Toxicological Evaluation of Kaempferol and Linearolactone as Treatments for Amoebic Liver Abscess Development in Mesocricetus auratus.

Several studies with kaempferol (KP) and linearolactone (LL) have demonstrated their antiparasitic activity. However, the toxicity of these treatments is unknown. Therefore, this study aimed to evaluate the possible toxicological effects of intraperitoneal (i.p.) administration of KP or LL on the amoebic liver abscess model (ALA) in Mesocricetus auratus. An ALA was induced in male hamsters with 1.5 × 105Entamoeba histolytica (E. histolytica) trophozoites inoculated in the left hepatic lobe. The lesion evolved for 4 days, and then KP (5 mg/kg body weight/day) or LL (10 mg/kg body weight/day) was administered for 4 consecutive days. Then, magnetic resonance imaging (MRI), paraclinical analyses, and necropsy for histopathological evaluation were performed. There was similar ALA inhibition by KP (19.42%), LL (28.16%), and metronidazole, the antiamoebic control (20.87%) (p ≤ 0.05, analysis of variance [ANOVA]). There were hepatic and renal biochemical alterations in all treatment groups, mainly for KP (aspartate aminotransferase: 347.5 ± 37.5 U/L; blood urea nitrogen: 19.4 ± 1.9 g/dL; p ≤ 0.05, ANOVA). Lesions found in the organs were directly linked to the pathology. In conclusion, KP and LL decreased ALA development and exerted fewer toxicological effects compared with metronidazole. Therefore, both compounds exhibit therapeutic potential as an alternative treatment of amoebiasis caused by E. histolytica. However, additional clinical studies in different contexts are required to reaffirm this assertion.

Discovery of Palindrome Dual PPARγ-GPR40 Agonists for Treating Type†2 Diabetes.

This work describes a first attempt of palindromic design for dual compounds that act simultaneously on peroxisome proliferator-activated receptor gamma (PPARγ) and G-protein-coupled receptor 40 (GPR40) for the treatment of type 2 diabetes. The compounds were synthesized by multi-step chemical reactions and the relative mRNA expression levels of PPARγ, GPR40, and GLUT-4 were measured in cultured C2 C12 muscle cells and RIN-m5 f ß-pancreatic cells. In addition, insulin secretion and GLUT-4 translocation were measured. Compound 2 displayed a moderate increase in the mRNA expression of PPARγ and GPR40. However, the translocation of the GLUT-4 transporter was 400 % with a similar effect to pioglitazone. The in vivo effect of compound 2 was determined at 25 mg/kg single dose using a normoglycemic and non-insulin dependent diabetes mellitus (NIDDM) rat models. Compound 2 showed basal plasma glucose in diabetic rats with feed intake, which is associated with the moderate release of insulin measured in cells. Surprisingly, the glucose does not decrease in normoglycemic rats. Compound 2 maintained significant interactions with the GPR40 and PPARγ receptors during molecular dynamics. Altogether, the results demonstrate that compound 2, with a palindromic design, simultaneously activates PPARγ and GPR40 receptors without inducing hypoglycemia.

Vasorelaxant Effect and Blood Pressure Reduction Potential of Pitaya Juice Concentrate (Stenocereus huastecorum) Associated with Calcium Channel Blockade.

Arterial hypertension is a highly prevalent chronic disease worldwide, with several etiologies and treatments that may eventually have side effects or result in patients developing tolerance. There is growing interest in traditional medicine and functional foods to isolate biomolecules that could be useful as coadjuvants for treating several aliments. Pitaya, a desert fruit endemic in Mexico, is a rich source of bioactive molecules (betalains and phenolic compounds). In this work, the vasorelaxation properties of pitaya juice concentrate and fraction one were investigated using aortic and mesenteric rings from rats. The incubation of rings with pitaya juice concentrate or fraction one induced significant vasorelaxation, independent of the endothelium, and showed resistance to potassium channel blockers. This vasorelaxation was associated with the transmembrane influx of extracellular calcium through the vascular smooth muscle cells, with an inhibitory effect on the voltage-dependent calcium channel currents. Also, 400 mg/mL of pitaya juice concentrate in spontaneous hypertensive rats reduced their blood pressure for 48 h. Phytochemical analyses showed that the primary compounds in F1 were glycosidic in nature, and could be a complex mixture of disaccharides, dimeric disaccharides, or even tetrasaccharides. The glycosidic compounds found in F1 primarily contributed to vasodilatation, establishing a voltage-dependent calcium channel inhibition as a possible molecular target.

