2 MAJOR STUDY ENDPOINTS

1. Mitochondrial Mass Per Cell (MMPC): Defined as, the ability of a compound to induce an increase in number of Mitochondrial organelles within human leukocytes (white blood cells).
2. Total Antioxidant Capacity (TAC): Defined as, the protective ability of a compound against free radical damage to human leukocytes.

ENDPOINTS

1. Mitochondrial Mass Per Cell:
   • Increases in MMPC were detected at 2 hours following treatment of cells with the TEN formula (see graphs below).
   • The 0.125 g/L dose of the TEN formula led to an 80-140% increase in MMPC in 3 different white blood cell populations. This shows the relative potency of the TEN formula, which is promising for future clinical studies where mitochondrial mass per cell will be tested in blood samples from human subjects before and after consuming the TEN formula.
   
   
2. Total Antioxidant Capacity:
   • The TEN formula showed robust antioxidant capacity. This is important since it is expected that many of the water-soluble compounds present in the TEN formula will be readily available following human consumption.
   • Graph below shown in Gallic acid equivalents.

STUDY ENDPOINTS

1. Mitochondrial Biogenesis/Mass: defined as, the ability of a compound to induce natural synthesis or an increase in volume of Mitochondrial organelles in the human body.
2. Mitochondrial Energetics/Function: defined as, the ability of a compound to induce ATP or energy synthesis in Mitochondrial organelles in the human body.
3. Study Participant Energy Questionnaire: defined as, a standardized testing method used to evaluate participant's physical energy, mental focus and mental function after ingesting a compound versus placebo.
4. Safety Parameters and Inflammatory Markers: defined as, a Complete Blood Count (CBC), Complete Metabolic Profile (CMP) and endogenous inflammatory cytokines. Used to evaluate the relative safety of a compound when ingested by humans over short and long term use.

TESTS PERFORMED

This pilot project compared several tests that evolve around:

• Total antioxidant capacity.
• Cellular antioxidant protection due to uptake of antioxidant compounds.
• Mitochondrial volume per cell:
  • Under normal culture conditions (unstressed).
  • Under oxidative stress.
  • Under inflamed conditions.
• Mitochondrial metabolic activity:
  • Under normal culture conditions (unstressed).
  • Under oxidative stress.
  • Under inflamed conditions.

PRODUCT HANDLING

The formula TEN contains a blend of water-soluble and water-insoluble/poorly soluble compounds. Therefore, this pilot testing compared the cellular effects of three different methods of handling the product for introduction into the liquid nutrient-rich medium in which the cells are grown in cell culture.

TOTAL ANTIOXIDANT CAPACITY

The three extracts shown in Table 1 were tested in the Folin-Ciocalteu assay (also known as the total phenolics assay). This assay makes use of the Folin-Ciocalteu reagent to measure antioxidants. The assay is performed by adding the FolinCiocalteu's phenol reagent to serial dilutions of each extract, thoroughly mixing, and incubating for 5 minutes. Sodium carbonate is then added, starting a chemical reaction producing a color. The reaction is allowed to continue for 30 minutes at 37°C. Optical absorbance is measured at 765nm in a colorimetric plate reader. Gallic acid is used as a reference standard, and the data reported in Gallic Acid Equivalents per gram product. Data from this assay helps interpret the subsequent Cellular Antioxidant Protection assay, as well as the assays for mitochondrial function. The results confirmed that TEN contains antioxidants. The antioxidant capacity was greater in the water-soluble fraction than in the ethanol fraction.

CELL-BASED ANTIOXIDANT PROTECTION (CAP-E) ASSAY

The rationaleiv behind the method that we use is important: It allows assessment of anti- oxidant potential in a method that is comparable to the ORAC antioxidant test, but only allows measurement of antioxidants that are able to cross the lipid bilayer cell membrane, enter the cells, and provide biologically meaningful antioxidant protection under conditions of oxidative stress. We developed the CAP-e bioassay specifically to work with natural products and ingredients.v The method has been used on multiple types of natural products and ingredients, published in the peer-reviewed scientific literature.vi vii viii ix x xi

As a model cell type, we use the red blood cell (RBC). This is an inert cell type, in contrast to other cell types. We developed this assay particularly to be able to assess antioxidants from complex natural products in a cell-based system.

