Investigating the Antibacterial Effects of Synthetic Gamma-Lactam Heterocycles on Methicillin-Resistant Staphylococcus aureus Strains and Assessing the Safety and Effectiveness of Lead Compound MFM514.

Methicillin-resistant Staphylococcus aureus (MRSA) continues to be one of the main causes of hospital-acquired infections in all regions of the world, while linezolid is one of the only commercially available oral antibiotics available against this dangerous gram-positive pathogen. In this study, the antibacterial activity from 32 analogues of synthetic gamma-lactam heterocycles against MRSA was determined. Amongst screened analogues for the minimum inhibitory concentration (MIC) assay, compound MFM514 displayed good inhibitory activity with MIC values of 7.8-15.6 µg/mL against 30 MRSA and 12 methicillin-sensitive S. aureus (MSSA) clinical isolates, while cytotoxicity evaluations displayed a mean inhibitory concentration (IC50) value of > 625 µg/mL, displaying a potential to becoming as a lead compound. In subsequent animal studies for MFM514, a single-dose oral acute toxicity test revealed an estimated mean lethal dose (LD50) value of <5000 mg/kg, while in the mice infection test, a mean effective dose (ED50) value of 29.39 mg/kg was obtained via oral administration. These results suggest that gamma-lactam carbon skeleton, particularly MFM514, is highly recommended to be evaluated further as a new safe and efficacious orally delivered antibacterial agent against MRSA.

Diversity of endophytic fungi isolated from different plant parts of Acacia mangium, and antagonistic activity against Ceratocystis fimbriata, a causal agent of Ceratocystis wilt disease of A. mangium in Malaysia.

Acacia mangium is an important wood for commercial products especially pulp and medium-density fibreboard. However, it is susceptible to Ceratocystis fimbriata infection, leading to Ceratocystis wilt. Therefore, the present work aimed to (i) establish the diversity of endophytic fungi in different plant parts of A. mangium,and (ii) evaluate the antifungal potentials of the isolated and identified endophytic fungi against C. fimbriata. Endophytic fungal identification was conducted by PCR amplification and sequencing of the internal transcribed spacer 1 (ITS1) and ITS4 regions of nuclear ribosomal DNA. A total of 66 endophytic fungi were successfully isolated from different parts of A. mangium; leaf (21), stem (13), petiole (12), root (9), flower (6), and fruit (5). The endophytic fungal isolates belonged to Ascomycota (95.5%) and Zygomycota (4.5%). For Ascomycota 13 genera were identified: Trichoderma (28.6%), Nigrospora (28.6%), Pestalotiopsis (12.7%), Lasiodiplodia (9.5%), Aspergillus (6.3%), Sordariomycetes (3%), and Neopestalotiopsis, Pseudopestalotiopsis, Eutiarosporella, Curvularia, Fusarium, Penicillium, and Hypoxylon each with a single isolate. For Zygomycota, only Blakeslea sp. (5%) was isolated. Against C. fimbriata, Trichoderma koningiopsis (AC 1S) from stem, Nigrospora oryzae (AC 7L) from leaf, Nigrospora sphaerica (AC 3F) from the flower, Lasiodiplodia sp. (AC 2 U) from fruit, Nigrospora sphaerica (AC 4P) from petiole, and Trichoderma sp. (AC 9R) from root exhibited strong inhibition for C. fimbriata between 58.33 to 69.23%. Thus, it can be concluded that certain endophytic fungi of A. mangium have the potential to be harnessed as anti-Ceratocystis agent in future biotechnological applications.

In Vitro Evaluations and In Vivo Toxicity and Efficacy Studies of MFM501 against MRSA.

