In Vitro and In Silico Anticoccidial Activity of Opuntia ficus-indica Seed Oil and Hexane Extract: Comprehensive GC-MS Profiling and Mechanistic Insights

In Vitro and In Silico Anticoccidial Activity of Opuntia ficus-indica Seed Oil and Hexane Extract: Comprehensive GC-MS Profiling and Mechanistic Insights | 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 Research Article In Vitro and In Silico Anticoccidial Activity of Opuntia ficus-indica Seed Oil and Hexane Extract: Comprehensive GC-MS Profiling and Mechanistic Insights Dalia Kellil, Rabah Arhab, Asma Kheddouma, Hamza Ahmed-Laloui, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7156619/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Oct, 2025 Read the published version in Acta Parasitologica → Version 1 posted 12 You are reading this latest preprint version Abstract The anticoccidial potential of Opuntia ficus-indica seed oil (OFI-SO) and its hexane extract from seed press cake (OFI-HexPC) was evaluated against Eimeria spp . using integrated in vitro and in silico approaches. Sporulated Eimeria oocysts were exposed to increasing concentrations (2–64 mg/mL) of OFI-SO and OFI-HexPC in Hank’s balanced salt solution. Oocyst viability was determined by microscopic counting, while membrane integrity was assessed by quantifying the release of 273 nm-absorbing substances. Toltrazuril (25 mg/mL) was used as a positive control. GC-MS profiling of both extracts revealed linoleic acid (54.2%) and oleic acid (25.7%) as the main bioactive fatty acids. GC–MS profiling revealed that linoleic acid (69.63%) was the major fatty acid in OFI-SO, while oleic acid (44.20%) predominated in OFI-HexPC. Both OFI-SO and OFI-HexPC significantly reduced oocyst counts and induced a dose-dependent increase in DNA release. In silico molecular docking was performed against three key Eimeria enzymes (EtDHODH, CDPK, and PKA). Identified fatty acids showed moderate binding affinities through hydrophobic interactions. Redocking validation (RMSD < 2 Å) confirmed the reliability of the docking protocol. ADMET predictions indicated favorable pharmacokinetics and safety profiles. These findings suggest that OFI-SO and OFI-HexPC act via a dual mechanism involving membrane disruption and enzymatic inhibition, supporting their potential as natural alternatives to synthetic anticoccidials in poultry production. Eimeria spp Anticoccidial activity Phytogenic compounds Opuntia ficus-indica in vitro GC-MS Molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Avian coccidiosis, an enteric parasitic disease caused by protozoa of the genus Eimeria , remains a major health and economic challenge in modern poultry production (Shirley et al., 2007 ; Chapman, 2014 ). Prevalent particularly in intensive farming systems, this parasitosis significantly compromises poultry growth, welfare, and productivity (Johnson, 1923 ; Taylor et al., 2022 ). Global economic losses associated with coccidiosis are estimated at approximately £10.4 billion annually (Blake et al., 2020 ), with recent assessments reporting losses of around £86.7 million per year in Algeria alone (Rahmani et al., 2024 ), underscoring its impact on national food security and farm profitability. Chemoprophylaxis using synthetic anticoccidials remains the primary strategy for disease control in poultry (Taylor et al., 2022 ). However, the continuous use of these compounds has led to the emergence of drug-resistant Eimeria strains and raised serious concerns regarding drug residues in poultry products and potential risks to human health (Abbas et al., 2011 ; Chapman et al., 2013 ; Noack et al., 2019 ). These limitations have intensified the search for effective, safe, and sustainable alternatives (Abbas et al., 2012 ). Phytogenic compounds derived from medicinal plants have gained considerable attention due to their diverse biological activities. Plant-based additives are known for their antimicrobial, antioxidant (Naidoo et al., 2008 ), antiviral (Bishop, 1995 ), antiparasitic, immunostimulant, and gut-modulating effects (Youn and Noh, 2001 ). Advances in phytochemical extraction and characterization techniques have reinforced the therapeutic value of many traditional medicinal plants (Gacem et al., 2020 ; Mahrose, 2021 ), supporting their use as alternatives to conventional antimicrobials in poultry production (Guèye, 1999 ). Opuntia ficus-indica , a drought-tolerant species widespread in arid and semi-arid regions (Giraldo-Silva et al., 2023 ), is a rich source of lipophilic bioactive compounds, including fatty acids, sterols, and tocopherols. Recent studies suggest its potential antiparasitic effects, including anticoccidial properties (Amrane-Abider et al., 2023a , b ). The present study aims to investigate the anticoccidial potential of OFI-SO and OFI-HexPC against Eimeria spp . An integrated approach combining in vitro evaluation of oocyst integrity and in silico molecular docking of identified fatty acids targeting key Eimeria enzymes was adopted. This dual strategy aims to explore the therapeutic relevance of these natural products and support their development as promising alternatives to conventional anticoccidials in poultry health management. Material and Method Plant Material and Extraction of Non-Polar Compounds Opuntia ficus-indica seed oil (OFI-SO) and the corresponding residual seed press cake were kindly provided by the NOPALTEC cooperative (Sidi-Fredj, Souk Ahras, Algeria), a local enterprise specialized in the processing of prickly pear-derived products. The plant material was harvested during the autumn season, corresponding to the full fruit maturity stage. Botanical identification was performed by the supplier’s technical staff, including a certified botanist. Product quality and traceability were verified by their internal R&D department. The oil was extracted by cold pressing of mature prickly pear seeds. The resulting seed press cake was air-dried, finely ground, sieved through a 1 mm mesh, and stored in amber glass bottles at room temperature until use. To isolate fatty acids and other non-polar constituents, OFI-HexPC was obtained by Soxhlet extraction using n-hexane at 100°C for 6 hours, following the protocol described by Kadda et al. (Kadda et al., 2023 ). The obtained extract was concentrated under reduced pressure using a Büchi rotary evaporator (Büchi, Switzerland), and subsequently stored at 4°C prior to analysis. Oocyst Collection and Identification Oocysts of Eimeria spp . were isolated from fresh fecal samples collected from naturally infected broiler chickens in the Baghai region (Khenchela Province, eastern Algeria), following the procedure described by Carvalho et al. (Carvalho et al., 2011 , 2025 ). The samples were processed for sporulation in a 2.5% potassium dichromate (K₂Cr₂O₇) solution under optimal conditions of aeration, temperature, and humidity. The concentration of sporulated oocysts (oocysts/mL) was determined using a Malassez counting chamber (Rahmani et al., 2021 ). For morphological identification, 100 sporulated oocysts were randomly selected and examined under a Leica microscope equipped with a digital camera. Image analysis was conducted using Motic Images Plus 2.0 software. Identification was based on morphometric parameters and compared with classical descriptions provided by Conway and McKenzie (Conway and McKenzie, 2007 ). Esterification and GC-MS Analysis Fatty acids from OFI-SO and OFI-HexPC were converted into fatty acid methyl esters (FAMEs) prior to gas chromatography–mass spectrometry (GC-MS) analysis. Briefly, 0.5 g of each sample was mixed with 5 mL of n-hexane, followed by the addition of 0.5 mL of 2 N KOH in methanol. After vortexing for 30 seconds, the mixture was centrifuged at 3000 rpm for 10 minutes. Then, 100 µL of the supernatant was collected and diluted with 1 mL of n-hexane. The resulting FAMEs were stored at − 20°C until further analysis. GC-MS analysis was performed using an Agilent 6890 Plus gas chromatograph coupled to an Agilent 5973 mass selective detector. The system was equipped with a splitless injector and an HP-5MS capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness; 5% phenyl–95% dimethylpolysiloxane). High-purity helium (N6) served as the carrier gas at a constant flow rate of 0.5 mL/min. The injector temperature was set to 250°C. The oven temperature was programmed as follows: initial temperature of 70°C (held for 5 min), ramped at 10°C/min to 130°C (held for 2 min), followed by 3°C/min to 220°C (held for 4 min), and finally ramped at 10°C/min to 280°C (held for 5 min). The mass spectrometer operated in electron impact ionization mode with a quadrupole analyzer. Compound identification was carried out by comparing retention times and mass spectra to entries in the NIST and PubChem spectral libraries. Anticoccidial activity The oocysticidal activity of OFI-SO and OFI-HexPC was evaluated in vitro according to the method described by Remmal et al (Remmal et al., 2011 ). Each extract was prepared in Hank’s Balanced Salt Solution (HBSS) supplemented with 0.2% agar to ensure homogeneous oocyst dispersion and tested at six increasing concentrations (2 to 64 mg/mL). HBSS alone served as the negative control. Each 1 mL reaction mixture contained 100 µL of purified Eimeria spp . oocyst suspension (1.34×10⁵ oocysts/mL for OFI-HexPC; 2.2×10⁵ oocysts/mL for OFI-SO), 700 µL of phosphate-buffered saline (PBS), and 200 µL of the test extract at the corresponding concentration. After 24 hours of incubation at room temperature, oocyst destruction was estimated by microscopic counting using a Malassez chamber. Samples were then centrifuged (300 ×g, 5 min, 4°C), and the absorbance of the supernatant was measured at 273 nm using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies) to assess oocyst lysis. Toltrazuril (Baycox 2.5%, 25 mg/mL, Bayer, Germany) was used as a positive control under the same test conditions. The lethal concentration (LC₅₀) for each treatment was determined by regression curve analysis. Molecular Docking and ADMET Prediction To analyze the interactions of the main compounds derived from OFI-SO and OFI-HexPC, as well as the positive control Toltrazuril and another anticoccidial drug, Diclazuril, with the active sites of three enzymes—calcium-dependent protein kinase (CDPK; PDB: 4YZB), cyclic AMP-dependent protein kinase (PKA; PDB: 4WB5), and Eimeria tenella dihydroorotate dehydrogenase (EtDHODH; PDB: 6AJE)—we performed molecular docking studies. Additionally, a comprehensive analysis of the ADMET properties (absorption, distribution, metabolism, excretion, and toxicity) of these compounds was carried out. The compounds examined include Toltrazuril, Diclazuril, linoleic acid, oleic acid, palmitic acid, and stearic acid. The three-dimensional structures of the enzymes were obtained from the Protein Data Bank (PDB) ( https://www.rcsb.org/ ). To ensure accuracy, ligands, ions, and water molecules were removed, and missing atoms in the crystallographic structures were added using Chimera (Pettersen et al., 2004 ). Active sites were identified by selecting amino acid residues within a 10 Å radius of the inhibitors. Energy minimization was performed with AutoDock Tools (Morris et al., 2009 ) to optimize structures and eliminate steric hindrance. The interactions between compounds and enzymes were modeled using Chimera and AutoDock Vina (Pettersen et al., 2004 ; Morris et al., 2009 ). Docking results were visualized using Discovery Studio Visualizer (Mohamed et al., 2023 ) to gain insights into molecular mechanisms. To confirm the reliability of the docking protocol, redocking validation was performed (Adawara et al., 2022 ). The success of redocking was evaluated by calculating the root mean square deviation (RMSD < 2 Å) (Aziz et al., 2023 ; Che et al., 2023 ) and analyzing docking scores, ensuring that the software could reproduce experimentally validated results, ΔG is utilized to evaluate the binding affinity between ligands and receptors, with a negative ΔG value indicating a favorable interaction. Reliable online tools such as SwissADME, ProTox 3.0, and ADMETlab 2.0 (Daina et al., 2017 ; Ganorkar et al., 2020 ; Xiong et al., 2021 ; Ganorkar and Vander Heyden, 2022 ) were used to assess the ADMET profiles, pharmacodynamic properties, therapeutic potential, and toxicity of the compounds. Statistical analysis All statistical analyses were performed using R software (R.4.4.3.; 2025). Data were initially subjected to analysis of variance (ANOVA) to assess the effects of extract types and concentrations on parasite count and absorbance. When ANOVA indicated significant differences (p < 0.05), post hoc multiple comparisons were conducted. Pearson correlation analysis and dose-response analysis using linear regression were also performed. The lethal concentration (LC50) was defined as the concentration that reduced the initial number of sporulated oocysts by 50%. Results and Discussion Oocysts identification Five Eimeria species were identified based on the morphological characteristics of sporulated oocysts: E. acervulina (39%), E. tenella (23%), E. mitis (15%), E. maxima (14%), and E. brunetti (9%). The predominance of E. acervulina and E. tenella aligns with previous findings reported in various regions of Algeria. Djemai et al. (Djemai et al., 