Freshly purified human RBC are washed repeatedly in physiological saline, and then exposed to the test products. During the incubation with a test product, any antioxidant compounds able to cross the cell membrane can enter the interior of the RBC. Then the RBC are washed to remove compounds that were not absorbed by the cells, and loaded with the DCF-DA dye, which turns fluorescent upon exposure to reactive oxygen species. Oxidation is triggered by addition of the peroxyl free radical generator AAPH. The fluorescence intensity is evaluated. The low fluorescence intensity of untreated control cells serve as a baseline, and RBC treated with AAPH alone serve as a positive control for maximum oxidative damage.

When we observe a reduced fluorescence intensity of RBC exposed to a test product and subsequently exposed to AAPH, this indicates that the test product contains antioxidants available to penetrate into the cells and protect these from oxidative damage.

The results showed that water-soluble antioxidants in TEN were capable of entering into and protecting the cells from oxidative stress-induced intracellular damage.

MITOCHONDRIAL METABOLIC ACTIVITY WHEN EXPOSED TO VARIOUS STRESSORS

The testing described below was performed on freshly isolated peripheral blood mononuclear cells from a healthy human blood donor. Serial dilutions of the test product (using all 3 handling methods in parallel) were added to cell cultures, where the cultures were tested for mitochondrial metabolic activity, using the MTT assay. Freshly purified cells were cultured for 24 hours in the presence of TEN, after which MTT reagents were added to allow a color formation to take place in proportion to mitochondrial function. In the MTT bioassay, chemical reactions trigger a specific color development based on cellular functions:

• When a reduction in color is measured, this is linked to a reduced cellular viability, either as a result of direct killing, or inhibition of mitochondrial function.
• When an increase in color is measured, this has multiple possible explanations: 1) increased cell numbers (growth); 2) increased mitochondrial mass; and 3) increased mitochondrial function (energy production).

Under the test conditions used for this project, no cellular growth was expected, therefore the changes are proportional to the combination of increased mitochondrial mass, and increased mitochondrial metabolic activity.

Culture conditions

One set of cell cultures was allowed to incubate under normal cell culture conditions ("Normal culture"), without any added stressors. The following four sets of cell cultures were made in parallel:

• Normal culture.
• Oxidative stress (induced by adding H2O2).
• Inflammation (induced by LPS (a highly inflammatory bacterial endotoxin)).

	The results showed that TEN was effective at very low doses. TEN provided mitochondrial support under normal healthy culture conditions, as well as under conditions of oxidative stress and inflammation.
	Cellular energy production. The lowest doses are shown for aqueous and ethanol handling. The horizontal black line shows the 100% value of cell cultures not treated with TEN.

MITOCHONDRIAL MASS PER CELL

The testing described below was performed on freshly purified white blood cells from a healthy human blood donor. Serial dilutions of the test product (using all 3 handling methods in parallel) were added to cell cultures, where the cultures were tested for mitochondrial volume after 2 hours incubation (to mimic timing proposed for clinical study). Untreated control samples were processed in parallel.

Cell types

Different cell types have different mitochondrial activity levels, both under normal and under stressed conditions. During flow cytometric analysis, separate analysis was performed on the following three cell types in the blood sample:

• Lymphocytes (Ly): Fairly inactive in the types of cell cultures used here;
• Monocytes (Mo): Moderately active cells, highly reactive to stressors;
• Polymorphonuclear cells (PMN): Highly active cells, extremely reactive to stressors.

Flow cytometry analysis of mitochondrial mass per cell. Electronic gates are set to define lymphocytes (Ly), monocytes (Mo), and polymorphonuclear (PMN) cells based on their forward scatter (size) and side scatter (granularity) properties. The green fluorescence intensity is measured for each cell type.

Culture conditions

One set of cell cultures was allowed to incubate under normal cell culture conditions ("Normal culture"), without any added stressors. The following four sets of cell cultures were made in parallel:

• Normal culture: Moderate increases in mitochondrial mass per cell when cells were treated with the aqueous as well as the ethanol-extract of TEN.
• Oxidative stress (induced by adding H2O2): Mild increases in mitochondrial mass per cell when Mo and PMN cells were treated with the ethanol-extract of TEN.
• Inflammation (induced by LPS (a highly inflammatory bacterial endotoxin)): TEN offered strong protection of mitochondrial mass per cell for all three cell types.

