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Daily BioBasics and Daily
Plus –Their potential to
inactivate oxygen radicals
in a cell-based test assay
with functional neutrophils
Peter C. Dartsch, Dartsch Scientific GmbH
Summary
Daily BioBasics and Daily Plus, two basic dietary
supplements of Life Plus International, Batesville,
USA, were tested for their antioxidant potential
using a cell-based test assay with functional
neutrophils. The supplements were tested in two
respects: (1) Use of the basic supplements and
(2) use of the basic supplements in combination
with Proanthenols 100 and OmeGold. All test
concentrations were adapted to the
recommended daily serving of each supplement
and were calculated for a distribution within 3.3
liters of blood fluid after an assumed complete
absorption of ingredients.
The results of the cell-based test assays
demonstrated that the daily recommended
servings of both basic supplements were able to
inactivate endogenous oxygen radicals in a
dose-dependent manner with a 90% inactivation
of radicals at the calculated blood fluid
concentration of 8 mg/ml. When combined
with Proanthenols 100 and OmeGold, a further
moderate increase of the radical-inactivating
efficacy of both basic supplements was the
result, demonstrating the synergistic action of
the combined formulations.
From the experimental results, Daily BioBasics
and Daily Plus – combined with Proanthenols 100
and OmeGold - can be recommended as a
protective combination when an excess of
oxygen radicals due to a metabolic and/or
cellular imbalance is generated in the body.
Background
The body constantly reacts with oxygen as
part of the energy producing processes of cells
(Droge 2002). As a consequence, most reactive
oxygen species (ROS) come from endogenous
sources as by-products of normal and necessary
metabolic reactions, such as energy generation
from mitochondria or the detoxification
reactions involving the liver cytochrome P-450
enzyme system (Lenaz et al. 1999; Kuhn 2003).
Exogenous sources include exposure to cigarette
smoke, environmental pollutants such as
emission from automobiles and industries,
consumption of alcohol in excess, exposure to
ionizing radiation, and bacterial, fungal or viral
infections. To counteract the effects of ROS in
vivo, there are distinct antioxidant enzymatic
mechanisms within the cells which inactivate ROS
under normal health conditions. Oxidative stress
and the associated damage to cellular lipids,
proteins and DNA occurs when these
52
compensatory mechanisms fail to detoxify
increasing loads of ROS. Elevated levels of ROS,
which contribute to a decline in cellular function,
have been reported to coincide with tissue
damage as well as a number of human disorders
and diseases (Halliwell 1994; Bergamini et al.
2004). By continuously neutralizing an excess of
exogenous and endogenous oxygen radicals by
the assistance of dietary components and
nutritional supplements, an improvement and
maintenance of the body’s function, vitality,
performance, and health may result.
Prompted by this background, the present
investigation was conducted to see whether the
beneficial effects of Daily BioBasics and Daily Plus
in respect to their radical-inactivating efficacy can
also be demonstrated in an experimental
cell-based test assay. Moreover, it was also
studied whether the addition of Proanthenols
100 and OmeGold to Daily BioBasics and Daily
Plus might enhance the efficacy of the basic
dietary supplements.
Material and methods
Basic dietary supplements
Daily BioBasics and its German equivalent,
Daily Plus, are multiple-vitamin-mineral products
which have a balance of vitamins, minerals,
amino acids, accessory nutrients, plant extracts,
and fibers and are recommended to stay at the
physical and mental best condition. Besides the
daily values of essential vitamins and minerals,
they contain phytonutrients concentrated from
35 fruits and vegetables, phytonutrients
concentrated from 20 different herbs, as well as
special greens such as spirulina and chlorella
algaes. In summary, Daily BioBasics and Daily Plus
are composed from a total of 81 herbs, fruits,
vegetables, vitamins, minerals and other key
nutrients with specific activity to help provide a
broad spectrum of nutrients, and help protect
against oxidative stress.
The recommended daily serving are two level
scoops which is equivalent to 26.4 g. When
considering a 100 % absorption and a distribution
within the blood fluid of 3.3 liters, a
concentration of 8 mg/ml results. Thus, a primary
stock solution of 100 mg/ml was made in
phosphate-buffered saline with calcium and
magnesium (PBS+): The powder of the product
was mixed with PBS+ and was pestled for 30
seconds at room temperature to obtain a good
solubility of the ingredients in the aqueous
buffer. Then, the suspension was centrifuged for
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3 min at 3,400 x g and the brownish supernatant
was taken for further dilution. It proved
necessary to make these working steps within 5
min, because otherwise the ingredients would
absorb the aqueous solution completely. Test
concentrations for both products were as
follows: 0 (= control) – 0.1 – 0.25 – 0.5 – 1 – 2.5 –
5 – 10 mg/ml.
Combined formulations with
Proanthenols 100 and OmeGold
In a second series of experiments it was
studied whether the addition of Proanthenols
100 and OmeGold to Daily BioBasics and Daily
Plus might enhance the radical-inactivating
efficacy of the basic dietary supplements.