2016 ) reported E. acervulina and E. maxima as the most prevalent species in poultry farms in the Jijel region. In Sétif, E. tenella was the most frequently detected species (68%), followed by E. mitis (10%) and E. acervulina (14%) (Sabrina, 2021 ). A similar distribution pattern was described by Rahmani et al. (Rahmani et al., 2021 ), who recorded E. acervulina (35%), E. tenella (30%), E. maxima (15%), E. brunetti (12%), and E. mitis (8%). Phytochemical Characterization of OFI-SO and OFI-HexPC by GC–MS GC–MS analysis of OFI-SO revealed that fatty acids constituted 91.33% of its total composition (Table 1 ; Fig. 1 ). Linoleic acid was the dominant component (69.63%), followed by palmitic (10.28%) and stearic (6.88%) acids. Several minor constituents were also detected, including palmitoleic (0.41%), gadoleic (0.47%), arachidic (0.90%), erucic (0.48%), behenic (0.49%), and lignoceric (0.34%) acids. The fatty acid profile of OFI-HexPC differed considerably (Table 2 ; Fig. 2 ), with oleic acid being the predominant fatty acid (44.20%), followed by palmitic (19.02%) and stearic (10.15%) acids. Minor quantities of gadoleic (2.65%), arachidic (2.99%), and nonadecenoic (2.11%) acids were identified, alongside trace amounts of erucic, behenic, and lignoceric acids. These findings are broadly consistent with previous analyses of Algerian Opuntia ficus-indica oils. Benattia et al. reported lower linoleic acid content (60.23%) and higher proportions of palmitic (14.2%) and oleic acids (13.35%) (Benattia et al., 2019 ). In contrast, Chafaa et al. found a higher linoleic acid level (74.24%) with a comparable stearic acid proportion (6.75%) (Chafaa et al., 2024 ). Abderrahmane et al. observed 62.63% linoleic acid and significantly more oleic acid (20.36%) (Bouaouich et al., 2023 ). Such variation is likely due to genetic background, environmental conditions, and extraction techniques. Outside Algeria, comparable values have been reported. An Iraqi variety displayed slightly higher linoleic acid content (72.90%) and greater palmitic acid proportion (15.12%) (Alsaad et al., 2019 ). A recent study from Saudi Arabia showed a fatty acid composition similar to the present work, reinforcing the general lipid stability of O. ficus-indica across arid regions (Alqurashi et al., 2022 ). In Tunisian samples, linoleic acid was again the major constituent (60.69%), followed by oleic (16.41%), palmitic (12.72%), stearic (3.20%), and palmitoleic (0.75%) acids(BORCHANI, 2022 ). In contrast, Moroccan OFI-HexPC was reported to contain only four main fatty acids: linoleic (37.21%), palmitic (29.9%), oleic (27.16%), and stearic (5.92%) (Kadda et al., 2023 ), highlighting the significant impact of regional and varietal factors on seed cake composition. Both OFI-SO and OFI-HexPC share several key fatty acids, though their relative abundances differ. Linoleic acid, a polyunsaturated fatty acid (PUFA), is widely recognized for its hypocholesterolemic, anti-inflammatory, and skin-protective properties, supporting its broad industrial relevance (Ramadan and Mörsel, 2003 ; Kadda et al., 2023 ). Palmitic acid, a saturated fatty acid, is associated with increased LDL cholesterol (Djeghim et al., 2024 ), whereas stearic acid has a neutral or potentially beneficial impact on lipid metabolism (Tong et al., 2020 ). These biochemical characteristics, combined with the high content of unsaturated fatty acids, suggest that both extracts may hold nutritional, pharmaceutical, and anticoccidial potential. Table 1 Volatile compounds identified by GC-MS in OFI-SO peak Retention Time RT (min) Area % Library/ID* Molecules Formule 1 24,53 0,41 Hexadecenoic acid Palmetoleic acid C16 :1(9) 2 25,05 10,28 Hexadecanoic acid Palmetic acid C16 :0 3 30,2 69,63 Octadecadienoic acid Linoleic acid C18 :2(9,12) 4 30,97 6,88 Octadecanoic acid Stearic acid C18 :0 5 35,66 0,47 Eicosenoic acid Gadolic acid C20 :1(9) 6 36,46 0,9 Eicosanoic acid Arachidic acid C20 :0 7 41,1 0,48 13-Docosenoic acid Erucic acid C22 :1(13) 8 41,62 0,49 Docosanoic acid Behenic acid C22 :0 9 47,27 0,34 Tetracosanoic acid Lignoceric acid C24 :0 10 54,32 1,45 Stigmastan 3.5diene *NIST 2 Table 2 Volatile compounds identified by GC-MS in OFI-HexPC PEAK RT Area % Library/ID* Molecules Formula 1 19,1 0,75 Nonanoic acid Nonanoic acid C9 :0 2 20,23 0,02 Dodecenoic acid Lauroleic acid C12 :1 3 23,47 0,08 Tetradecenoic acid Myristoleic acid C14 :1 4 24,53 0,86 Hexadecenoic acid Palmetoleic acid C16 :1(9) 5 25,05 14,86 Hexadecacanoic acid Palmetic acid C16 :0 6 28,1 0,2 Tridecanoic acid Tridecanoic acid C13 :0 7 30,37 44,2 Octadecenoic acid Oleic acid C18 :1 8 30,97 7,98 Octadecanoic acid Stearic acid C18 :0 9 32,97 0,23 Undecanoic acid Undecanoic acid C11 :0 10 34,76 0,68 Carbonic acid Carbonic acid C1 11 35,66 2,65 Eicosenoic acid Gadolic acid C20 :1(9) 12 36,46 2,99 Eicosanoic acid Arachidic acid C20 :0 13 37,19 2,11 Nonadecenoic acid Nonadecenoic acid C19 :1 14 41,1 0,55 13-Docosenoic acid Erucic acid C22 :1(13) 15 41,62 1,21 Docosanoic acid Behenic acid C22 :0 16 44,63 0,28 Butanoic acid Butanoic acid C4 17 47,27 0,22 Tetracosanoic acid Lignoceric acid C24 :0 18 49,52 0,11 Pentacosanoic acid Pentacosanoic acid C15 :0 19 54,32 0,11 Stigmastan 3.5diene *NIST 2 In Vitro Anticoccidial Activity of OFI-SO and OFI-HexPC In vitro assays revealed a clear, concentration-dependent destructive effect on Eimeria spp . oocysts following treatment with OFI-SO, OFI-HexPC, and the synthetic anticoccidial agent Toltrazuril (Fig. 3 ). Both extract type and concentration had statistically significant effects on oocyst counts and the release of absorbent intracellular material (p < 0.001), with a significant interaction between these factors (p < 0.001; Tables 3 and 4 ). This dose-dependent effect was further confirmed by the progressive increase in absorbance at 273 nm (Fig. 4 ), indicative of membrane disruption and cytoplasmic leakage. No such effects were observed in the negative control group (HBSS). Table 3 Two-way ANOVA results for Parasite Numbers Source df SS MS F P Significance Extract Type 2 901.45 450.72 3141.07 < 0.001 *** Concentration 6 70.61 11.77 82.02 < 0.001 *** Extract Type: Concentration 12 49.80 4.15 28.92 < 0.001 *** Residuals 42 6.03 0.14 NA df: Degree of Freedom, SS, Sum Square, MS: Mean Square, * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant Table 4 Two-way ANOVA results for Material Absorbance Source Df SS MS F p Significance Extract Type 2 0.51 0.26 277.65 < 0.001 *** Concentration 6 1.16 0.19 209.38 < 0.001 *** Extract Type: Concentration 12 0.55 0.05 50.05 < 0.001 *** Residuals 42 0.04 0.00 NA df: Degree of Freedom, SS, Sum Square, MS: Mean Square, * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant Post hoc comparisons demonstrated that OFI-HexPC consistently exhibited lower efficacy than both OFI-SO and Toltrazuril across all tested concentrations (p < 0.001). By contrast, OFI-SO showed similar levels of efficacy to Toltrazuril (p = 1.000), particularly at higher concentrations. Absorbance data confirmed these findings: while no significant differences were observed between treatments at low concentrations (0–4 mg/mL), OFI-HexPC’s performance was significantly reduced at 8–64 mg/mL compared to OFI-SO and the positive control (Tables 5 and 6 ). A strong negative correlation between oocyst counts and absorbance (Table 7 ; Fig. 5 ) supports a mechanism involving membrane damage. Table 5 Tukey’s HSD for Parasite Numbers across Extract Types at each Concentration Contrast Concentration Estimate SE Df t.ratio p.value EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C1 11.20 0.31 42 36.21 0.000 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C1 11.20 0.31 42 36.21 0.000 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C1 0.00 0.31 42 0.00 1.000 EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C2 9.10 0.31 42 29.42 0.000 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C2 9.10 0.31 42 29.42 0.000 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C2 0.00 0.31 42 0.00 1.000 EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C3 9.10 0.31 42 29.42 0.000 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C3 9.10 0.31 42 29.42 0.000 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C3 0.00 0.31 42 0.00 1.000 EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C4 8.30 0.31 42 26.84 0.000 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C4 8.30 0.31 42 26.84 0.000 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C4 0.00 0.31 42 0.00 1.000 EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C5 7.30 0.31 42 23.60 0.000 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C5 7.30 0.31 42 23.60 0.000 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C5 0.00 0.31 42 0.00 1.000 EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C6 5.60 0.31 42 18.11 0.000 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C6 5.60 0.31 42 18.11 0.000 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C6 0.00 0.31 42 0.00 1.000 EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C7 5.57 0.31 42 18.02 0.000 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C7 5.57 0.31 42 18.00 0.000 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C7 -0.01 0.31 42 -0.02 1.000 SE: Standard Error, df: Degree of Freedom Table 6 Tukey’s HSD for Material Absorbance across Extract Types at each Concentration Contrast Concentration Estimate SE Df t.ratio p.value EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C1 0.00 0.025 42 0.00 1.000 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C1 0.00 0.025 42 0.00 1.000 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C1 0.00 0.025 42 0.00 1.000 EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C2 -0.03 0.025 42 -1.21 0.700 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C2 -0.04 0.025 42 -1.41 0.497 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C2 -0.01 0.025 42 -0.20 1.000 EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C3 -0.02 0.025 42 -0.76 1.000 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C3 -0.04 0.025 42 -1.77 0.252 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C3 -0.03 0.025 42 -1.01 0.958 EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C4 0.25 0.025 42 10.08 0.000 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C4 0.22 0.025 42 9.07 0.000 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C4 -0.03 0.025 42 -1.01 0.958 EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C5 0.27 0.025 42 10.89 0.000 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C5 0.25 0.025 42 10.08 0.000 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C5 -0.02 0.025 42 -0.81 1.000 EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C6 0.48 0.025 42 19.15 0.000 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C6 0.43 0.025 42 17.13 0.000 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C6 -0.05 0.025 42 -2.02 0.151 EXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO) C7 0.47 0.025 42 19.08 0.000 EXTRACT 1(OFI-HexPC) - POSITIVE CONTROL C7 0.42 0.025 42 16.93 0.000 EXTRACT 2(OFI-SO) - POSITIVE CONTROL C7 -0.05 0.025 42 -2.15 0.112 SE: Standard Error, df: Degree of Freedom Table 7 Pearson correlation between Parasite Numbers and Material Absorbance by Extract Type Extract Type Pearson_r t_value df p_value CI_lower CI_upper EXTRACT 1(OFI-HexPC) -0.923 -10.493 19.000 0.000 -0.969 -0.818 EXTRACT 2(OFI-SO) -0.804 -5.903 19.000 0.000 -0.918 -0.571 POSITIVE CONTROL -0.890 -8.515 19.000 0.000 -0.955 -0.745 df: Degree of Freedom, CI: Confidence Interval The dose-response curves (Figs. 6 and 7 ) indicated that all tested compounds exhibited concentration-dependent effects on both parameters. Toltrazuril demonstrated the highest potency with an LC₅₀ of 47.00 mg/mL. Among the plant-derived compounds, OFI-SO showed slightly higher activity (LC₅₀ = 47.05 mg/mL) compared to OFI-HexPC (LC₅₀ = 50.09 mg/mL), confirming the superior activity of OFI-SO among the plant-derived treatments. These results align with prior investigations on Opuntia ficus-indica extracts, where peel and flower fractions demonstrated lower efficacy (LC₅₀ = 60.53 mg/mL and 66.04 mg/mL, respectively) (Amrane-Abider et al., 2023a , b ). By comparison, Pistacia lentiscus oil showed a substantially lower LC₅₀ (5.86 mg/mL), suggesting higher antiparasitic potency (Rahmani et al., 2021 ). Other plant-based products, such as olive leaf extracts, yielded varied results, with LC₅₀ values ranging from 14.88 to 194.92 mg/mL depending on the bioactive component (Debbou-Iouknane et al., 2021 ). Variability in experimental protocols, Eimeria species, and extract composition likely account for these differences. The potent activity observed for OFI-SO and OFI-HexPC is likely due to their high content of unsaturated fatty acids, particularly linoleic and oleic acids, as confirmed by GC–MS. These compounds are known to exhibit anti-protozoal activity, often through disruption of parasite membrane integrity and interference with ion transport (Ismaeil et al., 2025 ; Liu et al., 2025 ). The increased absorbance at 273 nm in treated groups suggests leakage of nucleotides and aromatic amino acids—typical indicators of cell lysis (Remmal et al., 2011 ). These findings are further supported by similar effects reported in other parasitic systems. PUFAs