Study On Antibacterial Activity Of Isoquinoline Alkaloids And Their Derivatives In Vitro

[Objective] To study antibacterial activity of isoquinoline alkaloid berberine and its derivatives, tetrandine and demethyl-tetrandrine in vitro, as well as the effect of combined with antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA) so as to develop new drugs with high effectiveness and low toxicity.[Methods] Based on foreign and domestic literatures, the derivatives of berberine dihydroberberine methomethylsulfate and 8-acetonyldihydroberberine were synthesized by improved method and their solubilities were studied, which provide a basis for continuous research. The antibacterial activities of berberine, dihydroberberine methomethylsulfate, 8-acetonyldihydroberberine, tetrandine and demethyl-tetrandrine were tested in vitro by using agar diffusion method, and their bacteriostatic activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans were evaluated by inhibition zone. The microdilution method was performed to determine minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against 10 strains of MRSA. The checkerboard microdilution method was performed to examine antibacterial activity of tested drugs combined with antibacterial agents against 10 strains of MRSA and the fractional inhibitory concentration index (FICI) was calculated. The synergistic action was also analyzed by the time-kill curve and Kirby-Bauer method.[Results] Dihydroberberine methomethylsulfate and 8-acetonyldihydroberberine were synthesized from berberine. Dihydroberberine methomethylsulfate was easy to dissolve in water, methanol and acetone with different pH value. Compared with berberine, 8-acetonyldihydroberberine was less soluble in water, but more soluble in methanol, especially in acetone solution with pH value of 7. Berberine, dihydroberberine methomethylsulfate,8-acetonyldihydroberberine, tetrandine and demethyl-tetrandrine showed good antimicrobial activity against S. aureus (including MRSA) and C. albicans at 2 mg/mL, with the average diameters of the inhibition zone were 10-22 mm. But these compounds had no activity against E. coli and P. aeruginosa. When tested with ten clinical SCCmec III typed MRSA isolates, the MIC/MBC ranges of berberine and 8-acetonyldihydroberberine were 32～128/64～256,32-128/128-512μg/mL, respectively. The MIC/MBC ranges of dihydroberberine methomethylsulfate, tetrandine and demethyl-tetrandine were 64～128/256～1024μg/mL. Combination of berberine and its derivatives with levofloxacin and azithromycin showed synergistic action against MRSA (FICI≤0.5), but combination of tested drugs with ampicillin and cephazolin showed indifference (2≥FICI>1). Combination of tetrandine and demethyl-tetrandrine with cephazolin could significantly decrease dosage of each drug which showed synergistic action against MRSA (FICI≤0.5), but combination of them with levofloxacin, azithromycin and ampicillin also showed indifference (2≥FICI>1). Compared with single drug group, the diameter of inhibition zone by disk diffusion method enlarged significantly in the group of tested drugs combined with other antibiotics. Thetime-kill curve confirmed the result of intensifying anti-MRSA effects by the tested drugs. [Conclusion] Berberine, dihydroberberine methomethylsulfate,8-acetonyldihydroberberine, tetrandine and demethyl-tetrandrine exhibited good antimicrobial activities against MRSA. Berberine, dihydroberberine methomethylsulfate and 8-acetonyl dihydroberberine combined with levofloxacin and azithromycin, as well as tetrandine and demethyl-tetrandrine combined with cefazolin have synergistic or additive effects against MRSA.