Antioxidant capacity of phenolic and related compounds: correlation among electrochemical, visible spectroscopy methods and structure-antioxidant activity

We investigated the antioxidant activity of phenylpropionic acids--caffeic (CAF), ferulic (FER), para-coumaric (COU) and cinnamic (CIN)--and phenolic acids and related compounds--gallic (GAL), methyl gallate (meGAL), vanillic (VAN) and gentisic (GEN)--using visible spectroscopy, inhibition of nitroblue tetrazolium (NBT) reduction, and electrochemical methods including cyclic voltammetry and potentiometry. In the spectroscopic assays, only CAF, GAL and meGAL were able to inhibit NBT reduction. The same compounds showed the lowest oxidation potentials (Epa) and the highest redox potentials deltaE) in the cyclic voltammetric and potentiometric studies, respectively. In addition, it was observed that the greater the number of hydroxyls linked to the aromatic ring, the greater was the antioxidant activity of the analysed compounds. The correlations of Spermann--used to compare the methods between themselves and the methods with the relationship structure-antioxidant activity--were r = -0.9762 for the cyclic voltammetric-potentiometric methods. r = 0.8333 for the inhibition of NBT reduction-potentiometric methods and r = -0.8095 for the inhibition of NBT reduction-cyclic voltammetric methods. The correlations for cyclic voltammetric, potentiometric and inhibition of NBT reduction methods-number of hydroxyls linked to the aromatic ring were r = -0.9636, 0.9636 and 0.9142, respectively. These findings indicate that the electrochemical methods together with spectroscopic studies are a good tool to evaluate the antioxidant activity of substances.     

A solution to the problems of cytolysis assays with additional applications to other immunological and biochemical assays

After cellular immunoassays are compared with classical bioassays, conventional methods and consequent problems of data analysis for cytolysis assays are reviewed and a new solution is proposed. This solution incorporates new methods, called dose-response surface assays and analysis (DRSA), which estimate cytolytic activity coefficients on a surface in a three-dimensional space with two dose variables (killers and targets) and one response variable (counts). These new methods based on dose-response surfaces are demonstrated to be more informative and reliable than classical methods based on dose-response curves. In a test of the methods' robustness (sensitivity of parameter estimates to changes in the dose levels of the assay design), cytolytic activity coefficients estimated by DRSA varied by less than or equal to 30% over a reduction of three to four orders of magnitude in the dose levels. This remarkable robustness should be compared with the corresponding figures of as much as 500% over less than 1 order of magnitude for previously published results of coefficients estimated by conventional methods. DRSA is distinguished from replot-of-plots methods such as those used for enzyme inhibition assays in biochemistry, and is recommended as a more efficient method that should replace replot-of-plot methods now antiquated by the advent of microcomputers. DRSA can be applied to any experimental system that requires an activity coefficient to be estimated on a dose-response surface in a space of greater than or equal to 3 dimensions (greater than or equal to 2 dose variables and one response variable), regardless of the mathematical model and statistical estimators used to analyze the dose-response interaction. Finally, DRSA is compared with the methods known as response surface methodology (RSM), and is described as a new class of methods to be added to those that constitute RSM.     

Enzyme microelectrophoresis of cell colonies

Mouse A9 cell colonies containing 1 000–2 000 cells can be electrophoresed in thin starch gels and enzyme activity can be detected for phosphohexose isomerase (PHI), malate dehydrogenase (MDH), lactate dehydrogenase (LDH), adenylate kinase (AK) and nucleoside phosphorylase (NP). The new methods developed here are techniques of manual micromanipulation of the cell colony samples and slight modifications of routine methods of starch gel electrophoresis.     

