Multiple on‐line HPLC coupled with biochemical detection methods to evaluate bioactive compounds in Danshen injection

On‐line high performance liquid chromatography (HPLC) coupled with three biochemical detection (BCD) methods was applied to evaluate bioactive components in Danshen injection. On‐line HPLC‐photo‐diode array–fluorescence detection based on the fluorogenic substrate 7‐acetoxy‐1‐methyl quinolinium iodide, was built to search acetylcholinesterase (AChE) inhibitors in Danshen injection. On‐line HPLC coupled with the scavenging assay of 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) and 2,2′‐azinobis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) free radicals was developed to screen antioxidants. The three active profiles were obviously different. Radical scavenging profiles revealed seven strong peaks in the chromatographic fingerprint possessing obvious free radical inhibition effects, while some minor peaks exhibited stronger AChE inhibition activities. The main radical scavengers and AChE inhibitors were identified by HPLC‐MS. Several unknown ingredients showing strong AChE inhibition activities needed further identification except protocatechuic aldehydrate, salvianolic acid H or I and lithospermic acid. The on‐line multiple on‐line HPLC‐BCD methods will provide powerful tools in the field of pharmacognosy for fast‐track identification of interesting and/or novel bioactive compounds.     

Novel 1,3,4-thiadiazole conjugates derived from protocatechuic acid: Synthesis,antioxidant activity,and computational and electrochemical studies

A series of 15 novel 1,3,4-thiadiazole amide derivatives containing a protocatechuic acid moiety were synthesized and structurally characterized. In addition, the corresponding imino (4) and amino (5) analogues of a phenyl-substituted 1,3,4-thiadiazole amide derivative 3a were prepared to compare the effects of the structural changes on the radical-scavenging activity. The obtained compounds were examined for their antioxidative potential by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. In addition, selected compounds were studied by density functional theory (DFT) and cyclic voltammetry experiments. The tested compounds showed high potential to scavenging DPPH radical and ABTS radical cation compared with the referent antioxidants ascorbic acid and nordihydroguaiaretic acid (NDGA). On the basis of the calculated thermodynamic parameters, it can be concluded that the sequential proton loss electron transfer (SPLET) mechanism represents the most probable reaction path in a polar solvent for DPPH radical–scavenging activity. On the other hand, the single electron transfer followed by proton transfer (SET-PT) can be a likely mechanistic pathway in the case of an ABTS radical cation.     

Effect of acid and base catalyzed hydrolysis on the yield of phenolics and antioxidant activity of extracts from germinated brown rice (GBR)

The influence of both acidic and basic hydrolysis on the yield, total phenolic content and antioxidative capacity of methanolic extract of germinated brown rice (GBR) was studied. Total phenolic content (TPC), total flavonoid content (TFC), 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical cation scavenging, and ferric reducing antioxidant power (FRAP) tests were used for the measurement of antioxidant ability. There was a significant difference p < 0.05) in the TPC and DPPH radical scavenging assay results when comparing neutral with acidic and basic catalysed hydrolysis. The yield of the crude extract was slightly higher in acidic hydrolysis than in basic hydrolysis p > 0.05). The TPC and TFC were highest in acidic hydrolysis. A significant correlation was observed between ABTS radical cation scavenging and FRAP. The antioxidant activity measured using DPPH radical scavenging assay showed high activity in acidic hydrolysis, while the ABTS radical cationscavenging activity and FRAP showed the highest values in basic hydrolysis. The samples were further evaluated using HPLC to determine the individual phenolic concentrations in different hydrolytic media contributing to the antioxidant effects. This study revealed that acidic and basic hydrolysis can improve the yield, phenolic content, and antioxidant activity of germinated brown rice.     

Screening and characterization of natural antioxidants in four Glycyrrhiza species by liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Licorice, derived from the dried roots and rhizomes of several species of genus Glycyrrhiza L. (Leguminosae family), has been traditionally used in herbal medicine for over 4000 years. In recent years, the interest in antioxidative constituents in licorice has greatly increased. In this work, a new method based on 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) spiking test combined with HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis was developed to screen and identify the antioxidants in licorice. The results of the method validation indicated that the developed method was reliable and repeatable. Compared with DPPH on-line method, the HPLC-Q-TOF MS/MS method combined with DPPH spiking test offered much higher sensitivity and resolution. Using this method, 35 radical scavengers were screened from four Glycyrrhiza species (G. inflata, G. glabra, G. pallidiflora and G. uralensis), and 21 of them were unambiguously or tentatively identified by HPLC-Q-TOF MS/MS. Among the 21 identified flavonoids, 10 compounds had been reported to possess antioxidative activities in the previous studies, and the radical scavenging activities of the other 11 compounds were reported for the first time. The effects of six purified flavonoids on DPPH radical and lipid peroxidation were evaluated for validation of the developed method. The results indicated that HPLC-Q-TOF MS/MS coupled with DPPH treatment is an efficient and powerful method to discover the potential antioxidative compounds from the complex natural product mixtures. In this study, the identified components with free radical scavenging activity, would help to explain the therapeutic benefit of licorice in the treatment of human disease associated with oxidative stress.     

