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Methods and results

In order to measure total antioxidant status of the samples we have used Randox TAS kit, commercially available. Shortly, the main principle of the method is the following: ABTS® (2,2'-Azino-di-[3-ethylbenzthiazoline sulphonate]) is incubated with a peroxidase (metmyoglobin) and H2O2 to produce the radical cation ABTS®*+. This has a relatively stable blue-green color, which is measured at 600 nm. Antioxidants in the added sample cause suppression of this color production to a degree which is proportional to their concentration.

In addition, for comparison, we have presented the results for a commercially available green tea extract, vitamin E and multivitamin tablets. The tested concentrations are 100 µg/mL, 200 µg/mL and 300 µg/mL, the equivalent of 1, 2 and 3 500 mg capsule content, respectively.

The results showed that the Energizer HPE 40 in the concentration of 200 µg/mL and 300 µg/mL reaches 100 % of the positive control (Fig. 1): it should be noted that the control is a solution of trolox (a water soluble analogue of vitamin E) and it is defined as 100% of antioxidant capacity. Interestingly, the concentration of trolox usually used in the kit is approximately 180 times higher than the recommended daily dose of vitamin E. Therefore, for comparison, we have inserted also the antioxidant capacity of 180-fold dilution of the control.

Due to the high antioxidant capacity of the Energizer HPE 40, it can be concluded that this method cannot accurately determine the percentage of its trolox activity for the concentrations of 200 µg/mL and 300 µg/mL.

The level of protein carbonylation (oxidative damage) was measured by using the OxyELISA kit from Millipore (S7250). The potential of the samples to inhibit protein carbonylation was tested in two systems:

1. in vitro oxidation of poly-L-arginine, in which all constitutive residues are a target for carbonylation (Fig.2), and
2. protein extract of exponentially growing Escherichia coli (Fig. 3).

Exponentially growing bacteria were harvested from a rich medium (LB for E. coli), centrifuged and re-suspended in the lysis buffer available in the OxyELISA kit supplemented with a mixture of protease inhibitors (Roche). Cells were broken by two freeze-thaw cycles, homogenized in a Dounce homogenizer, and centrifuged 20 min at 12,000 × g. The amount of protein in the supernatant was measured by the Lowry method.

Both, poly-L-arginine and the E.coli protein extracts were diluted to 10 µg/mL and loaded into wells (provided in the kit) and incubated over night at 4 °C to allow proteins to adsorb to the surface, followed by DHR derivatization of adsorbed proteins and detection of derivatized dinitrophenol (DNP)-carbonyl by a mouse DNP-specific monoclonal antibody conjugated to HRP. Subsequent incubation with enzyme substrate 3,3',5,5'-tetramethylbenzidine resulted in a colored product that was quantified using a micro plate reader with maximum absorbance at 450 nm.

Protein samples were oxidized by using hydrogen peroxide in the final concentration of 100 µM during 15 minutes at room temperature. The reaction was stopped by cooling the mixture to 4oC.

It should be noted that we have included also two very important controls in this experiment. One is a control without any sample added, that is a control for maximum oxidation and no protection. In this sample maximum carbonylation is achieved under the condition applied in these experiments. In addition, there is a control for zero oxidation where there is also no hydrogen peroxide added. This control serves as a minimum oxidation control under the condition applied in these experiments.

Several carbonylation tests of alpha-synuclein protein (Parkinson disease), tau protein (Alzheimer disease) and HeLa cells were also performed.

Alpha-synuclein protein (10 µg/mL) was oxidated with 100 µM hydrogen peroxide within 20 minutes at the room temperature, while tau protein and HeLa cells within 1 hour. The results (Fig. 4-6) show high capability of the Energizer HPE 40 (300 µg/mL) for the protection of utilized proteins and cells from the carbonylation, which is in agreement with high equivalent of the trolox activity (Fig. 1).
Only 25% of HeLa cells survived in the case of no protection, while 90% of them survived when they were protected with Energizer HPE 40.

