Evaluation of In Vitro Synergistic Effects of Tetracycline with Alkaloid-Related Compounds against Diarrhoeic Bacteria.

Diarrhoea remains an important public health concern, particularly in developing countries, and has become difficult to treat because of antibacterial resistance. The development of synergistic antimicrobial agents appears to be a promising alternative treatment against diarrhoeic infections. In this study, the combined effect of tetracycline together with either nitroxoline, sanguinarine, or zinc pyrithione (representing various classes of plant-based compounds) was evaluated in vitro against selected diarrhoeic bacteria (Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Shigella flexneri, Vibrio parahaemolyticus, and Yersinia enterocolitica). The chequerboard method in 96-well microtiter plates was used to determine the sum of the fractional inhibitory concentration indices (FICIs). Three independent experiments were performed per combination, each in triplicate. It was observed that the combination of tetracycline with either nitroxoline, sanguinarine, or zinc pyrithione produced synergistic effects against most of the pathogenic bacteria tested, with FICI values ranging from 0.086 to 0.5. Tetracycline-nitroxoline combinations produced the greatest synergistic action against S. flexneri at a FICI value of 0.086. The combinations of the agents tested in this study can thus be used for the development of new anti-diarrhoeic medications. However, studies focusing on their in vivo anti-diarrhoeic activity and safety are required before any consideration for utilization in human medicine.

Validation of Broth Macrodilution Volatilization Method for Testing of Essential Oils in Liquid and Vapor Phase: Chemical Composition, Cytotoxicity, and Antibacterial Effect of Indian Medicinal Plants against Pneumonia-Causing Pathogens.

Essential oils (EOs) have great potential in inhalation therapy for the treatment of respiratory infections. However, innovative methods for evaluation of antimicrobial activity of their vapors are still needed. The current study reports validation of the broth macrodilution volatilization method for assessment of the antibacterial properties of EOs and shows the growth-inhibitory effect of Indian medicinal plants against pneumonia-causing bacteria in liquid and vapor phase. Among all samples tested, Trachyspermum ammi EO exhibits the strongest antibacterial effect against Haemophilus influenzae, with minimum inhibitory concentrations of 128 and 256 µg/mL in the liquid and vapor phases, respectively. Furthermore, Cyperus scariosus EO is found to be nontoxic to normal lung fibroblasts assessed by modified thiazolyl blue tetrazolium bromide assay. Chemical analysis performed using gas chromatography-mass spectrometry identified α-citral, cyperotundone, and thymol as the main constituents of Cymbopogon citratus, C. scariosus, and T. ammi EOs, respectively. In addition, ß-cymene is identified as the major compound of T. ammi EO vapors when analyzed using solid-phase microextraction and gas-tight syringe sampling techniques. This study demonstrates the validity of the broth macrodilution volatilization method for antimicrobial screening of volatile compounds in the vapor phase and suggests the therapeutic potential of Indian medicinal plants in inhalation therapy.

Antibacterial Activity of Thymus vulgaris L. Essential Oil Vapours and Their GC/MS Analysis Using Solid-Phase Microextraction and Syringe Headspace Sampling Techniques.

While the inhalation of Thymus vulgaris L. essential oil (EO) is commonly approved for the treatment of mild respiratory infections, there is still a lack of data regarding the antimicrobial activity and chemical composition of its vapours. The antibacterial activity of the three T. vulgaris EOs against respiratory pathogens, including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes, was assessed in both liquid and vapour phases using the broth microdilution volatilisation (BMV) method. With the aim of optimising a protocol for the characterisation of EO vapours, their chemical profiles were determined using two headspace sampling techniques coupled with GC/MS: solid-phase microextraction (HS-SPME) and syringe headspace sampling technique (HS-GTS). All EO sample vapours exhibited antibacterial activity with minimum inhibitory concentrations (MIC) ranging from 512 to 1024 µg/mL. According to the sampling technique used, results showed a different distribution of volatile compounds. Notably, thymol was found in lower amounts in the headspace-peak percentage areas below 5.27% (HS-SPME) and 0.60% (HS-GTS)-than in EOs (max. 48.65%), suggesting that its antimicrobial effect is higher in vapour. Furthermore, both headspace sampling techniques were proved to be complementary for the analysis of EO vapours, whereas HS-SPME yielded more accurate qualitative results and HS-GTS proved a better technique for quantitative analysis.

