Abstract: The in vitro antibacterial efficacy of aqueous and methanolic extract of Quercus infectoria Olivier (Fagaceae) galls was tested against Cellulosimicrobium cellulans using extract concentration ranging from 0.25 to 4 mg mL-1. Both types of extract showed significant inhibition of C. cellulans growth with strong correlation between extract concentrations and degrees of antibacterial activity for concentrations ranging from 0.5 to 4 mg mL-1. Although, slight reduction of average diameter of inhibition zones after 24 h of incubation for aqueous extract (0.96 ± 0.148 cm) compared to methanolic extract (1.00 ± 0.182 cm), both extracts still attained the MIC value beginning at a concentration of 0.5 mg mL-1 but established higher concentration for the MBC at 2 mg mL-1. The antibacterial activity of methanolic extract was also significantly affected by the temperature with an optimum inhibition zone being obtained at 30 °C (1.38 ± 0.05 cm) and this was reduced to approximately 20% at temperatures of above 50 °C.
INTRODUCTION
Investigations of traditionally used plants for biologically active extracts had been well-documented. Recent studies have revealed that medicinal plants from various parts of the world could be rich sources of antibacterial and antimicrobial activities (Voravuthikunchai and Kitpipit, 2005; Cowan, 1999). Antibiotic-resistant bacteria (Voravuthikunchai et al., 2006), stimulation of toxin production (Cowan, 1999) and the recent upturn in consumer mistrust of synthetic additives have pushed the search for natural compounds from plants to replace antibiotics or artificial antimicrobials. Even though certain plants have been demonstrated to have effects against pathogenic bacteria, the majority of them have not yet been investigated for their antibacterial activities.
In this research, Quercus infectoria Olivier (Fagaceae) was studied in order to investigate its antibacterial properties. Quercus infectoria is a round-shaped abnormal growth found arising on young branches of the oak tree as a result of attack by the gall-wasp Adleria gallae-tinctoria (Samuelsson, 1999). Oak gall consists of gland (camata, fruit in cupola), hoof (trillo; sharp and stubby points covered with cupola) and cup (cupula; outside surface of oak gall). Research has shown that Q. infectoria is rich in bioactive compounds such as tannin (Haghi and Safaei, 2004), vitamins A and C, calcium, iron, fiber, protein and carbohydrates (Jalalpure et al., 2002) and has the ability to be an antimicrobial (Everest and Ozturk, 2005), antibacterial (Hamid et al., 2005) and antifungal agent (Yamunarani et al., 2005).
Cellulosimicrobium cellulans (previously identified as Oerskovia xanthineolytica or Brevibacterium fermentans or Arthrobacter luteus), a gram-positive bacterium belonging to the order Actinomycetales (Schumann et al., 2001) was selected to determine the antibacterial activity of Q. infectoria. Cellulosimicrobium cellulans is relatively a virulent and rarely associated with infections in humans (Rowlinson et al., 2006). However, it has been associated with the presence of foreign bodies and is generally found in immunocompromised patients (Kaur et al., 2004). Early case reports described meningitis and sepsis in infants and children due to infection by C. cellulans which at in the late 60s was known as B. fermentans (Tenover, 2001; Kailath et al., 1988). A few years later, other reports related C. cellulans to endocarditis, pyonephrosis, endophthalmitis, pneumonia, meningitis, parenteral nutrition-related septicemia, catheter-related septicemia and peritonitis (Maguire et al., 1996).
In this study, the screening of the antibacterial activities of methanolic and aqueous extracts of the galls of Q. infectoria against C. cellulans by the determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) are described. This study also investigated the effects of different solvents and temperatures on these activities.
MATERIALS AND METHODS
The experiment: This experiment was conducted at Plant Systematics and Microbial Laboratory, Biology Department, Universiti Putra Malaysia, Malaysia from July to September 2007.
Plant material: Quercus infectoria Olivier (Fagaceae) was purchased from the local herbal shop. The oak galls were washed with water, then surface sterilized with 10% sodium hypochlorite solution before they were rinsed with sterile distilled water and air dried at 30 °C. Then the samples were crushed into pieces and grinded into powder using a grinder before being sieved to get only a fine powder.
Preparation of plant extracts: Fifty grams of Q. infectoria fine powder were soaked in 125 mL of 80% (w/v) methanol or homogenized in sterile distilled water for preparing the methanolic and aqueous extracts, respectively. This mixture was then stirred for 24 h at 30 °C and filtered using Whatman No. 1 filter paper. Lastly, the filtrate was evaporated by using rotary evaporator (40-60 °C) until fully dried.
