Proposed mechanisms of action of herbal drugs and their biologically active constituents in the treatment of coughs: an overview.

Various medicinal plants find their use in cough treatment, based on traditions and long-term experience. Pharmacological principles of their action, however, are much less known. Herbal drugs usually contain a mixture of potentially active compounds, which can manifest diverse effects. Expectorant or antitussive effects, which can be accompanied by others, such as anti-inflammatory or antibacterial, are probably the most important in the treatment of coughs. The aim of this review is to summarize the current state of knowledge of the effects of medicinal plants or their constituents on cough, based on reliable pharmacological studies. First, a comprehensive description of each effect is provided in order to explain the possible mechanism of action in detail. Next, the results related to individual plants and substances are summarized and critically discussed based on pharmacological in vivo and in vitro investigation.

Flowering phenophases influence the antibacterial and anti-biofilm effects of Thymus vulgaris L. essential oil.

BACKGROUND: Essential oils are becoming increasingly popular in medicinal applications because of their antimicrobial effect. Thymus vulgaris L. (Lamiaceae) is a well-known and widely cultivated medicinal plant, which is used as a remedy for cold, cough and gastrointestinal symptoms. Essential oil content of thyme is responsible for its antimicrobial activity, however, it has been reported that the chemical composition of essential oils influences its biological activity. In order to explore flowering phenophases influence on the chemical composition of thyme essential oil and its antibacterial and anti-biofilm activity, plant materials were collected at the beginning of flowering, in full bloom and at the end of flowering periods in 2019. METHODS: Essential oils from fresh and dried plant materials were distilled and analyzed with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID). The antibacterial activity was performed by broth microdilution and thin layer chromatography-direct bioautography (TLC-DB) assays and the anti-biofilm effect by crystal violet assay, respectively. Scanning electron microscopy was applied to illustrate the cellular changes of bacterial cells after essential oil treatment. RESULTS: Thymol (52.33-62.46%) was the main component in the thyme essential oils. Thyme oil distilled from fresh plant material and collected at the beginning of flowering period exerted the highest antibacterial and anti-biofilm activity against Haemophilus influenzae, H. parainfluenzae and Pseudomonas aeruginosa. CONCLUSION: The different flowering periods of Thymus vulgaris influence the antibacterial and anti-biofilm activity of its essential oils, therefore, the collection time has to be taken into consideration and not only the full bloom, but the beginning of flowering period may provide biological active thyme essential oil.

Treatment of Benign Prostatic Hyperplasia by Natural Drugs.

Benign prostatic hyperplasia (BPH) is one of the most common urinary diseases affecting men, generally after the age of 50. The prevalence of this multifactorial disease increases with age. With aging, the plasma level of testosterone decreases, as well as the testosterone/estrogen ratio, resulting in increased estrogen activity, which may facilitate the hyperplasia of the prostate cells. Another theory focuses on dihydrotestosterone (DHT) and the activity of the enzyme 5α-reductase, which converts testosterone to DHT. In older men, the activity of this enzyme increases, leading to a decreased testosterone/DHT ratio. DHT may promote prostate cell growth, resulting in hyperplasia. Some medicinal plants and their compounds act by modulating this enzyme, and have the above-mentioned targets. This review focuses on herbal drugs that are most widely used in the treatment of BPH, including pumpkin seed, willow herb, tomato, maritime pine bark, Pygeum africanum bark, rye pollen, saw palmetto fruit, and nettle root, highlighting the latest results of preclinical and clinical studies, as well as safety issues. In addition, the pharmaceutical care and other therapeutic options of BPH, including pharmacotherapy and surgical options, are discussed, summarizing and comparing the advantages and disadvantages of each therapy.

Anti-inflammatory effect of lavender (Lavandula angustifolia Mill.) essential oil prepared during different plant phenophases on THP-1 macrophages.

