基质固相分散萃取-高效液相色谱-串联质谱法分析拟南芥中的赤霉素

建立了基质固相分散萃取-高效液相色谱-串联质谱法(MSPD-HPLC-MS/MS)同时测定拟南芥中3种赤霉素GA1, GA3和GA4的分析方法。将拟南芥样品与C18填料混合研磨制成MSPD柱,并采用80%冷甲醇洗脱。采用反相C18色谱柱进行分离,以0.05%甲酸水溶液和乙腈为流动相进行梯度洗脱,采用电喷雾离子源负离子模式(ESI~)电离,多反应监测模式检测。对样品前处理条件、色谱分离条件和质谱检测条件进行了优化,结果表明,在最优条件下,3种赤霉素在10～300 ng/g范围内均呈良好线性关系,相关系数(r2)均大于0.98,检出限在1.1～4.1 ng/g之间。在10～50 ng/g添加水平下,平均回收率范围为54.7%～102.6%,相对标准偏差（RSD,n=3)为3.2%～12.8%。该方法操作简单、灵敏度高、选择性好、回收率高,适合拟南芥中GA1、GA3、GA4含量的测定。     

Inhibitory effects of glycyrrhetic acid derivatives on 11 beta- and 3 alpha-hydroxysteroid dehydrogenases of rat liver.

Glycyrrhetic acid (GA), an aglycone of glycyrrhizin (GL), is a potent inhibitor of 11 beta- and 3 alpha-hydroxysteroid dehydrogenases. 11 beta-Hydroxysteroid dehydrogenase activity of rat liver microsomes was potently inhibited by GA, 3-deoxyglycyrrhetic acid (3-deoxyGA), 3-ketoglycyrrhetic acid (3-ketoGA), 3-epiglycyrrhetic acid (3-epiGA) and 11-deoxoglycyrrhetic acid (11-deoxoGA), with I50 values of 2-4 x 10(-7) M. However, 18 alpha-stereoisomers (I50 = 3-7 x 10(-6) M) of GA, 3-deoxyGA and 11-deoxoGA were one tenth less inhibitory on the enzyme activity than the corresponding 18 beta-isomers. On the other hand, 18 alpha-stereoisomers of GA, 3-deoxyGA and 11-deoxoGA inhibited 3 alpha-hydroxysteroid dehydrogenase activity of rat liver cytosol more potently than the corresponding 18 beta-isomers. I50 values of 18 alpha- and 18 beta-isomers were 2 and 7 x 10(-6) M, respectively, in the case of GA, 8 and 20 x 10(-6) M in 3-deoxyGA, 3 and 20 x 10(-6) M in 11-deoxoGA. These results indicate that the 18 beta-conformation of oleanane is important for the inhibition of 11 beta-hydroxysteroid dehydrogenase but on the contrary the 18 alpha-conformation is important for the inhibition of 3 alpha-hydroxysteroid dehydrogenase.     

Synthesis and bioactivity of the gibberellin, 18-hydroxy-GA1 (GA132)

As part of a study to confirm putative structural assignments to new gibberellins and to furnish sufficient quantities for biological investigations, a twenty step synthesis of 18-hydroxy GA1 from gibberellic acid (GA3) is described, allowing the confirmation of structure for a new gibberellin, GA132, that occurs in developing grains of barley (Hordeum vulgare). The early part of the sequence involved cleavage of the C(3)-C(4) bond in the A-ring of a 3-oxo intermediate. The ring was then reformed as part of a "domino" process involving the conjugate addition of alkoxide to an alpha-methylene lactone moiety followed by an intramolecular aldol reaction. The bioactivities of the new GA, and its 18-hydroxy-GA4 relative, have been confirmed in dwarf barley growth and alpha-amylase induction assays.     

高效液相色谱法测定谷子幼苗中的GA_1和GA_3

建立了同时测定谷子幼苗中赤霉素Ａ１（ＧＡ１）和赤霉素Ａ３（ＧＡ３）的高效液相色谱（ＨＰＬＣ）方法。采用ＯＤＳ Ｃ１８色谱柱,以磷酸盐缓冲液和甲醇为流动相,ＧＡ１和ＧＡ３在１０ｍｉｎ内获得良好分离。利用该方法对谷子的两个正常栽培品种及其矮秆突变体的幼苗进行了测定。测定结果表明,谷子幼苗中同时含有ＧＡ１和ＧＡ３两种活性赤霉素,它们各自的质量比是影响谷子株高的重要因素。     

