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new clinical study results july 2009 evaluation of cacti-neatm s diuretic effect in women showing water retention monocentric randomized placebo controlled in parallel double-blind format abstract the aim of the clinical study was to evaluate cacti-neatm s diuretic effect in women showing water retention forty-nine women aged between 20 and 60 and with a bmi between 20 and 25 were included in this monocentric randomized placebo controlled study conducted in parallel double-blind format during 4 weeks half of the volunteers consumed 2g of cacti-neatm each morning and the other half consumed a placebo the entire group followed a well-balanced diet the placebo as well as cacti-neatm were given in the form of capsules the efficiency of the tested product was evaluated on day 7 14 21 and on day 28 of the study the evaluation was made by measuring body composition parameters body fat mass body lean mass water mass body weight anthropometrical parameters waist hip thigh ankle calf circumferences and urinary parameters the blood pressure and the heart rate were controlled at d0 and d28 after 1 month of treatment a positive effect of cacti-neatm was observed in the per protocol population regarding body composition and weight control these results have been confirmed and reinforced positive effects of cactineatm on silhouette parameters were observed in a specific sub-group which corresponds to more than half of the total population contrary to usual diuretic substance cacti-neatm did not show any adverse effect on blood pressure urinary parameter nor mineral balance these results confirm cacti-neatm s efficiency for weight-management applications thanks to its beneficial effect on weight loss body composition and silhouette improvement key words weight-management body fat mass cacti-neatm diuretic water mass silhouette v 1.1 confidentiebsl m k g © 2 0 0 9 l 1

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i context cacti-neatm is a prickly pear fruit water extract those fruits are traditionally consumed throughout the world they are particularly enjoyed for their gentle sweet flavor subtle floral aroma and their nutritional benefits to produce cacti-neatm bio serae carefully selected prickly pear fruits with the highest betalains pigment content and especially indicaxanthin which is a unique pigment highly bioavailable from this original raw material bio serae developed an exclusive process design to preserve the nutritional benefits of the fruit and now offers an original ingredient with diuretic and antioxidant properties the diuretic properties of prickly pear fruit have been already demonstrated in in vivo studies on rats perfumi tacconi 1996 galati et al 2002 an in vivo study conducted in 2007 by bio serae laboratories s.a.s confirmed the diuretic properties of cacti-neatm on rats at the dosage of 240mg/kg/j during 7 days a significant increase of the urinary volume/24h was indeed observed without any modification of the urinary parameters these results are being published in phytotherapy research from these encouraging preliminary results bio serae recently invested on confirming them in human to reinforce cacti-neatm s scientific background based on the potential diuretic and weigh-management properties of the ingredient bio serae decided to conduct a one-month clinical study in women with normal bmi and showing water retention ii subjects method the aim of this clinical study was to evaluate the diuretic effect of cacti-neatm the study was conducted in france in 2009 from january to july on 49 female with a normal bmi body mass index between 20 and 25 and with water retention troubles at the day of inclusion the volunteers of the two groups were characterized as follows parameters at d0 average age average bmi average body weight kg body composition parameters body fat mass kg body lean mass kg body water mass kg silhouette parameters waist circumference cm hip circumference cm tight circumference cm calf circumference cm ankle circumference cm blood pressure systolic blood pressure mmhg diastolic blood pressure mmhg placebo 39.6 ± 0.8 22.9 ± 0.3 60.4 ± 0.8 18.3 ± 0.5 42.1 ± 0.7 31.1 ± 0.4 76.8 ± 1.3 96.5 ± 0.9 55.6 ± 0.4 34.0 ± 0.4 20.8 ± 0.2 115 ± 2,7 74.0 ± 1.5 cacti-neatm 40.1 ± 2.1 23.2 ± 0.9 61.3 ± 0.9 18.9 ± 0.6 42.3 ± 0.7 31.2 ± 0.4 76.5 ± 1.3 96.7 ± 1.0 55.9 ± 0.6 35.0 ± 0.4 21.6 ± 0.3 111.7 ± 1.9 69.7 ± 1.7 1 1 clinical study conducted by biofortis and coordinated by etap sa v1.1 confidentiabsl m k g © 2 0 0 9 l 2