New Broth Macrodilution Volatilization Method for Antibacterial Susceptibility Testing of Volatile Agents and Evaluation of Their Toxicity Using Modified MTT Assay In Vitro.

In this study, a new broth macrodilution volatilization method for the simple and rapid determination of the antibacterial effect of volatile agents simultaneously in the liquid and vapor phase was designed with the aim to assess their therapeutic potential for the development of new inhalation preparations. The antibacterial activity of plant volatiles (ß-thujaplicin, thymohydroquinone, thymoquinone) was evaluated against bacteria associated with respiratory infections (Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and their cytotoxicity was determined using a modified thiazolyl blue tetrazolium bromide assay against normal lung fibroblasts. Thymohydroquinone and thymoquinone possessed the highest antibacterial activity against H. influenzae, with minimum inhibitory concentrations of 4 and 8 µg/mL in the liquid and vapor phases, respectively. Although all compounds exhibited cytotoxic effects on lung cells, therapeutic indices (TIs) suggested their potential use in the treatment of respiratory infections, which was especially evident for thymohydroquinone (TI > 34.13). The results demonstrate the applicability of the broth macrodilution volatilization assay, which combines the principles of broth microdilution volatilization and standard broth macrodilution methods. This assay enables rapid, simple, cost- and labor-effective screening of volatile compounds and overcomes the limitations of assays currently used for screening of antimicrobial activity in the vapor phase.

Volatile Antimicrobial Agents and In Vitro Methods for Evaluating Their Activity in the Vapour Phase: A Review.

This review summarizes data on the in vitro antimicrobial effectiveness of volatile agents of plant origin and in vitro methods for evaluating their activity in the vapour phase. As a result of literature analysis, the antimicrobial efficacy of vapours from 122 different plant species and 19 pure compounds examined in 61 studies using different in vitro tests against a broad spectrum of microorganisms was identified and summarized. In addition, 11 different techniques found in the literature are described in detail. An original classification of methods based on the solid and liquid matrix volatilization principle is proposed because carrier medium/matrix selection is crucial for the volatilization of any agents tested. This review should be useful for medicinal, pharmaceutical, food, and agricultural experts working in areas related to the management of infectious diseases (especially respiratory and skin infections), food preservation (active packaging), and protection of agriculture products (controlled atmosphere). It may also stimulate the interest of pharmaceutical, cosmetic, food, and agriculture industries in the research and development of new antimicrobial agents of natural origin. Since several original apparatuses previously developed for antimicrobial susceptibility testing in the vapour phase are described in this review, labware manufacturers may also be interested in this topic. The review also provides specific guidelines and recommendations for researchers studying the antimicrobial activity of volatile agents. The article will therefore appeal to communities of industrial stakeholders, pharmacists, physicians, food experts, agriculturists, and researchers in related areas such as pharmacology, medicinal chemistry, microbiology, natural product chemistry, food preservation and plant protection.

Vapors of Volatile Plant-Derived Products Significantly Affect the Results of Antimicrobial, Antioxidative and Cytotoxicity Microplate-Based Assays.

Volatile plant-derived products were observed to exhibit broad spectrum of biological effects. However, due to their volatility, results of conventional microplate-based bioassays can be significantly affected by the vapors. With aim to demonstrate this phenomenon, antimicrobial, antioxidant, and cytotoxic activities of three essential oils (Alpinia elegans, Cinnamomum iners, and Xanthostemon verdugonianus), one supercritical CO2 extract (Nigella sativa), and four plant-derived compounds (capsaicin, caryophyllene oxide, 8-hydroxyquinoline, and thymoquinone) were evaluated in series of experiments including both ethylene vinyl acetate (EVA) Capmat sealed and nonsealed microplates. The results clearly illustrate that vapor transition to adjoining wells causes false-positive results of bioassays performed in nonsealed microtiter plates. The microplate layout and a duration of the assay were demonstrated as the key aspects defining level of the results affection by the vapors of volatile agents. Additionally, we reported biological activities and chemical composition of essential oils from A. elegans seeds and X. verdugonianus leaves, which were, according to our best knowledge, analyzed for the first time. Considering our findings, certain modifications of conventional microplate-based assays are necessary (e.g., using EVA Capmat as vapor barrier) to obtain reliable results when biological properties of volatile agents are evaluated.

