Essential Oils as Multicomponent Mixtures and Their Potential for Human Health and Well-Being.

Essential oils (EOs) and their individual volatile organic constituents have been an inherent part of our civilization for thousands of years. They are widely used as fragrances in perfumes and cosmetics and contribute to a healthy diet, but also act as active ingredients of pharmaceutical products. Their antibacterial, antiviral, and anti-inflammatory properties have qualified EOs early on for both, the causal and symptomatic therapy of a number of diseases, but also for prevention. Obtained from natural, mostly plant materials, EOs constitute a typical example of a multicomponent mixture (more than one constituent substances, MOCS) with up to several hundreds of individual compounds, which in a sophisticated composition make up the property of a particular complete EO. The integrative use of EOs as MOCS will play a major role in human and veterinary medicine now and in the future and is already widely used in some cases, e.g., in aromatherapy for the treatment of psychosomatic complaints, for inhalation in the treatment of respiratory diseases, or topically administered to manage adverse skin diseases. The diversity of molecules with different functionalities exhibits a broad range of multiple physical and chemical properties, which are the base of their multi-target activity as opposed to single isolated compounds. Whether and how such a broad-spectrum effect is reflected in natural mixtures and which kind of pharmacological potential they provide will be considered in the context of ONE Health in more detail in this review.

The Microbiome of the Medicinal Plants Achillea millefolium L. and Hamamelis virginiana L.

In the recent past many studies investigated the microbiome of plants including several medicinal plants (MP). Microbial communities of the associated soil, rhizosphere and the above-ground organs were included, but there is still limited information on their seasonal development, and in particular simultaneous investigations of different plant organs are lacking. Many studies predominantly addressed either the prokaryotic or fungal microbiome. A distinction of epi- and endophytic communities of above-ground plant organs has rarely been made. Therefore, we conducted a comprehensive investigation of the bacterial and fungal microbiome of the MP Achillea millefolium and studied the epi- and endophytic microbial communities of leaves, flower buds and flowers between spring and summer together with the microbiome of the associated soil at one location. Further, we assessed the core microbiome of Achillea from four different locations at distances up to 250 km in southern Germany and Switzerland. In addition, the bacterial and fungal epi- and endophytic leaf microbiome of the arborescent shrub Hamamelis virginiana and the associated soil was investigated at one location. The results show a generally decreasing diversity of both microbial communities from soil to flower of Achillea. The diversity of the bacterial and fungal endophytic leaf communities of Achillea increased from April to July, whereas that of the epiphytic leaf communities decreased. In contrast, the diversity of the fungal communities of both leaf compartments and that of epiphytic bacteria of Hamamelis increased over time indicating plant-specific differences in the temporal development of microbial communities. Both MPs exhibited distinct microbial communities with plant-specific but also common taxa. The core taxa of Achillea constituted a lower fraction of the total number of taxa than of the total abundance of taxa. The results of our study provide a basis to link interactions of the microbiome with their host plant in relation to the production of bioactive compounds.

Sweet spheres: succession and CAZyme expression of marine bacterial communities colonizing a mix of alginate and pectin particles.

Polysaccharide particles are important substrates and microhabitats for marine bacteria. However, substrate-specific bacterial dynamics in mixtures of particle types with different polysaccharide composition, as likely occurring in natural habitats, are undescribed. Here, we studied the composition, functional diversity and gene expression of marine bacterial communities colonizing a mix of alginate and pectin particles. Amplicon, metagenome and metatranscriptome sequencing revealed that communities on alginate and pectin particles significantly differed from their free-living counterparts. Unexpectedly, microbial dynamics on alginate and pectin particles were similar, with predominance of amplicon sequence variants (ASVs) from Tenacibaculum, Colwellia, Psychrobium and Psychromonas. Corresponding metagenome-assembled genomes (MAGs) expressed diverse alginate lyases, several colocalized in polysaccharide utilization loci. Only a single, low-abundant MAG showed elevated transcript abundances of pectin-degrading enzymes. One specific Glaciecola ASV dominated the free-living fraction, possibly persisting on particle-derived oligomers through different glycoside hydrolases. Elevated ammonium uptake and metabolism signified nitrogen as an important factor for degrading carbon-rich particles, whereas elevated methylcitrate and glyoxylate cycles suggested nutrient limitation in surrounding waters. The bacterial preference for alginate, whereas pectin primarily served as colonization scaffold, illuminates substrate-driven dynamics within mixed polysaccharide pools. These insights expand our understanding of bacterial niche specialization and the biological carbon pump in macroalgae-rich habitats.