Inhibition of multidrug-resistant MCF-7 breast cancer cells with combinations of clinical drugs and resin glycosides from Operculina hamiltonii.

The jalap roots, Operculina hamiltonii D.F. Austin & Staples (Convolvulaceae), are extensively commercialized as a depurative and laxative remedy in traditional medicine of the north and northeast regions of Brazil. The purification by recycling HPLC and structure elucidation of three new acyl sugars or resin glycosides are described here from a commercial product made of powdered roots. Three macrocyclic structures of a tetrasaccharide of (11S)-hydroxyhexadecanoic acid, operculinic acid C (1), the undescribed hamiltonins II and III (3 and 4), in addition to the known batatinoside III (5), presented a diastereoisomeric relationship as one residue of n-dodecanoic acid esterified the oligosaccharide core on a different position in each compound. Furthermore, hamiltonin IV (6) was characterized as an ester-type homodimer of acylated operculinic acid C with the same substitution pattern identified in hamiltonins II (3) and III (4) for each of the dimer subunits. All the isolated resin glycosides did not display any intrinsic cytotoxicity (IC50 > 25 µM). However, a combination of the individual isolated compounds 3-6 (1-50 µM) demonstrated an enhancement of cytotoxic effects with sublethal doses of vinblastine and podophyllotoxin (0.003 µM) in multidrug-resistant breast carcinoma epithelial cells (MCF-7/Vin).

Advances in the Properties of Incomptine A: Cytotoxic Activity and Downregulation of Hexokinase II in Breast Cancer Cell Lines.

Breast cancer treatments are limited by the cancer subtype and its selectivity towards tumor cells, hence the importance of finding compounds that increase the survival of healthy cells and target any subtype. Incomptine A (IA) is a sesquiterpene lactone with demonstrated cytotoxic activity. In this study, through in vitro assays, it was observed that IA has similar cytotoxic activity between the subtypes triple negative, HER2+, and luminal A of the breast cancer cell lines. IA cytotoxic activity is higher in cancer than in nontumorigenic cells, and its selectivity index for cancer cells is more than that of the drug doxorubicin. Molecular docking and its in silico comparison with the 2-Deoxyglucose inhibitor suggest that IA could bind to Hexokinase II (HKII), decreasing its expression. Since we did not find changes in the expression of the glycolytic pathway, we suppose that IA could affect the antiapoptotic function of HKII in cancer cells. The IA-HKII union would activate the voltage-gated anion channel 1 (VDAC1), resuming apoptosis. Therefore, we suggest that IA could be used against almost any subtype and that its cytotoxic effect could be due to the reactivation of apoptosis in breast cancer cells.

Linearolactone Induces Necrotic-like Death in Giardia intestinalis Trophozoites: Prediction of a Likely Target.

Linearolactone (LL) is a neo-clerodane type diterpene that has been shown to exert giardicidal effects; however, its mechanism of action is unknown. This work analyzes the cytotoxic effect of LL on Giardia intestinalis trophozoites and identifies proteins that could be targeted by this active natural product. Increasing concentrations of LL and albendazole (ABZ) were used as test and reference drugs, respectively. Cell cycle progression, determination of reactive oxygen species (ROS) and apoptosis/necrosis events were evaluated by flow cytometry (FCM). Ultrastructural alterations were analyzed by transmission electron microscopy (TEM). Ligand-protein docking analyses were carried out using the LL structure raised from a drug library and the crystal structure of an aldose reductase homologue (GdAldRed) from G. intestinalis. LL induced partial arrest at the S phase of trophozoite cell cycle without evidence of ROS production. LL induced pronecrotic death in addition to inducing ultrastructural alterations as changes in vacuole abundances, appearance of perinuclear and periplasmic spaces, and deposition of glycogen granules. On the other hand, the in silico study predicted that GdAldRed is a likely target of LL because it showed a favored change in Gibbs free energy for this complex.

