Genes cloning, sequencing and function identification of recombinant polyphenol oxidase isozymes for production of monomeric theaflavins from Camellia sinensis.

Theaflavins (TFs) are important quality compounds in black tea with a variety of biological activities. However, direct extraction of TFs from black tea is inefficient and costly. Therefore, we cloned two PPO isozymes from Huangjinya tea, termed HjyPPO1 and HjyPPO3. Both isozymes oxidized corresponding catechin substrates for the formation of four TFs (TF1, TF2A, TF2B, TF3), and the optimal catechol-type catechin to pyrogallol-type catechin oxidation rate of both isozymes was 1:2. In particular, the oxidation efficiency of HjyPPO3 was higher than that of HjyPPO1. The optimum pH and temperature of HjyPPO1 were 6.0 and 35 °C, respectively, while those of HjyPPO3 were 5.5 and 30 °C, respectively. Molecular docking simulation indicated that the unique residue of HjyPPO3 at Phe260 was more positive and formed a π-π stacked structure with His108 to stabilize the active region. In addition, the active catalytic cavity of HjyPPO3 was more conducive for substrate binding by extensive hydrogen bonding.

Molecular characterization of a feruloyl-CoA 6'-hydroxylase involved in coumarin biosynthesis in Clematis terniflora DC.

Coumarin is an important secondary metabolite that affects plant physiology. It is a lactone of cis-o-hydroxycinnamic acid and widely exists in medicinal plants. Clematis terniflora DC. is a plant belonging to Ranunculaceae and is rich in variety of coumarins. Feruloyl-CoA 6'-hydroxylase has been reported as a key enzyme in the formation of coumarin basic skeleton only in some common plants, however, its evidence in other species is still lacking especially for the biosynthesis of coumarins in C. terniflora. In the present study, we identified a feruloyl-CoA 6'-hydroxylase CtF6'H in C. terniflora, and functional characterization indicated that CtF6'H could hydroxylate feruloyl-CoA to 6-hydroxyferuloyl-CoA. Furthermore, the expression level of CtF6'H was differed among different tissues in C. terniflora, while under UV-B radiation, the level of CtF6'H was increased in the leaves. Biochemical characteristics and subcellular location showed that CtF6'H was mainly present in the cytosol. The crystal structure of CtF6'H was simulated by homology modeling to predict the potential residues affecting enzyme activity. This study provides the additional evidence of feruloyl-CoA 6'-hydroxylase in different plant species and enriches our understanding of biosynthetic mechanism of coumarin in C. terniflora.

The chloroplast GATA-motif of Mahonia bealei participates in alkaloid-mediated photosystem inhibition during dark to light transition.

Mahonia bealei and Mahonia fortunei are important plant resources in Traditional Chinese Medicine that are valued for their high levels of benzylisoquinoline alkaloids (BIAs). Although the phytotoxic activity of BIAs has been recognized, information is limited on the mechanism of action by which these compounds regulate photosynthetic activity. Here, we performed comparative chloroplast genome analysis to examine insertions and deletions in the two species. We found a GATA-motif located in the promoter region of the ndhF gene of only M. bealei. K-mer frequency-based diversity analysis illustrated the close correlation between the GATA-motif and leaf phenotype. We found that the GATA-motif significantly inhibits GUS gene expression in tobacco during the dark-light transition (DLT). The expression of ndhF was downregulated in M. bealei and upregulated in M. fortunei during the DLT. NDH-F activity was remarkably decreased and exhibited a significant negative correlation with BIA levels in M. bealei during the DLT. Furthermore, the NADPH produced through photosynthetic metabolism was found to decrease in M. bealei during the DLT. Taken together, our results indicate that this GATA-motif might act as the functional site by which BIAs inhibit photosynthetic metabolism through downregulating ndhF expression during the DLT.

Mitochondrial proteomic analysis reveals the regulation of energy metabolism and reactive oxygen species production in Clematis terniflora DC. leaves under high-level UV-B radiation followed by dark treatment.

