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beverage.
Reviewed by:
______________________ ____________________
Steve G. Carter Gitte Jensen Ph.D.
Analyst. Date: December 9, 2008.
1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113
E-mail: tech@holgernis.com • Web Site: www.holgernis.com
NIS/Zrii Initial exploratory in vitro work on the Zrii beverage - Report 1 2 of 11
Background
The Zrii beverage was formulated based on Ayurvedic principles, and contains a number
of ingredients with known antioxidant and anti-inflammatory properties. However, no
research is currently available to document exactly how this cocktail acts on specific
components of the human immune system.
NIS Labs has previously tested the Zrii beverage in the CAP-e antioxidant protection
assay. It was found that Zrii contained antioxidants able to enter into and protect cells
from oxidative damage. Thus, compounds in Zrii are bioavailable at the cellular level.
The subsequent research protocol, with data being reported here, undertook the next
important steps in creating a foundation of knowledge on how the specific product acts,
with a focus on mitochondrial function, cellular viability, and anti-inflammatory effects.
The data form this work serves as a basis for planning further cell-based and clinical
research, hopefully of sufficient significance to warrant further efforts towards publication.
Work performed
Summary of observations
1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113
E-mail: tech@holgernis.com • Web Site: www.holgernis.com
NIS/Zrii Initial exploratory in vitro work on the Zrii beverage - Report 1 3 of 11
We use lymphocyte viability testing to evaluate a safe working range for new products.
This is an important starting point, and leads to identification of the optimal working
range for a particular product in cell-based assays.
Occasionally, we discover that a product actually protects the survival of healthy immune
cells in culture. Within the circulating pool of normal healthy lymphocytes, some cells are
short-lived. At the time of blood draw, a small proportion of the lymphocytes may be on a
pathway to death. It is of interest to see when a product is able to extend the life of a
healthy cell in culture.
Freshly purified peripheral blood mononuclear cells from a healthy donor are used for this
initial viability testing. Cells are cultured overnight in the absence versus presence of
serial dilutions of test product. The following day, the cells are washed and stained with a
combination of two dyes that stain cells at different stages of cell death. Multiparameter
flow cytometry then allows analysis of a product’s impact on cellular viability versus
death.
Mitochondria are the intracellular organelles responsible for producing cellular energy.
Decreased mitochondrial function has been associated with inflammatory conditions,
ageing, and degenerative illnesses. This assay measures mitochondrial function, and
examines whether a test product contains compounds capable of protecting healthy
mitochondrial function, as well as protecting mitochondrial function in cells under
oxidative stress.
1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113
E-mail: tech@holgernis.com • Web Site: www.holgernis.com
NIS/Zrii Initial exploratory in vitro work on the Zrii beverage - Report 1 4 of 11
The testing was performed where each testing condition, including each serial dilution of
test product, was performed in triplicate. The experiment was repeated three times using
cells from different donors.
800
700
Mitotracker (MFI)
600
500
400
300
200
100
0
0 0.8 4 20 100 mL/L
Dose of Zrii added to cells in lab test
The graph above shows that Zrii provided a dose-dependent protection of mitochondrial
function, and is illustrated as the fluorescence intensity (MFI) of the dye MitoTracker in
the presence of oxidative stress.
PMN cells treated with Zrii were capable of maintaining and increasing mitochondrial
function. The presence of Zrii alone, in the absence of oxidative stress, caused a slight
increase in mitochondrial function.
When cells were exposed to oxidative stress, the presence of Zrii was protective of
mitochondrial function, and the cells were able to maintain mitochondrial function under
conditions of oxidative stress.
1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113
E-mail: tech@holgernis.com • Web Site: www.holgernis.com
NIS/Zrii Initial exploratory in vitro work on the Zrii beverage - Report 1 5 of 11
Oxidative damage can trigger premature cellular death by a mechanism called apoptosis
(programmed cell death). This death pathway can be monitored by highly specific cellular
markers. Protection from apoptosis can be monitored as delay or absence of these markers.
