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.

Towards a new application of amaranth seed oil as an agent against Candida albicans.

Amaranthus spp. (Amaranthaceae family), known as amaranth, are plants native of Central America, today produced in many parts of the world. due to their popularity popular as a health food. Because of its composition, amaranth can be considered to be attractive not only as a food but also for pharmaceutical and cosmetics uses. To date, antifungal activity of amaranth extracts has not been totally investigated, therefore the scope of this study was to evaluate the antifungal effect of the apolar fraction from Amaranthus cruentus L. seeds extract, alone and in association with antifungal drugs terbinafine, a common antifungal agent, which itself has only fungistatic effect on Candida albicans strains without exerting fungicidal activity. Our results demonstrate that this amaranth oil in combination with terbinafine has synergic fungistatic and fungicidal activity, with FICI of 0.466 and 0.496, respectively. No fungistatic and fungicidal activity of terbinafine alone at concentrations up to 64 µg/mL and amaranth oil alone at concentrations up to 2000 µg/mL, against all tested C. albicans strains, were observed. does not show activity towards Candida albicans strains but it can effectively potentiate the antifungal activity of terbinafine, a common antifungal agent which itself This result suggests the possible application of amaranth oil in the preparation of formulations with terbinafine for topical use.

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.

Antifungal activity of Mongolian medicinal plant extracts.

The in vitro antifungal activity of extracts obtained from 14 medicinal plants of the mongolian flora were investigated by measuring their minimal inhibitory concentration (MIC) against fungi cause of cutaneous diseases such as Candida species, dermatophytes and Malassezia furfur. Among the species examined, Stellaria dichotoma L., Scutellaria scordifolia L. Aquilegia sibirica Fisch. Et Schrenk. and Hyoscyamus niger L. extracts demonstrated antifungal activity against all studied fungi. In particular, S. scordifolia L. methanol extract, obtained at room temperature, showed the best activity against Candida spp., Malassezia furfur and dermatophytes with GMMIC50 values of 22 µg/mL, 64 µg/mL and 32 µg/mL, respectively. The flavones, luteolin and apigenin, identified in S. scordifolia extracts, and rutin identified in S. dichotoma and Hyoscyamus niger L. extracts, could be responsible of the observed antifungal activity.

Phytochemical and biological characterization of Italian "sedano bianco di Sperlonga" Protected Geographical Indication celery ecotype: A multimethodological approach.

Celery is a widely used vegetable known for its peculiar sensorial and nutritional properties. Here, the white celery (Apium graveolens L.) "sedano bianco di Sperlonga" PGI ecotype was investigated to obtain the metabolic profile of its edible parts (blade leaves and petioles) also related to quality, freshness and biological properties. A multi-methodological approach, including NMR, MS, HPLC-PDA, GC-MS and spectrophotometric analyses, was proposed to analyse celery extracts. Sugars, polyalcohols, amino acids, organic acids, phenols, sterols, fatty acids, phthalides, chlorophylls, tannins and flavonoids were detected in different concentrations in blade leaf and petiole extracts, indicating celery parts as nutraceutical sources. The presence of some phenols in celery extracts was here reported for the first time. Low contents of biogenic amines and mycotoxins confirmed celery quality and freshness. Regarding the biological properties, ethanolic celery extracts inhibited the oxidative-mediated DNA damage induced by tert-butylhydroperoxide and scavenged DPPH and ABTS radicals.

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.

A multi-methodological approach in the study of Italian PDO "Cornetto di Pontecorvo" red sweet pepper.

A multi-methodological approach was applied to study red sweet peppers (Capsicum annuum L.) ecotype "Cornetto di Pontecorvo" grown in a greenhouse or in open field. This approach includes morphological analysis, chemical composition determination, and biological activity evaluation of different extracts from pepper fruits. Untargeted analyses, namely NMR spectroscopy and mass spectrometry, allowed the comprehensive pepper metabolite profile of pepper pulp, peel and seeds hydroalcoholic and organic extracts to be determined, showing the presence of sugars, organic acids, amino acids and other secondary metabolites. Targeted analyses, such as HPLC-PDA, HPLC-TLC and spectrophotometric analyses allowed polyphenols, tannins, flavonoids and pigments content to be determined. Samples quality and freshness were verified by the low content of biogenic amines and mycotoxins, as determined using HPLC-FLD and HPLC-MS, respectively. Preliminary biological results demonstrated the ability of the organic extracts to inhibit α-amylase, a key enzyme in the control of glucose metabolism.