Clematis terniflora DC. is an important medicinal plant from the family Ranunculaceae. A previous study has shown that active ingredients in C. terniflora, such as flavonoids and coumarins, are increased under ultraviolet B radiation (UV-B) and dark treatment and that the numbers of genes related to the tricarboxylic acid cycle and mitochondrial electron transport chain (mETC) are changed. To uncover the mechanism of the response to UV-B radiation and dark treatment in C. terniflora, mitochondrial proteomics was performed. The results showed that proteins related to photorespiration, mitochondrial membrane permeability, the tricarboxylic acid cycle, and the mETC mainly showed differential expression profiles. Moreover, the increase in alternative oxidase indicated that another oxygen-consuming respiratory pathway in plant mitochondria was induced to minimize mitochondrial reactive oxygen species production. These results suggested that respiration and mitochondrial membrane permeability were deeply influenced to avoid energy consumption and maintain energy balance under UV-B radiation and dark treatment in C. terniflora leaf mitochondria. Furthermore, oxidative phosphorylation was able to regulate intracellular oxygen balance to resist oxidative stress. This study improves understanding of the function of mitochondria in response to UV-B radiation and dark treatment in C. terniflora. SIGNIFICANCE: C. terniflora was an important traditional Chinese medicine for anti-inflammatory. Previous study showed that the contents of coumarins which were the main active ingredient in C. terniflora were induced by UV-B radiation and dark treatment. In the present study, to uncover the regulatory mechanism of metabolic changes in C. terniflora, mitochondrial proteomics analysis of leaves was performed. The results showed that photorespiration and oxidative phosphorylation pathways were influenced under UV-B radiation and dark treatment. Mitochondria in C. terniflora leaf played a crucial role in energy mechanism and regulation of cellular oxidation-reduction to maintain cell homeostasis under UV-B radiation followed with dark treatment.

Transcriptomic analysis unravels the molecular response of Lonicera japonica leaves to chilling stress.

Lonicera japonica is not only an important resource of traditional Chinese medicine, but also has very high horticultural value. Studies have been performed on the physiological responses of L. japonica leaves to chilling, however, the molecular mechanism underlying the low temperature-induced leaves morphological changes remains unclear. In this study, it has been demonstrated that the ratio of pigments content including anthocyanins, chlorophylls, and carotenoids was significantly altered in response to chilling condition, resulting in the color transformation of leaves from green to purple. Transcriptomic analysis showed there were 10,329 differentially expressed genes (DEGs) co-expressed during chilling stress. DEGs were mainly mapped to secondary metabolism, cell wall, and minor carbohydrate. The upregulated genes (UGs) were mainly enriched in protein metabolism, transport, and signaling, while UGs in secondary metabolism were mainly involved in phenylpropaoids-flavonoids pathway (PFP) and carotenoids pathway (CP). Protein-protein interaction analysis illustrated that 21 interacted genes including CAX3, NHX2, ACA8, and ACA9 were enriched in calcium transport/potassium ion transport. BR biosynthesis pathway related genes and BR insensitive (BRI) were collectively induced by chilling stress. Furthermore, the expression of genes involved in anthocyanins and CPs as well as the content of chlorogenic acid (CGA) and luteoloside were increased in leaves of L. japonica under stress. Taken together, these results indicate that the activation of PFP and CP in leaves of L. japonica under chilling stress, largely attributed to the elevation of calcium homeostasis and stimulation of BR signaling, which then regulated the PFP/CP related transcription factors.

Use Chou's 5-steps rule to identify protein post-translational modification and its linkage to secondary metabolism during the floral development of Lonicera japonica Thunb.

