Rapid identification and evaluation of antioxidant compounds from extracts of Petasites japonicus by hyphenated-HPLC techniques

Gradient HPLC coupled to Diode Array Detector (DAD), MS/MS and NMR was applied to the rapid structure determination of major compounds of methanol extracts from leaves and roots of Petasites japonicus. The relative antioxidant capacities of the compounds were evaluated by an HPLC system with post-column on-line antioxidant detection based on 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical scavenging. Six compounds were successfully separated on a reverse-phase C(18) column and were identified as 5-caffeoylquinic acid (5-CQA), fukinolic acid (FA), 3,5-di-O-caffeoylquinic acid (3,5-DCQA), quercetin-3-O-(6″-acetyl)-β-glucopyranoside (QAG), 4,5-di-O-caffeoylquinic acid (4,5-DCQA) and kaempferol-3-O-(6″-acetyl)-β-glucopyranoside (KAG) by MS/MS and (1)H NMR data. Among these compounds, those containing a caffeoyl moiety (5-CQA, FA, 3,5- and 4,5-DCQA) showed relatively strong radical scavenging capacity, with 3,5-DCQA having the greatest radical scavenging capacity in leaf (23.09% of total antioxidant capacity) and root (26.47%) extracts. The relative radical scavenging portion of QAG was only 3.41% in the leaves and KAG did not show any radical scavenging activity. These results demonstrate that the hyphenated HPLC techniques can be successfully applied to rapidly identify structures and evaluate antioxidant activities without prior purification of compounds from plant tissues of P. japonicus.     

On-line high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry-chemiluminescence assay of radical scavengers in Epimedium

An on-line analysis method for the simultaneous detection and identification of radical scavenging compounds in plant extracts was developed by combining HPLC with hydrogen dioxide radical scavenging and HPLC-DAD-MS-CL system. The structural identification and activity characteristics of various constituents could be rapidly achieved by the on-line assay of UV, MS and CL in one run. In 4 species of Epimedium studied 32 compounds, including phenolic acids, 8-isopentenyl-flavonoid glycosides and flavonoid glycosides containing a ortho-hydroxyl group, were identified by comparison with authentic standards and published mass data. Among these compounds, phenolic acids and flavonoid glycosides containing an ortho-hydroxyl group could obviously inhibit CL, which suggested their strong radical scavenging activity. These four species each exhibited different active properties, which might correlate to their respective quality. The results indicated that the on-line HPLC-DAD-MS-CL system would be a potential method to rapidly and sensitively screen radical scavengers in herbal medicines, and could display an integrated fingerprint based on different detectors.     

Comparative analysis of radical scavenging and antioxidant activity of phenolic compounds present in everyday use spice plants by means of spectrophotometric and chromatographic methods

Comparative analysis of radical scavenging and antioxidant activities of phenolic compounds present in everyday use spice plants was carried out by means of spectrophotometric and chromatographic methods. Six spice plant samples, namely onion (Allium cepa), parsley (Petroselinum crispum) roots and leaves, celery (Apium graveolens) roots and leaves and leaves of dill (Anethum graveolens) were analyzed. Total amount of phenolic compounds and radical scavenging activity (RSA) was the highest in celery leaves and dill extracts and was the lowest in celery roots. Comparing commonly used spectrophotometric analysis of 2,2-diphenyl-1-picrylhydrazyl (DPPH) RSA of extracts with the results obtained using reversed-phase chromatographic separation with on-line post-column radical scavenging reaction detection, good correlation was obtained (R(2)=0.848). Studies using HPLC system with electrochemical detector showed that bioactive phytochemicals can be separated and antioxidant activities of individual compounds evaluated without the need of a complex HPLC system with reaction detector. The results obtained using electrochemical detection correlate with the RSA assayed using spectrophotometric method (R(2)=0.893).     

Screening of xanthine oxidase inhibitors in complex mixtures using online HPLC coupled with postcolumn fluorescence‐based biochemical detection

Xanthine oxidase (XO) catalyzes the metabolism of hypoxanthine and xanthine to uric acid, the overproduction and/or underexcretion of which could cause the incidence of hyperuricemia such as gout. Herein, the inhibition of XO is recognized as one of the therapeutic approaches to treat gout. In the present study, an off‐line fluorescence‐based microplate method was first developed for an XO assay in which the enzyme converted pterin to its fluorescent metabolite isoxanthopterin. Then, a postcolumn continuous XO assay as a means of bioactivity assessment was coupled to HPLC separation to establish the online HPLC with diode array detection, biochemical detection, and MS/MS system for the screening of XO inhibitors. The availability of the online system was first tested with a positive drug, allopurinol, a well‐known XO inhibitor, and subsequent analysis of Scutellaria baicalensis extract showed that two main bioactive compounds with XO inhibitory activities were observed, indicating that the developed online system was applicable to complex mixtures.     

