College of Science, King Saud University, under the name of “ Achillea fragrantissima shrub, Rafha, Riyadh”. Plant identification was performed by Prof. Najat Bukhari in the Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia. The plant was cleaned with running tap water first, then with sterile distilled water. The plant was then shade-dried, carefully powdered, and stored at 4°C until later usage. The infusion method was used to prepare the plant extract [40]. The fresh, dry plant components were crushed, and 30 g were soaked overnight in 300 ml of 100% ethyl alcohol in a closed container at room temperature, then transferred to another container overnight using a rotary shaker. The macerates were gravity-filtered twice, once through cotton and then through layers of tissue paper. Finally, the residual filtrates were dried overnight in cleaned metallic trays before being transferred to a clean, dry glass container and refrigerated until needed. Fourier transform infrared spectroscopy (FTIR) The functional groups were found using FTIR analysis. A spectrometer (Nicolet 6700, Thermo Fisher Scientific Inc., Waltham, MA, USA) with a beam splitter and detector (DTGS) loaded with OMNIC software was used to gather and analyze spectra in the 500–4000 cm1 scan range. The resulting IR spectra were utilized to analyze the functional groups found in the produced extract. The acquired wavelengths were evaluated by the Scientific Reaction Animation Image Builder (InstaNANO) https://instanano.com/all/characterization/ftir/ftir-functional-group-search/ to identify the relevant functional groups [ 41 ]. Gas Chromatography/Mass Spectrometry (GC/MS) The ethanol extract underwent GC/MS analysis. The carrier gas was helium, with a flow rate of 1 ml per minute. The Agilent 5977c GC/MSD (Agilent Inc., Santa Clara, California, USA) was used. A phenyl methyl siloxane column (30m, 250m, 0.25m) was utilized. The following settings were maintained throughout the GC run: time (90 minutes), volume (1 L), temperature (280°C; 250°C), ion source, and split ratio (20:1). Furthermore, the temperature was held at 40°C for 5 minutes before increasing to 280°C at 10°C/min and remaining there for another 5 minutes. For mass spectroscopy, an electron impact (70 eV) was created with a scan range of 35 to 780 m/z. The National Institute of Standards and Technology (NIST) mass spectral library ( https://chemdata.nist.gov/dokuwiki/doku.php?id=chemdata:start ) was used to identify the samples [42]. Scavenging activity assay The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay is widely used to assess the antioxidant activity of plant extracts. This assay evaluates bioactive compounds' ability to scavenge free radicals promptly and consistently [ 43 ]. A 0.1 mM DPPH solution in methanol with an initial absorbance of 0.9-1.0 at 517 nm was created. The plant extract or ascorbic acid, a common antioxidant, was produced in methanol at various quantities. Each sample was mixed with an equal volume of DPPH solution before being incubated for 30 minutes in the dark at room temperature. After incubation, the absorbance is measured at 517 nm using a UV-Vis spectrophotometer. The fraction of DPPH scavenging activity is calculated using the following formula: \(\:Scavenging\:\%=\frac{Ac-As}{Ac}\times\:100\) Here, “Ac” is the absorbance of DPPH solution alone without any samples, and “As” is the absorbance of DPPH mixed with either the plant extract or the ascorbic acid. Antimicrobial Activities The bacterial strains were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). These strains included Staphylococcus aureus (ATCC-25923), Bacillus subtilis (ATCC-6051), Escherichia coli (ATCC-11755), and Pseudomonas aeruginosa (ATCC-27584). All bacterial strains were cultured on Mueller-Hinton agar medium. To achieve a concentration of 1 µg/ml, the previously produced AFEE powder was dissolved in a 1:100 DMSO: distilled water solution. Mueller-Hinton agar growth media was made according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA). Briefly, 19 grams of Mueller-Hinton agar were dissolved in 500 ml of deionized water before being sterilized in an automated autoclave at 121°C for 15 minutes. After cooling to 70ºC, each Petri dish received approximately 10 ml of the medium. At room temperature, the medium was allowed to harden. The bacterial strain was plotted on three Petri dishes. Three discs were created, with three concentrations of the extract (10, 20, and 40 µl) inserted in each disc. Another disk was made for the control by adding only 0.2 µl of DMSO. Another Petri dish had cefalexin as a positive control (5 µg/ml). The plates were placed in an incubator at 37°C for 24 hours. After incubation, the areas of growth inhibition were measured in millimeters [ 44 ]. All experiments were conducted in duplicate. Minimum inhibitory concentrations (MIC) were determined using the following equation: $$\:MIC=\frac{Lowest\:Conc.\:inhibit\:the\:growth+Highest\:conc.\:allow\:the\:growth\:}{2}$$ Cell culture In the current study, the lung cancer cell line A594 was used to study the anticancer properties of A. fragrantissima ethanolic extract. A594 cell line was obtained from ATCC (Manassas, VA, USA). The cells were maintained in Dulbecco’s Modified Eagle's Medium (DMEM) supplemented with 1% Glutamax, 10% Fetal Bovine serum, and 1% 10000/1000 penicillin-streptomycin (Thermo Fisher Scientific Inc., GIBCO, Waltham, MA, USA). The cells were incubated in a 37ºC CO 2 incubator, and media were renewed every three days. Cytotoxicity assay In this assay, 5000 cells per well were seeded in a 96-well plate and adhered overnight in a humidified incubator at 37°C with 5% CO 2 . The following day, cells were treated with varied amounts of the plant extract, while untreated cells were the negative control. After 24 hours of incubation, add 10 µL of MTT solution (5 mg/ml in phosphate-buffered saline (PBS)) (Thermo Fisher Scientific Inc., Invitrogen, Waltham, MA, USA) to each well and incubate for 90 minutes. The formazan crystals will form a purple precipitate. The media was carefully removed, and the formazan crystals were solubilized with 100 µL of dimethyl sulfoxide (DMSO). The absorbance was measured at 540 nm with a microplate reader [ 21 ]. The IC50 was calculated by applying all the resulting optical densities and corresponding concentrations to the IC 50 Calculator of AAT Bioquest at https://www.aatbio.com/tools/ic50-calculator . Apoptosis/ Necrosis Assay The Annexin V/Propidium Iodide (PI) assay was used to identify and distinguish between apoptotic and necrotic cells based on phosphatidylserine externalization and membrane integrity [45]. Cells were planted into an appropriate culture plate and treated with the plant extract. After 24 hours of incubation, cells were extracted, including adherent and floating populations, and washed with cold PBS. The cells were resuspended in 100 µl of binding buffer at a concentration of 1×10 6 cells/ml. The FITC Annexin V Apoptosis Detection Kit with PI (Biolegend Co., San Diego, CA, USA) was used following the manufacturer's instructions. The mixture is incubated in the dark for 15 minutes at room temperature before being analyzed using a BD FACSCalibur™ Flow Cytometer (BD Co., Franklin Lakes, NJ, USA). Cell cycle assay The PI-based cell cycle test is a popular method for determining DNA content and cell distribution during the cell cycle. Cells were initially treated with the plant extract, then collected and fixed with cold 70% ethanol to retain their structure. After washing to remove any remaining ethanol, the cells were treated with a staining solution containing PI, RNase A to break down RNA, and a detergent to permeabilize the cell membrane. The samples were then analyzed using flow cytometry, with fluorescence intensity used to differentiate between cells in the G0/G1, S, and G2/M phases [ 46 ]. Western blotting To carry out the assay, treated cells were lysed in RIPA buffer (Santa Cruz Biotechnology Inc., Dallas, TX, USA) to extract total protein. The protein concentration was determined using the Bradford assay and a Nanodrop spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Equal amounts of protein were separated using SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk to prevent nonspecific binding. It was treated overnight with primary antibodies that targeted apoptosis (Bcl-2 and Caspase 3), cell cycle (Cyclin D1), and the housekeeping protein β-actin (Biolegend Co., San Diego, CA, USA). Following that, the membrane was incubated with suitable HRP-conjugated secondary antibodies and visualized with a c-digit scanner after staining with Luminol and oxidizing solutions (Thermo Fisher Scientific Inc., Waltham, MA, USA). The band intensities were measured by ImageJ densitometry software version 1.8.0_345 https://imagej.net/ij/ [47]. Statistical Analysis The data was statistically evaluated with SPSS version 22. The experiments were subjected to a one-way analysis of variance (ANOVA), and mean differences were analyzed using the Duncan test. At the 0.05 probability level, the mean values were separated using the least significant difference (LSD). All values in this study represent the average of at least three replicates. The data was reported as the mean with standard deviation (SD). Declarations Acknowledgments: The authors extend their appreciation to the King Salman Center for Disability Research for funding this work through Research Group no KSRG-2024-366. Author Contributions: Conceptualization, F.A. and M.S.E.; methodology, N.A.A. and M.S.E.; software, M.T.Y.; validation, F.A., M.T.Y., and T.A.; formal analysis, M.T.Y. and M.S.E.; investigation, B.K.A. and N.A.A.; resources, F.A. and N.A.A.; data curation, T.A. and M.S.E.; writing—original draft preparation, M.S.E., M.T.Y., and N.A.A.; writing—review and editing, F.A.; visualization, M.S.E., and M.T.Y.; supervision, F.A. and T.A.; project administration, F.A.; funding acquisition, F.A. All authors have read and agreed to the published version of the manuscript. Funding: This research project was supported by a grant from the King Salman Center for Disability Research through Research Group no KSRG-2024-366 Institutional Review Board Statement: This research does not require an ethical statement/clinical trial registration number, or informed consent. Informed Consent Statement: Not applicable. Data Availability Statement: The raw data supporting the conclusions of this article will be made available by the corresponding authors on request. Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References Elkordy, A. et al. Floristic diversity of Jabal Al-Ward, southwest Tabuk region, Kingdom of Saudi Arabia. Agronomy 12, 2626. (2022). https://doi.org/10.3390/agronomy12112626 Miraj, S. & Rafieian-Kopaei, Kiani, S. Melissa officinalis L: A review study with an antioxidant prospective. Evidence-Based Complement. Altern. Medicine . 22 , 385–394. https://doi.org/10.1177/2156587216663433 (2017). Saratale, R. G., Benelli, G., Kumar, G., Kim, D. S. & Saratale, G. D. 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Egypt. Acad. J. Biol. Sci. C Physiol. Mol. Biology . 16 , 387–401. https://doi.org/10.21608/eajbsc.2024.355267 (2024). Additional Declarations No competing interests reported. Supplementary Files gelimages.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6874992","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":491470250,"identity":"32bb4407-d0a8-4d58-b811-380ad2c220a5","order_by":0,"name":"Fatimah Al-Otibi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIie3PMQrCMBSA4VcK7RLJ+gS9Q6BQhA5exSLYxaFjB4dIoVOhV/EIKQW7RFwdOliEzj2AiEFdHEx1c8gfMgTy8RIAk+kPc7nNAZgglDzO9jAhwuIgFBnnvxG1mPyauOX20sfNxJN1h5AEIafVWU9ImDLBOuIfch9BRiHHFdOSOYQZClYR/0gctLJKEdATQtsn8Qq3Q+umCK17PcHXFDbiPlpcEVgPTME2ZVL9BaX0Zot95GW4jgcetizPybWZ0zxqT/0mmBa03mnJewu1nR/um0wmk+lDd56iR2KJqIotAAAAAElFTkSuQmCC","orcid":"","institution":"King Saud University","correspondingAuthor":true,"prefix":"","firstName":"Fatimah","middleName":"","lastName":"Al-Otibi","suffix":""},{"id":491470251,"identity":"c4803001-81ff-4d82-9bf4-dd10f0293f16","order_by":1,"name":"Mohamed Taha Yassin","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"Taha","lastName":"Yassin","suffix":""},{"id":491470252,"identity":"1bb792fd-3a9e-4aaa-8c2c-b427578406ee","order_by":2,"name":"Tarad Abalkhail","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Tarad","middleName":"","lastName":"Abalkhail","suffix":""},{"id":491470253,"identity":"9ca69a95-a353-40b0-b217-027b903cbde8","order_by":3,"name":"Buthainah Khalid Alsughair","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Buthainah","middleName":"Khalid","lastName":"Alsughair","suffix":""},{"id":491470254,"identity":"085dd8f1-478a-4bbb-ad08-d1aa4e57d245","order_by":4,"name":"Nourah A. Al-shammry","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Nourah","middleName":"A.","lastName":"Al-shammry","suffix":""},{"id":491470255,"identity":"4c76e99f-2b16-4c83-9275-35b15466aa39","order_by":5,"name":"Mohammad S. El-Wetidy","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"S.","lastName":"El-Wetidy","suffix":""}],"badges":[],"createdAt":"2025-06-11 21:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6874992/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6874992/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87738864,"identity":"c252ed77-1b57-4e34-be9d-6f8437cb81b9","added_by":"auto","created_at":"2025-07-28 13:00:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":485948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTIR results of the AFEE.\u003c/strong\u003e The results were obtained using a Nicolet 6700 FT-IR spectrometer over a range of 4000–500/cm.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6874992/v1/a3a21bb3a4b2fd7d45814355.jpg"},{"id":87738866,"identity":"c00f134e-75b4-4b22-ae43-16f1c5d6656c","added_by":"auto","created_at":"2025-07-28 13:00:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":173645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe scavenging activity of AFEE versus ascorbic acid.\u003c/strong\u003e DPPH assay was used to identify the antioxidant activity. All concentrations from the two materials were measured in triplicate, where the lines with dots express the mean scavenging percentage of all reads.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6874992/v1/09c13626ffc5358b4759b963.jpg"},{"id":87738871,"identity":"9444ec54-4f8e-4c14-beaf-7f945786a691","added_by":"auto","created_at":"2025-07-28 13:00:58","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":146161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxic activity of AFEE against A594 cells. \u003c/strong\u003eThe cancer cells were treated with different concentrations of AFEE (in triplicate) for 24 hours. The MTT assay was used to identify the cytotoxic and antiproliferative activities observed in treated cells compared to the control. *** indicates significant readings at \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6874992/v1/ce3805f622987e3b554034d1.jpg"},{"id":87740115,"identity":"47344fc8-64bc-4a2a-87fc-d22a278040c0","added_by":"auto","created_at":"2025-07-28 13:16:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1038014,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe apoptotic activity of AFEE against A594 cells. \u003c/strong\u003eThe cells were treated with different doses of AFEE for 24 hours, and then the cellular death was detected by Annexin V/Propidium iodide and a Flow cytometer. *** indicates significant readings at \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05. a) Dot blots of treated and untreated cells after staining with Annexin V/PI; b) Different cell death structures observed from the dot blots at all concentrations. c) the total apoptotic effect (early + late apoptosis) at all concentrations.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6874992/v1/8ceae6b3e3a0ee77ac4960dd.jpg"},{"id":87739785,"identity":"6761a1f6-c872-43c9-9fdb-559b0a70c1e1","added_by":"auto","created_at":"2025-07-28 13:08:58","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":640835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Cell cycle analysis of A594 treated with different doses of AFEE. \u003c/strong\u003eThe cells were treated with different doses of AFEE for 24 hours, and then the cell cycle was analyzed using Propidium iodide and a Flow cytometer. *** indicates significant readings at \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05. a) Histograms of treated and untreated cells after staining with PI; b) Different cell cycles expressed in treated cells and compared to the control cells.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6874992/v1/5089c90fa16cb94ae9df4cf5.jpg"},{"id":87738867,"identity":"bedc0bf3-9f4f-41e6-8f0e-ea58b3585bc8","added_by":"auto","created_at":"2025-07-28 13:00:58","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":707551,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe anticancer activity of AFEE against the cellular apoptotic and cellular arrest protein markers. \u003c/strong\u003eThe cells were treated with different doses of AFEE for 24 hours, and then western blotting was used to detect the apoptotic pathway proteins' expression of p53 and caspase 3, in addition to cyclin B as an indicator of G2/M phase arrest. *** indicates significant readings at \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05. a) Immunoblots of AFEE-treated and untreated cells compared to the housekeeping protein of β-actin; b) Band intensities calculated for all proteins and normalized to β-actin.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6874992/v1/d0f2bd4ea8e8d6a42108661f.jpg"},{"id":92546794,"identity":"7fd39399-93df-47ad-bd5d-edefea6ed513","added_by":"auto","created_at":"2025-09-30 21:01:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4418552,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6874992/v1/d12cec70-4c8b-42c5-9c1b-b3e95ef9b9bd.pdf"},{"id":87739781,"identity":"ce6a216a-b56f-4f73-bc41-20a4aa16e0ab","added_by":"auto","created_at":"2025-07-28 13:08:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":284009,"visible":true,"origin":"","legend":"","description":"","filename":"gelimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6874992/v1/4b9da310a1493061a406ef6f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Demonstrating the antioxidant, antibacterial, and anticancer properties of the Achillea fragrantissima shrub growing in Riyadh, Saudi Arabia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSaudi Arabia's flora is diverse, with herbs accounting for 70%, shrubs for 27%, and trees for only 3% [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Compounds obtained from numerous herbal treatments exhibited radical scavenging and antibacterial properties. They can be considered the human body's defense against pathogens and cellular oxidation. Because of their antibacterial, antiviral, anticancer, and antioxidant properties, these components are useful raw materials for producing many types of herbal medicine [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As a result of the extracted herbal active components, various studies have focused on developing antibacterial, antiviral, and anticancer medications with fewer side effects [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eAchillea\u003c/em\u003e is a genus of more than 130 flowering and perennial plants native to Europe and temperate Asia. These plants are recognized by their hairy, scented leaves and flat clusters of little flowers at the stem's tip. Because these blooms appear in various colors, several varieties make appealing garden plants [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eAchillea fragrantissima\u003c/em\u003e is a medicinal and aromatic shrub widely planted in Saudi Arabia. It has been stated to have medical applications and is utilized in traditional cultures and folkloric medicine among Saudis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. \u003cem\u003eA. fragrantissima\u003c/em\u003e has long been used to treat fever and chronic illnesses such as arthritis and diabetes, with anti-inflammatory, antioxidant, and antiproliferative effects on cancer cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eA. fragrantissima\u003c/em\u003e is a small, strongly scented, white-woolly shrub that grows to a height of 30\u0026ndash;60 cm. This yarrow is native to North Africa and the Middle East (including Egypt, Jordan, Palestine, Syria, Lebanon, Iraq, and Saudi Arabia). It flourishes in deserts and semi-deserts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Its ability to grow in arid environments underlines its potential application in sustainable agriculture, ecological restoration, and even extraterrestrial settlement, where resource-efficient medicinal plant production is critical.