Metabolomics-Guided Isolation of Anti-trypanosomal Metabolites from the Endophytic Fungus Lasiodiplodia theobromae.

Fungal endophytes offer diverse and unique secondary metabolites, making these organisms potential sources of promising drug leads. The application of high-resolution-liquid chromatography mass spectrometry and nuclear magnetic resonance-based metabolomics to fungal endophytes is practical in terms of dereplication studies and the mining of bioactive compounds. In this paper, we report the application of metabolomics in parallel with anti-trypanosomal assays to determine the ideal conditions for the medium-scale fermentation of the endophyte Lasiodiplodia theobromae. The 1H NMR comparison between the active versus inactive fractions identified several unique chemical fingerprints belonging to the active fractions. Furthermore, by integrating high-resolution-liquid chromatography mass spectrometry data with multivariate data analysis, such as orthogonal partial least squares-discriminant analysis (OPLS-DA) and the bioactivity results of the fractions of L. theobromae, the anti-trypanosomal agents were easily discerned. With available databases such as Antibase and Dictionary of Natural Products coupled to MZmine through in-house algorithms optimized in our laboratory, the predicted metabolites were readily identified prior to isolation. Fractionation was performed on the active fractions and three known compounds were isolated, namely, cladospirone B, desmethyl-lasiodiplodin, and R-(-)-mellein. Cladospirone B and desmethyl-lasiodiplodin were among the predicted compounds generated by the OPLS-DA S-plot, and these compounds exhibited good activity against Trypanosoma brucei brucei with minimum inhibitory concentrations of 17.8 µM and 22.5 µM, respectively.

An LCMS method for the assay of melittin in cosmetic formulations containing bee venom.

There is a growing interest in the potential of bee venom in cosmetics as a rejuvenating agent. Products currently on the market do not specify exactly their content of bee venom (BV). Therefore, we developed a method for the detection and quantification of melittin, as a marker of bee venom content, in selected commercial creams which contained BV according to their marketing claims, in order to gauge the relative quality of such formulations. A quantitative method was achieved following a rigorous extraction procedure involving sonication, liquid-liquid extraction and solid phase extraction since carryover of excipients was found to cause a rapid deterioration in the chromatographic performance. The method employed a standard additions approach using, as spiking standard, purified melittin isolated from bee venom and standardised by quantitative NMR. The aqueous extracts of the spiked creams were analysed by reversed phase LCMS on an LTQ Orbitrap mass spectrometer. The purity of the melittin spiking standard was determined to be 96.0%. The lowest measured mean melittin content in the creams was 3.19 ppm (±1.58 ppm 95% CI) while the highest was 37.21 ppm (±2.01 ppm 95% CI). The method showed adequate linearity (R (2) ≥ 0.98) and a recovery of 87.7-102.2% from a spiked blank cream. An assay precision of <20% RSD was achieved for all but one sample where the RSD value was 27.5%. The method was sensitive enough for use in routine assay of BV-containing cosmetic creams. Differences in the melittin content of the commercial products assayed were nearly tenfold.

The isolation of antiprotozoal compounds from Libyan propolis.

Propolis is increasingly being explored as a source of biologically active compounds. Until now, there has been no study of Libyan propolis. Two samples were collected in North East Libya and tested for their activity against Trypanosoma brucei. Extracts from both samples had quite high activity. One of the samples was fractionated and yielded a number of active fractions. Three of the active fractions contained single compounds, which were found to be 13-epitorulosal, acetyl-13-epi-cupressic acid and 13-epi-cupressic acid, which have been described before in Mediterranean propolis. Two of the compounds had a minimum inhibitory concentration value of 1.56 µg/mL against T. brucei. The active fractions were also tested against macrophages infected with Leishmania donovani, and again moderate to strong activity was observed with the compounds having IC50 values in the range 5.1-21.9 µg/mL.

Identification and development of the 1,4-benzodiazepin-2-one and quinazoline-2,4-dione scaffolds as submicromolar inhibitors of HAT.

A library of 1,4-benzodiazepines has been synthesised and evaluated for activity against Trypanosoma brucei, a causative parasite of Human African Trypanosomiasis (HAT). The most potent of these derivatives has an MIC value of 0.97 µM. Herein we report the design, synthesis and biological evaluation of the abovementioned compounds.

Synthesis and biological evaluation of 1,4-benzodiazepin-2-ones with antitrypanosomal activity.

A library of 1,4-benzodiazepines has been synthesized and evaluated against Trypanosoma brucei, a causative parasite of Human African trypanosomiasis. Benzodiazepines possessing a P2- transporter motif were found to have MIC values as low as 0.78 µM.

Metabolomic Profiling of the Synergistic Effects of Melittin in Combination with Cisplatin on Ovarian Cancer Cells.