However, mixing of primary stock solutions of
the combined formulations which represent the
daily recommended serving of all ingredients
proved to be difficult, because concentrations
> 100 mg/ml of both basic dietary supplements
resulted in a complete absorption of water within
2 minutes. Therefore, the most concentrated
combined primary stock solution was only
equivalent to a 80% absorption of the
ingredients after uptake of the daily
recommended serving. Since the primary stock
solution of OmeGold had to be made in 100%
dimethylsulfoxide, this organic reagent was also
added to the primary stock solutions of the basic
dietary supplements. However, the maximum
concentration of dimethylsulfoxide in the test
assays was 0.8 vol% which did not cause a cellular
irritation within the observation period of the
test assay.
Proanthenols 100 is a strong antioxidant
and based on the main ingredient, “Real OPCs”,
which are extracted from grape seeds and pine
bark. The recommended daily serving is one
tablet (550 mg) twice a day which is equivalent
to a total amount of 1,100 mg per day. When
considering a 100% absorption and a distribution
within the blood fluid of 3.3 liters, a
concentration of approximately 350 µg/ml
results. To yield a primary stock solution, the
tablets were crushed, mixed with PBS+ and were
pestled for 30 seconds at room temperature to
obtain a good solubility of the ingredients in the
aqueous solution.
OmeGold contains omega-3 fish oil
concentrate providing a source of the omega-3
polyunsaturates, DHA (docosahexaenoic acid)
and EPA (dicosapentaenoic acid) combined with
vitamin D and a special blend of antioxidant-rich
essential oils. In comparison to Proanthenols,
OmeGold provides oil-soluble antioxidants. The
recommended daily serving is one soft gel
capsule (1,450 mg) twice a day which is
equivalent to a total amount of 2,900 mg per
day. When considering a 100% absorption and a
distribution within the blood fluid of 3.3 liters, a
concentration of approximately 900 µg/ml
results. Because of the oil-soluble ingredients,
the primary stock solution had to be made in 100
% dimethylsulfoxide, analytical grade.
Inactivation of endogenous superoxide
anion radicals
The basic principle of the test assay has been
already described in detail elsewhere (Dartsch
2006). This cell-based test assay uses the
formation of intracellular superoxide anion
radicals of human phagocytic cells (functional
neutrophils) as a model to investigate the
efficacy of biologically active substances to
inactivate endogenously generated oxygen
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radicals. An overload of endogenous radicals
might be the result of oxidative stress or occurs
during complicated wound healing or
inflammatory processes (Ward et al. 1988;
Nathan 2002).
To perform the assay, human promyelocytic
HL 60 cells were differentiated to functional
neutrophils capable of undergoing an oxidative
or respiratory burst upon phorbol ester
stimulation after addition of 1.25%
dimethylsulfoxide to the culture medium for 6
days (Teufelhofer et al. 2003). The differentiated
cells were collected, washed twice with PBS+ by
resuspending and centrifugation (5 min at 200 x
g at room temperature) and finally resuspended
in PBS+ containing 15 mM glucose. This cell
suspension was added to the reaction mixture
consisting of PBS+ with glucose, phorbol ester,
WST-1 as tetrazolium dye, and the appropriate
test concentrations of the basic dietary
supplement or combined formulations. The
course of superoxide anion radical inactivation as
produced by the stimulated functional
neutrophils was monitored by the cleavage of
the tetrazolium dye to to yield a yellow-coloured
water-soluble formazan (Peskin and Winterbourn
2000). This colour change was monitored for 60
min at 37°C as a differential measurement of the
optical density at 450 nm – 690 nm using a
BioTek ELISA reader. The reading interval was 60
seconds with a vigorous shaking for 4 seconds
prior to each reading. For data analyses, the slope
of the linear regression curves of the monitored
optical density was calculated and expressed as
relative changes in comparison to untreated
controls. For further details, see Dartsch et al.
2006, 2008 and Dartsch 2010.
Results and discussion
Both basic dietary supplements, Daily
BioBasics and Daily Plus, were able to inactivate
endogenous superoxide anion radicals in a
dose-dependent manner (Fig. 1). At the
calculated blood fluid concentration of 8 mg/ml,
the radical-inactivating efficacy was
approximately 90%. However, there was a
difference between both supplements at lower
concentrations which is demonstrated by the
EC50, i.e. the concentration causing a 50% radical
inactivation. The EC50 was calculated to be
450 µg/ml for Daily BioBasics and 700 µg/ml for
Daily Plus. These values are equivalent to a 6%
and 9 % absorption of bioactive and
radical-inactivating ingredients.