BACKGROUND: Pseudomonas aeruginosa is the most common Gram-negative bacterium associated with nosocomial respiratory infections. Lavender essential oil is mainly used in aromatherapy, but it has several pharmacological and therapeutic properties. Furthermore, it possesses antifungal and antibacterial activities. The anti-inflammatory activity of essential oils may depend on the composition and the ratio of the compounds. The constitution of the essential oils extracted from the different stages of flowering period varies, which makes it plausible that the collection time of the flowers influences the anti-inflammatory effects. Different types of essential oils reduce inflammation acting similarly by modulating the activity and action of the NFκB signalling pathway, which is the major regulator of the transcription of pro-inflammatory cytokines. METHODS: Lavender essential oils were distilled from lavender plant cultivated in Hungary and the flowers were harvested at the beginning and at the end of flowering period. The experiments were carried out on THP-1 human monocyte/macrophage cell line as in vitro cell culture model for monitoring the effects of lavender essential oils and the main compound linalool on P. aeruginosa LPS stimulated inflammation. The mRNA and protein levels of four pro-inflammatory cytokines, IL-6, IL-1ß, IL-8 and TNFα were determined by Real Time PCR and ELISA measurements. The effects of essential oils were compared to the response to two NFκB inhibitors, luteolin and ACHP. RESULTS: Linalool and lavender essential oil extracted from plants at the beginning of flowering period were successful in decreasing pro-inflammatory cytokine production following LPS pretreatment. In case of IL-8 and IL-1ß lavender oil showed stronger effect compared to linalool and both of them acted similarly to NFκB inhibitors. Pretreatments with linalool and lavender essential oil/beginning of flowering period prevented pro-inflammatory cytokine production compared to LPS treatment alone. Although lavender essential oil/end of flowering period decreased IL-6, IL-1ß and IL-8 mRNA expression in case of LPS pretreatment, it was not capable to reduce cytokine secretion. CONCLUSION: Based on our results it has been proven that lavender essential oil extracted at the beginning of flowering period is a potent inhibitor of the synthesis of four pro-inflammatory cytokines IL-6, IL-8, IL-ß and TNFα of THP-1 cells. This supports the relevance of the collection of the lavender flowers from early blooming period for essential oil production and for the utilization as an anti-inflammatory treatment.

A Themed Issue in Honor of Professor K. Hüsnü Can Baser-Outstanding Contributions in the Fields of Pharmacognosy, Phytochemistry, Botany and Ethnopharmacology.

Dear Colleagues, [...].

Three chemotypes of thyme (Thymus vulgaris L.) essential oil and their main compounds affect differently the IL-6 and TNFα cytokine secretions of BV-2 microglia by modulating the NF-κB and C/EBPß signalling pathways.

BACKGROUND: The essential oils possess both antimicrobial and anti-inflammatory effects, therefore they can provide an effective treatment against infections. Essential oils are widely used as supportive ingredients in many diseases, especially in the acute and chronic diseases of the respiratory tract. Neuroinflammation is responsible for several diseases of the central nervous system. Some plant-derived bioactive molecules have been shown to have role in attenuating neuroinflammation by regulating microglia, the immune cells of the CNS. METHODS: In this study, the anti-inflammatory effect of three chemotypes of thyme essential oil and their main compounds (geraniol, thujanol and linalool) were examined on lipopolysaccharide-induced BV-2 microglia. Three different experimental setups were used, LPS pretreatment, essential oil pretreatment and co-treatments of LPS and essential oils in order to determine whether essential oils are able to prevent inflammation and can decrease it. The concentrations of the secreted tumour necrosis factor α (TNFα) and interleukin-6 (IL-6) proinflammatory cytokines were measured and we analysed by Western blot the activity of the cell signalling pathways, NF-κB and CCAAT-enhancer binding protein ß (C/EBPß) regulating TNFα and IL-6 proinflammatory cytokine expressions in BV-2 cells. RESULTS: Our results showed definite alterations in the effects of essential oil chemotypes and their main compounds at the different experimental setups. Considering the changes of IL-6 and TNFα secretions the best reduction of inflammatory cytokines could be reached by the pretreatment with the essential oils. In addition, the main compounds exerted better effects than essential oil chemotypes in case of LPS pretreatment. At the essential oil pretreatment experiment, the effect of linalool and geraniol was outstanding but there was no major difference between the actions of chemotypes and standards. Main compounds could be seen to have large inhibitory effects on certain cell signalling components related to the activation of the expression of proinflammatory cytokines. CONCLUSION: Thyme essential oils are good candidates to use in prevention of neuroinflammation and related neurodegeneration, but the exact ratio of the components has to be selected carefully.

Analyzing the Carotenoid Composition of Melilot (Melilotus officinalis (L.) Pall.) Extracts and the Effects of Isolated (All-E)-lutein-5,6-epoxide on Primary Sensory Neurons and Macrophages.

