Investigation of Hydrocolloid Plant Polysaccharides as Potential Candidates to Mimic the Functions of MUC5B in Saliva.

The successful substitution of complex physiological fluids, such as human saliva, remains a major challenge in drug development. Although there are a large number of saliva substitutes on the market, their efficacy is often inadequate due to short residence time in the mouth, unpleasant mouthfeel, or insufficient protection of the teeth. Therefore, systems need to be identified that mimic the functions of saliva, in particular the salivary mucin MUC5B and the unique physiological properties of saliva. To this end, plant extracts known to contain hydrocolloid polysaccharides and to have mucus-forming properties were studied to evaluate their suitability as saliva substitutes. The aqueous plant extracts of Calendula officinalis, Fucus sp. thalli, and lichenan from Lichen islandicus were examined for composition using a range of techniques, including GC-MS, NMR, SEC, assessment of pH, osmolality, buffering capacity, viscoelasticity, viscoelastic interactions with human saliva, hydrocolloid network formation, and in vitro cell adhesion. For this purpose, a physiologically adapted adhesive test was developed using human buccal epithelial cells. The results show that lichenan is the most promising candidate to mimic the properties of MUC5B. By adjusting the pH, osmolality, and buffering capacity with K2HPO4, it was shown that lichenan exhibited high cell adhesion, with a maximum detachment force that was comparable to that of unstimulated whole mouth saliva.

Larvicidal, Ovicidal, and Repellent Activities of Leucas stachydiformis (Hochst. ex Benth.) Briq Essential Oil against Anopheles arabiensis.

Larvicidal, ovicidal, and repellent activities of the essential oil extracted by hydrodistillation from the leaves of the endemic Ethiopian plant Leucas stachydiformis (Hochst. ex Benth.) Briq were investigated against Anopheles arabiensis, the dominant malaria vector species in Ethiopia with the objective of searching for a plant-based malaria vector control strategy from medicinal plants. The larvicidal effect was tested against the fourth instar An. arabiensis wild larvae whilst freshly laid ova of An. arabiensis were used to determine the ovicidal activity of the essential oil at concentrations ranging from 6.25 to 400 ppm. Concentrations of 41.6-366.7 µg/cm2 were used to evaluate the repellent activity of the essential oil on 3-5 days old adult female An. arabiensis. The oil composition of L. stachydiformis was also analyzed using GC-MS. The study revealed that the oil possesses the highest larvicidal activity at 400 ppm and 200 ppm after 24 h and 48 h of treatment. LC50 values for the fourth larval instar after 24 h and 48 h of treatment were 43.4 ppm and 34.2 ppm, respectively. After 72 h of exposure, the oil displayed 100% ovicidal activity at 400 ppm with an IH50 value of 32.2 ppm. In the repellency test, at concentrations of 366.7, 133.3, and 41.6 µg/cm2, the oil gave a total percentage protection of 67.9 ± 4.2%, 37.2 ± 2.8%, and 32 ± 2.2%, respectively, for 4 h. The highest concentration (366.7 µg/cm2) gave 100% protection up to 90 min. GC-MS analysis of the oil revealed the presence of 24 compounds representing 90.34% of the total oil with caryophyllene oxide, germacrene D, and trans-caryophyllene constituting more than 50% of its components. Results of the present study suggest that the essential oil of L. stachydiformis has the potential to be used for the control of An. arabiensis mosquitoes.

Natural Deep Eutectic Solvents for the Extraction of Spilanthol from Acmella oleracea (L.) R.K.Jansen.

