4-Hydroxybenzoic acid serves as an endogenous ring precursor for antroquinonol biosynthesis in Antrodia cinnamomea.

Antrodia cinnamomea, an endemic fungus species of Taiwan, has long been used as a luxurious dietary supplement to enhance liver functions and as a remedy for various cancers. Antroquinonol (AQ), identified from the mycelium of A. cinnamomea, is currently in phase II clinical trials in the USA and Taiwan for the treatment of non-small-cell lung cancer. In the previous studies, we have demonstrated that AQ and 4-acetylantroquinonol B (4-AAQB) utilize orsellinic acid, via polyketide pathway, as the ring precursor, and their biosynthetic sequences are similar to those of coenzyme Q. In order to test 4-hydroxybenzoic acid (4-HBA), synthesized via shikimate pathway, is the ring precursor of AQ analogs, the strategy of metabolic labeling with stable isotopes was applied in this study. Here we have confirmed that 4-HBA serves as the ring precursor for AQ but not a precursor of 4-AAQB. Experimental results indicated that A. cinnamomea preferentially utilizes endogenous 4-HBA via shikimate pathway for AQ biosynthesis. Exogenous tyrosine and phenylalanine can be utilized for AQ biosynthesis when shikimate pathway is blocked by glyphosate. The benzoquinone ring of 4-AAQB is synthesized only via polyketide pathway, but that of AQ is synthesized via both polyketide pathway and shikimate pathway. The precursor-products relationships diagram of AQ and 4-AAQB in A. cinnamomea are proposed based on the experimental findings.

Development of 17 novel microsatellite markers for Lycoris aurea and L. radiata (Amaryllidaceae) using next-generation sequencing.

PREMISE OF THE STUDY: Lycoris is an ornamental and medicinal plant. We developed microsatellite markers for L. aurea and L. radiata simultaneously by using a hybrid between these two species. METHODS AND RESULTS: Ion Torrent next-generation sequencing produced 1,784,504 reads. Testing 64 primer sets allowed for the development of 17 novel microsatellite markers: 16 for L. aurea, 10 for L. radiata, and nine common markers. Lycoris aurea had one to 12 alleles per locus and observed and expected heterozygosity levels of 0-0.923 and 0.038-0.809, respectively. Lycoris radiata had three to 12 alleles per locus and observed and expected heterozygosity levels of 0-0.909 and 0.127-0.797, respectively. Ten markers were cross-amplified for L. sprengeri. CONCLUSIONS: Hybrid sequencing can facilitate the cost-effective development of molecular markers for parental species. The markers developed here are useful for studying Lycoris population structure.

Biosynthesis of Antroquinonol and 4-Acetylantroquinonol B via a Polyketide Pathway Using Orsellinic Acid as a Ring Precursor in Antrodia cinnamomea.

Antroquinonol (AQ) and 4-acetylantroquinonol B (4-AAQB), isolated from the mycelium of Antrodia cinnamomea, have a similar chemical backbone to coenzyme Q (CoQ). Based on the postulation that biosynthesis of both AQ and 4-AAQB in A. cinnamomea starts from the polyketide pathway, we cultivated this fungus in a culture medium containing [U-13C]oleic acid, and then we analyzed the crude extracts of the mycelium using UHPLC-MS. We found that AQ and 4-AAQB follow similar biosynthetic sequences as CoQ. Obvious [13C2] fragments on the ring backbone were detected in the mass spectrum for [13C2]AQ, [13C2]4-AAQB, and their [13C2] intermediates found in this study. The orsellinic acid, formed from acetyl-CoA and malonyl-CoA via the polyketide pathway, was found to be a novel benzoquinone ring precursor for AQ and 4-AAQB. The identification of endogenously synthesized farnesylated intermediates allows us to postulate the routes of AQ and 4-AAQB biosynthesis in A. cinnamomea.

Analysis of aroma compounds and nutrient contents of mabolo (Diospyros blancoi A. DC.), an ethnobotanical fruit of Austronesian Taiwan.

Diospyros blancoi A. DC. is an evergreen tree species of high-quality wood. Mabolo, the fruit of this plant, is popular among the natives in Taiwan, but its potential in economic use has not been fully explored. Mabolo has a rich aroma. Of the 39 different volatile compounds isolated, its intact fruit and peel were found to both contain 24 compounds, whereas the pulp contained 28 compounds. The most important aroma compounds were esters and α-farnesene. Our data show that mabolo is rich in dietary fiber (3.2%), and the contents of other nutrients such as malic acid, vitamin B2, vitamin B3, folic acid, pantothenic acid, and choline chloride were 227.1 mg/100 g, 0.075 mg/100 g, 0.157 mg/100 g, 0.623 mg/100 g, 0.19 mg/100 g, and 62.52 mg/100 g, respectively. Moreover, it is rich in calcium and zinc; the contents of which were found to be 42.8 mg/100 g and 3.6 mg/100 g, respectively. Our results show that D. blancoi has the potential to be bred for a novel fruit.

The effects of Davallic acid from Davallia divaricata Blume on apoptosis induction in A549 lung cancer cells.