After incubation, cells were stained with a fluorescent probe that stains mitochondria in proportion to mitochondrial volume per cell. The overall sum of fluorescent output per cell is a measure of mitochondrial volume per cell. The Attune® acoustic aligning flow cytometer tracks cell numbers per volume unit, and provides fluorescence intensity measurements per cell.

On the following pages are two sets of graphs for each culture condition and cell type. The first graph shows the TEN-induced change for all three test fractions for all three doses tested.

The second graph serves to illustrate the effects of TEN when compared to untreated versus stressed cells.

Mitochondrial mass per lymphocyte (Ly) is shown as green fluorescence intensity. The graph on the left shows the average ± standard deviation of triplicate cultures. The graph on the right shows the same data plotted against the untreated cells (green dotted line).

Mitochondrial mass per monocyte (Mo) is shown as green fluorescence intensity. The graph on the left shows the average ± standard deviation of triplicate cultures for TEN-treated cells. The graph on the right shows the same data plotted against the untreated cells (green dotted line).

Mitochondrial mass per polymorphonuclear cell (PMN) is shown as green fluorescence intensity. The graph on the left shows the average ± standard deviation of triplicate cultures. The graph on the right shows the data plotted against untreated cells (green dotted line).

Mitochondrial mass per lymphocyte (Ly) is shown as green fluorescence intensity. The graph on the left shows the average ± standard deviation of triplicate cultures. The graph on the right shows the data plotted against untreated (green dotted line) and stressed cells (red dotted line).

Mitochondrial mass per monocyte (Mo) is shown as green fluorescence intensity. The graph on the left shows the average ± standard deviation of triplicate cultures. The graph on the right shows the data plotted against untreated (green dotted line) and stressed cells (red dotted line).

Mitochondrial mass per polymorphonuclear cell (PMN) is shown as green fluorescence intensity. The graph on the left shows the average ± standard deviation of triplicate cultures. The graph on the right shows the data plotted against untreated (green dotted line) and stressed cells (red dotted line).

Mitochondrial mass per lymphocyte (Ly) is shown as green fluorescence intensity. The graph on the left shows the average ± standard deviation of triplicate cultures. The graph on the right shows the data plotted against untreated (green dotted line) and inflamed cells (red dotted line).

Mitochondrial mass per monocyte (Mo) is shown as green fluorescence intensity. The graph on the left shows the average ± standard deviation of triplicate cultures. The graph on the right shows the data plotted against untreated (green dotted line) and inflamed cells (red dotted line).

Mitochondrial mass per polymorphonuclear cell (PMN) is shown as green fluorescence intensity. The graph on the left shows the average ± standard deviation of triplicate cultures. The graph on the right shows the data plotted against untreated (green dotted line) and inflamed cells (red dotted line).

This pilot project has generated results to support the effects of TEN at the cellular level, particularly under stress human culture conditions.

TEN supported mitochondrial metabolic activity under normal, oxidative stress and inflamed human culture conditions.

TEN supported healthy mitochondrial mass per cell under specific culture conditions: Mitochondrial mass per cell was mildly supported by TEN under normal conditions and strongly supported under inflamed conditions. The human cultures under oxidative stress showed some support by TEN for the more active cells (monocytes and PMN cells).

2.1 TESTS PERFORMED

This pilot project applied multiple tests to the increased understanding of TEN:

• Effect of product on intracellular levels of reduced glutathione under oxidative stress;
• Effect of product on mitochondrial membrane potential under normal and stressed culture conditions;
• Effect of product on free radical formation by human inflammatory cells under conditions of oxidative stress.

2.2 PRODUCT HANDLING

The energy formula TEN contains a blend of water-soluble and water-insoluble/poorly soluble compounds. Therefore, this pilot testing compared the cellular effects of three different methods of handling the product for introduction into cell culture.

4.1 INTRACELLULAR GLUTATHIONE LEVELS IN HUMAN CELLS

Glutathione (GSH) is an important antioxidant in plants, animals, fungi, and some bacteria. Glutathione is capable of preventing damage to important cellular components caused by damaging free radicals. Glutathione exists in two states: reduced (GSH) and oxidized (as the molecule glutathione disulfide, GSSG). In the reduced state, the thiol group of cysteine is able to donate a reducing equivalent to other molecules, such as reactive oxygen species, to neutralize the free radicals. We used an established protocoliv for flow cytometric evaluation of cellular protection by TEN, where product was applied to human peripheral blood mononuclear cells in the absence versus presence of free radical stress. ThiolTracker™ Violet is a bright intracellular thiol probe for reproducible detection of intracellular levels of reduced glutathione.v Since reduced glutathione represents the majority of intracellular free thiols in the cell; ThiolTracker™ Violet can be used to estimate the cellular level of reduced glutathione by flow cytometry.