Maytenus macrocarpa (Ruiz & Pav.) Briq.: Phytochemistry and Pharmacological Activity.

Maytenus macrocarpa (Celastraceae) is a tree native to Amazonia. Its roots, leaves, bark, and combinations of these are used in traditional medicine mainly to treat rheumatism and, to a lesser extent, to heal wounds and to combat bronchitis and diarrhea. To date, mainly triterpenes and dihydro-ß-agarofuran sesquiterpenes were isolated from M. macrocarpa. Extracts and selected pure compounds isolated from the leaves, roots, and stem bark showed antibacterial, antiviral, antiparasitic, anti-inflammatory, and cytotoxic activities in vitro. The aim of this review is to summarize the available ethnobotanical, phytochemical, and pharmacological information about this traditional Amazonian medicinal tree, as well as to attract the attention of phytochemists and pharmacognosists to this potentially interesting source of ethnopharmaceuticals.

Screening of in vitro antimicrobial activity of plants used in traditional Indonesian medicine.

CONTEXT: In many regions of Indonesia, there are numerous traditional herbal preparations for treatment of infectious diseases. However, their antimicrobial potential has been poorly studied by modern laboratory methods. OBJECTIVE: This study investigates in vitro antimicrobial activity of 49 ethanol extracts from 37 plant species used in Indonesian traditional medicine for treatment against Candida albicans, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. MATERIALS AND METHODS: The plants were collected from the Biopharmaca collection garden, Bogor, Indonesia. The plant material was dried, finely grounded, extracted using ethanol, concentrated, and the dried residue was dissolved in 100% DMSO. Antimicrobial activity was determined in terms of a minimum inhibitory concentration (MIC) using a broth microdilution method in 96-well microplates. RESULTS: The extract of Orthosiphon aristatus (Blume) Miq. (Lamiaceae) leaf produced the strongest antimicrobial effect, inhibiting the growth of C. albicans (MIC 128 µg/mL), S. aureus (MIC 256 µg/mL), E. faecalis (MIC 256 µg/mL) and P. aeruginosa (MIC 256 µg/mL). The leaf extract of Woodfordia floribunda Salisb. (Lythraceae) also exhibited significant effect against C. albicans (MIC 128 µg/mL), S. aureus (MIC 256 µg/mL) and E. faecalis (MIC 256 µg/mL). Rotheca serrata (L.) Steane & Mabb. (Lamiaceae) leaf extract inhibited the growth of S. aureus (MIC 256 µg/mL) and C. albicans (MIC 256 µg/mL). DISCUSSION AND CONCLUSIONS: The leaf extract of O. aristatus and W. floribunda exhibited a significant anti-candidal effect. Therefore, both of these plants can serve as prospective source materials for the development of new anti-candidal agents.

In vitro immunomodulatory activity, cytotoxicity and chemistry of some central European polypores.

Context Some mushrooms of the order Polyporales are known for their immunomodulatory actions. Objective The objective of this study is to evaluate the in vitro phagocytic and cytotoxic effects of extracts from polyporales native to Central Europe. Materials and methods The effects of ethanol extracts from 27 polypore species on opsonized zymosan-induced phagocytosis of isolated human neutrophils were tested by a chemiluminescence method. Colon epithelial cell lines, Caco-2 and HT-29, were used for cytotoxicity assays, and extracts were chemically characterized in terms of total phenolic and ß-glucan content. Results We observed phagocytosis or respiratory burst enhancing activity in 17 extracts, of which five species, namely Aurantiporus fissilis (Berk. & M.A. Curtis) H. Jahn ex Ryvarden, Trametes gibbosa (Pers.) Fr., Piptoporus betulinus (Bull.) P. Karst, Neolentinus lepideus (Fr.) Redhead & Ginns, Polyporus squamosus (Huds.) Fr., significantly increased phagocytosis in granulocytes by 205, 181, 158, 155 and 141%, respectively. The ß-glucan content of the three most potent extracts was 58, 42 and 74 mg/g, respectively, and the polyphenol content was 155.6, 133.5 and 155.2 µmol of gallic acid equivalent/g, respectively. Some extracts showed cytotoxic activity, with higher cytotoxicity in Caco-2 than in HT-29 cells. Pycnoporus cinnabarinus (Jacq.) P. Karst. extract was cytotoxic to both cell lines, with IC50 values of 81 and 31 µg/mL, respectively. Discussion and conclusion The most promising extracts were from N. lepideus and Polyporus squamosus, which are edible species and may be considered safe. Our findings support their use as culinary preparations or food supplements for various immunological gut disorders.

