Bioassay-guided identification of antithrombotic compounds from Cnidoscolus aconitifolius (Mill.) I. M. Jhonst.: molecular docking, bioavailability, and toxicity prediction.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Conventional antithrombotic therapy has reported hemorrhagic accidents. Ethnobotanical and scientific reports point to Cnidoscolus aconitifolius as an antithrombotic adjuvant. Previously, C. aconitifolius leaves ethanolic extract displayed antiplatelet, anticoagulant, and fibrinolytic activities. This work aimed to identify compounds from C. aconitifolius with in vitro antithrombotic activity through a bioassay-guided study. Antiplatelet, anticoagulant, and fibrinolytic tests guided the fractionation. Ethanolic extract was subjected to a liquid-liquid partitioning, followed by vacuum liquid, and size exclusion chromatography to obtain the bioactive JP10B fraction. The compounds were identified through UHPLC-QTOF-MS, and their molecular docking, bioavailability, and toxicological parameters were determined computationally. Kaempferol-3-O-glucorhamnoside and 15(S)-HPETE were identified; both showed affinity for antithrombotic targets, low absorption, and safety for human consumption. Further in vitro and in vivo evaluations will better understand their antithrombotic mechanism. This bioassay-guided fractionation demonstrated that C. aconitifolius ethanolic extract has antithrombotic compounds.Communicated by Ramaswamy H. Sarma.

Exploring the anti-virulence potential of plants used in traditional Mayan medicine to treat bacterial infections.

ETHNOPHARMACOLOGICAL RELEVANCE: While the antimicrobial activity of a number of plants used in traditional Mayan medicine against infectious diseases has been documented, their potential to inhibit quorum sensing (QS) as means of discovering novel anti-virulence agents remains unexplored. AIM OF THE STUDY: To evaluate the anti-virulence potential of plants used in traditional Mayan medicine by determining their inhibition of QS- regulated virulence factors in Pseudomonas aeruginosa. METHODS: A group of plants used in traditional Mayan medicine against infectious diseases was selected, and their methanolic extracts were evaluated at 10 mg/mL for their antibacterial and anti-virulence activity using the reference strain P. aeruginosa PA14WT. The broth microdilution method was used to determine antibacterial activity (MIC), while anti-virulence activity was evaluated by measuring the anti-biofilm effect and the inhibition of pyocyanin and protease activities. The most bioactive extract was fractionated using a liquid-liquid partition procedure and the semipurified fractions were evaluated at 5 mg/mL for antibacterial and anti-virulence activity. RESULTS: Seventeen Mayan medicinal plants traditionally used to treat infection-associated diseases were selected. None of the extracts exhibited antibacterial activity, whereas anti-virulence activity was detected in extracts of Bonellia flammea, Bursera simaruba, Capraria biflora, Ceiba aesculifolia, Cissampelos pareira and Colubrina yucatanensis. The most active extracts (74% and 69% inhibition) against biofilm formation were from C. aesculifolia (bark) and C. yucatanensis (root), respectively. Alternatively, the extracts of B. flammea (root), B. simaruba (bark), C. pareira (root), and C. biflora (root), reduced pyocyanin and protease production (50-84% and 30-58%, respectively). Fractionation of the bioactive root extract of C. yucatanensis allowed the identification of two semipurified fractions with anti-virulence activity. CONCLUSIONS: The anti-virulence activity detected in the crude extracts of B. flammea, B. simaruba, C. biflora, C. aesculifolia, C. pareira, and C. yucatanensis, confirms the efficacy and traditional use of these medicinal plants against infectious diseases. The activity of the extract and semipurified fractions of C. yucatanensis indicates the presence of hydrophilic metabolites capable of interfering with QS in P. aeruginosa. This study represents the first report of Mayan medicinal plants with anti-QS properties and suggests they represent an important source of novel anti-virulence agents.

Bee Species, Botanical Sources and the Chemical Composition of Propolis from Yucatan, Mexico.

