NIS/WW Report 1 1 of 11. Report 1. Testing of Willard s Water for antioxidant capacity and cellular uptake of selected nutrients.

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1 NIS/WW Report 1 1 of 11 October 18, Report for: John Willard WW Industries, Inc. PO Box 4040 Rapid City, South Dakota Phone: (888) Fax: (605) Web: Report 1. Testing of Willard s Water for antioxidant capacity and cellular uptake of selected nutrients. Reviewed by: Joni L. Beaman CLA (ASCP) Analyst Gitte S. Jensen, PhD. Date:

2 NIS/WW Report 1 2 of 11 Report 1. Testing of Willard s Water for antioxidant capacity and cellular uptake of selected nutrients. 1. Purpose The goal for this project was to perform testing of Willard s Water (WW) for antioxidant properties, as well as to examine whether WW helps cellular uptake of selected nutrients. This initial phase of work serves as a foundation for further exploration of the effect of WW on the mechanism of action at the cellular level. 2. Background Willard s Water (WW) has been documented to enhance the growth of plants and animals. There are also several pieces of information that suggest that uptake and/or retention of nutrients and drugs may be altered by WW. However, the exact underlying mechanisms of action remain obscure. WW is typically used in a highly dilute manner, ranging from 50 to 4,000 microliters per liter (250-20,000 fold). Cells, such as human blood and tissue cells, are sensitive to osmotic imbalances and ph. Cells are normally cultured at a 0.9% saline concentration (9 grams per liter) at a neutral ph ( ). The concentrated WW is highly alkaline. However, once diluted at least one thousand-fold, ph is only minimally affected, and ph approaches the neutral range. 3. Performed Work We performed testing of WW in terms of cellular uptake of nutrients and other compounds using our Cellular Antioxidant Protection (CAP-e) bioassay. The cell type used in this assay (erythrocytes) was specifically selected to help address the question of cellular uptake. Since the CAP-e test examined nutrient uptake as a function of antioxidant protection we also needed to know if WW possessed inherent antioxidant properties at the doses tested in the CAP-e assay. Therefore, we also tested the antioxidant capacity of WW using a simple Folin- Ciocalteu test, and a wide dose range of WW. This testing helped answer several fundamental questions about WW. This work, now that it is completed, sets the foundation for further discussion and planning of future work. Such future

3 NIS/WW Report 1 3 of 11 work could include evaluation of many other aspects of cellular functioning, energy production, activation in response to external signals, senescence, and cell death. Choice of nutrients for testing of whether Willard s Water facilitates cellular uptake The decision on which nutrients to include in the testing of nutrient uptake was not trivial. Smaller molecules of certain chemical structure are relatively easily assimilated by cells, whereas larger, complex molecules have no access. We did not foresee that WW would help assimilation of large molecules if the cells did not have specific uptake mechanisms for the specific molecule or compound. In contrast, we found it likely that WW might facilitate an increased uptake, and also possibly help retain the nutrient in the cells, for smaller, less complex molecules. We also included testing of lipophilic compounds which are normally carried in the blood stream on lipid particles, to see whether WW facilitates cellular uptake of lipid nutrients. We also tested a few natural products with complex chemical profiles. Based on these rationales we performed a comparison of the nutrients listed below in the testing: - Gallic Acid - Vitamin B2 (Riboflavin) - Green tea - Turmeric - Tocotrienol (a highly lipophilic Vitamin E compound, normally carried on lipid particles in the blood stream) - Trolox (a water-soluble vitamin E related compound) Gallic Acid is an antioxidant we normally use as a standard control compound in our CAP-e bioassay, hence we know the expected uptake well. Vitamin B2 (Riboflavin) is an antioxidant that has previously been tested in conjunction with WW.

