Infundibulicybe geotropa (Bull.) Harmaja is an edible mushroom found in Bolu province in northwestern Turkey. The chemical composition and bioactivity of these mushrooms has not been previously investigated. We examined the phenolic composition, elemental content, and antioxidant and antigenotoxic effects of methanol extracts of fruiting bodies. The phenolic compounds in the fungal samples were determined using high-performance liquid chromatography (HPLC), and element content was determined using atomic absorption spectrophotometry. Total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) were determined using the commercially available Rel assay kit. The antigenotoxic effects of the extract were determined using the MTT assay to assess cell viability and the alkaline single-cell gel electrophoresis assay (Comet assay). The total phenolic content (ppm) of I. geotropa was found to be catechin (361 ± 2.31), clorogenic acid (553.54 ± 5.06), and coumaric acid (9.93 ± 0.25). The TAS, TOS, and OSI of the extract were 1.854 ± 0.051 mmol/L, 30.385 ± 0.399 μmol/L, and 1.639 ± 0.067, respectively. The elemental levels were within “normal” range. In HT22 mouse hippocampal neuronal cells, the extract (100 and 200 μg/ml) showed no genotoxic potential and ameliorated hydrogen peroxide- (H2O2-) induced oxidative DNA damage. I. geotropa may be considered a good nutrient due to its phenolic constituents and antioxidant potential. In recent years, the search for natural sources of functional nutrients as a food supplement has become common. Thus, it is quite important to investigate mushrooms, as a natural source of nutrients which have demonstrated potent antioxidant, anti-inflammatory, antitumoral, immunomodulatory, cardioprotective, hepatoprotective, and neuroprotective properties –5. SinceIncreased production of free radicals in living organisms and insufficiency of endogenous antioxidants against this increase can result in deterioration of several molecular structures, including lipid, proteins, and nucleic acids. Metabolic and chronic diseases such as cardiovascular disorders, diabetes, cancer, cataracts, muscle degeneration, and neurological diseases such as Parkinson's disease and Alzheimer's disease, are associated with an imbalance between oxidative stress formation and endogenous antioxidant defense mechanisms –18. A gr2O2 radicals, methyl methanesulfonate, cyclophosphamide, and 2-amino-3-methylimidazoquinolone) in several cell lines, including Burkitt's lymphoma cells, Chinese hamster lung fibroblastic V79 cells, human laryngeal epidermoid carcinoma HEp2 cells, human hepatoma HepG2 cells, and human lymphocytes [The beneficial effects of edible mushrooms have been previously investigated. Mushrooms have been shown to scavenge reactive oxygen and reactive nitrogen species, chelate redox-active metals, inhibition lipid peroxidation, and protein carbonyl formation, increase the levels of endogenous antioxidants such as vitamin C and glutathione, and increase the activity of antioxidant enzymes such as glutathione reductase and catalase –23. Sevephocytes , 25. Morphocytes . Edible phocytes . MushrooInfundibulicybe contains 19 widespread species that are mostly edible. They grow saprotrophic on litter of leaves and needles or can be found in soils in forests, grassland, and alpine habitats. Unlike some other edible mushrooms, Infundibulicybe do not have a distinct or foul smell [Infundibulicybe geotropa (Bull.) Harmaja mushroom. We also assessed its antioxidant and antigenotoxic capacity in vitro. We also examined the heavy metal content of these mushrooms to evaluate the nutritional value of these mushrooms as a supplementary nutrient source in humans.The genus ul smell . HoweverInfundibulicybe geotropa samples were collected in Bolu province (Turkey). Morphological and ecological characteristics of the samples were noted and photographed in their natural habitats. After field studies, specimen were taken to the laboratory and confirmation of the mushrooms were made by mycological experts at Akdeniz University, Turkey. Micromorphological characteristics were observed by light microscopy using 3% KOH, Congo Red, and distilled water. The names of taxa and authors are quoted according to MycoBank (http://www.mycobank.org/) and Index Fungorum (http://www.indexfungorum.org/).Mushroom samples obtained during field studies were identified and dried at +40°C . Then, the samples were pulverized. Afterwards, 10 g of the pulverized samples was macerated (24 h), with 200 mL EtOH (ethanol), MeOH (methanol), and DCM (dichloromethane) using a magnetic stirrer (Gerhardt EV 14). The extracts obtained with the extraction process were condensed via filtration at 40°C with a rotary evaporator to prepare the samples for the experiments (Heidolph Laborota 4000 Rotary Evaporator). The extracts were stored at +4°C until the experiments were conducted.I. geotropa mushroom methanol extracts were analyzed using the Rel assay commercial assay kit  to determine the TAS, TOS, and OSI values. Trolox was used as the calibrator in the TAS analysis, and hydrogen peroxide was used as the calibrator in the TOS studies. To determine the OSI, the mmol unit of TAS and the μmol unit of the TOS were cross-converted, and the index value was expressed as percentage [rcentage , 30.μL of Reagent 1 was added to the wells on the 96-well plate. 12 μL of mushroom extract was placed on top. The absorbance was measured at 660 nm. Then, 30 μL of Reagent 2 was added and incubated at 37°C during 5 minutes. After incubation, the second absorbance was read at 660 nm. Standard 1 and Standard 2 included in the kit were measured in the same way. Processes for all mushroom extracts were repeated separately.The TAS assay kit contained Reagent 1 (Buffer), Reagent 2 , Standard 1 (1.00 mmol trolex Equiv./L), and Standard 2 (1.00 mmol trolex Equiv./L). 200 2O2 Equiv./L). For this dilution step, 5 μL of the Standard 2 was transferred to the Eppendorf and 1 mL of distilled water was added and vortexed. Then, 5 μL of this solution was placed into the Eppendorf and 1 ml of water was added and 20 mM H2O2 was prepared. This solution was reprepared each time. Then, 200 μL of Reagent 1 was first placed in the well on the Eliza plate and 30 μL of mushroom extract was added. The first absorbance was then read at 530 nm (first absorbance of the sample). After the measurement, 10 μL of Reagent 2 was added. It was then incubated for 5 min at 37°C and the second absorbance at 530 nm was read (second absorbance of the sample). The same procedure was repeated for Standard 2. Separate procedures were repeated for all mushroom samples [The TOS assay kit contained Reagent 1 (Assay buffer), Reagent 2 (Prochromogen solution), Standard 1 (Blank solution: distilled water), and Standard 2 (stock stabilized standard solution (SSSS): 800 mM HI. geotropa samples were determined using a SHIMADZU system HPLC device and a DAD detector [μL. Mobile phase A: 3% acetic acid and B: methanol was used, and the flow rate was adjusted to 0.8 mL per minute. Chromatographic separation was carried out with an Agilent Eclipse XDB-C18 column (250 × 4.6 mm id 5 μm) at 30°C.Phenolic compounds in methanol extracts of detector . The injI. geotropa mushroom samples were pulverized after the process of drying. One gram of dried sample was weighed and placed in 50 mL glass vials in 3 repeats. 10 mL concentrated HNO3 was added and left at room temperature for 24 h. The samples were heated on the heating plate until the formation of sediment on the surface. Then, 10 mL concentrated HCI was added to these flasks and the incineration process was repeated. Following the incineration, 20 mL diluted HCl was added to the samples and the product was filtered. The elemental content in the filtered samples was then determined with a PerkinElmer (AAS 400) atomic absorption spectrophotometer as previously described [escribed .5 cells/mL and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 1% glutamine, and 1% antibiotic/antifungal in an atmosphere containing 5% CO2 and 95% oxygen. All cell culture equipment were obtained from Invitrogen .HT22 mouse hippocampal neuronal cells  were cultured in a cell culture flask at a density of 1 × 104 cells/well. Stock solutions of compounds were prepared in 20 mM DMSO and diluted further in complete DMEM so that the final DMSO concentration was <0.5% (v/v). After 24 h, the cells were incubated with 50 μl of extracts at a range of concentrations (50–300 μg/ml) for 24 h/37°C in a humidified atmosphere containing 5% CO2 and 95% air. After this incubation, 20 μL of MTT (5 mg/mL) was added to each well and further incubated for 4 h at 37°C. The cells were dissolved in 100 μL of DMSO, and the plates were read at 570 nm using a scanning multiwell spectrophotometer. The validity of the MTT assay was confirmed by viable cell counts using Trypan blue [MTT assay was used to evaluate cell viability following exposure to mushroom methanol extracts as previously described . Brieflypan blue . The res2O2 (100 μM) was the positive control. After incubation with mushroom extract (100 and 200 μg/ml) for 24 h, cells were de-attached by trypsinisation for the Comet assay. For each treatment, the cell suspension were mixed with low-melting point agarose, of which 90 μL was added to 15 μL of the cell homogenate and placed on a microscope slide precoated with normal melting point agarose 1.0%. A coverslip was added, and the slides were placed on ice during 5 min. After solidification, the coverslips were removed and the slides were immersed in a lysis solution. The slides were kept frozen in the lysis solution (4°C) and protected from light for approximately 14 h. They were subsequently incubated in freshly prepared alkaline buffer for 20 min for DNA unwinding. Electrophoresis (20 min at 300 mA and 25 V) was carried out in the same buffer. The procedure was performed under dimmed light to prevent further DNA damage. After electrophoresis, the slides were rinsed with distilled water and neutralized in Tris 400 mM (pH 7.5) and left to dry overnight at room