Propolis is used by corbiculated bees to protect the bee hive; it is mostly used to seal cracks, to reduce or prevent microbial growth and to embalm invaders. Different factors have been reported to influence the chemical composition of propolis, including bee species and the flora surrounding the hive. Nevertheless, the majority of the studies are focused on propolis produced by Apis mellifera, while studies on the chemical composition of propolis produced by stingless bees are still limited. In this investigation, the chemical composition of 27 propolis samples collected in the Yucatan Peninsula from A. mellifera beehives, together with 18 propolis samples from six different species of stingless bees, were analyzed by GC-MS. Results showed that lupeol acetate and ß-amyrin were the characteristic triterpenes in propolis samples from A. mellifera, while grandiflorenic acid and its methyl ester were the main metabolites present in samples from stingless bees. Multivariate analyses were used to explore the relationship between bee species and botanical sources on the chemical composition of the propolis samples. Differences in body size and, therefore, foraging abilities, as well as preferences for specific botanical sources among bee species, could explain the observed variation in propolis chemical composition. This is the first report on the composition of propolis samples from the stingless bees Trigona nigra, Scaptotrigona pectoralis, Nannotrigona perilampoides, Plebeia frontalis and Partamona bilineata.

Activity of Semi-Synthetic Mulinanes against MDR, Pre-XDR, and XDR Strains of Mycobacterium tuberculosis.

Tuberculosis causes more than 1.2 million deaths each year. Worldwide, it is the first cause of death by a single infectious agent. The emergence of drug-resistant strains has limited pharmacological treatment of the disease and today, new drugs are urgently needed. Semi-synthetic mulinanes have previously shown important activity against multidrug-resistant (MDR) Mycobacterium tuberculosis. In this investigation, a new set of semi-synthetic mulinanes were synthetized, characterized, and evaluated for their in vitro activity against three drug-resistant clinical isolates of M. tuberculosis: MDR, pre-extensively Drug-Resistant (pre-XDR), and extensively Drug-Resistant (XDR), and against the drug-susceptible laboratory reference strain H37Rv. Derivative 1a showed the best anti-TB activity (minimum inhibitory concentration [MIC] = 5.4 µM) against the susceptible strain and was twice as potent (MIC = 2.7 µM) on the MDR, pre-XDR, and XDR strains and also possessed a bactericidal effect. Derivative 1a was also tested for its anti-TB activity in mice infected with the MDR strain. In this case, 1a produced a significant reduction of pulmonary bacilli loads, six times lower than the control, when tested at 0.2536 mg/Kg. In addition, 1a demonstrated an adjuvant effect by shortening second-line chemotherapy. Finally, the selectivity index of >15.64 shown by 1a when tested on Vero cells makes this derivative an important candidate for future studies in the development of novel antitubercular agents.

Using 13 C-NMR dereplication to aid in the identification of xanthones present in the stem bark extract of Calophyllum brasiliense.

INTRODUCTION: Xanthones are metabolites with a variety of biological properties. The Clusiaceae family, which until recently included the genus Calophyllum, is recognised for its production of monohydroxylated and polyhydroxylated xanthones. Presently, C. brasiliense is the only Calophyllum spp. known to occur in the Yucatan peninsula. OBJECTIVE: To use a combination of traditional phytochemical methods and carbon-13 nuclear magnetic resonance (13 C-NMR) dereplication analysis to identify xanthones in the stem bark of C. brasiliense. MATERIAL AND METHODS: Initial fractionation and purification of the stem bark extract of C. brasiliense produced macluraxanthone (1). Additional xanthones, together with chromanones and terpenoids, were identified using 13 C-NMR dereplication analysis in different semipurified fractions obtained from the low and medium polarity fractions of the stem bark extract of C. brasiliense. RESULTS: Initial identification of macluraxanthone (1) was confirmed by 13 C-NMR dereplication analysis; additionally, 13 C-NMR dereplication analysis allowed the identification of a number of monohydroxylated and polyhydroxylated xanthones, together with chromanones and terpenoids. CONCLUSION: This study confirms C. brasiliense as a rich source of xanthones and the 13 C-NMR dereplication analysis as a suitable method to quickly identify the presence of different families of secondary metabolites in semipurified fractions.

