Targeting the Antifungal Activity of Carbon Dots against Candida albicans Biofilm Formation by Tailoring Their Surface Functional Groups.

Carbon dots (CDs) are an emerging class of carbon nanoparticles, which for their characteristics have found applications in many fields such as catalysis, materials and biomedicine. Within this context, the application of CDs as antibacterial agents has received much attention in very recent years, while their use as antifungal nanoparticles has been scarcely investigated. Here we report a systematic investigation of the surface functional groups of CDs to study their influence on these nanoparticles' against Candida albicans. Three classes of CDs have been synthesised and fully characterized. A thorough in vitro and in vivo biological screening against C. albicans was performed to test their antifungal, antiadhesion and antibiofilm formation activities. Moreover, the interaction with C. albicans cells was investigated by microscopic analysis. Our results evidence how the presence of a positively polarised surface results crucial for the internalization into COS-7 cells. Positively charged nanoparticles were also able to inhibit adhesion and biofilm formation, to interact with the cellular membrane of C. albicans, and to increase the survival of G. mellonella infected larvae after the injection with positive nanoparticles. The antifungal activity of CDs and their extremely low toxicity may represent a new strategy to combat infections sustained by C.albicans.

Review Article title: Natural Compounds with Antimicrobial Activities in oral Candida infections during head and neck radiotherapy.

BACKGROUNDS: Oral colonization and infections are frequently observed in patients during and soon after radiation therapy (RT). Infective mucositis is a common side effect associated with cancer therapy, characterized by an inflammation of the oral mucous membranes with histological mucosal and submucosal changes. Ulcerative mucositis is responsible for significant pain, impairing the patient's nutritional intake and leading to local or systemic infections promoting mycosis due to several species of the genus Candida. According to international guidelines, treatment of candidiasis depends on the infection site and patient's condition. SUMMARY: Recently several studies have shown the protective role of natural compounds counteracting the activity of Candida biofilms. The aim of this review is to discuss the antimicrobial activities of natural compounds in fungal infections, especially Candida spp., during and soon after radiotherapy. Indeed new molecules are being discovered and assessed for their capacity to control Candida spp. growth and, probably in the future, will be used to treat oral candidiasis, overall, during radiotherapy. This review reports several preliminary data about preclinical and clinical evidence of their efficacy in the prevention and/or treatment of mucositis due to Radiotherapy with a brief description of the natural compounds with anti-Candida activities. KEY MESSAGES: The increase in the resistance to the available antifungal drugs related to Candida spp. infections increased as well as drug interactions, urging the development of innovative and more effective agents with antifungal action. Recent preclinical and clinical studies are identifying natural substances with anti-inflammatory and antifungal activity that could be tested in the prevention of candidiasis in patients undergoing radiotherapy. Further studies are needed to confirm these preliminary data.

Poly-(lactic-co-glycolic) Acid Nanoparticles Entrapping Pterostilbene for Targeting Aspergillus Section Nigri.

Poly-(lactic-co-glycolic) acid (PLGA) is a biodegradable, biosafe, and biocompatible copolymer. The Aspergillus section Nigri causes otomycosis localized in the external auditory canal. In this research, Aspergillus brasiliensis, a species belonging to the Nigri section, was tested. Coumarin 6 and pterostilbene loaded in poly-(lactic-co-glycolic) acid nanoparticles (PLGA-coumarin6-NPs and PLGA-PTB-NPs) were tested for fungal cell uptake and antifungal ability against A. brasiliensis biofilm, respectively. Moreover, the activity of PLGA-PTB-NPs in inhibiting the A. brasiliensis infection was tested using Galleria mellonella larvae. The results showed a fluorescence signal, after 50 nm PLGA-coumarin6-NPs treatment, inside A. brasiliensis hyphae and along the entire thickness of the biofilm matrix, which was indicative of an efficient NP uptake. Regarding antifungal activity, a reduction in A. brasiliensis biofilm formation and mature biofilm with PLGA-PTB-NPs has been demonstrated. Moreover, in vivo experiments showed a significant reduction in mortality of infected larvae after injection of PLGA-PTB-NPs compared to free PTB at the same concentration. In conclusion, the PLGA-NPs system can increase the bioavailability of PTB in Aspergillus section Nigri biofilm by overcoming the biofilm matrix barrier and delivering PTB to fungal cells.

