Direct Bioautography Research Articles

Molecular modelling and anticholinesterase activity of the essential oil from three chemotypes of Lippia alba (Mill.) N.E.Br. ex Britton & P. Wilson (Verbenaceae)

Lippia alba (Mill.) N.E. Brown (Verbenaceae), popularly known as “erva cidreira”, is one of the most used plants in Brazilian folk medicine. The species has several chemotypes and its volatile constituents have already been characterized, and present different chemical markers with known pharmacological properties, such as analgesic, sedative and antifungal properties. The objective of this study was to evaluate the anticholinesterase activity (AChE) of the essential oil of three chemotypes of Lippia alba and, by using molecular anchoring, determine the best receptor-ligand interaction energies of the main constituents present in the samples of oil. The essential oils were obtained via hydrodistillation (LA1 and LA2) and steam drag (LA3), and their volatile constituents determined using GC-MS. For the determination of anticholinesterase activity, direct bioautography and colorimetry assays based on Ellman's method were used. Molecular docking was performed using a multiple solution genetic algorithm and Merck molecular force field 94 (MMFF94) as the scoring function. In the main constituents of the oil samples, three chemotypes were identified for L. alba: LA1 is rich in citral, LA2 is rich in carvone and LA3 is rich in linalool. All L. alba chemotypes showed AChE enzyme inhibition with an IC50 of 3.57 μg/mL (LA1), 0.1 μg/mL (LA2) and 4.34 μg/mL (LA3). The molecular docking study complemented the results of the experiment and demonstrated significant interactions between the main constituents of the oils and the amino acid residues of the AChE enzyme. Irrespective of the chemotype, Lippia alba presents biotechnological potential for the discovery of anticholinesterase substances, with the chemotype LA2 (rich in carvone) being the most active.

Gram-negative rough mutants used as test bacteria can increase sensitivity of direct bioautography

Currently, the antimicrobial activity of essential oils (EOs) is an outstanding research field due to antibiotic resistance of microorganisms. Thin-layer chromatography‒direct bioautography (TLC‒DB) is an effective, fast method to find components with antimicrobial activity in a mixture of plant compounds, e.g., in EOs. The volatility and hydrophobic characters of EOs require special experimental conditions, and disc diffusion assay is not appropriate to explore the antimicrobial activity of them. The aim of this study was to use “R” mutants, which are more sensitive to synthetic antimicrobial drugs, in DB to increase the sensitivity of this method. Our hypothesis was that these mutants show sensitivity to some EOs (thyme, clove, and peppermint) as well. The chemical composition of our tested EOs was measured with gas chromatography‒mass spectrometry (GC‒MS). The main compounds (39.8% thymol, 78.8% eugenol, and 50.4% menthol) of EOs showed notable antibacterial activity in TLC‒DB. Based on our results, we suggest to use Salmonella minnesota Re595 rough strain as test bacterium in bioautography, because it showed the highest sensitivity to the tested antibiotics (gentamicin and cephalexin) and EOs. Furthermore, this rough mutant could make TLC‒DB more faster, because only 4 h incubation time was enough to detect the inhibition zones of the active compounds used in this study.

Silver-doped graphite oxide composites used as antimicrobial agents against Staphylococcus aureus, Escherichia coli and Tatumella terrea evaluated by direct TLC bioautography

Silver-doped graphite oxide composites presented mixed Ag and Ag2O crystalline phases accompanied by C/O and ID/IG average ratios of 2.13 and 1.16, associated with a good oxidation degree and high structural disorder or defects in the carbon material, respectively. The low-silver-doped GrO composite showed homogenous silver particle dispersion and a low particle size distribution (96 nm). However, high-silver-doped GrO composites generated materials with high relative crystallinity, silver particle agglomeration, and Ag metal phase promotion. At 3 and 5 mg ml−1 per chromatographic plate, the silver-doped graphite oxide composites were tested by direct TLC bioautography against Staphylococcus aureus, Escherichia coli, and Tatumella terrea microorganisms, where the minimum inhibitory concentration was 3 mg ml−1 per chromatographic plate. At 3.0 mg ml−1 per chromatographic plate, high-silver-doped GrO composites exhibited a 39%, 3.2-fold, and 83% higher retention factor (R f ) compared with the composites with low-silver-doped GrO composites against S. aureus, E. coli, and T. terra microorganisms, respectively. However, both composites showed similar inhibition capacities at 5.0 mg ml−1 per chromatographic plate against the three microorganisms. This behavior may be associated with both composites reaching the threshold limit. In general, the silver acetate amount used in the silver-doped GrO composites influenced the dispersion, crystalline phase promotion, particle size distribution, and the silver particle release capacity, which modified the electrostatic adsorption type between the composites and the bacterial cell walls.