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.

In vitro antifungal activity of extracts obtained from Hypericum perforatum adventitious roots cultured in a mist bioreactor against planktonic cells and biofilm of Malassezia furfur.

Xanthone-rich extracts from Hypericum perforatum root cultures grown in a Mist Bioreactor as antifungal agents against Malassezia furfur. Extracts of Hypericum perforatum roots grown in a bioreactor showed activity against planktonic cells and biofilm of Malassezia furfur. Dried biomass, obtained from roots grown under controlled conditions in a ROOTec mist bioreactor, has been extracted with solvents of increasing polarity (i.e. chloroform, ethyl acetate and methanol). The methanolic fraction was the richest in xanthones (2.86 ± 0.43 mg g(-1) DW) as revealed by HPLC. The minimal inhibitory concentration of the methanol extract against M. furfur planktonic cells was 16 µg mL(-1). The inhibition percentage of biofilm formation, at a concentration of 16 µg mL(-1), ranged from 14% to 39%. The results show that H. perforatum root extracts could be used as new antifungal agents in the treatment of Malassezia infections.

Chemical Composition and Bio-efficacy of Essential Oils from Italian Aromatic Plants: Mentha suaveolens, Coridothymus capitatus, Origanum hirtum and Rosmarinus officinalis.

The chemical composition, antifungal, antioxidant and cytotoxic activities of the essential oils (EOs) of mint (Mentha suaveolens), thyme (Coridothymus capitatus), oregano (Origanum hirtum) and rosemary (Rosmarinus officinalis) were investigated. The antifingal properties of the EOs were investigated againstfour species of Candida by a microdilution method. Cytotoxicity was tested on human keratinocyte (HaCaT) and lung cancer (A549) cell lines using the MTT test. DPPH· and ABTS·+ spectrophotometric assays and DPPH-- ABTS-+(HP)TLC-bioautographic assays were used to evaluate the antioxidant activity. The main compounds of thyme and oregano EOs were carvacrol and thymol, respectively; piperitenone oxide and y-terpinene were the most abundant compounds ofmint and rosemary EOs, respectively. All EOs showed activity against all Candida species in a range between 760 ± 290 to 3120 ± 0.0 µg/mL. Among the EOs, that of M suaveolens showed a stronger cytotoxic activity on HaCaT cells. Thyme, oregano and rosemary EQs exhibited important antioxidant activities by ABTS` assay compared with trolox.

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.

Evaluation of anti-Candida activity of Vitis vinifera L. seed extracts obtained from wine and table cultivars.

For the first time, grape seed extracts (GSEs), obtained from wine and table cultivars of Vitis vinifera L., cultured in experimental fields of Lazio and Puglia regions of Italy and grown in different agronomic conditions, have been tested on 43 Candida species strains. We demonstrated a significant correlation between the content of the flavan-3-ols in GSEs extracts, with a polymerization degree ≥ 4, and anti-Candida activity. Moreover, we demonstrated that GSEs, obtained from plants cultured with reduced irrigation, showed a content of polymeric flavan-3-ols >250 mg/g with geometric mean MIC values between 5.7 and 20.2 mg/L against Candida albicans reference strains. GSE, showing 573 mg/g of polymeric flavan-3-ols, has been tested in an experimental murine model of vaginal candidiasis by using noninvasive in vivo imaging technique. The results pointed out a significant inhibition of Candida albicans load 5 days after challenge. These findings indicate that GSEs with high content of polymeric flavan-3-ols can be used in mucosal infection as vaginal candidiasis.

Bioassay-guided fractionation of extracts from Hypericum perforatum in vitro roots treated with carboxymethylchitosans and determination of antifungal activity against human fungal pathogens.