temperature. The dry slides were rehydrated for 3 min in distilled water and then fixed for 10 min, rinsed three times in distilled water, and dried for at least 5 h. The dry slides were rehydrated for 3 min in distilled water, stained, and constantly shaken for 25 min. The slides submerged in the stop solution were rinsed again and immediately tagged for analysis. Comets were visualized using ethidium bromide staining (20 μg/ml for 30 s) and a fluorescent microscope . The slides were analyzed under blind conditions by at least two individuals. DNA damage was given as DNA damage index (DI). The OpenComet plugin was used for comet scoring. The results were reported as % tail DNA.The antigenotoxicity/genotoxicity of methanol extracts of edible mushrooms was evaluated using the alkaline single-cell gel electrophoresis (Comet) assay as previously described . Brieflyt test was performed using Microsoft Excel. Results were considered significant when p < 0.05.All experiments were performed 5 times, and results expressed as mean ± standard deviation unless otherwise stated. Statistical analysis using Student's I. geotropa mushroom ethanol extracts were determined using HPLC, and the findings are presented in I. geotropa: 361.49 ± 2.31 ppm catechin; 553.54 ± 5.06 ppm chlorogenic acid, and 9.93 ± 0.25 ppm coumaric acid. It is well-established that catechin is a potent antioxidant compound that is involved in the inhibition of free radicals. It has also been shown to have protective effects against skin, breast, prostate, and lung cancers in addition to its antioxidant properties [I. geotropa (former name Clitocybe geotrapa) showed that, apart from the named phenolic compounds identified in this study, protocatechuic acid, p-hydoxybenzoic acid, absisic acid, and cinnamic acid may also be present in the mushroom [I. geotropa may be consumed as a natural source for antioxidant phenols and may exert important health benefits.In this study, the phenolic compounds in operties . Coumarioperties . Anothermushroom . TherefoI. geotropa did not report the oxidative stress status of the mushroom species. Limited information is available in the literature on the TAS, TOS, and OSI values in other edible mushrooms. In the present study, TAS (mmol/L), TOS (μmol/L), and OSI values were determined using the I. geotropa ethanol extract. The findings are presented in Mushrooms have the potential to contain numerous antioxidant enzymes and reduced coenzymes such as phenolic compounds as an effective electron source in several different forms and types. Furthermore, they are rich in antioxidant vitamins A, C, and E, and other natural products that could metabolically contain and produce several elements with redox potential and strong antioxidant character. Previous studies on Tricholoma terreum, Coprinus micaceus, Pleurotus eryngii, Auricularia polytricha, A. auricular, Omphalotus olearius, Macrolepiota procera, Trametes versicolor, Geastrum pectinatum, and Fomitopsis pinicola were 0.38, 0.46, 1.93, 0.93, 1.010, 2.836, 2.823, 0.820, 1.278, and 1.57 mmol/L, respectively [I. geotropa in our study was significantly lower compared to the reported values for P. eryngii, O. olearius, and M. procera mushrooms as determined in previous studies. We also found that the mushroom had a higher TAS value when compared to the other abovementioned mushrooms. These differences may be attributed to the count and types of phenolic constituents, the release of secondary minerals produced by endogenous and exogenous factors, variation in antioxidant vitamin levels, as well as changes in the level of enzymatic/nonenzymatic antioxidant molecules capable of altering the total antioxidant capacity.As shown in ectively , 41. Theμmol/L. Previous reports on mushroom TOS values demonstrated that the TOS values of T. terreum, C. micaceus, O. olearius, M. procera, A. auricular, and T. versicolor were 16.76, 16.87, 8.26, 10.35, 23.91, and 2.03 μmol/L, respectively [I. geotropa was generally much higher when compared to the findings obtained in previous studies. Differences between mushroom TOS values were noteworthy since the analyses were carried out by different research groups at different times and with different species of mushroom collected in different locations. The differences among the TOS values are likely due to the differences between the regions where they were collected and the impact of the differences between the metabolic processes of the different mushroom species on their capacity to produce and accumulate oxidant compounds. It is suggested that mushrooms or other naturally occurring products with high TOS values should be cautiously consumed. It is considered that high TOS values in plants may be due to the impact of environmental and metabolic factors, which may stimulate the production of free radicals to protect against harmful endogenous factors in the environment such as environmental pollutants and certain microorganisms and parasites. Therefore, our findings highlight the importance of collecting mushrooms to be used as nutrients or in drug design studies in areas that are free of environmental toxic agents and heavy metal pollution and to enhance public awareness of those who work in the field.Our study also reported that the TOS value of the mushroom ethanol extract was 30.385 ± 0.399 ectively , 41. OurT. terreum, C. micaceus, O. olearius, M. procera, A. auricula, T. versicolor, G. pectinatum, and F. pinicola, respectively [I. geotropa (1.639 ± 0.067) in our study was observed to be lower than that of T. terreum, C. micaceus, A. auricula, and T. versicolor mushrooms. The higher TOS values observed in the present study coupled with a higher TAS in the mushroom samples led to the observation of a lower OSI value. Oxidative stress induced by oxidant molecules was prevented by TAS, which covers the whole enzymatic and nonenzymatic systems, resulting in low OSI values.The OSI value reflects the rate of inhibition of the oxidant compounds produced by mushrooms due to environmental and/or inherent factors by the antioxidant compounds present in the organism. Previous studies on the determination of OSI values reported that the OSI was 4.41, 3.67, 0.29, 0.37, 2.37, 2.17, 1.08, and 0.13 for ectively , 41. TheI. geotropa was determined and presented in Elements also play a role as cofactors in the functioning of certain enzymes, which are a part of several metabolic pathways and the endogenous antioxidant system. Given the cofactor properties of the elements and their potential effects on homeostasis , the identification of elemental content of mushrooms is of great importance. Mushrooms physiologically accumulate elements at different levels based on the elemental content of the substrate they utilize. In the present study, the elemental content of I. geotropa was 63.70 ± 8.88, while the Zn content was 61.24 ± 12.04, Cu content was 30.37 ± 1.23, Pb content was 7.00 ± 1.38 and Ni content was 1.12 ± 0.053 mg·kg−1. A previous study reported that the Fe content in I. geotropa mushroom collected in Muğla province (Turkey) was 662.0, Zn content was 130.4, Cu content was 65.6, Pb content was 3.2, and Ni content was 4.5 mg·kg−1 [I. geotropa mushroom collected in Eskişehir province (Turkey) was 516.7, Zn content was 86.6, Cu content was 82.4, Pb content was 1.22, and Ni content was 14.0 mg·kg−1 [I. geotropa collected in Bolu (Turkey) were lower. However, the Pb content was higher in the present study when compared to both abovementioned studies. The difference in the level of these elements stems from variation in the elemental soil content between the regions where the mushrooms were collected. Furthermore, it is considered that the high Pb content may be since the area where the mushrooms were collected in the present study was located near a busy highway.The Fe content in  mg·kg−1 . It was  mg·kg−1 . ComparaI. geotropa. This amount is much higher than an individual's daily iron requirement. Thus, it is considered that the consumption of the mushroom samples collected in suitable regions at certain intervals may be beneficial in meeting the physical Fe requirement, and the findings of the present study would be beneficial in making recommendations for the regular consumption of mushrooms collected in reliable localities.Fe is a very important element that is necessary in different amounts in different periods for the human body. Fe deficit leads to several diseases, especially anemia . Fe requirement is met by breast milk for the 6 postnatal months. Approximately 0.7-0.9 mg/day of Fe is utilized after 6 months . Between the ages of 1 and 6, the body needs twice as much iron and 1.17 mg/day Fe is needed between the ages of 6 and 15. Furthermore, male individuals aged 12-16 years require 1.82 mg/day of iron, while female individuals require 2.02 mg/day. During pregnancy, the Fe requirement is 1.14 mg/day, and an adult female needs approximately 2 mg/day of iron during menstruation and premenopausal period . In the −1 in most food products. However, cocoa products and peanuts may contain up to 3 to 10 mg·kg−1 Ni [I. geotropa mushroom is 1.12 mg and did not reach toxic levels. On the other hand, it was determined that the lowest and highest mushroom Pb content was 2.86-15.3 while the Zn and Cu contents were 29.8-158 and 6-187 mg·kg−1, respectively, in previous studies by our group [I. geotropa appeared to be within normal ranges identified in the literature. Thus, it was determined that the I. geotropa samples collected in Bolu (Turkey) possessed optimal element levels, and consumption of the mushroom is adequate for human health. However, controlled consumption and avoiding consumption at extreme levels are recommended.Ni content is lower than 0.5 mg·kg·kg−1 Ni . It can ur group , 42, 46.I. geotropa extract in neuronal cells. Our study investigated the effects of I. geotropa in HT22 cells. Our data shows that I. geotropa extracts did not significantly reduce cell viability at 50-300 μg/ml after 24 h  compared to nontreated control cells (Comet head only). After 24 h, treatment with I. geotropa extract (100 and 200 μg/ml) ameliorated the increase in DNA damage due to H2O2. The genoprotective effect of I. geotropa may be attributed to its phenolic content and high TAS levels. We hypothesize that I. geotropa extract may exert protection against H2O2 by either direct scavenging of free radicals and/or upregulation of endogenous antioxidant enzymes  and antioxidant concentrations  which detoxify H2O2. Other mushroom extracts  have been shown to protect against induced DNA damage in Raji cells [A. bisporus extracts have also been attributed to enhanced tyrosinase activity, which can enhance endogenous antioxidant defense mechanisms leading to increase glutathione levels [To the best of our knowledge, there are no previous reports of the toxicity of ter 24 h . Other m 2 mg/ml . We alsoet assay . This veji cells . Moreovee levels .A. blazei, was shown to be genotoxic to HTC rat hepatoma cells. However, this effect was likely due to the generation of cytotoxic metabolites in vitro [I. geotropa extracts in murine HT22 neuronal cells is encouraging and warrants further investigation to elucidate the modes of action underlying their antigenotoxic effects.While most studies have shown that edible mushrooms are safe and well tolerated, some studies have shown that they may exhibit some genotoxic potential. For example, extracts of in vitro . The genInfundibulicybe geotropa mushroom collected in Bolu province (Turkey) was determined. Catechin, chlorogenic acid, and coumaric acid were determined in the mushroom in the present study. It was considered that this mushroom species may be a natural resource, rich in the determined compounds. It was also found that the mushroom has a strong antioxidant potential. However, it has been suggested that fungi samples collected in more reliable regions containing lower oxidant levels may serve as a better antioxidant source. The extract also showed excellent protection against oxidative DNA damage and lacks genotoxic potential in vitro. The elemental content was at optimal levels as well, suggesting that this extract may be used as an antioxidant with little or no adverse effects. However, additional in vivo studies are necessary to confirm these assumptions.Mushrooms provide significant opportunities in the fields of pharmacological agent development, drug design, and medicine due to their structural properties and exhibit various metabolic activities in addition to their nutritional properties. In this study, the biological activity in


Staphylococcus aureus are a serious global threat, and with the emergence of antibiotic resistance, even more difficult to treat. One of the possible complications in antistaphylococcal therapy represents negative interactions of antibiotics with food. In this study, the in vitro interaction between oxacillin and crude palm seed oil from Astrocaryum vulgare, Cocos nucifera, and Elaeis guineensis against nine strains of S. aureus was determined using the checkerboard method. Lauric acid was identified as a major constituent of all tested oils by gas chromatography. The results showed strong concentration dependent antagonistic interactions between palm oils and oxacillin with values of fractional inhibitory concentrations indices ranging from 4.02 to 8.56 at concentrations equal or higher than 1024 µg/mL of the tested oils. Similarly, lauric acid in combination with oxacillin produced antagonistic action with fractional inhibitory concentration indices ranging from 4.01 to 4.28 at 1024 µg/mL. These findings suggest that interference between oxacillin and palm oils and their constituents can negatively affect the treatment of staphylococcal infections in humans and other animals.Infections caused by  Staphylococcus aureus, a gram-positive bacterium, occurs naturally on the skin and mucus membranes of healthy individuals and is a common cause of pneumonia, skin infections, and systemic infections in humans and other animals1. Its ability to resist a broad range of antibacterials in a short period makes it one of the most dangerous microorganisms influencing the global population, with strains resistant to beta-lactams, such as methicillin-resistant S. aureus (MRSA) being considered a high priority pathogen2. However, currently used antistaphylococcal antibiotics  are struggling against rising resistance, serious side effects, and relatively high costs3. Hence, steps toward elimination of bacteremia caused by multi-drug resistant strains are still needed. Among other possible options, combinatory treatment is valuable for eradicating evolution of resistance by targeting different sites of the bacterial cell5. For example, the combination of vancomycin and a beta-lactam antibiotic (oxacillin) is currently being used effectively in the treatment of MRSA6. A combination of beta-lactam antibiotic and beta-lactamase inhibitors, such as co-amoxiclav consisting of amoxicillin and clavulanic acid, has also been used for MRSA therapy7. However, combinations of certain antibiotics can produce negative interactions and undesirable side effects. Antagonistic action generally occurs in the treatment of infection when mixing bactericidal and bacteriostatic drugs8, as seen when using a combination of the slow acting bactericidal vancomycin and bacteriostatic clindamycin against S. aureus9. Generally faster acting agents, such as clindamycin or oxacillin, inhibit the function of vancomycin, which has a gradual onset of action and exhibits antibacterial properties only on replicating cells10.11. Such interactions are also known to occur when combining drugs with various foods and food products. Drug-food interactions are defined as changes in efficacy and/or toxicity of pharmaceutical drugs induced by the consumption of any food product, including functional foods and dietary supplements12. Various drug–food interactions , drug–nutrient interactions  and herb–drug interactions  have been described and reviewed16. The most well-known example of drug–food interaction is that of grapefruit juice that can inhibit the intestinal metabolism of more than 85 drugs by altering cytochrome P450 (CYP3A4) isoforms17. Grapefruit juice, combined with erythromycin, increases the bioavailability of the antibiotic in the small intestine18, thereby increasing the possibility of adverse effects, such as cardiac dysrhythmias19. In contrast, a combination of ampicillin and pomegranate methanol extract acts synergistically in vitro against MRSA20. Similarly, fatty acid methyl esters obtained from soybean, corn, and sunflower crude oils potentiate the antifungal effect of itraconazole in vitro21.Combining various drugs is the prevalent praxis used to obtain an increase of the desired effects, such as in anesthesia and pain management. However, enhancement of the undesired effects might also occur, limiting the therapeutic value22, coenzyme Q1023, and fatty acids24. These are carboxylic acids with long, unbranched carbon chains, some of which may contain double bonds25. In general, fatty acids are believed to be responsible for the antibacterial activity of palm oils. They usually occur in the form of triglycerides; their hydrolysis into free fatty acids is necessary for their antibacterial effect to be exerted28. The seeds of tropical palms such as tucuma (Astrocaryum vulgare), coconut (Cocos nucifera), and African oil (Elaeis guineensis) palm are one of the most economically important sources of plant oils, and are known to contain mainly fatty acids of medium-chain length (MCFAs), with a prevalence of lauric acid . Apart from various physiological functions, the LA produces a growth-inhibitory effect against algae29, fungi30, protozoa31, and both gram-negative34 and gram-positive bacteria35. It also inhibits the production of bacterial virulence factors such as beta-lactamases, a group of exoproteins inducing beta-lactams inactivation and moreover enhancing toxin production by S. aureus at subinhibitory antibiotic concentrations36; toxic shock syndrome toxins37; and hemolysins38. In addition to LA, tropical palms contain caproic (C6:0), caprylic (C8:0), and capric (C10:0) acids that also possess various antimicrobial properties41. In addition, MCFAs are known to be effective against S. aureus. Batovska et al.42 concluded that when these fatty acids are esterified with glycerol and create e.g., monolaurin, their direct antistaphylococcal effect is intensified. Ubgogu et al.43 observed a noticeable in vitro inhibitory effect of E. guineensis palm kernel oil against S. aureus. The mechanism of antimicrobial action of fatty acids is not fully known, the prime target seems to be the bacterial cell membrane together with various essential processes that occur within and at the membrane, including nutrient uptake or enzyme inhibition44. The amphiphilic nature of fatty acids enables them to act as detergents at high concentrations and aid the solubilization of the lipids in the membranes45. It has been experimentally proven that doses of LA equal to or higher than 100 µg/mL (≥ 500 µM) induce reversible morphological changes of lipid bilayers46, cause partial solubilization of the cell membrane, and interfere with metabolic regulation, leading to the inhibition of bacterial growth44. It is well known that bactericidal drugs are most potent with actively growing cells and that inhibition of growth, induced by a combination with a bacteriostatic drug, can result in reduction of drug efficacy8.Vegetable oils are essential components of animal nutrition and contain various biologically active constituents such as carotenoids, tocopherols42, lactic acid26, and gentamicin47. However, limited information is available regarding the possible negative interactions of palm oils with antimicrobial agents. Therefore, we decided to perform a screening test, focused on determining the combined effect of the chosen palm oils and free MCFAs (C6:0–C12:0) with representatives of all major antibiotic groups, namely beta-lactams , tetracyclines (tetracycline), glycopeptides (vancomycin), and aminoglycosides (gentamicin), against three reference strains of