In vitro antimicrobial activity of plants used in traditional medicine in Gurage and Silti Zones, south central Ethiopia.

BACKGROUND: To overcome the escalating problems associated with infectious diseases and drug resistance, discovery of new antimicrobials is crucial. The present study aimed to carry out in vitro antimicrobial analysis of 15 medicinal plant species selected according to their traditional medicinal uses in Gurage and Silti Zones, south central Ethiopia. METHODS: Ethanol extracts of various plant parts were investigated for their antimicrobial activity against 20 bacterial and one yeast strains. The minimum inhibitory concentration (MIC) was determined by broth microdilution method. RESULTS: Asparagus africanus, Guizotia schimperi, Lippia adoensis var. adoensis and Premna schimperi were active against Candida albicans, Enterococcus faecalis and Staphylococcus aureus at a concentration of 512 µg/ml or lower. Strong antibacterial activity (MIC≥128 µg/ml) was observed for G. schimperi extract against 17 resistant and sensitive Staphylococcus strains, at a concentration comparable to standard antibiotics. Moreover, this extract showed higher antibacterial activity for the test against S. aureus ATCC 33591, ATCC 33592, SA3 and SA5 strains (128-256 µg/ml) than oxacillin (512 µg/ml). CONCLUSIONS: The study revealed in vitro antibacterial activity of plants used in folk medicine in south central Ethiopia. The usefulness of these plants, in particular of G. schimperi, should be confirmed through further phytochemical and toxicity analyses.

In vitro combinatory antimicrobial effect of plumbagin with oxacillin and tetracycline against Staphylococcus aureus.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a bicyclic naphthoquinone naturally distributed among Plumbago species, has been reported to have antimicrobial activity against a wide range of microorganisms. In this study, plumbagin was examined for its combinatory antimicrobial effect with tetracycline or oxacillin against nine strains of Staphylococcus aureus, including its methicillin- and multidrug-resistant strains. Minimum inhibitory concentrations were determined through the broth microdilution method, whereas the combinatory effect was evaluated according to the sum of fractional inhibitory concentration (ΣFIC) indices. Additive interactions were obtained for both combinations against most of the strains tested. Synergy was obtained for combination with oxacillin against two out of seven strains (ΣFIC range 0.273-0.281), both were methicillin resistant. Our results proved plumbagin as a compound suitable for anti-Staphylococcal combinatory testing. Moreover, to the best of our knowledge, this is the first report of plumbagin synergy with oxacillin against S. aureus strains, including its resistant forms.

Anti-staphylococcal activity of soilless cultivated cannabis across the whole vegetation cycle under various nutritional treatments in relation to cannabinoid content.

Antibiotic resistance in staphylococcal strains and its impact on public health and agriculture are global problems. The development of new anti-staphylococcal agents is an effective strategy for addressing the increasing incidence of bacterial resistance. In this study, ethanolic extracts of Cannabis sativa L. made from plant parts harvested during the whole vegetation cycle under various nutritional treatments were assessed for in vitro anti-staphylococcal effects. The results showed that all the cannabis extracts tested exhibited a certain degree of growth inhibition against bacterial strains of Staphylococcus aureus, including antibiotic-resistant and antibiotic-sensitive forms. The highest antibacterial activity of the extracts was observed from the 5th to the 13th week of plant growth across all the nutritional treatments tested, with minimum inhibitory concentrations ranging from 32 to 64 µg/mL. Using HPLC, Δ9-tetrahydrocannabinolic acid (THCA) was identified as the most abundant cannabinoid in the ethanolic extracts. A homolog of THCA, tetrahydrocannabivarinic acid (THCVA), reduced bacterial growth by 74%. These findings suggest that the cannabis extracts tested in this study can be used for the development of new anti-staphylococcal compounds with improved efficacy.