Analytical and biological characterization of supercoiled plasmids purified by various chromatographic techniques

Supercoiled plasmids are an important component of gene-based delivery vehicles. A number of production methods for clinical applications have been developed, each resulting in very high-quality product with low levels of residual contaminants. There is, however, no consensus on the optimal methods to characterize plasmid quality, and further, to determine if these methods are predictive of either product stability or biological activity. We have produced two plasmids using four production purification methodologies based on PolyFlo and hydrophobic interaction chromatography (HIC), either alone or in tandem processes. In each case, the product was analyzed using standard molecular biological methods. We also performed a number of biophysical analyses such as dynamic light scattering (DLS), circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). Minimal differences were detected among the preparations based on the more standard molecular biological methods. Some small differences were detected, however, using biophysical techniques, particularly FTIR and DSC, which may reflect small variations in plasmid tertiary structure and thermal stability. Stability after heat exposure at 60 degrees C, exposure to fetal bovine serum and long-term storage at 4 degrees C varied between plasmids. One plasmid showed no difference in stability depending on the production process, but the other showed significant differences. Evaluation in vivo in models for gene immunization and gene therapy showed significant differences in the response depending on the method of purification. Preparations using a tandem process of PolyFlo used in two separation modes provided higher biological activity compared to a tandem HIC/PolyFlo process or either resin used alone in a single column process. These data indicate that the process by which supercoiled plasmids are made can influence plasmid stability and biological activity and emphasize the need for more rigorous methods to evaluate supercoiled plasmids as gene-delivery vehicles.     

Advantages and limitations of common testing methods for antioxidants

Abstract

Owing to the importance of antioxidants in the protection of both natural and man-made materials, a large variety of testing methods have been proposed and applied. These include methods based on inhibited autoxidation studies, which are better followed by monitoring the kinetics of oxygen consumption or of the formation of hydroperoxides, the primary oxidation products. Analytical determination of secondary oxidation products (e.g. carbonyl compounds) has also been used. The majority of testing methods, however, do not involve substrate autoxidation. They are based on the competitive bleaching of a probe (e.g. ORAC assay, β-carotene, crocin bleaching assays, and luminol assay), on reaction with a different probe (e.g. spin-trapping and TOSC assay), or they are indirect methods based on the reduction of persistent radicals (e.g. galvinoxyl, DPPH and TEAC assays), or of inorganic oxidizing species (e.g. FRAP, CUPRAC and Folin-Ciocalteu assays). Yet other methods are specific for preventive antioxidants. The relevance, advantages, and limitations of these methods are critically discussed, with respect to their chemistry and the mechanisms of antioxidant activity. A variety of cell-based assays have also been proposed, to investigate the biological activity of antioxidants. Their importance and critical aspects are discussed, along with arguments for the selection of the appropriate testing methods according to the different needs.     

Histochemical studies on the distribution of thiamine pyrophosphatase and enzymes related to carbohydrate metabolism in the intercalated neurons of the rat supraoptic nucleus

Histochemical studies have been conducted by applying hexokinase (HK), aldolase (AD), glyceraldehyde-3-phosphate dehydrogenase (G3), succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PD), and thiamine pyrophosphatase (TPPase) methods, as well as Nissl staining and Gomori's chrome-alum-hematoxylin-phloxine (CHP) methods to intercalated neurons of the supraoptic nucleus (SO) on Wistar strain rats. Intercalated neurons reacted weakly to the AD, G3, G6PD, and SDH tests, indicating that they belong to the category of ordinary neurons with low carbohydrate metabolism. Many fibrous astrocytes showing strong HK reactions surround neurosecretory neurons. However, they do not surround intercalated neurons with mild HK activity. These results indicate that the latter receive a poor supply of energy from glucose in the circulating blood in contrast to the former. Intercalated neurons are very rich in Nissl substance but lack CHP-positive material. They may have a high potential for synthesizing protein. The principal morphological features of the TPPase-positive Golgi material are peculiar and heterogeneous shape and poor development. These findings together with mild G6PD activity suggest that intercalated neurons are very likely to have poor synthesizing activity.     

Enzyme kinetics determined using calorimetry: a general assay for enzyme activity?

