Potential anti-Acanthamoeba and anti-adhesion activities of Annona muricata and Combretum trifoliatum extracts and their synergistic effects in combination with chlorhexidine against Acanthamoeba triangularis trophozoites and cysts.

Plants with medicinal properties have been used in the treatment of several infectious diseases, including Acanthamoeba infections. The medicinal properties of Cambodian plant extracts; Annona muricata and Combretum trifoliatum were investigated against Acanthamoeba triangularis. A total of 39 plant extracts were evaluated and, as a result, 22 extracts showed positive anti-Acanthamoeba activity. Of the 22 extracts, 9 and 4 extracts showed anti-Acanthamoeba activity against trophozoites and cysts of A. triangularis, respectively. The minimum inhibitory concentration of A. muricata and C. trifoliatum extracts against trophozoites and cysts was 500 and 1,000 µg/mL, respectively. The combination of A. muricata at 1/4×MIC with chlorhexidine at 1/8×MIC demonstrated a synergistic effect against trophozoites, but partial synergy against cysts. A 40% reduction in trophozoites and 60% of cysts adhered to the plastic surface treated with both extracts at 1/2×MIC were noted comparing to the control (P < 0.05). Furthermore, a reduction of 80% and 90% of trophozoites adhered to the surface was observed after pre-treatment with A. muricata and C. trifoliatum extracts, respectively. A 90% of cysts adhered to the surface was decreased with pre-treatment of A. muricata at 1/2×MIC (P < 0.05). A 75% of trophozoites and cysts from Acanthamoeba adhered to the surface were removed after treatment with both extracts at 4×MIC (P < 0.05). In the model of contact lens, 1 log cells/mL of trophozoites and cysts was significantly decreased post-treatment with both extracts compared to the control. Trophozoites showed strong loss of acanthopodia and thorn-like projection pseudopodia, while cysts demonstrated retraction and folded appearance treated with both extracts when observed by SEM, which suggests the potential benefits of the medicinal plants A. muricata and C. trifoliatum as an option treatment against Acanthamoeba infections.

Protective effects of Cambodian medicinal plants on tert­butyl hydroperoxide­induced hepatotoxicity via Nrf2­mediated heme oxygenase­1.

Liver diseases are considered to be primary contributors to morbidity and mortality rates in humans. Oxidative stress is critical in liver injury, and oxidant­induced liver injury may be caused by toxins, including tert­butyl hydroperoxide (t­BHP). The present study investigated the hepatoprotective activities of 64 crude ethanol extracts of Cambodian medicinal plants against t­BHP­induced cytotoxicity in human liver­derived HepG2 cells, and assessed their cytoprotective mechanism pertaining to the expression of heme oxygenase (HO)­1 and nuclear factor E2­related factor 2 (Nrf2). Protective effects in HepG2 cells were determined by MTT assay. Protein expression levels of HO­1 and Nrf2 were determined by western blotting and mRNA expression levels were determined by reverse transcription­quantitative polymerase chain reaction. Of the 64 extracts, 19 extracts exhibited high hepatoprotective activities: Ampelocissus martini, Bauhinia bracteata, Bombax ceiba, Borassus flabellifer, Cardiospermum halicacabum, Cayratia trifolia, Cinnamomum caryophyllus, Cyperus rotundus, Dasymaschalon lomentaceum, Ficus benjamina, Mangifera duperreana, Morinda citrifolia, Pandanus humilis, Peliosanthes weberi, Phyllanthus emblica, Quisqualis indica, Smilax glabra, Tinospora crispa and Willughbeia cochinchinensis, with half maximal effective concentrations ranging between 59.23 and 157.80 µg/ml. Further investigations revealed that, of these 19 extracts, HO­1 and Nrf2 were expressed in P. weberi and T. crispa expressed in a dose­dependent manner. In addition, the activities of reactive oxygen species were suppressed following treatment of these two extracts in t­BHP­induced HepG2 cells. These results indicated that, of the 64 Cambodian plants, P. weberi and T. crispa exhibited hepatoprotective effects on t­BHP­induced cytotoxicity in HepG2 cells, possibly by the induction of Nrf2­mediated expression of HO­1. Taken together, these results suggested that T. crispa or P. weberi may offer potential for therapeutic applications in liver disease characterized by oxidative stress.

