Potentiating Activity of Shikonin with Membrane-permeabilizing agents or ATPase inhibitors against Methicillin-resistant Staphylococcus aureus.

Shikonin (SKN), alkannin, and their derivatives are highly liposoluble naphthoquinone pigments isolated from the root of the plants belonging to the Boraginaceae family, including Lithospermum erythrorhizon Sieb. et Zucc, Alkanna tinctoria, Arnebia euchroma (Royle) Johnst, and Arnebia guttata Bunge. L. erythrorhizon is a well-known herbal crop in Republic of Korea used in traditional oriental medicine to treat burns, ulcers, hemorrhoids, infected crusts, bedsores, external wounds, and oozing dermatitis. In addition, L.erythrorhizon has been reported to exhibit wound healing, anti-inflammatory, antithrombotic, and antitumor effects, and antimicrobial activity. The aim of this study was to examine Shikonin antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The Shikonin was analyzed in combination with membrane-permeabilizing agents TRIS and Triton X-100, ATPase inhibitors Sodium azide and N,N'-dicyclohexylcarbodiimide, and Staphylococcus aureus-derived peptidoglycan; the effects on MRSA viability were evaluated by the broth microdilution method, time-kill test, and transmission electron microscopy. Addition of membrane-permeabilizing agents or ATPase inhibitors together with a low dose of Shikonin potentiated Shikonin anti-MRSA activity, as evidenced by the reduction of MRSA cell density by 75% compared to that observed when Shikonin was used alone; in contrast, addition of peptidoglycan blocked the antibacterial activity of Shikonin. The results indicate that the anti-MRSA effect of Shikonin is associated with its affinity to peptidoglycan, the permeability of the cytoplasmic membrane, and the activity of ATP-binding cassette (ABC) transporters. This study revealed the potential of Shikonin as an effective natural antibiotic and of its possible use to substantially reduce the use of existing antibiotic may also be important for understanding the mechanism underlying the anti-bacterial activity of natural compounds.