1H-NMR Metabolomic Study of the Mushroom Pleurotus djamor for the Identification of Nematocidal Compounds.

Due to the increasing populations of anthelmintic-resistant gastrointestinal nematodes and as a consequence of the adverse effects of synthetic drugs, this study focuses on the search for secondary metabolites with nematocidal activity from the edible mushroom Pleurotus djamor using The proton nuclear magnetic resonance (1H-NMR) metabolomics. The highest activity was shown by the ethyl acetate fractions of mycelium (EC50 290.8 µg/mL) and basidiomes (EC50 282.7 µg/mL). Principal component analysis (PCA) and hierarchical data analysis (HCA) of the 1H-NMR metabolic profiles data showed that the ethanolic extracts, the ethyl acetate, butanol, and water fractions from mycelium have different metabolic profiles than those from basidiomes, while low polarity (hexane) fractions from both stages of fungal development show similar profiles. Orthogonal partial least squares discriminant analysis (OPLS-DA) allowed the identification of signals in the 1H-NMR metabolic profile associated with nematocidal activity. The signals yielded via OPLS-DA and bidimensional NMR analysis allowed the identification of uracil as a component in the ethyl acetate fraction from basidiomes, with an EC50 of 237.7 µg/mL. The results obtained showed that chemometric analyses of the 1H-NMR metabolic profiles represent a viable strategy for the identification of bioactive compounds from samples with complex chemical profiles.

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.

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.

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.

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.

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.

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 natural and semisynthetic azorellane and mulinane diterpenoids.

The antituberculosis activity of 14 natural azorellane and mulinane diterpenoids isolated from Azorella compacta, Azorella madreporica, Mulinum crassifolium, and Laretia acaulis, together with eight semisynthetic derivatives, was evaluated against two Mycobacterium tuberculosis strains. The natural azorellanes azorellanol (3) and 17-acetoxy-13-alpha-hydroxyazorellane (6), and the semisynthetic mulinanes 13-hydroxy-mulin-11-en-20-oic-acid methyl ester (13) and mulinenic acid methyl ester (23), showed the strongest activity, with MIC values of 12.5 microg/mL against both strains. The methylated derivatives 13-hydroxy-mulin-11-en-20-oic-acid methyl ester (13), mulin-11,13-dien-20-oic acid methyl ester (15) and mulinenic acid methyl ester (23) proved to be more active than the parent compounds.

Leishmanicidal activity of Yucatecan medicinal plants on Leishmania species responsible for cutaneous leishmaniasis.

The leishmanicidal activity of 15 extracts and 4 pure metabolites obtained from Urechites andrieuxii, Colubrina greggii, Dorstenia contrajerva, and Tridax procumbens was evaluated using the newly developed MTS ({3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay, optimized for promastigotes of Leishmania major, Leishmania tropica, and Leishmania aethiopica, as well as for L. aethiopica axenic amastigotes. The assay was then used for calculating the percentage of viable stationary phase parasites after a 24-hr treatment with each plant extract or pure metabolite. The 3 most active samples, 2 from C. greggii (NCG-5C and DCG-3A) and 1 from T. procumbens (TPZ-2A), showed LD50 values of 62.4, 7.2, and 18.5 microg/ml, respectively, on stationary promastigotes, and of 94.2, 27.1, and 95.2 microg/ml, on amastigotes of L. aethiopica. Moreover, TPZ-2A and DCG-3A significantly reduced the percentage of infected monocyte-derived macrophages (THP-I). The percentage of infected cells decreased from 69.9% +/- 2.5% to 20.8% +/- 2% when the cells were treated with the DCG-3A fraction and to 14.9% +/- 0.5% when treated with TPZ-2A, without significantly decreasing the number of human cells. These findings indicate the presence of potentially bioactive metabolites in the roots of C. greggii and in T. procumbens and reflect the importance of pursuing the bioassay-guided purification of these metabolites.

Variation of leishmanicidal activity in four populations of Urechites andrieuxii.

Urechites andrieuxii Muell.-Arg. (Apocynaceae) is widely used in the Yucatan Peninsula for the treatment of cutaneous leishmaniasis. The influence of the environment in the variability of the leishmanicidal activity of the plant was evaluated using crude methanol extracts of roots from individuals belonging to four natural populations growing in the Yucatan Peninsula. The results of the growth inhibition test using three Leishmania spp. promastigotes showed a stronger leishmanicidal activity in populations of U. andrieuxii growing in more humid environments. Further evaluation against four human cancer cell lines and in the brine shrimp bioassay of both extracts from various parts of the plant and from the most active methanol root extracts, suggested that while the leaf extract appears to have selective toxicity against Leishmania parasites, the strong leishmanicidal activity detected in the root extracts of the plant might be due to its cytotoxicity.