Genome-Wide Investigation of 2,4-Diacetylphloroglucinol Protection Genes in Arabidopsis thaliana.

The compound 2,4-diacetylphloroglucinol (DAPG) is a well-known secondary metabolite produced by Pseudomonas spp. that are used as biocontrol agents. DAPG displays a remarkably broad spectrum of toxic activity against pathogens of plants. Yet high concentrations of DAPG may also have negative effect on plants, but the phytotoxicity of DAPG is not clearly understood. Here, we used genome-wide activation, tagging Arabidopsis plants as the model plant to investigate the plant response to DAPG. A total of 15 lines were selected as DAPG-tolerant plants from among 62,000 lines investigated. The DAPG-responsible genes were then identified via thermal asymmetric interlaced PCR and quantitative reverse transcription PCR, and the gene ontology analysis showed the distribution of these genes having different biological processes, cellular regulations, and molecular functional properties. Collectively, these findings suggest that plants may rely on several pathways to prevent DAPG phytotoxicity.

Polycyclic polyprenylated acylphloroglucinol derivatives from Hypericum scabrum.

Three new polycyclic polyprenylated acylphloroglucinol derivatives (PPAPs), hyperibrins E-G (1-3), along with seven known compounds were identified from the air-dried aerial parts of Hypericum scabrum. Their structures were determined by NMR spectroscopic methods, both experimental and calculated electronic circular dichroism (ECD) spectra and comparison with known compounds. Compound 1 was derived from an analogue of compound 2 by cyclization, while a retro-Claisen reaction transformed another analogue of compound 2 into compound 3. Compounds 3, 4, 5, and 10 showed obvious hepatoprotective activities against paracetamol-induced HepG2 cell damage.

Four species of arboreal folivore show differential tolerance to a secondary metabolite.

The marsupials that eat Eucalyptus in south-eastern Australia provide an example of animals with similar niche requirements occurring sympatrically. They certainly differ in size, ranging from about 1 kg in the greater glider (Petauroides volans) and the closely related common ringtail possum (Pseudocheirus peregrinus), to 4 kg (common brushtail possum, Trichosurus vulpecula) and up to 15 kg in the koala (Phascolarctos cinereus). All species, however, may eat considerable amounts of eucalypt foliage, often favouring the same species, and thus appear to compete for food. In order to better understand the degree of competition for food, we measured feeding by the greater glider in response to increasing concentrations of a specific group of eucalypt plant secondary metabolites (PSM), the sideroxylonals, and then compared it to results published for the other species. The greater glider was more resilient than the other species to increasing concentrations of sideroxylonals. We suggest this allows gliders to feed on leaves from the eucalypt subgenus, Symphyomyrtus, while its small size and gliding ability allow it to feed where koalas cannot, on the young leaves on top of the canopy. In contrast, the common ringtail possum is well adapted to feeding from species of the subgenus Eucalyptus, which do not produce sideroxylonals but contain less available nitrogen (AvailN) than do the symphyomyrtles. These 'nutritional niches' segregate the forest and along with other factors, such as generalist and specialist feeding strategies and differences in body size and requirements for shelter, presumably minimise competition between the marsupial species.

Polycyclic polyprenylated acylphloroglucinols and cytotoxic constituents of Hypericum androsaemum.

Two new polycyclic polyprenylated acylphloroglucinols (PPAPs), androforin A and hyperandrone A, together with twelve known compounds, were isolated from the aerial parts of Hypericum androsaemum. Their structures were established by detailed spectral analysis. In the cytotoxic assay, 1,4-O-diferuloylsecoisolariciresinol showed activities comparable with those of cisplatin, and acetyloleanolic acid exhibited moderate inhibitory effects against HL-60, SMMC-7721, A-549, MCF-7, and SW480 cancer cell lines.

Evaluation of Toxicity of Crude Phlorotannins and Phloroglucinol Using Different Model Organisms.

