Elsevier

Food Chemistry

Volume 132, Issue 1, 1 May 2012, Pages 544-548
Food Chemistry

Analytical Methods
Rapid antioxidant capacity screening in herbal extracts using a simple flow injection-spectrophotometric system

https://doi.org/10.1016/j.foodchem.2011.10.066Get rights and content

Abstract

A simple flow injection (FI)-spectrophotometric system for the screening of antioxidant capacity in herbal extracts was developed. The analysis was based on the color disappearance due to the scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical by antioxidant compounds. DPPHradical dot and ascorbic acid were used as reagent and antioxidant standard, respectively. Effects of the DPPHradical dot concentration, DPPHradical dot flow rate, and reaction coil length on sensitivity were studied. The optimized condition provided the linear range of 0.010–0.300 mM ascorbic acid with less than 5%RSD(n = 10). Detection limit and quantitation limit were 0.004 and 0.013 mM, respectively. Comparison of antioxidant capacity in some herbal extracts determined by the FI system and a standard method was carried out and no significant difference was obtained.

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 DPPHradical dot 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, DPPHradical dot assays have been most widely used. The DPPHradical dot 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 DPPHradical dot 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 DPPHradical dot assay. However, the flow-based methods based on the DPPHradical dot 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 DPPHradical dot 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 DPPHradical dot (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 DPPHradical dot 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 DPPHradical dot 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.

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