High-throughput serum metabolomics analysis of gouty arthritis rat treated by total saponins of Rhizoma Dioscoreae Makino by UPLC–Q/TOF–MS

Rhizoma Dioscoreae Makino (RDM) is effective in treating gouty arthritis (GA) and hyperuricacidemia, especially in promoting uric acid excretion and reducing the inflammatory reaction. Bioactive constituents in RDM are mainly steroidal saponins such as dioscin, trillin, protodioscin and protogracillin. However, the mechanism of its anti-GA action is still unclear, owing to the complex pathological and physiological characteristics of GA, and integration of RDM with multiple components, multiple targets and multiple pathways. Herein, a GA rat model was induced with monosodium urate (MSU), and RDM reduced inflammation of rat synovium tissue. Through metabolomics analysis, 35 potential biomarkers with significant changes involved in the pathogenesis of GA induced by MSU were identified, and perturbations were restored after RDM treatment. The most correlated pathways involved in d -galactose, d -mannose, d -glucose, myoinositol, Phosphatidylcholine (PC) (16:0/16:0), LysoPC (15:0), phosphatidic acid (PA) [18:1(9Z)/18:1(11Z)] and glutathione induced by MSU were galactose metabolism, inositol phosphate metabolism, glycerophospholipid metabolism and glutathione metabolism, and the derivations of all those biomarkers could be regulated by RDM treatment. RDM has a therapeutic effect on GA by intervening in changes in endogenous metabolisms and the related metabolic pathways.     

分散固相萃取-超高压液相色谱-串联质谱法测定茶叶中赤霉酸和α-萘乙酸

建立了分散固相萃取-超高压液相色谱-串联质谱分析茶叶中两种植物激素赤霉酸(GA3)与α-萘乙酸(NAA)含量的方法。样品经甲醇均质提取,采用弗罗里硅土、石墨化炭黑(GCB)、丙基乙二胺(PSA)和C18混合吸附剂分散萃取净化。采用HSS C18色谱柱(100mm×2.0mm,1.8μm),电喷雾电离(-),多反应监测模式扫描(MRM),UPLC-ESI(-)-MS/MS检测,外标法定量分析。GA3和NAA分别在0.05～5.0mg/kg与0.10～5.0mg/kg范围内线性关系良好,相关系数r≥0.9990,定量限分别为0.05与0.10mg/kg。GA3和NAA在0.1,0.5和1.0mg/kg水平上的添加回收率分别在85.0%～86.8%和82.9%～84.4%之间,精密度(RSD)≤4.5%。本方法操作简单、准确,适用于测定茶叶中GA3和NAA残留量。     

Mixed Langmuir monolayers of gramicidin A and fluorinated alcohols

Mixed monolayers of gramicidin A (GA) and three alcohols, differing in the degree of fluorination, namely C18OH, F18OH, and F8H10OH have been investigated by means of: surface manometry (pi-A isotherms) and Brewster angle microscopy (BAM) aiming at finding appropriate molecules for incorporating gramicidin A for a biosensor design. Our results proved that only the semifluorinated alcohol is appropriate material for this purpose since it forms miscible and homogeneous monolayers with GA within the whole concentration range. The experimental results have been supported by the calculations of van der Waals energy profiles using the Insight II program. Both the hydrogenated and perfluorinated alcohols were found to aggregate at higher surface pressures, which exclude their application for gramicidin-based biosensor construction.     

Inhibitory effects of glycyrrhetic acid and its related compounds on 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol.

Glycyrrhetic acid (GA), aglycone of glycyrrhizin (GL), inhibited potently (I50 = 7 x 10(-6) M) and non-competitively the activity of NAD(P)+-linked 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol. The inhibition was slightly weaker than that of indomethacin, a potent anti-inflammatory agent, but stronger than that of dexamethasone, another anti-inflammatory agent. GL, GA monoglucuronide, and 3-epi-glycyrrhetic acid also inhibited this enzyme activity, but did so less effectively (I50 = 5-8 x 10(-5) M). Carbenoxolone (GA 3-hemisuccinate) and 3-keto-glycyrrhetic acid showed potent inhibitory effects similar to GA, and 18 alpha-GA showed the most powerful inhibition of the activity.     

