2.3. TRAP Assay
Like the DPPH assay, the TRAP assay determines the IC50
of the extracts (Figure 4
). The TRAP assay again showed that most extracts had similar IC50
values, except those from the Maori potato, which had about a sevenfold higher IC50
value in comparison with pansy extract which had the lowest IC50
Four of the eight extracts, namely the 99N1/222 skin, pansies, lisianthus, and blueberry extracts were better antioxidants than the ascorbic acid standard, while the 99N1/222 flesh, red cabbage and red lettuce showed slightly less antioxidant activity than the standard.
This same assay was used to compare antioxidant activities of extracts and AVIs from the throat of the deep purple lisianthus, and from a cream lisianthus, after either 15 minutes or overnight (24 h) incubation at room temperature (Figure 4
After 15 minutes, the TRAP activity of the free anthocyanic solution was higher as compared with extracted AVIs, and no activity was seen in the extract from the non-pigmented lisianthus. Radical scavenging by the free anthocyanins was saturated after 15 minutes, with no further increase after 24 hours. In contrast, intact AVIs continued to scavenge the ABTS radical, showing markedly superior total antioxidant activity compared to free anthocyanins after 24 hours. Unfortunately, seasonal differences in the varieties of lisianthus available have made it impossible to extract sufficient quantities of AVIs to repeat this observation.
The three chemical assays evaluated measure primary antioxidant activity of the anthocyanic extracts, while the Comet assay can also measure secondary effects. However, the chemical assays are quick and simple methods of measuring potential antioxidant activity, whereas utilising oxidation of cellular components can be time consuming to perform and not practical where large numbers of samples are involved
The common feature of the three chemical assays is their direct measurement of free radical scavenging efficiency of the extracts. The DPPH assay is the quickest and easiest assay to perform, but it diverges from biological conditions the most, using an artificial DPPH radical and methanol as the solvent [18
]. This method is only able to measure direct reactions with the DPPH radical, which is dependent on the structure of an antioxidant compound and can only give a general indication of the radical scavenging abilities of antioxidants. However, it is a rapid and convenient method for screening many samples as well as not requiring expensive reagents or sophisticated equipment [22
]. The TRAP (Total Reactive Antioxidant Potential) assay is also relatively quick and easy to carry out, with the advantage over the DPPH assay of being in aqueous conditions. However, the TRAP assay still utilizes a non-biological ABTS cation radical [24
]. The TRAP and ORAC (Oxygen Radical Absorbance Capacity) are similar assays because they make use of the hydrogen atom transfer (HAT) reaction between an oxidant and a free radical. Both assays use AAPH [2,2′-azobis(2-amidino-propane) dihydrochloride] as a peroxyl radical generator, which is a commonly found free radical in the body [25
]. However, in the TRAP assay, the peroxyl radical does not directly interact with the antioxidant extract. In a pre-incubation step, before the addition of the antioxidant species, AAPH-generated peroxyl radicals oxidise ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate)] to generate the ABTS radical. The ability of an antioxidant to scavenge the pre-formed ABTS radical and the subsequent loss in absorbance at 734 nm, which is proportional to the antioxidant capacity of the antioxidant being tested[24
], forms the basis of the TRAP assay. As with the DPPH assay, the TRAP assay is a quick and easy method, convenient when high sample numbers are being tested, but uses a non-biological radical for measuring antioxidant activity.
The ORAC assay measures the degree and length of time the extracts take to inhibit the action of an oxidizing agent. It therefore takes into account the kinetics of the reaction, unlike the other two assays, as well as being performed at a physiological pH and producing a biologically relevant radical, the peroxyl radical [26
]. Since anthocyanin stability and therefore its antioxidant activity is sensitive to changes in temperature and pH, inappropriate conditions can greatly influence the result. The assay utilizes the fluorescent protein R-PE (R-phycoerythrin) as a detector of antioxidant activity. The peroxyl radicals generated by AAPH can either react with the antioxidant extract by removing a hydrogen atom from it or by damaging R-PE, resulting in a loss of fluorescence. The efficiency of the extract to inhibit the decline of R-PE fluorescence is measured [26
]. In contrast to the DPPH and TRAP assays, the ORAC assay measures the antioxidant activity of the extracts against the biologically relevant peroxyl radical, as well as taking into account the kinetics of the chain-breaking reactions [27
]. However, the ORAC assay does not measure the total antioxidant activity because other biologically relevant ROS exist, such as superoxide, the hydroxyl radical and singlet oxygen. Because different ROS have different reaction mechanisms, to completely determine antioxidant activity against a wide range of ROS, a more comprehensive set of assays need to be carried out [6
Far more biologically relevant is the Comet assay, which visualizes DNA damage in single cells arising from the exposure to various combinations of antioxidants and ROS [28
].. Cells are embedded in agarose coated slides and subjected to electrolysis, which causes the negatively charged DNA to migrate towards the anode. Damaged and fragmented DNA is able to migrate through the agarose faster, and can be visualized using ethidium bromide which intercalates within the DNA. An alkaline Comet assay was performed as opposed to the neutral Comet assay because this assay is able to detect single strand damage, due to denaturation of DNA, and therefore is more representative of actual DNA damage. Tail intensity represents the amount of DNA and, therefore, the degree of DNA damage, while the tail length gives an indication of fragment size since smaller fragments migrate faster and farther through the agarose [21
]. Combined, these parameters give the tail extent moment which takes into account both the extent of DNA damage and fragment size [30
The initial set of COMET assay results measure constitutive DNA damage in cells without exposure to an exogenous source of free radicals. They demonstrated that most of the extracts could protect the DNA from damage by endogenous free radicals generated by the cells during their 3 day incubation period. 99N1/222 skin offered the best protection of all of the extracts tested, while the red lettuce extract did not offer any significant protection relative to the controls. The remaining extracts, while showing significant improvement from the control, were in the middle ground in terms of their antioxidant capacities when comparing between extracts.
