Transcriptome-based mining and expression profiling of Pythium responsive transcription factors in Zingiber sp.

Transcription factors (TFs) fine-tune the host defense transcriptome in response to pathogen invasions. No information is available on Zingiber zerumbet (Zz) TFs involved in defense response against Pythium myriotylum. Here, we provide a global identification, characterization, and temporal expression profiling of Zz TFs following an incompatible interaction with P. myriotylum using a transcriptome sequencing approach. We identified a total of 903 TFs belonging to 96 families based on their conserved domains. Evolutionary analysis clustered the Zz TFs according to their phylogenetic affinity, providing glimpses of their functional diversities. High throughput expression array analysis highlighted a complex interplay between activating and repressing transcription factors in fine-tuning Zz defense response against P. myriotylum. The high differential modulation of TFs involved in cell wall fortification, lignin biosynthesis, and SA/JA hormone crosstalk allows us to envisage that this mechanism plays a central role in restricting P. myriotylum proliferation in Zz. This study lays a solid foundation and provides valuable resources for the investigation of the evolutionary history and biological functions of Zz TF genes involved in defense response.

Zerumbone suppresses the activation of inflammatory mediators in LPS-stimulated U937 macrophages through MyD88-dependent NF-κB/MAPK/PI3K-Akt signaling pathways.

Zerumbone (ZER), isolated mainly from the Zingiber zerumbet (Z. zerumbet) rhizomes was found to be effective against numerous inflammatory and immune disorders, however, the molecular and biochemical mechanisms underlying its anti-inflammatory and immunosuppressive properties have not been well studied. This study was carried out to examine the profound effects of ZER on inflammatory mediated MyD88-dependent NF-κB/MAPK/PI3K-Akt signaling pathways in LPS-stimulated U937 human macrophages. ZER significantly suppressed the up-regulation pro-inflammatory mediators, TNF-α, IL-1ß, PGE2, and COX-2 protein in LPS-induced human macrophages. Moreover, ZER significantly downregulated the phosphorylation of NF-κB (p65), IκBα, and IKKα/ß as well as restored the degradation of IκBα. ZER correspondingly showed remarkable attenuation of the expression of Akt, JNK, ERK, and p38 MAPKs phosphorylation in a concentration-dependent manner. ZER also diminished the expression of upstream signaling molecules TLR4 and MyD88, which are prerequisite for the NF-κB, MAPK and PI3K-Akt activation. Additionally, quantification of relative gene expression of TNF-α, IL-1ß, and COX-2 indicated that, at a higher dose (50µM), ZER significantly downregulated the elevated mRNA transcription levels of the stated pro-inflammatory markers in LPS-stimulated U937 macrophages. The strong suppressive effects of ZER on the activation of inflammatory markers in the macrophages via MyD88-dependent NF-κB/MAPK/PI3K-Akt signaling pathways suggest that ZER can be a preventive and potent therapeutic candidate for the management of various inflammatory-mediated immune disorders.

Protective effect of zerumbone reduces lipopolysaccharide-induced acute lung injury via antioxidative enzymes and Nrf2/HO-1 pathway.

Acute lung injury (ALI) is a serious disease with high morbidity and mortality rate. Although there are effective strategies for treatment of ALI; a widely accepted specific pharmacotherapy has not yet established. Zerumbone, the major active phytochemical compound from Zingiber zerumbet Smith, exhibits various beneficial biological and pharmacological activities, such as antioxidation, anti-inflammation, immunomodulation, and anti-cancer. We aimed to study the potential protective effects and mechanisms of zerumbone in mouse model of lipopolysaccharide (LPS)-induced ALI. Pretreatment with zerumbone inhibited the histopatholgical changes such as neutrophils infiltration, increased in alveolar barrier thickness, hemorrhage, and hyaline membrane formation occurred in lungs in LPS-induced ALI. In addition, not only LPS-induced activation of myeloperoxidase (MPO) and metallopeptidase-9 (MMP-9) was suppressed by zerumbone, but also lipid peroxidation in lungs was inhibited as well. Moreover, pretreatment with zerumbone reversed the antioxidative enzymes activities, including superoxide dismutase, catalase, and glutathione peroxidase, decreased by LPS and enhanced the expression of nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase (HO-1) induced by LPS. These results from present study suggested that the protective mechanisms of zerumbone on LPS-induced ALI were via up-regulation of antioxidative enzymes and Nrf2/HO-1 pathway.