Melilotus officinalis is known to contain several types of secondary metabolites. In contrast, the carotenoid composition of this medicinal plant has not been investigated, although it may also contribute to the biological activities of the drug, such as anti-inflammatory effects. Therefore, this study focuses on the isolation and identification of carotenoids from Meliloti herba and on the effect of isolated (all-E)-lutein 5,6-epoxide on primary sensory neurons and macrophages involved in nociception, as well as neurogenic and non-neurogenic inflammatory processes. The composition of the plant extracts was analyzed by high performance liquid chromatography (HPLC). The main carotenoid was isolated by column liquid chromatography (CLC) and identified by MS and NMR. The effect of water-soluble lutein 5,6-epoxide-RAMEB (randomly methylated-ß-cyclodextrin) was investigated on Ca2+-influx in rat primary sensory neurons induced by the activation of the transient receptor potential ankyrin 1 receptor agonist to mustard-oil and on endotoxin-induced IL-1ß release from isolated mouse peritoneal macrophages. (all-E)-Lutein 5,6-epoxide significantly decreased the percent of responsive primary sensory neurons compared to the vehicle-treated stimulated control. Furthermore, endotoxin-evoked IL-1ß release from macrophages was significantly decreased by 100 µM lutein 5,6-epoxide compared to the vehicle-treated control. The water-soluble form of lutein 5,6-epoxide-RAMEB decreases the activation of primary sensory neurons and macrophages, which opens perspectives for its analgesic and anti-inflammatory applications.

Effects of Thymus vulgaris L., Cinnamomum verum J.Presl and Cymbopogon nardus (L.) Rendle Essential Oils in the Endotoxin-induced Acute Airway Inflammation Mouse Model.

Thyme (TO), cinnamon (CO), and Ceylon type lemongrass (LO) essential oils (EOs) are commonly used for inhalation. However, their effects and mechanisms on inflammatory processes are not well-documented, and the number of in vivo data that would be important to determine their potential benefits or risks is low. Therefore, we analyzed the chemical composition and investigated the activity of TO, CO, and LO on airway functions and inflammatory parameters in an acute pneumonitis mouse model. The components of commercially available EOs were measured by gas chromatography-mass spectrometry. Airway inflammation was induced by intratracheal endotoxin administration in mice. EOs were inhaled during the experiments. Airway function and hyperresponsiveness were determined by unrestrained whole-body plethysmography on conscious animals. Myeloperoxidase (MPO) activity was measured by spectrophotometry from lung tissue homogenates, from which semiquantitative histopathological scores were assessed. The main components of TO, CO, and LO were thymol, cinnamaldehyde, and citronellal, respectively. We provide here the first evidence that TO and CO reduce inflammatory airway hyperresponsiveness and certain cellular inflammatory parameters, so they can potentially be considered as adjuvant treatments in respiratory inflammatory conditions. In contrast, Ceylon type LO inhalation might have an irritant effect (e.g., increased airway hyperresponsiveness and MPO activity) on the inflamed airways, and therefore should be avoided.

Antimicrobial Activity of Different Artemisia Essential Oil Formulations.

The extreme lipophilicity of essential oils (EOs) impedes the measurement of their biological actions in an aqueous environment. We formulated oil in water type Pickering Artemisia annua EO nanoemulsions (AEP) with surface-modified Stöber silica nanoparticles (20 nm) as the stabilizing agent. The antimicrobial activity of AEP and its effects on mature Candida biofilms were compared with those of Tween 80 stabilized emulsion (AET) and ethanolic solution (AEE) of the Artemisia EO. The antimicrobial activity was evaluated by using the minimum inhibitory concentrations (MIC90) and minimum effective concentrations (MEC10) of the compounds. On planktonic bacterial and fungal cells beside growth inhibition, colony formation (CFU/mL), metabolic activity, viability, intracellular ATP/total protein (ATP/TP), along with reactive oxygen species (ROS) were also studied. Artemisia annua EO nanoemulsion (AEP) showed significantly higher antimicrobial activity than AET and AEE. Artemisia annua EO nanoemulsions (AEP) generated superoxide anion and peroxides-related oxidative stress, which might be the underlying mode of action of the Artemisia EO. Unilamellar liposomes, as a cellular model, were used to examine the delivery efficacy of the EO of our tested formulations. We could demonstrate higher effectiveness of AEP in the EO components' donation compared to AET and AEE. Our data suggest the superiority of the AEP formulation against microbial infections.

Preparation, characterisation and microbiological examination of Pickering nano-emulsions containing essential oils, and their effect on Streptococcus mutans biofilm treatment.