Design, synthesis and biological evaluation of a series of iron and copper chelating deferiprone derivatives as new agents active against Candida albicans.

Candida albicans, in specific conditions, is responsible of severe invasive systemic candidiasis that are related to its ability to produce biofilm on biological and artificial surfaces. Many studies reported the role of iron in fungal growth and virulence and the ability of metal chelating agents to interfere with C. albicans metabolism, virulence and biofilm formation. Here we report the activity of 3-hydroxy-1,2-dimethyl-4(1H)-pyridinone (deferiprone) derivatives against C. albicans planktonic cells and biofilm. Some of the studied compounds (2b and 3b) were able to chelate Fe(III) and Cu(II), and showed an interesting activity on planktonic cells (MIC50 of 32 µg/mL and 16 µg/mL respectively) and on biofilm formation (BMIC50 of 32 µg/mL and 16 µg/mL respectively) in cultured ATCC 10,231C. albicans; this activity was reduced, in a concentration dependent way, by the addition of Fe(III) and Cu(II) to the culture media. Furthermore, the most active compound 3b showed a low toxicity on Galleria mellonella larvae.

Health Potential of Clery Strawberries: Enzymatic Inhibition and Anti-Candida Activity Evaluation.

Strawberries, belonging to cultivar Clery (Fragaria × ananassa Duchesne ex Weston) and to a graft obtained by crossing Clery and Fragaria vesca L., were chosen for a study on their health potential, with regard to the prevention of chronic and degenerative diseases. Selected samples, coming from fresh and defrosted berries, submitted to different homogenization techniques combined with thermal and microwave treatments, had been previously analyzed in their polyphenolic content and antioxidant capacity. In the present work, these homogenates were evaluated in relation to their enzymatic inhibition activity towards acetylcholinesterase and butyrylcholinesterase, α-amylase, α-glucosidase and tyrosinase. All these enzymes, involved in the onset of diabetes, and neurodegenerative and other chronic diseases, were modulated by the tested samples. The inhibitory effect on tyrosinase and cholinesterase was the most valuable. Antifungal activity against Candida albicans, recently shown to play a crucial role in human gut diseases as well as diabetes, rheumatoid arthritis and Alzheimer's disease, was also shown in vitro and confirmed by the in vivo text on Galleria mellonella. Overall, the obtained results confirm once again the health potential of strawberries; however, the efficacy is dependent on high quality products submitted to correct processing flow charts.

Antifungal Activity of Phenolic and Polyphenolic Compounds from Different Matrices of Vitis vinifera L. against Human Pathogens.

Phenolic compounds, the most widely distributed class of natural products in the plants, show several biological properties including antifungal activity. Phenolics contained in grapes can be classified in two main groups, flavonoids and non-flavonoids compounds. Variability and yield extraction of phenolic and polyphenolic compounds from different matrices of Vitis vinifera depends of cultivar, climate, soil condition and process technology. Unripe grapes, berry skins and seeds, leaves, canes and stems and not-fermented and fermented pomaces represent large reusable and valuable wastes from agricultural and agro-industrial processes. This review summarizes studies that examine the extraction method, chemical characterization, and antifungal activity of phenolic and polyphenolic compounds from edible and non-edible V. vinifera matrices against human fungal pathogens. In the world, around one billion people have fungal diseases related to skin, nail or hair and around 150 million have systemic diseases caused by fungi. Few studies on antifungal activity of plant extracts have been performed. This review provides useful information for the application of V. vinifera phenolics in the field of antifungals for human use.

Anti-Candida Biofilm Activity of Pterostilbene or Crude Extract from Non-Fermented Grape Pomace Entrapped in Biopolymeric Nanoparticles.