Screening and identification of antibacterial components in Artemisia argyi essential oil by TLC–direct bioautography combined with comprehensive 2D GC × GC-TOFMS

One of the primary components that contribute to Artemisia argyi 's effectiveness is essential oil, which has an exceptional antibacterial effect that has been well documented. The actual cause of its antibacterial activity and associated mechanism, however, are still not completely understood. For the first time, comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (2D GC × GC-TOFMS) and thin-layer chromatography-direct bioautography (TLC-DB) were employed to investigate its antibacterial components. The antibacterial properties of A. argyi essential oil were investigated, and the antibacterial activity of six compounds was evaluated, using Staphylococcus aureus (S. aureus) and Escherichia coli (E. coil) as test microorganisms. TLC-direct bioautography was used to screen two bioactive clusters. Following 2D GC × GC-TOFMS identification of bioactive clusters, six compounds were evaluated for in vitro antibacterial activity verification. All the components tested displayed antibacterial action. Results showed that α-terpineol and eugenol had high potent antibacterial activity (MIC＜0.62 mg/mL, IC50＜2.00 mg/mL). For complex essential oils from traditional Chinese medicine, this method is efficient for quick screening and identifying antibacterial compounds.

In vivo and ex vivo studies support the immunostimulant and immunoprotective effect of Damiana (Turnera diffusa Willd) in Almaco Jack (Seriola rivoliana)

Damiana (Turnera diffusa Willd) was evaluated in vitro for antioxidant and antibacterial activities against Staphylococcus aureus and Streptococcus pyogenes (as a preliminary screening assessment) by high‐performance thin‐layer chromatography (HPTLC)-Direct bioautography. A study was performed in vivo to evaluate the effects of Damiana enriched diets at 0.5 % on immune parameters in mucus and serum and gene expression in Almaco Jack (Seriola rivoliana) intestine after two and four weeks; an infection with Aeromonas hydrophila at 1x107 colony forming units (CFU) followed and an ex vivo study was carried out using head-kidney leukocytes. Ferric reducing ability of plasma (FRAP) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays showed high antioxidant activities in Damiana leaves; even in the ABTS assay, Damiana at 300 μg/mL showed similar activity to ascorbic acid – the standard control. Damiana exhibited strong in vitro antimicrobial activity against S. aureus and S. pyogenes. In vivo studies showed a strong enhancement of myeloperoxidase, nitric oxide, superoxide dismutase, and catalase activities in mucus and serum of S. rivoliana supplemented with Damiana; their immunological response enhanced after infection with A. hydrophila. IL-1β, TNF-α, and IL-10 gene expressions upregulated in the fish intestine challenged with the bacterium. Piscidin and macrophage (MARCO) receptor gene expression up-regulated at week 4 and down-regulated after infection. Intestinal histology results confirm that Damiana not cause inflammation or damage. Finally, the ex vivo study confirmed the immunostimulant and protective effects of Damiana through increased phagocytic, respiratory burst, myeloperoxidase activities and nitric oxide generation before and upon the bacterial encounter. These results support the idea that Damiana has the potential as an immunostimulant additive for diets in aquaculture by enhancing immune parameters and protecting Almaco Jack against A. hydrophila infections upon four weeks of supplementation.

Antibacterial effect of essential oils and their components against Xanthomonas arboricola pv. pruni revealed by microdilution and direct bioautographic assays.