Essential oils (EOs) are commonly applied in mouth care products like mouthwashes, mostly as an ethanolic solution or by usage of surfactants as solubilising agents. In this study, we present a formulation for preparation of Pickering nano-emulsions (PnE) of EOs as a novel form for application of EOs in mouth care. For the preparation of PnE, we have synthesised surface-modified silica nanoparticles with a mean diameter of 20 nm, as well as we have examined the effect of EOs concentration on PnE droplet size and stability. In vitro study of their effect on the Streptococcus mutans biofilm as the main pathogen of dental health problems has been performed. We have found that EOs in the PnE form has the highest effectiveness against biofilm formation. Diffusion through the biofilm model membrane was studied to explain this observation. We have found that PnEs have a better performance in the transportation of EOs trough model membrane than the ethanolic solutions and conventional emulsions (CEs).

Anti-Haemophilus Activity of Selected Essential Oils Detected by TLC-Direct Bioautography and Biofilm Inhibition.

Essential oils (EOs) are becoming increasingly popular in medical applications because of their antimicrobial effect. Direct bioautography (DB) combined with thin layer chromatography (TLC) is a screening method for the detection of antimicrobial compounds in plant extracts, for example, in EOs. Due to their lipophilic character, the common microbiological assays (etc. disk diffusion) could not provide reliable results. The aim of this study was the evaluation of antibacterial and anti-biofilm properties of the EO of cinnamon bark, clove, peppermint, thyme, and their main components against Haemophilus influenzae and H. parainfluenzae. Oil in water (O/W) type Pickering nano-emulsions stabilized with silica nanoparticles from each oil were prepared to increase their water-solubility. Samples with Tween80 surfactant and absolute ethanol were also used. Results showed that H. influenzae was more sensitive to the EOs than H. parainfluenzae (except for cinnamon bark oil). In thin layer chromatography-direct bioautography (TLC-DB) the ethanolic solutions of thyme oil presented the best activity against H. influenzae, while cinnamon oil was the most active against H. parainfluenzae. Pickering nano-emulsion of cinnamon oil inhibited the biofilm formation of H. parainfluenzae (76.35%) more efficiently than samples with Tween80 surfactant or absolute ethanol. In conclusion, Pickering nano-emulsion of EOs could inhibit the biofilm production effectively.

Antioxidant and antimicrobial properties of randomly methylated ß cyclodextrin - captured essential oils.

Free essential oils and their active components have a low physiochemical stability and low aqueous solubility which limit their applications as food preservatives and in packaging industry. The aim of this study was to characterize the physicochemical properties, antioxidant activities and antimicrobial activity of randomly methylated ß cyclodextrin (RAMEB) encapsulated thyme oil, lemon balm oil, lavender oil, peppermint oil and their active components that include thymol, citral, linalool, menthol and borneol. Inclusion complex formation of essential oils (EOs) and RAMEB were evaluated by several methods. Antioxidant capacities of RAMEB-EOs/components were reported to be more stable than free EOs/components (P < 0.05). Rapid SYBR green I/propidium iodide live/dead microbial cellular discrimination assay for Schizosaccharomyces pombe, Escherichia coli and Staphylococcus aureus showed similar results when compared with flow cytometry analysis (P < 0.01) suggesting that our novel microplate fluorescence method could be applied for the fast live/dead microbial discrimination in antimicrobial assays.

Antibacterial activity evaluation of selected essential oils in liquid and vapor phase on respiratory tract pathogens.