Methyl jasmonate enhances ursolic, oleanolic and rosmarinic acid production and sucrose induced biomass accumulation, in hairy roots of Lepechinia caulescens.

BACKGROUND: Ursolic (UA), oleanolic (OA) and rosmarinic (RA) acids are bioactive metabolites found in Lepechinia caulescens that have generated interest for their health benefits, which include antimicrobial, antioxidant, antimutagenic, gastroprotective, antidiabetic, antihypertensive and anti-inflammatory properties, among others. To date, very few attempts have been made to evaluate the potential for simultaneous production of these bioactive compounds, using a biotechnological approach. Hairy root cultures offer a biotechnology approach that can be used to study the factors affecting the biosynthesis and the production of UA, OA and RA. In the current study, we established hairy root cultures of L. caulescens and evaluated the effect of sucrose on biomass accumulation, and the effect of different concentrations and times of exposure of methyl jasmonate (MeJA), on the accumulation of UA, OA and RA. METHODS: Leaves from plants of L. caulescens were inoculated with Agrobacterium rhizogenes strain ATCC 15834. PCR of rolB gene confirmed the transgenic nature of hairy roots. Hairy roots were subcultured in semisolid MSB5 medium, supplemented with 15, 30, 45 or 60 g/L sucrose and after 4 weeks, dry weight was determined. The accumulation of UA, OA and RA of wild plants and hairy roots were determined by HPLC. Finally, the hairy roots were treated with 0, 100, 200 and 300 µM of MeJA and the content of bioactive compounds was analyzed, after 24, 48 and 72 h. RESULTS: High frequency transformation (75%) was achieved, using leaf explants from axenic seedlings, infected with A. rhizogenes. The hairy roots showed an enhanced linear biomass accumulation, in response to the increase in sucrose concentration. The hairy root cultures in MSB5 medium, supplemented with 45 g/L sucrose, were capable to synthesizing UA (0.29 ± 0.00 mg/g DW), OA (0.57 ± 0.00 mg/g DW) and RA (41.66 ± 0.31 mg/g DW), about two, seven and three times more, respectively, than in roots from wild plants. Elicitation time and concentration of MeJA resulted in significant enhancement in the production of UA, OA and RA, with treatments elicited for 24 h, with a concentration of 300 µM of MeJA, exhibiting greatest accumulation. CONCLUSION: This is the first report on development of hairy root cultures of L. caulescens. Future studies should aim towards further improving triterpenes and polyphenolic compound production in hairy roots of L. caulescens, for use in the pharmaceutical and biotechnological industry.

Anti-inflammatory and anti-hypersensitive effects of the chalcone isocordoin and its semisynthetic derivatives in mice.

Isocordoin (1), a chalcone isolated from different plants, has been found to present a range of interesting biological properties. This study aimed to evaluate the anti-hypersensitive and anti-inflammatory effects of isocordoin (1) and several natural and semisynthetic derivatives (2-10). Initial evaluation of (1), dihydroisocordoin (2) and six semisynthetic derivatives (3-8) in the inhibition of abdominal writhes induced by acetic acid model showed that only isocordoin dimethylether (5) caused more than 70% of inhibition. Further evaluation of 5 for its anti-oedematogenic activity and anti-hypersensitivity effect induced by carrageenan, lipopolysaccharide (LPS), bradykinin (BK), prostaglandin E2 (PGE2), and epinephrine showed that isocordoin dimethylether (5) presented a discrete inhibition of carrageenan- and LPS-induced hypersensitivity, and of carrageenan-induced paw oedema, and that it was able to significantly reduce both the oedema and hypersensitivity induced by BK. Furthermore, when tested in the PGE2 model, 5 interfered only with the paw-oedema, without showing any effect against the paw-hypersensitivity. Evaluation of the natural isocordoin (1), together with the semisynthetic derivatives isocordoin dimethylether (5), isocordoin methylether (9), and dihydroisocordoin methylether (10) in the BK-induced oedema and hypersensitivity showed that the monoalkylated derivatives 10 and 9 had the strongest antinociceptive activity. The results of this investigation indicate that both monoalkylation of the C-4' phenolic hydroxyl group and reduction of the double bond in the α,ß-unsaturated system of the chalcone skeleton favor activity.