Two techniques for determining enzyme kinetic constants using isothermal titration microcalorimetry are presented. The methods are based on the proportionality between the rate of a reaction and the thermal power (heat/time) generated. (i) An enzyme can be titrated with increasing amounts of substrate, while pseudo-first-order conditions are maintained. (ii) Following a single injection, the change in thermal power as substrate is depleted can be continuously monitored. Both methods allow highly precise kinetic characterization in a single experiment and can be used to measure enzyme inhibition. Applicability is demonstrated using a representative enzyme from each EC classification, including (i) oxidation-reduction activity of DHFR (EC 1.5.1.3); (ii) transferase activity of creatine phosphokinase (EC 2.7.3.2) and hexokinase (EC 2.7.1.1); (iii) hydrolytic activity of Helicobacter pylori urease (EC 3.5.1.5), trypsin (EC 3.4.21.4), and the HIV-1 protease (EC 3.4.21.16); (iv) lyase activity of heparinase (EC 4.1.1.7); and (v) ligase activity of pyruvate carboxylate (EC 6.4.1.1). This nondestructive method is completely general, enabling precise analysis of reactions in spectroscopically opaque solutions, using physiological substrates. Such a universal assay may have wide applicability in functional genomics.     

深度学习在药物活性预测研究中的应用

药物从研发到临床应用需要耗费较长的时间，研发期间的投入成本可高达十几亿元。而随着医药研发与人工智能的结合以及生物信息学的飞速发展，药物活性相关数据急剧增加，传统的实验手段进行药物活性预测已经难以满足药物研发的需求。借助算法来辅助药物研发，解决药物研发中的各种问题能够大大推动药物研发进程。传统机器学习方法尤其是随机森林、支持向量机和人工神经网络在药物活性方面能够达到较高的预测精度。深度学习由于具有多层神经网络，模型可以接收高维的输入变量且不需要人工限定数据输入特征，可以拟合较为复杂的函数模型，应用于药物研发可以进一步提高各个环节的效率。在药物活性预测中应用较为广泛的深度学习模型主要是深度神经网络（deep neural networks，DNN）、循环神经网络（recurrent neural networks，RNN）和自编码器（auto encoder，AE），而生成对抗网络（generative adversarial networks，GAN）由于其生成数据的能力常常被用来和其他模型结合进行数据增强。近年来深度学习在药物分子活性预测方面的研究和应用综述表明，深度学习模型的准确度和效率均高于传统实验方法和传统机器学习方法。因此，深度学习模型有望成为药物研发领域未来十年最重要的辅助计算模型。     

基因表达常规分析方法概述

在基因表达的分析中,RNA转录的稳态水平是检测细胞株和组织的基因表达活性的最方便的参数之一.本概述了用于检测mRNA丰度的常规分析方法,即Northern印迹、RNase保护试验和实时定量PCR技术,特别是为对数量不多的基因进行表达情况的测定时提供了较为实用的方法.     

Electron microscopic localization of monoamine oxidase in the rat liver

Summary Two methods have been employed to localize monoamine oxidase activity in the cells of rat liver, using either 2-(2′-benzothiazolyl)-5-stryl-3-(4′-phtalhydrazidyl) tetrazolium chloride (BSPT) or ferricyanide as electron acceptor. With both methods monoamine oxidase activity was found both in the inner and the outer mitochondral membrane, although the outer membrane appeared the most probable location. In addition the BSPT method but not the ferricyanide method, revealed monoamine oxidase activity in the endoplasmatic reticulum. The results obtained by the two methods have been compared and are discussed in view of available biochemical data on monoamine oxidase. Supported by research grants from the National Research Council of Canada (A 3651), The Swedish Medical Research Council (4145) and M. Bergwall's Foundation, Stockholm.     