Preparation of test extract solutions: A series of different Q. infectoria extract concentrations (0.25, 0.50, 0.75, 1, 2, 3 and 4 mg mL-1) were prepared by dissolving a known weight of the plant extract in 5% (v/v) dimethyl sulphoxide (DMSO) which acted as a solvent.
Preparation of microorganism culture: Cellulosimicrobium cellulans ATCC 21606 was obtained from the Plant Systematics and Microbial Laboratory, Biology Department, Universiti Putra Malaysia and maintained on Nutrient Agar slant. Preparation of the test strain culture was carried out by isolating a single colony of C. cellulans from the nutrient agar and transferred into 250 mL Nutrient Broth before being shaken at 180 rpm for 24 h.
Antibacterial assays: Agar well diffusion assay: The agar well diffusion was prepared by adding 1x105 cfu mL-1 C. cellulans culture into melt nutrient agar and homogenized slowly for a few seconds. The mixture was then poured into a petri dish and allowed to solidify prior to the preparation of 0.6 cm diameter wells made by using a sterilized cork borer. Twenty microliter of Q. infectoria extract solution in different concentration were transferred into each well and allowed to set. All the plates were incubated at 37 °C and the diameters of the inhibition zone surrounding each well were measured to the nearest millimeter at every 12 h internal. Sets of 5 replicates were used for each type of extract.
Determination of the Minimum Inhibitory Concentration (MIC): This process was conducted using microtiter plates and the same series of Q. infectoria extract concentrations as previously used in the antibacterial assays. Twenty microliter of Q. infectoria extract were mixed into 80 μL sterile Nutrient Broth before 1x105 cfu mL-1 C. cellulans culture was added. The microtiter plates were incubated at 37 °C for 24 h and the lowest Q. infectoria extract concentration that did not show any growth of C. cellulans after microscopic evaluations was determined as being the MIC.
Determination of the Minimum Bacterialcidal Concentration (MBC): All the microtiter wells used in the MIC determination which did not show any growth of C. cellulans after the incubation period were subcultures onto fresh Nutrient Agar plates and incubated further for 24 h at 37 °C. The least concentration with no C. cellulans growth was considered as being the MBC value.
Effects of temperature on antibacterial activity: The MBC concentration for Q. infectoria extract solution from the antibacterial assay experiment was used in this study. The six different temperatures tested in this experiment were 10, 30, 50, 70, 90 and 100 °C, respectively. Twenty microliter of Q. infectoria extract solution was heated to temperature required before being allowed to cool to room temperature and dispensed into the well made on a Nutrient Agar. Five replicates of each concentration were prepared and all the plates were incubated at 37 °C for 120 h before the diameters of the inhibition zones were measured.
Statistical analysis: The results obtained were expressed as Mean ± SEM. The data were analyzed using the Tukey test at the 5% significance level.
RESULTS AND DISCUSSION
The overall inhibitory concentration value of the methanolic and aqueous extracts from the galls of Q. infectoria against C. cellulans are shown in Table 1; with methanol 80% (v/v) as a negative control treatment with no inhibitory effect on the bacteria tested. Inhibition started to appear for extract for both solvents at 0.5 mg mL-1. However this inhibition zone can only be sustained for 24 h before it became cloudy and the bacteria colony started to grow. The outcome of this antibacterial assay was supported by the result of the MIC determination (Table 1) which showed that no C. cellulans could be seen under microscopic observation. In spite of this, values of MBC were much higher for both solvents being at 2 mg mL-1 Q. infectoria extract. This means that at 0.5 mg mL-1 concentration, aqueous and methanolic extracts of Q. infectoria could only be act as bacteriostatic agents rather than as bactericidal for C. cellulans. At this concentration, the bioactive compound was unable to eliminate C. cellulans or to sustain the activity for a long period thus allowing the bacteria to grow. Two possible explanations for this bacteriostatic effect are (i) the bioactive compound in the extract was not adequate to cause significant mortality to the bacteria (Basri and Fan, 2005) (ii) the sensitivity of the bioactive compound toward a certain type of solvent might cause or enhance the rate of deactivation or degradation (Matu and Staden, 2003).
After 24 h incubation, all concentrations except for 0.25 mg mL-1 for both solvents had shown drastically increases in the diameter of the inhibition zones with averages of 0.96 ± 0.148 and 1.00 ± 0.182 cm for the aqueous and methanolic extracts, respectively. This might be directly caused by immature bacteria being less resistant to antibacterial activities at this stage (Beukinga et al., 2004). However, slow drops in the inhibition zones diameters were observed after 48 h and after 120 h the sizes were reduced by about 3% for the aqueous and 2.2% for the methanolic extracts. The unsustainable antibacterial activities might due to the enzyme starting to degrade and the bacteria becoming much more dominant (Beukinga et al., 2004). Nonetheless, there were still strong correlations between extract concentrations and the diameters of the inhibition zones at this stage with correlation coefficients of 0.735 and 0.790 for the aqueous and the methanolic extracts, respectively. The concentration of the extract played an important role in the antibacterial activity in that a higher antibacterial activity would be obtained with a concentrated extract (de Boer et al., 2004; Sawangjaroen et al., 2004).