Metabolic profiling of Zingiber zerumbet following Pythium myriotylum infection: investigations on the defensive role of the principal secondary metabolite, zerumbone.

Induced biosynthesis of bioactive secondary metabolites constitutes one of the mechanisms of plant basal innate immunity to fungal infection. Metabolic changes were studied in rhizomes of Zingiber zerumbet, a wild congener of ginger, after infection with soft rot-causative necrotrophic phytopathogen, Pythium myriotylum, by gas chromatography-mass spectrometry (GC-MS) analysis. Infection triggered a considerable alteration in the relative content of zerumbone and α-caryophyllene (humulene) with enhancement in zerumbone content (81.59%) and that of α-caryophyllene (11.91%) compared to 9.97 and 1.11%, respectively, in uninfected rhizomes. While zerumbone is the principal secondary metabolite in Z. zerumbet, α-caryophyllene is its immediate precursor. Principal component analysis (PCA) identified the correlations between metabolite changes in Z. zerumbet rhizomes and P. myriotylum infection. Radial diffusion assay with zerumbone indicated a concentration-dependent P. myriotylum growth inhibition with 93.75% inhibition observed at 700 µg and 50% maximal effective concentration (EC50) value of 206 µg. Scanning electron microscopy (SEM) analysis revealed that the mechanistic basis of zerumbone's antagonistic action on P. myriotylum growth involved the induction of aberrant morphology including severe hyphal deformities and membrane disruption. Results are discussed highlighting the critical role played by sesquiterpenoid zerumbone in affording resistance in Z. zerumbet and could expedite the development of appropriate strategies for biocontrol of Pythium spp., thus reducing the usage of broad-spectrum fungicides.

Immunotoxicity activity of sesquiterpenoids from black galingale (Kaempferia parviflora Wall. Ex. Baker) against Aedes aegypti L.

The roots of black galingale (Kaempferia parviflora) were chloroform-extracted and the isolated two sesquiterpene and immunotoxicity effects were studied. The structures and stereochemistry of these compounds were established on the basis of analysis of spectra including UV, MS, (1)H-NMR, and (13)C-NMR as follows: 1 (4α-acetoxycadina-2,9-diene-1,8-dione), 2 (1α,3α,4ß-trihydroxy-9-cadinen-8-one). Compound 2 had a significant toxic effect against early fourth-stage larvae of Aedes aegypti L. with an LC(50) value of 0.7 µM and an LC(90) value of 3.8 µM. The results could be useful in search for newer, safer, and more effective natural immunotoxicity agents against A. aegypti.

Evaluation of allergenicity of constituents of myoga using the murine local lymph node assay.

Myoga (Zingiber Myoga Roscoe) is a perennial plant with a pungent smell from its flower buds. It is native to East Asia and has been reported to cause allergic contact dermatitis. The purpose of this study is to assess the allergenicity of myoga related to its major chemical components, alpha-pinene, beta-pinene, limonene, limonene oxide and beta-phellandrene, which are supposed to be the causative agents of contact dermatitis among myoga cultivators. We performed a toxicity study of the volatile constituents of myoga using the local lymph node assay (LLNA), in which limonene, limonene oxide and beta-phellandrene had positive responses and the EC3 was 35.8%, 8.22%, and 0.54%, respectively. EC3 for both alpha-pinene and beta-pinene was over 100%. Both chemicals failed to induce positive responses in the LLNA. While the maximization rating of limonene, limonene oxide and phellandrene were evaluated as moderate, extreme, and extreme respectively, alpha-pinene and beta-pinene were evaluated as weak in the previously reported GPMT. The usage of LLNA was also confirmed by comparing with previously reported GPMT results to detect the allergenicity of myoga constituents. The actual risk of humans developing an allergy to myoga constituents depends on many factors. The concentration of the compounds, the frequency and duration of exposure and the condition of the skin are supposed to be important factors.

