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2.3. CHARACTERISATION OF ANTIOXIDANT STATUS
The characterisation of antioxidants is based on different methods with different objectives. Most approaches detect the ability of antioxidants to scavenge free radicals, e.g. the TEAC (Trolox equivalent antioxidant activity) assay (Rice-Evans et al., 1996), ORAC (oxygen radical absorbance capacity) assay (Cao et al., 1997) or the DPPH radical (1,I-diphenyl-2-picryl-hydrazyl) assay (Sanchez-Moreno et al., 1998). Another assay which was originally established for detection of the ferric reducing ability of plasma (FRAP) (Benzie et al., 1996), is now also used to measure the reducing ability of food samples. (Frankel and Meyer, 2(00)
All of these assays deliver general information about specific radical scavenging ability of the sample but they all suffer from providing information on the biological target which should be protected. Contrasting results from different methods using the same molecules show that the activity is strongly dependent on the test system and on the substrate. 'There cannot be a short-cut approach to determining the activity of antioxidants'. (Frankel and Meyer, 2(00) Furthermore it is recommended to determine the antioxidant activity under various conditions of oxidation, to use several methods to detect different products of oxidation and the systems should be related to real systems (e.g. food and biological reactions).
It is presumed that the amounts of antioxidants are correlated with their activities.
Hence the philosophy is often that increasing the amounts of antioxidants in food should provide improved protection against "free radical attack" in cells. There are different analytical methods to determine the amount of certain antioxidants in plants and food.
The application of three analytical methods on different plant tissues for quantitation of antioxidants was one aim of this work. A photometric assay was used to determine the amount of anthocyanins, a fluorometric approach was chosen to detect ascorbic acid and an HPLC method was taken to separate carotenoids and detect their amount in certain plant tissues. These analytical methods are discussed below. (Kellner et al., 2004; Otto, 2000; Skoog and Leary, 1992)
selectivity, good reproducibility and accuracy (relative uncertainties of I to 3 %) and ease of operation. The limit of detection is dependent on the extinction coefficient. In our case, cyanidin-3-glucoside - the standard for anthocyanins - has an extinction coefficient of 28033 L mor1cm-1 (Wrolstad, 1982) which leads to a minimum detection of 3.5-10-7 M at an extinction of 0.01 according to the Lambert-Beer Law. Applications for qualitative analysis are limited because the number of absorption maxima and minima is relatively small. The information obtained from UVNIS spectroscopy can support identification of the molecular structure with the help of wavelength assignment tables, although there are now more informative techniques available, e.g. NMR spectroscopy. Nevertheless, UVNIS spectroscopy is a cheap and useful tool in combination with HPLC.
Fluorescence techniques are used for determination of inorganic and organic substances.
The concentration-intensity dependency of fluorescence spectra is more complicated than with UVNIS spectra. Fluorescence yield has also to be considered. In contrast to absorption techniques, fluorescence is directly proportional to the intensity of the excitation energy and the dynamic concentration range can cover three decades (10-7
-10-4 M). Fluorescence techniques are extremely sensitive (ppb range). However, the number of fluorescent substances is limited and the precision and accuracy is usually poorer than those of spectrophotometric methods.
There are some points which have to be considered with the fluorometric detection of ascorbic acid. The reaction of dehydroascorbic acid with o-phenylenediamine is time dependent and detection is made after 35 minutes reaction time. Hence there will always be a small range of fluorescence deviation due to the time-consuming handling of more than one sample per run. The sensitivity of the measurement can be improved by increasing the PMT (photomultiplier tube) voltage, but this also leads to a higher noise level. Therefore a compromise has to be found to reach the ideal signal-to-noise ratio. A calibration curve at a fixed PMT voltage covers a certain concentration range.
The sample points should be in that range of linearity. In our case the standard addition curve was carried out from 5 to 40 J.lglml ascorbic acid plus 2 g pepper (see Chapter 3.1.2.) using a PMT setting of 700.
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HPLC is a technique that is used for both qualitative and quantitative analysis. Ithas a good sensitivity, is adaptable to accurate quantitative determinations, is able to separate non-volatile species or thermally labile ones, and has a widespread applicability to substances of interest such as amino acids, proteins, nucleic acids. The reproducibility of retention times is less accurate than the wavelength precision in spectroscopy, but using standards for comparison provides useful information about the absence or presence of a substance. For quantitative analysis the peak height or area is taken. Both have disadvantages,e.g. the peak height is problematic in the case of alterations in the peak form, determination of the peak area is critical in case of peak broadening or very narrow peaks because of locating the exact positions for the beginning and the end of the peak. The separation and the quantitative yield can be maximised by choosing the right column temperature, flow rate, volume of injection, columns, and mobile phase.
An analysis by HPLC is much more time consuming than detection by a fluorometer or photometer. However, HPLC analysis allows the analysis of several compounds in one run in contrast to fluorescence or absorption spectroscopy. In the case of carotenoids the flow rate was chosen to be 1 ml/min. For applications to carotenoids, one has to bear in mind that the technique has some limitations. The columns used are PEEK with an upper pressure limit of 300 bar. Therefore the flow rate is limited. Any loss of sample during the preparation is compensated by using an internal standard in the extract. An external standard which is run before each set of samples is used to compensate any daily variations of the instrument. The peaks do not show a typical Gaussian form, which makes evaluation of peak area difficult, especially when there are overlapping peaks. Sometimes an error from manual integration, e.g. in the case of overlapping peaks, cannot be avoided, but it can be minimized if integration is always carried out in the same manner by the same person. The detector is a DAD (diode array detector). The carotenoids are measured at a fixed wavelength of 450 nm, which is not the absorption maximum for all detected molecules. Hence a small error has to be expected due to absorbance measurements beside the peak maximum.
The most time consuming part which introduces the largest variability, is the preparation of the sample. Frozen material is normally used with these methods and the
right time to read the weight. From my experience the biggest deviations of results come from the inhomogeneity of the sample powder and weighing errors.