Bacterial spot of stone fruits caused by Xanthomonas arboricola pv. pruni (Xap) is one of the most significant diseases of several Prunus species. Disease outbreaks can result in severe economic losses while the control options are limited. Antibacterial efficacy of essential oils (EOs) of thyme, cinnamon, clove, rosemary, tea tree, eucalyptus, lemon grass, citronella grass, and lemon balm was assessed against two Hungarian Xap isolates. The minimal inhibitory concentration (MIC) was determined by broth microdilution assay and for the identification of active EOs' components a newly introduced high-performance thin-layer chromatography (HPTLC)-Xap (direct bioautography) method combined with solid-phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS) was applied. All EOs inhibited both bacterium isolates, but cinnamon proved to be the most effective EO with MIC values of 31.25 µg/mL and 62.5 µg/mL, respectively. Compounds in the antibacterial HPTLC zones were identified as thymol in thyme, trans-cinnamaldehyde in cinnamon, eugenol in clove, borneol in rosemary, terpinen-4-ol in tea tree, citral (neral and geranial) in lemon grass and lemon balm, and citronellal and nerol in citronella grass. Regarding active compounds, thymol had the highest efficiency with a MIC value of 50 µg/mL. Antibacterial effects of EOs have already been proven for several Xanthomonas species, but to our knowledge, the studied EOs, except for lemon grass and eucalyptus, were tested for the first time against Xap. Furthermore, in case of Xap, this is the first report demonstrating that direct bioautography is a fast and suitable method for screening anti-Xap components of complex matrices, like EOs.

High-performance thin-layer chromatography – antibacterial assay first reveals bioactive clerodane diterpenes in giant goldenrod (Solidago gigantea Ait.)

The present work introduces a high-performance thin-layer chromatography (HPTLC)–direct bioautography method using the Gram-positive plant pathogenic bacterium, Rhodococcus fascians. The screening and isolation procedure comprised of a non-targeted high-performance thin-layer chromatography-effect-directed analysis (HPTLC–EDA) against Bacillus subtilis, B. subtilis subsp. spizizenii, R. fascians, and Aliivibrio fischeri, a targeted HPTLC–mass spectrometry (MS), and bioassay-guided column chromatographic (preparative flash and semi-preparative HPLC) fractionation and purification. The developed new separation methods enabled the discovery of four bioactive cis-clerodane diterpenes, solidagoic acid H (1), solidagoic acid E (2), solidagoic acid I (3), and solidagoic acid F (4), in the n-hexane extract of giant goldenrod (Solidago gigantea Ait.) leaf for the first time. These compounds were identified by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. The initially used HPTLC method (chloroform – ethyl acetate – methanol 15:3:2, V/V/V) was changed (to n-hexane – isopropyl acetate – methanol – acetic acid 29:20:1:1, V/V/V/V) to achieve the separation of the closely related isomer pairs (1–2 and 3–4). Compounds 1 and 3 exhibited moderate antibacterial activity against the Gram-positive B. subtilis subsp. spizizenii and R. fascians bacterial strains in microdilution assays with half-maximal inhibitory concentration (IC50) values in the range of 32.3–64.4 µg/mL. The mass spectrometric fragmentation of the isolated compounds was interpreted and their previously published NMR assignments lacking certain resonances were completed.

Separation and detection of apricot leaf triterpenes by high-performance thin-layer chromatography combined with direct bioautography and mass spectrometry

Prunus armeniaca leaf extract was screened for antibacterial compounds by high-performance thin-layer chromatography (HPTLC)-direct bioautography using a Gram-positive Bacillus subtilis bacterium. Six chromatographic zones exhibited characteristic bioactivity. Five of them also appeared after derivatization with vanillin-sulfuric acid reagent and could be characterized with HPTLC-electrospray ionization (ESI)-mass spectrometry (MS), suggesting the presence of triterpenoids and the fatty acids linolenic and palmitic acid. To confirm the identification of triterpenoids an HPTLC method using in situ pre-chromatographic derivatization with iodine was developed to separate the closely related triterpenoids. After development, the iodine could be eliminated from the chromatogram (verified by HPTLC-MS), making it suitable for the B. subtilis assay. Ursolic acid, oleanolic acid, betulinic acid, corosolic acid, and maslinic acid were discovered for the first time as antibacterial components of P. armeniaca leaves. Their presence was proved also by 2D-HPTLC combined with intermediate in situ derivatization by iodine.