such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (AA) have demonstrated inhibitory activity against Plasmodium falciparum , disrupting schizont development and inhibiting nucleic acid synthesis (Kumaratilake et al., 1992 ; Krugliak et al., 1995 ). In helminths like Schistosoma mansoni , AA induced tegumental disintegration through sphingomyelinase activation, resulting in exposure of parasitic antigens (El Ridi et al., 2012 ). Comparable membrane-disruptive effects were also noted in oocysts exposed to plant extracts, leading to loss of cytoplasmic mass and parasite death (Abbas et al., 2019 ). Taken together, these data suggest that OFI-SO and OFI-HexPC exert their anticoccidial effects through a combination of membrane disruption and intracellular destabilization. These preliminary findings justify further mechanistic investigation through molecular docking and in vivo validation. Molecular Docking Analysis To elucidate the molecular mechanisms underlying the anticoccidial activity observed in vitro, molecular docking simulations were conducted on three key enzymes: dihydroorotate dehydrogenase (EtDHODH; PDB: 6AJE), calcium-dependent protein kinase (CDPK; PDB: 4YZB), and cAMP-dependent protein kinase A (PKA; PDB: 4WB5). These targets are critical to pyrimidine biosynthesis, intracellular signaling, and host cell invasion, and are widely recognized as relevant targets for anticoccidial drug discovery (Haste et al., 2012 ; Sato et al., 2020 ; Aljedaie et al., 2021 ). The docking protocol was validated through redocking of the native ligands into their respective binding sites. The resulting RMSD values—1.018 Å for 4YZB, 0.861 Å for 4WB5, and 1.086 Å for 6AJE—demonstrated excellent overlap with crystallographic poses, confirming the reliability of the docking simulations. Docking results (Figs. 8 – 11 ) showed that Diclazuril exhibited the strongest binding affinities, particularly with CDPK (–8.2 kcal/mol), followed by Toltrazuril (–7.7 kcal/mol). Diclazuril formed multiple hydrogen bonds (e.g., TYR339, SER340, TYR98) and hydrophobic interactions (e.g., ILE342, LEU70, LEU80). Toltrazuril displayed strong interactions through hydrogen bonds (GLY317, SER287, THR95), hydrophobic contacts (CYS111, LEU148, LYS50), and halogen bonding, which may enhance its target binding stability. Among the fatty acids identified in OFI-SO and OFI-HexPC, linoleic, oleic, palmitic, and stearic acids displayed moderate binding energies (–5.4 to − 6.5 kcal/mol). These values are consistent with other phytochemicals such as apigenin (–6.44 kcal/mol) and artemisinin (–5.83 kcal/mol) (Aljedaie et al., 2021 ). The binding modes were predominantly stabilized by hydrophobic interactions within the active sites, suggesting a potential for partial enzymatic inhibition. Although the fatty acids exhibited weaker binding affinities than synthetic anticoccidials, their interaction profiles support a plausible mechanism of enzymatic interference, complementing the membrane-disruptive effects observed in vitro. In particular, linoleic acid demonstrated consistent binding behavior across all three targets and may significantly contribute to the dual anticoccidial action of OFI-SO and OFI-HexPC. These findings support the hypothesis that the anticoccidial effects of Opuntia ficus-indica seed-derived extracts arise from a synergistic mechanism involving both structural damage to oocyst membranes and targeted inhibition of key parasite enzymes. Analysis of ADMET Properties The ADMET analysis (Table 8 ) highlights that Diclazuril and Toltrazuril, used as reference anticoccidial drugs, display favorable pharmacokinetic properties but also present notable toxicological risks. Toltrazuril shows high respiratory toxicity and hormonal receptor interaction, while Diclazuril exhibits marked neurotoxicity, carcinogenic potential, and thyroid receptor binding. These toxic effects raise concerns about their safety for prolonged use in animals. In contrast, the fatty acids evaluated in this study, linoleic, oleic, palmitic, and stearic acids, derived from OFI-SO and OFI-HexPC, demonstrate a favorable ADMET profile. They do not show hepatotoxicity, neurotoxicity, respiratory toxicity, carcinogenic activity, or cytochrome P450 inhibition. While only palmitic acid crosses the blood-brain barrier, all compounds display acceptable bioavailability and pharmacokinetic behavior. In addition to their low toxicity, these fatty acids showed notable biological activity in molecular docking analyses, reinforcing their potential as natural, safer anticoccidial agents. Their ability to target specific molecular pathways, combined with good tolerability, supports their inclusion in the development of alternative therapeutic formulations. Table 8 Analysis of ADMET Properties and Toxicity Comparison: Toltrazuril, Diclazuril, and Fatty Acids Molcule Toltrazuril Diclazuril Linoleic Acid Oleic Acid Palmitic Acid Stearic Acid Physicochemical Properties MW 425.38 346.38 280.45 282.46 256.42 284.48 Csp3 0.17 0.2 0.72 0.83 0.94 0.94 NRB 5 3 14 15 14 16 NHA 7 4 2 2 2 2 NHD 1 1 1 1 1 1 TPSA 111.39 91.54 37.3 37.3 37.3 37.3 Lipophilicity Log P 3.64 2.75 2.75 4.57 4.19 4.67 Water Solubility Log S Moderately Moderately Moderately Moderately Moderately Moderately Pharmacokinetics HIA High High High High High High BBB No No No No Yes No Drug-likeness Lipinski 0 0 0 1 1 1 Ghose 0 0 0 1 0 1 Veber 0 0 0 1 1 1 Egan 0 0 0 1 0 1 Muegge 0 0 0 1 1 2 Medicinal Chemistry PAINS 0 0 0 0 0 0 Organ toxicity Hepatotoxicity Active (0.67) Active (0.53) Inactive (0.55) Inactive (0.55) Inactive (0.52) Inactive (0.52) Neurotoxicity Active (0.73) Active (0.88) Inactive (0.91) Inactive (0.91) Inactive (0.92) Inactive (0.92) Respiratory toxicity Active (0.75) Active (0.56) Inactive (0.84) Inactive (0.84) Inactive (0.85) Inactive (0.85) Toxicity end points Carcinogenicity Active (0.5) Active (0.5) Inactive (0.64) Inactive (0.64) Inactive (0.63) Inactive (0.63) THRβ Inactive (0.61) Active (0.66) Inactive (0.91) Inactive (0.78) Inactive (0.78) Inactive (0.9) Clinical toxicity Active (0.61) Active (0.58) Inactive (0.61) Inactive (0.61) Inactive (0.64) Inactive (0.64) Metabolism CYP1A2 Inactive (0.69) Inactive (0.64) Inactive (0.91) Inactive (0.91) Inactive (0.84) Inactive (0.84) CYP2C19 Inactive (0.78) Inactive (0.9) Inactive (0.98) Inactive (0.98) Inactive (0.98) Inactive (0.98) CYP2C9 Inactive (0.57) Inactive (0.65) Inactive (0.69) Inactive (0.69) Inactive (0.71) Inactive (0.71) CYP2D6 Inactive (0.8) Inactive (0.86) Inactive (0.88) Inactive (0.88) Inactive (0.88) Inactive (0.88) CYP3A4 Inactive (0.58) Inactive (0.88) Inactive (1.0) Inactive (1.0) Inactive (1.0) Inactive (1.0) CYP2E1 Inactive (0.99) Inactive (1.0) Inactive (0.99) Inactive (0.99) Inactive (0.99) Inactive (0.99 MW =Molecular Weight below 500, NHA =Num. H−bond Acceptors, NHD =Num. H−bond donors, NRB =Num. rotatable bonds, Fsp3 =Fraction Csp3 acceptors, HIA = Human Intestinal Absorption, CYP = Cytochrome P450, BBB = Blood−Brain Barrier, PAINS =Pan Assay Interference compounds or frequent hitters or promiscuous compounds, Log P : logarithm of the compound partition coefficient between n−octanol and water (− 2 to 5), TPSA = Polar surface area (0–140), THRβ =Thyroid hormone receptor bet Conclusion This study provides compelling evidence for the anticoccidial efficacy of OFI-SO and OFI-HexPC, based on integrated in vitro and in silico investigations. OFI-SO demonstrated a pronounced oocysticidal effect, comparable to that of the synthetic anticoccidial Toltrazuril. This activity is likely attributable to its high content of bioactive unsaturated fatty acids, particularly linoleic and oleic acids. Molecular docking analyses confirmed that these compounds are capable of interacting with key Eimeria enzymes, notably EtDHODH, CDPK, and PKA, suggesting a dual mode of action involving both membrane disruption and enzymatic inhibition. This mechanistic synergy enhances their potential as effective antiparasitic agents. Furthermore, ADMET predictions revealed favorable pharmacokinetic and safety profiles for the tested fatty acids, contrasting with the organ-specific toxicities commonly associated with synthetic anticoccidials. These data support the candidacy of OFI-SO and OFI-HexPC as natural, safer alternatives to conventional chemoprophylactic agents in poultry production. Future studies should focus on in vivo validation, dosage optimization, and large-scale application under commercial farming conditions to fully establish the practical utility of these phytogenic extracts. Declarations Ethics approval and consent to participate: All authors approved. Consent for publication: All authors agree to the publication. Conflicts of Interest: The authors declare no conflicts of interest. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding: This research was not funded Author Contribution The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Acknowledgement The authors would like to express their gratitude to the Ministry of Higher Education and Scientific Research and the General Directorate of Scientific Research and Technological Development (DGRSDT) for their support. The authors also gratefully acknowledge the NOPALTEC cooperative (Souk Ahras, Algeria) for providing the plant material and technical assistance. Special thanks are extended to the staff of the Parasitology Laboratory at the Biotechnology Research Center (C.R.Bt) for their valuable support in the collection and morphological identification of Eimeria spp. oocysts. We also acknowledge the Environmental Laboratory, represented by Dr. Cherb Nora, for her collaboration and technical contributions during the experimental procedures. Data availability statement: The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. References Abbas RZ, Abbas A, Raza MA, Khan MK, Saleemi MK, Saeed Z (2019) In vitro anticoccidial activity of Trachyspermum ammi (Ajwain) extract on oocysts of Eimeria species of Chicken. Advancements Life Sci 7:44–47 Abbas RZ, Colwell DD, Gilleard J (2012) Botanicals: an alternative approach for the control of avian coccidiosis. 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Bezghiche","email":"","orcid":"","institution":"Larbi Ben M’Hidi University","correspondingAuthor":false,"prefix":"","firstName":"Ramzi","middleName":"","lastName":"Bezghiche","suffix":""},{"id":490525415,"identity":"e44d3f84-f854-41a7-ae48-109bec9813f4","order_by":10,"name":"Houssam Eddine Bentounsi","email":"","orcid":"","institution":"Ali Mendjli","correspondingAuthor":false,"prefix":"","firstName":"Houssam","middleName":"Eddine","lastName":"Bentounsi","suffix":""},{"id":490525416,"identity":"1d44a25b-90f3-40eb-bd31-e106ce2ef5c9","order_by":11,"name":"Houria Bouteraa","email":"","orcid":"","institution":"University of Abbes Laghrour","correspondingAuthor":false,"prefix":"","firstName":"Houria","middleName":"","lastName":"Bouteraa","suffix":""},{"id":490525417,"identity":"bdcc0605-e728-453e-bc1d-2908260639ed","order_by":12,"name":"Abdelwahab Benhocine","email":"","orcid":"","institution":"University of Abbes Laghrour","correspondingAuthor":false,"prefix":"","firstName":"Abdelwahab","middleName":"","lastName":"Benhocine","suffix":""},{"id":490525418,"identity":"a0b15925-7363-48d9-ad18-8a5ffff40d7a","order_by":13,"name":"Mohammed Messaoudi","email":"","orcid":"","institution":"Nuclear Research Centre of Birine","correspondingAuthor":false,"prefix":"","firstName":"Mohammed","middleName":"","lastName":"Messaoudi","suffix":""}],"badges":[],"createdAt":"2025-07-18 10:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7156619/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7156619/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11686-025-01137-5","type":"published","date":"2025-10-16T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87555534,"identity":"b39ff813-4549-40c1-8b2e-51e1c9425f4e","added_by":"auto","created_at":"2025-07-25 07:03:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22233,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of GC-MS analysis of OFI-SO\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7156619/v1/f084705028e7b8b10e3021e8.png"},{"id":87556414,"identity":"a01f3235-20ad-435b-8ff9-b656ca1190c3","added_by":"auto","created_at":"2025-07-25 07:11:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30467,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of GC-MS analysis of OFI-HexPC\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7156619/v1/b0a9c70923723eb0d10d1902.png"},{"id":87556412,"identity":"24c2a87b-6e2c-49e6-9515-cec632877ece","added_by":"auto","created_at":"2025-07-25 07:11:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106957,"visible":true,"origin":"","legend":"\u003cp\u003eScatter plot of parasite numbers vs. concentration with trend lines showing the dose-response relationship for each extract type\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7156619/v1/d866d693af3481d54b1c33bc.png"},{"id":87556545,"identity":"238c5f3f-c667-4767-9736-80cc83c10123","added_by":"auto","created_at":"2025-07-25 07:19:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":127060,"visible":true,"origin":"","legend":"\u003cp\u003eScatter plot of material absorbance vs. concentration with trend lines showing the dose-response for each