Lonicera japonica Thunb. is widely used in traditional medicine systems of East Asian and attracts a large amount of studies on the biosynthesis of its active components. Currently, there is little understanding regarding the regulatory mechanisms behind the accumulation of secondary metabolites during its developmental stages. In this study, published transcriptomic and proteomic data were mined to evaluate potential linkage between protein modification and secondary metabolism during the floral development. Stronger correlations were observed between differentially expressed genes (DEGs) and their corresponding differentially abundant proteins (DAPs) in the comparison of juvenile bud stage (JBS)/third green stage (TGS) vs. silver flowering stage (SFS). Seventy-five and 76 cor-rDEGs and cor-rDAPs (CDDs) showed opposite trends at both transcriptional and translational levels when comparing their levels at JBS and TGS relative to those at SFS. CDDs were mainly involved in elements belonging to the protein metabolism and the TCA cycle. Protein-protein interaction analysis indicated that the interacting proteins in the major cluster were primarily involved in TCA cycle and protein metabolism. In the simple phenylpropanoids biosynthetic pathway of SFS, both phospho-2-dehydro-3-deoxyheptonate aldolase (PDA) and glutamate/aspartate-prephenate aminotransferase (AAT) were decreased at the protein level, but increased at the gene level. A confirmatory experiment indicated that protein ubiquitination and succinylation were more prominent during the early floral developmental stages, in correlation with simple phenylpropanoids accumulation. Taken together, those data indicates that phenylpropanoids metabolism and floral development are putatively regulated through the ubiquitination and succinylation modifications of PDA, AAT, and TCA cycle proteins in L. japonica.

Polygalaxanthone III downregulates inflammation in the lipopolysaccharide-stimulated RAW264.7 macrophages: A quantibody array analysis.

Polygala japonica Houtt. (PJ), a member of the Polygala L. family that is suggested to exhibit detoxification properties in traditional Chinese medicine, is often used to treat upper respiratory tract infections. The anti-inflammatory effects of four main components of PJ (POL, PS-XLIX, PS-E, and PS-F) were examined using the LPS(0.3 µg·mL-1)-stimulated RAW264.7 macrophage model. The levels of NO, ROS, and iNOS were examined to analyze the anti-inflammatory activity of POL. Additionally, the levels of extracellular inflammation-related cytokines and chemokines were measured using quantibody array. The KEGG pathway analysis was performed to examine the anti-inflammatory mechanism of POL. The levels of NO in the POL-pretreated group were significantly downregulated when compared with those in the PS-E-pretreated, PS-F-pretreated, and PS-XLIX-pretreated groups. POL significantly inhibited the changes of iNOS, ROS, and inflammatory factors caused by LPS stimulation (p < 0.001). The expression levels of IL21 and GM-CSF were examined using qPCR, while those of JAK-STAT signaling pathway-related proteins in the LPS-stimulated RAW264.7 macrophages were analyzed using western blotting. POL significantly downregulated the expression of IL-21 and GM-CSF. The anti-inflammatory mechanism of POL is mediated through the JAK-STAT pathway. Thus, this study demonstrated that POL is an anti-inflammatory component of PJ and elucidated its mechanism.

Isolation, Structural Elucidation, and Liquid Chromatography-Mass Spectrometry Analysis of Steroidal Glycosides from Polygonatum odoratum.

The rhizomes of Polygonatum odoratum represent a traditional Chinese medicine and functional food. A phytochemical investigation resulted in the isolation of eight steroidal glycosides (1-8), including two new compounds, polygonatumosides F (1) and G (2). The structures were elucidated by spectroscopic data and chemical reactions. Compound 7 showed antiproliferation activity against human hepatocellular carcinoma cell line HepG2 (IC50 of 3.2 µM). The chemical profile and contents of steroidal glycosides of P. odoratum rhizomes collected at different dates and geographical locations were also investigated, indicating that the rational harvest of P. odoratum in spring and autumn is preferable to obtain higher levels of steroidal glycosides. Compounds 1 and 7 showed the highest contents in all P. odoratum samples and have potential to serve as chemotaxonomic and chemical markers for quality control of this important plant material. 14-Hydroxylation may be a key step for the biosynthesis of compounds 1-7.