The Exometabolome of Two Model Strains of the Roseobacter Group: A Marketplace of Microbial Metabolites.

Recent studies applying Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) showed that the exometabolome of marine bacteria is composed of a surprisingly high molecular diversity. To shed more light on how this diversity is generated we examined the exometabolome of two model strains of the Roseobacter group, Phaeobacter inhibens and Dinoroseobacter shibae, grown on glutamate, glucose, acetate or succinate by FT-ICR-MS. We detected 2,767 and 3,354 molecular formulas in the exometabolome of each strain and 67 and 84 matched genome-predicted metabolites of P. inhibens and D. shibae, respectively. The annotated compounds include late precursors of biosynthetic pathways of vitamins B1, B2, B5, B6, B7, B12, amino acids, quorum sensing-related compounds, indole acetic acid and methyl-(indole-3-yl) acetic acid. Several formulas were also found in phytoplankton blooms. To shed more light on the effects of some of the precursors we supplemented two B1 prototrophic diatoms with the detected precursor of vitamin B1 HET (4-methyl-5-(ß-hydroxyethyl)thiazole) and HMP (4-amino-5-hydroxymethyl-2-methylpyrimidine) and found that their growth was stimulated. Our findings indicate that both strains and other bacteria excreting a similar wealth of metabolites may function as important helpers to auxotrophic and prototrophic marine microbes by supplying growth factors and biosynthetic precursors.

Rhodobacteraceae on the marine brown alga Fucus spiralis are abundant and show physiological adaptation to an epiphytic lifestyle.

Macroalgae harbour specific microbial communities on their surface that have functions related to host health and defence. In this study, the bacterial biofilm of the marine brown alga Fucus spiralis was investigated using 16S rRNA gene amplicon-based analysis and isolation of bacteria. Rhodobacteraceae (Alphaproteobacteria) were the predominant family constituting 23% of the epibacterial community. At the genus level, Sulfitobacter, Loktanella, Octadecabacter and a previously undescribed cluster were most abundant, and together they comprised 89% of the Rhodobacteraceae. Supported by a specific PCR approach, 23 different Rhodobacteraceae-affiliated strains were isolated from the surface of F. spiralis, which belonged to 12 established and three new genera. For seven strains, closely related sequences were detected in the 16S rRNA gene dataset. Growth experiments with substrates known to be produced by Fucus spp. showed that all of them were consumed by at least three strains, and vitamin B12 was produced by 70% of the isolates. Since growth of F. spiralis depends on B12 supplementation, bacteria may provide the alga with this vitamin. Most strains produced siderophores, which can enhance algal growth under iron-deficient conditions. Inhibiting properties against other bacteria were only observed when F. spiralis material was present in the medium. Thus, the physiological properties of the isolates indicated adaption to an epiphytic lifestyle.

Bacterial community dynamics during polysaccharide degradation at contrasting sites in the Southern and Atlantic Oceans.

