Phytochemical Investigation and In-Vitro Biological Activities evaluation of Selected Ethiopian Medicinal Plants and their Endophytes in Eastern Ethiopia

Abstract

The development of chemo-resistance by microbes, severe adverse effects associated with synthetic old antibiotics, unavailability of newer drugs, genetic variability of microbial strains and the re-emergence/emergence of opportunistic pathogens are among the challenges attributed to the crises of the current world medicine. This has led to the resurrection of the world research on natural sources, mainly medicinal plants and associated microbes (endophytes) to get possible non-toxic, affordable and effective novel natural drugs. This reseach project work intended to investigate phytochemical constituents, evaluate in vitro antibacterial (against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922), antifungal (against Candida albicans ATCC 10231) and antioxidant (via DPPH free radical and FRAP assays) activities of Gomphocarpus purpurascens (leaves), Gloriosa superba (tubers), Cadia purpurea (roots and leaves) and Caralluma speciosa (stems). These plants are among the top cited and ethnomedicinally valued plants found in the prehistoric Harla town and its surroundings, Dire Dawa, Ethiopia. We also proposed to conduct similar investigation on Portulaca oleracea, Rhynchosia erlangeri and Leucas stachydiformis. However, it was difficult to collect samples sufficient for the proposed study after even though repeatedly vistited their poposed habitat. Thus, investigation of these plants were excluded and replaced by some other medicinal plants. The results of those replaced plants which were published and shown in the list of publications of this reseach project. In this study, we also isolated possible endophytic bacteria and associated secondary metabolite from Gloriosa superba (stems, leaves and tubers). Themolecular binding capacity of the isolated compounds 65-69 was assessed against E. coli DNA gyraseB (PDB ID: 6F86), S. aureus pyruvate kinase (PDB ID: 3T07) and human peroxiredoxin 5 (PDB ID: 1HD2) enzymes using AutoDock vina version 4.2 software. The compounds 65-68, 70 and 79 were also docked against P. aeruginosa PqsA (5OE3) enzyme. The phytochemical investigation resulted in isolation of 17 compounds; six (65-70) from C. purpurea (65 and 66 from leaves, 68-70 from roots and 67 from both), five (71-75) from C. speciosa (stems), three (76-78) from G. superba (tubers), one (79) from G. purpurascens (leaves) and another one (80) from ethyl acetate extract of Escherichia coli (GST5) endophytic bacterium. The structures of these isolated compounds were established based on FT-IR, GC-MS and NMR analysis and comparison with reported data. Compound 65, the epoxy-lignan (68) and bis-(2-hydroxybutyl) phthalate (69) were found as new compounds. Based on the biochemical and MALDI-TOF MS analyses, thirty endophytic bacteria were isolated from leaves, stems and tubers of G. superba and classified into Bacillus spp. (66.7%), Escherichia spp. (26.7%), Providencia spp. (3.3%) and Corynebacterium spp. (3.3%). Calpurnine (67) and apigenin-7-O-neohesperidoside (66) displayed a better antibacterial activity against E. coli (18.5 ± 0.02 mm and 12.1 ± 0.1 mm, respectively) and P. aeruginosa (10.6 ± 0.01 mm and 14.5 ± 0.01 mm, respectively) at a maximum concentration (1000 μg/mL). All extracts and isolated compounds of G. superba, C. purpurea and G. purpurascens displayed better antibacterial activity against P. aeruginosa than chloramphenicol (7.2 ± 0.6 to 8.2 ± 0.6 mm) almost at all concentrations. The chloroform: methanol (1:1) stems extract of C. speciosa was found to give a promising antifungal activity against C. albicans with higher diameter of inhibition zone (17.17 ± 1.04 mm) recorded at the higher concentration (100, 000 μg/mL), compared to Ketoconazole (17.67 ± 2.52 at 10 μg/disc). The antioxidant activity data indicated that apigenin-7-O-neohesperidoside (66) and calpurnine (67) provided a good DPPH radical scavenging potential with IC50 values of 6.21 and 8.67 μg/mL, respectively, comparable to ascorbic acid (4.82 μg/mL) at similar concentrations. All the extracts of G. superba tubers and G. purpurascens leaves also exhibited a strong DPPH scavenging activity with IC50 values of 1.1-3.2 μg/mL equivalent to ascorbic acid (1.0-1.3 μg/mL). The molecular docking study revealed that compound 65, calpurnine (67) and compound 68 showed stronger binding affinity (-6.5, -7.4 and -7.3 kcal/mol, respectively) to 6F86 enzyme than chloramphenicol (-6.4 kcal/mol). Whereas all the compounds 65-69 showed a better binding energy to 3T07 and 1HD2 than chloramphenicol (-4.6 kcal/mol) and ascorbic acid (-4.5 kcal/mol), respectively. The compounds 65, 66, 67, 68 and 79 also recorded higher docking scores (-7.9, -10.9, -8.1, -7.8 and -10.3 kcal/mol, respectively) against 5OE3 than chloramphenicol (-7.0 kcal/mol). Apigenin-7-O-neohesperidoside (66) was found violated three rules of the Lipinski’s rule of five (with molecular weight > 500 g/mol, hydrogen-acceptor > 10 and hydrogen-donor > 5). Compound 68, bis-(2-methylheptyl)phthalate (70) and calotropin (79) were also found violated, each, one rule of the Lipinski’s rule of five (with hydrogen-donor > 5, LogP > 4.15 and molecular weight > 500 g/mol), respectively. Apigenin-7-O-neohesperidoside (66) and calotropin (79) were found as immunotoxic and mutagenic; and immunotoxic and cytotoxic isolates, respectively. Compounds 65 and 68 were also found as immunotoxic and mutagenic isolates, respectively. This finding provided an assertion to the possibility of exploration of endophytic bacteria and associated chemical constituents from their host plants in the context of Ethiopia as it was reported herein for the first time; and this would be a basis for similar works to be done in the future. Finally, it is recommended to do further instrumental analysis including the 2D-NMR and HRMS to fully confirm the structures of the newly isolated compounds 65, 68 and 69. The present study also recommends further molecular characterization of the isolated endophytic bacteria using 16S rRNA gene sequencing to identify them in strain level. Further extensive biochemical investigations would also be needed on the aforementioned plant species, by giving a special emphasis to the G. superba and G. purpurascens plants, to explore additional bioactive phytochemicals and endophytic microbes.