Determination of galacturonic acid content in pectin from fruit juices by liquid chromatographydiode array detection-electrospray ionization tandem mass spectrometry

A reversed-phase high-performance liquid chromatographic (HPLC) method is applied for the determination of galacturonic acid (GA) of pectins in different commercial fruit juices. The separation was carried out on a C₁₈ column using precolumn derivatization with p-aminobenzoic acid (p-ABA) and UV detection at 304 nm. The identification of GA was confirmed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in positive ion mode. The concentration of GA in the samples analyzed ranged from 12.9 ± 0.5 to 49.4 ± 0.5 mg_GA L⁻¹. Amongst the samples analyzed, mango juice was found to be richest in GA content, and therefore a good source of pectins. Detection and quantification limits of the described methodology were 1.2 and 3.9 mg L⁻¹, respectively. Quantitative GA recoveries in the beverages had a range between 90 and 98%. The results showed that the HPLC method proposed was precise and suitable for the identification and quantification of GA in commercial fruit juices.     

UHPLC‐MS method for determination of gambogic acid and application to bioavailability,pharmacokinetics, excretion and tissue distribution in rats

A sensitive ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC‐MS) method was developed for determination of gambogic acid (GA) in rat plasma, urine, bile and main tissues. GA was separated on an Agilent Zorbax XDB–C₁₈ column (50 × 2.1 mm, 1.8 µm) with gradient mobile phase at the flow rate of 0.2 mL/min. The detection was performed by negative electrospray ionization with multiple reaction monitoring mode. The calibration curves of GA were linear between 1.0 and 1000 ng/mL in rat plasma and bile and between 1.0 and 500 ng/mL in urine and tissues. The lowest limit of quantification for all matrices was 1.0 ng/mL. Both accuracy and precision of the assay were satisfactory. This validated method was firstly applied to bioavailability (BA), pharmacokinetics, excretion and tissue distribution in rats. The BAs of GA (40 and 80 mg/kg) in rats were 0.25 and 0.32%, respectively. GA was distributed extensively in rats after oral administration and exhibited the highest level in liver. GA reached the cumulative excretion amount of 25.3 ± 1.7 µg in bile and 0.275 ± 0.08 µg in urine after i.g. 80 mg/kg to rats at 24 h. The present results would be helpful for further clinical use of GA as a potential anticancer drug. Copyright © 2015 John Wiley & Sons, Ltd.     

高效液相色谱-串联质谱法测定葡萄中的吡效隆和赤霉素

采用高效液相色谱-串联质谱法（HPLC-MS/MS）建立了葡萄果实中吡效隆（CPPU）和赤霉素（GA3）两种植物生长调节剂残留量的定性定量分析方法。样品用0.5%甲酸水溶液和乙腈（体积比4:1）混合溶剂提取,经Strata-X固相萃取净化,使用Agilent SB-C18 色谱柱（50mm×2.1mm,1.8μm）,以0.5%甲酸水溶液-乙腈为流动相,流量0.3mL/min,采用电喷雾离子化源（ESI）多反应监测（MRM）模式下HPLC-MS/MS检测。在最佳检测条件下,得到吡效隆和赤霉素在2.0～100.0μg/L检测范围内线性关系良好,相关系数大于0.999;检出限(LOD)分别为0.3μg/L、0.5μg/L,定量限(LOQ)分别为1.0μg/L、2.0μg/L,回收率和精密度均能达到要求,表明该法准确,可用于葡萄中吡效隆和赤霉素残留量测定。     

18β-Glycyrrhetinic acid interaction with bovine serum albumin

The interaction of 18β-glycyrrhetinic acid (GA): The metabolite of glycyrrhizic acid which is the main active component of a commonly used traditional Chinese medicine (TCM) Glycyrrhiza Uralensis Fisch with bovine serum albumin (BSA) has been investigated. Fluorescence emission spectra of serum albumin in the presence of GA, recorded at the excitation wavelength 280 nm, clearly show that GA act as quencher and have different quenching mechanism at a pH below or above the isoelectric point (pI). The binding sites number n and apparent binding constant K were measured. The thermodynamic parameters ΔH°, ΔG°, ΔS° at different temperatures were calculated. The effects of some common metal ions on binding are considered. Synchronous fluorescence and UV–vis spectra were used to study protein conformation. Energy transfer between GA and HSA was calculated by Förster's theory and the binding site was suggested to be site II. The binding of monoammonium glycyrrhizinate (GL) to BSA is also compared.     