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definitions and reminder body composition brozek jk et al 1963 densitometric analysis of body composition revision of some quantitative assumptions ann ny acad sci;110 113-40 fat minerals proteins intracellular water extracellular water fat mass 100 90 80 70 60 50 40 30 20 10 0 25 10-30 6 16 lean mass 37 body weight fat mass lean mass body fat mass it corresponds to triglycerides stored in adipocytes whatever its anatomical localization may be this part of body composition is virtually deprived of water body lean mass it corresponds to the rest of the body mass and can be divided into 4 parts proteins minerals or calcic mass intracellular water water mass extracellular water to clarify we can say that the lean mass represents the sum of water bones and body organs excluding fat mass measure principle to evaluate body composition parameters we chose the bioelectrical impedance analysis on bodystat 1500 mdd the bio-electrical impedance measure is based on the ability of the hydrated tissues to conduct electrical energy the measure consists in controlling the flow of a multi-frequencies low intensity ac alternative current in the body by placing electrodes on the hand and the foot classical electrical formulas can be used to interpret the measures such as u z x i impedance is the resistance to the flow of the current in a given body as impedance z is proportional to the body liquid volume v we can combine the following equations to evaluate the liquid body volume z r x h s and v h x s then v r x h2 z r resistance of the medium h height of the body considered as a cylinder s section of the cylinder v body liquid volume v1.1 confidentiabsl m k g © 2 0 0 9 l 3

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body composition measurement the water mass the fat mass and the lean mass are calculated from the impedance using algorithms taking into consideration height body weight age and sex of subjects the measure of the impedance z which corresponds to the resistance to the flow of the current in a given body allows to extrapolate the water mass the higher is the fat mass rate the higher will be the resistance to electric current summary table of collected data collected data or measured parameters preparation of the patient patient codification randomization weighting and anthropometrical parameters measures clinical exam blood sampling urines collection logbook satisfaction questionnaire d-14 § § § § § d0 § § § § § d7 d14 d21 d28 § § § § § § § § § § § § § § general schedule of study during 4 weeks half of the volunteers daily consumed 2g of cacti-neatm in the form of capsule during breakfast while the other half of volunteers consumed in a similar way the placebo all along the study volunteers had to follow a well-balanced diet without any strict recommendation pre-inclusion d-14 clinical exam inclusion d0 clinical exam d7 d14 d21 d28 clinical exam cacti-neatm 2g/day 2 weeks 1 week 1 week 1 week 1 week blood sampling placebo 2g/day blood sampling on the day of the visit collection of the 24h urines weighting bio-impedance analysis bodystat 1500mdd anthropometrical measures v1.1 confidentiabsl m k g © 2 0 0 9 l 3

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statistical analysis of the results the efficiency analysis was realized on the per protocol population n=47 which only includes the volunteers who strictly respect the protocol of the study from the beginning to the end the evolutions of the parameters have been evaluated according to a variance analysis to allow the comparison of averages and study if the differences observed per group are statistically significant the intra-group evolutions have been evaluated by a « student » test the first level of risk is set up at 5 the test is thus significant when p 0.05 we then speak about significant trend when p is comprised between 0.05 and 0.10 iii results 1 per protocol population at the end of the study only volunteers who strictly respect the protocol of the study from the beginning to the end were included into the statistical analysis this per protocol population is composed of 47 volunteers only 2 volunteers did not respect the protocol and were withdrawn from the total population this population is divided as follows placebo group n=24 cacti-neatm group n=23 1.1 effect of cacti-neatm on body water mass kg per protocol population after the 4-weeks treatment a water mass reduction is observed in both groups cacti-neatm and placebo on the per protocol population cf table 1a table 1a effect of cacti-neatm on body water mass per protocol population average ± products placebo cacti-neatm d0 31.1 ± 0.4 31.2 ± 0.4 d7 30.9 ± 0.4 31.6 ± 0.4 d14 31.0 ± 0.4 31.5 ± 0.5 d21 31.0 ± 0.4 31.4 ± 0.5 d28 30.4 ± 0.3 31.0 ± 0.5 v1.1 confidentiabsl m k g © 2 0 0 9 l 4