Polymeric nanoparticle-based carriers are promising agents to deliver drugs to cells. Vitis vinifera phenolic compounds are known for their antifungal activity against Candida albicans. The aim of the present study was to investigate the antifungal activity of pterostilbene or crude extracts from non-fermented grape pomace, entrapped in poly(lactic-co-glycolic) acid nanoparticles (NPs), with diameters of 50 and 150 nm, on Candida biofilm. The fluorescent probe coumarin 6 was used to study the uptake of poly(lactic-co-glycolic)acid (PLGA) NPs in planktonic cells and biofilm. The green fluorescent signal of coumarin 6 was observed in Candida biofilm after 24 and 48 hours. Both pterostilbene and crude pomace extract entrapped in NPs exerted a significantly higher anti-biofilm activity compared to their free forms. The entrapment efficiency of both pterostilbene and crude pomace extract in PLGA NPs was ~90%. At 16 µg/mL, pterostilbene loaded in PLGA NPs reduced biofilm formation of 63% and reduced mature biofilm of 50%. Moreover, at 50 µg/mL, the pomace extract loaded in NPs reduced mature biofilm of 37%. These results strongly suggest that PLGA NPs are promising nanodevices for the delivery of antifungal drugs as the crude grape pomace extract, a by-product of white wine making.

Chitosan oligosaccharides affect xanthone and VOC biosynthesis in Hypericum perforatum root cultures and enhance the antifungal activity of root extracts.

KEY MESSAGE: Water-soluble chitosan oligosaccharides (COS) affect xanthone and volatile organic compound content, as well as antifungal activity against human pathogenic fungi of extracts obtained from Hypericum perforatum root cultures. Several studies have demonstrated the elicitor power of chitosan on xanthone biosynthesis in root cultures of H. perforatum. One of the major limitations to the use of chitosan, both for basic and applied research, is the need to use acidified water for solubilization. To overcome this problem, the elicitor effect of water-soluble COS on the biosynthesis of both xanthones and volatile organic compounds (VOCs) was evaluated in the present study. The analysis of xanthones and VOCs was performed by HPLC and GC-MS headspace analysis. The obtained results showed that COS are very effective in enhancing xanthone biosynthesis. With 400 mg L-1 COS, a xanthone content of about 30 mg g-1 DW was obtained. The antifungal activity of extracts obtained with 400 mg L-1 COS was the highest, with MIC50 of 32 µg mL-1 against Candida albicans and 32-64 µg mL-1 against dermatophytes, depending on the microorganism. Histochemical investigations suggested the accumulation of isoprenoids in the secretory ducts of H. perforatum roots. The presence of monoterpenes and sesquiterpenes was confirmed by the headspace analysis. Other volatile hydrocarbons have been identified. The biosynthesis of most VOCs showed significant changes in response to COS, suggesting their involvement in plant-fungus interactions.

In vitro biofilms and antifungal susceptibility of dermatophyte and non-dermatophyte moulds involved in foot mycosis.

Tinea pedis and onychomycosis are among the commonest fungal diseases in the world. Dermatophytes and, less frequently, non-dermatophyte moulds are aetiological agents of foot mycosis and are capable of forming biofilms. Fungal biofilm has demonstrated increasing drug resistance. This work aims to evaluate, in vitro, the ability to form biofilm and the susceptibility to antifungal drugs of sessile dermatophytes and non-dermatophyte moulds involved in foot mycosis. Thirty-six dermatophytes and non-dermatophyte moulds isolated from Tunisian patients with foot mycoses, and identified with MALDI-TOF have been tested. MICs of fluconazole, econazole, itraconazole, terbinafine and griseofulvin were carried out using CLSI broth microdilution method. The ability to form biofilm and antifungal activities of drugs against fungal biofilm formation has been quantified by Crystal Violet and Safranin Red staining. Biomass quantification revealed that all species studied were able to form biofilms in vitro after 72 hours. Fluconazole, econazole, itraconazole and terbinafine inhibited fungal growth with MIC values ranging from 0.031 to >64 µg mL-1 . The best antifungal activity has been obtained with terbinafine against Fusarium solani. Econazole showed the highest activity against fungal biofilm formation. These findings can help clinicians to develop the appropriate therapy of foot mycosis.

Anti-Dermatophyte and Anti-Malassezia Activity of Extracts Rich in Polymeric Flavan-3-ols Obtained from Vitis vinifera Seeds.

