The purple acid phosphatase GmPAP17 predominantly enhances phosphorus use efficiency in soybean.

Purple acid phosphatase (PAP) is an important plant acid phosphatase, which can secrete to the rhizosphere to decompose organophosphorus, promote phosphorus use efficiency, plant growth and development. However, little is known about the functions of intracellular PAP in plants, especially for soybean. Our previous study integrating QTL mapping and transcriptome analysis identified an promising low phosphorus (LP)-induced gene GmPAP17. Here, we determined that GmPAP17 was mainly expressed in roots and had a strong response to LP stress. Furthermore, and the relative expression in the root of LP tolerant genotypes NN94-156 was significantly greater than that of LP sensitive genotype Bogao after LP stress treatment. The overexpression of GmPAP17 significantly enhanced both acid phosphatase activity and growth performance of hairy roots under LP stress condition, it was vice versa for RNAi interference of GmPAP17, indicating that GmPAP17 plays an important role in P use efficiency. Moreover, yeast two-hybrid and bimolecular fluorescence complementation analysis showed that GmRAP2.2 was involved in the regulation network of GmPAP17. Taken together, our results suggest that GmPAP17 is a novel plant PAP that functions in the adaptation of soybean to LP stress, possibly through its involvement in P recycling in plants.

Multielements determination and metal transfer investigation in herb medicine Bupleuri Radix by inductively coupled plasma-mass spectrometry.

Bupleuri Radix is a famous traditional Chinese medicine (TCM) and an important raw material in TCM patent prescriptions. It is widely used in several countries, including China, Japan, South Korea, and America. However, the impact of heavy metal transfer rules on TCMs remains unknown. In this study, a total of 45 paired original medicines (OMs), decoction pieces (DPs), and vinegar-processed (VPs) samples were simultaneously determined via inductively coupled plasma-mass spectrometry after a microwave digestion. The concentrations of the elements were shown at three levels: (a) Al and Fe at the mg/g level; (b) Pb, Cu, Ba, Mn, Cr, and Ni at the mg/kg level; (c) Co, As, Cd, and Hg at µg/kg level. It is worth noting that the Cu levels were found to exceed the maximum concentration set by Chinese legislation (20.0 mg/kg). In addition, Mn, Ni, and Cu levels were higher in samples from the Gansu province than those from other provinces. The accumulation of the heavy metals decreased in the order of OMs > DPs > VPs; this was especially true for the Al and Fe levels. Furthermore, the results indicate that decocting the samples may reduce the intake of heavy metals. The element transfer ratios for decoctions were under 50% compared to herbal medicines and decreased in the order of Co > As > Mn > Hg > other metals. Our study strongly suggests that long-term and regular monitoring for heavy metals in the plant is necessary.

6'-Hydroxy Justicidin B Triggers a Critical Imbalance in Ca2+ Homeostasis and Mitochondrion-Dependent Cell Death in Human Leukemia K562 Cells.

Justicia procumbens (J. procumbens) is a traditional Chinese herbal medicine which was used for the treatment of fever, pain, and cancer. A compound 6'-hydroxy justicidin B (HJB) isolated from J. procumbens exhibits promising biological properties. However, the mechanism of action and the in vivo behavior of HJB remain to be elucidated. In this study, we investigated the mechanism of action of HJB on human leukemia K562 cells and its pharmacokinetic properties in rats. The results demonstrated that HJB significantly inhibited the proliferation of K562 cells and promoted apoptosis. Besides, HJB resulted in decreased mitochondrial membrane potential deltaPSIm, increased the level of the calcium homeostasis regulator protein TRPC6 and cytosolic calcium. The activity of caspase-8, caspase-9 and the expression of p53 were significantly increased after treatment with HJB. Additionally, HJB has rapid absorption rate and relative long elimination t1/2, indicating a longer residence time in vivo. The results indicate that HJB inhibited the proliferation of K562 cells and induced apoptosis by affecting the function of mitochondria and calcium homeostasis to activate the p53 signaling pathway. The pharmacokinetic study of HJB suggested it is absorbed well and has moderate metabolism in vivo. These results present HJB as a potential novel alternative to standard human leukemia therapies.

[Development of Tianma HPLC fingerprint and discriminant analysis].