Anti-HIV-1 protease activity of compounds from Boesenbergia pandurata

Searching for anti-HIV-1 protease (PR) inhibitors of Thai medicinal plants led to the isolation of a new cyclohexenyl chalcone named panduratin C (1) and chalcone derivatives (2-6) from the methanol extract of Boesenbergia pandurata rhizomes. The known compounds were identified to be panduratin A (2), hydroxypanduratin A (3), helichrysetin (4), 2',4',6'-trihydroxyhydrochalcone (5), and uvangoletin (6). The structures of all compounds were elucidated on the basis of chemical and spectroscopic methods. It was found that 3 possessed the most potent anti-HIV-1 PR activity with an IC50 value of 5.6 microM, followed by 2 (IC50 = 18.7 microM), whereas other compounds exhibited only mild activity. Structure-activity relationships of these compounds on anti-HIV-1 PR activity are summarized as follows: (1) hydroxyl moiety at position 4 conferred higher activity than methoxyl group; (2) prenylation of dihydrochalcone was essential for activity; (3) hydroxylation at position 4' reduced activity; and (4) introduction of double bond at C1' and C6' of chalcone gave higher activity. As regards active constituents contained in B. pandurata rhizomes, hydroxypanduratin A (3) and panduratin A (2) are active principles against HIV-1 PR.     

Validation of a specific prolylcarboxypeptidase activity assay and its suitability for plasma and serum measurements

Prolylcarboxypeptidase (PRCP, EC 3.4.16.2), a lysosomal carboxypeptidase, was discovered 45 years ago. However, research has been hampered by a lack of well-validated assays that are needed to measure low activities in biological samples. Two reversed-phase high-performance liquid chromatography (RP-HPLC) methods for quantifying PRCP activity in crude homogenates and plasma samples were optimized and validated. PRCP activity was determined by measuring the hydrolysis of N-benzyloxycarbonyl-l-proline (Z-Pro)-Phe. The enzymatically formed Z-Pro and Phe were measured independently under different HPLC conditions. The in-house methods showed good precision, linearity, accuracy, and specificity. Based on Michaelis–Menten constants, Z-Pro-Phe was chosen over Z-Pro-Ala as the substrate of preference. Cross-reactivity studies with dipeptidyl peptidases (DPPs) 2, 4, and 9 and prolyl oligopeptidase (PREP) confirmed the specificity of the PRCP activity assay. The average PRCP activity in plasma and serum of 32 healthy individuals was found to be 0.65 ± 0.02 and 0.72 ± 0.03 U/L, respectively. Both methods can be used to measure PRCP activity specifically in different biological samples and are well suited to evaluate PRCP inhibitors. These well-validated methods are valuable tools for studying PRCP’s role in cardiovascular diseases, stroke, inflammation, and metabolic syndrome.     

A novel training-free method for real-time prediction of femoral strain

Surrogate methods for rapid calculation of femoral strain are limited by the scope of the training data. We compared a newly developed training-free method based on the superposition principle (Superposition Principle Method, SPM) and popular surrogate methods for calculating femoral strain during activity. Finite-element calculations of femoral strain, muscle, and joint forces for five different activity types were obtained previously. Multi-linear regression, multivariate adaptive regression splines, and Gaussian process were trained for 50, 100, 200, and 300 random samples generated using Latin Hypercube (LH) and Design of Experiment (DOE) sampling. The SPM method used weighted linear combinations of 173 activity-independent finite-element analyses accounting for each muscle and hip contact force. Across the surrogate methods, we found that 200 DOE samples consistently provided low error (RMSE < 100 µε), with model construction time ranging from 3.8 to 63.3 h and prediction time ranging from 6 to 1236 s per activity. The SPM method provided the lowest error (RMSE = 40 µε), the fastest model construction time (3.2 h) and the second fastest prediction time per activity (36 s) after Multi-linear Regression (6 s). The SPM method will enable large numerical studies of femoral strain and will narrow the gap between bone strain prediction and real-time clinical applications.     

Distribution of some hydrolases in the rat kidney (author's transl)

