Biological and phytochemical analysis of three medicinally important plants: Prunus persica, Diospyros ianceifolia and Holboellia latifolia

The plants Prunus persica, Holboellia latifolia and Diospyros lanceifolia have been used as traditional medicines by several Indigenous communities. This includes the use of the roots of P. persica and leaves of H. latifolia and D. lanceifolia for treatment of skin infections and other ailments of a likely microbial origin. These plant parts have limited reported investigations on their phytochemistry and/or antimicrobial properties. Screening of the 70% aqueous ethanolic extracts of the roots of P. persica and leaves of H. latifolia and D. lanceifolia using the 3 - (4,5 - dimethyl dthiazol - 2 - yl) - 2,5 - diphenyltetrazolium bromide MTT microdilution assay showed promising activity against the human pathogens Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes and Salmonella typhimurium. The highest inhibitory activities were exhibited by the P. persica root extract, with MIC (minimum inhibitory concentration) values of 156 μg/mL for antibiotic susceptible S. aureus, methicillin resistant S. aureus (MRSA) and multidrug resistant S. aureus (MDRSA). The leaf extract of D. lanceifolia had MIC values of 156 μg/mL for S. aureus and P. aeruginosa, 625 μg/mL for MRSA and MDRSA, and 312 μg/mL for antibiotic susceptible E. coli. The leaf extract of H. latifolia had MIC values of 156 μg/mL for the susceptible S. aureus, 1250 μg/mL for MRSA and MDRSA and 625 μg/mL for S. typhimurium. The dried 70% aqueous ethanolic extracts of P. persica, H. latifolia and D. lanceifolia were partitioned against water and sequentially n - hexane, dichloromethane and ethyl acetate. The strongest antibacterial activity of the partitioned extracts was observed for the ethyl acetate partition of P. persica with MIC 312 μg/mL (susceptible as well as resistant strains of S. aureus) and n - hexane partition of P. persica (MIC 625 μg/mL for susceptible S. aureus, and 312 μg/mL for MRSA, MDRSA, susceptible E. coli and P. aeruginosa strains ). The n - hexane partition of D. lanceifolia showed antibacterial activity against susceptible S. aureus and P. aeruginosa with MIC s of 156 μg/mL, antibiotic sensitive E. coli , MRSA and MDRSA with MIC s of 312 μg/mL, and S. typhimurium with MIC 625 μg/mL. The dichloromethane partition of the H. latifolia extract showed activity against antibiotic sensitive as well as resistant strains of S. aureus (156 μg/mL for S. aureus and 1250 μg/mL for both, MRSA and MDRSA) as well as against P. aeruginosa (MIC 1250 μg/mL). The n - hexane and ethyl acetate partitions of P. persica were subjected to TLC bioautography guided isolation by normal phase chromatography, size exclusion chromatography and preparative TLC. This led to the isolation of α - cyanobenzyl benzoate, β - sitosterol and stigmast - 4 - en - 3 - one from the n - hexane partition and gallic acid and caffeic acid phenylethyl ester from the ethyl acetate partition. α - Cyanobenzyl benzoate, assayed by TLC bioautography (MIQ - minimum inhibitory quantity) and MTT microdilution tests (MIC), showed excellent antimicrobial properties (MIQ / M IC 78 μg /mL) against all tested S. aureus strains, MIQ /M IC of 312 μg/mL against β lac - E. coli , and MIQ /M IC of 612 μg /mL against P. aeruginosa . Gallic acid showed MIQ/M IC value s of 156 μg/mL against antibiotic susceptible S. aureus, MIQ/M IC of 312 μg/mL against MRSA and MIQ/M IC of 2500 μg/mL against β lac - E. coli as well as P. aeruginosa. Caffeic acid phenylethyl ester and β - sitosterol showed activity against MRSA and MDRSA (MIQ /M IC 625 μg /mL ), and activity against antibiotic sensitive S. aureus (MIQ/M IC 312 μg/mL) . Additionally, both compounds showed antibacterial activity against susceptible strains of E. coli , with a MIQ/MIC values for β - sitosterol of 2500 μg/mL and for caffeic acid phenylethyl ester 312 μg/mL. Stigmast - 4 - en - 3 - one was active against S. aureus, MRSA and β lac - E. coli with MIQ/M IC values of 2500 μg/mL, as well as P. aeruginosa with MIQ/M IC values of 1250 μg/mL. This is the first report of isolation of caffeic acid phenylethyl ester and stigmast - 4 - en - 3 - one from P. persica plant material, the second report of α - cyanobenzyl benzoate being present in the Prunus genus and the first report of antibacterial activity of stigmast - 4 - en - 3 - one (this compound was previously reported not active). TLC bioautography guided isolation of the n - hexane partition of D. lanceifolia led to the isolation of lupeol and plumbagin, whose structures were confirmed by spectral analyses. Lupeol showed antimicrobial activity by TLC bioautography against P. aeruginosa (MI Q 156 μg /mL) and E. coli (MI Q 312 μg /mL), against S. aureus and MRSA (MI Q 2500 μg /mL) and S. typhimurium (MI Q 625 μg/mL). Plumbagin, due to its very small amount, was tested only by the bioautography method against one microorganism, susceptible S. aureus, and showed excellent activity (MIQ 62 μg /mL ). G C - MS analysis of the n - hexane partition of D. lanceifolia also showed the presence of the quinones plumbagin and 7 - methyljuglone, which have been found in this and other Diospyros species and have both been previously reported for possessing antimicrobial activity. GS - MS analysis of H. latifolia showed the presence of hexanal and nonanal, which have been previously reported as volatile compounds from many plant species including those of medicinal value. In order to further investigate the phytochemistry of the antibacterially active extracts of P. persica, H. latifolia and D. lanceifolia, an L C - UV - MS metabolomics approach was used. The following compounds were tentatively identified from the P. persica ethanolic extract and/or dichloromethane and ethyl acetate partitions: afzelechin, caffeic acid phenylethyl ester, catechin, ent - epiafzelechin - (2 α O7',4 α 8') - ( - ) - ent - afzelechin, ent - epiafzelechin - (2α 7,4α 8) - epicatechin and/or ent - epiafzelechin - (2α 7,4α 8) - catechin and/or 3''' - hydroxyafzelechin - (2  7,4  8) - catechin, epicatechin, eriodictyol, gallic acid, gallocatechin, kaempferol, leucocyanidin, mandelic acid β - D - glucopyranoside, persicogenin , proanthocyanidin A1 or A2 type, quinic acid and taxifolin - 7 - O - rhamnoside. All compounds except mandelic acid β - D - glucopyranoside and taxifolin - 7 - O - rhamnoside were previously isolated from the Prunus genus. In the crude aqueous ethanolic extract of D. lanceifolia only one peak was identified with an acceptable degree of confidence, and it was identified as either ursolic, oleanolic or betulinic acid, which have all been previously reported from the Diospyros genus. In the n - hexane partition, scopoletin or isoscopoletin, epiisoshinalone or shinalone, and ricinoleic acid were tentatively identified. Those compounds were previously reported to be present in the Diospyros genus. No compounds could be confidently identified in the crude ethanolic extract or dichloromethane partition of H. latifolia, however some possible candidates were proposed. All of the compounds tentatively identified by the metabolomics approach, as well as the isolated compounds from P. persica roots and D. lanceifolia and H. latifolia leaves, align with the traditional applications of those plant parts by Indigenous communities.