S. aureus. Among the free MCFAs, only LA showed above mentioned synergism with gentamicin against chosen S. aureus strains; the interactions of all free MCFAs with tetracycline and vancomycin were indifferent, but beta-lactams, namely amoxicillin, ampicillin and especially oxacillin, showed results that were warranted further investigation with LA showing the strongest antagonistic interactions . In the present study, we evaluated in vitro combinatory effect of A. vulgare, C. nucifera, and E. guineensis seed crude oils and their main constituent LA with oxacillin, using the checkerboard microdilution method, against different strains of S. aureus.Besides the antimicrobial properties, LA is known to exhibit synergistic antistaphylococcal activity in combination with monolaurinA. vulgare, C. nucifera, and E. guineensis consisted mainly of 58.2, 53.88 and 52.24 mg/g of MCFAs, respectively. LA was a major constituent of the oils, present at a concentrations of 53.37 mg/g in A. vulgare, 45.24 mg/g in E. guineensis, and 41.31 mg/g in C. nucifera oil. This profile corresponded with the total saturated fatty acid composition, where A. vulgare is followed by C. nucifera and E. guineensis oil with values of 90.32, 82.35, and 80.21 mg/g, respectively. The contents of other MCFAs, namely caprylic, capric, and caproic acids, were 6.73, 5.29, and 0.55 mg/g in C. nucifera, 3.48, 3.27, and 0.25 mg/g in E. guineensis, and 2.47, 2.15, and 0.21 mg/g in A. vulgare oil, respectively. In addition to MCFAs, saturated fatty acids consisted of myristic, palmitic, and stearic acids. Their respective contents were 24.82, 5.41, and 1.89 mg/g in A. vulgare; 16.5, 9.05, and 2.92 mg/g in C. nucifera; and 15.85, 9.46, and 2.66 mg/g in E. guineensis oil.In the first part of the study, fatty acid composition of the tested oils was identified using GC-FID. As shown in Table E. guineensis oil (16.67 mg/g), and were present at relatively lower concentrations in C. nucifera and A. vulgare oil . The oleic acid content was the highest in all samples, ranging from 6.47 to 16.54 mg/g. In contrast, eicosenoic (0.05–0.15 mg/g) and palmitoleic acid (≤ 0.03 mg/g) were present in minor amounts.Although saturated fatty acids were the major constituents of all oils analysed, the composition of monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids was also determined. MUFAs, consisting of oleic, eicosenoic, and palmitoleic acids, were the most abundant in C. nucifera oil (5.8 mg/g) was double that of A. vulgare (2.9 mg/g) and E. guineensis (2.84 mg/g) oil. In C. nucifera oil, linoleic acid was the most abundant PUFA, similar to A. vulgare and E. guineensis oil at 4.79, 2.77, and 2.68 mg/g, respectively. The second most abundant PUFA in C. nucifera oil was α-linoleic acid (1.88 mg/g), which was also present in lower amounts in A. vulgare (0.06 mg/g) and E. guineensis (0.03 mg/g) oil. Compared to C. nucifera and A. vulgare oil (both 0.13 mg/g), arachidonic acid was the least abundant PUFA in E. guineensis oil (0.07 mg/g).PUFA content in S. aureus strains to oxacillin and hydrolyzed seed oils of A. vulgare, C. nucifera, and E. guineensis was determined using broth microdilution method to evaluate the suitable starting concentrations for combined effect . A. vulgare oil induced the strongest antistaphylococcal effect, with MIC values ranging from 240 to 356 µg/mL, followed by that of C. nucifera  and E. guineensis . The MIC values of oxacillin ranged from 0.72 to 56.89 µg/mL.In the first step, the susceptibility of the three tested S. aureus strains. All other combinations showed an indifferent relationship between tested agents, as shown in Table Further, analysis of the combined effect of tested seed crude oils and oxacillin was performed by the checkerboard method. The results showed some degree of antagonism in all tested strains at certain concentrations of combinations, with indices of fractional inhibitory concentration (FICI) ranging between 8.56 and 4.02. In addition, a consistently antagonistic, concentration-dependent effect of the selected palm oils at concentration of 2048 µg/mL, and in some cases 1024 µg/mL, was seen, when combined with oxacillin against all tested A. vulgare oil at a concentration 2048 µg/mL, when combined with oxacillin. At the same concentration, A. vulgare oil caused an adverse interaction in MRSA ATCC 43300 with a FICI of 5.78. This antagonistic effect was also observed at a concentration of 1024 µg/mL with the MSSA clinical isolate, SA1 (FICI 4.30), and the reference strain, ATCC 29213 (FICI 4.27). Indifferent relationships were displayed in all other combinations for both agents. Interestingly, a significant increase in MIC of oxacillin was observed when combined with A. vulgare oil at two lowest concentrations tested (32 and 16 µg/mL). In these cases, the MIC values rose by approximately 1/3 and 3/4, respectively, when compared to the MIC of the antibiotic alone, against MSSA ATTC 29213. In combination with oxacillin, C. nucifera and E. guineensis oils also exerted antagonistic properties in all tested strains, at a concentration of 2048 µg/mL (FICI 4.02–6.28). C. nucifera oil showed the strongest antagonistic effect for MRSA ATCC 43300 strain, followed by MRSA clinical isolate SA1 and MSSA ATCC 29213 strain . E. guineensis oil exerted similar antagonistic activity in MSSA ATCC 29213, MRSA clinical isolate SA1, and MRSA ATCC 43300 strain, with FICI values of 4.88, 4.83, and 4.02, respectively. The rise of oxacillin MIC, when combined with the lowest concentrations (16–64 µg/mL) of the tested oils, was even more pronounced with C. nucifera. It induced an increase of MIC in MSSA ATCC 29213 strain up to more than two times. A slight increase of oxacillin MIC was also detected in ATCC 29213 strain at a concentration of 0.32 µg/mL of E. guineensis oil.A strong antagonistic effect, manifested by very high FIC indices of 8.56  and 8.51 (MSSA ATCC 29213), was observed for S. aureus strains to oxacillin, LA, and a combination of oxacillin and LA was investigated using the same methodology as that used for palm oils. As shown in Table S. aureus strains at 256 µg/mL, except for MRSA ATCC 43300, where the MIC of LA decreased to 242 µg/mL.As mentioned above, GC-FID analysis revealed LA to be the predominant fatty acid in all tested oils. Based on this result, the susceptibility of nine S. aureus strains; the strongest antagonistic interaction was exhibited for the MRSA ATCC 43300 strain. Other combinations of concentrations revealed indifferent relationships, except for a combination of LA at 32 µg/mL with oxacillin against the S. aureus ATCC BAA 976 strain. In this case, the MIC of oxacillin increased four times, to 128 µg/mL, and the FICI value was 4.13. In all tested bacterial strains, rise of MIC values of tested antibiotics occurred when combined with LA at certain concentrations (16–256 µg/mL), up to two times with the average increase by nearly two-thirds.Subsequently, FICI values were calculated and the effect of combinations was analysed. Antagonistic mode of action was observed for LA at a concentration of 1024 µg/mL, when combined with oxacillin  in all tested A. vulgare, C. nucifera, and E. guineensis and LA influence the growth of S. aureus strains, the analysis of growth curves of three S. aureus strains, namely ATCC 29213, ATCC 43300, and clinical isolate SA1, was performed. According to Fig. S. aureus strains.To determine whether the hydrolyzed seed oils of S. aureus. As per the results of GC analysis, the oils used for antibacterial testing were of standard composition, as reported by other groups, who also identified LA as the major constituent of A. vulgare48, C. nucifera49, and E. guineensis oils50. In this study, the MIC values of LA against the tested S. aureus strains (242–256 µg/mL) were comparable to those reported by other authors. For example, Batovska et al.42 reported an MIC values for LA, measured by the macrodilution method against S. aureus strains at ≥ 125 µg/mL. Moreover, Nitbani et al.51 reported the antibacterial activity of LA isolated from C. nucifera oil against S. aureus. In contrast, Parsons et al.52 determined an MIC value of 50 µg/mL (250 µM) for LA against S. aureus, using the broth microdilution method. Similarly, Kelsey et al.53 observed MIC values equal to 50 µg/mL of LA against three different S. aureus strains, using turbidimetry with visual evaluation and ethanol as a solvent; however, these variations could be caused by the different methodologies and S. aureus strains used. To the best of our knowledge, there is only limited information on the antibacterial properties of palm oils rich in MCFAs. Free-fatty acids are known to have antibacterial activity in contrast with those bound to triglycerides27. As described by various authors previously52, the antistaphylococcal effect of fatty acids is induced by disruption of bacterial cell membrane resulting in its destabilization by inducing tubule formation on the lipid bilayer and cell lysis; thus, the antimicrobial activity of oils containing fatty acids is facilitated only upon hydrolysis of triglycerides28. In contrast, Ubgogu43 showed that E. guineensis oil exerted slight antibacterial effect on S. aureus, without getting hydrolyzed, using the disc diffusion technique. This result is the opposite of that observed in this study, where the oils acted as an antibacterial only after being hydrolyzed by the porcine pancreatic lipase. Rossato et al.54 also observed no antistaphylococcal activity of unhydrolyzed A. vulgare oil. Results of in vitro screening of C. nucifera oil and other MCFA-containing fats, with lipolytic enzymes that simulated gastric conditions in piglets, showed a significant change in suppression of gut microbiota 55. This finding suggests that MCFA-rich oils exert their antibacterial effects after enzymatic hydrolysis by lipases synthesized in the gastrointestinal tracts of humans and other animals.In our study, crude seed oils rich in MCFAs, hydrolyzed by porcine pancreatic lipase, and LA showed in vitro growth-inhibitory effect against reference strains