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.     

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.     

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.     

Separation and quantification of N-acetyl-l-cysteine and N-acetyl-cysteine-amide by HPLC with fluorescence detection

N-acetyl-l-cysteine (NAC) is a well-known antioxidant that is capable of facilitating glutathione (GSH) biosynthesis and replenishing intracellular GSH under oxidatively challenging circumstances. N-acetyl-cysteine-amide (NACA), the amide form of NAC, is a newly designed and synthesized thiol-containing compound which is believed to be more lipophilic and permeable through cell membranes than NAC. The metabolic and antioxidant effects of these compounds in vitro and in vivo are under investigation. However, an analytical method that can separate and quantify both compounds simultaneously is not yet available, to the best of our knowledge. Because of their structural similarities, the two compounds are difficult to separate using earlier HPLC methods which were designed for NAC quantification. Therefore, the goal of this work was to develop an HPLC method with fluorescence detection for simultaneous quantification of NAC and NACA in biological blood and tissue samples. A gradient HPLC program with fluorescence detection (lambda(ex) = 330 nm, lambda(em) = 376 nm) using N-(1-pyrenyl)maleimide (NPM) as the derivatizing agent was developed. The calibration curves were linear over a concentration range of 25-5000 nm (r(2) > 0.997). The coefficients of variation for within-run precision and between-run precision ranged from 0.67 to 5.23% and for accuracy ranged from 0.98 to 10.54%; the percentage relative recovery ranged from 94.5 to 102.8%. This new method provides satisfactory separation of NAC and NACA, along with other biological thiols, in 20 min with a 5 nm limit of detection (LOD) per 5 microL injection volume.     

Simultaneous determination of 19 constituents in Cimicifugae Rhizoma by HPLC–DAD and screening for antioxidants through DPPH free radical scavenging assay

Cimicifugae Rhizoma (sheng ma) is a well‐known traditional Chinese medicine, which has been demonstrated to possess anti‐inflammatory, antipyretic and analgesic activities. In the present study, a simple and efficient HPLC–DAD (high‐performance liquid chromatography coupled with diode array detection) method was developed and validated for simultaneous quantification of 19 chemical components (including 16 phenolic acids, one coumarin and two alkaloids) in Cimicifugae Rhizoma. The result indicated that this method could effectively evaluate the quality of Cimicifugae Rhizoma and provide a valuable reference for further study. Additionally, the antioxidant activity of Cimicifugae Rhizoma was evaluated by DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) radical scavenging assay. The results showed that the content of phenolic acids and antioxidant activity exhibited significant correlation coefficients.     

Optimization and validation of post-column assay for screening of radical scavengers in herbal raw materials and herbal preparations

On-line method, which combines HPLC distribution and post-column reaction, was designed for the search of individual antioxidants. Optimization of the assay was performed evaluating optimal ABTS(+) radical cation concentration in the reactor, reaction time, impact of flow rate, reaction coil length. HPLC-ABTS assay validation in this work was performed by assessing reference antioxidant negative peak areas in radical scavenging chromatogram. Sample free radical scavenging activity is expressed as trolox equivalent antioxidant capacity (TEAC). Optimized and validated method was applied in detection of compounds possessing free radical scavenging ability in complex mixtures. Antioxidant compounds were studied in perilla (Perilla frutescens (L.) Britton var. crispa f. viridis) herbal raw material and its preparations. The HPLC-separated antioxidant compounds were identified using HPLC-photodiode array coupled to mass spectrometer, using a reference mass for determining accurate masses. Radical scavenging characteristics of rosmarinic acid, which is the dominant phenolic compound in medicinal herbal raw material of perilla and its preparations, were confirmed by the calculated TEAC values. Compounds responsible for antioxidant effect in herbal raw materials and herbal preparations were identified, evaluated and compared.     

Comparison of pressurised fluid and ultrasonic extraction methods for analysis of plant antioxidants and their antioxidant capacity

The analytical method based on the high-performance liquid chromatography coupled with UV detection (HPLC/UV) for determination of selected antioxidants (i.e., esculetin, scopoletin, 7-hydroxycoumarine, rutin, xanthotoxin, 5-methoxypsoralen and quercetin) in plant material was developed. Pressurised fluid extraction (PFE) and ultrasonic extraction (USE) methods for the isolation of these compounds from ten real plant samples were used. Both extraction methods were optimised and compared to each other. For the proposed HPLC/UV method the LOQ values (limit of quantification) in the range from 22.7 (xanthotoxin) to 97.2 ng mL⁻¹ (rutin) were obtained. For all extracts the antioxidant capacity based on the reduction of free 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) was also determined. Results ranged from 82.04 to 94.43% of DPPH radical inhibition for PFE method and from 76.01 to 89.94% in the case of USE method.     

