Analytical MethodsRapid antioxidant capacity screening in herbal extracts using a simple flow injection-spectrophotometric system
Highlights
► We propose a rapid antioxidant capacity screening in herbal extracts. ► A simple flow injection (FI)-spectrophotometric method was developed. ► It is based on DPPH assay. ► The technique is simple, rapid, accurate (compared with standard method) and reproducible. ► It is useful in screening of large number of samples and for quality control.
Introduction
There is recent evidence that free radicals induce oxidative damage to biomolecules. This damage has been implicated in aging and in several human pathologies such as cancer, atherosclerosis, rheumatoid arthritis and other diseases (Huang et al., 2005, Kaur and Kapoor, 2001, Tang et al., 2005). Natural products such as fruits and vegetables have aroused considerable interest recently because of their potential beneficial effects on human health. It has been reported that they have antiviral, anti-allergic, antiplatelet, anti-inflammatory, anti-tumor and antioxidant activities (Valko et al., 2007). Companies have been established to extract antioxidant from natural products, pack these extracts and sell to the public, named as herbal extracts. For these companies there is a critical need for a quick and simple analysis method for quality control of finished products and of raw materials being extracted. Labeling legislation in the developed countries is now requiring the label to include the actual concentrations of active ingredients.
Over the past two decades, several methods have been used to determine the antioxidant activity in natural products, such as the thiobarbituric acid reactive substances (TBARS) assay (Aqil, Ahmad, & Mehmood, 2006), trolox equivalent antioxidant capacity (TEAC) assay (Iveković, Milardvić, Roboz, & Grabarić, 2005), total radical-trapping antioxidant parameter (TRAP) assay (Miller, Rigelhof, Marguart, Prakash, & Kanter, 2000), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical assay (Brand-Williams et al., 1995, Megalhães et al., 2009, Megalhães et al., 2006, Molyneux, 2004, Niederländer et al., 2008). TBARS assay is commonly used for the detection of lipid peroxidation and has been used to quantify oxidative damage (lipid peroxidation) in fish liver and gonad samples (Oakes & Kraak, 2003). It has been known to be dependent of the oxidation of a substrate, influenced by temperature, pressure, matrix, etc., and impractical for large numbers of samples (Böhm and Schlesier, 2004, Prakash, 2001). TEAC assay is a widely used in vitro assay for the determination of antioxidant activity of pure compounds (Böhm and Schlesier, 2004, Megalhães et al., 2009) and has been also applied in assaying food samples (Huang et al., 2005, Re et al., 1999). An advantage of the TEAC assay is that it is operationally simple. However, the TEAC values may not be the same for slow reactions, and it may take a long time to reach the endpoint. The evaluation of antioxidant capacity using TEAC can be troublesome or even impossible, but it can be used to provide a ranking order of antioxidants (Arts et al., 2004, Phipps et al., 2007). TRAP assay determines the overall ability of an antioxidant to trap free radicals. (Iveković et al., 2005, Kumaraswamy and Satish, 2008, Miller et al., 2000) The TRAP assay is often used for measurement of in vivo antioxidant capacity in serum or plasma but the assay is relatively complex and time consuming and requires a high degree of expertise and experience (Phipps, et al., 2007).
The DPPH assay is based on the reduction by antioxidants of the purple DPPH radical to the corresponding pale yellow and measured colorimetrically at 520 nm (Molyneux, 2004). Among these methods, DPPH assays have been most widely used. The DPPH assays are relatively simple and stable and the DPPH is available commercially in high purity. The DPPH radical decolourisation method is strongly consistent and the IC50 correlates with total phenolic content in herbal samples (Moraes-de-Souza, Oldoni, Regitano-d’Arce, & Alencar, 2008). Literatures suggested that the DPPH assay was an easy and accurate method with regard to measuring the antioxidant capacity of fruit and vegetable juices or extracts (Huang et al., 2005, Sánchez-Moreno, 2002, Sharma and Bhat, 2009). However, the batch DPPH assay has disadvantages, for example, it is time consuming and tedious, it uses high amounts of reagent, and requires strict adherence to reaction time limits (Prior, Wu, & Schaich, 2005).
