Bioassay-guided isolation and characterization of antibacterial compounds from Calotropis procera (Ait.)

Infectious diseases have been the leading cause of human mortality in the past and yet are challenging public health concerns worldwide. Thus, there is a huge demand for exploring new and effective antimicrobial leads from all sources to combat the infectious diseases. The medicinal plant Calotropis procera has been traditionally used in the treatment of various ailments, including infectious diseases. Although a wide array of biological activities and rich phytochemistry of C. procera are reported, a systematic investigation to isolate antibacterial compounds of the plant has not been reported so far. Considering the pressing need for new antibacterial leads, present bioassay-guided study was designed to explore antibacterial compounds of C. procera twigs, leaves, and flowers. The plant materials collected from the Dungun district of Terengganu, Malaysia, were extracted with n-hexane, dichloromethane, and methanol, applying a successive extraction method to prepare nine crude extracts. The obtained extracts were tested for their in-vitro antibacterial activity against a panel of ten Gram-positive and ten Gram-negative bacteria, applying the agar well diffusion (AWD) method. The methanolic extracts of leaves (LM) and flowers (FM) showing broader antibacterial effects were selected for further bioassay-guided studies applying different chromatographic techniques. The LM and FM yielded a total of eighteen compounds (1–18), which were elucidated by using different spectroscopic and spectrometric approaches, e.g., UV, IR, LCMS, 1D, and 2D NMR techniques along with a literature survey. The isolated compounds include seven flavonoids [kaempferol 3-O-robinoside (1), kaempferol 3-O-rutinoside (2), 8-bromoisorhamnetin 3-O-rutinoside (8), 8-chloroisorhamnetin 3-O-rutinoside (10), isorhamnetin 3-O-rutinoside (11), kaempferol 3-O-β-D-glucopyranoside (12), and quercetin (18)], seven cardenolides [calotoxin (3), frugoside (4), 15β-hydroxycalotropagenin (5), 12β-hydroxycalotropagenin (6), 19-carboxyl-2α,15β-dihydroxyuzarigenin (7), 12β-hydroxycoroglaucigenin (9), and calotropagenin (16)], two lignans [(+)-pinoresinol 4-O-(6ʹʹ-O-para-hydroxybenzoyl)-β-D-glucopyranoside (13) and (+)-pinoresinol 4-O-(6ʹʹ-O-vanillyl)-β-D-glucopyranoside (14)], and two benzene derivatives [benzyl β-primeveroside (15) and protocatechuic acid (17)]. All of the compounds were tested against Bacillus cereus, Staphylococcus epidermidis, and Proteus vulgaris at dose of 30 µg/well using the AWD method. The compounds (except 12) showed varied antibacterial potential (ZOI: 7–17.5 mm) against the tested bacteria. Conclusively, the present study justified the ethnopharmacological value of C. procera as a traditional remedy for infectious diseases. The study also pointed out that the antibacterial metabolites were largely accumulated in both the leaves and flowers compared to stems of the plant. Amongst the eighteen isolated phytochemicals, five new compounds (6–8, 10, and 13) showed strong antibacterial effects (ZOI of 11.5–17.5 mm) and were isolated for the first time from nature. Meanwhile, two known compounds (15 and 17) showing ZOI of 11–13 mm were reported for the first time from the genus Calotropis, whereas 5 producing ZOI of 12–17 mm against the tested G+ve bacteria and 12 (inactive in this study) were new for C. procera. Overall, the three isolated cardenolides (5, 7, and 9), one flavonoid (10), and two lignans (13 and 14) were among the potent compounds (ZOI: up to 17.5 mm), while S. epidermidis was the most sensitive strain. The three cardenolide aglycones (5, 7, and 9), two halogenated flavonoids (8 and 10), and the two lignans (13 and 14) could be emphasized for further antibacterial drug discovery research.