Tianma(the tuber of Gastrodia eleta) is a widely used and pricy Chinese herb. Its counterfeits are often found in herbal markets, which are the plant materials with similar macroscopic characteristics of Tianma. Moreover, the prices of Winter Tianma(cultivated Tianma) and Spring Tianma(mostly wild Tianma) have significant difference. However, it is difficult to identify the true or false, good or bad quality of Tianma samples. Thus, a total of 48 Tianma samples with different characteristics(including Winter Tianma, Spring Tianma, slice, powder, etc.) and 9 plant species 10 samples of Tianma counterfeits were collected and analyzed by HPLC-DAD-MS techniques. After optimizing the procedure of sample preparation, chromatographic and mass-spectral conditions, the HPLC chromatograms of all those samples were collected and compared. The similarities and Fisher discriminant analysis were further conducted between the HPLC chromatograms of Tianma and counterfeit, Winter Tianma and Spring Tianma. The results showed the HPLC chromatograms of 48 Tianma samples were similar at the correlation coefficient more than 0.848(n=48). Their mean chromatogram was simulated and used as Tianma HPLC fingerprint. There were 11 common peaks on the HPLC chromatograms of Tianma, in which 6 main peaks were chosen as characteristic peaks and identified as gastrodin, p-hydroxybenzyl alcohol, parishin A, parishin B, parishin C, parishin E, respectively by comparison of the retention time, UV and MS data with those of standard chemical compounds. All the six chemical compounds are bioactive in Tianma. However, the HPLC chromatograms of the 10 counterfeit samples were significantly different from Tianma fingerprint. The correlation coefficients between HPLC fingerprints of Tianma with the HPLC chromatograms of counterfeits were less than 0.042 and the characteristic peaks were not observed on the HPLC chromatograms of these counterfeit samples. It indicated the true or false Tianma can be identified by either the similarity or characteristic peaks on HPLC fingerprint. Comparing the Winter Tianma with Spring Tianma showed that the HPLC chromatograms of 15 winter Tianma samples and 11 spring Tianma samples were similar at the mean correlation coefficient of 0.908. But the intensity of the characteristic peaks were different between the two groups of Tianma samples, i.e. the intensity of gastrodin, paishin A and C in winter Tianma was lower than those in spring Tianma. The Winter Tianma and Spring Tianma could be discriminated by either the Fisher unstandardized discrimination function or Linear discriminant function, based on the peak areas of 11 common peaks on HPLC chromatograms as variate.

Cucumber Metallothionein-Like 2 (CsMTL2) Exhibits Metal-Binding Properties.

We identified a novel member of the metallothionein (MT) family, Cucumis sativus metallothionein-like 2 (CsMTL2), by screening a young cucumber fruit complementary DNA (cDNA) library. The CsMTL2 encodes a putative 77-amino acid Class II MT protein that contains two cysteine (Cys)-rich domains separated by a Cys-free spacer region. We found that CsMTL2 expression was regulated by metal stress and was specifically induced by Cd2+ treatment. We investigated the metal-binding characteristics of CsMTL2 and its possible role in the homeostasis and/or detoxification of metals by heterologous overexpression in Escherichia coli cells. Furthermore, we produced a deletion mutant form of the protein, CsMTL2m, that contained the two Cys-rich clusters but lacked the spacer region, in E. coli. We compared the metal-binding properties of CsMTL2 with those of CsMTL2m, the ß domain of human metallothionein-like protein 1 (HsMTXb), and phytochelatin-like (PCL) heterologously expressed in E. coli using metal-binding assays. We found that E. coli cells expressing CsMTL2 accumulated the highest levels of Zn2+ and Cd2+ of the four transformed cell types, with levels being significantly higher than those of control cells containing empty vector. E. coli cells expressing CsMTL2 had a higher tolerance for cadmium than for zinc ions. These findings show that CsMTL2 improves metal tolerance when heterologously expressed in E. coli. Future studies should examine whether CsMTL2 improves metal tolerance in planta.

Simultaneous determination of eight active components in the traditional Chinese Medicine Dan Deng Tong Nao capsules by HPLC-MS/MS.

