Initial screening of plants for possible active natural products typically begins by using crude aqueous or alcohol extraction and can be followed by various organic extraction methods. However, nearly all of the identified antimicrobial components from plants are aromatic or saturated organic compounds, they are often obtained through initial ethanol or methanol extraction [27
]. Therefore, in the present study, for the preparation of all the seven crude extracts, methanol was used as extraction solvent. For initial screening, these seven crude methanol extracts were tested against seven fungal and six bacterial strains covering a broad range of microbes. All the extracts showed low level of antifungal activity. However, antibacterial assay of the crude methanolic extracts showed that only two extracts; aerial parts of Q. dilatata
and roots of C. hierosolymitana
had antibacterial activity. The extract of aerial parts of Q. dilatata
was found highly effective against all the bacterial strains tested i.e., E. coli, B. subtilusS. aureus, M. luteus, B. bronchiseptica,
and S. setubal
while root extract of C. hierosolymitana
showed antibacterial activity against only three bacterial strains i.e., M. luteus, B. bronchiseptica,
and S. setubal.
Previously, there have been some reports of the antibacterial and antifungal activities of different species of Quercus
]. However, ours is the first report of antimicrobial activity of Q. dilatata
Based on the results of initial screening the methanol extract of Q. dilatata
was subjected to bio-guided fractionation by solubilisation in water and sequential partition with different solvents with increasing polarity; hexane, chloroform, ethyl acetate, acetone, ethanol and 50% methanol yielding six fractions. All the fractions were subjected to antibacterial assay. Four of the six partitioned fractions i.e. ethyl acetate, acetone, ethanol and 50% methanol showed antibacterial activity against all the bacterial strains tested whereas the hexane and chloroform extracts were inactive. Highest antibacterial activity was shown by ethanol fraction. Phytochemical analysis of partitioned fractions showed the presence of alkaloids in the three fractions in the order of, ethanol partitioned fraction > acetone partitioned fraction > 50% methanol partitioned fraction. Alkaloids are commonly known antibacterial agents [32
]. Therefore, our results suggested that the ethanol partitioned fraction contained maximum alkaloids which could be responsible for the highest antibacterial activity exhibited by this fraction. The polarity of a solvent plays an important role on composition of an extract and hence on its potential antibacterial activity. Similar results were reported by Berahou et al. [33
] that only ethyl acetate, butanol and aqueous phases of methanol extract of Quercus ilex
bark showed antibacterial activity against all the bacterial strains whereas the hexane and dichloromethane phases were almost inactive.
HPLC analysis of the most active partitioned fraction i.e., ethanol was done by isocratic RP-HPLC system consisting of Agilent 1200 series preparative pump coupled with UV diode array detector. HPLC has extensively been used for isolation and identification of active natural components components [34
]. In this study ethanol partition fraction was eluted with acetonitrile: methanol (70: 30) and seven peaks were observed at 230nm. These seven fractions were collected at different retention times. Only one of the seven fractions i.e. AM3 had antibacterial activity against all the bacterial strains tested. AM3 showed highest antibacterial activity against S. setubal
. RP- HPLC analysis of active fraction AM3 was done. When fraction AM3 was subjected to RP-HPLC analysis, three subfractions were collected at different retention times. Two of the three subfractions AM3b and AM3c showed antibacterial activity against all the bacterial strains tested.
Purified active subfractions were charaterized by comparing their absorption spectra with that of standard natural products. In the present study, four standard natural compounds used were ascorbic acid, quercitrin, gallic acid and rutin. All of them have been reprted previously from genus Quercus
i.e., ascorbic acid [37
], quercitrin [38
], gallic acid [29
] and rutin [39
]. The absorption spectra of the active fractions were different from that of the standard compounds previously isolated from the Quercus
genus suggesting that these are different compounds. However, structure elucidation is required to confirm their novelty. Antibacterial [15
] activity of genus Quercus
was previously reported, yet antibacterial activity of Q. dilatata
had never been checked before. Thus, this is the first report of identification and successful isolation of antibacterial compounds from this species. These compounds are effective against B. subtilis, M. leuteus, S. aureus E. coli, S. setubal
and B. bronchiseptica
. These bacterial strains are involved in causing many diseases like boils, cellulitis folliculitis, furuncles, carbuncles, pneumonia, meningitis, urinary tract infections etc. Furthermore, these compounds appear to be different from already known constituents of this genus and actually could be newly identified compoundsand hence can be potentially used against the above mentioned diseases.