Identification of candidate synovial membrane biomarkers after Achyranthes aspera treatment for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease whose main symptom is a heightened inflammatory response in synovial tissues. To verify the anti-arthritic activities of Achyranthes aspera and its possible therapy-related factors on the pathogenesis of RA, the saponins in A. aspera root were isolated and identified to treat the collagen-induced arthritis (CIA) rats. Phytochemical analysis isolated and identified methyl caffeate, 25-S-inokosterone, 25-S-inokosterone ß-D-glucopyranosyl 3-(O-ß-D-glucopyranosyloxy)-oleanolate, and ß-D-glucopyranosyl 3-(O-ß-D-galactopyranosyl (1→2)(O-ß-D-glucopyranosyloxy)-oleanolate as main compounds in the root of A. aspera. Proteomics was performed to determine the differentially expressed proteins in either inflamed or drug-treated synovium of CIA rats. Treatment resulted in dramatically decreased paw swelling, proliferation of inflammatory cells, and bone degradation. Fibrinogen, procollagen, protein disulfide-isomerase A3, and apolipoprotein A-I were all increased in inflamed synovial tissues and were found to decrease when administered drug therapy. Furthermore, Alpha-1-antiproteinase and manganese superoxide dismutase were both increased in drug-treated synovial tissues. The inhibition of RA progression shows that A. aspera is a promising candidate for future treatment of human arthritis. Importantly, the total saponins found within A. aspera are the active component. Finally, autoantigens such as fibrinogen and collagen could act as inducers of RA due to their aggravation of inflammation. Given this, it is possible that the vimentin and PDIA3 could be the candidate biomarkers specific to Achyranthes saponin therapy for rheumatoid arthritis in synovial membrane.

Acupuncture Therapy for the Treatment of Myelosuppression after Chemotherapy: A Literature Review over the Past 10 Years.

The aim of this study is to review current studies on the effect of acupuncture therapy on bone marrow suppression after chemotherapy. The authors of the present paper have searched related literature over the past 10 years at home and abroad, analyzing the features and the effects of acupuncture therapy (including acupuncture, moxibustion, point injection, point application, etc.) for treating myelosuppression after tumor chemotherapy. We also discuss the year of publication, document type, acupuncture therapy, acupoint selection, and adverse effects with figures. We analyzed 159 articles related to acupuncture therapy from 2004 to 2013, and the analysis revealed that point injection was the most frequently used therapy for clinical applications, and that Zusanli (ST36) was the most frequently used acupoint. The results showed that some problems regarding the design method, acupoint selection, and acupuncture intervention measure existed in those research studies. We hope to provide readers with an overall and objective understanding of acupuncture and moxibustion therapy for treating myelosuppression after tumor chemotherapy.

Experimental analysis on the main contents of Rhizoma gastrodiae extract and inter-transformation throughout the fermentation process of Grifola frondosa.

Gastrodin (GA), p-hydroxylbenzaldehyde (HBA), p-hydroxybenzyl alcohol (gastrodigenin, HA) and parishin not only are the major active ingredients of Rhizoma gastrodiae, but exist transformed relations from each other throughout the fermentation process of Grifola frondosa in this work. We had found that parishin (non-free gastrodin) almost could completely transformed into gastrodin (GA, free gastrodin) after R. gastrodiae alcohol extract was sterilized by moist heat (121 °C, 30 min), but before was added into submerged cultivation of G. frondosa. However, interestingly and importantly, gastrodin re-synthesized of parishin after R. gastrodiae alcohol extract's addition into submerged cultivation of G. frondosa. In addition, the reduction of p-hydroxylbenzaldehyde and p-hydroxybenzyl alcohol in G. frondosa fermentation process reconfirmed that the G. frondosa strain 51616 really could synthesize gastrodin into parishin by submerged fermentation. This paper firstly also reported G. frondosa's effects on R. gastrodiae.

UV-B induced changes in the secondary metabolites of Morus alba L. leaves.

Ultraviolet-B (UV-B) radiation is harmful to plants and human beings. Many secondary metabolites, like flavonoids, alkaloids, and lignin, are UV-B absorbing compounds, which can protect the genetic material of plants. Furthermore, they are active components of herbal drugs. UV-B radiation can activate the self-protective secondary metabolism system. The results of this paper provide a method to induce bioactive secondary metabolites from mulberry leaves (Morus alba L.) by UV-B irradiation in vitro. Five significantly different chromatographic peaks were found by HPLC fingerprint after induction, from which two active compounds were identified: One was chalcomoracin, a natural Diels-Alder type adduct with antibacterial activity; the other one was moracin N, which is a precursor of chalcomoracin. Their contents were 0.818 mg/g and 0.352 mg/g by dry weight, respectively.

