Effect of E-Beam Irradiation on Microbial Load, Stability of Active Components, and Anti-Inflammatory Activity of Cnidii Rhizoma and Alismatis Rhizoma.

To reduce microbial loads in medicinal herbs, Cnidii Rhizoma and Alismatis Rhizoma were subjected to electron-beam (e-beam) irradiation at doses (≤10 kGy) as permitted by the Korean Food Code. The effects of e-beam irradiation on the microbial load, stability of the active components, and anti-inflammatory activity of medicinal herbs were determined. We observed that the total aerobic bacteria (TAB; 4.0-7.0 log CFU/g), yeasts and molds (Y&M; 3.3-6.8 log CFU/g), and coliform counts (CC; 3.2-3.8 log CFU/g) in both herb samples were effectively reduced in a dose-dependent manner, resulting in acceptable levels of <3.0 log CFU/g in TAB and Y&M and negative in CC at 10 kGy irradiation. The concentration of the active components (0.87-4.22 mg/g) of Cnidii Rhizoma, including z-ligustilide, chlorogenic acid, senkyunolide A, and ferulic acid, in order of prevalence and those (0.86-2.76 mg/g) of Alismatis Rhizoma, including Alisol B acetate and Alisol B, were not changed at irradiation doses of ≤10 kGy. The extracts of e-beam irradiated Cnidii Rhizoma and Alismatis Rhizoma showed a reduced production of inflammation-related factors, such as nitric oxide, prostaglandin E2, interleukin (IL)-1ß, and IL-6, in a concentration-dependent manner, which was induced by lipopolysaccharide in RAW 264.7 cell. However, there was no significant difference observed at e-beam irradiation doses of 0, 1, 5, and 10 kGy. Thus, we confirm that e-beam irradiation up to 10 kGy was effective for the control of microbial load in Cnidii Rhizoma and Alismatis Rhizoma without causing considerable changes in their major active components and anti-inflammatory activity. The results show the potential of e-beam application for sanitization of medicinal herbs.

Dynamics of flavonol accumulation in leaf tissues under different UV-B regimes in Centella asiatica (Apiaceae).

MAIN CONCLUSION: A cumulative effect of UV-B doses on epidermal flavonol accumulation was observed during the first week of a time course study in Centella asiatica (Apiaceae). However, once flavonol levels had peaked, additional accumulation was possible only if higher daily UV-B irradiances were applied. We aimed to understand the dynamics of flavonol accumulation in leaf tissues using non-destructive spectroscopy and HPLC-mass spectrometry. When leaves that had grown without UV-B were given brief daily exposures to low-irradiance UV-B, they accumulated flavonols, predominantly kaempferol-3-O-ß-D-glucuronopyranoside and quercetin-3-O-ß-D-glucuronopyranoside, in their exposed epidermis, reaching a plateau after 7 days. More prolonged UV-B exposures or higher doses eventually augmented flavonol concentrations even in non-exposed tissues. If UV-B irradiance was subsequently reduced, leaves appeared to lose their ability to accumulate further flavonols in their epidermis even if the duration of daily exposure was increased. A higher irradiance level was then necessary to further increase flavonol accumulation. When subsequently acclimated to higher UV-B irradiances, mature leaves accumulated less flavonols than did developing ones. Our study suggests that levels of epidermal flavonols in leaves are governed primarily by UV-B irradiance rather than by duration of exposure.

[Comparative studies on photosynthetic characteristics of Notopterygium incisum and N. forbesii].

OBJECTIVE: To compare the photosynthetic characteristics of Notopterygium incisum and N. forbesii in order to provide basic data for introduction and cultivation of the two wild medicinal species. METHOD: The light-response, CO2-response and Chlorophy II fluorescence parameters of leaves at the booting stages between N. incisum and N. forbesii, were analyzed in situ by Li-6400 Portable Photosynthesis system under natural conditions. RESULT: 1) The light saturation point (LSP) was 1539 micromol x m(-2) x s(-1) for N. incisum and 1464 micromol x m(-2) x s(-1) for N. forbesii, the maximum net photosynthetic rate (Pmax) was 22.95 micromol x m(-2) x s(-1) for N. incisum and 19.65 micromol x m(-2) x s(-1) for N. forbesii, the apparent quantum yield (AQY) was 0.0509 for N. incisum and 0.0470 for N. forbesii, LSP, AQY and Pmax of N. incisum were significantly higher than those of N. forbesii; the light compensation point (LCP) was 17.92 micromol x m(-2) x s(-1) for N. incisum and 26.69 micromol x m(-2) x s(-1) for N. forbesii, LCP of N. incisum was significantly lower than that of N. forbesii. 2) The carbondioxide compensation point (CCP) were 33.41 micromol x mol(-1) for N. incisum and 37.82 micromol x mol(-1) for N. forbesii, the carbon dioxide saturation point (CSP) were 988 micromol x mol(-1) for N. incisum and 1150 micromol x mol(-1) for N. forbesii, CCP and CSP of N. incisum were significantly lower than N. forbesii; the carboxylation efficiency (CE) were 0.0591 for N. incisum and 0.0459 for N. forbesii; the maximum rate of RuBP regeneration (Jmax) were 28.18 micromol x m(-2) x s(-1) for N. incisum and 25.32 micromol x m(-2) x s(-1) for N. forbesii; the light respiration rate (Rd) were 1.971 micromol x m(-2) x s(-1) for N. incisum and 1.736 micromol x m(-2) x s(-1) for N. forbesii, CE, Jmax and Rd of N. incisum were higher than those of N. forbesii. 3) The primary light energy conversion of PS II (Fv/Fm) was 0.8213 for N. incisum and 0.8257 for N. forbesii, wihich didn't showed significant difference, between N. incisum and N. forbesii there was no photoinhibition. CONCLUSION: Both N. incisum and N. forbesii were C3 type plant, could perfectly acclimate to light condition. However, the weak light of N. incisum was absorbed significantly higher than that of N. forbesii, strong photosynthesis ability causes assimilation products accumulation of N. incisum obviously to be higher than that of N. forbesii.

