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Background: This paper aims to investigate the metabolic impact of walnut meal extracts rich in polyphenols (WMP) on high fat diet (HFD)-fed rats and to determine whether the lipid-lowering effects are related to modulations of the gut microbiota. SD rats were fed a standard chow diet or an HFD for 18 weeks. After 6 weeks, the HFD rats were supplemented with 750mg WMP/kg body weight and the vehicle for 12 weeks. The structure of gut microbiota was assessed by analyzing 16S rDNA sequences.Results: WMP suppressed the weight gain and visceral obesity. WMP treatment also improved lipid profiles and increased antioxidative activities. WMP fully reversed hepatic steatosis with upregulation of adipocytokines involved in lipid catabolism (e.g. adiponectin, PPAR-γ, visfatin, CEBPα), and increased the activity of lipoprotein lipase, hormone-sensitive lipase, in which were associated with glucose tolerance improved and insulin resistance mitigated. As revealed by 16S rDNA sequencing, WMP restored the diversity of intestinal flora reduced by HFD, dramatically reduced the abundance of Fusobacterium varium and Enterobacteriaceae, reversed and sharply raised the abundance of Lachnospiraceae UCG005 and Akkermansia decreased by HFD.Conclusion: Our findings demonstrated that WMP suppressed the weight gain and adiposity in HFD-fed rats, and fully reversed HFD diet-induced insulin resistance and hepatic steatosis while dramatically reduced the abundance of Fusobacteriaceae and Enterobacteriaceae, underscoring the gut-liver axis as a primary target of walnut polyphenols.

Walnut kernel was a traditional Chinese medicine, as well as a brain power boosting food. Walnut meal (WM) was prepared from walnut kernel by cold-press deoil, which was rich in polyphenols. In this study, we investigated the positive effects of walnut meal extracts on memory impairment of Alzheimer's disease (AD) mice induced by D-galactose. After 6 weeks of WMP treatment, the behavioral results showed that WMP could significantly improve the learning and memory of D-galactose Model (MOL) mice. The biochemical assay showed WMP could increase the amount of Acetylcholine (Ach) and reduce the oxidative stress and inflammation. Meanwhile, WMP improved the expression of neural stem cells, restored the number and the shape of neurons. The RNA-seq analysis revealed 284 differentially expressed genes in the hippocampus were regulated by WMP treatment, among which Gzma, Apol11b, H2-Q6 were up-regulated, while Rny1, Scgn, Col6a3 were down-regulated. Further analysis disclosed the differentially expressed genes were relevant to PI3K-Akt signaling pathway, FoxO signaling pathway, PPAR signaling pathway, neuroactive ligand-receptor interaction, cellular senescence, and particularly strongly interacted with the ribosomal family genes (Rpl35a, Rps27rt, Rpl3l, Rpl21, Rpl26). In addition, transcription factor Ep300 regulated genes of Cdkn1a, Spp1, and Tnfsf10 distinctly in the hippocampus, which were involved in inflammation and protein kinase. Transcription factor Pparg regulated genes of Angptl4, Fabp4, and Plin4, which were mainly expressed in PPAR (Peroxisome Proliferator-activated receptors) signaling pathway. This study might assist in identifying new targets to restore memory impairment in Alzheimer's disease.

Walnut meal (WM) is rich in polyphenols which exhibit multiple therapeutic effects. The purpose of this study was to investigate the therapeutic effects of walnut meal extracts (WMP) on glycolipid metabolism and liver transcriptomics in T2DM rats. A T2DM rat model was established by using a high-fat diet combined with streptozotocin. A 5-week WMP therapy showed the effects of decreasing water intake, excretion, fasting blood glucose, fasting insulin, and insulin resistance, increasing β-cell function and insulin sensitivity index; meanwhile regulating dysfunctional lipid metabolism and reducing inflammation; improving body weight, oral glucose tolerance test and insulin sensitivity; and increasing the activities of SOD and CAT while decreasing the MDA levels in the liver and serum of T2DM rats. Moreover, 10 key differentially expressed genes were identified by RNA-seq, including Gck, RT1-Ba, Fasn, Slc13a3, Cd74, Jun, Cyp4a1, Myh7b, Plin3, and Got1, and they were highly potentially related to glycolipid metabolism. Our results suggested that WMP exhibited the anti-diabetic effect and could regulate glycolipid metabolism in T2DM rats. This finding might assist in identifying potential therapeutic targets for T2DM prevention and intervention.

Ethnopharmacological relevance The diaphragma juglandis (DJ) comes from the wooden septum in the core of Juglans regia L, also known as the walnut septum. In Iranian traditional medicine, walnut distraction wood was widely used in the treatment of diabetes. However, there is a lack of research data on the mechanism of DJ against diabetes. Aim of the study: To explore the protective effect of diaphragma juglandis extract (DJE) on type 2 diabetic rats and the hypoglycemic mechanism of DJE. Material and methods Supplemented DJE and fed a high-fat diet for five weeks, and then injected low-dose STZ, successfully induced type 2 diabetic rats. Collected rat serum, liver, pancreas and feces to determine the biochemical parameters of serum and liver, analyze the pathological damages of pancreas and liver, and measure the changes of gut microbes in feces. Results DJE could inhibit the metabolic abnormalities of T2DM by improving insulin resistance, abnormal lipid metabolism, liver damage, oxidative stress, and reducing inflammation. DJE significantly held fasting blood glucose, glycosylated serum protein, serum low density lipoprotein, high density lipoprotein, oral glucose tolerance test, nitric oxide, superoxide dismutase and catalase, serum and liver triglycerides, total cholesterol, aspartate aminotransferase, alanine aminotransferase, malondialdehyde, lipopolysaccharide, fasting insulin and tumor necrosis factor-α and prevented the pathological damage of pancreas and liver. The 16SrRNA gene sequencing results showed that DJE intercepted the disorders of the fecal gut microbes, mainly including Lactobacillaceae, Rikenella, Pygmaiobacter, Oscillospiraceae and Klebsiella. Spearman correlation analysis showed that the changes of gut microbes were closely relative with biochemical parameters. Conclusion DJE might prevent type 2 diabetes and its complications and hold up the disorders of gut microbes.