Expression analysis of defense-related genes in Zingiber (Zingiberaceae) species with different levels of compatibility to the soft rot pathogen Pythium aphanidermatum.

Ginger (Zingiber officinale Roscoe) cultivars are susceptible to soft rot disease caused by Pythium aphanidermatum. We analyzed changes in transcript levels of 41 genes in the highly susceptible ginger cultivar varada, a less susceptible wild accession (wild ginger), and a Pythium aphanidermatum-resistant relative, Z. zerumbet, following treatment with Pythium aphanidermatum or one of three signaling molecules: salicylic acid (SA), jasmonic acid (JA), or ethylene (ET). The 41 studied genes were chosen because they are known to be involved in the hypersensitive response (HR), cell signaling, or host defense. Expression of most genes peaked within 24 h of Pythium aphanidermatum infection. Interestingly, the level of induction was typically manyfold higher in Z. zerumbet than in wild ginger. However, several HR genes that were significantly induced in wild ginger were not induced in Z. zerumbet. Most of the genes, including those involved in signaling, did not respond to any of the three signaling molecules in Z. zerumbet while several genes responded to all the three signaling molecules in varada. In wild ginger, a large proportion of the genes responded to ET, but not to SA or JA. These results suggest that different mechanisms govern the three pathosystems. Resistance in Z. zerumbet seems to be independent of HR and the tested signaling molecules, whereas both mechanisms appear to be activated in the tolerance reaction of wild ginger. This work revealed potential defense components of this understudied tropical taxa, and will contribute to the design of strategies for transgenic improvement of ginger.

Occupational allergic contact dermatitis from mioga (Zingiber mioga rosc.) in greenhouse cultivators.

UNLABELLED: Mioga (Zingiber mioga Rosc.) is a member of the ginger family (Zingiberaceae), which is native to tropical Asia. In Japan, the young flower buds are used as a spice, and hand dermatitis suspected as being an allergy to mioga has been recognized in mioga greenhouse cultivators. To investigate the extent of the problems and the causes of dermatitis, 20 householders cultivating mioga in their greenhouses were asked to participate in a questionnaire study. Consecutive patch tests were performed on some subjects with dermatitis. Self-reported questionnaires were distributed to the main cultivator in each household who attended a lecture of mioga cultivation methods held at an agriculture cooperative association in the area. Some subjects who answered as presenting or having had hand dermatitis were patch tested for mioga (as is), four kinds of mioga extracts, and three kinds of natural rubber gloves. RESULTS: 35 cultivators from 16 households answered the questionnaire. Eight of the 35 subjects (22.9 percent) answered that they had experienced hand dermatitis since they started mioga cultivation. Four of the 8 subjects were patch tested. Two of the 4 subjects showed allergic reactions to mioga (as is) and the extracts. The other two cases showed irritation to mioga (as is). The first two cases also showed allergic reactions to natural rubber gloves. To our knowledge, there is no previous report of allergic contact dermatitis from mioga.

Toxicity study of the volatile constituents of Myoga utilizing acute dermal irritation assays and the Guinea-pig Maximization test.

Myoga is a fragrant plant which is the special product of Japan and is cultivated throughout Japan. According to our earlier investigation (unpublished data) of myoga cultivators in Japan, 8 of 35 cultivators experienced contact dermatitis in the harvest season. The purpose of this study was to assess the allergenicity of myoga and its major volatile components. The volatile components of myoga were analyzed by gas chromatograph (GC). They included a-pinene, beta-pinene and R-(+)-limonene. We performed a toxicity study of each of the major fragrant components of myoga using acute dermal irritation assays and the Guinea-Pig Maximization test (GPMT) in order to probe the mechanism of allergic contact dermatitis. In acute dermal irritation assays, alpha-pinene, beta-pinene and limonene showed positive responses at concentrations of 4%; limonene oxide at 20% and myoga showed a positive response at concentrations of 100%. From the results of the GPMT, according to Kligman scores, limonene oxide was identified as an extreme skin sensitizer and myoga as a mild skin sensitizer. The results of the present study show that R-(+)-limonene is the most important allergen amongst the chemical components of myoga, and we consider it to be the reason why myoga cultivators experience allergic contact dermatitis.