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after the first week of treatment the water mass reduction observed in the cacti-neatm group becomes more important than the one observed in the placebo group cf graph 1a graph 1a effect of cacti-neatm on the water mass reduction between d7 d28 per protocol population water mass variation between d7 d28 0 -0,1 5 placebo cacti-neatm variation in kg -0,3 -0,45 -0,6 -0,75 -0,9 p=0.0151 p=0.0107 1.2 effect of cacti-neatm on the body weight kg per protocol population after the 4-weeks treatment a significant body weight reduction is observed in the cacti-neatm group essentially between d7 and d28 d14 and d28 and between d21 and d28 cf graph 1b in the placebo group no variation of body weight is observed during the study cf table 1b 1bbis table 1b effect of cacti-neatm on body weight per protocol population average ± products placebo cacti-neatm d0 60.4 ± 0.8 61.3 ± 0.9 d7 60.3 ± 0.8 61.4 ± 0.9 d14 60.3 ± 0.8 61.5 ± 1.0 d21 60.4 ± 0.8 61.3 ± 0.9 d28 60.3 ± 0.8 60.9 ± 1.0 table 1bbis body weight variation kg between d7 and d28 d14 and d28 and between d21 and d28 per protocol population average ± products placebo cacti-neatm d28-d7 -0.071 -0.43 p-value p=0.67 p=0.048 d28-d14 -0.025 -0.345 p-value p=0.85 p=0.02 d28-d21 -0.171 -0.326 p-value p=0.12 p=0.017 v1.1 confidentiabsl m k g © 2 0 0 9 l 5

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graph 1b effect of cacti-neatm on body weight reduction between d7 and d28 per protocol population body weight variation between d7 d28 placebo cacti-neatm 0,2 0 variation in kg -0,2 -0,4 -0,6 -0,8 ns p=0.0487 up to 3.3kg of body weight reduction observed in the cacti-neatm group 1.3 effect of cacti-neatm on the body composition per protocol population in the per protocol population the balance of the body composition is degraded in the placebo group this study shows that between d0 and d28 body fat mass of women in the placebo group significantly increases 3.4 whereas we do not observe any body fat mass variation on volunteers of the cacti-neatm group table 1c effect of cacti-neatm on body fat mass per protocol population average ± products placebo cacti-neatm d0 18.3 ± 0.5 18.9 ± 0.6 d7 18.5 ± 0.6 18.6 ± 0.7 d14 18.3 ± 0.6 18.9 ± 0.6 d21 18.5 ± 0.6 18.6 ± 0.6 d28 18.9 ± 0.6 18.9 ± 0.6 table 1cbis body fat mass variation kg between d7 and d28 d14 and d28 and between d21 and d28 per protocol population average ± products placebo cacti-neatm d28-d0 +0.629 -0.013 p-value p=0.023 p=0.957 d28-d7 +0.417 +0.296 p-value p=0.025 p=0.199 d28-d14 +0.579 +0.218 p-value p=0.004 p=0.331 d28-d21 +0.438 +0.168 p-value p=0.022 p=0.534 v1.1 confidentiabsl m k g © 2 0 0 9 l 6

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graph 1c effect of cacti-neatm on body fat mass per protocol population body fat mass variation between d0 d28 1 0,8 placebo cacti-neatm up to +3kg fat mass gain observed in the placebo group variation in kg 0,6 0,4 0,2 0 -0,2 -0,4 p=0.0239 ns note the lean mass consistently follows the water mass evolution in both groups cf table 1d and graphs 1d table 1d effect of cacti-neatm on body lean mass per protocol population average ± products placebo cacti-neatm d0 42.1 ± 0.7 42.3 ± 0.6 d7 41.8 ± 0.7 42.8 ± 0.7 d14 42.0 ± 0.7 42.6 ± 0.6 d21 42.0 ± 0.7 42.4 ± 0.6 d28 41.4 ± 0.6 42.0 ± 0.8 graphs 1d comparison of cacti-neatm effects on lean mass and water mass per protocol population lean mass variation between d0 and d28 44 43,5 43 31,5 32 water mass variation between d0 and d28 42,5 42 41,5 30,5 31 41 40,5 30 d0 d7 d14 d21 d28 d0 d7 d14 d21 d28 cacti-neatm seems to have a positive effect on body composition v1.1 confidentiabsl m k g © 2 0 0 9 l 7