Human peripheral blood mononuclear cells were treated with test products for 30 minutes, after which test products were removed. One set of cell cultures was handled using normal culture conditions (no stressors) while the other set of cell cultures was exposed to 1 mM H2O2 for 1 hour (oxidative stress culture conditions). Cells were then stained with ThiolTracker™ Violet and acquired by flow cytometry using an Attune® acoustic focusing cytometer. Data were analyzed for changes in fluorescence intensity, which reflects the reduced glutathione levels in the cells.

Cell types

During flow cytometric analysis, separate analysis was performed on the following two cell types in the blood sample:

• Lymphocytes (Ly): Fairly inactive in the types of cell cultures used here; Monocytes (Mono): Moderately active cells, highly reactive to stressors.

Figure 1. Flow cytometry analysis of cellular reduced glutathione levels. Electronic gates are set to define lymphocytes (Ly) and monocytes (Mono) based on their forward scatter (size) and side scatter (granularity) properties. The fluorescence intensity of the ThiolTracker™ Violet dye was measured for each cell type.

SYNOPSIS

Note: When reviewing this set of data, an increase in fluorescence intensity is a positive finding, and shows protection of reduced glutathione.

• Normal culture conditions
  • Treatment of cells with products led to some slight reduction in cellular reduced glutathione levels;
  • Monocytes were more sensitive to this effect, particularly at the 2 g/L dose of TEN Aqueous.
• Oxidative stress culture conditions
  • Pretreatment of cultures with TEN Aqueous and TEN Ethanol prior to exposure to 1 mM H2O2 led to protection from loss of reduced glutathione*;
    • For lymphocytes, protection was seen with the 3 highest doses of TEN Aqueous and the 0.4 and 0.08 g/L doses of TEN Ethanol*;
    • Monocytes showed a reduction in cellular reduced glutathione when exposed to the 2 g/L dose of TEN Aqueous but were protected from loss of reduced glutathione by the 0.4 g/L dose of both TEN Aqueous and TEN Ethanol.*
  • Immediately below is shown graphs for the protective results for 0.4 g/L TEN, specifically for the oxidative stress conditions.
  • On subsequent pages the complete dose curves are shown, followed by raw data graphs.

Figure 2. Effects of products on reduced glutathione levels in lymphocytes (left) and monocytes (right), shown as the percent change from control cultures. Oxidative stress culture conditions where cultures exposed to products prior to treatment with 1 mM H2O2 are shown as the percent change from cultures treated with H2O2 alone. The data shown here are for the 0.4 g/L dose of the aqueous and ethanol fractions of TEN, and also shows the corresponding ethanol control.

Figure 3. Effects of products on reduced glutathione levels in lymphocytes (percent change from control). Left: Normal cultures conditions where cellular glutathione levels in cultures exposed to products are shown as the percent change from untreated control cultures. Right: Oxidative stress culture conditions where cultures exposed to products prior to treatment with 1 mM H2O2 are shown as the percent change from cultures treated with H2O2 alone.

Figure 4. Effects of products on reduced glutathione levels in monocytes (percent change from control). Left: Normal cultures conditions where cellular glutathione levels in cultures exposed to products are shown as the percent change from untreated control cultures. Right: Oxidative stress culture conditions where cultures exposed to products prior to treatment with 1 mM H2O2 are shown as the percent change from cultures treated with H2O2 alone.

6.1.1 RAW DATA - NORMAL CULTURE CONDITIONS

6.1.1.1 LYMPHOCYTES

• An initial bar graph shows the most important data set for a particular bioassay, displayed as the percent change from control cultures;
• This is followed by a set of line graphs where all 4 doses of all 4 products are compared on single graphs, displayed as percent change from control data;
• Raw data is shown as bar graphs with any statistical significance indicated with asterisks. Each product is plotted separately with its appropriate control;
• For the reduced glutathione and mitochondrial membrane potential assays, several cell types were analyzed, and data is presented sequentially for individual cell populations (lymphocytes and monocytes for reduced glutathione; lymphocytes, monocytes and PMN cells for mitochondrial membrane potential).