Of the extracts that were able to offer some protection against hydrogen peroxide challenge, none were able to completely protect, indicated by the fact that there was still significantly more damage than in cells exposed to the respective extracts but not hydrogen peroxide. A possible explanation for this is that the Comet assay is performed in PBS, giving the extracts the opportunity to diffuse out of the cells, thus reducing intracellular protective effects. One interesting observation is that the Maori potato extracts, which were consistently the least protective antioxidant in the chemical assays, appear to be the most protective against ROS induced DNA damage at 0 ºC (table 4
). Perhaps a compound exists in the Maori potato which can not scavenge free radicals directly, but triggers the production of other compound within the cells which can. This tells us that the Comet assay is perhaps more reliable than the chemical assays because it is more biologically relevant, using living cells. Therefore, in addition to primary antioxidant activity such as ROS scavenging, the Comet assay is able to detect secondary or indirect antioxidant actions.
Also interesting is the observation that the second best antioxidant in the untreated group, red cabbage extract, now appears to be pro-oxidant. This indicates that, while red cabbage extract does not induce DNA damage directly, it contains other compounds which can enhance the deleterious effects of exogenous ROS in a non-enzymatic manner. Metal ions, for example, are able to synergistically increase the effects of ROS [31
Whereas performing the hydrogen peroxide challenge at 0 ºC showed direct scavenging of exogenous ROS by the extracts, performing the hydrogen peroxide challenge at 37 ºC also allowed enzymatic effects to be taken into account. Surprisingly the average tail extent moment seen in the 37 ºC hydrogen peroxide challenged controls was reduced back to around the same value as the non-hydrogen peroxide challenged controls. This may suggest that protective antioxidant enzymes such as catalase and glutathione peroxidase, which convert hydrogen peroxide into water and oxygen, as well as DNA repair enzymes, have sufficient protective capabilities to reduce ROS induced damage to background damage[32
]. To some degree this was expected in that the treatment at 0 °C was aimed at inactivating these enzymes, to be able to distinguish the antioxidant capacities of the extracts alone, without any contribution from antioxidant or repair enzymes, which is why increased damage was observed in the control at 0 °C. However, the treatment at 37 °C is perhaps more biologically representative of what is really happening in the human body. It was clear from the results that, just because an extract could protect from endogenous free radical damage, it does not mean that the same extract will protect against exogenous ROS.
Previous structure-activity relationships have suggested that antioxidant capacity varies considerably according to the pattern of substitution of the anthocyanin molecule, the presence of acyl groups, and the nature and positions of glycosyl groups [33
]. However, new analytical methods make it clear that many of the earlier studies were not identifying all possible anthocyanins. For example, Arapitsas and Turner [34
] analysed and tentatively identified anthocyanin species in red cabbage using HPLC/DAD-ESI/Qtrap MS. They used a pressurized liquid technique for extraction, used photodiode array detection to determine the UV/Vis spectral characteristic of the pigments. Electrospray ionization-linear ion trap mass spectrometry allowed the specific determination of the fragmentation patterns of the anthocyanins. They identified twenty four distinct anthocyanins (nine of them newly identified), all having cyanidin as aglycon, but presenting as mono- and/or di-glycoside, and acylated, or not, with aromatic and aliphatic acids.
In our studies, the TRAP assay revealed that AVIs and equimolar free anthocyanins have very similar antioxidant capacities in the short term, with the antioxidant potential of the AVIs only slightly less than that of the free anthocyanins. However, observation of the plate used in the TRAP assay after 24 hours showed that the AVIs, but not the free anthocyanins, had continued to scavenge the ABTS radical. These results show that although AVIs are slower in scavenging free radicals, they have greater total antioxidant activity based on an equimolar anthocyanin concentration. This may be due to the highly organised structure of the AVIs allowing free radicals to be delocalised across the many aromatic rings of the packed anthocyanins.
Although we have not done in vivo studies here, other researchers have reported that consumption of anthocyanins or anthocyanin-rich diets leads to increased serum antioxidant potential in both experimental animals and human subjects [35
]. Additionally, Ramirez-Tortosa [37
] reported that an anthocyanin-rich extract decreased hepatic lipid peroxidation in oxidatively-stressed rats. The present results with AVIs suggest that these structures may be of particular interest in such a model. The association of anthocyanins with plant cell walls, as in the kumara skin [38
], may also change in vivo properties and add another dimension to the complexity.