\u003c/p\u003e\u003cp\u003eCurrently, phrenological strategies using \u003cem\u003eA. fragrantissima\u003c/em\u003e pure extracts or active metabolites to overcome the resistance of more dangerous microbes are widespread. The use of \u003cem\u003eA. fragrantissima\u003c/em\u003e extracts in ethanol reduces metal ions to the base metal in a relatively short amount of time, at room temperature and pressure, and on a large scale [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. \u003cem\u003eA. fragrantissima\u003c/em\u003e has received attention for its powerful anticancer and antioxidant effects. This plant, which is high in bioactive substances including flavonoids, phenolics, and terpenoids, has strong free radical-scavenging activity, which helps protect cells from oxidative damage, a major cause in cancer development. Extracts of \u003cem\u003eA. fragrantissima\u003c/em\u003e have been proven in studies to induce apoptosis, decrease cancer cell growth, and restrict migration in a variety of cancer models, indicating their potential as a natural therapy [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Its antioxidant qualities enhance its anticancer benefits by lowering oxidative stress and regulating critical signaling pathways involved in tumor growth.\u003c/p\u003e\u003cp\u003e\u003cem\u003eA. fragrantissima's\u003c/em\u003e anti-inflammatory and neuroprotective qualities make it a promising candidate for rehabilitation and disability research, in addition to cancer research. Many chronic illnesses that cause disability, including arthritis, neurological disorders, and muscle deficits, have been related to oxidative stress and inflammation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eA. fragrantissima\u003c/em\u003e's traditional use in pain reduction and inflammatory control suggests that it has the potential to be used in the development of novel treatments in rehabilitation. For example, bioactive chemicals from this plant may aid in tissue regeneration, reduce muscle and joint inflammation, and promote neurological recovery after injuries or neurodegenerative disorders such as Parkinson's and Alzheimer's [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eA. fragrantissima's\u003c/em\u003e pharmacological capabilities and resilience in severe desert climates make it not only a prospective option for cancer prevention and therapy, but also a possible important species for future bioregenerative life support systems. Research into its bioactive chemicals and medical applications is consistent with the larger goals of inhabitation research, which emphasizes the importance of sustainable, self-sufficient ecosystems in maintaining human health in remote or severe locations. So, the study seeks to provide scientific justification for the traditional use of \u003cem\u003eA. fragrantissima\u003c/em\u003e, growing in Saudi Arabia, by examining its chemical composition and biological activity, as well as to investigate its potential as an antioxidant, antibacterial, and anticancer agent represents its rationale.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eFunctional groups analysis of AFEE\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe phytochemical properties of \u003cem\u003eA. fragrantissima\u003c/em\u003e ethanolic extract (AFEE) were analyzed using FTIR and GC/MS techniques. FTIR spectroscopy identified the presence of seven distinct functional Groups. A strong, broad peak at 3276 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was identified as the O-H stretching group, which indicated the presence of a carboxyl alcohol. Another broad peak was detected at 2985 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was translated as the N-H Stretching group, indicating an Amine salt, while a strong peak at 1733 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was more likely a C\u0026thinsp;=\u0026thinsp;O Stretching Aldehyde. Finally, two medium peaks at 1591 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1594 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were likely to be N-H Bending Amine, while the two medium peaks at 1249 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1026 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were identified as C-N Stretching Amine groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003ePhenolic compounds analysis of AFEE\u003c/em\u003e\u003c/p\u003e\u003cp\u003eGC/MS analysis revealed a variety of bioactive compounds in AFEE, including the fatty acid esters of cis-3-Hexenyl caproate (23.82%) and Ethyl (E)-3,4,4-trimethylpent-2-enoate (11.65%). Other major constituents included the di-alkyl ether E-1-Methoxy-4-hexene (11.67%), the long-chain fatty acids of Palmitic acid (10.06%), cis-vaccenic acid (8.67%), and Stearic acid (6%). Minor terpenoid constituents were reported and included R-Citronellene (0.4%), Norbornane (0.28%), Caryophyllene oxide (0.28%), and Norpatchoulenol (0.62%) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePhenolic constituents of AFEE.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhenolic compound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFormula\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMolecular weight\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eArea (Ab*s)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePeak area (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eClassification\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,5-dimethyl-2,3,4-Hexatriene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e108.094\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e117263\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBranched unsaturated hydrocarbons\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(1S,2S)-Cyclohexane-1,2-diyldimethanol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e144.115\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e33936\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePrimary alcohols\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5,9-dimethyl-4,8-Decadien-3-ol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e182.167\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16781\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMonoterpenoids\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCyclohexylacetone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e140.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e40075\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKetones\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-Dodecanone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e184.183\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e29956\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKetones\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-Pentyn-1-ol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84.058\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e30495\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePrimary alcohols\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-Methylthiazoline\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e7\u003c/sub\u003eNS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e101.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e45219\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eThiazoles\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCaproic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e116.084\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e35006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFatty acids\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003em-Cymene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e134.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e53866\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCumenes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e+-α-Thujone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e152.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e55361\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMonoterpenoids\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(1S)-1-methylcyclohex-2-en-1-ol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e112.089\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1106850\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e8.34\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTertiary alcohols\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3-Methyltetrahydrofuran\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e98.073\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e34770\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTetrahydrofurans\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN-Formylpiperidine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e113.084\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e60428\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePiperidines\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3,5-Dihydroxy-6-methyl-2H-pyran-4(3H)-one\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e144.042\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e242731\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1.83\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDihydropyranones\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR-Citronellene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e138.141\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e53685\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMonoterpenoids\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ecis-3-Hexenyl caproate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e198.162\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3163564\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e23.82\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFatty acid esters\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eE-1-Methoxy-4-hexene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e114.104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1549339\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e11.67\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDialkyl ethers\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(E)-3-Undecen-5-yne\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e11\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e150.141\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e145947\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eEnynes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEthyl (E)-3,4,4-trimethylpent-2-enoate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e170.131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1547363\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e11.65\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFatty acid esters\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1-(1-methylene-2-propenyl)-cyclopentanol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e138.104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e33680\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCyclopentanols\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4,4-Dimethyl-2-pentyne\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e96.094\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e343739\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAlkynes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFurfuryl amine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e7\u003c/sub\u003eNO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e97.053\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e34095\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAralkylamines\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3,5-Octadien-2-one\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e124.089\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e457042\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e3.44\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eUnsaturated ketones\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3-hydroxy-2(2-oxopropyl)-2-cyclohexene-1-one\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e152.084\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e105798\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKetones\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNorbornane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e138.141\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e37368\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMonoterpenoids\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAscaridole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e168.115\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e54515\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1,2-dioxanes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCaryophyllene oxide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e220.183\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e37679\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSesquiterpenoids\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8-Methoxy- [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] triazolo[4,3-a] pyridine-3-thiol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e181.031\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e101449\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTriazolopyridines\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD-glycero-D-gulo-Heptonic acid, delta-lactone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e208.058\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e50611\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMonosaccharides\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePalmitic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e256.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1336396\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e10.06\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFatty acids\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ecis-Vaccenic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e282.256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1150818\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e8.67\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFatty acids\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStearic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e36\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e284.272\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e796602\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFatty acids\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15-hydroxy-pentadecanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e258.219\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e45385\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFatty acids\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNorpatchoulenol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e190.172\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e82694\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTricyclic terpenoid\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-Palmitoylglycerol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e38\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e330.277\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e247888\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMonoacylglycerols\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eThe antibacterial activities of AFEE\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe antimicrobial activity of AFEE was evaluated using three different doses against various bacterial species, including \u003cem\u003eS. aureus, P. aeruginosa, B. subtilis\u003c/em\u003e, and \u003cem\u003eE. coli.\u003c/em\u003e The inhibition zone diameters were compared to erythromycin as a positive control. AFEE significantly inhibited the growth of the gram-positive species \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eB. subtilis\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). For \u003cem\u003eE. coli\u003c/em\u003e, only the highest dose of AFEE (40 \u0026micro;g/ml) was effective (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Conversely, \u003cem\u003eP. aeruginosa\u003c/em\u003e displayed the greatest resistance (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAntibacterial effects of AFEE (diameter of zone of inhibition (mm)).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDMSO\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCephalexin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10 \u0026micro;g/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e20 \u0026micro;g/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e40 \u0026micro;g/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMIC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;40 \u0026micro;g/ml\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedian (Min-Max)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003cp\u003e(18\u0026ndash;18)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12\u003c/p\u003e\u003cp\u003e(11\u0026ndash;12)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e% Inhibition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.45%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e13.26%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e15 \u0026micro;g/ml\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedian (Min-Max)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26\u003c/p\u003e\u003cp\u003e(25-26.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003cp\u003e(9\u0026ndash;10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e13\u003c/p\u003e\u003cp\u003e(13-13.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e% Inhibition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e29.36%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10.98%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e14.96%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;40 \u0026micro;g/ml\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedian (Min-Max)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003cp\u003e(19\u0026ndash;20)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e% Inhibition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e22.35%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e15 \u0026micro;g/ml\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedian (Min-Max)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27\u003c/p\u003e\u003cp\u003e(26-27.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13\u003c/p\u003e\u003cp\u003e(13-13.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15\u003c/p\u003e\u003cp\u003e(14-15.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e% Inhibition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30.49%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14.96%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e16.86%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e- value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e*Significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. MIC: Minimum Inhibition Concentration.