Abstract: The aqueous extract of Cryptolepis buchanani leaves was tested against food-borne pathogen bacteria (S. aureus ATCC 25923, E. coli ATCC 25922 and S. typhimurium ATCC 14028), nosocomial infection bacteria (K. pneumoniae ATCC 10031, P. vulgaris ATCC 13315 and Ps. aeruginosa ATCC 9721) and normal flora bacteria (L. plantarum ATCC 14917 and S. epidermidis ATCC 12228). The plant aqueous extract showed inhibitory effect against S. aureus ATCC 25923, E. coli ATCC 25922, S. typhimurium ATCC 14028, K. pneumoniae ATCC 10031, P. vulgaris ATCC 13315, B. subtilis ATCC 6633, L. plantarum ATCC 14917 and S. epidermidis ATCC 12228. The MICs (Minimal Inhibitory Concentrations) and MBCs (Minimal Bactericidal Concentrations) of this plant against all tested bacteria are in the range of 1-16 and 2-32 g L-1, respectively. In conclusion, C. buchanani leaves aqueous extract showed broad-spectrum antimicrobial activity against food-borne pathogen bacteria, nosocomial infection bacteria and some normal flora bacteria at low concentration. This may supported the used of C. buchanani aqueous extract as food-borne pathogen bacterial growth control additive and nosocomial infections treatment remedy.
INTRODUCTION
Food-borne infections are important public health concern worldwide (Busani et al., 2006). In food production countries to control the growth of bacterial in the product is important. The most common bacteria causing food-borne illness are Escherichia coli, Staphylococcus aureus, Samonella sp., Samonella typhimurium, Listeria monocytogenes, Clostridium botulinum, Vibrio vulnificus, Vibrio parahaemolyticus and others (Busani et al., 2006; Van et al., 2007; Gerner-Smidt and Whichard, 2008).
The application of medicinal plants in health care are in great interesting of topical countries (Kolawole, 2007). The development of medicinal plant as medicine consider the good way for natural resource management especially in topical countries that enrich of plant species. However, the developments of medicinal plant as remedy need scientific results support. Especially, the development of medicinal plants against food-borne pathogen bacteria that may give useful results that may indicate the efficacy of the plant in food additive applications.
Cryptolepis buchanani belongs to the family Periplocoideae and subfamily Asclepiadaceae (Paulo and Houghton, 2003). The plant has been used in Indian folkloric medicine (Ayurveda) for anti-diarrhoeal, anticulcerative, anti-inflammatory, blood purifier, cough treatment, curing rickets in children and antibacterial (Kaul et al., 2003). In Thailand, the alcoholic extract of stem of this plant has been used for treatment of inflammatory conditions such as arthritis, muscle and joint pain (Panthong et al., 1986; Laupattarakasem et al., 2003). Since, it has been used for antimicrobial in ayrveda folkloric medicine but still no scientific result to supported the used of C. buchanani. It would be interested to investigate the antimicrobial activity of C. buchanani aqueous extract whether the polar compound of it have antimicrobial activity or not. The objective of this study was to investigated antibacterial activity of C. buchanani leaves aqueous extract against food-borne pathogen, nosocomial infection and normal flora bacteria.
MATERIALS AND METHODS
Tested Bacteria
In this study gram positive (Staphylococcus aureus ATCC 25923, Staphylococcus
epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, Bacillus
subtillis ATCC 6633, Lactobacillus plantarum ATCC 14917) and gram
negative (Escherichia coli ATCC25922, Salmonella typhimurium ATCC
14028 Klebsiella pneumoniae ATCC 10031, Proteus vulgaris ATCC
13315, Pseudomonas aeruginosa ATCC 9721 ) bacteria were used. Among bacteria,
there are food-borne pathogenic bacteria (S. aureus ATCC 25923, E.
coli ATCC 25922 and S. typhimurium ATCC 14028), nosocomial infection
bacteria (K. pneumoniae ATCC 10031, P. vulgaris ATCC 13315 and
Ps. aeruginosa ATCC 9721) and normal flora bacteria (L. plantarum
ATCC 14917 and S. epidermidis ATCC 12228) were included in the susceptibility
test.