A simple analytical tool such as flow injection technique has been well known for reducing reagent and time consumption. Flow injection analysis (FIA) is an approach to chemical analysis that is accomplished by injecting a plug of sample into a flowing carrier stream. As the injected zone moves downstream, the sample solution disperses into the reagent, causing the reaction to occur. A flow through detector placed downstream records the desired physical parameter such as colorimetric absorbance or fluorescence (Tang, et al., 2005). The FIA can provide good reproducibility and rapid analysis of colorimetric methods. FIA methods typical reduce reagent consumption and waste and do not require completely color development which means the analysis can be done faster than batch methods. The DPPH batch method is a colorimetric method that may be adaptable to the FIA technique. Simple routine FIA colorimetric methods have been developed for the ABTS•+ assay but have not been developed for the DPPH assay. However, the flow-based methods based on the DPPH assay that have been developed are FIA with electron spin resonance (ESR) detector, and sequential injection analysis (SIA) and multisyringe flow injection analysis (MSFIA) using colorimetric detectors. These flow-based methods are generally more complicated, and require more expensive equipments.
Therefore, the aim of this work was to develop a simple FI-spectrophotometric system using a single line for the determination of antioxidant capacity based on DPPH assay in some herbal extract samples. The optimization of the FI system was carried out. Ascorbic acid was used as standard. The antioxidant capacity was calculated as ascorbic acid equivalent (AAE). The proposed system was employed to determine the antioxidant capacity in some crude herbal extracts and commercial Thai herb product samples. The comparison between the results obtained by the proposed system and those by the original batch method was performed.
Section snippets
Reagents and solutions
All the chemicals were dissolved in ethanol (⩾99% (Merck, Darmstadt, Germany)). A stock solution of DPPH (1.000 mM) was prepared and kept at 4 °C. It was found to be stable for a week if protected from light in a brown bottle and refrigerated. Working standard ascorbic acid solutions (0.010–0.300 mM) were freshly prepared by the dilution of the ascorbic acid stock solution (1.000 mM). For the batch method, a DPPH solution (0.050 mM) in ethanol was employed.
Samples
Twenty herbal samples (7 fresh-herb extract
Study of dynamic range
The concentration range of 0.005–1.000 mM ascorbic acid was used for this study. The absorbance of DPPH reagent (base line in Fig. 2A) was reduced when the ascorbic acid was injected into the reagent. The higher the ascorbic acid concentration, the lower the absorbance obtained and the higher the peak height. This is obviously because the ascorbic acid antioxidant reduced the colored radicals. Thus, the calibration curve used in this purpose was a plot of peak height against the ascorbic acid
Conclusions
A simple FI-spectrophotometric system for the determination of antioxidant capacity was setup. Ascorbic acid was used as the standard antioxidant. Thus, the antioxidant capacity was calculated as ascorbic acid equivalent (AAE). The linear range was 0.010–0.300 mM (y = 1.7087x + 0.0665, R2 = 0.9992). The proposed FI-spectrophotometric method provided accurate (compared with the standard method) and reproducible results (%RSD(n = 10) < 5%), and was a relatively rapid method and economic system. The
Acknowledgements
The authors acknowledge Mae Fah Luang University and Scientific and Technological Instrument Center (STIC) for laboratory facilities. The Natural Product Chemistry laboratory (Dr. S. Deachathai’s group) at Mae Fah Luang University was grateful for kindly providing the samples.
References (26)
- et al.
Antioxidant capacity of reaction products limits the applicability of the Trolox Equivalent Antioxidant Capacity (TEAC) assay
Food and Chemical Toxicology
(2004) - et al.
Use of a free radical method to evaluate antioxidant activity
Lebensmittel-Wissenschaft und-Technologie
(1995) - et al.
Phenolic compounds from the fruit of Garcinia dulcis
Phytochemistry
(2005) - et al.
A benzil and isoflavone derivatives from Derris scandens Benth
Phytochemistry
(2004) - et al.
Xanthones from Garcinia cowa Roxb. Latex
Phytochemistry
(2005) - et al.
Antioxidant activity assays on-line with liquid chromatography
Journal of Chromatography A
(2008) - et al.