Apoptosis is a carefully regulated process of cell death that occurs as a normal part of
cellular development. In contrast to necrosis, a form of cell death resulting from acute
cellular injury, apoptosis is carried out in an ordered process that is generally advantageous
during an organism's life cycle. An example of healthy apoptosis in an organism is the loss
of webbing between fingers in a human.
7AAD
1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113
E-mail: tech@holgernis.com • Web Site: www.holgernis.com
NIS/Zrii Initial exploratory in vitro work on the Zrii beverage - Report 1 6 of 11
Among freshly isolated human peripheral blood mononuclear cells (PBMC) and
polymorphonuclear (PMN) cells, a proportion is already on an apoptotic path. When
cultured in vitro, these cells will continue the apoptotic process. Early apoptosis is
reversible. By culturing freshly purified human cells under normal culture conditions (i.e.
no oxidative stress) with and without test product, and then staining for apoptotic cells, we
are comparing the ability of the test product to rescue cells already on the path to
apoptosis.
In parallel, H2O2 was added to trigger oxidative stress-induced apoptosis, and assess
whether the test product was able to protect the viability of cells under oxidative stress.
The testing was performed where all controls were performed in quadruplicate and each
serial dilution of test product was performed in duplicate.
More PMN cells are on an apoptotic pathway than PBMC cells. Also, PMN cells are easily
triggered by oxidative stress to produce ROS and thus escalate the stressful conditions.
This may be one reason that cultures of PMN cells showed clearer responses to Zrii than
PBMC cells. Similar trends were seen in both sets of cultures, but statistical significance
was only obtained for PMN cells. The graphs below show data from PMN cells.
EARLY APOPTOSIS
12 (NORMAL CULTURE CONDITIONS)
10
PERCENT CELLS
0
No product 0.16 0.8 4 20 mL/L
LATE APOPTOSIS
0.5
(NORMAL CULTURE CONDITIONS)
0.45
PERCENT CELLS
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
No product 0.16 0.8 4 20 mL/L
1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113
E-mail: tech@holgernis.com • Web Site: www.holgernis.com
NIS/Zrii Initial exploratory in vitro work on the Zrii beverage - Report 1 7 of 11
EARLY APOPTOSIS
12 (IN THE PRESENCE OF OXIDATIVE STRESS)
PERCENT CELLS 10
0
No product 0.16 0.8 4 20 mL/L
LATE APOPTOSIS
0.5 (IN THE PRESENCE OF OXIDATIVE STRESS)
0.45
0.4
PERCENT CELLS
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
No product 0.16 0.8 4 20 mL/L
The PMN cell is a highly active and migratory cell type. The migratory behavior of this
cell type is divided into at least two types: a) random migration and b) directed migration.
Random migration is part of normal immune surveillance, whereas directed migration
happens towards specific chemoattractants.
1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113
E-mail: tech@holgernis.com • Web Site: www.holgernis.com
NIS/Zrii Initial exploratory in vitro work on the Zrii beverage - Report 1 8 of 11
We have established a migration test where both types of migration are tested in parallel.
Furthermore, the directed migration is tested towards three distinctly different chemotactic
compounds:
Freshly purified PMN cells were set up cultures in double-chamber migration plates, where
the bottom chamber mimics tissue, and the top chamber mimics the blood stream. Cells
were plated in the top chambers with and without serial dilutions of test product, and
different chemo-attractants were present in the bottom chambers. Control wells included
cells un-exposed to test products and without chemo-attractant in the bottom wells, and
allowed evaluation of baseline random migration.
The assay was performed three times using freshly isolated cells from different healthy
human donors. Within each test, controls were performed in quadruplicate, and tests in
duplicate.
1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113
E-mail: tech@holgernis.com • Web Site: www.holgernis.com
NIS/Zrii Initial exploratory in vitro work on the Zrii beverage - Report 1 9 of 11
The four graphs below shows the increase in migratory behavior under the four different
test conditions.