Mulinane- and Azorellane-Type Diterpenoids: A Systematic Review of Their Biosynthesis, Chemistry, and Pharmacology.

Mulinane- and azorellane-type diterpenoids have unique tricyclic fused five-, six-, and seven-membered systems and a wide range of biological properties, including antimicrobial, antiprotozoal, spermicidal, gastroprotective, and anti-inflammatory, among others. These secondary metabolites are exclusive constituents of medicinal plants belonging to the Azorella, Laretia, and Mulinum genera. In the last 30 years, more than 95 mulinanes and azorellanes have been reported, 49 of them being natural products, 4 synthetics, and the rest semisynthetic and biotransformed derivatives. This systematic review highlights the biosynthetic origin, the chemistry, and the pharmacological activities of this remarkably interesting group of diterpenoids.

In vitro growth inhibition and bactericidal activity of spathulenol against drug-resistant clinical isolates of Mycobacterium tuberculosis

ABSTRACT Spathulenol was isolated from an extract of Azorella compacta Phil., Apiaceae, by various chromatographic method; identification of the chemical structure was confirmed by comparing its spectroscopic data with those reported in the literature. The anti-Mycobacterium tuberculosis activity of spathulenol was evaluated on MDR, pre-XDR, and XDR clinical isolates of M. tuberculosis, as well as on the reference susceptible strain H37Rv and its cytotoxic activity was evaluated on the Vero Cell Line. The anti-M. tuberculosis activity of spathulenol was twice as potent against the MDR, pre-XDR, and XDR clinical isolates (6.25 µg/ml) than on the susceptible H37Rv strain (12.5 µg/ml). Additionally, the anti-M. tuberculosis activity shown by spathulenol was established as bactericidal on drug-resistant and susceptible strains of M. tuberculosis. Finally, cytotoxic activity on the Vero cell line (CC50 = 95.7 µg/ml) indicated that spathulenol is a selective anti-M. tuberculosis compound, with a selective index of 15.31 against drug-resistant clinical isolates of M. tuberculosis.

Bactericidal Effect of the Leaf Extract from Musa spp. (AAB Group, Silk Subgroup), cv. "Manzano" Against Multidrug-Resistant Mycobacterium tuberculosis.

Air-dried leaves of a Musa spp. AAB, cv. "Manzano" plant, known as Ja'as in the Maya culture, were sequentially extracted with hexane, ethyl acetate, and methanol; the resulting extracts were investigated for their antimycobacterial activity against susceptible and drug-resistant strains of Mycobacterium tuberculosis (MTB) using the Microplate Alamar Blue Assay. Both the n-hexane extract (HE) and ethyl acetate extract (EE) showed potent activity against both strains of MTB, with the EE exhibiting the strongest activity and a Minimum Inhibitory Concentration of 12.5 and 6.25 µg/mL against susceptible and drug-resistant strains, respectively. Both extracts also demonstrated a mycobactericidal effect and a very good selectivity index when tested for cytotoxic activity on Vero monkey kidney cells, using the Sulforhodamine B assay. Our results demonstrate the efficiency and selectivity of Musa spp. AAB, cv. "Manzano" against MTB strains and support its traditional use as remedy against tuberculosis in Maya traditional medicine.

Genetic Transformation of Pentalinon andrieuxii Tissue Cultures.