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1.4 effect of cacti-neatm on bmi per protocol population during the study we observe that the bmi of volunteers in the cacti-neatm group tends to be reduced between d0 and d28 this variation becomes significant between d7 and d28 between d14 and d28 and between d21 and d28 cf table 1e at the same time no significant variation is observed on bmi in the placebo group between the different visits table 1e bmi variation between d7 and d28 d14 and d28 and between d21 and d28 per protocol population average ± products placebo cacti-neatm d28-d7 -0.017 -0.164 p-value p=0.729 p=0.046 d28-d14 0 -0.133 p-value p=1 p=0.021 d28-d21 -0.063 -0.127 p-value p=0.158 p=0.016 these noticeable variations were predictable since they are intimately linked to body weight variations 1.5 effect of cacti-neatm on anthropometrical parameters per protocol population in the per protocol population no significant variation has been observed in both groups 1.6 effect of cacti-neatm on blood pressure per protocol population blood pressure was measured at d0 and d28 blood pressure is evaluated by the mean of two measurements systolic blood pressure maximum blood pressure when heart contracts systole diastolic blood pressure maximum blood pressure when heart releases diastole concerning the systolic blood pressure no significant difference is observed between d0 and d28 whether in cacti-neatm group or placebo group table 1f effect of cacti-neatm on systolic blood pressure per protocol population average ± products placebo cacti-neatm d0 d28 115 ± 2.7 114.1 ± 2.1 111.7 ± 1.9 111.6 ± 1.9 concerning the diastolic blood pressure cacti-neatm does not induce any modification of the diastolic blood pressure between d0 and d28 whereas it significantly decreases in the placebo group table 1fbis effect of cacti-neatm on diastolic blood pressure per protocol population average ± products placebo cacti-neatm d0 74 ± 1.5 69.7 ± 1.7 d28 71.1 ± 1.5 69.1 ± 1.5 both results show that cacti-neatm does not induce any adverse effect on blood pressure unlike standard diuretic substances v1.1 confidentiabsl m k g © 2 0 0 9 l 8

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1.7 effect of cacti-neatm on urinary parameters per protocol population urinary rate of uric acid overall neither placebo nor cacti-nea tm induces significant changes of uric acid urinary rate during the study table 1g effect of cacti-neatm on urinary rate of uric acid per protocol population average ± products placebo cacti-neatm d0 1729.1 ± 104.8 1531.1 ± 125.7 d7 1485.9 ± 92.6 1401.7 ± 85.9 d14 1480.7 ± 106.5 1375.0 ± 94.4 d21 1594.2 ± 146.5 1507.9 ± 109.8 d28 1562.0 ± 138.5 1515.1 ± 99.3 potassium urinary rate overall neither placebo nor cacti-neatm induces significant changes in potassium urinary rate during the study table 1h effect of cacti-neatm on potassium urinary rate per protocol population average ± products placebo cacti-neatm d0 48.5 ± 3.7 46.6 ± 6.8 d7 39.7 ± 2.9 37.1 ± 2.5 d14 48.0 ± 4.6 38.5 ± 3.1 d21 45.6 ± 5.8 38.8 ± 3.3 d28 45.3 ± 5.2 46.3 ± 7.5 sodium urinary rate at the inclusion day d14 and d21 the sodium urinary rate is significantly different between the cacti-neatm group and the placebo group the sodium urinary rate in the cacti-neatm group is lower than the one in the placebo group besides those inter-group differences neither placebo nor cacti-neatm induces significant changes in the sodium urinary rate between d0 and d28 table 1i effect of cacti-neatm on the sodium urinary rate per protocol population average ± products placebo cacti-neatm d0 85.4 ± 7.4 68.5 ± 6.7 d7 66.0 ± 6.2 62.7 ± 4.1 d14 80.4 ± 6.0 61.9 ± 6.3 d21 74.8 ± 7.3 60.9 ± 3.1 d28 81.3 ± 7.1 68.9 ± 6.2 1.8 effect of cacti-neatm on heart rate per protocol population during the study cacti-neatm shows a positive effect by significantly reducing the heart rate between d0 and d28 5.3 p=0.209 jouven xp et al 2006 no significant effect is observed in the placebo group table 1j effect of cacti-neatm on heart rate per protocol population average ± products placebo cacti-neatm d0 74.8 ± 2.3 76.7 ± 2.3 d28 71.8 ± 2.3 72.6 ± 1.9 v1.1 confidentiabsl m k g © 2 0 0 9 l 9