Figure 5. Effects of products on reduced glutathione levels in lymphocytes cultured under normal culture conditions. Results are shown as the average ± standard deviation for each triplicate data set. Statistical significance when compared to untreated cultures is indicated by * p<0.05 and ** p<0.01.

6.1.1.2 MONOCYTES

Figure 6. Effects of products on reduced glutathione levels in monocytes cultured under normal culture conditions. Results are shown as the average ± standard deviation for each triplicate data set. Statistical significance when compared to untreated cultures is indicated by * p<0.05 and ** p<0.01.

6.1.2 RAW DATA - OXIDATIVE STRESS CULTURE CONDITIONS

6.1.2.1 LYMPHOCYTES

Figure 7. Effects of products on reduced glutathione levels in lymphocytes exposed to oxidative stress. Results are shown as the average ± standard deviation for each triplicate data set. Statistical significance when compared to cultures treated with H2O2 alone is indicated by * p<0.05 and ** p<0.01.

6.1.2.2 MONOCYTES

Figure 8. Effects of products on reduced glutathione levels in monocytes exposed to oxidative stress. Results are shown as the average ± standard deviation for each triplicate data set. Statistical significance when compared to cultures treated with H2O2 alone is indicated by * p<0.05 and ** p<0.01.

6.2 MITOCHONDRIAL METABOLIC MEMBRANE POTENTIAL WHEN EXPOSED TO VARIOUS STRESSORS

JC-1 is a cationic carbocyanine dye that accumulates in mitochondria. The dye exists as a monomer at low concentrations and yields green fluorescence, similar to fluorescein. At higher concentrations, the dye forms J-aggregates that exhibit a broad excitation spectrum and an emission maximum at ~590 nm. These characteristics make JC-1 a sensitive marker for mitochondrial membrane potential. Healthy mitochondria will fluoresce in the orange spectrum, whereas stressed mitochondrial will fluoresce in the green spectrum.

The testing was performed on freshly purified white blood cells from a healthy human blood donor. Serial dilutions of the test product (using all 3 handling methods in parallel) were added to cell cultures, where the cultures were tested for mitochondrial membrane potential after 2 hours incubation (to mimic timing proposed for clinical study). Untreated control samples were processed in parallel.

One set of cell cultures was handled using normal culture conditions (no stressors). Two additional sets of cell cultures were made in parallel, where:

• Oxidative stress was induced by adding H2O2;
• Inflammation was induced by adding LPS (a highly inflammatory bacterial endotoxin).

Flow cytometric analysis for the mean fluorescence intensity in both the green and red spectrum were evaluated and the ratio between red (healthy membrane potential) and green (compromised membrane potential) fluorescence was used as a measure of relative mitochondrial membrane potential.

Cell types

Different cell types have different mitochondrial activity levels, both under normal and under stressed conditions. During flow cytometric analysis, separate analysis was performed on the following three cell types in the blood sample:

• Lymphocytes (Ly): Fairly inactive in the types of cell cultures used here;
• Monocytes (Mo): Moderately active cells, highly reactive to stressors;
• Polymorphonuclear cells (PMN): Highly active cells, extremely reactive to stressors.

Figure 9. Flow cytometry analysis of mitochondrial membrane potential. Electronic gates are set to define lymphocytes (Ly), monocytes (Mono), and polymorphonuclear (PMN) cells based on their forward scatter (size) and side scatter (granularity) properties. Both green and red fluorescence intensity was measured for each cell type.

SYNOPSIS

Note: When reviewing this set of data, an increase in fluorescence intensity is a positive finding, and shows protection of the mitochondrial membrane potential.

• Normal culture conditions
  • Treatment of cells with products led to increases in mitochondrial membrane potential*;
    • Treatment with TEN Aqueous led to consistent increases in both lymphocyte and monocyte mitochondrial membrane potential.*
    • The effects of the TEN Ethanol is inconclusive because higher increases were seen in cultures exposed to ethanol alone (Ethanol control)*;
    • For PMN cells, the highest increases in mitochondrial membrane potential were seen in cultures treated with TEN Ethanol and TEN Blend.* The increases were seen with all 4 doses and they were higher than those seen with the Ethanol control.
• Oxidative stress culture conditions
  • Treatment of lymphocytes with all 4 doses of TEN Aqueous, TEN Ethanol and TEN Blend led to significant increases in mitochondrial membrane potential*;
  • Treatment of monocytes with TEN Blend and ethanol alone (Ethanol control) led to higher increases in mitochondrial membrane potential than treatment with either TEN Aqueous or TEN Ethanol*;
  • Treatment of PMN cells with TEN Aqueous led to decreased mitochondrial membrane potential at the two highest doses while the two lowest doses and all 4 doses of TEN Blend led to increases in mitochondrial membrane potential.*
• Inflammatory culture conditions
  • Treatment of lymphocytes with the two highest doses of TEN Aqueous led to significant increases in mitochondrial membrane potential*;
  • Treatment of monocytes with products did not lead to increased mitochondrial membrane potential;
  • Treatment of PMN cells with the two highest doses of TEN Aqueous and the highest dose of Ethanol control led to decreases in mitochondrial membrane potential while treatment with the two highest doses of TEN Blend and the three lowest doses of Ethanol control led to slight increases.*

Immediately below is shown graphs for the protective results for 0.4 g/L TEN, specifically for the oxidative stress conditions. On subsequent pages the complete dose curves are shown, followed by raw data graphs.

Figure 10. Effects of products on the mitochondrial membrane potential in lymphocytes under oxidative stress (left) and under inflammatory culture conditions (right), shown as the percent change from control cultures. The data shown here are for the 0.4 g/L dose of the aqueous and ethanol fractions of TEN, and also shows the corresponding ethanol control.

Figure 11. Effects of products on mitochondrial membrane potential in lymphocytes (percent change from control). Top left: Normal cultures conditions where cultures exposed to products are shown as the percent change from untreated control cultures. Top right: Oxidative stress culture conditions where cultures exposed to products are shown as the percent change from cultures treated with H2O2 alone. Bottom: Inflammatory culture conditions where cultures exposed to products are shown as the percent change from cultures treated with LPS alone.

Figure 12. Effects of products on mitochondrial membrane potential in monocytes (percent change from control). Top left: Normal cultures conditions where cultures exposed to products are shown as the percent change from untreated control cultures. Top right: Oxidative stress culture conditions where cultures exposed to products are shown as the percent change from cultures treated with H2O2 alone. Bottom: Inflammatory culture conditions where cultures exposed to products are shown as the percent change from cultures treated with LPS alone.

Figure 13. Effects of products on mitochondrial membrane potential in PMN cells (percent change from control). Top left: Normal cultures conditions where cultures exposed to products are shown as the percent change from untreated control cultures. Top right: Oxidative stress culture conditions where cultures exposed to products are shown as the percent change from cultures treated with H2O2 alone. Bottom: Inflammatory culture conditions where cultures exposed to products are shown as the percent change from cultures treated with LPS alone.

6.2.1 RAW DATA - NORMAL CULTURE CONDITIONS

6.2.1.1 LYMPHOCYTES

Figure 14. Effects of products on mitochondrial membrane potential in lymphocytes cultured under normal culture conditions. Results are shown as the average ± standard deviation for each triplicate data set. Statistical significance when compared to untreated cultures is indicated by * p<0.05 and ** p<0.01.

6.2.1.2 MONOCYTES

Figure 15. Effects of products on mitochondrial membrane potential in monocytes cultured under normal culture conditions. Results are shown as the average ± standard deviation for each triplicate data set. Statistical significance when compared to untreated cultures is indicated by * p<0.05 and ** p<0.01.

6.2.1.3 PMN CELLS

Figure 16. Effects of products on mitochondrial membrane potential in PMN cells cultured under normal culture conditions. Results are shown as the average ± standard deviation for each triplicate data set. Statistical significance when compared to untreated cultures is indicated by * p<0.05 and ** p<0.01.

6.2.2 RAW DATA - OXIDATIVE STRESS CULTURE CONDITIONS

6.2.2.1 LYMPHOCYTES

Figure 17. Effects of products on mitochondrial membrane potential in lymphocytes exposed to oxidative stress. Results are shown as the average ± standard deviation for each triplicate data set. Statistical significance when compared to untreated cultures is indicated by * p<0.05 and ** p<0.01.

6.2.2.2 MONOCYTES

Figure 18. Effects of products on mitochondrial membrane potential in monocytes exposed to oxidative stress. Results are shown as the average ± standard deviation for each triplicate data set. Statistical significance when compared to untreated cultures is indicated by * p<0.05 and ** p<0.01.