extract\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7156619/v1/0d7daddf953121f51f0b2882.png"},{"id":87555540,"identity":"adc24a2c-83a6-42c9-b7e2-6db0362c469e","added_by":"auto","created_at":"2025-07-25 07:03:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":106185,"visible":true,"origin":"","legend":"\u003cp\u003eScatter plots showing the correlation between parasite numbers and material absorbance for each extract type\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7156619/v1/581851f3535aa5a357442162.png"},{"id":87555541,"identity":"09d6b600-7d59-45ce-9791-3e0659bb996e","added_by":"auto","created_at":"2025-07-25 07:03:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":125838,"visible":true,"origin":"","legend":"\u003cp\u003eScatter plot showing the variation in parasite numbers according to extract concentration, illustrating the dose-response relationship for each extract type\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7156619/v1/79c5c1cea342f96db32b176a.png"},{"id":87555547,"identity":"d09cf1b5-d260-43a1-b5b6-1d8fd023fa35","added_by":"auto","created_at":"2025-07-25 07:03:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":115963,"visible":true,"origin":"","legend":"\u003cp\u003eScatter plot showing the variation in material absorbance according to extract concentration, illustrating the dose-response relationship for each extract type\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7156619/v1/1398604aa36c0f3a2053be6a.png"},{"id":87556547,"identity":"7688a555-52c9-4884-9a32-e9eaca369a3f","added_by":"auto","created_at":"2025-07-25 07:19:25","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":22871,"visible":true,"origin":"","legend":"\u003cp\u003eBinding Scores and Major Compounds Interacting with: (a) 4YZB: CDPK1 from Eimeria tenella, (b) 4WB5: human cAMP-dependent protein kinase A, and (c) 6AJE: DHODH\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7156619/v1/884de5f9279bda40ea73b537.png"},{"id":87556420,"identity":"60b04931-bab1-4bc1-b0a3-56e695949b91","added_by":"auto","created_at":"2025-07-25 07:11:25","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":164447,"visible":true,"origin":"","legend":"\u003cp\u003eThe interactions and binding modes of Toltrazuril, Diclazuril, and major compounds with 6AJE\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7156619/v1/48b28a8ee6967d1b33e9f2a2.png"},{"id":87555559,"identity":"b6868d59-7440-419f-8531-4f6c977aa31a","added_by":"auto","created_at":"2025-07-25 07:03:25","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":176808,"visible":true,"origin":"","legend":"\u003cp\u003eThe interactions and binding modes of Toltrazuril, Diclazuril, and major compounds with 4YZB\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-7156619/v1/6b7b9c5812a458f41bd782dc.png"},{"id":87555548,"identity":"03a13d90-009a-41b4-97d7-497214db9e1b","added_by":"auto","created_at":"2025-07-25 07:03:25","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":129132,"visible":true,"origin":"","legend":"\u003cp\u003eThe interactions and binding modes of Toltrazuril, Diclazuril, and major compounds with 4WB5\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-7156619/v1/d89edff878c378bb2334584e.png"},{"id":93955937,"identity":"0b747b75-a232-4e86-a93c-c7a2a2692cf9","added_by":"auto","created_at":"2025-10-20 16:07:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2510832,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7156619/v1/3fe03c4d-b772-412d-8cb3-015504972b7c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cem\u003eIn Vitro and In Silico\u003c/em\u003e Anticoccidial Activity of \u003cem\u003eOpuntia ficus-indica\u003c/em\u003e Seed Oil and Hexane Extract: Comprehensive GC-MS Profiling and Mechanistic Insights\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAvian coccidiosis, an enteric parasitic disease caused by protozoa of the genus \u003cem\u003eEimeria\u003c/em\u003e, remains a major health and economic challenge in modern poultry production (Shirley et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Chapman, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Prevalent particularly in intensive farming systems, this parasitosis significantly compromises poultry growth, welfare, and productivity (Johnson, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1923\u003c/span\u003e; Taylor et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Global economic losses associated with coccidiosis are estimated at approximately £10.4\u0026nbsp;billion annually (Blake et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), with recent assessments reporting losses of around £86.7\u0026nbsp;million per year in Algeria alone (Rahmani et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), underscoring its impact on national food security and farm profitability.\u003c/p\u003e\u003cp\u003eChemoprophylaxis using synthetic anticoccidials remains the primary strategy for disease control in poultry (Taylor et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the continuous use of these compounds has led to the emergence of drug-resistant \u003cem\u003eEimeria\u003c/em\u003e strains and raised serious concerns regarding drug residues in poultry products and potential risks to human health (Abbas et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Chapman et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Noack et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These limitations have intensified the search for effective, safe, and sustainable alternatives (Abbas et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePhytogenic compounds derived from medicinal plants have gained considerable attention due to their diverse biological activities. Plant-based additives are known for their antimicrobial, antioxidant (Naidoo et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), antiviral (Bishop, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), antiparasitic, immunostimulant, and gut-modulating effects (Youn and Noh, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Advances in phytochemical extraction and characterization techniques have reinforced the therapeutic value of many traditional medicinal plants (Gacem et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mahrose, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), supporting their use as alternatives to conventional antimicrobials in poultry production (Guèye, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eOpuntia ficus-indica\u003c/em\u003e, a drought-tolerant species widespread in arid and semi-arid regions (Giraldo-Silva et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), is a rich source of lipophilic bioactive compounds, including fatty acids, sterols, and tocopherols. Recent studies suggest its potential antiparasitic effects, including anticoccidial properties (Amrane-Abider et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003eb\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe present study aims to investigate the anticoccidial potential of OFI-SO and OFI-HexPC against \u003cem\u003eEimeria spp\u003c/em\u003e. An integrated approach combining in vitro evaluation of oocyst integrity and in silico molecular docking of identified fatty acids targeting key \u003cem\u003eEimeria\u003c/em\u003e enzymes was adopted. This dual strategy aims to explore the therapeutic relevance of these natural products and support their development as promising alternatives to conventional anticoccidials in poultry health management.\u003c/p\u003e"},{"header":"Material and Method","content":"\u003cp\u003e\u003cb\u003ePlant Material and Extraction of Non-Polar Compounds\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eOpuntia ficus-indica\u003c/em\u003e seed oil (OFI-SO) and the corresponding residual seed press cake were kindly provided by the NOPALTEC cooperative (Sidi-Fredj, Souk Ahras, Algeria), a local enterprise specialized in the processing of prickly pear-derived products. The plant material was harvested during the autumn season, corresponding to the full fruit maturity stage. Botanical identification was performed by the supplier’s technical staff, including a certified botanist. Product quality and traceability were verified by their internal R\u0026amp;D department.\u003c/p\u003e\u003cp\u003eThe oil was extracted by cold pressing of mature prickly pear seeds. The resulting seed press cake was air-dried, finely ground, sieved through a 1 mm mesh, and stored in amber glass bottles at room temperature until use. To isolate fatty acids and other non-polar constituents, OFI-HexPC was obtained by Soxhlet extraction using n-hexane at 100°C for 6 hours, following the protocol described by Kadda et al. (Kadda et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The obtained extract was concentrated under reduced pressure using a Büchi rotary evaporator (Büchi, Switzerland), and subsequently stored at 4°C prior to analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOocyst Collection and Identification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOocysts of \u003cem\u003eEimeria spp\u003c/em\u003e. were isolated from fresh fecal samples collected from naturally infected broiler chickens in the Baghai region (Khenchela Province, eastern Algeria), following the procedure described by Carvalho et al. (Carvalho et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The samples were processed for sporulation in a 2.5% potassium dichromate (K₂Cr₂O₇) solution under optimal conditions of aeration, temperature, and humidity.\u003c/p\u003e\u003cp\u003eThe concentration of sporulated oocysts (oocysts/mL) was determined using a \u003cem\u003eMalassez\u003c/em\u003e counting chamber (Rahmani et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For morphological identification, 100 sporulated oocysts were randomly selected and examined under a Leica microscope equipped with a digital camera. Image analysis was conducted using Motic Images Plus 2.0 software. Identification was based on morphometric parameters and compared with classical descriptions provided by Conway and McKenzie (Conway and McKenzie, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEsterification and GC-MS Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFatty acids from OFI-SO and OFI-HexPC were converted into fatty acid methyl esters (FAMEs) prior to gas chromatography–mass spectrometry (GC-MS) analysis. Briefly, 0.5 g of each sample was mixed with 5 mL of n-hexane, followed by the addition of 0.5 mL of 2 N KOH in methanol. After vortexing for 30 seconds, the mixture was centrifuged at 3000 rpm for 10 minutes. Then, 100 µL of the supernatant was collected and diluted with 1 mL of n-hexane. The resulting FAMEs were stored at − 20°C until further analysis.\u003c/p\u003e\u003cp\u003eGC-MS analysis was performed using an Agilent 6890 Plus gas chromatograph coupled to an Agilent 5973 mass selective detector. The system was equipped with a splitless injector and an HP-5MS capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness; 5% phenyl–95% dimethylpolysiloxane). High-purity helium (N6) served as the carrier gas at a constant flow rate of 0.5 mL/min. The injector temperature was set to 250°C.\u003c/p\u003e\u003cp\u003eThe oven temperature was programmed as follows: initial temperature of 70°C (held for 5 min), ramped at 10°C/min to 130°C (held for 2 min), followed by 3°C/min to 220°C (held for 4 min), and finally ramped at 10°C/min to 280°C (held for 5 min). The mass spectrometer operated in electron impact ionization mode with a quadrupole analyzer. Compound identification was carried out by comparing retention times and mass spectra to entries in the NIST and PubChem spectral libraries.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnticoccidial activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe oocysticidal activity of OFI-SO and OFI-HexPC was evaluated in vitro according to the method described by Remmal et al (Remmal et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Each extract was prepared in Hank’s Balanced Salt Solution (HBSS) supplemented with 0.2% agar to ensure homogeneous oocyst dispersion and tested at six increasing concentrations (2 to 64 mg/mL). HBSS alone served as the negative control.\u003c/p\u003e\u003cp\u003eEach 1 mL reaction mixture contained 100 µL of purified \u003cem\u003eEimeria spp\u003c/em\u003e. oocyst suspension (1.34×10⁵ oocysts/mL for OFI-HexPC; 2.2×10⁵ oocysts/mL for OFI-SO), 700 µL of phosphate-buffered saline (PBS), and 200 µL of the test extract at the corresponding concentration.\u003c/p\u003e\u003cp\u003eAfter 24 hours of incubation at room temperature, oocyst destruction was estimated by microscopic counting using a Malassez chamber. Samples were then centrifuged (300 ×g, 5 min, 4°C), and the absorbance of the supernatant was measured at 273 nm using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies) to assess oocyst lysis.