With a growing focus on green chemistry, the extraction of natural products with natural deep eutectic solvents (NADES), which are eutectic mixtures of hydrogen bond donors and acceptors, has become an ever-expanding field of research. However, the use of NADES for the extraction of spilanthol from Acmella oleracea (L.) R.K.Jansen has not yet been investigated. Therefore, in this study, 20 choline chloride-based NADES, and for comparison, ethanol, were used as green extraction agents for spilanthol from Acmella oleracea flower heads. The effects of time, water addition, and temperature on NADES extractions were investigated and analysed by HPLC-DAD quantification. Additionally, UHPLC-DAD-ESI-MSn results for dichloromethane extracts, as well as the isolation of spilanthol and other main constituents as reference compounds, are reported. The best green extraction results were achieved by choline chloride (ChCl) with 1,2-propanediol (P, 1:2 molar ratio, +20% water) at 244.58 µg/mL, comparable to yields with ethanol (245.93 µg/mL). Methylurea (MeU, 1:2, +20% water) also showed promising results as a hydrogen bond donor in combination with choline chloride (208.12 µg/mL). In further experiments with NADES ChCl/P (1:2) and ChCl/MeU (1:2), extraction time had the least effect on spilanthol extraction with NADES, while yield decreased with water addition over 20% and increased with extraction temperature up to 80 °C. NADES are promising extraction agents for the extraction of spilanthol, and these findings could lead to applicable extracts for medicinal purposes, due to their non-toxic constituents.

Highly oxidized flavones in Artemisia species - structure revisions and improved UHPLC-MSn analysis.

In course of our studies of the aerial parts of Artemisia abrotanum the major methoxyflavonol could be isolated. However, by NMR structural analysis it became obvious that the substitution pattern in ring B differs from reports for casticin (2). The position of methoxyl and hydroxyl groups are interchanged, i.e., the major flavone is actually chrysosplenetin (1). Three structures in A. abrotanum and A. frigida had to be revised. Use of pyridine-d5 instead of DMSO­d6 made the resolution of the B-ring 1H and 13C NMR signals possible and enabled correct structural assignment by 2D NMR experiments. Results from NMR structure elucidation for A. abrotanum were confirmed by LC-PDA-ESI-MSn analysis when a PFP (pentafluorophenyl) stationary phase with an optimized gradient elution was applied for separation of 1 and 2 instead of a corresponding C-18 phase. Electrospray mass spectrometry (positive and negative mode) with subsequent fragmentation (ESI-MSn) revealed distinctive mass spectral features of both compounds, especially at MS4 level. Several Artemisia extracts including A. annua were analysed on the PFP phase for the presence of 1 and 2.

Phytochemical Analysis of Pinus cembra Heartwood-UHPLC-DAD-ESI-MSn with Focus on Flavonoids, Stilbenes, Bibenzyls and Improved HPLC Separation.

The heartwood of the Swiss Stone Pine, Pinus cembra L., has been scarcely investigated for secondary metabolites for a long period of time. Considering age and relative simplicity of heartwood investigations dating back to the 1940s to 1960s, we conducted the first investigation of P. cembra heartwood by HPLC, using UHPLC-DAD-ESI-MSn and HPLC-DAD techniques in combination with isolation and NMR spectroscopy, with focus on stilbenes, bibenzyls and flavonoids. Analytical problems in the HPLC analysis of Pinus stilbenes and flavonoids on reversed stationary phases were also challenged, by comparing HPLC on pentafluorophenyl (PFP) and C18 stationary phases. Seven flavonoids (1, 2, 3, 7, 8, 11, 12), four stilbenes (4, 6, 10, 13), two bibenzyls (5, 9), three fatty acids (14, 16, 17) and one diterpenic acid (15) were detected in an ethanolic extract of Pinus cembra heartwood. HPLC comparison of reversed stationary phases in HPLC showed that the antifungal, antibacterial and chemosensitizing dihydropinosylvin monomethyl ether (9) and pinosylvin monomethyl ether (10) can be separated on PFP, but not on C18 material, when eluting with a screening gradient of 20-100% acetonitrile. Flavonoid separation showed additional benefits of combining analyses on different stationary phases, as flavonoids 7 and 8 could only be separated on one of two C18 stationary phases. Earlier phytochemical results for heartwood investigations were shown to be mostly correct, yet expandable. Substances 5 to 12 were found in alignment with these references, proving remarkable phytochemical analyses at the time. Evidence for the described presence of pinobanksin could not be found. Substances 1 to 4 and 13 have to our knowledge not yet been described for P. cembra.

Cycloartane Saponins from Astragalus glycyphyllos and Their In Vitro Neuroprotective, Antioxidant, and hMAO-B-Inhibiting Effects.

