Dayang Fredalina Basri

The potential of aqueous and acetone extracts of galls of Quercus infectoria as antibacterial agents

OBJECTIVE: To evaluate the antibacterial potential of aqueous and acetone extracts of galls of Quercus infectoria by determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values. MATERIALS AND METHODS: The extracts from the galls of Q. infectoria at 10 mg/ml were screened against three Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis and Bacillus subtilis) and three Gram-negative bacteria (Escherichia coli NCTC 12079 serotype O157:H7, Salmonella typhimurium NCTC 74 and Pseudomonas aeruginosa ATCC 27853). The MIC of the extracts were then determined using the twofold serial microdilution technique at a concentration ranging from 5 mg/ml to 0.0024 mg/ml. The MBC values were finally obtained from the MIC microtiter wells which showed no turbidity after 24 hrs of incubation by subculturing method. RESULTS: Out of the six bacterial species tested, S. aureus was the most susceptible. On the other hand, the extracts showed weak inhibitory effect against S. epidermidis, B. subtilis, S. typhimurium and P. aeruginosa while there was no inhibition zone observed for E. coli O157. The MIC values of the extracts ranged from 0.0781 mg/ml to 1.25 mg/ml whereas the MBC values ranged from 0.3125 mg/ml to 2.50 mg/ml. The MBC values of aqueous extract against S. aureus and S. typhimurium were higher than their MIC values. The MBC value of acetone extract against S. aureus was also higher than its MIC value. Interestingly, however, the MIC and MBC values of acetone extract against S. typhimurium were the same (1.25 mg/ml). CONCLUSION: The aqueous and acetone extracts displayed similarities in their antimicrobial activity on the bacterial species and as such, the galls of Quercus infectoria are potentially good source of antimicrobial agents.

In Vitro Antibacterial Activity of Galls of Quercus infectoria Olivier against Oral Pathogens

The galls of Quercus infectoria are commonly used in Malay traditional medicine to treat wound infections after childbirth. In India, they are employed traditionally as dental applications such as that in treatment of toothache and gingivitis. The aim of the present study was to evaluate the antibacterial activity of galls of Quercus infectoria Olivier against oral bacteria which are known to cause dental caries and periodontitis. Methanol and acetone extracts were screened against two Gram-positive bacteria (Streptococcus mutans ATCC 25175 and Streptococcus salivarius ATCC 13419) and two Gram-negative bacteria (Porphyromonas gingivalis ATCC 33277 and Fusobacterium nucleatum ATCC 25586). The screening test of antibacterial activity was performed using agar-well diffusion method. Subsequently, minimum inhibitory concentration (MIC) was determined by using twofold serial microdilution method at a concentration ranging between 0.01 mg/mL and 5 mg/mL. Minimum bactericidal concentration (MBC) was obtained by subculturing microtiter wells which showed no changes in colour of the indicator after incubation. Both extracts showed inhibition zones which did not differ significantly (P < 0.05) against each tested bacteria. Among all tested bacteria, S. salivarius was the most susceptible. The MIC ranges for methanol and acetone extracts were the same, between 0.16 and 0.63 mg/mL. The MBC value, for methanol and acetone extracts, was in the ranges 0.31–1.25 mg/mL and 0.31–2.50 mg/mL, respectively. Both extracts of Q. infectoria galls exhibited similar antibacterial activity against oral pathogens. Thus, the galls may be considered as effective phytotherapeutic agents for the prevention of oral pathogens.

Bacteriostatic antimicrobial combination: antagonistic interaction between epsilon-viniferin and vancomycin against methicillin-resistant Staphylococcus aureus.