Triterpene saponins from Panax notoginseng are widely used in the treatment of cardio-cerebrovascular diseases with increasing demand. Herein, key enzyme genes 3-hydroxy-3-methylglutaryl CoA reductase ( PnHMGR ) and squalene synthase ( PnSS ) involved in the triterpene saponins biosynthetic pathway were introduced into Panax notoginseng cells to enhance triterpene saponins productions. Overexpression of PnHMGR alone or co-overexpression of PnSS and PnHMGR could remarkably enhance the accumulation of total saponins compared with the control. The highest yield of total saponins was got from the co-overexpression of PnSS and PnHMGR cell line, which was 3-fold and 1.5-fold higher than those in control and the cell line overexpressed PnHMGR alone, respectively. In addition, the contents of six major monomer saponins (Re, Rb1, Rg1, Rh1, Rh2, and F1) and phytosterols were also increased in PnHMGR - PnSS transgenic cell lines. Our results provide a useful genetic engineering strategy to improve the contents of triterpene saponins in many medicinal plants.

Pressed degreased walnut meal (WM) was prepared from walnut kernel by cold-press deoil. It was rich in polyphenols with high activities of lowering lipids, antioxidation, anti-inflammation, and others, but the hypoglycemic activity has scarcely been reported. The purpose of this research was to investigate the preventive effect of the extracts of WM (WMP) on T2DM rats and the regulation of WMP on intestinal flora. Some results showed that WMP significantly held FBG, FINS and GSP, OGTT, the serum and hepatic TG, TC, HDL-C, LDL-C, AST, ALT, LPS, TNF-α, IL-6, and the hepatic MDA, CAT, SOD, as well as, prevented the pathological damage of the liver and the pancreas. The results of 16SrDNA sequencing demonstrated that WMP impeded the changes of intestinal flora in feces, mainly including Firmicutes, Bacteroidetes, and Proteobacteria. These indicated that WMP had promising effects on T2DM, which could be associated with the modulation of gut bacteria.

Transcription factors (TFs) are important regulative factors that mediate many life processes. The ERF family of TFs harbors a conserved AP2 domain, which plays vital roles in secondary metabolism in plants. However, few ERF genes in Panax notoginseng have been reported to date. In this study, a P. notoginseng ERF gene was cloned and designated as PnERF1. The cDNA of PnERF1 is 1113 bp long and encodes a protein consisting of 266 amino acids. Electrophoretic mobility shift assay (EMSA) demonstrated that the protein could bind to cis-element GCC-box, indicating that PnERF1 might bind to the promoter regions which contained GCC-box. The expression levels of some genes involved in triterpenoid saponin biosynthesis in PnERF1-transgenic cells were relatively higher than those in the wild cells. Correspondingly, the contents of total saponins were raised in the PnERF1-transgenic cell lines compared to the wild cell lines. Additionally, the contents of six major monomer saponins (Rg3, Rh1, Rd, Rg1, F1 and Re) were also increased in the transgenic cells. Such results suggest that the transcription factor PnERF1 is a positive regulator related to triterpenoid saponin biosynthesis in P. notoginseng.

Gallic acid is one of the important polyphenols in plants and it inhibits α-amylase. The interaction between gallic acid and α-amylase was investigated by fluorescence quenching spectroscopy, UV-vis spectroscopy, synchronous spectroscopy, and the three-dimensional fluorescence spectroscopy under mimic physiological conditions. The result of the emission quenching at different temperatures revealed that there are static quenching of intrinsic fluorescence of α-amylase induced by gallic acid and a complex of gallic acid-α-amylase was formed. The results obtained from the evaluation of three-dimensional fluorescence spectra, UV-vis spectra, and synchronous spectra suggested that the association between gallic acid and α-amylase did change the molecular conformation of α-amylase. Gallic acid can enter the primary substrate-binding pocket and alter the microenvironment around tryptophan and tyrosine residues.

OBJECTIVE(S) Accumulating evidence has suggested that oxidative stress and apoptosis are involved in the aging process. d‐galactose (gal) has been reported to cause symptoms of aging in mice, accompanied by liver and brain injuries. Our present work was to study the potential antioxidative and anti‐apoptotic effects of walnut and to explore how these effects act on mice in a d‐gal‐induced aging model. MATERIALS AND METHODS Aging mice were induced by subcutaneous injection of d‐gal (200 mg kg−1 d−1 for 8 weeks). Walnut samples were simultaneously administered to the d‐gal‐induced aging mice once daily by intragastric gavage. Finally, body weight, organ index, cognitive function, levels of antioxidative enzymes, and liver function were monitored. RESULTS The kernel pellicles of walnut could not only improve the learning and memory ability, and the organ index, but also significantly decrease body weight and normalize the levels of activity of antioxidative enzymes in aging mice. Further, the walnut seed coat would protect damages of hippocampus and liver in aging mice. HIGHLIGHTS In the current study, we investigated the effects of walnut kernels and walnut seed coats (WSCs) on d‐galactose‐induced aging mice. WSC was firstly found to have beneficial effects on d‐gal‐treated mouse's brain with learning and memory impairment, which probably through the underlying mechanism reduces oxidative damage and limits neuroinflammation. In addition, WSC had a protective effect on liver damage in d‐galactose sensing mice.