Pentalinon andrieuxii is a species used in Mayan traditional medicine due to its biological properties. Recent studies indicate that it produces a pentacyclic triterpene-denominated betulinic acid, which presents various biological activities: antibacterial, antifungal, antiplasmodial, anti-inflammatory, antimalarial, anticancer, leishmanicidal, and antiviral, as well as steroids and sterols with leishmanicidal properties. A recent study also reported the presence of urechitol A and B in the roots; these are secondary metabolites whose biochemical function is as yet unknown. This plant therefore represents a natural source of metabolites with potential application in the pharmaceutical industry. In this chapter, a protocol is described for obtaining transgenic plants, at the reporter gene of the ß-glucuronidase (GUS) via Agrobacterium tumefaciens from hypocotyl and root explants. The protocol established herein could be employed for the manipulation of the genes involved in the biosynthesis of isoprenoids or secondary metabolites of interest. To our knowledge, this is the first report of stable transformation of Pentalinon andrieuxii via Agrobacterium tumefaciens.

Effects of polyphenol removal methods on the in vitro exsheathment inhibitory activity of Lysiloma latisiliquum extracts against Haemonchus contortus larvae.

It has been suggested that the anthelmintic (AH) activity of tannin-rich forages is related to their tannin content. However, reports on tropical fodders such as Lysiloma latisiliquum describe the same AH activity after the addition of tannin-blocking agents, suggesting that the activity either depends on the method of tannin blocking/removal or is due to other type of secondary metabolites. This study compared both the effect of the drying process of the plant material and the effect of different polyphenol removal methods on the AH activity of L. latisiliquum acetone-water extracts when tested against Haemonchus contortus. The results showed that the extraction of oven-dried (OD) leaves of L. latisiliquum yielded a CT-free extract with high AH activity. However, polyphenol-free fractions showed similar or lower AH activity levels as of those original OD extract. HPLC analysis confirmed that common polyphenolic metabolites are not responsible for the AH activity of L. latisiliquum.

The use of 1 H-NMR Metabolomics to Optimise the Extraction and Preliminary Identification of Anthelmintic Products from the Leaves of Lysiloma latisiliquum.

INTRODUCTION: Tannin-rich forages are recognised as an important alternative for the control of gastrointestinal nematodes in small ruminants. Lysiloma latisiliquum, a forage commonly consumed by goats and sheep, has shown anthelmintic activity when tested against Haemonchus contortus. However, to date, the metabolites responsible for the activity are not known. OBJECTIVE: To use 1 H-NMR metabolomics in the extraction and identification of anthelmintic metabolites from L. latisiliquum. METHODOLOGY: Eight different solvent systems were compared for the optimum extraction of anthelmintic metabolites from L. latisiliquum. 1 H-NMR spectra of the tannin-free extracts were measured in methanol-d4 using trimethylsilylpropanoic acid (TSP) as internal standard. Extracts were also evaluated for their anthelmintic activity using the larval exsheathment inhibition assay against H. contortus. These data were correlated by multivariate analysis [principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA)] and analysed. To validate the results obtained after the OPLS-DA, a bioassay-guided isolation of bioactive metabolites was conducted. RESULTS: The PCA of the 1 H-NMR data allowed the identification of hydrophilic solvents as those best suited for the extraction of anthelmintics from L. latisiliquum and indicated that the bioactive metabolites are high-polarity, glycosylated products. Similarly, OPLS-DA of the data enabled the detection of activity-related signals, assigned to the glycosylated metabolites quercitrin and arbutin obtained from the bioassay-guided purification of the extract. CONCLUSION: The results of this investigation confirm metabolomics as a useful tool in the detection of bioactive metabolites in plants without previous phytochemical studies. Copyright © 2017 John Wiley & Sons, Ltd.

Chemometrics-enhanced high performance liquid chromatography-ultraviolet detection of bioactive metabolites from phytochemically unknown plants.