Stilbenoids have been considered as an alternative phytotherapeutic treatment against methicillin-resistant Staphylococcus aureus (MRSA) infection. The combined effect of ε-viniferin and johorenol A with the standard antibiotics, vancomycin and linezolid, was assessed against MRSA ATCC 33591 and HUKM clinical isolate. The minimum inhibitory concentration (MIC) value of the individual tested compounds and the fractional inhibitory concentration index (FICI) value of the combined agents were, respectively, determined using microbroth dilution test and microdilution checkerboard (MDC) method. Only synergistic outcome from checkerboard test will be substantiated for its rate of bacterial killing using time-kill assay. The MIC value of ε-viniferin against ATCC 33591 and johorenol A against both strains was 0.05 mg/mL whereas HUKM strain was susceptible to 0.1 mg/mL of ε-viniferin. MDC study showed that only combination between ε-viniferin and vancomycin was synergistic against ATCC 33591 (FICI 0.25) and HUKM (FICI 0.19). All the other combinations (ε-viniferin-linezolid, johorenol A-vancomycin, and johorenol A-linezolid) were either indifferent or additive against both strains. However, despite the FICI value showing synergistic effect for ε-viniferin-vancomycin, TKA analysis displayed antagonistic interaction with bacteriostatic action against both strains. As conclusion, ε-viniferin can be considered as a bacteriostatic stilbenoid as it antagonized the bactericidal activity of vancomycin. These findings therefore disputed previous report that ε-viniferin acted in synergism with vancomycin but revealed that it targets similar site in close proximity to vancomycins action, possibly at the bacterial membrane protein. Hence, this combination has a huge potential to be further studied and developed as an alternative treatment in combating MRSA in future.

Prevalence and antibiotic resistance of Salmonella Enteritidis and Salmonella Typhimurium in raw chicken meat at retail markets in Malaysia

Salmonellosis is one of the major food-borne diseases in many countries. This study was carried out to determine the occurrence of Salmonella spp., Salmonella Enteritidis, and Salmonella Typhimurium in raw chicken meat from wet markets and hypermarkets in Selangor, as well as to determine the antibiotic susceptibility profile of S. Enteritidis and S. Typhimurium. The most probable number (MPN) in combination with multiplex polymerase chain reaction (mPCR) method was used to quantify the Salmonella spp., S. Enteritidis, and S. Typhimurium in the samples. The occurrence of Salmonella spp., S. Enteritidis, and S. Typhimurium in 120 chicken meat samples were 20.80%, 6.70%, and 2.50%, respectively with estimated quantity varying from <3 to 15 MPN/g. The antibiogram testing revealed differential multi-drug resistance among S. Enteritidis and S. Typhimurium isolates. All the isolates were resistance to erythromycin, penicillin, and vancomycin whereas sensitivity was recorded for Amoxicillin/Clavulanic acid, Gentamicin, Tetracycline, and Trimethoprim. Our findings demonstrated that the retail chicken meat could be a source of multiple antimicrobial-resistance Salmonella and may constitute a public health concern in Malaysia.

Evaluation of the Combined Effects of Stilbenoid from Shorea gibbosa and Vancomycin against Methicillin-Resistant Staphylococcus aureus (MRSA)

The aim of this study is to determine the combined effects of stilbenoids from Shorea gibbosa and vancomycin against methicillin-resistant Staphylococcus aureus (MRSA). A total of nine pure compounds, five stilbenoid dimers ε-viniferin, ampelopsin A, balanocarpol, laevifonol and diptoindonesin G and four stilbenoid trimers α-viniferin, johorenol A, ampelopsin E and vaticanol G were evaluated for their antibacterial activities against ATCC 33591 and a HUKM clinical isolate. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for each active compound were determined using the serial microdilution and plate-streak techniques. The combined effect of stilbenoids with vancomycin against MRSA was evaluated using the checkerboard assay to determine their fractional inhibitory concentration (FIC) index values. The MIC value of α-viniferin on both MRSA strains was 100 μg/mL, whereas those of johorenol A on ATCC 33591 and HUKM strain were 100 μg/mL and 200 μg/mL, respectively. The MIC values of ampelopsin E and vaticanol G were higher than 400 μg/mL. Out of the five stilbenoid dimers, only ε-viniferin was capable of inhibiting the growth of both MRSA strains at MIC 400 μg/mL. The MBC value of ε-viniferin, α-viniferin and johorenol A showed bacteriostatic action against MRSA. The FIC index value of ε-viniferin and α-viniferin in combination with vancomycin showed an additive effect (0.5 < FIC ≤ 2.0) against both MRSA strains. Johorenol A-vancomycin combination was also additive against HUKM strain, but it showed synergistic interaction with vancomycin against ATCC 33591 (FIC < 0.5). Stilbenoid compounds from Shorea gibbosa have anti-MRSA activity and huge potential as an alternative phytotherapy in combating MRSA infections.