Most of the available histochemical methods and techniques (azodye, metal salt and indigogenic methods, cryostat, free-floating and lyophilized section techniques) and different modifications of these methods (different substrate concentrations, pH, temperature, incubation time e.g.) were applied to study the distribution of acid phosphatase (AcPB = after Barka and Anderson; AcPG = after Gomori), beta-glucuronidase (beta-Glu), aryl sulfatase (AS), beta-N-acetylglucosaminidase (NAG), acid 5'-nucleotidase (a5-Nucl), non-specific esterase (NE) and alkaline phosphatase (AlP) in the kidneys of rats of both sexes. The optimal conditions for the demonstration of these enzymes were established. As most important proved: the incubation of free-floating sections cut from "standard"-fixed (2 h in formol-calcium continued for another 18-22 h in the same fixative plus 0.88 M sucrose at 4 degrees C) kidney slices - only for AcPB and NE material fixed after Holt had to be used; the incubation for AlP and NE at 4 degrees C; final pH of the incubation medium for AcPB 5.5, AcPG 5.0 and NE 6.5; the use of Fast Garnet GBC Salt as coupler in the NE azo-dye reaction. Sex differences and for the female rats an increased activity during oestrus were established for all hydrolases studied. In particular the following results were obtained: AcPB, a5-Nucl and A1P are more intensive in male and AcPG in female S1 segments of the juxtamedullary nephrons in relation to the nephrons of the other parts of the cortex. In the medullary rays the NE and the a5-Nucl show a higher activity in the S2 segments of female rats demonstrate a more intensive activity for NAG and NE. This is true for AcPG and A1P in male rats. In the inner medulla a stronger beta-Glu activity in male rats and a stronger NAG activity in female rats is observed. The AcPB activity of the cortical distal tubules is higher in male rats.     

Evaluation of the effect of aqueous extract of Croton urucurana Baillon (Euphorbiaceae) on the hemorrhagic activity induced by the venom of Bothrops jararaca, using new techniques to quantify hemorrhagic activity in rat skin

Aqueous extracts of Croton urucurana (Sangra D'agua), a plant popularly considered a cicatrizant, were analyzed for anti-Bothrops jararaca venom activity. The plant extracts antagonized the hemorrhagic activity of the venom and proanthocyanidins were involved in this activity. Two new methods for the quantification of hemorrhagic activity evoked by bothropic venoms were employed. The first consists of graphic computer analysis of the hemorrhagic halo evoked in rats by dorsal intradermic administration of venom. The second method involves quantification of the hemoglobin present in the hemorrhagic halo. Based on the results, we suggest that these methods, easily implemented in the laboratory routine, allow for quantification of venom-induced hemorrhagic activity. In addition, this study demonstrates that the rich extracts of proanthocyanidins are powerful inhibitors of bothropic venom metalloproteinases.     

Selective ABTS radical-scavenging activity of prenylated flavonoids from Cudrania tricuspidata

The antioxidative properties of five prenylated flavonoids, including new flavanone (2), from the root bark of Cudrania tricuspidata were examined against the ABTS, DPPH, and hydroxyl radicals. In most of the assays to determine their antioxidative properties, the ABTS activity was strongly correlated with DPPH because both methods are responsible for the same chemical property of hydrogen- or electron-donation to the antioxidant. On the other hand, the prenylated flavonoids (1-5) acted differently with both methods; namely, all the prenylated flavonoids strongly scavenged the ABTS radical (IC(50) < 10 microM), while they were inactive against the DPPH radical (IC(50) > 300 microM). Even though isolated 5,7,2',4',-tetrahydroxy-6,5'-diprenylflavanone (3) showed weak reducing power (746 mV) by cyclic voltammetry when compared to quercetin (394 mV), both had similar ABTS activity (IC(50) < 5 microM).     

Evaluation of the composition and in vitro antimicrobial,antioxidant, and anti-inflammatory activities of Cilantro (Coriandrum sativum L. leaves) cultivated in Saudi Arabia (Al-Kharj)