Several human skin diseases are associated with fungi as dermatophytes and Malassezia. Skin mycoses are increasing and new alternatives to conventional treatments with improved efficacy and/or safety profiles are desirable. For the first time, the anti-dermatophytes and the anti-Malassezia activities of Vitis vinifera seed extracts obtained from different table and wine cultivars have been evaluated. Geometric minimal inhibitory concentration ranged from 20 to 97 µg/mL for dermatophytes and from 32 to 161 µg/mL for Malassezia furfur. Dried grape seed extracts analyzed by HPLC/DAD/ESI/MS showed different quali-quantitative compositions in terms of monomeric and polymeric flavan-3-ols. The minimal inhibitory concentrations for Trichophyton mentagrophytes and for M. furfur were inversely correlated with the amount of the polymeric fraction (r = -0.7639 and r = -0.7228, respectively). Differently, the antifungal activity against T. mentagrophytes was not correlated to the content of flavan-3-ol monomers (r = 0.2920) and only weakly correlated for M. furfur (r = -0.53604). These results suggest that extracts rich in polymeric flavan-3-ols, recovered from V. vinifera seeds, could be used for the treatment of skin fungal infections. Copyright © 2016 John Wiley & Sons, Ltd.

Exploring the anti-biofilm activity of cinnamic acid derivatives in Candida albicans.

Some compounds, characterized by phenylethenyl moiety, such as methyl cinnamate and caffeic acid phenethyl ester, are able to inhibit C. albicans biofilm formation. On these bases, and as a consequence of our previous work, we synthesized a series of cinnamoyl ester and amide derivatives in order to evaluate them for the activity against C. albicans biofilm and planktonically grown cells. The most active compounds 7 and 8 showed ⩾50% biofilm inhibition concentrations (BMIC50) of 2µg/mL and 4µg/mL respectively, against C. albicans biofilm formation; otherwise, 7 showed an interesting activity also against mature biofilm, with BMIC50 of 8µg/mL.

Xanthones from roots, hairy roots and cell suspension cultures of selected Hypericum species and their antifungal activity against Candida albicans.

KEY MESSAGE: Highest xanthone contents were found in Hypericum pulchrum and H. annulatum untransformed roots. The best anti- Candida activity was obtained for hairy roots extracts of H. tetrapterum clone 2 ATCC 15834. Extracts of root cultures, hairy roots and cell suspensions of selected Hypericum spp. were screened for the presence of xanthones and tested for their antifungal activity against Candida albicans strain ATCC 10231. At least one of the following xanthones, 5-methoxy-2-deprenylrheediaxanthone; 1,3,6,7-tetrahydroxyxanthone; 1,3,5,6-tetrahydroxyxanthone; paxanthone; kielcorin or mangiferin was identified in methanolic extracts of the untransformed root cultures. The highest total xanthone content, with five xanthones, was found in untransformed H. pulchrum and H. annulatum root cultures. Hairy roots and the controls of H. tetrapterum contained 1,7-dihydroxyxanthone, while hairy root cultures and the corresponding controls of H. tomentosum contained toxyloxanthone B, 1,3,6,7- and 1,3,5,6-tetrahydroxyxanthone. Two xanthones, cadensin G and paxanthone, were identified in cell suspension cultures of H. perforatum. Their content increased about two-fold following elicitation with salicylic acid. The anti-Candida activity of the obtained extracts ranged from MIC 64 to >256 µg ml(-1). Among the extracts of Hypericum untransformed roots, the best antifungal activity was obtained for extracts of H. annulatum grown under CD conditions. Extracts of hairy roots clones A4 and 7 ATCC15834 of H. tomentosum and clone 2 ATCC15834 of H. tetrapterum displayed inhibition of 90% of Candida growth with 256 µg ml(-1). Extracts from chitosan-elicitated cells did not show antifungal activity.

Activity of caffeic acid derivatives against Candida albicans biofilm.

The aim of this study was to evaluate the caffeic acid (1) and ester derivatives (2-10) against Candida albicans biofilm and to investigate whether these compounds are able to inhibit the biofilm formation or destroy pre-formed biofilm. Caffeic acid ester 7, cinnamic acid ester 8 and 3,4-dihydroxybenzoic acid ester 10 are more active than fluconazole, used as reference drug, both on biofilm in formation with MIC50 values of 32, 32 and 16µg/mL, respectively, and in the early stage of biofilm formation (4h) with MIC50 values of 64, 32 and 64µg/mL, respectively. These esters result also more active than fluconazole on mature biofilm (24h), especially 8 and 10 with MIC50 values of 64µg/mL.

Evaluation of the Antifungal and Antiproliferative Properties of the Lichen Roccella tinctoria DC. Extracts and Main Components.