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eAFEE is an antioxidant agent\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe Scavenging activity assay results revealed that showed significant free-radical scavenging of almost 38% by 12 \u0026micro;g/ml of AFEE and reached more than 48% at 100 \u0026micro;g/ml, in comparison to that of ascorbic acid (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eAFEE increased the cytotoxicity of A594 cells\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe MTT assay showed potential cytotoxicity activity that affected the growth and proliferation of A594 cells. The lowest significant inhibitory concentration was 12.5 \u0026micro;g/ml, which induced about 35% inhibition, whereas about 73% inhibition was obtained by 200 \u0026micro;g/ml (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The calculated IC\u003csub\u003e50\u003c/sub\u003e was 14.0.1 \u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eAFEE-triggered apoptosis of A594 cells\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, AFEE increased the cellular apoptosis of A594 cells at 20 \u0026micro;g/ml to 33.27% compared to the control (1.44%). Most cells (22.85%) underwent early apoptosis at a 20 \u0026micro;g/ml dose, while only 10.42% were processed to late apoptosis or complete cell death. At the highest concentration of 40 \u0026micro;g/ml, it was noticed that the total apoptotic effect seemed to decrease; however, as noticed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the number of dots was lower than those in the control, 10 \u0026micro;g/ml, and 20 \u0026micro;g/ml. This is because most cells shrieked and shifted to the debris area near the zero point, located outside the gated area of the FSC and SSC dot blot.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eAFEE induced G2/M phase arrest and increased cellular death\u003c/em\u003e\u003c/p\u003e\u003cp\u003eIn comparison to the untreated control cells, it was noticed that the G2/M phase levels increased significantly from 19.18\u0026ndash;27.97% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), 31.17% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and 36.77% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the cells treated with 10, 20, and 40 \u0026micro;g/ml of AFEE, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). On the other hand, a dramatic decrease in the G0/G1 phase levels was observed. Furthermore, the number of cells arrested at the Sub G stage increased significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), which indicated increased cellular death.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eEffect of AFEE on the cellular proteins integrated in the apoptotic and cell cycle pathways\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo confirm the apoptotic effect of AFEE in A594 cells, the expression of the apoptotic suppressor (Bcl-2) and apoptotic activator (caspase 3) was in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Bcl-2 levels decreased by 56.14%, while the expression levels of Caspase 3 increased by 66.27% at the dose of 40 \u0026micro;g/ml, compared to the control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe levels of Cyclin B increased up to 74.35% at the highest dose of AFEE, compared to the control, which might explain how AFEE impairs Cyclin B, causes its accumulation, and induces a cellular arrest at the G2/M phase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe current study investigated the phytochemical properties of \u003cem\u003eA. fragrantissima\u003c/em\u003e by FTIR and GC/MS analysis to evaluate the common functional groups and phenolic constituents, respectively. The FTIR spectrum confirmed that AFEE was rich in five groups of amine salts (N-H bending, N-H stretching, and C-N stretching), a strong peak of aldehyde (C\u0026thinsp;=\u0026thinsp;O stretching), and a strong peak of carboxylic/alcoholic group (O-stretching) at 3276 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, but no alkenes. Several studies on different plants found comparable chemical makeup of an \u003cem\u003eAsteraceae\u003c/em\u003e family member. FTIR examination confirmed the presence of alkyl halides, alkanes, alkenes, aldehydes, and amide groups in recent research on the aqueous extract of the leaves of \u003cem\u003eMatricaria chamonbmilla\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Another study confirmed the existence of aromatic compounds containing hydroxyl and carbonyl groups by FTIR examination of \u003cem\u003eCentaurea cyanus\u003c/em\u003e flower extract with additional acetyl, C\u0026thinsp;=\u0026thinsp;C, and C\u0026thinsp;=\u0026thinsp;O functional groups [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In another investigation, FTIR spectroscopy was used to determine the existence of the C-H stretch, C\u0026thinsp;=\u0026thinsp;O stretch, O-H stretch, and C-O stretch functional groups in the essential oils of \u003cem\u003eArnica montana, Echinacea purpurea, Calendula officinalis, Tagetes erecta\u003c/em\u003e, and \u003cem\u003eChamomilla recutita\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe chemical composition of AFEE was phytochemically examined by GC/MS analysis. The GC/MS analysis revealed the presence of multiple phenolic biomolecules, including phenolics, fatty acids, ketones, terpenoids, and antioxidants. The GC/MS results showed that the most represented compounds in the \u003cem\u003eA. fragrantissima\u003c/em\u003e extract were cis-3-Hexenyl caproate (23.82%), E-1-Methoxy-4-hexene (11.67%), Ethyl (E)-3,4,4-trimethylpent-2-enoate (11.65%), Palmitic acid (10.06%), cis-Vaccenic acid (8.67%), 1-methylcyclohex-2-en-1-ol (8.34%), and Octadecanoic acid (6%). Similar findings were reported by Qader \u003cem\u003eet al.\u003c/em\u003e (2018), in which the phytochemical constituents of the essential oils of \u003cem\u003eA. fragrantissima\u003c/em\u003e leaves were reported as m-cymene (0.8%) and α-Thujone (1.15%) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the study conducted by Alsohaili and Al-Fawwaz (2014), the GC/MS analysis of the \u003cem\u003eA. frangantissima\u003c/em\u003e essential oil revealed that the major constituents were Artemisia ketone (20%), α-Thujone (12%), Carvacrol (13%), p-Cymene (2%), and β-Sesquiphellandrene (15%), which is quite similar to the current findings [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Another study showed that the major fractions of the essential oil of \u003cem\u003eA. fragrantissima\u003c/em\u003e were (1S, 4S, 5R)-1-isopropyl-4-methylbicyclo [3.1.0] hexan-3-one (cis-thujone) and 3, 3, 6-trimethyl-1, 5-heptadien-4-one (artemisia ketone), whereas for \u003cem\u003eAchillea biebersteinii\u003c/em\u003e, cis-ascaridole and P-cymene were the most represented \u003cb\u003e[20].\u003c/b\u003e Furthermore, for the oil of \u003cem\u003eAchillea santolina\u003c/em\u003e, fragranyl acetate and 1,6-dimethyl-1,5-cyclooctadiene represented the highest concentrations, while chamazulene and b-pinene were more common in \u003cem\u003eAchillea millefolium\u003c/em\u003e \u003cb\u003e[20].\u003c/b\u003e A previous study analyzing the constituents of the essential oil of different members of the \u003cem\u003eAsteraceae\u003c/em\u003e family reported that ascaridole (43.22%), iso-ascaridole (37.17%), and 1,8-cineole (45.2%) were detected in \u003cem\u003eA. biebersteinii\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e21\u003c/span\u003e], chamazulene (52.60%) in \u003cem\u003eAchillea kellalensis\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], chrysanthenone (38.8%), trans-carveol (27.5%), neoiso-dihydrocarveol acetate (25.2%), filifolone (19.7%), apinene (11.8%), trans-piperitol (11.7%), (E)-caryophyllene (11.2%), (E)-nerolidol (10.8%), and lavandulyl acetate (26.19%) in \u003cem\u003eAchillea wilhelmsii\u003c/em\u003e \u003cb\u003e[23].\u003c/b\u003e All these reports and studies confirm the current findings about the robust phytochemical composition of different extracts of \u003cem\u003eA. fragrantissima.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAFEE showed substantial antioxidant activity, with 38% activity at 12 \u0026micro;g/ml and over 48% at 100 \u0026micro;g/ml, comparable to ascorbic acid. These findings are consistent with earlier research on other \u003cem\u003eAchillea\u003c/em\u003e species, emphasizing their potent antioxidant effects due to high phenolic and flavonoid content. For example, a study revealed that \u003cem\u003eAchillea atrata\u003c/em\u003e L. and \u003cem\u003eAchillea millefolium\u003c/em\u003e L. are rich in luteolin, apigenin, centaureidin, and nevadensin, which are known as their effective 2,2-diphenyl-picryl hydrazyl radical scavengers [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This shows that different species in the \u003cem\u003eAchillea\u003c/em\u003e genus have a similar bioactive profile, which contributes to their strong antioxidant effects. Comparable research from Saudi Arabia highlights the high antioxidant activity of natural medicinal herbs. Research of essential oils extracted from \u003cem\u003eA. fragrantissima\u003c/em\u003e grown in Saudi Arabia and Egypt discovered substantial radical-scavenging activity, with IC\u003csub\u003e50\u003c/sub\u003e values of 30.94 and 28.72 mg/L, respectively [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This highlights the importance of \u003cem\u003eA. fragrantissima\u003c/em\u003e as a prospective natural source of antioxidants. In the study conducted by Elsharkawy \u003cem\u003eet al.\u003c/em\u003e (2020), the antioxidant activity of \u003cem\u003eA. fragmentisma\u003c/em\u003e growing in different regions of Saudi Arabia significantly differed, where those from the Arar and Tabuk regions exhibited their scavenging activities at IC\u003csub\u003e50\u003c/sub\u003es of 0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 and 1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mg/ml, respectively [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These commonalities show that plants in this family share bioactive chemicals that provide variable antioxidant effects, which might be affected by weather parameters, and that supports their usage in traditional medicine. The observed antioxidant potential of AFEE can be attributable to its high phenolic and flavonoid content, as corroborated by prior phytochemical research on \u003cem\u003eA. fragrantissima\u003c/em\u003e. The inclusion of volatile oils, fatty acids, and aromatic components boosts its free radical scavenging activity. Given the growing interest in plant-based antioxidants for pharmaceutical and nutraceutical uses, \u003cem\u003eA. fragrantissima's\u003c/em\u003e high activity supports its promise as a natural alternative to synthetic antioxidants.