Two new guaiane-type sesquiterpenoids from the fruits of Daucus carota L.

Two new guaiane-type sesquiterpenoids containing an interesting epoxy unit, daucuside (1) and daucusol (2) were isolated from the fruits of Daucus carota L. Their chemical structures were elucidated on the basis of MS, NMR spectroscopic analyses coupled with chemical degradation and they were also evaluated for the cytotoxic effects against two human gastric cancer cell lines BGC-823 and AGS.

A new angeloylated triterpenoid saponin from the husks of Xanthoceras sorbifolia Bunge.

A new angeloylated oleanane-type triterpenoid saponin, sorbifoliaside (1), and a known saponin, xanifolia O54, were isolated from the husks of Xanthoceras sorbifolia Bunge. Their chemical structures were elucidated on the basis of various spectroscopic analyses coupled with chemical degradation. To our knowledge, compound 1 is the first example of a naturally occurring triterpenoid saponin with an angeloyl group at C-2 of the sugar moiety.

Triterpenoid saponins from Rubus ellipticus var. obcordatus.

Ten new triterpenoid saponins (1-10), named rubusides A-J, and 21 known saponins (11-31) were isolated from the roots of Rubus ellipticus var. obcordatus. The structures of 1-10 were established on the basis of spectroscopic analyses, mainly NMR and MS, and chemical degradations. The compounds demonstrated inhibitory activities against alpha-glucosidase with IC50 values in the range 0.65-3.09 mM.

Lanostane-type triterpenoids from the roots of Kadsura coccinea.

Seven new lanostane-type triterpenoids, seco-coccinic acids A-F (1- 6) and coccinilactone A (7), were isolated from the roots of Kadsura coccinea. Their structures were established on the basis of spectroscopic data analysis. The absolute configuration at C-24 of compound 5 was confirmed by the modified Mosher's method. The cell growth inhibitory effects of these compounds were determined in human leukemia HL-60 cells, and it was found that compounds 1, 2, 3, and 5 exhibited antiproliferative effects with GI 50 values ranging from 6.8 to 42.1 microM.

Structure and absolute configuration of clerodane diterpene glycosides and a rearranged cadinane sesquiterpene glycoside from the stems of Tinospora sinensis.

Three new clerodane diterpene glycosides, tinosposinensides A-C ( 1- 3), and a new rearranged cadinane sesquiterpene glycoside, tinosinenside ( 4), were isolated from the stems of Tinospora sinensis. The structure including the relative configuration was elucidated on the basis of spectroscopic analysis. The absolute configuration was determined by application of the modified Mosher's method and chemical transformation. The inhibitory activities of the isolated compounds against alpha-glucosidase are also described.

Pancreatic lipase-inhibiting triterpenoid saponins from fruits of Acanthopanax senticosus.

Sixteen triterpenoid saponins were isolated from the fruits of Acanthopanax senticosus, including a new compound, acanthopanaxoside E (1), which was established as 3-O-beta-D-glucuronopyranosyl echinocystic acid 28-O-beta-D-glucopyranoside on the basis of various spectroscopic analyses and chemical degradation. By using a pancreatic lipase-inhibiting assay system, the crude saponin fraction showed inhibitory activity on pancreatic lipase, which is a key enzyme in lipid digestion. Among the isolated compounds, silphioside F (2), copteroside B (3), hederagenin 3-O-beta-D-glucuronopyranoside 6'-O-methyl ester (4) and gypsogenin 3-O-beta-D-glucuronopyranoside (5) showed inhibitory activity toward pancreatic lipase with IC(50) values of 0.22, 0.25, 0.26 and 0.29 mM, respectively, and the free carboxylic acid groups in position 28 within their chemical structures were required for enhancement of pancreatic lipase inhibition.

A new isoflavone glucoside from Astragalus membranaceus var. mongholicus.

A new isoflavone glucoside (1) was isolated from the roots of Astragalus membranaceus var. mongholicus. Its structure was elucidated by spectroscopic methods.