The aim of this study was to individuate, by bioassay-guided fractionation, promising antifungal fractions and/or constituents from Hypericum perforatum subsp. angustifolium in vitro roots. Treatments with chitosan, O-carboxymethylchitosan (CMC) and its derivatives were used to improve xanthone production in the roots. The bioassay-guided fractionation of CMC-treated roots led to the individuation of an ethyl acetate fraction, containing the highest amount of xanthones (6.8%) and showing the best antifungal activity with minimal inhibitory concentration (MIC) values of 53.82, 14.18, and 36.52 µg/ml, against Candida spp., Cryptococcus neoformans and dermatophytes, respectively. From this fraction the prenylated xanthone, biyouxanthone D has been isolated and represented the 44.59% of all xanthones detected. For the first time in the present paper biyouxanthone D has been found in H. perforatum roots and tested against C. neoformans, dermatophytes, and Candida species. The xanthone showed the greatest antifungal activity against C. neoformans and dermatophytes, with MIC values of 20.16, 22.63 µg/ml. In conclusion, the results obtained in the present study demonstrated that CMC-treated Hpa in vitro root extracts represent a tool for the obtainment of promising candidates for further pharmacological and clinical studies.

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.

A three-step culture system to increase the xanthone production and antifungal activity of Hypericum perforatum subsp. angustifolium in vitro roots.

Hypericum perforatum is a well-known medicinal plant. Among all secondary metabolites produced by this species, xanthones are very interesting for their antifungal activity. In the present study, with the aim to improve xanthone production and antifungal activity of H. perforatum subsp. angustifolium (sin. Fröhlich) Borkh in vitro roots, a new methodology consisting of a three-step culture system, has been developed. Regenerated roots of H. perforatum were cultured in a three-step culture system: in the first step, to increase biomass, the roots were cultured in half-strength liquid Murashige and Skoog (MS) medium supplemented with 1 mg L(-1) indole butyric acid (IBA) and 1.5% sucrose. In the second and third steps, to stimulate secondary metabolism, the roots were cultured with 1.1 mg L(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 0.215 mg L(-1) kinetin (KIN), and 0.186 mg L(-1) 1-naphthalenacetic acid (NAA). In the third step, some of the roots were treated with chitosan. Xanthone production increased 2.7 times following the three-step method. The mean minimal inhibitory concentration (MIC) values were of 36.9, 26.7, and 65 µg mL(-1), against Candida species, Cryptococcus neoformans and dermatophytes, respectively. A positive correlation between xanthone accumulation and antifungal activity has been shown.

Root cultures of Hypericum perforatum subsp. angustifolium elicited with chitosan and production of xanthone-rich extracts with antifungal activity.

Hypericum perforatum is a well-known medicinal plant which contains a wide variety of metabolites, including xanthones, which have a wide range of biological properties, including antifungal activity. In the present study, we evaluated the capability of roots regenerated from calli of H. perforatum subsp. angustifolium to produce xanthones. Root biomass was positively correlated with the indole-3-butyric acid concentration, whereas a concentration of 1 mg l(-1) was the most suitable for the development of roots. High auxin concentrations also inhibited xanthone accumulation. Xanthones were produced in large amounts, with a very stable trend throughout the culture period. When the roots were treated with chitosan, the xanthone content dramatically increased, peaking after 7 days. Chitosan also induced a release of these metabolites into the culture. The maximum accumulation (14.26 ± 0.62 mg g(-1) dry weight [DW]) and release (2.64 ± 0.13 mg g(-1) DW) of xanthones were recorded 7 days after treatment. The most represented xanthones were isolated, purified, and spectroscopically characterized. Antifungal activity of the total root extracts was tested against a broad panel of human fungal pathogen strains (30 Candida species, 12 Cryptococcus neoformans, and 16 dermatophytes); this activity significantly increased when using chitosan. Extracts obtained after 7 days of chitosan treatment showed high antifungal activity (mean minimum inhibitory concentration of 83.4, 39.1, and 114 µg ml(-1) against Candida spp., C. neoformans, and dermatophytes, respectively). Our results suggest that root cultures can be considered as a potential tool for large-scale production of extracts with stable quantities of xanthones.