\u003c/p\u003e\u003cp\u003eIn the current study, three doses of AFEE were used to assess their antibacterial activities on the growth of different species (\u003cem\u003eS. aureus, P. aeruginosa, B. subtilis\u003c/em\u003e, and \u003cem\u003eE. coli\u003c/em\u003e). The inhibition zone diameter was measured and compared to the positive control of the antibiotic (Cephalaxin). A significant inhibition of the two gram-positive species (\u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eB. subtilis\u003c/em\u003e). Furthermore, the growth of \u003cem\u003eE. coli\u003c/em\u003e was affected only by the highest dose of AFEE (40 mg/ml). \u003cem\u003eP. aeruginosa\u003c/em\u003e was the most resistant species to the bactericidal effects of AFEE. Following these findings, a recent study found that \u003cem\u003eA. fragrantissima\u003c/em\u003e essential oil has antibacterial action against \u003cem\u003eBacillus cereus, E. coli, S. aureus, Aspergillus niger, Penicillium\u003c/em\u003e sp., and \u003cem\u003eRhizopus\u003c/em\u003e sp. in tomato media [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. A previous study discovered that aqueous and hydroethanolic extracts of \u003cem\u003eA. fragrantissima\u003c/em\u003e aerial parts were ineffective against \u003cem\u003eE. coli, MRSA, Streptococcus pneumoniae, Enterococcus faecalis, Shigella sonnei, P. aeruginosa, Candida albicans, Candida glabrata\u003c/em\u003e, and \u003cem\u003eSalmonella typhimurium\u003c/em\u003e, while \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e, \u003cem\u003eBacillus cereus\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e were more sensitive [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Other species in the \u003cem\u003eAsteraceae\u003c/em\u003e family demonstrated antibacterial activity. The antimicrobial activity of the essential oils of the \u003cem\u003eAchillea biebersteinii\u003c/em\u003e Afan. from Turkey was effective against \u003cem\u003eSalmonella enterica serovar typhimirium, S. saureus subsp., Yersinia pseudotuberculosis, Bacillus cereus, Enterobacter aerogenes, B. subtilis\u003c/em\u003e and \u003cem\u003eProteus vulgaris\u003c/em\u003e \u003cb\u003e[27].\u003c/b\u003e The gram-positive bacteria \u003cem\u003eB. subtilis, B. cereus, S. aureus\u003c/em\u003e, and \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e were more susceptible to the essential oil of \u003cem\u003eA. millefolium\u003c/em\u003e than the gram-negative bacteria S. \u003cem\u003etyphimurium, Salmonella enteritidis, Salmonella agona\u003c/em\u003e, and \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. \u003cem\u003eA. fragrantissima\u003c/em\u003e extracts' antimicrobial action could be attributed to their diverse chemical compositions of fatty acids, terpenoids, and ketones, besides, the sesquiterpene of β-Caryophyllene, which showed antibacterial, antifungal, and antioxidant effects against many gram-positive bacteria [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Palmitic and octadecanoic acids (stearic acid), which account for 10% and 6% of the current GC/MS study, respectively, have been shown to exhibit strong antibacterial effects against \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Furthermore, caproic acid has a strong antibacterial impact against \u003cem\u003eCampylobacter jejuni\u003c/em\u003e and \u003cem\u003eCampylobacter coli\u003c/em\u003e \u003cb\u003e[31].\u003c/b\u003e All these studies, in addition to the current findings, suggest the susceptibility of \u003cem\u003eA. fragrantissima\u003c/em\u003e as a possible antibacterial agent.\u003c/p\u003e\u003cp\u003eAFEE inhibited A594 cell proliferation in a dose-dependent manner, with an IC\u003csub\u003e50\u003c/sub\u003e of 14.01 \u0026micro;g/mL. These findings are consistent with prior research on \u003cem\u003eA. fragrantissima\u003c/em\u003e and other \u003cem\u003eAchillea\u003c/em\u003e species, which have shown strong anticancer activity because of their high bioactive content. Previous studies reported the cytotoxic effect of \u003cem\u003eA. fragrantissima\u003c/em\u003e against breast cancer cells of MCF-7 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], pancreatic cancer cells of MiaPaCa-2, prostate cancer cells of PC-3, and lung cancer cells of A594 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Furthermore, the essential oil extract of \u003cem\u003eA. fragrantissima\u003c/em\u003e demonstrated cytotoxic effects on breast (MCF7) and colorectal cancer (HCT-116) cells, with IC\u003csub\u003e50\u003c/sub\u003e values ranging from 0.51\u0026ndash;0.91 \u0026micro;g/ml [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Similarly, \u003cem\u003eAchillea membranacea\u003c/em\u003e extracts revealed high cytotoxic action against A2780 (ovarian cancer) and HT29 (colon cancer) cells with an IC\u003csub\u003e50\u003c/sub\u003e of around 12.99 and 14.02 \u0026micro;g/ml, respectively [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These findings indicate that distinct \u003cem\u003eAchillea\u003c/em\u003e species share bioactive chemicals responsible for their anticancer properties, such as flavonoids, sesquiterpenes, and phenolics. Comparable research from Saudi Arabia supports the medicinal herbs' high cytotoxic action against cancer cells. The observed cytotoxicity in A594 cells could be ascribed to the high concentration of flavonoids, terpenoids, and phenolic chemicals in AFEE, which have previously been shown to have anticancer effects. These findings indicate that Saudi Arabian medicinal plants, particularly those in the Asteraceae family, have potent anticancer characteristics that could be further investigated for therapeutic purposes.\u003c/p\u003e\u003cp\u003eAFEE's potential to induce apoptosis was validated by flow cytometry. Treatment with 20 \u0026micro;g/ml increased overall apoptosis to 33.27% compared to 1.44% in the control. Early apoptosis made up 22.85% of the total, with 10.42% proceeding to late apoptosis or full cell death. The highest dose of 40 \u0026micro;g/ml reduced total apoptotic % due to cell debris outside the gated area of the dot plot. This behavior is frequently seen in cells experiencing late-stage apoptosis or necrosis, in which cells shrink, lose integrity, and form apoptotic bodies \u003cb\u003e[34].\u003c/b\u003e To confirm the processes driving AFEE-induced apoptosis and cell cycle arrest, protein expression analysis was performed. AFEE treatment decreased Bcl-2 expression by 56.14% at 40 \u0026micro;g/ml compared to the control, indicating a dose-dependent effect. In contrast, caspase-3 expression increased by 66.27%, indicating apoptosis. Similar results were found for the aqueous extract of \u003cem\u003eA. fragrantissima\u003c/em\u003e against breast cancer (MCF7) and HepG2 cells by targeting apoptotic network indicators such as Caspase-3, Caspase-8, Caspase-9, Bcl-2, BAX, and Bcl-xl [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. On the other hand, other species from the \u003cem\u003eAsteraceae\u003c/em\u003e family showed similar behavior. The aqueous extract of \u003cem\u003eAchillea thracica\u003c/em\u003e seeds induced the cytotoxicity of HT1080 fibrosarcoma cells at an IC\u003csub\u003e50\u003c/sub\u003e of 9.85 g/l \u003cb\u003e[36].\u003c/b\u003e The hydroethanolic extract of \u003cem\u003eAchillea millefolium\u003c/em\u003e induced apoptosis in NCI-H460 (lung cancer) and HCT-15 (colorectal cancer), caused an increase in p53 and p21, and reduced XIAP expression levels [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The essential oils of \u003cem\u003eAchillea millefolium\u003c/em\u003e revealed similar findings on the Panc-1 and MCF-7 cancer cell lines [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Extracts from other species, including \u003cem\u003eAchillea membranacea\u003c/em\u003e and \u003cem\u003eAchillea Wilhelmsii C. Koch\u003c/em\u003e, were also apoptosis inducers against A2780 (ovarian cancer) and HeLa (cervical cancer) cells by altering the LIN28B and p53 genes expression \u003cb\u003e[12, 34].\u003c/b\u003e These findings demonstrate that AFEE exerts its anticancer effects by regulating critical apoptotic regulatory proteins, highlighting its potential as a natural cancer treatment.\u003c/p\u003e\u003cp\u003eCell cycle studies demonstrated that AFEE administration caused considerable G2/M phase arrest in A594 cells. Cells in the G2/M phase rose from 19.18% in untreated control cells to 27.97%, 31.17%, and 36.77% at 10, 20, and 40 \u0026micro;g/ml, respectively. This increase was followed by a significant decrease in the G0/G1 phase population and an increase in sub-G, indicating a high amount of cellular death. Cyclin B levels increased by 74.35% at 40 \u0026micro;g/ml, indicating that AFEE impairs mitotic progression and causes G2/M phase arrest. In comparable studies, \u003cem\u003eA. fragrantissima\u003c/em\u003e caused cycle arrest through G2-phase cell cycle arrest, which caused the elongation of the human chronic myeloid leukemia (K562) cells \u003cb\u003e[31].\u003c/b\u003e Also, the 48-hour exposure to the secondary metabolite, rupicoline from \u003cem\u003eAchillea grandifolia\u003c/em\u003e, induced a G2/M cell cycle arrest of U87MG and T98G Glioblastoma cells [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The observed G2/M arrest indicates that AFEE may interfere with cell division by targeting critical mitotic progression regulators, resulting in reduced cancer cell viability.