Phytochemical profiling, salt impurities removal and in vitro antibacterial evaluation of Calotropis procera twig, leaf and flower extracts

Abstract

• Hexane, dichloromethane and methanol leaf, twig, and flower extracts of Malaysian Calotropis procera (CP) have been studied first time against 10 Gram-positive and 9 Gram-negative bacteria, and methanolic extracts were found to be potent. • Different in vitro antibacterial methods were carried out and Bacillus sp. , Staphylococcus spp. Klebsiella pneumoniae and proteus vulgaris were found to be sensitives against extracts. • An easy, low-cost, and efficient trituration method was developed for the purification of crude extracts from salt impurities and it has removed salts and other undissolved impurities (approximately 38%). This purification strategy is very important prior to any biological activities especially antimicrobial. • Trituration method was found to be suitable for removing salts and undissolved impurities from crude extracts of plants growing near coastal and high salt concentration regions. • A new HPLC method was developed for the separation of constituents of methanolic C. procera extract and it shows the presence of different types of metabolites. Calotropis procera (family Apocynaceae) is a medicinal plant that is useful against microbial infections. The current work aimed to profile Malaysian C. procera for its phytochemical composition, develop a simple strategy to remove salt impurities from its crude extracts and assess its antibacterial activity in vitro . Ground twigs, leaves and flowers of the plant were extracted in n -hexane, dichloromethane and methanol by successive cold maceration method. Standard qualitative phytochemical tests were applied for phytochemical profiling of the extracts. The crude extracts were then subjected to clean-up procedure (trituration method) for removal of salt impurities. The antibacterial activity of the extracts (3 mg of 100 mg/mL/well) was assessed against 10 Gram-negative and nine Gram-positive bacteria by using the agar well diffusion method. Both minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the potent extracts were determined by the MTT assay and spread plate method, respectively. The potent methanolic extract was analysed by the HPLC for its metabolites pattern. The present findings confirmed the presence of flavonoids, terpenoids, steroids, phenolics, carbohydrates and resins in the crude extracts. The trituration method was effective in eliminating the salt impurities from the crude extracts. The methanolic (MeOH) extracts were more effective (ZOI; 8.5–12.5 mm) against Gram-positive bacteria when compared to the Gram-negative counterparts. The tested bacteria that were most sensitive to the extracts included Bacillus sp., Staphylococcus spp. Klebsiella pneumoniae and Proteus vulgaris . The MIC and MBC values of the MeOH extracts were in the range of 5–40 mg/mL against B. cereus, Staphylococcus spp., K. pneumoniae and P. vulgaris . The antibacterial potential of MeOH C. procera extracts could be attributed to its polar phytochemicals as indicated by the HPLC analyses. The current results warrant in-depth phytochemical and antimicrobial investigations of methanolic C. procera twig, leaf and flower extracts.