Trapped intermediate state of plant pyruvate phosphate dikinase indicates substeps in catalytic swiveling domain mechanism.

Pyruvate phosphate dikinase (PPDK) is an essential enzyme of both the C4 photosynthetic pathway and cellular energy metabolism of some bacteria and unicellular protists. In C4 plants, it catalyzes the ATP- and Pi -dependent formation of phosphoenolpyruvate (PEP) while in bacteria and protozoa the ATP-forming direction is used. PPDK is composed out of three distinct domains and exhibits one of the largest single domain movements known today during its catalytic cycle. However, little information about potential intermediate steps of this movement was available. A recent study resolved a discrete intermediate step of PPDK's swiveling movement, shedding light on the details of this intriguing mechanism. Here we present an additional structural intermediate that possibly represents another crucial step in the catalytic cycle of PPDK, providing means to get a more detailed understanding of PPDK's mode of function.

Litter mixture dominated by leaf litter of the invasive species, Flaveria bidentis, accelerates decomposition and favors nitrogen release.

In natural ecosystems, invasive plant litter is often mixed with that of native species, yet few studies have examined the decomposition dynamics of such mixtures, especially across different degrees of invasion. We conducted a 1-year litterbag experiment using leaf litters from the invasive species Flaveria bidentis (L.) and the dominant co-occurring native species, Setaria viridis (L.). Litters were allowed to decompose either separately or together at different ratios in a mothproof screen house. The mass loss of all litter mixtures was non-additive, and the direction and strength of effects varied with species ratio and decomposition stage. During the initial stages of decomposition, all mixtures had a neutral effect on the mass loss; however, at later stages of decomposition, mixtures containing more invasive litter had synergistic effects on mass loss. Importantly, an increase in F. bidentis litter with a lower C:N ratio in mixtures led to greater net release of N over time. These results highlight the importance of trait dissimilarity in determining the decomposition rates of litter mixtures and suggest that F. bidentis could further synchronize N release from litter as an invasion proceeds, potentially creating a positive feedback linked through invasion as the invader outcompetes the natives for nutrients. Our findings also demonstrate the importance of species composition as well as the identity of dominant species when considering how changes in plant community structure influence plant invasion.

Protective effect of quercetin in gentamicin-induced oxidative stress in vitro and in vivo in blood cells. Effect on gentamicin antimicrobial activity.

We have evaluated the effect of gentamicin and gentamicin plus quercetin on ROS production, endogenous antioxidant defenses (SOD and CAT) and lipid peroxidation in vitro on human leukocytes and in vivo on whole rat blood. Gentamicin generated ROS production in human leukocytes, produced a dual effect on both enzymes dosage-dependent and generated an increase in lipid peroxidation. Quercetin, in leukocytes stimulated by gentamicin, showed more inhibitory capacity in ROS production than the reference inhibitor (vitaminC) in mononuclear cells and a similar protective behavior at this inhibitor in polymorphonuclear cells. Quercetin, in both cellular systems, tend to level SOD and CAT activities, reaching basal values and could prevent lipidic peroxidation induced by gentamicin. The results in Wistar rats confirmed that therapeutic doses of gentamicin can induce oxidative stress in whole blood and that the gentamicin treatment plus quercetin can suppress ROS generation, collaborate with SOD and CAT and diminish lipid peroxidation. Finally, flavonoid and antibiotic association was evaluated on the antimicrobial activity in S. aureus and E. coli, showing that changes were not generated in the antibacterial activity of gentamicin against E. coli strains, while for strains of S. aureus a beneficial effect observes. Therefore, we have demonstrated that gentamicin could induce oxidative stress in human leukocytes and in whole blood of Wistar rats at therapeutic doses and that quercetin may to produce a protective effect on this oxidative stress generated without substantially modifying the antibacterial activity of gentamicin against E. coli strains, and it contributes to this activity against S. aureus strains.

Separation of isorhamnetin 3-sulphate and astragalin from Flaveria bidentis (L.) Kuntze using macroporous resin and followed by high-speed countercurrent chromatography.