A novel phenylpropenoyl sulfonic acid and a new chlorophyll from the leaves of Petasites formosanus KITAMURA

A novel phenylpropenoyl sulfonic acid, petasiformin-A (1), and a new chlorophyll, petasiphyll-A (2), were isolated from the leaves of Petasites formosanus KITAMURA (Compositae). Their structures were established by spectral and chemical transformation methods. Petasiformin-A (1) showed the significant antioxidative activity in DPPH radical scavenging assay.     

Mechanism and Cellular Kinetic Studies of the Enhancement of Antioxidant Activity by Using Surface‐Functionalized Gold Nanoparticles

The enhanced antioxidant activity of surface‐functionalized gold nanoparticles (AuNPs) synthesized by self‐assembly has attracted great attention, but little is known about the mechanism behind the enhanced activity. To address this challenge, the antioxidant activity of Au@PEG3SA (i.e., surface‐functionalization of spherical AuNPs with the antioxidant salvianic acid A) was used as an example to illustrate the mechanism of the enhanced activity. Evaluation of the antioxidant activity was performed in a radical‐scavenging reaction between Au@PEG3SA and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical. As expected, the rate constant for the reaction of Au@PEG3SA with DPPH was about nine times greater than that for the salvianic acid A monomer. A comparative analysis of the spectral characteristics of Au@PEG3SA and the salvianic acid A monomer further imply that the enhancement of the antioxidative reaction kinetics may be ascribed to the variation in the transition state for the DPPH‐radical scavenging reaction through π–π stacking interactions between and among adjacent groups on the surface of Au@PEG3SA. On the other hand, the kinetic enhancement of Au@PEG3SA on reactive‐oxygen‐species (ROS) scavenging can be observed in living cells and in vivo, which possibly provides new insight for the bioapplication of self‐assembly of surface‐functionalized AuNPs.     

甜樱桃色素的提取及抗氧化活性

采用溶剂浸提法从成熟甜樱桃中提取了甜樱桃色素,并通过C18柱分离纯化,HPLC／光电二极管阵列检测器分析色素的主要成分,同时测定了甜樱桃色素体外清除二苯代苦味肼基自由基（DPPH）和羟基自由基的活性．结果表明,甜樱桃色素含有新绿原酸和对-香豆酰奎宁酸两个主要成分,对DPPH和·OH自由基具有较强的清除能力,且随色素浓度的增大而增强．研究认为,甜樱桃色素是一种具有良好抗氧化活性的功能性天然色素．     

基于分子指纹对黄酮类化合物及其抗DPPH自由基活性的QSAR分析

应用定量构效关系（Quantitative structure activity relationship, QSAR）研究阐明黄酮类化合物（Flavonoid compounds, FCs）的子结构指纹（Substructure fingerprint）与1,1-二苯基-2-三硝基苯肼（1,1-Diphenyl-2-picrylhydrazyl, DPPH）自由基清除能力之间的关系,从而指导高效抗氧化物质的设计和发现。在PubMed数据库中收集77个具有明确抗氧化活性的黄酮类化合物,而在ChEMBL数据库中收集86个无抗DPPH活性的黄酮类化合物。这163个黄酮类化合物的子结构指纹由PubChem系统生成,然后通过卡方检验筛选出与黄酮类化合物的抗氧化活性显著相关的分子指纹,最后通过判别分析建立预测QSAR模型,并采用回代法和交叉验证法对已建立的模型进行准确性和稳健性的验证。结果表明,黄酮类化合物抗DPPH自由基活性与ESSSR环的计数、简单相邻原子的类型和简单的SMARTS模式等因素有关。此外,所建立的QSAR模型能较好地预测黄酮类化合物的DPPH自由基清除活性,可用于评价候选抗氧化剂的潜力。     