The bacterial degradation of polysaccharides is central to marine carbon cycling, but little is known about the bacterial taxa that degrade specific marine polysaccharides. Here, bacterial growth and community dynamics were studied during the degradation of the polysaccharides chitin, alginate and agarose in microcosm experiments at four contrasting locations in the Southern and Atlantic Oceans. At the Southern polar front, chitin-supplemented microcosms were characterized by higher fractions of actively growing cells and a community shift from Alphaproteobacteria to Gammaproteobacteria and Bacteroidetes. At the Antarctic ice shelf, chitin degradation was associated with growth of Bacteroidetes, with 24% higher cell numbers compared with the control. At the Patagonian continental shelf, alginate and agarose degradation covaried with growth of different Alteromonadaceae populations, each with specific temporal growth patterns. At the Mauritanian upwelling, only the alginate hydrolysis product guluronate was consumed, coincident with increasing abundances of Alteromonadaceae and possibly cross-feeding SAR11. 16S rRNA gene amplicon libraries indicated that growth of the Bacteroidetes-affiliated genus Reichenbachiella was stimulated by chitin at all cold and temperate water stations, suggesting comparable ecological roles over wide geographical scales. Overall, the predominance of location-specific patterns showed that bacterial communities from contrasting oceanic biomes have members with different potentials to hydrolyse polysaccharides.

Use of homeopathic preparations in experimental studies with abiotically stressed plants.

BACKGROUND: Experimental research on the effects of homeopathic treatments on impaired plants was last reviewed in 1990. OBJECTIVES: To compile a systematic review of the existing literature on basic research in homeopathy with abiotically stressed plants using predefined criteria. METHODS: The literature search was carried out on publications that reported experiments on homeopathy using abiotically stressed whole plants, seeds, plant parts and cells from 1920 to 2010. Outcomes had to be measured by established procedures and statistically evaluated. Using of a Manuscript Information Score (MIS) we identified those publications that provided sufficient information for proper interpretation (MIS≥5). A further evaluation was based on the use of adequate controls to investigate specific effects of homeopathic preparations and on the use of systematic negative control experiments. RESULTS: A total of 34 publications with abiotically stressed plants was identified, published between 1965 and 2010. The 34 publications described a total of 37 experimental studies. Twenty-two studies included statistics, 13 had a MIS≥5, 8 were identified with adequate controls and 4 with negative control experiments. Significant and reproducible effects with decimal and centesimal potencies were found, including dilution levels beyond Avogadro's number. One experimental model was independently assessed by another research team and yielded inverted results compared to the original trial. CONCLUSIONS: Abiotically stressed plant models seem to be a useful approach to investigate homeopathic basic research questions, but more experimentation and especially more independent replication trials are needed. Systematic negative control experiments should be implemented on a routine basis to exclude false-positive results.

Development of a test system for homeopathic preparations using impaired duckweed (Lemna gibba L.).

OBJECTIVES: A bioassay with arsenic-stressed duckweed (Lemna gibba L.) was developed to study potentially regulative effects of homeopathic preparations. We compared potentized substances (nine different potency levels between 17 x and 33 x ) with two controls (unsuccussed and succussed water) regarding their influence on number- and area-related growth rate and color of fronds (leaves). Screening included 11 potentized substances: Arsenicum album, gibberellic acid, nosode, arsenic(V), phosphorus, Conchae, Acidum picrinicum, Argentum nitricum, Crotalus horridus, Hepar sulfuris, and Mercurius vivus naturalis. DESIGN: Duckweed was stressed with arsenic(V) for 48 hours. Afterwards, plants grew in either potentized substances or water controls for 6 days. Growth rate and color of fronds were determined with a computerized image analysis system for different time intervals (days 0-2, 2-6, 0-6). A systematic negative control experiment with unsuccussed water was used to investigate the stability of the bioassay. All experiments were randomized and blinded. RESULTS: Arsenicum album and nosode potencies increased frond number-related growth rate compared to controls (succussed water controls or pooled water controls [succussed and unsuccussed], p < 0.05, t test). Regarding color classification, no effects were observed. CONCLUSIONS: The experimental setup with L. gibba stressed by arsenic(V) provides a valuable tool to investigate regulative effects of potentized substances. In order to verify the effects of Arsenicum album and nosode potencies, further independent replication experiments are necessary.

Investigation of arsenic-stressed yeast (Saccharomyces cerevisiae) as a bioassay in homeopathic basic research.