Potentiation of the in vitro antistaphylococcal effect of oxacillin and tetracycline by the anti-inflammatory drug diacetyl rhein.

BACKGROUND: The anti-inflammatory drug diacetyl rhein has been found to possess promising antistaphylococcal effects against various drug-resistant strains in our previous study. In the present work, we explored the in vitro combinatory interactions of diacetyl rhein with oxacillin and tetracycline against 13 standard strains and clinical isolates of Staphylococcus aureus, including those resistant to erythromycin, methicillin and tetracycline. METHODS: Minimum inhibitory concentrations were determined by broth microdilution assay, and the effects of combinations were evaluated according to the sum of fractional inhibitory concentrations (ΣFICs). RESULTS: Synergistic or additive effects were observed against all S. aureus strains (ΣFIC 0.258-1), whereas diacetyl rhein-oxacillin appeared to be the most effective combination, synergistically inhibiting the growth of 4 strains tested. CONCLUSION: To our best knowledge, this is the first report on a synergistic antibacterial effect of diacetyl rhein. Our results suggest this promising compound for further evaluation of its synergistic anti-infective potential as an agent with a combined anti-inflammatory and synergistic antibacterial action.

In vitro selective inhibitory effect of 8-hydroxyquinoline against bifidobacteria and clostridia.

8-Hydroxyquinoline (8HQ) inhibited Clostridium tertium, Clostridium clostridioforme, Clostridium difficile and Clostridium perfringens in vitro with MICs of 8, 16, 32 and 32 µg/mL, respectively. In contrast, MICs of most bifidobacteria (84%) were 512 µg/mL or higher. Thus, 8HQ could be used as anti-clostridial agent or in selective media for bifidobacteria isolation.

Distribution of isoflavones and coumestrol in neglected tropical and subtropical legumes.

BACKGROUND: Isoflavones and coumestrol from dietary legumes are plant constituents showing multiple beneficial effects on humans. Owing to their ability to bind with mammalian estrogenic receptors and thereby intervention in several kinds of hormone-related cancers, they have received much attention. Soybean (Glycine max) is currently the major source of isoflavonoids in human diet. However, dozens of tropical and subtropical leguminous species remain unexplored for their isoflavonoids content. RESULTS: We have analyzed 55 extracts from 41 tropical and subtropical legume species used either in human or animal diet by high-performance liquid chromatography for the content of soy isoflavones, biochanin A, daidzein, daidzin, formononetin, genistein, genistin, sissotrin, ononin and the coumestan coumestrol. Genistein and biochanin A were the most abundant compounds. The highest content of genistein was found in aerial parts of Andira macrothyrsa, seeds of Pachyrhizus tuberosus and aerial parts of Calopogonium mucunoides (598, 250 and 184 µg g(-1), respectively) and biochanin A in aerial parts of Cratylia argentea, C. mucunoides and flowers of A. macrothyrsa (76, 53 and 40 µg g(-1), respectively). CONCLUSION: None of the samples tested was richer overall source of soy isoflavones and coumestrol than soybean; nevertheless several species (C. mucunoides or A. macrothyrsa) may serve as a promising source of individual compounds.

Ethnobotanical inventory of medicinal plants used by Cashinahua (Huni Kuin) herbalists in Purus Province, Peruvian Amazon.

This study aims to document the diversity of medicinal plants used by the Cashinahua people (also known as Huni Kuin) of the Curanja River, as well as describe and compare their uses with pharmacological and phytochemical records from previously published studies. The ethnic has been studied to a limited extent from an ethnobotanical perspective. The study area is located in the Ucayali region, eastern Central Amazon, where ancestral knowledge is preserved due to the limited accessibility of the region. Between November 2010 and June 2015, a total of 11 months were spent on the survey, which included a short-term visit to complete voucher specimen collection and taxonomic identification. We conducted semi-structured interviews with 10 Cashinahua traditional healers and 10 midwives. Vernacular names, ethnomedicinal uses, plant parts used and forms of preparation and administration were recorded. Ethnopharmacological, pharmacological and phytochemical uses were checked through survey of the previously published papers indexed on Web of Science databases between 2018 and 2022. We obtained data on 467 plant taxa, among which we highlighted 79 species unreported or rarely cited for medicinal use or phytochemical analysis. These species were spread over 60 genera and 42 botanical families, with Acanthaceae being the most represented. Leaves were used the most frequently (93.56%). Among the 79 species, the most reported therapeutic activities involved pregnancy and birth disorders (13.84%), followed by poisonings, infections and infestations. The predominant application form was external (87%). Our study indicates that there are locally valuable species that have not yet been studied for their medical potential.