Phlorotannins have been proven to contain numerous bioactive compounds that have potential to be applied in variety industries, including cosmetics, functional foods, nutraceuticals, environmental management, and medicine. The larvicidal and growth-inhibiting properties of phlorotannins have been extensively studied in various organisms. However, the toxicity of the phloroglucinol oligomer of phlorotannin is unclear, especially in Artemia salina, Daphnia magna, Lactuca sativa, and Chlorella vulgaris, which are commonly used in many bioassays. Therefore, research using these four organisms should be designed to provide basic information about the toxic effects of phlorotannins and phloroglucinol. This study aimed to evaluate the larvicidal and inhibitory properties of phlorotannins and phloroglucinol on A. salina, D. magna, L. sativa, and C. vulgaris. Phlorotannin extract and phloroglucinol were administered at various concentrations to each test organism. The survival rate of A. salina nauplii and D. magna neonates was observed every 24 h to 72 h, whereas the L. sativa seed germination and inhibition rate of C. vulgaris were observed up to 96 h. The results showed that the 24 h LC50 of phlorotannin on A. salina and D. magna were 10.67 and 1.32 mg/mL, respectively. The germination inhibition of L. sativa was 53.3% with a seed growth of less than 4 mm after 96 h upon exposure to 1 mg/mL of phlorotannin. Freshwater and seawater C. vulgaris experienced yield inhibition of 39.47 and 43.46%, respectively, when 2 mg/mL of phlorotanin was added. These results indicate that phlorotannin affects the survival and growth of the test organisms, so its use as a pesticide, herbicide, and algaecide agent for environmental and aquaculture applications can be further studied.

Predicting mixture toxicity of seven phenolic compounds with similar and dissimilar action mechanisms to Vibrio qinghaiensis sp.nov.Q67.

The predictions of mixture toxicity for chemicals are commonly based on two models: concentration addition (CA) and independent action (IA). Whether the CA and IA can predict mixture toxicity of phenolic compounds with similar and dissimilar action mechanisms was studied. The mixture toxicity was predicted on the basis of the concentration-response data of individual compounds. Test mixtures at different concentration ratios and concentration levels were designed using two methods. The results showed that the Weibull function fit well with the concentration-response data of all the components and their mixtures, with all relative coefficients (Rs) greater than 0.99 and root mean squared errors (RMSEs) less than 0.04. The predicted values from CA and IA models conformed to observed values of the mixtures. Therefore, it can be concluded that both CA and IA can predict reliable results for the mixture toxicity of the phenolic compounds with similar and dissimilar action mechanisms.

Characterization of application scenario-dependent pharmacokinetics and pharmacodynamic properties of permethrin and hyperforin in a dynamic skin and liver multi-organ-chip model.

Microphysiological systems (MPS) aim to mimic the dynamic microenvironment and the interaction between tissues. While MPS exist for investigating pharmaceuticals, the applicability of MPS for cosmetics ingredients is yet to be evaluated. The HUMIMIC Chip2 ("Chip2″), is the first multi-organ chip technology to incorporate skin models, allowing for the topical route to be tested. Therefore, we have used this model to analyze the impact of different exposure scenarios on the pharmacokinetics and pharmacodynamics of two topically exposed chemicals, hyperforin and permethrin. The Chip2 incorporated reconstructed human epidermis models (EpiDerm™) and HepaRG-stellate spheroids. Initial experiments using static incubations of single organoids helped determine the optimal dose. In the Chip2 studies, parent and metabolites were analyzed in the circuit over 5 days after application of single and repeated topical or systemic doses. The gene expression of relevant xenobiotic metabolizing enzymes in liver spheroids was measured to reflect toxicodynamics effects of the compounds in liver. The results show that 1) metabolic capacities of EpiDerm™ and liver spheroids were maintained over five days; 2) EpiDerm™ model barrier function remained intact; 3) repeated application of compounds resulted in higher concentrations of parent chemicals and most metabolites compared to single application; 4) compound-specific gene induction e.g. induction of CYP3A4 by hyperforin depended on the application route and frequency; 5) different routes of application influenced the systemic concentrations of both parents and metabolites in the chip over the course of the experiment; 6) there was excellent intra- and inter-lab reproducibility. For permethrin, a process similar to the excretion in a human in vivo study could be simulated which was remarkably comparable to the in vivo situation. These results support the use of the Chip2 model to provide information on parent and metabolite disposition that may be relevant to risk assessment of topically applied cosmetics ingredients.

Petiolins J-M, prenylated acylphloroglucinols from Hypericum pseudopetiolatum var. kiusianum.

Four new prenylated acylphloroglucinols, petiolins J-M (1-4), were isolated from aerial parts of Hypericum pseudopetiolatum var. kiusianum, and the structures were elucidated by spectroscopic data and a single-crystal X-ray diffraction analysis. Petiolin J (1) exhibited antimicrobial activity.

Polycyclic polyprenylated acylphloroglucinols and chromone O-glucosides from Hypericum henryi subsp. uraloides.