This study investigated the response of arsenic-stressed yeast (Saccharomyces cerevisiae) towards homeopathically potentized Arsenicum album, a duckweed nosode, and gibberellic acid. The three test substances were applied in five potency levels (17x, 18x, 24x, 28x, 30x) and compared to controls (unsuccussed and succussed water) with respect to influencing specific growth parameters. Five independent experiments were evaluated for each test substance. Additionally, five water control experiments were analyzed to investigate the stability of the experimental setup (systematic negative control experiments). All experiments were randomized and blinded. Yeast grew in microplates over a period of 38 h in either potentized substances or water controls with 250 mg/l arsenic(V) added over the entire cultivation period. Yeast's growth kinetics (slope, Et50, and yield) were measured photometrically. The test system exhibited a low coefficient of variation (slope 1.2%, Et50 0.3%, yield 2.7%). Succussed water did not induce any significant differences compared to unsuccussed water. Data from the control and treatment groups were both pooled to increase statistical power. In this study with yeast, no significant effects were found for any outcome parameter or any homeopathic treatment. Since in parallel experiments arsenic-stressed duckweed showed highly significant effects after application of potentized Arsenicum album and duckweed nosode preparations from the same batch as used in the present study, some specific properties of this experimental setup with yeast must be responsible for the lacking response.

Effects of homeopathic arsenicum album, nosode, and gibberellic acid preparations on the growth rate of arsenic-impaired duckweed (Lemna gibba L.).

This study evaluated the effects of homeopathically potentized Arsenicum album, nosode, and gibberellic acid in a bioassay with arsenic-stressed duckweed (Lemna gibba L.). The test substances were applied in nine potency levels (17x, 18x, 21x-24x, 28x, 30x, 33x) and compared with controls (unsuccussed and succussed water) regarding their influence on the plant's growth rate. Duckweed was stressed with arsenic(V) for 48 h. Afterwards, plants grew in either potentized substances or water controls for 6 days. Growth rates of frond (leaf) area and frond number were determined with a computerized image analysis system for different time intervals (days 0-2, 2-6, 0-6). Five independent experiments were evaluated for each test substance. Additionally, five water control experiments were analyzed to investigate the stability of the experimental setup (systematic negative control experiments). All experiments were randomized and blinded. The test system exhibited a low coefficient of variation (approximately equal to 1%). Unsuccussed and succussed water did not result in any significant differences in duckweed growth rate. Data from the control and treatment groups were pooled to increase statistical power. Growth rates for days 0-2 were not influenced by any homeopathic preparation. Growth rates for days 2-6 increased after application of potentized Arsenicum album regarding both frond area (p < 0.001) and frond number (p < 0.001), and by application of potentized nosode (frond area growth rate only, p < 0.01). Potencies of gibberellic acid did not influence duckweed growth rate. The systematic negative control experiments did not yield any significant effects. Thus, false-positive results can be excluded with high certainty. To conclude, the test system with L. gibba impaired by arsenic(V) was stable and reliable. It yielded evidence for specific effects of homeopathic Arsenicum album preparations and it will provide a valuable tool for future experiments that aim at revealing the mode of action of homeopathic preparations. It may also be useful to investigate the influence of external factors (e.g., heat, electromagnetic radiation) on the effects of homeopathic preparations.

Effects of potentised substances on growth rate of the water plant Lemna gibba L.