D4020 resin offered the best dynamic adsorption and desorption capacity for total flavonoids based on the research results from ten kinds of macroporous resin. A column packed with D4020 resin was used to optimize the separation of total flavonoids from Flaveria bidentis (L.) Kuntze extracts. The content of flavonoids in the product was increased from 4.3 to 30.1% with a recovery yield of 90%. After the treatment with gradient elution on D4020 resin, the contents of isorhamnetin 3-sulfate and astragalin were increased from 0.49 to 8.70% with a recovery yield of 74.1% and 1.16 to 30.8%, with a recovery yield of 92.2%, respectively. Further purification was carried out by one-run high-speed countercurrent chromatography yielding 4.5 mg of isorhamnetin 3-sulfate at a high purity of 96.48% and yielding 24.4 mg of astragalin at a high purity of over 98.46%.

Determination of major components and fingerprint analysis of Flaveria bidentis (L.) Kuntze.

A sensitive high-performance liquid chromatography method coupled with photodiode array detection was developed for the simultaneous determination of six major constituents in Flaveria bidentis (L.) Kuntze: hyperoside, patuletin-3-O-glucoside, isorhamnetin 3-sulfate, astragalin, 6-methoxykaempferol-3-O-galactoside and α-terthienyl. The chemical fingerprint of Flaveria bidentis (L.) Kuntze leaves was established using raw materials of 12 batches in China. The chromatographic separations were obtained by using an Eclipse XDB-C18 reserved-phase column (150 × 4.6 mm i.d., 5 µm) using gradient elution with water (0.0125% trifluoroacetic acid, v/v) and acetonitrile at a flow rate of 1.0 mL/min, an operating temperature of 30°C and a detection wavelength of 360 nm. The new method was validated by linearity, limits of detection and quantification, precision, reproducibility, stability and recovery, and was also successfully applied to the simultaneous determination of components in Flaveria bidentis (L.) Kuntze. The results indicate that this multi-component determination method in combination with chromatographic fingerprint analysis is suitable for the quantitative analysis and identification of Flaveria bidentis (L.) Kuntze.

Separation of chlorogenic acid and concentration of trace caffeic acid from natural products by pH-zone-refining countercurrent chromatography.

Chlorogenic acid and caffeic acid were selected as test samples for separation by the pH-zone-refining countercurrent chromatography (CCC). The separation of these test samples was performed with a two-phase solvent system composed of methyl-tert-butyl-ether/acetonitrile/water at a volume ratio of 4:1:5 v/v/v where trifluoroacetic acid (TFA; 8 mM) was added to the organic stationary phase as a retainer and NH4 OH (10 mM) to the aqueous mobile phase as an eluter. Chlorogenic acid was successfully separated from Flaveria bidentis (L.) Kuntze (F. bidentis) and Lonicerae Flos by pH-zone-refining CCC, a slightly polar two-phase solvent system composed of methyl-tert-butyl-ether/acetonitrile/n-butanol/water at a volume ratio of 4:1:1:5 v/v/v/v was selected where TFA (3 mM) was added to the organic stationary phase as a retainer and NH4 OH (3 mM) to the aqueous mobile phase as an eluter. A 16.2 mg amount of chlorogenic acid with the purity of 92% from 1.4 g of F. bidentis, and 134 mg of chlorogenic acid at the purity of 99% from 1.3 g of crude extract of Lonicerae Flos have been obtained. These results suggest that pH-zone-refining CCC is suitable for the isolation of the chlorogenic acid from the crude extracts of F. bidentis and Lonicerae Flos.

In vivo prophylactic effects of oleanolic acid isolated from chloroform extract of Flaveria trinervia against ethanol induced liver toxicity in rats.

