Agarwood is mainly produced by trees in the species of Aquilaria (family Thymelaeaceae). These plants provide economically important natural products which are used for the production of incense, perfumes and traditional medicines in Asia . At least four species of agarwood trees are found in tropical rainforest areas of Thailand, namely Aquilaria crassna Pierre ex Lecomte, A. subintegra, A. malaccensis, and A. rugosa. Owing to over-exploitation, these plant species are nowadays considered as endangered species of Southeast Asia. Therefore, cultivation of agarwood is encouraged to reduce the harvest from wild populations. Since it takes about 10 years before the wood can produce valuable essential oil or resin, leaves from young agarwood are collected for production of healthy tea in Viet Nam, Combodia and Thailand.
The agarwood extract has been used as one of the active ingredients in several Thai traditional pharmaceutical preparations, such as “Krisanaglun” which is used as antispasmodic, antidiarrheal agent and cardiovascular function enhancer in fainted patient. Based on Thai folklore information, several parts of agarwood have been used for a long time in the treatment of infectious diseases such as diarrhoea, dysentery and skin diseases. Recently, the antibacterial activities of A. crassna leaf extract against enteric bacteria, such as Staphylococcus aureus, Clostoridium difficile, Peptostreptococcus anaerobius and Bacteroides fragilis, have been reported . However, its inhibitory effect on S. epidermidis, the pathogen known to cause skin disease and one of the most important opportunistic pathogens, has never been documented. In the present study, the antibacterial activity against S. epidermidis of the aqueous extract of A. crassna leaves and possible mechanism were investigated. The phytoconstiuents, antioxidant properties and acute toxicity of the extract were studied as well.
A. crassna leaves were collected from a cultivated field in Nakhon Ratchasima province, Thailand. The plant was identified by a botanist, Dr. Paul J. Grote, School of Biology, Suranaree University of Technology (SUT) and specimen of the plant has been kept at School of Pharmacology, SUT. The voucher specimen number is Pharm-Chu-005.
The leaves were oven dried at 50°C, and then cut into small pieces. Dried leaves (24 g) were extracted in boiling water (400 ml) for 30 min twice. The pooled extracts were filtered and concentrated at 40°C using a rotary evaporator under low pressure. The residue was freeze-dried in a lyophilizer. The extract with a total yield of 14.2% was stored at −20°C until used.
Phytochemical screening procedures were carried out according to the standard methods previously reported [4, 5]. Qualitative phytochemical compositions of the crude extract of Aquilaria crassna leaves were determined for the presence of alkaloids, flavonoids, tannins, saponins and cardiac glycosides.
Determination of total phenolic compounds
The amount of total phenolic compounds was measured by a method described by Matthaus . In brief, 5 mg of the extract was dissolved in 1 ml of distilled water. A 100 μl aliquot of this mixture was added to 2 ml of 2% Na2CO3 followed by 100 μl of Folin-Ciocalteau reagent in methanol (1:1 v/v). After 30 min of incubation, the absorbance was measured at 750 nm. The concentration was calculated using gallic acid as a standard. The results were expressed as milligrams gallic acid equivalents (GAE) per gram extract.
Determination of antioxidant activity
Scavenging effects on DPPH radicals
To measure antioxidant activity, the 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) radical scavenging assay was carried out according to the procedure described previously . The crude extract (100 μl; final concentration range from 0–50 μg/ml) was added to 4.0 ml of 50 μM DPPH in methanolic solution and the final volume was adjusted to 5.0 ml with water. After vortexing, the mixture was incubated for 30 min in the dark at room temperature. The decrease in absorbance at 517 nm was measured using a spectrophotometer. Antioxidant activity was expressed as IC50, which was defined as the concentration of the extract required to inhibit the formation of DPPH radicals by 50%.
ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) radical-scavenging activity of extract was carried out according to the procedure described previously . ABTS radical cation (ABTS•+) was produced by the reaction between 5 ml of 14 mM ABTS and 5 ml of 4.9 mM potassium persulfate (K2S2O8). The resulting solution was stored in the dark at room temperature for 16 h. Before used, the solution was diluted with ethanol to give an absorbance of 0.700 ± 0.020 at 734 nm. The plant extract (50 μl) at various concentrations were added to 950 μl of ABTS solution and mixed thoroughly. The reaction mixture was allowed to stand at room temperature for 6 min, the absorbance was measured at 734 nm and compared to the standard butylated hydroxytoluene (BHT).
Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was conducted according to procedure described by Dordevic et al. with minor modification. The FRAP reagent consists of 10 mM TPTZ (2,4,6-tripyridyl-striazine) in 40 mM HCl, 20 mM FeCl3, and 300 mM acetate buffer (pH 3.6) in proportions of 1:1:10 (v/v/v). Fifty μl of the sample was added to 1.5 ml of FRAP reagent (freshly prepared and warmed to 37°C before used). The absorbance was measured at 593 nm using a UV spectrophotometer after 4 min of incubation. A standard curve was constructed using FeSO4 solution. The results were expressed as μmol Fe2+/mg dry weight of plant material. All measurements were carried out in triplicate and the mean values were calculated.
Disc diffusion assay
The antibacterial activity of the crude extract was assayed against S. epidermidis (obtained from Thailand Institute of Scientific and Technological Research; TISTR 518) using disc diffusion method previously described . Briefly, 100 μl of bacteria (108 CFU/ml) was spread onto the Mueller-Hinton agar plate. The extract (2, 4 and 6 mg) was applied to filter paper discs (Whatman No. 1, 6 mm diameter) and then placed on the previously inoculated agar plate. After 24 h of incubation at 37°C, clear inhibition zones around the discs indicated the presence of antibacterial activity. The assay was carried out in triplicates. Vancomycin (30 μg) was used as a positive control.
Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
Determination of the minimum inhibitory concentration (MIC) against S. epidermidis (104 CFU/ml) was conducted by a two-fold serial dilution method in Mueller-Hinton broth (MHB). MIC was considered as the lowest concentrations of the agents that yielded no visible growth of microorganisms after 24 h of incubation at 37°C. The MBC determination was carried out by subculturing 100 μl from each tube from the MIC assay onto fresh substance-free MH agar plates. The MBC was defined as the lowest concentration of agent that produced no growth of subcultures.
The inhibitory effect of the extract on biofilm formation was examined by microscopic analysis. A culture of S. epidermidis was prepared in tryptic soy broth (TSB) at 37°C for 24 h. 30 μl aliquots of the culture were pipetted into each well of 24-well plates in the presence of 3 ml TSB and incubated at 37°C for 24 h to form biofilm. Thereafter, medium was replaced with fresh medium containing the extract of A. crassna leaves or vancomycin. After incubation of another 24 h, the medium was removed and each well was gently washed three times with phosphate buffer solution. The inhibitory effect on biofilm formation was observed by phase contrast microscopy.
Scanning electron microscopy
Scanning electron microscopy (SEM) was performed on S. epidermidis treated with MIC of A. crassna leaf extract. S. epidermidis was cultured to reach mid-log phase in MHB before use. Control and treated cells were prepared for morphological observation. The bacterial samples were washed five times with fresh media and then fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.2) at 4°C for 1 h, washed three times with phosphate buffer for 10 min and fixed with 1% osmium tetroxide for 2 h. This was followed by three washings in phosphate buffer for 10 min and subsequently dehydrated in a series of ethanol concentrations (30%, 50%, 70%, 90% and 95%), for 15 min each. The samples were subjected to 100% ethanol and CO2 to achieve the critical point and then coated with gold ion in a pressure metallic chamber. At the end of the process, the samples were submitted for analysis by SEM.
Transmission electron microscopy
Cellular damage of bacteria was examined using transmission electron microscopy (TEM). Bacterial cells treated with vehicle, vancomycin and the extract of A. crassna leaves were harvested after 24 h of incubation and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, for 2 h. The cells were washed three times with 0.05 M phosphate buffer (pH 7.2) and postfixed for 2 h with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.2) at room temperature. After washed twice in phosphate buffer, the cells were dehydrated through serial graded concentrations of ethanol (35, 70, 95 and 100%, respectively) for 15 min, then infiltrated and embedded in Spurr’s resin. Ultrathin sections were cut with a diamond knife using an ultramicrotome and then mounted on bare copper grids. Finally, specimens were counterstained with 2% (w/v) for 3 min and then with 0.25% (w/v) lead citrate solution for 2 min and examined with Tecnai G2 electron microscope (FEI, USA) operated at 120 kV.
Acute toxicity in mice
The acute toxicity test performed in this experiment was conducted by following the OECD guidelines for Testing of Chemicals (2001) . The aqueous extract of A. crassna leaves was prepared in the concentrations of 200 mg/ml and 750 mg/ml by dissolving in distilled water for dosing group of 2,000 and 15,000 mg/kg body weight respectively. All mice were monitored for clinical signs of toxicity at 0.5, 1 and 3 h after oral administration of the extract and once daily thereafter for 14 days. The organs of animals were removed immediately after sacrificed on day 14 and then examined macroscopically for pathological changes.
All experimental results were expressed as means ± standard deviation. The differences among groups in the acute toxicity test were analyzed by one way ANOVA followed by Student-Newman-Keuls test. The results with p value < 0.05 were considered as statistically significant differences.