In vivo activities of Baicalin against Chlamydia trachomatis

Our previous studies have shown that Baicalin could effectively inhibit Chlamydia trachomatis in vitro. In this study, Baicalin was tested for potential antichlamydial activity using a murine genital Chlamydia trachomatis infection model. It was demonstrated that Baicalin significantly reduced C. trachomatis loading in BALB/c mice that were vaginally infected with the pathogen. On the basis of these data and our previous observations, we concluded that further evaluation of Baicalin for prevention and treatment of sexually transmitted chlamydial infection is warranted.


INTRODUCTION
Chlamydia trachomatis is primarily a human pathogen associated with common sexually transmitted diseases and trachoma. Most developing countries that have the highest burden of chlamydial infections have limited capacity to effectively screen for chlamydial infections and treatment is thus largely based on symptomatic case ascertainment (Schachter, 1999;Behets et al., 2001).
Studies demonstrated that the majority of infected individuals do not seek treatment because they have no or very mild symptoms (Westrom and Mardh, 1983). Without proper treatment, about one-third of infected individuals develop long-term, devastating complications, such as infertility and chronic pelvic inflammatory pain syndrome (Laga et al., 1994). Infected individuals are also at increased risk of HIV acquisition, owing to ulcerative damages that occur in the epithelial tissues. The medical and financial burdens of these conditions call for the development of new strategies to effectively prevent C. trachomatis infection (Ridgway, 1997).
Although effective antimicrobial treatment is available, this has been largely unsuccessful in halting the spread of infection, most likely due to the high rate of asymptomatic infections which may persist for months to years. Multiple-*Corresponding author. E-mail: whhuanghao10@yahoo.com.cn. Tel: +86 27 85332365.
antibiotic resistant strains of Chlamydia have also been reported recently (Somani et al., 2000). Furthermore, it has been suggested that antibiotic treatment can result in persistent infections with aberrant forms of C. trachomatis that may be reactivated at a later date.
We have reported previously that Baicalin can effectively inhibit C. trachomatis in vitro (Huang et al., 2009). In the present investigation, we studied the in vivo therapeutic effects of Baicalin using a mouse experimental model of C. trachomatis -caused chlamydial genital infection.

MATERIALS AND METHODS
C. trachomatis serovar D (Department of Pathogenic Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China) was cultured in HeLa cells as described previously (Rasmussen et al., 1997). The infectivity of the chlamydia was expressed as the number of inclusion forming units. Baicalin (HPLC Content > 98.0%) from Chongqing Green Valley Bio-tech Co. LTD. (Chongqing, China). Medroxyprogesterone was used to make mice susceptible to infection (Amit et al., 2009). Eight-week-old Balb/c female mice were injected subcutaneously with the hormone (2.5 mg/mouse) 2 weeks before infection. The injection was repeated 7 days before infection. The progesteronetreated animals were given intravaginally either 15 ml of 1 mM Baicalin prepared in 50 mM Hepes buffer (pH 7.0) or the vehicle Hepes buffer (10 mice in each group). One hour later, the animals were infected intravaginally with C. trachomatis (2 × 10 7 IFUs per mouse). After infection, intravaginal administration of Baicalin or vehicle was repeated three times per day. 5, 11, 16 and 19 days after infection, vaginal swabs were taken. Infectious elementary Afr. J. Biotechnol.

RESULTS AND DISCUSSION
It is now widely accepted that C. trachomatis is the most prevalent sexually transmitted pathogen. Classical antibiotics are undesirable for long-term, prophylactic use because they frequently disrupt normal microflora and consequently increase the risk for bacterial vaginosis. Among non antibiotic reagents tested against C. trachomatis, only one offered partial protection in vivo; others were either ineffective or even worsened the infection in vivo although, in vitro, they demonstrated adverse effects on the pathogen. Baicalin, being a medicinal plant traditionally used in Oriental medicine, is a flavonoid derived from Scutellariae Radix and known to have various biological functions, including antimicrobial activities including antimicrobial, anti-inflammatory activities, and also shown to have the therapeutic potential for the treatment of atherosclerosis and restenosis (Gao et al.,1999). Previous studies showed that Baicalin can inhibit infection in vitro by several strains of Chlamydia pneumoniae (Liu et al., 2006). We previously also characterized the inhibition of C. trachomatis in cell culture by Baicalin (Huang et al., 2009).
The objective of the present study is to investigate whether Baicalin would also inhibit chlamydial infection in vivo. Here, we tested the effects of Baicalin on C. trachomatis infection in vivo using a MoPn vaginal infection model previously established in BALB/c mice. Table 1 apparently revealed that the majority of control vehicle-treated mice had higher bacterial loadings as compared with Baicalin-treated animals. The findings from this study further confirmed that Baicalin significantly reduced C. trachomatis loading in BALB/c mice that were vaginally infected with the pathogen and suggested that Baicalin deserved further evaluation as antichlamydial candidates for the prevention of sexually transmitted chlamydial infection.

ACKNOWLEDGMENT
This work was supported by the grant from the Hubei Provincial Nature Science Foundation of China (No. 2007ABA113).