High-performance thin-layer chromatography–direct bioautography combined with chemometrics for the distinction of goldenrod species

Thirteen root extract samples of four goldenrod (Solidago) species present in Europe were investigated by hyphenated high-performance thin-layer chromatography (HPTLC). Only S. virgaurea is native, whereas S. gigantea, S. canadensis, and S. graminifolia have been introduced from North America. The bioactive zones in the Aliivibrio fischeri bioautogram were identified as polyacetylenes, labdane diterpenes, or clerodane diterpenes by HPTLC coupled to high-resolution mass spectrometry, exploiting the two interfaces, heated electrospray ionization, and direct analysis in real time. Principal component analysis of the obtained bioprofiles enabled the discrimination of the Solidago species. Furthermore, chemometrics pointed to the discriminative components, the main bioactive markers of the species: Z,Z-matricaria ester from S. virgaurea, solidagenone from S. canadensis, solidagoic acid A, and a dialdehyde clerodane diterpene from S. gigantea, and Z-dehydromatricaria ester from S. graminifolia.

Schisandra rubriflora Fruit and Leaves as Promising New Materials of High Biological Potential: Lignan Profiling and Effect-Directed Analysis.

The effect-directed detection (EDD) of Schisandra rubriflora fruit and leaves extracts was performed to assess their pharmacological properties. The EDD comprised TLC—direct bioautography against Bacillus subtilis, a DPPH assay, as well as α-glucosidase, lipase, tyrosinase, and acetylcholinesterase (AChE) inhibition assays. The leaf extracts showed stronger antioxidant activity than the fruit extract as well as inhibition of tyrosinase and lipase. The fruit extract was found to be extremely active against B. subtilis and to inhibit α-glucosidase and AChE slightly more than the leaf extracts. UHPLC–MS/MS analysis was carried out for the bioactive fractions and pointed to the possible anti-dementia properties of the dibenzocyclooctadiene lignans found in the upper TLC fractions. Gomisin N (518 mg/100 g DW), schisanhenol (454 mg/100 g DW), gomisin G (197 mg/100 g DW), schisandrin A (167 mg/100 g DW), and gomisin O (150 mg/100 g DW) were the quantitatively dominant compounds in the fruit extract. In total, twenty-one lignans were found in the bioactive fractions.

Antibacterial Effect of Lemongrass (Cymbopogoncitratus) against the Aetiological Agents of Pitted Keratolyis.

Pitted keratolysis (PK) is a bacterial skin infection mostly affecting the pressure-bearing areas of the soles, causing unpleasant symptoms. Antibiotics are used for therapy, but the emergence of antiobiotic resistance, makes the application of novel topical therapeutic agents necessary. The antibacterial effects of 12 EOs were compared in the first part of this study against the three known aetiological agents of PK (Kytococcus sedentarius, Dermatophilus congolensis and Bacillus thuringiensis). The results of the minimal inhibitory concentration, minimal bactericidal concentration and spore-formation inhibition tests revealed that lemongrass was the most effective EO against all three bacterium species and was therefore chosen for further analysis. Seventeen compounds were identified with solid-phase microextraction followed by gas chromatography–mass spectrometry (HS-SPME/GC-MS) analysis while thin-layer chromatography combined with direct bioautography (TLC-BD) was used to detect the presence of antibacterially active compounds. Citral showed a characteristic spot at the Rf value of 0.47, while the HS-SPME/GC-MS analysis of an unknown spot with strong antibacterial activity revealed the presence of α-terpineol, γ-cadinene and calamenene. Of these, α-terpineol was confirmed to possess an antimicrobial effect on all three bacterium species associated with PK. Our study supports the hypothesis that, based on their spectrum, EO-based formulations have potent antibacterial effects against PK and warrant further investigation as topical therapeutics.

Therapeutic Values of Earthworm Species Extract from Azad Kashmir as Anticoagulant, Antibacterial, and Antioxidant Agents.

