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Commercial and Confidential Antioxidant and Immunomodulatory Activity: in vitro comparative study of three MultiplyPLUS preparations – Liquid, Freeze dried and Nutra dried Rochway 4th November 2008 A/Prof. Carol Morris Mr Paul Connellan Mr Dion Thompson Ms Kelly Winter Centre for Phytochemistry & Pharmacology Southern Cross University
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www.scu.edu.au/research/cp ABN 41995 651 524 TGA Licence No. 181048
BACKGROUND MultiplyPLUS (liquid preparation) has demonstrated significant immunomodulatory activity in vitro as previously reported (CPP Stage 1 & 2 Reports, dated 8/8/2008 & 7/10/08 respectively). It was agreed to now evaluate three preparations of MultiplyPLUS to determine if one of the preparations shows superior bioactivity. Samples of the Liquid, Freeze dried and Nutra dried MultiplyPLUS were tested for antioxidant and NK cytotoxic activity (NKCA). SUMMARY In vitro testing of the MultiplyPLUS preparations showed the following: 1. All three preparations (Liquid, Freeze dried & Nutra dried) showed significant
antioxidant and immunomodulatory activity.
2. Antioxidant capacity in decreasing order of potency (dry sample weight equivalent) –
3. Immunomodulatory capacity (as per NKCA) in decreasing order of potency (dry
sample weight equivalent) – Nutra dried, Freeze dried, Liquid.
MATERIALS AND METHODS Sample Preparation The samples received for testing were: CPR080280 MultiplyPLUS - fermented papaya & pomegranate probiotic liquid CPR080281 MultiplyPLUS - freeze dried CPR080282 MultiplyPLUS - Nutra dried The MultiplyPLUS preparations were tested over a range of concentrations for antioxidant activity (ORAC) and NKCA. The Freeze dried and Nutra dried samples were reconstituted in phosphate buffer (75mM pH7.4) for ORAC and phosphate buffered saline (PBS) for NKCA and cytotoxicity assays, while the Liquid preparation was diluted in these buffers for testing. A sample of the Liquid preparation was evaporated to dryness to determine the percentage dry weight (1.5% w/w). This enabled a direct comparison to be made between the liquid and dry preparations. Blood samples used for the NKCA assay were collected by venipuncture, from a healthy male donor, immediately prior to testing. Assays Cytotoxicity
The adenosine triphosphate (ATP) luminescent assay is used to determine any cytotoxic effects of extracts/compounds by using ATP quantitation as an indirect measure of cell proliferation. This assay was used to determine the levels at which the MultiplyPLUS preparations inhibit cell growth and as such provides important information on product concentrations to be used for further bioassay investigations. The cytotoxic effect of MultiplyPLUS was determined in vitro over a range of concentrations using the mouse lymphoblast cell line P388D1 over a 24 hour incubation.
P388D1 cells are particularly sensitive to cytotoxic substances, and are used as an initial indicator of cytotoxicity. Curcumin and chlorambucil are run as positive controls. ORAC The antioxidant capacity of MultiplyPLUS preparations was measured using the oxygen radical absorbance capacity (ORAC) assay. This assay has been widely accepted as a standard tool to measure the antioxidant activity in nutraceutical, pharmaceutical, food and natural products. This assay measures the ability of a sample to protect fluorescein from the peroxyl radicals induced by 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH) at 370C. Trolox, a water soluble analogue of vitamin E, is used as a reference. Results are expressed as μmol Trolox equivalents (TE) per gram of sample. Epicatechin is run as a positive control. Natural Killer Cell Cytotoxic Activity (NKCA) Natural killer (NK) cells are a subset of lymphocytes that are important in the body’s defence against viral infections and malignancy, participating in innate immunity and early defence. Impaired NK cell activity (NKCA) is associated with increased sensitivity to infection, and NKCA may have a role in the prevention of cancer and the control of cancer spread. Ficoll prepared suspensions of peripheral blood mononuclear cells (PBMCs) were used to test the in vitro effects of the MultiplyPLUS preparations on NK activity. PBMCs pre- incubated with MultiplyPLUS, were then incubated with K562 target cells. The percentage of dead target cells was then analysed using flow cytometry, with target cell controls being run to monitor spontaneous target cell death. NK cell cytotoxic activity was calculated as the difference in percentage dead target cells between the test and control samples for each extract. The effect of MultiplyPLUS on NKCA was determined in vitro over a range of concentrations by comparison to the PBS diluent control. A commercial mushroom proteoglycan concentrate (PSK) was run as a positive control at a final concentration of 10μg/mL.
RESULTS Cytotoxicity Overview The MultiplyPLUS preparations demonstrated IC50 values of at least 180μg/mL for the standard P388D1 cells tested. This is considered low level cytotoxicity. The chosen bioassay concentration upper limit of 25μg/mL for MultiplyPLUS showed negligible impact on cell viability (Fig.1). Concentration (ug/mL) Figure 1: Effect of MultiplyPLUS preparations at various concentrations on P388D1 cell growth
ORAC Overview The Liquid MultiplyPLUS showed a higher antioxidant capacity compared to the Freeze dried and Nutra dried preparations (Table1, Fig.2) on a dry weight equivalent basis. Table 1: Antioxidant capacity of MultiplyPLUS preparations. ORAC values expressed as μmol TE/g sample dry weight (n=12) MultiplyPLUS Preparation ORAC ± SEM Liquid /g 250.00 TE ol 200.00 Figure 2: Antioxidant activity for the various MultiplyPLUS preparations. Mean ORAC ± SEM (n=12)
NK Cell Cytotoxic Activity (NKCA) Overview 1. All three MultiplyPLUS preparations showed a significant stimulatory effect (p<0.05)
2. Mean maximal increases were Nutra dried (49.3% @ 5μg/mL), Freeze dried (43.5%
@ 5μg/mL) and Liquid preparation (38.9% @ 25 μg dry weight/mL).
Table 2: Relative NKCA activities of the MultiplyPLUS preparations compared to the PBS control. Values are mean ± SEM (n=2) Sample Final % Activity (μg/mL) Figure 3: Effect of MultiplyPLUS preparations at various concentrations on NKCA. Values are mean ± SEM (n=2) DISCUSSION All three MultiplyPLUS preparations showed significant antioxidant and immunomodulatory (NKCA) activity in vitro. In terms of antioxidant capacity, as determined by the hydrophilic ORAC assay, the Liquid preparation showed the highest activity at 387 μmol TE per gram (dry weight equivalent). Freeze dried appeared to have slightly more activity than the Nutra dried product with mean ORAC values of 223 and 182 respectively. However, in regard to immunomodulatory capacity, as determined by NKCA, it was Nutra dried which recorded the highest maximal activity of 49.3% stimulation at 5 μg/mL. Freeze dried and Liquid showed maxima of 43.5% (@ 5 μg/mL) and 38.9% (@ 25 μg/mL) respectively. Nutra dried displayed a marginally higher immunostimulatory potency compared to Freeze dried, and significantly higher than the Liquid preparation. In vitro testing is a useful, but not absolute, predictor of efficacy and mode of action in vivo. We have determined relative activities for the three MultiplyPLUS preparations in vitro, with all preparations showing significant but variable bioactivity. Confirmation of relative in vivo efficacy would need to be tested under clinical trial conditions.
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Commercial and Confidential 
Figure 3: Effect of MultiplyPLUS preparations at various concentrations on NKCA. Values are