Expanding the Study of the Cytotoxicity of Incomptines A and B against Leukemia Cells.

Heliangolide-type sesquiterpene lactones (HTSLs) are phytocompounds with several pharmacological activities including cytotoxic and antitumor activity. Both bioactivities are related to an α-methylene-γ-lactone moiety and an ester group on carbon C-8 in the sesquiterpene lactone (SL) structure. Two HTSLs, incomptines A (AI) and B (IB) isolated from Decachaeta incompta, were evaluated for their cytotoxic activity on three leukemia cell lines: HL-60, K-562, and REH cells. Both compounds were subjected to a molecular docking study using target proteins associated with cancer such as topoisomerase IIα, topoisomerase IIß, dihydrofolate reductase, methylenetetrahydrofolate dehydrogenase, and Bcl-2-related protein A1. Results show that IA and IB exhibit cytotoxic activity against all cell lines used. The CC50 value of IA was 2-4-fold less than etoposide and methotrexate, two anticancer drugs used as positive controls. The cytotoxic activity of IB was close to that of etoposide and methotrexate. The molecular docking analysis showed that IA and IB have important interaction on all targets used. These findings suggest that IA and IB may serve as scaffolds for the development of new treatments for different types of leukemia.

Understanding the Anti-Diarrhoeal Properties of Incomptines A and B: Antibacterial Activity against Vibrio cholerae and Its Enterotoxin Inhibition.

Incomptines A (IA) and B (IB) are two sesquiterpene lactones with antiprotozoal, antibacterial, cytotoxic, antitumor, spermicidal, and phytotoxic properties. The antibacterial activity of IA and IB against bacteria causing diarrhoea have been reported; however, no information is available regarding their antibacterial activity on Vibrio cholerae. In this work, both compounds were evaluated for their anti-diarrhoeal potential using the bacterium V. cholerae, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis on cholera toxin, and a cholera toxin-induced diarrhoea model in male Balb/c mice. In addition, a molecular docking study was carried out to understand the interaction of IA and IB with cholera toxin. In terms of antibacterial activity, IB was three times more active than IA on V. cholerae. In the case of SDS-PAGE analysis and the in silico study, IA was most effective, revealing its potential binding mode at a molecular level. In terms of anti-diarrhoeal activity, IA was 10 times more active than IB and racecadotril, an antisecretory drug used as positive control; the anti-diarrheal activity of IB was also closer than racecadotril. The results obtained from in vitro, in vivo, and computational studies on V. cholerae and cholera toxin support the potential of IA and IB as new anti-diarrhoeal compounds.

The entomopathogenic fungus Metarhizium anisopliae enhances Arabidopsis, tomato, and maize plant growth.

Species of the entomopathogenic fungi Metarhizium are used worldwide as biocontrol agents. Recently, other lifestyles have been associated with some Metarhizium species, which include their role as saprophytes, endophytes, and plant growth promoters. Herein, the effect of three Metarhizium anisopliae strains on the growth of Arabidopsis thaliana plantlets was evaluated using an in vitro split system. Arabidopsis fresh weight and total chlorophyll content significantly increased 7 days post-inoculation with the three Metarhizium anisopliae strains evaluated. The primary root length was promoted by all fungal strains without physical contact, whereas in direct contact primary root growth was inhibited. Volatile organic compounds identification revealed that during the interaction of Arabidopsis with Ma-20 and Ma-25 strains only ß-caryophyllene was produced, whereas in the Arabidopsis-Ma-28 interaction o-cymene was mainly emitted. The plant growth promoting effect induced by Metarhizium anisopliae strains was also achieved in Arabidopsis, tomato and maize plants grown in soil pots. Our results showed that three Metarhizium anisopliae strains were able to increase plant fresh weight, opening promising perspectives for field production, with the advantages of insect biocontrol and plant growth promotion induced by this species of fungus.