Melittin, the main peptide present in bee venom, has been proposed as having potential for anticancer therapy; the addition of melittin to cisplatin, a first line treatment for ovarian cancer, may increase the therapeutic response in cancer treatment via synergy, resulting in improved tolerability, reduced relapse, and decreased drug resistance. Thus, this study was designed to compare the metabolomic effects of melittin in combination with cisplatin in cisplatin-sensitive (A2780) and resistant (A2780CR) ovarian cancer cells. Liquid chromatography (LC) coupled with mass spectrometry (MS) was applied to identify metabolic changes in A2780 (combination treatment 5 µg/mL melittin + 2 µg/mL cisplatin) and A2780CR (combination treatment 2 µg/mL melittin + 10 µg/mL cisplatin) cells. Principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate data analysis models were produced using SIMCA-P software. All models displayed good separation between experimental groups and high-quality goodness of fit (R²) and goodness of prediction (Q²), respectively. The combination treatment induced significant changes in both cell lines involving reduction in the levels of metabolites in the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, purine and pyrimidine metabolism, and the arginine/proline pathway. The combination of melittin with cisplatin that targets these pathways had a synergistic effect. The melittin-cisplatin combination had a stronger effect on the A2780 cell line in comparison with the A2780CR cell line. The metabolic effects of melittin and cisplatin in combination were very different from those of each agent alone.

The Chemical Characterization of Nigerian Propolis samples and Their Activity Against Trypanosoma brucei.

Profiling of extracts from twelve propolis samples collected from eight regions in Nigeria was carried out using high performance liquid chromatography (LC) coupled with evaporative light scattering (ELSD), ultraviolet detection (UV) and mass spectrometry (MS), gas chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). Principal component analysis (PCA) of the processed LC-MS data demonstrated the varying chemical composition of the samples. Most of the samples were active against Trypanosoma b. brucei with the highest activity being in the samples from Southern Nigeria. The more active samples were fractionated in order to isolate the component(s) responsible for their activity using medium pressure liquid chromatography (MPLC). Three xanthones, 1,3,7-trihydroxy-2,8-di-(3-methylbut-2-enyl)xanthone, 1,3,7-trihydroxy-4,8-di-(3-methylbut-2-enyl)xanthone a previously undescribed xanthone and three triterpenes: ambonic acid, mangiferonic acid and a mixture of α-amyrin with mangiferonic acid (1:3) were isolated and characterised by NMR and LC-MS. These compounds all displayed strong inhibitory activity against T.b. brucei but none of them had higher activity than the crude extracts. Partial least squares (PLS) modelling of the anti-trypanosomal activity of the sample extracts using the LC-MS data indicated that high activity in the extracts, as judged from LCMS2 data, could be correlated to denticulatain isomers in the extracts.

Effect of Bee Venom and Its Fractions on the Release of Pro-Inflammatory Cytokines in PMA-Differentiated U937 Cells Co-Stimulated with LPS.

The venom of Apis mellifera (honey bee) has been reported to play a role in immunotherapy, but existing evidence to support its immuno-modulatory claims is insufficient. Four fractions from whole bee venom (BV) were separated using medium pressure liquid chromatography. Their ability to induce the production of cytokines TNFα, IL-1ß and IL-6 in phorbol-12-myristate-13-acetate (PMA)-treated U937 cells was assessed. The levels of the three cytokines produced by stimulation with the four fractions and crude BV without LPS were not significantly different from negative control values. However, co-stimulation of the cells with LPS and Fraction 4 (F-4) induced a 1.6-fold increase in TNF-α level (p < 0.05) compared to LPS alone. Likewise, LPS-induced IL-1ß production was significantly synergised in the presence of F-1 (nine-fold), F-2 (six-fold), F-3 (four-fold) and F-4 (two-fold) fractions, but was only slightly enhanced with crude BV (1.5-fold) relative to LPS. Furthermore, the LPS-stimulated production of IL-6 was not significantly increased in cells co-treated with F-2 and F-3, but the organic fraction (F-4) showed an inhibitory effect (p < 0.05) on IL-6 production. The latter was elucidated by NMR spectroscopy and found to contain(Z)-9-eicosen-1-ol. The effects observed with the purified BV fractions were more marked than those obtained with the crude sample.

Metabolomic Profiling of the Effects of Melittin on Cisplatin Resistant and Cisplatin Sensitive Ovarian Cancer Cells Using Mass Spectrometry and Biolog Microarray Technology.

