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T (61 2) 6622 3211 f (61 2) 6622 3459 military road lismore nsw 2480 po box 157 lismore nsw 2480 australia

Commercial and Confidential

Antioxidant and Immunomodulatory
Activity: in vitro comparative study of three

MultiplyPLUS preparations – Liquid, Freeze
dried and Nutra dried
4th November 2008

A/Prof. Carol Morris
Mr Paul Connellan
Mr Dion Thompson
Ms Kelly Winter
Centre for Phytochemistry & Pharmacology
Southern Cross University

T (61 2) 6622 3211 F (61 2) 6622 3459 Military Road Lismore NSW 2480 PO Box 157 Lismore NSW 2480 Australia 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

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.
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.
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.
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

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

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
/g 250.00
Figure 2: Antioxidant activity for the various MultiplyPLUS preparations.
Mean ORAC ± SEM (n=12)

NK Cell Cytotoxic Activity (NKCA)
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
Figure 3: Effect of MultiplyPLUS preparations at various concentrations on NKCA. Values are
mean ± SEM (n=2)
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
. We have determined relative activities for the three MultiplyPLUS preparations in
, 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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