High-performance liquid chromatographic separation and quantification of citric, lactic, malic, oxalic and tartaric acids using a post-column photochemical reaction and chemiluminescence detection

An HPLC method was developed for the determination of citric, lactic, malic, oxalic and tartaric acids by chemiluminescent detection following online irradiation with visible light. The organic acids were irradiated with visible light in the presence of Fe3+ and UO2(2+) to generate Fe2+, which was determined by measuring the chemiluminescence intensity in a luminol system in the absence of added oxidant. Factors affecting the photochemical and chemiluminescence reactions were optimised so that their contribution to the total band-broadening was negligible. The chromatographic separation was performed on a C18 column under isocratic reversed-phase conditions using 0.005 M H2SO4 mobile phase. The optimised method was validated with respect to linearity, precision, limits of detection and quantification, accuracy specificity and robustness. The applicability of the assay was demonstrated by analysing these compounds in real samples such as milk, fruit juices, soft drinks, wine and beer.     

Evaluation of the free radical scavenging capability of wheat extracts by capillary electrophoresis and multivariate curve resolution

In the current work, phenolic acids were analysed and their radical scavenging activity evaluated by CE, including the quantification of overlapping peaks by applying the multivariate curve resolution (MCR) method. To investigate radical scavenging activity, the solution of phenolic acids was treated with a stable free 2,2-diphenyl-1-picrylhydrazyl radical of different concentrations and the radical scavenging activity of the reaction mixture was monitored. The radical scavenging reaction was also applied to wheat bran extracts. The overlapping peaks on the electropherograms of both mixtures of standard compounds and extracts of different wheat bran varieties were resolved by using the MCR method, assuming that the overlapping compounds have different UV spectra. MCR enabled the insufficient resolution between syringic and ferulic acids to be effectively resolved.     

Method development and validation for the analysis of a new anti-cancer infusion solution via HPLC

A fast and simple HPLC method has been developed and validated for the quantification of a completely new anti-cancer drug during the manufacturing process. The combination of four compounds including α-ketoglutaric acid, hydroxymethylfurfural, N-acetyl-L-methionine and N-acetyl-L-selenomethionine, administered intravenously, is still in test phase but has already shown promising results in cancer therapy. HPLC separation was achieved on an RP-18 column with a gradient system. However, the highly different concentrations of the compounds required a variation in the detection wavelength within one run. In order to produce a chromatogram where peaks were comparable on a similar range scale, detection at absorption maxima for the two most concentrated components was avoided. After optimization of the gradient program it was possible to detect all four substances within 14 min in spite of their strongly different chemical structure. The method developed was validated for accuracy, repeatability, reproducibility and robustness in relation to temperature and pH of buffer. Linearity as well as the limit of detection and quantification were determined. This HPLC method was found to be precise, accurate and reproducible and can be easily used for in-line process control during the manufacture of the anti-tumour infusion solution.     

Profiling of different bioactive compounds in functional drinks by high-performance liquid chromatography

In the present work, an HPLC method is proposed to simultaneously detect and quantify water- and fat-soluble vitamins, phenolic compounds, carotenoids and chlorophylls in a single run, by using an ultradeactivated C18 column and gradient separation using trifluoroacetic acid, water and methanol. It is shown that the HPLC method provides baseline separation of all these compounds with good resolution values in 40 min. Moreover, other figures of merit of the method show a good linear response and low detection limits for all the compounds considered in the present study. Furthermore, the usefulness of this method is demonstrated via its successful application to the analysis of different beverages from different natural origin (orange, strawberry, apple, peach pineapple, plum and blackcurrant juices, soybean milk, beers) without the need of any previous sample preparation. A good correlation is also found by comparing the total phenol content (measured by Folin-Ciocalteu method) with the sum of total phenolic compounds obtained using the proposed HPLC method. By using statistical tools, the main compounds associated with antioxidant activity of the extracts (measured by 1,1-diphenyl-2-picrylhydrazyl radical scavenging) were assessed.     

几种天然黄酮类化合物对二苯代苦味肼自由基的清除作用

采用循环伏安法、示差脉冲伏安法对四种天然黄酮类化合物清除二苯代苦味肼自由基（DPPH）的能力进行了探讨.研究表明：此法可直接观测样品对DPPH的清除作用,而且灵敏度高、准确可靠,是一种检测自由基及物质对其清除作用的有效方法.     

