A large number of Chinese herbal drugs (CHDs) exhibit antibacterial activities both in vivo and in vitro, but until now little is known regarding their inhibitory mechanisms. Bacterial DNA gyrase is a proven target for antibacterial agents. Aim of this study was to investigate the in-vitro inhibitory effect of methanol extracts of CHDs against supercoiling activity of bacterial DNA gyrase. Fifteen CHDs were selected and extracted with methanol, respectively. Inhibitory effect of the extracts on DNA gyrase was tested using gel-based DNA supercoiling assay. Among fifteen CHDs tested, methanol extracts of Lonicerae Japonicae Flos (S2), Taraxaci Herba (S7), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (S8) demonstrated an obvious inhibitory effect against supercoiling activity of DNA gyrase, and the others were either less active or could not be determined with the present method. Moreover, it was likely that S7 and S8 inhibit gyrase in a concentration-dependent manner. In conclusion, DNA supercoiling assay is a promising method to study the inhibitory activity of CHDs on bacterial DNA gyrase. Some CHDs do have gyrase-inhibitory activity as proposed. Further investigations are needed to elucidate the inhibition mechanism of these CHDs on supercoiling activity of gyrase.
Chinese herbal drugs (CHDs) and formulas have been used for thousands of years in China to treat a wide variety of diseases and nowadays receive more and more recognitions worldwide. Modern researches have demonstrated that a large number of CHDs exhibit antibacterial activities both in vivo and in vitro [1,2]. For example, Lonicerae Japonicae Flos had in vitro obvious antibacterial activity against Staphylococcus aureus, Escherichia coli, Shigella flexneri and Branhamella catarrhlissuch [
Bacterial DNA gyrase, which belongs to type II topoisomerases, is highly conserved and essential for DNA replication and chromosome segregation, as well as for the maintenance of chromosome structure and DNA superhelicity [
Are there any CHDs that possess anti-bacterial activities targeting bacterial DNA gyrases, either? To present, there is no report on the gyrase inhibition of CHDs. Since CHDs contain tens of thousands of various components, it is very likely that some gyrase inhibitors can be found from them. Hence, we set out to conduct a comprehensive analysis of the inhibitory activity of methanol extracts of CHDs against bacterial DNA gyrase. The results, on one hand, will contribute to understanding of the antibacterial mechanism of CHDs; On the other hand, they may lead to development of novel therapeutic agents.
Fifteen CHDs (
2.0 g powder (through No. 3 sieve) of each CHD was weighed, added with 10 ml methanol and weighed again.
After ultrasonic extraction (power 120 W, frequency 40 kHz) for 30 min, the extract was allowed to cool down and weighed again; the loss of weight was replenished with methanol. The extract was filtrated and 2 ml of the successive filtrate was collected and evaporated to nearly dry. The residue was re-dissolved in methanol to obtain the expected concentration.
Over 70% of plasmid pBR322 bought from TAKARA (Dalian, China) was supercoiled. To obtain relaxed DNA, DNA topoisomerase I kit from TAKARA (Dalian, China) was used according to the manufacturer’s instruction with some modification. The reaction mixture (100 μl) containing 80 ng/μl pBR322, 0.3 U/μl DNA Topoisomerase I, 0.3% bovine serum albumin and 1 × reaction buffer was incubated at 37˚C for 60 min and terminated by cooling on ice. Relaxed pBR322 DNA was precipitated with ethanol and dissolved in sterile H2O.
Methanol extracts were tested for the inhibitory effect on supercoiling activity of bacterial DNA gyrase by using DNA gyrase kit from TopoGEN (Port Orange, USA) according to the manufacturer’s instruction with some modification. For each assay, the mixture (20 μl) contained 80 μg/μl relaxed pBR322, 0.075 U/μl DNA gyrase, 1 × reaction buffer and 2 μl methanol extract of CHD. As
negative control, gyrase was omitted. As positive control, methanol extract was substituted with sterile water. A blank control with 2 μl methanol was also included to evaluate the influence of the solvent methanol. The mixtures were incubated at 37˚C for 60 min and visualized by agarose gel. Semi-quantification of band intensity was performed using INFINITY-CAPT software Version 14.2 (VILBER LOURMAT, France). The inhibitory effect (IE) of different CHDs was defined as follows:
By searching literatures and the information database of traditional Chinese Medicine, fifteen potential antibacterial CHDs (S1 to S15) were selected, extracted and tested using DNA supercoiling assay.
The results are shown in
Furthermore, we investigated the concentration-effect relationships of S2, S5, S7 and S8. Interestingly, a higher inhibitory activity was observed when a higher concentration extract was added in the reaction mixture (
Our results showed that DNA supercoiling assay is a promising method to investigate the inhibitory effect of many CHDs on bacterial DNA gyrase. Some CHDs do inhibit the supercoiling activity of bacterial DNA gyrase in vitro as proposed. Further investigations will be carried out to identify which component/s is/are responsible for the gyrase inhibition of CHDs like S7 and S8. As DNA gyrase is exclusive to the prokaryotic kingdom and essential for the survival of bacteria, it appears to be an ideal target for antibacterial drugs [
As summarized in
Inhibitory activity *: “++”: strong inhibitory effect, IECHD < 0.5; “+”: week inhibitory effect, 0.5 ≤ IECHD < 0.9; “−”: no inhibitory effect, IECHD ≥ 0.9; “O”: not detectable.
infection to some extent? To draw a firm conclusion, more evidence is required. On the other side, S4, the socalled “antibiotic of TCM”, as well as S13 are widely used clinically to treat heat syndromes but exhibited little inhibitory activities on bacterial DNA gyrase, which might indicate both of them contain no gyrase inhibitor. In addition, comparison of chemical components of CHDs with gyrase inhibitory activity gave no insight into the relationship between chemical components and the activity.
In summary, in-vitro inhibitory effects of 15 CHDs on bacterial DNA gyrase were studied using DNA supercoiling assay and some interesting results were obtained. Further investigations will help to completely elucidate the inhibition mechanism of these CHDs against gyrase.
This work was sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Administration of TCM, PRC (No. 2005LHR20) and Guangdong Natural Science Foundation, PRC (No. 06301405). We thank Prof. Ruoting Zhan for his kindly help and Dr. Rui He for her critical comments on the manuscript.