The neoflavonoid latifolin isolated from MeOH extract of Dalbergia odorifera attenuates inflammatory responses by inhibiting NF-κB activation via Nrf2-mediated heme oxygenase-1 expression.

In Korea and China, the heartwood of Dalbergia odorifera T. Chen is an important traditional medicine used to treat blood disorders, ischemia, swelling, and epigastric pain. In this study, we investigated the inhibitory effects of latifolin, a major neoflavonoid component isolated from the MeOH extract of D. odorifera, on the inflammatory reaction of thioglycollate-elicited peritoneal macrophages exposed to lipopolysaccharide, with a particular focus on heme oxygenase-1 (HO-1) expression and nuclear factor-κB (NF-κB) signaling. Latifolin significantly inhibited the protein and mRNA expression of inducible nitric oxide synthase and COX-2, reduced NO, prostaglandins E2, tumor necrosis factor-α, and interleukin-1ß production in primary murine peritoneal macrophages exposed to lipopolysaccharide. Latifolin also suppressed inhibitor κB-α levels, NF-κB nuclear translocation, and NF-κB DNA-binding activity. Furthermore, latifolin upregulated HO-1 expression via nuclear transcription factor-E2-related factor 2 (Nrf2) nuclear translocation. In addition, using inhibitor tin protoporphyrin IX (SnPP), an inhibitor of HO-1, it was verified that the inhibitory effects of latifolin on the proinflammatory mediators and NF-κB DNA-binding activity were associated with the HO-1 expression. These results suggested that the latifolin-mediated up-regulation of HO-1 expression played a critical role in anti-inflammatory effects in macrophages. This study therefore identified potent therapeutic effects of latifolin, which warrants further investigation as a potential treatment for inflammatory diseases.

Secondary metabolites isolated from Castilleja rubra exert anti-inflammatory effects through NF-κB inactivation on lipopolysaccharide-induced RAW264.7 macrophages.

8-Epiloganin (1), mussaenoside (2), and 5-O-caffeoylshikimic acid (3) have been isolated from Castilleja rubra, and the anti-inflammatory properties of these metabolites in a cell culture system were investigated. Compounds 1-3 suppressed not only the production of nitric oxide (NO) and prostaglandin E2, but also the expression of inducible NO synthase and cyclooxygenase-2 induced by lipopolysaccharide (LPS) in the RAW264.7 murine macrophage cell line. Compounds 1-3 also inhibited the release of pro-inflammatory cytokines induced by LPS, namely, tumor necrosis factor-α and interleukin-1ß. The underlying mechanism of the anti-inflammatory action of compounds 1-3 was associated with downregulation of nuclear factor-κB.

Inhibitory effect of 9-hydroxy-6,7-dimethoxydalbergiquinol from Dalbergia odorifera on the NF-κB-related neuroinflammatory response in lipopolysaccharide-stimulated mouse BV2 microglial cells is mediated by heme oxygenase-1.

The heartwood of Dalbergia odorifera T. Chen (Leguminosae) is an important source of traditional Korean and Chinese medicines. 9-Hydroxy-6,7-dimethoxydalbergiquinol (HDDQ), a compound isolated from D. odorifera, has various biological activities. The aim of this study was to determine the efficacy of HDDQ in modulating the regulation of anti-inflammatory activity through the upregulation of heme oxygenase (HO)-1 in BV2 microglia. HDDQ inhibited the protein expression of inducible nitric oxide synthase (iNOS), iNOS-derived nitric oxide (NO), and the production of cyclooxygenase (COX)-2 and COX-2-derived prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-stimulated mouse BV2 microglia. HDDQ also reduced tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) production, and suppressed the phosphorylation and degradation of IκB-α and the nuclear translocation of p65 in mouse BV2 microglia in response to LPS. Furthermore, HDDQ upregulated HO-1 expression via nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) in mouse BV2 microglia. Using tin protoporphyrin (SnPP), an HO activity inhibitor, we verified that the inhibitory effects of HDDQ on the proinflammatory mediators NO, PGE2, TNF-α, and IL-1ß, and nuclear factor kappa B (NF-κB) DNA-binding activity are associated with the induction of HO-1 expression. Our data suggest that HDDQ has therapeutic potential against neurodegenerative diseases caused by neuroinflammation.