Quality assessment of radix salviae miltiorrhizae

This paper describes an improved quality assessment method for Radix Salviae Miltiorrhizae (Root of Salvia miltiorrhiza BGE.) which was established using chromatographic fingerprinting and quantification of multiple marker compounds in the crude drug. High-performance thin-layer chromatography (HPTLC) fingerprinting of water-soluble phenolics and nonpolar tanshinones was performed separately and the authentication of Radix Salviae Miltiorrhizae was achieved by comparing the fingerprints of the samples with those of the reference crude drug and by comparing the Rf values of the bands in TLC fingerprints with those of reference compounds. HPLC fingerprints were obtained by simultaneous separation of phenolics and diterpenoids in Radix Salviae Miltiorrhizae. The HPLC fingerprints of seven batches of samples from different regions of China showed similar chromatographic patterns, and seven peaks were selected as characteristic peaks. The relative retention time of these characteristic peaks in the HPLC fingerprints was established as an important parameter for the identification of this herbal medicine. The pharmacologically active marker compounds salvianolic acid B, rosmarinic acid, and tanshinone IIA in herbal medicine were quantitatively determined using reverse-phase HPLC techniques. The HPLC quantitation methods of the three marker compounds were validated and the measurement uncertainty, which is important for setting the proposed content limit of the marker compounds in herbal medicine, were further evaluated.     

Potent 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity of novel antioxidants, double-stranded tyrosine residues conjugating pyrocatechol

New potent antioxidants conjugating the catechol (=pyrocatechol; pyrCat) group to two N-termini of modified double-stranded tyrosine residues were synthesized and showed radical scavenging activity with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH radical, DPPH˙) as a free radical model, second-order rate constants for the DPPH˙ scavenging reaction, and the results from electron spin resonance (ESR) studies. It was found that the tyrosine (Tyr) residue and pyrCat containing new antioxidants developed in the study have about 3-20 times more potent antioxidative activity than Trolox, pyrCat, and L-ascorbic acid (VC). In order to elucidate the relationship between antioxidant activity and the molecular orbital states, and to design potent antioxidants we present an interesting approach using an absolute hardness (η)-absolute electronegativity (χ) diagram based on chemical hardness. It was shown that quantum chemicals were required to develop potent antioxidants.     

Spectrum–effect relationship between HPLC fingerprints and antioxidant activity of Angelica sinensis

Angelica sinensis is one of the most important traditional Chinese medicines and has antioxidant activities that greatly contribute to its pharmacological action. However, the compounds responsible for its antioxidant activity remain unknown. In this study, the fingerprints of 10 batches of A. sinensis collected from different locations in China were established with HPLC to identify the common peaks. The antioxidant activities of these 10 batches were evaluated with 2,2-diphenyl-1-picrylhydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and ferric-reducing antioxidant power assays. The spectrum–effect relationship between HPLC fingerprints and antioxidant effect of A. sinensis was examined by the partial-least-square regression analysis and the variable importance in projection method. Results showed that the antioxidant effect of A. sinensis results from the synergistic effect of various compounds, and peaks X3 and X7–X18 were the main substances responsible for antioxidant efficacy. This study successfully identified the spectrum–effect relationship between HPLC fingerprints and the antioxidant effect of A. sinensis. This relationship can provide methods for establishing the quality standards for A. sinensis and developing new and effective products of A. sinensis based on its antioxidant ingredients.     

Isolation and identification of radical scavengers in olive tree (Olea europaea) wood

Several extracts of Olea europaea wood (Picual olive cultivar) were obtained with solvents of different polarity and their antioxidant activities determined. The active compounds were detected in fractions of an ethyl acetate extract using HPLC with on-line radical scavenging detection. After applying different separation techniques, hydroxytyrosol, tyrosol, cycloolivil, 7-deoxyloganic acid, oleuropein and ligustroside were isolated and characterized. Hydroxytyrosol showed a higher activity than the natural antioxidant rosmarinic acid in scavenging the DPPH model radical. Cycloolivil and oleuropein showed stronger activities than the synthetic antioxidant BHT against the same radical. Ligustroside, tyrosol and 7-deoxyloganic acid showed little activity. The latter compound has not been previously identified in the genus Olea.     

Characterization of in vivo antioxidant constituents and dual‐standard quality assessment of Danhong injection