This work describes the use of Colubrina greggii as a model to investigate the use of chemometric analysis combined with data from a leishmanicidal bioassay, using Principal Component Analysis (PCA) and Orthogonal Projections to Latent Structures (O-PLS), to detect biologically active natural products in crude extracts from plants having little or no phytochemical information. A first analysis of the HPLC-UV profiles of the extract and its semi-purified fractions using both Principal Component Analysis (PCA) and Orthogonal Partial Least Squares (O-PLS) indicated that the components at tR 48.2, 48.7, 51.8min correlated with the variation in bioactivity. However, a further O-PLS analysis of the HPLC-UV profiles of fractions obtained through a final semi-preparative HPLC purification showed two components at tR 48.7 and 49.5min which correlated with the variation of the bioactivity in a high performance predictive model, with high determination coefficient, high correlation coefficient values (R(2) and Q(2)=0.99) and a low root mean square error (RMSE=0.018). This study demonstrates that the association of chemometric analysis with bioassay results can be an excellent strategy for the detection and isolation of bioactive metabolites from phytochemically unknown plant crude extracts.

Salvinorin A content in legal high products of Salvia divinorum sold in Mexico.

Salvia divinorum (Lamiaceae) is a herb native to Mexico where it is used by Mazatec shamans for spiritual and divination purposes. S. divinorum products are easily available to consumers and are used worldwide as legal highs because of the hallucinogenic effects caused mainly by salvinorin A. Highly popular videos and websites on the internet depicting the use of S. divinorum products have contributed to an increase in their consumption. Recent reports have highlighted the potential of these products to induce psychosis in consumers. In Mexico, dried leaf extracts of S. divinorum are sold in different strengths, claiming to correlate with increasing amounts of salvinorin A. In order to determine the variability of salvinorin A content between brands and to investigate possible correlation between brand strengths, this study sought to quantify salvinorin A in commercial products available in Mexico using an HPLC method. The HPLC analytical method showed a correlation coefficient R(2)>0.99, with LOD of 0.44 µg/mL and LOQ of 1.34 µg/mL. The retention time for salvinorin A was 23.09±0.95 min and the measured concentrations ranged between 8.32±0.65 and 56.52±3.77 mg/g dried leaf. The results for brand c did not show an agreement between the declared and the calculated amount of salvinorin A. Additionally, the emergence in Mexico of high strength salvia products (100×), the lack of regulation and the observed variability of salvinorin A content between brands of commercial legal highs products of S. divinorum could result in a health problem for consumers.

Phytochemical diversity of the essential oils of Mexican Oregano (Lippia graveolens Kunth) populations along an Edapho-climatic gradient.

Mexican oregano (Lippia graveolens) is an important aromatic plant, mainly used as flavoring and usually harvested from non-cultivated populations. Mexican oregano essential oil showed important variation in the essential-oil yield and composition. The composition of the essential oils extracted by hydrodistillation from 14 wild populations of L. graveolens growing along an edaphoclimatic gradient was evaluated. Characterization of the oils by GC-FID and GC/MS analyses allowed the identification of 70 components, which accounted for 89-99% of the total oil composition. Principal component and hierarchical cluster analyses divided the essential oils into three distinct groups with contrasting oil compositions, viz., two phenolic chemotypes, with either carvacrol (C) or thymol (T) as dominant compounds (contents >75% of the total oil composition), and a non-phenolic chemotype (S) dominated by oxygenated sesquiterpenes. While Chemotype C was associated with semi-arid climate and shallower and rockier soils, Chemotype T was found for plants growing under less arid conditions and in deeper soils. The plants showing Chemotype S were more abundant in subhumid climate. High-oil-yield individuals (>3%) were identified, which additionally presented high percentages of either carvacrol or thymol; these individuals are of interest, as they could be used as parental material for scientific and commercial breeding programs.

Further mulinane and azorellane diterpenoids isolated from Mulinum crassifolium and Azorella compacta.

The new mulinane diterpenoids 1 and 2 were isolated from the EtOAc extract of Mulinum crassifolium, while the rearranged mulinane 5, which was isolated for the first time from a natural source, was isolated from Azorella compacta. Compounds 1-2 were prepared by semi-synthesis thorough acetylation of the diterpene 17-acetoxymulinic acid (3). A mechanism of reaction was proposed, while the structures of the new compounds were elucidated on the basis of comprehensive spectroscopic analysis and computational methods.