4 NIS/WW Report 1 4 of 11 Green tea contains a complex chemical profile that includes a number of compounds that are able to be assimilated by live cells in the CAP-e test. Turmeric contains some water-soluble compounds but also a large proportion of lipophilic compounds. Without a carrier, turmeric only provides minimal protection in the CAP-e assay, and we suggest this is an interesting complex product to include in this initial testing. Trolox and tocotrienol reflect two vitamin E-related compounds with similar chemical structure but very different solubility properties. Product handling For the Folin-Ciocalteu antioxidant capacity assay, we used a broad dose range with fine increments. We prepared twelve 2-fold serial dilutions, and tested WW starting at 10mL/L, and going down to ml/l. For the CAP-e bioassay, WW was diluted 250-fold with phosphate-buffered saline (PBS, ph 7.4), and the ph was documented. Two further serial 10-fold dilutions were prepared in PBS. This allowed us to compare nutrient uptake into cells in the presence of WW at 250, 2,500, and 25,000-fold dilutions. The ph was documented to be 7.0 in all three dilutions of WW. For Tocotrienol, due to solubility issues in aqueous solutions, the initial reconstitution of tocotrienol was done in 95% ethanol followed by a 1:4 dilution in fetal calf serum (FCS). Subsequent dilutions were performed as described for the other 5 products. 4. Methodological details 4a. Antioxidant capacity using the Folin-Ciocalteu assay In order to look at inherent antioxidant capacity, we tested WW 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 Folin-Ciocalteu s phenol reagent to serial dilutions of extract, thoroughly mixing, and incubating for 5 minutes. Sodium carbonate is 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.

5 NIS/WW Report 1 5 of 11 4b. Cell-based Antioxidant Protection assay (CAP-e) The rationale behind the method that we use is important: It allows assessment of cellular antioxidant uptake and protection in a method that is comparable to the ORAC test, but only allows measurement of antioxidants that are able to cross the lipid bilayer cell membrane. As a model cell type, we use the red blood cell (RBC). This is an inert cell type, in contrast to other cell types such as polymorphonuclear (PMN) cells where pro-inflammatory compounds may induce the reactive oxidative burst, or anti-inflammatory compounds may perform cellular signaling and change the behavior of the PMN cell, at doses many times below levels of detection for antioxidants. We developed the CAP-e assay particularly to be able to assess antioxidant uptake from complex natural products in a cell-based system, without interference from other cellular processes involving free radicals. During our regular CAP-e bioassay, human RBC are washed repeatedly in PBS, and then exposed to the nutritional test products diluted in PBS. 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. If 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.

6 GAE (mg/l) NIS/WW Report 1 6 of 11 For the testing of whether WW helped facilitate nutrient uptake we performed parallel testing of the uptake of each separate nutrient, where the cell handling was either performed in PBS or in WW. Thus, testing of each nutrient was performed in 4 parallel methods: - PBS - WW diluted 250-fold - WW diluted 2,500-fold - WW diluted 25,000-fold Each nutrient was tested at 6 different doses. The comparison of the dose response curve for each nutrient, for each of the 4 handling methods described above, helped evaluate whether and to what extent WW altered the cellular uptake/retention of each nutrient. This testing did not directly distinguish between uptake and retention. If needed, future testing can be fine-tuned to help dissociate those two aspects. 5. Results 5a. Folin-Ciocalteu assay 2.1 Total Phenolics Assay WW ml/l Figure 1. Results of the Folin-Ciocalteu assay. A serial dilution series (2-fold) of WW was tested and results are presented above in gallic acid equivalents (GAE). These results show that WW has little to no antioxidants over the wide dose range tested.

7 % inhibition of oxidative damage NIS/WW Report 1 7 of 11 5b. Cellular Antioxidant Protection (CAP-e) Assay Below and on the following 3 pages are overlay graphs showing the cellular uptake and antioxidant protection for the 6 nutritional products using each of the 4 handling methods in the Cellular Antioxidant Protection Assay (CAP-e Assay). PBS was used as a baseline to compare each of the 3 dilutions of WW. 100 Gallic Acid/PBS Gallic Acid Gallic Acid/WW 250 Gallic Acid/WW2500 Gallic Acid/WW25, Figure 2. Results of the CAP-e assay comparing the cellular uptake of gallic acid reconstituted in PBS to gallic acid reconstituted in 3 dilutions of WW (1:250, 1:2,500 and 1:25,000). At the 4 highest doses of gallic acid, the cellular uptake of gallic acid worked as well or better in the presence of WW at 1:2,500 and 1:25,000, when compared to PBS.