Antioxidants and oxidative stress play a critical role in cardiovascular diseases. Danhong injection (DHI) is a well prescribed cardiovascular medication in China, but its detailed chemical basis and mechanisms of action remain unknown. To prove the antioxidant activity of DHI, its free radical scavenging capacity (RSC) was assessed by 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) spectrophotometric assay. The 50% radical scavenging activity value was 1:129.2 mL/mL, against 0.95 mm DPPH. To further identify the antioxidant compounds, modified thin‐layer chromatography combined with DPPH bioautography assay was used. Compared with vitamin C, 11 of 16 available compounds displayed strong antioxidant activity, which were also detected in rat serum after intravenous administration of DHI by ultra‐performance liquid chromatography–tandem mass spectrometry, except for hydroxysafflor yellow A. Therefore, 10 antioxidants remaining in the blood as key markers, and six other compounds as general markers, were employed to perform the quality control of DHI by ultra‐performance liquid chromatography–ultraviolet detection after systematic methodological validation. The analytical results indicate a high correlation (r = 0.9) between the total content of those antioxidants remaining in blood and RSC of DHI among 10 batches. Further, the antioxidant profiling and chemical marker quantification as dual‐standard quality assessment was successfully applied to evaluate Danshen and safflower injections. Copyright © 2012 John Wiley & Sons, Ltd.     

Assessing the antioxidative properties and chemical composition of Linaria vulgaris infusion

The ability of Linaria vulgaris (Scrophulariaceae) infusion to act as a scavenger of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, reactive oxygen species (superoxide radical, hydroxyl radical, hypochlorous acid (HOCl)) and nitric oxide was investigated. The obtained data indicate that the infusion has a good scavenging activity against superoxide radical and is a very potent nitric oxide and DPPH scavenger. In hydroxyl radical assay a pro-oxidant capacity was noticed, especially for concentrations higher than 31.25 microg mL(-1). No effect was found against HOCl. A phytochemical study of this extract was also performed. The HPLC/UV analysis allowed the identification and quantification of eight organic acids (oxalic, aconitic, citric, ketoglutaric, ascorbic, malic, shikimic and fumaric acids). The phenolic composition of the lyophilised infusion was also determined by HPLC/DAD and four compounds were quantified, but, despite its high content, only linarin was managed to be identified.     

Antimicrobial and antioxidative activities of bioactive constituents from Hydnophytum formicarum Jack

Hydnophytum formicarum Jack. (Rubiaceae) is a medicinal plant whose tubers possesses cardiovascular, anti-inflammatory and antiparasitic effects and have been used for the treatment of hepatitis, rheumatism and diarrhea. Herein we report the isolation of its active constituents and the testing of their antimicrobial activity against 27 strains of microorganisms using an agar dilution method and of their antioxidative activity using the DPPH and SOD assays. The results show that the crude hexane, dichloromethane, ethylacetate and methanol extracts exert such activities. Particularly, the crude ethyl acetate extract exhibits antigrowth activity against many Gram-positive and Gram-negative bacteria with MIC 256 microg/mL. Shewanella putrefaciens ATCC 8671 is completely inhibited at a lower MIC (128 microg/mL). Interestingly, Corynebacterium diphtheriae NCTC10356 is inhibited by all the tested extracts. Significantly, the ethyl acetate extract is also the most potent antioxidant, showing 83.31% radical scavenging activity with IC50 8.40 microg/mL in the DPPH assay. The other extracts display weak to moderate antioxidative activities, ranging from 28.60-56.80% radical scavenging. The SOD assay shows that methanol extract exhibits the highest activity (74.19% inhibition of superoxide radical). The dichloromethane and ethyl acetate extracts display comparable SOD activity. The promising bioactivities of the crude ethyl acetate extract guided the first isolation of bioactive flavonoid and phenolic compounds: isoliquiritigenin (2), protocatechualdehyde(3), butin (4) and butein (5) from this species. Their structures have been fully established by 1D and 2D NMR. In addition, stigmasterol was isolated from the crude hexane and dichloromethane extracts. The antimicrobial and cytotoxic activities of compounds 3-5 were evaluated. The tested compounds were inactive against HuCCA-1 and KB cell lines,showing ED50> 10 microg/mL. Protocatechualdehyde (3) completely inhibits the growth of Plesiomonas shigelloides with MIC 相似文献   

Separation and evaluation of free radical-scavenging activity of phenol components of Emblica officinalis extract by using an HPTLC-DPPH* method

A new procedure has been developed to separate and quantify the free radical-scavenging activity of individual compounds from an Emblica officinalis extract based on the combination of HPTLC with a diode array detector (DAD) and postchromatographic DPPH* radical derivatization. Free gallic and ellagic acids and emblicanins A and B in the E. officinalis extract were separated by TLC and identified. All the compounds of the extract were capable of scavenging of DPPH* radicals. It was established that the DPPH* scavenging activity of emblicanins A and B was 7.86 and 11.20 times more than that of ascorbic acid and 1.25 and 1.78 times more than gallic acid, respectively. From the estimated ID50 values, it can be seen that the increasing order of activity was emblicanin B > emblicanin A > gallic acid > ellagic acid > ascorbic acid. Probably, the antioxidant activity of E. officinalis extract is associated with the presence of hydrolyzable tannins having ascorbic acid-like action.     