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2 sub-group /fm at the end of the study the results analysis allows to highlight a specific sub-group particularly sensitive to cactineatm s diuretic and weight-management properties this sub-group is composed of women who tend to gain body fat mass between the pre-inclusion visit d-14 and the inclusion visit d0 this sub-group named sub-group/fm is composed of 27 volunteers which represents more than half of the per protocol population this sub-group is divided as follows placebo group n=12 cacti-neatm group n=15 2.1 effect of cacti-neatm on body water mass kg sub-group/fm similarly to the per protocol population a water mass reduction is observed in both groups cacti-neatm and placebo after 4 weeks of treatment cf table 2a table 2a effect of cacti-neatm on body water mass sub-group fm average ± products placebo cacti-neatm d0 30.6 ± 0.7 30.8 ± 0.5 d7 30.6 ± 0.6 31.3 ± 0.5 d14 30.5 ± 0.7 30.9 ± 0.5 d21 30.8 ± 0.7 31.0 ± 0.5 d28 30.2 ± 0.5 30.6 ± 0.5 in this sub-group/fm and after one week the water mass reduction observed in the cacti-neatm group is significant p=0.0094 while no significant effect is observed in the placebo group p=0,1621 cf graph 2a graph 2a water mass reduction effect of cacti-neatm between d7 and d28 sub-group/fm water mass reduction sub-group/fm between d7 d28 placebo 0 -0,2 cacti-neatm variation in kg -0,4 -0,6 -0,8 -1 ns p=0.0094 in the sub-group/fm cacti-neatm significantly reduces body water mass v1.1 confidentiabsl m k g © 2 0 0 9 l 10

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regarding the water mass variation and as already observed in the per protocol population the effects of cactineatm can be seen after a few days of treatment d7 these results suggest that the activity of cacti-neatm in the organism may imply a physiological adaptation which can be set up after one week 2.2 effect of cacti-neatm on body weight kg sub-group/fm in the sub-group/fm the average body weight loss observed in the cacti-neatm group is about 600g according to a significant trend between d0 and d28 cf graphs 2b this weight loss becomes significant between d7 and d28 whereas no significant effects is observed in the placebo group cf tables2b 2bbis table 2b effect of cacti-neatm on body weight sub-group/fm average ± products placebo cacti-neatm d0 59.8 ± 0.9 61.2 ± 1.4 d7 60.0 ± 0.9 61.3 ± 1.1 d14 59.9 ± 0.9 61.3 ± 1.1 d21 60.3 ± 0.9 61.0 ± 1.1 d28 60.1 ± 0.9 60.7 ± 1.1 table 2bbis body weight variation in kg between d0 and d28 and between d7 and d28 sub-group/fm average ± products placebo cacti-neatm d28-d0 +0.308 -0.573 p-value p=0.341 p=0.072 d28-d7 +0.100 -0.667 p-value p=0.69 p=0.033 graphs 2b effect of cacti-neatm on body weight reduction between d0 and d28 and between d7 and d28 subgroup/fm body weight variation sub-group/fm between d0 and d28 placebo 0,6 body weight variation sub-group/fm between d7 and d28 placebo 0,6 cacti-neatm cacti-neatm variation in kg variation in kg 0,2 -0,2 -0,6 -1 0,2 -0,2 -0,6 -1 ns p=0.0726 ns p=0.0334 the body weight loss observed with cacti-neatm in the per protocol population is confirmed and reinforced in the sub-group/fm beyond the quantitative aspect of this weight loss the effects of cacti-neatm are indeed more important in the sub-group/fm because of more significant results v1.1 confidentiabsl m k g © 2 0 0 9 l 11