Two new C(30)-epimeric polycyclic polyprenylated acylphloroglucinols (PPAPs), named uralodins B and C (1 and 2, resp.), were isolated from the aerial parts of Hypericum henryi subsp. uraloides together with two new chromone glucosides, urachromones A and B (3 and 4, resp.), as well as 16 known compounds. Their structures were established by extensive NMR techniques and MS analysis. The epimers 1 and 2 always behaved like a single compound when examined by TLC, and were separated by HPLC. Their configuration was distinguished by comparative analysis of the NMR data with known analogues together with the ROESY experiment. All the isolated PPAPs were evaluated for their cytotoxic activities against HepG2, SGC7901, HL-60, and K562 cell lines. Compound 1 showed modest cytotoxic activities against SGC7901 and HL-60 cell lines, and 2 showed modest cytotoxic activities against HepG2, SGC7901, HL-60, and K562 cell lines.

A genetically engineered Pseudomonas fluorescens strain possesses the dual activity against phytopathogenic fungi and insects.

A Pseudomonas fluorescens strain was isolated and showed antagonistic activity against phytopathogenic fungi and found to possess a gene responsible for production of antibiotic 2, 4-diacetylphloroglucinol. For the extension of biocontrol range, a gene for an Androctonus australis Hector insect toxin 1 (AaHIT1), one of the most toxic known insect-selective peptides, was designed and synthesized according to the preferred codon usage of Pseudomonas fluorescens, cloned and transformed into the strain by pSUP106 vector, a broad-host-range plasmid. Bioassays indicated that the engineered strain was able to produce AaHIT1 with insecticidal activity, in the same time retained the activity against plant pathogen. The experiments for nonplanted soil and rhizosphere colonization showed that, similar to the population of the wild-type strain, that of the engineered strain remained relatively constant in the first 10 d, and the subsequent 50 d, suggesting that AaHIT expression in the bacterial cell does not substantially impair its long-term colonization. It is first reported that a Pseudomonas fluorescens strain expressing an active scorpion neurotoxin has dual activity against phytopathogenic fungi and insects, attractive for agronomic applications.

Exploitation of Drosophila Choriogenesis Process as a Model Cellular System for Assessment of Compound Toxicity: the Phloroglucinol Paradigm.

Phloroglucinol (1,3,5 tri-hydroxy-benzene) (PGL), a natural phenolic substance, is a peroxidase inhibitor and has anti-oxidant, anti-diabetic, anti-inflammatory, anti-thrombotic, radio-protective, spasmolytic and anti-cancer activities. PGL, as a medicine, is administered to patients to control the symptoms of irritable bowel syndrome and acute renal colic, in clinical trials. PGL, as a phenolic substance, can cause cytotoxic effects. Administration of PGL up to 300 mg/kg (bw) is well tolerated by animals, while in cell lines its toxicity is developed at concentrations above the dose of 10 µg/ml. Furthermore, it seems that tumor or immortalized cells are more susceptible to the toxic power of PGL, than normal cells. However, studies of its cytotoxic potency, at the cellular level, in complex, differentiated and meta-mitotic biological systems, are still missing. In the present work, we have investigated the toxic activity of PGL in somatic epithelial cells, constituting the follicular compartment of a developing egg-chamber (or, follicle), which directs the choriogenesis (i.e. chorion assembly) process, during late oogenesis of Drosophila melanogaster. Our results reveal that treatment of in vitro growing Drosophila follicles with PGL, at a concentration of 0.2 mM (or, 25.2 µg/ml), does not lead to follicle-cell toxicity, since the protein-synthesis program and developmental pattern of choriogenesis are normally completed. Likewise, the 1 mM dose of PGL was also characterized by lack of toxicity, since the chorionic proteins were physiologically synthesized and the chorion structure appeared unaffected, except for a short developmental delay, being observed. In contrast, concentrations of 10, 20 or 40 mM of PGL unveiled a dose-dependent, increasing, toxic effect, being initiated by interruption of protein synthesis and disassembly of cell-secretory machinery, and, next, followed by fragmentation of the granular endoplasmic reticulum (ER) into vesicles, and formation of autophagic vacuoles. Follicle cells enter into an apoptotic process, with autophagosomes and large vacuoles being formed in the cytoplasm, and nucleus showing protrusions, granular nucleolus and condensed chromatin. PGL, also, proved able to induce disruption of nuclear envelope, activation of nucleus autophagy (nucleophagy) and formation of a syncytium-like pattern being produced by fusion of plasma membranes of two or more individual follicle cells. Altogether, follicle cell-dependent choriogenesis in Drosophila has been herein presented as an excellent, powerful and reliable multi-cellular, differentiated, model biological (animal) system for drug-cytotoxicity assessment, with the versatile compound PGL serving as a characteristic paradigm. In conclusion, PGL is a substance that may act beneficially for a variety of pathological conditions and can be safely used for differentiated somatic -epithelial- cells at clinically low concentrations. At relatively high doses, it could potentially induce apoptotic and autophagic cell death, thus being likely exploited as a therapeutic agent against a number of pathologies, including human malignancies.