Aims Current research aimed to explore the therapeutic values of different earthworms as antibacterial, anticoagulant, and antioxidant agents. Methods Ten different earthworms, i.e., Amynthas corticis, Amynthas gracilis, Pheretima posthuma, Eisenia fetida, Aporrectodea rosea, Allolobophora chlorotica, Aporrectodea trapezoides, Polypheretima elongata, Aporrectodea caliginosa, and Pheretima hawayana, were collected and screened for biological activities. Antibacterial effect analysis of earthworm species was done against fourteen bacterial pathogens, i.e., Escherichia coli, Serratia marcescens, Streptococcus pyogenes, Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa (1), Salmonella typhimurium, Shigella flexneri, Enterobacter amnigenus, Serratia odorifera, Pseudomonas aeruginosa (2), Staphylococcus warneri, and Lactobacillus curvatus, via agar well diffusion, crystal violet, MTT, agar disc diffusion, and direct bioautography assays. Antioxidant potential was evaluated through ABTS and DPPH assays. Lipolytic, proteolytic, and amylolytic assays were done for lipase, protease, and amylase enzymes confirmation. In vitro anticoagulant effects were examined in the blood samples by measuring prothrombin time. Results Results revealed that all earthworm extracts showed the inhibition of all tested bacterial pathogens except P. aeruginosa (1), P. aeruginosa (2), S. warneri, and L. curvatus. The maximum zone of inhibition of E. coli was recorded as 14.66 ± 0.57 mm by A. corticis, 25.0 ± 0.0 mm by P. posthuma, 20.0 ± 0.0 mm by E. fetida, and 20.0 ± 0.0 mm by A. trapezoid. Cell proliferation, biofilm inhibition, the synergistic effect of extracts along with antibiotics, and direct bioautography supported the results of agar well diffusion assay. Similarly, P. hawayana, A. corticis, A. caliginosa, and A. trapezoids increase the prothrombin time more efficiently compared to other earthworms. A. corticis, A. gracilis, A. rosea, A. chlorotica, P. elongata, and A. trapezoides showed maximum DPPH scavenging potential effect. Conclusions The coelomic fluid of earthworms possessed several bioactive compounds/enzymes/antioxidants that play an important role in the bacterial inhibition and act as anticoagulant agents. Therefore, the development of new therapeutic drugs from invertebrates could be effective and potential for the prevention of the emergence of multidrug-resistant bacteria.

Testing The Antibacterial Activity of Crude Extracts of Tithonia diversifolia Leaves, Flowers, and Roots in Various Solvent Fraction

Medicinal plants are natural sources of effective antimicrobials from natural products. Tithonia diversifolia ( Asteraceae ) is a conventional herb that can be used for the treatment of malaria, wounds, and other diseases with biological properties such as antioxidant, antimalarial, antidiabetic, antibacterial, and anti-hyperglycemic. Research on Tithonia diversifolia leaf extract as an antibacterial has been widely carried out. However, its use as a natural antibacterial is rarely studied, especially on flowers and roots. This study aimed to test the antibacterial activity of leaf, flower, and root extracts of the Tithonia diversifolia plant in various solvent fractions. The methods used in this research were Antibacterial Testing with Agar Diffusion and Direct Bioautography Methods, while the solvent fractions used were petroleum ether, chloroform, and ethyl acetate. The results showed that the crude extract of Tithonia diversifolia leaves, flowers, and roots for petroleum ether, chloroform, and ethyl acetate fractions contained antibacterial compounds. They could inhibit the growth of E. coli, P. aeruginosa, S. aureus , and B. subtilis bacteria, with inhibitory activity in chloroform fraction, which was better than in petroleum ether and ethyl acetate fractions. The results of the bioautography test showed that the inhibition was good on gram-positive bacteria with the highest number of spots in the chloroform fraction for the crude leaf and flower extract samples.

Composition, Anti-MRSA Activity and Toxicity of Essential Oils from Cymbopogon Species.