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2.3 effect of cacti-neatm on body composition sub-group/fm as observed in the per protocol population the body fat mass increases in the placebo group 3.2 whereas no significant variation is observed in the cacti-neatm group in the placebo group the body fat increases according to a significant trend between d0 and d28 this body fat gain becomes significant between d7 and d28 in the placebo group table 2c effect of cacti-neatm on body fat mass sub-group/fm average ± products placebo cacti-neatm d0 18.8 ± 0.8 19.0 ± 0.7 d7 18.9 ± 0.8 18.5 ± 0.8 d14 18.9 ± 0.9 19.1 ± 0.7 d21 19.0 ± 0.9 18.3 ± 0.8 d28 19.5 ± 0.8 18.7 ± 0.8 table 2cbis body fat mass variation in kg between d0 and d28 and between d7 and d28 sub-group/fm average ± products placebo cacti-neatm d28-d0 +0.717 -0.307 p-value p=0,065 p=0,268 d28-d7 +0.608 +0.213 p-value p=0.027 p=0.463 graphs 2c anti-fat mass effect of cacti-neatm between d0 and d28 and between d7 and d28 sub-group/fm body fat mass variation sub-group/fm between d0 d28 placebo 1,2 0,9 body fat mass variation sub-group/fm between d7 d28 placebo 1,2 0,9 cacti-neatm cacti-neatm variation in kg 0,6 0,3 0 variation in kg p=0.0653 0,6 0,3 0 -0,3 -0,6 -0,9 -0,3 -0,6 -0,9 ns p=0.0273 ns at the same time and just like for the per protocol population the lean mass consistently follows the water mass evolution in both groups cf table 2d table 2d effect of cacti-neatm on body lean mass sub-group/fm average ± products placebo cacti-neatm d0 41.0 ± 0.9 42.2 ± 0.7 d7 41.1 ± 0.9 42.8 ± 0.6 d14 41.0 ± 0.9 42.2 ± 0.7 d21 41.3 ± 0.9 42.3 ± 0.7 d28 40.6 ± 0.7 42.0 ± 0.8 v1.1 confidentiabsl m k g © 2 0 0 9 l 12

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graphs 2d comparison of cacti-neatm s effect on body lean mass and body water mass sub-group/fm body lean mass variation kg between d0 and d28 sub-group/fm body water mass variation kg between d0 and d28 sub-group/fm 43,5 43 42,5 42 41,5 41 40,5 40 39,5 32 31,5 31 30,5 30 29,5 d0 d7 d14 d21 d28 d0 d7 d14 d21 d28 the positive effect of cacti-neatm on body composition balance is confirmed on this more sensitive subgroup/fm 2.4 effect of cacti-neatm on bmi sub-group/fm during this study and as already observed in the per protocol population the body mass index of volunteers included in the cacti-neatm group decreases between d0 and d28 according to a significant trend this evolution becomes significant between d7 and d28 between d14 and d28 and between d21 and d28 cf table 1e on the contrary in the placebo group average bmi significantly increases between d0 and d21 p=0.0394 and between d14 and d21 p=0;0073 table 2e effect of cacti-neatm on bmi kg/m² sub-group/fm average ± products placebo cacti-neatm d0 23.2 ± 0.4 22.8 ± 0.4 d7 23.2 ± 0.4 22.8 ± 0.4 d14 23.2 ± 0.4 22.9 ± 0.4 d21 23.4 ± 0.4 22.7 ± 0.4 d28 23.3 ± 0.4 22.6 ± 0.4 v1.1 confidentiabsl m k g © 2 0 0 9 l 13

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table 2ebis bmi variation between d7 and d28 d14 and d28 and between d21 and dj28 sub-group/fm products placebo cacti-neatm d28-d0 +0.133 -0.220 p-value 0.309 0.077 d28-d7 +0.050 -0.251 p-value p=0.614 p=0.033 d28-d14 +0.117 -0.159 p-value p=0.1411 p=0.039 d28-d21 -0.075 -0.135 p-value p=0.378 p=0.046 in the sub-group/fm the combined effects of cacti-neatm on body composition and weight-loss induce a significant reduction of the bmi on the contrary the bmi tends to increase in the placebo group 2.5 effect of cacti-neatm on anthropometrical parameters sub-group/fm contrary to the results observed in the per protocol population cacti-neatm induces in the sub-group/fm a positive effect on some anthropometrical parameters while no significant variation is observed in the placebo group of this sub-group between d0 and d28 the ankle circumference is significantly reduced in the cacti-neatm group the calf circumference is also reduced between d7 and d28 according to a significant trend in the cacti-neatm group and the waist circumference is significantly reduced between d14 and d28 cf graphs 2e,2f et 2g as for the other anthropometrical parameters monitored during the study hip thigh no significant variation is observed in both groups v1.1 confidentiabsl m k g © 2 0 0 9 l 14

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