Isolation and characterization of four novel parsley proteins that interact with the transcriptional regulators CPRF1 and CPRF2.

The common plant regulatory factors (CPRFs) from parsley are transcription factors with a basic-leucine-zipper motif that bind to cis-regulatory elements frequently found in promoters of light-regulated genes. Proposed to function in concert with members of other transcription factor families, CPRFs regulate the transcriptional activity of many target genes. Here, we report that, in contrast to CPRF2, which operates as a transcriptional activator, CPRF1 functions as repressor in vivo. Two-hybrid screens using CPRF1 and CPRF2 as "baits" resulted in the isolation of four novel parsley proteins which interact with either CPRF1 or CPRF2 in vivo. Three of these factors represent new parsley bZIP factors, designated CPRF5-CPRF7, whereas the fourth, named CPRF1-interacting protein (CIP), shows no homology to any other known protein. CPRF5 and CIP specifically interact with CPRF1, whilst CPRF6 and CPRF7 exclusively form heterodimers with CPRF2. CPRF5, CPRF6 and CPRF7 are transcription factors that exhibit sequence-specific DNA-binding as well as transactivation abilities, whereas the function of CIP remains elusive. The newly isolated CPRFs and CIP are constitutively localized in the nucleus in parsley protoplasts. Furthermore, mRNA accumulation studies revealed that the expression of these novel bZIP genes and CIP is not altered by exposure to light. We discuss the possible roles of the newly identified proteins in CPRF1- and CPRF2-dependent target gene expression.

Glutathione and a UV light-induced glutathione S-transferase are involved in signaling to chalcone synthase in cell cultures.

UV irradiation stimulates expression of the gene encoding the key enzyme chalcone synthase (CHS), which leads to the generation of protective flavonoids in parsley cell cultures. CHS transcripts increase after 3 to 4 hr, and early genes are involved in the signal transduction to the CHS promoter. By using the fluorescent differential display technique in a large-scale screening, several early UV light-induced genes were isolated. Of these, a novel glutathione S-transferase (PcGST1) is induced within 2 hr and precedes CHS expression. Overexpression of PcGST1 in transformed cell lines containing a CHS promoter/luciferase reporter (CHS-LUC) affected the onset of LUC transcription. Supplementing these cell lines with glutathione immediately stimulated CHS-LUC expression within 2 hr in dark-incubated cells and resulted in a biphasic induction profile in UV-irradiated cells. Our data indicate the involvement of glutathione and PcGST1 in early events of a UV light-dependent signal transduction pathway to CHS. In this context, the oxidative status of a cell acts as a central regulating element.

Transactivation properties of parsley proline-rich bZIP transcription factors.

Light-responsive chalcone synthase (CHS) gene activation requires LRUCHS, a light regulatory promoter unit including the MYB recognition element MRECHS and the ACGT-containing element ACECHS. ACECHS is bound by the parsley basic region/leucine zipper (bZIP) factors CPRF1 and 4. Factors containing the bZIP domain exist in animals, plants and yeast, and recognize DNA sequence-specifically after formation of homo- or heterodimers. To determine the potential role of CPRFs in the regulation of CHS promoter activity, we investigated the functions of distinct CPRF domains in a homologous co-transfection system. The proline-rich domains of CPRF1 and CPRF4 activate transcription, indicating that CPRF1 and CPRF4 have transactivating properties. Over-expression of the CPRF1 bZIP domain caused a reduction of LRUCHS-mediated light inducibility, and point mutations throughout ACECHS affected both responsiveness to UV-containing white light and transactivation by CPRF1:VP16. The data suggest that a CPRF1-containing bZIP heterodimer interacts with ACECHS in vivo. We discuss regulatory steps in light-induced CHS transcription that may be influenced by CPRF1 and/or related bZIP factors.

UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley.

The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells.