Efficient Plant Regeneration System from Leaf Explant Cultures of Daphne genkwa via Somatic Embryogenesis.

This study aimed to establish an efficient plant regeneration system from leaf-derived embryogenic structure cultures of Daphne genkwa. To induce embryogenic structures, fully expanded leaf explants of D. genkwa were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.1, 0.5, 1, 2, and 5 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. After 8 weeks of incubation, the highest frequency of embryogenic structure formation reached 100% when the leaf explants were cultivated on MS medium supplemented with 0.1 to 1 mg·L-1 2,4-D. At higher concentrations of 2,4-D (over 2 mg·L-1 2,4-D), the frequency of embryogenic structure formation significantly declined. Similar to 2,4-D, indole butyric acid (IBA) and α-naphthaleneacetic acid (NAA) treatments were also able to form embryogenic structures. However, the frequency of embryogenic structure formation was lower than that of 2,4-D. In particular, the yellow embryonic structure (YES) and white embryonic structure (WES) were simultaneously developed from the leaf explants of D. genkwa on culture medium containing 2,4-D, IBA, and NAA, respectively. Embryogenic calluses (ECs) were formed from the YES after subsequent rounds of subculture on MS medium supplemented with 1 mg·L-1 2,4-D. To regenerate whole plants, the embryogenic callus (EC) and the two embryogenic structures (YES and WES) were transferred onto MS medium supplemented with 0.1 mg·L-1 6-benzyl aminopurine (BA). The YES had the highest plant regeneration potential via somatic embryo and shoot development compared to the EC and WES. To our knowledge, this is the first successful report of a plant regeneration system via the somatic embryogenesis of D. genkwa. Thus, the embryogenic structures and plant regeneration system of D. genkwa could be applied to mass proliferation and genetic modification for pharmaceutical metabolite production in D. genkwa.

Metabolic Discrimination between Adventitious Roots and Standard Medicinal Part of Atractylodes macrocephala Koidz. Using FT-IR Spectroscopy.

This study aims to examine the metabolic discrimination between in vitro grown adventitious roots and the standard medicinal parts of Atractylodes macrocephala. To achieve this goal, firstly, in vitro culture conditions of adventitious roots such as indole-3-butyric acid (IBA) concentrations, types of media, inorganic salt strength of culture medium, and elicitor types and concentrations were optimized. The optimal culture conditions for proliferation of adventitious roots was found to consist of Murashige and Skoog (MS) medium containing 5 mg L-1 IBA. Whole cell extracts from adventitious roots and the standard medicinal parts of A. macrocephala were subjected to Fourier transform infrared spectroscopy (FT-IR). Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) from FT-IR spectral data showed that adventitious roots and standard medicinal parts were clearly distinguished in the PCA and PLS-DA score plot. Furthermore, the overall metabolite pattern from adventitious roots was changed depending on the dose-dependent manner of chemicals. These results suggest that FT-IR spectroscopy can be applied as an alternative tool for the screening of higher metabolic root lines and for discriminating metabolic similarity between in vitro grown adventitious roots and the standard medicinal parts. In addition, the adventitious roots proliferation system established in this study can be directly applied as an alternative means for the commercial production of A. macrocephala.

Proteome Changes Reveal the Protective Roles of Exogenous Citric Acid in Alleviating Cu Toxicity in Brassica napus L.

Citric acid (CA), as an organic chelator, plays a vital role in alleviating copper (Cu) stress-mediated oxidative damage, wherein a number of molecular mechanisms alter in plants. However, it remains largely unknown how CA regulates differentially abundant proteins (DAPs) in response to Cu stress in Brassica napus L. In the present study, we aimed to investigate the proteome changes in the leaves of B. L. seedlings in response to CA-mediated alleviation of Cu stress. Exposure of 21-day-old seedlings to Cu (25 and 50 µM) and CA (1.0 mM) for 7 days exhibited a dramatic inhibition of overall growth and considerable increase in the enzymatic activities (POD, SOD, CAT). Using a label-free proteome approach, a total of 6345 proteins were identified in differentially treated leaves, from which 426 proteins were differentially expressed among the treatment groups. Gene ontology (GO) and KEGG pathways analysis revealed that most of the differential abundance proteins were found to be involved in energy and carbohydrate metabolism, photosynthesis, protein metabolism, stress and defense, metal detoxification, and cell wall reorganization. Our results suggest that the downregulation of chlorophyll biosynthetic proteins involved in photosynthesis were consistent with reduced chlorophyll content. The increased abundance of proteins involved in stress and defense indicates that these DAPs might provide significant insights into the adaptation of Brassica seedlings to Cu stress. The abundances of key proteins were further verified by monitoring the mRNA expression level of the respective transcripts. Taken together, these findings provide a potential molecular mechanism towards Cu stress tolerance and open a new route in accelerating the phytoextraction of Cu through exogenous application of CA in B. napus.

Quinic acid esters from Erycibe obtusifolia with antioxidant and tyrosinase inhibitory activities.

AbstractA new quinic acid derivative, 3-O-syringoylquinic acid methyl ester (1), along with eight known quinic acid derivatives (2-9), three coumarins (10-12), one phenylpropanoid (13), three feruloyltyramine derivatives (14-16), one lignan (17) and two isoflavones (18-19) were isolated from an ethyl acetate-soluble fraction of the roots and stems of Erycibe obtusifolia. The structure was elucidated on the basis of spectroscopic methods such as 1D and 2D-NMR, including HR-ESI-MS spectrometry. All of these compounds were investigated for their 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and inhibitory effects on mushroom tyrosinase. Compounds 2-9, quinic acid derivatives with caffeoyl moiety, showed significant DPPH radical scavenging activity. Moreover, compounds 2 and 5-10 showed weak mushroom tyrosinase inhibitory effects.

Phenolic amides from Tribulus terrestris and their inhibitory effects on nitric oxide production in RAW 264.7 cells.

A new phenolic amide, named cis-terrestriamide (7), together with ten known compounds (1-6, 8-11), were isolated from the methanolic extract of the fruits of Tribulus terrestris. The structure of 7 was elucidated on the basis of extensive analyses of 1D and 2D nuclear magnetic resonance spectroscopic and high resolution mass spectrometry data. Compounds 1, 2, 5, 6, 8, 9, and 11 exhibited inhibitory effects on the lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 cells, with IC50 values of 18.7-49.4 µM.

Proteome characterization of copper stress responses in the roots of sorghum.

Copper (Cu) is a important micronutrient for plants, but it is extremely toxic to plants at high concentration and can inactivate and disturb protein structures. To explore the Cu stress-induced tolerance mechanism, the present study was conducted on the roots of sorghum seedlings exposed to 50 and 100 µM CuSO4 for 5 days. Accumulation of Cu increased in roots when the seedlings were treated with the highest concentration of Cu2+ ions (100 µM). Elevated Cu concentration provoked notable reduction of Fe, Zn, Ca, and Mn uptake in the roots of sorghum seedlings. In the proteome analysis, high-throughput two-dimensional polyacrylamide gel electrophoresis combined with MALDI-TOF-TOF MS was performed to explore the molecular responses of Cu-induced sorghum seedling roots. In two-dimensional silver-stained gels, 422 protein spots were identified in the 2-D gel whereas twenty-one protein spots (≥1.5-fold) were used to analyze mass spectrometry from Cu-induced sorghum roots. Among the 21 differentially expressed proteins, 10 proteins were increased, while 11 proteins were decreased due to the intake of Cu ions by roots of sorghum. Abundance of most of the identified proteins from the roots that function in stress response and metabolism was remarkably enhanced, while proteins involved in transcription and regulation were severely reduced. Taken together, these results imply insights into a potential molecular mechanism towards Cu stress in C4 plant, sorghum.

Chemical Constituents Isolated from Bletilla striata and Their Inhibitory Effects on Nitric Oxide Production in RAW 264.7 Cells.

Bioassay-guided fractionation of the MeOH extract of the tubers of Bletilla striata led to the isolation of two new C-methylated flavan-3-ols, bletillanols A (1) and B (2), along with ten known compounds (3 - 12). Their structures were determined by using extensive spectroscopic analysis including 1D-, 2D-NMR, and circular dichroism data. All of the isolated compounds were tested for their inhibitory potential on the nitric oxide generation in LPS-stimulated RAW 264.7 cells.

Leaf proteome characterization in the context of physiological and morphological changes in response to copper stress in sorghum.

Copper (Cu) is an essential micronutrient required for normal growth and development of plants; however, at elevated concentrations in soil, copper is also generally considered to be one of the most toxic metals to plant cells due to its inhibitory effects against many physiological and biochemical processes. In spite of its potential physiological and economical significance, molecular mechanisms under Cu stress has so far been grossly overlooked in sorghum. To explore the molecular alterations that occur in response to copper stress, the present study was performed in ten-day-old Cu-exposed leaves of sorghum seedlings. The growth characteristics were markedly inhibited, and ionic alterations were prominently observed in the leaves when the seedlings were exposed to different concentrations (0, 100, and 150 µM) of CuSO4. Using two-dimensional gels with silver staining, 643 differentially expressed protein spots (≥1.5-fold) were identified as either significantly increased or reduced in abundance. Of these spots, a total of 24 protein spots (≥1.5-fold) from Cu-exposed sorghum leaves were successfully analyzed by MALDI-TOF-TOF mass spectrometry. Of the 24 differentially expressed proteins from Cu-exposed sorghum leaves, 13 proteins were up-regulated, and 11 proteins were down-regulated. The abundance of most identified protein species, which function in carbohydrate metabolism, stress defense and protein translation, was significantly enhanced, while that of another protein species involved in energy metabolism, photosynthesis and growth and development were severely reduced. The resulting differences in protein expression patterns together with related morpho-physiological processes suggested that these results could help to elucidate plant adaptation to Cu stress and provide insights into the molecular mechanisms of Cu responses in C4 plants.

Morpho-Physiological and Proteome Level Responses to Cadmium Stress in Sorghum.

Cadmium (Cd) stress may cause serious morphological and physiological abnormalities in addition to altering the proteome in plants. The present study was performed to explore Cd-induced morpho-physiological alterations and their potential associated mechanisms in Sorghum bicolor leaves at the protein level. Ten-day-old sorghum seedlings were exposed to different concentrations (0, 100, and 150 µM) of CdCl2, and different morpho-physiological responses were recorded. The effects of Cd exposure on protein expression patterns in S. bicolor were investigated using two-dimensional gel electrophoresis (2-DE) in samples derived from the leaves of both control and Cd-treated seedlings. The observed morphological changes revealed that the plants treated with Cd displayed dramatically altered shoot lengths, fresh weights and relative water content. In addition, the concentration of Cd was markedly increased by treatment with Cd, and the amount of Cd taken up by the shoots was significantly and directly correlated with the applied concentration of Cd. Using the 2-DE method, a total of 33 differentially expressed protein spots were analyzed using MALDI-TOF/TOF MS. Of these, treatment with Cd resulted in significant increases in 15 proteins and decreases in 18 proteins. Major changes were absorbed in the levels of proteins known to be involved in carbohydrate metabolism, transcriptional regulation, translation and stress responses. Proteomic results revealed that Cd stress had an inhibitory effect on carbon fixation, ATP production and the regulation of protein synthesis. Our study provides insights into the integrated molecular mechanisms involved in responses to Cd and the effects of Cd on the growth and physiological characteristics of sorghum seedlings. We have aimed to provide a reference describing the mechanisms involved in heavy metal damage to plants.

Myelin oligodendrocyte glycoprotein (MOG35-55)-induced experimental autoimmune encephalomyelitis is ameliorated in interleukin-32 alpha transgenic mice.

Multiple sclerosis (MS), also known as disseminated sclerosis or encephalomyelitis disseminate, is an inflammatory disease in which myelin in the spinal cord and brain are damaged. IL-32α is known as a critical molecule in the pathophysiology of immune-mediated chronic inflammatory disease such as rheumatoid arthritis, chronic pulmonary disease, and cancers. However, the role of IL-32α on spinal cord injuries and demyelination is poorly understood. Recently, we reported that the release of proinflammatory cytokines were reduced in IL-32α-overexpressing transgenic mice. In this study, we investigated whether IL-32α plays a role on MS using experimental autoimmune encephalomyelitis (EAE), an experimental mouse model of MS, in human IL-32α Tg mice. The Tg mice were immunized with MOG35-55 suspended in CFA emulsion followed by pertussis toxin, and then EAE paralysis of mice was scored. We observed that the paralytic severity and neuropathology of EAE in IL-32α Tg mice were significantly decreased compared with that of non-Tg mice. The immune cells infiltration, astrocytes/microglials activation, and pro-inflammatory cytokines (IL-1ß and IL-6) levels in spinal cord were suppressed in IL-32α Tg mice. Furthermore, NG2 and O4 were decreased in IL-32α Tg mice, indicating that spinal cord damaging was suppressed. In addition, in vitro assay also revealed that IL-32α has a preventive role against Con A stimulation which is evidenced by decrease in T cell proliferation and inflammatory cytokine levels in IL-32α overexpressed Jurkat cell. Taken together, our findings suggested that IL-32α may play a protective role in EAE by suppressing neuroinflammation in spinal cord.

Chemical constituents from Belamcanda chinensis and their inhibitory effects on nitric oxide production in RAW 264.7 macrophage cells.

Belamcanda chinensis (L.) DC., which belongs to the family of Iridaceae, has been used as a folk medicine for the treatment of inflammation, asthma, tonsillitis, and many other throat disorders. Bioactivity-guided purification of the methylene chloride-soluble fraction of the rhizomes of B. chinensis based on the inhibition of nitric oxide production led to the identification of seventeen known compounds. Their structures were elucidated on the basis of extensive spectroscopic measurement such as NMR and ESI-MS. All of the isolated compounds were evaluated for their inhibitory effects on nitric oxide production in LPS-induced RAW 264.7 macrophage cells.

Profiling of mitochondrial proteome in wheat roots.

Mitochondria are important organelles for cellular respiration within the eukaryotic cell and have many important functions including vitamin synthesis, amino acid metabolism and photorespiration. To investigate the mitochondrial proteome of the roots of wheat seedlings, a systematic and targeted analysis were carried out on the mitochondrial proteome from 15 day-old wheat seedling root material. Mitochondria were isolated by Percoll gradient centrifugation; and extracted proteins were disassociated and analyzed by Tricine SDS-PAGE couple to LTQ-FTICR mass spectrometry. From the isolated the sample, 184 proteins were identified which is composed of 140 proteins as mitochondria and 44 proteins as other subcellular proteins that are predicted by the freeware sub-cellular predictor. The identified proteins in mitochondria were functionally classified into 12 classes using the ProtFun 2.2 servers based on biological processes. Proteins were shown to be involved in amino acid biosynthesis (17.1%), biosynthesis of cofactors (6.4%), cell envelope (11.4%), central intermediary metabolism (10%), energy metabolism (20%), fatty acid metabolism (0.7%), purines and pyrimidines (5.7%), regulatory functions (0.7%), replication and transcription (1.4%), translation (22.1%), transport and binding (1.4%), and unknown (2.8%). These results indicate that many of the protein components present and functions of identifying proteins are common to other profiles of mitochondrial proteins performed to date. These results are provided the extensive and noble clues, to our knowledge, of mitochondrial proteins from wheat roots.

Anti-inflammatory effect of tricin 4'-O-(threo-ß-guaiacylglyceryl) ether, a novel flavonolignan compound isolated from Njavara on in RAW264.7 cells and in ear mice edema.

Although recent study has shown tricin 4'-O-(threo-ß-guaiacylglyceryl) ether (TTGE), an isolated compound from Njavara rice, to have the most potent anti-inflammatory effects, the action mechanism has not been fully understood. Here, we examined the effect of TTGE on the inflammation and elucidated the potential mechanism. We demonstrated that TTGE significantly inhibited LPS-induced NO and ROS generation in RAW264.7 cells, which was correlated with the down-regulating effect of TTGE on the iNOS and COX-2 expression via NF-κB and STAT3. TPA-induced ear edema was also efficiently inhibited by the TTGE treatment. TTGE blocked the induction of iNOS and COX-2 through the regulation of NF-κB and STAT3, which could explain the reduced TPA-induced edema symptoms. Moreover, the introduction of ERK inhibitor abrogated the anti-inflammatory effect of TTGE via the recovery of NF-κB and STAT3 signalings. Taken together, these results suggest that TTGE has anti-inflammatory properties through down-regulation of NF-κB and STAT3 pathways.

Acceleration of the development of Alzheimer's disease in amyloid beta-infused peroxiredoxin 6 overexpression transgenic mice.

The amyloid beta (Aß) peptide in the brains of patients with Alzheimer's disease (AD) is cytotoxic to neurons and has a central role in the pathogenesis of the disease. Peroxiredoxin 6 (Prdx6) is an antioxidant protein and could act as a cytoprotective protein. However, the role of Prdx6 in neurodegenerative disease has not been studied. Thus, the roles and action mechanisms in the development of AD were examined. Aß1-42-induced memory impairment in Prdx6 transgenic mice was worse than C57BL/6 mice, and the expression of amyloid precursor protein cleavage, C99, ß-site APP-cleaving enzyme 1, inducible nitric oxide synthase, and cyclooxygenase-2 was greatly increased. In addition, the astrocytes and microglia cells of Aß-infused Prdx6 transgenic mice were more activated, and Aß also significantly increased lipid peroxidation and protein carbonyl levels, but decreased glutathione levels. Furthermore, we found that translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus was increased in Aß-infused Prdx6 transgenic mice. These results suggest that the overexpression of Prdx6 could accelerate the development of AD through increased amyloidogenesis through independent PLA2 activation and Nrf2 transcription.

Novel iridoids from the flowers of Campsis grandiflora.

A non-glycosidic iridoid, campsinol (1), and two iridoid glucosides, 7-O-(Z)-p-coumaroylcachineside V (2) and 7-O-(E)-p-coumaroylcachineside I (3), were isolated from the fresh flowers of Campsis grandiflora along with five known iridoid glycosides, ixoroside (4), campsiside (5), cachineside I (6), 5-hydroxycampenoside (7), and 5-hydroxycampsiside (8), and two known phenylpropanoid glycosides, acteoside (9) and leucosceptoside A (10). The structures of these compounds were determined based on the NMR and Mass spectroscopic data and other chemical evidences.

Obovatol attenuates LPS-induced memory impairments in mice via inhibition of NF-κB signaling pathway.

Neuroinflammation and accumulation of ß-amyloid are critical pathogenic mechanisms of Alzheimer's disease (AD). In the previous study, we have shown that systemic lipopolysaccharide (LPS) caused neuroinflammation with concomitant increase in ß-amyloid and memory impairments in mice. In an attempt to investigate anti-neuroinflammatory properties of obovatol isolated from Magnolia obovata, we administered obovatol (0.2, 0.5 and 1.0 mg/kg/day, p.o.) to animals for 21 days before injection of LPS (0.25 mg/kg, i.p.). We found that obovatol dose-dependently attenuates LPS-induced memory deficit in the Morris water maze and passive avoidance tasks. Consistent with the results of memory tasks, the compound prevented LPS-induced increases in Aß1₋42 formation, ß- and γ-secretases activities and levels of amyloid precursor protein, neuronal ß-secretase 1 (BACE1), and C99 (a product of BACE1) in the cortex and hippocampus. The LPS-mediated neuroinflammation as determined by Western blots and immunostainings was significantly ameliorated by the compound. Furthermore, LPS-induced nuclear factor (NF)-κB DNA binding activity was drastically abolished by obovatol as shown by the electrophoretic mobility shift assay. The anti-neuroinflammation and anti-amyloidogenesis by obovatol were replicated in in vitro studies. These results show that obovatol mitigates LPS-induced amyloidogenesis and memory impairment via inhibiting NF-κB signal pathway, suggesting that the compound might be plausible therapeutic intervention for neuroinflammation-related diseases such as AD.

Naphthoquinones from Catalpa ovata and their inhibitory effects on the production of nitric oxide.

Bioassay-guided fractionation of a CH2Cl2-soluble fraction of the stems of Catalpa ovata led to isolation of a new naphthoquinone, 4-hydroxy-2-(2-methoxy-3-hydroxy-3-methyl-but-1-enyl)-4-hydro-1H-naphthalen-1-one (10), together with nine known compounds, catalponol (1), catalponone (2), catalpalactone (3), alpha-lapachone (4), 9-hydroxy-alpha-lapachone (5), 4,9-dihydroxy-alpha-lapachone (6), 9-methoxy-alpha-lapachone (7), 4-oxo-alpha-lapachone (8), and 9-methoxy-4-oxo-alpha-lapachone (9). The structures were elucidated on the basis of spectroscopic analyses. The inhibitory effects of these isolates on lipopolysaccharide-induced NO synthesis in RAW 264.7 cells were evaluated. Among them, catapalactone (3), 9-hydroxy-alpha-lapachone (5) and 4,9-dihydroxy-alpha-lapachone (6) exhibited potent inhibitory effects, with IC(50) values of 9.80, 4.64 and 2.73 microM, respectively.

Neurite outgrowth effect of 4-O-methylhonokiol by induction of neurotrophic factors through ERK activation.

Compounds isolated from Magnolia officinalis such as magnolol, honokiol and obovatol exhibit several pharmacological effects on CNS including depressant, anxiolytic and anticonvulsant effects, as well as neuroprotective effects against chemical and heat damages. Recently, honokiol was found to have a neurotrophic effect in fetal rat cortical neurons. In the present study, we show that 4-O-methylhonokiol, a novel compound from Magnolia officinalis, promotes neurite outgrowth in a concentration- dependent manner in rat embryonic neuronal cells. In parallel with the neurite outgrowth activity, the expression of neurite outgrowth marker proteins is also increased by treatment with 4-O-methylhonokiol. We also found that 4-O-methylhonokiol promotes the release of NGF and BDNF into cell culture medium. In addition, lower concentration of 4-O-methylhonokiol (1 and 2 lM) further enhanced neurite outgrowth and expression of neurite outgrowth marker proteins in the presence of NGF (50 ng/ml) or BDNF (10 ng/ml). Subsequently, we found that 4-O-methylhonokiol activates ERK in a concentration- dependent manner. However, the neurite outgrowth activity and the NGF and BDNF release induced by 4-O-methylhonokiol are suppressed by an ERK-specific inhibitor. These results suggest that 4-O-methylhonokiol has the ability to induce neurite outgrowth via the increase of neurotrophic factor levels through ERK activation.

A new furofuran lignan from Isodon japonicus.

A new sesamin type furofuran lignan, (-)-sesamin-2,2'-diol (1), along with two known flavonoids (2 and 3) and three phenolic compounds (4-6) were isolated from the aerial parts of Isodon japonicus. The structures of these compounds were determined by analysis of spectroscopic data (1D-, 2D-NMR, HRMS and CD) and by comparison of the data with those of related metabolites.

Inhibitory effect of proanthocyanidin on ultraviolet B irradiation-induced melanogenesis.

Repetitive exposure of the skin to ultraviolet (UV) radiation induces various adverse effects, including skin thickening, wrinkle formation, inflammation, and pigmentation. Various natural and synthetic compounds were studied to determine whether they might prevent UV induction of these adverse effects. In particular, naturally occurring antioxidants were used for regulating skin damage induced by UV radiation since several antioxidants were found to inhibit photoaging through prevention of collagen synthesis via inhibition of matrix metalloproteinases (MMP) and/or decrease of melanin synthesis. The L values in pigmented skin were lower at 4 wk (52.97 +/- 2.09) than at the start of this study (0 wk, 62.89 +/- 0.56) in the control. In the proanthocyanidin mixture group, the L value was increased (56.83 +/- 1.71) similar to the control (52.97 +/- 2.09). Proanthocyanidin also suppressed the expression levels of tyrosinase by 20-40%, and blocked the expression of MITF, TRP-1, and TRP-2, which are factors implicated in the control of melanogenesis. Taken together, these data indicate that proanthocyanidin may be useful to attenuate UVB-induced melanogenesis.