Plant Sample and Extraction
C. buchanani was collected in on May 2008 from Pattani Province,
Thailand. The plant was identified by Department of Biology, Faculty of Science,
Mahasarakham University, Thailand.
The leaves of plant were air dried and ground into powder. Ten gram of plant’s leaves dried powder was boiled in 500 mL water and the residue of extraction where repeated the boiling process for 3 times. The water was filtrated, pooled and spray-dried. The yield of extraction was 1.2-2.5% of dried weight of dried grind plant powder.
Anti-Microbial Susceptibility Test
The anti-microbial susceptibility test of C. buchanani was screened
using agar diffusion susceptibility test as described in the standard guideline
(Lorian, 1996). The plant extract spray-dried powder was
dissolved in sterile water at concentration of 125, 250 and 500 g L-1.
All test bacteria were cultured overnight on a Tryptic Soy Agar (TSA) slant at 37°C. Bacteria were washed from the surface of the agar slant with sterile Normal Saline Solution (NSS) (0.9% NaCl), which was then adjusted to match the turbidity of a standard Mcfarland No. 0.5 (108 colony-forming units (cfu) mL-1) before used as the starter solution. Twenty milliliter of Mueller Hinton Agar (MHA) was put in cultivation plates and swabbed with starter solution on the agar surface, by using swab cotton.
The plant solution were filled in sterile stainless steel cylinders (6 mm internal diameter and 10 mm height) that were placed on the inoculated agar surface. After pre-diffusion at room temperature for 1 h, the plates were incubated at 37°C for 19 h. The NSS filled in the cylinder was used as control and a 10 mg L-1 gentamicin sulphate (Sigma Chemical Co., St. Louis, USA) solution was used as standard in same cultivation plate.
MICs and MBCs Determination Using Agar Dilution and Broth Macro Dilution
Methods
MICs of C. buchanani aqueous extract was determined by the agar dilution
method (Merck) while MBCs were determined by the broth macro-dilution method
(Lorian, 1996) and using gentamicin sulphate as reference
antibiotics (Sigma Chemical Co., St. Louis, USA). Briefly, 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 tubes were incubated at 37°C and
the MICs were recorded after 24 h of incubation. The MIC was defined as the
lowest concentration of plant extract or gentamicin sulphate at which the microorganism
tested did not showed visible growth, while MBC was defined as the minimum bactericidal
concentration with negative subcultures on the agar medium.
RESULTS
In this study, the plant’s leaves were extracted using aqueous system with 1.2-2.5% yield of dried grind plant’s leaves powder. Cryptolepis buchanani aqueous extract was tested against 10 bacterial strains. There are selected food-borne pathogen bacteria (S. aureus ATCC 25923, E. coli ATCC 25922 and S. typhimurium ATCC 14028), nosocomial infection bacteria (K. pneumoniae ATCC 10031, P. vulgaris ATCC 13315 and Ps. aeruginosa ATCC 9721) and normal flora bacteria (L. plantarum ATCC 14917 and S. epidermidis ATCC 12228) were include in the susceptibility test.
The agar diffusion susceptibility test showed the inhibition zone of C. buchanani aqueous extract against 8 out of 10 tested strains. There are S. aureus ATCC 25923, E. coli ATCC 25922, S. typhimurium ATCC 14028, K. pneumoniae ATCC 10031, P. vulgaris ATCC 13315, B. subtilis ATCC 6633, L. plantarum ATCC 14917 and S. epidermidis ATCC 12228 were inhibited by C. buchanani aqueous extract (Table 1). The MICs and MBCs of this plant against all tested bacteria are in the range of 1-16 and 2-32 g L-1, respectively (Table 2).