Random migration
1.50
Increased migratory activity
1.00
0.50
0.0001 0.001 0.01 0.1 1 10 mL/L
1.00
0.50
0.0001 0.001 0.01 0.1 1 10 mL/L
In these graphs, the value “1.00” is the baseline, i.e. the level of migratory activity without
Zrii-treatment of the cells. Values higher than “1.00”indicatesthatZrii induced an increase
in migratory behavior. Values lower than “1.00” indicates that Zrii reduced the migration.
As can be seen from the graphs on the next page, Zrii did not have a clear effect on IL-8
directed migration. In contrast, Zrii had a strong, pronounced anti-inflammatory effect
on LTB4 directed migration.
1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113
E-mail: tech@holgernis.com • Web Site: www.holgernis.com
NIS/Zrii Initial exploratory in vitro work on the Zrii beverage - Report 1 10 of 11
0.50
0.0001 0.001 0.01 0.1 1 10 mL/L
1.00
0.50
0.0001 0.001 0.01 0.1 1 10 mL/L
The trend to an anti-inflammatory effect, seen at the lowest dose of Zrii, led to testing
across an extremely wide dose range of Zrii. Data from this experiment is shown in a
separate graph below. The dashed line shows the baseline level of PMN migration when
no test product was added.
LTB4 directed migration
1.20
0.20
0.00001 0.0001 0.001 0.01 0.1 1 10 100 1,000 10,000 100000
nL/L
1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113
E-mail: tech@holgernis.com • Web Site: www.holgernis.com
NIS/Zrii Initial exploratory in vitro work on the Zrii beverage - Report 1 11 of 11
Conclusion
Zrii provided a protective effect in both the testing of apoptosis and mitochondrial
function, when PMN cells were used. PBMC showed a similar trend, but much less
pronounced.
Zrii supported two important aspects of PMN immune function, namely random migration
and migration towards the bacterial peptide f-MLP.
Zrii strongly inhibited PMN migration towards the inflammatory mediator LTB4.
Thus, Zrii supported cellular viability and energy formation, as well as key aspects of the
innate (immediate) anti-bacterial immune response. However, the antioxidant capacity and
the anti-inflammatory properties of Zrii would indicate that Zrii may boost the immune
defense mechanisms while protecting the body’s own cells from the resulting oxidative
stress.
Future research
Zrii’s anti-inflammatory effects are very promising. The low dose required to inhibit PMN
cell migration has not been explored to its fullest yet – even at a dose of 10 femtoliter per
liter culture showed a strong effect, and we have not yet been able to identify the lowest
active dose.
1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113
E-mail: tech@holgernis.com • Web Site: www.holgernis.com
Zrii International
RESULTS
Palatability
Palatability scores are given in Table 3. The product was well accepted and
palatability of the AMALAKI Liquid Dietary Supplement was significantly higher than
glucose (72±7 vs. 46±8 mm)(p<0.02).
Glycemic Index
The GI values of the foods are tabulated in Table 3. The GI value of the
AMALAKI Liquid Dietary Supplement was significantly lower than the glucose GI (100).
Using the classification of Brand-Miller, the AMALAKI Liquid Dietary Supplement is
classified as a low GI food.
The blood glucose responses are shown on the pages labeled GIL-9002 with the
graphs showing a comparison of the total and incremental blood glucose values in response
to the test food and mean of the three glucose controls with significance determined by
paired t-test.
Mean fasting blood glucose was identical before each test meal within each series.
The blood glucose responses are not described in detail here but can be viewed on the
analysis pages.
There was no significant effect of order on the iAUC values after the repeated glucose
meals. The mean within-subject coefficient of variation (CV) of the iAUC values after the 3
repeated glucose tests was 20.2±4.1%.
GI Labs, 36 Lombard St, Suite 100, Toronto, Ontario, Canada M5C 2X3