Antimicrobial Activity of Curcuma longa Aqueous Extract

MATERIALS AND METHODS

Plant Collection Identification and Extraction
The plant material was brought from local market at Mahasarakham Province during September, 2008. The plant was identified by Department of Biology, Faculty of Sciences, Mahasarakham University, Thailand. The root of C. longa were washed with water and cut into small pieces and dried in oven at 40°C for 48 h. After the drying process C. longa was ground to powder. Ten gram powder of dried plant were boiled in 500 mL of distilled water and spray dried. The yields of extraction were in the range of 3.7-4% of dried weight plant powder. The extraction and antimicrobial activity test were conducted from November 2008-February 2009 at Department of Chemistry, Faculty of Sciences, Mahasarakham University, Thailand.

Microorganisms
Five strain of gram positive (Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, Bacillus subtillis ATCC 6633, Lactobacillus plantarum ATCC 14917) and five strains of gram negative (Escherichia coli ATCC25922, Salmonella typhimurium ATCC 14028 Klebsiella pneumonia ATCC 10031 Proteus vulgaris ATCC 13315, Pseudomonas aeruginosa ATCC 9721) bacteria were used as test organisms.

Antimicrobial Susceptibility Test
Agar Diffusion Method
The agar diffusion method was conducted as standard method (Lorian, 1996) and as described in previous report (Sittiwet and Poungpronpitag, 2008). Briefly, the solution of plant extract were prepared the solution of plant extract was prepared at concentration 125, 250 and 500 g L^-1 using sterile distilled water. The solutions were putted into the stainless cylinder (6 mm internal diameter and 10 mm height) which place on the inoculated Mueller-Hinton agar surface. After pre-incubation for 1 h the plates were incubated at 37°C for 19 h. The clear diameters of inhibition zones were observed.

Agar Dilution and Broth Macro Dilution Method
The Minimum Inhibitory Concentrations (MICs) were determined using agar dilution method and confirmed by result of broth macro-dilution method while Minimum Bactericidal Concentration (MBCs) were determined using broth macro-dilution method according to standard guideline (Lorian, 1996). The agar dilution method was using plant extract concentration in range of 0.5-256 g L^-1 in Mueller-Hinton agar and spot with 0.5 McFarland bacterial suspension. The MICs of the plant was recorded by observed no growth of bacteria on the agar surface at each concentration after incubated at 37°C for 24 h. While broth macro-dilution method, plant solution were prepared in sterile water at concentration 256 g L^-1 (range 0.5-256 g L^-1). Two fold serial dilutions in 3 mL of Mueller-Hinton broth were made and then 3 mL of bacterial suspension of was added to give final inoculums of 0.5x10⁶ cfu mL^-1. The solutions were incubated at 37°C for 24 h. The MICs were recorded by observed the lowest concentration that showed no visible growth of bacteria while MBCs were recorded as the lowest concentration that showed no growth of bacteria after subculture on agar medium.

RESULTS

The root of C. longa has been used as remedy in many countries as an insect repellant, antimicrobial (Rudrappa and Bais, 2008), antidiabetic (Mohamed et al., 2009) rheumatism, bodyache, skin diseases, intestinal worms, diarrhea, intermittent fever, hepatic disorders, biliousness, urinary discharges, dyspepsia, inflammations, constipation, leukoderma, amenorrhoea and colic inflammatory disorders (Villegas et al., 2008).

Table 1:	Inhibition zone diameters of C. longa root aqueous extract against various bacteria
Data are Mean±SD (n = 3); nz: No inhibition zone

Table 2:	The MICs and MBCs of C. longa root aqueous extract against various bacteria
nd: Not determine

Curcumin is a major constituent of C. longa rhizomes. It has been reported about antimicrobial activity of C. longa against various bacteria such as Pseumonas aeruginosa (Rudrappa and Bais, 2008; Nagi et al., 1999), Aeromonas hydrophila (Harikrishnan and Balasundaram, 2008), Helicobacer pylori (Zaidi et al., 2009), Escherichia coli O157:H7 (Gupta and Ravishankar, 2005), Listeria monocytogenes and Salmonella typhimurium DT104 and methicillin-resistant Staphylococcus aureus (Kim et al., 2005).

In this study, the aqueous extract of root part of C. longa was evaluated. The results revealed that the aqueous extract of root part of C. longer showed inhibitory against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC25923, Krebsilla pneumoniae ATCC and Staphylococcus epidermidis ATCC, respectively (Table 1). The MICs of aqueous extract of root part of C. longer were in the range of 4-16 g L^-1, while the MBCs were in the range of 16-32 g L^-1 as shown in Table 2. The results give additional information for antimicrobial activity of C. longa. In comparison C. longa root aqueous extract exhibited the inhibitory effect against E. coli ATCC 25922, S. aureus ATCC 25923, S. epidermidis ATCC 12228 and K. pneumoniae ATCC 10031. The result did not showed inhibitory effect Ps. aeruginosa compared with previous reported by Rudrappa and Bais (2008) and Negi et al. (1999) maybe because of curcumin showed weak inhibitory effect against Ps aeruginosa and the inconsistency of natural abundance of curcumin in each season. However, in this study did not supply confirmation data for L. monocytogenes, H. pylori and A. hydrophila.

In conclusion, the aqueous extract of root part of C. longa showed good inhibitory effect on 4 out of 10 pathogenic bacteria which tested in this study.

DISCUSSION

As traditional medicine C. longa or turmeric has been extensively used for centuries to treat a diversity of disorders including antimicrobial (Rudrappa and Bais, 2008), antidiabetic (Mohamed et al., 2009) rheumatism, bodyache, skin diseases, intestinal worms, diarrhea, intermittent fever, hepatic disorders, biliousness, urinary discharges, dyspepsia, inflammations, constipation, leukoderma, amenorrhoea, colic inflammatory disorders, colorectal cancer (Villegas et al., 2008) and diabetes (Mohamed et al., 2009).

The present study investigated the antibacterial activity of C. longa aqueous extract against various bacteria. The results indicate that the C. longa aqueous extract showed antimicrobial activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC25923, Krebsilla pneumoniae ATCC 10031 and Staphylococcus epidermidis ATCC 12228 at low concentration. Previous study show antimicrobial activity of C. longa in both essential oil which is non polar and aqueous extract which is polar compound. It has been reported the chemical composition of essential oil from C. longa consist of ar-turmerone (Martins et al., 2001) while aqueous extract mainly consist of curcumin (Rudrappa and Bais, 2008).

The antimicrobial activity of C. longa has been reported in previous study. It was found that the C. longa have inhibitory effect against Pseumonas aeruginosa (Rudrappa and Bais, 2008; Negi et al., 1999), Aeromonas hydrophila (Harikrishnan and Balasundaram, 2008), Helicobacer pylori (Zaidi et al., 2009), Escherichia coli O157:H7 (Gupta and Ravishankar, 2005), Listeria monocytogenes and Salmonella typhimurium DT104 and methicillin-resistant Staphylococcus aureus (Kim et al., 2005). In this study, the results showed antimicrobial activity of C. longa aqueous extract against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC25923, Krebsilla pneumoniae ATCC 10031 and Staphylococcus epidermidis ATCC 12228. The result from this study may supported the antimicrobial activity and somehow the confirmation of antimicrobial activity of C. longa. Moreover, it may support the use of C. longa for antimicrobial treatment disease or prevention of bacteria growth.

ACKNOWLEDGMENT

This study was partially financial supported from Faculty of Science, Mahasarakham University, Thailand.