Free-radical scavenging properties and reducing power of grape cane extracts from 11 selected grape cultivars widely grown in China

Total phenolic contents (TPC), total flavonoid contents (TFC), related antioxidative and antiradical capabilities of grape cane extracts from 11 varieties (five V. vinifera cultivars and six Chinese wild grapes) widely grown in China were evaluated. Antioxidant properties were determined as DPPH and ABTS radical-scavenging abilities, superoxide anion and hydroxyl radical and hydrogen peroxide scavenging assays, as well as reducing power. Phenolic profiles of the extracts were characterized by using high-performance liquid chromatography (HPLC) techniques. All extracts exhibited strong antioxidant and effective free radical inhibition activities (EC(50) values), which generally correlated negatively with TPC (r = -0.804 to -0.918) and TFC (r = -0.749 to -0.888). In comparison with gallic acid, Trolox and tert-butylhydroquinone (positive controls), most grape cane extracts showed more efficient scavenging effects toward different reactive oxygen species. HPLC analysis revealed the presence of (+)-catechin, (-)-epicatechin, and trans-resveratrol as major phenolic components in the extracts. These results suggest that grape cane extracts may serve as a potential source of natural antioxidant for food and pharmaceutical application.     

Bioassay‐guided Isolation and Antioxidant Activity of Sulfur‐containing Compounds from Clinacanthus nutans

Clinacanthus nutans has been used in traditional herbal medicine for cancer prevention, but the specific bioactive compounds responsible for the observed activities have not been explored. Different polar solvents such as methanol, chloroform, ethyl acetate, and hexane were used for the extraction. The extracts, fractions, and isolated compounds were subjected to DPPH and ferric reducing antioxidant potential (FRAP) assays. Methanol extracts show significant free‐radical scavenging activity of 69.09% in DPPH and 56.49% FRAP. Purification of MeOH extracts afforded the fraction FB28 and two new sulfur‐containing compounds, named clinamide D and E ( 1 , 2 ). Compound ( 1 ) proved to be more active with an IC₅₀ value for DPPH radical scavenging of 118.27 ± 0.01 µg/mL and reduction of Fe³⁺–TPTZ complex of 386.24 ± 0.02, higher than that of the standard ascorbic acid. Sulfur‐containing compounds isolated from C. nutans is a potential natural antioxidant.     

Screening of antioxidant activity and volatile compounds composition of Chamerion angustifolium (L.) Holub ecotypes grown in Lithuania

Since biological activity of medicinal plants is dependent on cultivation area, climatic conditions, developmental stage, genetic modifications and other factors, it is important to study flora present in different growing sites and geographical zones. This study was focused on screening of antioxidant activity of C. angustifolium harvested in six different locations in Lithuania. The total contents of phenolic compounds, flavonoids and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity were evaluated by spectrophotometric methods. A correlation between radical scavenging activity and total phenolic compounds content was observed (correlation coefficient 0.98). HPLC with online post-column DPPH radical scavenging reaction detection was used for the separation of extracts. Oenothein B, rutin and one unidentified compound were predominant. Volatile compounds were analysed using solid-phase microextraction coupled with gas chromatography–mass spectrometry. Based on the analysis of volatiles, all samples were classified into two chemotypes: (I) with predominant α- and β-caryophyllenes and (II) with predominant anethole.     

On‐Line Radical Scavenging Detection and Characterization of Antioxidants from Artemisia herba‐alba

Four caffeoylquinic acid (CQA) derivatives, 5‐O‐caffeoylquinic acid ( 1 ), 3,5‐di‐O‐caffeoylquinic acid ( 3 ), 4,5‐di‐O‐caffeoylquinic acid ( 4 ), and 3,4,5‐tri‐O‐caffeoylquinic acid ( 5 ), have been isolated from Artemisia herba‐alba growing wild in Algeria, using the on‐line HPLC? DAD? DPPH radical‐scavenging detection technique as guidance. In the course of the purification work, the non‐frequent (E)‐2‐(β‐D ‐glucopyranosyloxy)‐4‐methoxycinnamic acid ( 2 ) has also been isolated. The CQAs showed fair‐to‐good antioxidant activities determined by the DPPH^. scavenging assay. The structures of the five isolated compounds were determined by spectroscopic methods. The on‐line HPLC? DAD? DPPH technique allowed for a rapid pinpointing of antioxidants in the studied plant, accomplishing the facile guided isolation of the target molecules. Algerian A. herba‐alba could be an interesting source of natural antioxidants that deserve further work.