OBJECTIVES: This study investigated, whether the growth rate of Lemna gibba L. (duckweed) can be influenced by the application of homeopathic potencies of gibberellic acid, kinetin, argentum nitricum, and lemna minor. METHODS: Duckweed was grown in either potencies (14x-30x, decimal steps) or water controls (unsuccussed and succussed) over seven days. Frond (leaf-like structure) growth was measured using a non-destructive image analysis system. Growth rates were calculated for three time intervals (0-7, 0-3, 3-7 days). Five to six independent, randomized and blinded experiments were analysed for each of the four tested substances. Water control experiments were performed repeatedly to test the reliability of the experimental set-up (systematic negative controls). RESULTS: The systematic negative control experiments did not yield any significant effects. Hence, false positive results could be excluded. The test system had a low coefficient of variation (1.5%). Out of the four tested substances gibberellic acid had the most pronounced effect (p=0.0002, F-test) on the main outcome parameter frond growth rate (r(area) day 0-7). Potency levels 15x, 17x, 18x, 23x and 24x reduced growth rate of Lemna gibba (p<0.05 against the pooled water control, LSD test). CONCLUSIONS: Lemna gibba may be considered as a suitable test organism for further studies on the efficacy of homeopathic potencies. Evidence accumulates, that adjacent potency levels may strongly differ in their biological activity. Potential consequences for therapeutical application might be worth investigating.

Duckweed (Lemna gibba L.) as a test organism for homeopathic potencies.

OBJECTIVES: A bioassay with duckweed (Lemna gibba L.) was used to study the effects of homeopathic potencies on the plant's growth rate. Screening included 12 substances: argentum nitricum, copper sulfate, gibberellic acid, 3-indole acetic acid, kinetin, lactose, lemna minor, methyl jasmonate, metoxuron, phosphorus, potassium nitrate, and sulfur. Each substance was tested in the potency range 14x-30x. Controls were unsuccussed and succussed water. DESIGN: In randomized and blinded experiments, duckweed was grown in either potentized substances or water controls over 7 days. Frond (leaf) growth was measured regularly with a computerized image analysis system and growth rates were calculated for different time intervals (day 0-7, 0-3, 3-7). Additionally, a water control run with unsuccussed water as the only test substance was performed to determine the variability of the bioassay. RESULTS: For the water control run, the between-group coefficient of variance for groups of five replicates was 0.87% for the frond area-related average specific growth rate r(area) compared to 1.60% for the frond number-related average specific growth rate r(num). Thus, the former is the preferred parameter to be used. Of twelve tested substances, potentized argentum nitricum, phosphorus, and kinetin significantly (p<0.05, analysis of variance F-test) affected the main parameter: frond area-related average specific growth rate (day 0-7). Segmented area growth rates (day 0-3 or 3-7) were affected by potentized argentum nitricum, gibberellic acid, lactose, and phosphorus. CONCLUSIONS: The described experimental set-up with L. gibba as test organism appears to be a promising new model system to investigate effects of potentized substances. Yet larger sets of replication experiments with selected test substances and systematic negative controls are necessary to verify the effects found.

Effects of potentised substances on growth kinetics of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

BACKGROUND: Homeopathic potencies are used as specific remedies in complementary medicine. Since the mode of action is unknown, the presumed specificity is discussed controversially. OBJECTIVE: This study investigated the effects of potentised substances on two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe, in a stable and reliable test system with systematic negative controls. MATERIALS AND METHODS: Yeast cells were cultivated in either potentised substances or water controls in microplates and their growth kinetics were measured photometrically. Water control runs were performed repeatedly to investigate the stability of the experimental set-up (systematic negative controls). RESULTS: 4 out of 14 screened substances seem to have affected the growth curve parameters slope or yield. Out of these substances, azoxystrobin and phosphorus were chosen for 8 further replication experiments, which partly confirmed the results of the screening. On the average of all experiments, azoxystrobin affected the slope of the growth curve of Saccharomyces cerevisiae (p < 0.05), and phosphorus affected the slope of the growth curve of Schizosaccharomyces pombe (p < 0.05). No effects were seen in the water control runs. In addition, significant interactions between treatment with potentised substances and experiment number were observed in all experiments with potentised substances (p < 0.01), but not in the water control runs. CONCLUSIONS: Both yeast species reacted to certain potentised substances by changing their growth kinetics. However, the interactions found point to additional factors of still unknown nature, that modulate the effects of potentised substances. This stable test system with yeasts may be suitable for further studies regarding the efficacy of homeopathic potencies.