Josphat C. Matasyoh

Endophytes as Producers of Peptides: An Overview About the Recently Discovered Peptides from Endophytic Microbes

An endophyte is a fungus or bacterium that lives within a plant in a symbiotic relationship. Extensive colonization of the plant tissue by endophytes creates a barrier effect, where they outcompete and prevent pathogenic organisms from taking hold. This happens by producing secondary metabolites that inhibit the growth of the competitors or pathogens. In this way they play a very important role in the plant defence mechanisms. The metabolites produced by these endophytes fall within a wide range of classes of compounds that include peptides which are the focus of this review. Peptides are increasingly being selected for drug development because they are specific for their targets and have a higher degree of interactions. There have been quite a number of endophytic peptides reported in the recent past indicating that endophytes can be used for the production of peptide based drugs. Molecular screening for NRPS, which shows peptide producing capability, has also shown that endophytes are potential producers of peptides. The presence of NRPS also offers the possibility of genetic modifications which may generate peptides with high pharmacological activities. This review, therefore, aims to show the current status of peptides isolated from endophytic bacteria and fungi in the recent decade. Endophytes as potential sources of peptides according to NRPS studies will also be discussed.

Laxitextines A and B, Cyathane Xylosides from the Tropical Fungus Laxitextum incrustatum

Bioassay-guided fractionation of the mycelial extract of a basidiomycete culture collected in Kenya led to the isolation of two new cyathane diterpenoids named laxitextines A (1) and B (2). The producer strain was characterized by detailed taxonomic studies based on rDNA using the 5.8S gene region, the internal transcribed spacer 2 (ITS2), and part of the large subunit that identified the fungus as Laxitextum incrustatum. The structures of 1 and 2 were elucidated by NMR spectroscopic and mass spectrometric analyses. Both compounds exhibited moderate activities against Gram-positive bacteria Bacillus subtilis (DSM 10), Staphylococcus aureus (DSM 346), and methicillin-resistant Staph. aureus (DSM 1182). The two compounds also showed variable antiproliferative activities against mouse fibroblast (L929) and selected human cell lines (breast cancer MCF-7, epidermoid carcinoma A431, and umbilical vein endothelial HUVEC). The IC50 values with respect to the MCF-7 cell line for compounds 1 and 2 were 2.3 and 2.0 μM, respectively.

Monochlorinated calocerins A-D and 9-oxostrobilurin derivatives from the basidiomycete Favolaschia calocera

Eight previously undescribed compounds were isolated and characterised from the supernatant and mycelium of a culture of the basidiomycete Favolaschia calocera originating from Kakamega equatorial rainforest in Kenya. These were: 9- oxostrobilurins A, G, K and I and the four monochlorinated calocerins A, B, C and D. The calocerins extend our knowledge of halogenated compounds obtained from natural sources. Four further known compounds were also identified: strobilurin G, favolon, pterulinic acid and 2,3 -dihydro-1-benzoxepin derivative. The four oxostrobilurins exhibited prominent antifungal and cytotoxic activities while the four calocerins only showed cytotoxic activity.

Synthesis and Spin Trapping Stereochemistry of the Chiral Spin Trap, 5,5-Dimethyl-3-phenylpyrroline-1-oxide

A chiral spin trap, 5,5‐dimethyl‐3‐phenylpyrroline‐1‐oxide (5), and several hydroxylamines were synthesized. The structures and conformations of these compounds were investigated mainly by 1H NMR spectroscopy. This spin trap was used to trap carbon‐ and oxygen‐centered radicals. The corresponding hydrogen spin adduct was prepared by oxidizing the hydroxylamine to the nitroxide radical. By the combination of 1H NMR and EPR results, it was shown that the conformation with the phenyl group of C‐3 at the equatorial position is exclusively populated and its lifetime is long compared with the EPR time‐scale. Addition of carbon‐centered radicals to 5 leads to trans adducts whereas oxygen‐centered radicals formed cis isomers. These could be confirmed by the investigation of the monovalent oxidation products of the hydroxylamines. The preference for these conformers is explained by the competition between steric and stereoelectronic effects. Despite the racemic nature of 5 and the formation of a new chiral center(s) in the spin adducts, the presence of different diastereomers would not be observed by EPR whereas 1H NMR studies of some of the nitrones showed clear evidence of diastereomeric mixtures. However, the spin adduct of sec‐hydroxybutyl radical showed some linewidth effects which could be attributed to the presence of two groups of diastereomers that were resolved by ENDOR spectroscopy. In general, the spin adducts of 5 are closely related to those of the well known DMPO, but the presence of a phenyl substituent at the 3‐position results in a variation of the β‐H coupling constants. In contrast to DMPO, 5 can scavenge short‐lived radicals in aqueous and non‐aqueous solutions.