Redox and non-redox mechanism of in vitro cyclooxygenase inhibition by natural quinones.

In this study, ten anthra-, nine naphtho-, and five benzoquinone compounds of natural origin and five synthetic naphthoquinones were assessed, using an enzymatic in vitro assay, for their potential to inhibit cyclooxygenase-1 and -2 (COX-1 and COX-2), the key enzymes of the arachidonic acid cascade. IC50 values comparable with COX reference inhibitor indomethacin were recorded for several quinones (primin, alkannin, diospyrin, juglone, 7-methyljuglone, and shikonin). For some of the compounds, we suggest the redox potential of quinones as the mechanism responsible for in vitro COX inhibition because of the quantitative correlation with their pro-oxidant effect. Structure-relationship activity studies revealed that the substitutions at positions 2 and 5 play the key roles in the COX inhibitory and pro-oxidant actions of naphthoquinones. In contrast, the redox mechanism alone could not explain the activity of primin, embelin, alkannin, and diospyrin. For these four quinones, molecular modeling suggested similar binding modes as for conventional nonsteroidal anti-inflammatory drugs (NSAIDs).

Essential oils in the ranunculaceae family: chemical composition of hydrodistilled oils from Consolida regalis, Delphinium elatum, Nigella hispanica, and N. nigellastrum seeds.

In this study, we analyzed the chemical composition of volatile oils hydrodistilled from seeds of Consolida regalis, Delphinium elatum, Nigella hispanica, and N. nigellastrum using GC and GC/MS. In C. regalis, octadecenoic (77.79%) and hexadecanoic acid (8.34%) were the main constituents. Similarly, the oils from D. elatum and N. hispanica seeds consisted chiefly of octadecadienoic (42.83 and 35.58%, resp.), hexadecanoic (23.87 and 28.59%, resp.), and octadecenoic acid (21.67 and 19.76%, resp.). Contrastingly, the monoterpene hydrocarbons α-pinene (34.67%) and ß-pinene (36.42%) were the main components of N. nigellastrum essential oil. Our results confirm the presence of essential oils in the family Ranunculaceae and suggest chemotaxonomical relationships within the representatives of the genera Consolida, Delphinium, and Nigella. In addition, the presence of various bioactive constituents such as linoleic acid, (-)-ß-pinene, squalene, or carotol in seeds of D. elatum, N. hispanica, and N. nigellastrum indicates a possible industrial use of these plants.

In Vitro Growth-Inhibitory Synergistic Effect of Zinc Pyrithione in Combination with Gentamicin against Bacterial Skin Pathogens of Livestock.

Bacterial skin diseases of livestock could be a serious global threat, especially in association with overcoming bacterial resistance. Combinatory action of antimicrobial agents proves to be an effective strategy to overcome the problem of increasing antibiotic resistance of microorganisms. In this study, the in vitro combined effect of zinc pyrithione with gentamicin against bacterial skin pathogens of livestock (Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae) was evaluated according to the sum of fractional inhibitory concentration indices (FICI) obtained by checkerboard method. The results showed that a combination of zinc pyrithione with gentamicin produced a strong synergistic effect (p < 0.001) against all tested streptococcal strains (with FICI values ranging from 0.20 to 0.42). Compared to that, only three out of eight S. aureus strains were highly susceptible to the combination of antimicrobial agents at single concentration (0.25 µg/mL) of zinc pyrithione with range of FICI 0.35−0.43. These findings suggest that interference between agents tested in this study can be used for the development of future veterinary pharmaceutical preparations for the treatment of bacterial skin infections of livestock.