In the present study, liquid chromatography-mass spectrometry (LC-MS) was employed to characterise the metabolic profiles of two human ovarian cancer cell lines A2780 (cisplatin-sensitive) and A2780CR (cisplatin-resistant) in response to their exposure to melittin, a cytotoxic peptide from bee venom. In addition, the metabolomics data were supported by application of Biolog microarray technology to examine the utilisation of carbon sources by the two cell lines. Data extraction with MZmine 2.14 and database searching were applied to provide metabolite lists. Principal component analysis (PCA) gave clear separation between the cisplatin-sensitive and resistant strains and their respective controls. The cisplatin-resistant cells were slightly more sensitive to melittin than the sensitive cells with IC50 values of 4.5 and 6.8 µg/mL respectively, although the latter cell line exhibited the greatest metabolic perturbation upon treatment. The changes induced by melittin in the cisplatin-sensitive cells led mostly to reduced levels of amino acids in the proline/glutamine/arginine pathway, as well as to decreased levels of carnitines, polyamines, adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+). The effects on energy metabolism were supported by the data from the Biolog assays. The lipid compositions of the two cell lines were quite different with the A2780 cells having higher levels of several ether lipids than the A2780CR cells. Melittin also had some effect on the lipid composition of the cells. Overall, this study suggests that melittin might have some potential as an adjuvant therapy in cancer treatment.

Chemical and Antimicrobial Profiling of Propolis from Different Regions within Libya.

Extracts from twelve samples of propolis collected from different regions of Libya were tested for their activity against Trypanosoma brucei, Leishmania donovani, Plasmodium falciparum, Crithidia fasciculata and Mycobacterium marinum and the cytotoxicity of the extracts was tested against mammalian cells. All the extracts were active to some degree against all of the protozoa and the mycobacterium, exhibiting a range of EC50 values between 1.65 and 53.6 µg/ml. The toxicity against mammalian cell lines was only moderate; the most active extract against the protozoan species, P2, displayed an IC50 value of 53.2 µg/ml. The extracts were profiled by using liquid chromatography coupled to high resolution mass spectrometry. The data sets were extracted using m/z Mine and the accurate masses of the features extracted were searched against the Dictionary of Natural Products (DNP). A principal component analysis (PCA) model was constructed which, in combination with hierarchical cluster analysis (HCA), divided the samples into five groups. The outlying groups had different sets of dominant compounds in the extracts, which could be characterised by their elemental composition. Orthogonal partial least squares (OPLS) analysis was used to link the activity of each extract against the different micro-organisms to particular components in the extracts.

Infectivity of macrophages and the histopathology of cutaneous lesions, liver and spleen is attenuated by leaf extract of Vernonia amygdalina in Leishmania major infected BALB/c mice.

Preliminary investigation of the in vitro and in vivo efficacies of different extracts from the leaves of Vernonia amygdalina (VA), a plant widely used in Nigeria was evaluated in Balb/C mice infected with a laboratory strain of Leishmania major (L. major). The ability of the methanol, hexane and aqueous extracts of the plant to suppress the infection rate and its cytotoxicity on macrophages and L929 cells were determined in the in vitro study. The in vivo study evaluated time course of infection, lesion progression and the histopathology of cutaneous lesions, liver and spleen after inoculation with metacyclic promastigotes. Methanolic extract of VA containing high levels of flavanoids, was the most potent extract as it showed the highest suppression on infectivity and viability of intracellular amastigotes at a concentration lower than that which elicited cytotoxicity on macrophages. Treatment of infected mice with methanolic extract of VA showed delayed onset of disease with a significant reduction in lesion size and attenuation of the histopathological outcome characterised by intact epidermis and less tissue destruction in skin, spleen and liver. In conclusion, these results demonstrate that VA has potent antileishmanial properties which warrants further investigation into the immunological mechanism.

Side-on binding of p-sulphonatocalix[4]arene to the dinuclear platinum complex trans-[{PtCl(NH3)2}2mu-dpzm]2+ and its implications for anticancer drug delivery.

The utility of p-sulphonatocalix[4]arene (s-CX[4]) as a drug delivery vehicle for multinuclear platinum anticancer agents, using trans-[{PtCl(NH(3))(2)}(2)mu-dpzm](2+) (di-Pt; where dpzm=4,4'-dipyrazolylmethane) as a model complex, has been examined using (1)H nuclear magnetic resonance, electrospray ionisation mass spectrometry, molecular modelling and in vitro growth inhibition assays. s-CX[4] binds di-Pt in a side-on fashion in a ratio of 1:1, with the dpzm ligand of the metal complex located within the s-CX[4] cavity with binding further stabilised by ion-ion interactions and hydrogen bonding between the metal complex am(m)ine groups and the s-CX[4] sulphate groups. Partial encapsulation of di-Pt within the cavity does not prevent binding of 5'-guanosine monophosphate to the metal complex. When bound to two individual guanosine molecules, di-Pt also remains partially bound by s-CX[4]. The cytotoxicity of free di-Pt and s-CX[4] and their host guest complex was examined using in vitro growth inhibition assays in the A2780 and A2780cis human ovarian cancer cell lines. Free di-Pt has an IC(50) of 100 and 60 microM, respectively, in the cell lines, which is significantly less active than cisplatin (1.9 and 8.1 microM, respectively). s-CX[4] displays no cytotoxicity at concentrations up to 1.5mM and does not affect the cytotoxicity of di-Pt, probably because its low binding constant to the metal complex (6.8 x 10(4)M(-1)) means the host-guest complex is mostly disassociated at biologically relevant concentrations.