Antilymphoma Effect of Incomptine A: In Vivo, In Silico, and Toxicological Studies.

Incomptine A (IA) is a sesquiterpene lactone isolated from Decachaeta incompta that induces apoptosis, reactive oxygen species production, and a differential protein expression on the U-937 (diffuse histiocytic lymphoma) cell line. In this work, the antitumor potential of IA was investigated on Balb/c mice inoculated with U-937 cells and through the brine shrimp lethality (BSL) test. Furthermore, IA was subjected to molecular docking study using as targets proteins associated with processes of cancer as apoptosis, oxidative stress, and glycolytic metabolism. In addition to determining the potential toxicity of IA in human, its acute toxicity was performed in mice. Results reveals that IA showed high antilymphoma activity and BSL with an EC50 of 2.4 mg/kg and LC50 16.7 µg/mL, respectively. The molecular docking study revealed that IA has strong interaction on all targets used. In the acute oral toxicity, IA had a LD50 of 149 mg/kg. The results showed that the activities of IA including antilymphoma activity, BSL, acute toxicity, and in silico interactions were close to the methotrexate, an anticancer drug used as positive control. These findings suggest that IA may serve as a candidate for the development of a new drug to combat lymphoma.

Incomptine A Induces Apoptosis, ROS Production and a Differential Protein Expression on Non-Hodgkin's Lymphoma Cells.

Sesquiterpene lactones are of pharmaceutical interest due their cytotoxic and antitumor properties, which are commonly found within plants of several genera from the Asteraceae family such as the Decachaeta genus. From Decachaeta incompta four heliangolide, namely incomptines A-D have been isolated. In this study, cytotoxic properties of incomptine A (IA) were evaluated on four lymphoma cancer cell lines: U-937, Farage, SU-DHL-2, and REC-1. The type of cell death induced by IA and its effects on U-937 cells were analyzed based on its capability to induce apoptosis and produce reactive oxygen species (ROS) through flow cytometry with 4',6-diamidino-2-phenylindole staining, dual annexin V/DAPI staining, and dichlorofluorescein 2',7'-diacetate, respectively. A differential protein expression analysis study was carried out by isobaric tags for relative and absolute quantitation (iTRAQ) through UPLC-MS/MS. Results reveal that IA exhibited cytotoxic activity against the cell line U-937 (CC50 of 0.12 ± 0.02 µM) and the incubation of these cells in presence of IA significantly increased apoptotic population and intracellular ROS levels. In the proteomic approach 1548 proteins were differentially expressed, out of which 587 exhibited a fold-change ≥ 1.5 and 961 a fold-change ≤ 0.67. Most of these differentially regulated proteins are involved in apoptosis, oxidative stress, glycolytic metabolism, or cytoskeleton structuration.

Analysis of Volatile Molecules Present in the Secretome of the Fungal Pathogen Candida glabrata.

Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis are the four most common human fungal pathogens isolated that can cause superficial and invasive infections. It has been shown that specific metabolites present in the secretomes of these fungal pathogens are important for their virulence. C. glabrata is the second most common isolate world-wide and has an innate resistance to azoles, xenobiotics and oxidative stress that allows this fungal pathogen to evade the immune response and persist within the host. Here, we analyzed and compared the C. glabrata secretome with those of C. albicans, C. parapsilosis, C. tropicalis and the non-pathogenic yeast Saccharomyces cerevisiae. In C. glabrata, we identified a different number of metabolites depending on the growth media: 12 in synthetic complete media (SC), 27 in SC-glutamic acid and 23 in rich media (YPD). C. glabrata specific metabolites are 1-dodecene (0.09 ± 0.11%), 2,5-dimethylundecane (1.01 ± 0.19%), 3,7-dimethyldecane (0.14 ± 0.15%), and octadecane (0.4 ± 0.53%). The metabolites that are shared with C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and S. cerevisiae are phenylethanol, which is synthesized from phenylalanine, and eicosane and nonanoic acid (identified as trimethylsilyl ester), which are synthesized from fatty acid metabolism. Phenylethanol is the most abundant metabolite in all fungi tested: 26.36 ± 17.42% (C. glabrata), 46.77 ± 15.58% (C. albicans), 49.76 ± 18.43% (C. tropicalis), 5.72 ± 0.66% (C. parapsilosis.) and 44.58 ± 27.91% (S. cerevisiae). The analysis of C. glabrata's secretome will allow us to further our understanding of the possible role these metabolites could play in its virulence.

Linearolactone and Kaempferol Disrupt the Actin Cytoskeleton in Entamoeba histolytica: Inhibition of Amoebic Liver Abscess Development.

Linearolactone (1) and kaempferol (2) have amebicidal activity in in vitro studies. The type of cell death induced by 1 and 2 and their effects on the virulence of E. histolytica were analyzed by transmission and confocal electron microscopy, reactive oxygen species (ROS) production, and apoptosis, detected by flow cytometry with dichlorofluorescein 2',7'-diacetate and annexin-V binding, respectively, and confirmed by TUNEL. The interaction of 1 and 2 with actin was analyzed by docking, and the in vivo amoebicidal activity was established with the Mesocricetus auratus model; amebic liver abscess (ALA) development was evaluated by magnetic resonance (MR) and validated post mortem. In vitro, compounds 1 and 2 caused chromatin condensation, intracellular ROS, and loss of actin structures. Coupling analysis showed that they bind to the allosteric and catalytic sites of actin with binding energies of -11.30 and -8.45 kcal/mol, respectively. Treatments with 1 and 2 induced a decrease in ALA formation without toxic effects on the liver and kidney. Thus, compound 1, but not 2, was able to induce apoptosis-like effects in E. histolytica trophozoites by intracellular production of ROS that affected the actin cytoskeleton structuration. In vivo, compound 1 was more active than compound 2 to reduce the development of ALA.

A Review of the Ephedra genus: Distribution, Ecology, Ethnobotany, Phytochemistry and Pharmacological Properties.

Ephedra is one of the largest genera of the Ephedraceae family, which is distributed in arid and semiarid regions of the world. In the traditional medicine from several countries some species from the genus are commonly used to treat asthma, cold, flu, chills, fever, headache, nasal congestion, and cough. The chemical constituents of Ephedra species have been of research interest for decades due to their contents of ephedrine-type alkaloids and its pharmacological properties. Other chemical constituents such as phenolic and amino acid derivatives also have resulted attractive and have provided evidence-based supporting of the ethnomedical uses of the Ephedra species. In recent years, research has been expanded to explore the endophytic fungal diversity associated to Ephedra species, as well as, the chemical constituents derived from these fungi and their pharmacological bioprospecting. Two additional aspects that illustrate the chemical diversity of Ephedra genus are the chemotaxonomy approaches and the use of ephedrine-type alkaloids as building blocks in organic synthesis. American Ephedra species, especially those that exist in Mexico, are considered to lack ephedrine type alkaloids. In this sense, the phytochemical study of Mexican Ephedra species is a promising area of research to corroborate their ephedrine-type alkaloids content and, in turn, discover new chemical compounds with potential biological activity. Therefore, the present review represents a key compilation of all the relevant information for the Ephedra genus, in particular the American species, the species distribution, their ecological interactions, its ethnobotany, its phytochemistry and their pharmacological activities and toxicities, in order to promote clear directions for future research.