and clinical isolates of S. aureus, can be found in literature56. However, the findings of these studies markedly vary, depending on the class of antimicrobial agent used. For example, Kitahara et al.47 observed synergistic interaction of LA at 50 µg/mL with gentamicin  and imipenem (0.13–0.25) and indifferent interactions of LA (50 µg/mL) with ampicillin and oxacillin, against MRSA clinical isolates. Similarly, Hess et al.57 observed indifferent interactions of the LA-ampicillin or vancomycin combination against S. aureus biofilms and a synergistic interaction between LA and streptomycin. We observed indifferent antistaphylococcal action of oxacillin and LA (50 µg/mL); however, increasing the amount (≥ 1024 µg/mL) of LA, produced strong antagonism in the presence of the antibiotic. The reason for the lack of antagonism in Kitahara et al.47 and Hess et al.57 could be the concentration of LA used in the study, which probably did not reach a sufficient value for the antagonism to be exerted. There is no information on the interaction of palm oils rich in MCFAs as their antibacterial activity was not confirmed in the study58 because hydrolysis of oils was not performed26.Studies on the combinatory effect of MCFAs and their esters with different organic acids, inorganic compounds, and antibiotics against various bacteria, including A. vulgare/C. nucifera/E. guineensis oil. It can be assumed that while the LA temporarily prevents the growth of bacterial cells, oxacillin is not able to properly exert its activity. However, it is not just the membrane lipid structure that can change under the influence of exogenous lipids but the protein structure as well. Other factors including membrane strain may account for the organization of membrane proteins59. It can be hypothesized that under increased membrane strain, such as after the treatment of lipid bilayers with high concentrations of LA46, membrane protein function is altered60. Such a circumstance can affect the antibacterial activity of oxacillin, which strongly depends on its ability to inhibit bacterial cell wall synthesis by preferentially binding to penicillin-binding proteins (PBPs) that are located inside the bacterial cell wall61. Therefore, oxacillin probably becomes ineffective after change in PBP function, induced by LA. As membrane protein stability also depends on membrane energetics, LA can reduce membrane fluidity and disrupt the electron transport system, perhaps by restricting the movement of carriers within the membrane44. The eventual impairment of membrane electron carriers can lead to a change in the intracellular and extracellular pH, which can cause the precipitation of PBPs62 and make them to lose the ability to interact with oxacillin. In addition, the change in extracellular pH can affect chemical structure of oxacillin as this drug is highly unstable in acidic environments63. Moreover, S. aureus is known to produce persisters, which are representing a fraction of the bacterial population that exhibits tolerance to antibiotics in response to various stresses64. According to the finding of Peyrusson et al.65, S. aureus in the presence of high concentration of various antibiotics including oxacillin showed a biphasic killing manner, meaning that a bulk of the bacterial population was susceptible and rapidly killed while a subpopulation with a slower killing rate was persisting for a much longer period of time, in addition showing the reversibility of the phenotype after antibiotic removal. Therefore, another possible explanation of antagonism between oxacillin and LA/LA-rich palm oils, can be in inducing persister cells of S. aureus in the presence of high concentrations of tested compounds.From the results presented in this study, it is not possible to infer by which mechanism the antagonism between LA, tested oils and oxacillin occurs. Nevertheless, the detected prolongation of the generation time resulting in the decrease of specific growth rate arising with increasing concentrations of LA and LA-rich tested palm oils indicates that a possible mechanism underlying the antagonistic interactions between tested compounds might lie in the halting of cell division caused by LA/66. For this reason, coadministration of a drug with specific foods is noted in medical leaflets. Generally, food intake can influence the effectiveness of an antibiotic67. Ingestion of food, dietary fiber, or milk reduces the bioavailability of most antibiotics, including some penicillins68. For example, minerals in milk and cheese create complexes with antibiotics that decrease their absorption69, and as seen in the case of isoxazolyl penicillins, when administered shortly before or after a meal, delayed gastric emptying and increased acidity interfere with their absorption70. The consumption of coconut oil and related products is currently growing among certain populations, for the claimed health benefits associated with cardiovascular disease and weight loss71. On the other hand, recommendations of lowering intake of saturated fatty acids and replacing them with unsaturated fatty acids exist in order to reduce risk of atherosclerosis and type-2 diabetes73. The average concentration of LA in human serum of healthy adult male and female blood donors, ages ranging from 18 to 55 years, was found to be < 10 µg/mL74. It has, however, been proven that higher (14.2–140 g/day) intakes of MCFAs in diet may result in higher concentrations of high density lipoprotein cholesterol than found with long-chain fatty acids, highlighting the importance of considering chain length when measuring the effect of dietary saturated fatty acids on lipid profile75. Moreover, in a high-carbohydrate, high-fat diet, the increases in systolic blood pressure and diastolic stiffness in the heart were inhibited in mice with diet enriched by virgin coconut oil, composed predominantly of LA, at concentrations of 200 g/kg76. Our results showing strong in vitro antagonistic effect of oxacillin with LA or LA-rich palm oils at concentrations ≥ 1024 µg/mL  suggest that simultaneous administration of these agents can negatively affect their pharmacological properties. The recommended intake of fats in the diet of men is estimated to be around 65 g/day 77. According to FAO78, average energy requirement of an adult female is 2410 kcal/day and of an adult men is 3100 kcal/day. Counting daily intake of fats as 30% of total energy requirement, the daily intake of fats can be estimated to be 80 g/day for women and 103 g/day for men. Studies on coconut oil supplementation in diet usually focus on the addition of the oil in range 20–50 g/day81. Nevertheless, there have been reports on even higher daily consumption of coconut oil reaching up to 80 g/day84. Therefore, the observed antagonistic action between LA-rich oils/LA may be important especially in high-fat diets. Thus, our results showing strong in vitro antagonistic effect of oxacillin with LA or LA-rich palm oils suggest that simultaneous administration of these agents in high, but still reachable concentrations can negatively affect their pharmacological properties in the treatment of S. aureus. The risk of antagonistic interactions between oxacillin and LA-rich oils might be primarily important to systemic application, as their antibacterial effect is attributed to fatty acids unleashed from triglycerides only, therefore the topical application of LA-rich oils such as A. vulgare/C. nucifera/E. guineensis should not influence the antibacterial activity of oxacillin. But, according to Verallo-Rowell et al.85, 5 mL of extra virgin coconut oil applied two times a day on the affected areas that include the test sites is able to decolonise skin from S. aureus in adults with atopic dermatitis. This discrepancies between the theoretical background and practice can be debited to the lipolytic activity of skin microbiota, including staphylococci86, highlighting the importance of possible negative effect of LA-rich oils on topical treatment of S. aureus. Moreover, LA was previously tested in vitro at concentration of 0.24–500 μg/mL to evaluate its antibacterial properties against various bacteria causing inflammatory acne vulgaris, including S. aureus, proposing it as an promising remedy in the treatment of staphylococcal skin infections87. However, the mentioned tested concentrations of LA were not high enough for the antagonism with oxacillin to be exerted. Nevertheless, these hypotheses must be confirmed by further in vitro and in vivo tests and clinical trials because physiological processes can also induce changes in antibacterial activity of tested compounds.Food-drug interactions are a major threat to safe and effective oral pharmacotherapy and can result in decreased bioavailability of a drug, which predisposes the patient to treatment failure, increases the risk of adverse events, and may even precipitate toxicitiesA. vulgare, C. nucifera, and E. guineensis oils when combined with oxacillin in higher amounts against various strains of S. aureus. The strongest antagonism was observed for A. vulgare oil, which contains the highest amount of LA. This compound was identified as the main agent responsible for antagonistic antistaphylococcal action of all oils assayed. To the best of our knowledge, this is the first study to report the antagonistic interactions between these agents. The mechanism underlying the antagonistic action of tested agents probably acts at the cellular level and is linked to the cell membranes. These findings suggest that interference between oxacillin and palm oils and their constituents can negatively affect the treatment of staphylococcal infections in humans and animals. However, these assumptions are based on in vitro tests and the negative interactions of the above-mentioned combinations should be confirmed by in vivo trials.In summary, this in vitro study revealed a concentration-dependent antagonistic effect between C. nucifera and E. guineensis oils, and oxacillin sodium monohydrate were obtained from Sigma-Aldrich . A. vulgare oil was purchased from Sweet Natural