Microporenic Acids A–G, Biofilm Inhibitors, and Antimicrobial Agents from the Basidiomycete Microporus Species

The need for effective compounds to combat antimicrobial resistance and biofilms which play important roles in human infections continues to pose a major health challenge. Seven previously undescribed acyclic diterpenes linked to isocitric acid by an ether linkage, microporenic acids A-G (1-7), were isolated from the cultures of a hitherto undescribed species of the genus Microporus (Polyporales, Basidiomycota) originating from Kenyas Kakamega forest. Microporenic acids D and E (4 and 5) showed antimicrobial activity against a panel of Gram positive bacteria and a yeast, Candida tenuis. Moreover, microporenic acids A and B (1 and 2) demonstrated dose-dependent inhibition of biofilm formation by Staphylococcus aureus. Compound 1 further showed significant activity against Candida albicans and Staphylococcus aureus preformed biofilms.

Anti-Trypanosomatid Elemanolide Sesquiterpene Lactones from Vernonia lasiopus O. Hoffm

Sleeping sickness or human African trypanosomiasis (HAT) is a neglected tropical disease (NTD) threatening millions of peoples’ lives with thousands infected. The disease is endemic in poorly developed regions of sub-Saharan Africa and is caused by the kinetoplastid “protozoan” parasite Trypanosoma brucei. The parasites are transmitted to humans through bites of infected tsetse flies of the genus Glossina. The few available drugs for treatment of this disease are highly toxic, difficult to administer, costly and unavailable to poor rural communities bearing the major burden of this infection. Therefore, the search for new efficacious, safe and affordable drugs is of high importance. Vernonia lasiopus O. Hoffm., an indigenous African plant of the Asteraceae family, has been extensively reported to be used ethno-medicinally as a treatment for malaria. Its crude extracts obtained with solvents of different polarity were screened in vitro for anti-protozoal activity and the dichloromethane extract was found to be particularly active against Trypanosoma brucei rhodesiense (IC50 = 0.17 µg/mL). Bioassay-guided chromatographic fractionation of the dichloromethane extract led to the isolation and identification of six elemanolide type sesquiterpene lactones: 8-desacylvernolide, vernolepin, vernomenin, vernodalol, vernodalin and 11,13-dihydrovernodalin. All these elemanolide sesquiterpene lactones showed in vitro anti-trypanosomal activity. They were also tested for cytotoxicity against mammalian cells (L6 cell line). Vernolepin, the main component in the extract, was also the most potent with an IC50 value of 0.05 µg/mL against T.b. rhodesiense trypomastigotes. This compound showed a selectivity index of 14.5, which makes it an interesting candidate for in vivo tests and determination of its mechanism of action.

SYNTHESIS AND STEREOCHEMISTRY OF THE SPIN ADDUCTS OF A NEW CHIRAL SPIN TRAP, 3,5-DIPHENYL-5-METHYLPYRROLINE-1-OXIDE

The synthesized spin trap 3,5‐diphenyl‐5‐methylpyrroline‐1‐oxide (3), its derivatives and spin adducts were studied using NMR, ESR and ENDOR spectroscopic methods. The structure of 3 was determined using x‐ray analysis. The nitrone 3 was found to have a rigid structure. Two species were observed in the EPR and ENDOR spectra of the spin adducts of 3, but not in the NMR spectra of its diamagnetic derivatives. These two species were attributed to those formed when the addends attack carbon‐2 from both the re and si sides, resulting in trans (I) and cis (II) adducts in relation to the phenyl substituent in position 3. Therefore, these two species differ only through the substitution on carbon‐2, whereby there are different steric interactions of the substituents within both species. The lack of observation of these two species in NMR spectra could be interpreted in terms of unfavourable relaxation times on the NMR time‐scale. These species are observed in EPR and ENDOR spectra mainly because of their different β‐C—H dihedral angles, which do not play a role in NMR spectroscopy. Both species have the ability to trap stereoselectively carbon‐ and oxygen‐centred radicals, which occupy pseudo‐equatorial and pseudo‐axial positions, respectively. The occupation of oxygen‐centred addends in the pseudo‐axial positions is stabilized by the ‘anomeric effects.’ This nitrone 3 was found to have the ability to scavenge short‐lived free radicals in both aqueous and non‐aqueous solutions.