\u003c/p\u003e\u003cp\u003eToltrazuril (Baycox 2.5%, 25 mg/mL, Bayer, Germany) was used as a positive control under the same test conditions. The lethal concentration (LC₅₀) for each treatment was determined by regression curve analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular Docking and ADMET Prediction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo analyze the interactions of the main compounds derived from OFI-SO and OFI-HexPC, as well as the positive control Toltrazuril and another anticoccidial drug, Diclazuril, with the active sites of three enzymes—calcium-dependent protein kinase (CDPK; PDB: 4YZB), cyclic AMP-dependent protein kinase (PKA; PDB: 4WB5), and \u003cem\u003eEimeria tenella\u003c/em\u003e dihydroorotate dehydrogenase (EtDHODH; PDB: 6AJE)—we performed molecular docking studies. Additionally, a comprehensive analysis of the ADMET properties (absorption, distribution, metabolism, excretion, and toxicity) of these compounds was carried out. The compounds examined include Toltrazuril, Diclazuril, linoleic acid, oleic acid, palmitic acid, and stearic acid.\u003c/p\u003e\u003cp\u003eThe three-dimensional structures of the enzymes were obtained from the Protein Data Bank (PDB) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To ensure accuracy, ligands, ions, and water molecules were removed, and missing atoms in the crystallographic structures were added using Chimera (Pettersen et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Active sites were identified by selecting amino acid residues within a 10 Å radius of the inhibitors. Energy minimization was performed with AutoDock Tools (Morris et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) to optimize structures and eliminate steric hindrance. The interactions between compounds and enzymes were modeled using Chimera and AutoDock Vina (Pettersen et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Morris et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Docking results were visualized using Discovery Studio Visualizer (Mohamed et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) to gain insights into molecular mechanisms.\u003c/p\u003e\u003cp\u003eTo confirm the reliability of the docking protocol, redocking validation was performed (Adawara et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The success of redocking was evaluated by calculating the root mean square deviation (RMSD \u0026lt; 2 Å) (Aziz et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Che et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and analyzing docking scores, ensuring that the software could reproduce experimentally validated results, ΔG is utilized to evaluate the binding affinity between ligands and receptors, with a negative ΔG value indicating a favorable interaction.\u003c/p\u003e\u003cp\u003eReliable online tools such as SwissADME, ProTox 3.0, and ADMETlab 2.0 (Daina et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ganorkar et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xiong et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ganorkar and Vander Heyden, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) were used to assess the ADMET profiles, pharmacodynamic properties, therapeutic potential, and toxicity of the compounds.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using R software (R.4.4.3.; 2025). Data were initially subjected to analysis of variance (ANOVA) to assess the effects of extract types and concentrations on parasite count and absorbance. When ANOVA indicated significant differences (p \u0026lt; 0.05), post hoc multiple comparisons were conducted. Pearson correlation analysis and dose-response analysis using linear regression were also performed. The lethal concentration (LC50) was defined as the concentration that reduced the initial number of sporulated oocysts by 50%.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cb\u003eOocysts identification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFive \u003cem\u003eEimeria\u003c/em\u003e species were identified based on the morphological characteristics of sporulated oocysts: \u003cem\u003eE. acervulina\u003c/em\u003e (39%), \u003cem\u003eE. tenella\u003c/em\u003e (23%), \u003cem\u003eE. mitis\u003c/em\u003e (15%), \u003cem\u003eE. maxima\u003c/em\u003e (14%), and \u003cem\u003eE. brunetti\u003c/em\u003e (9%). The predominance of \u003cem\u003eE. acervulina\u003c/em\u003e and \u003cem\u003eE. tenella\u003c/em\u003e aligns with previous findings reported in various regions of Algeria. Djemai et al. (Djemai et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) reported \u003cem\u003eE. acervulina\u003c/em\u003e and \u003cem\u003eE. maxima\u003c/em\u003e as the most prevalent species in poultry farms in the Jijel region. In S\u0026eacute;tif, \u003cem\u003eE. tenella\u003c/em\u003e was the most frequently detected species (68%), followed by \u003cem\u003eE. mitis\u003c/em\u003e (10%) and \u003cem\u003eE. acervulina\u003c/em\u003e (14%) (Sabrina, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A similar distribution pattern was described by Rahmani et al. (Rahmani et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), who recorded \u003cem\u003eE. acervulina\u003c/em\u003e (35%), \u003cem\u003eE. tenella\u003c/em\u003e (30%), \u003cem\u003eE. maxima\u003c/em\u003e (15%), \u003cem\u003eE. brunetti\u003c/em\u003e (12%), and \u003cem\u003eE. mitis\u003c/em\u003e (8%).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhytochemical Characterization of OFI-SO and OFI-HexPC by GC\u0026ndash;MS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGC\u0026ndash;MS analysis of OFI-SO revealed that fatty acids constituted 91.33% of its total composition (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Linoleic acid was the dominant component (69.63%), followed by palmitic (10.28%) and stearic (6.88%) acids. Several minor constituents were also detected, including palmitoleic (0.41%), gadoleic (0.47%), arachidic (0.90%), erucic (0.48%), behenic (0.49%), and lignoceric (0.34%) acids.\u003c/p\u003e\u003cp\u003eThe fatty acid profile of OFI-HexPC differed considerably (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), with oleic acid being the predominant fatty acid (44.20%), followed by palmitic (19.02%) and stearic (10.15%) acids. Minor quantities of gadoleic (2.65%), arachidic (2.99%), and nonadecenoic (2.11%) acids were identified, alongside trace amounts of erucic, behenic, and lignoceric acids.\u003c/p\u003e\u003cp\u003eThese findings are broadly consistent with previous analyses of Algerian \u003cem\u003eOpuntia ficus-indica\u003c/em\u003e oils. Benattia et al. reported lower linoleic acid content (60.23%) and higher proportions of palmitic (14.2%) and oleic acids (13.35%) (Benattia et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In contrast, Chafaa et al. found a higher linoleic acid level (74.24%) with a comparable stearic acid proportion (6.75%) (Chafaa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Abderrahmane et al. observed 62.63% linoleic acid and significantly more oleic acid (20.36%) (Bouaouich et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Such variation is likely due to genetic background, environmental conditions, and extraction techniques.\u003c/p\u003e\u003cp\u003eOutside Algeria, comparable values have been reported. An Iraqi variety displayed slightly higher linoleic acid content (72.90%) and greater palmitic acid proportion (15.12%) (Alsaad et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A recent study from Saudi Arabia showed a fatty acid composition similar to the present work, reinforcing the general lipid stability of \u003cem\u003eO. ficus-indica\u003c/em\u003e across arid regions (Alqurashi et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In Tunisian samples, linoleic acid was again the major constituent (60.69%), followed by oleic (16.41%), palmitic (12.72%), stearic (3.20%), and palmitoleic (0.75%) acids(BORCHANI, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast, Moroccan OFI-HexPC was reported to contain only four main fatty acids: linoleic (37.21%), palmitic (29.9%), oleic (27.16%), and stearic (5.92%) (Kadda et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), highlighting the significant impact of regional and varietal factors on seed cake composition.\u003c/p\u003e\u003cp\u003eBoth OFI-SO and OFI-HexPC share several key fatty acids, though their relative abundances differ. Linoleic acid, a polyunsaturated fatty acid (PUFA), is widely recognized for its hypocholesterolemic, anti-inflammatory, and skin-protective properties, supporting its broad industrial relevance (Ramadan and M\u0026ouml;rsel, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Kadda et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Palmitic acid, a saturated fatty acid, is associated with increased LDL cholesterol (Djeghim et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), whereas stearic acid has a neutral or potentially beneficial impact on lipid metabolism (Tong et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These biochemical characteristics, combined with the high content of unsaturated fatty acids, suggest that both extracts may hold nutritional, pharmaceutical, and anticoccidial potential.\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\u003eVolatile compounds identified by GC-MS in OFI-SO\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=\"char\" char=\".\" 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=\"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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003epeak\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRetention Time RT (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eArea %\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLibrary/ID*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMolecules\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFormule\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e24,53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHexadecenoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePalmetoleic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC16 :1(9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e25,05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10,28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHexadecanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePalmetic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC16 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30,2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e69,63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOctadecadienoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLinoleic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC18 :2(9,12)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30,97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6,88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOctadecanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStearic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC18 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e35,66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEicosenoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGadolic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC20 :1(9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e36,46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEicosanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eArachidic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC20 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e41,1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13-Docosenoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eErucic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC22 :1(13)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e41,62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDocosanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBehenic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC22 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e47,27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTetracosanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLignoceric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC24 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e54,32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1,45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStigmastan 3.5diene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e*NIST 2\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\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\u003eVolatile compounds identified by GC-MS in OFI-HexPC\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=\"char\" char=\".