The results are presented in Figures 1 and 2.

It can be concluded that Energizer HPE 40 increases the concentration of reduced glutathione (GSH) in incubated lenses and reduces, or more precisely, prevents the formation of toxic protein carbonyls which are developed under the influence of glucose.

The quantity of extracted glutathione (GSH), which is the most important antioxidant in the human cells, significantly increases up to 6 hours from taking 1 capsule of the Energizer HPE 40 (Fig. 1). This process is less intensive in saliva, which means that GSH binds to or deposit poorly in saliva.

The amount of polyphenols in the urine and saliva also increases over time (Fig. 2) together with the concentration of flavonoids (Fig. 3). The concentration of flavonoids in the urine is several times higher than in the saliva. Presumably, it can be concluded that saliva does not bind flavonoids.

The amount of total antioxidants, presented as Trolox equivalent, is similar in urine and saliva (Fig. 4). According to TAC, Energizer HPE 40 has the strongest antioxidative efficacy 6 hours after taking 1 capsule. From the saliva samples it can be concluded that antioxidants probably accumulate in all organs and in salivary glands. The augmentation of TAC concentration after 3 and 6 hours is a direct result of the antioxidants release from the analyzed preparation.

Lipid degradation, measured as lipid peroxidation, is reduced in the urine and saliva 6 hours after taking analyzed preparation (Fig. 5). Lipid peroxides in the urine are extracted from many organs, and in saliva only from salivary glands. This analysis has shown that Energizer HPE 40 positively influences on the organism in the sense of lipids protection from reactive oxygen species (oxidative stress) which damage their structure, and thus, the function of cell organelles.

The next analysis shows that the total amount of oxidants from urine (reactive oxygen species + other oxidative compounds), which are primary harmful for the normal organism functioning, decreases from the moment of taking 1 capsule of the Energizer HPE 40 (Fig. 6). These results indirectly indicate that Energizer HPE 40 is a strong and efficient antioxidant, which reduces the activity of various harmful oxidizing substances.

Catalase activity has also been analyzed. Catalase is one of the most important enzyme for the protection from reactive oxygen species. It is one of the most potent catalysts known and the reactions it catalyses are crucial to life. Catalase catalyses conversion of Hydrogen Peroxide (H2O2), powerful and potentially harmful oxidizing agent, to water and molecular oxygen. Catalase also uses Hydrogen Peroxide to oxidize toxins including phenols, formic acid, formaldehyde and alcohols. Hydrogen peroxide is a harmful by-product of many normal metabolic processes, and to prevent damage, it must be quickly converted into other, less dangerous substances.
During this 6-hours analysis, continuously increased catalase activity has been observed (Fig. 7). This clearly indicates that the tested preparation has a positive effect on the catalase activity in saliva, and thus, probably in other parts of the organism.

Superoxide radical (O2¯) is a very harmful form of reactive oxygen species released in oxidative stress. Energizer HPE 40 reduces superoxide concentration, thereby showing a direct antioxidant efficacy in saliva (Fig. 8).

Nitric oxide (NO) is a free radical formed from the conversion of L-Arginine amino acid. It is now known to play important functional roles in a variety of physiological systems. Within the vasculature, NO induces vasodilatation, inhibits platelet aggregation, prevents neutrophil/platelet adhesion to endothelial cells, inhibits smooth muscle cell proliferation and migration, regulates programmed cell death (apoptosis) and maintains endothelial cell barrier function. NO generated by neurons acts as a neurotransmitter, whereas NO generated by macrophages in response to invading microbes acts as an antimicrobial agent. In the past 10 years, NO has established itself as a polyvalent molecule which also plays a decisive role in regulating multiple functions within the female as well as the male reproductive system.
It has been shown that the Energizer HPE 40 has practically no influence on the variation of NO concentration in saliva, i.e., it does not affect on the activity of this extremely important radical on the normal organism functioning (Fig. 9). This result is especially important in the context of simultaneous positive influence of the analyzed preparation on the superoxide radical neutralization.