As shown in Figs. 2 and 3 for an additional
series of experiments which allow a direct
comparison between the basic dietary
supplement and after the addition of
Proanthenols 100 and OmeGold, a moderate
increased radical-inactivating efficacy of the
combined formulations was observed. When
calculating the difference between the basic
supplement and the combined formulation it
turned out that the maximum increase was in
the range of 10 to 15% (Fig. 4). This increase was
not statistically significant when applying
Student’s t-test, possibly due to the very high
radical inactivation activity of Daily BioBasics and
Daily Plus alone. However, the addition of
Proanthenols 100 and OmeGold did not cause a
Fig. 1: Dose-dependent radical-inactivating effect of Daily BioBasics and
Daily Plus in the cell-based test assay with functional neutrophils which
generate superoxide anion radicals upon stimulation. The calculated blood
fluid concentration is marked by the arrows. Data represent mean value ±
standard deviation of three experiments.
reduced efficacy of both basic dietary
supplements due to an antagonistic effect of the
ingredients, but actually represented a synergistic
mode of action.
For comparative reasons, the EC50 of some
plant extracts, supplements, vitamins or enzymes
as obtained in this cell-based test assay with
functional neutrophils are as follows: Superoxide
dismutase: 2.5 µg/ml – Vitamin C: 200 µg/ml –
Daily BioBasics: 450 µg/ml – Acerola: 500 µg/ml –
Black cumin extract: 600 µg/ml – Daily Plus: 700
µg/ml – Dandelion leaves: 1,000 µg/ml – Black
chokeberry extract: 2,000 µg/ml.
The radical-inactivating efficacy can be
checked by a variety of methods without cells or
by use of specific cell types. For example, the
Oxygen Radical Absorbance Capacity (ORAC) is a
cell-free method of measuring antioxidant
capacities in biological samples in vitro (Cao et al.
1993; Ou et al. 2001). However, it does not take
the effect on the cellular level or after generation
of an excess of endogenous oxygen radicals into
account.
In this study, the endogenous superoxide
radicals generated by functional neutrophils are
used as a model to evaluate the radicalinactivating efficacy of the dietary supplements
and test compounds. The functional neutrophils
are also a model for testing the
anti-inflammatory potential of drugs, because
endogenous radicals are known to play a
emarkable role during not only during a
metabolic imbalance but also during complicated
wound healing or inflammatory processes
(Nathan 2002). Thus, this study not only checked
Fig. 2: Dose-dependent radical-inactivating effect of Daily BioBasics as basic
dietary supplement and of Daily BioBasics in combination with
Proanthenols 100 and OmeGold in the cell-based test assay with functional
neutrophils which generate superoxide anion radicals upon stimulation. The
calculated blood fluid concentration is marked by the arrows. Data
represent mean value ± standard deviation of three experiments.
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Fig. 3: Dose-dependent radical-inactivating effect of Daily Plus as basic
dietary supplement and of Daily Plus in combination with Proanthenols
100 and OmeGold in the cell-based test assay with functional neutrophils
which generate superoxide anion radicals upon stimulation. The calculated
blood fluid concentration is marked by the arrows. Data represent mean
value ± standard deviation of three experiments.
the radical-inactivating efficacy in general, but
also the potential to inactivate endogenously
generated radicals during metabolic imbalance or
health problems related to a local excess of
Fig. 4: Calculated difference between combined formulation and basic
dietary supplement for Daily BioBasics and Daily Plus. A positive column
represents an increased radical-inactivating efficacy of the combined
formulation. The calculated blood fluid concentration is marked by the
arrows. Data represent mean value ± standard error of the mean of three
experiments as depicted in Figs. 2 and 3.
oxygen radicals.
As can be seen from the experimental data
presented here, both basic dietary supplements
are able to activate endogenous radicals in a
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dose-dependent manner. Even when considering
an absorption of about 10% of the ingredients,
approximately 50% of the oxygen radicals will be
inactivated. Moreover, Daily Biobasics is more
efficient than Daily Plus which might be related
to its slightly different ingredients concentration.
Although both supplements are very effective
when tested without the addition of other
formulations, their efficacy is even slightly
enhanced by the addition of Proanthenols 100
and OmeGold. This demonstrates a synergistic
mode of action – and not an unwanted
antagonistic effect – after application of the
combined formulations. It should be noted that
the measured effect of endogenous radical
inactivation is only one aspect of the many
functions of the nutrients included in these
formulations in biological systems.
Thus, Daily BioBasics and Daily Plus –
combined with Proanthenols 100 and OmeGold –
can be recommended as a protective
combination when an excess of oxygen radicals
due to a metabolic and/or cellular imbalance is
generated in the body.
Author’s address:
Prof. Dr. rer. nat. habil. Peter C. Dartsch
Dartsch Scientific GmbH – Institut für
zellbiologische Testsysteme
Oskar-von-Miller-Straße 10
D-86956 Schongau/Oberbayern, Germany
Tel: +49 (0) 8861 256-5250
Fax: +49 (0) 8861-256-7162
Email: [email protected]