Research of antimicrobial potentialities of an endemic plant of the genus -Calycotome-

Abstract

The main objective of this research is the phytochemical characterization, the quantification of phenolic compounds, as well as the evaluation of the antioxidant and the antimicrobial potentials of certain extracts and fractions of leaves and flowers from the endemic plant Calycotome spinosa (L.) Link. Besides, the purification, the structural identification, and the investigation of the antioxidant and the antimicrobial capacities of isolated bioactive compounds have also been elucidated. As a result, many interesting chemical groups have been detected in this plant such as polyphenols, flavonoids, alkaloids, and sugars. The extracts (MeOH and aqueous) and the fractions (CHCl3, EtOAc, and n-BuOH) of the leaves and the flowers have high total polyphenols contents (TPC) compared to their flavonoids contents. Indeed, the EtOAc fraction of leaves exhibited the highest amount of TPC (107.75 ± 2.09 mg GAE / g DE), followed by the MeOH extract of the same part (98.72 ± 2.47 mg GAE / g DE), however, the MeOH extract of flowers had the lowest amount of these compounds (24.63 ± 0.35 mg GAE / g DE). Besides, it was shown that the same extract and the same fraction of leaves revealed the strongest free radical scavenging activity (DPPH; IC50 = 45.25 ± 1.8 and 41.04 ± 0.15 µg / mL, respectively), in addition to a remarkable reducing power (FRAP; EC50 = 763.73 ± 0.32 and 780.04 ± 1.36 µg / mL, consecutively), due to their, relatively, good content in phenolic compounds compared to the other tested extracts. The previous biological activity was accompanied by antimicrobial activity. Therefore, the diverse extracts displayed excellent antimicrobial activity, where the Gram+ test bacteria were more sensitive to these extracts than the Gram- ones. Thus, S. aureus was more sensitive to the MeOH extract of leaves with an inhibition zone (IZ) of 20.00 ± 0.28 mm, in contrast, bacterial strains ; B. subtilis and S. abony showed sensitivities to the MeOH extract and the EtOAc fraction of leaves with IZs of (16.00 ± 0.50 and 13 ± 0.65 mm) and (12 ± 0.29 and 16.00 ± 1.53 mm), respectively. the leaves’ MeOH extract was, furthermore, bactericidal against B. subtilis, S. aureus and S. abony with MICs ≤ 0.125 mg/ disc and a MBC of 1.00 mg/disc, whereas, Gram- bacteria; E. coli, P. aeruginosa, and K. pneumoniae were less susceptible. Moreover, two phytopathogenic fungal species of Alternaria were sensitive to all the tested extracts (MeOH and Aqueous) with variable inhibition percentages (IP %), however, no antifungal activity was observed against the yeast ‘Candida albicans’ and the other four tested fungal isolates; Penicillium sp.1, Penicillium sp.2, Aspergillus sp., and Rhizopus sp. From all these results, it was concluded that the leaves MeOH extract was the most efficient; therefore, it was selected for LC-ESI-MS2 analysis and for the separation of bioactive molecules. This separation using column chromatography(CC) led to a very interesting result; it is in fact the highlight, for the first time, of two new molecules from the plant, subject of study; 5-hydroxyindoline (4) and D-Pinitol (5), together with three well-known glucosidic flavonoids; Chrysin-7-O-(β-D-glucopyranoside) (1), chrysin-7-O-β-D-(6″-acetyl) glucopyranoside (2), and apigenin-7-O-β-D-glucopyranoside (3). The chemical structures of these isolated compounds have been elucidated based on spectroscopic analysis data and mass spectrometry; comprising a new approach 1D-NMR, 2D-NMR with LIT-ESI-MSn. The new compound, 5- Hydroxyindoline (4), demonstrated a very strong antibacterial potential against S. aureus (16 ± 0.5 mm), P. aeruginosa (9.83 ± 0.29 mm), and S. abony (8 ± 0.28 mm), as well it revealed a pertinent antioxidant capacity using four different methods (DPPH : IC50 < 10 μg / mL ; TAC = 985.54 ± 0.13 mg AAE / g DE ; FRAP : EC50 = 344.82 ± 0.02 μg / mL ; ABTS: IC50 = 7.8 ± 0.43 μg / mL), followed by the compound apigenin-7-O-β-D-glucopyranoside (3) which, also, exhibited remarkable antioxidant power, but lower than that of the new compound (4). These interesting results confirm that the aerial part of C. spinosa (L.) Link, in particular the leaves, usually used in traditional medicine in Algeria, can be considered as a source of substances with very effective antioxidant and antimicrobial activities.