\u003c/p\u003e\u003cp\u003eThis study offers a complete phytochemical characterization of \u003cem\u003eA. fragrantissima\u003c/em\u003e, revealing its rich composition of phenolics, fatty acids, ketones, and terpenoids. It focuses on the plant's powerful antioxidant, antibacterial, and anticancer capabilities, as well as its substantial cytotoxic effects on A594 lung cancer cells. Unlike prior studies that focused on \u003cem\u003eA. fragrantissima\u003c/em\u003e from other countries, this study looks particularly at specimens produced in Saudi Arabia, providing region-specific insights into its bioactive potential. The findings show that \u003cem\u003eA. fragrantissima\u003c/em\u003e from Saudi Arabia has high antioxidant and antibacterial activity, implying that the country's particular climatic conditions may influence its chemical composition. These findings lend credence to the plant's prospective pharmacological and nutraceutical applications, notably as a natural alternative to synthetic antioxidants and antibacterial agents. The chemical composition, antioxidant, antibacterial, and anticancer properties of the ethanolic extract from the aerial parts of \u003cem\u003eAchillea fragrantissima\u003c/em\u003e have important implications for disability research, particularly in the treatment of chronic illnesses, infections, and cancer-related disabilities. Chronic illnesses, including diabetes, arthritis, and cancer, are frequently connected with oxidative stress, which can worsen impairment by destroying cells and tissues. The study emphasizes AFEE's powerful antioxidant action, which may help buffer oxidative stress and improve the quality of life for those with disabilities caused by these illnesses. Furthermore, infections, particularly those produced by antibiotic-resistant bacteria, can result in serious consequences and disability. The study reveals that AFEE has substantial antibacterial action against both gram-positive and gram-negative bacteria, including S. aureus and E. coli. This shows that AFEE may be a natural alternative to infection control, lowering the likelihood of disability caused by severe or recurring infections. On the other hand, cancer and its therapies, such as chemotherapy and radiation, frequently cause physical disabilities owing to tissue damage, discomfort, and functional impairments. The study found that AFEE causes apoptosis and cell cycle arrest in A594 cells, showing its potential as a natural anticancer agent. By targeting cancer cells, AFEE may lessen the need for chemotherapy treatments, which frequently result in impairment, thereby improving patient outcomes.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe findings emphasized \u003cem\u003eA. fragrantissima\u003c/em\u003e's potential antibacterial efficacy against harmful bacteria, hinting that its active metabolites may be a hidden source of powerful bioactive compounds. This work successfully characterized the complex phytochemical composition of \u003cem\u003eA. fragrantissima\u003c/em\u003e from Saudi Arabia, indicating a high phenolic content and powerful bioactive properties. The plant extract exhibits substantial antioxidative, antibacterial, and anticancer activities, indicating its potential utility in both traditional and modern medicine. Its strong cytotoxic activity and induction of apoptosis in lung cancer cells highlight its potential as a natural anticancer medication, prompting further investigation into its therapeutic applications. Given these findings, \u003cem\u003eA. fragrantissima\u003c/em\u003e from Saudi Arabia could be researched further for pharmaceutical development, particularly for natural antibacterial and anticancer formulations. Future research should analyze the phytochemical content of different regions and conduct in vivo studies to assess the clinical potential.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003ePlant Material and Extract Preparation\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eA. fragrantissima\u003c/em\u003e aerial parts were obtained in March 2023 from the Rafha region of the Kingdom of Saudi Arabia, located at 29\u0026deg;38\u0026prime;19\u0026Prime;N 43\u0026deg;30\u0026prime;5\u0026Prime;E. The collection and handling of the plant material were according to the International Union for Conservation of Nature (IUCN) Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. The plant was deposited in the herbarium of the College of Science, King Saud University, under the name of \u0026ldquo;\u003cem\u003eAchillea fragrantissima\u003c/em\u003e shrub, Rafha, Riyadh\u0026rdquo;. Plant identification was performed by Prof. Najat Bukhari in the Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia.\u003c/p\u003e\u003cp\u003eThe plant was cleaned with running tap water first, then with sterile distilled water. The plant was then shade-dried, carefully powdered, and stored at 4\u0026deg;C until later usage. The infusion method was used to prepare the plant extract \u003cb\u003e[40].\u003c/b\u003e The fresh, dry plant components were crushed, and 30 g were soaked overnight in 300 ml of 100% ethyl alcohol in a closed container at room temperature, then transferred to another container overnight using a rotary shaker. The macerates were gravity-filtered twice, once through cotton and then through layers of tissue paper. Finally, the residual filtrates were dried overnight in cleaned metallic trays before being transferred to a clean, dry glass container and refrigerated until needed.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFourier transform infrared spectroscopy (FTIR)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe functional groups were found using FTIR analysis. A spectrometer (Nicolet 6700, Thermo Fisher Scientific Inc., Waltham, MA, USA) with a beam splitter and detector (DTGS) loaded with OMNIC software was used to gather and analyze spectra in the 500\u0026ndash;4000 cm1 scan range. The resulting IR spectra were utilized to analyze the functional groups found in the produced extract. The acquired wavelengths were evaluated by the Scientific Reaction Animation Image Builder (InstaNANO) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://instanano.com/all/characterization/ftir/ftir-functional-group-search/\u003c/span\u003e\u003cspan address=\"https://instanano.com/all/characterization/ftir/ftir-functional-group-search/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e to identify the relevant functional groups [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eGas Chromatography/Mass Spectrometry (GC/MS)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe ethanol extract underwent GC/MS analysis. The carrier gas was helium, with a flow rate of 1 ml per minute. The Agilent 5977c GC/MSD (Agilent Inc., Santa Clara, California, USA) was used. A phenyl methyl siloxane column (30m, 250m, 0.25m) was utilized. The following settings were maintained throughout the GC run: time (90 minutes), volume (1 L), temperature (280\u0026deg;C; 250\u0026deg;C), ion source, and split ratio (20:1). Furthermore, the temperature was held at 40\u0026deg;C for 5 minutes before increasing to 280\u0026deg;C at 10\u0026deg;C/min and remaining there for another 5 minutes. For mass spectroscopy, an electron impact (70 eV) was created with a scan range of 35 to 780 m/z. The National Institute of Standards and Technology (NIST) mass spectral library (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chemdata.nist.gov/dokuwiki/doku.php?id=chemdata:start\u003c/span\u003e\u003cspan address=\"https://chemdata.nist.gov/dokuwiki/doku.php?id=chemdata:start\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to identify the samples \u003cb\u003e[42].\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eScavenging activity assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe DPPH (2,2-diphenyl-1-picrylhydrazyl) assay is widely used to assess the antioxidant activity of plant extracts. This assay evaluates bioactive compounds' ability to scavenge free radicals promptly and consistently [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. A 0.1 mM DPPH solution in methanol with an initial absorbance of 0.9-1.0 at 517 nm was created. The plant extract or ascorbic acid, a common antioxidant, was produced in methanol at various quantities. Each sample was mixed with an equal volume of DPPH solution before being incubated for 30 minutes in the dark at room temperature. After incubation, the absorbance is measured at 517 nm using a UV-Vis spectrophotometer. The fraction of DPPH scavenging activity is calculated using the following formula:\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Scavenging\\:\\%=\\frac{Ac-As}{Ac}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eHere, \u0026ldquo;Ac\u0026rdquo; is the absorbance of DPPH solution alone without any samples, and \u0026ldquo;As\u0026rdquo; is the absorbance of DPPH mixed with either the plant extract or the ascorbic acid.\u003c/p\u003e\u003cp\u003e\u003cem\u003eAntimicrobial Activities\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe bacterial strains were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). These strains included \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (ATCC-25923), \u003cem\u003eBacillus subtilis\u003c/em\u003e (ATCC-6051), \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC-11755), and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (ATCC-27584). All bacterial strains were cultured on Mueller-Hinton agar medium.\u003c/p\u003e\u003cp\u003eTo achieve a concentration of 1 \u0026micro;g/ml, the previously produced AFEE powder was dissolved in a 1:100 DMSO: distilled water solution. Mueller-Hinton agar growth media was made according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA). Briefly, 19 grams of Mueller-Hinton agar were dissolved in 500 ml of deionized water before being sterilized in an automated autoclave at 121\u0026deg;C for 15 minutes. After cooling to 70\u0026ordm;C, each Petri dish received approximately 10 ml of the medium. At room temperature, the medium was allowed to harden. The bacterial strain was plotted on three Petri dishes. Three discs were created, with three concentrations of the extract (10, 20, and 40 \u0026micro;l) inserted in each disc. Another disk was made for the control by adding only 0.2 \u0026micro;l of DMSO. Another Petri dish had cefalexin as a positive control (5 \u0026micro;g/ml). The plates were placed in an incubator at 37\u0026deg;C for 24 hours. After incubation, the areas of growth inhibition were measured in millimeters [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. All experiments were conducted in duplicate. Minimum inhibitory concentrations (MIC) were determined using the following equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:MIC=\\frac{Lowest\\:Conc.\\:inhibit\\:the\\:growth+Highest\\:conc.