The prophylactic effects of oleanolic acid (OA) isolated from chloroform extract (CE) of Flaveria trinervia against ethanol induced liver toxicity was investigated using rats. CE and OA at three different doses were tested by administering orally to the ethanol treated animals during the last week of the 7 weeks study. Silymarin was used as the standard reference. The substantially elevated serum enzymatic levels of serum glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, alkaline phosphatase and bilirubin in ethanol treated animals were restored towards normalcy by treatment of CE and OA. In vivo antioxidant and in vitro free radical scavenging activities were also positive for all the three concentrations of CE and OA. However, OA at 150 mg/kg showed significant activity when compared to the other two doses. Biochemical observations in support with histopathological examinations revealed that CE and OA possess hepatoprotective action against ethanol induced hepatotoxicity in rats.

Preparative separation of bioactive compounds from essential oil of Flaveria bidentis (L.) Kuntze using steam distillation extraction and one step high-speed counter-current chromatography.

In order to utilize and control the invasive weed, bioactive compounds from essential oil of Flaveria bidentis (L.) Kuntze were studied. Steam distillation extraction and one step high-speed counter-current chromatography were applied to separate and purify the caryophyllene oxide, 7,11-dimethyl-3-methylene-1,6,10-dodecatriene, and caryophyllene from essential oil of Flaveria bidentis (L.) Kuntze. The two-phase solvent system containing n-hexane/acetonitrile/ethanol (5:4:3, v/v/v) was selected for the one step separation mode according to the partition coefficient values (K) of the target compounds and the separation factor (α). The purity of each isolated fraction after a single high-speed counter-current chromatography run was determined by high performance liquid chromatography. A 3.2 mg of caryophyllene oxide at a purity of 92.6%, 10.4 mg of 7,11-dimethyl-3-methylene-1,6,10-dodecatriene at a purity of 99.1% and 5.7 mg of caryophyllene at a purity of 98.8% were obtained from 200 mg essential oil of Flaveria bidentis (L.) Kuntze. The chemical structures of these components were identified by GC-MS, (1) H-NMR, and (13) C-NMR.

Quercetin 3,7,3',4'-tetrasulphated isolated from Flaveria bidentis inhibits tissue factor expression in human monocyte.

Sulphated esters of the flavonoids sulphated quercetin 3,7,3',4'-tetrasulphated (QTS) and quercetin 3-acetyl-7,3,4'-trisulphate (ATS), isolated from Flaveria bidentis, have demonstrated anticoagulant and antiplatelet properties. In this study, we examined if both compounds affected the expression of the procoagulant tissue factor (TF) induced by lipopolysaccharide (LPS) on human monocyte. Monocytes were pretreated with different concentrations of each flavonoid (0.1-500 µM), followed by a 4h incubation with LPS in order to induce TF expression. Results of the TF expression showed different behaviors for the two flavonoids studied. A slight inhibitory effect on the TF expression was detected at a QTS concentration of 0.1 µM, but from 1 µM onwards a significant inhibitory effect that remained up to 500 µM could be observed. In contrast, ATS induced a poor inhibitory effect on TF expression at all concentrations tested. These results suggest that QTS has another antithrombotic property, to be added to its already renowned ability as an anticoagulant and antiplatelet compound.

Isolation of bioactive components from Flaveria bidentis (L.) Kuntze using high-speed counter-current chromatography and time-controlled collection method.

Semipreparative high-speed counter-current chromatography (HSCCC) by time-controlled collection method was successfully applied for isolation and purification of α-terthienyl, 5-(3-buten-1-ynyl)-2,2'-bithienyl, and 5-(3-penten-1-ynyl)-2,2'-bithienyl from Flaveria bidentis (L.) Kuntze for the first time. The two-phase solvent system composed of n-hexane and acetonitrile at the volume ratio of 1:1 (v/v) was used for the semipreparative HSCCC. The 5.2 mg α-terthienyl, 2.2 mg 5-(3-buten-1-ynyl)-2,2'-bithienyl, and 4.3 mg 5-(3-penten-1-ynyl)-2,2'-bithienyl with the purity of 99.9, 90.2, and 92.1% were produced from 265.6 mg crude extract, respectively, and 5-(3-penten-1-ynyl)-2,2'-bithienyl was first isolated from Flaveria bidentis (L.) Kuntze. The structures of the separated compounds were identified by electrospray-ionization mass spectrometry and proton and carbon nuclear magnetic resonance ((1)H- and (13)C-NMR).

