Isolation and characterization of plant bioactive compounds and assessment of their use in veterinary science

The research activity of the PhD project in Veterinary Science was aimed at the isolation and structural characterization of secondary metabolites from medicinal plants and the evaluation of their use in veterinary science. The selected plants are species used in folk medicine or in the veterinary medicine, particularly plants used as antiparasitic and antimicrobial The chemical-biological investigation was carried out on the following plants: • Psiadia punctulata (DC.) Vatke of the Asteraceae family it is found in tropical arid areas from the grasslands and shrubs in arid pastures to the edge of the forest, South-Eastern Africa, and Middle East, some plants of this family are widely used as antimicrobial; • Hypoestes forskaolii (Vahl) Roem. & Schult, herbaceous plant belonging to the Acanthaceae family, widely distributed throughout the southern region of Saudi Arabia, some plants of this family are widely used as a natural anthelmintic; • Trichilia maynasiana C.DC belonging to the family of the Meliaceae is a tree widely distributed in tropical areas of South Africa used in folk medicine to treat various anti-inflammatory diseases; • Vernonia nigritiana Oliv. & Hiern. (Asteraceae) is an herbaceous plant or shrub widely distributed in West Africa, the leaves and other parts of the plant are traditionally used against skin inflammations, rheumatism, fever, headache and impairment. In veterinary medicine many plants of Vernonia genus are used for bacterial infections and intestinal worms. The isolation of secondary metabolites was achieved by chromatographic techniques such as column chromatography, thin layer chromatography, HPLC (High Pressure Liquid Chromatography). The structural characterization of all the isolates was performed by spectroscopic techniques, nuclear magnetic resonance experiments monodimensional (1H-NMR, 13C-NMR) and two-dimensional (COSY, HSQC, HMBC, ROESY), and mass spectrometry. The investigation of Psiadia punctulata led to the isolation of 30 compounds including diterpenes and flavones, of which 13 were new natural compounds. The antimicrobial activity of the dicloromethan extract was tested against Gram-positive bacteria (Staphylococcus aureus), Gram-negative (Pseudomonas aeruginosa) and dimorphic fungus Candida albicans. The results of this study highlighted that the dicloromethan extract showed antibacterial activity against S. aureus (MIC50 = 100 ug/mL), whereas no activity was detected against Gram-negative bacteria. Moreover, the extract showed antifungal activity against Candida albicans (MIC2 = 50 μg/mL and MIC0 = 130 μg/mL). The nine fractions A-I obtained after a silica gel column separation were tested for their antimicrobial activity at doses ranged from 20 to 100 μg/ml. The A,B and I fractions were found to be inactive on all species, instead six fractions (C-H) showed a remarkable activity. All the compounds isolated from active fractions were tested for their antimicrobial activity. Compound 3, 3',4',5,7-tetramethoxyflavone, at a concentration of 40 g/mL was able to inhibit by 50% the production of biofilm of the S. aureus and 90% for Candida albigans. The phytochemical investigation of H. forskaolii leaves led to the isolation of 13 compounds belonging to the class of diterpenes fusicoccane and isopimarane, among these 4 of fusicoccane diterpenes are new natural compounds. The n-hexane, chloroform, chloroform: methanol 9:1 and methanolic extracts of Hypoestes forskaolii were tested fot in vitro anthelmintic activity via Egg hacth Assay on different species of gastrointestinal netamathodes that naturally parasitize small ruminants. Polar extracts have not been able to show significant ovicidal activity (less than 50% of hatching), while the n-hexane extract at a concentration of 1 mg/ml showed moderate inhibition of 30% hatching. All the isolated compounds were also tested in vitro for the antiproliferative activity on HeLa and Jurkat U973 cell lines, all compounds did not significantly affect growth and cell viability. The phytochemical investigation of Trichilia maynasiana led to the isolation of nine compounds of which two are new natural compounds. These compounds belonging to the class of tirucallane and secoguaiane terpenes. The phytochemical study of V. nigritiana led to the isolation of seven secondary metabolites including two sesquiterpene lactones, never described before.