BACKGROUND: The increasing number of multidrug-resistant bacteria and the fact of antibiotic resistance is leading to a continuous need for discovering alternative treatments against infections, e.g. in the case of respiratory tract diseases. Essential oils (EOs), because of their volatility, can easily reach both the upper and lower parts of the respiratory tract via inhalation. Therefore, the aim of the present study was the antibacterial evaluation of clove, cinnamon bark, eucalyptus, thyme, scots pine, peppermint, and citronella EOs against respiratory tract pathogens such as Streptococcus pneumoniae, S. mutans, S. pyogenes, Haemophilus influenzae, H. parainfluenzae, and Moraxella catarrhalis. Furthermore, we wanted to compare the antibacterial effect of these EOs in two different test systems to provide data for the development of an appropriate product formulation. METHODS: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined with in vitro vapor phase test (VPT) and broth macrodilution test (BDT). The chemical and percentage compositions of the EOs were determined by GC-MS and GC-FID analysis. RESULTS: Among the EOs, thyme was the most effective against S. mutans (MIC: 0.04 mg/mL in BDT, but cinnamon bark and clove oils also presented high inhibition in liquid medium with MIC values of 0.06 mg/mL and 0.1 mg/mL against S. pneumoniae and S. pyogenes, respectively. M. catarrhalis was the most sensitive to thyme EO (MIC: 0.09 mg/mL). Cinnamon bark EO was the most effective against Haemophilus spp. (MIC: 0.06 mg/mL). In the VPT, cinnamon bark was the most effective oil against all investigated pathogens with MIC values in the range of 15.62-90 µl/L. Surprisingly, the eucalyptus and scots pine showed weak activity against the test bacteria in both test systems. CONCLUSIONS: The EO of thyme, clove and cinnamon bark may provide promising antibacterial activity against respiratory tract pathogens either in liquid medium or in vapor phase. However, their effect is lower than that of the reference antibiotics. The combination of EOs and antibiotics may be beneficial in the alternative treatment of respiratory tract diseases. In vivo studies are necessary to calculate the effective dose of EOs in patients and determine their possible side effects and toxicity.

Layer chromatography-bioassays directed screening and identification of antibacterial compounds from Scotch thistle.

The antibacterial profiling of Onopordum acanthium L. leaf extract and subsequent targeted identification of active compounds is demonstrated. Thin-layer chromatography (TLC) and off-line overpressured layer chromatography (OPLC) coupled with direct bioautography were utilized for investigation of the extract against eight bacterial strains including two plant and three human pathogens and a soil, a marine and a probiotic human gut bacteria. Antibacterial fractions obtaining infusion-transfusion OPLC were transferred to HPLC-MS/MS analysis that resulted in the characterization of three active compounds and two of them were identified as, linoleic and linolenic acid. OPLC method was adopted to preparative-scale flash chromatography for the isolation of the third active compound, which was identified after a further semi-preparative HPLC purification as the germacranolide sesquiterpene lactone onopordopicrin. Pure onopordopicrin exhibited antibacterial activity that was specified as minimal inhibitory concentration in the liquid phase as well.

Comparative Histological and Phytochemical Study of Fallopia species.

Fallopia species which belong to the Polygonaceae family have several data related to their use in the Asian herbal medicine. In this work, some histological and phytochemical parameters of Fallopia japonica, F. sachalinensis, and F. x bohemica were analysed and compared. Rhizome and leaf samples were collected before, during, and after the flowering period at 3 habitats in Szombathely and 4 habitats in Baranya County, Hungary. The main histological characteristics of the stem, leaf and petiole were studied by light microscopy in cross section. Total tannin and anthraquinone contents were determined according to the official methods of the Hungarian Pharmacopoeia VIIIth (equal to the European Pharmacopoeia 6th). No species-specific markers were found in any plant part. In the rhizome, the highest tannin content was measured in Japanese knotweed, followed by Bohemian and giant knotweed in each period. The tannin content measured in each plant was higher in the leaves than in the rhizomes except F. japonica. The rhizome of F. japonica had the highest anthraquinone content before the flowering period, followed by F. x bohemica and F. sachalinensis. According to earlier and our preliminary data, Fallopia taxa are of great therapeutic promise in the future.

A modified bioautographic method for antibacterial component screening against anaerobic and microaerophilic bacteria.

Direct bioautography is a useful method to identify antimicrobial compounds with potential therapeutic importance. Because of technical limitations till now, it has been applied only for aerobic bacteria. In this work we present the modification of the original method by which antimicrobial screening of bacteria requiring modified atmosphere became feasible by direct bioautography. Here we demonstrate its applicability by testing three anaerobic Clostridium perfringens and three microaerophilic Campylobacter jejuni strains against two essential oils, clove and thyme. Antimicrobial component profiles of clove and thyme essential oils against these two medically important pathogenic bacteria were compared and significant differences were revealed in their inhibition capacities. Linalool, a component of thyme essential oil exerted a more expressed antibacterial activity against C. perfringens than against C. jejuni. Our results demonstrate that direct bioautography is not only suitable for testing aerobic bacteria, but by applying the presently described modified version it can also contribute to the quest to find novel antimicrobial agents against multidrug resistant anaerobic and microaerophilic bacteria.

Detection and Identification of Antibacterial and Antioxidant Components of Essential Oils by TLC-Biodetection and GC-MS.