A new cytotoxic polycyclic polyprenylated acylphloroglucinol from Garcinia nujiangensis screened by the LC-PDA and LC-MS.

A new polycyclic polyprenylated acylphloroglucinol (1), nujiangefolin D, together with five known analogues (2-6), were isolated from the fruits of Garcinia nujiangensis. Compound 1 was screened by the LC-MS and LC-PDA. The structure of 1 was elucidated on the basis of extensive spectroscopic techniques including 1 D and 2 D NMR and MS analyses. The compounds isolated were evaluated for their cytotoxic activities against three cancer cell lines, 1 showed moderate cytotoxic activity against Hela, PANC-1, and MDA-MB-231 cell lines with IC50 values of 5.6 ± 0.1, 9.1 ± 0.2, and 8.3 ± 0.2 µM, respectively. The antitumor mechanism was explained via virtual docking of 1 to the main sites in the human serine/threonine-protein kinase mTOR (mTOR) crystal structure (PDB code: 4DRI). Furthermore, 1 may inhibit Hela cell proliferation through mTOR by the western blotting analysis. Taken together, 1 may be a potential mTOR inhibitor used for the treatment of cervical cancer.

Assessment of the ecotoxicological impact of natural and synthetic ß-triketone herbicides on the diversity and activity of the soil bacterial community using omic approaches.

The emergence of pesticides of natural origin appears as an environmental-friendly alternative to synthetic pesticides for managing weeds. To verify this assumption, leptospermone, a natural ß-triketone herbicide, and sulcotrione, a synthetic one, were applied to soil microcosms at 0× (control), 1× or 10× recommended field dose. The fate of these two herbicides (i.e. dissipation and formation of transformation products) was monitored to assess the scenario of exposure of soil microorganisms to natural and synthetic herbicides. Ecotoxicological impact of both herbicides was explored by monitoring soil bacterial diversity and activity using next-generation sequencing of 16S rRNA gene amplicons and soil metabolomics. Both leptospermone and sulcotrione fully dissipated over the incubation period. During their dissipation, transformation products of natural and synthetic ß-triketone were detected. Hydroxy-leptospermone was almost completely dissipated by the end of the experiment, while CMBA, the major metabolite of sulcotrione, remained in soil microcosms. After 8 days of exposure, the diversity and structure of the soil bacterial community treated with leptospermone was significantly modified, while less significant changes were observed for sulcotrione. For both herbicides, the diversity of the soil bacterial community was still not completely recovered by the end of the experiment (45 days). The combined use of next-generation sequencing and metabolomic approaches allowed us to assess the ecotoxicological impact of natural and synthetic pesticides on non-target soil microorganisms and to detect potential biomarkers of soil exposure to ß-triketones.

Synthesis and antibiotic activity of novel acylated phloroglucinol compounds against methicillin-resistant Staphylococcus aureus.

The rise in antibiotic resistance among pathogenic microorganisms has created an imbalance in the drugs available for treatment, in part due to the slow development of new antibiotics. Cystic fibrosis (CF) patients are highly susceptible to antibiotic-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Phloroglucinols and related polyketide natural products have demonstrated antimicrobial activity against a number of Gram-positive bacteria including S. aureus. In this study, we investigated a series of acylated phloroglucinol derivatives to determine their potential as lead compounds for the design of novel therapeutics. To assess the activity of these compounds, we determined the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively), the minimum biofilm inhibitory and biofilm eradication concentration (MBIC and MBEC, respectively), and evaluated hemolytic activity, as well as their interaction with clinically relevant antibiotics. Of the 12 compounds tested against MRSA and methicillin-susceptible strains, four showed MIC values ranging from 0.125 to 8 µg ml-1 and all of them were bactericidal. However, none of the compounds were able to eradicate biofilms at the concentrations tested. Three of the four did not display hemolytic activity under the conditions tested. Further studies on the interactions of these compounds with clinically relevant antibiotics showed that phlorodipropanophenone displayed synergistic activity when paired with doxycycline. Our results suggest that these acylated phloroglucinols have potential for being further investigated as antibacterial leads.

Tomoeones A-H, cytotoxic phloroglucinol derivatives from Hypericum ascyron.