In this work, phytochemical analysis on different extracts of Roccella tinctoria DC. was reported using different techniques with respect to the past. Twenty volatile and three non-volatile compounds were identified, some of which were found in this species for the first time. The methanolic extracts and their non-volatile components were then evaluated for their antitumor effects in cancerous A549 and Mz-ChA-1 cells and for their tolerability in non-cancerous BEAS-2B and H69 cells, showing IC50 values from 94.6 µg/mL to 416.4 µg/mL, in general. The same extracts and compounds were also tested for their antifungal effects in Candida albicans, with only compound 2 being active, with an MIC50 value of 87 µg/mL. In addition, they were tested for their anti-Candida adhesion activity, anti-Candida biofilm formation, and anti-Candida mature biofilm inhibition, with efficacy percentages generally above 50% but not for all of them. Lastly, the DF3 extract and compounds 1-2 were tested in vivo according to the Galleria mellonella survival assay, showing positive mortality rates above 50% at different concentrations. All these biological assays were conducted on this species for the first time. Comparisons with other lichens and compounds were also presented and discussed.

Solid lipid nanoparticles as effective reservoir systems for long-term preservation of multidose formulations.

Cosmetic multidose preparations, as well as pharmaceutical ones, are at risk of contamination by microorganisms, due to their high water content. Besides the risk of contamination during manufacturing, multidose cosmetic preparations may be contaminated by consumers during their use. In this paper, the results of the utilization of nanoparticles as reservoir systems of parabens, the most used class of preservatives, were reported. Two different systems, solid lipid nanoparticles (SLN) made of pure precirol and nanostructured lipid carriers (NLC) made of precirol and almond oil, containing three parabens as single molecules or as a mixture, were prepared and tested. All the systems were characterized for size, polydispersion index, zeta potential and encapsulation efficiency. Release experiments, carried out in steady state and sink conditions, allowed to evidence that both SLN and NLC were able to act as reservoir systems. The antimicrobial activity of the systems was tested against Candida albicans ATCC 10231 with repeat insult tests. The results of the release experiments and the antimicrobial tests showed very low water concentration of parabens still maintaining their antimicrobial activity.

Synergistic Effect of Plant Compounds in Combination with Conventional Antimicrobials against Biofilm of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida spp.

Bacterial and fungal biofilm has increased antibiotic resistance and plays an essential role in many persistent diseases. Biofilm-associated chronic infections are difficult to treat and reduce the efficacy of medical devices. This global problem has prompted extensive research to find alternative strategies to fight microbial chronic infections. Plant bioactive metabolites with antibiofilm activity are known to be potential resources to alleviate this problem. The phytochemical screening of some medicinal plants showed different active groups, such as stilbenes, tannins, alkaloids, terpenes, polyphenolics, flavonoids, lignans, quinones, and coumarins. Synergistic effects can be observed in the interaction between plant compounds and conventional drugs. This review analyses and summarises the current knowledge on the synergistic effects of plant metabolites in combination with conventional antimicrobials against biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. The synergism of conventional antimicrobials with plant compounds can modify and inhibit the mechanisms of acquired resistance, reduce undesirable effects, and obtain an appropriate therapeutic effect at lower doses. A deeper knowledge of these combinations and of their possible antibiofilm targets is needed to develop next-generation novel antimicrobials and/or improve current antimicrobials to fight drug-resistant infections attributed to biofilm.

Biofilm Inhibition of Inula viscosa (L.) Aiton and Globularia alypum L. Extracts Against Candida Infectious Pathogens and In Vivo Action on Galleria mellonella Model.

The increasing importance of fungal infections has fueled the search for new beneficial alternatives substance from plant extracts. The current study investigates the antifungal and antibiofilm activity of Inula viscosa (L.) Aiton and Globularia alypum (L.) leaves extracts against Candida both in vitro and in vivo. The inhibition of planktonic and sessile Candida albicans and Candida glabrata growth using both leaf extracts are evaluated. Moreover; an in vivo infection model using Galleria mellonella larvae; infected and treated with the extracts are performed. All extracts show fungicidal activity; with a minimum fungicidal concentration (MFC) ranging from 128 to 512 µg mL-1 against the two selected strains of Candida. In particular, the best results are obtained with methanolic extract of I. viscosa and G. alypum with an MFC value of 128 µg mL-1 . The extracts are capable to prevent 90% of biofilm development at minor concentrations ranging from 100.71 ± 2.49 µg mL-1 to 380.4 ± 0.92 µg mL-1 . In vivo, tests on Galleria mellonella larvae show that the extracts increase the survival of the larvae infected with Candida. The attained results reveal that I. viscosa and G. alypum extracts may be considered as new antifungal agents and biofilm inhibiting agents for the pharmaceutical and agro-food field.