Utility of the TBARS assay in detecting oxidative stress in white sucker (Catostomus commersoni) populations exposed to pulp mill effluent
Aquatic Toxicology
(2003) - et al.
Antioxidant activity applying an improved ABTS radical cation decolorization assay
Free Radical Biology & Medicine
(1999) - et al.
DPPH antioxidant assay revisited
Food Chemistry
(2009) - et al.
Determination of the antioxidant capacity of different food natural products with a new developed flow injection spectrofluorometry detecting hydroxyl radicals
Talanta
(2005)
Free radicals and antioxidants in normal physiological functions and human disease
The International Journal of Biochemistry & Cell Biology
Antioxidant and free radical scavenging properties of twelve traditionally used Indian medicinal plants
Turkish Journal of Biology
Methods to evaluate the antioxidant activity
Production Practices and Quality Assessment of Food Crops
Cited by (21)
Flow-based food analytical methods
2020, Innovative Food AnalysisQuantitative fingerprint and quality control analysis of Compound Liquorice Tablet combined with antioxidant activities and chemometrics methods
2019, PhytomedicineCitation Excerpt :GE and PPCE, as principal herbs in CLT, mainly contains flavonoids, triterpene saponins and alkaloids, which have been suggested to be main antioxidant active constituents and typically evaluated during quality control of CLT (Koolen et al., 2017; Puente-Garza et al., 2017; Zahari et al., 2016; Zhang et al., 2015a, 2015b). To investigate the relationship between CLT antioxidant activity and chemical fingerprinting, rapid total antioxidant activity assay (Arribas et al., 2013; Llorent-Martinez et al., 2011; Mrazek et al., 2012) and partial least squares (PLS) model (Shimamoto and Tubino, 2016) were performed to establish a fingerprint–efficacy relationship. According to the theory of HM, drug compatibility (Pei-Wu in Chinese), referring to the relationships between drugs such as mutual reinforcement, restraint and inhibition (Qiu, 2007; Wang et al., 2012).
Bio-active nanoemulsions enriched with gold nanoparticle, marigold extracts and lipoic acid: In vitro investigations
2014, Colloids and Surfaces B: BiointerfacesCitation Excerpt :Natural preparations from herbal sources have been used to fight against diseases since ancient time [1–7]. There is a great interest for the use of plant extracts or herbal oils due to their beneficial effects on human health [8]. In recent years, there is a considerable growth in natural product industries containing not only food and beverages, but also pharmaceuticals and cosmetics [9].
Isolation and characterization of bioactive compounds from plant resources: The role of analysis in the ethnopharmacological approach
2014, Journal of Pharmaceutical and Biomedical AnalysisCitation Excerpt :The stable and coloured radical reagent can be added post-column to the HPLC eluate by an extra pump system and individual radical scavenging activity can be monitored by a UV-vis detector as a negative peak, due to the conversion of radicals to their uncoloured reduced form. Mrazek et al. [87] determined the antioxidant properties of twenty herbal samples by means of conventional and simple flow injection (FI)-spectrophotometric DPPH antioxidant assays. Both methods gave accurate and reproducible results but FI resulted faster and thus more suitable for antioxidants screening of large number of samples.
Rapid high-throughput assay to assess scavenging capacity index using DPPH
2013, Food ChemistryCitation Excerpt :However, AAI does not reflect the stoichiometry of the reaction and lacks theoretical meaning and, while AAU does reflect a stoichoimetric relationship, is not intuitive and is restricted to pure compounds. DPPH assay has been carried out using many different organic solvents (Brand-Williams et al., 1995; Cheng, Moore, & Yu, 2006; Mrazek, Watla-iad, Deachathai, & Suteerapataranon, 2012; Scherer & Godoy, 2009). Curiously, the reaction can be completed in seconds when semi-aqueous medium is used, at a physiological pH, and the in vivo predictability of the antioxidant properties of the analysed pure compound samples is improved (Bartasiute, Westerink, Verpoorte, & Niederländer, 2007).
Chemical analysis of antioxidant capacity: Mechanisms and techniques
2020, Chemical Analysis of Antioxidant Capacity: Mechanisms and Techniques