\" 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=\"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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePEAK\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eArea %\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLibrary/ID*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMolecules\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFormula\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e19,1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNonanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNonanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC9 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20,23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDodecenoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLauroleic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC12 :1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e23,47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTetradecenoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMyristoleic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC14 :1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e24,53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHexadecenoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePalmetoleic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC16 :1(9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e25,05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14,86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHexadecacanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePalmetic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC16 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e28,1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTridecanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTridecanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC13 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30,37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e44,2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOctadecenoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOleic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC18 :1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30,97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7,98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOctadecanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStearic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC18 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e32,97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUndecanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUndecanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC11 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e34,76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCarbonic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCarbonic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e35,66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2,65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEicosenoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGadolic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC20 :1(9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e36,46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2,99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEicosanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eArachidic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC20 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e37,19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2,11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNonadecenoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNonadecenoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC19 :1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e41,1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13-Docosenoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eErucic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC22 :1(13)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e41,62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1,21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDocosanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBehenic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC22 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e44,63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eButanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eButanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e47,27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTetracosanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLignoceric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC24 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e49,52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePentacosanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePentacosanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC15 :0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e54,32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0,11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStigmastan 3.5diene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e*NIST 2\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn Vitro Anticoccidial Activity of OFI-SO and OFI-HexPC\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn vitro assays revealed a clear, concentration-dependent destructive effect on \u003cem\u003eEimeria spp\u003c/em\u003e. oocysts following treatment with OFI-SO, OFI-HexPC, and the synthetic anticoccidial agent Toltrazuril (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Both extract type and concentration had statistically significant effects on oocyst counts and the release of absorbent intracellular material (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with a significant interaction between these factors (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This dose-dependent effect was further confirmed by the progressive increase in absorbance at 273 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), indicative of membrane disruption and cytoplasmic leakage. No such effects were observed in the negative control group (HBSS).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTwo-way ANOVA results for Parasite Numbers\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" 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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003edf\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSignificance\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtract Type\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e901.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e450.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3141.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e***\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e70.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e82.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e***\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtract Type: Concentration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e49.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e28.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e***\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eResiduals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003edf: Degree of Freedom, SS, Sum Square, MS: Mean Square, * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ns\u0026thinsp;=\u0026thinsp;not significant\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTwo-way ANOVA results for Material Absorbance\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" 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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDf\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ep\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSignificance\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtract Type\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e277.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e***\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e209.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e***\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtract Type: Concentration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e50.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e***\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eResiduals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003edf: Degree of Freedom, SS, Sum Square, MS: Mean Square, * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ns\u0026thinsp;=\u0026thinsp;not significant\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePost hoc comparisons demonstrated that OFI-HexPC consistently exhibited lower efficacy than both OFI-SO and Toltrazuril across all tested concentrations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). By contrast, OFI-SO showed similar levels of efficacy to Toltrazuril (p\u0026thinsp;=\u0026thinsp;1.000), particularly at higher concentrations. Absorbance data confirmed these findings: while no significant differences were observed between treatments at low concentrations (0\u0026ndash;4 mg/mL), OFI-HexPC\u0026rsquo;s performance was significantly reduced at 8\u0026ndash;64 mg/mL compared to OFI-SO and the positive control (Tables\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). A strong negative correlation between oocyst counts and absorbance (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) supports a mechanism involving membrane damage.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTukey\u0026rsquo;s HSD for Parasite Numbers across Extract Types at each Concentration\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eContrast\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEstimate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDf\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003et.ratio\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e36.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e36.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e29.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e29.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e29.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e29.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e26.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e26.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e23.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e23.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e18.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e18.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e18.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e18.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eSE: Standard Error, df: Degree of Freedom\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTukey\u0026rsquo;s HSD for Material Absorbance across Extract Types at each Concentration\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eContrast\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEstimate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDf\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003et.ratio\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-1.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.700\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-1.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.497\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-1.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.252\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.958\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e10.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.958\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e10.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e10.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e19.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e17.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-2.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.151\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - EXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e19.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO) - POSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-2.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.112\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eSE: Standard Error, df: Degree of Freedom\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePearson correlation between Parasite Numbers and Material Absorbance by Extract Type\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtract Type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePearson_r\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003et_value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003edf\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ep_value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCI_lower\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCI_upper\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 1(OFI-HexPC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-0.923\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-10.493\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-0.969\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.818\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEXTRACT 2(OFI-SO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-0.804\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-5.903\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-0.918\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.571\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePOSITIVE CONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-0.890\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-8.515\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-0.955\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.745\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003edf: Degree of Freedom, CI: Confidence Interval\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe dose-response curves (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) indicated that all tested compounds exhibited concentration-dependent effects on both parameters. Toltrazuril demonstrated the highest potency with an LC₅₀ of 47.00 mg/mL. Among the plant-derived compounds, OFI-SO showed slightly higher activity (LC₅₀ = 47.05 mg/mL) compared to OFI-HexPC (LC₅₀ = 50.09 mg/mL), confirming the superior activity of OFI-SO among the plant-derived treatments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese results align with prior investigations on \u003cem\u003eOpuntia ficus-indica\u003c/em\u003e extracts, where peel and flower fractions demonstrated lower efficacy (LC₅₀ = 60.53 mg/mL and 66.04 mg/mL, respectively) (Amrane-Abider et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003eb\u003c/span\u003e). By comparison, Pistacia lentiscus oil showed a substantially lower LC₅₀ (5.86 mg/mL), suggesting higher antiparasitic potency (Rahmani et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Other plant-based products, such as olive leaf extracts, yielded varied results, with LC₅₀ values ranging from 14.88 to 194.92 mg/mL depending on the bioactive component (Debbou-Iouknane et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Variability in experimental protocols, Eimeria species, and extract composition likely account for these differences.