A B S T R A C TObjective: To investigate the antibacterial activities of crude ethanol extracts of 12 Philippine medicinal plants. Methods: Crude ethanol extracts from 12 Philippine medicinal plants were evaluated for their antibacterial activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, extended spectrum b-lactamase-producing, carbapenem-resistant Enterobacteriaceae and metallo-b-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii. Results: The leaf extracts of Psidium guajava, Phyllanthus niruri, Ehretia microphylla and Piper betle (P. betle) showed antibacterial activity against the Gram-positive methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. P. betle showed the highest antibacterial activity for these bacteria in the disk diffusion (16-33 mm inhibition diameter), minimum inhibitory concentration (19-156 mg/mL) and minimum bactericidal concentration (312 mg/mL) assays. P. betle leaf extracts only showed remarkable antibacterial activity for all the Gram-negative multidrug-resistant bacteria (extended spectrum b-lactamase-producing, carbapenem-resistant Enterobacteriaceae and metallo-b-lactamase-producing) in the disk diffusion (17-21 mm inhibition diameter), minimum inhibitory concentration (312-625 mg/mL) and minimum bactericidal concentration (312-625 mg/mL) assays.Conclusions: P. betle had the greatest potential value against both Gram-negative and Gram-positive multidrug-resistant bacteria. Favorable antagonistic activities were also exhibited by the ethanol extracts of Psidium guajava, Phyllanthus niruri and Ehretia microphylla.

Piper betle L. has traditionally been used in alternative medicine in different countries for various therapeutic purposes, including as an anti-infective agent. However, studies reported in the literature are mainly on its activities on drug susceptible bacterial strains. This study determined the antimicrobial activities of its ethanol, methanol, and supercritical CO2 extracts on clinical isolates of multiple drug resistant bacteria which have been identified by the Infectious Disease Society of America as among the currently more challenging strains in clinical management. Assay methods included the standard disc diffusion method and the broth microdilution method for the determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC) of the extracts for the test microorganisms. This study revealed the bactericidal activities of all the P. betle leaf crude extracts on methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), extended spectrum β-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and metallo-β-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii, with minimum bactericidal concentrations that ranged from 19μg/ml to 1250 μg/ml. The extracts proved to be more potent against the Gram positive MRSA and VRE than for the Gram negative test bacteria. VRE isolates were more susceptible to all the extracts than the MRSA isolates. Generally, the ethanol extracts proved to be more potent than the methanol extracts and supercritical CO2 extracts as shown by their lower MICs for both the Gram positive and Gram negative MDRs. MTT cytotoxicity assay showed that the highest concentration (100 μg/ml) of P. betle ethanol extract tested was not toxic to normal human dermal fibroblasts (HDFn). Data from the study firmly established P. betle as an alternative source of anti-infectives against multiple drug resistant bacteria.

This study isolated and identified the antimicrobial compounds of Philippine Piper betle L. leaf ethanol extracts by thin layer chromatography- (TLC-) bioautography and gas chromatography-mass spectrometry (GC-MS). Initially, TLC separation of the leaf ethanol extracts provided a maximum of eight compounds with R f values of 0.92, 0.86, 0.76, 0.53, 0.40, 0.25, 0.13, and 0.013, best visualized when inspected under UV 366 nm. Agar-overlay bioautography of the isolated compounds demonstrated two spots with R f values of 0.86 and 0.13 showing inhibitory activities against two Gram-positive multidrug-resistant (MDR) bacteria, namely, methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. The compound with an R f value of 0.86 also possessed inhibitory activity against Gram-negative MDR bacteria, namely, carbapenem-resistant Enterobacteriaceae-Klebsiella pneumoniae and metallo-β-lactamase-producing Acinetobacter baumannii. GC-MS was performed to identify the semivolatile and volatile compounds present in the leaf ethanol extracts. Six compounds were identified, four of which are new compounds that have not been mentioned in the medical literature. The chemical compounds isolated include ethyl diazoacetate, tris(trifluoromethyl)phosphine, heptafluorobutyrate, 3-fluoro-2-propynenitrite, 4-(2-propenyl)phenol, and eugenol. The results of this study could lead to the development of novel therapeutic agents capable of dealing with specific diseases that either have weakened reaction or are currently not responsive to existing drugs.

Eichhornia crassipes (Mart.) Solms. plantlets were grown in 0.1 and 1.0 ppm treatment solutions of Hoagland's hydroponic solutions modified with Hg(NO₃)2 in order to examine the specific cellular and biochemical mechanisms involved in the tolerance of this plant exposed to mercury. This study assessed the responses of chloroplast pigments, i.e., carotenoids and chlorophylls, and evaluated the enzymatic and nonenzymatic antioxidant systems. Inductively coupled plasma-atomic emission spectrometry (ICP-AES) revealed varying Hg(2+) levels in the young and mature leaf tissues, with greater amounts of Hg(2+) found in the tissues of the young leaves. Total chlorophyll levels, notably those of chlorophyll a, chlorophyll b, and carotenoids, showed significant elevation in young leaf tissues, while a decrease in their levels was observed in mature leaf tissues in comparison to those of the control plants. These results lend support to the protective role of increased chlorophyll and carotenoid levels in the photosynthetic apparatus of young E. crassipes leaves in the presence of Hg(2+). The antioxidant responses of Hg-treated E. crassipes plants were also measured, revealing a highly significant increase in catalase units, catalase and ascorbate peroxidase activities, and mercury-binding thiols in leaves from Hg-treated plants. Moreover, substantial differences in the degree of oxidative injury between the cells in leaves from the control and Hg-treated plants were evidenced by the lipid peroxidation activities monitored. The Hg-treatment-induced significant decrease in malondialdehyde (MDA) levels was observed in 0.1-ppm Hg(NO₃)2-exposed plants, while a highly significant increase in MDA levels was noted in 1.0-ppm Hg(NO₃)2-exposed plants. The high degree of lipid peroxidation at 1.0-ppm Hg treatment was evidently counteracted by the compensatory protective mechanism brought about by the increased levels in chloroplast pigments and the enhanced activities of the antioxidant systems. E. crassipes responded to mercury treatments by enhancing the synthesis of chlorophyll and carotenoid pigments, enzymatic, and nonenzymatic antioxidant substances, concomitantly increasing the antioxidative activities, thus rendering E. crassipes capable of tolerating Hg-induced stress. The potential of E. crassipes as a phytoremediator is evident.

Storm events are common in tropical regions and predicted to increase in frequency and intensity attributed to climate change. The iconic giant clams thriving in shallow coral reef areas in the IndoPacific region could experience salinity fluctuations during these events characterized by extreme precipitation. Given the limited ecotoxicological studies on the effects of osmotic stress in tridacnids, this study investigated the acclimatization potential to different salinities i.e., 18, 25, and 35% (control) for 14 days in juvenile Tridacna gigas. Results showed significant decrease in the chlorophyll a concentrations in giant clams at 18%. This may be caused by the marked increase in number of degenerating zooxanthellae. Chlorophyll c and carotenoid concentrations were also significantly reduced at 18%. The zooxanthellae density decreased significantly on Day 1 in both low salinity treatments, although bleaching was not observed. Symbiont cell enlargement was also observed at both low salinity treatments, affecting pigment concentrations at 18% but not at 25%. This study shows that zooxanthellae in giant clams were affected at 18 and 25%, but exposure to the latter displayed acclimation response as indicated by recovery in the various measured parameters after 14 days of exposure.