Glutathione S -transferases (GSTs) are multifunctional proteins that are encoded by a large gene family. Plant GSTs are involved in host defense against both biotic and abiotic stresses as well as are involved in some physiological processes under normal conditions. In the present study, a novel tau GST gene, LrGSTU5 , was isolated from Lilium regale Wilson. The deduced protein LrGSTU5 shared a high identity with AcGSTU1 from Allium cepa . In addition, there was relatively high gene expression of LrGSTU5 in the roots but the only minimal expression in both the stem and leaf of L. regale under normal conditions. The gene expression of LrGSTU5 was obviously up-regulated after treatment with signaling molecules including salicylic acid and ethylene as well as after inoculation with Fusarium oxysporum . Furthermore, in order to verify the function of LrGSTU5 , a constitutive plant expression vector of LrGSTU5 was constructed and transferred into tobacco ( Nicotiana tabacum L. cv Xanthi). Compared to the wild type (WT), several diseases resistance-related genes, including osmotin , PR1b , chitinase , and MnSOD , were up-regulated in the transgenic lines. Moreover, three important antioxidant enzymes, GST, superoxide dismutase, and ascorbate peroxidase, exhibited significantly higher activities in the T1 transgenic lines than in the WT after inoculation with F. oxysporum . Meanwhile, the rate of superoxidate anion production in the transgenic tobacco lines was significantly lower than in the WT. The antifungal activity of three LrGSTU5 transgenic tobacco lines was estimated through in vivo inoculation, and the results showed that the transgenic tobacco plants enhanced the resistance to F. oxysporum .

An indirect enzyme-linked immunosorbent assay (ELISA) capable of detecting walnut proteins in commercial products was developed. Polyclonal antibodies against soluble walnut protein were used. Specific epitopes of walnut protein were analyzed for cross activity with peanut, soybean, sesame, rice, wheat, jujube, and defatted milk protein. Epitopes of walnut proteins had no cross activity. The indirect ELISA was highly specific for soluble walnut proteins. Recovery values from walnut protein solutions ranged from 88.47 to 115%, and low coefficients of variation (

Panax notoginseng has been extensively used as a traditional Chinese medicine. In the current study, molecular cloning and characterization of PnbHLH1 transcription factor were explored in Panax notoginseng. The full length of the PnbHLH1 gene obtained by splicing was 1430 bp, encoding 321 amino acids. Prokaryotic expression vector pET-28a-PnbHLH1 was constructed and transferred into the BL21 prokaryotic expression strain. An electrophoretic mobility shift assay of PnbHLH1 protein binding to E-box cis-acting elements verified that PnbHLH1 belonged to the bHLH class transcription factor which could interact with the promoter region of the E-box core sequence. The expression levels of key genes involved in the biosynthesis of triterpenoid saponins in PnbHLH1 transgenic cells were higher than those in the wild cells. Similarly, the total saponin contents were increased in the PnbHLH1 transgenic cell lines compared with the wild cell lines. Such results suggest that the PnbHLH1 transcription factor is a positive regulator in the biosynthesis of triterpenoid saponins in Panax notoginseng.

Solid-state fermentation (SSF) of walnut protein meal (WPM) by Bacillus subtilis was optimized for the maximum degree of hydrolysis (DH) and reducing power (RP) using response surface methodology (RSM). Optimum fermentation conditions were fermentation time of 82.01 h, inoculum concentration of 10.40, and water content of 1.50 mL/g. Optimized values were 41.80% and 0.78 for DH and RP, respectively. Walnut peptides (WP) were ultrafiltration membrane fractionated. The WPs-II (molecular weight

This study was aimed at the chemical composition and inhibitory effects of walnut polyphenols (WP) in vitro lipase activity and on obesity in obese mice models. More than 20 individual phenolics such as gallotannins, ellagitannins, flavonoids and phenolic acids were identified in WP. Studying the interactions between porcine pancreatic lipase (PL) and WP were based on fluorescence quenching and an enzymatic assay. The addition of WP to lipase caused a reduction of protein fluorescence intensity at 310 K. In addtion, we found that the effect of WP on PL was dependent on reaction medium and substrate used and the half maximal inhibitory concentration of WP was determined to be 163 μg/mL. At the beginning of the experiment, mice were divided into 3 groups, one of them served as normal control group (NCG), the second as hyperlipidemia control group (HCG), the last as walnut polyphenol-treated group (WTG). After 8 weeks of treatment, we investigated the effects of WP on weight gain, food intake and biochemical indexes in obese mice. The results showed that WP could significantly decrease body weight in obese mice (-13.52%, P < 0.05). However, there was no remarkable difference in food intake among three groups (1760.2–1823.3 g). In addition, WP could significantly decrease the TG, TC and LDL-cholesterol (LDL-C) concentrations and increase the HDL-cholesterol (HDL-C) concentration when compared to the HCG (-36.12%, -31.27%, -73.3%, and +59.72%, respectively, P < 0.01). The levels of hepatic TG and TC were significantly decrease in WTG when compared to the HCG (-27.72% and -48.43%, respectively, P < 0.01). In conclusion, these results suggest that WP could be a potentially therapeutic alternative in the treatment of obesity caused by a high-fat diet.