Astragalus glycyphyllos (Fabaceae) is used in the traditional medicine of many countries against hepatic and cardiac disorders. The plant contains mainly flavonoids and saponins. From a defatted methanol extract from its overground parts, a new triterpenoid saponin, 3-O-[α-L-rhamnopyranosyl-(1→2)]-ß-D-xylopyranosyl]-24-O-α-L-arabinopyranosyl-3ß,6α,16ß,24(R),25-pentahydroxy-20R-cycloartane, together with the rare saponin astrachrysoside A, were isolated using various chromatography methods. The compounds were identified via extensive high resolution electrospray ionisation mass spectrometry (HRESIMS) and NMR analyses. Both saponins were examined for their possible antioxidant and neuroprotective activity in three different in vitro models. Rat brain synaptosomes, mitochondria, and microsomes were isolated via centrifugation using Percoll gradient. They were treated with the compounds in three different concentrations alone, and in combination with 6-hydroxydopamine or tert-butyl hydroperoxide as toxic agents. It was found that the compounds had statistically significant dose-dependent in vitro protective activity on the sub-cellular fractions. The compounds exhibited a weak inhibitory effect on the enzyme activity of human recombinant monoamine oxidase type B (hMAO-B), compared to selegiline.

Three cuticular amides in the tripartite symbiosis of leafcutter ants.

Cuticular hydrocarbons (CHCs) play various roles in insects' chemical ecology. As leafcutter ants live in a specific symbiosis with fungi, they harvest and with different bacteria, some of these CHCs might be associated with a mutualistic function within this symbiosis. To obtain a more precise picture in that respect we compared the CHC profiles of the leafcutter ants, Atta sexdens, Atta cephalotes, and Acromyrmex octospinosus inhabited by mutualistic bacteria with the profiles of Polyrhachis dives and Messor aciculatus by GC-EI-MS analysis and 28 other ant species by data from the literature. We were able to identify three alkyl amides (hexadecanamide, hexadecenamide, and tetradecanamide), occurring only in the CHC profiles of leafcutter ants inhabited by symbiotic bacteria. Our results lead to the conclusion that those alkyl amides could have a function in the tripartite symbiosis of leafcutter ants.

Chemical composition and antimicrobial activity of essential oils made from Lavandula x intermedia from Hvar (Croatia).

The aim of the study was to investigate the essential oil (EO) of Lavandula x intermedia cv. Bila, which has not been studied before. The EOs were distilled from plants collected in two consecutive years on the island of Hvar (Croatia) and in the Karst (Slovenia) and analysed for chemical composition and antimicrobial activity. The main component of EOs was linalool, but the EOs from Hvar had higher contents of Z-ß-ocimene and borneol + lavandulol than the EOs from Karst, in which camphor, linalyl acetate and 1,8-cineole predominated. The antimicrobial activity was evaluated using the minimum inhibitory concentration and proved that the EOs were effective against Candida spp. Studies have shown that the composition of L. x intermedia EO depends on the variety and the climatic and geographical characteristics of the plant growth. The antimicrobial activity of EO is also influenced by the type and strain of microorganisms involved in the research.

Amaryllidaceae Alkaloids from Clivia miniata (Lindl.) Bosse (Amaryllidaceae): Isolation, Structural Elucidation, and Biological Activity.

Clivia miniata (Amaryllidaceae) is an herbaceous evergreen flowering plant that is endemic to South Africa and Swaziland and belongs to one of the top-10 traded medicinal plants in informal medicine markets in South Africa. The species has been reported as the most important component of a traditional healer's pallet of healing plants. Eighteen known Amaryllidaceae alkaloids (AAs) of various structural types, and one undescribed alkaloid of homolycorine-type, named clivimine B (3), were isolated from Clivia miniata. The chemical structures of the isolated alkaloids were elucidated by a combination of MS, HRMS, 1D and 2D NMR techniques and by comparison with literature data. Compounds isolated in a sufficient quantity, and not tested previously, were evaluated for their in vitro acetylcholinesterase (AChE; E.C. 3.1.1.7) and butyrylcholinesterase (BuChE; E.C. 3.1.1.8) inhibition activities.

Structural analysis of unusual alkaloids isolated from Narcissus pseudonarcissus cv. Carlton.