BackgroundIn the present study, we explored the composition of Cilantro (Coriandrum sativum L. leaves) essential oil (CEO) cultivated in Saudi Arabia (Al-Kharj) and explored its antioxidant, antimicrobial, and anti-inflammatory effects in vitro.MethodsGas chromatography-mass spectroscopy was used to detect the CEO composition. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH)-induced free radical and ferric chloride scavenging methods were used to determine the antioxidant activity. Antimicrobial activity was investigated using the well diffusion method. Anti-inflammatory activity was evaluated using egg albumin and trypsin-induced inflammation methods.ResultsForty-six compounds representing 90.17% of the total aroma were identified in the CEO; the major constituents were found to be 1-decanol (17.85%), decanal (11.04%), trans-2-dodecen-1-ol (7.87%), menthone (6.71%), 2-decen-1-ol, trans- (5.44%), dodecanal (4.76%), trans-tetradec-2-enal (3.14%), sedanolide (3.02), and thymol (3.01%). DPPH-induced free radical and ferric chloride scavenging assays demonstrated low antioxidant effects of CEO, and the antioxidant activity was observed at a high CEO concentration. The antimicrobial activity of CEO was assessed against 5 microorganisms (bacteria and fungi) by using well diffusion methods; CEO was found to possess excellent antimicrobial activity against all microorganisms, except Escherichia coli. Moreover, CEO demonstrated strong anti-inflammatory activity against egg albumin- and trypsin-induced inflammation.ConclusionThe essential oil extracted from C. sativum chemotype grown in Al-Kharj region of Saudi Arabia possesses low antioxidant potential, superior antimicrobial activity, and outstanding anti-inflammatory effects.     

Influence of the peptide chain length of new elongated tuftsin analogs on phagocytosis process

In this paper the synthesis of the following elongated tuftsin analogs: Arg-Thr-Lys-Pro-Arg (1), Pro-Arg-Thr-Lys-Pro-Arg(II), Lys-Pro-Arg-Thr-Lys-Pro-Arg(III) and Thr-Lys-Pro-Arg-Thr-Lys-Pro-Arg(IV) by classical and solid-phase methods are described. The obtained peptides were tested for their biological activity: restoration of the phagocytosis of defected granulocytes from blood of children with acute lymphoblastic leukemia (ALL).     

Chiral separation principles in chromatographic and electromigration techniques

Almost half of the drugs in use today are chiral. It is well established that the pharmacological activity is mostly restricted to one of the enantiomers (eutomer). There can be qualitative and quantitative differences in the activity of the enantiomers. In many cases, the inactive enantiomer (distomer) shows unwanted side effects or even toxic effects. Even if the side effects are not that drastic, the distomer has to be metabolized and this represents an unnecessary burden for the organism. Therefore, the development of methods for the separation of enantiomers, both on analytical and preparative scale, has become increasingly important. Chromatographic techniques such as thin layer chromatography (TLC), gas chromatography (GC), supercritical fluid chromatography (SFC), and above all high-performance liquid chromatography (HPLC) have been used for enantiomer separation for about two decades. More recently, electromigration techniques, such as capillary electrophoresis and capillary electrochromatography, have been shown to be powerful alternatives to chromatographic methods. This review gives a short overview of different chiral separation principles and their application. Several new developments are discussed.     

Structure-function studies of human granulocyte-macrophage colony-stimulating factor. Identification of residues required for activity

Human granulocyte-macrophage colony stimulating factor (hGM CSF), a protein containing 127 amino acids, was chemically synthesized by using automated stepwise solid-phase methods. The unpurified synthetic hGM-CSF had the same range of actions on hemopoietic cells as the purified recombinant protein. The structural requirements for the activities of synthetic hGM-CSF were examined by the design and synthesis of fragments and analogs. The synthetic fragment, hGM-CSF (54-127), containing all four of the cysteine residues found in the intact protein, lacked detectable activity. Assays of fragments shortened at the N terminus showed that the residues 1-13 were not required for activity, but that the integrity of residues 14-25, particularly residues 16, 17, and 18, was critical for biologic activity. The 14-25 region is predicted to form the first alpha-helix in hGM-CSF. Synthetic peptides within the N-terminal 53 residue region lacked detectable activity. The synthetic analog hGM-CSF (1-121), which lacks the C-terminal 6 residues, had similar activity to hGM-CSF (1-127) indicating that residues 122-127 are not required for activity. An analog, [Ala88] hGM-CSF (14-96), which lacks the hydrophobic C-terminal region and 2 cysteine residues, had low but readily detectable activity suggesting that residues 14-96 are sufficient for detectable synthetic hGM-CSF activity, although the presence of residues 97-121 are required for full activity. No dissociation of the multiple biological activities of hGM-CSF was detected.