Tyrosinase is a key rate-limiting enzyme in melanin synthesis which can be blocked to reduce melasma and freckles by inhibiting the activity of tyrosinase. Salidroside showed certain inhibitory effects on tyrosinase by enzyme kinetics with inhibition of 33.04%. Besides it was also a competitive inhibitor of tyrosinase which served as a substrate analogue to combine with enzyme to produce quinones with stronger light absorption. Furthermore, docking results implied that inhibitory mechanisms might be attributed to the weak interactions between salidroside and tyrosinase such as hydrogen bonds and van del waals. Salidroside is a main kind of plant flavonoids from Rhodiola rosea, walnut and so on. Studying inhibitory mechanism of salidroside on tyrosinase is not only helpful for developing anti-freckle products in which salidroside is one of the most important components, but also beneficial for making better use of plant flavonoids to seek for a new generation of natural skin care cosmetics.

In this article, the effect of the solvents (water, methanol, ethanol, chloroform, N-butanol and ethyl acetate) on the extraction yields (EY), total flavonoids content (TFC), total phenols content (TPC) form walnut shell and the inhibitory effect of extracts on pancreatic lipase were analysed. The kinetics of enzyme inhibition and was investigated. The inhibitory effect of extracts on adipogenesis of 3T3-L1 preadipocytes was also investigated. The highest EY, TFC and TPC were achieved with N-butanol (4.54%), Ethyl acetate (80.40 mg QEs/g extract) and ethyl acetate (200.40 mg GAE/g extract). The order of inhibitory activity was methanol extract (ME) > ethyl acetate extract (EAE) > ethanol extract (EE) > water extract (WE) > N-butanol extract (NBE) > chloroform extract (CE), the inhibition ratios were 82.51%, 73.13%, 70.49%, 69.42%, 65.57% and 60.66%, respectively under the concentration of 500 μg/mL. The IC50 values of WE, ME, EE, CE, NBE and EAE were 371.35, 308.91, 407.57, 196.50, 247.79 and 254.27 μg/mL respectively. The inhibitory types of walnut shell extracts were noncompetitive. All the extracts were non-toxic to 3T3-L1 preadipocytes. The ME, EE, CE, NBE and EAE have inhibitory effect on triglyceride accumulation of 3T3-L1 cells.

Walnut shell is a waste generated in the walnut (Juglans regia L.) harvest, containing natural compounds with antioxidant properties. In this connection, the effect of the solvent (water, chloroform, methanol, ethanol, ethyl acetate and N-butanol) on the extraction yields, total flavonoids content and antioxidant properties was analyzed. Total flavonoids content of extracts was determined by NaNO2-Al(NO3)3 method. Extract antioxidant activity was measured by hydroxyl, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide anion radical scavenging capacity and the total antioxidant capacity and reducing power assay. The highest extraction yield was achieved with N-butanol (4.54%) and the greatest total flavonoids content shown by the samples extracted with ethyl acetate. The highest ability to scavenging DPPH, hydroxyl and superoxide radicals was ethyl acetate extract (EC50=81.03μg/mL), methanol extract (EC50=131.35 μg/mL) and chloroform extract (EC50=176.35μg/mL) respectively under the concentration of 500 μg/mL. The methanol extract showed the greatest total antioxidant activity and reducing power. The results showed that different solvent had significant effect on total flavonoids content and antioxidant activity. The result obtained demonstrated the potential of walnut shell as a source of antioxidant.

The basic leucine zipper (bZIP) proteins are ubiquitous in plants and play important roles in plant defense responses. In this study, based on an expressed sequence tag from a suppression subtractive hybridization cDNA library of Lilium regale Wilson during Fusarium oxysporum f. sp. lilii infection, a novel bZIP transcription factor gene LrbZIP1 was isolated from L. regale root using the rapid amplification of cDNA ends method. The predicted protein of LrbZIP1 with 142 amino acid residues contains a basic domain signature and a leucine zipper motif. The quantitative reverse transcription-PCR (qRT-PCR) analysis showed that the transcription level of LrbZIP1 was higher in roots of L. regale than in young stems and leaves. Moreover, the expression of LrbZIP1 was up-regulated in the incompatible interaction between L. regale and F. oxysporum f. sp. lilii as well as after treatments with stress-related signaling molecules. To verify the function of LrbZIP1, a constitutive expression vector of LrbZIP1 was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi). The results of Southern blotting and qRT-PCR analyses demonstrated that the LrbZIP1 was integrated into genome of the tobacco transformants and highly expressed. Under normal conditions, the T1 transgenic tobacco lines showed higher antioxidant enzyme activities and transcription levels of several resistance-related genes than the wild type. Moreover, the T1 transgenic tobacco plants showed strong resistance to F. oxysporum f. sp. lilii infection.

Evidence suggests that dietary walnuts are able to induce improvements in memory and learning functions. In addition, polyphenols have been shown to modulate critical neuronal signalling pathways involved in processes of learning and memory. The aim of our present work was to study the effect of polyphenol extracts from walnut testa (42%) on learning and memory functions in hypercholesterolemia mice based on obesity, hypercholesterolemia and oxidative stress. At the beginning of the experiment, mice were divided into 3 groups, one of them served as normol control group (NCG), the second as hypercholesterolemia control group (HCG), the last as walnut polyphenol-treated group (WTG). After 8 weeks of treatment, we investigated the performance of C57BL/6J mice in Morris water maze test. The results showed that the escape latency was significant increase in HCG, when compared to NCG and WTG. In addition, the number of crossings was significant decrease in HCG, when compared to NCG and WTG (–47.35% and –43.35%, respectively, P 0.05]. On the other hand, walnut polyphenol (WP) significantly decreased serum total triglycerides, cholesterol and malondialdehyde (MDA) level (–36.31%, –31.48% and –21.51%, respectively, P < 0.01) and increased superoxide dismutase (SOD) activity (+48.39%, P < 0.01). Administration of WP significantly decreased MDA level (–36.86%, P < 0.01) and increased SOD activity (+17.08%, P < 0.01) in brain tissues. In conclusion, walnut polyphenol was able to improve learning and memory functions.