Nanoscale pore morphology and distribution of lacustrine shale reservoirs:Examples from the Upper Triassic Yanchang Formation,Ordos Basin

Pore structure plays an important role in the gas storage and flow capacity of shale gas reservoirs. Fieldemission environmental scanning electron microscopy(FE-SEM) in combination with low-pressure carbon dioxide gas adsorption(CO2GA),nitrogen gas adsorption(N2GA),and high-pressure mercury intrusion(HPMI) were used to study the nanostructure pore morphology and pore-size distributions(PSDs) of lacustrine shale from the Upper Triassic Yanchang Formation,Ordos Basin. Results show that the pores in the shale reservoirs are generally nanoscale and can be classified into four types: organic,interparticle,intraparticle,and microfracture. The interparticle pores between clay particles and organic-matter pores develop most often,l with pore sizes that vary from several to more than 100 nm. Mercury porosimetry analysis shows total porosities ranging between 1.93 and 7.68%,with a mean value of 5.27%. The BET surface areas as determined by N2 adsorption in the nine samples range from 10 to 20 m2/g and the CO2 equivalent surface areas(2 nm)vary from 18 to 71 m2/g. Together,the HPMI,N2 GA,and CO2 GA curves indicate that the pore volumes are mainly due to pores 100 nm in size. In contrast,however,most of the specific surface areas are provided by the micropores. The total organic carbon(TOC) and clay minerals are the primary controls of the structures of nanoscale pores(especially micropores and mesopores). Micropores are predominantly determined by the content of the TOC,and mesopores are possibly related to the content of clay minerals,particularly the illite-montmorillonite mixed-layer content.     

High Pressure Liquid Chromatography Assay for Determination of 18-β-Glycyrrhetinic Acid in Plasma

Abstract

This report presents a useful high pressure chromatography assay for determination of the proposed chemopreventive agent 18-β-glycerrhetinic acid (GA) in murine and human plasma. Drug was released from plasma proteins through precipitation with a mixture of sodium bisulfate and sodium chloride after which it was extracted with acetonitrile. Standard calibration curves of GA covered the concentration range of 2.5 to 120 μg/ml. The lower limit of detection was 0.5 μg/ml. No endogenous plasma constituents from plasma were found to interfere with the determination of GA. Recover of GA from plasma was greater than 95% over a concentration range of 20 to 100 μg/ml. Linearity of the assay was excellent; within-run precision showed a C.V. of 4.2% at 25 μg/ml, 5.6% at 20 μg/ml and 3.4% at 120 μg/ml. Between-run assay precision and accuracy was also considered to be excellent. The specificity, sensitivity and reproducibility of the procedure are adequate for proposed clinical pharmacology studies of this agent.     

Group additivity methods without group values

The conventional group additivity (GA) formalism may be identically reduced to a stoichiometric and thermochemical analysis of a special class of reactions referred to as GA reactions, that is, reactions that preserve the type and number of groups. Within this approach, the performance (error) of a GA scheme is determined by the stoichiometry and enthalpy changes of the GA reactions. That is, the lower the enthalpy changes of the GA reactions, the better the performance of a GA scheme. Ideally, an exact GA scheme would imply any conceivable GA reaction to be precisely thermoneutral, that is, have a zero enthalpy change. A somewhat surprising result is that, additionally, the performance of GA methods is influenced by a purely stoichiometric factor of GA reactions. These findings do not improve the performance of a given GA scheme. Rather, it is an interpretation that leads to a deeper understanding of the performance of a GA scheme and may be used in designing more accurate GA schemes.     

Antibacterial Activity of Novel 18β-Glycyrrhetinic Hydrazide or Amide Derivatives

Through a facile structural modification on the natural bioactive ingredient 18β-glycyrrhetinic acid(GA), a series of novel GA hydrazide or amide derivatives was obtained, and their final molecular frameworks were characterized by NMR and HRMS analysis. Antibacterial bioassays revealed that some of the GA hydrazide or amide derivatives were able to suppress the growth of three tested plant pathogens. Particularly, compound 3c exhibited excellent in vitro activity against Xanthomonas oryzae pv. Oryzae (Xoo), Pseudomonas syringae pv. actinidiae(Psa), and Xanthomonas axonopodis pv. citri(Xac), providing the EC₅₀ values of 5.89, 16.1, and 3.64 μg/mL, respectively. The data were better than those of the positive controls thiodiazole copper(92.7, 77.8, and 89.9 μg/mL, respectively) and bismerthiazol(31.1, 125.6, and 77.4 μg/mL, respectively). In addition, in vivo experiments suggested that, compared with thiodiazole copper(41.93% and 39.73%, respectively), compound 3c exerted prominently curative and protective activities against rice bacterial leaf blight at 200 μg/mL with the control effects of 52.36% and 51.40%, respectively. Given these obtained results, GA hydrazide or amide derivatives could serve as the feasible leads for exploring highly bioactive substrates.     

