Antimicrobial activities of Linum usitatissimum extracts on selected oral pathogens : an in vitro study

Abstract

Despite the tremendous advancement in medical field, oral infections are still considered as one of the major public health problems suffered by human population causing a burden to health care system worldwide. Apart from using synthetic antibiotics, antimicrobial agents from natural sources are suggested as an alternative for infectious diseases treatment. Flaxseed (Linum usitatissimum) has been shown to demonstrate some antimicrobial properties. However, its effects towards oral pathogens are still limited. Hence, present study aimed to investigate the antimicrobial effects of L. usitatissimum extracts against selected oral pathogens (Streptococcus pyogenes, Streptococcus mutans, Lactobacillus casei, Enterococcus faecalis and Candida albicans) using disc diffusion and broth dilution method as well as to examine their synergistic effect when combined with current antibiotics. Besides that, this study also aimed to study their mode of action on the cell membrane using the most effective L. usitatissimum extract. Non-polar, semi polar and polar extracts of L. usitatissimum; n-hexane, dichloromethane (DCM) and methanol (MeOH), were prepared by sequential extraction using soxhlet extraction method while aqueous extract was prepared using maceration technique at room temperature. All the extracts were qualitatively screened through GC-MS (n-Hexane, DCM and MeOH extracts) and LC-MS (MeOH and aqueous extracts) to detect the presence of bioactive compounds. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) of the extracts were determined against selected strains. Synergistic effect was tested upon combination of extracts with antibiotics (ampicillin and chlorhexidine) against S. pyogenes, S. mutans, L. casei and E. faecalis while nystatin was tested in combination against C. albicans using checker board titration method. There were three modes of action tested, which included time-killing, crystal violet assays and leakage of cellular metabolites at 260 nm and 280 nm. All of the tests were done in triplicate. GC-MS screening of methanolic extract recorded the presence of fatty acid; myristic acid, palmitic acid, linoleic acid, linolenic acid and stearic acid while LC-MS screening demonstrated the presence of linolenic acid, secoisolariciresinol diglycoside, (-)-secoisolariciresinol-4-O-β-D-glucopyranoside, E-p-Coumaric acid, and flavone-5,7-dihydroxy-4′-O-α-D-glucoside which may have contributed greatly to the inhibitory activities of the methanol plant extract. Methanolic extract demonstrated significant antimicrobial activity (p<0.01) against all tested oral pathogens except for C. albicans which is comparable with the zone of inhibition exhibited by penicillin. The MIC value of methanolic extract against all of tested microorganisms was found at concentration between 15.63 mg/mL and 62.5 mg/mL while for their MBC/MFC value between 125 mg/mL and 500 mg/mL. The Fractional Inhibitory Concentration (FIC) index upon combination of MeOH extract with antibiotics were mainly indifferent except for the combination therapy of C. albicans (MeOH extract + nystatin) and L. casei (MeOH + ampicillin) showed synergistic and additive effects, respectively. The antimicrobial action on the cell membrane was time and dose-dependent. The present study was able to clarify the crucial role of methanolic extract as a potent antimicrobial agent of the flaxseed extract and proven to act on the cell membrane of microorganisms tested particularly against oral pathogens.