Pathogenesis-related (PR) proteins play key roles in plant responses to pathogens and abiotic stresses. In this study, nine novel PR genes were isolated from Lilium regale Wilson, which is a wild lily species of China with high-level resistance to the soilborne fungal pathogen Fusarium oxysporum f. sp. lilii, and homology analysis classified them into the PR10 family. These novel LrPR10s were clustered together with PR10s from monocotyledons in a phylogenetic tree, moreover, phylogenetic analysis divided the nine LrPR10s into two groups. The main-chain conformation and folding patterns of the LrPR10s were highly conserved with other plant PR10s. The expression patterns of the nine LrPR10s in L. regale during normal development were examined by QRT-PCR, and the transcription levels of the LrPR10s were relatively high in roots. Furthermore, QRT-PCR analysis indicated that the expression levels of LrPR10-1, LrPR10-2, LrPR10-5, LrPR10-6, and LrPR10-7 in L. regale roots were up-regulated by two or more stress-related signaling molecules including salicylic acid, jasmonic acid, ethylene, and H2O2, while the other four LrPR10s were repressed by these four signaling molecules. In addition, five members of the LrPR10 gene family including LrPR10-2, LrPR10-4, LrPR10-5, LrPR10-6, LrPR10-7, and LrPR10-9 were strongly induced by F. oxysporum in resistant L. regale compared with the susceptible Lilium Oriental hybrid ‘Siberia’. The other four LrPR10s were down-regulated by F. oxysporum infection. In summary, our results indicate that the members of PR10 gene family are involved in L. regale defense responses against F. oxysporum f. sp. lilii.

14-3-3 proteins exist in all eukaryotes and act as important regulators to modulate diverse signaling pathways associated with a wide range of biological processes. Besides, they are involved in molecular networks protecting plant from damages caused by the biotic and abiotic stresses, and it has been reported that many 14-3-3s play an important role in defense response against pathogens. To isolate 14-3-3 genes involved in self-defense during pathogenic infection, a novel gene Lr14-3-3 was isolated from the root of Lilium regale Wilson which is a kind of wild lily species with high resistance to phytopathogens, viruses, and drought. The Lr14-3-3 was expressed at relatively low level in root, stem, and leaf of L. regale under normal development, by contraries, Lr14-3-3 was significantly up-regulated in an incompatible interaction between L. regale and Fusarium oxysporum as well as in a compatible interaction between susceptible Lilium Oriental Hybrid ‘Siberia’ and F. oxysporum. However, the transcription level of Lr14-3-3 in L. regale was evidently higher than that in ‘Siberia’ during F. oxysporum infection. Moreover, the gene expression of Lr14-3-3 was responded to ethylene treatment. Furthermore, in order to verify the function of Lr14-3-3, the constitutive plant expression vector of Lr14-3-3 was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi). The following quantitative reverse transcription-PCR (QRT-PCR) analysis demonstrated that the Lr14-3-3 gene was steadily expressed in the T1 transgenic tobacco lines, and compared with the WT, the disease resistance-related genes including N gene, osmotin, MYC, and NADPH oxidase gene were up-regulated in the transgenic lines. Moreover, the antioxidant enzymes, superoxide dismutase (SOD), glutathione S-transferase (GST), and ascorbate peroxidase (APX), showed significantly higher activities in the T1 transgenic lines than that in WT. The antifungal activity of Lr14-3-3 transgenic tobacco was performed in vitro plates and in vivo inoculation, respectively, and the results showed that the transgenic tobacco plants evidently resisted the infection of Botryosphaeria dothidea, Phomopsis sp., and F. oxysporum.

Background Due to Rhizoma gastrodiae (Tianma) from different producing areas have vital difference in quality and physiological efficacy. This study focused on the classification and identification of Tianma from different producing areas using Fourier Transform Infrared (FT-IR) Spectroscopy coupled with chemometrics. Frequencies at 1800–600 cm −1 were exploited for both classification and identification. Principal component analysis (PCA) and Partial least squares-Discriminant analysis (PLS-DA) were used for classification and identification analysis of Tianma from different producing areas. Results Fourier Transform Infrared (FT-IR) Spectroscopy coupled with Principal component analysis (PCA) and Partial least squares-Discriminant analysis (PLS-DA) can successfully classify and identify Tianma from different producing areas. Conclusion Taken together, the proposed methodology is a useful tool to identify Tianma from different producing areas.

Conjugated linoleic acid (CLA) was prepared by alkali isomerization using walnut oil as raw materials, KOH as catalyst and propylene glycol as solvent. The kinetics of alkali isomerization of linoleic acid from walnut oil converted into CLA was experimentally studied under the reaction conditions of 393~453 K,and which the initial mass ratio of propylene glycol, walnut oil and KOH was 3:2:1. The mechanism of alkali isomerization was proposed, and the mechanism model was put forward through theoretical derivation.The rate constants were determined, the mechanism model was verified,and the kinetics parameters were obtained.The results showed that alkali isomerization reaction was first order the concentration of linoleic acid, which the apparent activation energy was 67.22 kJ·mol-1and the pre-exponential constant was 8.963×105min-1.

