A Novel Approach to Estimate In Vitro Antibacterial Potency of Chinese Medicine Using a Concentration-Killing Curve Method
Abstract
The antibacterial pharmacodynamics against E. coli of Chinese medicine (CM) Rhizoma coptidis (Coptis Root) and its formula Sanhuang, and the control antibiotics enoxacin, were analyzed by a concentration-killing curve (CKC) approach, and the novel parameters BC50 and r for antibacterial potency were proposed. Using the agar plate method, about 400 cells of E. coli were evenly inoculated into LB agar plates containing a series of different concentrations of CM or antibiotic, and after a 24 hour incubation at 37°C, all the viable colonies were enumerated. This resulted in a sigmoid concentration-killing curve that could be closely fitted (R2 > 0.9) with the function: 
, in which N0, BC50 and r represent meaningfully inoculums size, median bactericidal concentration, and bactericidal intensity, respectively. N modeled the survival of colony-forming units on each plate (CFU/plate) in a concentration series x of the drug. The CKC was symmetrical about its single inflexion (BC50, N0/2). Therefore theoretically, 2BC50 can replace MBC (minimum bactericidal concentration). 
, the drug concentration at which only one colony survived, was the least critical value of MBC in CKC. The parameters 2BC50 and BC1 agreed more closely with the definition of MBC, and were little affected by either the biochemical basis of the antibacterial or the inoculum's size (200–400 CFU/plate), and were determined by a multi-point curve. As a result, these were more accurate, reproducible and practical as metrics than was the endpoint of MBC. The two-dimensional CKC, involving BC50 and r, captures the intrinsic dynamics of the antibacterial effect of CM/strain versus concentration, and it is consistent with the Logistic equation of the bacterial growth curve in the format. This verified approach has considerable value as a tool for the accurate and proper administration of CM. The CKC of CM, different from that of antibiotics, is likely to be the resultant force of each ingredient in certain CM, which provides a clue to solve the problem of antibiotic resistance.