Sesquiterpene Lactones from Vernonia cinerascens Sch. Bip. and Their in Vitro Antitrypanosomal Activity

In the endeavor to obtain new antitrypanosomal agents, particularly sesquiterpene lactones, from Kenyan plants of the family Asteraceae, Vernonia cinerascens Sch. Bip. was investigated. Bioactivity-guided fractionation and isolation in conjunction with LC/MS-based dereplication has led to the identification of vernodalol (1) and isolation of vernodalin (2), 11β,13-dihydrovernodalin (3), 11β,13-dihydrovernolide (4), vernolide (5), 11β,13-dihydrohydroxyvernolide (6), hydroxyvernolide (7), and a new germacrolide type sesquiterpene lactone vernocinerascolide (8) from the dichloromethane extract of V. cinerascens leaves. Compounds 3–8 were characterized by extensive analysis of their 1D and 2D NMR spectroscopic and HR/MS spectrometric data. All the compounds were evaluated for their in vitro biological activity against bloodstream forms of Trypanosoma brucei rhodesiense and for cytotoxicity against the mammalian cell line L6. Vernodalin (2) was the most active compound with an IC50 value of 0.16 µM and a selectivity index of 35. Its closely related congener 11β,13-dihydrovernodalin (3) registered an IC50 value of 1.1 µM and a selectivity index of 4.2.

Aethiopinolones A–E, New Pregnenolone Type Steroids from the East African Basidiomycete Fomitiporia aethiopica

A mycelial culture of the Kenyan basidiomycete Fomitiporia aethiopica was fermented on rice and the cultures were extracted with methanol. Subsequent HPLC profiling and preparative chromatography of its crude extract led to the isolation of five previously undescribed pregnenolone type triterpenes 1–5, for which we propose the trivial name aethiopinolones A–E. The chemical structures of the aethiopinolones were determined by extensive 1D- and 2D-NMR, and HRMS data analysis. The compounds exhibited moderate cytotoxic effects against various human cancer cell lines, but they were found devoid of significant nematicidal and antimicrobial activities.

Toxicity Studies on Anti-fungal Essential Oils Extracted from Selected Aromatic Plants from Mabira and Kakamega Forests, East Africa

Aim: In a bid to explore for grain bio-preservatives, essential oils extracted from Cymbopogon citratus, Rosmarinus officinalis, Monanthataxis littoralis and Aframomum angustifolium, that were earlier established to have anti-mold activity, were evaluated for their biosafety by determining the oral LD50. Methods: The essential oils were extracted by hydro-distillation from aromatic plants collected from Kakamega and Mabira forests in Kenya and Uganda, respectively. Acute oral toxicity was established using mice by determining the LD50; after which sub-acute toxicity studies were performed. The animals were observed for behavioural changes; and the gross and Original Research Article Nakavuma et al.; EJMP, 14(2): 1-14, 2016; Article no.EJMP.25185 2 histopathological effects, if any, on the intestinal mucosa, spleen, lungs, liver, kidney and heart were noted. Results: The oral LD50 for Cymbopogon citratus, Rosmarinus officinalis, Monanthataxis littoralis and Aframomum angustifolium essential oils were established as 7,046.90; 4,723.33; 13,335.82; and 17,539.82 (mg/kg body weight), respectively. In all cases, increased breathing rates were observed, however Rosemary also caused lethargy and convulsions. Grossly, no changes were seen in the liver, kidney, lungs, heart and spleen from both the control and the treated mice except for Monanthataxis littoralis where the lungs and liver seemed changed; and the urinary bladder distended. However, the latter effects were noted at higher doses than the established oral LD50. Histopathologically, thickened intestinal mucosa lining; tubular degeneration and proteinuria in the kidneys; vascular congestion, focal necrosis and hydropic degeneration of hepatocytes in the liver, were encountered. Conclusion: Basing on the oral LD50 in mice, all oils were safe and can be explored further as antimold grain preservatives. However, Rosmarinus officinalis was marginally safe as per the OECD guidelines. The histopathological effects of Monanthataxis littoralis essential oil need to be investigated further.