Biodiesel has attracted considerable interest in recent years as an alternative, biodegradable and nonpolluting transportation fuel. Conventional alkaline process for biodiesel production are energy consuming and generate undesirable by-products such as soaps, that make difficult the separation and purification of biodiesel. Ionic liquids as environmentally benign chemical solvent, had been used in diverse chemical reactions. Especially, they could be applied in biodiesel production. Preparation of biodiesel catalyzed by ionic liquids have many merits, such as no corrosion to equipment, no pollution to environment, and reusability. Ionic liquids can improve the catalytic activity and they have wide utilization prospect in biodiesel preparation. In this paper, the advances in the Brønsted ionic liquids catalysts and their application in biodiesel production through esterification of fatty acids were reviewed. In addition, the prospect for the application of the ionic liquids to catalyze biodiesel production was also stated. Since the cost of ionic liquid may be an issue, there are some challenges to be faced, such as the production of ionic liquids with low cost, easy recovery and with the possibility of reutilization of the catalyst for several cycles.

Biodiesel has attracted considerable interest in recent years as an alternative, biodegradable and nonpolluting transportation fuel. Conventional alkaline process for biodiesel production are energy consuming and generate undesirable by-products such as soaps, that make difficult the separation and purification of biodiesel. Ionic liquids as environmentally benign chemical solvent, had been used in diverse chemical reactions. Especially, they could be applied in biodiesel production. Preparation of biodiesel catalyzed by ionic liquids have many merits, such as no corrosion to equipment, no pollution to environment, and reusability. Ionic liquids can improve the catalytic activity and they have wide utilization prospect in biodiesel preparation. In this paper, the advances in the Brønsted ionic liquids catalysts and their application in biodiesel production through transesterification of oils and fats were reviewed. In addition, the prospect for the application of the ionic liquids to catalyze biodiesel production was also stated. Since the cost of ionic liquid may be an issue, there are some challenges to be faced, such as the production of ionic liquids with low cost, easy recovery and with the possibility of reutilization of the catalyst for several cycles.

BACKGROUND Up to now, there has been no report on the taxonomic and functional analysis of the microbial community in fermenting puer tea by pyrosequencing. In this study, metagenomic pyrosequencing was first used in fermenting puer tea to delineate a relatively comprehensive overview of the microbial taxonomy while also preliminarily characterising the functional ontologies of microbial genes present in puer tea pile fermentation. RESULTS A total of 251 738 pyrosequencing reads (9197 contigs and 145 402 singletons) were generated by pyrosequencing. Taxonomic analysis revealed three dominant bacterial phyla, Proteobacteria (23.56%), Actinobacteria (23.35%) and Firmicutes (11.37%), and one dominant eukaryotic phylum, Ascomycota (30.47%). A total of 58 664 hits were categorised into 28 functional subsystems based on the SEED database. Moreover, two categories, ‘metabolism of terpenoids and polyketides’ and ‘biosynthesis of other secondary metabolites’, were selectively analysed and 69 enzyme genes were presented in 16 pathways. CONCLUSION The dominant microbes of puer tea fermentation were bacteria in the present study, and yeasts rather than moulds accounted for the overwhelming majority of Eukaryota. The analysis of functional genes and metabolic pathways will be helpful for further study of the mechanism of puer tea fermentation at molecular level. © 2013 Society of Chemical Industry

Adulteration of walnut oil with sunflower oil is a major issue for the walnut oil industry. In this paper, the potential use of total synchronous fluorescence (TSyF) spectra to differentiate walnut oil from sunflower oil and synchronous fluorescence spectra combined with multivariate analysis to assess the adulteration of walnut oil is demonstrated. TSyF spectra were acquired by varying the excitation wavelength in the region 250–700 nm and the wavelength interval (Δλ) in the region from 10 to 100 nm. TSyF contour plots for walnut oil, in contrast to sunflower oil, show an extra fluorescence region in the excitation wavelength lower than 280 nm. Fifty-one oil mixtures were prepared by adulterating walnut oil with sunflower oil at varying levels (0–100 %). The partial least-squares regression model was used for the quantification of adulteration using wavelength intervals of 20, 40, 60 and 80 nm. This technique is useful for the detection of sunflower oil in walnut oil at levels down to 0.3 % (v/v) in just 2.5 min using an 80-nm wavelength interval.

Glutathione S-transferases (GSTs) are ubiquitous enzymes in animals and plants, and they are multifunctional proteins encoded by a large gene family. GSTs are involved in response to the oxidative stress including drought, salt, heavy metals, and so on. Under oxidative stress, the excessive reactive oxygen species (ROS) induce an increase in GST levels, and then the GSTs metabolize the toxic products of lipid peroxidation, damaged DNA and other molecules. Previously, a full-length cDNA of a novel zeta GST gene, PpGST, was characterized from fruit of Pyrus pyrifolia Nakai cv Huobali. In the present study, a constitutive plant expression vector of PpGSTwas constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi) to verify the function of PpGST. As a result, the PpGSTgene was successfully integrated into the genome of the transgenic tobacco lines and expressed as expected in the transformants through Southern blotting and quantitative reverse transcription-polymerase chain reaction analysis. Growth of T1 generation plants of PpGST transgenic lines and WT under non-stressful conditions was similar, however, the transgenic tobacco lines showed relatively normal growth under drought, NaCl, and cadmium (Cd) stresses. Furthermore, the T1 transgenic tobacco lines showed significantly slower superoxide anion production rate than the WT under abiotic stress. Simultaneously, the MDA content of each T1 transgenic tobacco plant was only slightly increased and significantly lower than that of the WT under drought, salt and Cd stress. Together with the GST activity of the transgenic tobacco lines, which was significantly increased under stressful conditions, as compared with that in WT, overexpression of PpGSTin tobacco enhanced the tolerance of transgenic tobacco lines to oxidative damage caused by drought, NaCl, and Cd stresses.