Many of the essential oils obtained from medicinal plants possess proven antimicrobial activity and are suitable for medicinal purposes and applications in the food industry. The aim of the present work was the chemical analysis of 19 essential oils (EOs) from seven different Cymbopogon species (C. nardus, C. citratus, C winterianus, C. flexuosus, C. schoenanthus, C. martinii, C. giganteus). Five different chemotypes were established by GC/MS and TLC assay. The EOs, as well as some reference compounds, i.e., citronellol, geraniol and citral (neral + geranial), were also tested for their antimicrobial and antibiofilm activity against methicillin-resistant Staphylococcus aureus (MRSA) by the microdilution method and direct bioautography. The toxicity of EOs was evaluated by Danio rerio ‘Zebrafish’ model assay. All examined EOs showed moderate to high activity against MRSA, with the highest activity noted for C. flexuosus—lemongrass essential oil, both in microdilution and direct autobiography method. Significant difference in the toxicity of the examined EOs was also detected.

New method for screening anti-Leishmania compounds in plants extracts by HPTLC-bioautography

Leishmania genus is responsible for leishmaniasis, a group of diseases affecting 12 million people in the tropical and subtropical zone. Currently, the few drugs that are available to treat this disease are expensive and cause many side effects. Searching for new therapeutics from plant species seems to be a promising path. This work proposes an original HPTLC test against parasites, in particular on Leishmania infantum, to screen new molecules from plant extracts. The technique uses protozoa transformed to express the luciferase gene to observe the bioautogram in bioluminescence. We have developed two different test protocols based on the two dimorphic stages of the parasite. The free promastigote stage, and an intracellular stage parasitizing macrophage cells called the amastigote stage. These two stages only survive under extremely different conditions which required the development of two very different test protocols. For the promastigote free stage of the protozoa, the direct bioautography technique was chosen while for the intracellular amastigote stage, bioautography by immersion (agar overlay) was required. Amphotericine B was chosen as the reference compound for this assay. The development of each of these two tests made it possible to clearly detect areas of activity on the bioautogram, allowing a rapid and inexpensive screening of the antiparasitic properties of molecules in natural extracts.

In Vitro Qualitative Phytochemical Screening, Tlc-Bioautography and Spot Screening of Bistorta Amplexicaulis (D.DON) Greene Extracts

The biological activities of Bistorta amplexicaulis (D.Don) Greene rhizome and leaves extracts were evaluated. In vitro antibacterial activity, direct bioautography, and spot screening of TLC developed bands were investigated against seven bacterial pathogens. Screening of phytochemical constituents was also done through both qualitative and thin layer chromatographic methods. DMSO extracts of rhizome indicated high sensitivity (12.33 ± 1.52 and 11.33 ± 0.57 mm) against Escherichia coli. Whereas methanolic and acetonic extracts of rhizome indicated significant inhibition (11.66 ± 1.15 and 11.33 ± 0.57 mm) of S. marcesscens. All leaf extracts revealed low sensitivity against E. coli. TLC-bioautography and spot screening methods showed the significant use of B. amplexicaulis as an antibacterial agent. Antioxidant activity indicated that acetone and DMSO extracts of rhizome and methanolic leaf extract have maximum scavenging potential. Among the screened phytochemicals, terpenoids, phenols, and quinones were detected in all extracts indicating the potential use of B. amplexicaulis as both antibacterial and antioxidant agents. Bangladesh J. Bot. 50(3): 613-622, 2021 (September)

Nanomole-scaled high-throughput chemistry plus direct bioautography on the same chromatography plate for drug discovery

The powerful fusion of on-surface synthesis and effect-directed analysis was introduced as novel tool for synthetic drug discovery, all on the same high-performance thin-layer chromatography plate. Precise automated sample application allowed both, high-throughput chemistry of 60 reactions at once and reaction miniaturization down to the 15-nmol scale. The antibiotic activity of all on-surface synthesized compounds was evaluated on the same surface via the Gram-positive Bacillus subtilis bioassay. For one product, synthesis (reaction, purification and identification) took 5.3 min and semi-quantitative biological evaluation took 2.8 min. Out of 60 on-surface reactions 10 products (17%) were identified to be more active than a well-known antibiotic reference. The concept was transferred to the Gram-negative Aliivibrio fischeri bioassay. For the first time, a new analytical platform was shown for a streamlined workflow at the most miniaturized scale from synthesis, purification, identification and quantification to semi-quantitative biological activity evaluation (all on the same chromatography plate).

In Vitro Antibacterial and Antibiofilm Activity of Hungarian Honeys against Respiratory Tract Bacteria.