8 % inhibition of oxidative damage NIS/WW Report 1 8 of B2 Riboflavin Riboflavin/PBS B2 Riboflavin/WW 250 Riboflavin/ww 2500 Riboflavin/WW 25, Figure 3. Results of the CAP-e assay comparing the cellular uptake of vitamin B2 (riboflavin) reconstituted in PBS to vitamin B2 reconstituted in 3 dilutions of WW (1:250, 1:2,500 and 1:25,000). Vitamin B2 is an antioxidant, but did not perform well in the CAP-e assay, since it was not absorbed by red blood cells in any clear dose dependent fashion. At the dose range from g/l of vitamin B2, the two higher dilutions of WW (1:2,500 and 1:25,000) mediated a mild cellular uptake of vitamin B2, in contrast to PBS and WW 1:250. At the highest dose, it actually seemed to induce oxidative stress in the cells. The highest dilution of WW (WW 1:250) seemed to moderate this oxidative stress.

9 NIS/WW Report 1 9 of 11 Green Tea/H₂O 100 % inhibition of oxidative damage Green Tea/H₂O/PBS 80 Green Tea/H₂0/WW 250 Green Tea/H₂O/WW Green Tea/H₂O/WW 25, Figure 4. Results of the CAP-e assay comparing the cellular uptake of green tea reconstituted in PBS to green tea reconstituted in 3 dilutions of WW (1:250, 1:2,500 and 1:25,000). For the middle two doses of green tea, the WW 2,500-fold and 25,000-fold dilutions performed as well or better than the PBS. At higher doses where there was plenty of green tea compounds, and at low doses, we did not demonstrate an advantage mediated by WW. 70 Turmeric % inhibition of oxidative damage 50 Turmeric PBS Turmeric/WW Turmeric/WW 2500 Turmeric/WW 25, Figure 5. Results of the CAP-e assay comparing the cellular uptake of turmeric reconstituted in PBS to turmeric reconstituted in 3 dilutions of WW (1:250, 1:2,500 and 1:25,000). The cellular uptake of turmeric was higher in WW 2,500-fold and 25,000-fold dilutions than the PBS for all except the lowest dose of turmeric.

10 % inhibition of oxidative damage % inhibition of oxidative damage NIS/WW Report 1 10 of Tocotrienol Tocotri /EtOH/FCS/PBS Tocotri /EtOH/FCS/WW 250 Tocotri /EtOH/FCS/WW 2500 Tocotri /EtOH/FCS/WW 25, Figure 6. Results of the CAP-e assay comparing the cellular uptake of tocotrienol reconstituted in PBS to tocotrienol reconstituted in 3 dilutions of WW (1:250, 1:2,500 and 1:25,000). The cellular absorption of tocotrienols worked better in WW 2,500-fold and 25,000-fold dilutions than the PBS in all except the lowest two doses of tocotrienol. * Red data points indicate cell lysing. Cell lysing can happen at higher doses of test products that for various reasons are not well tolerated by the live cells. Lysing can be caused by unfavorable ph, salt concentration and many other factors. 70 Trolox Trolox/PBS Trolox/WW 250 Trolox/WW 2500 Trolox/WW 25, Figure 7. Results of the CAP-e assay comparing the cellular uptake of Trolox reconstituted in PBS to Trolox reconstituted in 3 dilutions of WW (1:250, 1:2,500 and 1:25,000). For the three highest doses of Trolox, the cellular uptake was slightly higher when performed in the presence of WW 25,000 when compared to PBS.

11 NIS/WW Report 1 11 of Conclusions WW did not demonstrate inherent antioxidant activity when tested in the total phenolics assay. o This result is important for the interpretation of the CAP-e results (below). For five of the six nutritional products (i.e. all except vitamin B2) that we tested in the Cellular Antioxidant Protection (CAP-e) assay, the 2,500- and 25,000-fold dilutions of WW enhanced antioxidant uptake and cellular protection at one or more doses of product. Interestingly, this was not the case for the 250-fold dilution of WW. The effect of increasing uptake/retention was demonstrated across a wide range of different types of compounds. o The effect on simple compounds such as gallic acid and water-soluble antioxidants in green tea were minimally affected by WW, and any effect seen was more pronounced at lower dilutions of WW. o The effect on cellular uptake of compounds from products with limited solubility in water was greater in the presence of highly diluted WW. Most pronounced was the increased cellular uptake of antioxidants from Turmeric and tocotrienols, which are both products with limited or no solubility in water. Because WW did not demonstrate antioxidant capacity itself, this data suggests a role for WW in increasing the uptake/retention of nutrients into cells.

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