Biopolymer- and Natural Fiber-Based Biomimetic Tissues to Realize Smart Cosmeceuticals and Nutraceuticals Using an Innovative Approach.

More sustainable and smart cosmeceuticals and nutraceuticals are necessary due to the ecological transition. In this study, a pullulan-based water solution containing chitin nanofibril-nano-lignin (CN-LG) complexes that encapsulate fish collagen polypeptide, allantoin and nicotinamide was electrospun onto a nonwoven substrate made of bamboo fibers to obtain a smart nanostructured bilayer system for releasing active molecules onto the skin or other body tissues. Infrared spectroscopy was used to characterize the composition of the bilayer system before and after rapid washing of the sample with distilled water and liquids mimicking physiological fluids. The viability of keratinocytes was studied as well as the antioxidant activity, protective activity towards UV light, metalloproteinase release of aged fibroblasts and the inhibitor activity against collagen degradation. Immunomodulatory tests were performed to investigate the anti-inflammatory activity of the bilayer system as well as its indirect antimicrobial activity. The results indicate that the bilayer system can be used in the production of innovative sustainable cosmeceuticals. In general, the adopted strategy can be extended to several smart treatments for fast release that can be commercialized as solid products, thus avoiding the use of preservatives and water.

Thymol-Functionalized Hyaluronic Acid as Promising Preservative Biomaterial for the Inhibition of Candida albicans Biofilm Formation.

Hyaluronic acid (HA) is a naturally occurring biopolymer that has been employed for a plethora of medicinal applications. Nevertheless, as HA is a natural polysaccharide, it can be a substrate able to promote microbial growth and proliferation. Biopolymer-drug conjugates have gained attention over the years to overcome drawbacks of each single component. Within this context, thymol (Thy), a phenolic compound occurring in essential oils (EOs) extracted from Thymus and Origanum, has been largely studied for its antimycotic applications. However, it is characterized by a low water solubility and moderate cytotoxicity. Herein, we report an innovative HA-thymol conjugate (HA-Thy) biomaterial to circumvent the drawbacks of free thymol use by providing the polymer conjugate with the beneficial properties of both components. Preliminary biological tests evidenced the decrease of thymol cytotoxicity for the HA-Thy conjugate, paired with a promising antibiofilm formation activity against Candida albicans, similar to pure thymol, highlighting its potential application as a preservative biomaterial in formulations.

Protective Effect of Procyanidin-Rich Grape Seed Extract against Gram-Negative Virulence Factors.

Biofilm formation and lipopolysaccharide (LPS) are implicated in the pathogenesis of gastrointestinal (GI) diseases caused by Gram-negative bacteria. Grape seeds, wine industry by-products, have antioxidant and antimicrobial activity. In the present study, the protective effect of procyanidin-rich grape seed extract (prGSE), from unfermented pomace of Vitis vinifera L. cv Bellone, on bacterial LPS-induced oxidative stress and epithelial barrier integrity damage has been studied in a model of Caco-2 cells. The prGSE was characterized at the molecular level using HPLC and NMR. The in vitro activity of prGSE against formation of biofilm of Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli was investigated. In vivo, prGSE activity using infected Galleria mellonella larvae has been evaluated. The results show that the prGSE, if administered with LPS, can significantly reduce the LPS-induced permeability alteration. Moreover, the ability of the extract to prevent Reactive Oxygen Species (ROS) production induced by the LPS treatment of Caco-2 cells was demonstrated. prGSE inhibited the biofilm formation of E. coli and S. Typhimurium. In terms of in vivo activity, an increase in survival of infected G. mellonella larvae after treatment with prGSE was demonstrated. In conclusion, grape seed extracts could be used to reduce GI damage caused by bacterial endotoxin and biofilms of Gram-negative bacteria.