An high-performance liquid chromatography-tandem mass spectrometry method has been optimised and established for the quality evaluation of the traditional Chinese medicine Dan Deng Tong Nao capsules through simultaneous determination of the following eight active components: danshensu, salvianolic acid A, salvianolic acid C, puerarin, scutellarin, apigenin, 3,4-dihydroxybenzaldehyde, and ferulic acid. All of the analytes were separated on a Waters Xbridge™ C18 column (4.6 × 150 mm, 3.5 µm particle size) with a mobile phase consisting of methanol/acetonitrile (50 : 50, v/v) and water containing 0.1 % formic acid. All of the compounds showed good linearity (R2 > 0.997). The recoveries, measured at three concentration levels, varied from 94.94 to 107.3 %. The validated method was successfully applied to evaluate the eight active components in Dan Deng Tong Nao capsules collected from different production batches. The results suggested that the method established in this study could be considered a good approach to controlling the quality of Dan Deng Tong Nao capsules and other related botanical drugs.

Metabolism of chamaechromone in vitro with human liver microsomes and recombinant human drug-metabolizing enzymes.

Chamaechromone is a major component in the dried roots of Stellera chamaejasme with antihepatitis B virus and insecticidal activity. In this study, metabolic profiles of chamaechromone were investigated in human liver microsomes. One monohydroxide and two monoglucuronides of chamaechromone were identified. The enzyme kinetics for both hydroxylation and glucuronidation were fitted to the Michaelis-Menten equation. The hydroxylation of chamaechromone was inhibited by α-naphthoflavone, and predominantly catalyzed by recombinant human cytochrome P450 1A2, whereas the glucuronidation was inhibited by quercetin, 1-naphthol, and fluconazole, and mainly catalyzed by recombinant human UDP-glucuronosyltransferase 1A3, 1A7, 1A9, and 2B7.

Metabolites characterization of chamaechromone in vivo and in vitro by using ultra-performance liquid chromatography/Xevo G2 quadrupole time-of-flight tandem mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Stellera chamaejasme L. (Thymelaeaceae) was a toxic perennial herb and widely used as pesticide and dermatological agents in China. Chamaechromone was a major component in the dried roots of Stellera chamaejasme with anti-HBV and insecticidal activity. Analysis of metabolic profile in vivo and in vitro plays a pivotal role to unravel how TCM works. And the metabolites of chamaechromone might influence the effects and toxicity of Stellera chamaejasme. Moreover, the metabolic routes of chamaechromone provide an important basis for toxicological safety evaluation. Until now, little is known about the metabolism of chamaechromone. The current study was designed to characterize the whole metabolic pathways of chamaechromone in vitro and in vivo. MATERIALS AND METHODS: Twenty-four rats were randomly divided into four groups, including two oral administration groups (100mgkg(-1)), one intravenous injection group (5 mgkg(-1)), and one control group. The metabolites in rat urine and feces and bile were identified by UPLC/Q-TOF MS analysis and ß-glucuronidase hydrolysis. Moreover, the possible metabolic mechanism was further confirmed by Phase I and Phase II metabolism and catechol-O-methyltransferase methylation in rat liver S9 fraction and degradation in rat intestinal bacteria. RESULTS: A total of 24 metabolites from chamaechromone were detected and identified in vivo and in vitro, 20 of which were novel. And the major metabolic processes were hydroxylation, methylation, glucuronation, acetylation, dehydroxylation and degradation. CONCLUSIONS: The present study revealed the whole metabolic pathways of chamaechromone in rat through both in vitro and in vivo experiments for the first time. And chamaechromone could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of chamaechromone. In addition, the results of this work have showed the feasibility of the UPLC/Q-TOF-MS approach for rapid and reliable characterization of metabolites.

De novo assembly of the peanut (Arachis hypogaea L.) seed transcriptome revealed candidate unigenes for oil accumulation pathways.

Peanuts are one of the most important edible oil crops in the world. In order to survey key genes controlling peanut oil accumulation, we analyzed the seed transcriptome in different developmental stages of high- and low-oil peanut varieties. About 54 million high quality clean reads were generated, which corresponded to 4.85 Gb total nucleotides. These reads were assembled into 59,236 unique sequences. Differential mRNA processing events were detected for most of the peanut Unigenes and found that 15.8% and 18.0% of the Unigenes were differentially expressed between high- and low-oil varieties at 30 DAF and 50 DAF, respectively. Over 1,500 Unigenes involved in lipid metabolism were identified, classified, and found to participate in FA synthesis and TAG assembly. There were seven possible metabolic pathways involved in the accumulation of oil during seed development. This dataset provides more sequence resource for peanut plant and will serve as the foundation to understand the mechanisms of oil accumulation in oil crops.

