Lab Results

Report 1: Initial exploratory in vitro work on the Zrii beverage.

Test types:

Protection from apoptosis Protection of mitochondrial function Testing of PMN migration (random, directed)

Performed:

November 2008. Reviewed by:

______________________ Steve G. Carter Analyst.

____________________ Gitte Jensen Ph.D. Date: December 9, 2008.

1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113 E-mail: [email protected] • Web Site: www.holgernis.com

NIS/Zrii

Initial exploratory in vitro work on the Zrii beverage - Report 1

2 of 11

Report 1: Initial exploratory in vitro work on the Zrii beverage. Purpose The purpose of this study was to evaluate a panel of biological/immunological effects that can be induced in cultures of human immune cells, when exposed to the Zrii beverage.

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 During this study we tested the following: 0. Baseline tests for starting work on new test products; 1. Evaluation of protection of mitochondrial function in the absence versus presence of oxidative stress (CAP-m); 2. Evaluation of protection of cellular viability (protection from programmed cell death (apoptosis)) in the absence versus presence of oxidative stress (CAP-a); 3. Evaluation of effects on anti-inflammatory versus immune surveillance behavior of the PMN cell.

Summary of observations We found that Zrii:

1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113 E-mail: [email protected] • Web Site: www.holgernis.com

NIS/Zrii

Initial exploratory in vitro work on the Zrii beverage - Report 1

3 of 11

•

Increased mitochondrial function in PMN cells;

•

Maintained cell viability under normal and induced oxidative stress;

•

Increased the migration of PMN cells (considered part of normal immune surveillance);

•

Increased the migration of PMN cells towards a bacterial peptide f-MLP (considered a model for one important aspect of our anti-bacterial immune defense mechanisms);

•

Reduced the migration of PMN cells towards LTB4 inflammatory mediators.

Methodological details for Report 1: 0. Baseline tests for starting in vitro work on new test products. 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.

1. Evaluation of protection of mitochondrial function in the absence versus presence of oxidative stress. 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: [email protected] • Web Site: www.holgernis.com

NIS/Zrii

Initial exploratory in vitro work on the Zrii beverage - Report 1

4 of 11

Freshly purified human peripheral blood mononuclear cells (PBMC) and polymorphonuclear leucocytes (PMN) were cultured in the absence versus presence of serial dilutions of test product, either without further treatment or in the presence of oxidative stress. After incubation, the level of mitochondrial function was measured by staining with the marker MitoTracker-Red, which becomes fluorescent as a result of, and in proportion to, mitochondrial function. Therefore, the resulting fluorescence intensity is proportional to mitochondrial function, and is evaluated by flow cytometry. A decrease in fluorescence intensity reflects a reduction of mitochondrial function, as mitochondria are damaged by oxidative stress. If the presence of test product protects mitochondrial function, then higher fluorescence intensities will be observed. 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. ZRII protected mitochondrial function under conditions of oxidative stress

Mitotracker (MFI)

800 700 600 500 400 300 200 100 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: [email protected] • Web Site: www.holgernis.com

NIS/Zrii

Initial exploratory in vitro work on the Zrii beverage - Report 1

5 of 11

2. Evaluation of protection from programmed cell death (apoptosis) in the absence versus presence of oxidative stress. 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.

The human vascular anticoagulant, annexin V, is a Ca2+-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine. In normal viable cells, phosphatidylserine is located on the cytoplasmic surface of the cell membrane. However, in apoptotic cells, PS is translocated from the inner to the outer leaflet of the plasma membrane, thus exposing PS to the external cellular environment. Annexin V labeled with a fluorophore can identify apoptotic cells by binding to phosphatidylserine exposed on the outer leaflet. We use AnnexinV-FITC to label apoptotic cells. Co-staining with PI or 7AAD, which only stains cells at a late phase of cell death, allows us to distinguish early and late apoptosis. Cells staining only with PI or 7AAD are necrotic cells.

7AAD

1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113 E-mail: [email protected] • 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.

PERCENT CELLS

12

EARLY APOPTOSIS (NORMAL CULTURE CONDITIONS)

10 8 6 4 2 0

PERCENT CELLS

No product

0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

0.16

0.8

4

20 mL/L

LATE APOPTOSIS (NORMAL CULTURE CONDITIONS)

No product

0.16

0.8

4

20 mL/L

The dose of 4 mL/L Zrii resulted in statistically significant inhibition of apoptosis under normal culture conditions (i.e. no oxidative stress) (P<0.01).

1437 Esplanade Ave, Klamath Falls, OR 97601 • Telephone (541) 884-0112 • Fax (541) 884-0113 E-mail: [email protected] • Web Site: www.holgernis.com

NIS/Zrii

Initial exploratory in vitro work on the Zrii beverage - Report 1

PERCENT CELLS

12

7 of 11

EARLY APOPTOSIS (IN THE PRESENCE OF OXIDATIVE STRESS)

10 8 6 4 2 0

PERCENT CELLS

No product

0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

0.16

0.8

4

20 mL/L

LATE APOPTOSIS (IN THE PRESENCE OF OXIDATIVE STRESS)

No product

0.16

0.8

4

20 mL/L

The dose of 4 mL/L Zrii resulted in statistically significant inhibition of apoptosis under culture conditions where oxidative stress was introduced (P<0.01).

3. Evaluation of effects on immune surveillance, anti-bacterial, and inflammatory behavior of the PMN cell. 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: [email protected] • 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: i) ii) iii)

bacterial peptide f-Met-Leu-Phe; the inflammatory cytokine Interleukin-8 (IL-8); and the inflammatory mediator Leukotriene B4 (LTB4).

Previously, we have found interesting evidence during evaluation of several natural products that some products may specifically reduce directed PMN migration towards the inflammatory mediators IL-8 and/or LTB4 while allowing PMN migration towards bacterial peptide as part of the normal anti-bacterial immune defense. We hoped to see a similar effect with Zrii. 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: [email protected] • 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 Increased migratory activity

1.50

1.00

0.50 0.0001

0.001

0.01

0.1

1

10 mL/L

1

10 mL/L

f-MLP directed migration Increased migratory activity

1.50

1.00

0.50 0.0001

0.001

0.01

0.1

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. Zrii-treatment of PMN cells resulted in a dose-dependent increase in random and f-MLP directed migration. Thus, the data showed that Zrii supported two aspects of PMN cell behavior in vitro, associated with a healthy immune defense in vivo. 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: [email protected] • Web Site: www.holgernis.com

NIS/Zrii

Initial exploratory in vitro work on the Zrii beverage - Report 1

10 of 11

IL-8 directed migration Increased migratory activity

1.50

1.00

0.50 0.0001

0.001

0.01

0.1

1

10 mL/L

1

10 mL/L

LTB4 directed migration Increased migratory activity

1.50

1.00

0.50 0.0001

0.001

0.01

0.1

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: [email protected] • 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: [email protected] • 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. Table 3. Palatability, glycemic index, and GI category Test Meal

Abbr

Glycemic Index 100

GI Category*

Gluc

Palatability (mm) 46±8

Glucose Control AMALAKI Liquid Dietary Supplement

Zrii

72±7**

43.9 ± 5.8**

Low

High

*Category from GI Factor (Brand-Miller). ** statistically different from glucose (p<0.05).

Blood Glucose Response 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. Repeated Glucose Trials 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%. The tests appeared to be technically satisfactory, as judged by the average withinsubject variation of glycemic responses for the repeated glucose tests. GI Labs, 36 Lombard St, Suite 100, Toronto, Ontario, Canada M5C 2X3