Narciindole A, the first representative of Amaryllidaceae alkaloids with an indol-3-ylmethanone framework, was isolated from bulbs of Narcissus pseudonarcissus (L.) cv. Carlton, together with carltonine D and carltonine E, which share the same unusual structural motif as dimeric carltonine C (reported in 2020), exhibiting atropisomerism. Unambiguous structure elucidations have been achieved by NMR spectroscopy, HRMS, and comparison with literature data of related alkaloids. Furthermore, the chirality of known alkaloids with a galanthindole biaryl core was revised using optical rotation. Last, but not least, a biosynthetic pathway for dimeric carltonine-type alkaloids was proposed. Unfortunately, in terms of biological activity, the isolated alkaloids showed only moderate inhibition of human acetylcholinesterase and/or butyrylcholinesterase.

Rhodiola rosea Reduces Intercellular Signaling in Campylobacter jejuni.

Campylobacter jejuni is a major foodborne pathogen and the leading cause of bacterial gastroenteritis, i.e., campylobacteriosis. Besides searching for novel antimicrobials, identification of new targets for their action is becoming increasingly important. Rhodiola rosea has long been used in traditional medicine. Ethanolic extracts from the roots and rhizomes of the plant contain a wide range of bioactive compounds with various pharmacological activities. In this study, cultivated plant materials have been used, i.e., "Mattmark" and "Rosavine". Through optimized protocols, we obtained fractions of the initial ethanolic extracts rich in most important bioactive compounds from R. rosea, including salidroside, rosavins, proanthocyanidins (PACs), and flavonoids. The antimicrobial activity in relation to the chemical composition of the extracts and their fractions was studied with an emphasis on C. jejuni AI-2-mediated intercellular signaling. At concentration 15.625 mg/L, bioluminescence reduction rates varied from 27% to 72%, and the membrane remained intact. Fractions rich in PACs had the strongest antimicrobial effect against C. jejuni, with the lowest minimal inhibitory concentrations (MICs) (M F3 40%: 62.5 mg/L; R F3 40%: 250 mg/L) and the highest intercellular signaling reduction rates (M F3 40%: 72%; R F3 40%: 65%). On the other hand, fractions without PACs were less effective (MICs: M F5 PVP: 250 mg/L; R F5 PVP: 1000 mg/L and bioluminescence reduction rates: M F5 PVP: 27%; R F5 PVP: 43%). Additionally, fractions rich in flavonoids had strong antimicrobial activity (MICs: M F4 70%: 125 mg/L; R F4 70%: 250 mg/L and bioluminescence reduction rates: M F4 70%: 68%; R F4 70%: 50%). We conclude that PACs and flavonoids are crucial compound groups responsible for the antimicrobial activity of R. rosea roots and rhizomes in C. jejuni.

Campylobacter jejuni Biofilm Control with Lavandin Essential Oils and By-Products.

The food industry is constantly struggling with one of the most prevalent biofilm-forming and food-borne pathogenic bacteria, Campylobacter jejuni. Different approaches are used to control biofilms in the food production chain, but none is fully effective. In this study, we aim to produce and determine the chemical profile of essential oils (EOs), ethanolic extracts of flowers prior to distillation (EFs), and ethanolic extracts of post-distillation waste material (EWMs) from Lavandula × intermedia 'Bila', 'Budrovka' St Nicholas and 'Budrovka', which were further used to reduce C. jejuni intercellular signaling, adhesion, and biofilm formation, as well as to test their antioxidant activity. Glycosides of hydroxycinnamic acids were the major constituents of both types of lavandin ethanolic extract, while linalool, linalyl acetate, 1,8-cineol, and camphor were the major compounds found in lavandin EOs. Tested EOs showed the best antibacterial activity with a minimal inhibitory concentration of 0.25 mg/mL. Lavandin EFs proved more effective in reducing C. jejuni intercellular signaling and adhesion compared to lavandin EOs and EWMs, while lavandin EOs showed a slightly better effect against biofilm formation. Interestingly, the best antioxidant activity was determined for lavandin EWMs. A positive and moderate correlation was found between the reduction of C. jejuni intercellular signaling and adhesion, as well as between adhesion and biofilm formation. These findings mean novel bacterial targets are of interest for biofilm control with alternative natural agents throughout the whole food production chain.