Pharmacodynamic Interaction of Quercus infectoria Galls Extract in Combination with Vancomycin against MRSA Using Microdilution Checkerboard and Time-Kill Assay

The galls of Quercus infectoria Olivier possess astringent properties which helps in the tightening of the vaginal epithelium in the post-natal period. The present study aimed to observe the time-kill kinetics of the acetone and methanol extracts of gall of Q. infectoria in combination with vancomycin against two methicillin-resistant Staphylococcus aureus (MRSA) strains; ATCC 33591 and MU 9495 (laboratory-passaged strain). Minimum inhibitory concentration (MIC) of the extracts were determined using microdilution technique whereas the checkerboard and time-kill kinetics were employed to verify the synergistic effects of treatment with vancomycin. The FIC index value of the combinations against both MRSA strains showed that the interaction was synergistic (FIC index <0.5). Time-kill assays showed the bactericidal effect of the combination treatment at 1/8XMIC of the extract and 1/8XMIC of vancomycin, were respectively at 7.2 ± 0.28 hr against ATCC 33591 compared to complete attenuation of the growth of the same strain after 8 hr of treatment with vancomycin alone. In conclusion, the combination extracts of Q. infectoria with vancomycin were synergistic according to FIC index values. The time-kill curves showed that the interaction was additive with a more rapid killing rate but, which did not differ significantly with vancomycin.

Evaluation of Analgesic Activity of the Methanol Extract from the Galls of Quercus infectoria (Olivier) in Rats

The present study aims to investigate the analgesic activity of the methanol extract of the galls of Quercus infectoria in rats using hot plate and tail-flick methods. The extract was administered intraperitoneally at a dose of 20 mg/kg while morphine sulfate and sodium salicylate (10 mg/kg) served as standards. The methanol extract exhibited significant analgesic activity in the tail-flick model (P < 0.05) by increasing the reaction time of the rats to 8.0 sec at 30 min after treatment in comparison to control (4.4 sec). Morphine sulfate produced a reaction time of 11.9 sec in the same test. At the peak of activity (30 min), the extract produced maximum possible analgesia (MPA) of 34.2%, whilst morphine sulfate achieved a peak MPA of 70.9%. No analgesic effects have been observed using sodium salicylate in the tail-flick model. In the same model, the extract and sodium salicylate demonstrated comparable reaction times. Tail-flick is a better method to evaluate analgesic activity as no significant results were observed for all treatments using hot plate with the exception of morphine sulfate, which showed significant results only at 45 and 60 min after treatment. In conclusion, the methanol extract of the galls of Quercus infectoria displayed analgesic activity.

Leaves Extract from Canarium odontophyllum Miq. (Dabai) Exhibits Cytotoxic Activity against Human Colorectal Cancer Cell HCT 116