Traditional or folk medicinal herbs continue to be prescribed in the treatment of various diseases and conditions in many cultures. Recent scientific efforts have focused on the potential roles of extracts of traditional herbs as alternative and complementary medications for cancer treatment. In Taiwan, Davallia divaricata Blume has been traditionally employed in folk medicine for therapy of lung cancer, davallic acid being the major active compound of D. divaricata Blume. In this study, we investigated the inhibitory activity of davallic acid on the proliferation of A549 lung cancer cells. Davallic acid was extracted from D. divaricata Blume, and its effects on cell viability, cell cycle distribution, ROS level, and apoptotic protein expression in A549 cells were determined. Davallic acid significantly induced reactive oxygen species (ROS) generation as well as caspase-3, -8, and -9 activation, thereby repressing A549 cell growth and elevating apoptotic activity. Since lung cancer has a high incidence of recurrence, these results indicate that davallic acid may have the potential to be a natural anti-lung cancer compound, and may provide a basis for further study of its use in combating cancer.

Resveratrol upregulates Nrf2 expression to attenuate methylglyoxal-induced insulin resistance in Hep G2 cells.

Oxidative stress can result in insulin resistance, a primary cause of type-2 diabetes. Methylglyoxal (MG), a highly reactive dicarbonyl metabolite generated during glucose metabolism, has also been confirmed to cause pancreatic injury and induce inflammation, thereby resulting in insulin resistance. Recently, resveratrol has been reported to exert antioxidant properties, protecting cells from the generation of reactive oxygen species (ROS). The aim of this study was to evaluate resveratrol activation of nuclear factor erythroid 2-related factor 2 (Nrf2) to attenuate MG-induced insulin resistance in Hep G2 cells. Therefore, the molecular signaling events affecting resveratrol-mediated heme oxygenase-1 (HO-1) and glyoxalase expression levels were further investigated in this study. Our findings indicated that resveratrol activated the extracellular signal-regulated kinase (ERK) pathway but not the p38 or c-Jun N-terminal kinase (JNK) pathways, subsequently leading to Nrf2 nuclear translocation and elevation of HO-1 and glyoxalase expression levels. Moreover, resveratrol significantly elevated glucose uptake and protected against MG-induced insulin resistance in Hep G2 cells. In contrast, depletion of Nrf2 by small interfering RNA (si-RNA) resulted in the abrogation of HO-1 and glyoxalase expression in the MG-treated resveratrol group in Hep G2 cells. Administration of an appropriate chemopreventive agent, such as resveratrol, may be an alternative strategy for protecting against MG-induced diabetes.

Scopoletin attenuates allergy by inhibiting Th2 cytokines production in EL-4 T cells.

Scopoletin is an antioxidant found in certain weedy plants. It exerts anti-inflammatory, anti-allergic, and anti-diabetic activities. It remains unknown whether scopoletin regulates T helper (Th) cells, including Th 1 and Th 2 cells. We found that scopoletin significantly inhibited phorbol myristate acetate (PMA)/ionomycin-induced interleukin-4 (IL-4), IL-5, and IL-10 production in EL-4 T cells. In addition, scopoletin significantly enhanced the inhibitory action of PMA/ionomycin on interferon-γ (IFN-γ) expression. In EL-4 T cells, PMA/ionomycin treatment markedly increased the expression of nuclear factor of activated T cells (NFAT) and GATA-3; in contrast, scopoletin significantly down-regulated expressions of these transcription factors. Furthermore, this downregulation depended on protein kinase C (PKC) attenuation. This leads us to suggest that the anti-allergic properties of scopoletin might be mediated by the downregulation of cytokine expression in Th 2 cells.

Lactic fermentation and antioxidant activity of Zingiberaceae plants in Taiwan.

The present study evaluated functional properties of lactic-fermented ginger products. Three Zingiberaceae species were used as the substrate for fermentation using three lactic acid bacteria. The fermentation process ended 35-40 h after inoculation and reached a pH value of 3.5-4.0. Total antioxidant performances were 68-75%, and were best observed using Bifidobacterium longum as the starter in three ginger samples. DPPH scavenging was on average 70%, with free radical anion scavenging and peroxide removal effects of 30.6% and 43.7%, respectively. The product acceptance survey showed that the 100% fermented juice without a mixture with non-fermented ginger juice obtained the highest score in overall performance. The lactic-fermented Vanoverberghia and Hedychium ginger species retained an antioxidant activity and DPPH scavenging activity of on average 70%. This study may suggest a new way of ginger food processing with high functionality. Also, it may help to popularize the growing and processing of endemic ginger plants in Taiwan.

Antioxidant and antimicrobial activity of Zingiberaceae plants in Taiwan.

The rhizomes of the Zingiberaceae family are a vegetable widely used in many Asian countries, and their medicinal functions have been broadly discussed and accepted in many traditional recipes. In this study, 18 species of five genus of Zingiberaceae plants from Taiwan area were collected and analyzed for their functional properties. Methanolic extracts of the plants were analyzed for their total phenol compounds, alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) scavenging activity, and reducing power. Antimicrobial activity of these samples was also determined. The results showed that the total phenol compounds of the Alpinia genus averaged 17, 30 mg/g for Curcumas, and the highest, 36.5 mg/g for Vanoverberghia sasakiana. Antioxidant performances were best observed in Vanoverberghia and Hedychium, both 89%, and DPPH scavenging activity followed similar trends. Particularly, Zingiber oligophyllum, considered as a traditional medicinal plant used in Taiwan exhibited low DPPH scavenging activity and reducing power. Most Zingiberaceae plant extracts exhibited antimicrobial activity against all tested food microorganisms. Hedychium and Vanoverberghia, did not show antimicrobial activities on Escherichia coli and Vibrio parahaemolyticus. This study is a positive demonstration of the utility of screening Taiwan's endemic Zingiberaceous plants for their food and medicinal uses.