Separation and purification of isorhamnetin 3-sulphate from Flaveria bidentis (L.) Kuntze by counter-current chromatography comparing two kinds of solvent systems.

The first preparative separation of a flavonoid sulphate isorhamnetin 3-sulphate from Flaveria bidentis (L.) Kuntze by counter-current chromatography (CCC) was presented. Two kinds of solvent systems were used. A conventional organic/aqueous solvent system n-butanol-ethyl acetate-water (4:1:5, v/v) was used, yielding isorhamnetin 3-sulphate 2.0 mg with a purity of 93.4% from 83 mg of pre-enriched crude extract obtained from 553 mg ethanol extract by macroporous resin. A one-component organic/salt-containing system composed of n-butanol-0.25% sodium chloride aqueous solution (1:1, v/v) was also used, and the LC column packed with macroporous resin has been employed for desalination of the target compound purified from CCC. As a result, 2.1 mg of isorhamnetin 3-sulphate with a purity of over 97% has been isolated from 402 mg of crude extract without pre-enrichment. Compared with the conventional organic/aqueous system, the one-component organic/salt-containing aqueous system was more suitable for the separation of isorhamnetin 3-sulphate, and purer target compound was obtained from the crude extract without pre-enrichment using the new solvent system. The chemical structure was confirmed by ESI-MS and (1)H, (13)C NMR. In summary, our results indicated that CCC using one-component organic/salt-containing aqueous solution is very promising and powerful for high-throughput purification of isorhamnetin 3-sulphate from Flaveria bidentis (L.) Kuntze.

Antinociceptive activity and acute toxicity study of Flaveria trinervia whole plant extracts using mice.

The antinociceptive activity of Flaveria trinervia extracts by tail-flicking and writhing methods was evaluated using mice. The abdominal writhing method was carried out by administering petroleum ether, chloroform and methanol extracts orally. After 1 h of incubation, 0.6% acetic acid (10 mL kg(-1)) was administered intraperitoneally. After 5 min, each group of mice was observed for the amount of writhing for a duration of 20 min. Acetyl salicylic acid was used as the standard reference. The tail-flick method was carried out using a thermal model and the maximum possible analgesia was calculated. The LD50 values of petroleum ether, chloroform and methanol extracts were 700, 700 and 500 mg kg⁻¹, respectively. The methanol extract at 50 mg kg⁻¹ showed significant antinociceptive activity. The petroleum ether and chloroform extracts showed moderate antinociceptive activity at a dose of 70 mg kg⁻¹. The methanol extract showed more significant antinociceptive activity than the other extracts but was less effective than the standard. This investigation finds support for the ethnomedicinal claims of F. trinervia.

Separation of patuletin-3-O-glucoside, astragalin, quercetin, kaempferol and isorhamnetin from Flaveria bidentis (L.) Kuntze by elution-pump-out high-performance counter-current chromatography.

Flaveria bidentis (L.) Kuntze is an annual alien weed of Flaveria Juss. (Asteraceae) in China. Bioactive compounds, mainly flavonol glycosides and flavones from F. bidentis (L.) Kuntze, have been studied in order to utilize this invasive weed, Analytical high-performance counter-current chromatography (HPCCC) was successfully used to separate patuletin-3-O-glucoside, a mixture of hyperoside (quercetin-3-O-galactoside) and 6-methoxykaempferol-3-O-galactoside, astragalin, quercetin, kaempferol and isorhamnetin using two runs with different solvent system. Ethyl acetate-methanol-water (10:1:10, v/v) was selected by analytical HPCCC as the optimum phase system for the separation of patuletin-3-O-glucoside, a mixture of hyperoside and 6-methoxykaempferol-3-O-galactoside, and astragalin. A Dichloromethane-methanol-water (5:3:2, v/v) was used for the separation of quercetin, kaempferol and isorhamnetin. The separation was then scaled up: the crude extract (ca 1.5 g) was separated by preparative HPCCC, yielding 12 mg of patuletin-3-O-glucoside at a purity of 98.3%, yielding 9 mg of a mixture of hyperoside and 6-methoxykaempferol-3-O-galactoside constituting over 98% of the fraction, and 16 mg of astragalin (kaempferol-3-O-glucoside) at a purity of over 99%. The pump-out peaks are isorhanetin (98% purity), kaemferol (93% purity) and quercitin (99% purity). The chemical structure of patuletin-3-O-glucoside and astragalin were confirmed by MS and ¹H, ¹³C NMR.