\\:allow\\:the\\:growth\\:}{2}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCell culture\u003c/em\u003e\u003c/p\u003e\u003cp\u003eIn the current study, the lung cancer cell line A594 was used to study the anticancer properties of \u003cem\u003eA. fragrantissima\u003c/em\u003e ethanolic extract. A594 cell line was obtained from ATCC (Manassas, VA, USA). The cells were maintained in Dulbecco\u0026rsquo;s Modified Eagle's Medium (DMEM) supplemented with 1% Glutamax, 10% Fetal Bovine serum, and 1% 10000/1000 penicillin-streptomycin (Thermo Fisher Scientific Inc., GIBCO, Waltham, MA, USA). The cells were incubated in a 37\u0026ordm;C CO\u003csub\u003e2\u003c/sub\u003e incubator, and media were renewed every three days.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCytotoxicity assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eIn this assay, 5000 cells per well were seeded in a 96-well plate and adhered overnight in a humidified incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The following day, cells were treated with varied amounts of the plant extract, while untreated cells were the negative control. After 24 hours of incubation, add 10 \u0026micro;L of MTT solution (5 mg/ml in phosphate-buffered saline (PBS)) (Thermo Fisher Scientific Inc., Invitrogen, Waltham, MA, USA) to each well and incubate for 90 minutes. The formazan crystals will form a purple precipitate. The media was carefully removed, and the formazan crystals were solubilized with 100 \u0026micro;L of dimethyl sulfoxide (DMSO). The absorbance was measured at 540 nm with a microplate reader [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The IC50 was calculated by applying all the resulting optical densities and corresponding concentrations to the IC\u003csub\u003e50\u003c/sub\u003e Calculator of AAT Bioquest at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.aatbio.com/tools/ic50-calculator\u003c/span\u003e\u003cspan address=\"https://www.aatbio.com/tools/ic50-calculator\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eApoptosis/ Necrosis Assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe Annexin V/Propidium Iodide (PI) assay was used to identify and distinguish between apoptotic and necrotic cells based on phosphatidylserine externalization and membrane integrity \u003cb\u003e[45].\u003c/b\u003e Cells were planted into an appropriate culture plate and treated with the plant extract. After 24 hours of incubation, cells were extracted, including adherent and floating populations, and washed with cold PBS. The cells were resuspended in 100 \u0026micro;l of binding buffer at a concentration of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/ml. The FITC Annexin V Apoptosis Detection Kit with PI (Biolegend Co., San Diego, CA, USA) was used following the manufacturer's instructions. The mixture is incubated in the dark for 15 minutes at room temperature before being analyzed using a BD FACSCalibur\u0026trade; Flow Cytometer (BD Co., Franklin Lakes, NJ, USA).\u003c/p\u003e\u003cp\u003e\u003cem\u003eCell cycle assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe PI-based cell cycle test is a popular method for determining DNA content and cell distribution during the cell cycle. Cells were initially treated with the plant extract, then collected and fixed with cold 70% ethanol to retain their structure. After washing to remove any remaining ethanol, the cells were treated with a staining solution containing PI, RNase A to break down RNA, and a detergent to permeabilize the cell membrane. The samples were then analyzed using flow cytometry, with fluorescence intensity used to differentiate between cells in the G0/G1, S, and G2/M phases [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eWestern blotting\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo carry out the assay, treated cells were lysed in RIPA buffer (Santa Cruz Biotechnology Inc., Dallas, TX, USA) to extract total protein. The protein concentration was determined using the Bradford assay and a Nanodrop spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Equal amounts of protein were separated using SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk to prevent nonspecific binding. It was treated overnight with primary antibodies that targeted apoptosis (Bcl-2 and Caspase 3), cell cycle (Cyclin D1), and the housekeeping protein β-actin (Biolegend Co., San Diego, CA, USA). Following that, the membrane was incubated with suitable HRP-conjugated secondary antibodies and visualized with a c-digit scanner after staining with Luminol and oxidizing solutions (Thermo Fisher Scientific Inc., Waltham, MA, USA). The band intensities were measured by ImageJ densitometry software version 1.8.0_345 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.net/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003cb\u003e[47].\u003c/b\u003e\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eThe data was statistically evaluated with SPSS version 22. The experiments were subjected to a one-way analysis of variance (ANOVA), and mean differences were analyzed using the Duncan test. At the 0.05 probability level, the mean values were separated using the least significant difference (LSD). All values in this study represent the average of at least three replicates. The data was reported as the mean with standard deviation (SD).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe authors extend their appreciation to the King Salman Center for Disability Research for funding this work through Research Group no KSRG-2024-366.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, F.A. and M.S.E.; methodology, N.A.A. and M.S.E.; software, M.T.Y.; validation, F.A., M.T.Y., and T.A.; formal analysis, M.T.Y. and M.S.E.; investigation, B.K.A. and N.A.A.; resources, F.A. and N.A.A.; data curation, T.A. and M.S.E.; writing—original draft preparation, M.S.E., M.T.Y., and N.A.A.; writing—review and editing, F.A.; visualization, M.S.E., and M.T.Y.; supervision, F.A. and T.A.; project administration, F.A.; funding acquisition, F.A. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research project was supported by a grant from the King Salman Center for Disability Research through Research Group no KSRG-2024-366\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThis research does not require an ethical statement/clinical trial registration number, or informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe raw data supporting the conclusions of this article will be made available by the corresponding authors on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eElkordy, A. et al. Floristic diversity of Jabal Al-Ward, southwest Tabuk region, Kingdom of Saudi Arabia. \u003cem\u003eAgronomy\u003c/em\u003e 12, 2626. (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/agronomy12112626\u003c/span\u003e\u003cspan address=\"10.3390/agronomy12112626\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMiraj, S. \u0026amp; Rafieian-Kopaei, Kiani, S. \u003cem\u003eMelissa officinalis\u003c/em\u003e L: A review study with an antioxidant prospective. \u003cem\u003eEvidence-Based Complement. Altern. 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Biology\u003c/em\u003e. \u003cb\u003e16\u003c/b\u003e, 387\u0026ndash;401. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21608/eajbsc.2024.355267\u003c/span\u003e\u003cspan address=\"10.21608/eajbsc.2024.355267\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Achillea fragrantissima, FTIR, GC/MS, antibacterial properties, anticancer agent","lastPublishedDoi":"10.21203/rs.3.rs-6874992/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6874992/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eAchillea fragrantissima\u003c/em\u003e is a known medicinal plant in traditional Saudi culture. The current study aimed to evaluate the chemical composition of \u003cem\u003eA. fragrantissima\u003c/em\u003e and its biological activity as a possible antioxidant, antibacterial, and anticancer agent. The phenolic content of \u003cem\u003eA. fragrantissima\u003c/em\u003e was studied using Fourier transform infrared spectroscopy (FTIR) and gas chromatography/mass spectrometry (GC/MS). The antibacterial activity was investigated using the well-diffusion method. The anticancer was investigated against A594 lung cancer cells by MTT, annexin V/Propidium iodide assay, Cell cycle analysis, and Western blotting. The chemical analysis showed that \u003cem\u003eA. fragrantissima\u003c/em\u003e includes volatile oils, fatty acids, and other aromatic chemicals. \u003cem\u003eA. fragrantissima\u003c/em\u003e ethanolic extract (AFEE) had a 38% scavenging of free radicals at 12 \u0026micro;g/ml and 48% at 100 \u0026micro;g/ml (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Using a 40% dose, the antibacterial test revealed substantial efficacy against all species, particularly \u003cem\u003eS. aureus\u003c/em\u003e. The cytotoxic effect showed dose-dependent growth inhibition of A594 cells with an IC\u003csub\u003e50\u003c/sub\u003e of 14.01 \u0026micro;g/ml. At 20 \u0026micro;g/ml, AFEE caused 33.27% of cellular apoptosis. At 40 \u0026micro;g/ml, G2/M phase arrest increased to 36.77% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Mechanistically, AFEE reduced the anti-apoptotic protein Bcl-2 by 56.14% and increased caspase-3 by 66.27% while boosting Cyclin B levels by 74.35%. These findings emphasize AFEE's potential as an antioxidant, antibacterial, and anticancer agent.\u003c/p\u003e","manuscriptTitle":"Demonstrating the antioxidant, antibacterial, and anticancer properties of the Achillea fragrantissima shrub growing in Riyadh, Saudi Arabia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 13:00:53","doi":"10.21203/rs.3.rs-6874992/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"57319129-9873-4403-9807-3bf727072b6c","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52180846,"name":"Biological sciences/Cancer"},{"id":52180847,"name":"Biological sciences/Microbiology"},{"id":52180848,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-09-30T20:53:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-28 13:00:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6874992","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6874992","identity":"rs-6874992","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}