Honey is a rich source of carbohydrates, while minor compounds such as amino acids and polyphenols contribute to its health-promoting effects. Honey is one of the oldest traditional remedies applied for microbial infections, due to its antibacterial, anti-inflammatory, and antioxidant properties. The aim of this study was to investigate the antibacterial and antibiofilm effects of Hungarian black locust, linden, and sunflower honeys against the most common biofilm-forming respiratory tract pathogens Haemophilus spp., Pseudomonas aeruginosa, and Streptococcus pneumoniae. The unifloral character of all three honey types was confirmed by melissopalynological analysis. The antibacterial activity of each honey sample against each bacterium strain was proven with agar well diffusion assay and thin layer chromatography—direct bioautography. Kinetics and mechanisms of antibacterial action were clarified with time-kill assay and membrane degradation study. The anti-biofilm activity was evidenced using crystal violet assay. In each assay, linden honey was the most effective, followed by sunflower and black locust honey. In addition, each honey sample had greater potential to suppress respiratory tract bacteria, compared to major sugar components. In conclusion, honey in general and linden honey in particular, can have a role in the treatment of respiratory tract infections caused by biofilm-forming bacteria.

Diffusible Compounds Produced by Hanseniaspora osmophila and Gluconobacter cerinus Help to Control the Causal Agents of Gray Rot and Summer Bunch Rot of Table Grapes

Gray and summer bunch rot are important diseases of table grapes due to the high economic and environmental cost of their control with synthetic fungicides. The ability to produce antifungal compounds against the causal agents Botrytis, Aspergillus, Penicillium, and Rhizopus of two microorganisms isolated from table grapes and identified as Hanseniaspora osmophila and Gluconobacter cerinus was evaluated. In dual cultures, both biocontrol agents (together and separately) inhibited in vitro mycelial growth of these pathogens. To identify the compounds responsible for the inhibitory effect, extractions were carried out with organic solvents from biocontrol agents separately. Through dual cultures with pathogens and pure extracts, only the hexane extract from H. osmophila showed an inhibitory effect against Botrytis cinerea. To further identify these compounds, the direct bioautography technique was used. This technique made it possible to determine the band displaying antifungal activity at Rf = 0.05–0.2. The compounds present in this band were identified by GC-MS and compared to the NIST library. The most abundant compounds, not previously reported, corresponded to alkanes, ketones, alcohols, and terpenoids. H. osmophila and G. cerinus have the potential to control the causal agents of gray and summer bunch rot of table grapes.

The effect of extractive lacto-fermentation on the bioactivity and natural products content of Pittosporum angustifolium (gumbi gumbi) extracts

Pittosporum angustifolium, known as gumbi gumbi, is a native Australian plant, which has traditionally been used as an Aboriginal medicine. This study investigates the effect of different solvents and extractive fermentation on the content and natural products composition of Pittosporum angustifolium extracts, and compares their antioxidant activity, in vitro α-amylase inhibition, and anti-inflammatory properties. Anti-inflammatory activity of the extracts was determined by measuring the inhibition of nitric oxide (NO) production. Extracts were characterised with FTIR-ATR spectroscopy, and screened for antioxidant activities and α-amylase inhibitory activity via High-performance thin-layer chromatography (HPTLC)-Effect-directed analysis (EDA) with direct bioautography. HPTLC combined with chemical derivatization and bioassays was used for EDA screening. The results show that lactic acid fermentation of gumbi gumbi leaves boosts the antioxidant activity in extracts by increasing the total phenolic content, but does not affect (increase or decrease) α-amylase inhibitory activity or nitrogen scavenging/anti-inflammatory activity. Analysis of the ATR-FTIR spectra from the band at RF = 0.85 that inhibits α-amylase, suggests that fatty acid esters are responsible for the enzyme inhibition; both saturated fatty acid esters in unfermented extracts and unsaturated fatty acid esters in fermented extracts. The ATR-FTIR spectra of the polyphenolics in fermented extracts (RF = 0.15-0.20) suggests the presence of soluble lignin fragments (i.e. lignins depolymerized into monomers and oligomers during the fermentation process).