Plants used for the treatment of diarrhoea from Mexican flora with amoebicidal and giadicidal activity, and their phytochemical constituents.

ETHNOPHARMACOLOGICAL RELEVANCE: Flora of the Mexican region is represented by approximately 30,000 vascular plant species, many of which are used as traditional medicines based on knowledge compiled and refined since ancient times (e.g. Cruz-Badiano and Florentino Codexes). The traditional use of plants as medicines, including the treatment of infectious diseases such as diarrhoea, is generally practiced in communities that are geographically isolated or in human settlements where health services are scarce. AIM OF THE STUDY: The aim of this review is to evaluate current research advances in the ethnopharmacology and phytochemistry of Mexican medicinal plants exhibiting antiprotozoal activity, used to treat diarrhoea, and to identify the gaps in this research area for future studies. METHODS: The literature study and compilation of information relied on books and scientific journals from leading electronic databases including Scopus, Springer, SciFinder, ISI Web of Science, PubMed, and Google Scholar; the topics searched for were antiprotozoal activity, followed by Entamoeba histolytica or Giardia lamblia. Ethnopharmacological data were obtained from books regarding medicinal plants and their uses in Mexico as well as from the government databases of "Comisiòn Nacional para el Conocimiento y Uso de la Biodiversidad (CONABIO)" as "NaturaLista" and "EncicloVida". RESULTS: A total of 80 plant species used in the Mexican traditional medicine for the treatment of diarrhoea have been evaluated as potential sources of antiprotozoal agents against E. histolytica or G. lamblia. From these samples, 150 compounds have been isolated and screened in vitro, including specialized metabolites such as flavonoids and terpenoids as well as semisynthesized derivatives. However, in vivo pharmacological studies on these substances are limited. Additional pharmacological and molecular studies of the most active compounds have also been summarized. CONCLUSION: Research performed in the past 25 years on specialized metabolites derived from plants with antiprotozoal activity has yielded relevant findings whose results provide evidence-based support for the use of these plants in the traditional medicine of Mexico to treat diarrhoea. Toxicological and clinical trials of standardized extracts and bioactive compounds are proposed as priority future works in this research area. In addition, in vivo assays are required, of more extracts and/or pure compounds. The optimization of the pharmacological properties of the bioactive specialized metabolites through semisynthetic derivatives and computational methods could aid in developing new antiprotozoal phytomedicines and novel drugs for the treatment of these types of infections. Furthermore, elucidation of the mechanism of action of these bioactive compounds through pharmacological and molecular studies are also necessary.

Further galphimines from a new population of Galphimia glauca.

Both DNA barcoding and phylogenetic data of the studied botanical material suggested the existence a new population of Galphimia glauca. Their leaves afforded three new nor-3,4-seco-friedelanes named galphimines M-O, together with known galphimines D, E, G, and I. Galphimines M and N possess bicyclic orthoacetates which are the first examples of orthoesters found in the Malpighiaceae family, while galphimine O has a 27,20-δ-lactone moiety. The structures elucidation followed from spectroscopic means and the absolute configuration followed from single crystal X-ray diffraction analyses. Tests for antibacterial and antifungal activities of galphimines N and M showed no promising effects.

The Ustilago maydis null mutant strains of the RNA-binding protein UmRrm75 accumulate hydrogen peroxide and melanin.

Ustilago maydis is a dimorphic fungus that has emerged as a model organism for the study of fungal phytopathogenicity and RNA biology. In a previous study, we isolated the U. maydis UmRrm75 gene. The deletion of the UmRrm75 gene affected morphogenesis and pathogenicity. UmRrm75 gene encodes a protein containing three RNA recognition motifs. Here we determined that UmRrm75 has chaperone activity in Escherichia coli using the transcription anti-termination assay. Subsequently, we analyzed the growth of ΔUmRrm75 mutants at 15 °C and 37 °C, observing that mutant strains had reduced growth in comparison to parental strains. UmRrm75 gene expression was induced under these non-optimal temperatures. ΔUmRrm75 mutant colonies displayed a dark-brown color at 28 °C, which was confirmed to be melanin based on spectroscopic analysis and spectrometric data. Furthermore, ΔUmRrm75 mutant strains showed the presence of peroxisomes, and increased H2O2 levels, even at 28 °C. The ΔUmRrm75 mutant strains displayed a higher expression of redox-sensor UmYap1 gene and increased catalase activity than the parental strains. Our data show that deletion of the UmRrm75 gene results in higher levels of H2O2, increased melanin content, and abiotic stress sensitivity.