Botanicals . LA and A. vulgare, C. nucifera, and E. guineensis oils were dispersed in dimethyl sulfoxide, which was previously proposed for sparingly soluble substances88, including the LA89 and palm oil90; and emulsified using Tween 80 , that is a common procedure of hydrophobic sample preparation91, not influencing the antibacterial properties of tested compounds when used in recommended concentrations93. Complete dissolution of LA was achieved by heating (70 °C for 10 min) it in an ultrasonic-bath. Analysed plant oils were selected based on the high content of LA as described in literature96. Hydrolysis of oils, to induce antimicrobial properties, was achieved by adding 5 × 10–3 mg/mL of porcine pancreatic lipase  and shaking for 1 h in a water-bath heated to 37 °C. Degree of hydrolysis of oils was dependent on the lipolytic activity of the enzyme. One unit of porcine pancreatic lipase hydrolyzes 1.0 microequivalent of fatty acid from triacetin in 1 h at pH 7.4, at 37 °C.LA, S. aureus strains were tested. Five reference strains including methicillin sensitive (MSSA) ATCC 29213, MRSA ATCC 33591, ATCC 33592, ATCC 43300 and ATCC BAA 976 were purchased from Oxoid . Three clinical isolates of drug resistant S. aureus  and one epidemic MRSA strain (EMRSA-15) were obtained from the Motol University Hospital . Oxacillin, gentamicin, tetracycline, and penicillin were used as markers of the antibiotic resistance as it has been previously determined99. Based on the MIC values, clinical isolates were identified to be resistant to: SA1—resistant to gentamicin (MIC 8 µg/mL) and tetracycline (MIC 8 µg/mL); SA2—resistant to oxacillin (MIC 68 µg/mL), gentamicin (MIC 16 µg/mL) and tetracycline (MIC 8 µg/mL); SA3—resistant to gentamicin (MIC 8 µg/mL) and penicillin (MIC 18.67 µg/mL); EMRSA-15—resistant to oxacillin (MIC 99.56 µg/mL) and penicillin (MIC 16 µg/mL). The clinical isolates were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), as described previously by Rondevaldova et al.98. Bacterial stocks were stored at 4 °C on Müller–Hinton agar . Working cultures were maintained in Müller-Hinton broth at 37 °C for 24 h before testing.In this study, nine A. vulgare, C. nucifera, and E. guineensis, alkaline trans-methylation of fatty acids was carried out as described by Raes et al.100. Analysis of methyl esters was performed using gas chromatography (GC) with the HP 6890 chromatograph , equipped with a 60 m DB-23 capillary column (60 m × 0.25 mm × 0.25 μm) and a flame-ionization detector (FID); split injections were performed using an Agilent autosampler. A total of 1 µL of standards in hexane were injected in split mode (1:40 ratio) into the injector, heated to 230 °C. The column temperature was initially set at 120 °C for 6 min then programmed to 170 °C at a rate of 15 °C/min. The temperate gradient was further configured to 210 °C at the rate of 3 °C/min and held for 13.5 min. Finally, the temperature was programmed to 230 °C at the rate of 40 °C/min and held for 7 min. Nitrogen was used as the carrier gas, at a flow rate of 0.8 mL/min. Supelco 37 Component FAME Mix, PUFA 1, PUFA 2, PUFA 3, trans-vaccenic acid, and a mixture of conjugated isomers of linoleic acid  were used as standards. Fatty acids were identified based on retention times with respect to standards.To evaluate fatty acid composition of oils obtained from 101, the antibacterial activities of oxacillin; oils extracted from A. vulgare, C. nucifera, and E. guineensis; and LA were evaluated in vitro by the broth microdilution method, modified as per the recommendations of Cos et al.102, for effectively assessing the anti-infective potential of the natural products. Antistaphylococcal effect of a combination of oxacillin/LA or oxacillin/palm oil was tested in vitro using the microdilution broth checkerboard method, as described in the Clinical Microbiology Procedures Handbook103. The determination of MIC of oxacillin, palm oils and LA, as well as oxacillin/LA or oxacillin/A. vulgare oil/C. nucifera oil/E. guineensis oil combinatory effect evaluation by FICI was performed in 96-well microtiter plates. For the testing of combinatory effects, eight twofold serial dilutions of oxacillin were placed in the horizontal rows of the plate and were subsequently cross-diluted vertically by eight twofold serial dilutions of the test compound , resulting in 64 different combinations of concentrations. The initial concentration for palm oils was 4096 µg/mL and for LA 2048 µg/mL; the starting concentration of antibiotic was adjusted according to the tested strain. The microtiter plate assay was performed using the automatic pipetting platform, Freedom EVO 100 equipped with a four-channel liquid handling arm . Cation-adjusted Müller-Hinton broth, equilibrated to a final pH of 7.6 with Trizma base  was used as growth medium. Buffering the culture media was performed to ensure the stability of oxacillin that is known to decrease under the low pH conditions63. Inoculation of the plates was carried out using bacterial suspensions, at a final density of 5 × 105 CFU/mL, standardized using Densi-La-Meter II by adjusting turbidity of the microorganism suspension to 0.5 McFarland standard. Next, incubation at 37 °C for 24 h was performed. Evaluation of bacterial growth was performed spectrophotometrically using multimode reader Cytation 3  at 405 nm. MIC values were expressed as the lowest compound concentrations that resulted in ≥ 80% growth reduction compared to that of the agent-free growth control. The lipase added to the Müller-Hinton broth, in concentrations ranging from 0.005 to 9.77 × 10–6 mg/mL did not affect the growth of any strain of S. aureus tested when assayed as a negative control. FICI values were determined as ΣFIC, derived from the equation,ABUsing guidelines of the Clinical and Laboratory Standards Institute104—synergy (ΣFIC ≤ 0.5), indifference (ΣFIC > 0.5–4), and antagonism (ΣFIC > 4). All compounds and their combinations were tested in three independent experiments, each carried out in triplicate; MIC values and FICs presented in this paper are average values.According to the value of FIC, three different effects could be observed according to OddsS. aureus growth, standardised microdilution assay was used105. Briefly, the determination in 96-well microtiter plates where eight two-fold serial dilutions of tested compound (LA or A. vulgare/C. nucifera/E. guineensis oil emulsion cleaved by porcine pancreatic lipase as described previously) starting at concentration 4096 µg/mL in cation-adjusted Müller-Hinton broth was performed. Next, the plates were inoculated with bacterial suspensions at a final density of 5 × 105 CFU/mL, standardized using Densi-La-Meter II by adjusting turbidity of the microorganism suspension to 0.5 McFarland standard, same as in case of MIC and FIC determination. Subsequently, incubation at 37 °C was performed, measuring the absorbance of each well spectrophotometrically by multimode reader Cytation 3  at 405 nm every half-to-one hour for 14 h and after 24 h.To evaluate the influence of tested palm oils and LA concentrations on parameters of the


The effectiveness of the two metabolites was confirmed in a hamster model of cutaneous Leishmaniasis by Leishmania braziliensis and in Balb/c mice infected by Trypanosoma cruzi. In vitro, 3,5-dimethoxystilbene was the most active against L. braziliensis amastigotes, with a median lethal concentration (LC50) of 4.18 μg/ml (17.40 μM) and a selectivity index of 3.55, but showed moderate activity for T. cruzi, with a median effective concentration (EC50) value of 27.7 μg/ml (115.36 μM). Flavanone pinostrobin, meanwhile, showed high activity against L. braziliensis, with an EC50 of 13.61 μg/ml (50.39 μM), as well as for T. cruzi, with an EC50 of 18.2 μg/ml (67.38 μM). The animal model assay of cutaneous Leishmaniasis showed that 50% of the hamsters treated with pinostrobin were definitively cured the cutaneous ulcer, and 40% showed an improvement, with a reduction in the size of the of 84–87%. Moreover, Balb/c mice experimentally infected with T. cruzi and treated for 25 days with pinostrobin experienced a reduction in their parasitemia by 71%. These results demonstrate the high potential of C. brunnea Amshoff against cutaneous Leishmaniasis and American trypanosomiasis and indicate the pharmacological potential of waste from the wood industry, which has tons of potentially useful chemicals for the development of new medicines.Through bioguided  The WHO recently announced the 21 neglected diseases, including Leishmaniasis and trypanosomiasis, responsible for high mortality and morbidity rates, mainly in developing countries. For the management of such diseases, there are few drugs available, and those that are available have increased resistance, unpleasant side effects, and usually involve long and expensive treatments . For thiNatural products are a significant source of new molecules and drugs. However, there are severe limitations related to the small amounts of compounds isolated from plants; this is a severe problem for molecular optimization by chemical transformation to establish structure-activity correlations and for carrying out clinical trials, as well as for commercialization. Due to this, various bioactive natural molecules remain laboratory curiosities. Proof of this is the fact that, between 1981 and 2014, only 10 drugs were approved for antiparasitic use, two of which are natural products (unmodified in structure), five were derived from a natural product, and three were made by total synthesis using a pharmacophore from a natural product .Thus, our primary focus for finding antiparasitic extracts and molecules was rich sources such as the sawdust and bark of the timber industry. These plant materials represent a valuable fraction of Latin American biodiversity, including the Amazon Forest from Colombia, Brazil, Ecuador, and Perú—that are frequently used in traditional medicine. Thousands of tons of by-products also contain hundreds to thousands of kilograms of molecules potentially useful in human