Previously we have discovered a synthetically derived pyrrolidone alkaloid, MFM501, exhibiting good inhibitory activity against 53 MRSA and MSSA isolates with low cytotoxicity against three normal cell-lines with IC50 values at >625 µg/ml. Time-kill assay, scanning electron microscopy (SEM) analysis, in vivo oral acute toxicity test, and mice peritonitis model were carried out in this study. In the time-kill study, MFM501 showed a less than 3 log10 decrease in bacterial colony concentration value (CFU/ml) which represented a bacteriostatic action while displaying a time-dependent inhibitory mechanism. Following that, SEM analysis suggested that MFM501 may exert its inhibitory activity via cytoplasmic membrane disruption. Moreover, MFM501 showed no toxicity effect on treated mice at an estimated median acute lethal dose (LD50) value of more than 300 mg/kg and less than 2000 mg/kg. For the efficacy test, a mean effective dose (ED50) of 87.16 mg/kg was obtained via a single dose oral administration. Our data demonstrated that MFM501 has the potential to be developed further as a new, safe, and effective oral-delivered antibacterial agent against MRSA isolates.

In vitro inhibitory and cytotoxic activity of MFM 501, a novel codonopsinine derivative, against Methicillin-Resistant Staphylococcus aureus clinical isolates.

28 new pyrrolidine types of compounds as analogues for natural polyhydroxy alkaloids of codonopsinine were evaluated for their anti-MRSA activity using MIC and MBC value determination assay against a panel of S. aureus isolates. One pyrrolidine compound, MFM 501, exhibited good inhibitory activity with MIC value of 15.6 to 31.3 µg/mL against 55 S. aureus isolates (43 MRSA and 12 MSSA isolates). The active compound also displayed MBC values between 250 and 500 µg/mL against 58 S. aureus isolates (45 MRSA and 13 MSSA isolates) implying that MFM 501 has a bacteriostatic rather than bactericidal effect against both MRSA and MSSA isolates. In addition, MFM 501 showed no apparent cytotoxicity activity towards three normal cell lines (WRL-68, Vero, and 3T3) with IC50 values of >625 µg/mL. Selectivity index (SI) of MFM 501 gave a value of >10 suggesting that MFM 501 is significant and suitable for further in vivo investigations. These results suggested that synthetically derived intermediate compounds based on natural products may play an important role in the discovery of new anti-infective agents against MRSA.

Antimicrobial compounds from Alpinia conchigera.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Alpinia conchigerahas been used as a condiment in the northern states of Peninsular Malaysia and occasionally in folk medicine in the east coast to treat fungal infections. In some states of Peninsular Malaysia, the rhizomes are consumed as a post-partum medicine and the young shoots are prepared into a vegetable dish. This study aimed to investigate the chemical constituents of the pseudostems and rhizomes of Malaysian Alpinia conchigera and to evaluate the antimicrobial activity of the dichloromethane (DCM) extracts of the pseudostems, rhizomes and the isolated compounds against three selected fungi and five strains of Staphylococcus aureus. MATERIALS AND METHODS: The dried and ground pseudostems (0.8kg) and rhizomes (1.0kg) were successively extracted in Soxhlet extractor using n-hexane, dichloromethane (DCM) and methanol. The n-hexane and DCM extracts of the pseudostem and rhizome were subjected to isolation and purification using column chromatography on silica gel using a stepwise gradient system (n-hexane to methanol). Briefly, a serial two fold dilutions of the test materials dissolved in DMSO were prepared prior to addition of 100µl overnight microbial suspension (108 cfu/ml) followed by incubation at 37°C (bacteria) or 26°C (dermatophytes and candida) for 24h. The highest concentration of DMSO remaining after dilution (5%, v/v) caused no inhibition to bacterial/candida/dermatophytes' growth. Antibiotic cycloheximide was used as reference for anticandidal and antidermatophyte comparison while oxacilin was used as reference for antibacterial testing. DMSO served as negative control. Turbidity was taken as indication of growth, thus the lowest concentration which remains clear after macroscopic evaluation was taken as the minimum inhibitory concentration (MIC). RESULTS: The isolation of n-hexane and DCM extracts of the rhizomes and pseudostems of Alpinia conchigera via column chromatography yielded two triterpenes isolated as a mixture of stigmasterol and ß-sitosterol: caryophyllene oxide, chavicol acetate 1, p-hydroxy cinnamaldehyde 2, 1'S-1'-acetoxychavicol acetate 3, trans-p-coumaryl diacetate 4, 1'S-1'-acetoxyeugenol acetate 5, 1'-hydroxychavicol acetate 6, p-hydroxycinnamyl acetate 7 and 4-hydroxybenzaldehyde. The DCM extract of the rhizome of Alpinia conchigera indicated potent antifungal activity against Candida albicans, Microsporum canis and Trycophyton rubrum with MIC values of 625µg/ml, 156µg/ml and 156µg/ml, respectively. It also showed significant inhibitory activity with MIC values between 17.88 and 35.75µg/ml against the mutant Staphylococci isolates MSSA, MRSA and Sa7. Amongst the isolated compounds, the lowest inhibition observed were of 1'S-1'-acetoxyeugenol against the dermatophytes (MIC 313µg/ml) followed by trans-p-coumaryl diacetate against both dermatophytes and candida (MIC 625µg/ml). The compound p-hydroxycinnamyl acetate strongly inhibited Staphylococcusaureus strain VISA (MIC 39µg/ml) followed by trans-p-coumaryl diacetate and 1'-hydroxychavicol acetate with MIC value of 156µg/ml. CONCLUSION: In conclusion, the observed antibacterial, anticandidal and antidermatophyte activity of the extracts and compounds obtained from the rhizome confirm the traditional use of Alpinia cochigera rhizome in the treatment of skin infection.