Polygalacturonase-inhibiting proteins (PGIPs) are multifunctional proteins related to plant autoimmunity and belong to the plant extracellular leucine-rich repeat (eLRR) protein superfamily. PGIPs play a role in host defense in many plants. In the present study, a novel PGIP gene, PpPGIP was isolated from Pyrus pyrifolia Nakai cv Huobali. The nucleotide sequence of PpPGIP was highly homologous with PGIPs from other plant species and the protein encoded by PpPGIP has several conserved LRR domains. The putative protein PpPGIP was closely clustered with several PGIPs from horticultural plants on the phylogenetic tree. The constructed homology model of PpPGIP indicated that the main-chain conformation and the folding patterns of PpPGIP were highly similar to structural features of PvPGIP2 from Phaseolus vulgaris. The expression levels of PpPGIP in healthy tissue and organ of ‘Huobali’ were analyzed with RT-PCR, and PpPGIP accumulated a little in young leaves, but PpPGIP was expressed abundantly in the pericarp of ‘Huobali’ fruits. Furthermore, in order to verify the function of PpPGIP, the constitutive plant expression vector of PpPGIP was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi). The Southern blot and real-time PCR analyses demonstrated that the PpPGIP gene was integrated into the genome of the tobacco transformants and highly expressed in the transgenic lines. The antifungal activity of PpPGIP was detected in vitro plates, and the crude protein extract of transgenic tobacco plants inhibited the hyphal growth of Phomopsis sp., Alternaria sp., Penicillium sp., and Aspergillus niger in different degrees.

β-1,3-glucanases, one of the well known families of pathogenesis-related (PR) proteins in responding to pathogen infection or some environmental stresses, are hydrolytic enzymes abundant in plants. The plant β-1,3-glucanases are considered to have a broad range of biological functions in the normal physiological process as well as in the defence response against pathogens. Some plant β-1,3-glucanases were studied in detail as antifungal proteins in vivo or in vitro. To investigate the β-1,3-glucanase genes involved in self-defence during fruit development, a novel gene PpGlu was isolated from the fruit of Pyrus pyrifolia Nakai cv Huobali, which is a kind of local sand pear of China with high resistance to biotic and abiotic stresses. The putative protein sequence of PpGlu was highly homologous with β-1,3-glucanases from other plant species. Bioinformatics analyses indicated that the catalytic and other conserved amino acid residues occupied highly conserved positions in the primary structure of the putative protein PpGlu and other plant β-1,3-glucanases. Moreover, PpGlu had rather conserved main-chain conformation and the folding patterns of plant β-1,3-glucanases. The transcripts of PpGlu was dramatically induced in pericarps of ‘Huobali’ fruits by removing the bags for 2 days, but rapidly declined after the fruits were debagged for 1 week. Furthermore, a constitutive plant expression vector of PpGlu was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi) to verify the function of PpGlu. The PpGlu gene was successfully integrated into the genome of the transgenic tobacco lines and expressed as expect in the transformants by Southern blotting and quantitative reverse transcription-polymerase chain reaction (QRT-PCR) analyses. The antifungal activity of PpGlu was detected in vitro plates, and the crude protein extract of transgenic tobacco lines inhibited the hyphal growth of the following three fungi, Phomopsis sp., Alternaria sp. and Fusarium sp. at different degrees.

Thaumatin-like proteins (TLPs) belong to the pathogenesis-related proteins 5 family, and members of TLP gene family accumulate in plants in response to pathogen infection or environmental stress. The biological functions of plant TLPs are believed to be diverse, and they are involved in host defense against both the biotic and abiotic stresses as well as the other physiological processes. Lots of TLPs were well studied as antifungal proteins in vivo or in vitro. To isolate TLP genes involved in self-defense during fruit development, a novel gene PpTLP was isolated from the fruit of Pyrus pyrifolia Nakai cv Huobali which is a kind of local sand pear with high resistance to biotic and abiotic stresses in Yunnan province of China. The nucleotide sequence of PpTLP was highly homologous with TLPs from other plant species, and the protein encoded by PpTLP has 16 conserved cysteine residues and 5 amino acid residues related with antifungal activity of plant TLPs, an arginine residue, a glutamic acid residue, and 3 aspartic acid residues. What is more, the putative protein PpTLP has the highly conserved main-chain conformation and the folding patterns as other plant TLPs with antifungal activity. PpTLP was abundantly expressed in pericarp and leaf of P. pyrifolia Nakai cv Huobali. Furthermore, in order to verify the function of PpTLP, the constitutive plant expression vector of PpTLP was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi). The Southern blot and quantitative reverse transcription-polymerase chain reaction (QRT–PCR) analyses demonstrated that the PpTLP gene was integrated into the genome of the tobacco transformants and highly expressed in the transgenic lines. The antifungal activity of PpTLP was detected in vitro plates, and the crude protein extract of transgenic tobacco plants inhibited the hyphal growth of Sclerotinia sclerotiorum, Phomopsis sp., Phytophthora parasitica var. nicotianae, and Alternaria sp. in different degrees.