Globularia alypum L. and Related Species: LC-MS Profiles and Antidiabetic, Antioxidant, Anti-Inflammatory, Antibacterial and Anticancer Potential.

Species from the genus Globularia L. have been used as healing agents for various ailments, with utilization of Globularia alypum L. being most frequently reported. The aim of this study was to evaluate the antidiabetic, antioxidant, anti-inflammatory, antibacterial and anticancer potential of G. alypum and three related species, G. punctata Lapeyr., G. cordifolia L. and G. meridionalis (Podp.) O.Schwarz, in relation to their phytochemical compositions. Globularin and verbascoside were identified using LC-PDA-ESI-MSn as the major metabolites of G. alypum with known biological activities. G. alypum demonstrated the greatest α-glucosidase inhibitory activity and DPPH radical scavenging activity (IC50 = 17.25 µg/mL), while its anti-inflammatory activity was not significantly different from those of related species. All investigated species showed considerable antibacterial activity against methicillin-resistant Staphylococcus aureus in the broth microdilution method (MIC = 1.42-3.79 mg/mL). G. punctata also showed antibacterial activities against Escherichia coli (MIC = 1.42 mg/mL), Bacillus subtilis (MIC = 1.89 mg/mL), B. cereus (MIC = 2.84 mg/mL) and Enterococcus faecalis (MBC = 5.68 mg/mL). G. punctata, G. cordifolia and G. meridionalis showed greater anticancer potential than G. alypum. Obtained results indicate investigated Globularia species could serve as sources of diverse bioactive molecules, with G. punctata having the greatest antibacterial potential.

Flavonoids as Inhibitors of Bacterial Efflux Pumps.

Flavonoids are widely occurring secondary plant constituents, and are abundant in vegetable and fruit diets as well as herbal medicines. Therapeutic treatment options for bacterial infections are limited due to the spread of antimicrobial resistances. Hence, in a number of studies during the last few years, different classes of plant secondary metabolites as resistance-modifying agents have been carried out. In this review, we present the role of flavonoids as inhibitors of bacterial efflux pumps. Active compounds could be identified in the subclasses of chalcones, flavan-3-ols, flavanones, flavones, flavonols, flavonolignans and isoflavones; by far the majority of compounds were aglycones, although some glycosides like kaempferol glycosides with p-coumaroyl acylation showed remarkable results. Staphylococcus aureus NorA pump was the focus of many studies, followed by mycobacteria, whereas Gram-negative bacteria are still under-investigated.

UHPLC Analysis of Reynoutria japonica Houtt. Rhizome Preparations Regarding Stilbene and Anthranoid Composition and Their Antimycobacterial Activity Evaluation.

Reynoutria japonica Houtt. is a critical invasive alien plant in Europe and North America with a drastic impact on native flora. However, R. japonica has medicinal potential, especially as a source of stilbenes. In order to explore the potential of simple extractions of R. japonica, we conducted qualitative and quantitative analyses of fresh R. japonica rhizome infusion, decoction, and macerates with ethanol by UHPLC-DAD-ESI-MSn and UHPLC-DAD, with a focus on major constituent groups of stilbenes and anthranoids. Since R. japonica rhizome extracts showed antimicrobial potential in the past, we also evaluated the antimycobacterial effect of raw R. japonica extracts for the first time against Mycobacterium smegmatis. Of thirty-four characterized substances, six were stilbenes and twelve anthranoids. The main constituents, four trans-stilbenes and eight anthranoids, were quantified in a validated UHPLC-DAD method. The 38% ethanol macerate showed high stilbene (155.078 mg/100 g fluid extract) and low anthranoid content (5.420 mg/100 g fluid extract), while decoction showed the highest anthranoids. Antimycobacterial testing gave good results for all macerates (MIC 256 µg/mL) and trans-resveratrol (64 µg/mL). Extraction and enrichment of stilbenes from fresh plant material by simple extraction methods with food-grade solvents might encourage consideration of wild harvest of rhizomes over classic means of eradication of R. japonica.

Efflux Pump Inhibition and Resistance Modulation in Mycobacterium smegmatis by Peucedanum ostruthium and Its Coumarins.