Synthesis of glycyrrhetic acid derivatives

11-Deoxyglycyrrhetinic acid (DGA) (2) was produced by Clemmensen reduction of the C-11 carbonyl of 18β-glycyrrhetic acid (GA) (1). Four derivatives of GA and DGA (3a–3d) were synthesized. Their structures were elucidated using spectral data (IR, mass, ¹H, ¹³C NMR). __________ Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 153–155, March–April, 2008.     

Gallic acid‐loaded electrospun cellulose acetate nanofibers as potential wound dressing materials

Gallic acid (GA)–loaded cellulose acetate (CA) nanofiber mats with 10 to 40 wt.% GA contents (based on the weight of CA) were fabricated by electrospinning. The effects of GA contents and applied potential on the morphology and the average diameters of fibers were studied. The electrospun fiber mats containing 20 and 40 wt.% GA were investigated for their potential use as carrier of GA in wound dressing application. The GA‐loaded CA films were prepared by solvent casting technique for use in comparative studies. Determination of the release characteristics of GA from the GA‐loaded fiber mats and films was carried out by the total immersion and the transdermal diffusion through a pig skin method in acetate buffer solution (pH 5.5) or normal saline (pH 7.0) at either 32 or 37°C, respectively. In the total immersion method, the maximum amounts of the GA released from the fiber mats containing 20 and 40 wt.% GA in the acetate buffer were approximately 97% and 71% (based on the weight of initial GA), while those of the GA released into the normal saline were approximately 96% and 81%, respectively. Lower values were observed in the experiments of the transdermal diffusion through a pig skin method. The corresponding GA‐loaded CA films showed the lower amounts of GA released into media. The as‐loaded and the as‐released GA remained its antioxidant activity as investigated by 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) assay. Lastly, the GA‐loaded CA fiber mats exhibited antibacterial activity against Staphylococcus aureus, which showed the potential for use as wound dressing materials.     

Gambogic acid-encapsulated polymeric micelles improved therapeutic effects on pancreatic cancer

Monomethyl poly(ethylene glycol)-poly(ε-caprolactone)-poly(trimethylene carbonate) (MPEG-P(CL-ran-TMC)) copolymer was synthesized, which could encapsulate GA by a single-step solid dispersion and form nano-sized micelles. The MPEG-P(CL-ran-TMC) based nano-formulation of GA could improve the anti-tumor effect in vivo, which may serve as a candidate for pancreatic cancer therapy.     

Microwave synthesis of gibberellin acid 3 magnetic molecularly imprinted polymer beads for the trace analysis of gibberellin acids in plant samples by liquid chromatography-mass spectrometry detection

In this study, novel GA3 magnetic molecularly imprinted polymer (mag-MIP) beads were synthesized by a microwave irradiation method, and the beads were applied for the trace analysis of gibberellin acids (GAs) in plant samples including rice and cucumber coupled with high performance liquid chromatography-mass spectrometry (HPLC-MS). The microwave synthetic procedure was optimized in detail. In particular, the interaction between GA3 and functional monomers was further studied for the selection of the optimal functional monomers during synthesis. It can be seen that the interaction between GA3 and acrylamide (AM) finally selected was stronger than that between GA3 and other functional monomers. GA3 mag-MIP beads were characterized by a series of physical tests. GA3 mag-MIP beads had a porous and homogeneous surface morphology with stable chemical, thermal and magnetic properties. Moreover, GA3 mag-MIP beads demonstrated selective and specific absorption behavior for the target compounds during unsaturated extraction, which resulted in a higher extraction capacity (～708.4 pmol for GA3) and selectivity than GA3 mag-non-imprinted polymer beads. Finally, an analytical method of GA3 mag-AM-MIP bead extraction coupled with HPLC-MS detection was established and applied for the determination of trace GA1, GA3, GA4 and GA7 in rice and cucumber samples. It was satisfactory that GA4 could be actually found to be 121.5 ± 1.4 μg kg(-1) in real rice samples by this novel analytical method. The recoveries of spiked rice and cucumber samples were found to be 76.0-109.1% and 79.9-93.6% with RSDs of 2.8-8.8% and 3.1-7.7% (n = 3), respectively. The proposed method is efficient and applicable for the trace analysis of GAs in complicated plant samples.