The fact that C. cellulans was categorized as a gram-positive
bacteria (Schumann et al., 2001) has some contribution towards
the effectiveness of Q. infectoria extract as a bacteriostatic
or bactericidal agent. Generally plant extracts are much more active against
gram-positive bacteria than against gram-negative bacteria (Lin et
al., 1999; Cimanga et al., 2002) and this was demonstrated
by the positive effects of several plants extracts on other gram-positive
bacteria such as S. epidermidis, Bacillus subtilis (Fatima
et al., 2001) and Pseudomonas aeruginosa (Nimri et al.,
1999).
| Table 1: | Antibacterial activity of the Q. infectoria extract prepared using different solvents against C. cellulans |
Means in each column
with same superscript letter are not significantly different amongst
themselves when Tukey tests were used at 5% significance level. |
|
The type of solvent used in the extract preparation also greatly influenced the bioactive compound extraction (Pinelo et al., 2005). Due to the difference in the degree of polarity between aqueous and methanol, a difference in antibacterial activity was expected. Although the means of the inhibition zones diameter for all concentrations after 120 h, incubation were larger for the methanolic when compared to the aqueous extract; analysis of variance (ANOVA; p = 0.05) showed no significant difference in size between these two types of solvents. The similarity in the antimicrobial activity of both extracts suggested that these extracts may have high total tannin contents as tannin is a major compound in Q. infectoria (Jalalpure et al., 2002) which is soluble in water, alcohol and acetone (Basri and Fan 2005). Tannin is a form of phenolic acid (Chung et al., 1998) and alcoholic solvents have been commonly employed to extract phenolics from natural sources even though alcoholic solvents are not highly selective for phenols because it is able to yield high quantities of total extract compared to other types of solvent (Spigno et al., 2006). This explained why the methanolic extract showed higher extraction capability compared to the aqueous extract and similar outcomes had been reported in comparisons of extraction capabilities between acetone and aqueous to extract bioactive compounds from Euclea natalensis (Lall and Meyer, 2000). The intermediate polarity solvents such as methylene dichloride, tetrahydrofuran, ethyl acetate and acetone were able to extract much higher quantities of plant compounds than polar (aqueous and methanol) or non-polar solvents such as hexane (Eloff et al., 2005). However, the use of alcohol and water mixture gave better phenolic constituents extraction than other solvent systems (Yilmaz and Toledo, 2006).
Two milligrams per milliliters of methanolic extract was selected to
be studied further on the effects of temperature based on the results
obtained from the antibacterial assay done previously. The largest inhibition
was shown at 30 °C, followed by 50 and 10 °C, while almost similar
size of inhibition zone was obtained for temperatures 70, 90 and 100 °C
(Table 2). The treatment at 30 °C did preserve the
antibacterial activity with the highest diameter of inhibition zone (1.38
± 0.05 cm) compared to the other temperatures. This was in accordance
with earlier reports that most plant extracts released considerable heat
and their effects were most optimum at this temperature (28-30 °C)
(Baysale et al., 2003). Thus, although the time and temperature
of extraction are important parameters to be optimized in order to minimize
the energy cost of the process and to
| Table 2: | Diameter of growth inhibition zone for C. cellulans after 120 h incubation with 2 mg mL-1 Q. infectoria methanolic extract heated to different temperatures |
Means in each column
with same superscript letter(s) are not significantly different
amongst themselves when Tukey tests were used at 5% significance
level.*Diameter of the inhibition zone was included 0.6 cm of the
well diameter and expressed as the mean ± SD; (N = 5) |
|
enhance both the solubility of the solute and the diffusion coefficient (Eloff et al., 2005), they may also cause certain phenolic compounds to be denatured (Pinelo et al., 2005).
In conclusion, this study had demonstrated the ability of Q. infectoria extract to act as a bactericidal agent. However, this is much dependent on the concentration applied, the temperature and type of solvent used in the extraction process. The potential of Q. infectoria extract in pharmacology as an antibacterial agent is enormous, but further biochemical analysis is required to prove this.
ACKNOWLEDGMENTS
The research grant from the Ministry of Higher Education Malaysia through Fundamental Research Grant Scheme (No. 01-01-07-114FR) and all staff of the PS and M Laboratory, Biology Department, Universiti Putra Malaysia are gratefully acknowledged.