Flourensia cernua: Hexane Extracts a Very Active Mycobactericidal Fraction from an Inactive Leaf Decoction against Pansensitive and Panresistant Mycobacterium tuberculosis.

The efficacy of decoction in extracting mycobactericidal compounds from Flourensia cernua (Hojasé) leaves and fractionation with solvents having ascending polarity was compared with that of (i) ethanol extraction by still maceration, extraction with a Soxhlet device, shake-assisted maceration, or ultrasound-assisted maceration, followed by fractionation with n-hexane, ethyl acetate, and n-butanol; (ii) sequential extraction with n-hexane, ethyl acetate, and n-butanol, by still maceration, using a Soxhlet device, shake-assisted maceration, or ultrasound-assisted maceration. The in vitro mycobactericidal activity of each preparation was measured against drug-sensitive (SMtb) and drug-resistant (RMtb) Mycobacterium tuberculosis strains. The results of which were expressed as absolute mycobactericidal activity (AMA). These data were normalized to the ΣAMA of the decoction fraction set. Although decoction was inactive, the anti-RMtb normalized ΣAMA (NAMA) of its fractions was comparable with the anti-RMtb NAMA of the still maceration extracts and significantly higher than the anti-SMtb and anti-RMtb NAMAs of every other ethanol extract and serial extract and fraction. Hexane extracted, from decoction, material having 55.17% and 92.62% of antituberculosis activity against SMtb and RMtb, respectively. Although the mycobactericidal activity of decoction is undetectable; its efficacy in extracting F. cernua active metabolites against M. tuberculosis is substantially greater than almost all pharmacognostic methods.

The in vitro secretome of Mycosphaerella fijiensis induces cell death in banana leaves.

The hemibiotrophic filamentous fungus Mycosphaerella fijiensis causes the banana foliar disease known as black Sigatoka, responsible for major worldwide losses in the banana fruit industry. In this work the in vitro secretome of M. fijiensis was characterized. Native and denaturant polyacrylamide gel protease assays showed the M. fijiensis secretome contains protease activity capable of degrading gelatin. Necrotic lesions on leaves were produced by application of the in vitro secretome to the surface of one black Sigatoka-resistant banana wild species, one susceptible cultivar and the non-host plant Carica papaya. To distinguish if necrosis by the secretome is produced by phytotoxins or proteins, the latter ones were precipitated with ammonium sulfate and applied in native or denatured forms onto leaves of the same three plant species. Proteins applied in both preparations were able to produce necrotic lesions. Application of Pronase, a commercial bacterial protease suggested that the necrosis was, at least in part, caused by protease activity from the M. fijiensis secretome. The ability to cause necrotic lesions between M. fijiensis secreted- and ammonium sulfate-precipitated proteins, and purified lipophilic or hydrophilic phytotoxins, was compared. The results suggested that leaf necrosis arises from the combined action of non-host specific hydrolytic activities from the secreted proteins and the action of phytotoxins. This is the first characterization of the M. fijiensis protein secretome produced in vitro but, more importantly, it is also the first time the M. fijiensis secretome has been shown to contain virulence factors capable of causing necrosis to its natural host.

Antituberculosis activity of alkylated mulinane diterpenoids.

Natural azorellane and mulinane diterpenoids show antituberculosis activity, which is increased by methylation of their free carboxyl group. We have systematically investigated the effect of alkylation in this class of diterpenoids and found that the profile of bioactivity is relatively unaffected by the introduction of short alkyl groups, both linear and branched. In this investigation, three semisynthetic diterpenoids, 13 hydroxy-mulin-11-en-20-oic acid n-propyl ester (3) and the n-propyl (19) and n-butyl (20) esters of isomulinic acid, showed the strongest antituberculosis activity (MIC=6.25 microg/mL) against a drug-resistant strain of Mycobacterium tuberculosis.