Growth of the C4 dicot Flaveria bidentis: photosynthetic acclimation to low light through shifts in leaf anatomy and biochemistry.

In C(4) plants, acclimation to growth at low irradiance by means of anatomical and biochemical changes to leaf tissue is considered to be limited by the need for a close interaction and coordination between bundle sheath and mesophyll cells. Here differences in relative growth rate (RGR), gas exchange, carbon isotope discrimination, photosynthetic enzyme activity, and leaf anatomy in the C(4) dicot Flaveria bidentis grown at a low (LI; 150 micromol quanta m(2) s(-1)) and medium (MI; 500 micromol quanta m(2) s(-1)) irradiance and with a 12 h photoperiod over 36 d were examined. RGRs measured using a 3D non-destructive imaging technique were consistently higher in MI plants. Rates of CO(2) assimilation per leaf area measured at 1500 micromol quanta m(2) s(-1) were higher for MI than LI plants but did not differ on a mass basis. LI plants had lower Rubisco and phosphoenolpyruvate carboxylase activities and chlorophyll content on a leaf area basis. Bundle sheath leakiness of CO(2) (phi) calculated from real-time carbon isotope discrimination was similar for MI and LI plants at high irradiance. phi increased at lower irradiances, but more so in MI plants, reflecting acclimation to low growth irradiance. Leaf thickness and vein density were greater in MI plants, and mesophyll surface area exposed to intercellular airspace (S(m)) and bundle sheath surface area per unit leaf area (S(b)) measured from leaf cross-sections were also both significantly greater in MI compared with LI leaves. Both mesophyll and bundle sheath conductance to CO(2) diffusion were greater in MI compared with LI plants. Despite being a C(4) species, F. bidentis is very plastic with respect to growth irradiance.

Inhibitory effects of sulphated flavonoids isolated from Flaveria bidentis on platelet aggregation.

Flaveria bidentis is a plant species that has as major constituents sulphated flavonoids in the highest degree of sulphatation. Among them, quercetin 3,7,3',4'-tetrasulphate (QTS) and quercetin 3-acetyl-7,3',4'-trisulphate (ATS) are the most important constituents. Both showed anticoagulant properties. The objective of the present study was to evaluate the effects of these flavonoids on human platelet aggregation in comparison with the well-known inhibitor quercetin (Qc) by using several agonists. Platelet-rich plasma (PRP) or washed human platelets (WP) were incubated with different concentrations of the flavonoids to be tested (1 to 1000 microM, final concentration), and the platelet aggregation was induced by using adenosine 5'-diphosphate (ADP), epinephrine (EP), collagen, arachidonic acid (AA) and ristocetin as agonists. QTS (500 microM) and Qc (250 microM) markedly inhibited platelet aggregation with all the aggregant agents, except ristocetin, whereas ATS (1000 microM) showed only slight antiplatelet effects. In addition, QTS and Qc antagonized the aggregation of PRP or WP induced by U-46619, a mimetic thromboxane A2 (TxA2) receptor agonist. Challenged with collagen or arachidonic acid, the thromboxane B2 (TxB2) formation was also inhibited by the flavonoids, mainly by QTS and Qc, in WP. These results demonstrate that QTS and in minor extension ATS induce a deleterious effect on the production of TxA2, as judged by TxB2 formation, in stimulated WP and a marked interference on the TxA2 receptor according to the profile of inhibition of the agonist-induced platelet aggregation when using ADP, EP, AA and collagen and confirmed with U-46619.