[Estrogenicity of adzuki bean Phaseolus angularis wight. and its effect on proesterone receptor level in human breast cancer MCF-7 cells].

OBJECTIVE: To explore the estrogenic activity of ethanol extract from adzuki bean Phaseolus angularis and its effect on progesterone receptor (PR) level of human breast cancer MCF-7 cells. METHOD: The modified MCF-7 cell proliferation assay was used to evaluate the estrogenic activity of adzuki bean extract. And its effect on PR mRNA and protein were addressed using RT-PCR measurements and western blotting analysis respectively in which pure estrogen receptor antagonist ICI 182,780 was employed as the tool. RESULT: The estrogenic activity of adzuki bean extract at various concentrations (10-200 microg x mL(-1)), expressed as proliferative effect (PE) relative to that of solvent control, was examined. The results indicated that the extract of adzuki bean was able to induce MCF-7 cell growth with a maximum at 100 microg x mL(-1). The results of RT-PCR and western blotting showed that PR mRNA and protein could be significantly induced by adzuki bean extract in MCF-7 cells. Moreover, the specific estrogen receptor antagonist ICI 182,780 could block these reactions. CONCLUSION: Ethanol extract of adzuki bean has the estrogen-like activities through the estrogen response pathway.

[Research on cytokines gene express of rats alcoholic liver disease affected by tea polyphenol].

OBJECTIVE: To evaluate the effect of tea polyphenol(TP) on the rat with alcoholic liver damage. METHOD: Rats were divided into 3 groups, in which 2 groups were stomach perfused with alcohol to result in ALD, and 1 group of them stomach perfused with TP simultaneously. Another group was normal control groups (stomach perfused with drinking water). In the end of 12 weeks, the liver specimen of each rat was observed by anglicizing its tissue damage, and all data collected was performed by statistical analysis in quantum and semi-quantum. Meanwhile cytokines gene express of each group is determined. RESULT: In the end of 12 weeks, alcoholic hepatitis appeared in rat liver. Hepatic injury in alcohol group and TP group were found, but could not be found in normal group. Compared with pure alcohol group, alcoholic liver damage mainly showing with steatosis in TP group were slight, in addition showing liver cellular swelling with small area, with less spot and focal necrosis, none bridging necrosis. Steatosis were slight relatively, mega-bubble steatosis were less found. Collagen deposition of TP group were less than those of pure alcohol group. Gene expression of. cytokine have diversity statistically such as IL-3, IL-4, IL-1R2, IL-6R, IL-7R2, IL-3Ra, IL-R1, IL-13, IL-1R1, IL-7R2, EPO-R, LIFR, IL-1R2, IL-5R2, CSF1, CD27, IL-6R. CONCLUSION: TP is able to attenuate alcoholic liver damage. It's mechanism is possibly due to modulating cytokines gene expression of cytokine.

[Effect of tea polyphenols on alcoholic liver injury].

OBJECTIVE: To reproduce an experimental model of alcoholic liver disease in rats and to investigate the preventive and treatment effects of tea polyphenols on alcoholic liver disease. METHODS: 68 male Sprague-Dawley rats were randomly divided into 3 groups: alcohol group (gastrically infused with 56% of ethanol once a day with a dose of 7 g/kg body weight for 4, 12 and 24 weeks), tea polyphenols group (gastric infusion with alcohol same as in the alcohol group and with tea polyphenols at 0.25 g/kg bw) and control group (gastric infusion with normal saline). At the end of 4, 12 and 24 weeks, blood samples were collected and then the rats were sacrificed. Liver samples were obtained for routine histological examination and the degree of hepatic steatosis and alcoholic hepatitis were examined. Blood specimens were used for evaluation of alanine transaminase (ALT) and aspartate aminotransferase (AST). RESULTS: (1) The levels of the two transaminases were elevated with the increase of the duration of ethanol feeding and the difference is significant. TP significantly mitigated the increase of ALT and AST activities induced by the alcohol. (2) Histological changes of the liver injury indicated that piecemeal or focal necrosis of hepatocytes was present in the centrilobular area. As fibrosis advanced, broader septa were formed with central-central and centra-portal bridging necrosis. In the TP infusion group, the severity of the pathological changes was significantly milder. CONCLUSION: The results of this study revealed that TP mitigated the development of alcoholic liver disease, and TP may be a potential drug for treatment of alcoholic liver disease.