Antibiotic resistance is a growing problem and may become the next major global health crisis if no timely actions are taken. Mycobacterial infections are widespread and, due to antibiotic resistance, also hard to treat and a major cause of mortality. Natural compounds have the potential to increase antibiotic effectiveness due to their resistance modulatory and antimicrobial effects. In this study, Peucedanum ostruthium extracts, fractions, and isolated compounds were investigated regarding their antimicrobial and resistance-modulatory effects as well as efflux pump inhibition in Mycobacterium smegmatis. P. ostruthium extracts were found to have anti-mycobacterial potential and resistance modulating effects on ethidium bromide activity. The major antibacterial effect was attributed to ostruthin, and we found that the more lipophilic the substrate, the greater the antimicrobial effect. Imperatorin caused potent modulatory effects by interfering with the action of the major LfrA efflux pump in M. smegmatis. The plant P. ostruthuim has a complex effect on M. smegmatis, including antibacterial, efflux pump inhibition, resistance modulation, and membrane permeabilization, and its major constituents, ostruthin and imperatorin, have a distinct role in these effects. This makes P. ostruthium and its coumarins promising therapeutics to consider in the fight against drug-resistant mycobacteria.

Antibiofilm Potential of Lavandula Preparations against Campylobacter jejuni.

New approaches for the control of Campylobacter jejuni biofilms in the food industry are being studied intensively. Natural products are promising alternative antimicrobial substances to control biofilm production, with particular emphasis on plant extracts. Dried flowers of Lavandula angustifolia were used to produce essential oil (LEO), an ethanol extract (LEF), and an ethanol extract of Lavandula postdistillation waste material (LEW). The chemical compositions determined for these Lavandula preparations included seven major compounds that were selected for further testing. These were tested against C. jejuni for biofilm degradation and removal. Next-generation sequencing was used to study the molecular mechanisms underlying LEO actions against C. jejuni adhesion and motility. Analysis of LEO revealed 1,8-cineol, linalool, and linalyl acetate as the main components. For LEF and LEW, the main components were phenolic acid glycosides, with flavonoids rarely present. The MICs of the Lavandula preparations and pure compounds against C. jejuni ranged from 0.2 mg/ml to 1 mg/ml. LEO showed the strongest biofilm degradation. The reduction of C. jejuni adhesion was ≥1 log10 CFU/ml, which satisfies European Food Safety Authority recommendations. Lavandula preparations reduced C. jejuni motility by almost 50%, which consequently can impact biofilm formation. These data are in line with the transcriptome analysis of C. jejuni, which indicated that LEO downregulated genes important for biofilm formation. LEW also showed good antibacterial and antibiofilm effects, particularly against adhesion and motility mechanisms. This defines an innovative approach using alternative strategies and novel targets to combat bacterial biofilm formation and, hence, the potential to develop new effective agents with biofilm-degrading activities. IMPORTANCE The Lavandula preparations used in this study are found to be effective against C. jejuni, a common foodborne pathogen. They show antibiofilm properties at subinhibitory concentrations in terms of promoting biofilm degradation and inhibiting cell adhesion and motility, which are involved in the initial steps of biofilm formation. These results are confirmed by transcriptome analysis, which highlights the effect of Lavandula essential oil on C. jejuni biofilm properties. We show that the waste material from the hydrodistillation of Lavandula has particular antibiofilm effects, suggesting that it has potential for reuse for industrial purposes. This study highlights the need for efforts directed toward such innovative approaches and alternative strategies against biofilm formation and maintenance by developing new naturally derived agents with antibiofilm activities.

Palladium-catalysed synthesis of arylnaphthoquinones as antiprotozoal and antimycobacterial agents.