pharmacology, agrochemicals, cosmetics, and other types of chemical industries.Clathrotropis brunnea Amshoff against Leishmania and Trypanosoma parasites. Moreover, a bioguided assay isolated and identified two molecules that have high activity against L. braziliensis and Trypanosoma cruzi in both in vitro and in vivo studies. The flavanone pinostrobin was isolated and identified and demonstrated high efficacy in a hamster model of L. braziliensis in the topical application of an ointment at 6% for 28 days, as well as in a mouse model for acute/undetermined T. cruzi infection in oral administration at a dose of 50 mg/kg/day for 25 days. However, purification of the active substance does not appear to be a necessary process, since the extracts exhibit a level of activity akin to that of the pure substances.In this paper, we present the general procedures followed to detect bioactive molecules in the abundant wastes from L. (V) braziliensis strain MHOM/CO/88/UA301-EGFP  were obtained from Charles River (United Kingdom) and then bred in-house under specific pathogen-free conditions. Animals were maintained as described previously  and their identification was made by Hernán Darío Cañola at the Colegio Mayor de Antioquia, Laboratorio de Maderas; a voucher was deposited under number M1.SIU 25032020.Sawdust from C. brunnea Amshoff (SQB-2) sawdust was extracted with ethanol (5 L) and concentrated under reduced pressure to obtain 79.5 g of dry extract. Five grams of the crude extract was then separated using Sephadex® LH-20  with 100% methanol as eluent. Thus, seven fractions were obtained, which were submitted to antiparasitic assays.Approximately 1 kg of To identify the compounds responsible of the activity, an aliquot of the fraction SQB2-S2 (450 mg) was separated by repeated preparative thin layer chromatographies in silica gel 60 F 254, 0.5 mm  using petroleum ether/ethyl acetate (4.5:0.5 v/v) to obtain two pure and leishmanicidal compounds, known as SQB-2-S2-1-3  and SQB-2-S2-1-4 .To obtain enough pinostrobin for assays on animal models of antiparasitic activities, 13 g of crude extract was dissolved in 30 ml of 100% methanol and then filtrated. After evaporation, the extract was separated over silica gel in a glass column with petroleum ether/ethyl acetate , providing a total of 14 fractions. Then, 224.6 mg from fractions 4–7 were purified again over silica gel and petroleum ether/ethyl acetate  to yield 84.1 mg of pinostrobin. By repeated column chromatography, approximately 600 mg of the pure compound were obtained for the leishmanicidal and trypanocidal assays.1H and 13C NMR spectra were recorded on a Fourier 300 spectrometer , operating at 300 MHz for 1H and 75 MHz for 13C NMR, using CDCl3  as the solvent and tetramethylsilane as an internal standard. Chemical shifts (δ) are reported in ppm, and the coupling constants (J) in Hz.Mass spectrometry: UHR ESI–QqTOF, positive ion mode .1H NMR (CDCl3) δ (ppm): 12.08  7.48 , 6,12 , 5.46 , 3.85 , 3.11 , 2.85 .13C NMR (CDCl3) δ (ppm), APT: 195.85 , 168.02 (s), 164.18 (s), 162.83 , 138.41 (s), 128.93 (d), 126.20 (d) 103.5 (s), 95.18 (d), 94.31 (d), 79.23 (d), 55.34 (q), 43.40 (t).16H15O4 (M+H): 271.0964HR-MS (M+H): 271.0888, calcd for C1H NMR (CDCl3) δ (ppm): 7.56 , 7.43 , 7.33 , 7.16 , 7.14 , 6.75 , 6.47 , 3.89 .13C NMR (CDCl3), δ ppm, APT: 161.02 (s), 139.40 (s), 137.17 (s), 129.25 (d), 128.76 (d), 128.71 (d), 127.81 (d), 126.65 (d), 104.62 (d), 100.02 (d), 55.43 (q).16H15O4 (M+H): 271.0964HR-MS (M+H): 241.1145, calcd for Cin vitro biological assays, a stock solution of 200 μg/ml in complete RPMI-1640 medium was prepared.The extracts, fractions, and molecules were dissolved in DMSO . Before the The crude extract solution at 1 mg/ml in methanol was used to develop the HPLC method. A C8 reverse phase column kept at 25°C  allowed a good separation pattern of the sample. The gradient mixture of water/methanol was 90:10–50:50 (10 min), 50:50 (5 min), 50:50–20:80 (5 min), 20:80–100% methanol (5 min), 100% methanol (5 min), 100% methanol–90:10 (5 min), and 90:10 (5 min). At 1 ml/min, 10 μL of the sample was injected. DAD detection of the compound showed a maximum absorption wavelength of 215 and 290 nm; detection at 290 nm was selected to avoid noise interferences from the UV absorption of the solvents at low wavelengths .r = 0.9953.Pure pinostrobin (1.0 mg/ml (3.70 mM) stock solution was prepared, and from this solution, concentrations of 10, 20, 30, and 40 μg/ml  each were injected by triplicate, through which an equation was obtained. The calibration curve gave the equation Y = 40,008.12506 * X and The overlapped chromatograms of the standard and the crude extract showed good resolution of the compound peak, with a retention time of 17.6 min. The crude extract was prepared at the concentrations of 2.47 and 1.57 mg/ml in methanol, and 10 μL of each solution was injected in duplicate. The active fraction SQB-2-S2 was also quantified, and 10 μL of 0.110 mg/ml was injected in duplicate.2 atmosphere, and after 72 h of incubation, 10 μL/well of MTT solution (0.5 mg/ml) was added. Plates were incubated again at 37°C for 3 h. The enzymatic reaction was stopped by adding 100 μL/well of dimethyl sulfoxide. Plates were read at 570 nm in a spectrophotometer . Cell viability was determined based on the quantity of formazan produced according to the intensity of the color (absorbance) registered as optical density (OD). Cells cultured in the absence of compounds were used as the control of viability , while doxorubicin was used as a control for cytotoxicity. Nonspecific absorbance was corrected by subtracting the absorbance (OD) of the blank (RPMI-1640 plus 0.2% DMSO). Assays were conducted in duplicate, with each concentration in triplicate , as described elsewhere . Brieflyiplicate .L. braziliensis. Briefly, U-937 human cells  in enriched RPMI-1640 and 0.1 μg/ml of PMA were dispensed on 24-well tissue culture plates and incubated at 37°C and 5% CO2. After 72 h of incubation, the cells were infected with 5-day-old growing promastigotes in a 15:1 parasite per cell ratio. The plates were incubated at 34°C and 5% CO2 for 3 h; then, the cells were washed twice with PBS. One milliliter of fresh enriched RPMI-1640 was added into each well, and the plates were incubated again for 24 h. Then, the culture medium was replaced by fresh enriched RPMI-1640 medium containing each extract/fraction/metabolite tested at four serial dilutions . The plates were incubated again at 37°C and 5% CO2 for 72 h. Then, the cells were removed from the bottom plate with 100 µL of a solution of trypsin/EDTA (250 mg), and the cells were centrifuged at 1,100 rpm for 10 min at 4°C, following which, the supernatant was discarded and the cells were washed with PBS. Next, the cells were suspended in 500 μL of PBS and were read at 488 nm (exciting) and 525 nm (emitting) over an argon laser of a flow cytometer , counting 10.000 events. Infected cells were determined according to the green fluorescence events corresponding to the parasites . As a control, infected cells exposed to amphotericin B (AMB) were used, and infected cells not exposed to any compound or drug were used to control for 100% infection. Nonspecific fluorescence was corrected by subtracting the fluorescence of the unstained cells. Determinations were done in triplicate in at least two independent experiments.The activity of the extracts, fractions, and pure metabolites was evaluated by flow cytometry in intracellular amastigotes of T. cruzi in a 5:1 parasites/cell ratio. After infection, 100 μL of each extract/fraction/metabolite tested at four serial dilutions  were added to each well, and the plates were incubated for 72 h at 37°C and 5% CO2. Lastly, the effect of each compound and each concentration on the viability of the intracellular amastigotes were determined by measuring the β-gal activity by the colorimetric method after adding 100 μM chlorophenol red-β-d-galactopyranoside , and nonidet P-400.1%. After 3 h of incubation at room temperature, the plates were read at 570 nm in a spectrophotometer , and absorbance was recorded as OD. The antitrypanosomal activity of benznidazole (BNZ) was used as a positive control, while RPMI-1640 medium was used as a negative control. Nonspecific absorbance was corrected by subtracting the OD of the blank sample. Determinations were done in triplicate in at least two independent experiments  were placed into each well of the 96-wells cell culture plates. The cells were then infected with epimastigotes (24 h of growing) of eriments .An aqueous solution of pinostrobin was prepared using Tween 80 5% and mineral oil 5%. In turn, the ointment was prepared based on a mixture of lanolin and petrolatum (2:1 w/w), and glycerin 1% and pinostrobin were subsequently incorporated into the base ointment until a final concentration of 6%.M. auratus) were infected with 25 × 107 stationary growth phase promastigotes of L. (V) braziliensis in the dorsum as described by others (n = 6) was treated topically with 40 mg of an ointment formulation of 6% 5-hydroxy-7-methoxy flavanone for 28 days (defined at convenience according to the available quantity). The second group of hamsters (n = 3) was treated with intralesional meglumine antimoniate (MA) at 200 μg/three times per week for 4 weeks. The hamsters were monitored before treatment (TD0), at the end of the treatment (TD28), and every month post-treatment for a total of three months. The size (width and length) of the ulcer and the bodyweight were obtained at these times during the study. Since it has been shown that parasites can persist in scars  were inoculated i.p with 100 μL blood containing 100 trypomastigotes of T. cruzi obtained from an infected mouse donor on day 24 of infection. After infection, clinical monitoring for health status and body weight was carried out weekly. Parasitemia was monitored daily by blood concentration using microhematocrit and parasite counting in the Neubauer chamber. Once the infection was confirmed (day 24 after inoculation), mice were aleatory distributed into three treatment groups (n = 5). One