It was found that C. buchanani showed broad-spectrum antibacterial activity against 8 out of 10 tested strains. It showed very good inhibitory effect on food-borne pathogen bacteria (S. aureus, E. coli and S. typhimurium) and nosocomial infection bacteria (K. pneumoniae, P. vulgaris and E. coli) at low concentration. However, it also showed inhibitory effect on normal flora bacteria such as L. plantarum and S. epidermidis.
| Table 1: | Inhibition zone diameters of C. buchanani aqueous extract against various bacteria |
| Data are expressed as Mean±SD (n = 3); nz: No inhibition zone | |
| Table 2: | The MICs and MBCs of C. buchanani aqueous extract against various bacteria |
| nd: Not determine | |
In conclusion, the aqueous extract of C. buchanani leaves showed anti-microbial against food-borne pathogenic bacteria and nosocomial infection bacteria at low concentration. This suggests the possibility to use C. buchanani aqueous extract as the food-borne pathogen bacteria growth control additive in food.
DISCUSSION
The food-borne pathogen bacteria contaminations are the big problem in livestock production (pigs, chickens, cattle and aquatic animals) countries. There is a report about the food-borne pathogen bacteria contaminated in Thailand (Padungtod et al., 2008) and Vietnam especially in Vietnam also found the antibiotic resistant strains (Van et al., 2007). Therefore, the growth of food-borne pathogen bacteria should be control. However, using of antibiotic may cause adverse effect and in most countries the amount of antibiotic applies in food is under control.
It is well known that some plant have anti-microbial activity and has been apply as the remedy for local people even before the chemical medicine was existed. As the fact that in topical area have a variety of plants species, development the plant extract as the anti-food-borne pathogen bacteria is one useful possibility.
Cryptolepis buchanani was used in ayurvedic Indian folkloric medicine for the treatment of diarrhea, ulcer, inflammation, cough, infection and others (Kaul et al., 2003). It has been reported anti-inflammatory activity (Laupattarakasem et al., 2006) and immunopotentiating properties (Kaul et al., 2003). It was also reported chemical component such as nicotinoyl glucoside (Dutta et al., 1980), cryptosin (Venkateswara et al., 1987, 1989) and buchananine (pyridine alkaloid) (Dutta et al., 1978). This study was aimed to evaluate the efficacy of C. buchanani aqueous extract anti-bacterial activity.
C. buchanani has been used in Indian folkloric antimicrobial remedy (Kaul et al., 2003) but its antimicrobial activity never been reported. This study was evaluated the possibility to used this plant as antifood-borne pathogen bacteria. The plant’s leaves aqueous extract was tested against 10 selected bacteria with 3 strains of food-borne pathogen bacteria (E. coli ATCC25922, S. typhimurium ATCC 14028 and Ps. aeruginosa ATCC 9721), 4 strains of nosocomial infection bacteria (S. aureus ATCC 25923, M. luteus ATCC 9341, K. pneumoniae ATCC 10031 and P. vulgaris ATCC 13315), 2 strains of normal flora bacteria (S. epidermidis ATCC 12228 and L. plantarum ATCC 14917) and B. subtillis ATCC 6633. The results showed inhibitory effect of C. buchanani leaves aqueous extract against S. aureus ATCC 25923, E. coli ATCC 25922, S. typhimurium ATCC 14028, K. pneumoniae ATCC 10031, P. vulgaris ATCC 13315, B. subtilis ATCC 6633, L. plantarum ATCC 14917 and S. epidermidis ATCC 12228 with low concentrations. The evidence supports the used of C. buchanani aqueous extract as antifood-borne pathogen bacteria. It also showed the good antimicrobial activity against nosocomial infection bacteria (K. pneumoniae ATCC 10031 and P. vulgaris ATCC 13315) even though the plant aqueous extract cannot inhibit growth of Ps. aeruginosa ATCC 9721.
In conclusion, C. buchanani aqueous extract showed broad spectrum inhibitory effect on growth of all food-borne pathogen bacteria and some of nosocomial bacteria. This may support use of C. buchanani as antimicrobial remedy. However, it’s active compound should also been studied to evaluated the mechanism of action and those toxicity of the plant as well as to supported the safety of used this plant extract as the food additive.
ACKNOWLEDGMENT
This study was partially financial supported from Faculty of Science, Mahasarakham University, Thailand. Authors declared that there are no conflicts of interest in this study.