\u003c/p\u003e\u003cp\u003eThe potent activity observed for OFI-SO and OFI-HexPC is likely due to their high content of unsaturated fatty acids, particularly linoleic and oleic acids, as confirmed by GC\u0026ndash;MS. These compounds are known to exhibit anti-protozoal activity, often through disruption of parasite membrane integrity and interference with ion transport (Ismaeil et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The increased absorbance at 273 nm in treated groups suggests leakage of nucleotides and aromatic amino acids\u0026mdash;typical indicators of cell lysis (Remmal et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThese findings are further supported by similar effects reported in other parasitic systems. PUFAs such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (AA) have demonstrated inhibitory activity against \u003cem\u003ePlasmodium falciparum\u003c/em\u003e, disrupting schizont development and inhibiting nucleic acid synthesis (Kumaratilake et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Krugliak et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). In helminths like \u003cem\u003eSchistosoma mansoni\u003c/em\u003e, AA induced tegumental disintegration through sphingomyelinase activation, resulting in exposure of parasitic antigens (El Ridi et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Comparable membrane-disruptive effects were also noted in oocysts exposed to plant extracts, leading to loss of cytoplasmic mass and parasite death (Abbas et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTaken together, these data suggest that OFI-SO and OFI-HexPC exert their anticoccidial effects through a combination of membrane disruption and intracellular destabilization. These preliminary findings justify further mechanistic investigation through molecular docking and in vivo validation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular Docking Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the molecular mechanisms underlying the anticoccidial activity observed in vitro, molecular docking simulations were conducted on three key enzymes: dihydroorotate dehydrogenase (EtDHODH; PDB: 6AJE), calcium-dependent protein kinase (CDPK; PDB: 4YZB), and cAMP-dependent protein kinase A (PKA; PDB: 4WB5). These targets are critical to pyrimidine biosynthesis, intracellular signaling, and host cell invasion, and are widely recognized as relevant targets for anticoccidial drug discovery (Haste et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sato et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Aljedaie et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe docking protocol was validated through redocking of the native ligands into their respective binding sites. The resulting RMSD values\u0026mdash;1.018 \u0026Aring; for 4YZB, 0.861 \u0026Aring; for 4WB5, and 1.086 \u0026Aring; for 6AJE\u0026mdash;demonstrated excellent overlap with crystallographic poses, confirming the reliability of the docking simulations.\u003c/p\u003e\u003cp\u003eDocking results (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e) showed that Diclazuril exhibited the strongest binding affinities, particularly with CDPK (\u0026ndash;8.2 kcal/mol), followed by Toltrazuril (\u0026ndash;7.7 kcal/mol). Diclazuril formed multiple hydrogen bonds (e.g., TYR339, SER340, TYR98) and hydrophobic interactions (e.g., ILE342, LEU70, LEU80). Toltrazuril displayed strong interactions through hydrogen bonds (GLY317, SER287, THR95), hydrophobic contacts (CYS111, LEU148, LYS50), and halogen bonding, which may enhance its target binding stability.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmong the fatty acids identified in OFI-SO and OFI-HexPC, linoleic, oleic, palmitic, and stearic acids displayed moderate binding energies (\u0026ndash;5.4 to \u0026minus;\u0026thinsp;6.5 kcal/mol). These values are consistent with other phytochemicals such as apigenin (\u0026ndash;6.44 kcal/mol) and artemisinin (\u0026ndash;5.83 kcal/mol) (Aljedaie et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The binding modes were predominantly stabilized by hydrophobic interactions within the active sites, suggesting a potential for partial enzymatic inhibition.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlthough the fatty acids exhibited weaker binding affinities than synthetic anticoccidials, their interaction profiles support a plausible mechanism of enzymatic interference, complementing the membrane-disruptive effects observed in vitro. In particular, linoleic acid demonstrated consistent binding behavior across all three targets and may significantly contribute to the dual anticoccidial action of OFI-SO and OFI-HexPC.\u003c/p\u003e\u003cp\u003eThese findings support the hypothesis that the anticoccidial effects of \u003cem\u003eOpuntia ficus-indica\u003c/em\u003e seed-derived extracts arise from a synergistic mechanism involving both structural damage to oocyst membranes and targeted inhibition of key parasite enzymes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of ADMET Properties\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe ADMET analysis (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) highlights that Diclazuril and Toltrazuril, used as reference anticoccidial drugs, display favorable pharmacokinetic properties but also present notable toxicological risks. Toltrazuril shows high respiratory toxicity and hormonal receptor interaction, while Diclazuril exhibits marked neurotoxicity, carcinogenic potential, and thyroid receptor binding. These toxic effects raise concerns about their safety for prolonged use in animals.\u003c/p\u003e\u003cp\u003eIn contrast, the fatty acids evaluated in this study, linoleic, oleic, palmitic, and stearic acids, derived from OFI-SO and OFI-HexPC, demonstrate a favorable ADMET profile. They do not show hepatotoxicity, neurotoxicity, respiratory toxicity, carcinogenic activity, or cytochrome P450 inhibition. While only palmitic acid crosses the blood-brain barrier, all compounds display acceptable bioavailability and pharmacokinetic behavior. In addition to their low toxicity, these fatty acids showed notable biological activity in molecular docking analyses, reinforcing their potential as natural, safer anticoccidial agents. Their ability to target specific molecular pathways, combined with good tolerability, supports their inclusion in the development of alternative therapeutic formulations.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAnalysis of ADMET Properties and Toxicity Comparison: Toltrazuril, Diclazuril, and Fatty Acids\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\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMolcule\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eToltrazuril\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDiclazuril\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLinoleic Acid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eOleic Acid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePalmitic Acid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eStearic Acid\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003ePhysicochemical Properties\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMW\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e425.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e346.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e280.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e282.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e256.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e284.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCsp3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNRB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNHA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNHD\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\u003e1\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\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTPSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e111.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e91.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e37.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e37.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e37.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e37.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLipophilicity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLog P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWater Solubility\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLog S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eModerately\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eModerately\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eModerately\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eModerately\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eModerately\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eModerately\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePharmacokinetics\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHIA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBBB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eDrug-likeness\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLipinski\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\u003e0\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\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGhose\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\u003e0\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\u003e1\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\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVeber\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\u003e0\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\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEgan\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\u003e0\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\u003e1\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\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMuegge\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\u003e0\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\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedicinal Chemistry\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePAINS\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\u003e0\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\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eOrgan toxicity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHepatotoxicity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eActive (0.67)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eActive (0.53)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInactive (0.55)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eInactive (0.55)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInactive (0.52)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eInactive (0.52)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNeurotoxicity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eActive (0.73)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eActive (0.88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInactive (0.91)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eInactive (0.91)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInactive (0.92)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eInactive (0.92)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRespiratory toxicity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eActive (0.75)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eActive (0.56)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInactive (0.84)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eInactive (0.84)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInactive (0.85)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eInactive (0.85)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eToxicity end points\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCarcinogenicity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eActive (0.