group was treated with pinostrobin at 40 mg/kg/day for 25 days (defined at convenience according to the available quantity); the second group was treated with BNZ at 100 mg/kg/day for 25 days; the third group remained without treatment (control). All mice were treated with 100 µL of the corresponding drugs or vehicle administered orally. At the end of the treatment and every month post-treatment for a total of three months, the parasitemia and the bodyweight were recorded. The state of health was monitored daily. At the end of the study, mice were euthanized for necropsy, and samples were obtained for histology analysis.Cytotoxicity was determined according to the mortality percentages obtained for each experimental condition, i.e., extracts/fractions/molecules, AMB, BNZ, doxorubicin, and culture medium (viability control), using the following equation:The trypanocidal activity was calculated according to a decrease in the OD using the following equation:The leishmanicidal activity was calculated by a reduction in the AFU (Active Fluorescent Units) using the following equation:50, while the percentages of the reduction of parasites data were used to calculate the EC50 by the linear regression model Probit using Graphpad Prism software.These percentages of cell mortality were used to calculate the LC50 or HC50 values, using the follow scale: Toxic/hemolytic, LC50/HC50 < 100 μg/ml (extracts or fractions) or μM (pure compounds); moderately toxic, LC50/HC50 > 100 μg/ml or μM and <200 μM or μg/mL; potentially nontoxic, LC50/HC50 > 200 μg/ml or μM.The cytotoxicity was defined according to the LCin vitro leishmanicidal and trypanocidal activity were graded as high, moderate, or low according to the EC50 values, as follows: High activity when EC50 < 25 μg/ml or μM; moderate activity when EC50 = 25–50 μg/ml or μM; low activity when EC50 > 50 μg/ml or μM. The selectivity index (SI) was calculated by dividing the cytotoxic activity and the antiparasitic activity using the following formula: SI = LC50/EC50.The in vivo leishmanicidal effectiveness was determined based on the reduction in the size of the ulcer in response to the treatment. For this, lesion sizes before and after treatment for every individual were compared. At the end of the study, the clinical outcome was recorded as either cure , improvement (reduction in the lesion size by at least 20%), relapse , or failure (an increase in the lesion size). Similarly, the trypanocidal effectiveness in the mouse model was determined by the reduction in parasitemia after treatment. The clinical outcome was graded cure when the parasitemia was reduced by 100%, improvement if it was reduced by any percentage lower than 100%, and failure when parasitemia increased.The The experimental procedures followed the practices, procedures and requirements for the safe handling and use of biohazardous materials for research activities specified in the Biosafety Manual of the research groups responsible for the present research.The procedures involving animals were in compliance with the Colombian Law of Use and care of Animals in experimentation (Ley 84 del 27 Diciembre de 1989) and had the approval of the Ethical Committee for the Experimentation in animals of the Universidad de Antioquia .C. brunnea Amshoff showed high activity against the amastigotes of L. braziliensis (EC50 = 15.4 μg/ml) and T. cruzi (EC50 = 25.0 μg/ml)- but also exhibited remarkable cytotoxicity for U-937 cells that serve as hosts of both parasites, with an LC50 of 10.3 g/ml; therefore, the SI values were low, i.e., 0.67 and 0.4 . Given the high activity shown by the SQB-2-S2 fraction for both parasites, it was decided to continue the active metabolites isolation phase with this fraction.One crude extract (SQB-2), seven fractions (SQB-2-S0 to SQB-2-S6), and two metabolites (SQB-2-S2-1-3 and SQB-2-S2-1-4) were obtained. The raw ethanolic extract of  and 0.4 . The de-L. braziliensis, with an LC50 of 4.2 μg/ml (17.4 μΜ) and an SI of 3.6, but showed moderate activity for T. cruzi, with an EC50 value of 27.7 μg/ml (115.36 μΜ). The second molecule was the flavanone pinostrobin, with an EC50 of 13.6 μg/ml (50.4 μΜ) for L. braziliensis and 18.2 μg/ml (67.4 μΜ) for T. cruzi. On the other hand, the activity shown by pinostrobin in L. braziliensis was similar to the extract from which it was previously obtained, and for T. cruzi, it increased by 27% since the EC50 values decreased from 25.0 to 18.2 μg/ml (67.4 μΜ).Through repeated chromatographic processes, two substances were obtained; the first, corresponding to the 3,5-dimethoxystilbene, was the most active against the amastigotes of Alpinia katsumadai  responded favorably to the treatment with pinostrobin ointment (6%), three of these hamsters (50%) were completely cured, and in the other two hamsters, their lesion decreased by 87.7% and 84.1%. The remaining hamster, although cured by the end of the treatment, had a reactivation one month later .No evident signs of toxicity were observed in the animals at the end of the study. The mean weight in the treated group decreased slightly, with a loss of 4.3 g on average at the end of the study .The parasitological evaluation showed a reduction in the levels of parasitemia in those animals treated with pinostrobin (SQB-2-S2-1-4) at the end of treatment and during the subsequent follow-up. The number of blood parasites decreased by 21.6%, and after 30, 60, and 90 days of follow-up, it was reduced by 52.0%, 66.4%, and 90.4%, respectively, in comparison to parasitemia before the treatment .In the group treated with BNZ, animals were complete cured by the end of the therapy and remained so during the post-treatment follow-up. In contrast, in the nontreated mice, there was an increase in parasitemia. Nevertheless, there was a significant reduction in parasitemia in terms of the number of parasites in blood on day 30 post-treatment, which is a normal process considering that at that time of infection (approximately 120 days after inoculation), parasites enter into a latency period. The reduction in parasitemia in the animal group treated with pinostrobin in comparison to the parasitemia showed before treatment was 21.6% at the end of treatment and 52.0%, 66.4% and 90.4 at 30, 60, and 90 days’ post-treatment, respectively.Clinical monitoring was performed daily alongside the administration of the substances. Body weight was recorded to monitor losses and to adjust the compound volumes based on changes in weights. None of the studied mice showed alterations with the administration of the treatments, nor did they show severe clinical signs, which allowed completing the therapeutic scheme for all groups. The evolution of body weight gain remained stable throughout the trial, increasing as expected . At the The search for new antiparasitic drugs, especially leishmanicidal and trypanocidal compounds, is necessary for many countries worldwide due to the low number of drugs available, the microbial resistance and side effects, and various other factors. However, the pharmaceutical industry does not invest in such drug discovery processes because of the few gains that can be made. For this reason, countries with high parasitosis rates should be implementing efforts to find new molecules and to carry out their development into medicines. Furthermore, the WHO recommends the examination of topical formulations for skin leishmaniasis , as wellC. brunnea Amshoff, which could eventually allow access to large amounts of bioactive molecules, and thus are potential sources for the development of new medicines.Natural products are a source of medicines and innovative structures. However, restrictions in the discovery and development of new drugs are low concentrations found in plants and the lack of abundant and renewable raw materials for their production. The discovery of new bioactive molecules can take, as a basis, the existence of rich plant biodiversity in many countries affected by parasitosis, specially Leishmaniasis, and trypanosomiasis, and specifically in Asia, Africa, and Latin America. Timber industry residues, including sawdust, bark, branches, and foliage, are a good alternative, as they are produced in tons and belong to plant families rich in secondary metabolites. For this reason, in this work, we analyzed the leishmanicidal and trypanocidal effects of wood tree sawdust L. braziliensis and T. cruzi . In this way, the extract of C. brunnea Amshoff sawdust was analyzed firstly as leishmanicidal, and after in vitro assays and purification, an active chromatographic fraction against L. braziliensis was detected, with an EC50 of 13.32 μg/ml and an LC50 of 37.16 μg/ml , including 16 μg/ml . Again, L. braziliensis-infected hamsters, once daily for 28 days, allowed to totally cure the disease in three out of five hamsters and improvement in the two remaining hamsters observed, with a reduction in the size of the ulcer by 84.1% and 87.7%  and showed in vitro activity against L. amazonensis and L. braziliensis, with an EC50 of 22.7 and 11.3 μg/ml, respectively in the latter parasite, similar to the value found by us (13.6 × 0.28 μg/ml), thus reconfirming the leishmanicidal activity observed  against L. amazonensis amastigotes as well as the analog flavanone tectochrysin.  and pterostilbene  have shown some in vitro activity. The last molecule was reported against the promastigotes and amastigotes of L. amazonensis, showing an EC50 of 17.7 and 33.2 µM, respectively. However, its mechanism of action did not affect the Sub-G0 phase or cells in the G0–G1 phase, nor did it influence the mitochondrial membrane potential (ΔΨ), which was observed for piceatannol and the structural analog .FT, supervision, and reports evaluation; FT, FE, EC, and JG performed isolation compounds; SR, NA, and JM carried out biological assays. RA ointment preparation, while FE, WQ, and GE carried out the structural analysis. FE, SR, WQ, and FT wrote the manuscript.Authors thank Minciencias (Colombia) for the financial support through the grant 111571249866.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.