6.2.2.3 PMN CELLS

Figure 19. Effects of products on mitochondrial membrane potential in PMN cells exposed to oxidative stress. Results are shown as the average ± standard deviation for each triplicate data set. Statistical significance when compared to untreated cultures is indicated by * p<0.05 and ** p<0.01.

6.2.3 RAW DATA - INFLAMMATORY CULTURE CONDITIONS

6.2.3.1 LYMPHOCYTES

Figure 20. Effects of products on mitochondrial membrane potential in lymphocytes cultured under inflammatory culture conditions. Results are shown as the average ± standard deviation for each triplicate data set. Statistical significance when compared to untreated cultures is indicated by * p<0.05 and ** p<0.01.

6.2.3.2 MONOCYTES

Figure 21. Effects of products on mitochondrial membrane potential in monocytes cultured under inflammatory culture conditions. Results are shown as the average ± standard deviation for each triplicate data set. Statistical significance when compared to untreated cultures is indicated by * p<0.05 and ** p<0.01.

6.2.3.3 PMN CELLS

Figure 22. Effects of products on mitochondrial membrane potential in PMN cells cultured under inflammatory culture conditions. Results are shown as the average ± standard deviation for each triplicate data set. Statistical significance when compared to untreated cultures is indicated by * p<0.05 and ** p<0.01.

6.3 EFFECT ON FREE RADICAL FORMATION BY INFLAMMATORY CELLS

Many natural products with antioxidant capacity also reduce the ROS formation in inflammatory cells.vi vii However, other products may actually increase the ROS formation, despite antioxidant capacity, and this may indicate an interesting cooperation between support of antimicrobial defense mechanisms and antioxidant capacity.viii

Thus, natural products may affect PMN cell ROS formation by three different mechanisms (see figure below):

1. Neutralizing ROS by direct antioxidant affect;
2. Triggering an anti-inflammatory cellular signal, leading to reduced ROS formation;
3. Triggering an immune reaction, leading to enhanced ROS formation.

Human polymorphonuclear (PMN) cells are used for testing effects of a product on ROS formation. This cell type constitutes approximately 70% of the white blood cells in humans. PMN cells produce high amounts of ROS upon certain inflammatory stimuli.

Freshly purified human PMN cells were exposed to the test products. During the incubation with a test product, any antioxidant compounds able to cross the cell membrane can enter the interior of the PMN cells, and compounds that trigger a signaling event can do so. Then the cells were washed, loaded with the DCF-DA dye, which turns fluorescent upon exposure to ROS. Formation of ROS was triggered by addition of H2O2. The fluorescence intensity of the PMN cells was evaluated by flow cytometry. The low fluorescence intensity of untreated control cells served as a baseline and PMN cells treated with H2O2 alone served as a positive control.

If the fluorescence intensity of PMN cells exposed to an extract, and subsequently exposed to H2O2, is reduced compared to H2O2 alone, this indicates that a test product has anti- inflammatory effects.

In contrast, if the fluorescence intensity of PMN cells exposed to a test product is increased compared to H2O2 alone, this indicates that a test product has pro-inflammatory effects by enhancing this aspect of anti-microbial immune defense mechanisms.

The testing is a direct extension on the previously performed cellular antioxidant protection (CAP-e) assay and was performed in triplicate on cells from a healthy blood donor.

SYNOPSIS

• Treatment of PMN cells with the two highest doses of TEN Aqueous (0.4 and 2.0 g/L) led to a strong reduction in the formation of damaging reactive oxygen species by human inflammatory cells (decreases of 50% and 60% respectively)*;
• Treatment of PMN cells with the highest dose of TEN Ethanol (2.0 g/L) also reduced formation of damaging reactive oxygen species by human inflammatory cells (50% reduction)*.

Figure 25. Intracellular free radical formation in cultures where inflammation was induced by addition of H2O2. Untreated (UT) negative control cultures and H2O2-treated positive control cultures were analyzed in hexaplicate at the beginning and at the end of the flow cytometry acquisition. The thin blue line connects the two set of H2O2-treated controls and show only a very minimal change in cellular function over the time of data acquisition. The cell cultures treated with test products before inducing inflammation by addition of H2O2 were performed in triplicate. For all data sets, the average + standard deviation is shown. Statistical significance between a test sample and H2O2-treated control cell cultures is indicated by * p<0.05 and ** p<0.01.