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Antioxidant Methods in Food Integration: Preliminary Results of a Scientific Study

Dr. Alberto Fiorito, Surgeon, La Spezia ; Dr. Sauro Lorenzini, University of Siena, Department of Clinical Medicine and Immunological Science, Section of Rheumatology: Director Prof. Roberto Marcolongo.

1. Introduction

Since the beginning of 1970s we have understood how our organism is submitted to the action of both endogen and hexogen free radicals. In physiologic conditions some of these free radicals perform a useful role in our body; but when free radicals production overcomes antioxidant defence ability, it generates an oxidative stress evidenced in many human pathologies and which sometimes significantly contributes to their pathogenesis. The potential damage caused by an excess of free radicals is controlled by a series of antioxidant defence mechanisms. Radicalic reactions seem to take part in a number of diseases with different aetiology and pathogenesis, such as immune-inflammatory, neoplastic, dismetabolic, degenerative illnesses affecting various organs and systems (1,2,3,4,5).

Particularly in rheumatic pathologies, the role of radicals and lipoperoxidation in causing phlogosis is recognised; hence the interest in investigating on substances which could have a scavenger and antilipoperoxidant effect (6,7,8,9).

We thought interesting to examine an integrator like OxyLift, particularly assessing its scavenger effect on Reactive Oxygen Species (ROS).

The integrator OxyLift is composed of bioavailable minerals ready to be used directly by the cell, enzymes, amino acids, and it can release nascent H2 and O2, that is not coming from breathing.

We have analysed OxyLift’s scavenger action on RLO global production (chemiluminescence).

2. Materials and methods

In our in vitro works, the integrator OxyLift (International Biolife) has been used. The performed experiments focussed on the assessment of OxyLift’s scavenger effect at various dosages by chemiluminescence.

Chemiluminescence (10) is a means of assessing the scavenger action on ROS pool, which are produced by PMN stimulated with Zymosan (10 mg · ml -1 phosphate swab without Ca 2+ and Mg2+; Sigma) opsonized according to Bellavite method (11). Polimorphonucleated (PMN) have been taken from peripheral venous blood samples of healthy subjects by means of polymorphoprep (Nycomed) that, after being centrifuged, forms a density gradient on which blood cells split.

Purity (> 90%) and vitality (> 95%) of cell population have been checked by examining a smear and performing the Tripan-Blue exclusion test. Then, 100 ml luminol (2 mg in 10 ml NaOH 0,01M afterwards diluted 1 : 10 with PBS; Sigma) and 10 l stimulator (Zymosan) have been added to a rate (100 ml) of a PMN suspension, 10 6. ml -1 PBS. This preparation has been introduced into the chemiluminometer (Berthold Multi-biolumat LB 9505C ) at 37°C ; kinetic reaction has been monitored for 40 minutes (see Diagram 1).

PMN phagocyte Zymosan (unicellular fungus), during phagocytosis the so-called “respiratory burst” takes place releasing ROS. This reaction provokes a small release of energy which is amplified by added luminol and revealed for 40’ by the luminometer. Released chemiluminescence, monitored in time, originates a curve and the software carries out the integral of the curve itself, mathematically translating the noticed luminescence (see Graphic 1).

Diagram 1

SAMPLE 1 100 l cell suspension + 100μl luminol + 10 l (basal) Zymosan

SAMPLE 2 100 l cell suspension + 10μl PBS + 100 l luminol + 10 l (basal 1) Zymosan

SAMPLE 3 100 l cell suspension + 10μl OxyLift*(Solution 1) + 100 l luminol + 10 l Zymosan

SAMPLE 4 100 l cell suspension + 10μl OxyLift*(Solution 2) + 100 l luminol + 10 l Zymosan

SAMPLE 5 100 l cell suspension + 10μl OxyLift*(Solution 3) + 100 l luminol + 10 l Zymosan

SAMPLE 6 100 l cell suspension + 10μl OxyLift*(Solution 4) + 100 l luminol + 10 l Zymosan

Diagram 2

OxyLift*. Solutions have been prepared by adding some drops of this product to 50 ml PBS swab; 1 drop corresponds to 50μl about. Four solutions have been prepared:

Solution 1 – 1 drop OxyLift (~50μl) + 50 ml PBS swab

Solution 2 – 3 drops OxyLift (~150μl) + 50 ml PBS swab

Solution 3 – 5 drops OxyLift (~250μl) + 50 ml PBS swab

Solution 4 – 8 drops OxyLift (~400μl) + 50 ml PBS swab

From these solutions, the 10 μl shown in methodology and added to the cells have been taken.

3. Statistical analysis

Statistical analysis has been carried out using the Anova RM One Way Test and the Student Newman Keuls Test for multiple comparisons. A p<0,05 has been considered as significant.

RESULTS

In Table 1 and Graphic 1 are reported respectively: in the first, the average values of percentage inhibition referred to a 0 inhibition value noticed in ROS basal production, in lack of solutions containing OxyLift; in the second, the average of shots per minute (s.p.m.) expressed in the 40’ of chemiluminescence by PMN stimulated with Zymosan (Anova, P=0,00394).

From both data representations, we notice an inhibition of ROS due to the presence of OxyLift, and this results to be dose-dependent (Graphic 1, Student Newman Keuls p<0,05).

GRAPHIC 1

Graphic 1 shows luminescent shots per minute proportional to the ROS produced by PMN stimulated with Zymosan. Values reported above the columns represent the average of 5 tests (* p<0,05 Student Newman Keuls).

Column 1 BASAL

Column 2 BASAL 1 (Sample 2)

Column 3 Sample 3

Column 4 Sample 4

Column 5 Sample 5

Column 6 Sample 6

Table 1

CHEMILUMINESCENCE, stimulation with Zymosan

OxyLift inhibition %

SAMPLE 2 ---

SAMPLE 3 13,4 ± 7,14

SAMPLE 4 16,7 ± 11,2

SAMPLE 5 30,6 ± 2,3

SAMPLE 6 37,5 ± 6,0

Table 1 shows the same data reported in the graphic, but they are expressed in inhibition percentage compared with the basal of s.p.m. (shots/minute) produced in 40’ of chemiluminescence of prepared samples (see Diagram 1). Referred values are the result of 5 tests carried out with chemiluminescence.

4. Discussion

On the basis of these results, we notice that the integrator OxyLift can limit the ROS produced by stimulated human PMN, significantly and dose-dependently. When referred to man, this food integrator could contribute to a scavenger action, strengthening defence systems against free radicals. Thus, the integrator OxyLift is supposed to have a potential role in helping the therapy of many diseases in which phlogosis and lipoperoxidation caused by ROS have an important pathogenetic role. Originality of data consists in highlighting OxyLift’s scavenger action on the ROS produced in vitro by PMN of human peripheral blood.

Abstract

The Authors report the results of a first study on the use of a food integrator with supposed andioxidant properties. Leukocytes have been stimulated with Zymosan to produce ROS. Signal has been suitably amplified and recorded by chemiluminescence. These results give evidence for a statistically significant activity of the tested product.

Bibliografy

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