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eActive (0.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInactive (0.64)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eInactive (0.64)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInactive (0.63)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eInactive (0.63)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTHRβ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInactive (0.61)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eActive (0.66)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInactive (0.91)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eInactive (0.78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInactive (0.78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eInactive (0.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eClinical toxicity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eActive (0.61)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eActive (0.58)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInactive (0.61)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eInactive (0.61)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInactive (0.64)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eInactive (0.64)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003eMetabolism\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCYP1A2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInactive (0.69)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInactive (0.64)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInactive (0.91)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eInactive (0.91)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInactive (0.84)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eInactive (0.84)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCYP2C19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInactive (0.78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInactive (0.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInactive (0.98)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eInactive (0.98)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInactive (0.98)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eInactive (0.98)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCYP2C9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInactive (0.57)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInactive (0.65)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInactive (0.69)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eInactive (0.69)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInactive (0.71)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eInactive (0.71)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCYP2D6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInactive (0.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInactive (0.86)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInactive (0.88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eInactive (0.88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInactive (0.88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eInactive (0.88)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCYP3A4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInactive (0.58)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInactive (0.88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInactive (1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eInactive (1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInactive (1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eInactive (1.0)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCYP2E1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInactive (0.99)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInactive (1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInactive (0.99)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eInactive (0.99)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInactive (0.99)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eInactive (0.99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003cb\u003eMW\u003c/b\u003e =Molecular Weight below 500, \u003cb\u003eNHA\u003c/b\u003e=Num. H\u0026minus;bond Acceptors, \u003cb\u003eNHD\u003c/b\u003e=Num. H\u0026minus;bond donors, \u003cb\u003eNRB\u003c/b\u003e=Num. rotatable bonds, \u003cb\u003eFsp3\u003c/b\u003e =Fraction Csp3 acceptors, \u003cb\u003eHIA\u003c/b\u003e= Human Intestinal Absorption, \u003cb\u003eCYP\u003c/b\u003e= Cytochrome P450, \u003cb\u003eBBB\u003c/b\u003e = Blood\u0026minus;Brain Barrier, \u003cb\u003ePAINS\u003c/b\u003e=Pan Assay Interference compounds or frequent hitters or promiscuous compounds, \u003cb\u003eLog P\u003c/b\u003e: logarithm of the compound partition coefficient between n\u0026minus;octanol and water (\u0026minus; 2 to 5), \u003cb\u003eTPSA\u003c/b\u003e= Polar surface area (0\u0026ndash;140), \u003cb\u003eTHRβ\u003c/b\u003e =Thyroid hormone receptor bet\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides compelling evidence for the anticoccidial efficacy of OFI-SO and OFI-HexPC, based on integrated in vitro and in silico investigations. OFI-SO demonstrated a pronounced oocysticidal effect, comparable to that of the synthetic anticoccidial Toltrazuril. This activity is likely attributable to its high content of bioactive unsaturated fatty acids, particularly linoleic and oleic acids.\u003c/p\u003e\u003cp\u003eMolecular docking analyses confirmed that these compounds are capable of interacting with key \u003cem\u003eEimeria\u003c/em\u003e enzymes, notably EtDHODH, CDPK, and PKA, suggesting a dual mode of action involving both membrane disruption and enzymatic inhibition. This mechanistic synergy enhances their potential as effective antiparasitic agents.\u003c/p\u003e\u003cp\u003eFurthermore, ADMET predictions revealed favorable pharmacokinetic and safety profiles for the tested fatty acids, contrasting with the organ-specific toxicities commonly associated with synthetic anticoccidials. These data support the candidacy of OFI-SO and OFI-HexPC as natural, safer alternatives to conventional chemoprophylactic agents in poultry production.\u003c/p\u003e\u003cp\u003eFuture studies should focus on in vivo validation, dosage optimization, and large-scale application under commercial farming conditions to fully establish the practical utility of these phytogenic extracts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate:\u003c/h2\u003e\u003cp\u003eAll authors approved.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003cp\u003eAll authors agree to the publication.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis research was not funded\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to express their gratitude to the Ministry of Higher Education and Scientific Research and the General Directorate of Scientific Research and Technological Development (DGRSDT) for their support. The authors also gratefully acknowledge the NOPALTEC cooperative (Souk Ahras, Algeria) for providing the plant material and technical assistance. Special thanks are extended to the staff of the Parasitology Laboratory at the Biotechnology Research Center (C.R.Bt) for their valuable support in the collection and morphological identification of Eimeria spp. oocysts. We also acknowledge the Environmental Laboratory, represented by Dr. Cherb Nora, for her collaboration and technical contributions during the experimental procedures.\u003c/p\u003e\u003ch2\u003eData availability statement:\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbas RZ, Abbas A, Raza MA, Khan MK, Saleemi MK, Saeed Z (2019) In vitro anticoccidial activity of Trachyspermum ammi (Ajwain) extract on oocysts of Eimeria species of Chicken. Advancements Life Sci 7:44\u0026ndash;47\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbbas RZ, Colwell DD, Gilleard J (2012) Botanicals: an alternative approach for the control of avian coccidiosis. 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Vet Parasitol 96:257\u0026ndash;263. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0304-4017(01)00385-5\u003c/span\u003e\u003cspan address=\"10.1016/S0304-4017(01)00385-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-parasitologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"actp","sideBox":"Learn more about [Acta Parasitologica](http://link.springer.com/journal/11686)","snPcode":"11686","submissionUrl":"https://submission.springernature.com/new-submission/11686/3","title":"Acta Parasitologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Eimeria spp, Anticoccidial activity, Phytogenic compounds, Opuntia ficus-indica, in vitro, GC-MS, Molecular docking","lastPublishedDoi":"10.21203/rs.3.rs-7156619/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7156619/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe anticoccidial potential of \u003cem\u003eOpuntia ficus-indica\u003c/em\u003e seed oil (OFI-SO) and its hexane extract from seed press cake (OFI-HexPC) was evaluated against \u003cem\u003eEimeria spp\u003c/em\u003e. using integrated in vitro and in silico approaches.\u003c/p\u003e\u003cp\u003eSporulated Eimeria oocysts were exposed to increasing concentrations (2\u0026ndash;64 mg/mL) of OFI-SO and OFI-HexPC in Hank\u0026rsquo;s balanced salt solution. Oocyst viability was determined by microscopic counting, while membrane integrity was assessed by quantifying the release of 273 nm-absorbing substances. Toltrazuril (25 mg/mL) was used as a positive control.\u003c/p\u003e\u003cp\u003eGC-MS profiling of both extracts revealed linoleic acid (54.2%) and oleic acid (25.7%) as the main bioactive fatty acids.\u003c/p\u003e\u003cp\u003eGC\u0026ndash;MS profiling revealed that linoleic acid (69.63%) was the major fatty acid in OFI-SO, while oleic acid (44.20%) predominated in OFI-HexPC. Both OFI-SO and OFI-HexPC significantly reduced oocyst counts and induced a dose-dependent increase in DNA release.\u003c/p\u003e\u003cp\u003eIn silico molecular docking was performed against three key \u003cem\u003eEimeria\u003c/em\u003e enzymes (EtDHODH, CDPK, and PKA). Identified fatty acids showed moderate binding affinities through hydrophobic interactions. Redocking validation (RMSD\u0026thinsp;\u0026lt;\u0026thinsp;2 \u0026Aring;) confirmed the reliability of the docking protocol. ADMET predictions indicated favorable pharmacokinetics and safety profiles.\u003c/p\u003e\u003cp\u003eThese findings suggest that OFI-SO and OFI-HexPC act via a dual mechanism involving membrane disruption and enzymatic inhibition, supporting their potential as natural alternatives to synthetic anticoccidials in poultry production.\u003c/p\u003e","manuscriptTitle":"In Vitro and In Silico Anticoccidial Activity of Opuntia ficus-indica Seed Oil and Hexane Extract: Comprehensive GC-MS Profiling and Mechanistic Insights","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-25 07:03:20","doi":"10.21203/rs.3.rs-7156619/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-20T09:56:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-18T16:58:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-11T11:01:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-02T03:10:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95078254378137911193222411056860970505","date":"2025-07-25T15:57:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68097578408674482990323683505722386135","date":"2025-07-24T21:16:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299900236060170778050571836042079145174","date":"2025-07-23T09:15:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55402246128906042571248033493048783714","date":"2025-07-23T08:18:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-23T07:13:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-19T04:47:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-19T04:47:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Parasitologica","date":"2025-07-18T10:16:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-parasitologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"actp","sideBox":"Learn more about [Acta Parasitologica](http://link.springer.com/journal/11686)","snPcode":"11686","submissionUrl":"https://submission.springernature.com/new-submission/11686/3","title":"Acta Parasitologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"193c1897-0cd3-4dc1-85da-03550b1d8fee","owner":[],"postedDate":"July 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T16:00:16+00:00","versionOfRecord":{"articleIdentity":"rs-7156619","link":"https://doi.org/10.1007/s11686-025-01137-5","journal":{"identity":"acta-parasitologica","isVorOnly":false,"title":"Acta Parasitologica"},"publishedOn":"2025-10-16 15:57:14","publishedOnDateReadable":"October 16th, 2025"},"versionCreatedAt":"2025-07-25 07:03:20","video":"","vorDoi":"10.1007/s11686-025-01137-5","vorDoiUrl":"https://doi.org/10.1007/s11686-025-01137-5","workflowStages":[]},"version":"v1","identity":"rs-7156619","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7156619","identity":"rs-7156619","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}