Colorectal cancer is the second most common cancer occurs in Malaysia after breast cancer. Bioactive compounds that reside in Canarium odontophyllum leaves could probably have the potential to being develop as an alternative cytotoxic agent against colorectal cancer. The objective of this study is to evaluate the cytotoxic effect of acetone, methanol and water extracts from the leaves of C. odontophyllum against human HCT 116 colorectal carcinoma cells using MTT assay. Result showed that the percentage yield of the extraction from C. odontophyllum leaves was the highest when distilled water was used as an extraction solvent followed by methanol extract and acetone extract. Phytochemical screening revealed the presence of flavonoid, tannin and terpenoid in the three extracts. All the extracts demonstrated cytotoxic effect after 24-hr treatment with acetone extract at IC50 value of 0.08 ± 0.003 mg/mL against HCT 116 cells compared to methanol and aqueous extracts with IC50 values of 0.10 ± 0.011 mg/mL and 0.40 ± 0.162 mg/mL, respectively. In conclusion, this preliminary study of the C. odontophyllum leaves extract against HCT116 cells provides an insight of its promising anticancer property.

Determination of the post-antibiotic effect (PAE) of combinations of extracts from galls of Quercus infectoria with vancomycin against methicillin-resistant Staphylococcus aureus (MRSA)

Post-antibiotic effect (PAE) is one of the pharmacodynamic parameters that can be defined as the time it takes for the microorganisms to regain its normal growth after the complete removal of the antimicrobial agent. PAE on 2 strains of methicillin-resistant staphylococcus aureus (MRSA)- Staphyloccous were induced by galls of Quercus infectoria in combination with vancomycin. The determination of minimum inhibitory concentration (MIC) and PAE were carried out on two strains of S. aureus with vancomycin, methanol and acetone extracts. The test for fractional inhibitory concentration (FIC) index was done to verify the type of interaction of the combinations using checkerboard assay. The FIC value obtained for methanol and acetone extract with vancomycin against both strains of MRSA indicated the interaction of these combinations as synergistic. The combination of methanol and acetone extract with vancomycin significantly enhanced the PAE for both MRSA strains compared to the PAE when these agents were used singly. Both combinations of methanol extract with vancomycin and acetone extract with vancomycin gave slightly higher PAE values for reference strain, MRSA ATCC 33591 compared to the passaged strain, Mu 9495. The longer PAE of extracts from galls of Quercus infectoria in combination with vancomycin in comparison to that of singly tested extracts and antibiotic could have some potential implications for the timing of doses during therapy with antimicrobial combinations against MRSA. Key words: Post antibiotic effect, combination, Quercus infectoria, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin.

Prevalence, virulence genes and antimicrobial resistance profiles of Salmonella serovars from retail beef in Selangor, Malaysia

The aim of the present study was to investigate the prevalence of Salmonella spp., Salmonella Enteritidis and Salmonella Typhimurium in retail beef from different retail markets of Selangor area, as well as, to assess their pathogenic potential and antimicrobial resistance. A total of 240 retail beef meat samples (chuck = 60; rib = 60; round = 60; sirloin = 60) were randomly collected. The multiplex polymerase chain reaction (mPCR) in combination with the most probable number (MPN) method was employed to detect Salmonella spp., S. Enteritidis and S. Typhimurium in the meat samples. The prevalence of Salmonella spp., S. Enteritidis and S. Typhimurium in 240 beef meat samples were 7.50, 1.25, and 0.83%, respectively. The microbial loads of total Salmonella was found in the range of <3 to 15 MPN/g. Eight different serovars of Salmonella were identified among the 23 isolates, and S. Agona was the predominant serovar (26.09%). Interestingly, all the Salmonella isolates were resistant to penicillin, erythromycin and vancomycin, but the sensitivity was observed for tetracycline, gentamicin and amoxicillin/clavulanic acid. All 23 isolates were resistant to at least three antibiotics. Two S. Typhimurium isolates (8.70%) exhibited the highest multiple antibiotic resistance (MAR) index value of 0.56 which shown resistance to nine antibiotics. PCR analysis of virulence genes showed that all Salmonella isolates (100%) were positive for the invA gene. Meanwhile, pefA was only identified in S. Enteritidis and S. Typhimurium. The findings in this study indicate that retail beef products tested were widely contaminated with multi-drug resistant (MDR) Salmonella and various virulence genes are present among the isolated Salmonella serovars.