Chemical constituents and antimicrobial activity of the leaf and rhizome oils of Alpinia pahangensis Ridl., an endemic wild ginger from peninsular Malaysia.

The essential oils from the leaves and rhizomes of Alpinia pahangensis Ridl., collected from Pahang, Peninsular Malaysia, were obtained by hydrodistillation, and their chemical compositions were determined by GC and GC/MS analyses. The major components of the rhizome oil were γ-selinene (11.60%), ß-pinene (10.87%), (E,E)-farnesyl acetate (8.65%), and α-terpineol (6.38%), while those of the leaf oil were ß-pinene (39.61%), α-pinene (7.55%), and limonene (4.89%). The investigation of the antimicrobial activity of the essential oils using the broth microdilution technique revealed that the rhizome oil of A. pahangensis inhibited five Staphylococcus aureus strains with minimum inhibitory concentration (MIC) values between 0.08 and 0.31 µg/µl, and four selected fungi with MIC values between 1.25 and 2.50 µg/µl.

Inhibitory and resistance-modifying potential of plant-based alkaloids against methicillin-resistant Staphylococcus aureus (MRSA).

Increased prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has become a major threat to the health sector worldwide due to their virulence, limited therapeutic options and their distribution in both hospital and community settings. Discovery and development of new anti-MRSA agents as alternatives to the very few antibiotics left in the armamentarium are, thus, urgently required. Recently, an efflux mechanism in MRSA has been identified as one of the main contributors of resistance towards various structurally unrelated antibiotics. The potential of reserpine (a phytoalkaloid) as efflux pump inhibitor (EPI) against various microbes remains limited as the concentration needed for inhibition is toxic to humans. This study therefore aimed to evaluate 13 alkaloid compounds as potential inhibitory agents and/or potential EPIs against a panel of three MRSA isolates which not only differ in their susceptibility to vancomycin (amongst the last drugs available to treat serious MRSA infection), but also exhibited active efflux activity. Results indicated berberine's moderate inhibitiory activity against two MRSA isolates scoring a minimum inhibitory concentration (MIC) value of 125 microg/ml. Notable efflux inhibitory activity (ranging from two- to eightfold Ethidium Bromide MIC reduction) meanwhile was detected from quinine, piperine and harmaline using reserpine as the positive control. Findings from this study support the opinion that a vast number of potential phytocompounds with pharmacological potential await discovery. Therapeutic application of these compounds, however, warrants further investigation to ascertain their pharmacodynamics and safety aspects.