The binding properties on alizarin to human serum albumin (HSA) have been studied for the first time using fluorescence spectroscopy in combination with UV-visible absorbance spectroscopy. The results showed that alizarin strongly quenched the intrinsic fluorescence of HSA through a static quenching procedure, and non-radiation energy transfer occurred within the molecules. The number of binding sites was 1, and the efficiency of Förster energy transfer provided a distance of 1.83 nm between tryptophan and alizarin binding site. ΔH(θ), ΔS(θ) and ΔG(θ) were obtained based on the quenching constants and thermodynamic theory (ΔH(θ)0, ΔS(θ)0 and ΔG(θ)0). These results indicated that hydrophobic and electrostatic interactions are the main binding forces in the alizarin-HSA system. In addition, the results obtained from synchronous fluorescence spectra and three-dimensional fluorescence spectra showed that the binding of alizarin with HSA could induce conformational changes in HSA.

The binding properties on theasinesin to human serum albumin (HSA) have been studied for the first time using fluorescence spectroscopy in combination with UV–vis absorbance spectroscopy. The results showed that theasinesin strongly quenched the intrinsic fluorescence of HSA through a static quenching procedure, and non-radiation energy transfer happened within molecules. The number of binding site was 1, and the efficiency of Forster energy transfer provided a distance of 4.64 nm between tryptophan and theasinesin binding site. At 298, 310 and 323 K, the quenching constants of HSA–theasinesin system were 2.55×10 3 , 2.16×10 3 and 1.75×10 3 mol L −1 . Δ H θ , Δ S θ and Δ G θ were obtained based on the quenching constants and thermodynamic theory (Δ H θ S θ >0 and Δ G θ

A thermostable alkaline phosphatase with high specific activity and thermal resistance was purified from a novel species of Thermus sp. named as Thermus yunnanensis sp. nov. The enzyme contains a single peptide with a molecular mass of about 52 kDa on SDS-PAGE analysis and appears to be a homodimer in solution with the molecular mass of 104 kDa. The optimal pH and temperature for its activities are pH 8.0-10.0 and 70-80 degrees C, respectively. The catalytic activities of the enzyme are metal ion dependent, and Mg2+, Zn2+ and Co2+ are the main activators. Among these, Co2+ is the most active stimulator and has unique activation effect at high temperature. Metal binding analysis showed the binding of Mg2+ at the metal binding site was easy to loss in the thermoinactivation, and Co2+ was apt to bind at that site and kept the favorable configuration of catalysis, which would result high activation in the incubation with Co2+ at high temperature. According to this study, a model was proposed for the explanation of the activation and the results of actual experiments demonstrated the validity of the model.

The sequences of the 16S rRNA genes from 38 strains of the family Thermaceae were compared by alignment analysis. The genus-specific and species-specific base substitutions or base deletions (signature positions) were found in three hypervariable regions (in the helices 6, 10 and 17). The differentiation of secondary structures of the high variable regions in the 5' end (38-497) containing several signature positions further supported the concept. Based on the comparisons of the secondary structures in the segments of 16S rRNAs, a key to the species of the family Thermaceae was proposed.

A rosy-pigmented Gram-negative, thermophilic bacterium with an optimum growth temperature of about 55 degrees C was isolated from Tengchong hot springs in Yunnan province, China. Its growth scarcely occurred below 40 degrees C or above 70 degrees C. Phylogenetic and secondary structural analyses of 16S rRNA and DNA-DNA hybridization showed that the organism represented a new species of the genus Meiothermus. This new species could be distinguished easily from other species of the genus Meiothermus by the following phenotypic characteristics: rosy pigment, expanded body, sucrose and maltose were not utilized, gelatin and starch were not hydrolyzed. On the basis of the above data, the name Meiothermus rosaceus sp. nov. was proposed for the species represented by the strain RH9901(T)(CCTCC-AB200291).

Rhizoma Gastrodiae (Tianma) of different variants and different geographical origins has vital difference in quality and physiological efficacy. This paper focused on the classification and identification of Tianma of six types (two variants from three different geographical origins) using three dimensional synchronous fluorescence spectroscopy (3D-SFS) coupled with principal component analysis (PCA). 3D-SF spectra of aqueous extracts, which were obtained from Tianma of the six types, were measured by a LS-50B luminescence spectrofluorometer. The experimental results showed that the characteristic fluorescent spectral regions of the 3D-SF spectra were similar, while the intensities of characteristic regions are different significantly. Coupled these differences in peak intensities with PCA, Tianma of six types could be discriminated successfully. In conclusion, 3D-SFS coupled with PCA, which has such advantages as effective, specific, rapid, non-polluting, has an edge for discrimination of the similar Chinese herbal medicine. And the proposed methodology is a useful tool to classify and identify Tianma of different variants and different geographical origins.

Up to now, there has been no report on the taxonomic and functional analysis of the microbial community in fermenting puer tea by pyrosequencing. In this study, metagenomic pyrosequencing was first used in fermenting puer tea to delineate a relatively comprehensive overview of the microbial taxonomy while also preliminarily characterising the functional ontologies of microbial genes present in puer tea pile fermentation.A total of 251 738 pyrosequencing reads (9197 contigs and 145 402 singletons) were generated by pyrosequencing. Taxonomic analysis revealed three dominant bacterial phyla, Actinobacteria (30.08%), Proteobacteria (24.47%) and Firmicutes (20.23%), and one dominant eukaryotic phylum, Ascomycota (15.21%) [corrected]. A total of 58 664 hits were categorised into 28 functional subsystems based on the SEED database. Moreover, two categories, 'metabolism of terpenoids and polyketides' and 'biosynthesis of other secondary metabolites', were selectively analysed and 69 enzyme genes were presented in 16 pathways.The dominant microbes of puer tea fermentation were bacteria in the present study, and yeasts rather than moulds accounted for the overwhelming majority of Eukaryota. The analysis of functional genes and metabolic pathways will be helpful for further study of the mechanism of puer tea fermentation at molecular level.