MATERIALS AND METHODS

Plant Material
Malvastrum coromandelianum Garcke (Malvaceae) was identified by Royal Forest department of Thailand and cultivated on January 2006 in open field at Singburi province, Thailand. Aerial part of plant was collected in the beginning of June 2006 and the following extraction processes were finished in the end of June 2006. The antimicrobial investigation in this article was done on December of 2006.

Extraction
The extraction method was modified from Chinese traditional method (Lang and Wai, 2003). Dried aerial parts were collected and 10 kg of dried plant was extracted by boiling in 100 L of water for 30 min. The extraction was repeated twice. The pooled filtrates were spray-dried. The yield of spray-dried extract powder was 8-10% w/w of raw material. In this study used one batch of extraction and HPLC chemical finger print was determined for each experiment to confirm the same quality of ME.

Tested microorganisms Twelve clinical isolates of S. aureus (supplied by Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand) were included in this study. The tested isolates were divided into 2 groups, 6 of each, according to susceptibility to methicillin where MRSA referred to methicillin-resistant strains and MSSA referred to methicillin-susceptible strains. S. aureus ATCC 25923 and S. aureus ATCC 29213 were also used as reference strains.

Antimicrobial Assay
MICs of crude water extract of ME were determined by agar dilution method (Merck) (Lorian, 1996) while MBCs were determined by broth macro-dilution method were (Lorian, 1996) and reference antibiotics Oxacillin (Sigma Chemical Co., St.Louis, USA). Inoculates were prepared in the same medium at density adjusted to 0.5 McFarland turbidity standard (10⁸ colony-forming units (cfu mL^-1) and two fold dilution for the broth macro-dilution procedure. The inoculated tube were incubated at 37°C and the MICs were recorded after 24 h of incubation. The MIC was defined as the lowest concentration of ME or Oxacillin at which the microorganism tested did not showed visible growth while MBC was defined as the minimum bactericidal concentration with negative subcultures on agar medium. Values were means of triplicate.

RESULTS AND DISCUSSION

Methicillin-resistant S. aureus (MRSA) is increased and caused of nosocomial infection problem in many countries (Abrahamian and Snyder, 2007). Recently, only few antimicrobial agent including vancomycin and teicoplanin are still effective against MRSA. Thus, this pathogen can cause serious infection in various body systems in patients in particular the ICU patients.

The screening of plant extract to inhibit growth of MRSA is during interesting. Agar dilution and broth macro dilution were selected to evaluated in vitro antimicrobial activity tests in this study. Due to the red brown color of ME the MICs from only broth macro dilution was difficult to observed.

Table 1:	Minimum Inhibitory Concentration (MICs) and Minimum Bactericidal Concentration (MBCs) of ME against strains of S. aureus, standard antibacterial agent: oxacillin

Therefore, agar dilution method was used to determine MICs of ME while MBCs was from the broth macro dilution method. The water boiling extraction methods which have been used for thousands of years in China to prepare herbal remedies (Lang and Wai, 2003) was used in this study. Furthermore, water was no toxic drug vehicle and less interfering effect on experiment. The record of using of plant in family Malvaceae as medicinal plant has been report such as in Turkey (Abu-Shanab et al., 2003), New Zealand (Bloor, 1995) and Iran (Sharhidi Bonjar, 2004). The plant in Malvaceae has been tested against MRSA for Althaea officinalis and showed no inhibition effect against MRSA (Abu-Shanab et al., 2006).

Crude water extract of M. coromandelianum Garcke showed antibacterial activity against MSSA (MICs = 2.5-5 mg mL^-1, MBCs = 10-80 mg mL^-1) and MRSA (MICs = 2.5-5 mg mL^-1, MBCs = 10-20 mg mL^-1) as well as S. aureus ATCC 25923 and S. aureus ATCC 29213. Although ME did not showed bactericidal activity to all tested strains of MRSA (MRSA No. 6; MBC >160 mg mL^-1) but it did show inhibitory effect at considerable low concentration for crude water extract against most of tested MRSA (Table 1).

This study is the first reported of antibacterial activity of M. coromandelianum water extract. In addition the results from this study provide very useful information concerning new natural drug discovery.

ACKNOWLEDGMENTS

This study has been partly supported by grant year 2005 from graduate school of Chulalongkorn University, Bangkok, Thailand. The authors would like to show their appreciations to the staff of the Royal Forest department of Thailand and of the Government Pharmaceutical Organization of Thailand for their co-operations.