Components of cinnamon bark, rosemary, clove and thyme essential oils were screened for antioxidant and antibacterial activity utilizing thin-layer chromatography (TLC) coupled with the DPPH(.) test and direct bioautography using Bacillus subtilis cells. The compounds in the active chromatographic zones were identified by solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) after their elution. Seven antibacterial components were found: cinnamaldehyde and eugenol in cinnamon bark oil, 1,8-cineole, camphor, borneol and α-terpineol in rosemary oil, eugenol in clove oil and thymol in thyme oil. Only two of them, thymol and eugenol displayed a free radical scavenging effect.

Essential Oils and Their Vapors as Potential Antibacterial Agents against Respiratory Tract Pathogens.

Increasing appearance of antibiotic-resistant pathogens, which could be one of the major causes of respiratory tract infections, has again drawn attention to natural substances and alternative treatments. Therefore, the aim of the present study was the antibacterial evaluation of cinnamon bark, clove, thyme, citronella, peppermint, Scots pine, and eucalyptus essential oils (EOs) against respiratory tract pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa with in vitro vapor phase (VP) and tube dilution (TD) techniques. The chemical and percentage compositions of the EOs were determined by GC-FID and GC-MS analysis. Among the EOs, cinnamon bark was the most effective against all the investigated pathogens (MIC: 31.25-125 µL/L) in the VP assay, but clove oil presented the best inhibition against MRSA in liquid medium (MIC: 0.1 mg/mL). Thyme oil also showed antibacterial activity against MRSA and the antibiotic-sensitive strain of P. aeruginosa in both methods. In higher concentration, we found that peppermint oil was effective only in vapor form; contrarily, eucalyptus oil was more efficient in liquid medium. Surprisingly, Scots pine did not show any activity in our test systems. These results suggest that EOs could be promising solutions for the problem of antibiotic resistance due to their multiple composition and complex mode of action. However, more in vivo studies are necessary to calculate the effective dose of EOs in patients and determine their possible side effects and toxicity.

TLC-Direct Bioautography and LC/MS as Complementary Methods in Identification of Antibacterial Agents in Plant Tinctures from the Asteraceae Family.

Matricaria recutita L. (chamomile) and Achillea millefolium L. (yarrow) are very common herbs growing in meadows, pathways, crop fields, and home gardens. Preparations from these plants, e.g., infusions or alcohol extracts, are widely used as remedies. Both chamomile and yarrow have anti-inflammatory, analgesic, antimicrobial, and antioxidant properties. Most microbiological assays used today give information only on activity of whole extracts and do not provide information on the composition and activity of individual components. This problem can be solved by using TLC with direct microbiological detection, i.e., TLC-direct bioautography (TLC-DB), followed by LC/MS of active fractions. The aim of our study was chemical and microbiological screening of plant components of chamomile and yarrow tinctures using derivatization reagents and TLC-DB against eight bacterial strains: Staphylococcus epidermidis, S. aureus, methicillin-resistant S. aureus, Escherichia coli, Pseudomonas syringae pv. maculicola, Xanthomonas campestis pv. vesicatoria, Aliivibrio fischeri, and Bacillus subtilis. The identity of compounds exhibiting the widest range of activity (apigenin and α-linolenic acid) was confirmed by LC/MS.

TLC-Direct Bioautography as a Bioassay Guided Method for Investigation of Antibacterial Compounds in Hypericum perforatum L.

Fast high-throughput TLC-direct bioautography (DB) is an effect-directed analysis method that enables searching for biologically active (e.g., antimicrobial) substances in complex mixtures like plant extracts. The principle of the method is that separation and detection of biological properties of given mixture components is performed directly on a TLC plate. In searching for antibacterial activity, the developed plate is immersed in a bacterial broth, and bacteria grow directly on its layer during a proper incubation time. Inhibition zones are formed in places where antimicrobial components are located. The active compounds can be further identified using spectroscopic techniques. The aim of our study was investigation of plant components of Hypericum perforatum L. tincture by TLC-DB using nine bacterial strains: Micrococcus luteus, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, methicillin-resistant S. aureus, S. epidermidis, Pseudomonas syringae pv. maculicola, Xanthomonas campestris pv. vesicatoria, and Aliivibrio fischeri. Compounds showing the widest range of antimicrobial activity were isolated using semipreparative TLC and identified as apigenin, 3,8'-biapigenin, quercetin, kaempferol, and linolenic acid by TLC, HPLC-diode array detection, and HPLC/MS/MS techniques.