Phloroglucinol derivatives tomoeones A-H (1-8) and three known compounds were isolated from leaves of Hypericum ascyron. Their structures were established based on spectroscopic analyses. They are all acylphloroglucinol derivatives possessing a spiro skeleton with geminal isoprenyl groups and a monoterpene moiety, and they are stereoisomers to each other at C-4 and C-13. They appear to be a class of phloroglucinol derivatives. Cytotoxicities of the isolated phloroglucinol derivatives against human tumor cell lines, including multidrug-resistant (MDR) cancer cell lines, were evaluated. Tomoeone F (6) demonstrated significant cytotoxicity against KB cells with an IC50 value of 6.2 microM. Compound 6 was also cytotoxic against MDR cancer cell lines (KB-C2 and K562/Adr), which was more potent than doxorubicin.

Influence of hyperforin on the morphology of internal organs and biochemical parameters, in experimental model in mice.

BACKGROUND AND AIMS: Hyperforin (HY) is used to treat depression and skin irritation and has been shown a number of pharmacological activities. The literature does no cite data on changes that may occur in the body after HY intake (ethylene diammonium salt - EDS) in long-term administration. From this point of view, the present work is a key to determining the pharmacotoxicological profile of the HY-EDS, in long-term administration. MATERIALS AND METHODS: In present research, the influence of toxic doses of HY-EDS was investigated on the biochemical serum parameters and the histopathological changes in internal organs on the experimental mice model. For acute toxicity determination, the HY-EDS was tested in doses of 2000-5000 mg÷kg, administered once per day orally. For subacute toxicity, the HY-EDS was tested in three groups of mice, in doses of 50, 75 and 100 mg÷kg÷day, administered once daily, for 28 consecutive days. RESULTS AND CONCLUSIONS: As concern acute toxicity, a lethal effect has not occurred at any of the two tested doses and HY-EDS was classified as Class V toxic: median lethal dose (LD50) >5000 mg÷kg, p.o. After 14 days of follow-up in acute toxicity, the experimental results showed a statistically significant increase of aspartate transaminase (AST) and alanine transaminase (ALT), compared to the control group. There were no changes in creatinine and serum glucose compared to the control group. After the administration of repeated doses, it was observed an increase of serum transaminases and alkaline phosphatase. Histological examination revealed that the liver injuries were in an initial stage, making them reversible in case of HY-EDS treatment discontinuation. There was no evidence of kidney damage to any of the doses of HY-EDS.

New Phloroglucinol Derivatives from the Fruit Tree Syzygium jambos and Their Cytotoxic and Antioxidant Activities.

Seven new phloroglucinol derivatives (1-7) were isolated from the fruit tree Syzygium jambos together with four known triterpenoids (8-11) and two known flavones (12 and 13). According to the spectroscopic analyses (infrared, electrospray ionization mass spectrometry (ESIMS), high-resolution ESIMS, 1D and 2D nuclear magnetic resonance), the structures of compounds 1-7 were elucidated as jambone A (1), jambone B (2), jambone C (3), jambone D (4), jambone E (5), jambone F (6), and jambone G (7). All the isolates were determined for their cytotoxic activities on melanoma cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and compounds 10 and 11 showed potent activities. Moreover, compounds 1, 2, 4-7, 12, and 13 exhibited weak antioxidant activities under ferric-reducing antioxidant power and 2,2-diphenyl-1-picryhydrazyl radical-scavenging assays.

Eucalyptone from Eucalyptus globulus.

A new cariostatic compound named eucalyptone was isolated from the leaves of Eucalyptus globulus. The structure of this compound was elucidated by spectroscopic methods.

Evaluation of the anti-tumour action and acute toxicity of kosins from Hagenia abyssinica.

The kosins are phloroglucinol derivatives isolated from female flowers of Hagenia abyssinica (Rosaceae) and were tested for possible cytotoxic activity in vitro and in vivo against a panel of three transplantable murine adenocarcinomas of the colon of varying growth characteristics and morphology (MAC system). Significant reductions in colony formation were observed in vitro in MAC 15A tumour following 1, 3, 6 and 24 h exposure to all kosins (alpha-kosin, kosotoxin and protokosin). The kosins (kosotoxin and protokosin) were also found to be cytotoxic against MAC tumour cells in vivo in some cases. Kosotoxin was subjected to preliminary toxicity studies in mice. It showed no observable toxicity up to 200 mg kg-1 orally and was found to be toxic at doses in excess of 50 mg kg-1 (i.p.). A single dose of 100 mg kg-1 (i.p.) was lethal for 100% of the animals.