Amarisolide F, an Acylated Diterpenoid Glucoside and Related Terpenoids from Salvia amarissima.

Nine terpenoids were isolated from the leaves and flowers of Salvia amarissima, including a new acylated diterpenoid glucoside, amarisolide F (1), a new neo-clerodane diterpenoid, amarissinin D (2), which was isolated as an acetyl derivative (2a), and four known diterpenoids. The structure of amarisolide F (1) was elucidated by NMR and MS data analyses, as well as its methanolysis products 7 and 8, which also constituted new diterpenoids, named amarissinin E and 8- epi-amarissinin E, respectively. The absolute configuration of compound 7 was established by single-crystal X-ray diffraction. The cytotoxicity and anti-MDR effect of 1 in three phenotypes of the MCF-7 cell lines were assayed. Compound 1 was 2-3.6-fold more active than amarissinins A (3) and B (4), but several orders of magnitude less active than teotihuacanin (6) and reserpine.

The Arabidopsis-Trichoderma interaction reveals that the fungal growth medium is an important factor in plant growth induction.

Trichoderma spp colonizes the plant rhizosphere and provides pathogen resistance, abiotic stress tolerance, and enhance growth and development. We evaluated the Arabidopsis-Trichoderma interaction using a split system in which Trichoderma atroviride and Trichoderma virens were grown on PDA or MS medium. Arabidopsis growth was significantly increased at 3 and 5 days post-inoculation with both Trichoderma species, when the fungal strains were grown on PDA in split interaction. The analysis of DR5:uidA reporter line revealed a greater auxin accumulation in root tips when the fungi were grown on PDA in a split interaction. The root hair-defective phenotype of Arabidopsis rhd6 mutant was reverted with both Trichoderma species, even in split interactions. At 12 °C, Trichoderma species in split interactions were able to mitigate the effects of cold stress on the plant, and also Trichoderma induced the AtERD14 expression, a cold related gene. Volatile organic compounds analysis revealed that Trichoderma strains produce mainly sesquiterpenes, and that the type and abundance of these compounds was dependent on the fungal strain and the culture medium. Our results show that fungal nutrition is an important factor in plant growth in a split interaction.

neo-Clerodane Diterpenoids from Salvia polystachya Stimulate the Expression of Extracellular Matrix Components in Human Dermal Fibroblasts.

Eleven neo-clerodane diterpenoids (1-11) including the new analogues 1, 2, and 10, and 3',5,6,7-tetrahydroxy-4'-methoxyflavone (12) were isolated from the aerial parts of Salvia polystachya. Polystachyne G (1) and 15-epi-polystachyne G (2) were isolated as an epimeric mixture, containing a 5-hydroxyfuran-2(5H)-one unit in the side chain at C-12 of the neo-clerodane framework. Polystachyne H (10) contains a 1(10),2-diene moiety and a tertiary C-4 hydroxy group. The structures of these compounds were established by analysis of their NMR spectroscopic and MS spectrometric data. The absolute configurations of compounds 3, 4, and 10 were determined through single-crystal X-ray diffraction analysis. The antibacterial, antifungal, and phytotoxic activities of the diterpenoids were determined. In addition, the stimulatory effect of the expression of extracellular matrix components of nine of the isolates (1-8 and 11) was assayed. Compounds 1-4, 8, and 11 increased the expression of the genes codifying for type I, type III, and type V collagens and for elastin.