Malaria and tuberculosis are still among the leading causes of death in low-income countries. The 1,4-naphthoquinone (NQ) scaffold can be found in a variety of anti-infective agents. Herein, we report an optimised, high yield process for the preparation of various 2-arylnaphthoquinones by a palladium-catalysed Suzuki reaction. All synthesised compounds were evaluated for their in-vitro antiprotozoal and antimycobacterial activity. Antiprotozoal activity was assessed against Plasmodium falciparum (P.f.) NF54 and Trypanosoma brucei rhodesiense (T.b.r.) STIB900, and antimycobacterial activity against Mycobacterium smegmatis (M.s.) mc2 155. Substitution with pyridine and pyrimidine rings significantly increased antiplasmodial potency of our compounds. The 2-aryl-NQs exhibited trypanocidal activity in the nM range with a very favourable selectivity profile. (Pseudo)halogenated aryl-NQs were found to have a pronounced effect indicating inhibition of mycobacterial efflux pumps. Cytotoxicity of all compounds towards L6 cells was evaluated and the respective selectivity indices (SI) were calculated. In addition, the physicochemical parameters of the synthesised compounds were discussed.

Antimicrobial and Efflux Pump Inhibitory Activity of Carvotacetones from Sphaeranthus africanus Against Mycobacteria.

Carvotacetones (1-7) isolated from Sphaeranthus africanus were screened for their antimycobacterial and efflux pump (EP) inhibitory potential against the mycobacterial model strains Mycobacterium smegmatis mc2 155, Mycobacterium aurum ATCC 23366, and Mycobacterium bovis BCG ATCC 35734. The minimum inhibitory concentrations (MICs) of the carvotacetones were detected through high-throughput spot culture growth inhibition (HT-SPOTi) and microbroth dilution assays. In order to assess the potential of the compounds 1 and 6 to accumulate ethidium bromide (EtBr) in M. smegmatis and M. aurum, a microtiter plate-based fluorometric assay was used to determine efflux activity. Compounds 1 and 6 were analyzed for their modulating effects on the MIC of EtBr and the antibiotic rifampicin (RIF) against M. smegmatis. Carvotacetones 1 and 6 had potent antibacterial effects on M. aurum and M. bovis BCG (MIC ≤ 31.25 mg/L) and could successfully enhance EtBr activity against M. smegmatis. Compound 1 appeared as the most efficient agent for impairing the efflux mechanism in M. smegmatis. Both compounds 1 and 6 were highly effective against M. aurum and M. bovis BCG. In particular, compound 1 was identified as a valuable candidate for inhibiting mycobacterial efflux mechanisms and as a promising adjuvant in the therapy of tuberculosis or other non-tubercular mycobacterial infections.

Targeting fish spoilers Pseudomonas and Shewanella with oregano and nettle extracts.

To control Pseudomonas and Shewanella as important psychrotrophic spoilage bacteria in fish meat, we used ethanolic extracts of oregano (Origanum vulgare subsp. vulgare) and nettle (Urtica dioica), with phytochemical characterisation of the extracts and their bioactive compounds. Liquid chromatography coupled with photodiode array detection and electrospray ionisation-mass spectrometry was used for qualitative compositional determination of the extracts. Four main compounds were identified in the oregano extract, with rosmarinic acid the most abundant, followed by three glycosylated phenolics, one of which is reported for the first time in O. vulgare: 4'-O-ß-d-glucopyranosyl-3',4'-dihydroxybenzyl-4-hydroxybenzoate. Six main compounds were identified in the nettle extract, as caffeoylmalic acid and five flavonoid glycosides. These oregano and nettle ethanolic extracts showed in-vitro antimicrobial activities against selected Pseudomonas and Shewanella strains in broth and fish meat homogenate when evaluated at two inoculum concentrations. The antimicrobial activities were more pronounced for the nettle extract at the lower inoculum concentration, and for both the Shewanella strains. Growth inhibition in the fish meat homogenate was evaluated at 3.13 mg/mL and 1.56 mg/mL at 5 °C. Again, the nettle extract showed greater antimicrobial activity, which was seen as the lowest maximum growth rate, followed by the oregano extract, which was inhibitory only at 3.13 mg/mL. Finally, the extracts were applied to fish meat that was then stored at 5 °C for 9 days. Evaluation here was for the counts of the mesophilic, psychrotrophic, Pseudomonas and H2S producers. These confirmed the better antimicrobial effects of the nettle extract, especially against the H2S-producing bacteria, which included Shewanella. Both of the extracts were rich in glycosides of flavonoids and phenolic acids. The enzymatic activities of the Pseudomonas and Shewanella spoilage bacteria and their actions on the phenolic glycosides from natural sources will be further investigated.