Effects of tea polyphenols on hepatic fibrosis in rats with alcoholic liver disease.

BACKGROUND: As an anti-oxidation agent, tea polyphenols may have the effect of anti-fibrosis. This study was designed to observe the effect of tea polyphenols on hepatic fibrosis in rats with alcoholic liver disease and to explore the related mechanisms. METHODS: Sixty healthy Sprague-Dawley rats were divided randomly into normal control group, single alcohol group, and three alcohol groups given different doses of tea polyphenols. Alcohol or isovolumic normal saline and corresponding doses of tea polyphenols were given daily to the rats separately. The rats were sacrificed at the end of the 24th week. Masson staining was performed to observe liver fibrosis, serum endotoxin, and oxidant and anti-oxidant activity. RESULTS: Hepatic fibrosis was less severe in the rats of the alcohol groups given tea polyphenols than in the single alcohol group. Tea polyphenols increased the serum anti-oxidant capacity and decreased the endotoxin level. CONCLUSION: Tea polyphenols show anti-fibrosis effect in rats with alcoholic liver disease, and the mechanism may be related to the clearance of overall oxidant and decrease of the endotoxin level.

[Studies of soil embryological characteristics of the Salvia miltiorrhiza of Zhongjiang].

OBJECTIVE: To study the characteristics of the SiO2/R2O3 in non-zonal purple soil in the light of the difference between the high yield and low yield of Salvia miltiorrhiza. METHOD: Both the high yield and low yield drugs developed from the same parental material (k1cy) were chosen in the ecology area in Zhongjiang county, Sichuan Province. By using the air-dried drugs as sample, quadrat sampling method was used to calculat the production. Gravimetric method, volumetric method, and colorimetric method and neutralization titration were used to measure the content of adhesive particle colloids element in the soil. And T-test was used for the variable-difference analysis. RESULT: There was a sharp contrast between the production of high yield and low yield drugs (P < 0.001). The characteristic value of the high yield soil embryology was much higher than that of the low yield soil. CONCLUSION: The characteristic value of the purple soil embryology is one of the important factors of the soil adaptability of the drug and its quality. The weathering degree of the soil is closely related to the yield of the drug.

In vitro TGF-beta1 antagonistic activity of ursolic and oleanolic acids isolated from Clerodendranthus spicatus.

The mechanism of action of the aerial parts of Clerodendranthus spicatus (Thunb.) C.Y. Wu, [syn. Orthosiphon aristatus (Blume) Miq,] a medicinal plant used in China to treat human renal disease, was investigated. The aqueous and methanol crude extracts exhibited dose-dependent inhibitory activity on 125I-TGF-beta1 binding to its receptor in Balb/c 3T3 cells. Subsequent bioassay-guided fractionation led to identification of two known triterpenoidal constituents, ursolic and oleanolic acids. Ursolic and oleanolic acids inhibited the binding of 125I-TGF-beta1 to its receptor with IC 50 values of 6.9 +/- 0.8 and 21.0 +/- 2.3 microM, respectively. The results suggest that TGF-beta1 antagonistic activity is responsible, at least in part, for the therapeutic efficacy of this plant to treat humans with renal disease.

[Effect of tea polyphenol on liver fibrosis in rats and related mechanism].

OBJECTIVE: To investigate the effect of tea polyphenol (TP)'s on liver fibrosis in rats and its possible mechanism. METHOD: Rats were divided into 5 groups, 4 groups of which were stomach perfused with alcohol resulting in alcoholic liver fibrosis, and 3 groups of which were stomach perfused with TP simultaneously. Another group was normal control one. Histological change of rat liver was investigated and quantitative analysis was made in 24 weeks, and rat liver anti-oxidation index and serum endotoxin were determined at the same time. RESULT: Liver fibrosis in TP group was slight, and anti-oxidize index and endotoxin level were markedly improved in comparison with those of alcohol groups. CONCLUSION: Tea polyphenol can protect hepatocytes from fibrosis. Its mechanism is possibly due to cleaning up overfull lipid per-oxidation and reducing the level of endotoxin.