Functionalized graphene quantum dots loaded with free radicals combined with liquid chromatography and tandem mass spectrometry to screen radical scavenging natural antioxidants from Licorice and Scutellariae

A novel screening method was developed for the detection and identification of radical scavenging natural antioxidants based on a free radical reaction combined with liquid chromatography with tandem mass spectrometry. Functionalized graphene quantum dots were prepared for loading free radicals in the complex screening system. The detection was performed with and without a preliminary exposure of the samples to specific free radicals on the functionalized graphene quantum dots, which can facilitate charge transfer between free radicals and antioxidants. The difference in chromatographic peak areas was used to identify potential antioxidants. This is a novel approach to simultaneously evaluate the antioxidant power of a component versus a free radical, and to identify it in a vegetal matrix. The structures of the antioxidants in the samples were identified using tandem mass spectrometry and comparison with standards. Fourteen compounds were found to possess potential antioxidant activity, and their free radical scavenging capacities were investigated. The order of scavenging capacity of 14 compounds was compared according to their free radical scavenging rate. 4′,5,6,7‐Tetrahydroxyflavone (radical scavenging rate: 0.05253 mL mg^?1 s^?1) showed the strongest capability for scavenging free radicals.     

On-Line HPLC with Biochemical Detection for Screening Bioactive Compounds in Complex Matrixes

On-line high-performance liquid chromatography (HPLC) coupled with biochemical detection (BCD) has been developed to screen compounds showing antioxidant action, enzyme inhibition and receptor affinity in complex matrixes. This review summarizes HPLC methods combining different post-column detection methods, such as diode-array detection (DAD), mass spectrometry (MS), chemiluminescence (CL) and nuclear magnetic resonance, for antioxidant screening. The methods based on a single relatively stable reagent such as DPPH^• and ABTS^•+ were the most popular. Oxygen free radical scavengers mainly depended on post-column CL detection. The on-line hyphenated HPLC–BCD systems based on post-column UV/DAD fluorescence and MS detection were also widely applied to screen enzyme- and receptor-active compounds. These strategies provide a convenient tool for quick identification and quantification of active compounds in complex matrixes.

     

Simultaneous determination of four 5-hydroxy polymethoxyflavones by reversed-phase high performance liquid chromatography with electrochemical detection

Accumulating evidence has suggested the potential health-promoting effects of 5-hydroxy polymethoxyflavones (5-OH-PMFs) naturally existing in citrus genus. However, research efforts are hampered by the lack of reliable and sensitive methods for their determination in plant materials and biological samples. Using reversed-phase high performance liquid chromatography (HPLC) equipped with electrochemical (EC) detection, we have developed a fast and highly sensitive method for quantification of four 5-OH-PMFs, namely 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone, 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone, 5-hydroxy-6,7,4′-trimethoxyflavone, and 5-hydroxy-6,7,8,4′-tetramethoxyflavone. The method was fully validated in terms of linearity, accuracy and precision. The limit of detection (LOD) was determined as being between 0.65 and 1.8 ng/mL (ppb), demonstrating an over 160 times higher sensitivity in comparison with the previously reported method using UV detection. The recovery rate of the method was between 96.17% and 110.82%, and the precision for the retention times and peak areas was all below 13%. The method was successfully used to quantify 5-OH-PMFs with a wide range of abundance in the citrus products and preparations, such as orange juice, citrus peel, and dried tangerine peel. The quantification method for 5-OH-PMFs developed herein could be useful for the nutritional and pharmacological studies of these compounds in future.     

Evaluation and comparison of two improved techniques for the on-line detection of antioxidants in liquid chromatography eluates

Two methods for the on-line detection in HPLC eluates of analytes possessing radical scavenging activity were improved and compared. The instrumental set-up of the method that is based on on-line inhibition of luminol chemiluminescence (CL) by antioxidants was improved using better quality syringe pumps, employing a diode array detector, and introducing a mixing/neutralisation coil and a pulse damper. Sensitivity of the HPLC-CL detection increased by a factor of 4. Post-column neutralisation of eluates improved compatibility of this detection method with acidified HPLC eluents. The second method, which is based on the post-column quenching of 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH*), was improved by readjusting composition and flow-rate of the reagent, mounting an additional pulse damper and detecting unreacted DPPH* with a detector equipped with a tungsten lamp. Purging of the DPPH* solution with He gas prior to analysis was introduced. This led to 30-fold better detection limits. The improved methods were compared with respect to limits of detection, the radical scavenging mechanism involved, compatibility with common HPLC solvents and pH range, and some technical aspects. The techniques described have high potential for the rapid identification of radical scavengers in complex samples like plant extracts.